Pettitt, S.J.
Shao, N.
Zatreanu, D.
Frankum, J.
Bajrami, I.
Brough, R.
Krastev, D.B.
Roumeliotis, T.I.
Choudhary, J.S.
Lorenz, S.
Rust, A.
de Bono, J.S.
Yap, T.A.
Tutt, A.N.
Lord, C.J.
(2023). A HUWE1 defect causes PARP inhibitor resistance by modulating the BRCA1-∆11q splice variant. ,
Vol.42
(36),
pp. 2701-2709.
show abstract
Although PARP inhibitors (PARPi) now form part of the standard-of-care for the treatment of homologous recombination defective cancers, de novo and acquired resistance limits their overall effectiveness. Previously, overexpression of the BRCA1-∆11q splice variant has been shown to cause PARPi resistance. How cancer cells achieve increased BRCA1-∆11q expression has remained unclear. Using isogenic cells with different BRCA1 mutations, we show that reduction in HUWE1 leads to increased levels of BRCA1-∆11q and PARPi resistance. This effect is specific to cells able to express BRCA1-∆11q (e.g. BRCA1 exon 11 mutant cells) and is not seen in BRCA1 mutants that cannot express BRCA1-∆11q, nor in BRCA2 mutant cells. As well as increasing levels of BRCA1-∆11q protein in exon 11 mutant cells, HUWE1 silencing also restores RAD51 nuclear foci and platinum salt resistance. HUWE1 catalytic domain mutations were also seen in a case of PARPi resistant, BRCA1 exon 11 mutant, high grade serous ovarian cancer. These results suggest how elevated levels of BRCA1-∆11q and PARPi resistance can be achieved, identify HUWE1 as a candidate biomarker of PARPi resistance for assessment in future clinical trials and illustrate how some PARPi resistance mechanisms may only operate in patients with particular BRCA1 mutations..
Bland, P.
Saville, H.
Wai, P.T.
Curnow, L.
Muirhead, G.
Nieminuszczy, J.
Ravindran, N.
John, M.B.
Hedayat, S.
Barker, H.E.
Wright, J.
Yu, L.
Mavrommati, I.
Read, A.
Peck, B.
Allen, M.
Gazinska, P.
Pemberton, H.N.
Gulati, A.
Nash, S.
Noor, F.
Guppy, N.
Roxanis, I.
Pratt, G.
Oldreive, C.
Stankovic, T.
Barlow, S.
Kalirai, H.
Coupland, S.E.
Broderick, R.
Alsafadi, S.
Houy, A.
Stern, M.-.
Pettit, S.
Choudhary, J.S.
Haider, S.
Niedzwiedz, W.
Lord, C.J.
Natrajan, R.
(2023). SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response. ,
Vol.55
(8),
pp. 1311-1323.
show abstract
SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population..
Wan, M.S.
Muhammad, R.
Koliopoulos, M.G.
Roumeliotis, T.I.
Choudhary, J.S.
Alfieri, C.
(2023). Mechanism of assembly, activation and lysine selection by the SIN3B histone deacetylase complex. ,
Vol.14
(1),
p. 2556.
show abstract
Lysine acetylation in histone tails is a key post-translational modification that controls transcription activation. Histone deacetylase complexes remove histone acetylation, thereby repressing transcription and regulating the transcriptional output of each gene. Although these complexes are drug targets and crucial regulators of organismal physiology, their structure and mechanisms of action are largely unclear. Here, we present the structure of a complete human SIN3B histone deacetylase holo-complex with and without a substrate mimic. Remarkably, SIN3B encircles the deacetylase and contacts its allosteric basic patch thereby stimulating catalysis. A SIN3B loop inserts into the catalytic tunnel, rearranges to accommodate the acetyl-lysine moiety, and stabilises the substrate for specific deacetylation, which is guided by a substrate receptor subunit. Our findings provide a model of specificity for a main transcriptional regulator conserved from yeast to human and a resource of protein-protein interactions for future drug designs..
Yadav, D.
Verma, S.
Choudhary, J.
Kaur, H.
Tiwari, P.
Singh, S.
Kushwaha, P.
Dubey, P.K.
Chandra, P.
Singh, S.P.
(2023). Nanohybrid Comprising Gold Nanoparticles – MoS2 Nanosheets for Electrochemical Sensing of Folic Acid in Serum Samples. Electroanalysis,
Vol.35
(4).
show abstract
AbstractFolic acid (FA) deficiency is associated with several clinical conditions such as megaloblastic anemia, neuropsychiatric, and pregnancy‐related syndromes, this makes FA an important metabolite to be monitored. We have fabricated an electrochemical biosensor based on gold nanoparticles decorated molybdenum disulfide nanosheets (AuNPs−MoS2NSs) nanocomposite as a transducer matrix for specific and rapid electrochemical detection of FA. Differential pulse voltammetry (DPV) studies displayed a rapid analytical response of the fabricated AuNPs−MoS2NSs/GCE sensor probe towards FA in a wide concentration range of 0.001–100 μM with a very low detection limit of 0.72±0.03 nM. The selectivity of the fabricated sensor probe has been examined in the presence of interferents such as dopamine, uric acid, ascorbic acid, glucose, and urea. The clinical potential of the fabricated biosensor was established by monitoring FA in human serum samples. The developed AuNPs−MoS2NSs/GCE sensor probe showed high reproducibility and stability, indicating its promise for FA detection in clinical settings..
Frankish, A.
Carbonell-Sala, S.
Diekhans, M.
Jungreis, I.
Loveland, J.E.
Mudge, J.M.
Sisu, C.
Wright, J.C.
Arnan, C.
Barnes, I.
Banerjee, A.
Bennett, R.
Berry, A.
Bignell, A.
Boix, C.
Calvet, F.
Cerdán-Vélez, D.
Cunningham, F.
Davidson, C.
Donaldson, S.
Dursun, C.
Fatima, R.
Giorgetti, S.
Giron, C.G.
Gonzalez, J.M.
Hardy, M.
Harrison, P.W.
Hourlier, T.
Hollis, Z.
Hunt, T.
James, B.
Jiang, Y.
Johnson, R.
Kay, M.
Lagarde, J.
Martin, F.J.
Gómez, L.M.
Nair, S.
Ni, P.
Pozo, F.
Ramalingam, V.
Ruffier, M.
Schmitt, B.M.
Schreiber, J.M.
Steed, E.
Suner, M.-.
Sumathipala, D.
Sycheva, I.
Uszczynska-Ratajczak, B.
Wass, E.
Yang, Y.T.
Yates, A.
Zafrulla, Z.
Choudhary, J.S.
Gerstein, M.
Guigo, R.
Hubbard, T.J.
Kellis, M.
Kundaje, A.
Paten, B.
Tress, M.L.
Flicek, P.
(2023). GENCODE: reference annotation for the human and mouse genomes in 2023. Nucleic acids research,
Vol.51
(D1),
pp. D942-D949.
show abstract
Abstract
GENCODE produces high quality gene and transcript annotation for the human and mouse genomes. All GENCODE annotation is supported by experimental data and serves as a reference for genome biology and clinical genomics. The GENCODE consortium generates targeted experimental data, develops bioinformatic tools and carries out analyses that, along with externally produced data and methods, support the identification and annotation of transcript structures and the determination of their function. Here, we present an update on the annotation of human and mouse genes, including developments in the tools, data, analyses and major collaborations which underpin this progress. For example, we report the creation of a set of non-canonical ORFs identified in GENCODE transcripts, the LRGASP collaboration to assess the use of long transcriptomic data to build transcript models, the progress in collaborations with RefSeq and UniProt to increase convergence in the annotation of human and mouse protein-coding genes, the propagation of GENCODE across the human pan-genome and the development of new tools to support annotation of regulatory features by GENCODE. Our annotation is accessible via Ensembl, the UCSC Genome Browser and https://www.gencodegenes.org..
Shcherbakova, L.
Pardo, M.
Roumeliotis, T.
Choudhary, J.
(2023). Identifying and characterising Thrap3, Bclaf1 and Erh interactions using cross-linking mass spectrometry. ,
Vol.6,
pp. 260-260.
show abstract
Background: Cross-linking mass spectrometry (XL-MS) is a powerful technology capable of yielding structural insights across the complex cellular protein interaction network. However, up to date most of the studies utilising XL-MS to characterise individual protein complexes’ topology have been carried out on over-expressed or recombinant proteins, which might not accurately represent native cellular conditions. Methods: We performed XL-MS using MS-cleavable crosslinker disuccinimidyl sulfoxide (DSSO) after immunoprecipitation of endogenous BRG/Brahma-associated factors (BAF) complex and co-purifying proteins. Data are available via ProteomeXchange with identifier PXD027611. Results: Although we did not detect the expected enrichment of crosslinks within the BAF complex, we identified numerous crosslinks between three co-purifying proteins, namely Thrap3, Bclaf1 and Erh. Thrap3 and Bclaf1 are mostly disordered proteins for which no 3D structure is available. The XL data allowed us to map interaction surfaces on these proteins, which overlap with the non-disordered portions of both proteins. The identified XLs are in agreement with homology-modelled structures suggesting that the interaction surfaces are globular. Conclusions: Our data shows that MS-cleavable crosslinker DSSO can be used to characterise in detail the topology and interaction surfaces of endogenous protein complexes without the need for overexpression. We demonstrate that Bclaf1, Erh and Thrap3 interact closely with each other, suggesting they might form a novel complex, hereby referred to as TEB complex. This data can be exploited for modelling protein-protein docking to characterise the three-dimensional structure of the complex. Endogenous XL-MS might be challenging due to crosslinker accessibility, protein complex abundance or isolation efficiency, and require further optimisation for some complexes like the BAF complex to detect a substantial number of crosslinks..
Russell, A.J.
Sanderson, T.
Bushell, E.
Talman, A.M.
Anar, B.
Girling, G.
Hunziker, M.
Kent, R.S.
Martin, J.S.
Metcalf, T.
Montandon, R.
Pandey, V.
Pardo, M.
Roberts, A.B.
Sayers, C.
Schwach, F.
Choudhary, J.S.
Rayner, J.C.
Voet, T.
Modrzynska, K.K.
Waters, A.P.
Lawniczak, M.K.
Billker, O.
(2023). Regulators of male and female sexual development are critical for the transmission of a malaria parasite. ,
Vol.31
(2),
pp. 305-+.
show abstract
Malaria transmission to mosquitoes requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei, the transcription factor AP2-G is required and sufficient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify genes essential for the formation of either male or female sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of ten mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in the determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain as well as the protein interactors of a female-determining zinc-finger protein indicate that germ-granule-like ribonucleoprotein complexes complement transcriptional processes in the regulation of both male and female development of a malaria parasite..
Lau, R.
Yu, L.
Roumeliotis, T.I.
Stewart, A.
Pickard, L.
Riisanes, R.
Gurel, B.
de Bono, J.S.
Choudhary, J.S.
Banerji, U.
(2023). Unbiased differential proteomic profiling between cancer-associated fibroblasts and cancer cell lines. Journal of proteomics,
Vol.288,
pp. 104973-104973.
Newey, A.
Yu, L.
Barber, L.J.
Choudhary, J.S.
Bassani-Sternberg, M.
Gerlinger, M.
(2023). Multifactorial Remodeling of the Cancer Immunopeptidome by IFNγ. Cancer research communications,
Vol.3
(11),
pp. 2345-2357.
show abstract
Abstract
IFNγ alters the immunopeptidome presented on HLA class I (HLA-I), and its activity on cancer cells is known to be important for effective immunotherapy responses. We performed proteomic analyses of untreated and IFNγ-treated colorectal cancer patient-derived organoids and combined this with transcriptomic and HLA-I immunopeptidomics data to dissect mechanisms that lead to remodeling of the immunopeptidome through IFNγ. IFNγ-induced changes in the abundance of source proteins, switching from the constitutive to the immunoproteasome, and differential upregulation of different HLA alleles explained some, but not all, observed peptide abundance changes. By selecting for peptides which increased or decreased the most in abundance, but originated from proteins with limited abundance changes, we discovered that the amino acid composition of presented peptides also influences whether a peptide is upregulated or downregulated on HLA-I through IFNγ. The presence of proline within the peptide core was most strongly associated with peptide downregulation. This was validated in an independent dataset. Proline substitution in relevant core positions did not influence the predicted HLA-I binding affinity or stability, indicating that proline effects on peptide processing may be most relevant. Understanding the multiple factors that influence the abundance of peptides presented on HLA-I in the absence or presence of IFNγ is important to identify the best targets for antigen-specific cancer immunotherapies such as vaccines or T-cell receptor engineered therapeutics.
Significance:
IFNγ remodels the HLA-I–presented immunopeptidome. We showed that peptide-specific factors influence whether a peptide is upregulated or downregulated and identified a preferential loss or downregulation of those with proline near the peptide center. This will help selecting immunotherapy target antigens which are consistently presented by cancer cells.
.
Jones, I.
Dent, L.
Higo, T.
Roumeliotis, T.
Arias Garcia, M.
Shree, H.
Choudhary, J.
Pedersen, M.
Bakal, C.
(2023). Characterization of proteome-size scaling by integrative omics reveals mechanisms of proliferation control in cancer. ,
Vol.9
(4),
p. eadd0636.
show abstract
Almost all living cells maintain size uniformity through successive divisions. Proteins that over and underscale with size can act as rheostats, which regulate cell cycle progression. Using a multiomic strategy, we leveraged the heterogeneity of melanoma cell lines to identify peptides, transcripts, and phosphorylation events that differentially scale with cell size. Subscaling proteins are enriched in regulators of the DNA damage response and cell cycle progression, whereas super-scaling proteins included regulators of the cytoskeleton, extracellular matrix, and inflammatory response. Mathematical modeling suggested that decoupling growth and proliferative signaling may facilitate cell cycle entry over senescence in large cells when mitogenic signaling is decreased. Regression analysis reveals that up-regulation of TP53 or CDKN1A/p21CIP1 is characteristic of proliferative cancer cells with senescent-like sizes/proteomes. This study provides one of the first demonstrations of size-scaling phenomena in cancer and how morphology influences the chemistry of the cell..
Burns, J.
Wilding, C.P.
Krasny, L.
Zhu, X.
Chadha, M.
Tam, Y.B.
Ps, H.
Mahalingam, A.H.
Lee, A.T.
Arthur, A.
Guljar, N.
Perkins, E.
Pankova, V.
Jenks, A.
Djabatey, V.
Szecsei, C.
McCarthy, F.
Ragulan, C.
Milighetti, M.
Roumeliotis, T.I.
Crosier, S.
Finetti, M.
Choudhary, J.S.
Judson, I.
Fisher, C.
Schuster, E.F.
Sadanandam, A.
Chen, T.W.
Williamson, D.
Thway, K.
Jones, R.L.
Cheang, M.C.
Huang, P.H.
(2023). The proteomic landscape of soft tissue sarcomas. ,
Vol.14
(1),
p. 3834.
show abstract
Soft tissue sarcomas (STS) are rare and diverse mesenchymal cancers with limited treatment options. Here we undertake comprehensive proteomic profiling of tumour specimens from 321 STS patients representing 11 histological subtypes. Within leiomyosarcomas, we identify three proteomic subtypes with distinct myogenesis and immune features, anatomical site distribution and survival outcomes. Characterisation of undifferentiated pleomorphic sarcomas and dedifferentiated liposarcomas with low infiltrating CD3 + T-lymphocyte levels nominates the complement cascade as a candidate immunotherapeutic target. Comparative analysis of proteomic and transcriptomic profiles highlights the proteomic-specific features for optimal risk stratification in angiosarcomas. Finally, we define functional signatures termed Sarcoma Proteomic Modules which transcend histological subtype classification and show that a vesicle transport protein signature is an independent prognostic factor for distant metastasis. Our study highlights the utility of proteomics for identifying molecular subgroups with implications for risk stratification and therapy selection and provides a rich resource for future sarcoma research..
Rozowsky, J.
Gao, J.
Borsari, B.
Yang, Y.T.
Galeev, T.
Gürsoy, G.
Epstein, C.B.
Xiong, K.
Xu, J.
Li, T.
Liu, J.
Yu, K.
Berthel, A.
Chen, Z.
Navarro, F.
Sun, M.S.
Wright, J.
Chang, J.
Cameron, C.J.
Shoresh, N.
Gaskell, E.
Drenkow, J.
Adrian, J.
Aganezov, S.
Aguet, F.
Balderrama-Gutierrez, G.
Banskota, S.
Corona, G.B.
Chee, S.
Chhetri, S.B.
Cortez Martins, G.C.
Danyko, C.
Davis, C.A.
Farid, D.
Farrell, N.P.
Gabdank, I.
Gofin, Y.
Gorkin, D.U.
Gu, M.
Hecht, V.
Hitz, B.C.
Issner, R.
Jiang, Y.
Kirsche, M.
Kong, X.
Lam, B.R.
Li, S.
Li, B.
Li, X.
Lin, K.Z.
Luo, R.
Mackiewicz, M.
Meng, R.
Moore, J.E.
Mudge, J.
Nelson, N.
Nusbaum, C.
Popov, I.
Pratt, H.E.
Qiu, Y.
Ramakrishnan, S.
Raymond, J.
Salichos, L.
Scavelli, A.
Schreiber, J.M.
Sedlazeck, F.J.
See, L.H.
Sherman, R.M.
Shi, X.
Shi, M.
Sloan, C.A.
Strattan, J.S.
Tan, Z.
Tanaka, F.Y.
Vlasova, A.
Wang, J.
Werner, J.
Williams, B.
Xu, M.
Yan, C.
Yu, L.
Zaleski, C.
Zhang, J.
Ardlie, K.
Cherry, J.M.
Mendenhall, E.M.
Noble, W.S.
Weng, Z.
Levine, M.E.
Dobin, A.
Wold, B.
Mortazavi, A.
Ren, B.
Gillis, J.
Myers, R.M.
Snyder, M.P.
Choudhary, J.
Milosavljevic, A.
Schatz, M.C.
Bernstein, B.E.
Guigó, R.
Gingeras, T.R.
Gerstein, M.
(2023). The EN-TEx resource of multi-tissue personal epigenomes & variant-impact models. ,
Vol.186
(7),
pp. 1493-1511.e40.
show abstract
Understanding how genetic variants impact molecular phenotypes is a key goal of functional genomics, currently hindered by reliance on a single haploid reference genome. Here, we present the EN-TEx resource of 1,635 open-access datasets from four donors (∼30 tissues × ∼15 assays). The datasets are mapped to matched, diploid genomes with long-read phasing and structural variants, instantiating a catalog of >1 million allele-specific loci. These loci exhibit coordinated activity along haplotypes and are less conserved than corresponding, non-allele-specific ones. Surprisingly, a deep-learning transformer model can predict the allele-specific activity based only on local nucleotide-sequence context, highlighting the importance of transcription-factor-binding motifs particularly sensitive to variants. Furthermore, combining EN-TEx with existing genome annotations reveals strong associations between allele-specific and GWAS loci. It also enables models for transferring known eQTLs to difficult-to-profile tissues (e.g., from skin to heart). Overall, EN-TEx provides rich data and generalizable models for more accurate personal functional genomics..
Zhong, Q.
Chatterjee, S.
Choudhary, J.S.
Frankel, G.
(2022). EPEC-induced activation of the Ca2+ transporter TRPV2 leads to pyroptotic cell death. Molecular microbiology,
Vol.117
(2),
pp. 480-492.
show abstract
The enteropathogenic Escherichia coli (EPEC) type III secretion system effector Tir, which mediates intimate bacterial attachment to epithelial cells, also triggers Ca2+ influx followed by LPS entry and caspase-4-dependent pyroptosis, which could be antagonized by the effector NleF. Here we reveal the mechanism by which EPEC induces Ca2+ influx. We show that in the intestinal epithelial cell line SNU-C5, Tir activates the mechano/osmosensitive cation channel TRPV2 which triggers extracellular Ca2+ influx. Tir-induced Ca2+ influx could be blocked by siRNA silencing of TRPV2, pre-treatment with the TRPV2 inhibitor SET2 or by growing cells in low osmolality medium. Pharmacological activation of TRPV2 in the absence of Tir failed to initiate caspase-4-dependent cell death, confirming the necessity of Tir. Consistent with the model implicating activation on translocation of TRPV2 from the ER to plasma membrane, inhibition of protein trafficking by either brefeldin A or the effector NleA prevented TRPV2 activation and cell death. While infection with EPECΔnleA triggered pyroptotic cell death, this could be prevented by NleF. Taken together this study shows that while integration of Tir into the plasma membrane activates TRPV2, EPEC uses NleA to inhibit TRPV2 trafficking and NleF to inhibit caspase-4 and pyroptosis..
Krastev, D.B.
Li, S.
Sun, Y.
Wicks, A.J.
Hoslett, G.
Weekes, D.
Badder, L.M.
Knight, E.G.
Marlow, R.
Pardo, M.C.
Yu, L.
Talele, T.T.
Bartek, J.
Choudhary, J.S.
Pommier, Y.
Pettitt, S.J.
Tutt, A.N.
Ramadan, K.
Lord, C.J.
(2022). The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin. Nature cell biology,
.
show abstract
Poly (ADP-ribose) polymerase (PARP) inhibitors elicit antitumour activity in homologous recombination-defective cancers by trapping PARP1 in a chromatin-bound state. How cells process trapped PARP1 remains unclear. Using wild-type and a trapping-deficient PARP1 mutant combined with rapid immunoprecipitation mass spectrometry of endogenous proteins and Apex2 proximity labelling, we delineated mass spectrometry-based interactomes of trapped and non-trapped PARP1. These analyses identified an interaction between trapped PARP1 and the ubiquitin-regulated p97 ATPase/segregase. We found that following trapping, PARP1 is SUMOylated by PIAS4 and subsequently ubiquitylated by the SUMO-targeted E3 ubiquitin ligase RNF4, events that promote recruitment of p97 and removal of trapped PARP1 from chromatin. Small-molecule p97-complex inhibitors, including a metabolite of the clinically used drug disulfiram (CuET), prolonged PARP1 trapping and enhanced PARP inhibitor-induced cytotoxicity in homologous recombination-defective tumour cells and patient-derived tumour organoids. Together, these results suggest that p97 ATPase plays a key role in the processing of trapped PARP1 and the response of tumour cells to PARP inhibitors..
Schiavoni, F.
Zuazua-Villar, P.
Roumeliotis, T.I.
Benstead-Hume, G.
Pardo, M.
Pearl, F.M.
Choudhary, J.S.
Downs, J.A.
(2022). Aneuploidy tolerance caused by BRG1 loss allows chromosome gains and recovery of fitness. ,
Vol.13
(1),
p. 1731.
show abstract
Aneuploidy results in decreased cellular fitness in many species and model systems. However, aneuploidy is commonly found in cancer cells and often correlates with aggressive growth, suggesting that the impact of aneuploidy on cellular fitness is context dependent. The BRG1 (SMARCA4) subunit of the SWI/SNF chromatin remodelling complex is frequently lost in cancer. Here, we use a chromosomally stable cell line to test the effect of BRG1 loss on the evolution of aneuploidy. BRG1 deletion leads to an initial loss of fitness in this cell line that improves over time. Notably, we find increased tolerance to aneuploidy immediately upon loss of BRG1, and the fitness recovery over time correlates with chromosome gain. These data show that BRG1 loss creates an environment where karyotype changes can be explored without a fitness penalty. At least in some genetic backgrounds, therefore, BRG1 loss can affect the progression of tumourigenesis through tolerance of aneuploidy..
Llorca-Cardenosa, M.J.
Aronson, L.I.
Krastev, D.B.
Nieminuszczy, J.
Alexander, J.
Song, F.
Dylewska, M.
Broderick, R.
Brough, R.
Zimmermann, A.
Zenke, F.T.
Gurel, B.
Riisnaes, R.
Ferreira, A.
Roumeliotis, T.
Choudhary, J.
Pettitt, S.J.
de Bono, J.
Cervantes, A.
Haider, S.
Niedzwiedz, W.
Lord, C.J.
Chong, I.Y.
(2022). SMG8/SMG9 Heterodimer Loss Modulates SMG1 Kinase to Drive ATR Inhibitor Resistance. ,
Vol.82
(21),
pp. 3962-3973.
show abstract
UNLABELLED: Gastric cancer represents the third leading cause of global cancer mortality and an area of unmet clinical need. Drugs that target the DNA damage response, including ATR inhibitors (ATRi), have been proposed as novel targeted agents in gastric cancer. Here, we sought to evaluate the efficacy of ATRi in preclinical models of gastric cancer and to understand how ATRi resistance might emerge as a means to identify predictors of ATRi response. A positive selection genome-wide CRISPR-Cas9 screen identified candidate regulators of ATRi resistance in gastric cancer. Loss-of-function mutations in either SMG8 or SMG9 caused ATRi resistance by an SMG1-mediated mechanism. Although ATRi still impaired ATR/CHK1 signaling in SMG8/9-defective cells, other characteristic responses to ATRi exposure were not seen, such as changes in ATM/CHK2, γH2AX, phospho-RPA, or 53BP1 status or changes in the proportions of cells in S- or G2-M-phases of the cell cycle. Transcription/replication conflicts (TRC) elicited by ATRi exposure are a likely cause of ATRi sensitivity, and SMG8/9-defective cells exhibited a reduced level of ATRi-induced TRCs, which could contribute to ATRi resistance. These observations suggest ATRi elicits antitumor efficacy in gastric cancer but that drug resistance could emerge via alterations in the SMG8/9/1 pathway. SIGNIFICANCE: These findings reveal how cancer cells acquire resistance to ATRi and identify pathways that could be targeted to enhance the overall effectiveness of these inhibitors..
Sialana, F.J.
Roumeliotis, T.I.
Bouguenina, H.
Chan Wah Hak, L.
Wang, H.
Caldwell, J.
Collins, I.
Chopra, R.
Choudhary, J.S.
(2022). SimPLIT: Simplified Sample Preparation for Large-Scale Isobaric Tagging Proteomics. ,
Vol.21
(8),
pp. 1842-1856.
show abstract
Large scale proteomic profiling of cell lines can reveal molecular signatures attributed to variable genotypes or induced perturbations, enabling proteogenomic associations and elucidation of pharmacological mechanisms of action. Although isobaric labeling has increased the throughput of proteomic analysis, the commonly used sample preparation workflows often require time-consuming steps and costly consumables, limiting their suitability for large scale studies. Here, we present a simplified and cost-effective one-pot reaction workflow in a 96-well plate format (SimPLIT) that minimizes processing steps and demonstrates improved reproducibility compared to alternative approaches. The workflow is based on a sodium deoxycholate lysis buffer and a single detergent cleanup step after peptide labeling, followed by quick off-line fractionation and MS2 analysis. We showcase the applicability of the workflow in a panel of colorectal cancer cell lines and by performing target discovery for a set of molecular glue degraders in different cell lines, in a 96-sample assay. Using this workflow, we report frequently dysregulated proteins in colorectal cancer cells and uncover cell-dependent protein degradation profiles of seven cereblon E3 ligase modulators (CRL4CRBN). Overall, SimPLIT is a robust method that can be easily implemented in any proteomics laboratory for medium-to-large scale TMT-based studies for deep profiling of cell lines..
Sanchez-Garrido, J.
Ruano-Gallego, D.
Choudhary, J.S.
Frankel, G.
(2022). The type III secretion system effector network hypothesis. Trends in microbiology,
Vol.30
(6),
pp. 524-533.
Beykou, M.
Arias-Garcia, M.
Roumeliotis, T.I.
Choudhary, J.S.
Moser, N.
Georgiou, P.
Bakal, C.
(2022). Proteomic characterisation of triple negative breast cancer cells following CDK4/6 inhibition. ,
Vol.9
(1),
p. 395.
show abstract
When used in combination with hormone treatment, Palbociclib prolongs progression-free survival of patients with hormone receptor positive breast cancer. Mechanistically, Palbociclib inhibits CDK4/6 activity but the basis for differing sensitivity of cancer to Palbociclib is poorly understood. A common observation in a subset of Triple Negative Breast Cancers (TNBCs) is that prolonged CDK4/6 inhibition can engage a senescence-like state where cells exit the cell cycle, whilst, remaining metabolically active. To better understand the senescence-like cell state which arises after Palbociclib treatment we used mass spectrometry to quantify the proteome, phosphoproteome, and secretome of Palbociclib-treated MDA-MB-231 TNBC cells. We observed altered levels of cell cycle regulators, immune response, and key senescence markers upon Palbociclib treatment. These datasets provide a starting point for the derivation of biomarkers which could inform the future use CDK4/6 inhibitors in TNBC subtypes and guide the development of potential combination therapies..
Mullineaux-Sanders, C.
Kozik, Z.
Sanchez-Garrido, J.
Hopkins, E.G.
Choudhary, J.S.
Frankel, G.
(2022). Citrobacter rodentium Infection Induces Persistent Molecular Changes and Interferon Gamma-Dependent Major Histocompatibility Complex Class II Expression in the Colonic Epithelium. Mbio,
Vol.13
(1).
show abstract
Mucosal surfaces respond to infection by mounting an array of metabolic, inflammatory, and tissue repair responses. While these have been well studied during acute infection, less is known about tissue recovery after pathogen clearance..
Kalesh, K.
Wei, W.
Mantilla, B.S.
Roumeliotis, T.I.
Choudhary, J.
Denny, P.W.
(2022). Transcriptome-Wide Identification of Coding and Noncoding RNA-Binding Proteins Defines the Comprehensive RNA Interactome of Leishmania mexicana. Microbiology spectrum,
Vol.10
(1).
show abstract
Advances in proteomics and mass spectrometry have revealed the mRNA-binding proteins in many eukaryotic organisms, including the protozoan parasites
Leishmania
spp., the causative agents of leishmaniasis, a major infectious disease in over 90 tropical and subtropical countries. However, in addition to mRNAs, which constitute only 2 to 5% of the total transcripts, many types of non-coding RNAs participate in crucial biological processes.
.
Randzavola, L.O.
Mortimer, P.M.
Garside, E.
Dufficy, E.R.
Schejtman, A.
Roumelioti, G.
Yu, L.
Pardo, M.
Spirohn, K.
Tolley, C.
Brandt, C.
Harcourt, K.
Nichols, E.
Nahorski, M.
Woods, G.
Williamson, J.C.
Suresh, S.
Sowerby, J.M.
Matsumoto, M.
Santos, C.X.
Kiar, C.S.
Mukhopadhyay, S.
Rae, W.M.
Dougan, G.J.
Grainger, J.
Lehner, P.J.
Calderwood, M.A.
Choudhary, J.
Clare, S.
Speak, A.
Santilli, G.
Bateman, A.
Smith, K.G.
Magnani, F.
Thomas, D.C.
(2022). EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling. ,
Vol.11.
show abstract
EROS (essential for reactive oxygen species) protein is indispensable for expression of gp91phox, the catalytic core of the phagocyte NADPH oxidase. EROS deficiency in humans is a novel cause of the severe immunodeficiency, chronic granulomatous disease, but its mechanism of action was unknown until now. We elucidate the role of EROS, showing it acts at the earliest stages of gp91phox maturation. It binds the immature 58 kDa gp91phox directly, preventing gp91phox degradation and allowing glycosylation via the oligosaccharyltransferase machinery and the incorporation of the heme prosthetic groups essential for catalysis. EROS also regulates the purine receptors P2X7 and P2X1 through direct interactions, and P2X7 is almost absent in EROS-deficient mouse and human primary cells. Accordingly, lack of murine EROS results in markedly abnormal P2X7 signalling, inflammasome activation, and T cell responses. The loss of both ROS and P2X7 signalling leads to resistance to influenza infection in mice. Our work identifies EROS as a highly selective chaperone for key proteins in innate and adaptive immunity and a rheostat for immunity to infection. It has profound implications for our understanding of immune physiology, ROS dysregulation, and possibly gene therapy..
Feng, H.
Lane, K.A.
Roumeliotis, T.I.
Jeggo, P.A.
Somaiah, N.
Choudhary, J.S.
Downs, J.A.
(2022). PBAF loss leads to DNA damage-induced inflammatory signaling through defective G2/M checkpoint maintenance. ,
.
show abstract
The PBRM1 subunit of the PBAF (SWI/SNF) chromatin remodeling complex is mutated in ∼40% of clear cell renal cancers. PBRM1 loss has been implicated in responses to immunotherapy in renal cancer, but the mechanism is unclear. DNA damage-induced inflammatory signaling is an important factor determining immunotherapy response. This response is kept in check by the G2/M checkpoint, which prevents progression through mitosis with unrepaired damage. We found that in the absence of PBRM1, p53-dependent p21 up-regulation is delayed after DNA damage, leading to defective transcriptional repression by the DREAM complex and premature entry into mitosis. Consequently, DNA damage-induced inflammatory signaling pathways are activated by cytosolic DNA. Notably, p53 is infrequently mutated in renal cancer, so PBRM1 mutational status is critical to G2/M checkpoint maintenance. Moreover, we found that the ability of PBRM1 deficiency to predict response to immunotherapy correlates with expression of the cytosolic DNA-sensing pathway in clinical samples. These findings have implications for therapeutic responses in renal cancer..
Koliopoulos, M.G.
Muhammad, R.
Roumeliotis, T.I.
Beuron, F.
Choudhary, J.S.
Alfieri, C.
(2022). Structure of a nucleosome-bound MuvB transcription factor complex reveals DNA remodelling. ,
Vol.13
(1),
p. 5075.
show abstract
Genes encoding the core cell cycle machinery are transcriptionally regulated by the MuvB family of protein complexes in a cell cycle-specific manner. Complexes of MuvB with the transcription factors B-MYB and FOXM1 activate mitotic genes during cell proliferation. The mechanisms of transcriptional regulation by these complexes are still poorly characterised. Here, we combine biochemical analysis and in vitro reconstitution, with structural analysis by cryo-electron microscopy and cross-linking mass spectrometry, to functionally examine these complexes. We find that the MuvB:B-MYB complex binds and remodels nucleosomes, thereby exposing nucleosomal DNA. This remodelling activity is supported by B-MYB which directly binds the remodelled DNA. Given the remodelling activity on the nucleosome, we propose that the MuvB:B-MYB complex functions as a pioneer transcription factor complex. In this work, we rationalise prior biochemical and cellular studies and provide a molecular framework of interactions on a protein complex that is key for cell cycle regulation..
Oberoi, J.
Guiu, X.A.
Outwin, E.A.
Schellenberger, P.
Roumeliotis, T.I.
Choudhary, J.S.
Pearl, L.H.
(2022). HSP90-CDC37-PP5 forms a structural platform for kinase dephosphorylation. ,
Vol.13
(1),
p. 7343.
show abstract
Activation of client protein kinases by the HSP90 molecular chaperone system is affected by phosphorylation at multiple sites on HSP90, the kinase-specific co-chaperone CDC37, and the kinase client itself. Removal of regulatory phosphorylation from client kinases and their release from the HSP90-CDC37 system depends on the Ser/Thr phosphatase PP5, which associates with HSP90 via its N-terminal TPR domain. Here, we present the cryoEM structure of the oncogenic protein kinase client BRAFV600E bound to HSP90-CDC37, showing how the V600E mutation favours BRAF association with HSP90-CDC37. Structures of HSP90-CDC37-BRAFV600E complexes with PP5 in autoinhibited and activated conformations, together with proteomic analysis of its phosphatase activity on BRAFV600E and CRAF, reveal how PP5 is activated by recruitment to HSP90 complexes. PP5 comprehensively dephosphorylates client proteins, removing interaction sites for regulatory partners such as 14-3-3 proteins and thus performing a 'factory reset' of the kinase prior to release..
Chabanon, R.M.
Morel, D.
Eychenne, T.
Colmet-Daage, L.
Bajrami, I.
Dorvault, N.
Garrido, M.
Meisenberg, C.
Lamb, A.
Ngo, C.
Hopkins, S.R.
Roumeliotis, T.I.
Jouny, S.
Hénon, C.
Kawai-Kawachi, A.
Astier, C.
Konde, A.
Del Nery, E.
Massard, C.
Pettitt, S.J.
Margueron, R.
Choudhary, J.S.
Almouzni, G.
Soria, J.-.
Deutsch, E.
Downs, J.A.
Lord, C.J.
Postel-Vinay, S.
(2021). PBRM1 Deficiency Confers Synthetic Lethality to DNA Repair Inhibitors in Cancer. ,
Vol.81
(11),
pp. 2888-2902.
show abstract
Inactivation of Polybromo 1 (PBRM1), a specific subunit of the PBAF chromatin remodeling complex, occurs frequently in cancer, including 40% of clear cell renal cell carcinomas (ccRCC). To identify novel therapeutic approaches to targeting PBRM1-defective cancers, we used a series of orthogonal functional genomic screens that identified PARP and ATR inhibitors as being synthetic lethal with PBRM1 deficiency. The PBRM1/PARP inhibitor synthetic lethality was recapitulated using several clinical PARP inhibitors in a series of in vitro model systems and in vivo in a xenograft model of ccRCC. In the absence of exogenous DNA damage, PBRM1-defective cells exhibited elevated levels of replication stress, micronuclei, and R-loops. PARP inhibitor exposure exacerbated these phenotypes. Quantitative mass spectrometry revealed that multiple R-loop processing factors were downregulated in PBRM1-defective tumor cells. Exogenous expression of the R-loop resolution enzyme RNase H1 reversed the sensitivity of PBRM1-deficient cells to PARP inhibitors, suggesting that excessive levels of R-loops could be a cause of this synthetic lethality. PARP and ATR inhibitors also induced cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) innate immune signaling in PBRM1-defective tumor cells. Overall, these findings provide the preclinical basis for using PARP inhibitors in PBRM1-defective cancers. SIGNIFICANCE: This study demonstrates that PARP and ATR inhibitors are synthetic lethal with the loss of PBRM1, a PBAF-specific subunit, thus providing the rationale for assessing these inhibitors in patients with PBRM1-defective cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/11/2888/F1.large.jpg..
Shcherbakova, L.
Pardo, M.
Roumeliotis, T.
Choudhary, J.
(2021). Identifying and characterising Thrap3, Bclaf1 and Erh interactions using cross-linking mass spectrometry. ,
Vol.6,
p. 260.
show abstract
Background: Cross-linking mass spectrometry (XL-MS) is a powerful technology capable of yielding structural insights across the complex cellular protein interaction network. However, up to date most of the studies utilising XL-MS to characterise individual protein complexes' topology have been carried out on over-expressed or recombinant proteins, which might not accurately represent native cellular conditions. Methods: We performed XL-MS using MS-cleavable crosslinker disuccinimidyl sulfoxide (DSSO) after immunoprecipitation of endogenous BRG/Brahma-associated factors (BAF) complex and co-purifying proteins. Data are available via ProteomeXchange with identifier PXD027611. Results: Although we did not detect the expected enrichment of crosslinks within the BAF complex, we identified numerous crosslinks between three co-purifying proteins, namely Thrap3, Bclaf1 and Erh. Thrap3 and Bclaf1 are mostly disordered proteins for which no 3D structure is available. The XL data allowed us to map interaction surfaces on these proteins, which overlap with the non-disordered portions of both proteins. The identified XLs are in agreement with homology-modelled structures suggesting that the interaction surfaces are globular. Conclusions: Our data shows that MS-cleavable crosslinker DSSO can be used to characterise in detail the topology and interaction surfaces of endogenous protein complexes without the need for overexpression. We demonstrate that Bclaf1, Erh and Thrap3 interact closely with each other, suggesting they might form a novel complex, hereby referred to as TEB complex. This data can be exploited for modelling protein-protein docking to characterise the three-dimensional structure of the complex. Endogenous XL-MS might be challenging due to crosslinker accessibility, protein complex abundance or isolation efficiency, and require further optimisation for some complexes like the BAF complex to detect a substantial number of crosslinks..
Garcia, L.R.
Tenev, T.
Newman, R.
Haich, R.O.
Liccardi, G.
John, S.W.
Annibaldi, A.
Yu, L.
Pardo, M.
Young, S.N.
Fitzgibbon, C.
Fernando, W.
Guppy, N.
Kim, H.
Liang, L.-.
Lucet, I.S.
Kueh, A.
Roxanis, I.
Gazinska, P.
Sims, M.
Smyth, T.
Ward, G.
Bertin, J.
Beal, A.M.
Geddes, B.
Choudhary, J.S.
Murphy, J.M.
Aurelia Ball, K.
Upton, J.W.
Meier, P.
(2021). Ubiquitylation of MLKL at lysine 219 positively regulates necroptosis-induced tissue injury and pathogen clearance. Nature communications,
Vol.12
(1),
pp. 3364-?.
show abstract
Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system..
van Strien, J.
Haupt, A.
Schulte, U.
Braun, H.-.
Cabrera-Orefice, A.
Choudhary, J.S.
Evers, F.
Fernandez-Vizarra, E.
Guerrero-Castillo, S.
Kooij, T.W.
Páleníková, P.
Pardo, M.
Ugalde, C.
Wittig, I.
Wöhlbrand, L.
Brandt, U.
Arnold, S.
Huynen, M.A.
(2021). CEDAR, an online resource for the reporting and exploration of complexome profiling data. Biochimica et biophysica acta (bba) - bioenergetics,
Vol.1862
(7),
pp. 148411-148411.
Frankish, A.
Diekhans, M.
Jungreis, I.
Lagarde, J.
Loveland, J.E.
Mudge, J.M.
Sisu, C.
Wright, J.C.
Armstrong, J.
Barnes, I.
Berry, A.
Bignell, A.
Boix, C.
Carbonell Sala, S.
Cunningham, F.
Di Domenico, T.
Donaldson, S.
Fiddes, I.T.
García Girón, C.
Gonzalez, J.M.
Grego, T.
Hardy, M.
Hourlier, T.
Howe, K.L.
Hunt, T.
Izuogu, O.G.
Johnson, R.
Martin, F.J.
Martínez, L.
Mohanan, S.
Muir, P.
Navarro, F.C.
Parker, A.
Pei, B.
Pozo, F.
Riera, F.C.
Ruffier, M.
Schmitt, B.M.
Stapleton, E.
Suner, M.-.
Sycheva, I.
Uszczynska-Ratajczak, B.
Wolf, M.Y.
Xu, J.
Yang, Y.T.
Yates, A.
Zerbino, D.
Zhang, Y.
Choudhary, J.S.
Gerstein, M.
Guigó, R.
Hubbard, T.J.
Kellis, M.
Paten, B.
Tress, M.L.
Flicek, P.
(2021). GENCODE 2021. ,
Vol.49
(D1),
pp. D916-D923.
show abstract
The GENCODE project annotates human and mouse genes and transcripts supported by experimental data with high accuracy, providing a foundational resource that supports genome biology and clinical genomics. GENCODE annotation processes make use of primary data and bioinformatic tools and analysis generated both within the consortium and externally to support the creation of transcript structures and the determination of their function. Here, we present improvements to our annotation infrastructure, bioinformatics tools, and analysis, and the advances they support in the annotation of the human and mouse genomes including: the completion of first pass manual annotation for the mouse reference genome; targeted improvements to the annotation of genes associated with SARS-CoV-2 infection; collaborative projects to achieve convergence across reference annotation databases for the annotation of human and mouse protein-coding genes; and the first GENCODE manually supervised automated annotation of lncRNAs. Our annotation is accessible via Ensembl, the UCSC Genome Browser and https://www.gencodegenes.org..
Chatterjee, S.
Lekmeechai, S.
Constantinou, N.
Grzybowska, E.A.
Kozik, Z.
Choudhary, J.S.
Berger, C.N.
Frankel, G.
Clements, A.
(2021). The type
III
secretion system effector
EspO
of enterohaemorrhagic
Escherichia coli
inhibits apoptosis through an interaction with
HAX
‐1. Cellular microbiology,
Vol.23
(9).
Ruano-Gallego, D.
Sanchez-Garrido, J.
Kozik, Z.
Núñez-Berrueco, E.
Cepeda-Molero, M.
Mullineaux-Sanders, C.
Naemi-Baghshomali Clark, J.
Slater, S.L.
Wagner, N.
Glegola-Madejska, I.
Roumeliotis, T.I.
Pupko, T.
Fernández, L.Á.
Rodríguez-Patón, A.
Choudhary, J.S.
Frankel, G.
(2021). Type III secretion system effectors form robust and flexible intracellular virulence networks. Science,
Vol.371
(6534).
show abstract
Decrypting pathogen effector networks
Many disease-causing bacteria use a molecular syringe to inject dozens of their proteins, called effectors, into intestinal cells, blocking key immune responses. Ruano-Gallego
et al.
used the mouse pathogen
Citrobacter rodentium
to model effector function in vivo. They found that effectors work together as a network, allowing the microbe great flexibility in maintaining pathogenicity. An artificial intelligence platform correctly predicted colonization outcomes of alternative networks from the in vivo data. However, the host was able to bypass the obstacles erected by different effector networks and activate complementary immune responses that cleared the pathogen and induced protective immunity.
Science
, this issue p.
eabc9531
.
Choudhary, J.
Dheeman, S.
Sharma, V.
Katiyar, P.
Karn, S.K.
Sarangi, M.K.
Chauhan, A.K.
Verma, G.
Baliyan, N.
(2021). Insights of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) pandemic: a current review. Biological procedures online,
Vol.23
(1).
show abstract
AbstractCOVID-19, a pandemic of the 21st century caused by novel coronavirus SARS-CoV-2 was originated from China and shallowed world economy and human resource. The medical cures via herbal treatments, antiviral drugs, and vaccines still in progress, and studying rigorously. SARS-CoV-2 is more virulent than its ancestors due to evolution in the spike protein(s), mediates viral attachment to the host’s membranes. The SARS-CoV-2 receptor-binding spike domain associates itself with human angiotensin-converting enzyme 2 (ACE-2) receptors. It causes respiratory ailments with irregularities in the hepatic, nervous, and gastrointestinal systems, as reported in humans suffering from COVID-19 and reviewed in the present article. There are several approaches, have been put forward by many countries under the world health organization (WHO) recommendations and some trial drugs were introduced for possible treatment of COVID-19, such as Lopinavir or Ritonavir, Arbidol, Chloroquine (CQ), Hydroxychloroquine (HCQ) and most important Remdesivir including other like Tocilizumab, Oritavancin, Chlorpromazine, Azithromycin, Baricitinib, etc. RT-PCR is the only and early detection test available besides the rapid test kit (serodiagnosis) used by a few countries due to unreasonable causes. Development of vaccine by several leader of pharmaceutical groups still under trial or waiting for approval for mass inoculation. Management strategies have been evolved by the recommendations of WHO, specifically important to control COVID-19 situations, in the pandemic era. This review will provide a comprehensive collection of studies to support future research and enhancement in our wisdom to combat COVID-19 pandemic and to serve humanity..
Cutts, E.
Taylor, G.
Pardo, M.
Yu, L.
Wills, J.
Choudhary, J.
Vannini, A.
Wood, A.
(2021). A commercial antibody to the human condensin II subunit NCAPH2 cross-reacts with a SWI/SNF complex component. ,
.
show abstract
Condensin complexes compact and disentangle chromosomes in preparation for cell division. Commercially available antibodies raised against condensin subunits have been widely used to characterise their cellular interactome. Here we have assessed the specificity of a polyclonal antibody (Bethyl A302-276A) that is commonly used as a probe for NCAPH2, the kleisin subunit of condensin II, in mammalian cells. We find that, in addition to its intended target, this antibody cross-reacts with one or more components of the SWI/SNF family of chromatin remodelling complexes in an NCAPH2-independent manner. This cross-reactivity, with an abundant chromatin-associated factor, is likely to affect the interpretation of protein and chromatin immunoprecipitation experiments that make use of this antibody probe..
Peck, B.
Bland, P.
Mavrommati, I.
Muirhead, G.
Cottom, H.
Wai, P.T.
Maguire, S.L.
Barker, H.E.
Morrison, E.
Kriplani, D.
Yu, L.
Gibson, A.
Falgari, G.
Brennan, K.
Farnie, G.
Buus, R.
Marlow, R.
Novo, D.
Knight, E.
Guppy, N.
Kolarevic, D.
Susnjar, S.
Milijic, N.M.
Naidoo, K.
Gazinska, P.
Roxanis, I.
Pancholi, S.
Martin, L.-.
Holgersen, E.M.
Cheang, M.C.
Noor, F.
Postel-Vinay, S.
Quinn, G.
McDade, S.
Krasny, L.
Huang, P.
Daley, F.
Wallberg, F.
Choudhary, J.S.
Haider, S.
Tutt, A.N.
Natrajan, R.
(2021). 3D Functional Genomics Screens Identify CREBBP as a Targetable Driver in Aggressive Triple-Negative Breast Cancer. Cancer research,
.
show abstract
Triple-negative breast cancers (TNBC) are resistant to standard-of-care chemotherapy and lack known targetable driver gene alterations. Identification of novel drivers could aid the discovery of new treatment strategies for this hard-to-treat patient population, yet studies using high-throughput and accurate models to define the functions of driver genes in TNBC to date have been limited. Here, we employed unbiased functional genomics screening of the 200 most frequently mutated genes in breast cancer, using spheroid cultures to model in vivo-like conditions, and identified the histone acetyltransferase CREBBP as a novel tumor suppressor in TNBC. CREBBP protein expression in patient tumor samples was absent in 8% of TNBCs and at a high frequency in other tumors, including squamous lung cancer, where CREBBP-inactivating mutations are common. In TNBC, CREBBP alterations were associated with higher genomic heterogeneity and poorer patient survival and resulted in upregulation and dependency on a FOXM1 proliferative program. Targeting FOXM1-driven proliferation indirectly with clinical CDK4/6 inhibitors (CDK4/6i) selectively impaired growth in spheroids, cell line xenografts, and patient-derived models from multiple tumor types with CREBBP mutations or loss of protein expression. In conclusion, we have identified CREBBP as a novel driver in aggressive TNBC and identified an associated genetic vulnerability in tumor cells with alterations in CREBBP and provide a preclinical rationale for assessing CREBBP alterations as a biomarker of CDK4/6i response in a new patient population. SIGNIFICANCE: This study demonstrates that CREBBP genomic alterations drive aggressive TNBC, lung cancer, and lymphomas and may be selectively treated with clinical CDK4/6 inhibitors..
Barreiro-Alonso, A.
Lamas-Maceiras, M.
Lorenzo-Catoira, L.
Pardo, M.
Yu, L.
Choudhary, J.S.
Cerdán, M.E.
(2021). HMGB1 Protein Interactions in Prostate and Ovary Cancer Models Reveal Links to RNA Processing and Ribosome Biogenesis through NuRD, THOC and Septin Complexes. ,
Vol.13
(18),
p. 4686.
show abstract
This study reports the HMGB1 interactomes in prostate and ovary cancer cells lines. Affinity purification coupled to mass spectrometry confirmed that the HMGB1 nuclear interactome is involved in HMGB1 known functions such as maintenance of chromatin stability and regulation of transcription, and also in not as yet reported processes such as mRNA and rRNA processing. We have identified an interaction between HMGB1 and the NuRD complex and validated this by yeast-two-hybrid, confirming that the RBBP7 subunit directly interacts with HMGB1. In addition, we describe for the first time an interaction between two HMGB1 interacting complexes, the septin and THOC complexes, as well as an interaction of these two complexes with Rab11. Analysis of Pan-Cancer Atlas public data indicated that several genes encoding HMGB1-interacting proteins identified in this study are dysregulated in tumours from patients diagnosed with ovary and prostate carcinomas. In PC-3 cells, silencing of HMGB1 leads to downregulation of the expression of key regulators of ribosome biogenesis and RNA processing, namely BOP1, RSS1, UBF1, KRR1 and LYAR. Upregulation of these genes in prostate adenocarcinomas is correlated with worse prognosis, reinforcing their functional significance in cancer progression..
van der Weyden, L.
Harle, V.
Turner, G.
Offord, V.
Iyer, V.
Droop, A.
Swiatkowska, A.
Rabbie, R.
Campbell, A.D.
Sansom, O.J.
Pardo, M.
Choudhary, J.S.
Ferreira, I.
Tullett, M.
Arends, M.J.
Speak, A.O.
Adams, D.J.
(2021). CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation. Communications biology,
Vol.4
(1),
pp. 395-?.
show abstract
Melanoma represents ~5% of all cutaneous malignancies, yet accounts for the majority of skin cancer deaths due to its propensity to metastasise. To develop new therapies, novel target molecules must to be identified and the accessibility of cell surface proteins makes them attractive targets. Using CRISPR activation technology, we screened a library of guide RNAs targeting membrane protein-encoding genes to identify cell surface molecules whose upregulation enhances the metastatic pulmonary colonisation capabilities of tumour cells in vivo. We show that upregulated expression of the cell surface protein LRRN4CL led to increased pulmonary metastases in mice. Critically, LRRN4CL expression was elevated in melanoma patient samples, with high expression levels correlating with decreased survival. Collectively, our findings uncover an unappreciated role for LRRN4CL in the outcome of melanoma patients and identifies a potential therapeutic target and biomarker..
Steinberg, J.
Southam, L.
Roumeliotis, T.I.
Clark, M.J.
Jayasuriya, R.L.
Swift, D.
Shah, K.M.
Butterfield, N.C.
Brooks, R.A.
McCaskie, A.W.
Bassett, J.H.
Williams, G.R.
Choudhary, J.S.
Wilkinson, J.M.
Zeggini, E.
(2021). A molecular quantitative trait locus map for osteoarthritis. Nature communications,
Vol.12
(1),
pp. 1309-?.
show abstract
Osteoarthritis causes pain and functional disability for over 500 million people worldwide. To develop disease-stratifying tools and modifying therapies, we need a better understanding of the molecular basis of the disease in relevant tissue and cell types. Here, we study primary cartilage and synovium from 115 patients with osteoarthritis to construct a deep molecular signature map of the disease. By integrating genetics with transcriptomics and proteomics, we discover molecular trait loci in each tissue type and omics level, identify likely effector genes for osteoarthritis-associated genetic signals and highlight high-value targets for drug development and repurposing. These findings provide insights into disease aetiopathology, and offer translational opportunities in response to the global clinical challenge of osteoarthritis..
Zhu, F.
Collins, M.O.
Harmse, J.
Choudhary, J.S.
Grant, S.G.
Komiyama, N.H.
(2020). Cell-type-specific visualisation and biochemical isolation of endogenous synaptic proteins in mice. The european journal of neuroscience,
Vol.51
(3),
pp. 793-805.
show abstract
In recent years, the remarkable molecular complexity of synapses has been revealed, with over 1,000 proteins identified in the synapse proteome. Although it is known that different receptors and other synaptic proteins are present in different types of neurons, the extent of synapse diversity across the brain is largely unknown. This is mainly due to the limitations of current techniques. Here, we report an efficient method for the purification of synaptic protein complexes, fusing a high-affinity tag to endogenous PSD95 in specific cell types. We also developed a strategy, which enables the visualisation of endogenous PSD95 with fluorescent-protein tag in Cre-recombinase-expressing cells. We demonstrate the feasibility of proteomic analysis of synaptic protein complexes and visualisation of these in specific cell types. We find that the composition of PSD95 complexes purified from specific cell types differs from those extracted from tissues with diverse cellular composition. The results suggest that there might be differential interactions in the PSD95 complexes in different brain regions. We have detected differentially interacting proteins by comparing data sets from the whole hippocampus and the CA3 subfield of the hippocampus. Therefore, these novel conditional PSD95 tagging lines will not only serve as powerful tools for precisely dissecting synapse diversity in specific brain regions and subsets of neuronal cells, but also provide an opportunity to better understand brain region- and cell-type-specific alterations associated with various psychiatric/neurological diseases. These newly developed conditional gene tagging methods can be applied to many different synaptic proteins and will facilitate research on the molecular complexity of synapses..
Carson, D.
Barry, R.
Hopkins, E.G.
Roumeliotis, T.I.
García-Weber, D.
Mullineaux-Sanders, C.
Elinav, E.
Arrieumerlou, C.
Choudhary, J.S.
Frankel, G.
(2020). Citrobacter rodentium induces rapid and unique metabolic and inflammatory responses in mice suffering from severe disease. Cellular microbiology,
Vol.22
(1),
pp. e13126-?.
show abstract
The mouse pathogen Citrobacter rodentium is used to model infections with enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC). Pathogenesis is commonly modelled in mice developing mild disease (e.g., C57BL/6). However, little is known about host responses in mice exhibiting severe colitis (e.g., C3H/HeN), which arguably provide a more clinically relevant model for human paediatric enteric infection. Infection of C3H/HeN mice with C. rodentium results in rapid colonic colonisation, coinciding with induction of key inflammatory signatures and colonic crypt hyperplasia. Infection also induces dramatic changes to bioenergetics in intestinal epithelial cells, with transition from oxidative phosphorylation (OXPHOS) to aerobic glycolysis and higher abundance of SGLT4, LDHA, and MCT4. Concomitantly, mitochondrial proteins involved in the TCA cycle and OXPHOS were in lower abundance. Similar to observations in C57BL/6 mice, we detected simultaneous activation of cholesterol biogenesis, import, and efflux. Distinctly, however, the pattern recognition receptors NLRP3 and ALPK1 were specifically induced in C3H/HeN. Using cell-based assays revealed that C. rodentium activates the ALPK1/TIFA axis, which is dependent on the ADP-heptose biosynthesis pathway but independent of the Type III secretion system. This study reveals for the first time the unfolding intestinal epithelial cells' responses during severe infectious colitis, which resemble EPEC human infections..
Barry, R.
Ruano-Gallego, D.
Radhakrishnan, S.T.
Lovell, S.
Yu, L.
Kotik, O.
Glegola-Madejska, I.
Tate, E.W.
Choudhary, J.S.
Williams, H.R.
Frankel, G.
(2020). Faecal neutrophil elastase-antiprotease balance reflects colitis severity. Mucosal immunology,
Vol.13
(2),
pp. 322-333.
show abstract
Given the global burden of diarrheal diseases on healthcare it is surprising how little is known about the drivers of disease severity. Colitis caused by infection and inflammatory bowel disease (IBD) is characterised by neutrophil infiltration into the intestinal mucosa and yet our understanding of neutrophil responses during colitis is incomplete. Using infectious (Citrobacter rodentium) and chemical (dextran sulphate sodium; DSS) murine colitis models, as well as human IBD samples, we find that faecal neutrophil elastase (NE) activity reflects disease severity. During C. rodentium infection intestinal epithelial cells secrete the serine protease inhibitor SerpinA3N to inhibit and mitigate tissue damage caused by extracellular NE. Mice suffering from severe infection produce insufficient SerpinA3N to control excessive NE activity. This activity contributes to colitis severity as infection of these mice with a recombinant C. rodentium strain producing and secreting SerpinA3N reduces tissue damage. Thus, uncontrolled luminal NE activity is involved in severe colitis. Taken together, our findings suggest that NE activity could be a useful faecal biomarker for assessing disease severity as well as therapeutic target for both infectious and chronic inflammatory colitis..
Jackman, M.
Marcozzi, C.
Barbiero, M.
Pardo, M.
Yu, L.
Tyson, A.L.
Choudhary, J.S.
Pines, J.
(2020). Cyclin B1-Cdk1 facilitates MAD1 release from the nuclear pore to ensure a robust spindle checkpoint. The journal of cell biology,
Vol.219
(6).
show abstract
How the cell rapidly and completely reorganizes its architecture when it divides is a problem that has fascinated researchers for almost 150 yr. We now know that the core regulatory machinery is highly conserved in eukaryotes, but how these multiple protein kinases, protein phosphatases, and ubiquitin ligases are coordinated in space and time to remodel the cell in a matter of minutes remains a major question. Cyclin B1-Cdk is the primary kinase that drives mitotic remodeling; here we show that it is targeted to the nuclear pore complex (NPC) by binding an acidic face of the kinetochore checkpoint protein, MAD1, where it coordinates NPC disassembly with kinetochore assembly. Localized cyclin B1-Cdk1 is needed for the proper release of MAD1 from the embrace of TPR at the nuclear pore so that it can be recruited to kinetochores before nuclear envelope breakdown to maintain genomic stability..
Khan, Y.A.
Jungreis, I.
Wright, J.C.
Mudge, J.M.
Choudhary, J.S.
Firth, A.E.
Kellis, M.
(2020). Evidence for a novel overlapping coding sequence in POLG initiated at a CUG start codon. Bmc genetics,
Vol.21
(1),
pp. 25-?.
show abstract
BACKGROUND: POLG, located on nuclear chromosome 15, encodes the DNA polymerase γ(Pol γ). Pol γ is responsible for the replication and repair of mitochondrial DNA (mtDNA). Pol γ is the only DNA polymerase found in mitochondria for most animal cells. Mutations in POLG are the most common single-gene cause of diseases of mitochondria and have been mapped over the coding region of the POLG ORF. RESULTS: Using PhyloCSF to survey alternative reading frames, we found a conserved coding signature in an alternative frame in exons 2 and 3 of POLG, herein referred to as ORF-Y that arose de novo in placental mammals. Using the synplot2 program, synonymous site conservation was found among mammals in the region of the POLG ORF that is overlapped by ORF-Y. Ribosome profiling data revealed that ORF-Y is translated and that initiation likely occurs at a CUG codon. Inspection of an alignment of mammalian sequences containing ORF-Y revealed that the CUG codon has a strong initiation context and that a well-conserved predicted RNA stem-loop begins 14 nucleotides downstream. Such features are associated with enhanced initiation at near-cognate non-AUG codons. Reanalysis of the Kim et al. (2014) draft human proteome dataset yielded two unique peptides that map unambiguously to ORF-Y. An additional conserved uORF, herein referred to as ORF-Z, was also found in exon 2 of POLG. Lastly, we surveyed Clinvar variants that are synonymous with respect to the POLG ORF and found that most of these variants cause amino acid changes in ORF-Y or ORF-Z. CONCLUSIONS: We provide evidence for a novel coding sequence, ORF-Y, that overlaps the POLG ORF. Ribosome profiling and mass spectrometry data show that ORF-Y is expressed. PhyloCSF and synplot2 analysis show that ORF-Y is subject to strong purifying selection. An abundance of disease-correlated mutations that map to exons 2 and 3 of POLG but also affect ORF-Y provides potential clinical significance to this finding..
Chen, Y.-.
Roumeliotis, T.I.
Chang, Y.-.
Chen, C.-.
Han, C.-.
Lin, M.-.
Chen, H.-.
Chang, G.-.
Chang, Y.-.
Wu, C.-.
Lin, M.-.
Hsieh, M.-.
Wang, Y.-.
Chen, Y.-.
Jonassen, I.
Ghavidel, F.Z.
Lin, Z.-.
Lin, K.-.
Chen, C.-.
Sheu, P.-.
Hung, C.-.
Huang, K.-.
Yang, H.-.
Lin, P.-.
Yen, T.-.
Lin, Y.-.
Wang, J.-.
Raghav, L.
Lin, C.-.
Chen, Y.-.
Wu, P.-.
Lai, C.-.
Weng, S.-.
Su, K.-.
Chang, W.-.
Tsai, P.-.
Robles, A.I.
Rodriguez, H.
Hsiao, Y.-.
Chang, W.-.
Sung, T.-.
Chen, J.-.
Yu, S.-.
Choudhary, J.S.
Chen, H.-.
Yang, P.-.
Chen, Y.-.
(2020). Proteogenomics of Non-smoking Lung Cancer in East Asia Delineates Molecular Signatures of Pathogenesis and Progression. Cell,
Vol.182
(1),
pp. 226-244.e17.
show abstract
Lung cancer in East Asia is characterized by a high percentage of never-smokers, early onset and predominant EGFR mutations. To illuminate the molecular phenotype of this demographically distinct disease, we performed a deep comprehensive proteogenomic study on a prospectively collected cohort in Taiwan, representing early stage, predominantly female, non-smoking lung adenocarcinoma. Integrated genomic, proteomic, and phosphoproteomic analysis delineated the demographically distinct molecular attributes and hallmarks of tumor progression. Mutational signature analysis revealed age- and gender-related mutagenesis mechanisms, characterized by high prevalence of APOBEC mutational signature in younger females and over-representation of environmental carcinogen-like mutational signatures in older females. A proteomics-informed classification distinguished the clinical characteristics of early stage patients with EGFR mutations. Furthermore, integrated protein network analysis revealed the cellular remodeling underpinning clinical trajectories and nominated candidate biomarkers for patient stratification and therapeutic intervention. This multi-omic molecular architecture may help develop strategies for management of early stage never-smoker lung adenocarcinoma..
Cano-Gamez, E.
Soskic, B.
Roumeliotis, T.I.
So, E.
Smyth, D.J.
Baldrighi, M.
Willé, D.
Nakic, N.
Esparza-Gordillo, J.
Larminie, C.G.
Bronson, P.G.
Tough, D.F.
Rowan, W.C.
Choudhary, J.S.
Trynka, G.
(2020). Single-cell transcriptomics identifies an effectorness gradient shaping the response of CD4+ T cells to cytokines. Nature communications,
Vol.11
(1),
pp. 1801-?.
show abstract
Naïve CD4+ T cells coordinate the immune response by acquiring an effector phenotype in response to cytokines. However, the cytokine responses in memory T cells remain largely understudied. Here we use quantitative proteomics, bulk RNA-seq, and single-cell RNA-seq of over 40,000 human naïve and memory CD4+ T cells to show that responses to cytokines differ substantially between these cell types. Memory T cells are unable to differentiate into the Th2 phenotype, and acquire a Th17-like phenotype in response to iTreg polarization. Single-cell analyses show that T cells constitute a transcriptional continuum that progresses from naïve to central and effector memory T cells, forming an effectorness gradient accompanied by an increase in the expression of chemokines and cytokines. Finally, we show that T cell activation and cytokine responses are influenced by the effectorness gradient. Our results illustrate the heterogeneity of T cell responses, furthering our understanding of inflammation..
Lee, Y.-.
Khan, K.
Armfield-Uhas, K.
Srikanth, S.
Thompson, N.A.
Pardo, M.
Yu, L.
Norris, J.W.
Peng, Y.
Gripp, K.W.
Aleck, K.A.
Li, C.
Spence, E.
Choi, T.-.
Kwon, S.J.
Park, H.-.
Yu, D.
Heo, W.D.
Mooney, M.R.
Baig, S.M.
Wentzensen, I.M.
Telegrafi, A.
McWalter, K.
Moreland, T.
Roadhouse, C.
Ramsey, K.
Lyons, M.J.
Skinner, C.
Alexov, E.
Katsanis, N.
Stevenson, R.E.
Choudhary, J.S.
Adams, D.J.
Kim, C.-.
Davis, E.E.
Schwartz, C.E.
(2020). Mutations in FAM50A suggest that Armfield XLID syndrome is a spliceosomopathy. Nature communications,
Vol.11
(1),
pp. 3698-?.
show abstract
Intellectual disability (ID) is a heterogeneous clinical entity and includes an excess of males who harbor variants on the X-chromosome (XLID). We report rare FAM50A missense variants in the original Armfield XLID syndrome family localized in Xq28 and four additional unrelated males with overlapping features. Our fam50a knockout (KO) zebrafish model exhibits abnormal neurogenesis and craniofacial patterning, and in vivo complementation assays indicate that the patient-derived variants are hypomorphic. RNA sequencing analysis from fam50a KO zebrafish show dysregulation of the transcriptome, with augmented spliceosome mRNAs and depletion of transcripts involved in neurodevelopment. Zebrafish RNA-seq datasets show a preponderance of 3' alternative splicing events in fam50a KO, suggesting a role in the spliceosome C complex. These data are supported with transcriptomic signatures from cell lines derived from affected individuals and FAM50A protein-protein interaction data. In sum, Armfield XLID syndrome is a spliceosomopathy associated with aberrant mRNA processing during development..
ENCODE Project Consortium,,
Snyder, M.P.
Gingeras, T.R.
Moore, J.E.
Weng, Z.
Gerstein, M.B.
Ren, B.
Hardison, R.C.
Stamatoyannopoulos, J.A.
Graveley, B.R.
Feingold, E.A.
Pazin, M.J.
Pagan, M.
Gilchrist, D.A.
Hitz, B.C.
Cherry, J.M.
Bernstein, B.E.
Mendenhall, E.M.
Zerbino, D.R.
Frankish, A.
Flicek, P.
Myers, R.M.
(2020). Perspectives on ENCODE. Nature,
Vol.583
(7818),
pp. 693-698.
show abstract
The Encylopedia of DNA Elements (ENCODE) Project launched in 2003 with the long-term goal of developing a comprehensive map of functional elements in the human genome. These included genes, biochemical regions associated with gene regulation (for example, transcription factor binding sites, open chromatin, and histone marks) and transcript isoforms. The marks serve as sites for candidate cis-regulatory elements (cCREs) that may serve functional roles in regulating gene expression1. The project has been extended to model organisms, particularly the mouse. In the third phase of ENCODE, nearly a million and more than 300,000 cCRE annotations have been generated for human and mouse, respectively, and these have provided a valuable resource for the scientific community..
Zhong, Q.
Roumeliotis, T.I.
Kozik, Z.
Cepeda-Molero, M.
Fernández,
Shenoy, A.R.
Bakal, C.
Frankel, G.
Choudhary, J.S.
(2020). Clustering of Tir during enteropathogenic E coli infection triggers calcium influx-dependent pyroptosis in intestinal epithelial cells. Plos biology,
Vol.18
(12),
pp. e3000986-?.
show abstract
Clustering of the enteropathogenic Escherichia coli (EPEC) type III secretion system (T3SS) effector translocated intimin receptor (Tir) by intimin leads to actin polymerisation and pyroptotic cell death in macrophages. The effect of Tir clustering on the viability of EPEC-infected intestinal epithelial cells (IECs) is unknown. We show that EPEC induces pyroptosis in IECs in a Tir-dependent but actin polymerisation-independent manner, which was enhanced by priming with interferon gamma (IFNγ). Mechanistically, Tir clustering triggers rapid Ca2+ influx, which induces lipopolysaccharide (LPS) internalisation, followed by activation of caspase-4 and pyroptosis. Knockdown of caspase-4 or gasdermin D (GSDMD), translocation of NleF, which blocks caspase-4 or chelation of extracellular Ca2+, inhibited EPEC-induced cell death. IEC lines with low endogenous abundance of GSDMD were resistant to Tir-induced cell death. Conversely, ATP-induced extracellular Ca2+ influx enhanced cell death, which confirmed the key regulatory role of Ca2+ in EPEC-induced pyroptosis. We reveal a novel mechanism through which infection with an extracellular pathogen leads to pyroptosis in IECs..
Abascal, F.
Acosta, R.
Addleman, N.J.
Adrian, J.
Afzal, V.
Ai, R.
Aken, B.
Akiyama, J.A.
Jammal, O.A.
Amrhein, H.
Anderson, S.M.
Andrews, G.R.
Antoshechkin, I.
Ardlie, K.G.
Armstrong, J.
Astley, M.
Banerjee, B.
Barkal, A.A.
Barnes, I.H.
Barozzi, I.
Barrell, D.
Barson, G.
Bates, D.
Baymuradov, U.K.
Bazile, C.
Beer, M.A.
Beik, S.
Bender, M.A.
Bennett, R.
Bouvrette, L.P.
Bernstein, B.E.
Berry, A.
Bhaskar, A.
Bignell, A.
Blue, S.M.
Bodine, D.M.
Boix, C.
Boley, N.
Borrman, T.
Borsari, B.
Boyle, A.P.
Brandsmeier, L.A.
Breschi, A.
Bresnick, E.H.
Brooks, J.A.
Buckley, M.
Burge, C.B.
Byron, R.
Cahill, E.
Cai, L.
Cao, L.
Carty, M.
Castanon, R.G.
Castillo, A.
Chaib, H.
Chan, E.T.
Chee, D.R.
Chee, S.
Chen, H.
Chen, H.
Chen, J.-.
Chen, S.
Cherry, J.M.
Chhetri, S.B.
Choudhary, J.S.
Chrast, J.
Chung, D.
Clarke, D.
Cody, N.A.
Coppola, C.J.
Coursen, J.
D’Ippolito, A.M.
Dalton, S.
Danyko, C.
Davidson, C.
Davila-Velderrain, J.
Davis, C.A.
Dekker, J.
Deran, A.
DeSalvo, G.
Despacio-Reyes, G.
Dewey, C.N.
Dickel, D.E.
Diegel, M.
Diekhans, M.
Dileep, V.
Ding, B.
Djebali, S.
Dobin, A.
Dominguez, D.
Donaldson, S.
Drenkow, J.
Dreszer, T.R.
Drier, Y.
Duff, M.O.
Dunn, D.
Eastman, C.
Ecker, J.R.
Edwards, M.D.
El-Ali, N.
Elhajjajy, S.I.
Elkins, K.
Emili, A.
Epstein, C.B.
Evans, R.C.
Ezkurdia, I.
Fan, K.
Farnham, P.J.
Farrell, N.P.
Feingold, E.A.
Ferreira, A.-.
Fisher-Aylor, K.
Fitzgerald, S.
Flicek, P.
Foo, C.S.
Fortier, K.
Frankish, A.
Freese, P.
Fu, S.
Fu, X.-.
Fu, Y.
Fukuda-Yuzawa, Y.
Fulciniti, M.
Funnell, A.P.
Gabdank, I.
Galeev, T.
Gao, M.
Giron, C.G.
Garvin, T.H.
Gelboin-Burkhart, C.A.
Georgolopoulos, G.
Gerstein, M.B.
Giardine, B.M.
Gifford, D.K.
Gilbert, D.M.
Gilchrist, D.A.
Gillespie, S.
Gingeras, T.R.
Gong, P.
Gonzalez, A.
Gonzalez, J.M.
Good, P.
Goren, A.
Gorkin, D.U.
Graveley, B.R.
Gray, M.
Greenblatt, J.F.
Griffiths, E.
Groudine, M.T.
Grubert, F.
Gu, M.
Guigó, R.
Guo, H.
Guo, Y.
Guo, Y.
Gursoy, G.
Gutierrez-Arcelus, M.
Halow, J.
Hardison, R.C.
Hardy, M.
Hariharan, M.
Harmanci, A.
Harrington, A.
Harrow, J.L.
Hashimoto, T.B.
Hasz, R.D.
Hatan, M.
Haugen, E.
Hayes, J.E.
He, P.
He, Y.
Heidari, N.
Hendrickson, D.
Heuston, E.F.
Hilton, J.A.
Hitz, B.C.
Hochman, A.
Holgren, C.
Hou, L.
Hou, S.
Hsiao, Y.-.
Hsu, S.
Huang, H.
Hubbard, T.J.
Huey, J.
Hughes, T.R.
Hunt, T.
Ibarrientos, S.
Issner, R.
Iwata, M.
Izuogu, O.
Jaakkola, T.
Jameel, N.
Jansen, C.
Jiang, L.
Jiang, P.
Johnson, A.
Johnson, R.
Jungreis, I.
Kadaba, M.
Kasowski, M.
Kasparian, M.
Kato, M.
Kaul, R.
Kawli, T.
Kay, M.
Keen, J.C.
Keles, S.
Keller, C.A.
Kelley, D.
Kellis, M.
Kheradpour, P.
Kim, D.S.
Kirilusha, A.
Klein, R.J.
Knoechel, B.
Kuan, S.
Kulik, M.J.
Kumar, S.
Kundaje, A.
Kutyavin, T.
Lagarde, J.
Lajoie, B.R.
Lambert, N.J.
Lazar, J.
Lee, A.Y.
Lee, D.
Lee, E.
Lee, J.W.
Lee, K.
Leslie, C.S.
Levy, S.
Li, B.
Li, H.
Li, N.
Li, S.
Li, X.
Li, Y.I.
Li, Y.
Li, Y.
Li, Y.
Lian, J.
Libbrecht, M.W.
Lin, S.
Lin, Y.
Liu, D.
Liu, J.
Liu, P.
Liu, T.
Liu, X.S.
Liu, Y.
Liu, Y.
Long, M.
Lou, S.
Loveland, J.
Lu, A.
Lu, Y.
Lécuyer, E.
Ma, L.
Mackiewicz, M.
Mannion, B.J.
Mannstadt, M.
Manthravadi, D.
Marinov, G.K.
Martin, F.J.
Mattei, E.
McCue, K.
McEown, M.
McVicker, G.
Meadows, S.K.
Meissner, A.
Mendenhall, E.M.
Messer, C.L.
Meuleman, W.
Meyer, C.
Miller, S.
Milton, M.G.
Mishra, T.
Moore, D.E.
Moore, H.M.
Moore, J.E.
Moore, S.H.
Moran, J.
Mortazavi, A.
Mudge, J.M.
Munshi, N.
Murad, R.
Myers, R.M.
Nandakumar, V.
Nandi, P.
Narasimha, A.M.
Narayanan, A.K.
Naughton, H.
Navarro, F.C.
Navas, P.
Nazarovs, J.
Nelson, J.
Neph, S.
Neri, F.J.
Nery, J.R.
Nesmith, A.R.
Newberry, J.S.
Newberry, K.M.
Ngo, V.
Nguyen, R.
Nguyen, T.B.
Nguyen, T.
Nishida, A.
Noble, W.S.
Novak, C.S.
Novoa, E.M.
Nuñez, B.
O’Donnell, C.W.
Olson, S.
Onate, K.C.
Otterman, E.
Ozadam, H.
Pagan, M.
Palden, T.
Pan, X.
Park, Y.
Partridge, E.C.
Paten, B.
Pauli-Behn, F.
Pazin, M.J.
Pei, B.
Pennacchio, L.A.
Perez, A.R.
Perry, E.H.
Pervouchine, D.D.
Phalke, N.N.
Pham, Q.
Phanstiel, D.H.
Plajzer-Frick, I.
Pratt, G.A.
Pratt, H.E.
Preissl, S.
Pritchard, J.K.
Pritykin, Y.
Purcaro, M.J.
Qin, Q.
Quinones-Valdez, G.
Rabano, I.
Radovani, E.
Raj, A.
Rajagopal, N.
Ram, O.
Ramirez, L.
Ramirez, R.N.
Rausch, D.
Raychaudhuri, S.
Raymond, J.
Razavi, R.
Reddy, T.E.
Reimonn, T.M.
Ren, B.
Reymond, A.
Reynolds, A.
Rhie, S.K.
Rinn, J.
Rivera, M.
Rivera-Mulia, J.C.
Roberts, B.S.
Rodriguez, J.M.
Rozowsky, J.
Ryan, R.
Rynes, E.
Salins, D.N.
Sandstrom, R.
Sasaki, T.
Sathe, S.
Savic, D.
Scavelli, A.
Scheiman, J.
Schlaffner, C.
Schloss, J.A.
Schmitges, F.W.
See, L.H.
Sethi, A.
Setty, M.
Shafer, A.
Shan, S.
Sharon, E.
Shen, Q.
Shen, Y.
Sherwood, R.I.
Shi, M.
Shin, S.
Shoresh, N.
Siebenthall, K.
Sisu, C.
Slifer, T.
Sloan, C.A.
Smith, A.
Snetkova, V.
Snyder, M.P.
Spacek, D.V.
Srinivasan, S.
Srivas, R.
Stamatoyannopoulos, G.
Stamatoyannopoulos, J.A.
Stanton, R.
Steffan, D.
Stehling-Sun, S.
Strattan, J.S.
Su, A.
Sundararaman, B.
Suner, M.-.
Syed, T.
Szynkarek, M.
Tanaka, F.Y.
Tenen, D.
Teng, M.
Thomas, J.A.
Toffey, D.
Tress, M.L.
Trout, D.E.
Trynka, G.
Tsuji, J.
Upchurch, S.A.
Ursu, O.
Uszczynska-Ratajczak, B.
Uziel, M.C.
Valencia, A.
Biber, B.V.
van der Velde, A.G.
Van Nostrand, E.L.
Vaydylevich, Y.
Vazquez, J.
Victorsen, A.
Vielmetter, J.
Vierstra, J.
Visel, A.
Vlasova, A.
Vockley, C.M.
Volpi, S.
Vong, S.
Wang, H.
Wang, M.
Wang, Q.
Wang, R.
Wang, T.
Wang, W.
Wang, X.
Wang, Y.
Watson, N.K.
Wei, X.
Wei, Z.
Weisser, H.
Weissman, S.M.
Welch, R.
Welikson, R.E.
Weng, Z.
Westra, H.-.
Whitaker, J.W.
White, C.
White, K.P.
Wildberg, A.
Williams, B.A.
Wine, D.
Witt, H.N.
Wold, B.
Wolf, M.
Wright, J.
Xiao, R.
Xiao, X.
Xu, J.
Xu, J.
Yan, K.-.
Yan, Y.
Yang, H.
Yang, X.
Yang, Y.-.
Yardımcı, G.G.
Yee, B.A.
Yeo, G.W.
Young, T.
Yu, T.
Yue, F.
Zaleski, C.
Zang, C.
Zeng, H.
Zeng, W.
Zerbino, D.R.
Zhai, J.
Zhan, L.
Zhan, Y.
Zhang, B.
Zhang, J.
Zhang, J.
Zhang, K.
Zhang, L.
Zhang, P.
Zhang, Q.
Zhang, X.-.
Zhang, Y.
Zhang, Z.
Zhao, Y.
Zheng, Y.
Zhong, G.
Zhou, X.-.
Zhu, Y.
Zimmerman, J.
Moore, J.E.
Purcaro, M.J.
Pratt, H.E.
Epstein, C.B.
Shoresh, N.
Adrian, J.
Kawli, T.
Davis, C.A.
Dobin, A.
Kaul, R.
Halow, J.
Van Nostrand, E.L.
Freese, P.
Gorkin, D.U.
Shen, Y.
He, Y.
Mackiewicz, M.
Pauli-Behn, F.
Williams, B.A.
Mortazavi, A.
Keller, C.A.
Zhang, X.-.
Elhajjajy, S.I.
Huey, J.
Dickel, D.E.
Snetkova, V.
Wei, X.
Wang, X.
Rivera-Mulia, J.C.
Rozowsky, J.
Zhang, J.
Chhetri, S.B.
Zhang, J.
Victorsen, A.
White, K.P.
Visel, A.
Yeo, G.W.
Burge, C.B.
Lécuyer, E.
Gilbert, D.M.
Dekker, J.
Rinn, J.
Mendenhall, E.M.
Ecker, J.R.
Kellis, M.
Klein, R.J.
Noble, W.S.
Kundaje, A.
Guigó, R.
Farnham, P.J.
Cherry, J.M.
Myers, R.M.
Ren, B.
Graveley, B.R.
Gerstein, M.B.
Pennacchio, L.A.
Snyder, M.P.
Bernstein, B.E.
Wold, B.
Hardison, R.C.
Gingeras, T.R.
Stamatoyannopoulos, J.A.
Weng, Z.
(2020). Expanded encyclopaedias of DNA elements in the human and mouse genomes. Nature,
Vol.583
(7818),
pp. 699-710.
show abstract
AbstractThe human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes..
Mudge, J.M.
Jungreis, I.
Hunt, T.
Gonzalez, J.M.
Wright, J.C.
Kay, M.
Davidson, C.
Fitzgerald, S.
Seal, R.
Tweedie, S.
He, L.
Waterhouse, R.M.
Li, Y.
Bruford, E.
Choudhary, J.S.
Frankish, A.
Kellis, M.
(2019). Discovery of high-confidence human protein-coding genes and exons by whole-genome PhyloCSF helps elucidate 118 GWAS loci. Genome research,
Vol.29
(12),
pp. 2073-2087.
show abstract
The most widely appreciated role of DNA is to encode protein, yet the exact portion of the human genome that is translated remains to be ascertained. We previously developed PhyloCSF, a widely used tool to identify evolutionary signatures of protein-coding regions using multispecies genome alignments. Here, we present the first whole-genome PhyloCSF prediction tracks for human, mouse, chicken, fly, worm, and mosquito. We develop a workflow that uses machine learning to predict novel conserved protein-coding regions and efficiently guide their manual curation. We analyze more than 1000 high-scoring human PhyloCSF regions and confidently add 144 conserved protein-coding genes to the GENCODE gene set, as well as additional coding regions within 236 previously annotated protein-coding genes, and 169 pseudogenes, most of them disabled after primates diverged. The majority of these represent new discoveries, including 70 previously undetected protein-coding genes. The novel coding genes are additionally supported by single-nucleotide variant evidence indicative of continued purifying selection in the human lineage, coding-exon splicing evidence from new GENCODE transcripts using next-generation transcriptomic data sets, and mass spectrometry evidence of translation for several new genes. Our discoveries required simultaneous comparative annotation of other vertebrate genomes, which we show is essential to remove spurious ORFs and to distinguish coding from pseudogene regions. Our new coding regions help elucidate disease-associated regions by revealing that 118 GWAS variants previously thought to be noncoding are in fact protein altering. Altogether, our PhyloCSF data sets and algorithms will help researchers seeking to interpret these genomes, while our new annotations present exciting loci for further experimental characterization..
Moens, T.G.
Niccoli, T.
Wilson, K.M.
Atilano, M.L.
Birsa, N.
Gittings, L.M.
Holbling, B.V.
Dyson, M.C.
Thoeng, A.
Neeves, J.
Glaria, I.
Yu, L.
Bussmann, J.
Storkebaum, E.
Pardo, M.
Choudhary, J.S.
Fratta, P.
Partridge, L.
Isaacs, A.M.
(2019). C9orf72 arginine-rich dipeptide proteins interact with ribosomal proteins in vivo to induce a toxic translational arrest that is rescued by eIF1A. Acta neuropathologica,
Vol.137
(3),
pp. 487-500.
show abstract
A GGGGCC hexanucleotide repeat expansion within the C9orf72 gene is the most common genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense repeat-containing transcripts undergo repeat-associated non-AUG-initiated translation to produce five dipeptide proteins (DPRs). The polyGR and polyPR DPRs are extremely toxic when expressed in Drosophila neurons. To determine the mechanism that mediates this toxicity, we purified DPRs from the Drosophila brain and used mass spectrometry to identify the in vivo neuronal DPR interactome. PolyGR and polyPR interact with ribosomal proteins, and inhibit translation in both human iPSC-derived motor neurons, and adult Drosophila neurons. We next performed a screen of 81 translation-associated proteins in GGGGCC repeat-expressing Drosophila to determine whether this translational repression can be overcome and if this impacts neurodegeneration. Expression of the translation initiation factor eIF1A uniquely rescued DPR-induced toxicity in vivo, indicating that restoring translation is a potential therapeutic strategy. These data directly implicate translational repression in C9orf72 repeat-induced neurodegeneration and identify eIF1A as a novel modifier of C9orf72 repeat toxicity..
Roumeliotis, T.I.
Weisser, H.
Choudhary, J.S.
(2019). Evaluation of a Dual Isolation Width Acquisition Method for Isobaric Labeling Ratio Decompression. J proteome res,
Vol.18
(3),
pp. 1433-1440.
show abstract
Isobaric labeling is a highly precise approach for protein quantification. However, due to the isolation interference problem, isobaric tagging suffers from ratio underestimation at the MS2 level. The use of narrow isolation widths is a rational approach to alleviate the interference problem; however, this approach compromises proteome coverage. We reasoned that although a very narrow isolation window will result in loss of peptide fragment ions, the reporter ion signals will be retained for a significant portion of the spectra. On the basis of this assumption, we have designed a dual isolation width acquisition (DIWA) method, in which each precursor is first fragmented with HCD using a standard isolation width for peptide identification and preliminary quantification, followed by a second MS2 HCD scan using a much narrower isolation width for the acquisition of quantitative spectra with reduced interference. We leverage the quantification obtained by the "narrow" scans to build linear regression models and apply these to decompress the fold-changes measured at the "standard" scans. We evaluate the DIWA approach using a nested two species/gene knockout TMT-6plex experimental design and discuss the perspectives of this approach..
Hopkins, E.G.
Roumeliotis, T.I.
Mullineaux-Sanders, C.
Choudhary, J.S.
Frankel, G.
(2019). Intestinal Epithelial Cells and the Microbiome Undergo Swift Reprogramming at the Inception of Colonic Citrobacter rodentium Infection. Mbio,
Vol.10
(2).
show abstract
We used the mouse attaching and effacing (A/E) pathogen Citrobacter rodentium, which models the human A/E pathogens enteropathogenic Escherichia coli and enterohemorrhagic E. coli (EPEC and EHEC), to temporally resolve intestinal epithelial cell (IEC) responses and changes to the microbiome during in vivo infection. We found the host to be unresponsive during the first 3 days postinfection (DPI), when C. rodentium resides in the caecum. In contrast, at 4 DPI, the day of colonic colonization, despite only sporadic adhesion to the apex of the crypt, we observed robust upregulation of cell cycle and DNA repair processes, which were associated with expansion of the crypt Ki67-positive replicative zone, and downregulation of multiple metabolic processes (including the tricarboxylic acid [TCA] cycle and oxidative phosphorylation). Moreover, we observed dramatic depletion of goblet and deep crypt secretory cells and an atypical regulation of cholesterol homeostasis in IECs during early infection, with simultaneous upregulation of cholesterol biogenesis (e.g., 3-hydroxy-3-methylglutaryl-coenzyme A reductase [Hmgcr]), import (e.g., low-density lipoprotein receptor [Ldlr]), and efflux (e.g., AbcA1). We also detected interleukin 22 (IL-22) responses in IECs (e.g., Reg3γ) on the day of colonic colonization, which occurred concomitantly with a bloom of commensal Enterobacteriaceae on the mucosal surface. These results unravel a new paradigm in host-pathogen-microbiome interactions, showing for the first time that sensing a small number of pathogenic bacteria triggers swift intrinsic changes to the IEC composition and function, in tandem with significant changes to the mucosa-associated microbiome, which parallel innate immune responses.IMPORTANCE The mouse pathogen C. rodentium is a widely used model for colonic infection and has been a major tool in fundamental discoveries in the fields of bacterial pathogenesis and mucosal immunology. Despite extensive studies probing acute C. rodentium infection, our understanding of the early stages preceding the infection climax remains relatively undetailed. To this end, we apply a multiomics approach to resolve temporal changes to the host and microbiome during early infection. Unexpectedly, we found immediate and dramatic responses occurring on the day of colonic infection, both in the host intestinal epithelial cells and in the microbiome. Our study suggests changes in cholesterol and carbon metabolism in epithelial cells are instantly induced upon pathogen detection in the colon, corresponding with a shift to primarily facultative anaerobes constituting the microbiome. This study contributes to our knowledge of disease pathogenesis and mechanisms of barrier regulation, which is required for development of novel therapeutics targeting the intestinal epithelium..
Zhang, W.
Chronis, C.
Chen, X.
Zhang, H.
Spalinskas, R.
Pardo, M.
Chen, L.
Wu, G.
Zhu, Z.
Yu, Y.
Yu, L.
Choudhary, J.
Nichols, J.
Parast, M.M.
Greber, B.
Sahlén, P.
Plath, K.
(2019). The BAF and PRC2 Complex Subunits Dpf2 and Eed Antagonistically Converge on Tbx3 to Control ESC Differentiation. Cell stem cell,
Vol.24
(1),
pp. 138-152.e8.
show abstract
BAF complexes are composed of different subunits with varying functional and developmental roles, although many subunits have not been examined in depth. Here we show that the Baf45 subunit Dpf2 maintains pluripotency and ESC differentiation potential. Dpf2 co-occupies enhancers with Oct4, Sox2, p300, and the BAF subunit Brg1, and deleting Dpf2 perturbs ESC self-renewal, induces repression of Tbx3, and impairs mesendodermal differentiation without dramatically altering Brg1 localization. Mesendodermal differentiation can be rescued by restoring Tbx3 expression, whose distal enhancer is positively regulated by Dpf2-dependent H3K27ac maintenance and recruitment of pluripotency TFs and Brg1. In contrast, the PRC2 subunit Eed binds an intragenic Tbx3 enhancer to oppose Dpf2-dependent Tbx3 expression and mesendodermal differentiation. The PRC2 subunit Ezh2 likewise opposes Dpf2-dependent differentiation through a distinct mechanism involving Nanog repression. Together, these findings delineate distinct mechanistic roles for specific BAF and PRC2 subunits during ESC differentiation..
Capalbo, L.
Bassi, Z.I.
Geymonat, M.
Todesca, S.
Copoiu, L.
Enright, A.J.
Callaini, G.
Riparbelli, M.G.
Yu, L.
Choudhary, J.S.
Ferrero, E.
Wheatley, S.
Douglas, M.E.
Mishima, M.
D'Avino, P.P.
(2019). The midbody interactome reveals unexpected roles for PP1 phosphatases in cytokinesis. Nature communications,
Vol.10
(1),
pp. 4513-?.
show abstract
The midbody is an organelle assembled at the intercellular bridge between the two daughter cells at the end of mitosis. It controls the final separation of the daughter cells and has been involved in cell fate, polarity, tissue organization, and cilium and lumen formation. Here, we report the characterization of the intricate midbody protein-protein interaction network (interactome), which identifies many previously unknown interactions and provides an extremely valuable resource for dissecting the multiple roles of the midbody. Initial analysis of this interactome revealed that PP1β-MYPT1 phosphatase regulates microtubule dynamics in late cytokinesis and de-phosphorylates the kinesin component MKLP1/KIF23 of the centralspindlin complex. This de-phosphorylation antagonizes Aurora B kinase to modify the functions and interactions of centralspindlin in late cytokinesis. Our findings expand the repertoire of PP1 functions during mitosis and indicate that spatiotemporal changes in the distribution of kinases and counteracting phosphatases finely tune the activity of cytokinesis proteins..
Hillier, C.
Pardo, M.
Yu, L.
Bushell, E.
Sanderson, T.
Metcalf, T.
Herd, C.
Anar, B.
Rayner, J.C.
Billker, O.
Choudhary, J.S.
(2019). Landscape of the Plasmodium Interactome Reveals Both Conserved and Species-Specific Functionality. Cell reports,
Vol.28
(6),
pp. 1635-1647.e5.
show abstract
Malaria represents a major global health issue, and the identification of new intervention targets remains an urgent priority. This search is hampered by more than one-third of the genes of malaria-causing Plasmodium parasites being uncharacterized. We report a large-scale protein interaction network in Plasmodium schizonts, generated by combining blue native-polyacrylamide electrophoresis with quantitative mass spectrometry and machine learning. This integrative approach, spanning 3 species, identifies >20,000 putative protein interactions, organized into 600 protein clusters. We validate selected interactions, assigning functions in chromatin regulation to previously unannotated proteins and suggesting a role for an EELM2 domain-containing protein and a putative microrchidia protein as mechanistic links between AP2-domain transcription factors and epigenetic regulation. Our interactome represents a high-confidence map of the native organization of core cellular processes in Plasmodium parasites. The network reveals putative functions for uncharacterized proteins, provides mechanistic and structural insight, and uncovers potential alternative therapeutic targets..
Liess, A.K.
Kucerova, A.
Schweimer, K.
Yu, L.
Roumeliotis, T.I.
Diebold, M.
Dybkov, O.
Sotriffer, C.
Urlaub, H.
Choudhary, J.S.
Mansfeld, J.
Lorenz, S.
(2019). Autoinhibition Mechanism of the Ubiquitin-Conjugating Enzyme UBE2S by Autoubiquitination. Structure (london, england : 1993),
Vol.27
(8),
pp. 1195-1210.e7.
show abstract
Ubiquitin-conjugating enzymes (E2s) govern key aspects of ubiquitin signaling. Emerging evidence suggests that the activities of E2s are modulated by posttranslational modifications; the structural underpinnings, however, are largely unclear. Here, we unravel the structural basis and mechanistic consequences of a conserved autoubiquitination event near the catalytic center of E2s, using the human anaphase-promoting complex/cyclosome-associated UBE2S as a model system. Crystal structures we determined of the catalytic ubiquitin carrier protein domain combined with MD simulations reveal that the active-site region is malleable, which permits an adjacent ubiquitin acceptor site, Lys+5, to be ubiquitinated intramolecularly. We demonstrate by NMR that the Lys+5-linked ubiquitin inhibits UBE2S by obstructing its reloading with ubiquitin. By immunoprecipitation, quantitative mass spectrometry, and siRNA-and-rescue experiments we show that Lys+5 ubiquitination of UBE2S decreases during mitotic exit but does not influence proteasomal turnover of this E2. These findings suggest that UBE2S activity underlies inherent regulation during the cell cycle..
Verma, S.
Choudhary, J.
Singh, K.P.
Chandra, P.
Singh, S.P.
(2019). Uricase grafted nanoconducting matrix based electrochemical biosensor for ultrafast uric acid detection in human serum samples. International journal of biological macromolecules,
Vol.130,
pp. 333-341.
Frankish, A.
Diekhans, M.
Ferreira, A.-.
Johnson, R.
Jungreis, I.
Loveland, J.
Mudge, J.M.
Sisu, C.
Wright, J.
Armstrong, J.
Barnes, I.
Berry, A.
Bignell, A.
Carbonell Sala, S.
Chrast, J.
Cunningham, F.
Di Domenico, T.
Donaldson, S.
Fiddes, I.T.
García Girón, C.
Gonzalez, J.M.
Grego, T.
Hardy, M.
Hourlier, T.
Hunt, T.
Izuogu, O.G.
Lagarde, J.
Martin, F.J.
Martínez, L.
Mohanan, S.
Muir, P.
Navarro, F.C.
Parker, A.
Pei, B.
Pozo, F.
Ruffier, M.
Schmitt, B.M.
Stapleton, E.
Suner, M.-.
Sycheva, I.
Uszczynska-Ratajczak, B.
Xu, J.
Yates, A.
Zerbino, D.
Zhang, Y.
Aken, B.
Choudhary, J.S.
Gerstein, M.
Guigó, R.
Hubbard, T.J.
Kellis, M.
Paten, B.
Reymond, A.
Tress, M.L.
Flicek, P.
(2019). GENCODE reference annotation for the human and mouse genomes. Nucleic acids research,
Vol.47
(D1),
pp. D766-D773.
show abstract
The accurate identification and description of the genes in the human and mouse genomes is a fundamental requirement for high quality analysis of data informing both genome biology and clinical genomics. Over the last 15 years, the GENCODE consortium has been producing reference quality gene annotations to provide this foundational resource. The GENCODE consortium includes both experimental and computational biology groups who work together to improve and extend the GENCODE gene annotation. Specifically, we generate primary data, create bioinformatics tools and provide analysis to support the work of expert manual gene annotators and automated gene annotation pipelines. In addition, manual and computational annotation workflows use any and all publicly available data and analysis, along with the research literature to identify and characterise gene loci to the highest standard. GENCODE gene annotations are accessible via the Ensembl and UCSC Genome Browsers, the Ensembl FTP site, Ensembl Biomart, Ensembl Perl and REST APIs as well as https://www.gencodegenes.org..
Dunstan, R.A.
Pickard, D.
Dougan, S.
Goulding, D.
Cormie, C.
Hardy, J.
Li, F.
Grinter, R.
Harcourt, K.
Yu, L.
Song, J.
Schreiber, F.
Choudhary, J.
Clare, S.
Coulibaly, F.
Strugnell, R.A.
Dougan, G.
Lithgow, T.
(2019). The flagellotropic bacteriophage YSD1 targets Salmonella Typhi with a Chi‐like protein tail fibre. Molecular microbiology,
Vol.112
(6),
pp. 1831-1846.
show abstract
SummaryThe discovery of a Salmonella‐targeting phage from the waterways of the United Kingdom provided an opportunity to address the mechanism by which Chi‐like bacteriophage (phage) engages with bacterial flagellae. The long tail fibre seen on Chi‐like phages has been proposed to assist the phage particle in docking to a host cell flagellum, but the identity of the protein that generates this fibre was unknown. We present the results from genome sequencing of this phage, YSD1, confirming its close relationship to the original Chi phage and suggesting candidate proteins to form the tail structure. Immunogold labelling in electron micrographs revealed that YSD1_22 forms the main shaft of the tail tube, while YSD1_25 forms the distal part contributing to the tail spike complex. The long curling tail fibre is formed by the protein YSD1_29, and treatment of phage with the antibodies that bind YSD1_29 inhibits phage infection of Salmonella. The host range for YSD1 across Salmonella serovars is broad, but not comprehensive, being limited by antigenic features of the flagellin subunits that make up the Salmonella flagellum, with which YSD1_29 engages to initiate infection..
Gentzel, M.
Pardo, M.
Subramaniam, S.
Stewart, A.F.
Choudhary, J.S.
(2019). Proteomic navigation using proximity-labeling. Methods (san diego, calif.),
Vol.164-165,
pp. 67-72.
show abstract
The identification of bona fide protein-protein interactions and the mapping of proteomes was greatly enhanced by protein tagging for generic affinity purification methods and analysis by mass spectrometry (AP-MS). The high quality of AP-MS data permitted the development of proteomic navigation by sequential tagging of identified interactions. However AP-MS is laborious and limited to relatively high affinity protein-protein interactions. Proximity labeling, first with the biotin ligase BirA, termed BioID, and then with ascorbate peroxidase, termed APEX, permits a greater reach into the proteome than AP-MS enabling both the identification of a wider field and weaker protein-protein interactions. This additional reach comes with the need for stringent controls. Proximity labeling also permits experiments in living cells allowing spatiotemporal investigations of the proteome. Here we discuss proximity labeling with accompanying methodological descriptions for E. coli and mammalian cells..
Bianco, J.N.
Bergoglio, V.
Lin, Y.-.
Pillaire, M.-.
Schmitz, A.-.
Gilhodes, J.
Lusque, A.
Mazières, J.
Lacroix-Triki, M.
Roumeliotis, T.I.
Choudhary, J.
Moreaux, J.
Hoffmann, J.-.
Tourrière, H.
Pasero, P.
(2019). Overexpression of Claspin and Timeless protects cancer cells from replication stress in a checkpoint-independent manner. Nature communications,
Vol.10
(1),
pp. 910-?.
show abstract
Oncogene-induced replication stress (RS) promotes cancer development but also impedes tumor growth by activating anti-cancer barriers. To determine how cancer cells adapt to RS, we have monitored the expression of different components of the ATR-CHK1 pathway in primary tumor samples. We show that unlike upstream components of the pathway, the checkpoint mediators Claspin and Timeless are overexpressed in a coordinated manner. Remarkably, reducing the levels of Claspin and Timeless in HCT116 cells to pretumoral levels impeded fork progression without affecting checkpoint signaling. These data indicate that high level of Claspin and Timeless increase RS tolerance by protecting replication forks in cancer cells. Moreover, we report that primary fibroblasts adapt to oncogene-induced RS by spontaneously overexpressing Claspin and Timeless, independently of ATR signaling. Altogether, these data indicate that enhanced levels of Claspin and Timeless represent a gain of function that protects cancer cells from of oncogene-induced RS in a checkpoint-independent manner..
Berger, C.N.
Crepin, V.F.
Roumeliotis, T.I.
Wright, J.C.
Serafini, N.
Pevsner-Fischer, M.
Yu, L.
Elinav, E.
Di Santo, J.P.
Choudhary, J.S.
Frankel, G.
(2018). The Citrobacter rodentium type III secretion system effector EspO affects mucosal damage repair and antimicrobial responses. Plos pathogens,
Vol.14
(10),
pp. e1007406-?.
show abstract
Infection with Citrobacter rodentium triggers robust tissue damage repair responses, manifested by secretion of IL-22, in the absence of which mice succumbed to the infection. Of the main hallmarks of C. rodentium infection are colonic crypt hyperplasia (CCH) and dysbiosis. In order to colonize the host and compete with the gut microbiota, C. rodentium employs a type III secretion system (T3SS) that injects effectors into colonic intestinal epithelial cells (IECs). Once injected, the effectors subvert processes involved in innate immune responses, cellular metabolism and oxygenation of the mucosa. Importantly, the identity of the effector/s triggering the tissue repair response is/are unknown. Here we report that the effector EspO ,an orthologue of OspE found in Shigella spp, affects proliferation of IECs 8 and 14 days post C. rodentium infection as well as secretion of IL-22 from colonic explants. While we observed no differences in the recruitment of group 3 innate lymphoid cells (ILC3s) and T cells, which are the main sources of IL-22 at the early and late stages of C. rodentium infection respectively, infection with ΔespO was characterized by diminished recruitment of sub-mucosal neutrophils, which coincided with lower abundance of Mmp9 and chemokines (e.g. S100a8/9) in IECs. Moreover, mice infected with ΔespO triggered significantly lesser nutritional immunity (e.g. calprotectin, Lcn2) and expression of antimicrobial peptides (Reg3β, Reg3γ) compared to mice infected with WT C. rodentium. This overlapped with a decrease in STAT3 phosphorylation in IECs. Importantly, while the reduced CCH and abundance of antimicrobial proteins during ΔespO infection did not affect C. rodentium colonization or the composition of commensal Proteobacteria, they had a subtle consequence on Firmicutes subpopulations. EspO is the first bacterial virulence factor that affects neutrophil recruitment and secretion of IL-22, as well as expression of antimicrobial and nutritional immunity proteins in IECs..
Mayer, L.
Jasztal, M.
Pardo, M.
Aguera de Haro, S.
Collins, J.
Bariana, T.K.
Smethurst, P.A.
Grassi, L.
Petersen, R.
Nurden, P.
Favier, R.
Yu, L.
Meacham, S.
Astle, W.J.
Choudhary, J.
Yue, W.W.
Ouwehand, W.H.
Guerrero, J.A.
(2018). Nbeal2 interacts with Dock7, Sec16a, and Vac14. ,
Vol.131
(9),
pp. 1000-1011.
show abstract
Mutations in NBEAL2, the gene encoding the scaffolding protein Nbeal2, are causal of gray platelet syndrome (GPS), a rare recessive bleeding disorder characterized by platelets lacking α-granules and progressive marrow fibrosis. We present here the interactome of Nbeal2 with additional validation by reverse immunoprecipitation of Dock7, Sec16a, and Vac14 as interactors of Nbeal2. We show that GPS-causing mutations in its BEACH domain have profound and possible effects on the interaction with Dock7 and Vac14, respectively. Proximity ligation assays show that these 2 proteins are physically proximal to Nbeal2 in human megakaryocytes. In addition, we demonstrate that Nbeal2 is primarily localized in the cytoplasm and Dock7 on the membrane of or in α-granules. Interestingly, platelets from GPS cases and Nbeal2-/- mice are almost devoid of Dock7, resulting in a profound dysregulation of its signaling pathway, leading to defective actin polymerization, platelet activation, and shape change. This study shows for the first time proteins interacting with Nbeal2 and points to the dysregulation of the canonical signaling pathway of Dock7 as a possible cause of the aberrant formation of platelets in GPS cases and Nbeal2-deficient mice..
Gozdecka, M.
Meduri, E.
Mazan, M.
Tzelepis, K.
Dudek, M.
Knights, A.J.
Pardo, M.
Yu, L.
Choudhary, J.S.
Metzakopian, E.
Iyer, V.
Yun, H.
Park, N.
Varela, I.
Bautista, R.
Collord, G.
Dovey, O.
Garyfallos, D.A.
De Braekeleer, E.
Kondo, S.
Cooper, J.
Göttgens, B.
Bullinger, L.
Northcott, P.A.
Adams, D.
Vassiliou, G.S.
Huntly, B.J.
(2018). UTX-mediated enhancer and chromatin remodeling suppresses myeloid leukemogenesis through noncatalytic inverse regulation of ETS and GATA programs. Nature genetics,
Vol.50
(6),
pp. 883-894.
show abstract
The histone H3 Lys27-specific demethylase UTX (or KDM6A) is targeted by loss-of-function mutations in multiple cancers. Here, we demonstrate that UTX suppresses myeloid leukemogenesis through noncatalytic functions, a property shared with its catalytically inactive Y-chromosome paralog, UTY (or KDM6C). In keeping with this, we demonstrate concomitant loss/mutation of KDM6A (UTX) and UTY in multiple human cancers. Mechanistically, global genomic profiling showed only minor changes in H3K27me3 but significant and bidirectional alterations in H3K27ac and chromatin accessibility; a predominant loss of H3K4me1 modifications; alterations in ETS and GATA-factor binding; and altered gene expression after Utx loss. By integrating proteomic and genomic analyses, we link these changes to UTX regulation of ATP-dependent chromatin remodeling, coordination of the COMPASS complex and enhanced pioneering activity of ETS factors during evolution to AML. Collectively, our findings identify a dual role for UTX in suppressing acute myeloid leukemia via repression of oncogenic ETS and upregulation of tumor-suppressive GATA programs..
Steinberg, J.
Brooks, R.A.
Southam, L.
Bhatnagar, S.
Roumeliotis, T.I.
Hatzikotoulas, K.
Zengini, E.
Wilkinson, J.M.
Choudhary, J.S.
McCaskie, A.W.
Zeggini, E.
(2018). Widespread epigenomic, transcriptomic and proteomic differences between hip osteophytic and articular chondrocytes in osteoarthritis. Rheumatology (oxford),
Vol.57
(8),
pp. 1481-1489.
show abstract
Objectives: To identify molecular differences between chondrocytes from osteophytic and articular cartilage tissue from OA patients. Methods: We investigated genes and pathways by combining genome-wide DNA methylation, RNA sequencing and quantitative proteomics in isolated primary chondrocytes from the cartilaginous layer of osteophytes and matched areas of low- and high-grade articular cartilage across nine patients with OA undergoing hip replacement surgery. Results: Chondrocytes from osteophytic cartilage showed widespread differences to low-grade articular cartilage chondrocytes. These differences were similar to, but more pronounced than, differences between chondrocytes from osteophytic and high-grade articular cartilage, and more pronounced than differences between high- and low-grade articular cartilage. We identified 56 genes with significant differences between osteophytic chondrocytes and low-grade articular cartilage chondrocytes on all three omics levels. Several of these genes have known roles in OA, including ALDH1A2 and cartilage oligomeric matrix protein, which have functional genetic variants associated with OA from genome-wide association studies. An integrative gene ontology enrichment analysis showed that differences between osteophytic and low-grade articular cartilage chondrocytes are associated with extracellular matrix organization, skeletal system development, platelet aggregation and regulation of ERK1 and ERK2 cascade. Conclusion: We present a first comprehensive view of the molecular landscape of chondrocytes from osteophytic cartilage as compared with articular cartilage chondrocytes from the same joints in OA. We found robust changes at genes relevant to chondrocyte function, providing insight into biological processes involved in osteophyte development and thus OA progression..
Barreiro-Alonso, A.
Lamas-Maceiras, M.
García-Díaz, R.
Rodríguez-Belmonte, E.
Yu, L.
Pardo, M.
Choudhary, J.S.
Cerdán, M.E.
(2018). Delineating the HMGB1 and HMGB2 interactome in prostate and ovary epithelial cells and its relationship with cancer. Oncotarget,
Vol.9
(27),
pp. 19050-19064.
show abstract
High Mobility Group B (HMGB) proteins are involved in cancer progression and in cellular responses to platinum compounds used in the chemotherapy of prostate and ovary cancer. Here we use affinity purification coupled to mass spectrometry (MS) and yeast two-hybrid (Y2H) screening to carry out an exhaustive study of HMGB1 and HMGB2 protein interactions in the context of prostate and ovary epithelia. We present a proteomic study of HMGB1 partners based on immunoprecipitation of HMGB1 from a non-cancerous prostate epithelial cell line. In addition, HMGB1 and HMGB2 were used as baits in yeast two-hybrid screening of libraries from prostate and ovary epithelial cell lines as well as from healthy ovary tissue. HMGB1 interacts with many nuclear proteins that control gene expression, but also with proteins that form part of the cytoskeleton, cell-adhesion structures and others involved in intracellular protein translocation, cellular migration, secretion, apoptosis and cell survival. HMGB2 interacts with proteins involved in apoptosis, cell motility and cellular proliferation. High confidence interactors, based on repeated identification in different cell types or in both MS and Y2H approaches, are discussed in relation to cancer. This study represents a useful resource for detailed investigation of the role of HMGB1 in cancer of epithelial origins, as well as potential alternative avenues of therapeutic intervention..
Pollard, D.J.
Berger, C.N.
So, E.C.
Yu, L.
Hadavizadeh, K.
Jennings, P.
Tate, E.W.
Choudhary, J.S.
Frankel, G.
(2018). Broad-Spectrum Regulation of Nonreceptor Tyrosine Kinases by the Bacterial ADP-Ribosyltransferase EspJ. Mbio,
Vol.9
(2).
show abstract
Tyrosine phosphorylation is key for signal transduction from exogenous stimuli, including the defense against pathogens. Conversely, pathogens can subvert protein phosphorylation to control host immune responses and facilitate invasion and dissemination. The bacterial effectors EspJ and SeoC are injected into host cells through a type III secretion system by enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), Citrobacter rodentium, and Salmonella enterica, where they inhibit Src kinase by coupled amidation and ADP-ribosylation. C. rodentium, which is used to model EPEC and EHEC infections in humans, is a mouse pathogen triggering colonic crypt hyperplasia (CCH) and colitis. Enumeration of bacterial shedding and CCH confirmed that EspJ affects neither tolerance nor resistance to infection. However, comparison of the proteomes of intestinal epithelial cells isolated from mice infected with wild-type C. rodentium or C. rodentium encoding catalytically inactive EspJ revealed that EspJ-induced ADP-ribosylation regulates multiple nonreceptor tyrosine kinases in vivo Investigation of the substrate repertoire of EspJ revealed that in HeLa and A549 cells, Src and Csk were significantly targeted; in polarized Caco2 cells, EspJ targeted Src and Csk and the Src family kinase (SFK) Yes1, while in differentiated Thp1 cells, EspJ modified Csk, the SFKs Hck and Lyn, the Tec family kinases Tec and Btk, and the adapter tyrosine kinase Syk. Furthermore, Abl (HeLa and Caco2) and Lyn (Caco2) were enriched specifically in the EspJ-containing samples. Biochemical assays revealed that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, controls immune responses and the Src/Csk signaling axis.IMPORTANCE Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) strains cause significant mortality and morbidity worldwide. Citrobacter rodentium is a mouse pathogen used to model EPEC and EHEC pathogenesis in vivo Diarrheal disease is triggered following injection of bacterial effectors, via a type III secretion system (T3SS), into intestinal epithelial cells (IECs). While insights into the role of the effectors were historically obtained from pathological, immunologic, or cell culture phenotypes, subtle roles of individual effectors in vivo are often masked. The aim of this study was to elucidate the role and specificity of the ADP-ribosyltransferase effector EspJ. For the first time, we show that the in vivo processes affected by a T3SS effector can be studied by comparing the proteomes of IECs extracted from mice infected with wild-type C. rodentium or an espJ catalytic mutant. We show that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, regulates the host immune response in vivo..
Bakos, G.
Yu, L.
Gak, I.A.
Roumeliotis, T.I.
Liakopoulos, D.
Choudhary, J.S.
Mansfeld, J.
(2018). An E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules. Nature communications,
Vol.9
(1),
pp. 4776-?.
show abstract
Covalent modifications of proteins with ubiquitin and ubiquitin-like molecules are instrumental to many biological processes. However, identifying the E3 ligase responsible for these modifications remains a major bottleneck in ubiquitin research. Here, we present an E2-thioester-driven identification (E2~dID) method for the targeted identification of substrates of specific E2 and E3 enzyme pairs. E2~dID exploits the central position of E2-conjugating enzymes in the ubiquitination cascade and provides in vitro generated biotinylated E2~ubiquitin thioester conjugates as the sole source for ubiquitination in extracts. This enables purification and mass spectrometry-based identification of modified proteins under stringent conditions independently of the biological source of the extract. We demonstrate the sensitivity and specificity of E2-dID by identifying and validating substrates of APC/C in human cells. Finally, we perform E2~dID with SUMO in S. cerevisiae, showing that this approach can be easily adapted to other ubiquitin-like modifiers and experimental models..
Raouf, R.
Lolignier, S.
Sexton, J.E.
Millet, Q.
Santana-Varela, S.
Biller, A.
Fuller, A.M.
Pereira, V.
Choudhary, J.S.
Collins, M.O.
Moss, S.E.
Lewis, R.
Tordo, J.
Henckaerts, E.
Linden, M.
Wood, J.N.
(2018). Inhibition of somatosensory mechanotransduction by annexin A6. Sci signal,
Vol.11
(535).
show abstract
Mechanically activated, slowly adapting currents in sensory neurons have been linked to noxious mechanosensation. The conotoxin NMB-1 (noxious mechanosensation blocker-1) blocks such currents and inhibits mechanical pain. Using a biotinylated form of NMB-1 in mass spectrometry analysis, we identified 67 binding proteins in sensory neurons and a sensory neuron-derived cell line, of which the top candidate was annexin A6, a membrane-associated calcium-binding protein. Annexin A6-deficient mice showed increased sensitivity to mechanical stimuli. Sensory neurons from these mice showed increased activity of the cation channel Piezo2, which mediates a rapidly adapting mechano-gated current linked to proprioception and touch, and a decrease in mechanically activated, slowly adapting currents. Conversely, overexpression of annexin A6 in sensory neurons inhibited rapidly adapting currents that were partially mediated by Piezo2. Furthermore, overexpression of annexin A6 in sensory neurons attenuated mechanical pain in a mouse model of osteoarthritis, a disease in which mechanically evoked pain is particularly problematic. These data suggest that annexin A6 can be exploited to inhibit chronic mechanical pain..
Gialitakis, M.
Tolaini, M.
Li, Y.
Pardo, M.
Yu, L.
Toribio, A.
Choudhary, J.S.
Niakan, K.
Papayannopoulos, V.
Stockinger, B.
(2017). Activation of the Aryl Hydrocarbon Receptor Interferes with Early Embryonic Development. Stem cell reports,
Vol.9
(5),
pp. 1377-1386.
show abstract
The transcriptional program of early embryonic development is tightly regulated by a set of well-defined transcription factors that suppress premature expression of differentiation genes and sustain the pluripotent identity. It is generally accepted that this program can be perturbed by environmental factors such as chemical pollutants; however, the precise molecular mechanisms remain unknown. The aryl hydrocarbon receptor (AHR) is a widely expressed nuclear receptor that senses environmental stimuli and modulates target gene expression. Here, we have investigated the AHR interactome in embryonic stem cells by mass spectrometry and show that ectopic activation of AHR during early differentiation disrupts the differentiation program via the chromatin remodeling complex NuRD (nucleosome remodeling and deacetylation). The activated AHR/NuRD complex altered the expression of differentiation-specific genes that control the first two developmental decisions without affecting the pluripotency program. These findings identify a mechanism that allows environmental stimuli to disrupt embryonic development through AHR signaling..
Jia, Y.
Marq, J.
Bisio, H.
Jacot, D.
Mueller, C.
Yu, L.
Choudhary, J.
Brochet, M.
Soldati‐Favre, D.
(2017). Crosstalk between
PKA
and
PKG
controls
pH
‐dependent host cell egress of
Toxoplasma gondii. The embo journal,
Vol.36
(21),
pp. 3250-3267.
Bayés, À.
Collins, M.O.
Reig-Viader, R.
Gou, G.
Goulding, D.
Izquierdo, A.
Choudhary, J.S.
Emes, R.D.
Grant, S.G.
(2017). Evolution of complexity in the zebrafish synapse proteome. Nature communications,
Vol.8,
pp. 14613-?.
show abstract
The proteome of human brain synapses is highly complex and is mutated in over 130 diseases. This complexity arose from two whole-genome duplications early in the vertebrate lineage. Zebrafish are used in modelling human diseases; however, its synapse proteome is uncharacterized, and whether the teleost-specific genome duplication (TSGD) influenced complexity is unknown. We report the characterization of the proteomes and ultrastructure of central synapses in zebrafish and analyse the importance of the TSGD. While the TSGD increases overall synapse proteome complexity, the postsynaptic density (PSD) proteome of zebrafish has lower complexity than mammals. A highly conserved set of ∼1,000 proteins is shared across vertebrates. PSD ultrastructural features are also conserved. Lineage-specific proteome differences indicate that vertebrate species evolved distinct synapse types and functions. The data sets are a resource for a wide range of studies and have important implications for the use of zebrafish in modelling human synaptic diseases..
Thomas, D.C.
Clare, S.
Sowerby, J.M.
Pardo, M.
Juss, J.K.
Goulding, D.A.
van der Weyden, L.
Storisteanu, D.
Prakash, A.
Espéli, M.
Flint, S.
Lee, J.C.
Hoenderdos, K.
Kane, L.
Harcourt, K.
Mukhopadhyay, S.
Umrania, Y.
Antrobus, R.
Nathan, J.A.
Adams, D.J.
Bateman, A.
Choudhary, J.S.
Lyons, P.A.
Condliffe, A.M.
Chilvers, E.R.
Dougan, G.
Smith, K.G.
(2017). Eros is a novel transmembrane protein that controls the phagocyte respiratory burst and is essential for innate immunity. The journal of experimental medicine,
Vol.214
(4),
pp. 1111-1128.
show abstract
The phagocyte respiratory burst is crucial for innate immunity. The transfer of electrons to oxygen is mediated by a membrane-bound heterodimer, comprising gp91phox and p22phox subunits. Deficiency of either subunit leads to severe immunodeficiency. We describe Eros (essential for reactive oxygen species), a protein encoded by the previously undefined mouse gene bc017643, and show that it is essential for host defense via the phagocyte NAPDH oxidase. Eros is required for expression of the NADPH oxidase components, gp91phox and p22phox Consequently, Eros-deficient mice quickly succumb to infection. Eros also contributes to the formation of neutrophil extracellular traps (NETS) and impacts on the immune response to melanoma metastases. Eros is an ortholog of the plant protein Ycf4, which is necessary for expression of proteins of the photosynthetic photosystem 1 complex, itself also an NADPH oxio-reductase. We thus describe the key role of the previously uncharacterized protein Eros in host defense..
Weisser, H.
Choudhary, J.S.
(2017). Targeted Feature Detection for Data-Dependent Shotgun Proteomics. J proteome res,
Vol.16
(8),
pp. 2964-2974.
show abstract
Label-free quantification of shotgun LC-MS/MS data is the prevailing approach in quantitative proteomics but remains computationally nontrivial. The central data analysis step is the detection of peptide-specific signal patterns, called features. Peptide quantification is facilitated by associating signal intensities in features with peptide sequences derived from MS2 spectra; however, missing values due to imperfect feature detection are a common problem. A feature detection approach that directly targets identified peptides (minimizing missing values) but also offers robustness against false-positive features (by assigning meaningful confidence scores) would thus be highly desirable. We developed a new feature detection algorithm within the OpenMS software framework, leveraging ideas and algorithms from the OpenSWATH toolset for DIA/SRM data analysis. Our software, FeatureFinderIdentification ("FFId"), implements a targeted approach to feature detection based on information from identified peptides. This information is encoded in an MS1 assay library, based on which ion chromatogram extraction and detection of feature candidates are carried out. Significantly, when analyzing data from experiments comprising multiple samples, our approach distinguishes between "internal" and "external" (inferred) peptide identifications (IDs) for each sample. On the basis of internal IDs, two sets of positive (true) and negative (decoy) feature candidates are defined. A support vector machine (SVM) classifier is then trained to discriminate between the sets and is subsequently applied to the "uncertain" feature candidates from external IDs, facilitating selection and confidence scoring of the best feature candidate for each peptide. This approach also enables our algorithm to estimate the false discovery rate (FDR) of the feature selection step. We validated FFId based on a public benchmark data set, comprising a yeast cell lysate spiked with protein standards that provide a known ground-truth. The algorithm reached almost complete (>99%) quantification coverage for the full set of peptides identified at 1% FDR (PSM level). Compared with other software solutions for label-free quantification, this is an outstanding result, which was achieved at competitive quantification accuracy and reproducibility across replicates. The FDR for the feature selection was estimated at a low 1.5% on average per sample (3% for features inferred from external peptide IDs). The FFId software is open-source and freely available as part of OpenMS ( www.openms.org )..
Collins, M.O.
Woodley, K.T.
Choudhary, J.S.
(2017). Global, site-specific analysis of neuronal protein S-acylation. Scientific reports,
Vol.7
(1),
pp. 4683-?.
show abstract
Protein S-acylation (palmitoylation) is a reversible lipid modification that is an important regulator of dynamic membrane-protein interactions. Proteomic approaches have uncovered many putative palmitoylated proteins however, methods for comprehensive palmitoylation site characterization are lacking. We demonstrate a quantitative site-specific-Acyl-Biotin-Exchange (ssABE) method that allowed the identification of 906 putative palmitoylation sites on 641 proteins from mouse forebrain. 62% of sites map to known palmitoylated proteins and 102 individual palmitoylation sites are known from the literature. 54% of palmitoylation sites map to synaptic proteins including many GPCRs, receptors/ion channels and peripheral membrane proteins. Phosphorylation sites were also identified on a subset of peptides that were palmitoylated, demonstrating for the first time co-identification of these modifications by mass spectrometry. Palmitoylation sites were identified on over half of the family of palmitoyl-acyltransferases (PATs) that mediate protein palmitoylation, including active site thioester-linked palmitoyl intermediates. Distinct palmitoylation motifs and site topology were identified for integral membrane and soluble proteins, indicating potential differences in associated PAT specificity and palmitoylation function. ssABE allows the global identification of palmitoylation sites as well as measurement of the active site modification state of PATs, enabling palmitoylation to be studied at a systems level..
Berger, C.N.
Crepin, V.F.
Roumeliotis, T.I.
Wright, J.C.
Carson, D.
Pevsner-Fischer, M.
Furniss, R.C.
Dougan, G.
Dori-Bachash, M.
Yu, L.
Clements, A.
Collins, J.W.
Elinav, E.
Larrouy-Maumus, G.J.
Choudhary, J.S.
Frankel, G.
(2017). Citrobacter rodentium Subverts ATP Flux and Cholesterol Homeostasis in Intestinal Epithelial Cells In Vivo. Cell metabolism,
Vol.26
(5),
pp. 738-752.e6.
show abstract
The intestinal epithelial cells (IECs) that line the gut form a robust line of defense against ingested pathogens. We investigated the impact of infection with the enteric pathogen Citrobacter rodentium on mouse IEC metabolism using global proteomic and targeted metabolomics and lipidomics. The major signatures of the infection were upregulation of the sugar transporter Sglt4, aerobic glycolysis, and production of phosphocreatine, which mobilizes cytosolic energy. In contrast, biogenesis of mitochondrial cardiolipins, essential for ATP production, was inhibited, which coincided with increased levels of mucosal O2 and a reduction in colon-associated anaerobic commensals. In addition, IECs responded to infection by activating Srebp2 and the cholesterol biosynthetic pathway. Unexpectedly, infected IECs also upregulated the cholesterol efflux proteins AbcA1, AbcG8, and ApoA1, resulting in higher levels of fecal cholesterol and a bloom of Proteobacteria. These results suggest that C. rodentium manipulates host metabolism to evade innate immune responses and establish a favorable gut ecosystem..
Fang, H.
Klages, N.
Baechler, B.
Hillner, E.
Yu, L.
Pardo, M.
Choudhary, J.
Brochet, M.
(2017). Multiple short windows of calcium-dependent protein kinase 4 activity coordinate distinct cell cycle events during Plasmodium gametogenesis. Elife,
Vol.6.
show abstract
Malaria transmission relies on the production of gametes following ingestion by a mosquito. Here, we show that Ca2+-dependent protein kinase 4 controls three processes essential to progress from a single haploid microgametocyte to the release of eight flagellated microgametes in Plasmodium berghei. A myristoylated isoform is activated by Ca2+ to initiate a first genome replication within twenty seconds of activation. This role is mediated by a protein of the SAPS-domain family involved in S-phase entry. At the same time, CDPK4 is required for the assembly of the subsequent mitotic spindle and to phosphorylate a microtubule-associated protein important for mitotic spindle formation. Finally, a non-myristoylated isoform is essential to complete cytokinesis by activating motility of the male flagellum. This role has been linked to phosphorylation of an uncharacterised flagellar protein. Altogether, this study reveals how a kinase integrates and transduces multiple signals to control key cell-cycle transitions during Plasmodium gametogenesis..
Oshota, O.
Conway, M.
Fookes, M.
Schreiber, F.
Chaudhuri, R.R.
Yu, L.
Morgan, F.J.
Clare, S.
Choudhary, J.
Thomson, N.R.
Lio, P.
Maskell, D.J.
Mastroeni, P.
Grant, A.J.
(2017). Transcriptome and proteome analysis of Salmonella enterica serovar Typhimurium systemic infection of wild type and immune-deficient mice. Plos one,
Vol.12
(8),
pp. e0181365-?.
show abstract
Salmonella enterica are a threat to public health. Current vaccines are not fully effective. The ability to grow in infected tissues within phagocytes is required for S. enterica virulence in systemic disease. As the infection progresses the bacteria are exposed to a complex host immune response. Consequently, in order to continue growing in the tissues, S. enterica requires the coordinated regulation of fitness genes. Bacterial gene regulation has so far been investigated largely using exposure to artificial environmental conditions or to in vitro cultured cells, and little information is available on how S. enterica adapts in vivo to sustain cell division and survival. We have studied the transcriptome, proteome and metabolic flux of Salmonella, and the transcriptome of the host during infection of wild type C57BL/6 and immune-deficient gp91-/-phox mice. Our analyses advance the understanding of how S. enterica and the host behaves during infection to a more sophisticated level than has previously been reported..
Modrzynska, K.
Pfander, C.
Chappell, L.
Yu, L.
Suarez, C.
Dundas, K.
Gomes, A.R.
Goulding, D.
Rayner, J.C.
Choudhary, J.
Billker, O.
(2017). A Knockout Screen of ApiAP2 Genes Reveals Networks of Interacting Transcriptional Regulators Controlling the Plasmodium Life Cycle. Cell host & microbe,
Vol.21
(1),
pp. 11-22.
show abstract
A family of apicomplexa-specific proteins containing AP2 DNA-binding domains (ApiAP2s) was identified in malaria parasites. This family includes sequence-specific transcription factors that are key regulators of development. However, functions for the majority of ApiAP2 genes remain unknown. Here, a systematic knockout screen in Plasmodium berghei identified ten ApiAP2 genes that were essential for mosquito transmission: four were critical for the formation of infectious ookinetes, and three were required for sporogony. We describe non-essential functions for AP2-O and AP2-SP proteins in blood stages, and identify AP2-G2 as a repressor active in both asexual and sexual stages. Comparative transcriptomics across mutants and developmental stages revealed clusters of co-regulated genes with shared cis promoter elements, whose expression can be controlled positively or negatively by different ApiAP2 factors. We propose that stage-specific interactions between ApiAP2 proteins on partly overlapping sets of target genes generate the complex transcriptional network that controls the Plasmodium life cycle..
Invergo, B.M.
Brochet, M.
Yu, L.
Choudhary, J.
Beltrao, P.
Billker, O.
(2017). Sub-minute Phosphoregulation of Cell Cycle Systems during Plasmodium Gamete Formation. Cell reports,
Vol.21
(7),
pp. 2017-2029.
show abstract
The transmission of malaria parasites to mosquitoes relies on the rapid induction of sexual reproduction upon their ingestion into a blood meal. Haploid female and male gametocytes become activated and emerge from their host cells, and the males enter the cell cycle to produce eight microgametes. The synchronized nature of gametogenesis allowed us to investigate phosphorylation signaling during its first minute in Plasmodium berghei via a high-resolution time course of the phosphoproteome. This revealed an unexpectedly broad response, with proteins related to distinct cell cycle events undergoing simultaneous phosphoregulation. We implicate several protein kinases in the process, and we validate our analyses on the plant-like calcium-dependent protein kinase 4 (CDPK4) and a homolog of serine/arginine-rich protein kinases (SRPK1). Mutants in these kinases displayed distinct phosphoproteomic disruptions, consistent with differences in their phenotypes. The results reveal the central role of protein phosphorylation in the atypical cell cycle regulation of a divergent eukaryote..
Pardo, M.
Yu, L.
Shen, S.
Tate, P.
Bode, D.
Letney, B.L.
Quelle, D.E.
Skarnes, W.
Choudhary, J.S.
(2017). Myst2/Kat7 histone acetyltransferase interaction proteomics reveals tumour-suppressor Niam as a novel binding partner in embryonic stem cells. Scientific reports,
Vol.7
(1),
pp. 8157-?.
show abstract
MYST histone acetyltransferases have crucial functions in transcription, replication and DNA repair and are hence implicated in development and cancer. Here we characterise Myst2/Kat7/Hbo1 protein interactions in mouse embryonic stem cells by affinity purification coupled to mass spectrometry. This study confirms that in embryonic stem cells Myst2 is part of H3 and H4 histone acetylation complexes similar to those described in somatic cells. We identify a novel Myst2-associated protein, the tumour suppressor protein Niam (Nuclear Interactor of ARF and Mdm2). Human NIAM is involved in chromosome segregation, p53 regulation and cell proliferation in somatic cells, but its role in embryonic stem cells is unknown. We describe the first Niam embryonic stem cell interactome, which includes proteins with roles in DNA replication and repair, transcription, splicing and ribosome biogenesis. Many of Myst2 and Niam binding partners are required for correct embryonic development, implicating Myst2 and Niam in the cooperative regulation of this process and suggesting a novel role for Niam in embryonic biology. The data provides a useful resource for exploring Myst2 and Niam essential cellular functions and should contribute to deeper understanding of organism early development and survival as well as cancer. Data are available via ProteomeXchange with identifier PXD005987..
Fernández, E.
Collins, M.O.
Frank, R.A.
Zhu, F.
Kopanitsa, M.V.
Nithianantharajah, J.
Lemprière, S.A.
Fricker, D.
Elsegood, K.A.
McLaughlin, C.L.
Croning, M.D.
Mclean, C.
Armstrong, J.D.
Hill, W.D.
Deary, I.J.
Cencelli, G.
Bagni, C.
Fromer, M.
Purcell, S.M.
Pocklington, A.J.
Choudhary, J.S.
Komiyama, N.H.
Grant, S.G.
(2017). Arc Requires PSD95 for Assembly into Postsynaptic Complexes Involved with Neural Dysfunction and Intelligence. Cell reports,
Vol.21
(3),
pp. 679-691.
show abstract
Arc is an activity-regulated neuronal protein, but little is known about its interactions, assembly into multiprotein complexes, and role in human disease and cognition. We applied an integrated proteomic and genetic strategy by targeting a tandem affinity purification (TAP) tag and Venus fluorescent protein into the endogenous Arc gene in mice. This allowed biochemical and proteomic characterization of native complexes in wild-type and knockout mice. We identified many Arc-interacting proteins, of which PSD95 was the most abundant. PSD95 was essential for Arc assembly into 1.5-MDa complexes and activity-dependent recruitment to excitatory synapses. Integrating human genetic data with proteomic data showed that Arc-PSD95 complexes are enriched in schizophrenia, intellectual disability, autism, and epilepsy mutations and normal variants in intelligence. We propose that Arc-PSD95 postsynaptic complexes potentially affect human cognitive function..
Steinberg, J.
Ritchie, G.R.
Roumeliotis, T.I.
Jayasuriya, R.L.
Clark, M.J.
Brooks, R.A.
Binch, A.L.
Shah, K.M.
Coyle, R.
Pardo, M.
Le Maitre, C.L.
Ramos, Y.F.
Nelissen, R.G.
Meulenbelt, I.
McCaskie, A.W.
Choudhary, J.S.
Wilkinson, J.M.
Zeggini, E.
(2017). Integrative epigenomics, transcriptomics and proteomics of patient chondrocytes reveal genes and pathways involved in osteoarthritis. Scientific reports,
Vol.7
(1),
pp. 8935-?.
show abstract
Osteoarthritis (OA) is a common disease characterized by cartilage degeneration and joint remodeling. The underlying molecular changes underpinning disease progression are incompletely understood. We investigated genes and pathways that mark OA progression in isolated primary chondrocytes taken from paired intact versus degraded articular cartilage samples across 38 patients undergoing joint replacement surgery (discovery cohort: 12 knee OA, replication cohorts: 17 knee OA, 9 hip OA patients). We combined genome-wide DNA methylation, RNA sequencing, and quantitative proteomics data. We identified 49 genes differentially regulated between intact and degraded cartilage in at least two -omics levels, 16 of which have not previously been implicated in OA progression. Integrated pathway analysis implicated the involvement of extracellular matrix degradation, collagen catabolism and angiogenesis in disease progression. Using independent replication datasets, we showed that the direction of change is consistent for over 90% of differentially expressed genes and differentially methylated CpG probes. AQP1, COL1A1 and CLEC3B were significantly differentially regulated across all three -omics levels, confirming their differential expression in human disease. Through integration of genome-wide methylation, gene and protein expression data in human primary chondrocytes, we identified consistent molecular players in OA progression that replicated across independent datasets and that have translational potential..
Schlaffner, C.N.
Pirklbauer, G.J.
Bender, A.
Choudhary, J.S.
(2017). Fast, Quantitative and Variant Enabled Mapping of Peptides to Genomes. Cell systems,
Vol.5
(2),
pp. 152-156.e4.
show abstract
Current tools for visualization and integration of proteomics with other omics datasets are inadequate for large-scale studies and capture only basic sequence identity information. Furthermore, the frequent reformatting of annotations for reference genomes required by these tools is known to be highly error prone. We developed PoGo for mapping peptides identified through mass spectrometry to overcome these limitations. PoGo reduced runtime and memory usage by 85% and 20%, respectively, and exhibited overall superior performance over other tools on benchmarking with large-scale human tissue and cancer phosphoproteome datasets comprising ∼3 million peptides. In addition, extended functionality enables representation of single-nucleotide variants, post-translational modifications, and quantitative features. PoGo has been integrated in established frameworks such as the PRIDE tool suite and OpenMS, as well as a standalone tool with user-friendly graphical interface. With the rapid increase of quantitative high-resolution datasets capturing proteomes and global modifications to complement orthogonal genomics platforms, PoGo provides a central utility enabling large-scale visualization and interpretation of transomics datasets..
Yeung, A.T.
Hale, C.
Lee, A.H.
Gill, E.E.
Bushell, W.
Parry-Smith, D.
Goulding, D.
Pickard, D.
Roumeliotis, T.
Choudhary, J.
Thomson, N.
Skarnes, W.C.
Dougan, G.
Hancock, R.E.
(2017). Exploiting induced pluripotent stem cell-derived macrophages to unravel host factors influencing Chlamydia trachomatis pathogenesis. Nature communications,
Vol.8,
pp. 15013-?.
show abstract
Chlamydia trachomatis remains a leading cause of bacterial sexually transmitted infections and preventable blindness worldwide. There are, however, limited in vitro models to study the role of host genetics in the response of macrophages to this obligate human pathogen. Here, we describe an approach using macrophages derived from human induced pluripotent stem cells (iPSdMs) to study macrophage-Chlamydia interactions in vitro. We show that iPSdMs support the full infectious life cycle of C. trachomatis in a manner that mimics the infection of human blood-derived macrophages. Transcriptomic and proteomic profiling of the macrophage response to chlamydial infection highlighted the role of the type I interferon and interleukin 10-mediated responses. Using CRISPR/Cas9 technology, we generated biallelic knockout mutations in host genes encoding IRF5 and IL-10RA in iPSCs, and confirmed their roles in limiting chlamydial infection in macrophages. This model can potentially be extended to other pathogens and tissue systems to advance our understanding of host-pathogen interactions and the role of human genetics in influencing the outcome of infections..
Roumeliotis, T.I.
Williams, S.P.
Gonçalves, E.
Alsinet, C.
Del Castillo Velasco-Herrera, M.
Aben, N.
Ghavidel, F.Z.
Michaut, M.
Schubert, M.
Price, S.
Wright, J.C.
Yu, L.
Yang, M.
Dienstmann, R.
Guinney, J.
Beltrao, P.
Brazma, A.
Pardo, M.
Stegle, O.
Adams, D.J.
Wessels, L.
Saez-Rodriguez, J.
McDermott, U.
Choudhary, J.S.
(2017). Genomic Determinants of Protein Abundance Variation in Colorectal Cancer Cells. Cell reports,
Vol.20
(9),
pp. 2201-2214.
show abstract
Assessing the impact of genomic alterations on protein networks is fundamental in identifying the mechanisms that shape cancer heterogeneity. We have used isobaric labeling to characterize the proteomic landscapes of 50 colorectal cancer cell lines and to decipher the functional consequences of somatic genomic variants. The robust quantification of over 9,000 proteins and 11,000 phosphopeptides on average enabled the de novo construction of a functional protein correlation network, which ultimately exposed the collateral effects of mutations on protein complexes. CRISPR-cas9 deletion of key chromatin modifiers confirmed that the consequences of genomic alterations can propagate through protein interactions in a transcript-independent manner. Lastly, we leveraged the quantified proteome to perform unsupervised classification of the cell lines and to build predictive models of drug response in colorectal cancer. Overall, we provide a deep integrative view of the functional network and the molecular structure underlying the heterogeneity of colorectal cancer cells..
Scanlan, E.
Yu, L.
Maskell, D.
Choudhary, J.
Grant, A.
(2017). A quantitative proteomic screen of the Campylobacter jejuni flagellar-dependent secretome. Journal of proteomics,
Vol.152,
pp. 181-187.
show abstract
UNLABELLED: Campylobacter jejuni is the leading cause of bacterial gastroenteritis in the world. A number of factors are believed to contribute to the ability of C. jejuni to cause disease within the human host including the secretion of non-flagellar proteins via the flagellar type III secretion system (FT3SS). Here for the first time we have utilised quantitative proteomics using stable isotope labelling by amino acids in cell culture (SILAC), and label-free liquid chromatography-mass spectrometry (LC/MS), to compare supernatant samples from C. jejuni M1 wild type and flagella-deficient (flgG mutant) strains to identify putative novel proteins secreted via the FT3SS. Genes encoding proteins that were candidates for flagellar secretion, derived from the LC/MS and SILAC datasets, were deleted. Infection of human CACO-2 tissue culture cells using these mutants resulted in the identification of novel genes required for interactions with these cells. This work has shown for the first time that both CJM1_0791 and CJM1_0395 are dependent on the flagellum for their presence in supernatants from C. jejuni stains M1 and 81-176. BIOLOGICAL SIGNIFICANCE: This study provides the most complete description of the Campylobac er jejuni secretome to date. SILAC and label-free proteomics comparing mutants with or without flagella have resulted in the identification of two C. jejuni proteins that are dependent on flagella for their export from the bacterial cell..
Tay, C.L.
Jones, M.L.
Hodson, N.
Theron, M.
Choudhary, J.S.
Rayner, J.C.
(2016). Study of Plasmodium falciparum DHHC palmitoyl transferases identifies a role for PfDHHC9 in gametocytogenesis. Cellular microbiology,
Vol.18
(11),
pp. 1596-1610.
show abstract
Palmitoylation is the post-translational reversible addition of the acyl moiety, palmitate, to cysteine residues of proteins and is involved in regulating protein trafficking, localization, stability and function. The Aspartate-Histidine-Histidine-Cysteine (DHHC) protein family, named for their highly conserved DHHC signature motif, is thought to be responsible for catalysing protein palmitoylation. Palmitoylation is widespread in all eukaryotes, including the malaria parasite, Plasmodium falciparum, where over 400 palmitoylated proteins are present in the asexual intraerythrocytic schizont stage parasites, including proteins involved in key aspects of parasite maturation and development. The P. falciparum genome includes 12 proteins containing the conserved DHHC motif. In this study, we adapted a palmitoyl-transferase activity assay for use with P. falciparum proteins and demonstrated for the first time that P. falciparum DHHC proteins are responsible for the palmitoylation of P. falciparum substrates. This assay also reveals that multiple DHHCs are capable of palmitoylating the same substrate, indicating functional redundancy at least in vitro. To test whether functional redundancy also exists in vivo, we investigated the endogenous localization and essentiality of a subset of schizont-expressed PfDHHC proteins. Individual PfDHHC proteins localized to distinct organelles, including parasite-specific organelles such as the rhoptries and inner membrane complex. Knock-out studies identified individual DHHCs that may be essential for blood-stage growth and others that were functionally redundant in the blood stages but may have functions in other stages of parasite development. Supporting this hypothesis, disruption of PfDHHC9 had no effect on blood-stage growth but reduced the formation of gametocytes, suggesting that this protein could be exploited as a transmission-blocking target. The localization and stage-specific expression of the DHHC proteins may be important for regulating their substrate specificity and thus may provide a path for inhibitor development..
Bode, D.
Yu, L.
Tate, P.
Pardo, M.
Choudhary, J.
(2016). Characterization of Two Distinct Nucleosome Remodeling and Deacetylase (NuRD) Complex Assemblies in Embryonic Stem Cells. Molecular & cellular proteomics : mcp,
Vol.15
(3),
pp. 878-891.
show abstract
Pluripotency and self-renewal, the defining properties of embryonic stem cells, are brought about by transcriptional programs involving an intricate network of transcription factors and chromatin remodeling complexes. The Nucleosome Remodeling and Deacetylase (NuRD) complex plays a crucial and dynamic role in the regulation of stemness and differentiation. Several NuRD-associated factors have been reported but how they are organized has not been investigated in detail. Here, we have combined affinity purification and blue native polyacrylamide gel electrophoresis followed by protein identification by mass spectrometry and protein correlation profiling to characterize the topology of the NuRD complex. Our data show that in mouse embryonic stem cells the NuRD complex is present as two distinct assemblies of differing topology with different binding partners. Cell cycle regulator Cdk2ap1 and transcription factor Sall4 associate only with the higher mass NuRD assembly. We further establish that only isoform Sall4a, and not Sall4b, associates with NuRD. By contrast, Suz12, a component of the PRC2 Polycomb repressor complex, associates with the lower mass entity. In addition, we identify and validate a novel NuRD-associated protein, Wdr5, a regulatory subunit of the MLL histone methyltransferase complex, which associates with both NuRD entities. Bioinformatic analyses of published target gene sets of these chromatin binding proteins are in agreement with these structural observations. In summary, this study provides an interesting insight into mechanistic aspects of NuRD function in stem cell biology. The relevance of our work has broader implications because of the ubiquitous nature of the NuRD complex. The strategy described here can be more broadly applicable to investigate the topology of the multiple complexes an individual protein can participate in..
Maggiore, L.
Yu, L.
Omasits, U.
Rossi, O.
Dougan, G.
Thomson, N.R.
Saul, A.
Choudhary, J.S.
Gerke, C.
(2016). Quantitative proteomic analysis of Shigella flexneri and Shigella sonnei Generalized Modules for Membrane Antigens (GMMA) reveals highly pure preparations. International journal of medical microbiology,
Vol.306
(2),
pp. 99-108.
Wright, J.C.
Mudge, J.
Weisser, H.
Barzine, M.P.
Gonzalez, J.M.
Brazma, A.
Choudhary, J.S.
Harrow, J.
(2016). Improving GENCODE reference gene annotation using a high-stringency proteogenomics workflow. Nature communications,
Vol.7,
pp. 11778-?.
show abstract
Complete annotation of the human genome is indispensable for medical research. The GENCODE consortium strives to provide this, augmenting computational and experimental evidence with manual annotation. The rapidly developing field of proteogenomics provides evidence for the translation of genes into proteins and can be used to discover and refine gene models. However, for both the proteomics and annotation groups, there is a lack of guidelines for integrating this data. Here we report a stringent workflow for the interpretation of proteogenomic data that could be used by the annotation community to interpret novel proteogenomic evidence. Based on reprocessing of three large-scale publicly available human data sets, we show that a conservative approach, using stringent filtering is required to generate valid identifications. Evidence has been found supporting 16 novel protein-coding genes being added to GENCODE. Despite this many peptide identifications in pseudogenes cannot be annotated due to the absence of orthogonal supporting evidence..
Weisser, H.
Wright, J.C.
Mudge, J.M.
Gutenbrunner, P.
Choudhary, J.S.
(2016). Flexible Data Analysis Pipeline for High-Confidence Proteogenomics. J proteome res,
Vol.15
(12),
pp. 4686-4695.
show abstract
Proteogenomics leverages information derived from proteomic data to improve genome annotations. Of particular interest are "novel" peptides that provide direct evidence of protein expression for genomic regions not previously annotated as protein-coding. We present a modular, automated data analysis pipeline aimed at detecting such "novel" peptides in proteomic data sets. This pipeline implements criteria developed by proteomics and genome annotation experts for high-stringency peptide identification and filtering. Our pipeline is based on the OpenMS computational framework; it incorporates multiple database search engines for peptide identification and applies a machine-learning approach (Percolator) to post-process search results. We describe several new and improved software tools that we developed to facilitate proteogenomic analyses that enhance the wealth of tools provided by OpenMS. We demonstrate the application of our pipeline to a human testis tissue data set previously acquired for the Chromosome-Centric Human Proteome Project, which led to the addition of five new gene annotations on the human reference genome..
Petryszak, R.
Keays, M.
Tang, Y.A.
Fonseca, N.A.
Barrera, E.
Burdett, T.
Füllgrabe, A.
Fuentes, A.M.
Jupp, S.
Koskinen, S.
Mannion, O.
Huerta, L.
Megy, K.
Snow, C.
Williams, E.
Barzine, M.
Hastings, E.
Weisser, H.
Wright, J.
Jaiswal, P.
Huber, W.
Choudhary, J.
Parkinson, H.E.
Brazma, A.
(2016). Expression Atlas update—an integrated database of gene and protein expression in humans, animals and plants. Nucleic acids research,
Vol.44
(D1),
pp. D746-D752.
Steinberg, J.
Ritchie, G.R.
Roumeliotis, T.I.
Jayasuriya, R.L.
Brooks, R.A.
Binch, A.L.
Shah, K.M.
Coyle, R.
Pardo, M.
Le Maitre, C.L.
Ramos, Y.F.
Nelissen, R.G.
Meulenbelt, I.
McCaskie, A.W.
Choudhary, J.S.
Mark Wilkinson, J.
Zeggini, E.
(2016). Integrative epigenomics, transcriptomics and proteomics of patient chondrocytes reveal genes and pathways involved in osteoarthritis. ,
.
show abstract
ABSTRACTBackgroundOsteoarthritis (OA) is a common disease characterized by cartilage degeneration and joint remodeling. The underlying molecular changes underpinning disease progression are incompletely understood, but can be characterized using recent advances in genomics technologies, as the relevant tissue is readily accessible at joint replacement surgery. Here we investigate genes and pathways that mark OA progression, combining genome-wide DNA methylation, RNA sequencing and quantitative proteomics in isolated primary chondrocytes from matched intact and degraded articular cartilage samples across twelve patients with OA undergoing knee replacement surgery.ResultsWe identify 49 genes differentially regulated between intact and degraded cartilage at multiple omics levels, 16 of which have not previously been implicated in OA progression. Using independent replication datasets, we replicate statistically significant signals and show that the direction of change is consistent for over 90% of differentially expressed genes and differentially methylated CpG probes. Three genes are differentially regulated across all 3 omics levels: AQP1, COL1A1 and CLEC3B, and all three have evidence implicating them in OA through animal or cellular model studies. Integrated pathway analysis implicates the involvement of extracellular matrix degradation, collagen catabolism and angiogenesis in disease progression. All data from these experiments are freely available as a resource for the scientific community.ConclusionsThis work provides a first integrated view of the molecular landscape of human primary chondrocytes and identifies key molecular players in OA progression that replicate across independent datasets, with evidence for translational potential..
Röst, H.L.
Sachsenberg, T.
Aiche, S.
Bielow, C.
Weisser, H.
Aicheler, F.
Andreotti, S.
Ehrlich, H.-.
Gutenbrunner, P.
Kenar, E.
Liang, X.
Nahnsen, S.
Nilse, L.
Pfeuffer, J.
Rosenberger, G.
Rurik, M.
Schmitt, U.
Veit, J.
Walzer, M.
Wojnar, D.
Wolski, W.E.
Schilling, O.
Choudhary, J.S.
Malmström, L.
Aebersold, R.
Reinert, K.
Kohlbacher, O.
(2016). OpenMS: a flexible open-source software platform for mass spectrometry data analysis. Nature methods,
Vol.13
(9),
pp. 741-748.
show abstract
High-resolution mass spectrometry (MS) has become an important tool in the life sciences, contributing to the diagnosis and understanding of human diseases, elucidating biomolecular structural information and characterizing cellular signaling networks. However, the rapid growth in the volume and complexity of MS data makes transparent, accurate and reproducible analysis difficult. We present OpenMS 2.0 (http://www.openms.de), a robust, open-source, cross-platform software specifically designed for the flexible and reproducible analysis of high-throughput MS data. The extensible OpenMS software implements common mass spectrometric data processing tasks through a well-defined application programming interface in C++ and Python and through standardized open data formats. OpenMS additionally provides a set of 185 tools and ready-made workflows for common mass spectrometric data processing tasks, which enable users to perform complex quantitative mass spectrometric analyses with ease..
Blohmke, C.J.
Darton, T.C.
Jones, C.
Suarez, N.M.
Waddington, C.S.
Angus, B.
Zhou, L.
Hill, J.
Clare, S.
Kane, L.
Mukhopadhyay, S.
Schreiber, F.
Duque-Correa, M.A.
Wright, J.C.
Roumeliotis, T.I.
Yu, L.
Choudhary, J.S.
Mejias, A.
Ramilo, O.
Shanyinde, M.
Sztein, M.B.
Kingsley, R.A.
Lockhart, S.
Levine, M.M.
Lynn, D.J.
Dougan, G.
Pollard, A.J.
(2016). Interferon-driven alterations of the host's amino acid metabolism in the pathogenesis of typhoid fever. The journal of experimental medicine,
Vol.213
(6),
pp. 1061-1077.
show abstract
Enteric fever, caused by Salmonella enterica serovar Typhi, is an important public health problem in resource-limited settings and, despite decades of research, human responses to the infection are poorly understood. In 41 healthy adults experimentally infected with wild-type S. Typhi, we detected significant cytokine responses within 12 h of bacterial ingestion. These early responses did not correlate with subsequent clinical disease outcomes and likely indicate initial host-pathogen interactions in the gut mucosa. In participants developing enteric fever after oral infection, marked transcriptional and cytokine responses during acute disease reflected dominant type I/II interferon signatures, which were significantly associated with bacteremia. Using a murine and macrophage infection model, we validated the pivotal role of this response in the expression of proteins of the host tryptophan metabolism during Salmonella infection. Corresponding alterations in tryptophan catabolites with immunomodulatory properties in serum of participants with typhoid fever confirmed the activity of this pathway, and implicate a central role of host tryptophan metabolism in the pathogenesis of typhoid fever..
Hodson, N.
Invergo, B.
Rayner, J.C.
Choudhary, J.S.
(2015). Palmitoylation and palmitoyl-transferases in Plasmodium parasites. Biochemical society transactions,
Vol.43
(2),
pp. 240-245.
show abstract
Protein post-translational modifications (PTM) are commonly used to regulate biological processes. Protein S-acylation is an enzymatically regulated reversible modification that has been shown to modulate protein localization, activity and membrane binding. Proteome-scale discovery on Plasmodium falciparum schizonts has revealed a complement of more than 400 palmitoylated proteins, including those essential for host invasion and drug resistance. The wide regulatory affect on this species is endorsed by the presence of 12 proteins containing the conserved DHHC–CRD (DHHC motif within a cysteine-rich domain) that is associated with palmitoyl-transferase activity. Genetic interrogation of these enzymes in Apicomplexa has revealed essentiality and distinct localization at cellular compartments; these features are species specific and are not observed in yeast. It is clear that palmitoylation has an elaborate role in Plasmodium biology and opens intriguing questions on the functional consequence of this group of acylation modifications and how the protein S-acyl transferases (PATs) orchestrate molecular events..
Jasuja, N.D.
Sharma, S.
Choudhary, J.
Joshi, S.C.
(2015). Essential Oil and Important Activities ofThuja orientalisandThuja occidentalis. Journal of essential oil bearing plants,
Vol.18
(4),
pp. 931-949.
Peltier, J.
Shaw, H.A.
Couchman, E.C.
Dawson, L.F.
Yu, L.
Choudhary, J.S.
Kaever, V.
Wren, B.W.
Fairweather, N.F.
(2015). Cyclic diGMP Regulates Production of Sortase Substrates of Clostridium difficile and Their Surface Exposure through ZmpI Protease-mediated Cleavage. Journal of biological chemistry,
Vol.290
(40),
pp. 24453-24469.
Kunowska, N.
Rotival, M.
Yu, L.
Choudhary, J.
Dillon, N.
(2015). Identification of protein complexes that bind to histone H3 combinatorial modifications using super-SILAC and weighted correlation network analysis. Nucleic acids research,
Vol.43
(3),
pp. 1418-1432.
show abstract
The large number of chemical modifications that are found on the histone proteins of eukaryotic cells form multiple complex combinations, which can act as recognition signals for reader proteins. We have used peptide capture in conjunction with super-SILAC quantification to carry out an unbiased high-throughput analysis of the composition of protein complexes that bind to histone H3K9/S10 and H3K27/S28 methyl-phospho modifications. The accurate quantification allowed us to perform Weighted correlation network analysis (WGCNA) to obtain a systems-level view of the histone H3 histone tail interactome. The analysis reveals the underlying modularity of the histone reader network with members of nuclear complexes exhibiting very similar binding signatures, which suggests that many proteins bind to histones as part of pre-organized complexes. Our results identify a novel complex that binds to the double H3K9me3/S10ph modification, which includes Atrx, Daxx and members of the FACT complex. The super-SILAC approach allows comparison of binding to multiple peptides with different combinations of modifications and the resolution of the WGCNA analysis is enhanced by maximizing the number of combinations that are compared. This makes it a useful approach for assessing the effects of changes in histone modification combinations on the composition and function of bound complexes..
Mehla, K.
Magotra, A.
Choudhary, J.
Singh, A.K.
Mohanty, A.K.
Upadhyay, R.C.
Srinivasan, S.
Gupta, P.
Choudhary, N.
Antony, B.
Khan, F.
(2014). Genome-wide analysis of the heat stress response in Zebu (Sahiwal) cattle. Gene,
Vol.533
(2),
pp. 500-507.
Collins, M.O.
Wright, J.C.
Jones, M.
Rayner, J.C.
Choudhary, J.S.
(2014). Confident and sensitive phosphoproteomics using combinations of collision induced dissociation and electron transfer dissociation. Journal of proteomics,
Vol.103,
pp. 1-14.
Wen, B.
Li, G.
Wright, J.C.
Du, C.
Feng, Q.
Xu, X.
Choudhary, J.S.
Wang, J.
(2014). The OMSSAPercolator: An automated tool to validate OMSSA results. Proteomics,
Vol.14
(9),
pp. 1011-1014.
show abstract
Protein identification by MS/MS is an important technique in proteome studies. The Open Mass Spectrometry Search Algorithm (OMSSA) is an open‐source search engine that can be used to identify MS/MS spectra acquired in these experiments. Here, we present a software tool, termed OMSSAPercolator, which interfaces OMSSA with Percolator, a post‐search machine learning method for rescoring database search results. We demonstrate that it outperforms the standard OMSSA scoring scheme, and provides reliable significant measurements. OMSSAPercolator is programmed using JAVA and can be readily used as a standalone tool or integrated into existing data analysis pipelines. OMSSAPercolator is freely available and can be downloaded at http://sourceforge.net/projects/omssapercolator/..
Bayés, À.
Collins, M.O.
Galtrey, C.M.
Simonnet, C.
Roy, M.
Croning, M.D.
Gou, G.
van de Lagemaat, L.N.
Milward, D.
Whittle, I.R.
Smith, C.
Choudhary, J.S.
Grant, S.G.
(2014). Human post-mortem synapse proteome integrity screening for proteomic studies of postsynaptic complexes. Molecular brain,
Vol.7
(1).
Pettit, L.J.
Browne, H.P.
Yu, L.
Smits, W.K.
Fagan, R.P.
Barquist, L.
Martin, M.J.
Goulding, D.
Duncan, S.H.
Flint, H.J.
Dougan, G.
Choudhary, J.S.
Lawley, T.D.
(2014). Functional genomics reveals that Clostridium difficileSpo0A coordinates sporulation, virulence and metabolism. Bmc genomics,
Vol.15
(1).
Purcell, S.M.
Moran, J.L.
Fromer, M.
Ruderfer, D.
Solovieff, N.
Roussos, P.
O’Dushlaine, C.
Chambert, K.
Bergen, S.E.
Kähler, A.
Duncan, L.
Stahl, E.
Genovese, G.
Fernández, E.
Collins, M.O.
Komiyama, N.H.
Choudhary, J.S.
Magnusson, P.K.
Banks, E.
Shakir, K.
Garimella, K.
Fennell, T.
DePristo, M.
Grant, S.G.
Haggarty, S.J.
Gabriel, S.
Scolnick, E.M.
Lander, E.S.
Hultman, C.M.
Sullivan, P.F.
McCarroll, S.A.
Sklar, P.
(2014). A polygenic burden of rare disruptive mutations in schizophrenia. Nature,
Vol.506
(7487),
pp. 185-190.
Mousnier, A.
Schroeder, G.N.
Stoneham, C.A.
So, E.C.
Garnett, J.A.
Yu, L.
Matthews, S.J.
Choudhary, J.S.
Hartland, E.L.
Frankel, G.
(2014). A New Method To Determine
In Vivo
Interactomes Reveals Binding of the Legionella pneumophila Effector PieE to Multiple Rab GTPases. Mbio,
Vol.5
(4).
show abstract
ABSTRACT
Legionella pneumophila
, the causative agent of Legionnaires’ disease, uses the Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effectors into host cells, where they subvert host cell signaling. The function and host cell targets of most effectors remain unknown. PieE is a 69-kDa Dot/Icm effector containing three coiled-coil (CC) regions and 2 transmembrane (TM) helices followed by a fourth CC region. Here, we report that PieE dimerized by an interaction between CC3 and CC4. We found that ectopically expressed PieE localized to the endoplasmic reticulum (ER) and induced the formation of organized smooth ER, while following infection PieE localized to the
Legionella
-containing vacuole (LCV). To identify the physiological targets of PieE during infection, we established a new purification method for which we created an A549 cell line stably expressing the
Escherichia coli
biotin ligase BirA and infected the cells with
L. pneumophila
expressing PieE fused to a BirA-specific biotinylation site and a hexahistidine tag. Following tandem Ni
2+
nitrilotriacetic acid (NTA) and streptavidin affinity chromatography, the effector-target complexes were analyzed by mass spectrometry. This revealed interactions of PieE with multiple host cell proteins, including the Rab GTPases 1a, 1b, 2a, 5c, 6a, 7, and 10. Binding of the Rab GTPases, which was validated by yeast two-hybrid binding assays, was mediated by the PieE CC1 and CC2. In summary, using a novel, highly specific strategy to purify effector complexes from infected cells, which is widely applicable to other pathogens, we identified PieE as a multidomain LCV protein with promiscuous Rab GTPase-binding capacity.
IMPORTANCE
The respiratory pathogen
Legionella pneumophila
uses the Dot/Icm type IV secretion system to translocate more than 300 effector proteins into host cells. The function of most effectors in infection remains unknown. One of the bottlenecks for their characterization is the identification of target proteins. Frequently used
in vitro
approaches are not applicable to all effectors and suffer from high rates of false positives or missed interactions, as they are not performed in the context of an infection. Here, we determine key functional domains of the effector PieE and describe a new method to identify host cell targets under physiological infection conditions. Our approach, which is applicable to other pathogens, uncovered the interaction of PieE with several proteins involved in membrane trafficking, in particular Rab GTPases, revealing new details of the
Legionella
infection strategy and demonstrating the potential of this method to greatly advance our understanding of the molecular basis of infection.
.
Pardo, M.
Choudhary, J.S.
(2012). Assignment of Protein Interactions from Affinity Purification/Mass Spectrometry Data. Journal of proteome research,
Vol.11
(3),
pp. 1462-1474.
Kirov, G.
Pocklington, A.J.
Holmans, P.
Ivanov, D.
Ikeda, M.
Ruderfer, D.
Moran, J.
Chambert, K.
Toncheva, D.
Georgieva, L.
Grozeva, D.
Fjodorova, M.
Wollerton, R.
Rees, E.
Nikolov, I.
van de Lagemaat, L.N.
Bayés, À.
Fernandez, E.
Olason, P.I.
Böttcher, Y.
Komiyama, N.H.
Collins, M.O.
Choudhary, J.
Stefansson, K.
Stefansson, H.
Grant, S.G.
Purcell, S.
Sklar, P.
O'Donovan, M.C.
Owen, M.J.
(2012). De novo CNV analysis implicates specific abnormalities of postsynaptic signalling complexes in the pathogenesis of schizophrenia. Molecular psychiatry,
Vol.17
(2),
pp. 142-153.
Sebastian, S.
Brochet, M.
Collins, M.O.
Schwach, F.
Jones, M.L.
Goulding, D.
Rayner, J.C.
Choudhary, J.S.
Billker, O.
(2012). A Plasmodium Calcium-Dependent Protein Kinase Controls Zygote Development and Transmission by Translationally Activating Repressed mRNAs. Cell host & microbe,
Vol.12
(1),
pp. 9-19.
Jones, M.L.
Collins, M.O.
Goulding, D.
Choudhary, J.S.
Rayner, J.C.
(2012). Analysis of Protein Palmitoylation Reveals a Pervasive Role in Plasmodium Development and Pathogenesis. Cell host & microbe,
Vol.12
(2),
pp. 246-258.
Wright, J.C.
Collins, M.O.
Yu, L.
Käll, L.
Brosch, M.
Choudhary, J.S.
(2012). Enhanced Peptide Identification by Electron Transfer Dissociation Using an Improved Mascot Percolator. Molecular & cellular proteomics,
Vol.11
(8),
pp. 478-491.
Wilson, C.H.
Crombie, C.
van der Weyden, L.
Poulogiannis, G.
Rust, A.G.
Pardo, M.
Gracia, T.
Yu, L.
Choudhary, J.
Poulin, G.B.
McIntyre, R.E.
Winton, D.J.
March, H.N.
Arends, M.J.
Fraser, A.G.
Adams, D.J.
(2012). Nuclear receptor binding protein 1 regulates intestinal progenitor cell homeostasis and tumour formation. The embo journal,
Vol.31
(11),
pp. 2486-2497.
Chaudhuri, R.R.
Yu, L.
Kanji, A.
Perkins, T.T.
Gardner, P.P.
Choudhary, J.
Maskell, D.J.
Grant, A.J.
(2011). Quantitative RNA-seq analysis of the Campylobacter jejuni transcriptome. Microbiology,
Vol.157
(10),
pp. 2922-2932.
show abstract
C
ampylobacter jejuni is the most common bacterial cause of foodborne disease in the developed world. Its general physiology and biochemistry, as well as the mechanisms enabling it to colonize and cause disease in various hosts, are not well understood, and new approaches are required to understand its basic biology. High-throughput sequencing technologies provide unprecedented opportunities for functional genomic research. Recent studies have shown that direct Illumina sequencing of cDNA (RNA-seq) is a useful technique for the quantitative and qualitative examination of transcriptomes. In this study we report RNA-seq analyses of the transcriptomes of C. jejuni (NCTC11168) and its rpoN mutant. This has allowed the identification of hitherto unknown transcriptional units, and further defines the regulon that is dependent on rpoN for expression. The analysis of the NCTC11168 transcriptome was supplemented by additional proteomic analysis using liquid chromatography-MS. The transcriptomic and proteomic datasets represent an important resource for the Campylobacter research community..
Bayés, À.
van de Lagemaat, L.N.
Collins, M.O.
Croning, M.D.
Whittle, I.R.
Choudhary, J.S.
Grant, S.G.
(2011). Characterization of the proteome, diseases and evolution of the human postsynaptic density. Nature neuroscience,
Vol.14
(1),
pp. 19-21.
Brosch, M.
Saunders, G.I.
Frankish, A.
Collins, M.O.
Yu, L.
Wright, J.
Verstraten, R.
Adams, D.J.
Harrow, J.
Choudhary, J.S.
Hubbard, T.
(2011). Shotgun proteomics aids discovery of novel protein-coding genes, alternative splicing, and “resurrected” pseudogenes in the mouse genome. Genome research,
Vol.21
(5),
pp. 756-767.
show abstract
Recent advances in proteomic mass spectrometry (MS) offer the chance to marry high-throughput peptide sequencing to transcript models, allowing the validation, refinement, and identification of new protein-coding loci. We present a novel pipeline that integrates highly sensitive and statistically robust peptide spectrum matching with genome-wide protein-coding predictions to perform large-scale gene validation and discovery in the mouse genome for the first time. In searching an excess of 10 million spectra, we have been able to validate 32%, 17%, and 7% of all protein-coding genes, exons, and splice boundaries, respectively. Moreover, we present strong evidence for the identification of multiple alternatively spliced translations from 53 genes and have uncovered 10 entirely novel protein-coding genes, which are not covered in any mouse annotation data sources. One such novel protein-coding gene is a fusion protein that spans the Ins2 and Igf2 loci to produce a transcript encoding the insulin II and the insulin-like growth factor 2–derived peptides. We also report nine processed pseudogenes that have unique peptide hits, demonstrating, for the first time, that they are not just transcribed but are translated and are therefore resurrected into new coding loci. This work not only highlights an important utility for MS data in genome annotation but also provides unique insights into the gene structure and propagation in the mouse genome. All these data have been subsequently used to improve the publicly available mouse annotation available in both the Vega and Ensembl genome browsers (http://vega.sanger.ac.uk)..
Pagliuca, F.W.
Collins, M.O.
Lichawska, A.
Zegerman, P.
Choudhary, J.S.
Pines, J.
(2011). Quantitative Proteomics Reveals the Basis for the Biochemical Specificity of the Cell-Cycle Machinery. Molecular cell,
Vol.43
(3),
pp. 406-417.
Mansfeld, J.
Collin, P.
Collins, M.O.
Choudhary, J.S.
Pines, J.
(2011). APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment. Nature cell biology,
Vol.13
(10),
pp. 1234-1243.
Pagliuca, F.W.
Collins, M.O.
Choudhary, J.S.
(2011). Coordinating cell cycle progression via cyclin specificity. Cell cycle,
Vol.10
(24),
pp. 4195-4196.
Pardo, M.
Lang, B.
Yu, L.
Prosser, H.
Bradley, A.
Babu, M.M.
Choudhary, J.
(2010). An Expanded Oct4 Interaction Network: Implications for Stem Cell Biology, Development, and Disease. Cell stem cell,
Vol.6
(4),
pp. 382-395.
Pickard, D.
Toribio, A.L.
Petty, N.K.
van Tonder, A.
Yu, L.
Goulding, D.
Barrell, B.
Rance, R.
Harris, D.
Wetter, M.
Wain, J.
Choudhary, J.
Thomson, N.
Dougan, G.
(2010). A Conserved Acetyl Esterase Domain Targets Diverse Bacteriophages to the Vi Capsular Receptor of
Salmonella enterica
Serovar Typhi. Journal of bacteriology,
Vol.192
(21),
pp. 5746-5754.
show abstract
ABSTRACT
A number of bacteriophages have been identified that target the Vi capsular antigen of
Salmonella enterica
serovar Typhi. Here we show that these Vi phages represent a remarkably diverse set of phages belonging to three phage families, including
Podoviridae
and
Myoviridae
. Genome analysis facilitated the further classification of these phages and highlighted aspects of their independent evolution. Significantly, a conserved protein domain carrying an acetyl esterase was found to be associated with at least one tail fiber gene for all Vi phages, and the presence of this domain was confirmed in representative phage particles by mass spectrometric analysis. Thus, we provide a simple explanation and paradigm of how a diverse group of phages target a single key virulence antigen associated with this important human-restricted pathogen.
.
Jones, M.L.
Collins, M.O.
Choudhary, J.
Rayner, J.C.
(2010). A comprehensive survey of protein palmitoylation in late blood-stage Plasmodium falciparum. Malaria journal,
Vol.9
(S2).
Jones, M.L.
Collins, M.O.
Choudhary, J.S.
Rayner, J.C.
(2010). A comprehensive survey of protein palmitoylation in late blood-stage Plasmodium falciparum. Malaria journal,
Vol.9
(S2).
Tee, W.-.
Pardo, M.
Theunissen, T.W.
Yu, L.
Choudhary, J.S.
Hajkova, P.
Surani, M.A.
(2010). Prmt5 is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency. Genes & development,
Vol.24
(24),
pp. 2772-2777.
show abstract
Prmt5, an arginine methyltransferase, has multiple roles in germ cells, and possibly in pluripotency. Here we show that loss of Prmt5 function is early embryonic-lethal due to the abrogation of pluripotent cells in blastocysts. Prmt5 is also up-regulated in the cytoplasm during the derivation of embryonic stem (ES) cells together with Stat3, where they persist to maintain pluripotency. Prmt5 in association with Mep50 methylates cytosolic histone H2A (H2AR3me2s) to repress differentiation genes in ES cells. Loss of Prmt5 or Mep50 results in derepression of differentiation genes, indicating the significance of the Prmt5/Mep50 complex for pluripotency, which may occur in conjunction with the leukemia inhibitory factor (LIF)/Stat3 pathway..
Sharma, J.
(2010). Structural characterisation of photosystem II. ,
.
Brosch, M.
Choudhary, J.
(2010). Scoring and Validation of Tandem MS Peptide Identification Methods. ,
,
pp. 43-53.
Fernández, E.
Collins, M.O.
Uren, R.T.
Kopanitsa, M.V.
Komiyama, N.H.
Croning, M.D.
Zografos, L.
Armstrong, J.D.
Choudhary, J.S.
Grant, S.G.
(2009). Targeted tandem affinity purification of PSD‐95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins. Molecular systems biology,
Vol.5
(1).
Brosch, M.
Yu, L.
Hubbard, T.
Choudhary, J.
(2009). Accurate and Sensitive Peptide Identification with Mascot Percolator. Journal of proteome research,
Vol.8
(6),
pp. 3176-3181.
Lawley, T.D.
Croucher, N.J.
Yu, L.
Clare, S.
Sebaihia, M.
Goulding, D.
Pickard, D.J.
Parkhill, J.
Choudhary, J.
Dougan, G.
(2009). Proteomic and Genomic Characterization of Highly Infectious
Clostridium difficile
630 Spores. Journal of bacteriology,
Vol.191
(17),
pp. 5377-5386.
show abstract
ABSTRACT
Clostridium difficile
, a major cause of antibiotic-associated diarrhea, produces highly resistant spores that contaminate hospital environments and facilitate efficient disease transmission. We purified
C. difficile
spores using a novel method and show that they exhibit significant resistance to harsh physical or chemical treatments and are also highly infectious, with <7 environmental spores per cm
2
reproducibly establishing a persistent infection in exposed mice. Mass spectrometric analysis identified ∼336 spore-associated polypeptides, with a significant proportion linked to translation, sporulation/germination, and protein stabilization/degradation. In addition, proteins from several distinct metabolic pathways associated with energy production were identified. Comparison of the
C. difficile
spore proteome to those of other clostridial species defined 88 proteins as the clostridial spore “core” and 29 proteins as
C. difficile
spore specific, including proteins that could contribute to spore-host interactions. Thus, our results provide the first molecular definition of
C. difficile
spores, opening up new opportunities for the development of diagnostic and therapeutic approaches.
.
Coba, M.P.
Pocklington, A.J.
Collins, M.O.
Kopanitsa, M.V.
Uren, R.T.
Swamy, S.
Croning, M.D.
Choudhary, J.S.
Grant, S.G.
(2009). Neurotransmitters Drive Combinatorial Multistate Postsynaptic Density Networks. Science signaling,
Vol.2
(68).
show abstract
Analysis of protein phosphorylation patterns provides insight into the organization of molecular networks at the postsynaptic density..
Pickard, D.
Thomson, N.R.
Baker, S.
Wain, J.
Pardo, M.
Goulding, D.
Hamlin, N.
Choudhary, J.
Threfall, J.
Dougan, G.
(2008). Molecular Characterization of the
Salmonella enterica
Serovar Typhi Vi-Typing Bacteriophage E1. Journal of bacteriology,
Vol.190
(7),
pp. 2580-2587.
show abstract
ABSTRACT
Some bacteriophages target potentially pathogenic bacteria by exploiting surface-associated virulence factors as receptors. For example, phage have been identified that exhibit specificity for Vi capsule producing
Salmonella enterica
serovar Typhi. Here we have characterized the Vi-associated E1-typing bacteriophage using a number of molecular approaches. The absolute requirement for Vi capsule expression for infectivity was demonstrated using different Vi-negative
S. enterica
derivatives. The phage particles were shown to have an icosahedral head and a long noncontractile tail structure. The genome is 45,362 bp in length with defined capsid and tail regions that exhibit significant homology to the
S. enterica
transducing phage ES18. Mass spectrometry was used to confirm the presence of a number of hypothetical proteins in the Vi phage E1 particle and demonstrate that a number of phage proteins are modified posttranslationally. The genome of the Vi phage E1 is significantly related to other bacteriophages belonging to the same serovar Typhi phage-typing set, and we demonstrate a role for phage DNA modification in determining host specificity.
.
Brosch, M.
Swamy, S.
Hubbard, T.
Choudhary, J.
(2008). Comparison of Mascot and X!Tandem Performance for Low and High Accuracy Mass Spectrometry and the Development of an Adjusted Mascot Threshold. Molecular & cellular proteomics,
Vol.7
(5),
pp. 962-970.
Collins, M.O.
Yu, L.
Campuzano, I.
Grant, S.G.
Choudhary, J.S.
(2008). Phosphoproteomic Analysis of the Mouse Brain Cytosol Reveals a Predominance of Protein Phosphorylation in Regions of Intrinsic Sequence Disorder. Molecular & cellular proteomics,
Vol.7
(7),
pp. 1331-1348.
Emes, R.D.
Pocklington, A.J.
Anderson, C.N.
Bayes, A.
Collins, M.O.
Vickers, C.A.
Croning, M.D.
Malik, B.R.
Choudhary, J.S.
Armstrong, J.D.
Grant, S.G.
(2008). Evolutionary expansion and anatomical specialization of synapse proteome complexity. Nature neuroscience,
Vol.11
(7),
pp. 799-806.
Collins, M.O.
Choudhary, J.S.
(2008). Mapping multiprotein complexes by affinity purification and mass spectrometry. Current opinion in biotechnology,
Vol.19
(4),
pp. 324-330.
Collins, M.O.
Yu, L.
Choudhary, J.S.
(2007). Analysis of protein phosphorylation on a proteome‐scale. Proteomics,
Vol.7
(16),
pp. 2751-2768.
show abstract
AbstractPhosphorylation, the most intensively studied and common PTM on proteins, is a complex biological phenomenon. Its complexity manifests itself in the large numbers of proteins that attach it, remove it and recognise it as a protein code. Since the first report of protein phosphorylation on vitellin 100 years ago, a wide variety of biochemical and analytical chemical approaches have been developed to enrich and detect protein phosphorylation. The last 5 years have witnessed a renaissance in methodologies capable of characterising protein phosphorylation on a proteome‐scale. These technological advances have allowed identification of hundreds to thousands of phosphorylation sites in a proteome and have resulted in a profound paradigm shift. For the first time, using quantitative MS, the topology and significance of global phosphorylation networks may be investigated, marking a new era of cell signalling research. This review addresses recent technological advances in the purification of phosphorylated proteins and peptides and current MS‐based strategies used to qualitatively and quantitatively probe these enriched phosphoproteomes. In addition, we review the application of complementary array‐based technologies to derive signalling networks from kinase–substrate interactions and discuss future challenges in the field..
Hiran, B.L.
Choudhary, J.
Paliwal, S.N.
Choudhary, P.R.
(2007). Homo and copolymerization of N-(phenylamino)maleimide: Synthesis and characterization. Journal of the indian chemical society,
Vol.84
(11),
pp. 1153-6.
Collins, M.O.
Husi, H.
Yu, L.
Brandon, J.M.
Anderson, C.N.
Blackstock, W.P.
Choudhary, J.S.
Grant, S.G.
(2006). Molecular characterization and comparison of the components and multiprotein complexes in the postsynaptic proteome. Journal of neurochemistry,
Vol.97
(s1),
pp. 16-23.
show abstract
AbstractCharacterization of the composition of the postsynaptic proteome (PSP) provides a framework for understanding the overall organization and function of the synapse in normal and pathological conditions. We have identified 698 proteins from the postsynaptic terminal of mouse CNS synapses using a series of purification strategies and analysis by liquid chromatography tandem mass spectrometry and large‐scale immunoblotting. Some 620 proteins were found in purified postsynaptic densities (PSDs), nine in AMPA‐receptor immuno‐purifications, 100 in isolates using an antibody against the NMDA receptor subunit NR1, and 170 by peptide‐affinity purification of complexes with the C‐terminus of NR2B. Together, the NR1 and NR2B complexes contain 186 proteins, collectively referred to as membrane‐associated guanylate kinase‐associated signalling complexes. We extracted data from six other synapse proteome experiments and combined these with our data to provide a consensus on the composition of the PSP. In total, 1124 proteins are present in the PSP, of which 466 were validated by their detection in two or more studies, forming what we have designated the Consensus PSD. These synapse proteome data sets offer a basis for future research in synaptic biology and will provide useful information in brain disease and mental disorder studies..
Collins, M.O.
Yu, L.
Coba, M.P.
Husi, H.
Campuzano, I.
Blackstock, W.P.
Choudhary, J.S.
Grant, S.G.
(2005). Proteomic Analysis of in Vivo Phosphorylated Synaptic Proteins. Journal of biological chemistry,
Vol.280
(7),
pp. 5972-5982.
Collins, M.O.
Yu, L.
Husi, H.
Blackstock, W.P.
Choudhary, J.S.
Grant, S.G.
(2005). Robust Enrichment of Phosphorylated Species in Complex Mixtures by Sequential Protein and Peptide Metal-Affinity Chromatography and Analysis by Tandem Mass Spectrometry. Science's stke,
Vol.2005
(298).
show abstract
Reversible protein phosphorylation mediated by kinases, phosphatases, and regulatory molecules is an essential mechanism of signal transduction in living cells. Although phosphorylation is the most intensively studied of the several hundred known posttranslational modifications on proteins, until recently the rate of identification of phosphorylation sites has remained low. The use of tandem mass spectrometry has greatly accelerated the identification of phosphorylation sites, although progress was limited by difficulties in phosphoresidue enrichment techniques. We have improved upon existing immobilized metal-affinity chromatography (IMAC) techniques for capturing phosphopeptides, to selectively purify phosphoproteins from complex mixtures. Combinations of phosphoprotein and phosphopeptide enrichment were more effective than current single phosphopeptide purification approaches. We have also implemented iterative mass spectrometry-based scanning techniques to improve detection of phosphorylated peptides in these enriched samples. Here, we provide detailed instructions for implementing and validating these methods together with analysis by tandem mass spectrometry for the study of phosphorylation at the mammalian synapse. This strategy should be widely applicable to the characterization of protein phosphorylation in diverse tissues, organelles, and in cell culture..
Choudhary, J.
Grant, S.G.
(2004). Proteomics in postgenomic neuroscience: the end of the beginning. Nature neuroscience,
Vol.7
(5),
pp. 440-445.
Gan, J.-.
Yu, L.
Wu, J.
Xu, H.
Choudhary, J.S.
Blackstock, W.P.
Liu, W.-.
Xia, Z.-.
(2004). The Three-Dimensional Structure and X-Ray Sequence Reveal that Trichomaglin Is a Novel S-like Ribonuclease. Structure,
Vol.12
(6),
pp. 1015-1025.
Woods, A.J.
Roberts, M.S.
Choudhary, J.
Barry, S.T.
Mazaki, Y.
Sabe, H.
Morley, S.J.
Critchley, D.R.
Norman, J.C.
(2002). Paxillin Associates with Poly(A)-binding Protein 1 at the Dense Endoplasmic Reticulum and the Leading Edge of Migrating Cells. Journal of biological chemistry,
Vol.277
(8),
pp. 6428-6437.
Bell, A.W.
Ward, M.A.
Blackstock, W.P.
Freeman, H.N.
Choudhary, J.S.
Lewis, A.P.
Chotai, D.
Fazel, A.
Gushue, J.N.
Paiement, J.
Palcy, S.
Chevet, E.
Lafrenière-Roula, M.
Solari, R.
Thomas, D.Y.
Rowley, A.
Bergeron, J.J.
(2001). Proteomics Characterization of Abundant Golgi Membrane Proteins. Journal of biological chemistry,
Vol.276
(7),
pp. 5152-5165.
Choudhary, J.S.
Blackstock, W.P.
Creasy, D.M.
Cottrell, J.S.
(2001). Interrogating the human genome using uninterpreted mass spectrometry data. Proteomics,
Vol.1
(5),
pp. 651-667.
Choudhary, J.S.
Blackstock, W.P.
Creasy, D.M.
Cottrell, J.S.
(2001). Matching peptide mass spectra to EST and genomic DNA databases. Trends in biotechnology,
Vol.19
(10),
pp. S17-S22.
Rowley, A.
Choudhary, J.S.
Marzioch, M.
Ward, M.A.
Weir, M.
Solari, R.C.
Blackstock, W.P.
(2000). Applications of Protein Mass Spectrometry in Cell Biology. Methods,
Vol.20
(4),
pp. 383-397.
Husi, H.
Ward, M.A.
Choudhary, J.S.
Blackstock, W.P.
Grant, S.G.
(2000). Proteomic analysis of NMDA receptor–adhesion protein signaling complexes. Nature neuroscience,
Vol.3
(7),
pp. 661-669.
Zheleva, D.
Sharma, J.
Panico, M.
Morris, H.R.
Barber, J.
(1998). Isolation and Characterization of Monomeric and Dimeric CP47-Reaction Center Photosystem II Complexes. Journal of biological chemistry,
Vol.273
(26),
pp. 16122-16127.
Sharma, J.
Panico, M.
Barber, J.
Morris, H.R.
(1997). Characterization of the Low Molecular Weight Photosystem II Reaction Center Subunits and Their Light-induced Modifications by Mass Spectrometry. Journal of biological chemistry,
Vol.272
(7),
pp. 3935-3943.
Sharma, J.
Panico, M.
Shipton, C.A.
Nilsson, F.
Morris, H.R.
Barber, J.
(1997). Primary Structure Characterization of the Photosystem II D1 and D2 Subunits. Journal of biological chemistry,
Vol.272
(52),
pp. 33158-33166.
Sharma, J.
Panico, M.
Barber, J.
Morris, H.R.
(1997). Purification and Determination of Intact Molecular Mass by Electrospray Ionization Mass Spectrometry of the Photosystem II Reaction Center Subunits. Journal of biological chemistry,
Vol.272
(52),
pp. 33153-33157.
CHOUDHARY, J.
PRASAD, S.
(1982). KINETICS OF OXIDATION OF GLYCOLIC ACID BY QUINQUEVALENT VANADIUM IN SULFURIC-ACID MEDIUM. Journal of the indian chemical society,
Vol.59
(5),
pp. 638-3.
Wright, J.C.
Choudhary, J.S.
DecoyPyrat: Fast Non-redundant Hybrid Decoy Sequence Generation for Large Scale Proteomics. Journal of proteomics & bioinformatics,
Vol.09
(06).
Bayés, À.
Collins, M.O.
Croning, M.D.
van de Lagemaat, L.N.
Choudhary, J.S.
Grant, S.G.
Comparative Study of Human and Mouse Postsynaptic Proteomes Finds High Compositional Conservation and Abundance Differences for Key Synaptic Proteins. Plos one,
Vol.7
(10),
pp. e46683-e46683.
Perkins, T.T.
Kingsley, R.A.
Fookes, M.C.
Gardner, P.P.
James, K.D.
Yu, L.
Assefa, S.A.
He, M.
Croucher, N.J.
Pickard, D.J.
Maskell, D.J.
Parkhill, J.
Choudhary, J.
Thomson, N.R.
Dougan, G.
A Strand-Specific RNA–Seq Analysis of the Transcriptome of the Typhoid Bacillus Salmonella Typhi. Plos genetics,
Vol.5
(7),
pp. e1000569-e1000569.
Brochet, M.
Collins, M.O.
Smith, T.K.
Thompson, E.
Sebastian, S.
Volkmann, K.
Schwach, F.
Chappell, L.
Gomes, A.R.
Berriman, M.
Rayner, J.C.
Baker, D.A.
Choudhary, J.
Billker, O.
Phosphoinositide Metabolism Links cGMP-Dependent Protein Kinase G to Essential Ca2+ Signals at Key Decision Points in the Life Cycle of Malaria Parasites. Plos biology,
Vol.12
(3),
pp. e1001806-e1001806.
McMahon, A.C.
Barnett, M.W.
O'Leary, T.S.
Stoney, P.N.
Collins, M.O.
Papadia, S.
Choudhary, J.S.
Komiyama, N.H.
Grant, S.G.
Hardingham, G.E.
Wyllie, D.J.
Kind, P.C.
SynGAP isoforms exert opposing effects on synaptic strength. Nature communications,
Vol.3
(1).
Petty, N.K.
Feltwell, T.
Pickard, D.
Clare, S.
Toribio, A.L.
Fookes, M.
Roberts, K.
Monson, R.
Nair, S.
Kingsley, R.A.
Bulgin, R.
Wiles, S.
Goulding, D.
Keane, T.
Corton, C.
Lennard, N.
Harris, D.
Willey, D.
Rance, R.
Yu, L.
Choudhary, J.S.
Churcher, C.
Quail, M.A.
Parkhill, J.
Frankel, G.
Dougan, G.
Salmond, G.P.
Thomson, N.R.
Citrobacter rodentium is an Unstable Pathogen Showing Evidence of Significant Genomic Flux. Plos pathogens,
Vol.7
(4),
pp. e1002018-e1002018.
Shariff, M.
Choudhary, J.
Zahoor, S.
Deb, M.
Characterization of Streptococcus pneumoniae isolates from India with special reference to their sequence types. The journal of infection in developing countries,
Vol.7
(02),
pp. 101-109.
show abstract
Introduction: Streptococcus pneumoniae is a major cause of mortality and morbidity in young children and the elderly. In the present study we evaluated antimicrobial susceptibilities, serotypes, and sequence types of pneumococcal isolates recovered in New Delhi, India. Methodology: A total of 126 clinical isolates of Streptococcus pneumoniae were investigated. They were subjected to disk diffusion susceptibility testing, broth microdilution testing, serotyping and multilocus sequence typing. Results: Broth microdilution assay showed that 5%, 20% and 23% of the isolates exhibited resistance to penicillin, erythromycin and ciprofloxacin, respectively. Serotypes19, 1 and 6 were more frequently isolated. Thirty per cent of the strains were comprised of serotypes 1, 3, 5, 19A and 7F, which are not included in the seven-valent vaccine. Fifty-nine isolates were typed using multilocus sequence typing. Thirty new sequence types were encountered in this study. Only one clonal complex with 4 isolates was seen; 11 clonal complexes and 96 sequence types (STs) were observed among 115 Indian isolates. Only 18 of the 96 STs were found globally, of which only 4 STs were found in many countries with larger numbers. Conclusions: This study identifies the non-vaccine serotypes of Streptococcus pneumoniae circulating in India. It is important that an appropriate vaccine which covers all serotypes is used in the region..
Barzine, M.
Freivalds, K.
Wright, J.C.
Opmanis, M.
Rituma, D.
Ghavidel, F.Z.
Jarnuczak, A.J.
Celms, E.
Cerans, K.
Jonassen, I.
Lace, L.
Vizcaíno, J.A.
Choudhary, J.
Brazma, A.
Viksna, J.
Using Deep Learning to Extrapolate Protein Expression Measurements. Proteomics,
,
pp. e2000009-e2000009.
show abstract
MOTIVATION: Mass spectrometry (MS) based quantitative proteomics experiments typically assay a subset of up to 60% of the ∼20,000 human protein coding genes. Computational methods for imputing the missing values using RNA expression data usually allow only for imputations of proteins measured in at least some of the samples. In silico methods for comprehensively estimating abundances across all proteins are still missing. RESULTS: We propose a novel method using deep learning to extrapolate the observed protein expression values in label-free MS experiments to all proteins, leveraging gene functional annotations and RNA measurements as key predictive attributes. We tested our method on four datasets, including human cell lines and human and mouse tissues. Our method predicts the protein expression values with average R2 scores between 0.46 and 0.54, which is significantly better than predictions based on correlations using the RNA expression data alone. Moreover, we demonstrate that the derived models can be "transferred" across experiments and species. For instance, the model derived from human tissues gave a R2 = 0.51 when applied to mouse tissue data. We conclude that protein abundances generated in label free MS experiments can be computationally predicted using functional annotated attributes and can be used to highlight aberrant protein abundance values. This article is protected by copyright. All rights reserved..
More, K.R.
Kaur, I.
Giai Gianetto, Q.
Invergo, B.M.
Chaze, T.
Jain, R.
Huon, C.
Gutenbrunner, P.
Weisser, H.
Matondo, M.
Choudhary, J.S.
Langsley, G.
Singh, S.
Chitnis, C.E.
Phosphorylation-Dependent Assembly of a 14-3-3 Mediated Signaling Complex during Red Blood Cell Invasion by Plasmodium falciparum Merozoites. Mbio,
Vol.11
(4).
show abstract
Red blood cell (RBC) invasion by Plasmodium merozoites requires multiple steps that are regulated by signaling pathways. Exposure of P. falciparum merozoites to the physiological signal of low K+, as found in blood plasma, leads to a rise in cytosolic Ca2+, which mediates microneme secretion, motility, and invasion. We have used global phosphoproteomic analysis of merozoites to identify signaling pathways that are activated during invasion. Using quantitative phosphoproteomics, we found 394 protein phosphorylation site changes in merozoites subjected to different ionic environments (high K+/low K+), 143 of which were Ca2+ dependent. These included a number of signaling proteins such as catalytic and regulatory subunits of protein kinase A (PfPKAc and PfPKAr) and calcium-dependent protein kinase 1 (PfCDPK1). Proteins of the 14-3-3 family interact with phosphorylated target proteins to assemble signaling complexes. Here, using coimmunoprecipitation and gel filtration chromatography, we demonstrate that Pf14-3-3I binds phosphorylated PfPKAr and PfCDPK1 to mediate the assembly of a multiprotein complex in P. falciparum merozoites. A phospho-peptide, P1, based on the Ca2+-dependent phosphosites of PKAr, binds Pf14-3-3I and disrupts assembly of the Pf14-3-3I-mediated multiprotein complex. Disruption of the multiprotein complex with P1 inhibits microneme secretion and RBC invasion. This study thus identifies a novel signaling complex that plays a key role in merozoite invasion of RBCs. Disruption of this signaling complex could serve as a novel approach to inhibit blood-stage growth of malaria parasites.IMPORTANCE Invasion of red blood cells (RBCs) by Plasmodium falciparum merozoites is a complex process that is regulated by intricate signaling pathways. Here, we used phosphoproteomic profiling to identify the key proteins involved in signaling events during invasion. We found changes in the phosphorylation of various merozoite proteins, including multiple kinases previously implicated in the process of invasion. We also found that a phosphorylation-dependent multiprotein complex including signaling kinases assembles during the process of invasion. Disruption of this multiprotein complex impairs merozoite invasion of RBCs, providing a novel approach for the development of inhibitors to block the growth of blood-stage malaria parasites..
Zou, X.
Koh, G.C.
Nanda, A.S.
Degasperi, A.
Urgo, K.
Roumeliotis, T.I.
Agu, C.A.
Badja, C.
Momen, S.
Young, J.
Amarante, T.D.
Side, L.
Brice, G.
Perez-Alonso, V.
Rueda, D.
Gomez, C.
Bushell, W.
Harris, R.
Choudhary, J.S.
Ambrose, J.C.
Arumugam, P.
Baple, E.L.
Bleda, M.
Boardman-Pretty, F.
Boissiere, J.M.
Boustred, C.R.
Brittain, H.
Caulfield, M.J.
Chan, G.C.
Craig, C.E.
Daugherty, L.C.
de Burca, A.
Devereau, A.
Elgar, G.
Foulger, R.E.
Fowler, T.
Furió-Tarí, P.
Giess, A.
Hackett, J.M.
Halai, D.
Hamblin, A.
Henderson, S.
Holman, J.E.
Hubbard, T.J.
ibáñez, K.
Jackson, R.
Jones, L.J.
Kasperaviciute, D.
Kayikci, M.
Kousathanas, A.
Lahnstein, L.
Lawson, K.
Leigh, S.E.
Leong, I.U.
Lopez, J.F.
Maleady-Crowe, F.
Mason, J.
McDonagh, E.M.
Moutsianas, L.
Mueller, M.
Murugaesu, N.
Need, A.C.
O’Donovan, P.
Odhams, C.A.
Orioli, A.
Patch, C.
Pereira, M.B.
Perez-Gil, D.
Polychronopoulos, D.
Pullinger, J.
Rahim, T.
Rendon, A.
Riesgo-Ferreiro, P.
Rogers, T.
Ryten, M.
Savage, K.
Sawant, K.
Scott, R.H.
Siddiq, A.
Sieghart, A.
Smedley, D.
Smith, K.R.
Smith, S.C.
Sosinsky, A.
Spooner, W.
Stevens, H.E.
Stuckey, A.
Sultana, R.
Tanguy, M.
Thomas, E.R.
Thompson, S.R.
Tregidgo, C.
Tucci, A.
Walsh, E.
Watters, S.A.
Welland, M.J.
Williams, E.
Witkowska, K.
Wood, S.M.
Zarowiecki, M.
Jiricny, J.
Skarnes, W.C.
Nik-Zainal, S.
A systematic CRISPR screen defines mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage. Nature cancer,
Vol.2
(6),
pp. 643-657.
Abdi, A.
Yu, L.
Goulding, D.
Rono, M.K.
Bejon, P.
Choudhary, J.
Rayner, J.
Proteomic analysis of extracellular vesicles from a Plasmodium falciparum Kenyan clinical isolate defines a core parasite secretome. Wellcome open research,
Vol.2,
pp. 50-50.
show abstract
BACKGROUND:Many pathogens secrete effector molecules to subvert host immune responses, to acquire nutrients, and/or to prepare host cells for invasion. One of the ways that effector molecules are secreted is through extracellular vesicles (EVs) such as exosomes. Recently, the malaria parasite P. falciparum has been shown to produce EVs that can mediate transfer of genetic material between parasites and induce sexual commitment. Characterizing the content of these vesicles may improve our understanding of P. falciparum pathogenesis and virulence. METHODS:Previous studies of P. falciparum EVs have been limited to long-term adapted laboratory isolates. In this study, we isolated EVs from a Kenyan P. falciparum clinical isolate adapted to in vitro culture for a short period and characterized their protein content by mass spectrometry (data are available via ProteomeXchange, with identifier PXD006925). RESULTS:We show that P. falciparum extracellular vesicles ( PfEVs) are enriched in proteins found within the exomembrane compartments of infected erythrocytes such as Maurer's clefts (MCs), as well as the secretory endomembrane compartments in the apical end of the merozoites, suggesting that these proteins play a role in parasite-host interactions. Comparison of this novel clinically relevant dataset with previously published datasets helps to define a core secretome present in Plasmodium EVs. CONCLUSIONS:P. falciparum extracellular vesicles contain virulence-associated parasite proteins. Therefore, analysis of PfEVs contents from a range of clinical isolates, and their functional validation may improve our understanding of the virulence mechanisms of the parasite, and potentially identify targets for interventions or diagnostics..
Fierro-Monti, I.
Wright, J.C.
Choudhary, J.S.
Vizcaíno, J.A.
Identifying individuals using proteomics: are we there yet?. Frontiers in molecular biosciences,
Vol.9.
show abstract
Multi-omics approaches including proteomics analyses are becoming an integral component of precision medicine. As clinical proteomics studies gain momentum and their sensitivity increases, research on identifying individuals based on their proteomics data is here examined for risks and ethics-related issues. A great deal of work has already been done on this topic for DNA/RNA sequencing data, but it has yet to be widely studied in other omics fields. The current state-of-the-art for the identification of individuals based solely on proteomics data is explained. Protein sequence variation analysis approaches are covered in more detail, including the available analysis workflows and their limitations. We also outline some previous forensic and omics proteomics studies that are relevant for the identification of individuals. Following that, we discuss the risks of patient reidentification using other proteomics data types such as protein expression abundance and post-translational modification (PTM) profiles. In light of the potential identification of individuals through proteomics data, possible legal and ethical implications are becoming increasingly important in the field..
Yadav, R.
Saini, A.
Choudhary, J.
Sardana, S.
Ohlan, A.
Singh, K.
Singh, S.P.
High‐performance flexible supercapacitor based on morphology tuned polypyrrole/molybdenum disulfide nanocomposites. Energy storage,
.
show abstract
AbstractPolypyrrole (PPy) is the most studied material among the conducting polymers for high‐performance supercapacitors. However, due to morphology collapse, polypyrrole exhibits poor cycling ability and lower charge storage capacity, limiting its real‐world applications. To address these issues herein, the binary nanocomposites of PPy with MoS2 (molybdenum disulfide) NSs (nanostructures) in two different morphologies, that is, cenosphere (PPyC) and nanotubes (PPyNTs), have been prepared through in situ polymerization technique. The electrochemical performances of PPyC/MoS2 and PPyNTs/MoS2 nanocomposite are compared to define the most favorable morphology when PPy nanostructures are utilized as the electrode materials for creating flexible supercapacitors. The rational design of PPyNTs/MoS2 nanocomposite consisting of dense wrapping of PPyNTs on MoS2 leads to high specific capacitance (Csp) up to 481 F/g at 0.5 A/g. After 2000 charge‐discharge cycles, a decline of 5.6% in the specific capacitance of PPyNTs/MoS2 nanocomposite was observed, primarily attributed to the presence of chemically active sites on MoS2 nanosheets. This led to a strong alignment with the nitrogen group of pyrrole rings in the polymer chain to form a robust interconnected framework between tubular PPy and sheet‐like MoS2. This outstanding performance of PPyNTs/MoS2 nanocomposite resulted in designing futuristic flexible supercapacitors..