Publications

Therapies that target estrogen signaling have made a very considerable contribution to reducing mortality from breast cancer. However, resistance to tamoxifen remains a major clinical problem. Here we have used a genome-wide functional profiling approach to identify multiple genes that confer resistance or sensitivity to tamoxifen. Combining whole-genome shRNA screening with massively parallel sequencing, we have profiled the impact of more than 56,670 RNA interference reagents targeting 16,487 genes on the cellular response to tamoxifen. This screen, along with subsequent validation experiments, identifies a compendium of genes whose silencing causes tamoxifen resistance (including BAP1, CLPP, GPRC5D, NAE1, NF1, NIPBL, NSD1, RAD21, RARG, SMC3, and UBA3) and also a set of genes whose silencing causes sensitivity to this endocrine agent (C10orf72, C15orf55/NUT, EDF1, ING5, KRAS, NOC3L, PPP1R15B, RRAS2, TMPRSS2, and TPM4). Multiple individual genes, including NF1, a regulator of RAS signaling, also correlate with clinical outcome after tamoxifen treatment.

The endothelial ETS transcription factor Erg plays an important role in homeostasis and angiogenesis by regulating many endothelial functions including survival and junction stability. Here we show that Erg regulates endothelial cell (EC) migration. Transcriptome profiling of Erg-deficient ECs identified ∼ 80 genes involved in cell migration as candidate Erg targets, including many regulators of Rho- GTPases. Inhibition of Erg expression in HUVECs resulted in decreased migration in vitro, while Erg overexpression using adenovirus caused increased migration. Live-cell imaging of Erg-deficient HUVECs showed a reduction in lamellipodia, in line with decreased motility. Both actin and tubulin cytoskeletons were disrupted in Erg-deficient ECs, with a dramatic increase in tubulin acetylation. Among the most significant microarray hits was the cytosolic histone deacetylase 6 (HDAC6), a regulator of cell migration. Chromatin immunoprecipitation (ChIP) and transactivation studies demonstrated that Erg regulates HDAC6 expression. Rescue experiments confirmed that HDAC6 mediates the Erg-dependent regulation of tubulin acetylation and actin localization. In vivo, inhibition of Erg expression in angiogenic ECs resulted in decreased HDAC6 expression with increased tubulin acetylation. Thus, we have identified a novel function for the transcription factor Erg in regulating HDAC6 and multiple pathways essential for EC migration and angiogenesis.

Next generation sequencing has enabled systematic discovery of mutational spectra in cancer samples. Here, we used whole genome sequencing to characterize somatic mutations and structural variation in a primary acral melanoma and its lymph node metastasis. Our data show that the somatic mutational rates in this acral melanoma sample pair were more comparable to the rates reported in cancer genomes not associated with mutagenic exposure than in the genome of a melanoma cell line or the transcriptome of melanoma short-term cultures. Despite the perception that acral skin is sun-protected, the dominant mutational signature in these samples is compatible with damage due to ultraviolet light exposure. A nonsense mutation in ERCC5 discovered in both the primary and metastatic tumors could also have contributed to the mutational signature through accumulation of unrepaired dipyrimidine lesions. However, evidence of transcription-coupled repair was suggested by the lower mutational rate in the transcribed regions and expressed genes. The primary and the metastasis are highly similar at the level of global gene copy number alterations, loss of heterozygosity and single nucleotide variation (SNV). Furthermore, the majority of the SNVs in the primary tumor were propagated in the metastasis and one nonsynonymous coding SNV and one splice site mutation appeared to arise de novo in the metastatic lesion.

Next generation DNA sequencing (NGS) technologies have revolutionized the pace at which whole genome and exome sequences can be generated. However, despite these advances, many of the methods for targeted resequencing, such as the generation of high-depth exome sequences, are somewhat limited by the relatively large amounts of starting DNA that are normally required. In the case of tumour analysis this is particularly pertinent as many tumour biopsies often return submicrogram quantities of DNA, especially when tumours are microdissected prior to analysis. Here, we present a method for exome capture and resequencing using as little as 50 ng of starting DNA. The sequencing libraries generated by this minimal starting amount (MSA-Cap) method generate datasets that are comparable to standard amount (SA) whole exome libraries that use three micrograms of starting DNA. This method, which can be performed in most laboratories using commonly available reagents, has the potential to enhance large scale profiling efforts such as the resequencing of tumour exomes.

Epidemiological studies have provided strong evidence for a role of endogenous sex steroids in the etiology of breast cancer. Our aim was to identify common variants in genes involved in sex steroid synthesis or metabolism that are associated with hormone levels and the risk of breast cancer in premenopausal women.

Cancer is caused by mutations in oncogenes and tumor suppressor genes, resulting in the deregulation of processes fundamental to the normal behavior of cells. The identification and characterization of oncogenes and tumor suppressors has led to new treatment strategies that have significantly improved cancer outcome. The advent of next generation sequencing has allowed the elucidation of the fine structure of cancer genomes, however, the identification of pathogenic changes is complicated by the inherent genomic instability of cancer cells. Therefore, functional approaches for the identification of novel genes involved in the initiation and development of tumors are critical. Here we report the first whole human genome in vivo RNA interference screen to identify functionally important tumor suppressor genes. Using our novel approach, we identify previously validated tumor suppressor genes including TP53 and MNT, as well as several novel candidate tumor suppressor genes including leukemia inhibitory factor receptor (LIFR). We show that LIFR is a key novel tumor suppressor, whose deregulation may drive the transformation of a significant proportion of human breast cancers. These results demonstrate the power of genome wide in vivo RNAi screens as a method for identifying novel genes regulating tumorigenesis.

Caspases have been extensively studied as critical initiators and executioners of cell death pathways. However, caspases also take part in non-apoptotic signalling events such as the regulation of innate immunity and activation of nuclear factor-κB (NF-κB). How caspases are activated under these conditions and process a selective set of substrates to allow NF-κB signalling without killing the cell remains largely unknown. Here, we show that stimulation of the Drosophila pattern recognition protein PGRP-LCx induces DIAP2-dependent polyubiquitylation of the initiator caspase DREDD. Signal-dependent ubiquitylation of DREDD is required for full processing of IMD, NF-κB/Relish and expression of antimicrobial peptide genes in response to infection with Gram-negative bacteria. Our results identify a mechanism that positively controls NF-κB signalling via ubiquitin-mediated activation of DREDD. The direct involvement of ubiquitylation in caspase activation represents a novel mechanism for non-apoptotic caspase-mediated signalling.

The p63 transcription factor (TP63) is critical in development, growth and differentiation of stratifying epithelia. This is highlighted by the severity of congenital abnormalities caused by TP63 mutations in humans, the dramatic phenotypes in knockout mice and de-regulation of TP63 expression in neoplasia altering the tumour suppressive roles of the TP53 family. In order to define the normal role played by TP63 and provide the basis for better understanding how this network is perturbed in disease, we used chromatin immunoprecipitation combined with massively parallel sequencing (ChIP-seq) to identify >7500 high-confidence TP63-binding regions across the entire genome, in primary human neonatal foreskin keratinocytes (HFKs). Using integrative strategies, we demonstrate that only a subset of these sites are bound by TP53 in response to DNA damage. We identify a role for TP63 in transcriptional regulation of multiple genes genetically linked to cleft palate and identify AP-2alpha (TFAP2A) as a co-regulator of a subset of these genes. We further demonstrate that AP-2gamma (TFAP2C) can bind a subset of these regions and that acute depletion of either TFAP2A or TFAP2C alone is sufficient to reduce terminal differentiation of organotypic epidermal skin equivalents, indicating overlapping physiological functions with TP63.

Acral melanoma is a rare melanoma subtype with distinct epidemiological, clinical and genetic features. To determine if acral melanoma cell lines are representative of this melanoma subtype, six lines were analysed by whole-exome sequencing and array comparative genomic hybridisation. We demonstrate that the cell lines display a mutation rate that is comparable to that of published primary and metastatic acral melanomas and observe a mutational signature suggestive of UV-induced mutagenesis in two of the cell lines. Mutations were identified in oncogenes and tumour suppressors previously linked to melanoma including BRAF, NRAS, KIT, PTEN and TP53, in cancer genes not previously linked to melanoma and in genes linked to DNA repair such as BRCA1 and BRCA2. Our findings provide strong circumstantial evidence to suggest that acral melanoma cell lines and acral tumours share genetic features in common and that these cells are therefore valuable tools to investigate the biology of this aggressive melanoma subtype. Data are available at: http://rock.icr.ac.uk/collaborations/Furney_et_al_2012/.

Clozapine has markedly superior clinical properties compared to other antipsychotic drugs but the side effects of agranulocytosis, weight gain and diabetes limit its use. The reason why clozapine is more effective is not well understood. We studied messenger RNA (mRNA) gene expression in the mouse brain to identify pathways changed by clozapine compared to those changed by haloperidol so that we could identify which changes were specific to clozapine. Data interpretation was performed using an over-representation analysis (ORA) of gene ontology (GO), pathways and gene-by-gene differences. Clozapine significantly changed gene expression in pathways related to neuronal growth and differentiation to a greater extent than haloperidol; including the microtubule-associated protein kinase (MAPK) signalling and GO terms related to axonogenesis and neuroblast proliferation. Several genes implicated genetically or functionally in schizophrenia such as frizzled homolog 3 (FZD3), U2AF homology motif kinase 1 (UHMK1), pericentriolar material 1 (PCM1) and brain-derived neurotrophic factor (BDNF) were changed by clozapine but not by haloperidol. Furthermore, when compared to untreated controls clozapine specifically regulated transcripts related to the glutamate system, microtubule function, presynaptic proteins and pathways associated with synaptic transmission such as clathrin cage assembly. Compared to untreated controls haloperidol modulated expression of neurotoxic and apoptotic responses such as NF-kappa B and caspase pathways, whilst clozapine did not. Pathways involving lipid and carbohydrate metabolism and appetite regulation were also more affected by clozapine than by haloperidol.

We conducted a genome-wide association study of male breast cancer comprising 823 cases and 2,795 controls of European ancestry, with validation in independent sample sets totaling 438 cases and 474 controls. A SNP in RAD51B at 14q24.1 was significantly associated with male breast cancer risk (P = 3.02 × 10(-13); odds ratio (OR) = 1.57). We also refine association at 16q12.1 to a SNP within TOX3 (P = 3.87 × 10(-15); OR = 1.50).

The mammary primordium forms during embryogenesis as a result of inductive interactions between its constitutive tissues, the mesenchyme and epithelium, and represents the earliest evidence of commitment to the mammary lineage. Previous studies of embryonic mouse mammary epithelium indicated that, by mid-gestation, these cells are determined to a mammary cell fate and that a stem cell population has been delimited. Mammary mesenchyme can induce mammary development from simple epithelium even across species and classes, and can partially restore features of differentiated tissue to mouse mammary tumours in co-culture experiments. Despite these exciting properties, the molecular identity of embryonic mammary cells remains to be fully characterised.

Capan-1 is a well-characterised BRCA2-deficient human cell line isolated from a liver metastasis of a pancreatic adenocarcinoma. Here we report a genome-wide assessment of structural variations and high-depth exome characterization of single nucleotide variants and small insertion/deletions in Capan-1. To identify potential somatic and tumour-associated variations in the absence of a matched-normal cell line, we devised a novel method based on the analysis of HapMap samples. We demonstrate that Capan-1 has one of the most rearranged genomes sequenced to date. Furthermore, small insertions and deletions are detected more frequently in the context of short sequence repeats than in other genomes. We also identify a number of novel mutations that may represent genetic changes that have contributed to tumour progression. These data provide insight into the genomic effects of loss of BRCA2 function.

Integrating transcriptomic sequencing with conventional cytogenetics, we identified WWTR1 (WW domain-containing transcription regulator 1) (3q25) and CAMTA1 (calmodulin-binding transcription activator 1) (1p36) as the two genes involved in the t(1;3)(p36;q25) chromosomal translocation that is characteristic of epithelioid hemangioendothelioma (EHE), a vascular sarcoma. This WWTR1/CAMTA1 gene fusion is under the transcriptional control of the WWTR1 promoter and encodes a putative chimeric transcription factor that joins the amino terminus of WWTR1, a protein that is highly expressed in endothelial cells, in-frame to the carboxyl terminus of CAMTA1, a protein that is normally expressed only in brain. Thus, CAMTA1 expression is activated inappropriately through a promoter-switch mechanism. The gene fusion is present in virtually all EHEs tested but is absent from all other vascular neoplasms, demonstrating it to be a disease-defining genetic alteration. A sensitive and specific break-apart fluorescence in situ hybridization assay was also developed to detect the translocation and will assist in the evaluation of this diagnostically challenging neoplasm. The chimeric WWTR1/CAMTA1 transcription factor may represent a therapeutic target for EHE and offers the opportunity to shed light on the functions of two poorly characterized proteins.

Background: Genomic copy number changes and regional alterations in epigenetic states have been linked to grade in breast cancer. However, the relative contribution of specific alterations to the pathology of different breast cancer subtypes remains unclear. The heterogeneity and interplay of genomic and epigenetic variations means that large datasets and statistical data mining methods are required to uncover recurrent patterns that are likely to be important in cancer progression.Results: We employed ridge regression to model the relationship between regional changes in gene expression and proliferation. Regional features were extracted from tumour gene expression data using a novel clustering method, called genomic distance entrained agglomerative (GDEC) clustering. Using gene expression data in this way provides a simple means of integrating the phenotypic effects of both copy number aberrations and alterations in chromatin state. We show that regional metagenes derived from GDEC clustering are representative of recurrent regions of epigenetic regulation or copy number aberrations in breast cancer. Furthermore, detected patterns of genomic alterations are conserved across independent oestrogen receptor positive breast cancer datasets. Sequential competitive metagene selection was used to reveal the relative importance of genomic regions in predicting proliferation rate. The predictive model suggested additive interactions between the most informative regions such as 8p22-12 and 8q13-22.Conclusions: Data-mining of large-scale microarray gene expression datasets can reveal regional clusters of coordinate gene expression, independent of cause. By correlating these clusters with tumour proliferation we have identified a number of genomic regions that act together to promote proliferation in ER+ breast cancer. Identification of such regions should enable prioritisation of genomic regions for combinatorial functional studies to pinpoint the key genes and interactions contributing to tumourigenicity.

Markers at the pericentriolar material 1 gene (PCM1) have shown genetic association with schizophrenia in both a University College London (UCL) and a USA-based case-control sample. In this paper we report a statistically significant replication of the PCM1 association in a large Scottish case-control sample from Aberdeen. Resequencing of the genomic DNA from research volunteers who had inherited haplotypes associated with schizophrenia showed a threonine to isoleucine missense mutation in exon 24 which was likely to change the structure and function of PCM1 (rs370429). This mutation was found only as a heterozygote in 98 schizophrenic research subjects and controls out of 2246 case and control research subjects. Among the 98 carriers of rs370429, 67 were affected with schizophrenia. The same alleles and haplotypes were associated with schizophrenia in both the London and Aberdeen samples. Another potential aetiological base pair change in PCM1 was rs445422, which altered a splice site signal. A further mutation, rs208747, was shown by electrophoretic mobility shift assays to create or destroy a promoter transcription factor site. Five further non-synonymous changes in exons were also found. Genotyping of the new variants discovered in the UCL case-control sample strengthened the evidence for allelic and haplotypic association (P=0.02-0.0002). Given the number and identity of the haplotypes associated with schizophrenia, further aetiological base pair changes must exist within and around the PCM1 gene. PCM1 protein has been shown to interact directly with the disrupted-in-schizophrenia 1 (DISC1) protein, Bardet-Biedl syndrome 4, and Huntingtin-associated protein 1, and is important in neuronal cell growth. In a separate study we found that clozapine but not haloperidol downregulated PCM1 expression in the mouse brain. We hypothesize that mutant PCM1 may be responsible for causing a subtype of schizophrenia through abnormal cell division and abnormal regeneration in dividing cells in the central nervous system. This is supported by our previous finding of orbitofrontal volumetric deficits in PCM1-associated schizophrenia patients as opposed to temporal pole deficits in non-PCM1-associated schizophrenia patients. Caution needs to be exercised in interpreting the actual biological effects of the mutations we have found without further cell biology. However, the DNA changes we have found deserve widespread genotyping in multiple case-control populations.

The clinical and pathological heterogeneity of breast cancer has instigated efforts to stratify breast cancer sub-types according to molecular profiles. These profiling efforts are now being augmented by large-scale functional screening of breast tumour cell lines, using approaches such as RNA interference. We have developed ROCK ( rock.icr.ac.uk ) to provide a unique, publicly accessible resource for the integration of breast cancer functional and molecular profiling datasets. ROCK provides a simple online interface for the navigation and cross-correlation of gene expression, aCGH and RNAi screen data. It enables the interrogation of gene lists in the context of statistically analysed functional genomic datasets, interaction networks, pathways, GO terms, mutations and drug targets. The interface also provides interactive visualisations of datasets and interaction networks. ROCK collates data from a wealth of breast cancer molecular profiling and functional screening studies into a single portal, where analysed and annotated results can be accessed at the level of a gene, sample or study. We believe that portals such as ROCK will not only afford researchers rapid access to profiling data, but also aid the integration of different data types, thus enhancing the discovery of novel targets and biomarkers for breast cancer.

FLIGHT (http://flight.icr.ac.uk/) is an online resource compiling data from high-throughput Drosophila in vivo and in vitro RNAi screens. FLIGHT includes details of RNAi reagents and their predicted off-target effects, alongside RNAi screen hits, scores and phenotypes, including images from high-content screens. The latest release of FLIGHT is designed to enable users to upload, analyze, integrate and share their own RNAi screens. Users can perform multiple normalizations, view quality control plots, detect and assign screen hits and compare hits from multiple screens using a variety of methods including hierarchical clustering. FLIGHT integrates RNAi screen data with microarray gene expression as well as genomic annotations and genetic/physical interaction datasets to provide a single interface for RNAi screen analysis and data-mining in Drosophila.

Human serine racemase (hSR) is a cytosolic pyridoxal-5'-phosphate dependent enzyme responsible for production of D-serine in the central nervous system. D-Serine acts as an endogenous coagonist of N-methyl-D-aspartate receptor ion channels. Increased levels of D-serine have been linked to amyotrophic lateral sclerosis and Alzheimer's disease, indicating that SR inhibitors might be useful tools for investigation or treatment of neurodegenerative diseases. However, despite hSR's promise as a therapeutic target, relatively few specific inhibitors have been identified, which is due in part to the lack of a three-dimensional structure of the enzyme. Here, we present a strategy for the generation and screening of random hSR mutants. From a library of randomly mutated hSR variants, twenty-seven soluble mutants were selected, expressed, and evaluated for enzymatic activity. Taking three carefully characterized mutants as an example, we show how this strategy can be used to pinpoint structurally and functionally important residues. In particular, we identify S84 and P111 as residues crucial for hSR activity and C217 and K221 as residues important for binding of the Mg2+ cofactor as well as for overall stability of the enzyme.

Endocrine therapy is well established for the treatment of breast cancer, and antiangiogenic agents are showing considerable promise. Targeting the vascular endothelial growth factor (VEGF) and estrogen receptor (ER) signaling pathways concomitantly may provide enhanced therapeutic benefit in ER-positive breast cancer. Therefore, the effects of the VEGF receptor (VEGFR) tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK) were investigated using human breast cancer cell lines engineered to express aromatase. As expected in this system, estrogen (E2) or androstenedione induced a proliferative response and increased ER-mediated transcription in ER-positive cell lines expressing aromatase. However, surprisingly, in the presence of androstenedione, PTK/ZK suppressed both the androstenedione-stimulated proliferation and ER-mediated transcription. PTK/ZK alone and in the presence of E2 had no observable effect on proliferation or ER-mediated transcription. These effects result from PTK/ZK having previously unrecognized antiaromatase activity and PTK/ZK being a competitive aromatase inhibitor. Computer-assisted molecular modeling showed that PTK/ZK could potentially bind directly to aromatase. The demonstration that PTK/ZK inhibits aromatase and VEGFR indicates that agents cross-inhibiting two important classes of targets in breast cancer could be developed.

To identify therapeutic targets in ovarian clear cell carcinomas, a chemoresistant and aggressive type of ovarian cancer.

D-serine plays a key role in glutamatergic neurotransmission in mammalian brain as a co-agonist of N-methyl-D-aspartate receptors. The enzyme responsible for D-serine biosynthesis, serine racemase (SR), is therefore a promising target for treatment of neuropathologies related to glutamate receptor excitotoxicity, such as stroke or Alzheimer's disease. Much of the experimental work to date has been performed on mouse serine racemase, which shares a high level of sequence identity with its human ortholog. In this work, we report the synthesis of a human SR gene variant optimized for heterologous expression in Escherichia coli and describe the expression and purification of active recombinant human SR. This strategy may be of general interest to researchers wishing to express mammalian proteins in a bacterial system. Furthermore, we conduct a thorough analysis of the kinetics and inhibitor-sensitivity of the recombinant enzyme, and we provide the first direct comparison of human and mouse SR based on our kinetic data. The orthologs behave similarly overall and exhibit identical inhibition profiles, validating the use of mouse models in SR research.

Members of the protein kinase family are amongst the most commonly mutated genes in human cancer, and both mutated and activated protein kinases have proved to be tractable targets for the development of new anticancer therapies The MoKCa database (Mutations of Kinases in Cancer, http://strubiol.icr.ac.uk/extra/mokca) has been developed to structurally and functionally annotate, and where possible predict, the phenotypic consequences of mutations in protein kinases implicated in cancer. Somatic mutation data from tumours and tumour cell lines have been mapped onto the crystal structures of the affected protein domains. Positions of the mutated amino-acids are highlighted on a sequence-based domain pictogram, as well as a 3D-image of the protein structure, and in a molecular graphics package, integrated for interactive viewing. The data associated with each mutation is presented in the Web interface, along with expert annotation of the detailed molecular functional implications of the mutation. Proteins are linked to functional annotation resources and are annotated with structural and functional features such as domains and phosphorylation sites. MoKCa aims to provide assessments available from multiple sources and algorithms for each potential cancer-associated mutation, and present these together in a consistent and coherent fashion to facilitate authoritative annotation by cancer biologists and structural biologists, directly involved in the generation and analysis of new mutational data.

L-selectin is a cell adhesion molecule that tethers leukocytes to the luminal walls of venules during inflammation and enables them to roll under the force of blood flow. Clustering of L-selectin during rolling is thought to promote outside-in signals that lead to integrin activation and chemokine receptor expression, ultimately contributing to leukocyte arrest. Several studies have underscored the importance of the L-selectin cytoplasmic tail in functionally regulating adhesion and signaling. Interestingly, the L-selectin tail comprises only 17 amino acids, and yet it is thought to bind simultaneously to several proteins. For example, constitutive association of calmodulin (CaM) and ezrin/radixin/moesin (ERM) to L-selectin confers resistance to proteolysis and microvillar positioning, respectively. In this report we found that recombinant purified CaM and ERM bound non-competitively to the same tail of L-selectin. Furthermore, molecular modeling supported the possibility that CaM, L-selectin, and moesin could form a heterotrimeric complex. Finally, using fluorescence lifetime imaging microscopy to measure fluorescence resonance energy transfer, it was shown that CaM, L-selectin, and ERM could interact simultaneously in vivo. Moreover, L-selectin clustering promoted CaM/ERM interaction in cis (i.e. derived from neighboring L-selectin tails). These results highlight a novel intracellular event that occurs as a consequence of L-selectin clustering, which could participate in transducing signals that promote the transition from rolling to arrest.

A series of small molecule, ATP-competitive phosphoinositide 3-kinase inhibitors have been examined in homology models of the four class I isoforms, p110alpha, p110beta, p110delta and p110gamma. This analysis provided an insight into the mode of binding of these inhibitors to the hinge and to other key regions of the ATP binding site in each of the four subtypes. Significantly, residues were identified that differ between these proteins, and which help explain the isoform-selective inhibition profiles of the compounds.

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis, biological activity, and further profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer.

The covalent attachment of ubiquitin to target proteins influences various cellular processes, including DNA repair, NF-kappaB signalling and cell survival. The most common mode of regulation by ubiquitin-conjugation involves specialized ubiquitin-binding proteins that bind to ubiquitylated proteins and link them to downstream biochemical processes. Unravelling how the ubiquitin-message is recognized is essential because aberrant ubiquitin-mediated signalling contributes to tumour formation. Recent evidence indicates that inhibitor of apoptosis (IAP) proteins are frequently overexpressed in cancer and their expression level is implicated in contributing to tumorigenesis, chemoresistance, disease progression and poor patient-survival. Here, we have identified an evolutionarily conserved ubiquitin-associated (UBA) domain in IAPs, which enables them to bind to Lys 63-linked polyubiquitin. We found that the UBA domain is essential for the oncogenic potential of cIAP1, to maintain endothelial cell survival and to protect cells from TNF-alpha-induced apoptosis. Moreover, the UBA domain is required for XIAP and cIAP2-MALT1 to activate NF-kappaB. Our data suggest that the UBA domain of cIAP2-MALT1 stimulates NF-kappaB signalling by binding to polyubiquitylated NEMO. Significantly, 98% of all cIAP2-MALT1 fusion proteins retain the UBA domain, suggesting that ubiquitin-binding contributes to the oncogenic potential of cIAP2-MALT1 in MALT lymphoma. Our data identify IAPs as ubiquitin-binding proteins that contribute to ubiquitin-mediated cell survival, NF-kappaB signalling and oncogenesis.

Ubiquitin-mediated inactivation of caspases has long been postulated to contribute to the regulation of apoptosis. However, detailed mechanisms and functional consequences of caspase ubiquitylation have not been demonstrated. Here we show that the Drosophila Inhibitor of Apoptosis 1, DIAP1, blocks effector caspases by targeting them for polyubiquitylation and nonproteasomal inactivation. We demonstrate that the conjugation of ubiquitin to drICE suppresses its catalytic potential in cleaving caspase substrates. Our data suggest that ubiquitin conjugation sterically interferes with substrate entry and reduces the caspase's proteolytic velocity. Disruption of drICE ubiquitylation, either by mutation of DIAP1's E3 activity or drICE's ubiquitin-acceptor lysines, abrogates DIAP1's ability to neutralize drICE and suppress apoptosis in vivo. We also show that DIAP1 rests in an "inactive" conformation that requires caspase-mediated cleavage to subsequently ubiquitylate caspases. Taken together, our findings demonstrate that effector caspases regulate their own inhibition through a negative feedback mechanism involving DIAP1 "activation" and nondegradative polyubiquitylation.

Background: Understanding the molecular control of cell lineages and fate determination in complex tissues is key to not only understanding the developmental biology and cellular homeostasis of such tissues but also for our understanding and interpretation of the molecular pathology of diseases such as cancer. The prerequisite for such an understanding is detailed knowledge of the cell types that make up such tissues, including their comprehensive molecular characterisation. In the mammary epithelium, the bulk of the tissue is composed of three cell lineages, namely the basal/myoepithelial, luminal epithelial estrogen receptor positive and luminal epithelial estrogen receptor negative cells. However, a detailed molecular characterisation of the transcriptomic differences between these three populations has not been carried out.Results: A whole transcriptome analysis of basal/myoepithelial cells, luminal estrogen receptor negative cells and luminal estrogen receptor positive cells isolated from the virgin mouse mammary epithelium identified 861, 326 and 488 genes as highly differentially expressed in the three cell types, respectively. Network analysis of the transcriptomic data identified a subpopulation of luminal estrogen receptor negative cells with a novel potential role as non-professional immune cells. Analysis of the data for potential paracrine interacting factors showed that the basal/myoepithelial cells, remarkably, expressed over twice as many ligands and cell surface receptors as the other two populations combined. A number of transcriptional regulators were also identified that were differentially expressed between the cell lineages. One of these, Sox6, was specifically expressed in luminal estrogen receptor negative cells and functional assays confirmed that it maintained mammary epithelial cells in a differentiated luminal cell lineage.Conclusion: The mouse mammary epithelium is composed of three main cell types with distinct gene expression patterns. These suggest the existence of a novel functional cell type within the gland, that the basal/myoepithelial cells are key regulators of paracrine signalling and that there is a complex network of differentially expressed transcription factors controlling mammary epithelial cell fate. These data will form the basis for understanding not only cell fate determination and cellular homeostasis in the normal mammary epithelium but also the contribution of different mammary epithelial cell types to the etiology and molecular pathology of breast disease.

The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85alpha subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.

The PI3Ks (phosphatidylinositol 3-kinases) regulate cellular signalling networks that are involved in processes linked to the survival, growth, proliferation, metabolism and specialized differentiated functions of cells. The subversion of this network is common in cancer and has also been linked to disorders of inflammation. The elucidation of the physiological function of PI3K has come from pharmacological studies, which use the enzyme inhibitors Wortmannin and LY294002, and from PI3K genetic knockout models of the effects of loss of PI3K function. Several reports have shown that LY294002 is not exclusively selective for the PI3Ks, and could in fact act on other lipid kinases and additional apparently unrelated proteins. Since this inhibitor still remains a drug of choice in numerous PI3K studies (over 500 in the last year), it is important to establish the precise specificity of this compound. We report here the use of a chemical proteomic strategy in which an analogue of LY294002, PI828, was immobilized onto epoxy-activated Sepharose beads. This affinity material was then used as a bait to fish-out potential protein targets from cellular extracts. Proteins with high affinity for immobilized PI828 were separated by one-dimensional gel electrophoresis and identified by liquid chromatography-tandem MS. The present study reveals that LY294002 not only binds to class I PI3Ks and other PI3K-related kinases, but also to novel targets seemingly unrelated to the PI3K family.

The promising antitumor activity of 17-allylamino-17-demethoxygeldanamycin (17AAG) results from inhibition of the molecular chaperone heat shock protein 90 (HSP90) and subsequent degradation of multiple oncogenic client proteins. Gene expression microarray and proteomic analysis were used to profile molecular changes in the A2780 human ovarian cancer cell line treated with 17AAG. Comparison of results with an inactive analogue and an alternative HSP90 inhibitor radicicol indicated that increased expression of HSP72, HSC70, HSP27, HSP47, and HSP90beta at the mRNA level were on-target effects of 17AAG. HSP27 protein levels were increased in tumor biopsies following treatment of patients with 17AAG. A group of MYC-regulated mRNAs was decreased by 17AAG. Of particular interest and novelty were changes in expression of chromatin-associated proteins. Expression of the heterochromatin protein 1 was increased, and expression of the histone acetyltransferase 1 and the histone arginine methyltransferase PRMT5 was decreased by 17AAG. PRMT5 was shown to be a novel HSP90-binding partner and potential client protein. Cellular protein acetylation was reduced by 17AAG, which was shown to have an antagonistic interaction on cell proliferation with the histone deacetylase inhibitor trichostatin A. This mRNA and protein expression analysis has provided new insights into the complex molecular pharmacology of 17AAG and suggested new genes and proteins that may be involved in response to the drug or be potential biomarkers of drug action.

Understanding how pathogens acquire resistance to drugs is important for the design of treatment strategies, particularly for rapidly evolving viruses such as HIV-1. Drug treatment can exert strong selective pressures and sites within targeted genes that confer resistance frequently evolve far more rapidly than the neutral rate. Rapid evolution at sites that confer resistance to drugs can be used to help elucidate the mechanisms of evolution of drug resistance and to discover or corroborate novel resistance mutations. We have implemented standard maximum likelihood methods that are used to detect diversifying selection and adapted them for use with serially sampled reverse transcriptase (RT) coding sequences isolated from a group of 300 HIV-1 subtype C-infected women before and after single-dose nevirapine (sdNVP) to prevent mother-to-child transmission. We have also extended the standard models of codon evolution for application to the detection of directional selection. Through simulation, we show that the directional selection model can provide a substantial improvement in sensitivity over models of diversifying selection. Five of the sites within the RT gene that are known to harbor mutations that confer resistance to nevirapine (NVP) strongly supported the directional selection model. There was no evidence that other mutations that are known to confer NVP resistance were selected in this cohort. The directional selection model, applied to serially sampled sequences, also had more power than the diversifying selection model to detect selection resulting from factors other than drug resistance. Because inference of selection from serial samples is unlikely to be adversely affected by recombination, the methods we describe may have general applicability to the analysis of positive selection affecting recombining coding sequences when serially sampled data are available.

Many human cancers involve up-regulation of the phosphoinositide 3-kinase PI3Kalpha, with oncogenic mutations identified in both the p110alpha catalytic and the p85alpha regulatory subunits. We used crystallographic and biochemical approaches to gain insight into activating mutations in two noncatalytic p110alpha domains-the adaptor-binding and the helical domains. A structure of the adaptor-binding domain of p110alpha in a complex with the p85alpha inter-Src homology 2 (inter-SH2) domain shows that oncogenic mutations in the adaptor-binding domain are not at the inter-SH2 interface but in a polar surface patch that is a plausible docking site for other domains in the holo p110/p85 complex. We also examined helical domain mutations and found that the Glu545 to Lys545 (E545K) oncogenic mutant disrupts an inhibitory charge-charge interaction with the p85 N-terminal SH2 domain. These studies extend our understanding of the architecture of PI3Ks and provide insight into how two classes of mutations that cause a gain in function can lead to cancer.

FLIGHT (www.flight.licr.org) is a new database designed to help researchers browse and cross-correlate data from large-scale RNAi studies. To date, the majority of these functional genomic screens have been carried out using Drosophila cell lines. These RNAi screens follow 100 years of classical Drosophila genetics, but have already revealed their potential by ascribing an impressive number of functions to known and novel genes. This has in turn given rise to a pressing need for tools to simplify the analysis of the large amount of phenotypic information generated. FLIGHT aims to do this by providing users with a gene-centric view of screen results and by making it possible to cluster phenotypic data to identify genes with related functions. Additionally, FLIGHT provides microarray expression data for many of the Drosophila cell lines commonly used in RNAi screens. This, together with information about cell lines, protocols and dsRNA primer sequences, is intended to help researchers design their own cell-based screens. Finally, although the current focus of FLIGHT is Drosophila, the database has been designed to facilitate the comparison of functional data across species and to help researchers working with other systems navigate their way through the fly genome.

pSTIING (http://pstiing.licr.org) is a new publicly accessible web-based application and knowledgebase featuring 65 228 distinct molecular associations (comprising protein-protein, protein-lipid, protein-small molecule interactions and transcriptional regulatory associations), ligand-receptor-cell type information and signal transduction modules. It has a particular major focus on regulatory networks relevant to chronic inflammation, cell migration and cancer. The web application and interface provide graphical representations of networks allowing users to combine and extend transcriptional regulatory and signalling modules, infer molecular interactions across species and explore networks via protein domains/motifs, gene ontology annotations and human diseases. pSTIING also supports the direct cross-correlation of experimental results with interaction information in the knowledgebase via the CLADIST tool associated with pSTIING, which currently analyses and clusters gene expression, proteomic and phenotypic datasets. This allows the contextual projection of co-expression patterns onto prior network information, facilitating the identification of functional modules in physiologically relevant systems.

iASPP is one of the most evolutionarily conserved inhibitors of p53, whereas ASPP1 and ASPP2 are activators of p53. We show here that, in addition to the DNA-binding domain, the ASPP family members also bind to the proline-rich region of p53, which contains the most common p53 polymorphism at codon 72. Furthermore, the ASPP family members, particularly iASPP, bind to and regulate the activity of p53Pro72 more efficiently than that of p53Arg72. Hence, escape from negative regulation by iASPP is a newly identified mechanism by which p53Arg72 activates apoptosis more efficiently than p53Pro72.

In systems biology, biologically relevant quantitative modelling of physiological processes requires the integration of experimental data from diverse sources. Recent developments in high-throughput methodologies enable the analysis of the transcriptome, proteome, interactome, metabolome and phenome on a previously unprecedented scale, thus contributing to the deluge of experimental data held in numerous public databases. In this review, we describe some of the databases and simulation tools that are relevant to systems biology and discuss a number of key issues affecting data integration and the challenges these pose to systems-level research.

Protein expression and de novo synthesis in normal and prostate cancer cell lines derived from the same patient were compared by proteomic analysis, and the effects of INFalpha and INFgamma (INF=interferon) determined. The expressions of several INF-inducible proteins, including MxA, Nmi, PA28a and IFP53, were downregulated in the cancer cells. INFgamma induced a more than twofold increase or decrease in the synthesis rates of almost twice as many proteins in the cancer cell line. The positive regulator of INF-induced transcription ISGF3gamma was upregulated in the cancer cells and inversely regulated by INFalpha and INFgamma in the normal and cancer cells. Moreover, ISGF3gamma's induction by INFgamma in the cancer cells was more enhanced by simultaneous stimulation with EGF, than its induction in the normal cells. In all, 31 differentially regulated proteins were identified by mass spectrometry analysis, several of which are involved in chaperone-assisted protein folding in the endoplasmic reticulum (ER) or in regulated protein degradation. Our results suggest that the exclusion of proteins by the ER quality control system, crosstalk between the EGF- and INF-induced signalling pathways and the regulation of INF-inducible genes are all altered in the prostate cancer cells. The combination of upregulated activity in the growth-promoting PI3K/Akt pathway, suppression of Nmi and overexpression of hnRNP-K and c-myc proteins may explain why the prostate cancer cells were found to be more resistant to the growth inhibitory effects of INFgamma.

Dictyostelium, the only known non-metazoan organism to employ SH2 domain:phosphotyrosine signaling, possesses STATs (signal transducers and activators of transcription) and protein kinases with orthodox SH2 domains. Here, however, we describe a novel Dictyostelium STAT containing a remarkably divergent SH2 domain. Dd-STATb displays a 15 amino acid insertion in its SH2 domain and the conserved and essential arginine residue, which interacts with phosphotyrosine in all other known SH2 domains, is substituted by leucine. Despite these abnormalities, Dd-STATb is biologically functional. It has a subtle role in growth, so that Dd-STATb-null cells are gradually lost from the population when they are co-cultured with parental cells, and microarray analysis identified several genes that are either underexpressed or overexpressed in the Dd-STATb null strain. The best characterised of these, discoidin 1, is a marker of the growth-development transition and it is overexpressed during growth and early development of Dd-STATb null cells. Dimerisation of STAT proteins occurs by mutual SH2 domain:phosphotyrosine interactions and dimerisation triggers STAT nuclear accumulation. Despite its aberrant SH2 domain, the Dd-STATb protein sediments at the size expected for a homodimer and it is constitutively enriched in the nucleus. Moreover, these properties are retained when the predicted site of tyrosine phosphorylation is substituted by phenylalanine. These observations suggest a non-canonical mode of activation of Dd-STATb that does not rely on orthodox SH2 domain:phosphotyrosine interactions.

In the current study, the protein expression maps (PEMs) of 26 breast cancer cell lines and three cell lines derived from normal breast or benign disease tissue were visualised by high resolution two-dimensional gel electrophoresis. Analysis of this data was performed with ChiClust and ChiMap, two analytical bioinformatics tools that are described here. These tools are designed to facilitate recognition of specific patterns shared by two or more (a series) PEMs. Both tools use PEMs that were matched by an image analysis program and locally written programs to create a match table that is saved in an object relational database. The ChiClust tool uses clustering and subclustering methods to extract statistically significant protein expression patterns from a large series of PEMs. The ChiMap tool calculates a differential value (either as percentage change or a fold change) and represents these graphically. All such differentials or just those identified using ChiClust can be submitted to ChiMap. These methods are not dependent on any particular commercial image analysis program, and the whole software package gives an integrated procedure for the comparison and analysis of a series of PEMs. The ChiClust tool was used here to order the breast cell lines into groups according to biological characteristics including morphology in vitro and tumour forming ability in vivo. ChiMap was then used to highlight eight major protein feature-changes detected between breast cancer cell lines that either do or do not proliferate in nude mice. Mass spectrometry was used to identify the proteins. The possible role of these proteins in cancer is discussed.

The technique of fluorescent two-dimensional (2D) difference gel electrophoresis for differential protein expression analysis has been evaluated using a model breast cancer cell system of ErbB-2 overexpression. Labeling of paired cell lysate samples with N-hydroxy succinimidyl ester-derivatives of fluorescent Cy3 and Cy5 dyes for separation on the same 2D gel enabled quantitative, sensitive, and reproducible differential expression analysis of the cell lines. SyproRuby staining was shown to be a highly sensitive and 2D difference gel electrophoresis-compatible method for post-electrophoretic visualization of proteins, which could then be picked and identified by matrix-assisted laser-desorption ionization mass spectroscopy. Indeed, from these experiments, we have identified multiple proteins that are likely to be involved in ErbB-2-mediated transformation. A triple dye labeling methodology was used to identify proteins differentially expressed in the cell system over a time course of growth factor stimulation. A Cy2-labeled pool of samples was used as a standard with all Cy3- and Cy5-labeled sample pairs to facilitate cross-gel quantitative analysis. DeCyder (Amersham Biosciences, Inc.) software was used to distinguish clear statistical differences in protein expression over time and between the cell lines.

It is only recently that quantitative studies of differential proteome analysis (DPA) have become possible. In this paper the issues involved in quantitative DPA are discussed and novel tools to select features for identification by mass spectrometry (MS) are described. The problem of comparing two sets of gels on a global level is explored as well as how to find specific protein features that differentiate two sets of two-dimensional electrophoresis gels. The concept of a 'virtual' gel, derived from gene expression data, is introduced. The virtual gel enables the co-analysis of data from gene and protein expression. We discuss the value of such an approach, and consider what new information can be gained by using gene and protein expression together. These tools are illustrated by analysis of data from tandem gene and protein expression experiments. Features that are highlighted by the above methods are putative candidates for MS identification. Tools are described that integrate the process of feature selection, cutting, and MS analysis.

The protein complement of breast cells consists of many thousands of proteins. Recent developments in 2D gel electrophoresis technology have made studies requiring the quantitative analysis of a differential proteome, such as comparison between normal and malignant cells or investigation of drug effects on cells, truly feasible. Computer software plays a central part in the comparisons between multiple gels required for such experiments. In addition, software tools allow patterns of coexpression of proteins to be studied, offering potential insights into protein regulation, interactions, and functions, especially when combined with complementary data on gene expression. In this paper, the technology and limitations of 2D gel-based proteomics are reviewed. Techniques for comparing sets of gels at a global level as well as identifying specific protein features that differentiate gels are discussed. Our own experience of studying the breast cell proteome is used to illustrate the difficulties and achievements of differential proteomics.

Normal somatic cells undergo a finite number of divisions and then cease dividing whereas cancer cells are able to proliferate indefinitely. To identify the underlying mechanisms that limit the mitotic potential, a two-dimensional differential proteome analysis of replicative senescence in serially passaged rat embryo fibroblasts was undertaken. Triplicate independent two-dimensional gels containing over 1200 spots each were run, curated, and analyzed. This revealed 49 spots whose expression was altered more than 2-fold. Of these, 42 spots yielded positive protein identification by mass spectrometry comprising a variety of cytoskeletal, heat shock, and metabolic proteins, as well as proteins involved in trafficking, differentiation, and protein synthesis, turnover, and modification. These included gelsolin, a candidate tumor suppressor for breast cancer, and alpha-glucosidase II, a member of the family of glucosidases that includes klotho; a defect in klotho expression in mice results in a syndrome that resembles human aging. Changes in expression of TUC-1, -2, -4, and -4 beta, members of the TUC family critical for neuronal differentiation, were also identified. Some of the identified changes were also shown to occur in two other models of senescence, premature senescence of REF52 cells and replicative senescence of mouse embryo fibroblasts. The majority of these candidate proteins were unrecognized previously in replicative senescence. They are now implicated in a new role.

Phosphoinositide 3-kinases (PI3Ks) are dual specificity lipid and protein kinases. While the lipid-dependent PI3K downstream signaling is well characterized, little is known about PI3K protein kinase signaling and structural determinants of lipid substrate specificity across the various PI3K classes. Here we show that sequences C-terminal to the PI3K ATP-binding site determine the lipid substrate specificity of the class IA PI3Kalpha (p85/p110alpha). Transfer of such activation loop sequences from class II PI3Ks, class III PI3Ks, and a related mammalian target of rapamycin (FRAP) into p110alpha turns the lipid substrate specificity of the resulting hybrid protein into that of the donor protein, while leaving the protein kinase activity unaffected. All resulting hybrids lacked the ability to produce phosphatidylinositol 3,4,5-trisphosphate in intact cells. Amino acid substitutions and structure modeling showed that two conserved positively charged (Lys and Arg) residues in the activation loop are crucial for the functionality of class I PI3Ks as phosphatidylinositol 4,5-bisphosphate kinases. By transient transfecion of 293 cells, we show that p110alpha hybrids, although unable to support lipid-dependent PI3K signaling, such as activation of protein kinase B/Akt and p70(S6k), retain the capability to associate with and phosphorylate insulin receptor substrate-1, with the same specificity and higher efficacy than wild type PI3Kalpha. Our data lay the basis for the understanding of the class I PI3K substrate selectivity and for the use of PI3Kalpha hybrids to dissect PI3Kalpha function as lipid and protein kinase.

There is an urgent need for the development of new drugs to treat Chagas' disease, which is caused by the protozoan parasite Trypanosoma cruzi. The enzyme dihydrofolate reductase (DHFR) has been a very successful drug target in a number of diseases and we decided to investigate it as a potential drug target for Chagas' disease. A homology model of the enzyme was used to search the Cambridge Structural Database using the program DOCK 3.5. Compounds were then tested against the enzyme and the whole parasite. Compounds were also screened against the related parasite, Trypanosoma brucei.

Fas ligand (FasL) induces apoptosis through its cell surface receptor Fas. T lymphocytes and natural killer cells sort newly synthesised FasL to secretory lysosomes but, in cell types with conventional lysosomes, FasL appears directly on the plasma membrane. Here, we define a proline-rich domain (PRD) in the cytoplasmic tail of FasL that is responsible for sorting FasL to secretory lysosomes. Deletion of this PRD results in cell surface expression of FasL in cells with secretory lysosomes. Positively charged residues flanking the PRD are crucial to the sorting motif and changing the charge of these residues causes mis-sorting to the plasma membrane. In cells with conventional lysosomes, this motif is not recognised and FasL is expressed at the plasma membrane. The FasL PRD is not required for endocytosis in any cell type, as deletion mutants lacking this motif are endocytosed efficiently to the lysosomal compartment. Endogenous FasL cannot internalise extracellular antibody, demonstrating that FasL does not transit the plasma membrane en route to the secretory lysosomes. We propose that an interaction of the PRD of FasL with an SH3-domain-containing protein, enables direct sorting of FasL from the Golgi to secretory lysosomes.

The class II phosphoinositide 3-kinases (PI3K) PI3K-C2alpha and PI3K-C2beta are two recently identified members of the large PI3K family. Both enzymes are characterized by the presence of a C2 domain at the carboxy terminus and, in vitro, preferentially utilize phosphatidylinositol and phosphatidylinositol 4-monophosphate as lipid substrates. Little is understood about how the catalytic activity of either enzyme is regulated in vivo. In this study, we demonstrate that PI3K-C2alpha and PI3K-C2beta represent two downstream targets of the activated epidermal growth factor (EGF) receptor in human carcinoma-derived A431 cells. Stimulation of quiescent cultures with EGF resulted in the rapid recruitment of both enzymes to a phosphotyrosine signaling complex that contained the EGF receptor and Erb-B2. Ligand addition also induced the appearance of a second, more slowly migrating band of PI3K-C2alpha and PI3K-C2beta immunoreactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both PI3K enzymes can utilize Ca(2+) as an essential divalent cation in lipid kinase assays and since the catalytic activity of PI3K-C2alpha is refractory to the inhibitor wortmannin, these properties were used to confirm the recruitment of each PI3K isozyme to the activated EGF receptor complex. To examine this interaction in greater detail, PI3K-C2beta was chosen for further investigation. EGF and platelet-derived growth factor also stimulated the association of PI3K-C2beta with their respective receptors in other cells, including epithelial cells and fibroblasts. The use of EGF receptor mutants and phosphopeptides derived from the EGF receptor and Erb-B2 demonstrated that the interaction with recombinant PI3K-C2beta occurs through E(p)YL/I phosphotyrosine motifs. The N-terminal region of PI3K-C2beta was found to selectively interact with the EGF receptor in vitro, suggesting that it mediates the association of this PI3K with the receptor. However, the mechanism of this interaction remains unclear. We conclude that class II PI3K enzymes may contribute to the generation of 3' phosphoinositides following the activation of polypeptide growth factor receptors in vivo and thus mediate certain aspects of their biological activity.

Epidemiological studies have shown an increased risk of breast cancer in obligate ataxia telangiectasia (A-T) heterozygotes. We analyzed 100 samples from young breast cancer patients for mutations in ataxia-telangiectasia mutated (ATM), the gene responsible for the autosomal recessive condition, A-T, to determine whether A-T heterozygosity predisposes such individuals to develop breast cancer. These patients were selected from families with a moderate or absent family history of breast cancer and included a subset of 16 radiosensitive patients. Forty-four germline sequence variants were detected by fluorescent chemical cleavage of mismatch of RT-PCR products. These included seven rare variants found in nine patients (three described for the first time), but no truncating mutations. Although three variants were detected in the radiosensitive subset, this was not statistically significant compared to the nonradiosensitive group. One variant, G2765S, is likely to be a missense mutation, but the other six variants probably represent rare polymorphisms. However, five of the seven rare germline variants detected showed loss of heterozygosity of the wild-type ATM allele for one or more markers close to the ATM locus in matched tumor DNA. This high rate of somatic inactivation of ATM may indicate either that these rare variants play a role in breast cancer development or alternatively that a neighboring tumor suppressor gene is important for tumorigenesis. We found germline truncating ATM mutations to be rare in these young breast cancer patients and therefore they are unlikely to play a role in the etiology of their disease. Genes Chromosomes Cancer 26:286-294, 1999.

Normal human luminal and myoepithelial breast cells separately purified from a set of 10 reduction mammoplasties by using a double antibody magnetic affinity cell sorting and Dynabead immunomagnetic technique were used in two-dimensional gel proteome studies. A total of 43,302 proteins were detected across the 20 samples, and a master image for each cell type comprising a total of 1,738 unique proteins was derived. Differential analysis identified 170 proteins that were elevated 2-fold or more between the two breast cell types, and 51 of these were annotated by tandem mass spectrometry. Muscle-specific enzyme isoforms and contractile intermediate filaments including tropomyosin and smooth muscle (SM22) alpha protein were detected in the myoepithelial cells, and a large number of cytokeratin subclasses and isoforms characteristic of luminal cells were detected in this cell type. A further 134 nondifferentially regulated proteins were also annotated from the two breast cell types, making this the most extensive study to date of the protein expression map of the normal human breast and the basis for future studies of purified breast cancer cells.

Hoechst is developing flavopiridol, a synthetic flavonoid based on an extract from an Indian plant, for the potential treatment of cancer. Flavopiridol, a cyclin-dependent kinase inhibitor, arrests cell division and causes apoptosis in non-small lung cancer cells [283660]. A phase II trial, in collaboration with the National Cancer Institute, has commenced at the University of Chicago Medical Center, which involves patients with high or intermediate-grade lymphoma or multiple myeloma [272937], [277372]. In ex vivo experiments with tumor cells from refractory chronic lymphoblastic leukemia, dose-dependent CDK2 inhibition associated with apoptotic changes was seen at concentrations greater than 100 nM of flavopiridol. In vitro pharmacokinetic studies have shown that flavopiridol undergoes hepatic biotransformation to its corresponding glucoronide by uridine diphosphate glucoronosyltransferases [283791]. Flavopiridol inhibits CDK with an IC50 value of 0.4 mM [285707]. Preclinical toxicology studies in rats and dogs demonstrated dose-related leukopenia and drug-related lesions in the thymus, spleen and bone marrow. The gastrointestinal and bone marrow toxicity was dose-limiting [178579]. Hoechst Marion Roussel expects to launch flavopiridol in the year 2001, with potential sales in excess of DM 750 million [288651].

The three-dimensional structure of the Sorghum bicolor seed protein gamma-thionin SIalpha1 has been determined by 2D 1H nuclear magnetic resonance (NMR) spectroscopy. The secondary structure of this 47-residue antifungal protein with four disulphide bridges consists of a three-stranded antiparallel sheet and one helix. The helix is tethered to the sheet by two disulphide bridges which link two successive turns of the helix to alternate residues i, i+2 in one strand. Possible binding sites for antifungal activity are discussed. The same fold has been observed previously in several scorpion toxins.

The cDNA for a human Class II phosphoinositide 3-kinase (PI 3-kinase C2beta) with a C2 domain was cloned from a U937 monocyte cDNA library and the enzyme expressed in mammalian and insect cells. Like other Class II PI 3-kinases in vitro, PI 3-kinase C2beta utilizes phosphatidylinositol (PI) and PI 4-monophosphate but not PI 4, 5-biphosphate as substrates in the presence of Mg2+. Remarkably, and unlike other PI 3-kinases, the enzyme can use either Mg-ATP or Ca-ATP to generate PI 3-monophosphate. PI 3-kinase C2beta, like the Class I PI 3-kinases, but unlike PI 3-kinase C2alpha, is sensitive to low nanomolar levels of the inhibitor wortmannin. The enzyme is not regulated by the small GTP-binding protein Ras. The C2 domain of the enzyme bound anionic phospholipids such as PI and phosphatidylserine in vitro, but did not co-operatively bind Ca2+ and phospholipids. Deletion of the C2 domain increased the lipid kinase activity suggesting that it functions as a negative regulator of the catalytic domain. Although presently it is not known whether PI 3-kinase C2beta is regulated by Ca2+ in vivo, our results suggest a novel role for Ca2+ ions in phosphate transfer reactions.

SH2 domains have a fundamental role in signal transduction. These domains interact with proteins containing phosphorylated tyrosine residues and, in doing so, mediate the interactions of proteins involved in tyrosine kinase signalling. The issue of specificity in SH2 domain interactions is therefore of great interest in terms of understanding tyrosine kinase signal-transduction pathways and in the discovery of drugs to inhibit them. Water molecules are found at the interfaces of many complexes, however, to date little attention has been paid to their role in dictating specificity.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that have been implicated in signal transduction through tyrosine kinase- and heterotrimeric G-protein-linked receptors. We report herein the cloning and characterization of p110delta, a novel class I PI3K. Like p110alpha and p110beta, other class I PI3Ks, p110delta displays a broad phosphoinositide lipid substrate specificity and interacts with SH2/SH3 domain-containing p85 adaptor proteins and with GTP-bound Ras. In contrast to the widely distributed p110alpha and beta, p110delta is exclusively found in leukocytes. In these cells, p110alpha and delta both associate with the p85alpha and beta adaptor subunits and are similarly recruited to activated signaling complexes after treatment with the cytokines interleukin 3 and 4 and stem cell factor. Thus, these class I PI3Ks appear not to be distinguishable at the level of p85 adaptor selection or recruitment to activated receptor complexes. However, distinct biochemical and structural features of p110delta suggest divergent functional/regulatory capacities for this PI3K. Unlike p110alpha, p110delta does not phosphorylate p85 but instead harbors an intrinsic autophosphorylation capacity. In addition, the p110delta catalytic domain contains unique potential protein-protein interaction modules such as a Pro-rich region and a basic-region leucine-zipper (bZIP)-like domain. Possible selective functions of p110delta in white blood cells are discussed.

The generation of phosphatidylinositide 3-phosphates has been observed in a variety of cellular responses. The enzymes that mediate synthesis are the phosphoinositide 3-kinases (PI3-Ks) that form a family of structurally diverse enzymes with distinct substrate specificities. In this paper, we describe the cloning of a novel human PI3-K, namely PI3-K-C2 alpha, which contains a C-terminal C2 domain. This enzyme can be assigned to the class II PI3-Ks, which was defined by characterization of the Drosophila 68D enzyme and includes the recently described murine enzymes m-cpk and p170. Despite the overall similarity in the amino acid sequence of the murine and human enzymes, which suggests that they are encoded by closely related genes, these molecules show marked sequence heterogeneity at their N-termini. Biochemical analysis of recombinant PI3-K-C2 alpha demonstrates a restricted lipid substrate specificity. As reported for other members of this class, the enzyme only phosphorylates PtdIns and PtdIns4P when the lipids are presented alone. However, when lipids were presented together with phosphatidylserine acting as a carrier, phosphorylation of PtdIns(4,5)P2 was also observed. The catalytic activity of PI3-K-C2 alpha is refractory to concentrations of wortmannin and LY294002 which inhibit the PI3-K activity of other family members. The comparative insensitivity of PI3-K-C2 alpha to these inhibitors suggests that their use should be reevaluated in the study of PI3-Ks.

Perforin is a secreted protein synthesized by activated cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. It is a key component of the lytic machinery of these cells, being able to insert into the plasma membrane of targeted cells, forming a pore which leads to their destruction. Here we analyse the synthesis, processing and intracellular transport of perforin in the NK cell line YT. Perforin is synthesized as a 70 kDa inactive precursor which is cleaved at the C-terminus to yield a 60 kDa active form. This proteolytic cleavage occurs in an acidic compartment and can be inhibited by incubation of the cells in ammonium chloride, concanamycin A, leupeptin and E-64. The increased lytic activity of the cleaved form can be demonstrated by killing assays in which cleavage of the pro-piece is inhibited. Epitope mapping reveals that cleavage of the pro-piece occurs at the boundary of a C2 domain, which we show is able to bind phospholipid membranes in a calcium-dependent manner. We propose that removal of the pro-piece, which contains a bulky glycan, allows the C2 domain to interact with phospholipid membranes and initiate perforin pore formation.

Phosphoinositide 3-kinases (PI3-kinases) have been shown to be recruited to cell surface receptor signal complexes whose formation is triggered by growth factors, cytokines and other ligands. PI3-kinases are also involved in protein sorting phenomena. A number of PI3-kinase isotypes have been characterised in several laboratories. Here the relations between the PI3-kinases, PI4-kinases and PI5-kinases and other potential phosphoinositide kinases are analysed. A study of the relation of structure to function for sequence motifs defined through the use of homology searches and protein modelling techniques is described and used to assign the family of phosphoinositide kinases to subgroups.

Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations, wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA-dependent protein kinase (DNA-PKcs). We report here the identification of the site of interaction of wortmannin on the catalytic subunit of PI 3-kinase, p110alpha. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110alpha. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant p110alpha were mapped with anti-wortmannin and anti-p110alpha peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes.

Src-homology 3 (SH3) domains are small protein modules that bind to proline-rich motifs and mediate the formation of signalling complexes. SH3 domains have been implicated in the assembly of the phagocyte NADPH oxidase complex, a multicomponent enzyme responsible for the production of antimicrobial oxidants. Two components of the NADPH oxidase, p67phox and p47phox, each contain two SH3 domains and we have previously shown that the SH3 domain near the carboxyl terminus of p67phox interacts with a proline-rich region of p47phox. In order to gain an insight into the specificity of this interaction, a structural model of the p67phox SH3 domain has been produced using the known structure of the c-abl SH3 domain as a template. The model suggests that the proline-rich ligand of p47phox can bind to the SH3 domain in either of two orientations. In each orientation, the key residues of the SH3 domain that contact the ligand have been identified and altered by site-directed mutagenesis. The ability of the mutated SH3 domains to associate with p47phox from cell lysates was tested and the results provide the first evidence for the binding of a full-length protein to an SH3 domain in a reversed orientation.

Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor-based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interacted with liposomes containing phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol-4-phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide-PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically bound liposomes containing phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X-linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy.

The relationships between the antigen-binding specificities of four human monoclonal anti-DNA antibodies and the structural aspects of the combining sites of two of these were examined. Competition ELISAs were used to examine the reactivities of two IgM MAbs (WRI-176 and RT-79) and two IgG mAbs (D5 and B3) to a wide range of polynucleotides. The mAbs WRI-176 and RT-79 were found to bind predominantly ssDNA, with a preference for poly (dT), whilst D5 and B3 bound components of both ss- and dsDNA, and Z-DNA. The mAb B3 also exhibited a preference for A(T) rich nucleotides. Computer models were generated for the Fv regions of WRI-176 and B3. Models for RT-79 and D5 were not generated as the structure of the long CDR-H3 loops in these mAbs could not be predicted. The B3 combining site contains a groove flanked by three arginines at positions CDR-L1-27A, CDR-L2-54 and CDR-H2-53. Using interactive molecular graphics, B-DNA was docked into the B3 antigen combining site along the plane of the VH/VL interface, whilst Z-DNA was best-fitted at approximately 90 degrees to this direction. The models provide a hypothesis to explain the ability of a single autoantibody to bind two different antigens. In addition, aspects of the base specificity of B3 may be explained. The model of the WRI-176 Fv region revealed a relatively flat surface, on which a large number of hydrophobic and aromatic residues were present. Trp-H52, in particular, is prominent on the surface. This may participate in ssDNA binding through base stacking interactions. The models allow identification of potential targets for site-directed mutagenesis.

Phosphoinositide (PI) 3-kinases have been characterized as enzymes involved in receptor signal transduction in mammalian cells and in a complex which mediates protein trafficking in yeast. PI 3-kinases linked to receptors with intrinsic or associated tyrosine kinase activity are heterodimeric proteins, consisting of p85 adaptor and p110 catalytic subunits, which can generate the 3-phosphorylated forms of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 as potential second messengers. Yeast Vps34p kinase, however, has a substrate specificity restricted to PtdIns and is a PtdIns 3-kinase. Here the molecular characterization of a new human PtdIns 3-kinase with extensive sequence homology to Vps34p is described. PtdIns 3-kinase does not associate with p85 and phosphorylates PtdIns, but not PtdIns4P or PtdIns(4,5)P2. In vivo PtdIns 3-kinase is in a complex with a cellular protein of 150 kDa, as detected by immunoprecipitation from human cells. Protein sequence analysis and cDNA cloning show that this 150 kDa protein is highly homologous to Vps15p, a 160 kDa protein serine/threonine kinase associated with yeast Vps34p. These results suggest that the major components of the yeast Vps intracellular trafficking complex are conserved in humans.

Germline mutations within one of six codons of the RET proto-oncogene account for the majority of cases of multiple endocrine neoplasia (MEN) type 2A and type 2B and familial medullary thyroid carcinoma (FMTC). MEN 2A and FMTC mutations characterised thus far occur exclusively in the cysteine-rich domain of the extracellular region of RET. We now report a missense mutation in the intracellular tyrosine kinase domain of RET in the germline of a family with FMTC that does not have a cysteine codon mutation. In this family, the mutation, which alters GAG (Glu) to GAC (Asp) at codon 768, segregates with the FMTC phenotype. The same mutation was also detected in sporadic MTC but not in corresponding constitutional DNA, confirming that it is likely to be of pathological significance rather than a rare polymorphism.

Deficiencies in a tyrosine kinase, designated Btk, cause X-linked agammaglobulinemia (XLA) in man, a hereditary defect of B-cell differentiation. Mutations in the newly found PH domain located at the N-terminus of Btk have been shown to be the direct cause of XLA, and here two new mutations, T33P and V64F, are presented. Btk is thus far the only protein in which mutations of the PH domain have been found to cause a disease. The three-dimensional structure of the Btk PH domain was modeled on the basis of the dynamin PH structure. Despite a relatively low sequence similarity the Btk PH domain seems to have the same two beta-sheet structure observed in the known structures. The model was used to interpret the structural basis for disease in five independent point mutations and in an insertion in patients with XLA. The mutated residues F25, V64, and V113, and possibly residue(s) around Q103, could form a binding site, since these amino acids are located close to each other on the surface of the molecule.

Src homology 2 (SH2) domains are small protein modules of approximately 100 amino acids that are found in many proteins involved in intracellular signal transduction. They mediate protein-protein interactions and modulate enzyme activity by their ability to bind to specific sequence patterns that contain a phosphorylated tyrosine. As the three-dimensional structures of the phosphatidylinositol (PI) 3-kinase, Lck, Src and Abl SH2 domains have been shown to be similar, we have modelled other SH2 domains that show distinct sequence specificity to allow comparative analysis of SH2-phosphopeptide interactions. The SH2 domains of PLC gamma-Nterm., Nck, Grb2, GAP and Abl have been model-built with high-affinity phosphopeptides fitted into the putative binding sites. For each SH2 domain a detailed analysis of the peptide-protein interaction was performed. It is apparent that specificity is mainly conferred by three to five residues downstream from the phosphotyrosine residue (Y*), especially, although not exclusively, peptide position Y* + 3. The SH2 pocket that binds the Y* + 3 residue is mainly composed of three sections: part of strand beta E going into loop EF, part of alpha B and loop BG. The residues that constitute the Y* + 3 binding pocket show variability that seems to determine which amino acid binds preferentially. Residue position beta E4 seems to play a vital role in the SH2 specificity. This study shows that the development of modelling protocols for SH2 domains whose structure has not been determined can prove very useful in predicting which residues are involved in conferring the affinity and binding specificity of these domains towards distinct phosphotyrosine-containing sequences.

An unexpected structural similarity is described between the pleckstrin homology (PH) domain and verotoxin. This similarity has escaped detection primarily due to the differences in topology that exist between the two proteins. By comparing this result with two previously reported similarities for the PH domain, one with the lipocalins and another with the FK506 binding protein, we discuss the problems of measuring and assessing structural similarities.

The pleckstrin homology (PH) domain is a region of approximately 100 amino acids, defined by sequence similarity, that has been found in about 60 proteins, many of which are involved in signal transduction downstream of cell surface receptors; the function of PH domains is unknown. The only clue to the function of PH domains is the circumstantial evidence that they may link beta gamma subunits of G proteins to second messenger systems. Knowledge of the three-dimensional structures of PH domains should help to elucidate the roles they play in the proteins that contain them.

The dithiol trypanothione, novel to trypanosomatids and analogous to glutathione in mammalian systems, has been shown to interact with anti-trypanocidal trivalent arsenical drugs forming a stable adduct, MelT. This adduct is a competitive inhibitor of the flavoprotein trypanothione reductase, responsible for maintaining intracellular trypanothione in the reduced form. Since trypanothione reductase and the analogous glutathione reductase both contain catalytically active sulphydryl groups we have examined the ability of several arsenicals to differentially inhibit these enzymes. Melarsen oxide [p-(4,6-diamino-s-triazin-2-yl)aminophenylarsenoxide] potently inhibits both enzymes in two stages, the first being essentially complete within 1 min, the second being time dependent, exhibiting saturable pseudo-first-order kinetics with kinact of 14.3 x 10(-4) s-1 and 1.06 x 10(-4) s-1 and Ki of 17.2 microM and 9.6 microM for trypanothione reductase and glutathione reductase, respectively. Inhibition requires prior reduction of the enzyme by NADPH and can be reversed by excess dithiols or prevented by MelT in the case of trypanothione reductase. In both cases a time-dependent loss of the characteristic charge-transfer absorbance band at 530 nm is observed upon addition of arsenical to pre-reduced enzyme, which with excess NADPH leads to a spectrum resembling the EH4 form and is accompanied by an increased ability to reduce molecular oxygen. A model for inhibition is proposed where, first, free arsenical and previously reduced enzyme immediately establish an equilibrium with an inactive monothioarsane enzyme-inhibitor complex involving the interchange cysteine distal to the FAD; second, a subsequent rearrangement about the sulphur-arsenic bond leads to the binding of the arsenical to the charge-transfer cysteine, proximal to the FAD, forming a more stable dithioarsane complex. Molecular modelling suggests that the differences in kinetic behaviour of the two enzymes can be attributed to structural features of their respective disulphide-binding sites. Incubation of reduced trypanothione reductase with excess dihydrotrypanothione and melarsen oxide prevents direct inhibition of the enzyme, suggesting that dihydrotrypanothione acts as a protectant in vivo, preventing the direct modification of trypanothione reductase by sequestering the arsenical as MelT.

The sequence of the major soluble protein component of the cuticle of filarial nematodes is homologous to that of bovine glutathione peroxidase, for which an X-ray structure is available. Due to the high degree of sequence identity (42%), it has been possible to build an apparently reliable three-dimensional model of the gp29 cuticular protein from Brugia spp. that will aid studies of the molecule both as a target immunogen and secreted enzyme. The modelled core of the gp29 structure is conserved compared to the bovine enzyme, consisting of a beta-sheet surrounded by alpha-helices. Experimental data showed that Brugia spp. gp29 has four subunits, and a tetrameric form of gp29 has also been modelled. The two N-linked glycosylation sites per subunit were predicted to lie on the surface of the tetramer. Most of the variation in amino acid sequence compared to that of mammalian enzymes, occurs in the surface loops, several of which are larger and more exposed in gp29. Deglycosylated gp29 was demonstrated to be immunogenic in human infection, and six likely B-cell epitopes have been predicted on the basis of a high protrusion index and sequence variability.

Using a variety of techniques, including sequence alignment, secondary structure prediction, molecular mechanics and molecular dynamics, we have constructed a model for the three-dimensional structure of P-450arom (human aromatase) based on that of P-450cam, the only cytochrome P-450 enzyme for which the crystal structure is known. The predicted structure is found to be in good agreement with current experimental data; both direct, from site-directed mutagenesis studies, and indirect, from the consideration of the structures and activities of known substrates and inhibitors.

The pyruvate-stimulated adenylate cyclase from Brevibacterium liquefaciens produces up to 450 microM cyclic AMP in the culture medium when the bacterium is grown on glucose and alanine. In this paper we report the cloning, expression and sequencing of the gene for this enzyme. Residues were identified, within the C-terminal domain, which are conserved in adenylate and guanylate cyclase sequences from eukaryotes and in the adenylate cyclase of the prokaryote Rhizobium meliloti. We have also identified a sequence of 30 residues near the N-terminus of the protein which is homologous to part of the regulatory domain of the cellular homologues of the oncogenes fes and fps; this sequence is also present in the avian Fujinami sarcoma virus fps gene.

A three-dimensional structure for human cytochrome P450IA1 was predicted based on the crystal coordinates of cytochrome P450cam from Pseudomonas putida. As there was only 15% residue identity between the two enzymes, additional information was used to establish an accurate sequence alignment that is a prerequisite for model building. Twelve representative eukaryotic sequences were aligned and a net prediction of secondary structure was matched against the known alpha-helices and beta-sheets of P450cam. The cam secondary structure provided a fixed main-chain framework onto which loops of appropriate length from the human P450IA1 structure were added. The model-built structure of the human cytochrome conformed to the requirements for the segregation of polar and nonpolar residues between the core and the surface. The first 44 residues of human cytochrome P450 could not be built into the model and sequence analysis suggested that residues 1-26 formed a single membrane-spanning segment. Examination of the sequences of cytochrome P450s from distinct gene families suggested specific residues that could account for the differences in substrate specificity. A major substrate for P450IA1, 3-methyl-cholanthrene, was fitted into the proposed active site and this planar aromatic molecule could be accommodated into the available cavity. Residues that are likely to interact with the haem were identified. The sequence similarity between 59 eukaryotic enzymes was represented as a dendrogram that in general clustered according to gene family. Until a crystallographic structure is available, this model-building study identifies potential residues in cytochrome P450s important in the function of these enzymes and these residues are candidates for site-directed mutagenesis.

Methods to predict the three-dimensional structure of a protein from its sequence are reviewed. The approaches to derive information about the local conformation from the local sequence include hydrophobicity plots, secondary structure prediction and the identification of short, functional sequence motifs. The most reliable method of tertiary structure prediction is model building from the experimentally determined coordinates of a protein with an homologous sequence. This approach is illustrated by a prediction of the three-dimensional structure of human cytochrome P450-IA1. If no known homologous structure is available, then the only approach is to suggest models for the tertiary fold of proteins by packing together predicted secondary structures. A three-dimensional model for the dimerisation of the transmembrane alpha-helices of neu, a tyrosine kinase growth factor receptor, is described. In general, structure prediction can suggest approaches for regulating protein activity that may lead to new pharmaceutical therapies for cancer.

The enzyme cytochrome P450(17 alpha) catalyses two key steps in the biosynthesis of the androgens from pregnanes: the 17 alpha hydroxylation step and the subsequent 17-20 lyase reaction. Using a variety of techniques, including sequence alignment, secondary structure prediction, molecular mechanics and molecular dynamics, we have constructed a model for the three-dimensional structure of P450(17 alpha) based on that of P450cam, the only cytochrome P450 enzyme for which the crystal structure is known. The model suggests the possibility of two modes of binding of steroid substrates at the active site, perhaps reflecting the dual functionality of the enzyme.

An important consideration in the design of vaccines to prevent HIV-1 infection effective against different strains is the amino acid sequence conservation of antigenic determinants. Even one amino acid change can destroy the antigenicity of a site for the antibody or T-cell receptor. The comparisons of predicted T- and B-cell epitopes between human HIV-1, HIV-2 and monkey SIVMAC AIDS viruses are presented. The three major gene products (env, gag and pol) were examined. A number of epitopes were identical between strains of HIV-1. Our analysis highlights the problem of designing an effective HIV-1 and HIV-2 vaccine and also the problem of testing human vaccines in monkey models.

The catalytic residues of an enzyme are defined as the amino acids directly involved in chemical catalysis. They mainly act as a general acid--base, electrophilic or nucleophilic catalyst or they polarize and stabilize the transition state. An analysis of the structural features of 36 catalytic residues in 17 enzymes of known structure and with defined mechanism is reported. Residues that bind metal ions (Zn2+ and Cu2+) are considered separately. The features examined are: residue type, location in secondary structure, separation between the residues, accessibility to solvent, intra-protein electrostatic interactions, mobility as evaluated from crystallographic temperature factors, polarity of the environment and the sequence conservation between homologous enzymes of residues that were sequentially or spatially close to the catalytic residue. In general the environment of catalytic residues is similar to that of polar side chains that have low accessibility to solvent. Two algorithms have been developed to identify probable catalytic residues. Scanning an alignment of homologous enzyme sequences for peaks of sequence conservation identifies 13 out of the 16 catalytic residues with 50 residues overpredicted. When the conservation of the spatially close residues is used instead, a different set of 13 residues are identified with 47 residues overpredicted. A combination of the two algorithms identifies 11 residues with 36 residues overpredicted.

Criteria for the design of peptide vaccines to prevent AIDS are presented. The best vaccine candidates contain both B and T lymphocyte-defined epitopes in regions conserved in sequence between viral isolates. We propose that attention should focus on proteins specified by the gag and, possibly, pol genes in addition to the env gene envelope glycoproteins being actively studied. The predictions of B- and T-epitopes are refined by consideration of secondary structure prediction and inter-isolate sequence variability to suggest peptides from env, gag and pol that would be the best vaccine candidates.

The prediction of protein secondary structure (alpha-helices, beta-sheets and coil) is improved by 9% to 66% using the information available from a family of homologous sequences. The approach is based both on averaging the Garnier et al. (1978) secondary structure propensities for aligned residues and on the observation that insertions and high sequence variability tend to occur in loop regions between secondary structures. Accordingly, an algorithm first aligns a family of sequences and a value for the extent of sequence conservation at each position is obtained. This value modifies a Garnier et al. prediction on the averaged sequence to yield the improved prediction. In addition, from the sequence conservation and the predicted secondary structure, many active site regions of enzymes can be located (26 out of 43) with limited over-prediction (8 extra). The entire algorithm is fully automatic and is applicable to all structural classes of globular proteins.