Division of Structural Biology
The work in the Division of Structural Biology is focused on structural and biochemical studies of proteins and complexes of importance in the aetiology and treatment of cancer. Our research activities, which utilise the methodologies of X-ray crystallography, electron microscopy and a variety of biochemical/biophysical techniques, can be subdivided into the following biological areas:
- Signal transduction processes
- Mechanisms and consequences of protein phosphorylation and protein ubiquitination
- Cell cycle control
- Chromatin remodelling and modification
- DNA damage recognition, signalling and repair
- Regulatory mechanisms in methyltransferase enzymes
- Proteasome structure
- Structure-based drug design
- Gene transcription and regulation
Cell cycle control and signal transduction processes
Virtually every cellular function is regulated by post-translational modifications such as reversible protein phosphorylation, methylation and ubiquitin-dependent events. Such processes regulate the activities of signal transduction cascades that ultimately lead to changes in gene expression and control of the cell cycle and apoptosis. Disregulation of these pathways has important implications for the development of cancer. Our interests are concerned with understanding the structural mechanisms by which the post-translational modification of proteins regulates signal transduction pathways and the cell cycle.
Professor David Barford's laboratory investigates the structures and mechanisms of protein kinases, phosphatases and E3 ubiquitin ligases to understand these proteins at a fundamental level, and also to facilitate rational drug design programmes.
In the cell cycle field a major interest is focussed on understanding the molecular mechanism and architecture of the anaphase promoting complex or cyclosome (APC/C), a large multi-subunit E3 protein ubiquitin ligase that functions to regulate cell cycle transitions by targeting key cell cycle proteins, such as cyclin and securin for degradation by the ubiquitin proteasome system.
To understand the mechanism of RAS post-translational modifications, necessary for the signalling function of RAS, we have initiated a programme to determine the crystal structures of the RAS converting enzyme (RCE1), a CAAX motif protease, and isoprenyl cysteine methyl transferase (ICMT), a cysteinyl methyltransferase.
DOCK proteins are guanine nucleotide exchange factors of Rac and Cdc42 RhoGTPases and have been implicated in the activation of Rac and Cdc42 in cell migration, morphogenesis and phagocytosis, and as important components of tumour cell movement and invasion.
DNA damage recognition, signalling and repair
The recognition and repair of DNA damage is essential to protect cell viability and prevent mutations that may lead to cancer. Professor Dale Wigley's group are studying how DNA damage within nucleosomes is detected and made accessible to repair proteins by enzyme complexes called Chromatin Remodellers. These multi-subunit systems translocate nucleosomes along DNA to expose the damage and facilitate repair. Other related complexes also acetylate histones and/or swap them for other histone variants as a part of the DNA damage response.
Once the DNA damage has been recognised and exposed it needs to be repaired and a second focus of the group is studying the structure and mechanisms of the enzymes that repair double-strand breaks in DNA in both bacteria and humans. In bacteria, the broken DNA ends are processed by either the RecBCD or AddAB complexes – multi-subunit protein machines that combine nuclease and helicase activities to process the DNA ends and eventually load RecA protein onto the DNA to initiate homologous recombination. In humans, similar enzyme activities are involved in processing DNA ends but are contained within the Dna2/BLM/RPA complex. These enzymes are assisted by a variety of other proteins including the Mre11/Rad50/Nbs1 complex and BRCA2 to eventually load the Rad51 recombinase onto the DNA to initiate homologous recombination.
Dr Ed Morris applies the techniques of electron microscopy and single particle reconstruction techniques to understand the structure and function of large proteins of medical and scientific interest. Current focus includes the proteasome, the inositol 1,4,5 tris-phosphate receptor and the signalosome.
Structure-based drug design
Dr Rob van Montfort's team is responsible for the hit generation for the ICR's drug discovery projects using high-throughput and fragment screening approaches on a variety of cancer targets involved in reversible protein phosphorylation, stress response, chromatin modification and targets involving specific protein-protein interactions.
In addition, the team supports the design and improvement of the hits to potent and selective inhibitors with a range of assay and biophysical techniques as well as iterative protein-ligand crystallography.
Gene transcription and regulation
The eukaryotic nuclear genome is transcribed by the multisubunit enzymes RNA polymerase (Pol) I, II and III, which catalyze DNA-dependent RNA synthesis. In particular, RNA Pol III transcribes genes encoding short, untranslated RNAs such as tRNAs, 5S rRNA, the spliceosomal U6 snRNA, the signal recognition particle 7SL RNA, and short regulatory RNAs. Because of the central role of RNA Pol III transcripts in basal cellular processes, the level of RNA Pol III transcription is a critical determinant of cell growth and, as a consequence, its deregulation has a profound impact on cancer development.
Dr Alessandro Vannini's team focuses on the molecular mechanisms underlying RNA Pol III gene transcription and its regulation. Our goal is to better understand the molecular basis of RNA Pol III recruitment at its target genes and the subsequent formation of transcriptionally active pre-initiation complexes. This process is tightly regulated in normal cells by tumour suppressor proteins, such as Rb and p53, and oncogenes, such as c-Myc, but this regulation is often lost during tumorigenesis. An additional layer of regulation is provided by Maf1-dependendt upstream kinases which assemble at RNA Pol III loci across the eukaryotic genome and act as central nuclear coordinators of events which happen at chromatin level, such as DNA damage repair, chromosomal condensation and cohesion and ribosome biogenesis.
Mechanisms and functions of ADP-ribosylation
ADP-ribosylation describes the covalent attachment of ADP-ribose, obtained from NAD+, onto substrates and is catalysed by ADP-ribosyltransferases. Numerous members of this enzyme family represent promising anti-cancer targets.
The transfer of a single ADP-ribose unit is achieved by mono-ADP-ribosyl transferases while the processive generation of poly(ADP-ribose) chains is catalysed by poly(ADP-ribose)polymerases (PARPs). Four PARP enzymes (PARP1, PARP2, Tankyrase and Tankyrase 2) are gaining an increasing interest as potential targets in anti-cancer strategies, due to their involvement in processes critical to the function and survival of cancer cells. At the same time, the biological roles of many ADP-ribosyltransferases remain unknown.
Dr Sebastian Guettler’s team studies the mechanisms of action of ADP-ribosyltransferases, using an approach that combines biochemistry, structural biology and functional studies in mammalian cells. The group aims to understand how ADP-ribosyltransferases are regulated, what determines their substrate specificities, the nature of their catalysis products and the implications of these factors in the biological functions of these enzymes. A better understanding of the mechanisms and roles of ADP-ribosylation has the potential to open up novel strategies for therapeutic intervention.