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2017 MCB/LISCB/CRUK project short-list Structural investigation of the role of G-quadruplexes in Bcl-x alternative splicing Dr. Cyril Dominguez Targeting nucleocytoplasmic shuttling in a hepatocellular carcinoma therapeutic strategy Dr. Aude Echalier Exploring the role of cortactin phosphorylation in cancer cell division and migration Professor Andrew Fry Structural analysis of the bacterial DNA replication machinery Professor Meindert Lamers Structural and functional studies of early spliceosomal complexes using electron microscopy Dr. Olga Makarova Self-guided nano-robots to spy on nano-machines switching genes in living cells Dr. Andrey Revyakin Cryo-Electron Microscopy of Histone Deacetylase Complexes Professor John Schwabe Triggering the innate immune system: Structural analysis of the C1 complex and its mechanism of activation Professor Russell Wallis Structural investigation of the role of G-quadruplexes in Bcl-x alternative splicing Supervisors: Dr. Cyril Dominguez / Professor Ian Eperon Department of Molecular and Cell Biology Email: [email protected] The Bcl-x protein is of major importance in cell death and its overexpression is associated with cancer (1). Although strategies to target this protein for cancer treatment have led to the development of numerous inhibitors, many being in phase I and phase II clinical trials, issues of specificity, toxicity and resistance have been described (2). The Bcl-x pre-mRNA can be alternatively spliced to produce two different protein isoforms, Bcl-x L and Bcl-x S , with antagonistic functions (3). It would greatly facilitate cancer treatments if damaged cancer cells were more likely to die, and this could be accomplished by enforcing the usage of the x S splice site. We have recently shown that a specific RNA structure, called a G-quadruplex, regulates Bcl-x alternative splicing (4). By testing a number of G-quadruplex stabilizers, we found one small molecule that induces the production of the Bcl-x S isoform and consequently cell death. In this project, we aim to further characterize the effect of this molecule on alternative splicing and cancer. First, we will investigate the structural details of the interactions made between the Bcl-x pre-mRNA and GQC-05, using Nuclear Magnetic Resonance, X-ray Crystallography, and Small Angle X-ray scattering (SAXS). Second, using single-molecule methods, we will investigate the stoichiometry of GQC-05 bound to individual molecules of Bcl-x premRNA, and how GQC-05 affects the binding of splicing factors to the Bcl-x premRNA. This project will allow us to derive a structural model for the contribution of Gquadruplexes and G-quadruplex stabilizers in Bcl-x alternative splicing regulation. References: 1. Yip and Reed. Oncogene, 27, 6398-6406 (2008). 2. Besbes et al., OncoTarget, 6, 12862-71 (2015) 3. Boise et al., Cell, 74, 597-608 (1993) 4. Weldon et al., Nat. Chem. Biol., Advance Online Publication, doi:10.1038/nchembio.2228 Targeting nucleocytoplasmic therapeutic strategy shuttling in a hepatocellular carcinoma Supervisor: Dr. Aude Echalier Department of Molecular and Cell Biology Email: [email protected] Pro-proliferative signalling pathways can be activated by the aberrant nucleocytoplasmic shuttling of tumor suppressor proteins. Cell cycle progression is driven by the activity of cyclin-dependent kinases, CDKs. CDK activity is controlled by natural CDK inhibitors, CKIs of the INK/KIP families. CDKN1B of the KIP family, also known as p27KIP1, regulates cell cycle CDKs by binding to and inhibiting the binary complex that they form with their cognate cyclin subunit. Via its inhibitory activity on CDKs, CDKN1B acts as a tumour suppressor protein in the nucleus. Misregulation of CDKN1B is recurrently observed in cancer patients, with its aberrant cytoplasmic localisation and subsequent cytoplasmic degradation. One of the key protagonists in the CRM1-dependent nuclear export of CDKN1B is CSN5 (JAB1) (1). CSN5, a key regulator of the ubiquitin system binds directly to and promotes CDKN1B nuclear export in a number of cancer types, including in hepatocellular carcinoma (HCC), the second most common cause of cancer mortalities worldwide (CR-UK). The frequent overexpression of CSN5 in HCC (36%; COSMIC) is negatively correlated with CDKN1B levels and patient prognosis (2). We propose to develop tool compounds that disrupt the CSN5/CDKN1B interaction, exploiting our knowledge and experience on these two targets. The hypothesis is that compounds binding to CDKN1B-binding site on CSN5 will rebalance CDKN1B distribution, elevate its nuclear protein levels and have an antiproliferative effect on HCC cell lines, as previously shown in CSN5 depletion studies (3). This project suits a highly motivated candidate, eager to learn different techniques, including biochemistry, mammalian cell culture, X-ray crystallography, NMR. The student will join a well-established and vibrant lab and work in collaboration with colleagues at the University of Leicester and in the Cancer Research UK network. References: 1- Tomoda K, Kubota Y, Arata Y, Mori S, Maeda M, Tanaka T, Yoshida M, YonedaKato N, Kato JY. The cytoplasmic shuttling and subsequent degradation of p27Kip1 mediated by Jab1/CSN5 and the COP9 signalosome complex. J Biol Chem. 2002 Jan 18;277(3):2302-10. 2- Qin J, Wang Z, Wang Y, Ma L, Ni Q, Ke J. JAB1 expression is associated with inverse expression of p27(kip1) in hepatocellular carcinoma. Hepatogastroenterology. 2010 May-Jun;57(99-100):547-53. 3- Lee YH, Judge AD, Seo D, Kitade M, Gómez-Quiroz LE, Ishikawa T, Andersen JB, Kim BK, Marquardt JU, Raggi C, Avital I, Conner EA, MacLachlan I, Factor VM, Thorgeirsson SS. Molecular targeting of CSN5 in human hepatocellular carcinoma: a mechanism of therapeutic response. Oncogene. 2011 Oct 6;30(40):4175-84. Exploring the role of cortactin phosphorylation in cancer cell division and migration Supervisor: Professor Andrew Fry Department of Molecular and Cell Biology Email: [email protected] Applications are invited from highly motivated and enthusiastic candidates for a PhD studentship aimed at understanding novel molecular events in cancer cell division and migration. Cortactin is an F-actin binding protein that stimulates actin polymerization. It is frequently overexpressed in human cancer and promotes migration and invasion, particularly of breast, melanoma and colorectal cancer cells [1]. Cortactin also has a role in regulating cell proliferation and promotes disassembly of the microtubulebased primary cilium that forms in quiescent cells. The primary cilium is an antennaelike structure that transduces external signals to control proliferation but which must be disassembled for cell division. We have discovered that cortactin is phosphorylated by the cell cycle-regulated Nek6 kinase [2]. We therefore wish to first test whether this regulates the ability of cortactin to promote F-actin polymerization. Second, as the phosphorylation sites lie close to sites that undergo acetylation, and deacetylation of cortactin contributes to cilia disassembly, then we will investigate potential cross-talk between phosphorylation and acetylation in regulating cortactin function. Finally, we will explore how phosphorylation of cortactin by Nek6 might promote proliferation, migration and invasion of human cancer cells in vivo. This exciting multidisciplinary cell biology project will contribute to the CRUK Leicester Cancer Centre strategy both in terms of exploring cancer cell mechanisms and testing whether Nek6 would make an appropriate target for development of novel anti-cancer drugs. It will also provide training in cutting-edge techniques from quantitative microscopy to gene-editing, involve collaboration with academic and clinical research colleagues in Leicester and other academic institutions, and give opportunities for involvement in diverse public engagement events. References: 1. MacGrath SM & Koleske AJ (2012) Cortactin in cell migration and cancer. Journal of Cell Science 125: 1621-1626 2. Fry AM, O’Regan L, Sabir SR and Bayliss R (2012) Cell cycle regulation by the NEK family of protein kinases. Journal of Cell Science 125: 4423-4433 Structural analysis of the bacterial DNA replication machinery Supervisor: Professor Meindert Lamers Department of Molecular and Cell Biology Email: [email protected] Faithful replication of genomic DNA is essential to all organisms. However, DNA replication is greatly complicated by the two strand of the DNA that run in opposite direction. Therefore, as the replication machinery is moving along the DNA, one strand is copied in one continuous stretch of more than 100,000 base pairs while the other strand is synthesized in a discontinuous manner with fragments sizes of up to 1000 base pairs. The simultaneous replication of both strands is performed by a large assembly of 12 different proteins called the DNA polymerase III holoenzyme. This large complex catalyzes several events such as DNA unwinding by the DNA helicase, RNA primer synthesis by the primase, clamp loading by the clamp loader complex, DNA synthesis by the polymerase, and proofreading by the exonuclease. Structures of the individual proteins are known, but no structural information is available on the complete holoenzyme. With modern cryo-EM techniques and biochemical trapping of stable states we are now able for the first time to study the structure of the intact holoenzyme. The aim of the PhD project is therefore to study the structural features of the complete DNA polymerase III holoenzyme. For this you will be using different structural techniques, such as cryo electron microscopy, protein crystallography, small angle X-ray scattering as well as diverse biochemical techniques. Relevant publications: Fernandez-Leiro R, Conrad J, Scheres HWS and Lamers MH. (2015) Cryo-EM structures of the E. coli replicative DNA polymerase reveal dynamic interactions with clamp, exonuclease and τ. eLife 4:e11134 Rock JM, Lang UF, Chase MR, Ford CB, Gerrick ER, Gawande R, Coscolla M, Gagneux S, Fortune SM and Lamers MH. (2015) Replication fidelity in M. tuberculosis is mediated by an ancestral prokaryotic proofreader. Nature Genetics 47, 677-681 Toste Rêgo A, Holding A, Kent H and Lamers MH. (2013) Architecture of the Pol IIIclamp-exonuclease complex reveals key roles of the exonuclease subunit in processive DNA synthesis and repair. EMBO J. 32, 1334-43 Structural and functional studies of early spliceosomal complexes using electron microscopy Supervisor: Dr. Olga Makarova Department of Molecular and Cell Biology Email: [email protected] Gene expression in eukaryotes involves the removal of non-coding sequences, introns, from the genes' transcripts. This is accomplished by the highly intricate molecular machine called the spliceosome. The spliceosome assembles on every intron in a step-wise manner: 1) recognising intron's 5' and 3' ends (splice sites, SS); 2) brining SS in close proximity; 3) performing two trans-esterification reactions that remove intron from the final product, mRNA. The spliceosome for its function requires hard-core factors including five small nuclear RNP particles (snRNPs) and about 200 essential proteins that made the spliceosome known as the most complex molecular machine in the cell. Interestingly, the splicing factors are much abounded in the cell as compared, e.g., to histone modifying enzymes. In humans as in other higher eukaryotes, splicing or intron removal is regulated by specific sequences, enhancers and silencers and mutations in these sequences were identified as being responsible for many degenerative diseases as well as cancer. The novel emerging gene therapies use complementary oligo nucleotides to block their function and these were shown to be extremely effective, e.g., in muscular dystrophy. Although the benefits of the oligo-approach are widely accepted, understanding the molecular mechanisms of action for these regulatory sequences is controversial and being disputed by many researchers in the field. This project aims to investigate the function of regulatory sequences and mechanism of spliceosome assembly at the molecular level by isolating spliceosomal complexes formed in the presence or absence of the regulatory elements. The isolated complexes will be subjected to analysis by mass-spectrometry (MS) to determine their protein composition and their structure will be determined using electron microscopy (EM). The structural analysis of the complexes will provide new essential information about molecular mechanisms of function for these regulatory elements. This should promote further insights into the mechanisms of alternative splicing and provide novel targets for future gene therapy. The objectives of the project will include 1) isolation of complexes formed on substrate pre-mRNAs using affinity purification and density glycerol centrifugation; 2) identification of protein composition using MS; 3) visualisation of complexes using EM (negative staining and cryo); 4) classification of images into representative views - class averages; 5) determination of structures using dedicated software; 6) analysis of differences in complexes formed on substrate pre-mRNAs in the presence or absence of regulatory elements. Relevant publications: • • • • Makarov, E. M., Owen, N., Bottrill, A., and Makarova, O. V. (2012) Functional mammalian spliceosomal complex E contains SMN complex proteins in addition to U1 and U2 snRNPs. Nucleic Acids Res 40, 2639-52. Makarova, O. V., Makarov, E. M., Urlaub, H., Will, C. L., Gentzel, M., Wilm, M., and Luhrmann, R. (2004) A subset of human 35S U5 proteins, including Prp19, function prior to catalytic step 1 of splicing. EMBO J 23, 2381-91. Boehringer, D., Makarov, E. M., Sander, B., Makarova, O. V., Kastner, B., Luhrmann, R., and Stark, H. (2004) Three-dimensional structure of a pre-catalytic human spliceosomal complex B. Nat Struct Mol Biol 11, 463-8. Makarov, E. M., Makarova, O. V., Urlaub, H., Gentzel, M., Will, C. L., Wilm, M., and Luhrmann, R. (2002) Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome. Science 298, 2205-8. Self-guided nano-robots to spy on nano-machines switching genes in living cells Supervisors: Dr. Andrey Revyakin/ Professor Ian Eperon Department of Molecular and Cell Biology Email: [email protected] A living cell is a vibrant, crowded ‘molecular city’ in which the seemingly chaotic movement of its citizens (protein molecules) leads to formation of amazingly complex structures, allowing the cell to function within the ‘country’ of the organism. The ‘molecular city government’ resides in the cell’s nucleus, and the ‘code of law’ is written in the cell’s DNA. This code is constantly read and copied by special molecular ‘nano-machines’ about 20-30 nm in size, each comprised of hundreds of protein molecules. These nano-machines (RNA polymerases and spliceosomes) assemble on genes, crawl along the strands of DNA, constantly lose and acquire parts, and interact with other nano-machines. Thus, to understand the decision making process of the ‘city government’ (for instance, to make the cell differentiate into another cell type), we have to track the assembly and the movement of the nanomachines on genes inside living cells, in real time. However, because the nanomachines are tiny, and the ‘molecular city’ is crowded, the resolution of traditional diffraction-limited optical microscopes (~500 nm) is not sufficient to capture the movements on DNA against the noise and chaos of the ‘city life’. In this challenging, low-risk/high-reward project you will create a fundamentally new type of a microscope, with the aim to directly visualize the nano-machines being assembled in real-time and moving on genes inside living cells. To succeed, you will need hands-on and trouble-shooting skills in laser optics, protein and DNA purification, opto-mechanics, silicon-based surface chemistry, digital image processing, modelling of stochastic processes, molecular cloning, basic programming (Python, Matlab, CUDA), radioactivity-based assays, basic CAD (Autodesk Inventor), bio-conjugation, rapid prototyping (3D printing), metal end-milling, basic electronics and ultracentrifugation. Cryo-Electron Microscopy of Histone Deacetylase Complexes Supervisor: Professor John Schwabe Department of Molecular and Cell Biology Email: [email protected] The Schwabe group is seeking to understand the molecular mechanisms that underlie the epigentic reprogramming of the genome during cellular differentiation and development. At least five large, multiprotein Histone Deacetylase (HDAC) complexes are key players. They trigger the programmed repression of regions of the genome which is one of the critical first steps in epigenetic regulation. Interestingly these complexes also play a role in the expression of active genes. HDAC’s 1, 2 and 3 are incorporation into specific complexes and this is fundamental to their function since this directs both substrate specificity as well as regulating the enzymatic activity of the HDAC. HDAC inhibitors are currently used to treat some cancers. The Schwabe group have been successful in expressing and purifying the core components of many HDAC complexes in HEK293F cells in sufficient quantity for structural studies. We have led the field in using crystallographic studies to explore the assembly of the SMRT complex (SMRT:HDAC3:TBL1:GPS2), the NuRD complex (HDAC1:MTA1:RBBP4) and the MiDAC complex (HDAC1:MIDEAS:DNTTIP1). These studies led to the unexpected and breakthrough discovery that HDAC complexes are regulated by small molecule inositol phosphates. There has recently been a revolution in cryo-electron microscopy, which means that large protein and protein complex structures that previously could not be crystallised can now be solved at atomic resolution. The aim of this PhD project is to solve the structure of histone deacetylase complexes such as the CoREST complex using cryo-electron microscopy. The core CoREST complex consists three proteins, HDAC1, LSD1 and CoREST. It contains two enzymatic activities: a histone deacetylase HDAC1 (targeting Histone H3 K9ac and others) and a histone demethylase LSD1 (primarily targeting Histone H3 K4me1/me2). CoREST has been shown to be a promising drug target in multiple cancers including neuroblastoma, melanoma, breast cancer, PML and more recently colon cancer. The overall aim is to fully understand the structure of the different HDAC complexes, their interaction with chromatin and their interaction with other components involved in transcription. An understanding of the structure of the different HDAC complexes may lead to the design of novel and more specific histone deacetylase inhibitors. Recent publications: • • Millard CJ, Varma N, Saleh A, Morris K, Watson PJ, Bottrill AR, Fairall L, Smith CJ & Schwabe JW (2016) The structure of the core NuRD repression complex provides insights into its interaction with chromatin. eLife 5: e13941 Watson PJ, Millard CJ, Riley AM, Robertson NS, Wright LC, Godage HY, Cowley SM, Jamieson AG, Potter BVL & Schwabe JWR (2016) Insights into the activation mechanism of class I HDAC complexes by inositol phosphates. Nature Communications 7: 11262 Triggering the innate immune system: Structural analysis of the C1 complex and its mechanism of activation Supervisor: Professor Russell Wallis Department of Molecular and Cell Biology/ Department of Infection, Immunity and Inflammation Email: [email protected] The C1 complex is a large multiprotein assembly present in blood that binds to antibodies on pathogens to activate key immune and inflammatory pathways. It is important for maintaining health and preventing disease. For example, a deficiency of the complex leads to increased susceptibility to infections and autoimmune diseases such as systemic lupus erythmatosus, whereas overactivity can lead to activation on our own cells leading to tissue damage. The C1 complex is formed from a large bouquet-shaped protein, called C1q, and two proteases, C1r and C1s. The aim of this project is to understand the structure of the complex and the mechanism that leads to activation on pathogens. We will use a combination of structural approaches including single-particle cryo-electron microscopy, X-ray crystallography and smallangle scattering to investigate the complex. Work will be carried out in the Leicester Institute of Structural and Chemical Biology (LISCB) fully equipped with state-of-the art technology for structural analysis, under the supervision of Professors Russell Wallis and Peter Moody (Leicester) and Dr Corinne Smith at the University of Warwick. Relevant publications: Venkatraman Girija, U., et al., Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation. Proc Natl Acad Sci U S A, 2013.