Download 2017 MCB/LISCB/CRUK project short-list Structural investigation of

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.