Research

The research group ‘Structural biology of cancer-related protein targets’ was established in Leicester in July 2014, after moving from the Centre de Biochimie Structurale in Montpellier (France). The goal of our research activities is to find new targeted therapeutic ways to control abnormal cell proliferation observed in cancers. This activity is carried out in the Departments of Biochemistry (HoD. Prof. J. Schwabe) and of Cancer Studies (HoD. Prof. C. Pritchard) and in the context of the Cancer Research UK Leicester Centre (https://www2.le.ac.uk/partnership/lcrc/cruk). Our research is currently developed in the following themes: (i) Regulation of protein degradation; (ii) Cancer-related protein interactomes; (iii) Cell cycle CDKs: inhibitor design and chemical biology

News

December 2015 -  A fully-funded PhD studentship is available in the group to work on the regulation of the ubiquitin-like molecule NEDD8 in collaboration with Prof M. Searle (Nottingham) and Dr D. Xirodimas (Montpellier, France). The project description can be found on the following page (University of Leicester section); http://www.birmingham.ac.uk/research/activity/mrc-impact/complex-diseases.aspx . Deadline: January 10th

November 2015 - Two PhD studentship projects are available in the group. To read about the projects, check eligibility criteria and application procedure, go to ;

http://www2.warwick.ac.uk/fac/cross_fac/mibtp/pgstudy/phdopportunities/histone_2a

http://www2.warwick.ac.uk/fac/cross_fac/mibtp/pgstudy/phdopportunities/ubiquitin

November 2015 - Welcome to Chitra! Chitra has joined our group jointly with that of Prof R. Bayliss and M. Carr to work on the Cancer Research UK Accelerator Award

October 2015 - An exciting research associate position in X-ray crystallography is now opened in our group jointly with Richard Bayliss and Mark Carr. Deadline - 26th October 2015

October 2015 - Welcome to Sun a Jung, David Brown and Austin Burroughs who have joined to group for their 3rd year undergraduate projects!

July 2015 - We are welcoming interest from self-funded students to carry out their PhD in the group. Contact us to discuss projects linked to cancer therapeutic targets and molecules and funding sources

June 2015 - Grant success - Wellcome Trust Seed Award in Science. A two-year research technician post is associated with this grant and will be advertised soon

June 2015 - Julie Fournier, student at the Ecole Nationale Supérieur de Chimie de Montpellier has joined the group for a three-month internship

January 2015 - Grant success - MRC DTG studentship awarded to the group. The studentship will be advertised in due course

January 2015 - Kirsty Ford, Cancer Cell and Molecular Biology MSc student is joining us. Welcome Kirsty! Kirsty will be working on CSN5.

January 2015 - Nina Banji has joined the lab. A very warm welcome Nina!

December 2014 - Melissa Birol, PhD student in my group in Montpellier was viva-ed. Well done and good luck in your new adventures in Yale!

August 2014 - Our paper on the COP9 activity regulation and topology has been published! Good work with Rado Enchev, Matthias Peter, Florian Stengel and Ruedi Aebersold! Follow the link to download it: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105688

July 2014 - I joined the Departments of Biochemistry and of Cancer Studies at the University of Leicester. Very exciting time!

Regulation of protein ubiquitylation:

 

Control of the protein degradation is an aspect central to most cellular processes. In the context of the ubiquitin-proteasome system (UPS), most proteins are targeted to the 26S proteasome for degradation by a post-translational modification, named ubiquitylation that corresponds to a covalent linkage of ubiquitin molecules to the target protein. Protein ubiquitylation function is not limited to the control of protein turn-over, but intervenes in many signalling pathways by altering the modified protein subcellular localisation, partner, activity, etc… The last protagonist in this transfer reaction is an ubiquitin ligase. The cullin-RING-ubiquitin ligases (CRLs) are a predominant ubiquitin ligase family. They are large multiprotein complexes, made of several subunits among which are the scaffolding protein, cullin and the RING subunit carrying the enzymatic activity. The activity of these CRLs is regulated by cycles of neddylation/deneddylation of the cullin subunit (covalent attachment of an ubiquitin-like molecule, Nedd8).

 

Figure 1: The ubiquitin-proteasome system and cullin-RING-ubiquitin ligases (CRLs).

This research theme, involving structural biology, analytical biochemistry and biophysics of cell signalling proteins/complexes involved in cancers, focuses on the multiprotein complex COP9 signalosome (CSN) that is responsible for the regulation of CRLs by deneddylation.

This is a ubiquitous complex well conserved in eukaryotes, composed of 8 subunits (CSN1 to 8) that is yet well understood.

Figure 2: The COP9 regulates the activity of a major E3 ubiquitin ligase family

The group investigates, in particular, the molecular regulation of this complex and its interactions with substrates by a multidisciplinary approach.

The subunit 5 of the CSN complex (CSN5) carries the catalytic activity i.e. the isopeptidase activity that allows hydrolysis of the isopeptide bond between Nedd8 and cullin. We are interested in understanding why CSN5 displays the isopeptidase activity in the context of the CSN complex and is void of it in its stand-alone form. To answer this question, we used X-ray crystallography, molecular dynamics simulations (AMBER) and enzymatic activity assays.

Figure 3 : The zinc-binding site and the Ins-1 region of CSN5 and of AMSH-LP

Recent structural work on CSN5 and on CSN5 complexed with CSN6 brought to light the basis of CSN5 enzymatic activity regulation (the CSN5/CSN6 pair corresponds to the paralog Rpn11(POH1)/Rpn8(Mov34) pair in the proteasome lid).

Figure 4: CSN5-CSN6 heterodimer architecture explored by experimental and in silico approaches.

Further to that work, we are investigating the determinant of CSN5 activity regulation with the COP9. We are developing new substrates and assays to follow this isopeptidase activity, as well as pursuing the identification of inhibitors of CSN5/COP9 catalytic activity.

 

Our investigations extended to revisiting the architecture of the COP9 using cross-linking mass spectrometry in collaboration with Profs. M. Peter and R. Aebersold (ETH Zurich), yielding a refined model of the multisubunit assembly.

 

Regulation of protein ubiquitylation:Figure 5: Cross-linking study of the COP9 architecture. Panel A: Contact points between the eight subunits; Panel B: Refined model of the COP9.

If you are interested to join our efforts to understand the molecular basis of the COP9 regulation and join us or work with us, please contact Aude.

 

Collaborations:

Dr. Andrew Jamieson (University of Leicester. Department of Chemistry)

Prof. Sylvie Urbé (University of Liverpool)

Prof. Michael Clague (University of Liverpool)

Dr. L. Pintard (Institut J. Monod, Paris)

Dr. F.-X. Claret (MD Anderson, Texas)

UPStream European ITN (http://www.upstreamproject.eu/Aude-Echalier.html)

COST Proteostasis

 

 

Associated publications.

Echalier A§, Pan Y*, Birol M*, Tavernier N, Pintard L, Hoh F, Ebel C, Galophe N, Claret FX, Dumas C (2013) Insights into the regulation of the human COP9 signalosome catalytic subunit, CSN5/Jab1. PNAS 110 (4) 1273-8.

§ : sole corresponding author ; * : equal contribution

 

Birol M, Enchev RI, Padilla A, Stengel F, Aebersold R, Betzi S, Yang Y, Hoh F, Peter M, Dumas C, Echalier A§. Structural and biochemical characterization of the Cop9 signalosome CSN5/CSN6 heterodimer. PLoS One. 2014 Aug 21;9(8):e105688.

§: corresponding author

 

Birol M & Echalier A§. Structure and Function of MPN (Mpr1/Pad1 N-terminal) Domain- Containing Proteins. Curr Protein Pept Sci. 2014;15(5):504-17.

§: corresponding author

 

 

CNRS press release :

http://www.cnrs.fr/insb/recherche/parutions/articles2013/a-echalier.html

 

CSN5 interactome: function and molecular basis

The functions of the Cop9 linked to many pathways important to cellular homeostasis, including cell cycle, gene expression, immunity and DNA repair, appear altered in several human pathologies, mostly in cancers and it is therefore essential to study its regulation. In addition, a remarkable aspect of the Cop9 resides in its modularity (Fig.6). Several CSN subunits are found in distinct smaller complexes than the Cop9, some of which formed by a subgroup of CSN subunits and others with proteins exogenous to the COP9.

Figure 6: Schematic representation of the COP9 modularity and interactome

Although some of these sub-complexes are implicated in cellular proliferation, their exact role, preponderance in cellulo and activity are unclear. Interestingly, the catalytic subunit appears to interact with more than 80 different partners from various cellular regulatory pathways (such as CDK2, p27Kip1, thioredoxin, c-Jun; Fig.7). Particularly interesting to us is the tractable complex that CSN5 forms with the natural inhibitor of cyclin-dependent kinases (CDKs), p27Kip1 (CDKN1B). Indeed this direct association is linked to p27Kip1 nuclear export and increased turn-over in many cancers. Specific depletion of CSN5 increases p27Kip1 nuclear localisation and stability in cells. The effect of CSN5 on protein sub-cellular localisation and stability does not appear to be limited to p27Kip1 and extend to other CSN5 interactors. Some partners are probably not directly binding to CSN5, but it is now clear that many of them are. However, only few interactions have been characterised and their relevance in the context of the COP9 and of the UPS and other pathways is unclear.

Figure 7: CSN5 interactome. Panel A. Interactome3D output centered on CSN5 ; Panel B. List of CSN5 interactors for which we have an active interest.

Our aim is to answer the following questions: What is the CSN5/COP9 interactome and what is its functional role in context of the regulation of the ubiquitin-proteasome system? This project rests on the interrogation of a protein interaction network to better understand the regulation of protein degradation and will bring essential elements to specifically understand how the Cop9 is inserted in signalling networks implicated in the UPS.

To do so, using our specific biological and technological expertise, we are: (i) reconstituting the CSN5-centred network, (ii) characterising this interactome at the molecular level and (iii) we will evaluate the effects of this protein-protein interaction on the Cop9 and on the UPS.

 

In the context of this work, we have a special interest in proteins that either play a role in cell proliferation or in transcriptional regulation.

 

 

If you are interested to join our efforts to understand the role and the structural basis of CSN5/COP9 interactome and join us or work with us, please contact Aude.

 

Collaborations:

Dr. M. Piechaczyk (IGMM, Montpellier)

Prof. F. Sobott (University of Antwerp)

Prof. P. Aloy (IRB, Barcelona)

Dr. F.-X. Claret (MD Anderson, Texas)

UPStream European ITN (http://www.upstreamproject.eu/Aude-Echalier.html)

COST Proteostasis


Cell cycle control by CDKs: chemical biology and structure-guided inhibitor design

The timely controlled catalytic activity of some cyclin-dependent kinases contributes to the progression through the phases of the cell division cycle to the mitosis. The functions and regulations of cell cycle have been extensively studied. Despite that, we have an active interest in the link between their structural, functional properties and identities (ie what specific determinants make a given CDK its properties? This is an important question to (i) design ways to comprehend the specific function of a CDK, given the fact that genetic studies can be limited due to the functional redundancy of CDKs and (ii) design more selective ways to inhibit a CDK. Following are two examples of the work that we have contributed in these areas.

 

We have been working in collaboration with Dr. D. Fisher’s team (IGMM), leader in this project, on developing some chemical/biochemical tools to elucidate the role of CDK1/CDK2 and their functional redundancy. Using a multidisciplinary approach, our data supports the view that CDK2 has a rate-limiting role in DNA replication. Further exploring this role, we designed, tested and structurally characterised inhibitor-resistant-CDK2 mutants that confirmed that CDK2 activity is rate-limiting in replication.

The strength of this work resides in the use of a broad multidisciplinary approach that spans from bio-informatics, mathematical modelling of enzymology data, structural biology and functional analysis on Xenopus egg extracts. This work has been published in Chemistry & Biology:

Echalier, A.*, Cot, E.*, Camasses, A. *, Hodimont, E., Hoh, F., Jay, P., Sheinerman, F. Krasinska, L. and Fisher, D. An integrated chemical biology approach provides insight into CDK2 functional redundancy and inhibitor sensitivity. (2012). Chem. & Biol. 19(8), 1028-1040. * equal contribution

 

This work has been supported by (for our contribution): the Université Montpellier I, the CNRS, and the INSERM.

Publications

Birol M, Enchev RI, Padilla A, Stengel F, Aebersold R, Betzi S, Yang Y, Hoh F, Peter M, Dumas C, Echalier A. Structural and biochemical characterization of the Cop9 signalosome CSN5/CSN6 heterodimer. PLoS One. 2014 Aug 21;9(8):e105688.

Echalier A, Hole AJ, Lolli G, Endicott JA, Noble ME. An inhibitor's-eye view of the ATP-binding site of CDKs in different regulatory states. ACS Chem Biol. 2014 Jun 20;9(6):1251-6.

Birol M, Echalier A. Structure and Function of MPN (Mpr1/Pad1 N-terminal) Domain- Containing Proteins. Curr Protein Pept Sci. 2014;15(5):504-17.

Carbain B, Paterson DJ, Anscombe E, Campbell AJ, Cano C, Echalier A, Endicott JA, Golding BT, Haggerty K, Hardcastle IR, Jewsbury PJ, Newell DR, Noble ME, Roche C, Wang LZ, Griffin RJ. 8-Substituted O(6)-cyclohexylmethylguanine CDK2 inhibitors: using structure-based inhibitor design to optimize an alternative binding mode. J Med Chem. 2014 Jan 9;57(1):56-70.

Echalier, A.), Pan, Y.*, Birol, M.*, Tavernier, N., Pintard, L., Hoh, F., Ebel, C., Galophe, N., Claret, F.-X., Dumas, C. Insights into the regulation of the human Cop9 signalosome catalytic subunit, CSN5/Jab1. (2013). PNAS. 110(4), 1273-1278.

) Corresponding author. * equal contribution

Echalier, A. ), Noble, M.E. and Endicott, J.A. (2010). Recent developments in cyclin-dependent kinase biochemical, structural and inhibitor-design studies. BBA. 1804(3):511-519. ) Corresponding author

Takaki, T.*, Echalier, A.*, Brown, N.*, Hunt, T., Endicott, J.A. and Noble, M. (2009). The structure of CDK4/cyclin D3 has implications for models of CDK activation. PNAS. 106(11), 4171-4176. *these authors contributed equally to his paper

Merckx, A.*, Echalier, A.*, Langford, K., Sicard, A., Langsley, G., Joore, J., Doerig, C., Noble, M. and Endicott, J. (2008). Structures of P. falciparum protein kinase 7 identify an activation motif and leads for inhibitor design. Structure. 16(2), 228-238. * these authors contributed equally to this paper

Bettayeb, K., Sallam, H., Ferandin, Y., Popowycz, F., Fournet, G., Hassan, M., Echalier, A., Bernard, P., Endicott, J., Joseph, B. and Meijer L. (2008). N-&-N, a new class of cell death-inducing kinase inhibitors derived from the purine roscovitine. Mol. Cancer Ther. 7(9), 2713-2724.

Bettayeb, K., Oumata, N., Echalier, A., Ferandin, Y., Endicott, J.A., Galons, H. and Meijer, L. (2008). CR8, a potent and selective, roscovitine-derived inhibitor of cyclin-dependent kinases. Oncogene. 27(44), 5797-5807.

Echalier, A.*, Bettayeb, K.*, Ferandin, Y., Lozach, O., Clément, M., Valette, A., Liger, F., Marquet, B., Morris, J.C., Endicott, J.A., Joseph, B. and Meijer, L. (2008). Meriolins (3-(pyrimidin-4-yl)-7-azaindoles): synthesis, kinase inhibitory activity, cellular effects, and structure of a CDK2/cyclin A/meriolin complex. J. Med. Chem. 51(4), 737-751. * these authors contributed equally to this paper

Echalier, A., Brittain, T., Wright, J., Boycheva, S., Mortuza, G.B., Fülöp, V. and Watmough, N.J. (2008). Redox-linked structural changes associated with the formation of a catalytically competent form of the diheme cytochrome c peroxidase from Pseudomonas aeruginosa. Biochemistry. 47(7), 1947-1956.

Brittain, T., Boyd, P.D.W., Fülöp, V. and Echalier, A. (2008). The mechanism of action of di-heme peroxidases: a theoretical study. Inorg. Biochem: Res. Prog. Chapter 8, 225-242. (Hughes. J.G. and Robinson A.J. Eds.), Nova Science, N.Y., U.S.A.

Bettayeb, K., Tirado, O.M., Marionneau-Lambot, S., Ferandin, Y., Lozach, O., Morris, J.C., Mateo-Lozano, S., Drueckes, P., Schächtele, C., Kubbutat, M.H., Liger, F., Marquet, B., Joseph, B., Echalier, A., Endicott, J.A., Notario, V. and Meijer, L. (2007). Meriolins, a new class of cell death inducing kinase inhibitors with enhanced selectivity for cyclin-dependent kinases. Cancer Res. 67(17), 8325-8334.

Griffin, R.J., Henderson, A., Curtin, N.J., Echalier, A., Endicott, J.A., Hardcastle, I.R., Newell, D.R., Noble, M.E.M., Wang, L.-Z., and Golding, B.T. (2006). Searching for cyclin-dependent kinase inhibitors using a new variant of the Cope elimination. J. Am. Chem. Soc. 128(18), 6012-6013.

Pettigrew, G.W., Echalier, A. and Pauleta, S.R. (2006). Structure and mechanism in the bacterial dihaem cytochrome c peroxidases. J. Inorg. Biochem. 100(4), 551-567.

Echalier, A., Goodhew, C.F., Pettigrew, G.W. and Fülöp,V. (2006). Activation and catalysis of the di-heme cytochrome c peroxidase from Paracoccus pantotrophus. Structure. 14(1), 107-117.

Echalier, A.*, Trivelli, X.*, Corbier, C., Rouhier , N., Walker, O., Tsan, P., Jacquot, J.P., Aubry, A., Krimm, I. and Lancelin, J.M. (2005). Crystal structure and solution NMR dynamics of a D (type II) peroxiredoxin glutaredoxin and thioredoxin dependent: a new insight into the peroxiredoxin oligomerism. Biochemistry. 44(6):1755-67. * these authors contributed equally to this paper

Echalier, A., Glazer, R. L., Fülöp, V. and Geday, M. A. (2004). Assessing crystallization droplets contents using birefringence. Acta Cryst. D60, 696-702.

Echalier, A., Goodhew, C. F., Pettigrew, G. W. and Fülöp, V. (2004). Crystallization and preliminary X-ray diffraction analysis of a di-haem cytochrome c peroxidase from Paracoccus denitrificans. Acta Cryst. D60, 331-333.

Blewett, A. M., Lloyd, A. J., Echalier, A., Fülöp, V., Dowson, C. G., Bugg, T. D. H. and Roper, D. I. (2004). Expression, purification, crystallization and preliminary characterization of uridine 5’-diphospho-N-acetylmuramoyl L-alanyl-D-glutamate:lysine ligase (MurE) from Streptococcus pneumoniae 110K/70. Acta Cryst. D60, 359-361

Josse D., Ebel C., Stroebel D., Fontaine A., Borges F., Echalier, A., Baud D., Renault F., Le Maire M., Chabrieres E. and Masson P. (2002). Oligomeric states of the detergent-solubilized human serum paraoxonase (PON1). J. Biol. Chem. 277(36), 33386-97.

Echalier, A., Corbier, C., Rouhier, N., Jacquot, J.-P. and Aubry, A. (2002). Crystallization and preliminary X-ray data of a bifunctional peroxiredoxin from poplar. Acta Cryst. D58, 1501-1503.

Job/internship opportunities

All available positions will be advertised at the University's recruitment website. If you are interested in working in our laboratory, please contact Aude.

 

PhD students

Students that are interested in doing their PhD with us are welcome to contact Aude for more information. A variety of PhD projects is available in the fields of precision medicine, structural biology, biochemistry and chemical biology. Prospective students (including self-funded) are welcome to contact Aude for informal enquiries.

November 2015 - Two PhD studentship projects are available in the group. To read about the projects, check eligibility criteria and application procedure, go to ;

http://www2.warwick.ac.uk/fac/cross_fac/mibtp/pgstudy/phdopportunities/histone_2a

http://www2.warwick.ac.uk/fac/cross_fac/mibtp/pgstudy/phdopportunities/ubiquitin

 

Postdocs

Postdocs with a competitive CV and an interest in the field of cancer biology, structural/chemical biology are welcome to contact Aude to discuss projects and writing fellowships to join the lab.

 

 

Internships

Undergraduate students interested to work in the lab over the summer can contact Aude.


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Contact Details

Department of Molecular and Cell Biology
Henry Wellcome Building
Lancaster Road
Leicester
LE1 9HN (Postal)

LE1 7HB (Sat Nav/Online maps)

T:  +44(0)116 229 7038
F:  +44(0)116 229 7123
MolCellBiol@le.ac.uk

Redfearn Lecture 2015

Professor Jean Beggs FRS

Wellcome Trust Centre for Cell Biology

University of Edinburgh

"Functional interactions between splicing, transcription and chromatin"

January 22, 2015