Faites connaître cette offre !
Reference : UMR5089-JULMAR-001
Workplace : TOULOUSE
Date of publication : Tuesday, August 27, 2019
Scientific Responsible name : Julien Marcoux
Type of Contract : PhD Student contract / Thesis offer
Contract Period : 36 months
Start date of the thesis : 1 November 2019
Proportion of work : Full time
Remuneration : 2 135,00 € gross monthly
Description of the thesis topic
Context. Multiprotein complexes play various key roles in diverse biological processes. Among them, the 26S proteasome is a very well conserved multi-subunit protein assembly that presents structural and functional heterogeneities. Its main function, through the ubiquitin-proteasome system, is to determine the turnover rate of intracellular proteins, but specific other functions, such as the generation of functional peptides, were recently described1-2 resulting from the association with different regulators (19S, PA28αβ, PA28γ, PA200). Its dysregulation can be cytotoxic and has been associated to several neurodegenerative diseases and cancers. We are aware of three main ways to tune its proteolytic activity: the interaction of the 20S catalytic core with dedicated regulators, the replacement of the standard (Std) catalytic subunits (β1, β2 and β5) by inducible immunological subunits that form the immunoproteasome (i20S) upon inflammation for instance, and finally by post-translational modifications (PTM). Three proteasome inhibitors targeting its chymotrypsin-like activity have been approved by the FDA since 2003 in the treatment of multiple myeloma3-5. However, a better understanding of proteasome regulation would allow more specific therapies that target subtypes of the proteasome, avoiding the unnecessary shutdown of downstream pathways that may cause deleterious side-effects4-5. Specific inhibition of the immunoproteasome was recently shown to reduce graft rejection6 and suppress the progression of colorectal cancer in mice7.
Objectives This project aims at better understanding the complex interplay between the three distinct proteasome regulation pathways. To do so, we will first characterize in-depth the proteasome through the definition of its proteoform repertoire, associated regulators and partners, in the context of Inflammatory Bowel Diseases (IBD). Second, we will investigate the molecular basis explaining the preferential interaction between 20S subtypes and their different regulators. Finally we will screen new potential specific activators or inhibitors of the different 20S proteasome subtypes.
Strategies. The complexity of the proteasome complexes composition is a real challenge and widely-used bottom-up proteomics approaches are not able to correlate this structural diversity with specific functions. Although very powerful for protein identification and quantification, they do not account for PTM combination leading to various proteoforms8. To tackle this loss of information we will analyze these proteins entirely rather than after enzymatic digestion. We will employ state-of-the-art mass spectrometers to realize this top-down approach and have access to the many combinations of proteoforms present in a cellular extract and identify regulators that are specific to certain subtypes of the proteasome. We will study this proteasome heterogeneity in the context IBD as we know that the i20S will be recruited, at least partially, upon inflammation. We will use another innovative approach, Hydrogen-Deuterium Exchange (HDX) MS, to compare the conformations of the Std vs. i20S proteasomes in the absence and presence of the PA28αβ and PA28γ regulators. The same method will also be used to identify binding interfaces of newly described Proteasome Interacting Proteins (PIP)9-10 and study the binding of specific and non-specific inhibitors to the Std vs. i20S. Finally, we will set out to identify new activators or inhibitors of the different proteasomes using a high-throughput workflow based on the chemical, fragment and peptide-based libraries available at the institute (PICT Platform). We are confident that these innovative structural MS methods11-12 will shed a new light on our understanding of the proteasome regulation by studying how its molecular and structural diversity can explain its different functions. Furthermore, we will undoubtedly gather structural information that will be useful for the design of new specific inhibitors.
1 Liepe J et al. (2016) “A large fraction of HLA class I ligands are proteasome-generated spliced peptides” Science 354(6310):354-358
2 Dasgupta S et al. (2016) Analysis of the Yeast Peptidome and Comparison with the Human Peptidome” Plos One 11(9):e0163312
3 Bonvini P et al. (2007) "Bortezomib-mediated 26S proteasome inhibition causes cell-cycle arrest and induces apoptosis in CD-30+ anaplastic large cell lymphoma" Leukemia 21(4):838–42.
4 Kuhn DJ et al. (2007) “Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma” Blood 110(9):3281-90.
5 Kupperman E et al. (2010) “Evaluation of the proteasome inhibitor MLN9708 in preclinical models of human cancer” Cancer Res 70(5):1970-80.
6 Li J et al. (2018) “Immunoproteasome inhibition prevents chronic antibody-mediated allograft rejection in renal transplantation” Kidney Int. 93(3):670-680.
7 Koerner J et al. (2018) “Inhibition and deficiency of the immunoproteasome subunit LMP7 suppress the development and progression of colorectal carcinoma in mice” Oncotarget. 8(31):50873-50888.
8 Smith LM et al. (2013) “Proteoform: a single term describing protein complexity” Nature 10(3):186-187.
9 Jonik-Nowak et al. (2018) “PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28γ” PNAS 115(28):E6477-86.
10 Sbardella et al. (2018) “The insulin-degrading enzyme is an allosteric modulator of the 20S proteasome and a potential competitor of the 19S” Cell Mol Life Sci 75(18):3441-3456.
11 Marcoux J and Robinson CV (2013) “Twenty years of gas phase structural biology” Structure 21(9):1541-1550.
12 Marcoux J and Cianférani S (2015) “Towards integrative structural mass spectrometry: benefits from hybrid approaches” Methods 89:4-12.
The principal objective of the host laboratory (IPBS, Toulouse) is to characterize fundamental mechanisms that control the functions of biological systems, by analyzing the structure-function relationship of macromolecules in order to develop pharmacological applications. In this context, the team of Proteomics and Mass Spectrometry of Biomolecules led by Odile Burlet-Schiltz has spent the last 15 years using proteomics to investigate the proteasome complexes that are a therapeutic target of choice because of their implications in many pathologies. A better description of the different forms of the proteasome will lead, in the long run, to more specific treatments generating less side effects.
This project originates from the wish of the team and the institute to develop innovating Structural Mass Spectrometry techniques in order to bring a new point of view on the proteasome system. In this context, the team obtained a grant from the Région Occitanie (within the frame of the CPER 2015-2020) that allowed the acquisition of a state-of-the-art instrument to realize this kind of experiment (Synapt G2Si with automated HDX-MS).
The PhD co-supervisor (Julien Marcoux) spent the last 12 years using mass spectrometry as a tool for the structural characterization of soluble and membrane proteins & protein complexes. His expertise will be supported by a decade of experience in proteasome purification and proteomics analysis in the team. Recent work include the use of in-vivo cross-linking and proteomics to describe the heterogeneity of proteasome complexes (in terms of catalytic subunits but also interacting activators) in different human cell lines. This pioneering work will strongly benefit from the complementary methods developed by Julien Marcoux, to provide a more structural point of view on this complex system. Our team also counts with highly experienced engineers who will support the PhD student, in terms of sample preparation, instrument handling/maintenance and data analysis. Finally, the long-term presence of skilled bioinformaticians in the team, is a very strong asset that enabling to efficiently develop new tools (VisioProt-MS, HDX-Viewer), which could otherwise constitute a bottleneck in our workflows.
Our collaborators (Audrey Ferrand & Frédérick Barreau) at the Research Institute of Digestive Health (IRSD, INSERM U1220, Toulouse), who will provide us biological samples, are also internationally renowned in the field of colonic pathophysiology, IBDs and fully committed to this collaboration. They are specialized in the inflammation of digestive tissues and have developed models to mimic inflammation on colorectal epithelial cells with cocktails of pro-inflammatory cytokines. They also have regular access to biopsies from patients with different IBD (including ulcerative colitis and Crohn's disease) or colorectal cancer (with and without inflammation). Because part of their research is focused on epithelial regeneration, they also developed 3D cell culture models (intestinal organoids)73 providing sufficient amount of healthy and inflamed cells for our top-down and bottom-up proteomics study. Clinicians (D. Bonnet, A. Breton and E. Mas), in their research groups, are largely involved in the group research, providing critical clinical inputs and comments on the different basic science projects.
Constraints and risks
No particular Health & Safety risk is associated with this project.
We talk about it on Twitter!