PhD in proteomics and structural MS M/F 3 years

Job Description

Thesis Subject

Background
The human proteasome is a multi-protein complex essential for protein degradation and cellular homeostasis. Its deregulation is associated with neurodegenerative diseases, inflammation and cancer. Since 2003, three FDA-approved inhibitors have targeted its catalytic sites to treat multiple myeloma and mantle cell lymphoma, but they cause severe side effects by fully blocking proteasomal activity. Developing selective inhibitors of specific proteasome subtypes offers a promising alternative.

Proteasome heterogeneity stems from alternative subunits, interactions with regulators and post-translational modifications. Its catalytic core (std20S) consists of 14 subunits, while the immunoproteasome (i20S), induced by interferon-γ, enhances antigen presentation. Human cells contain mixtures of std20S, i20S and intermediate complexes. Due to the low specificity of first-generation inhibitors and the involvement of i20S in various diseases, selective inhibitors have emerged, showing promising results in graft rejection, cancer and autoimmune diseases.

Proteasome subtypes interact with activators such as 19S, PA28αβ, PA28γ and PA200, which facilitate substrate access. Although the 20S alone has low activity, these activators can considerably increase its function. PA28αβ and PA28γ increase 20S activity up to 50-fold and form hybrid complexes involved in antigen processing, the oxidative stress response, cancer and autoimmune diseases.

Objectives
This PhD project, funded by the ANR (Proteasom-inhib), aims to develop a 3rd generation of inhibitors specific to PA28-associated 20S proteasome subtypes, in an auto-inflammatory context. Targeting these subtypes via their activators could significantly reduce the toxicity observed with 1st and 2nd generation drugs.

Introduction
The proteasome is a macromolecular machine responsible for the degradation of intracellular proteins, an essential element of cell homeostasis. In humans, the 20S particle consists of two outer rings formed from seven different α subunits (α1-7) and two inner rings formed from seven different β subunits (β1-7). The two β rings form a proteolytic chamber, while the α rings, located above and below, gate access to this chamber and provide a binding interface for regulator proteins. Catalytic activity is carried out by three β subunits: β1, which has caspase-like activity, β2, which has trypsin-like activity, and β5, which has chymotrypsin-like activity [1]. These subunits are part of the ubiquitously expressed form of the proteasome, termed the 'constitutive proteasome' (c20S).

In addition to the constitutive proteasome, which is found in all tissue types, other forms have been identified, including the immunoproteasome (i20S). This form is permanently expressed in immune cells and medullary thymic epithelial cells [2], and can be induced by the pro-inflammatory cytokine interferon (IFN)-γ in many other cell types [3]. The immunoproteasome replaces β1, β2 and β5 with the catalytically active immune subunits β1i, β2i and β5i. Intermediate proteasomes β1-β2-β5i and β1i-β2-β5i also exist, which combine constitutive and immune catalytic subunits [4].

The immunoproteasome plays an important role in the function of the immune system by altering the antigenic peptide repertoire upon infection or inflammation. Antigenic peptides are presented by major histocompatibility complexes (MHC) on the cell surface for recognition by T lymphocytes [3]. The immunoproteasome generates peptides that are longer, more uniform in length and more hydrophobic at their C-terminal ends. These properties are favourable for binding to MHC class I molecules, leading to increased peptide presentation and activation of the immune system. Beyond peptide presentation, the immunoproteasome is involved in inducing pro-inflammatory cytokines, maintaining the proliferation of T cells and degrading oxidised proteins [4]. Due to its major involvement in the immune response, the immunoproteasome is implicated in autoimmune diseases such as rheumatoid arthritis [5] and inflammatory bowel disease (IBD) [6]. Moreover, studies have shown that specifically inhibiting the immunoproteasome reduces chronic rejection of allogeneic organ transplants.

Beyond catalytic subunit variation, proteasome heterogeneity is further increased by the presence of regulators, which dock into pockets formed by the α-rings of the 20S proteasome, thereby modifying its activity and functionality [7]. Notable examples include the heptameric activators PA28αβ, often associated with i20S [2], and PA28γ, which associates with c20S. Cytosolic PA28αβ not only increases the catalytic activity of the immunoproteasome: it also strengthens antigen presentation and thus the immune response. PA28γ, found in the nucleus, regulates the degradation of a small subset of proteins, including cell cycle regulators and the tumour suppressor p53 [8]. By a mechanism opposite to that of PA28αβ, which promotes inflammation through increased antigen presentation, PA28γ degrades MHC-I peptides in cancer, thereby facilitating evasion of immunosurveillance [8].

Three first-generation proteasome inhibitors have been approved for the treatment of multiple myeloma; however, they cause significant side effects due to their lack of specificity for a particular proteasome subtype [3]. Studies have been conducted on 2nd generation inhibitors targeting the catalytic sites of the immunoproteasome, but targeting a single catalytic subunit proved insufficient to achieve therapeutic goals [9]. Developing inhibitors of proteasome subtypes that specifically target the regulators PA28αβ and PA28γ constitutes a promising alternative.

Aim and objectives
This project aims to develop a 3rd generation of inhibitors targeting the i20S-PA28αβ and c20S-PA28γ complexes, due to their respective involvement in autoimmune diseases and cancer. The objective is to find drugs that are selective enough to reduce the side effects observed with 1st generation drugs, and which, by not targeting catalytic sites, are more selective than 2nd generation drugs.

The first objective is to assess the ability of a series of previously identified inhibitors to reduce proteasome activity in vitro, in order to identify the most effective and most selective molecules. Promising candidates would then be assessed in cellulo for their effectiveness and toxicity within cells. The most selective, most effective and least toxic inhibitors will be retained for further experiments. Next, any off-target effects of the inhibitors on protein abundance will be detected by proteome-wide analysis. This will determine whether major cellular pathways are up- or down-regulated after treatment. To further verify specificity and gain insight into the location of binding, another objective is to identify conformotypic peptides, i.e. peptides specific for bound and unbound states, which reflect binding of the drug to its target, but also to off-targets. Finally, the mode of action of the drugs will be studied to observe potential effects on proteasome disassembly, as well as inhibitor stoichiometry and the location of binding.

Conclusion
This project aims to develop highly selective and effective third-generation inhibitors of the i20S-PA28αβ and c20S-PA28γ complexes. Selective modulation of c20S-PA28γ would offer a more targeted approach to inhibiting proteasome activity in cancer, while inhibiting i20S-PA28αβ would be of great benefit to patients with autoimmune and inflammatory diseases such as rheumatoid arthritis and IBD. In conclusion, the results of this PhD project will provide essential knowledge to support the development of more selective and clinically relevant proteasome-targeting drugs.

References
[1] Kniepert, A. and Groettrup, M. (2014) 'The unique functions of tissue-specific proteasomes', Trends in Biochemical Sciences, 39(1), pp. 17–24. Available at: https://doi.org/10.1016/j.tibs.2013.10.004.
[2] Morozov, A.V. and Karpov, V.L. (2018) 'Biological consequences of structural and functional proteasome diversity', Heliyon, 4(10), p. e00894. Available at: https://doi.org/10.1016/j.heliyon.2018.e00894.
[3] Dafun, A.S. et al. (2023) 'Establishing 20S Proteasome Genetic, Translational and Post-Translational Status from Precious Biological and Patient Samples with Top-Down MS', Cells, 12, p. 844. Available at: https://doi.org/10.3390/cells12060844.
[4] Guillaume, B. et al. (2010) 'Two abundant proteasome subtypes that uniquely process some antigens presented by HLA class I molecules', Proceedings of the National Academy of Sciences of the United States of America, 107(43), pp. 18599–18604. Available at: https://doi.org/10.1073/pnas.1009778107.
[5] Muchamuel, T. et al. (2009) 'A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis', Nature Medicine, 15(7), pp. 781–787. Available at: https://doi.org/10.1038/nm.1978.
[6] Visekruna, A. et al. (2006) 'Proteasome-mediated degradation of IκBα and processing of p105 in Crohn disease and ulcerative colitis', Journal of Clinical Investigation, 116(12), pp. 3195–3203. Available at: https://doi.org/10.1172/JCI28804.
[7] Fabre, B. et al. (2014) 'Label-Free Quantitative Proteomics Reveals the Dynamics of Proteasome Complexes Composition and Stoichiometry in a Wide Range of Human Cell Lines', Journal of Proteome Research, 13(6), pp. 3027–3037. Available at: https://doi.org/10.1021/pr500193k.
[8] Boulpicante, M. et al. (2020) 'Tumors escape immunosurveillance by overexpressing the proteasome activator PSME3', Oncoimmunology, 9(1), p. 1761205. Available at: https://doi.org/10.1080/2162402X.2020.1761205.
[9] Basler, M. and Groettrup, M. (2020) 'Recent insights how combined inhibition of immuno/proteasome subunits enables therapeutic efficacy', Genes & Immunity, 21(5), pp. 273–287. Available at: https://doi.org/10.1038/s41435-020-00109-1.

Your Work Environment

The Institute of Pharmacology and Structural Biology (IPBS) is a joint research unit of the CNRS and the University of Toulouse, internationally recognised for its work in molecular biology, structural biology, immunology, microbiology and cancer research. Based in Toulouse, the institute brings together more than 250 scientific and technical staff across 18 research teams and hosts state-of-the-art technological platforms in proteomics, biophysics, imaging and functional exploration. Its research aims to identify and characterise new therapeutic targets in the fields of cancer, infectious diseases and inflammatory diseases, while fostering innovation, international collaborations and training through research.
The recruited student will be co-supervised by two researchers within the Proteomics and Mass Spectrometry of Biomolecules team (Marcoux/Gonzalez).
The project aims to express and purify proteasome complexes that will be used to identify new inhibitors.
The position is located in a sector covered by the protection of scientific and technical potential (PPST) and therefore requires, in accordance with regulations, that your arrival be authorised by the competent authority of the French Ministry of Higher Education and Research (MESR).

Constraints and risks

No particular risk for this position.

Compensation and benefits

Compensation

2300 € gross monthly

Annual leave and RTT

44 jours

Remote Working practice and compensation

Pratique et indemnisation du TT

Transport

Prise en charge à 75% du coût et forfait mobilité durable jusqu’à 300€

About the offer

About the CNRS

The CNRS is a major player in fundamental research on a global scale. The CNRS is the only French organization active in all scientific fields. Its unique position as a multi-specialist allows it to bring together different disciplines to address the most important challenges of the contemporary world, in connection with the actors of change.

CNRS

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