Structural Mass Spectrometry to study the interplay between proteasome complexes regulation mechanisms – ProteasoRegMS

In this project, the proteasome complexes are analyzed either directly from cell lysates or after immunopurification with an antibody that recognizes the alpha2 subunit (present in all types of proteasome). The interactions with potential partners can be stabilized using reversible formaldehyde crosslinking.
We use bottom-up proteomics for large-scale identification and quantification of the proteins present in the samples. Statistical tests are then employed to highlight proteins that are up- or down-regulated within one sample/condition.
Top-down proteomics is used to precisely determine and semi-quantify the proteoforms of the 20S core particle.
We use Hydrogen-deuterium exchange coupled to mass spectrometry, to probe the dynamics and solvent accessibilty of the 20S proteasome regulators/partners and identify which regions are being stabilized or destabilized upon interaction with regulators/partners/drugs or upon replacement of catalytic subunits.
The drug-screening is using an enzymatic assays based on fluorogenic peptides and differential scanning fluorimetry.

WP1 was initiated by the ANR-funded PhD student, who first got used to the different cell culture, biochemistry and MS-based pipelines. We established the feasibility of our approach in Caco2 cells, by showing the overexpression of proteasome immuno-catalytic subunits upon Interferon-gamma stimulation. We have not seen any similar immunoproteasome induction using other tested cytokines so far.
WP2 is almost complete and the results obtained were published in December 2020 in Nature Communications. In this work, we describe for the first time the use of HDX-MS to study proteasome complex assemblies. By comparing the data obtained on proteasomes containing different catalytic subunits (std20S vs. i20S), we highlighted fine intramolecular perturbations starting from these subunits and gradually reaching the surface of the complex. These results explain how the introduction of a different isoform into the complex can promote interaction with specific regulators. Moreover, the comparison of these proteasomes with or without regulators has made it possible to highlight a reverse regulation mechanism, in which the contact of a regulator at the proteasome surface is propagated to the catalytic sites. Besides revealing key aspects on the allosteric regulation of the 20S proteasome, this article sets the basis for the HDX-MS analysis of 20S complexes. We took advantage of this expertise to study another proteasome subtype: the spermatoproteasome that is specifically expressed in germ cells. HDX-MS analysis revealed differences in dynamics between the C-ter of a4/a4s subunits, which were further supported by molecular dynamics simulations. We also investigated the benefit of using ion-mobility MS coupled to HDX-MS in order to improve our pipeline and get better sequence coverages on the 20S proteasome.
The std20S and i20S were also immunopurified from HEK cells to set up a new way to semi-quantify 20S proteoforms using Top-Down MS.

Oustanding feature
• Setup of the methodological conditions for the Top-Down and Bottom-up proteomics analysis of proteasome interactors from Caco2 cells, enabling the further analysis of samples derived from IBD-patients.
• First HDX-MS analysis of std20S and i20S proteasomes alone and in complex with the PA28aß or PA28gamma regulators (Lesne et al, 2020 Nature Communications).
• HDX-MS comparison of the std20S and spermatoproteasome (Zivkovic et al, In preparation).
• Top-Down MS semi-quantification of 20S proteasome proteoforms (Dafun Sanchez et al, In preparation, a).
• Review on Structural MS analysis of membrane proteins (Dafun Sanchez and Marcoux, 2021 BBA Proteins and Proteomics, In revision) and vulgarization article on Structural MS (Marcoux, 2021 L’actualité Chimique).
• Use of Ion-Mobility MS to increase the sequence coverage of large multiprotein complexes in HDX-MS analysis (Dafun Sanchez et al, In preparation, b)

Perspectives:
WP1: We are now ready to start working on cell lysates obtained from IBD patient-derived biopsies and organoïds that will be soon provided by our collaborator.
WP2: The last experiment, consisting in the HDX-MS analysis of Proteasome Interacting Proteins (PIP: IDE and PIP30) should be straightforward since the proof of concept on the proteasome is now established.
WP3 has not started yet and is still scheduled at the end of the project. The HDX-MS analysis of 20S proteasome with and without inhibitors, will be conducted together with the PIPs and will also benefit from our acquired expertise. The enzymatic screening and the Differential Scanning Fluorimetry screening of drugs, will be achieved in mid and late 2022, respectively.

Articles:
1. Lesne J*, Locard-Paulet M*, Parra J, Zivkovic D, Menneteau T, Bousquet MP, Burlet-Schiltz O, Marcoux J (2020) “Conformational maps of human 20S proteasomes reveal PA28- and immuno-dependant inter-ring crosstalks” Nature Communications 11(1):6140.
2. Sanchez Dafun A and Marcoux J (2021) “Structural Mass Spectrometry of Membrane Proteins” BBA Proteins and Proteomics In Revision
3. Marcoux J (2021) “Peser le protéasome pour sonder sa structure” L’Actualité Chimique In Revision
4. Zivkovic D, Sanchez Dafun A, Lise S, Menneteau T, Toste-Rêgo A, Da Fonseca P, Pineau C, Burlet-Schiltz O, Marcoux J*, Bousquet-Dubouch MP* (2021) “Structural and functional study of the proteasome complexes in spermatogenesis using modern mass spectrometry approaches” In Preparation

Talks:
1. “Hydrogen-deuterium eXchange coupled to Mass Spectrometry highlights a reciprocal crosstalk between the inner and outer rings of the 20S proteasome”, Symposium on Structural Proteomics, Göttingen, Germany, 11/2019
2. “Study of the largest and most heterogeneous macromolecular complex by HDX-MS: bringing new important mechanistic insights in proteasome regulation”, Albert Heck FAQ Meeting, Utrecht, The Netherlands (webinar), 01/2021
3. “Structural MS in Proteomics”, British Mass Spectrometry Society – Virtual Biomacromolecular Structure Special Interest Group, United Kingdom, 03/2021
4. “Study of the largest and most heterogeneous macromolecular complex by HDX-MS: bringing new important mechanistic insights in proteasome regulation”, SMAP 2019, Strasbourg, 09/2019
5. “Hydrogen-deuterium eXchange coupled to Mass Spectrometry highlights a reciprocal crosstalk between the inner and outer rings of the 20S proteasome” Integrative Structural Biology, Toulouse, France, 10/2019
6. “Study of the largest and most heterogeneous macromolecular complex by HDX-MS: bringing new important mechanistic insights in proteasome regulation”, Centre de Biologie Structurale, Montpellier, 01/2020

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 described resulting from the association with different regulators (19S, PA28aß, PA28?, 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 (ß1, ß2 and ß5) 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 myeloma. 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-effects. Specific inhibition of the immunoproteasome was recently shown to reduce graft rejection and suppress the progression of colorectal cancer in mice.

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 regulator-associated 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 proteoforms. 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 of IBDs 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 PA28aß and PA28? regulators. The same method will also be used to identify binding interfaces of newly described Proteasome Interacting Proteins (PIP) and study the binding of specific and non-specific inhibitors to the Std vs. i20S. Finally, our specific ligand screening will be performed using a high-throughput workflow based on the chemical, fragment and peptide-based libraries available at the institute (PICT Platform).

Originality:
We are confident that these innovative structural MS methods 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, our screening strategy targeting regulator associated proteasomes is cutting-edge and very promising.