The role of the proteasome activator PA200 in myofibroblast differentiation and fibrosis of the lung – PA200_in_IPF

We will first investigate the regulation of PA200 in cells and ex vivo tissue of IPF patients in detail. Development of novel genetically engineered cell lines will then allow depletion, mutation or activation of PA200 in fibroblasts and these tools will be combined with unbiased omic analysis (RNA, DNA and protein) and novel methods of the Bousquet lab for isolation and quantitative analysis of PA200/proteasome complexes and their substrates. We will thereby decipher the molecular function of PA200/proteasome complexes in myofibroblast differentiation with a particular focus on chromatin remodeling. Moreover, with our in vitro drug screening approach, we aim to find novel lead compounds that specifically inhibit PA200/proteasome complexes which might then prove useful for therapeutic targeting of PA200 in IPF pathogenesis.

• WP 1.1 & 1.2: As PA200 was not identified in PCLS samples from IPF patients, we have focused our analysis on isolated primary human lung fibroblasts (phLF) that were isolated from donors and IPF patients. Using this model, we confirmed that TGF-ß1 upregulates PA200 also in ex vivo mouse lung cultures, even before myofibrogenesis is induced.
• WP 1.3: The proteomics analysis of the composition and interactome of PA200/proteasome was achieved, using cultures obtained from 5 healthy donor- and 5 IPF-derived phLF lines with or without TGF-ß1 treatment (48 hours). We were able to identify the full proteasome interactome with and without TGF-ß1 treatment. We observed changes in the proteasome core composition, as well as in the regulator recruitment to 20S, when both IPF and healthy samples are treated with TGF-ß1. TGF-ß1 treatment seems to trigger the enrichment of ribosomal and ribonuclear proteins in the PA200 interactome.
• WP 2: The construction of PA200-depleted, PA200-mutated and PA200-overexpressing cells is ongoing (task 2.1). We have set up and validated the methodological conditions for the identification of proteasome substrates on bovine testis using the protocol from Wolf-Levy et al. (Nature Biotech. 2018 Oct 22;10.1038/nbt.4279, doi: 10.1038/nbt.4279) in Bousquet’s laboratory. This protocol is thus ready to be implemented to identify PA200 substrates by comparing peptides that are enriched in PA200-overexpressing fibroblasts compared to PA200-depleted or PA200-mutated cells (task 2.3).
• WP3: the production of PA200 and 20S proteins for the screening assay is performed and we have received the drug library (task 3.1). We also set up a collaboration with the PICT-ICEO platform for the high-throughput screening assay that is scheduled before the end of 2022 (task 3.2).

We are pleased with the current progress of the project and with the recruitment of our research engineer. WP1 is complete and we have set up the basis to achieve the objectives of WP3. WP2 takes a little bit longer than expected but the method to identify proteasome substrates has been settled in Bousquet’s lab and is ready to be used on the fibroblastic cells that overexpress PA200 (construction ongoing).

D. Zivkovic, A. Sanchez Dafun, T. Menneteau, A. Schahl, S. Lise, C. Kervarrec, A. Toste-Rêgo, P. Da Fonseca, M. Chavent, C. Pineau, O. Burlet-Schiltz, J. Marcoux* and MP Bousquet* (2022). Proteasome complexes experience profound structural and functional rearrangements throughout spermatogenesis. Proc Natl Acad Sci U S A. 119(15): e2116826119. DOI: 10.1073/pnas.2116826119 * :co-corresponding authors.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with no effective therapies beyond lung transplantation. Novel therapeutic approaches based on a mechanistic understanding of IPF development are thus urgently needed. The pathogenesis of IPF involves aberrant wound healing in response to continuous or repeated damage of the alveolar epithelium and subsequent activation of myofibroblasts. These activated myofibroblasts are key drivers for fibrosis as they produce excessive extracellular matrix proteins which deposition then contributes to impaired lung function and finally death in IPF patients. Dysregulation of the proteasome system – a central gatekeeper of protein homeostasis - is emerging as a novel pathomechanism for IPF. While application of catalytic proteasome inhibitors is hampered due to their toxic side effects, proteasome function might be modulated more specifically by interference with defined proteasome complexes. In the proposed project, we aim to explore the role and regulation of the alternative proteasome complexes containing the proteasome activator PA200.
PA200 has been proposed to be involved in the degradation of acetylated histones and chromatin remodeling. The exact cellular function of PA200, however, is not understood yet. Disease related regulation or dysfunction of PA200 has not been observed so far. Our own published data revealed upregulation of PA200 in IPF lung tissue in activated myofibroblasts and unraveled PA200 to function as a negative regulator of myofibroblast differentiation. Our unpublished data indicate that PA200 regulates chromatin remodeling which may contribute to IPF disease pathogenesis.
To test this hypothesis, the project combines the translational expertise of the groups of Ju¨rgen Behr from the university clinics of the Ludwigs-Maximilians Universität and Silke Meiners at the Comprehensive Pneumology Center in Munich with the unique expertise of Marie-Pierre Bousquet at the Institute of Pharmacology and Structural Biology in Toulouse on mass-spec based analysis of proteasome complexes.
We will first investigate the regulation of PA200 in cells and ex vivo tissue of IPF patients in detail. Development of novel genetically engineered cell lines will then allow depletion, mutation or activation of PA200 in fibroblasts and these tools will be combined with unbiased omic analysis (RNA, DNA and protein) and novel methods of the Bousquet lab for isolation and quantitative analysis of PA200/proteasome complexes and their substrates. We will thereby decipher the molecular function of PA200/proteasome complexes in myofibroblast differentiation with a particular focus on chromatin remodeling. Moreover, with our in vitro drug screening approach, we aim to find novel lead compounds that specifically inhibit PA200/proteasome complexes which might then prove useful for therapeutic targeting of PA200 in IPF pathogenesis.