Biology subjects

Bousquet, M.-P.

Publications and source records attributed to Bousquet, M.-P..

5 recordsLinked to original sources

bioRxiv2025

Revealing the unexpected interplay between the Proteasome Activator PA200 and the immunoproteasome

The proteasome activator PA200 binds to the catalytic core of the proteasome, the 20S, and activates its proteolytic activities. The cellular function of PA200 is poorly understood and appears to be cell type and differentiation specific. Recent evidence suggests that PA200 not only binds to the standard 20S (s20S) proteasome but also to the specialized immunoproteasome (i20S) which plays a key role in anti-viral and anti-tumor immunity. We here investigated the interaction of PA200 and the immunoproteasome in detail. We show the very first cryo-EM structures of the singly- and doubly-capped i20S-PA200 complexes that revealed no major difference regarding the first binding event of PA200 to the i20S vs. the s20S. However, first PA200 binding triggered a subtle and long range allosteric bending of the i20S barrel which was not seen in the s20S-PA200 complexes. This resulted in major structural rearrangements in the opposite unbound ring - the displacement of atoms up to 5.4 [A] and the increase in its outer diameter - thereby increasing the occupancy of the second PA200 binding site. Mass photometry confirmed higher occupancy of PA200 to the i20S versus the s20S. Binding of PA200 to the i20S enhanced proteasomal activation compared to the s20S. Co-expression of PA200 and the i20S in cells and tissues, however, is restricted but their interaction is favored upon co-expression. The expression of PA200 and the catalytic subunits of the i20S is differentially regulated depending on the cellular context. Our data also suggest that PA200 has the potential to regulate i20S gene expression whereas the i20S has no effects on PA200 expression. Overall, this work sheds new light on the interaction of PA200 with the i20S from a structural, mechanistic and cellular point of view. Importantly, we identify PA200 as a key regulator of the i20S whenever PA200 and the catalytic subunits of the i20S are co-expressed in the same cell.

biochemistry↗

bioRxiv2024

The proteasome activator PA200 regulates tumor cell invasion

Proteasome activator 200 (PA200) is upregulated in non-small cell lung cancer (NSCLC) and linked to poor prognosis. We previously demonstrated that the overexpression of PA200 in NSCLC is associated with immune evasion and reduced responsiveness to immune checkpoint inhibitors. The cell autonomous function of PA200 in cancer growth, however, is not solved. We here demonstrate that deletion of PA200 in two distinct lung cancer cell lines induced cell-specific alterations in proteasome composition and activities with a minor direct impact on overall proteasome activity. Deficiency of PA200 in lung cancer cells did not consistently alter tumor cell growth in vitro and in vivo. However, we observed concerted inhibition of tumor cell migration and invasion with conserved downregulation of the integrin ITGB3 and transcriptional dysregulation of multiple cell adhesion and ECM regulators. Our transcriptome profiling revealed a striking disparity in the transcriptional response to PA200 deletion in the two lung cancer cell lines. Together with our PA200 interactome analysis that uncovered an unexpected cell-dependent profile of PA200-interacting proteins, our data indicate that the function of PA200 is cell-specific and depends on the cellular context. In conclusion, we here demonstrate that PA200 cell-autonomously regulates the invasive capacities of tumor cells thereby potentially promoting lung cancer spread and metastasis formation. This mechanism might add to PA200-related immune evasion and may contribute to the observed poor prognosis of PA200-overexpressing lung cancer patients.

cancer biology↗

bioRxiv2021

Interactome analyses reveal profound proteasome structural and functional rearrangements throughout mammalian spermatogenesis

During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their path to spermatozoa. To achieve this, a succession of processes requiring high proteolytic activity are in part orchestrated by the proteasome. The spermatoproteasome (s20S) is specific to the developing gametes, in which the gamete-specific 4s subunit replaces the 4 isoform found in the constitutive proteasome (c20S). Although the s20S is conserved across species and was shown to be crucial for germ cell development, its mechanism, function and structure remain incompletely characterized. Here, we used advanced mass spectrometry (MS) methods to map the composition of proteasome complexes and their interactomes throughout spermatogenesis. We observed that the s20S becomes highly activated as germ cells enter meiosis, mainly through a particularly extensive 19S activation and, to a lesser extent, PA200 binding. Additionally, the proteasome population shifts from c20S (98%) to s20S (>82-92%) during differentiation, presumably due to the shift from 4 to 4s expression. We demonstrated that s20S, but not c20S, interacts with components of the meiotic synaptonemal complex, where it may localize via association with the PI31 adaptor protein. In vitro, s20S preferentially binds to 19S, and displays higher trypsin- and chymotrypsin-like activities, both with and without PA200 activation. Moreover, using MS methods to monitor protein dynamics, we identified significant differences in domain flexibility between 4 and 4s. We propose that these differences induced by 4s incorporation result in significant changes in the way the s20S interacts with its partners, and dictate its role in germ cell differentiation.

biochemistry↗

bioRxiv2020

Phosphoproteomics identifies PI3K inhibitor-selective adaptive responses in pancreatic cancer cell therapy and resistance

The PI3K pathway is highly active in human cancers. The four class I isoforms of PI3K are activated by distinct mechanisms leading to a common downstream signaling. Their downstream redundancy is thought to be responsible for treatment failures of PI3K inhibitors. We challenged this concept, by mapping the differential phosphoproteome evolution in response to PI3K inhibitors with different isoform selectivity patterns in pancreatic cancer, a disease currently without effective therapy. In this cancer, the PI3K signal was shown to control cell proliferation. We compared the effects of LY294002 that inhibit with equal potency all class I isoenzymes and downstream mTOR with the action of inhibitors with higher isoform-selectivity towards PI3K, PI3K{beta} or PI3K{gamma} (namely A66, TGX-221 and AS-252424). A bioinformatics global pathway analysis of phosphoproteomics data allowed us to identify common and specific signals activated by PI3K inhibitors supported by the biological data. AS-252424 was the most effective treatment and induced apoptotic pathway activation as well as the highest changes in global phosphorylation-regulated cell signal. However, AS-252424 treatment induced re-activation of Akt, therefore decreasing the treatment outcome on cell survival. Reversely, AS-252424 and A66 combination treatment prevented p-Akt reactivation and led to synergistic action in cell lines and patient organoids. The combination of clinically approved -selective BYL-719 with {gamma}-selective IPI-549 was more efficient than single molecule treatment on xenograft growth. Mapping unique adaptive signaling responses to isoform-selective PI3K inhibition will help to design better combinative treatments that prevent the induction of selective compensatory signals. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/313833v3_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@32004eorg.highwire.dtl.DTLVardef@b22f25org.highwire.dtl.DTLVardef@352a98org.highwire.dtl.DTLVardef@7f97a1_HPS_FORMAT_FIGEXP M_FIG C_FIG SignificanceHalf of all human cancers show increased PI3K activity but the mutational pattern of the PI3K pathway is not sufficient to predict sensitivity to PI3K inhibitors. By identifying for the first time specific signaling induced by PI3K inhibitors with different isoform-selectivity patterns, we provide insight in how to handle heterogeneity of PI3K expression in tumor samples for the choice of available PI3K-targetting drugs. Our work provides a roadmap to target the PI3K pathway, balancing the use of isoform-selective PI3K inhibitors with personalized information extending beyond the specific mutational status.

cancer biology↗

bioRxiv2020

Conformational maps of human 20S proteasomes reveal PA28- and immuno-dependant inter-ring crosstalks

Here, we used for the first time Hydrogen-Deuterium eXchange coupled to Mass Spectrometry (HDX-MS) to investigate conformational differences between the human standard 20S (std20S) and immuno 20S (i20s) proteasomes alone or in complex with PA28{beta} or PA28{gamma} activators. Their solvent accessibility was analyzed through a dedicated bioinformatic pipeline including stringent statistical analysis and 3D visualization. These data confirmed the existence of allosteric differences between the std20S and i20S at the surface of the -ring triggered from inside the catalytic {beta}-ring. Additionally, binding of the PA28 regulators to the 20S proteasomes modified solvent accessibility due to conformational changes of the {beta}-rings. This work is not only a proof-of-concept that HDX-MS can be used to get structural insights on large multi-protein complexes in solution, it also demonstrates that the binding of the std20S or i20S subtype to any of its PA28 activator triggers allosteric changes that are specific to this 20S/PA28 pair.

biophysics↗