Marie-Pierre Bousquet-Dubouch

Two abundant proteasome subtypes that uniquely process some antigens presented by HLA class I molecules

Most antigenic peptides presented by MHC class I molecules result from the degradation of intracellular proteins by the proteasome. In lymphoid tissues and cells exposed to IFNγ, the standard proteasome is replaced by the immunoproteasome, in which all of the standard catalytic subunits β1, β2, and β5 are replaced by their inducible counterparts β1i, β2i, and β5i, which have different cleavage specificities. The immunoproteasome thereby shapes the repertoire of antigenic peptides. The existence of additional forms of proteasomes bearing a mixed assortment of standard and inducible catalytic subunits has been suggested. Using a new set of unique subunit-specific antibodies, we have now isolated, quantified, and characterized human proteasomes that are intermediate between the standard proteasome and the immunoproteasome. They contain only one (β5i) or two (β1i and β5i) of the three inducible catalytic subunits of the immunoproteasome. These intermediate proteasomes represent between one-third and one-half of the proteasome content of human liver, colon, small intestine, and kidney. They are also present in human tumor cells and dendritic cells. We identified two tumor antigens of clinical interest that are processed exclusively either by intermediate proteasomes β5i (MAGE-A3271–279) or by intermediate proteasomes β1i-β5i (MAGE-A10254–262). The existence of these intermediate proteasomes broadens the repertoire of antigens presented to CD8 T cells and implies that the antigens presented by a given cell depend on their proteasome content.

Pharmacological targeting of the protein synthesis mTOR/4E‐BP1 pathway in cancer‐associated fibroblasts abrogates pancreatic tumour chemoresistance

Pancreatic ductal adenocarcinoma (PDAC) is extremely stroma‐rich. Cancer‐associated fibroblasts (CAFs) secrete proteins that activate survival and promote chemoresistance of cancer cells. Our results demonstrate that CAF secretome‐triggered chemoresistance is abolished upon inhibition of the protein synthesis mTOR/4E‐BP1 regulatory pathway which we found highly activated in primary cultures of α‐SMA‐positive CAFs, isolated from human PDAC resections. CAFs selectively express the sst1 somatostatin receptor. The SOM230 analogue (Pasireotide) activates the sst1 receptor and inhibits the mTOR/4E‐BP1 pathway and the resultant synthesis of secreted proteins including IL‐6. Consequently, tumour growth and chemoresistance in nude mice xenografted with pancreatic cancer cells and CAFs, or with pieces of resected human PDACs, are reduced when chemotherapy (gemcitabine) is combined with SOM230 treatment. While gemcitabine alone has marginal effects, SOM230 is permissive to gemcitabine‐induced cancer cell apoptosis and acts as an antifibrotic agent. We propose that selective inhibition of CAF protein synthesis with sst1‐directed pharmacological compounds represents an anti‐stromal‐targeted therapy with promising chemosensitization potential.

Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins

An affinity purification strategy was developed to characterize human proteasome complexes diversity as well as endogenous proteasome-interacting proteins (PIPs). This single step procedure, initially used for 20 S proteasome purification, was adapted to purify all existing physiological proteasome complexes associated to their various regulatory complexes and to their interacting partners. The method was applied to the purification of proteasome complexes and their PIPs from human erythrocytes but can be used to purify proteasomes from any human sample as starting material. The benefit of in vivo formaldehyde cross-linking as a stabilizer of protein-protein interactions was studied by comparing the status of purified proteasomes and the identified proteins in both protocols (with or without formaldehyde cross-linking). Subsequent proteomics analyses identified all proteasomal subunits, known regulators, and recently assigned partners. Moreover other proteins implicated at different levels of the ubiquitin-proteasome system were also identified for the first time as PIPs. One of them, the ubiquitin-specific protease USP7, also known as HAUSP, is an important player in the p53-HDM2 pathway. The specificity of the interaction was further confirmed using a complementary approach that consisted of the reverse immunoprecipitation with HAUSP as a bait. Altogether we provide a valuable tool that should contribute, through the identification of partners likely to affect proteasomal function, to a better understanding of this complex proteolytic machinery in any living human cell and/or organ/tissue and in different cell physiological states.

Analysis of the Processing of Seven Human Tumor Antigens by Intermediate Proteasomes

We recently described two proteasome subtypes that are intermediate between the standard proteasome and the immunoproteasome. They contain only one (β5i) or two (β1i and β5i) of the three inducible catalytic subunits of the immunoproteasome. They are present in tumor cells and abundant in normal human tissues. We described two tumor antigenic peptides that are uniquely produced by these intermediate proteasomes. In this work, we studied the production by intermediate proteasomes of tumor antigenic peptides known to be produced exclusively by the immunoproteasome (MAGE-A3114–122, MAGE-C242–50, MAGE-C2336–344) or the standard proteasome (Melan-A26–35, tyrosinase369–377, gp100209–217). We observed that intermediate proteasomes efficiently produced the former peptides, but not the latter. Two peptides from the first group were equally produced by both intermediate proteasomes, whereas MAGE-C2336–344 was only produced by intermediate proteasome β1i-β5i. Those results explain the recognition of tumor cells devoid of immunoproteasome by CTL recognizing peptides not produced by the standard proteasome. We also describe a third antigenic peptide that is produced exclusively by an intermediate proteasome: peptide MAGE-C2191–200 is produced only by intermediate proteasome β1i-β5i. Analyzing in vitro digests, we observed that the lack of production by a given proteasome usually results from destruction of the antigenic peptide by internal cleavage. Interestingly, we observed that the immunoproteasome and the intermediate proteasomes fail to cleave between hydrophobic residues, despite a higher chymotrypsin-like activity measured on fluorogenic substrates. Altogether, our results indicate that the repertoire of peptides produced by intermediate proteasomes largely matches the repertoire produced by the immunoproteasome, but also contains additional peptides.

Subcellular Distribution and Dynamics of Active Proteasome Complexes Unraveled by a Workflow Combining in Vivo Complex Cross-Linking and Quantitative Proteomics

Through protein degradation, the proteasome plays fundamental roles in different cell compartments. Although the composition of the 20S catalytic core particle (CP) has been well documented, little is known about the composition and dynamics of the regulatory complexes that play a crucial role in its activity, or about how they associate with the CP in different cell compartments, different cell lines, and in response to external stimuli. Because of difficulties performing acceptable cell fractionation while maintaining complex integrity, it has been challenging to characterize proteasome complexes by proteomic approaches. Here, we report an integrated protocol, combining a cross-linking procedure on intact cells with cell fractionation, proteasome immuno-purification, and robust label-free quantitative proteomic analysis by mass spectrometry to determine the distribution and dynamics of cellular proteasome complexes in leukemic cells. Activity profiles of proteasomes were correlated fully with the composition of protein complexes and stoichiometry. Moreover, our results suggest that, at the subcellular level, proteasome function is regulated by dynamic interactions between the 20S CP and its regulatory proteins—which modulate proteasome activity, stability, localization, or substrate uptake—rather than by profound changes in 20S CP composition. Proteasome plasticity was observed both in the 20S CP and in its network of interactions following IFNγ stimulation. The fractionation protocol also revealed specific proteolytic activities and structural features of low-abundance microsomal proteasomes from U937 and KG1a cells. These could be linked to their important roles in the endoplasmic reticulum associated degradation pathway in leukemic cells.

Label-Free Quantitative Proteomics Reveals the Dynamics of Proteasome Complexes Composition and Stoichiometry in a Wide Range of Human Cell Lines

The proteasome is the main proteolytic system involved in intracellular proteins homeostasis in eukaryotes. Although the structure of proteasome complexes has been well characterized, the distribution of its activators and associated proteins are less studied. Here, we determine the composition and the stoichiometry of proteasome complexes and their associated proteins in a wide range of human cell lines using a one-step affinity purification method and a label-free quantitative proteomic approach. We show that proteasome complexes are highly dynamic protein assemblies, the activity of which being regulated at different levels by variations in the stoichiometry of bound regulators, in the composition of catalytic subunits and associated proteins, and in the rate of the 20S catalytic core complex assembly.

Proteasome inhibitor-induced apoptosis in acute myeloid leukemia: A correlation with the proteasome status

The proteasome plays a critical role in the regulation of many cellular processes, including the cell cycle and tumor growth. The proteasome inhibitor bortezomib has recently been approved for the treatment of relapsed and refractory multiple myeloma. In this study, we investigated the induction of apoptosis by proteasome inhibitors in several human acute myeloid leukemia (AML) cell lines and in primary cells from patients. We demonstrate that these drugs induce a high level of apoptosis in the KG1a cell line, in which the therapeutic drug daunorubicin is poorly active, compared to other AML cell lines. In parallel, we found that significantly different levels of apoptosis were induced in primary cells from patients depending on the FAB-based differentiation status of these cells. Moreover, the level of 20S proteasome in KG1a cells was also high compared to other AML cell lines, suggesting a relationship between the high sensitivity to proteasome inhibitors and an elevated amount of 20S proteasome. In good accordance, we identified two groups of patient cells expressing high and low levels of 20S proteasome, with respective high and low sensitivity to proteasome inhibitors. Further comparison of the proteasome status in KG1a and U937 cells also suggests that a high proportion of the 19S regulatory complex in U937 cells compared to the 20S core complex may explain an increased proteasome activity. Altogether, our results suggest that various AML subtypes may present different responses to proteasome inhibitors, that these molecules can be potentially considered as interesting therapeutic alternatives for these pathologies, and that the amount of 20S proteasome in AML cells may be predictive of the cellular response to these inhibitors.

Chronic ethanol feeding affects proteasome-interacting proteins.

Studies on alcoholic liver injury mechanisms show a significant inhibition of the proteasome activity. To investigate this phenomenon, we isolated proteasome complexes from the liver of rats fed ethanol chronically, and from the liver of their pair‐fed controls, using a non‐denaturing multiple centrifugations procedure to preserve proteasome‐interacting proteins (PIPs). ICAT and MS/MS spectral counting, further confirmed by Western blot, showed that the levels of several PIPs were significantly decreased in the isolated ethanol proteasome fractions. This was the case of PA28α/β proteasome activator subunits, and of three proteasome‐associated deubiquitinases, Rpn11, ubiquitin C‐terminal hydrolase 14, and ubiquitin carboxyl‐terminal hydrolase L5. Interestingly, Rpn13 C‐terminal end was missing in the ethanol proteasome fraction, which probably altered the linking of ubiquitin carboxyl‐terminal hydrolase L5 to the proteasome. 20S proteasome and most 19S subunits were however not changed but Ecm29, a protein known to stabilize the interactions between the 20S and its activators, was decreased in the isolated ethanol proteasome fractions. It is proposed that ethanol metabolism causes proteasome inhibition by several mechanisms, including by altering PIPs and proteasome regulatory complexes binding to the proteasome.

Deciphering preferential interactions within supramolecular protein complexes: the proteasome case

In eukaryotic cells, intracellular protein breakdown is mainly performed by the ubiquitin–proteasome system. Proteasomes are supramolecular protein complexes formed by the association of multiple sub‐complexes and interacting proteins. Therefore, they exhibit a very high heterogeneity whose function is still not well understood. Here, using a newly developed method based on the combination of affinity purification and protein correlation profiling associated with high‐resolution mass spectrometry, we comprehensively characterized proteasome heterogeneity and identified previously unknown preferential associations within proteasome sub‐complexes. In particular, we showed for the first time that the two main proteasome subtypes, standard proteasome and immunoproteasome, interact with a different subset of important regulators. This trend was observed in very diverse human cell types and was confirmed by changing the relative proportions of both 20S proteasome forms using interferon‐γ. The new method developed here constitutes an innovative and powerful strategy that could be broadly applied for unraveling the dynamic and heterogeneous nature of other biologically relevant supramolecular protein complexes.

Scaled-down purification protocol to access proteomic analysis of 20S proteasome from human tissue samples: comparison of normal and tumor colorectal cells.

The proteasome is a proteolytic complex that constitutes the main pathway for degradation of intracellular proteins in eukaryotic cells. It regulates many physiological processes and its dysfunction can lead to several pathologies like cancer. To study the 20S proteasome structure/activity relationship in cells that derive from human biopsy samples, we optimized an immuno-purification protocol for the analysis of samples containing a small number of cells using magnetic beads. This scaled-down protocol was used to purify the cytoplasmic 20S proteasome of adjacent normal and tumor colorectal cells arising from tissue samples of several patients. Proteomic analyses based on two-dimensional gel electrophoresis (2DE) and mass spectrometry showed that the subunit composition of 20S proteasomes from these normal and tumor cells were not significantly different. The proteasome activity was also assessed in the cytoplasmic extracts and was similar or higher in tumor colorectal than in the corresponding normal cells. The scaled-down 20S proteasome purification protocol developed here can be applied to any human clinical tissue samples and is compatible with further proteomic analyses.