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.

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-A3_271–279) or by intermediate proteasomes β1i-β5i (MAGE-A10_254–262). 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.

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.

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.

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.