Identification de nouveaux types de protéasome et leur implication dans l’apprêtement de peptides antigéniques présentés par les molécules CMH de classe I
(2011)
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- Authors
- Supervisors
- Van Den Eynde, Benoît
- Abstract
- (en) Cytolytic T Lymphocytes recognize peptides derived from intracellular proteins and presented by MHC class I molecules. Most of these peptides result from the degradation of cytosolic proteins by the proteasome. The composition in ß-catalytic subunits determines the type of proteasome. Until now, two proteasome types have been described. The standard proteasome (ß1ß2ß5) is present in most cell types, while the immunoproteasome (ß1iß2iß5i) is present in dendritic cells and in cells exposed to IFN-. Some antigenic peptides are processed differentially by the two proteasome types and thus, the set of peptides expressed at the cell surface is dependent on the type of proteasome present inside the cell. This view should be refined by the existence of other proteasome types. The ß-catalytic inducible subunits are incorporated following a cooperative mechanism to form a fully immunoproteasome. Theoritically, they can also form intermediate proteasomes characterized by a mix of constitutive and inducible ß-catalytic subunits. Experimentally, a heterogeneity in catalytic subunits composition was observed in rat muscle, rodent heart and in human colon and liver. The lack of catalytic subunit-specific antibodies recognizing native proteasome particles has prevented the isolation of intermediate proteasomes. Here we produced a panel of unique subunit-specific antibodies to allowing to isolate intermediate proteasomes in their native conformation. With the help of these antibodies, we identified two new types of 20S proteasome which contain only one or two catalytic immunosubunits. These intermediate proteasomes contain either ß1iß2ß5i (double intermediate) or ß1ß2ß5i (single intermediate). They were found in eight human tumor cell lines of different histological types. They represent 10-20% of the total proteasome content of these tumor cells. In normal human tissues, we observed high proportions of intermediate proteasomes : these represented between one-third and one-half of the proteasome content of liver, kidney, small bowel, and colon. Intermediate proteasomes were also abundant in monocyte-derived dendritic cells, mainly the proteasome ß5i which represents about 40% of the proteasome content of dendritic cells. To study their function in antigen processing, we produced 293 cell lines expressing only intermediate proteasomes. When tested on fluorogenic substrates, the proteolytic activities of the intermediate proteasomes were different from those of the standard proteasome and the immunoproteasome. We identified three tumor antigens produced exclusively by the proteasome ß5i (MAGE-A3271-279), or by the proteasome ß1i-ß5i (MAGE-A10254-262 and MAGE-C2191-200). We also studied the ability of intermediate proteasomes to produce six antigenic peptides (MAGE-C241-49, MAGE-C2336-344, MAGE-A3114-122, gp100209-217, tyrosinase369-377, Melan-A26-35) that are known to be processed differentially by the standard proteasome and the immunoproteasome. Both intermediate proteasomes showed a processing activity similar to that of the immunoproteasome with regard to all but one peptides. The latter peptide, derived from MAGE-C2336-344 and presented by HLA-A2 is produced by the double but not the single intermediate proteasome. We studied cleavage sites within precursor peptides by mass spectrometry, and observed that the intermediate proteasomes, like the immunoproteasome, were characterized by a poor ability to cleave between hydrophobic residues. Surprisingly, we also observed stronger cleavages after aspartic acids by the intermediate proteasome ß5i as compared to the standard proteasome, despite the presence of subunit ß1, which is classically associated with the caspase-like activity, in both proteasome types. Interesting questions for further studies will be to analyse if the modulation of proteasome content also affects other proteasome functions such as the activation of transcription factor, the production of inflammatory cytokines, or the degradation of oxidized proteins.
- Affiliations
Citations
Guillaume, B. (2011). Identification de nouveaux types de protéasome et leur implication dans l’apprêtement de peptides antigéniques présentés par les molécules CMH de classe I. https://hdl.handle.net/2078.5/149048