Production d'un peptide antigénique par l'insulin-degrading enzyme
(2012)
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- Authors
- Supervisors
- Van den Eynde, Benoît
- Abstract
- (en) Most antigenic peptides recognized by CD8 T lymphocytes are produced through degradation of intracellular proteins by the proteasome. However, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. MAGE-A3168-176 is a tumor antigenic peptide presented by HLA-A1 and widely used for vaccination of melanoma patients. We observed that proteasome and tripeptidyl peptidase inhibitors failed to block presentation of the antigen by tumor cells. However, processing of this peptide occured in the cytosol because TAP-inhibition prevented its presentation. To characterize the cytosolic peptidase producing MAGE-A3168-176 we setup an in vitro digestion assay using a 20-mer precursor peptide encompassing the sequence of the antigenic peptide. We observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. This production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. This inhibitor also blocked the presentation of MAGE-A3168-176 by tumor cells. Processing of the antigenic peptide was observed in HLA-A1 cells electroporated by the 20-mer precursor or by a precursor modified at the C- and N-terminus to exclude exopeptidase activity. With both precusors, the processing was inhibited by treating cells with o-phenanthroline concluding to a major role of a cytosolic metalloendopeptidase. One such enzyme is insulin-degrading enzyme (IDE). We observed that depletion of IDE abolished the capacity of a cytosolic fraction to produce the antigenic peptide. Furthermore, recombinant IDE was able to produce the peptide in vitro from the 20-mer peptide. Using a siRNA approach we observed that a reduction of IDE expression in tumor cells resulted in a reduced capacity of MAGE-A3168-176 presentation. Lastly, half-life studies revealed that in melanoma cells proteasome and IDE act as parallel ways for the degradation of MAGE-A3 contributing together to a different set of peptides.
- Affiliations
Citations
Parmentier, N. (2012). Production d’un peptide antigénique par l’insulin-degrading enzyme. https://hdl.handle.net/2078.5/158380