PROTEASOMES CAN EITHER GENERATE OR DESTROY MHC CLASS-I EPITOPES - EVIDENCE FOR NONPROTEASOMAL EPITOPE GENERATION IN THE CYTOSOL

Proteasomes have been implicated in the production of the majority of peptides that associate with MC class I molecules, We used two differe nt proteasome inhibitors, the peptide aldehyde N-acetyl-L-leucyl-L-leu cyl-L-norleucinal (LLnL) and the highly specific inhibitor lactacystin , to examine the role of proteasomes in generating peptide epitopes as sociated with HLA-A0201. Neither LLnL nor lactacystin was able to com pletely block the expression of the HLA-A0201). Furthermore, the effe cts of LLnL and lactacystin on the expression of different categories of specific epitopes, TAP independent vs TAP dependent and derived fro m either cytosolic or membrane proteins, were assessed. As predicted, presentation of two TAP-dependent epitopes was blocked by LLnL and lac tacystin, while a TAP-independent epitope that is processed in the end oplasmic reticulum was unaffected by either inhibitor, Surprisingly, b oth LLnL and lactacystin increased rather than inhibited the expressio n of a cytosolically transcribed and TAP-dependent peptide from the in fluenza A virus M1 protein. Mass spectrometric analyses of in vitro pr oteasome digests of a synthetic 24 mer containing this epitope reveale d no digestion products of any length that included the intact epitope . Instead, the major species resulted from cleavage sites within the e pitope, Although cleavage at these sites was inhibitable by LLnL and l actacystin, epitope-containing species were still not produced. We con clude that proteasomes may in some cases actually destroy epitopes tha t would otherwise be destined for presentation by class I molecules. T hese results suggest that some epitopes are generated by nonproteasoma l proteases in the cytosol.