CytosolNonproteasomal Epitope Generation in the MHC Class I Epitopes: Evidence for Proteasomes Can Either Generate or Destroy

Proteasomes have been implicated in the production of the majority of peptides that associate with MHC class I molecules. We used two different proteasome inhibitors, the peptide aldehyde N -acetyl- L -leucyl- L -leucyl- L -norleucinal (LLnL) and the highly specific inhibitor lactacystin, to examine the role of proteasomes in generating peptide epitopes associated with HLA-A*0201. Neither LLnL nor lactacystin was able to completely block the expression of the HLA-A*0201. Furthermore, the effects of LLnL and lactacystin on the expression of different categories of specific epitopes, TAP independent vs TAP dependent and derived from either cytosolic or membrane proteins, were assessed. As predicted, presentation of two TAP-dependent epitopes was blocked by LLnL and lactacystin, while a TAP-independent epitope that is processed in the endoplasmic reticulum was unaffected by either inhibitor. Surprisingly, both LLnL and lactacystin increased rather than inhibited the expression of a cytosolically transcribed and TAP-dependent peptide from the influenza A virus M1 protein. Mass spectrometric analyses of in vitro proteasome digests of a synthetic 24 mer containing this epitope revealed no digestion products of any length that included the intact epitope. Instead, the major species resulted from cleavage sites within the epitope. Although cleavage at these sites was inhibitable by LLnL and lactacystin, epitope-containing species were still not produced. We conclude that proteasomes may in some cases actually destroy epitopes that would otherwise be destined for presentation by class I molecules. These results clone AHIII12-2 at an E:T cell ratio of 14:1, CTL clone AT1-15 was used at an E:T cell ratio of 40:1, CTL lines recognizing TYR 368–376 were used at an E:T cell ratio of 12:1, and gp100 154–162 was used at an E:T cell ratio of 10:1. The lymphoblastoid transfectant line C1R.A2.1.M1 was used as a target for the CTL line LSGM1S recognizing M1 58–66 at an E:T cell ratio of 40:1. The results are representative of at least three experiments. complexes. These representative independent

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