Cells adapted to the proteasome inhibitor 4-hydroxy- 5-iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone require enzymatically active proteasomes for continued survival

Abstract

The proteasome is the primary protease used by cells for degrading proteins and generating peptide ligands for class I molecules of the major histocompatibility complex. Based on the properties of cells adapted to grow in the presence of the proteasome inhibitor 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone (NLVS), it was proposed that proteasomes can be replaced by alternative proteolytic systems, particularly a large proteolytic complex with a tripeptidyl peptidase II activity. Here we show that NLVS-adapted cells retain sensitivity to a number of highly specific proteasome inhibitors with regard to antigenic peptide generation, accumulation of polyubiquitinated proteins, degradation of p53, and cell viability. In addition, we show that in the same assays (with a single minor exception), NLVS-adapted cells are about as sensitive as nonselected cells to Ala-Ala-Phe-chloromethylketone, a specific inhibitor of tripeptidyl peptidase II activity. Based on these findings, we conclude that proteasomes still have essential proteolytic functions in adapted cells that are not replaced by Ala-Ala-Phe-chloromethylketone-sensitive proteases.

The Final N-Terminal Trimming of a Subaminoterminal Proline-Containing HLA Class I-Restricted Antigenic Peptide in the Cytosol Is Mediated by Two Peptidases

“…Proteolytic activity was monitored by the increased fluorescence resulting from the release of the AMC group (excitation/emission 380/440 nm). A total of 4 nmol peptide RU1 [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] was incubated with 12 l fractions 29, 31, and 32, respectively, for 20 min at 37°C. The digestion was stopped with 2% TFA and analyzed by MS as above.…”

“…Earlier work, using the precursor peptide RU1 24 -47 , demonstrated that the exact C terminus of the antigenic peptide was directly produced by purified standard proteasome, but that the N terminus always carried additional 3 (RU1 [31][32][33][34][35][36][37][38][39][40][41][42] ) and 5 (RU1 29 -42 ) aa (16) (data not shown). This suggested that other peptidases might be necessary to trim the N-terminal extensions to the final nonamer RU1 34 -42 .…”

“…Two major peaks of activity were detected in fractions 29 -33 and 38 -45. The activity of the first peak was able to digest the N terminus of RU1 [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] , resulting in the enrichment of a fragment corresponding to RU1 34 -47 , which displays the exact N terminus of the antigenic peptide (Fig. 3A, inset).…”

“…Fractions are separated by the vertical narrow line, and the fractions containing the relevant activity are marked by the thick horizontal line (fractions 10 -14). B, An aliquot of fraction 12 was incubated with the precursor peptide RU1 29 -47 , resulting in the production of a fragment lacking the 3 N-terminal aa (RU1 [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] ). In each panel, the filled box corresponds to the sequence VPYGSFKHVDTRLQ, as described in Fig.…”

“…We also searched for proteolytic activities in membranes enriched in ER. Although we observed a detectable proteolytic activity against one of the proteasomal products, RU1 [31][32][33][34][35][36][37][38][39][40][41][42] , it could apparently not rescue the loss of CTL recognition of tumor cells resulting from the inhibition of proteasome and TPP II/PSA activities. Our data suggest that the production of RU1 34 -42 /HLA-B51 is a cytosolic process, involving the proteasome to generate the exact C terminus of the antigenic peptide and TPP II and PSA to trim the N-terminal extensions produced by the proteasome.…”