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

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.

蛋白酶体（proteasome）是细胞降解蛋白质、为主要组织相容性复合体（major histocompatibility complex, MHC）I类分子生成肽配体的核心蛋白酶。基于对可在蛋白酶体抑制剂4-羟基-5-碘-3-硝基苯乙酰-亮氨酰-亮氨酰-亮氨醛-乙烯基砜（NLVS）存在下增殖的细胞的特性研究，曾有假说提出蛋白酶体可被其他蛋白水解系统替代，尤其是一类具备三肽肽酶II（tripeptidyl peptidase II）活性的大型蛋白水解复合物。
本研究证实，经NLVS适应性驯化的细胞，在抗原肽生成、多泛素化蛋白积累、p53蛋白降解以及细胞活力等多个维度，仍对多种高特异性蛋白酶体抑制剂保持敏感性。此外，本研究还发现，在上述实验体系中（仅存在一处微小例外），NLVS驯化细胞对丙氨酰-丙氨酰-苯丙氨酰氯甲基酮（Ala-Ala-Phe-chloromethylketone，一种特异性三肽肽酶II活性抑制剂）的敏感性与未经过筛选的细胞大致相当。
基于上述研究结果，我们认为在适应性驯化细胞中，蛋白酶体仍发挥着不可或缺的蛋白水解功能，该功能无法被对丙氨酰-丙氨酰-苯丙氨酰氯甲基酮敏感的蛋白酶所替代。