Importance of the different proteolytic sites of the proteasome and the efficacy of inhibitors varies with the protein substrate
Importance of the different proteolytic sites of the proteasome and the efficacy of inhibitors varies with the protein substrate
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DOI：
10.1074/jbc.m509043200
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发表时间：
2006-03-31
影响因子：
4.8
通讯作者：
Goldberg, AL
中科院分区：
文献类型：
--
作者：
Kisselev, AF;Callard, A;Goldberg, AL
哺乳动物蛋白酶体中不同的蛋白水解点在蛋白质降解中的相对重要性尚未有系统的研究。然而，人们普遍认为，抑制凝乳酶样位点是蛋白酶体抑制剂在研究和癌症治疗中的主要靶点，反映了蛋白质分解的抑制程度。在这里，我们证明了选择性地灭活类糜蛋白酶位点仅使26个S蛋白酶体对模型蛋白质的降解减少了11-50%，并且仅轻微地减少了HeLa细胞中蛋白质的分解。Caspase样位点的失活使模型蛋白的分解减少了12-22%，胰酶样位点的分解减少了3-35%。这些不同位点的相对贡献取决于蛋白质底物，类胰酶位点的重要性取决于底物中碱性残基的含量。需要同时抑制类糜蛋白酶和类半胱氨酸天冬氨酸酶或类胰蛋白酶，才能将降解率降低50%。因此，1)所有三种类型的活性位点都对蛋白质的分解有显著的贡献，2)它们的相对重要性因底物的不同而有很大的不同，3)凝乳酶样活性的测定高估了蛋白质降解的实际减少量，以及4)需要抑制多个活性位点才能显著减少蛋白质的降解。
The relative importance of the different proteolytic sites in mammalian proteasomes in protein degradation has not been studied systematically. Nevertheless, it is widely assumed that inhibition of the chymotrypsin-like site, the primary target of the proteasome inhibitors used in research and cancer therapy, reflects the degree of inhibition of protein breakdown. Here we demonstrate that selective inactivation of the chymotrypsin-like site reduced degradation of model proteins by pure 26 S proteasomes by only 11 - 50% and decreased only slightly the breakdown of proteins in HeLa cells. Inactivation of the caspase-like site decreased breakdown of model proteins by 12 - 22% and of the trypsin-like site by 3 - 35%. The relative contributions of these different sites depended on the protein substrate, and the importance of the trypsin-like sites depended on the substrate's content of basic residues. Simultaneous inhibition of the chymotrypsin-like and the caspase-or trypsin-like sites was needed to reduce degradation by > 50%. Thus, 1) all three types of active sites contribute significantly to protein breakdown, 2) their relative importance varies widely with the substrate, 3) assaying the chymotrypsin-like activity overestimates the actual reduction in protein degradation, and 4) inhibition of multiple sites is required to markedly decrease proteolysis.