Bogyo M, Shin S, McMaster J S, Ploegh H L
Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.
Chem Biol. 1998 Jun;5(6):307-20. doi: 10.1016/s1074-5521(98)90169-7.
The proteasome is a multicatalytic protease complex responsible for most cytosolic protein breakdown. The complex has several distinct proteolytic activities that are defined by the preference of each for the carboxyterminal (P1) amino acid residue. Although mutational studies in yeast have begun to define substrate specificities of individual catalytically active beta subunits, little is known about the principles that govern substrate hydrolysis by the proteasome.
A series of tripeptide and tetrapeptide vinyl sulfones were used to study substrate binding and specificity of the proteasome. Removal of the aromatic amino-terminal cap of the potent tripeptide vinyl sulfone proteasome inhibitor 4-hydroxy-3-iodo-2-nitrophenyl-leucinyl-leucinyl-leucine vinyl sulfone resulted in the complete loss of binding and inhibition. Addition of a fourth amino acid (P4) to the tri-leucine core sequence fully restored inhibitory potency. 125I-labeled peptide vinyl sulfones were also used to examine inhibitor binding and to determine the correlation of subunit modification with inhibition of peptidase activity. Changing the amino acid in the P4 position resulted in dramatically different profiles of beta-subunit modification.
The P4 position, distal to the site of hydrolysis, is important in defining substrate processing by the proteasome. We observed direct correlations between subunit modification and inhibition of distinct proteolytic activities, allowing the assignment of activities to individual beta subunits. The ability of tetrapeptides, but not tripeptide vinyl sulfones, to act as substrates for the proteasome suggests there could be a minimal length requirement for hydrolysis by the proteasome. These studies indicate that it is possible to generate inhibitors that are largely specific for individual beta subunits of the proteasome by modulation of the P4 and carboxy-terminal vinyl sulfone moieties.
蛋白酶体是一种多催化蛋白酶复合体，负责大多数胞质蛋白的降解。该复合体具有几种不同的蛋白水解活性，每种活性由其对羧基末端（P1）氨基酸残基的偏好来定义。尽管酵母中的突变研究已开始确定各个具有催化活性的β亚基的底物特异性，但关于蛋白酶体控制底物水解的原理知之甚少。
使用一系列三肽和四肽乙烯砜来研究蛋白酶体的底物结合和特异性。强效三肽乙烯砜蛋白酶体抑制剂4-羟基-3-碘-2-硝基苯基-亮氨酰-亮氨酰-亮氨酸乙烯砜的芳香族氨基末端帽被去除后，结合和抑制作用完全丧失。在三亮氨酸核心序列中添加第四个氨基酸（P4）可完全恢复抑制效力。125I标记的肽乙烯砜也用于检查抑制剂结合情况，并确定亚基修饰与肽酶活性抑制之间的相关性。改变P4位置的氨基酸会导致β亚基修饰的显著不同模式。
水解位点远端的P4位置对于蛋白酶体定义底物加工很重要。我们观察到亚基修饰与不同蛋白水解活性抑制之间存在直接相关性，从而能够将活性分配给各个β亚基。四肽而非三肽乙烯砜作为蛋白酶体底物的能力表明，蛋白酶体水解可能存在最小长度要求。这些研究表明，通过调节P4和羧基末端乙烯砜部分，有可能生成对蛋白酶体各个β亚基具有高度特异性的抑制剂。
