Substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes
Substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes
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DOI：
10.1016/s1074-5521(98)90169-7
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发表时间：
1998-06-01
影响因子：
--
通讯作者：
Ploegh, HL
中科院分区：
文献类型：
--
作者：
Bogyo, M;Shin, S;Ploegh, HL
背景：蛋白酶体是导致大多数胞质蛋白分解的多催化蛋白酶复合物。该复合物具有几种不同的蛋白水解活性，这些活性是由每个氨基酸残基的偏好（P1）氨基酸残基所定义的。尽管酵母中的突变研究已开始定义单个催化活性P亚基的底物特异性，但对由蛋白酶体控制底物水解的原理知之甚少。分子：一系列三肽和四肽乙烯基乙烯基磺基磺酮用于研究底物的结合和特异性和特异性结合和特异性和特异性结合和特异性蛋白酶体。去除有效的三肽乙烯基磺酸盐蛋白酶体抑制剂4-羟基-3-碘-2-硝基苯基 - 核核糖素糖苷 - 杜氨基 - 葡萄蛋白乙烯基磺基磺基磺基磺基磺基磺基的芳族氨基末端帽，导致结合和抑制作用的完全丧失。将第四个氨基酸（P4）添加到三亮氨酸核心序列完全恢复的抑制效力。 （125）L标记的肽乙烯基磺酮还用于检查抑制剂结合，并确定亚基修饰与抑制肽酶活性的相关性，在P4位置改变氨基酸，导致β-亚基修饰的概况极大地不同。 ：P4位置位于水解部位的远端，对于定义蛋白酶体的底物处理很重要。我们观察到亚基修饰与抑制不同蛋白水解活性之间的直接相关性，从而使活动分配给单个β亚基。四肽（而不是三肽的能力）充当蛋白酶体底物的能力表明，蛋白酶体水解的长度可能最少。这些研究表明，可以通过调节P4和羧基末端乙烯基磺基磺基部分来产生主要针对蛋白酶体单个β亚基的抑制剂。
Background: 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 carboxy terminal (P1) amino acid residue. Although mutational studies in yeast have begun to define substrate specificities of individual catalytically active p subunits, little is known about the principles that govern substrate hydrolysis by the proteasome.Results: 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. (125)l-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,Conclusions: 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.