The active sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor processing
The active sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor processing
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DOI：
10.1074/jbc.272.40.25200
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发表时间：
1997-10-03
影响因子：
4.8
通讯作者：
Wolf, DH
中科院分区：
文献类型：
--
作者：
Heinemeyer, W;Fischer, M;Wolf, DH
在真核生物中，26s蛋白酶体是参与泛素介导的蛋白质降解的中心蛋白酶，具有重要的调节功能。该复合物的蛋白水解核心是20s蛋白酶体，它是一个圆柱形颗粒，两个外环分别由7个不同的α型亚基组成，两个内环由7个不同的β型亚基组成。在古细菌中，20s蛋白酶体祖先的蛋白水解活性位点位于14个统一的β亚基中。它们的n端苏氨酸残基通过前体加工释放，为肽键水解执行亲核攻击。通过对酿酒酵母20s蛋白酶体β型蛋白的定向突变分析，我们确定了三个携带活性位点的亚基，分别负责不同的肽水解活性：pre - 4亚基中两个潜在活性位点苏氨酸残基的突变排除了其对三种肽酶活性的催化作用。活性位点突变体中Pre4前体的不完全加工形式表明，非活性蛋白酶体β型亚基的成熟是由活性亚基发挥作用的，并发生在完全组装的颗粒中。这种反式作用的蛋白质水解活性也可能解释了活性位点突变的Pre2亚基的加工中间体不能进行自催化成熟的原因。
The 26 S proteasome is the central protease involved in ubiquitin-mediated protein degradation and fulfills vital regulatory functions in eukaryotes, The proteolytic core of the complex is the 20 S proteasome, a cylindrical particle with two outer rings each made of 7 different alpha-type subunits and two inner rings made of 7 different beta-type subunits, In the archaebacterial 20 S proteasome ancestor proteolytically active sites reside in the 14 uniform beta-subunits. Their N-terminal threonine residues, released by precursor processing, perform the nucleophilic attack for peptide bond hydrolysis. By directed mutational analysis of 20 S proteasomal beta-type proteins of Saccharomyces cerevisiae, we identified three active site-carrying subunits responsible for different peptidolytic activities as follows: Pre3 for post-glutamyl hydrolyzing, Pup1 for trypsin-like, and Pre2 for chymotrypsin-like activity, Double mutants harboring only trypsin-like or chymotrypsin-like activity were viable, Mutation of two potentially active site threonine residues in the Pre4 subunit excluded its catalytic involvement in any of the three peptidase activities, The generation of different, incompletely processed forms of the Pre4 precursor in active site mutants suggested that maturation of non-active proteasomal beta-type subunits is exerted by active subunits and occurs in the fully assembled particle, This trans-acting proteolytic activity might also account for processing intermediates of the active site mutated Pre2 subunit, which was unable to undergo autocatalytic maturation.