K63-specific deubiquitination by two JAMM/MPN plus complexes: BRISC-associated Brcc36 and proteasomal Poh1
K63-specific deubiquitination by two JAMM/MPN plus complexes: BRISC-associated Brcc36 and proteasomal Poh1
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DOI：
10.1038/emboj.2009.27
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发表时间：
2009-03-18
期刊：
影响因子：
11.4
通讯作者：
Cohen, Robert E.
中科院分区：
文献类型：
--
作者：
Cooper, Eric M.;Cutcliffe, Colleen;Cohen, Robert E.
HeLa 细胞提取物中存在一种不寻常的去泛素化 (DUB) 活性，该活性对于裂解 K63 连接的多聚泛素链但不是 K48 连接的多聚泛素链具有高度特异性。该活性对 N-乙基-马来酰亚胺和泛素醛都不敏感，表明它缺乏活性位点半胱氨酸残基，凝胶过滤实验表明它存在于高分子量（类似于 600 kDa）复合物中。使用生化方法，我们发现 K63 特异性 DUB 活性通过七个色谱步骤与三个多亚基复合物共同分级：26S 蛋白酶体的 19S (PA700) 部分、COP9 信号体 (CSN) 和包含 JAMM/MPN + 含有结构域的蛋白 Brcc36 的新型复合物。当我们分析各个复合物时，我们发现该活性是 PA700 和 Brcc36 异肽酶复合物 (BRISC) 固有的，但 CSN 相关活性完全是由于与 Brcc36 的相互作用。这些复合物均不会裂解 K6、K11、K29、K48 或 α 连接的多聚泛素，但它们会裂解混合连接链内的 K63 连接。我们的结果表明，K63 连接的多聚泛素的特异性是 JAMM/MPN + DUB 家族的共同特性。
An unusual deubiquitinating (DUB) activity exists in HeLa cell extracts that is highly specific for cleaving K63-linked but not K48-linked polyubiquitin chains. The activity is insensitive to both N-ethyl-maleimide and ubiquitin aldehyde, indicating that it lacks an active site cysteine residue, and gel filtration experiments show that it resides in a high molecular weight (similar to 600 kDa) complex. Using a biochemical approach, we found that the K63-specific DUB activity co-fractionated through seven chromatographic steps with three multisubunit complexes: the 19S (PA700) portion of the 26S proteasome, the COP9 signalosome (CSN) and a novel complex that includes the JAMM/MPN + domain-containing protein Brcc36. When we analysed the individual complexes, we found that the activity was intrinsic to PA700 and the Brcc36 isopeptidase complex (BRISC), but that the CSN-associated activity was due entirely to an interaction with Brcc36. None of the complexes cleave K6, K11, K29, K48 or alpha-linked polyubiquitin, but they do cleave K63 linkages within mixed-linkage chains. Our results suggest that specificity for K63-linked polyubiquitin is a common property of the JAMM/MPN + family of DUBs.