Nonhydrolyzable diubiquitin analogues are inhibitors of ubiquitin conjugation and deconjugation.
Nonhydrolyzable diubiquitin analogues are inhibitors of ubiquitin conjugation and deconjugation.
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DOI：
10.1021/bi0007019
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发表时间：
2000-07
期刊：
影响因子：
2.9
通讯作者：
Luming Yin;Bryan A. Krantz;N. Russell;Seema S. Deshpande;Keith D. Wilkinson
中科院分区：
文献类型：
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作者：
Luming Yin;Bryan A. Krantz;N. Russell;Seema S. Deshpande;Keith D. Wilkinson
一系列不可水解的泛素二聚体类似物已被合成和评价为泛素依赖性过程的抑制剂。通过将含有末端半胱氨酸（G76 C）的泛素与在位置11（（76-11）Ub（2））、29（（76-29）Ub（2））、48（（76-48）Ub（2））或63（（76-63）Ub（2））处含有半胱氨酸的泛素交联来合成二聚体类似物。半胱氨酸G76 C（（76-76）Ub（2））的头对头二聚体用作对照。这些类似物是在天然聚泛素链中观察到的不同链键的模拟物。所有类似物对UCH-L3的催化结构域和UBP假基因表现出弱抑制。在没有泛素的情况下，异肽酶T仅被通过残基29连接的二聚体抑制。在0.5 μ M泛素存在下，异肽酶T被几种二聚体类似物抑制，其中（76-29）Ub（2）二聚体显示K（i）为1.8 nM。然而，USP 14，酵母Ubp 6的人类同源物，在测试的浓度下没有被抑制。泛素二聚体的一些类似物也可作为网织红细胞组分II催化的泛素缀合和去缀合的选择性抑制剂。（76-76）Ub（2）和（76-11）Ub（2）不抑制泛素的结合，而（76-29）Ub（2）、（76-48）Ub（2）和（76-63）Ub（2）是有效的结合抑制剂。这种特异性与细胞形成K29-、K48-和K63-连接的多聚泛素链的已知能力一致。虽然（76-11）Ub（2）、（76-29）Ub（2）和（76-63）Ub（2）抑制了泛素从总缀合物库中的释放，但（76-48）Ub（2）和（76-76）Ub（2）未显示出显著的抑制作用。异肽酶T显示出特异性地分解在网织红细胞裂解物系统中形成的两种缀合物（假定为具有26和17 kDa质量的二和三泛素）。所有二聚体类似物均不同程度地抑制了该活性。26和17 kDa缀合物的解缀合的抑制剂选择性与对异肽酶T观察到的相似。观察结果表明，这两个共轭蛋白质的网织红细胞裂解物的特异性底物的异肽酶T的裂解物。
A series of nonhydrolyzable ubiquitin dimer analogues has been synthesized and evaluated as inhibitors of ubiquitin-dependent processes. Dimer analogues were synthesized by cross-linking ubiquitin containing a terminal cysteine (G76C) to ubiquitin containing cysteine at position 11 ((76-11)Ub(2)), 29 ((76-29)Ub(2)), 48 ((76-48)Ub(2)), or 63 ((76-63)Ub(2)). A head-to-head dimer of cysteine G76C ((76-76)Ub(2)) served as a control. These analogues are mimics of the different chain linkages observed in natural polyubiquitin chains. All analogues showed weak inhibition toward the catalytic domain of UCH-L3 and a UBP pseudogene. In the absence of ubiquitin, isopeptidase T was inhibited only by the dimer linked through residue 29. In the presence of 0.5 microM ubiquitin, isopeptidase T was inhibited by several of the dimer analogues, with the (76-29)Ub(2) dimer exhibiting a K(i) of 1.8 nM. However, USP14, the human homologue of yeast Ubp6, was not inhibited at the concentrations tested. Some analogues of ubiquitin dimer also acted as selective inhibitors of conjugation and deconjugation of ubiquitin catalyzed by reticulocyte fraction II. (76-76)Ub(2) and (76-11)Ub(2) did not inhibit the conjugation of ubiquitin, while (76-29)Ub(2), (76-48)Ub(2), and (76-63)Ub(2) were potent inhibitors of conjugation. This specificity is consistent with the known ability of cells to form K29-, K48-, and K63-linked polyubiquitin chains. While (76-11)Ub(2), (76-29)Ub(2), and (76-63)Ub(2) inhibited release of ubiquitin from a pool of total conjugates, (76-48)Ub(2) and (76-76)Ub(2) showed no significant inhibition. Isopeptidase T was shown to specifically disassemble two conjugates (assumed to be di- and triubiquitin with masses of 26 and 17 kDa) formed in the reticulocyte lysate system. This activity was inhibited differentially by all dimer analogues. The inhibitor selectivity for deconjugation of the 26 and 17 kDa conjugates was similar to that observed for isopeptidase T. The observations suggest that these two conjugated proteins of the reticulocyte lysate are specific substrates for isopeptidase T in lysates.