Communication - Preferential interaction of sentrin with a ubiquitin-conjugating enzyme, Ubc9
Communication - Preferential interaction of sentrin with a ubiquitin-conjugating enzyme, Ubc9
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DOI：
10.1074/jbc.272.45.28198
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发表时间：
1997-11-07
影响因子：
4.8
通讯作者：
Yeh, ETH
中科院分区：
文献类型：
--
作者：
Gong, LM;Kamitani, T;Yeh, ETH
前哨蛋白是一种泛素样分子，已被证明与Fas和肿瘤坏死因子受体1(TNFR1)、PML、RAD51、Rad52和RanGAP1的死亡结构域相互作用。我们以前已经报道过，Sent rin可以以类似于蛋白质泛素化的方式与其他蛋白质结合(Kamitani，T.，Nguyen，H.P.和Yeh，E.T.H.(1997)J.Biol)。化学。272、14001-14004)。此外，保守的C-末端Gly-Gly残基是发生致死作用所必需的。为了筛选在胎盘定位中起作用的酶，采用酵母双杂交系统，以Sent rin为诱饵，筛选了人胎盘c DNA文库。发现一个强正相互作用克隆含有编码泛素结合酶Ubc9的c DNA插入片段。Sent rin与Ubc9的相互作用需要Sent rin的泛素结构域和C-末端的Gly-Gly残基。这种相互作用似乎是特异的，因为Sentrin只能与UbcH5B弱相互作用，而不能与HHR6B、UbcH6或E2-EPF相互作用。体外翻译的Sent蛋白可被GST-UBC9融合蛋白沉淀，但不能被谷胱甘肽S转移酶沉淀。在体外结合试验中也可以鉴定出对β-巯基乙醇敏感的Ubc9-Sentrin偶联物。丝氨酸取代了Ubc9保守的半胱氨酸残基，取消了Ubc9-Sentrin结合物的形成。综上所述，Ubc9是一个很有可能成为信号转导途径中关键结合酶的候选基因。
Sentrin is a ubiquitin-like molecule that has been shown to interact with the death domains of Fas and tumor necrosis factor receptor 1 (TNFR1), PML, Rad51, Rad52, and RanGAP1. We have reported previously that sentrin can be conjugated to other proteins in a manner analogous to protein ubiquitination (Kamitani, T., Nguyen, H. P., and Yeh, E. T. H. (1997) J. Biol. Chem. 272, 14001-14004). Furthermore, the conserved C-terminal Gly-Gly residues are required for sentrinization to occur. To identify enzymes which play a role in sentrinization, the yeast two-hybrid system was used to screen a human placenta cDNA library using sentrin as bait. A strong positive interacting clone was found to contain a cDNA insert encoding the ubiquitin-conjugating enzyme, Ubc9. The interaction between sentrin and Ubc9 required the ubiquitin domain and the C-terminal Gly-Gly residues of sentrin. This interaction appears to be specific because sentrin could only interact weakly with UbcH5B, but could not interact with HHR6B, UbcH6 nor E2-EPF. In vitro translated sentrin could be precipitated by a GST-Ubc9 fusion protein, but not by glutathione S-transferase. A beta-mercaptoethanol-sensitive Ubc9-sentrin conjugate could also be identified in the in vitro binding assay. Substitution of the conserved cysteine residue of Ubc9 by serine abolished the formation of the Ubc9-sentrin conjugate. Taken together, Ubc9 is a strong candidate to be the key conjugating enzyme in the sentrinization pathway.