A high throughput screen to identify substrates for the ubiquitin ligase Rsp5
A high throughput screen to identify substrates for the ubiquitin ligase Rsp5
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DOI：
10.1074/jbc.m502197200
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发表时间：
2005-08-19
影响因子：
4.8
通讯作者：
Rotin, D
中科院分区：
文献类型：
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作者：
Kus, B;Gajadhar, A;Rotin, D
泛素蛋白连接酶（E3s）与各种人类疾病有关，是治疗干预的有吸引力的靶点。虽然大多数细胞蛋白被泛素化，但对于这些蛋白质的很大一部分，泛素化不能直接与特定的E3联系起来，而且大多数E3酶的底物是未知的。我们已经开发了一种荧光法来检测体外泛素化，它比传统的泛素化检测更定量，更有效，更敏感。通过利用基因组研究获得的丰富的纯化蛋白，我们筛选了数百种纯化的酵母蛋白进行泛素化，并确定了之前报道的酵母E3连接酶Rsp5的新底物。这些底物的相关性在体内得到了证实，其中一些底物与Rsp5基因相互作用，一些底物在体内被Rsp5泛素化。结合这种灵敏的测定和纯化底物的可用性，将能够鉴定任何纯化的E3酶的底物。
Ubiquitin-protein ligases (E3s) are implicated in various human disorders and are attractive targets for therapeutic intervention. Although most cellular proteins are ubiquitinated, ubiquitination cannot be linked directly to a specific E3 for a large fraction of these proteins, and the substrates of most E3 enzymes are unknown. We have developed a luminescent assay to detect ubiquitination in vitro, which is more quantitative, effective, and sensitive than conventional ubiquitination assays. By taking advantage of the abundance of purified proteins made available by genomic efforts, we screened hundreds of purified yeast proteins for ubiquitination, and we identified previously reported and novel substrates of the yeast E3 ligase Rsp5. The relevance of these substrates was confirmed in vivo by showing that a number of them interact genetically with Rsp5, and some were ubiquitinated by Rsp5 in vivo. The combination of this sensitive assay and the availability of purified substrates will enable the identification of substrates for any purified E3 enzyme.