Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region
Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region
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DOI：
10.1073/pnas.96.16.9112
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发表时间：
1999-08-03
影响因子：
11.1
通讯作者：
Horinouchi, S
中科院分区：
文献类型：
--
作者：
Kudo, N;Matsumori, N;Horinouchi, S
核输出抑制剂来普霉素B（LMB）的细胞靶点已被鉴定为CRMI（exportin 1），CRMI是蛋白质核输出信号的进化上保守的受体。然而，LMB抑制CRM 1的机制尚不清楚，在一株对LMB表现出极高抗性的裂殖酵母突变株中，CRM 1在Cys-529处被Ser单氨基酸取代，命名为crm 1-K1，该突变基因赋予野生型S. 1H-1 NMR分析表明LMB通过Michael型加成结合N-乙酰基-L-半胱氨酸甲酯，这与LMB通过其α，β-不饱和δ-内酯共价结合Cys-529的巯基的想法一致。当HeLa细胞与生物素化的LMB一起培养时，共价结合的唯一细胞蛋白是CRM 1，N-乙基马来酰亚胺（NEM），一种烷化剂，对CRM 1介导的核输出的抑制可能是由NEM中的亲电结构与Cys-529的巯基共价结合引起的，因为crm 1-K1突变体显示Rev核输出信号输出的正常速率-这些结果表明，单个半胱氨酸残基决定了LMB的敏感性，并被LMB选择性烷基化，导致CRM 1失活。
The cellular target of leptomycin B (LMB), a nuclear export inhibitor, has been identified as CRMI (exportin 1), an evolutionarily conserved receptor for the nuclear export signal of proteins. However, the mechanism by which LMB inhibits CRM1 still remains unclear, CRM1 in a Schizo-saccharomyces pombe mutant showing extremely high resistance to LMB had a single amino acid replacement at Cys-529 with Ser. The mutant gene, named crm1-K1, conferred LMB resistance on wild-type S. pombe, and Crm1-K1 no longer bound biotinylated LMB,H-1 NMR analysis showed that LMB bound N-acetyl-L-cysteine methyl ester through a Michael-type addition, consistent with the idea that LMB binds covalently via its alpha,beta-unsaturated delta-lactone to the sulfhydryl group of Cys-529, When HeLa cells were cultured with biotinylated LMB, the only cellular protein bound covalently was CRM1, Inhibition by N-ethylmaleimide (NEM), an alkylating agent, of CRM1-mediated nuclear export probably was caused by covalent binding of the electrophilic structure in NEM to the sulthydryl group of Cys-529, because the crm1-K1 mutant showed the normal rate for the export of Rev nuclear export signal-bearing proteins in the presence of not only LMB but also NEM, These results show that the single cysteine residue determines LMB sensitivity and is selectively alkylated by LMB, leading to CRM1 inactivation.