微囊藻毒素在体内和体外与蛋白磷酸酶1和2A的结合

In vivo and in vitro binding of microcystin to protein phosphatases 1 and 2A.

作者信息

Runnegar M, Berndt N, Kong S M, Lee E Y, Zhang L

机构信息

University of Southern California Center for Liver Diseases, University of Southern California School of Medicine, Los Angeles 90033, USA.

出版信息

Biochem Biophys Res Commun. 1995 Nov 2;216(1):162-9. doi: 10.1006/bbrc.1995.2605.

DOI:10.1006/bbrc.1995.2605

PMID:7488083

Abstract

The hepatotoxic microcystins (Mcyst) are potent inhibitors of the ser/thr protein phosphatases (PP1 and PP2A) with IC50's of 0.1-1.0 nM. Mcyst and other PP inhibitors like okadaic acid or calyculin A interact with the C-terminal region of PP1 and PP2A. Using [125I]-Mcyst and antibodies specific for PP1 and PP2A, we show by immunoprecipitation and autoradiography, that in hepatocytes Mcyst forms secondary covalent bonds with both PP1 and PP2A catalytic subunits. We demonstrate that the bond resulted from the reaction between the electrophilic alpha, beta unsaturated carbonyl of the methyldehydroalanine residue of Mcyst and the thiol of Cys 273 located in the C-terminal of PP1 (Cys 266 in PP2A), since site-directed mutagenesis of Cys 273 to Ala in PP1 alpha led to complete loss of ability for the formation of a covalent Mcyst-PP1 adduct.

摘要

肝毒性微囊藻毒素（Mcyst）是丝氨酸/苏氨酸蛋白磷酸酶（PP1和PP2A）的强效抑制剂，其半数抑制浓度（IC50）为0.1 - 1.0 nM。Mcyst以及其他蛋白磷酸酶抑制剂，如冈田酸或花萼海绵诱癌素A，与PP1和PP2A的C末端区域相互作用。利用[125I] - Mcyst以及针对PP1和PP2A的特异性抗体，我们通过免疫沉淀和放射自显影表明，在肝细胞中Mcyst与PP1和PP2A催化亚基均形成二级共价键。我们证明该键是由Mcyst的甲基脱氢丙氨酸残基的亲电α,β - 不饱和羰基与位于PP1 C末端的Cys 273（PP2A中为Cys 266）的巯基之间的反应形成的，因为将PP1α中的Cys 273定点突变为丙氨酸导致形成共价Mcyst - PP1加合物的能力完全丧失。