Arimura Takuro, Inagaki Natsuko, Hayashi Takeharu, Shichi Daisuke, Sato Akinori, Hinohara Kunihiko, Vatta Matteo, Towbin Jeffrey A, Chikamori Taishiro, Yamashina Akira, Kimura Akinori
Department of Molecular Pathogenesis, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Bunkyo-Ku, Tokyo, Japan.
Cardiovasc Res. 2009 Jul 1;83(1):80-8. doi: 10.1093/cvr/cvp119. Epub 2009 Apr 17.
Z-band alternatively spliced PDZ-motif protein (ZASP)/Cypher is a Z-disc component of which several dilated cardiomyopathy (DCM)-associated mutations have been reported. Most of the mutations were found in exons 4 and 10 of ZASP/Cypher gene LDB3 and both exons were expressed preferentially in the heart. The aim of this study was to investigate the functional alteration of ZASP/Cypher caused by the DCM-associated mutations.
The yeast-two-hybrid method was used to identify the protein bound to a domain encoded by exon 4 of LDB3. Interaction of ZASP/Cypher with the binding protein was investigated in relation to the functional alterations caused by LDB3 mutations. Localization of the ZASP/Cypher-binding protein was examined at the cellular level in rat cardiomyocytes. Phosphoglucomutase 1 (PGM1), a metabolic enzyme involved in glycolysis and gluconeogenesis, was identified as a protein interacting with ZASP/Cypher. PGM1 bound to ZASP/Cypher at the domains encoded by exons 4 and 10. Two LDB3 mutations in exon 4 (Ser189Leu and Thr206Ile) and another mutation in exon 10 (Ile345Met) reduced the binding to PGM1. PGM1 showed diffuse localization in the cytoplasm of rat cardiomyocytes under standard culture conditions, and distribution at the Z-discs was observed under stressed culture conditions. Binding of endogenous PGM1 and ZASP/Cypher was found to be enhanced by stress in rat cardiomyocytes.
ZASP/Cypher anchors PGM1 to Z-disc under conditions of stress. The impaired binding of PGM1 to ZASP/Cypher might be involved in the pathogenesis of DCM.
Z带交替剪接的PDZ基序蛋白（ZASP）/Cypher是一种Z盘成分，已有数种与扩张型心肌病（DCM）相关的突变报道。大多数突变位于ZASP/Cypher基因LDB3的外显子4和10中，且这两个外显子在心脏中优先表达。本研究旨在探讨由DCM相关突变引起的ZASP/Cypher功能改变。
采用酵母双杂交法鉴定与LDB3外显子4编码结构域结合的蛋白。研究了ZASP/Cypher与结合蛋白的相互作用，并分析了LDB3突变引起的功能改变。在大鼠心肌细胞的细胞水平上检测了ZASP/Cypher结合蛋白的定位。磷酸葡萄糖变位酶1（PGM1）是一种参与糖酵解和糖异生的代谢酶，被鉴定为与ZASP/Cypher相互作用的蛋白。PGM1在由外显子4和10编码的结构域处与ZASP/Cypher结合。外显子4中的两个LDB3突变（Ser189Leu和Thr206Ile）以及外显子10中的另一个突变（Ile345Met）降低了与PGM1的结合。在标准培养条件下，PGM1在大鼠心肌细胞的细胞质中呈弥漫性定位，在应激培养条件下观察到其在Z盘处分布。在大鼠心肌细胞中，应激可增强内源性PGM1与ZASP/Cypher的结合。
在应激条件下，ZASP/Cypher将PGM1锚定到Z盘。PGM1与ZASP/Cypher结合受损可能参与了DCM的发病机制。
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