Roles of Cypher in Cardiac Muscle

描述(申请人提供)：Cypher，一种新型的横纹肌特异体 细胞骨架蛋白有两种剪接异构体，Cypher 1和Cypher 2。 两种Cypher亚型的纯合子在围产期死亡，并伴有严重的缺陷 横纹肌。明显的心力衰竭表现为血液充血。 肝脏和心腔，伴有脑室扩张。传输 电子显微镜研究表明，突变体的横纹肌 老鼠的Z线严重紊乱。这项提案将测试Cypher是一种 关键连接蛋白，起到调节结构完整性和 Z线上与α-肌动蛋白2相互作用的信号通路， α-心脏肌动蛋白、蛋白激酶C和其他潜在的Z线蛋白。密码2可能是 与L部分冗余，和/或可能成为L的拮抗者 功能。具体目标是： 1)利用噬菌体展示系统鉴定Cypher的蛋白伴侣。 全长Cypher1和Cyph2将作为诱饵筛选噬菌体 展示成人心脏c DNA文库。 2)确定两种Cypher剪接异构体的生物学功能，Cypher L 和Cypher 2。我们将通过 创建小鼠线条，其中每个亚型被选择性地消融。 3)测试Cypher无法与其结合的生理后果 α-肌动蛋白-2通过产生小鼠品系，在其中必需的氨基酸 与α-肌动蛋白-2结合的Cypher Leu78突变为Lys。 4)为了进一步研究Cypher L与PKC的相互作用机制，我们 将结合诱变技术进行广泛的体外结合研究 确定Cypher L LIM结构域中的关键残基 PKC绑定需要。 对于AIMS 2和AIMS 3中的每个小鼠模型，详细的生化和细胞 由此产生的心肌表型分析，并综合 将进行心脏功能的生理学评估。

DESCRIPTION (provided by applicant): Cypher, a novel striated muscle specific cytoskeletal protein, has two splice isoforms, Cypher 1 and Cypher 2. Mice homozygous for both Cypher isoforms die perinatally with severe defects in striated muscle. Apparent heart failure is suggested by blood congestion in liver, and cardiac chambers, accompanied by ventricular dilation. Transmission Electronic Microscopy (TEM) studies indicated that striated muscle in mutant mice has severely disorganized Z-lines. This proposal will test the Cypher is a critical linker protein which serves to mediate structural integrity and signaling pathways at the Z-line via its interactions with alpha-actinin 2, alpha-cardiac actin, PKC, and other potential Z-line proteins. Cypher 2 may be partially redundant with Cypher l, and/or may act as an antagonist of Cypher l function. The Specific Aims are: 1) To identify protein partners of Cypher utilizing the phage display system. Full length Cypherl and Cypher2 will be utilized as baits to screen a phage display adult heart cDNA library. 2) To determine the biological function of two Cypher splice isoforms, Cypher l and Cypher 2. We will investigate the function of Cypher 1 and Cypher 2 by creating mouse lines in which each isoform is selectively ablated. 3) To test physiological consequences of the inability of Cypher to bind to alpha-actinin-2 by generating mouse lines in which the essential amino acid for Cypher binding to alpha-actinin-2, Leu78, is mutated to Lys. 4) To further investigate mechanisms of interaction of Cypher l with PKC, we will perform extensive in vitro binding studies in combination with mutagenesis to determine critical residues within the Cypher l LIM domains which are required for PKC binding. For each mouse model in Aims 2 and 3, detailed biochemical and cellular analyses of resulting cardiac muscle phenotypes, and comprehensive physiological assessment of cardiac function will be performed.