EFFECT OF CARDIOMYOPATHY MUTATIONS ON MYOSIN AND ACTIN

描述(申请人提供)：β-心肌肌球蛋白重链和α-心脏肌动蛋白的点突变都会导致家族性肥厚型心肌病(FHC)或扩张型心肌病(DCM)。为了找出这两种疾病的主要病因的确切机制，我们建议对突变的心肌肌球蛋白和肌动蛋白进行广泛的动力学、力学和结构分析。目的1通过比较转基因小鼠过表达获得的α-或β-小鼠心肌肌球蛋白重链(MHC)亚型中的R403Q突变，来检验异型骨架对功能的影响。突变的MHC将在N端被His标记，以便于金属螯合亲和层析分离。突变的肌球蛋白将通过稳态和瞬时动力学进行酶学表征，并通过速度和平均力的测量进行力学表征。野生型和突变型肌球蛋白异构体之间的结构差异将通过计算机对通过电子冷冻显微镜获得的肌球蛋白复合体的3D重建的晶体结构进行匹配来研究。类似的分析将扩展到导致FHC(G741 R，R453C)和DCM(S532和F764)的点突变。为了更好地了解人类肌球蛋白点突变的功能后果，AIM 2将分析从表达人β-心肌肌球蛋白(R403Q)基因的转基因兔中分离出的肌球蛋白。同时，还将探索几种在体外表达人β-心肌肌球蛋白的新策略，包括使用果蝇S2表达系统，以及添加伴侣以增加横纹肌肌球蛋白亚型的产量。目标3将寻求表征导致FHC或DCM的肌动蛋白点突变的影响。在杆状病毒/昆虫细胞系统中表达的α-心脏肌动蛋白使我们能够研究正确骨架中突变的影响，而不是在目前使用的酵母肌动蛋白系统中。肌动蛋白内在丝状结构的改变及其与肌球蛋白的相互作用，将通过许多与肌球蛋白突变相同的方法进行评估。该提案的总体目标是阐明FHC和DCM中涉及的突变如何影响肌球蛋白和肌动蛋白的机械性能，并确定原始突变的影响与最终疾病表型之间是否存在关联。

DESCRIPTION (provided by applicant): Point mutations in both the beta-cardiac myosin heavy chain and in alpha-cardiac actin lead to either familial hypertrophic cardiomyopathy (FHC) or dilated cardiomyopathy (DCM). To arrive at a definitive mechanism for the primary cause of these two diseases, we propose to undertake an extensive kinetic, mechanical, and structural analysis of mutated cardiac myosins and actins. Aim 1 will examine the effect of isoform backbone on function by comparing the R403Q mutation in an alpha- or a beta-murine cardiac myosin heavy chain (MHC) isoform obtained by over-expression in transgenic mice. Mutated MHCs will be HIS-tagged at the N terminus to facilitate isolation by metal chelate affinity chromatography. The mutant myosins will be characterized enzymatically by steady-state and transient kinetics, and mechanically by measurements of velocity and average force. Structural differences between wildtype and mutant myosin isoforms will be investigated by computer-based fitting of crystal structures into 3D-reconstructions of actomyosin complexes obtained by electron cryomicroscopy. Similar analyses will be extended to point mutations leading to FHC (G741 R, R453C) and DCM (S532 and F764). For a better understanding of the functional consequences of a point mutation in human myosin, Aim 2 will analyze myosin isolated from transgenic rabbits that express a human beta-cardiac myosin (R403Q) gene. In parallel, several new strategies for expression of human beta-cardiac myosin in vitro will be explored, including use of the Drosophila S2 expression system, and the addition of chaperones to increase the yield of striated muscle myosin isoforms. Aim 3 will seek to characterize the effect of point mutations in actin that lead to FHC or DCM. alpha-cardiac actin expressed in the baculovirus/insect cell system allows us to investigate the effect of mutations in the correct backbone, rather than in the currently used yeast actin system. Alterations to actin's intrinsic filamentous structure, and to its interactions with myosin, will be assessed by many of the same approaches as described for the myosin mutations.The overall goal of the proposal is to elucidate how mutations implicated in FHC and in DCM affect the mechanical performance of myosin and actin, and to determine if any correlation can be made between the effect of the primary mutation and the ultimate disease phenotype.