BIOCHEMISTRY OF CONTRACTILE PROTEINS

描述（由申请人提供）：肌球蛋白II磷酸化是平滑肌和非肌肉细胞的主要调节机制。在平滑肌（动脉、静脉、胃肠道、子宫等）中，它控制收缩-松弛循环，并与血管生成有关。在非肌肉细胞中，它调节基本的细胞功能，包括：运动、细胞骨架结构的动力学、细胞分裂（细胞质分裂）和分泌。肌球蛋白II磷酸化的改变涉及心血管系统的几种疾病，如高血压、脑血管和冠状血管痉挛，以及其他中空器官的疾病，如支气管哮喘、早产和勃起功能障碍。肌球蛋白II磷酸化在癌细胞运动和转移中也很重要。因此，了解调节肌球蛋白II磷酸化的机制对于治疗这些疾病至关重要，并且对我们的社会具有相当大的健康益处。控制肌球蛋白II磷酸化的两个关键酶：肌球蛋白激酶（通常是肌球蛋白轻链激酶）和肌球蛋白磷酸酶（MP）。在恒定的[Ca2+]i下，调节肌球蛋白II磷酸化的主要因素是MP的调节。激活（与环核苷酸水平增加有关）和抑制（与上面列出的许多疾病有关）都有记录。RhoA/ rho激酶对在抑制中起重要作用。本提案的目的是建立以肌球蛋白磷酸酶靶亚基（MYPT1）为中心的MP调控的分子基础。Specific Aim 1的目的是描述MYPT1的几个重要相互作用，重点是底物的结合，以及调控的分子机制。生化分析将使用分离的MP全酶和纯化的MYPT1和片段。A7r5细胞系（来源于大鼠主动脉）将在接下来的两个目标中作为模型系统来测试几个假设。在sa2中，环核苷酸的作用，首先是cGMP，随后是cAMP，将被研究。目的是建立活化MP的分子基础。单个同工异构体的作用将通过RNA干扰（siRNA）来探测。最终SA表征了A7r5细胞中的MYPT1亚型。同种异构体的亚细胞定位将被确定，单个同种异构体的推测作用将被siRNA评估。这三个目标运用了生物化学、分子生物学和细胞生物学的综合技术。如果成功，他们将在分子水平上建立对肌肉和非肌肉细胞功能必不可少的过程的理解，并将促进药物干预。

DESCRIPTION (provided by applicant): Myosin II phosphorylation is a major regulatory mechanism in smooth muscle and non-muscle cells. In smooth muscle (arteries, veins, gastrointestinal tract, uterus, etc.) it controls the contraction-relaxation cycle and is implicated in angiogenesis. In non-muscle cells it regulates essential cell functions including: motility, dynamics of cytoskeletal structure, cell division (cytokinesis) and secretion. An alteration of myosin II phosphorylation is involved in several disorders of the cardiovascular system, e.g. hypertension, cerebral and coronary vasospasm, and of other hollow organs, e.g. bronchial asthma, preterm labor and erectile dysfunction. Myosin II phosphorylation also is important in cancer cell motility and metastasis. Thus, understanding the mechanisms regulating myosin II phosphorylation is essential in treatment of these disorders and is of considerable health benefit to our society. Two key enzymes control myosin II phosphorylation: a myosin kinase (usually myosin light chain kinase) and a myosin phosphatase (MP). At constant [Ca2+]i the major factor modulating myosin II phosphorylation is regulation of MP. Both activation (associated with increased cyclic nucleotide levels) and inhibition (implicated in many of the disorders listed above) are documented. The RhoA/Rho-kinase pair is important in inhibition. Objectives of this proposal are to establish the molecular basis for regulation of MP as centered on the myosin phosphatase target subunit (MYPT1). The intent of Specific Aim 1 is to characterize several important interactions of MYPT1, with emphasis on binding of substrates, and the molecular mechanism(s) of regulation. Biochemical analyses will use isolated MP holoenzyme and purified MYPT1 and fragments. The A7r5 cell line (derived from rat aorta) will be used in the next 2 Aims as a model system to test several hypotheses. In SA 2 the effects of cyclic nucleotides, initially cGMP and subsequently cAMP, will be investigated. The objective is to establish the molecular basis for activation of MP. The role of individual isoforms will be probed by RNA interference (siRNA). The final SA characterizes MYPT1 isoforms in A7r5 cells. Subcellular localizations of isoforms will be determined and putative roles of individual isoforms assessed by siRNA. These 3 aims use combined techniques of biochemistry, molecular biology and cell biology. If successful, they will establish an understanding at a molecular level of a process essential for function in muscle and non-muscle cells, and will facilitate pharmacological intervention.