BIOCHEMISTRY OF CONTRACTILE PROTEINS

描述（由申请方提供）：肌球蛋白II磷酸化是平滑肌和非肌肉细胞中的主要调节机制。平滑肌（动脉、静脉、胃肠道、子宫等）它控制收缩-舒张周期并与血管生成有关。在非肌肉细胞中，它调节基本的细胞功能，包括：运动性、细胞骨架结构的动力学、细胞分裂（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，例如高血压、脑和冠状动脉血管痉挛，以及其他中空器官的疾病，例如支气管哮喘、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是重要的。因此，了解肌球蛋白II磷酸化的调节机制是治疗这些疾病的关键，对我们的社会有相当大的健康益处。两个关键酶控制肌球蛋白II磷酸化：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[Ca 2 +]i下，调节肌球蛋白II磷酸化的主要因素是MP的调节。激活（与增加的环核苷酸水平相关）和抑制（与上文列出的许多疾病有关）都有记录。RhoA/Rho-激酶对在抑制中是重要的。本提案的目的是建立以肌球蛋白磷酸酶靶亚基（MYPT 1）为中心的MP调节的分子基础。具体目标1的目的是表征MYPT 1的几种重要相互作用，重点是底物的结合和调控的分子机制。生化分析将使用分离的MP全酶和纯化的MYPT 1和片段。A7 r5细胞系（来自大鼠主动脉）将在接下来的2个目标中用作模型系统，以检验几个假设。在SA 2中，将研究环核苷酸（最初为cGMP，随后为cAMP）的影响。目的是建立激活MP的分子基础。将通过RNA干扰（siRNA）探测单个同种型的作用。最终SA表征A7 r5细胞中的MYPT 1同种型。将确定亚型的亚细胞定位，并通过siRNA评估单个亚型的推定作用。这三个目标使用生物化学，分子生物学和细胞生物学的组合技术。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至关重要的过程的理解，并将促进药理学干预。

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.