Specific inhibition of myosin-Va and - Vb

描述（由申请人提供）： 肌球蛋白是细胞内运输所必需的，编码肌球蛋白的基因突变已被证明会导致几种遗传性疾病。非常规肌球蛋白的特异性细胞功能的表征由于几个原因而落后于它们的鉴定。首先，在相关肌球蛋白家族中存在大量冗余。几个小组已经通过表达肌球蛋白的货物结合或尾部结构域确定了特定的功能;然而，这种方法无法检测到依赖于运动结构域先前定位的任何功能，并且可以从高水平表达产生非特异性效应。第二，研究了突变体。虽然基因消融的方法是非常强大的，无效突变体往往只照亮的初始过程中，一个给定的肌球蛋白是必不可少的发展，排除了后来的生理功能的鉴定。本研究的最终目的是了解肌球蛋白-Va和-Vb在多种细胞功能中的作用。为此，已经开发了一种化学遗传策略，其通过使用定点诱变扩大肌球蛋白的ATP结合位点来实现选择性、特异性和时间控制。这使肌球蛋白对特定ADP类似物的抑制敏感，同时仍允许其水解细胞ATP。急性应用的类似物，然后导致紧密结合肌动蛋白，阻止运动的致敏突变肌球蛋白。本申请中提出的实验将使用HeLa、PC 12、MDCK和B16细胞系以及来自转基因和敲入突变小鼠的原代细胞中的特异性抑制来检验肌球蛋白-Va和肌球蛋白-Vb在基于微管的运动性的起始、维持、拮抗或终止中起作用的假设。

DESCRIPTION (provided by applicant): Myosins are essential for intracellular transport, and mutations in the genes encoding them have been shown to cause several inherited diseases. The characterization of the specific cellular functions of unconventional myosins has lagged behind their identification for several reasons. First, there is a great deal of redundancy within families of related myosins. Several groups have identified specific functions by expressing the cargo-binding, or tail, domains of myosins; however, this approach fails to detect any functions dependent upon prior positioning by the motor domain and can yield nonspecific effects from high levels of expression. Second, the study of mutants has been employed. Although genetic ablation approaches are very powerful, null mutants often only illuminate the initial process for which a given myosin is essential during development, precluding the identification of later physiological functions. The ultimate goal of this research is to understand the roles of myosin-Va and -Vb in multiple cellular functions. To this end, a chemical-genetic strategy has been developed that achieves selectivity, specificity, and temporal control by enlarging the ATP-binding site of the myosin using site-directed mutagenesis. This sensitizes the myosin to inhibition by a specific ADP analog while still allowing it to hydrolyze cellular ATP. The acute application of the analog then causes tight binding to actin, arresting the movement of the sensitized mutant myosin. The experiments proposed in this application will test the hypothesis that myosin-Va and myosin-Vb function in initiation, maintenance, antagonism, or termination of microtubule-based motility, using specific inhibition in HeLa, PC 12, MDCK, and B16 cell lines as well as primary cells from transgenic and knock-in mutant mice.