Specific inhibition of myosin-Va and - Vb

特异性抑制肌球蛋白-Va 和-Vb

• 批准号: 6822465
• 负责人: JOHN A MERCER
• 金额: $ 28.93万
• 依托单位: MC LAUGHLIN RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6822465
• 关键词: HeLa cells; MDCK cell; PC12 cells; adenosine triphosphate; binding sites; cell biology; cell motility; chemical genetics; confocal scanning microscopy; fluorescence microscopy; genetically modified animals; green fluorescent proteins; immunoelectron microscopy; inhibitor /antagonist; laboratory mouse; membrane transport proteins; myosins; polymerase chain reaction; protein structure function; site directed mutagenesis; tissue /cell culture

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.

描述（由申请人提供）： 肌球蛋白对于细胞内运输至关重要，编码肌球蛋白的基因突变已被证明会导致多种遗传性疾病。由于多种原因，非常规肌球蛋白的特定细胞功能的表征落后于其鉴定。首先，相关肌球蛋白家族内部存在大量冗余。一些研究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任何依赖于运动域先前定位的功能，并且可能会因高水平表达而产生非特异性效应。其次，采用了突变体的研究。尽管基因消融方法非常强大，但无效突变体通常只能阐明特定肌球蛋白在发育过程中所必需的初始过程，从而排除了后续生理功能的识别。这项研究的最终目标是了解肌球蛋白-Va 和-Vb 在多种细胞功能中的作用。为此，我们开发了一种化学遗传策略，通过定点诱变扩大肌球蛋白的 ATP 结合位点，实现选择性、特异性和时间控制。这使得肌球蛋白对特定 ADP 类似物的抑制敏感，同时仍允许其水解细胞 ATP。然后，类似物的急性应用会导致与肌动蛋白紧密结合，从而阻止致敏突变肌球蛋白的运动。本申请中提出的实验将测试肌球蛋白-Va 和肌球蛋白-Vb 在基于微管的运动的启动、维持、拮抗或终止中发挥作用的假设，使用 HeLa、PC 12、MDCK 和 B16 细胞系中的特异性抑制作为以及来自转基因和敲入突变小鼠的原代细胞。