Expression Studies of Other Unconventional Myosins

其他非常规肌球蛋白的表达研究

基本信息

批准号：
10929093
负责人：
James Sellers
金额：
$ 77.93万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

We have begun to analyze the structure and stepping pattern of myosin-6, a processive myosin which moves in the opposite direction on actin compared to other myosins. EM studies show that the angle between the two heads is more variable than in most myosins and that when bound to actin, the motors can be spaced at 13 actins (preferred) or 11 or 15 actin monomers apart. Optical trapping and EM studies demonstrate that the molecule can sometimes take an "inchworm" like step where the two heads occupy closely spaced binding sites. Myosin 7a is an actin based motor protein essential for vision and hearing. Mutations of myosin 7a cause Usher syndrome type 1, the most common and severe form of deaf blindness in humans. The molecular mechanisms that governs its mechanochemistry remain poorly understood, primarily due to the difficulty of purifying stable, intact protein. Here, we recombinantly produce the complete human myosin 7a holoenzyme in insect cells and characterize its biochemical and motile properties. Unlike the Drosophila ortholog which primarily associates with calmodulin, we found that human myosin 7a utilizes a unique combination of light chains including regulatory light chain, calmodulin, and calmodulin like protein 4 (CALML4). Our results further reveal that CALML4 does not function as a Ca2+ sensor but plays a crucial role in maintaining the lever arms structural functional integrity. Using our recombinant protein system, we purified two myosin 7a splicing isoforms which have been shown to be differentially expressed along the cochlear tonotopic axis. We show that they possess distinct mechano enzymatic properties despite differing by only 11 amino acids at their N termini. Using single molecule in vitro motility assays, we demonstrate that human myosin 7a exists as an autoinhibited monomer and can move processively along actin when artificially dimerized or bound to cargo adaptor proteins such as MyRIP. These results suggest that myosin 7a can serve multiple roles in the sensory systems such as a transporter or an anchor/force sensor. Furthermore, our research highlights that human myosin 7a has evolved unique regulatory elements that enable precise tuning of its mechanical properties suitable for mammalian auditory functions. In collaboration with John Hammer we have begun to study myosin 19 a mitochondrial associated myosin to complement ongoing cell biological studies.

我们已经开始分析肌球蛋白6的结构和步进模式，肌球蛋白6是一种肌球蛋白，与其他肌球蛋白相比，肌动蛋白的方向相反。 EM研究表明，两个头部之间的角度比大多数肌球蛋白更可变，并且在与肌动蛋白界定时，电动机可以在13个肌动蛋白（首选）或11或15个肌动蛋白单体分开。光学诱捕和EM研究表明，该分子有时可以像两个头一样占据紧密间隔的结合位点的“ Inch虫”。肌球蛋白7a是一种基于肌动蛋白的运动蛋白，对视觉和听力必不可少。肌球蛋白7a的突变导致usher综合征1型，这是人类聋失明的最常见和严重形式。控制其机械化学的分子机制仍然很少理解，这主要是由于难以纯化稳定的完整蛋白质。在这里，我们重组在昆虫细胞中重组产生完整的人肌球蛋白7a全酶，并表征其生化和运动特性。与主要与钙调蛋白相关的果蝇直系同源物不同，我们发现人肌球蛋白7a利用了独特的轻链组合，包括调节轻链，钙调蛋白和钙蛋白（如蛋白4（Calml4））（Calml4）。我们的结果进一步表明，Calml4不充当CA2+传感器，而是在维持杠杆臂结构功能完整性方面起着至关重要的作用。使用我们的重组蛋白系统，我们纯化了两个肌球蛋白7a剪接同工型，这些剪接同工型已被证明沿着人工耳蜗轴差异表达。我们表明，尽管在其N末端只有11个氨基酸，但它们具有独特的机械酶特性。使用单分子在体外运动测定中，我们证明了人肌球蛋白7a作为自身抑制的单体存在，并且当人为地二聚体或与货物适配蛋白（如myrip）（如myrip）时，可以沿肌动蛋白进行过程移动。这些结果表明，肌球蛋白7a可以在转运蛋白或锚/力传感器等感觉系统中发挥多种作用。此外，我们的研究强调，人肌球蛋白7a已经发展了独特的调节元素，可以精确调整其机械性能适用于适合哺乳动物的听觉功能。通过与约翰·哈默（John Hammer）合作，我们已经开始研究肌球蛋白19 A肌球蛋白相关的肌球蛋白，以补充正在进行的细胞生物学研究。