Structural-Functional Basis of Actin Cytoskeleton Dynamics

项目摘要： 长期目标是了解控制肌动蛋白细胞骨架的结构和功能机制 健康和疾病的动态。在第四个周期，该赠款将填补以下领域的重要知识空白： 在这方面，我们将继续努力，在以往重点领域的基础上，同时根据该领域最近的事态发展，扩大到新的领域。 该实验室与挪威的一个小组合作，最近发现了肌动蛋白的专用N- Aim-1将重点研究肌动蛋白N-末端乙酰转移酶（Naa 80）的作用机制和功能 乙酰化（N-乙酰化）和同种型变化。Aim-1a将确定N-乙酰化的机理， 包括Naa 80-肌动蛋白相互作用的生物化学和细胞研究，以及一系列的晶体结构， 中间反应步骤，并使用不同的肌动蛋白同种型。Aim-1b将检验肌球蛋白 活性强烈地受肌动蛋白同种型变化和N-乙酰化的支配。这将解决一个持久的 缺乏领域;大多数肌球蛋白研究使用α-骨骼肌动蛋白，忽略了差异的事实， 在肌动蛋白亚型中，N-末端集中，其也被乙酰化并形成肌球蛋白的一部分， 结合位点Aim-1c将探讨肌动蛋白N-乙酰化和profilin/actin异构体变异在 函数。形成蛋白是细胞中最重要的肌动蛋白丝延伸因子， 发现肌动蛋白N-乙酰化对纤维伸长有深远的影响。进一步假设肌动蛋白 集中在N-末端的同种型变异将对α-淀粉酶活性具有同样强的影响。 Aim-2提出了解决肌动蛋白成核领域持续存在的问题的新策略， 这个实验室的长期利益。Aim-2a将并行研究Tmods和Lmods，尽管它们具有 共同褶皱演化出不同的活动--分别为尖端封盖和成核。源 这两个亚家族之间的功能差异将被研究，重点是Lmod 3， 在线状体肌病中。提出了一种创新的策略，确定Tmod的结构， 端最后，在体外和细胞中研究了特异性Tmod和原肌球蛋白亚型如何与 彼此组装形态和功能不同的肌动蛋白网络。Aim-2B将解决长期的 WASP家族成核促进因子如何与分支相互作用并激活分支的一个长期存在的问题 Arp 2/3复合物的形成。这些计划建立在执行生物化学和结构的能力之上 研究杆状病毒表达的Arp 2/3复合物和亚复合物。广泛的初步和 已发表的工作提供了科学的前提和支持的可行性。

Project Summary: The long-term goal is to understand the structural and functional mechanisms that control actin cytoskeleton dynamics in health and disease. In its fourth cycle, this grant addresses important gaps of knowledge in previous focus areas, while also expanding into new areas in response to recent developments in the field. Building upon the recent discovery by this lab in collaboration with a group in Norway of actin's dedicated N- terminal acetyltransferase (Naa80), Aim-1 will focus on the mechanism and function of actin N-terminal acetylation (N-acetylation) and isoform variations. Aim-1a will determine the mechanism of N-acetylation, including biochemical and cellular studies of the Naa80-actin interaction and a series of crystal structures of intermediate reaction steps, and using different actin isoforms. Aim-1b will test the hypothesis that myosin activity is strongly dictated by actin isoform variations and N-acetylation. This will resolve an enduring deficiency in the field; most myosin studies have used α-skeletal actin, overlooking the fact that differences among actin isoforms concentrate at the N-terminus, which is also acetylated and forms part of the myosin- binding site. Aim-1c will explore the role of actin N-acetylation and profilin/actin isoform variations on formin function. Formins are the most important actin filament elongation factors in cells, and this group has found that actin N-acetylation has a profound effect on filament elongation. It is further postulated that actin isoform variations that concentrate at the N-terminus will have an equally strong effect on formin activity. Aim-2 proposes new strategies to tackle persisting questions in the area of actin nucleation, which constitutes a long-standing interest of this lab. Aim-2a will study in parallel Tmods and Lmods, which despite having a common fold have evolved different activities – pointed-end capping and nucleation, respectively. The source of the functional differences between these two subfamilies will be studied with a focus on Lmod3, implicated in nemaline myopathy. An innovative strategy is proposed to determine the structure of Tmod at the pointed end. It is finally investigated, in vitro and in cells, how specific Tmod and Tropomyosin isoforms interact with each other to assemble morphologically and functionally distinct actin networks. Aim-2b will tackle the long- standing problem of how WASP-family Nucleation Promoting Factors interact with and activate branch formation by the Arp2/3 complex. The plans build upon the ability to perform biochemical and structural studies on the baculovirus-expressed Arp2/3 complex and subcomplexes. Extensive preliminary and published work provide the scientific premise and support feasibility.