Structure and function of the Plasmodium myosin XIV-actin glideosome

疟原虫肌球蛋白 XIV 肌动蛋白滑胶体的结构和功能

• 批准号: 9363011
• 负责人: DORIT HANEIN
• 金额: $ 73.75万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-11 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9363011
• 关键词: Actin-Binding Protein; Actins; Actomyosin; Affect; Baculoviridae; Baculoviruses; Binding Sites; Biochemical; Biological; Biological Assay; Biophysics; Blood; Cell membrane; Cells; Cessation of life; Complex; Cryoelectron Microscopy; Crystallization; Crystallography; Culicidae; Disease; Drug Targeting; Erythrocytes; Filament; Goals; Grant; Human; Imagery; In Vitro; Insecta; Institutes; Integral Membrane Protein; Invaded; Kinetics; Knowledge; Laboratories; Length; Life Cycle Stages; Light; Malaria; Membrane; Microfilaments; Molecular; Molecular Motors; Motor; Motor Activity; Muscle; Myosin ATPase; Nucleotides; Parasites; Phosphorylation; Plasmodium; Plasmodium falciparum; Preclinical Drug Evaluation; Process; Property; Protein Isoforms; Proteins; Protomer; Regulation; Resistance; Resolution; Role; Structure; System; Tail; Techniques; Universities; Vermont; Virulent; Work; cell motility; coronin protein; global health; image reconstruction; malaria infection; milligram; monomer; next generation; parasite invasion; polymerization; profilin; protein expression; reconstitution; small molecule; small molecule inhibitor

Malaria is a blood-borne disease caused by apicomplexan parasites of the genus Plasmodium, which causes more than a half million deaths per year. The life cycle alternates between a mosquito and a human stage; in the latter stage merozoites invade red blood cells, a process that occurs in seconds. Invasion into and egress from an infected host cell are powered by a multi-protein assembly called the glideosome, the core of which is the class XIV myosin motor PfMyoA, making it a primary target against malaria. This motor is anchored via its light chain subunit MTIP (myosin tail interacting protein) to integral membrane proteins in a double-membraned flattened complex called the inner membrane complex (IMC), which lies ~25nm below the plasma membrane. The Plasmodium actin isoform (PfAct1) that interacts with PfMyoA is quite divergent in sequence from, and much more dynamic than, muscle actin. Despite the importance of the parasite motor, knowledge of its structure, function, and regulation has been limited primarily because PfMyoA to date has not been expressed in a heterologous system. The Trybus laboratory has, however, recently discovered how to express milligram quantities of this motor using the baculovirus/insect cell expression system. They have also expressed Plasmodium actin, which allows actomyosin interactions to be studied with native isoforms. The Plasmodium motor and actin will be characterized by a combination of state-of-the-art biochemical, biophysical, and high resolution structural biological techniques. This is a multiple PI R01 grant: Trybus (protein expression, biochemical/biophysical assays of Plasmodium myosin and actin, University of Vermont), Anne Houdusse (crystallography, Institute Curie) and Dorit Hanein and Niels Volkmann (high resolution cryo- electron microscopy and image reconstruction, Sanford Burnham Prebys Institute). In Aim 1 we will determine how PfMyoA motor activity is regulated in the glideosome, and the mechanism by which small molecules inhibit activity. Unloaded and loaded ensemble in vitro motility assays and transient kinetics will be used to assess function. The goal of Aim 2 is to crystallize the Plasmodium falciparum class XIV myosin for structure-function studies, and to determine the site of binding of small molecule inhibitors. Aim 3 seeks to understand how the unique properties of Plasmodium actin and its interaction with Plasmodium actin-binding proteins regulate actin dynamics and affect its ability to interact with PfMyoA. In Aim 4 we will determine the structure of Plasmodium actin filaments, alone or decorated with PfMyoA, at 5Å resolution or better by high-resolution cryo-electron microscopy. Taken together, these studies will establish the molecular basis for Plasmodium glideosome activity.

疟疾是一种由疟原虫属顶复门寄生虫引起的血液传播疾病， 每年造成超过 50 万人死亡。生命周期在蚊子和蚊子之间交替 人类阶段；在后期，裂殖子侵入红细胞，这一过程在几秒钟内发生。 受感染宿主细胞的入侵和流出是由称为“ 滑翔体，其核心是 XIV 类肌球蛋白运动 PfMyoA，使其成为对抗的主要目标 疟疾。该马达通过其轻链亚基 MTIP（肌球蛋白尾部相互作用蛋白）锚定到积分 双膜扁平复合物中的膜蛋白，称为内膜复合物 (IMC)，位于质膜下方~25nm。疟原虫肌动蛋白异构体 (PfAct1) 与 PfMyoA 的相互作用在序列上与肌肉肌动蛋白非常不同，并且比肌肉肌动蛋白更具动态性。 尽管寄生虫运动很重要，但对其结构、功能和调节的了解已经深入人心。 受到限制主要是因为迄今为止 PfMyoA 尚未在异源系统中表达。这 然而，Trybus 实验室最近发现了如何表达该电机的毫克量 使用杆状病毒/昆虫细胞表达系统。他们还表达了疟原虫肌动蛋白， 允许研究肌动球蛋白与天然亚型的相互作用。疟原虫马达和肌动蛋白将 其特点是结合了最先进的生物化学、生物物理和高分辨率 结构生物学技术。这是多项 PI R01 资助：Trybus（蛋白质表达， 疟原虫肌球蛋白和肌动蛋白的生物化学/生物物理测定，佛蒙特大学），安妮 Houdusse（晶体学，居里研究所）以及 Dorit Hanein 和 Niels Volkmann（高分辨率冷冻- 电子显微镜和图像重建，桑福德伯纳姆普雷比斯研究所）。在目标 1 中，我们将 确定 PfMyoA 运动活动如何在滑胶体中受到调节，以及小体的机制 分子抑制活性。卸载和加载整体体外运动测定和瞬态动力学 将用于评估功能。目标 2 的目标是结晶 XIV 类恶性疟原虫 肌球蛋白用于结构功能研究，并确定小分子抑制剂的结合位点。 目标 3 旨在了解疟原虫肌动蛋白的独特特性及其与疟原虫的相互作用 疟原虫肌动蛋白结合蛋白调节肌动蛋白动力学并影响其与 PfMyoA 相互作用的能力。 在目标 4 中，我们将确定单独的或用 PfMyoA 修饰的疟原虫肌动蛋白丝的结构， 通过高分辨率冷冻电子显微镜以 5Å 分辨率或更好的分辨率进行观察。综合起来，这些研究将 建立疟原虫滑胶体活性的分子基础。