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.

疟疾是由疟原虫属的顶复门寄生虫引起的血液传播疾病， 每年造成50多万人死亡生命周期在蚊子和 人类阶段;在后一阶段，裂殖子侵入红细胞，这一过程在几秒钟内发生。 入侵和离开受感染的宿主细胞是由一种多蛋白质组装提供动力的， 滑体，其核心是XIV类肌球蛋白马达PfMyoA，使其成为抗 疟疾该马达通过其轻链亚基MTIP（肌球蛋白尾相互作用蛋白）锚定到整合的 双膜扁平复合物中的膜蛋白，称为内膜复合物 (IMC)位于质膜下约25nm处。疟原虫肌动蛋白同种型（PfAct1）， 与PfMyoA相互作用的蛋白质在序列上与肌肉肌动蛋白相当不同，并且比肌肉肌动蛋白更具动态性。 尽管寄生虫马达的重要性，但对其结构、功能和调节的了解， 主要是因为PfMyoA迄今尚未在异源系统中表达。的 然而，特里布斯实验室最近发现了如何表达这种马达的毫克数量。 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同种型的相互作用。疟原虫的马达和肌动蛋白 其特征在于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一项多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（结晶学，居里研究所）和Dorit Hanein和Niels Mülmann（高分辨率低温， 电子显微镜和图像重建，Sanford Burnham Prebys Institute）。在目标1中， 确定PfMyoA运动活动如何在滑体中调节，以及小分子运动调节的机制。 分子抑制活性。体外运动试验和瞬时动力学中的卸载和加载集合 将用于评估功能。目标2的目标是使第XIV类恶性疟原虫结晶 肌球蛋白的结构-功能研究，并确定小分子抑制剂的结合位点。 目的3旨在了解疟原虫肌动蛋白的独特性质及其与 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，我们将确定疟原虫肌动蛋白丝的结构，单独或用PfMyoA修饰， 高分辨率冷冻电子显微镜的分辨率为5 μ m或更高。这些研究将 建立疟原虫滑动体活性的分子基础。