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寄生虫引起的血源性疾病 每年造成超过500万人死亡。蚊子和一个蚊子之间的生命周期替代品 人类舞台；在后期，梅罗寄生虫入侵了红细胞，这一过程在几秒钟内发生。 受感染宿主细胞的入侵和出口由称为The The的多蛋白组件提供动力 GLIDEOSOMS，其核心是XIV肌球蛋白电动机PfMyoA，使其成为反对的主要目标 疟疾。该电动机通过其轻链亚基MTIP（肌球蛋白尾巴相互作用蛋白）锚定为积分 双膜扁平复合物中的膜蛋白称为内膜复合物 （IMC），位于质膜下方约25nm。疟原虫肌动蛋白同工型（PFACT1） 与PFMYOA相互作用的序列与肌肉肌动蛋白的序列相差很大，而且动态性得多。 尽管寄生虫发动机很重要，但其结构，功能和调节的了解 之所以有限，主要是因为迄今尚未在异源系统中表达PFMYOA。这 但是，Trybus实验室最近发现了如何表达该电机的毫克数量 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， 允许肌动球蛋白相互作用与天然同工型研究。疟原虫和肌动蛋白将 以最先进的生化，生物物理和高分辨率结合的特征 结构生物学技术。这是多个pi r01赠款：trybus（蛋白质表达， 佛蒙特大学的生物化学/生物物理测定法，佛蒙特大学），安妮 Houdusse（晶体学，研究所）和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·普雷比学院）。在目标1中，我们将 确定如何调节pfmyoa运动活性在格莱德体中，以及小的机制 分子抑制活性。卸载和加载的整体体外运动评估和瞬态动力学 将用于评估功能。目标2的目的是使恶性疟原虫XIV结晶 用于结构功能研究的肌球蛋白，并确定小分子抑制剂的结合位点。 AIM 3试图了解疟原虫肌动蛋白的独特特性及其与之相互作用 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AIM 4中，我们将单独确定单独或用pfmyoa装饰的疟原虫肌动蛋白丝的结构， 在5Å分辨率或通过高分辨率冷冻电子显微镜以更好的分辨率。两者一起，这些研究将 建立疟原虫滑翔体活性的分子基础。