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.

疟疾是一种血源性疾病，由疟原虫属的顶复合体寄生虫引起