Structure and function of the Plasmodium myosin XIV-actin glideosome.

人类疟疾感染是由疟原虫属单细胞寄生虫引起的，是全球主要 健康挑战。尽管在过去15年中取得了显著进展，但全世界仍有超过4亿人死亡 每年，大多数是5岁以下的儿童。最近获得许可的第一种疟疾疫苗预示着 在努力控制疟疾的新时代，但RTS的疗效相对温和，S的疫苗意味着 如果世卫组织到2030年将发病率降低90%的目标是 意识到了。疟疾寄生虫在其复杂的人类和蚊子生命周期中都是能动的。他们移动了一个 这一过程被称为滑动运动，这是它们接触、交叉和进入宿主组织和细胞的能力的基础。 滑行是由寄生虫肌球蛋白马达提供动力的，这种马达的破坏会杀死具有传染性的寄生虫。朝向 开发寄生虫肌动球蛋白马达作为可用药的靶点，我们的合作团队一直在努力 描述基本的XIV类单头肌球蛋白马达PfMyoA，滑翔运动的核心。我们是在 第一个表征和结晶PfMyoA的人，证明了它的功能是由N-末端唯一调节的 重链磷酸化。我们首先展示了PfMyoA及其基本轻链的基本作用 在推动红细胞(RBC)入侵的阶段，负责所有疟疾的发病。从那以后我们就 使用PfMyoA突变体，利用活细胞成像揭示了红细胞入侵所必需的能量障碍。这些 基金会专业地定位了我们的团队，以扩展对疟疾生命周期和 探索更多的疟原虫肌球蛋白及其细胞作用，这是这项竞赛的共同目标 更新。我们建议(目标1)通过比较结构来定义PfMyoB和PfMyoA的细胞角色， 功能特性，以及重链磷酸化在体外和体内的作用。目标2建议 确定了结合袋、作用机理和对两种寄生虫的影响 PfMyoA ATPase活性的分子抑制剂。AIM 3研究了另外两种基本的疟原虫肌球蛋白 (PfMyoF和K)，它们作为发动机和未来潜在的可用药靶标几乎都没有研究过。PfMyoF PfMyoK可能扮演着过程运输者的角色，而PfMyoK可能在性发育过程中发挥作用， 运动区插入真核生物VI类电机中典型的逆方向性。我们将使用一种综合的 强调体外功能分析的方法(运动力和整体作用力分析，光学陷阱分析，稳定的 状态和瞬时动力学)和结构研究(X射线结晶学和低温电子显微镜)以及活细胞 方法(包括超分辨率成像)和马达遗传研究(有条件 在疟原虫生命周期的几个阶段)。完工后，我们将拥有 对基本疟原虫的功能有了前所未有的深入了解 肌球蛋白作为可用药的靶点，揭示了肌球蛋白产生的结构基础 力和运动，并发现了对疟疾寄生虫细胞生物学的基本见解。

Malaria infection in humans, caused by single-celled parasites from the genus Plasmodium, is a major global health challenge. Despite marked progress in the last 15 years, more than 400 million deaths occur worldwide annually, the majority being children under age 5. Recent licensure of the first ever malaria vaccine heralds a new era in efforts to control malaria, but the relatively modest efficacy of the RTS,S vaccine means that complementary approaches will be essential if the WHO's goal of a 90% reduction in rates by 2030 is to be realized. Malaria parasites are motile throughout their complex human and mosquito lifecycle. They move by a process called gliding motility, which underpins their ability to reach, cross, and enter host tissues and cells. Gliding is powered by a parasite actomyosin motor the disruption of which kills the infectious parasite. Towards development of the parasite actomyosin motor as a druggable target, our collaborative team has worked to characterize the essential class XIV single-headed myosin motor PfMyoA, the core of gliding motility. We were the first to characterize and crystallize PfMyoA, demonstrating that its function is uniquely tuned by N-terminal heavy chain phosphorylation. We were the first to show the essential role of PfMyoA and its essential light chain in powering red blood cell (RBC) invasion, the stage responsible for all malaria pathogenesis. We have since used PfMyoA mutants to reveal the energetic barriers necessary for RBC invasion using live cell imaging. These foundations expertly position our team to extend investigation of gliding motility across the malaria lifecycle and explore additional Plasmodium myosins and their cellular roles, which are the combined aims of this competitive renewal. We propose (Aim 1) to define the cellular roles of PfMyoB versus PfMyoA by comparing structures, functional properties, and the role of heavy chain phosphorylation in vitro and in vivo. Aim 2 proposes to determine the binding pocket, mechanism of action, and impact on the parasite of two first-in-class small molecule inhibitors of PfMyoA ATPase activity. Aim 3 investigates two other essential Plasmodium myosins (PfMyoF and K), which are virtually unstudied both as motors and potential future druggable targets. PfMyoF likely plays a role as a processive transporter, while PfMyoK likely functions during sexual development, with motor domain inserts typical of reverse-directionality in eukaryotic class VI motors. We will use an integrative approach highlighting in vitro functional assays (motility and ensemble force assays, optical trap assays, steady- state and transient kinetics) and structural studies (X-ray crystallography and cryo-EM) together with live cell approaches (including super resolution imaging) and genetic investigation of motors (conditional knockouts/substitutions) in several stages of the Plasmodium parasite lifecycle. At completion we will have developed a previously unattainable depth of understanding into the function of the essential Plasmodium myosins as druggable targets, revealed profound insights into the structural basis by which myosins produce force and motion, and discovered fundamental insights into malaria parasite cell biology.