Chaperones and Proteases of the Plasmodium falciparum Parasite

恶性疟原虫寄生虫的伴侣和蛋白酶

• 批准号: RGPIN-2014-05393
• 负责人: Houry, Walid
• 金额: $ 3.42万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=630010
• 关键词: Chaperones; Proteases; Plasmodium; falciparum; Parasite

Protein homeostasis in the cell is regulated by a wide array of quality control systems consisting of molecular chaperones and proteases. These systems assist newly-translated proteins in folding to their native state and also ensure that misfolded proteins and short-lived regulatory proteins are targeted for degradation. Understanding the cellular roles and mechanisms of function of chaperones and proteases will provide critical insights into the basic physical principles that govern protein folding in the cell. The eukaryotic protozoan parasite Plasmodium falciparum is the causative agent of malaria. It harbors two organelles of prokaryotic origin: the mitochondrion and the apicoplast. The apicoplast is a non-photosynthetic plastid derived from an ancient red algal endosymbiont. Hence, the apicoplast is considered a vestigial plastid and studies on the apicoplast should provide critical insights into the evolution of the plant chloroplast-specific processes. The parasite has a large number of molecular chaperones and proteases. Importantly, several of these chaperones and proteases reside in the apicoplast and are shown to be critical for the development of a functional organelle. To this end, we started a trans-disciplinary research program aimed at biochemically and biophysically characterizing the chaperones and proteases in the P. falciparum apicoplast. Our studies in the current proposed project will specifically concentrate on the Caseinolytic (Clp) chaperone-protease complexes. These complexes are highly conserved across species and perform essential quality control functions by degrading regulatory and misfolded proteins. Initial studies from my group have shown that a PfClpCRP chaperone-protease complex exists in the parasite apicoplast. PfClpC is an ATPase and a member of the AAA+ superfamily, and, hence, contains in its sequence conserved Walker A and B motifs. PfClpC is proposed to form a hexameric complex. PfClpP and PfClpR are paralogs that we propose form a cylindrically-shaped oligomeric protease containing a proteolytic chamber for degradation. While PfClpP has the Ser-His-Asp catalytic triad, these residues are mutated in PfClpR rendering PfClpR the inactive subunit in the PfClpRP oligomer. We propose that the mechanism of function of the PfClpCRP complex involves the binding and unfolding of target proteins by PfClpC ATPase chaperone, which then translocates them into the PfClpRP cylindrical protease for degradation. The proposed project will concentrate on the structural and functional characterization of PfClpCRP. The project has two specific aims:1. Biochemical and biophysical studies on PfClpCRP. We will initially reconstitute a PfClpRP complex from purified protein components. The oligomeric state, activity, and proteolytic specificity of the complex will be assessed using biophysical approaches. Similarly, PfClpC will be characterized. The ATPase and unfoldase activity of the chaperone will be investigated using model substrates. Subsequently, the mechanism of function of the complete PfClpCRP complex will be investigated.2. Structural studies on PfClpCRP. Earlier, we had obtained the X-ray crystal structure of PfClpP and PfClpR, and negative stain electron microscopy (EM) 2D images of the separate PfClpP and PfClpR heptamers. We will continue our structural efforts with the long term goal of obtaining a high resolution structure of the PfClpCRP complex by combining X-ray and electron microscopy approaches.Such a comprehensive study of the PfClpCRP system in P. falciparum apicoplast will provide valuable insights into the mechanism of function of this essential chaperone-protease system and the principles that govern protein homeostasis in this vestigial plastid.

细胞中的蛋白质稳态由一系列由分子伴侣和蛋白酶组成的质量控制系统调节。这些系统帮助新翻译的蛋白质折叠到其天然状态，并确保错误折叠的蛋白质和短寿命的调节蛋白质被靶向降解。了解分子伴侣和蛋白酶在细胞中的作用和功能机制，将为研究细胞中蛋白质折叠的基本物理原理提供重要的见解。真核原生动物寄生虫恶性疟原虫是疟疾的病原体。它含有两个原核起源的细胞器：细胞器和顶质体。顶质体是一种起源于古老红藻内共生体的非光合质体。因此，顶质体被认为是一种退化的质体，对顶质体的研究应该为植物叶绿体特定过程的进化提供重要的见解。寄生虫有大量的分子伴侣和蛋白酶。重要的是，这些分子伴侣和蛋白酶中的几种存在于顶质体中，并且被证明对于功能性细胞器的发育至关重要。为此，我们开始了一项跨学科的研究计划，旨在生物化学和生物病理学特征的伴侣蛋白和蛋白酶在恶性疟原虫顶质体。我们的研究在目前提出的项目将特别集中在酪蛋白溶解（CLP）伴侣蛋白酶复合物。这些复合物在物种间高度保守，并通过降解调节和错误折叠的蛋白质来执行必要的质量控制功能。我的小组的初步研究表明，PfClpCRP伴侣蛋白酶复合物存在于寄生虫顶质体中。PfClpC是一种ATP酶，是AAA+超家族的成员，因此在其序列中含有保守的步行者A和B基序。提出PfClpC形成六聚体复合物。PfClpP和PfClpR是旁系同源物，我们提出形成含有用于降解的蛋白水解室的圆柱形寡聚蛋白酶。虽然PfClpP具有Ser-His-Asp催化三联体，但这些残基在PfClpR中突变，使得PfClpR成为PfClpRP寡聚体中的无活性亚基。我们认为PfClpCRP复合物的功能机制涉及PfClpC ATP酶伴侣蛋白与靶蛋白的结合和解折叠，然后将其转移到PfClpRP圆柱形蛋白酶中进行降解。拟议的项目将集中在PfClpCRP的结构和功能表征。该项目有两个具体目标：1。PfClpCRP的生化和生物物理研究。我们将首先从纯化的蛋白质组分重构PfClpRP复合物。将使用生物物理方法评估复合物的寡聚状态、活性和蛋白水解特异性。类似地，将表征PfClpC。将使用模型底物研究伴侣蛋白的ATP酶和解折叠酶活性。随后，将研究完整PfClpCRP复合物的功能机制。2. PfClpCRP的结构研究。早些时候，我们已经获得了PfClpP和PfClpR的X射线晶体结构，以及单独的PfClpP和PfClpR七聚体的负染色电子显微镜（EM）2D图像。我们将继续我们的长期目标，通过结合X-射线和电子显微镜的方法，获得一个高分辨率的PfClpCRP复合物的结构的结构的努力。PfClpCRP系统在恶性疟原虫apicoplast这样一个全面的研究将提供有价值的见解，这一重要的伴侣蛋白酶系统的功能机制和原则，在这个退化的质体蛋白质稳态。