Chaperones and Proteases of the Plasmodium falciparum Parasite

细胞内的蛋白质动态平衡由分子伴侣和蛋白水解酶组成的一系列质量控制系统来调节。这些系统帮助新翻译的蛋白质折叠到它们的自然状态，并确保错误折叠的蛋白质和短暂的调节蛋白质成为降解的目标。了解伴侣蛋白和蛋白水解酶在细胞中的作用和作用机制，将有助于我们深入了解控制细胞内蛋白质折叠的基本物理原理。真核原虫原虫恶性疟原虫是疟疾的病原体。它有两个原核起源的细胞器：线粒体和质外体。顶生质体是一种非光合体，来源于一种古老的红藻内共生体。因此，质外体被认为是一种残留的质体，对质外体的研究将为植物叶绿体特有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水解酶。重要的是，其中几种伴侣蛋白和蛋白水解酶存在于质外体中，并被证明对功能细胞器的发育至关重要。为此，我们启动了一项跨学科的研究计划，旨在从生化和生物物理的角度研究恶性疟原虫质外体中的伴侣和蛋白水解酶。在目前拟议的项目中，我们的研究将专门集中在酪蛋白溶解(CLP)伴侣-蛋白酶复合体上。这些复合体在物种间高度保守，并通过降解调节蛋白和错误折叠的蛋白质来执行基本的质量控制功能。我的团队的初步研究表明，寄生虫的质外体中存在一种PfClpCRP伴侣-蛋白酶复合体。PfClpC是一种ATPase，是AAA超家族的成员，因此在其序列中包含保守的Walker A和B基序。PfClpC被认为是形成一个六聚体络合物。PfClpP和PfClpR是我们建议形成的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中发生突变，使PfClpR成为PfClpRP寡聚体中的非活性亚基。我们认为，PfClpCRP复合体的作用机制涉及PfClpC ATPase伴侣蛋白与靶蛋白的结合和去折叠，然后将其移位到PfClpRP圆柱形酶中进行降解。拟议的项目将集中于PfClpCRP的结构和功能特征。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中重建PfClpRP复合体。将使用生物物理方法评估该复合体的低聚状态、活性和蛋白降解专一性。同样，将对PfClpC进行表征。将使用模型底物来研究伴侣蛋白的ATPase和Unfoldase活性。随后，将对完整的PfClpCRP复合体的作用机制进行研究。PfClpCRP的结构研究在此之前，我们已经获得了PfClpP和PfClpR的X射线晶体结构，以及PfClpP和PfClpR七聚体的负染色电子显微镜(EM)2D图像。我们将继续我们的结构努力，长期目标是通过X射线和电子显微镜相结合的方法获得PfClpCRP复合体的高分辨率结构。对恶性疟原虫质外体中PfClpCRP系统的全面研究将为了解这个重要的伴侣-蛋白酶系统的功能机制和控制这个残留的质体中蛋白质稳态的原理提供有价值的见解。

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.