Chaperones and Proteases of the Plasmodium falciparum Parasite

细胞中的蛋白质稳态受到一系列由分子伴侣和蛋白酶组成的质量控制系统的调节。这些系统帮助新翻译的蛋白质折叠至其天然状态，并确保错误折叠的蛋白质和短寿命的调节蛋白成为降解的目标。了解分子伴侣和蛋白酶的细胞作用和功能机制将为了解控制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。它含有两种原核细胞器：线粒体和顶质体。顶端质体是一种源自古老红藻内共生体的非光合质体。因此，顶端质体被认为是一种退化质体，对顶端质体的研究应该为植物叶绿体特异性过程的进化提供重要的见解。该寄生虫具有大量的分子伴侣和蛋白酶。重要的是，其中一些伴侣和蛋白酶存在于顶质体中，并且被证明对于功能性细胞器的发育至关重要。为此，我们启动了一项跨学科研究计划，旨在从生化和生物物理角度表征恶性疟原虫顶端质体中的分子伴侣和蛋白酶。我们在当前拟议项目中的研究将特别集中于酪蛋白分解 (Clp) 分子伴侣-蛋白酶复合物。这些复合物在物种间高度保守，并通过降解调节蛋白和错误折叠蛋白来执行基本的质量控制功能。我的小组的初步研究表明，寄生虫顶质体中存在 PfClpCRP 分子伴侣-蛋白酶复合物。 PfClpC 是一种 ATP 酶，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 被提议形成六聚复合物。 PfClpP 和 PfClpR 是旁系同源物，我们建议它们形成圆柱形寡聚蛋白酶，含有用于降解的蛋白水解室。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 寡聚物中的失活亚基。我们提出 PfClpCRP 复合物的功能机制涉及 PfClpC ATP 酶分子伴侣对靶蛋白的结合和解折叠，然后将其易位到 PfClpRP 圆柱形蛋白酶中进行降解。拟议的项目将集中于 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。我们首先将从纯化的蛋白质成分中重建 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分子伴侣的 ATP 酶和解折叠酶活性。随后对PfClpCRP完整复合物的作用机制进行研究。 2． PfClpCRP 的结构研究。早些时候，我们获得了 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。我们将继续我们的结构努力，长期目标是通过结合 X 射线和电子显微镜方法获得 PfClpCRP 复合物的高分辨率结构。对恶性疟原虫顶端质体中 PfClpCRP 系统的这种全面研究将为了解这种重要的伴侣蛋白-蛋白酶系统的功能机制以及控制这种退化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.