ROLE OF CHAPERONES IN MAINTAINING THE ASPARAGINE REPEAT-RICH PROTEOME OF P. FALCI

项目摘要/摘要 恶性疟原虫是一种致命的人类病原体，每年导致100多万人死亡。 恶性疟原虫的蛋白质组不成比例地富含天冬酰胺重复序列；四分之一的蛋白质含有 诸如此类的重复。蛋白质中ASN重复序列的存在会导致淀粉样原纤维的形成。在整个生活史中，P. 恶性疟原虫经历了巨大的温度波动，这可以促进淀粉样原纤维的形成。监护人或 热休克蛋白，如Hsp110、Hsp70和Hsp40，存在于生命和影响的所有王国中 淀粉样原纤维形成。然而，它们的生物学作用和作用机制仍不清楚。 拟议的职业发展计划旨在从根本上深入了解监护人在 维持恶性疟原虫富含ASN重复序列的蛋白质组，同时建立独立的学术生涯 在一所大学里。候选人将在生物化学、蛋白质生物物理学和 寄生虫学将在Dr。 丹尼尔·戈德伯格是研究疟疾寄生虫红细胞内阶段的先驱和领导者。这个 该计划将在华盛顿大学传染病学部医学系进行 医学院，生物医学科学领域的领先机构。 在最近的一篇论文中，候选人报告了一种新的可调节的荧光亲和力(RFA)的发展 标签，用于研究恶性疟原虫蛋白酶体亚基Rpn6中28个残基的ASN重复序列。ASN 即使在热休克下，重复序列也不会影响蛋白质的功能、稳定性或相互作用；这表明 存在确保含有ASN重复序列的蛋白质正确折叠的活性过程。在.期间 在指导阶段，候选人将：1)研究PfHsp110在寄生虫蛋白平衡中的作用 PfHsp110基因的寄生虫系已经通过生化和显微方法进行了RFA标记。 淀粉样蛋白形成蛋白也将在这些寄生虫系中表达，以研究PfHsp110如何影响 原纤维形成。2)通过补充标记的RFA来确定作用机制和特异性 带有PfHsp110的突变体和来自其他生物的同源物的寄生虫。PfHsp110还将在 将研究异种哺乳动物系统及其对哺乳动物细胞中纤维形成的影响。 生化和显微方法。在奖项的独立阶段，候选人将阐明 寄生虫Hsp70、Hsp70样蛋白和Hsp40分子伴侣在维持ASN重复序列稳定丰富中的作用 蛋白质组。这些伴侣被候选人发现与PfHsp110相关。 研究计划中概述的生化、显微和细胞生物学方法方面的培训 使候选人能够以独立的身份开始全面而富有成效的研究计划 研究员。拟议的研究将揭示恶性疟原虫伴侣功能的基本方面， 这将为治疗疟疾和其他蛋白质错折叠疾病提供新的药物靶点。

Project Summary/Abstract Plasmodium falciparum is a deadly human pathogen that is responsible for over a million deaths every year. The proteome of P. falciparum is disproportionately rich in repeats of asparagine; one in four proteins contain such repeats. The presence of Asn repeats in a protein leads to amyloid fibril formation. During its life cycle, P. falciparum undergoes large temperature fluctuations that can promote amyloid fibril formation. Chaperones or heat shock proteins, such as Hsp110, Hsp70 and Hsp40, are present in all kingdoms of life and influence amyloid fibril formation. However, their biological roles and mechanism of action remains poorly defined. The proposed career development plan aims to gain fundamental insights into the function of chaperones in maintaining the Asn repeat-rich proteome of P. falciparum, while establishing an independent academic career in a university setting. The candidate will build on his strong foundation in biochemistry, protein biophysics and parasitology to develop into an independent researcher in P. falciparum biology under the mentorship of Dr. Daniel Goldberg, a pioneer and leader in the study of the intraerythrocytic stages of the malaria parasite. The plan will be carried out in the Department of Medicine, Division of Infectious Diseases at Washington University School of Medicine, a leading institution in biomedical science. In a recent paper, the candidate reported the development of a novel regulatable, fluorescent, affinity (RFA) tag that was used to study a 28-residue Asn repeat in the P. falciparum proteasome subunit, Rpn6. The Asn repeat did not affect the protein function, stability or interactions even under heat shock; suggesting the presence of an active process that ensures proper folding of Asn repeat containing proteins. During the mentored phase, the candidate will: 1) Examine the function of PfHsp110 in parasite proteostasis using the parasite lines where PfHsp110 gene has been RFA tagged via biochemical and microscopic approaches. Amyloid forming proteins will also be expressed in these parasite lines to study how the PfHsp110 influences fibril formation. 2) Determine the mechanism of action and specificity by complementing the RFA tagged parasite with mutants of PfHsp110 and homologs from other organisms. PfHsp110 will also be expressed in a heterologous mammalian system and its effect on fibril formation in mammalian cells will be studied using biochemical and microscopic approaches. In the independent phase of the award the candidate will elucidate the roles of Hsp70, Hsp70-like and Hsp40 chaperones of the parasite in maintaining a stable Asn repeat-rich proteome. These chaperones were discovered by the candidate to be associated with PfHsp110. Training in biochemical, microscopic and cell biological approaches as outlined in the research plan has equipped the candidate to embark on a comprehensive and fruitful research program as an independent researcher. The proposed studies will reveal fundamental aspects of chaperone function in P. falciparum, which will furnish new drug targets against malaria as well as other protein misfolding diseases.