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

项目总结/摘要 恶性疟原虫是一种致命的人类病原体，每年造成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 这样的重复。蛋白质中Asn重复序列的存在导致淀粉样原纤维的形成。在其生命周期中，P。 恶性疟原虫经历大的温度波动，这可以促进淀粉样纤维形成。监护人或 热休克蛋白，如Hsp 110、Hsp 70和Hsp 40，存在于所有生命界和影响界中 淀粉样纤维形成。然而，它们的生物学作用和作用机制仍不明确。 拟议的职业发展计划旨在从根本上了解伴侣在以下方面的作用： 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 在大学里候选人将建立在他在生物化学，蛋白质生物物理学和 寄生虫学发展成为一个独立的研究员在恶性疟原虫生物学博士的指导下。 丹尼尔戈德堡，疟疾寄生虫红细胞内阶段研究的先驱和领导者。的 该计划将在华盛顿大学医学系传染病系实施 医学院，生物医学科学的领先机构。 在最近的一篇论文中，该候选人报告了一种新型可调控的荧光亲和力（RFA）的发展。 标签，用于研究恶性疟原虫蛋白酶体亚基Rpn 6中的28个残基Asn重复序列。的Asn 重复并不影响蛋白质的功能，稳定性或相互作用，即使在热休克;表明 存在确保含Asn重复序列的蛋白质正确折叠的活性过程。期间 指导阶段，候选人将：1）使用 其中PfHsp 110基因已经通过生物化学和显微镜方法被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PfHsp 110如何影响 原纤维形成。2)通过补充RFA标记确定作用机制和特异性 具有PfHsp 110突变体和来自其他生物体的同源物的寄生虫。PfHsp 110也将以一种 异源哺乳动物系统及其对哺乳动物细胞中纤维形成的影响将使用 生物化学和微观方法。在奖项的独立阶段，候选人将阐明 Hsp 70、Hsp 70-like和Hsp 40分子伴侣在维持一个稳定的富含Asn重复序列的 蛋白质组这些分子伴侣被发现的候选人与PfHSP 110。 研究计划中概述的生化、显微镜和细胞生物学方法的培训， 装备候选人走上全面和富有成效的研究计划作为一个独立的 研究员拟议的研究将揭示恶性疟原虫伴侣蛋白功能的基本方面， 这将为治疗疟疾和其他蛋白质错误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.