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

描述(由申请人提供)： 摘要 PI：Muralidharan，Vasant Project：1K99AI099156-01标题：伴侣在维持黄曲霉富含天冬酰胺重复序列蛋白质组中的作用注册号：3400178 = 注意：此摘要摘自应用程序，未经SRA校对。如果应用程序扫描过程有问题，提取的文本可能不正确或不完整。 = 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；每四种蛋白质中就有一种含有这种重复序列。蛋白质中ASN重复序列的存在会导致淀粉样原纤维的形成。在其生命周期中，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。伴侣或热休克蛋白，如Hsp110、Hsp70和Hsp40，存在于所有生命王国中，并影响淀粉样原纤维的形成。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获得对伴侣在维持恶性疟原虫富含ASN重复的蛋白质组中的作用的基本见解，同时在大学环境中建立独立的学术生涯。这位候选人将在他在生物化学、蛋白质生物物理学和寄生虫学方面的坚实基础上，在丹尼尔·戈德堡博士的指导下发展成为恶性疟原虫生物学的独立研究人员。丹尼尔·戈德伯格博士是研究疟疾寄生虫红细胞内阶段的先驱和领导者。该计划将在华盛顿大学医学院传染病学部医学部实施，这是生物医学科学的领先机构。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)标签的开发，该标签用于研究恶性疟原虫蛋白酶体亚单位Rpn6中的28个残基ASN重复。即使在热休克下，ASN重复序列也不会影响蛋白质的功能、稳定性或相互作用；这表明存在一个确保含有ASN重复序列的蛋白质正确折叠的活跃过程。在指导阶段，候选人将：1)使用PfHsp110基因已通过生化和显微方法标记的寄生虫系来检测PfHsp110在寄生虫蛋白稳定中的功能。淀粉样蛋白也将在这些寄生虫系中表达，以研究PfHsp110如何影响纤维形成。2)用PfHsp110的突变体和其他生物的同源物来补充RFA标记的寄生虫，以确定其作用机制和特异性。PfHsp110也将在异源哺乳动物系统中表达，并将使用生化和显微方法研究其对哺乳动物细胞中纤维形成的影响。在奖项的独立阶段，候选人将阐明寄生虫的Hsp70、Hsp70样和Hsp40伴侣在维持稳定的ASN重复序列丰富的蛋白质组中的作用。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具备了作为独立研究员开始全面和富有成效的研究计划的能力。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。相关性疟疾是一种致命的疾病，困扰着全世界数亿人。导致疟疾最致命形式的寄生虫--恶性疟原虫--正迅速产生抗药性，因此必须确定杀死这种寄生虫的新策略。这项拟议的研究将调查新的药物靶点，以了解它们在寄生虫生物学中的作用，并开发更好的药物来对抗它们。

DESCRIPTION (provided by applicant): Abstract PI: MURALIDHARAN, VASANT Project: 1K99AI099156-01 Title: ROLE OF CHAPERONES IN MAINTAINING THE ASPARAGINE REPEAT-RICH PROTEOME OF P. FALCI Accession Number: 3400178 ================== NOTICE: THIS ABSTRACT WAS EXTRACTED FROM APPLICATION AND HAS NOT BEEN PROOFED BY AN SRA.WHEN THERE ARE PROBLEMS WITH THE APPLICATION SCANNING PROCESS, THE EXTRACTED TEXT MAY BE INCORRECT OR INCOMPLETE. ================== 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 carrid 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 th 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. Relevance Malaria is a deadly disease that afflicts hundreds of millions of people worldwide. The parasite that causes the deadliest form of malaria, Plasmodium falciparum, is rapidly gaining drug resistance making it imperative to identify new strategies to kill this parasite. The proposed research will investigate new classes of drug targets to understand their role in parasite biology and develop better drugs against them.