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

伴侣在维持 P. FALCI 富含天冬酰胺重复蛋白质组中的作用

基本信息

批准号：
8616715
负责人：
Vasant Muralidharan
金额：
$ 24.9万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-01 至 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8616715
关键词：
Affect Affinity Affinity Chromatography Amino Acids Amyloid Amyloid Fibrils Animal Model Anopheles Genus Asparagine Award Binding Biochemical Biological Biological Models Biology Biophysics Cells Cessation of life Communicable Diseases Complement Complex Culicidae Development Development Plans Disease Drug Targeting Drug resistance Ensure Exposure to Falciparum Malaria Fever Foundations Genes Genomics Glutamine Guanine Nucleotide Exchange Factors Heat shock proteins Heat-Shock Response Homologous Gene Human In Vitro Insect Vectors Institution Knock-out Lead Life Life Cycle Stages Malaria Mammalian Cell Masks Medicine Mentors Mentorship Microscopic Microscopy Molecular Chaperones Nucleotides Organism Paper Parasites Parasitology Pharmaceutical Preparations Phase Phenotype Plasmodium falciparum Play Process Property Protein Biochemistry Proteins Proteome Reporting Research Research Personnel Role Science Specificity Staging Stress System Temperature Training Universities Washington Yeasts abstracting amyloid fibril formation amyloidogenesis asexual career career development extreme temperature insight killings medical schools member multicatalytic endopeptidase complex mutant novel pathogen prevent programs protein function protein misfolding protein protein interaction

项目摘要

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.

项目概要/摘要恶性疟原虫是一种致命的人类病原体，每年导致超过一百万人死亡。恶性疟原虫的蛋白质组中天冬酰胺的重复序列异常丰富。四分之一的蛋白质含有这样的重复。蛋白质中 Asn 重复序列的存在会导致淀粉样原纤维的形成。在其生命周期中，P. 恶性疟原虫经历较大的温度波动，可以促进淀粉样原纤维的形成。监护人或热休克蛋白，例如 Hsp110、Hsp70 和 Hsp40，存在于所有生命和影响领域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然不明确。拟议的职业发展计划旨在对监护人的功能有基本的了解维持恶性疟原虫富含 Asn 重复的蛋白质组，同时建立独立的学术生涯在大学环境中。候选人将建立在生物化学、蛋白质生物物理学和在寄生虫学博士的指导下发展成为恶性疟原虫生物学的独立研究员。丹尼尔·戈德堡（Daniel Goldberg），疟疾寄生虫红细胞内阶段研究的先驱和领导者。这计划将在华盛顿大学医学系传染病科进行医学院是生物医学科学领域的领先机构。在最近的一篇论文中，该候选人报告了一种新型可调节荧光亲和力 (RFA) 的开发该标签用于研究恶性疟原虫蛋白酶体亚基 Rpn6 中的 28 个残基 Asn 重复序列。阿斯恩即使在热激下重复也不影响蛋白质功能、稳定性或相互作用；建议存在确保含有 Asn 重复序列的蛋白质正确折叠的活性过程。期间在指导阶段，候选人将： 1) 使用以下方法检查 PfHsp110 在寄生虫蛋白质稳态中的功能 PfHsp110 基因已通过生化和显微镜方法进行 RFA 标记的寄生虫品系。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RFA标记确定作用机制和特异性带有 PfHsp110 突变体和其他生物体同源物的寄生虫。 PfHsp110 也将表示为将使用异源哺乳动物系统及其对哺乳动物细胞中原纤维形成的影响进行研究生物化学和显微方法。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 重复序列中的作用蛋白质组。候选者发现这些伴侣与 PfHsp110 相关。研究计划中概述的生物化学、显微镜和细胞生物学方法的培训已使候选人能够作为独立的人开展全面且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，这将为对抗疟疾以及其他蛋白质错误折叠疾病提供新的药物靶点。