MECHANISMS FOR SPECIFICATION OF HSP40 FUNCTION

HSP40 功能的规范机制

基本信息

批准号：
7359623
负责人：
DOUGLAS M CYR
金额：
$ 3.75万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-02-01 至 2009-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7359623
关键词：
ATP phosphohydrolase Accounting Address Award Binding Binding Sites Biochemical Biogenesis Biological Biological Assay Brazil Cell Survival Cell physiology Cells Chimera organism Chimeric Proteins Collaborations Cytosol Data Elements Exhibits Family Family member Goals Housing In Vitro Inherited Insulinase Laboratories Mass Spectrum Analysis Modeling Molecular Molecular Chaperones Molecular Conformation Monitor Partner in relationship Peptide Phage Display Library Peptides Pheromone Principal Investigator Prion Diseases Prions Process Protein Region Protein-Folding Disease Proteins Research Research Personnel Resolution Ribosomes Roentgen Rays Scanning Site Specific qualifier value Specificity Stress Structural Models Structure Substrate Specificity Synchrotrons Testing To specify United States National Institutes of Health Work Yeasts crosslink in vivo member model design mutant parent grant polypeptide programs protein aggregation protein folding protein function protein metabolism protein protein interaction research study trait transmission process

项目摘要

The proposed research will be conducted by Dr. Carlos Ramos, primarily in Campinas, Brazil, at the Brazilian Synchrotron Laboratory (LNLS), in collaboration with Dr. Douglas Cyrof the Department of Cell Biolog at UNC-Chapel Hill. The parent grant held by Dr. Cyr GM R01 NIH R01GM56981 is focused on understanding the mechansims by which Hsp40 proteins regulate Hsp70 action in cell stress. This FIRCA award seeks to extend the aims of the parent grant and previous work of Dr. Ramos to study the mechansims by which Type I and Type II Hsp40s specify the cellular functions of Hsp70. One goal of this proposal is to determine the structural elements that control the quaternary structure of Type I and Type II Hsp40s. Dr. Cyr has demonstrated that Type I and Type II Hsp40s exhibit differences in substrate specificity and protein folding activity that control the cellular function of Hsp70. However, the mechansim for Hsp40 action as a protein folding factor is unknown. Dr. Ramos has demonstrated that Type I and Type II Hsp40 proteins and have grossly different quaternary strucutres, which may account for the functional differences exhibited by these co-chaperones. In this aims we set of defined to experiments to define the features that control the quaternary strucuture. To accomlish this goal the Ramos laboratory will utilize small angle X-ray scattering, cross-linking and and analytic ultracentrafugation to study the quaternary strucutres of Hsp40s. At present we have high resolution structural information on fragments of Hsp70 and Hsp40, but there is little information that describes contacts formed between Hsp70 and Hsp40 during the process of protein folding. Since the field has not have been able to obtain high resolution structural data to describe Hsp70/Hsp40 interactions, as a second goal we propose to utilize SASX and cross-linking/mass spectroscopy approaches to build such models. Then we will test the predictions made from these models by designing mutants and testing there activity in in vivo and in vitro functional assays. These data will determine the mechanism by which Type I and Type II Hsp40s interact with Hsp70 to suppress protein aggregation and facilitate protein folding/assembly.

拟议的研究将由卡洛斯·拉莫斯 (Carlos Ramos) 博士主要在巴西坎皮纳斯的巴西同步加速器实验室 (LNLS) 与细胞系 Douglas Cyrof 博士合作北卡罗来纳大学教堂山分校的生物学家。 Cyr GM R01 NIH R01GM56981 博士持有的家长补助金重点关注了解 Hsp40 蛋白在细胞应激中调节 Hsp70 作用的机制。这个FIRCA 该奖项旨在扩展拉莫斯博士家长资助和之前工作的目标，以研究 I 型和 II 型 Hsp40 指定 Hsp70 细胞功能的机制。该提案的一个目标是确定控制四级结构的结构元素 I 型和 II 型 Hsp40。 Cyr 博士证明，I 型和 II 型 Hsp40 在以下方面表现出差异：控制 Hsp70 细胞功能的底物特异性和蛋白质折叠活性。然而， Hsp40 作为蛋白质折叠因子的作用机制尚不清楚。拉莫斯博士已经证明，I 型和 II 型 Hsp40 蛋白，并且具有截然不同的四级结构，这可能是这些共伴侣表现出的功能差异。为了这个目标，我们定义了一组实验来定义控制四元结构的特征。为了实现这一目标，拉莫斯实验室将利用小角度 X 射线散射、交联和分析超速离心来研究四元 Hsp40 的结构。目前我们拥有 Hsp70 和 Hsp40 片段的高分辨率结构信息，但还存在描述蛋白质加工过程中 Hsp70 和 Hsp40 之间形成的接触的信息很少折叠式的。由于该领域尚未能够获得高分辨率的结构数据来描述 Hsp70/Hsp40 相互作用，作为第二个目标，我们建议利用 SASX 和交联/质量光谱学方法来构建此类模型。然后我们将测试这些模型的预测通过设计突变体并在体内和体外功能测定中测试其活性。这些数据将确定 I 型和 II 型 Hsp40 与 Hsp70 相互作用抑制蛋白质的机制聚集并促进蛋白质折叠/组装。