MECHANISMS FOR SPECIFICATION OF HSP40 FUNCTION

描述（由申请人提供）：拟议的研究将由Carlos Ramos博士进行，主要在巴西坎皮纳斯，在巴西同步加速器实验室（LNLS），与北卡罗来纳大学教堂山分校细胞生物学系的Douglas Cyr博士合作。由Cyr GM R01 NIH R01GM56981博士持有的父母资助重点是了解Hsp40蛋白在细胞应激中调节Hsp70作用的机制。此次FIRCA奖旨在扩展Ramos博士之前研究I型和II型hsp40指定Hsp70细胞功能的机制的目的。本提案的一个目标是确定控制I型和II型hsp40的四级结构的结构元件。Cyr博士已经证明I型和II型hsp40在底物特异性和控制Hsp70细胞功能的蛋白质折叠活性方面表现出差异。然而，Hsp40作为蛋白质折叠因子的作用机制尚不清楚。Ramos博士已经证明I型和II型Hsp40蛋白具有截然不同的四级结构，这可能解释了这些共同伴侣蛋白所表现出的功能差异。在此目的下，我们设置了定义实验来确定控制四元结构的特征。为了实现这一目标，Ramos实验室将利用小角度x射线散射、交联和分析超离心来研究Hsp40s的四级结构。目前我们对Hsp70和Hsp40片段有高分辨率的结构信息，但对蛋白质折叠过程中Hsp70和Hsp40之间形成的接触信息描述甚少。由于该领域尚未能够获得描述Hsp70/Hsp40相互作用的高分辨率结构数据，作为第二个目标，我们建议利用SASX和交联/质谱方法来构建此类模型。然后，我们将通过设计突变体并在体内和体外功能分析中测试其活性来测试从这些模型中做出的预测。这些数据将确定I型和II型Hsp40s与Hsp70相互作用抑制蛋白质聚集并促进蛋白质折叠/组装的机制。

DESCRIPTION (provided by applicant): 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 Cyr of 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 mechanisms 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 mechanisms 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 mechanism 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 structures, 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 structure. To accomplish this goal the Ramos laboratory will utilize small angle X-ray scattering, cross-linking and analytic ultracentrifugation to study the quaternary structures 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 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.