MECHANISMS FOR SPECIFICATION OF HSP40 FUNCTION

拟议的研究将由卡洛斯·拉莫斯博士进行，主要在巴西的坎皮纳斯，在 巴西同步加速器实验室(LNLS)，与细胞部的Douglas Cyr博士合作 北卡罗来纳大学教堂山分校的Biolog。由Cyr GM R01 NIH R01GM56981博士持有的家长资助重点是 了解Hsp40蛋白调节Hsp70在细胞应激中作用的机制。本FIRCA 该奖项旨在扩大父母资助的目标和拉莫斯博士之前的工作，以研究 I型和II型Hsp40决定Hsp70细胞功能的机制。 这项建议的目标之一是确定控制第四纪结构的结构元素 I型和II型Hsp40s。Cyr博士已经证明了I型和II型Hsp40在 控制Hsp70细胞功能的底物特异性和蛋白质折叠活性。然而， Hsp40作为一种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了I型 和II型Hsp40蛋白，并具有非常不同的四级结构，这可能解释了 这些辅助伴侣表现出功能上的差异。在这个目标中，我们定义了一套实验来 定义控制第四系结构的特征。为了实现这一目标，拉莫斯实验室将 利用小角X射线散射、交联和分析超速离心法研究第四系 Hsp40s的结构。 目前我们有关于Hsp70和Hsp40片段的高分辨率结构信息，但有 描述Hsp70和Hsp40在蛋白质过程中形成的接触的信息很少 折叠。由于野外一直无法获得高分辨率的构造数据来描述 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 光谱学方法来建立这样的模型。然后，我们将测试从这些模型做出的预测 通过设计突变体并在体内和体外功能分析中测试它们的活性。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 聚集和促进蛋白质折叠/组装。

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.