MECHANISMS FOR SPECIFICATION OF HSP40 FUNCTION

描述（由申请方提供）：拟定研究将由卡洛斯拉莫斯博士（主要位于巴西坎皮纳斯的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道格拉斯西尔博士合作进行。Cyr GM R 01 NIH R 01 GM 56981博士持有的母基金专注于了解Hsp 40蛋白在细胞应激中调节Hsp 70作用的机制。这个FIRCA奖旨在扩大父母补助金的目的和以前的工作博士拉莫斯研究的机制，其中I型和II型热休克蛋白40指定的细胞功能的热休克蛋白70。该提议的一个目标是确定控制I型和II型Hsp 40四级结构的结构元件。Cyr博士已经证明，I型和II型Hsp 40在控制Hsp 70细胞功能的底物特异性和蛋白质折叠活性方面表现出差异。然而，Hsp 40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Hsp 40蛋白具有明显不同的四级结构，这可能是这些辅助分子伴侣所表现出的功能差异的原因。在这个目标中，我们定义了一组实验来定义控制四级结构的特征。为了实现这一目标，拉莫斯实验室将利用小角X射线散射、交联和分析超浓缩来研究HSP 40的四级结构。目前我们已经获得了Hsp 70和Hsp 40片段的高分辨率结构信息，但对Hsp 70和Hsp 40在蛋白质折叠过程中形成的接触的描述还很少。由于该领域还没有能够获得高分辨率的结构数据来描述热休克蛋白70/热休克蛋白40的相互作用，作为第二个目标，我们建议利用SASX和交联/质谱方法来建立这样的模型。然后，我们将通过设计突变体并在体内和体外功能测定中测试其活性来测试从这些模型做出的预测。这些数据将确定I型和II型Hsp 40与Hsp 70相互作用以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.