MECHANISMS FOR SPECIFICATION OF HSP40 FUNCTION

描述(由申请人提供)：拟进行的研究将由卡洛斯·拉莫斯博士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，主要在巴西坎皮纳斯的巴西同步加速器实验室(LNLS)进行。由Cyr GM R01 NIH R01GM56981博士持有的父母资助重点是了解Hsp40蛋白调节Hsp70在细胞应激中作用的机制。FIRCA的这一奖项旨在扩大父母资助和Ramos博士之前的工作的目标，以研究I型和II型Hsp40指定Hsp70细胞功能的机制。这项建议的目标之一是确定控制I型和II型Hsp40的四元结构的结构元素。Cyr博士已经证明，I型和II型Hsp40在底物专一性和控制Hsp70细胞功能的蛋白质折叠活性方面存在差异。然而，Hsp40作为一种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蛋白具有非常不同的四级结构，这可能是这些辅助伴侣呈现出功能差异的原因。在这个目的中，我们定义了一组实验，以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X射线散射、交联和分析超速离心法来研究HSP40的四元结构。目前，我们已经获得了Hsp70和Hsp40片段的高分辨率结构信息，但很少有信息描述Hsp70和Hsp40在蛋白质折叠过程中形成的接触。由于该领域一直无法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/质谱学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分析中测试它们的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Hsp40与Hsp70相互作用以抑制蛋白质聚集和促进蛋白质折叠/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.