Cellular Mechanisms of Aggregation of Abnormal Proteins

描述（由申请人提供）：泛素蛋白酶体机制和分子伴侣的失效可能导致突变或受损多肽的聚集，从而导致各种毁灭性疾病。然而，特殊的分子机制已经发展成为减轻蛋白质毒性的最后一道防线，通过将错误折叠的蛋白质聚集体/低聚物运输到中心体定位的聚集体。本提案的主要目的是揭示聚合体形成的机制。提出的工作是基于我们的酵母模型的蛋白质聚集与扩展的聚谷氨酰胺（polyQ）结构域。过去，该模型被用于识别HD小鼠模型中抑制polyQ聚集、改善运动表现和减少神经元萎缩的小分子。通过该模型，我们已经确定(a)酵母和哺乳动物细胞中的聚集体形成都需要将聚集体靶向信号转移到底物蛋白上，例如huntingtin外显子1的脯氨酸富集区（p区），(b) sh3结构域蛋白Boi2作为p区聚集体靶向信号的识别元件，(c)酵母14-3-3蛋白Bmh1和Cdc48/VCP-Ufd1-Nlp4复合物的成分在聚集体形成中发挥重要作用。我们还建立了一个聚集体形成的细胞培养模型，以研究酵母中发现的聚集体成分与哺乳动物细胞的相关性。在目标1中，我们将阐明聚合体机制如何识别小聚q聚集体/低聚物。我们将确定sh3结构域蛋白Boi2在酵母中识别聚集物中的作用，并将阐明Boi2同源物是否在哺乳动物细胞中识别亨廷顿蛋白和其他含多q的病理蛋白。在Aim 2中，我们将建立14-3-3蛋白Bmh1在酵母聚集体形成早期阶段的功能，并将测试14-3-3蛋白是否在哺乳动物细胞聚集体形成中起普遍作用。这些实验将有助于阐明聚集体机制如何区分异常多肽的小聚集体/低聚物与可溶性单体。在Aim 3中，使用生化和遗传方法，我们将确定参与聚合体形成的新成分。作为这项工作的结果，我们计划获得足够的信息来理解聚合体的形成机制。许多破坏性疾病，包括主要的神经退行性疾病，都是由异常蛋白的积累引起的。这些异常物种倾向于聚集，在这里我们将建立蛋白质聚集的细胞机制。这项工作将揭示生物体如何试图保护自己免受亨廷顿舞蹈症和某些其他疾病的影响。

DESCRIPTION (provided by applicant): Failure of the ubiquitin proteasome machinery and molecular chaperones may result in aggregation of mutant or damaged polypeptides leading to various devastating diseases. However, special molecular machinery has evolved as a last line of defense to relieve proteotoxicity by transporting misfolded protein aggregates/oligomers to the centrosome-localized aggresome. The main objective of this proposal is to uncover the mechanisms of aggresome formation. The proposed work is based on our yeast model of aggregation of proteins with expanded polyglutamine (polyQ) domains. In the past, this model was used to identify small molecules that suppress polyQ aggregation, improve motor performance and reduce neuronal atrophy in a mouse model of HD. With this model, we have established that (a) aggresome formation both in yeast and mammalian cells requires transferable aggresome-targeting signals on substrate proteins, e.g. the proline-rich region (P-region) of exon 1 of huntingtin, (b) an SH3-domain protein Boi2 serves as a recognition element for the P-region aggresome- targeting signal, (c) a yeast 14-3-3 protein Bmh1 and components of the Cdc48/VCP-Ufd1-Nlp4 complex play an essential role in aggresome formation. We also established a cell culture model of aggresome formation to investigate the relevance for mammalian cells of aggresome components identified in yeast. In Aim 1 we will clarify how the aggresome machinery recognizes small polyQ aggregates/oligomers. We will establish the role of the SH3-domain protein Boi2 in the recognition of the aggregates in yeast, and will clarify whether Boi2 homologs function in mammalian cells to recognize huntingtin and other polyQ-containing pathological proteins. In Aim 2 we will establish the function of the 14-3-3 protein Bmh1 in early stages of aggresome formation in yeast, and will test whether 14-3-3 proteins play a general role in aggresome formation in mammalian cells. These experiments will help to clarify how the aggresome machinery distinguishes small aggregates/oligomers of abnormal polypeptides from soluble monomers. In Aim 3 using both biochemical and genetic approaches we will identify novel components involved in aggresome formation. As a result of this work, we plan to obtain sufficient information for understanding the mechanism of aggresome formation. PUBLIC HEALTH RELEVANCE Many devastating diseases, including major neurodegenerative disorders, are caused by accumulation of abnormal proteins. These abnormal species tend to aggregate, and here we will establish cellular mechanisms of protein aggregation. This work will uncover how organisms try to protect themselves from development of Hungtington's and certain other diseases.