Novel functions for gp78 in ER-associated degradation

描述(由申请人提供)： 内质网相关降解(ERAD)是一种通过泛素蛋白酶体途径将错误折叠、未组装和一些正常蛋白质作为目标进行降解的过程。Erad对许多正常的细胞过程是必不可少的，其功能障碍导致阿尔茨海默氏症和帕金森氏病、囊性纤维化和α-抗胰蛋白酶缺乏症相关的病理条件。虽然已知ERAD涉及底物泛素化、逆转录易位和蛋白酶体降解，但这些事件的分子机制尚不清楚。为了开始阐明这些分子机制，我们发现自分泌运动因子受体，也被称为gp78，是ERAD所必需的内质网驻留泛素连接酶(E3)，现在已经发现了另外两个与gp78相互作用的蛋白：Valosin包含蛋白(VCP)和泛素。VCP与gp78的相互作用似乎激活了VCP-多泛素结合，促进了多泛素蛋白的降解。前人的研究表明，VCP是逆转易位的一个重要因素。Gp78与VCP和泛素相互作用的发现为阐明ERAD的分子机制提供了新的途径。我们假设gp78活化的VCP多聚泛素结合偶联底物在ERAD过程中泛素化、逆转录易位和降解。为了验证这一假说，我们提出了以下具体目标：(1)表征gp78与VCP、Ubc7、泛素和Sec61通道的相互作用；(2)阐明gp78激活VCP多泛素相互作用的机制；(3)评价gp78激活的VCP-多泛素相互作用在ERAD底物逆转移位和降解中的作用。这些研究将促进目前对gp78和VCP如何参与ERAD过程中底物泛素化、逆转录易位和降解的理解。拟议的工作与该实验室了解ERAD的分子机制及其对疾病发病机制的影响的长期目标是一致的。

DESCRIPTION (provided by applicant): Endoplasmic reticulum-associated degradation (ERAD) is a process by which misfolded, unassembled, and some normal proteins are targeted for degradation by the ubiquitin proteasome pathway. ERAD is essential for many normal cellular processes and its dysfunctions contribute to the pathological conditions associated with Alzheimers' and Parkinson's diseases, cystic fibrosis, and al-antitrypsin-deficiency. While it is known that ERAD involves substrate ubiquitination, retrotranslocation, and proteasomal degradation, the molecular mechanisms for these events are not clear. To begin elucidating these molecular mechanisms, we have discovered that autocrine motility factor receptor, also known as gp78, is an ER-resident ubiquitin ligase (E3) required for ERAD and have now identified two additional gp78-interacting proteins: valosin-containing protein (VCP) and ubiquitin. Interaction of VCP with gp78 appears to activate VCP-polyubiquitin binding and facilitate polyubiquitinated protein degradation. Previous studies by others have shown that VCP is an essential factor for retrotranslocation. The finding of gp78 interactions with VCP and ubiquitin provides a novel avenue for elucidating the molecular mechanisms in ERAD. We hypothesize that gp78-acivated VCPpolyubiquitin binding couples substrate ubiquitination, retrotranslocation, and degradation during ERAD. To test the hypothesis, we propose the following specific aims: (1) to characterize gp78 interaction with VCP, ubc7, ubiquitin, and sec61 channel; (2) to elucidate the mechanisms by which gp78 activates VCPpolyubiquitin interaction; and (3) to evaluate the roles of gp78-activated VCP-polyubiquitin interaction in retrotranslocation and degradation of ERAD substrates. These studies will advance the current understanding of how gp78 and VCP is involved in substrate ubiquitination, retrotranslocation, and degradation during ERAD. The proposed work is consistent with this laboratory's long-term goal of understanding the molecular mechanisms of ERAD and its implications for disease pathogenesis.