Novel functions for gp78 in ER-associated degradation

描述（由申请人提供）： 内质网相关降解（ERAD）是一个过程，其中错误折叠，未组装，和一些正常的蛋白质被靶向降解的泛素蛋白酶体途径。ERAD对于许多正常细胞过程是必不可少的，并且其功能障碍导致与阿尔茨海默病和帕金森病、囊性纤维化和α 1-抗胰蛋白酶缺乏症相关的病理状况。虽然已知ERAD涉及底物泛素化、逆转录转位和蛋白酶体降解，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，也称为gp 78，是ERAD所需的ER-驻留泛素连接酶（E3），并且现在已经鉴定了另外两种与gp 78相互作用的蛋白质：含valosin-containing蛋白（VCP）和泛素。VCP与gp 78的相互作用似乎激活VCP-多聚泛素结合并促进多聚泛素化蛋白降解。其他人的先前研究表明VCP是逆易位的必要因素。gp 78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gp 78活化的VCPpolyubiquitin结合偶联底物在ERAD过程中的泛素化、逆转位和降解。为了验证这一假设，我们提出了以下具体目标：（1）表征gp 78与VCP，ubc 7，泛素和sec 61通道的相互作用;（2）阐明gp 78激活VCP-聚泛素相互作用的机制;（3）评估gp 78激活的VCP-聚泛素相互作用在ERAD底物的逆转录和降解中的作用。这些研究将推进目前的理解gp 78和VCP是如何参与底物泛素化，retrotranslocation，并在ERAD降解。拟议的工作是符合本实验室的长期目标，了解ERAD的分子机制及其对疾病发病机制的影响。

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.