? 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. ? ?
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