This project is oriented towards determining factors that regulate the fate of transmembrane proteins on the cell surface the secretory pathway and at other organelles including mitochondria. One aspect of this project relates to determining the role of the ubiquitin system in mitochondrial membrane dynamics and how this in turn relates to cellular energetics in cancer. This project was begun in yeast and is now being expanded to mammalian systems. In preliminary studies we have determined that multiple ubiquitin ligases play roles in determining the fate of a critical protein involved in mitochondrial fusion, which is an essential step in preserving oxidative phosphorylation and mitochondrial DNA inheritance. The exact mechanisms whereby these two ligase function is under investigation in the laboratory. A major aspect of this project relates to degradation from the endoplasmic reticulum. Together with collaborators we are stdying E2s and E3s critical to this process. This work has implications for Parkinson's disease where the ubiquitin ligase Parkin is believed to play a critical role in degrading a misfolded protein from the ER. We now have preliminary evidence that other E3s plays roles in this process as well. gp78, also known as the tumor autocrine motility factor receptor (AMFR), was discovered by our laboratory to be a ubiquitin ligase resident to the endoplasmic reticulum. We have recently determined that gp78 plays essential roles in the degradation of multiple substrates functioning together with an E2 known as MmUBC7 (Ube2g2). We have now determined that multiple domains within gp78 function together to mediate its ubiquitin ligase activity. These include its RING finger, a ubiquitin-binding Cue domain and a novel region that specifically recruits Ube2g2 independent of the RING finger, referred to as the G2BR. Moreover, we now know that expressing the G2BR in in isolation can block ERAD and induce ER stress and are exploring the potential for such expression or other means of blocking the interaction between the gp78 and Ube2g2 may represent a means to target cells that are predisposed to ER stress, such as multiple myeloma cells, to undergo apoptosis. This work is further being complemented by structural studies being carried out in collaboration with Dr. R. Andrew Byrd oriented towards understanding the solution structure of the G2BR bound to Ube2g2. gp78 levels are correlated with the metastatic potential of tumors including melanomas and lung cancers. We have now determined (in work not yet submitted for publication) that knocking down gp78 levels results in a decrease in migration of cells in response to AMF as well as to to other stimuli and an inhibition of migration in in vitro wound healing type experiments. In vivo studies in mice using knockdowns of gp78 and reexpression have now determined that gp78 does in fact play an important role in the metastatic potential of tumors and that this potential for metastasis requires intact ubiquitin ligase function of this protein. Further studies are underway to chacacterize the molecular basis for this activity as well as to identify substrates that might be playing critical roles in enhancing metastasis in response to gp78.
