Determining the Fate of Transmembrane Proteins

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

One aspect of this project relates to determining the role of the ubiquitin system in mitochondrial protein turnover including membrane dynamics and how this in turn relates to cellular energetics in cancer and to apoptosis. Related to this is our interest in understanding mitochondrial protein quality control and how the ubiquitin system is involved in this. This project was begun in yeast and expanded to mammalian systems. In yeast we have determined that the E3 SCF-Mdm30 plays a critical role in targeting the mitofusin, Fzo1, for degradation and that this degradation is integral to the process of mitochondrial outer membrane fusion. In mammals we have established a pathway leading from cell stress to activation of JNK, which leads to phosphorylation of mammalian mitofusin 2 (Mfn2). This in turn leads to the recruitment of the large HECT domain ubiquitin ligase Huwe1. Mfn2 is then ubiquitinated and degraded by the 26S proteasome leading to mitochondrial fragmentation and cell death. Thus, we have established a new pathway from cell stress to degradation of a specific mitochondrial protein to apoptosis. Our mitochondrial quality control studies in yeast use temperature sensitive (ts) alleles of proteins to tease out requirements for their degradation. This work has resulted in the discovery of proteins of the ubiquitin system that were not previously thought to be involved in degradation of mitochondrial proteins. Consequently, we have elucidated a novel quality control pathway for mitochondrial outer membrane proteins. These studies are now expanding to include studies to determine the role of mitochondrial matrix proteases in degradation of ts proteins. Our quality control studies in mammalian cells are at an earlier stage, but should result in the identification of proteins involved in targeting proteins for degradation from each of the compartments of mitochondria. A major aspect of this project relates to degradation from the endoplasmic reticulum (endoplasmic reticulum (ER)-associated degradation; ERAD). Together with collaborators we are studying E2s and E3s critical to this process. gp78/RNF45, also known as the tumor autocrine motility factor receptor (AMFR), was discovered by our laboratory to be a ubiquitin ligase resident to the ER. We have determined that gp78 plays essential roles in the degradation of multiple substrates functioning together with an E2 that we first characterized, originally known as MmUBC7 and now referred to as UBE2G2. We have 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 isolation can block ERAD and induce ER stress. We are exploring the potential for such expression or other means of blocking the interaction between the gp78 and UBE2G2 as a means to target cells that are predisposed to ER stress, such as multiple myeloma cells, to undergo apoptosis. gp78 levels are correlated with the metastatic potential of tumors including melanomas and lung cancers. Xenograft studies using knockdowns of gp78 followed by its re-expression have now determined that gp78 does, in fact, play an important role in the metastatic potential of multiple different sarcomas and that this potential for metastasis requires intact its ubiquitin ligase activity. We have also determined that gp78 targets a metastasis suppressor, KAI1 (CD82), for degradation in sarcomas. This provides at least a partial explanation for our findings. We have generated mouse models to study gp78, studies are underway to determine the role of this ubiquitin ligase in breast cancer including in the metastasis of spontaneously arising tumors. Our work in animals is being complemented by studies with our collaborator, who is looking at the role of gp78 in aggressiveness of human breast cancers and the relationship of this to race. In particular, we have strong evidence suggesting gp78 is predictive of poor outcome in breast cancer specifically in women of African-American descent. gp78 has been reported as being responsible for the targeting of the rate-limiting enzyme in cholesterol synthesis, HMG-CoA reductase, for degradation. We determined that this was an artifact. On the other hand, gp78 does target Insig-1, which is a major negative regulator of sterol and fatty acid biosynthesis. This potentially makes gp78 an attractive target for treatment of hypercholesterolemia and reinforces its potential role in cancer where lipid biosynthesis is critical for tumor growth. Studies in mouse models are ongoing to evaluate the in vivo significance of these findings. At the same time, we are exploring the potential to identify deubiquitinating enzymes (DUBs) involved in regulating critical regulators of cholesterol and fatty acid biosynthesis in the endoplasmic reticulum and Golgi. These represent potentially important targets, and may potentially be more druggable then ligases. Our studies on DUBs have now expanded to include assessment of cellular roles of a Sars-CoV-2 enzyme that has both deubiquitinating and deISGylating activity (Nsp3). We are currently studying how Nsp3 affects interferon responses and cellular metabolic pathways. Our studies have thus far uncovered unexpected activities of this protease towards cellular targets.