Regulation of Protein Degradation at the Endoplasmic Reticulum

Abstract

Endoplasmic reticulum (ER) associated degradation (ERAD) is important for removing damaged or misfolded proteins at the ER membrane, and is central to the physiological regulation of proteins such as HMG-CoA Reductase (HMGCR), the rate limiting enzyme in cholesterol biosynthesis. Under sterol rich conditions, HMGCR is rapidly degraded through ERAD, however, when sterols are limited, HMGCR is stabilised through decreased degradation and increased transcription by the transcription factor SREBP2. As well as being tightly regulated by sterol levels, the cholesterol synthesis pathway is highly oxygen dependent; however, how oxygen levels affect the regulation of this pathway is largely unknown. Here, using a combination of fluorescent reporters, genetic approaches, and cellular assays, I determine the role of hypoxia in the regulation of cholesterol synthesis, and also characterise the ubiquitination machinery for an ERAD pathway involving the degradation of misfolded cytosolic proteins. Under low sterol conditions, I show that low oxygen availability overrides the stabilisation of HMGCR. This is not dependent upon the canonical mammalian hypoxia response, driven by hypoxia-inducible factors (HIFs), but instead relies on SREBP2. I find that SREBP2 protein levels are reduced in hypoxia which are partially rescued by proteasome inhibition, suggesting that SREBP2 protein degradation occurs when oxygen is scarce. Recently, using forward genetic screens to probe for the ERAD machinery required to degrade a model misfolded protein, the CL1 degron, the Nathan group identified that two ER E3 ligases, MARCH6 and TRC8, were also potentially involved in the degradation of SREBP2. Here I show that the ubiquitin conjugating enzyme UBE2J2, is involved in this pathway, and identify a role for the ER membrane complex (EMC) in regulating the membrane insertion of UBE2J2. My data also shows that in hypoxia, depletion of MARCH6 and TRC8 partially rescues HMGCR levels, consistent with a role for SREBP2 protein degradation in regulating cholesterol synthesis. Together, these studies demonstrate the importance of hypoxia in the regulation of cholesterol synthesis, which could have broad implications for our understating of how solid cancerous tumours adapt to their hypoxic and nutrient deprived environments.