OS-9 regulates hypoxia-inducible factor 1a in the endoplasmic reticulum

Hypoxia-inducible factor 1 (HIF-1) is the master regulator of oxygen homeostasis and plays critical roles in cells’ responses to multiple signaling by activating proangiogenic progress and adaptive metabolism. This critical transcription factor is also involved in pathologic processes such as erythrocytosis, inflammation, and tumor progression. Therefore, understanding HIF-1 regulation will provide better strategies for management of diseases. HIF-1 is an alpha/beta heterodimer and its regulation is mainly through post-translational regulation of HIF-1alpha. Oxygen-dependent hydroxylationubiquitination- proteasomal degradation of HIF-1alpha represents the canonical mechanism underlying oxygen sensing. Ubiquitination-independent degradation (UIP) induced by cancer chemotherapeutics also has been observed, but its mechanism is still unknown. TS20 cells have been used in studying UIP due to the instability of the ubiquitin activating enzyme E1 at the restrictive temperature. In the first part of this study, we have investigated the molecular basis of the temperature sensitive phenotype of the TS20 cells. We demonstrate that there are two point mutations on E1 protein that cause the instability of E1 protein under restrictive temperature which further leads to the inhibition of the ubiquitination process. This study validates an essential model cell line to study the ubitination-dependent and –independent degradation of proteins. OS-9, which has been found as an HIF-1alpha partner that regulates HIF-1alpha stability, is a component of endoplasmic reticulum (ER)-associated protein control system which removes misfolded glycoproteins in the ER-associated degradation (ERAD) pathway. In the second part of this study, we demonstrate that HIF-1alpha is under the surveillance of the ER associated protein quality control system where OS-9 directly interacts with HIF-1alpha in ER-derived vesicles and promotes its degradation by the proteasome. These findings indicate a novel mechanism that regulates HIF-1alpha stability and suggests that the ER protein quality control system appears to participate in the surveillance of nuclear proteins. Tunicamycin, an antibiotic that inhibits N-linked glycosylation, is also an ER-stress inducer. In the 3rd part of this study we that tunicaymcin decreases HIF-1alpha levels and functions by a mechanism that is independent of ubiquitination system. We further demonstrate that hypoxia induced doxorubicin resistance in renal carcinoma cells is reduced by tunicamycin treatment, indicating that tunicamycin may serve as a HIF-1alpha inhibitor that sensitizes cells to tumor therapy by inhibition of hypoxic adaptation in tumor cells. Taken together, our data indicate that an OS-9 participated ER function is involved in the UIP degradation of HIF-1alpha which may form part of the quality control system of HIF-1alpha.Ph.D., Biological Sciences -- Drexel University, 201