Hypoxia inhibits disulfide bond formation and protein folding in the endoplasmic reticulum

Poorly oxygenated (hypoxic) tumor cells limit the success of current cancer therapy due to treatment resistance and biological adaptation, which stimulates metastasis, angiogenesis and altered metabolism. This adaptation is governed by oxygen sensing pathways, such as the hypoxia inducible factors (HIFs) and the unfolded protein response (UPR). Whereas the oxygen dependency of HIF regulation is well understood, it currently remains unknown how and why hypoxia activates the UPR. The UPR represents a coordinated regulation of transcription and translation aimed to alleviate endoplasmic reticulum (ER) stress. The ER is a compartment where maturation of newly synthesized proteins destined for the extracellular space occurs. Other stresses that activate the UPR, such as lack of glycosylation, result in accumulation of misfolded or unfolded proteins in the ER that tend to aggregate. This activates the ER stress sensors PERK, IRE1 and ATF6 and downstream signaling important for restoration of ER homeostasis and survival. We hypothesized that one or more ER localized protein maturation steps depend on molecular oxygen, and hypoxia consequently activates the UPR via accumulation of improperly folded proteins in the ER. We hence monitored the maturation steps of several secreted or membrane-destined proteins during normoxia and anoxia using a pulse-chase assay. We found that oligosaccharide modifications, including ER localized N-linked glycosylation and glycan trimming, Golgi-transport and Golgi-localized complex glycosylation, occurred independent of oxygen availability. In contrast, oxygen was strictly required for disulfide bond formation in all proteins investigated. This defect in disulfide bond formation during hypoxia was completely reversible upon reintroduction of oxygen. During hypoxia, we also observed an immediate accumulation of reduced Ero1L, an oxidoreductase responsible for oxidative folding of client proteins in the ER. This is consistent with a requirement for oxygen in the ultimate reoxidation of Ero1L following introduction of a disulfide bond. Finally, transcriptome analysis of 3 cancer cell lines of various origins (breast, prostate, colon) demonstrated specific upregulation of oxidoreductases, protein disulfide isomerases and chaperones in the ERome during hypoxic conditions, consistent with an effort to restore the capacity for the ER to introduce disulfide bonds in client proteins. These results demonstrate that oxygen is the required oxidizing agent in vivo for disulfide bond formation. The limited capacity to mature proteins bound for the extracellular space during hypoxia has profound implications for the expression of secreted and membrane-bound proteins that confer metastasis, angiogenesis and metabolism.