Abstract
Low levels of oxygen (hypoxia) occurs in many (patho)physiological situations. Adaptation to hypoxia is in part mediated by proteins expressed in the extracellular space that mature in the endoplasmic reticulum (ER) prior to traversing the secretory pathway. The majority of such ER cargo proteins require disulfide bonds for structural stability. Disulfide bonds are formed co- and posttranslationally in a redox relay that requires a terminal electron acceptor such as oxygen. We have previously demonstrated that some ER cargo proteins such as low-density lipoprotein receptor (LDLR) and influenza hemagglutinin (Flu-HA) are unable to complete disulfide bond formation in the absence of oxygen, limiting their ability to pass ER quality control and their ultimate expression. Here, using radioactive pulse-chase immunoprecipitation analysis, we demonstrate that hypoxia-induced ER cargo proteins such as carbonic anhydrase 9 (CA9) and vascular endothelial growth factor A (VEGF-A) complete disulfide bond formation and mature with similar kinetics under hypoxia and normoxia. A global in silico analysis of ER cargo revealed that hypoxia-induced proteins on average contain fewer free cysteines and shorter-range disulfide bonds in comparison to other ER cargo proteins. These data demonstrate the existence of alternative electron acceptors to oxygen for disulfide bond formation in cellulo. However, the ability of different proteins to utilize an oxygen-independent pathway for disulfide bond formation varies widely, contributing to differential gene expression in hypoxia. The superior ability of hypoxia-induced proteins such as VEGF-A and CA9 to mature in hypoxia may be conferred by a simpler disulfide architecture.
Highlights
Most notably, the HIF family of hypoxia-inducible transcription factors are stabilized and activated to induce genes that contribute to metabolic adaptation
Both HIF and unfolded protein response (UPR) contribute to hypoxia tolerance and malignant cancer phenotypes [2]. These aggressive phenotypes are in part mediated by induction of secreted proteins such as vascular endothelial growth factor A (VEGF-A) that stimulate angiogenesis [13], and membrane-bound proteins such as carbonic anhydrase 9 (CA9) that contribute to cellular pH homeostasis [5, 14, 15]
VEGF-A and CA9 are induced by HIF and UPR during hypoxia through both transcriptional and translational mechanisms, but little is known about the oxygen dependencies of protein maturation, which affects expression in the extracellular space
Summary
The HIF family of hypoxia-inducible transcription factors are stabilized and activated to induce genes that contribute to metabolic adaptation. Three endoplasmic reticulum (ER) stress sensors are activated by the accumulation of unfolded or misfolded proteins in the ER, leading to translational repression and transcriptional upregulation of proteins involved in protein maturation, autophagy, and redox homeostasis [2, 10,11,12] Both HIF and UPR contribute to hypoxia tolerance and malignant cancer phenotypes [2]. It has been demonstrated using proteins from yeast and of human origin that molecular oxygen can serve as a terminal electron acceptor for disulfide bond formation in solution reconstituted with substrates, PDI and ERO1-Lα [22,23,24] This role for molecular oxygen in disulfide bond formation is in line with the demonstration that hypoxia is a strong physiological activator of the UPR in mammalian cells [7,8,9], which suggests that proteins misfold in the absence of oxygen. This work suggested that an oxygen-independent pathway for disulfide bond formation exists, but it cannot fully support productive protein maturation and expression
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