Oxygen-dependent changes in binding partners and post-translational modifications regulate the abundance and activity of HIF-1α/2α

Abstract

Cellular adaptation to low-oxygen environments is mediated in part by the hypoxia-inducible factors (HIFs). Like other transcription factors, the stability and transcriptional activity of HIFs—and consequently, the hypoxic response—are regulated by post-translational modifications (PTMs) and changes in protein-protein interactions. Our current understanding of PTM-mediated regulation of HIFs is primarily based on in vitro protein fragment–based studies typically validated in fragment-expressing cells treated with hypoxia-mimicking compounds. Here, we used immunoprecipitation-based mass spectrometry to characterize the PTMs and binding partners for full-length HIF-1α and HIF-2α under normoxic (21% oxygen) and hypoxic (1% oxygen) conditions. Hypoxia substantially altered the complexity and composition of the HIFα protein interaction networks, particularly for HIF-2α, with the hypoxic networks of both isoforms being enriched for mitochondrial proteins. Moreover, both HIFα isoforms were heavily covalently modified. We identified ~40 PTM sites composed of 13 different types of modification on both HIFα isoforms, including multiple cysteine modifications and an unusual phosphocysteine. More than 80% of the PTMs identified were not previously known and about half exhibited oxygen dependency. We further characterized an evolutionarily conserved phosphorylation of Ser31 in HIF-1α as a regulator of its transcriptional function, and we propose functional roles for Thr406, Thr528, and Ser581 in HIF-2α. These data will help to delineate the different physiological roles of these closely related isoforms in fine-tuning the hypoxic response.

Systems approaches to understand oxygen sensing: how multi-omics has driven advances in understanding oxygen-based signalling

“…The activity of HIF, although primarily controlled by the activity and availability of the HIF-α subunits by PHDs and VHL, can be modulated and tuned by other modifying enzymes and interacting proteins [3][4][5]84]. Unbiased MS experiments have revealed that the PTM landscape and interaction partners if HIF transcription factors are in fact extremely complex [82,85]. The phosphosite plus repository of post-translational modifications of individual proteins either identified from MS or throughput approaches, reveals that HIF-1α, HIF-2α, HIF-3α and HIF-1β have 73, 39, 20 and 29 unique PTMs, respectively [86,87].…”

“…However, this is likely to be an underestimation of the real level of PTMs present. Recent detailed analysis specifically analysing phosphorylation of HIF-1α and HIF-2α identified 41 and 39 different phosphosites on HIF-1α and HIF-2α respectively, including evidence of non-canonical phosphorylation of cystine residues [ 85 ]. Critically this study examined changes to the phosphorylation landscape on HIF-1α and HIF-2α in response to both normal oxygen and hypoxic conditions in cervical carcinoma cells [ 85 ].…”

“…Recent detailed analysis specifically analysing phosphorylation of HIF-1α and HIF-2α identified 41 and 39 different phosphosites on HIF-1α and HIF-2α respectively, including evidence of non-canonical phosphorylation of cystine residues [ 85 ]. Critically this study examined changes to the phosphorylation landscape on HIF-1α and HIF-2α in response to both normal oxygen and hypoxic conditions in cervical carcinoma cells [ 85 ]. Interestingly, more than 50% of the mapped phosphorylation sites on the HIF-α subunits were differentially observed at different oxygen tensions [ 85 ].…”

“…Critically this study examined changes to the phosphorylation landscape on HIF-1α and HIF-2α in response to both normal oxygen and hypoxic conditions in cervical carcinoma cells [ 85 ]. Interestingly, more than 50% of the mapped phosphorylation sites on the HIF-α subunits were differentially observed at different oxygen tensions [ 85 ]. Further analysis may reveal that the dynamic nature of this combinatory PTM code of HIF can alter its activity in response to specific stimuli or timing, as seen in the combinatorial modification of the NF-κB transcription factor in response to different stimuli [ 88 ].…”

Detection of Hypoxia in Cancer Models: Significance, Challenges, and Advances

“…Both HIF-1 and HIF-2 heterodimers recognize and bind to the 5′-ACGTG-3′ enhancer sequence, resulting in the transcriptional regulation of more than a thousand genes [ 10 , 11 ]. The abundance and activity of both HIF-1α and HIF-2α can also be enhanced due to post-translational modifications [ 12 ]. HIF-regulated genes have been associated with angiogenesis, apoptosis, cell proliferation, cell survival, metabolism, invasion, metastasis, altered pH, and chemoresistance [ 5 ].…”