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.

细胞对低氧环境的适应部分是由缺氧诱导因子（HIF）介导的。与其他转录因子一样，HIF 的稳定性和转录活性（以及由此产生的缺氧反应）受到翻译后修饰 (PTM) 和蛋白质-蛋白质相互作用的变化的调节。我们目前对 PTM 介导的 HIF 调节的理解主要基于基于体外蛋白质片段的研究，这些研究通常在用模拟缺氧化合物处理的片段表达细胞中进行验证。在这里，我们使用基于免疫沉淀的质谱法来表征全长 HIF-1α 和 HIF-2α 在常氧（21% 氧气）和低氧（1% 氧气）条件下的 PTM 和结合伴侣。缺氧显着改变了 HIFα 蛋白相互作用网络的复杂性和组成，特别是 HIF-2α，两种亚型的缺氧网络都富集了线粒体蛋白。此外，两种 HIFα 亚型均经过大量共价修饰。我们在两种 HIFα 同工型上鉴定了约 40 个 PTM 位点，由 13 种不同类型的修饰组成，包括多个半胱氨酸修饰和不寻常的磷酸半胱氨酸。超过 80% 的已鉴定 PTM 以前未知，大约一半表现出氧依赖性。我们进一步表征了 HIF-1α 中 Ser ³¹的进化保守磷酸化作为其转录功能的调节剂，并提出了 HIF-2α 中 Thr ⁴⁰⁶ 、 Thr ⁵²⁸和 Ser ^{581 的}功能作用。这些数据将有助于描述这些密切相关的亚型在微调缺氧反应中的不同生理作用。