Hif-α non-canonical regulation by PTMs and new interaction partners

All human cells need oxygen for energy production by oxidative phosphorylation. If the oxygen levels are low, cells switch from oxidative phosphorylation to glycolysis. The Hif1α subunit of the transcription factor HIF-1 is essential for this regulatory switch. The switch is regulated by the oxygen level-dependent formation of hydroxyproline on the Hif1α subunit, which in turn is recognized by the VHL-E3 ligase leading to Hif1α's degradation by the ubiquitin-proteasome system. In recent years other pathways for the regulation of Hif1α emerged, including other degradation pathways or the modulation of translation, leading to higher expression of Hif1α.
As one of the metabolic master regulators for metabolic switching from hypoxic to normoxic conditions and vice versa, Hif1α is important for disease progression prediction. Hif1α has been related to shifting the metabolic programs on the inner parts of solid tumors, like gliomas, lung tumors, and colon carcinomas, by modulating the tumors ability to survive treatment by chemotherapeutic.
Ubiquitination is one of the major cellular signaling pathways. Unlike other signaling pathways, ubiquitin can transmit different signals depending on the target protein's modification by attaching a single ubiquitin moiety or forming a ubiquitin chain, consisting of several ubiquitins connected covalently to each other. Ubiquitin modification is catalyzed by a multi-enzyme cascade and can be negatively regulated by deubiquitinating enzymes, which cut the ubiquitin modification from the target or disassembling the ubiquitin chains.
Using a newly developed screening technology, Prisma, we identified a significant number of new interaction partners of Hif1α, many of which belong to the ubiquitin signaling system and the ubiquitin-proteasome degradation system. The screen identified three E3 ligases, which have not been reported as interactors before, connecting Hif1α degradation to new degradation pathways. The characterization of these new degradation pathways for Hif1α will allow targeting Hif1α by different means.
A second class of proteins related to ubiquitin signaling is a number of deubiquitinases, which were also identified as Hif1α interactors. Nine different hydrolases were identified on the screen, pointing to a complicated negative regulation of Hif1α. Deubiquitinases are single-molecule enzymes, and recent inhibitor development allows the direct targeting of several of these enzymes, allowing the modulation of ubiquitin signaling.
The Prisma screening technology allows the identification of the interaction site within Hif1α and can quantify the interactome changes induced by small post-translational modifications like phosphorylations, acetylations, or methylations. The Prisma screen with Hif1α revealed several phosphorylation sites, which are necessary for the recruitment of deubiquitinases to a specific site in Hif1α. The proposed project will study the impact of these PTMs on the function and regulation of Hif1α stability and the impact on metabolic switching.
Post-translational modifications are often understudied concerning diseases, as the detection of PTMs in disease material is difficult and can only be done using protein-based technologies. We will investigate the presence of the different known Hif1α modifications using targeted proteomics on the Luxembourgish colon carcinoma cohort. Here we will focus our effortson measuring the PTMs important for ubiquitin signaling and characterize their presence in the samples in the normoxic and hypoxic parts of the tumor.