Oxygen-dependent bond formation with FIH regulates the activity of the client protein OTUB1

Protein:protein interactions are the basis of molecular communication and are usually of transient non-covalent nature, while covalent interactions other than ubiquitination are rare. For cellular adaptations, the cellular oxygen and peroxide sensor factor inhibiting HIF (FIH) confers oxygen and oxidant stress sensitivity to the hypoxia inducible factor (HIF) by asparagine hydroxylation. We investigated whether FIH contributes to hypoxia adaptation also through other mechanisms and identified a hypoxia sensitive, likely covalent, bond formation by FIH with several client proteins, including the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1). Biochemical analyses were consistent with a co-translational amide bond formation between FIH and OTUB1, occurring within mammalian and bacterial cells but not between separately purified proteins. Bond formation is catalysed by FIH and highly dependent on oxygen availability in the cellular microenvironment. Within cells, a heterotrimeric complex is formed, consisting of two FIH and one covalently linked OTUB1. Complexation of OTUB1 by FIH regulates OTUB1 deubiquitinase activity. Our findings reveal an alternative mechanism for hypoxia adaptation with remarkably high oxygen sensitivity, mediated through covalent protein-protein interactions catalysed by an asparagine modifying dioxygenase.