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

Abstract

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.

HIF hydroxylase inhibitors decrease cellular oxygen consumption depending on their selectivity

“…A possible explanation is that the sensitivity of FIH to DM‐NOFD depends on its protein substrate. We have previously shown that the hypoxia‐sensitivity of FIH‐dependent OTUB1 HD formation is much higher than the published sensitivity of HIF‐α asparagine hydroxylation, further supporting that FIH binding of cofactors and cosubstrates, and probably also of cosubstrate mimetics, depends on the protein substrate. It would be of interest to compare the DM‐NOFD sensitivity of FIH‐dependent HIF‐α asparagine hydroxylation and HD formation within the same cell lysates.…”

“…To date, it has been difficult to determine the target selectivity of HIs in cells because of the lack of a widely‐available and easily applicable readout, allowing for the efficient quantification of their HIF‐α prolyl and asparaginyl hydroxylase inhibition activities. We have previously shown that FIH forms a stable, likely covalent, heterodimeric complex with the deubiquitinase OTUB1, which is FIH hydroxylase activity‐dependent and can be detected by immunoblotting . Hence, the formation of the FIH‐OTUB1 heterodimer (HD) complex can be used as a specific read‐out for FIH inhibition.…”

“…We have previously shown that FIH forms a stable, likely covalent, heterodimeric complex with the deubiquitinase OTUB1, which is FIH hydroxylase activity-dependent and can be detected by immunoblotting. 15 Hence, the formation of the FIH-OTUB1 heterodimer (HD) complex can be used as a specific read-out for FIH inhibition. It is established that the stabilization of HIF-1α and HIF-2α is regulated through prolyl-4-hydroxylation by the PHDs and not through FIH.…”

“…The HD represents a (likely) covalently conjugated FIH-OTUB1 protein complex which we previously described. 15 B, Quantification of HIF-1α protein levels described in (A) normalized to α-tubulin levels and to HIF-1α levels detected in hypoxia (red broken line). Shown normoxia values were derived from the immunoblots containing the DFX samples.…”

“…[9][10][11][12] FIH also hydroxylates the deubiquitinase (DUB) ovarian tumor domain-containing ubiquitin aldehyde binding protein 1 (OTUB1), 13,14 with which it forms a covalently linked protein complex in an oxygen-dependent manner by affecting the OTUB1 DUB activity. 15 The HIF transcription factor was originally identified through the investigations of the regulation of erythropoietin (Epo) gene expression. 16 Epo is the essential hormone for the regulation of red blood cell formation, affecting oxygen transport in the blood.…”