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

Christina Pickel,Julia Günter,Amalia Ruiz-Serrano,Patrick Spielmann,Jacqueline-Alba Fabrizio,Witold Wolski,Daniel J Peet,Roland H Wenger,Carsten C Scholz

doi:10.1016/j.redox.2019.101265

Christina Pickel, Julia Günter + Show 7 more

Open Access

https://doi.org/10.1016/j.redox.2019.101265

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Journal: Redox Biology	Publication Date: Jul 2, 2019
Citations: 17	License type: cc-by

Affiliation: University of Adelaide, University of Zurich

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.

Highlights

Cellular oxygen sensing is of vital importance for cells and tissues in order to adapt to hypoxic conditions when cellular oxygen demand exceeds its supply [1]
We recently demonstrated that factor inhibiting hypoxia inducible factor (HIF) (FIH) interacts with the deubiquitinase (DUB) ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1) and hydroxylates it on asparagine 22 (N22), regulating cellular energy metabolism [17,18]
We demonstrate that this formation has functional consequences for OTUB1 deubiquitinase activity and is highly oxygen sensitive but relatively slow, indicating a role in chronic hypoxia adaptation

Summary

Introduction

Cellular oxygen sensing is of vital importance for cells and tissues in order to adapt to hypoxic conditions when cellular oxygen demand exceeds its supply [1]. The best characterized cellular oxygen sensors are the prolyl-4-hydroxylase domain (PHD) proteins 1–3 and factor inhibiting HIF (FIH) [2]. PHDs hydroxylate two different prolines and FIH hydroxylates one asparagine residue of HIFα subunits [2]. Besides molecular oxygen, these enzymes require Fe2+ and ascorbate or other reducing agents as co-factors and 2-oxoglutarate as co-substrate in order to reduce molecular oxygen and oxidize the substrate protein (hydroxylation) and 2-oxoglutarate (conversion to succinate) [3,4]. Proline-4-hydroxylation of HIFα leads to its proteasomal degradation whereas asparagine hydroxylation inhibits its interaction with the transcriptional co-activators p300 and CBP, attenuating HIF-dependent gene transactivation [2]. While in higher organisms the only known reaction of 2-oxoglutarate-dependent dioxygenases is hydroxylation [5], in lower organisms they catalyse ring expansion, rearrangement, desaturation, halogenation and epoxidation [6]

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