Selective Hypoxia-Sensitive Oxomer Formation by FIH Prevents Binding of the NF-κB Inhibitor IκBβ to NF-κB Subunits

Abstract

Pharmacologic inhibitors of cellular hydroxylase oxygen sensors are protective in multiple preclinical in vivo models of inflammation. However, the molecular mechanisms underlying this regulation are only partly understood, preventing clinical translation. We previously proposed a new mechanism for cellular oxygen sensing: oxygen-dependent, (likely) covalent protein oligomer (oxomer) formation. Here, we report that the oxygen sensor factor inhibiting HIF (FIH) forms an oxomer with the NF-κB inhibitor β (IκBβ). The formation of this protein complex required FIH enzymatic activity and was prevented by pharmacologic inhibitors. Oxomer formation was highly hypoxia-sensitive and very stable. No other member of the IκB protein family formed an oxomer with FIH, demonstrating that FIH-IκBβ oxomer formation was highly selective. In contrast to the known FIH-dependent oxomer formation with the deubiquitinase OTUB1, FIH-IκBβ oxomer formation did not occur via an IκBβ asparagine residue, but depended on the amino acid sequence VAERR contained within a loop between IκBβ ankyrin repeat domains 2 and 3. Oxomer formation prevented IκBβ from binding to its primary interaction partners p65 and c-Rel, subunits of NF-κB, the master regulator of the cellular transcriptional response to pro-inflammatory stimuli. We therefore propose that FIH-mediated oxomer formation with IκBβ contributes to the hypoxia-dependent regulation of inflammation.

Keywords:

• HIF1AN
• HIF
• hydroxylase inhibitor
• inflammation
• oxygen sensor

Acknowledgments

We thank Prof. Daniel J. Peet (Department of Molecular and Biomedical Science, University of Adelaide, Australia) for the gifts of plasmids and Dr. Anja Bremm (Institute of Biochemistry II, Goethe-University, Frankfurt am Main, Germany) for providing FIH KO HEK293 cells.

Disclosure statement

The authors report there are no competing interests to declare.

Data availability statement

All underlying raw data have been made available on https://dataverse.harvard.edu/. The data has obtained the DOI 10.7910/DVN/XYXNPA and is accessible under https://dataverse.harvard.edu/privateurl.xhtml?token=29762520-1072-418d-873a-5a110caa04cf. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE⁶⁰ partner repository with the dataset identifier PXD047442.

Additional information

Funding

No funding was received.