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Updated: Jun 28, 2025
Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
Published on: August 2, 2018
Selective Hypoxia-Sensitive Oxomer Formation by FIH Prevents Binding of the NF-κB Inhibitor IκBβ to NF-κB Subunits
Yulia L Volkova1, Agnieszka E Jucht1, Nina Oechsler2
1Institute of Physiology, University of Zurich, Zurich, Switzerland.
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.
Insights
Factor Inhibiting HIF (FIH) forms a novel oxygen-dependent complex with IκBβ, inhibiting NF-κB signaling. This discovery sheds light on hypoxia-dependent inflammation regulation and potential therapeutic targets.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Biology
Background:
- Pharmacologic inhibitors of cellular hydroxylase oxygen sensors show protective effects in preclinical inflammation models.
- Understanding the molecular mechanisms of oxygen sensing is crucial for clinical translation.
- A novel mechanism of cellular oxygen sensing involving oxygen-dependent protein oligomer (oxomer) formation was previously proposed.
Purpose of the Study:
- To investigate the formation and function of oxygen-dependent protein oligomers (oxomers) in cellular oxygen sensing.
- To identify novel interactions of the oxygen sensor Factor Inhibiting HIF (FIH).
- To elucidate the role of FIH-mediated oxomer formation in the regulation of inflammation.
Main Methods:
- Investigated the interaction between FIH and IκBβ using biochemical assays.
- Assessed the requirement of FIH enzymatic activity for oxomer formation.
- Utilized pharmacologic inhibitors to block oxomer formation.
- Determined the selectivity of FIH-IκBβ oxomer formation among IκB protein family members.
- Mapped the specific amino acid sequence responsible for FIH-IκBβ oxomer formation.
- Examined the impact of oxomer formation on NF-κB signaling pathway components.
Main Results:
- FIH forms a highly selective, hypoxia-sensitive, and stable oxomer with IκBβ, requiring FIH enzymatic activity.
- This FIH-IκBβ oxomer formation is distinct from FIH-OTUB1 interaction and depends on a specific VAERR sequence in IκBβ.
- Oxomer formation prevents IκBβ from binding to NF-κB subunits p65 and c-Rel.
- No other IκB protein family member formed an oxomer with FIH.
Conclusions:
- FIH-mediated oxomer formation with IκBβ represents a novel mechanism for cellular oxygen sensing.
- This interaction plays a role in the hypoxia-dependent regulation of inflammation by inhibiting NF-κB signaling.
- The FIH-IκBβ oxomer pathway offers potential therapeutic targets for inflammatory diseases.
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