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

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 ß (IκBß). 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-IκBß oxomer formation was highly selective. In contrast to the known FIH-dependent oxomer formation with the deubiquitinase OTUB1, FIH-IκBß oxomer formation did not occur via an IκBß asparagine residue, but depended on the amino acid sequence VAERR contained within a loop between IκBß ankyrin repeat domains 2 and 3. Oxomer formation prevented IκBß 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 IκBß contributes to the hypoxia-dependent regulation of inflammation.

Oxomer- and Reporter Gene-Based Analysis of FIH Activity in Cells.

Cellular and tissue adaptations to oxygen deprivation (hypoxia) are necessary for both normal physiology and disease. Responses to hypoxia are initiated by the cellular oxygen sensors prolyl-4-hydroxylase domain (PHD) proteins 1-3 and factor inhibiting HIF (FIH). These enzymes regulate the transcription factor hypoxia-inducible factor (HIF) in a hypoxia-sensitive manner. FIH also regulates proteins outside the HIF pathway, including the deubiquitinase OTUB1. Numerous preclinical analyses have demonstrated that treatment with HIF hydroxylase inhibitors is beneficial and protective in many hypoxia-associated diseases. However, clinically available HIF hydroxylase inhibitors increase erythropoietin (EPO) gene expression and red blood cell production, which can be detrimental in hypoxia-associated conditions, such as ischemia/reperfusion injury of the heart or chronic inflammation. Our understanding of the relevance of FIH in (patho)physiology is only in its infancy, but FIH activity does not govern erythropoietin expression. Therefore, it is of prime interest to assess the relevance of FIH activity in (patho)physiology in detail, as it may contribute to developing novel therapeutic options for treating hypoxia-associated diseases that do not affect Epo regulation. Here, we describe specific protocols for two different methods to assess FIH enzymatic activity within cells, using a HIF-dependent firefly luciferase-reporter gene and an oxomer-dependent assay. Oxomers are oxygen-dependent stable protein oligomers formed by FIH, for example, with the deubiquitinase OTUB1. Oxomer formation directly depends on FIH activity, providing a suitable cellular readout for an easy-to-use analysis of FIH enzymatic activity in cellulo. These techniques permit an analysis of FIH activity toward HIF and outside the HIF pathway, allowing the investigation of FIH activity under different (patho)physiological conditions and assessment of novel (putative) inhibitors.

The Asparagine Hydroxylase FIH: A Unique Oxygen Sensor.

Significance: Limited oxygen availability (hypoxia) commonly occurs in a range of physiological and pathophysiological conditions, including embryonic development, physical exercise, inflammation, and ischemia. It is thus vital for cells and tissues to monitor their local oxygen availability to be able to adjust in case the oxygen supply is decreased. The cellular oxygen sensor factor inhibiting hypoxia-inducible factor (FIH) is the only known asparagine hydroxylase with hypoxia sensitivity. FIH uniquely combines oxygen and peroxide sensitivity, serving as an oxygen and oxidant sensor. Recent Advances: FIH was first discovered in the hypoxia-inducible factor (HIF) pathway as a modulator of HIF transactivation activity. Several other FIH substrates have now been identified outside the HIF pathway. Moreover, FIH enzymatic activity is highly promiscuous and not limited to asparagine hydroxylation. This includes the FIH-mediated catalysis of an oxygen-dependent stable (likely covalent) bond formation between FIH and selected substrate proteins (called oxomers [oxygen-dependent stable protein oligomers]). Critical Issues: The (patho-)physiological function of FIH is only beginning to be understood and appears to be complex. Selective pharmacologic inhibition of FIH over other oxygen sensors is possible, opening new avenues for therapeutic targeting of hypoxia-associated diseases, increasing the interest in its (patho-)physiological relevance. Future Directions: The contribution of FIH enzymatic activity to disease development and progression should be analyzed in more detail, including the assessment of underlying molecular mechanisms and relevant FIH substrate proteins. Also, the molecular mechanism(s) involved in the physiological functions of FIH remain(s) to be determined. Furthermore, the therapeutic potential of recently developed FIH-selective pharmacologic inhibitors will need detailed assessment. Antioxid. Redox Signal. 37, 913-935.

OTUB1 regulates lung development, adult lung tissue homeostasis, and respiratory control.

OTUB1 is one of the most highly expressed deubiquitinases, counter-regulating the two most abundant ubiquitin chain types. OTUB1 expression is linked to the development and progression of lung cancer and idiopathic pulmonary fibrosis in humans. However, the physiological function of OTUB1 is unknown. Here, we show that constitutive whole-body Otub1 deletion in mice leads to perinatal lethality by asphyxiation. Analysis of (single-cell) RNA sequencing and proteome data demonstrated that OTUB1 is expressed in all lung cell types with a particularly high expression during late-stage lung development (E16.5, E18.5). At E18.5, the lungs of animals with Otub1 deletion presented with increased cell proliferation that decreased saccular air space and prevented inhalation. Flow cytometry-based analysis of E18.5 lung tissue revealed that Otub1 deletion increased proliferation of major lung parenchymal and mesenchymal/other non-hematopoietic cell types. Adult mice with conditional whole-body Otub1 deletion (wbOtub1del/del ) also displayed increased lung cell proliferation in addition to hyperventilation and failure to adapt the respiratory pattern to hypoxia. On the molecular level, Otub1 deletion enhanced mTOR signaling in embryonic and adult lung tissues. Based on these results, we propose that OTUB1 is a negative regulator of mTOR signaling with essential functions for lung cell proliferation, lung development, adult lung tissue homeostasis, and respiratory regulation.