Article ; Online: Oxomer- and Reporter Gene-Based Analysis of FIH Activity in Cells.
Methods in molecular biology (Clifton, N.J.)
2024 Volume 2755, Page(s) 249–264
Abstract: 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 ... ...
| Abstract | 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. |
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| MeSH term(s) | Humans ; Genes, Reporter ; Erythropoietin/genetics ; Mixed Function Oxygenases ; Hypoxia ; Oxygen ; Deubiquitinating Enzymes |
| Chemical Substances | Erythropoietin (11096-26-7) ; Mixed Function Oxygenases (EC 1.-) ; Oxygen (S88TT14065) ; Deubiquitinating Enzymes (EC 3.4.19.12) |
| Language | English |
| Publishing date | 2024-02-06 |
| Publishing country | United States |
| Document type | Journal Article |
| ISSN | 1940-6029 |
| ISSN (online) | 1940-6029 |
| DOI | 10.1007/978-1-0716-3633-6_18 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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