Characterization of ankyrin repeat-containing proteins as substrates of the asparaginyl hydroxylase factor inhibiting hypoxia-inducible transcription factor.

摘要: The hypoxia‐inducible transcription factors (HIFs) are essential mediators of the genomic response to oxygen deficiency (hypoxia) in multicellular organisms. HIFs regulated by four oxygen‐sensitive hydroxylases—three prolyl hydroxylases and one asparaginyl hydroxylase. These all members 2‐oxoglutarate (2OG)–dependent dioxygenase superfamily convey changes cellular concentration HIF‐alpha (α) subunit, leading potent accumulation activity hypoxia versus degradation repression normoxia. HIF‐α hydroxylation is catalyzed factor‐inhibiting HIF‐1 (FIH‐1) directly regulates proteins. Recent work has demonstrated that, addition hydroxylating HIF‐α, FIH‐1 can also hydroxylate ankyrin domains a wide range This paper presents vitro cell‐based techniques for preliminary characterization domain–containing proteins as substrates interacting Strategies presented expression purification from mammalian or bacterial cells. Similar proteins, ankyrin‐containing examined purified expressed bacteria overexpressed cells form synthetic peptides. Specific conditions efficient compared with Escherichia coli detailed. Hydroxylation rapidly inferred, utilizing described CO2 capture assay. Finally, substrate non‐substrate interactions using affinity pull‐down assays co‐immunoprecipitation assays. Together, these methods rapid well suited potential therapeutically relevant oxygen‐sensing enzyme FIH‐1.