Abstract
The asparaginyl hydroxylase, Factor Inhibiting HIF (FIH), is a cellular dioxygenase. Originally identified as oxygen sensor in the cellular response to hypoxia, where FIH acts as a repressor of the hypoxia inducible transcription factor alpha (HIF-α) proteins through asparaginyl hydroxylation, FIH also hydroxylates many proteins that contain ankyrin repeat domains (ARDs). Given FIH’s promiscuity and the unclear functional effects of ARD hydroxylation, the biological relevance of HIF-α and ARD hydroxylation remains uncertain. Here, we have employed evolutionary and enzymatic analyses of FIH, and both HIF-α and ARD-containing substrates, in a broad range of metazoa to better understand their conservation and functional importance. Utilising Tribolium castaneum and Acropora millepora, we provide evidence that FIH from both species are able to hydroxylate HIF-α proteins, supporting conservation of this function beyond vertebrates. We further demonstrate that T. castaneum and A. millepora FIH homologs can also hydroxylate specific ARD proteins. Significantly, FIH is also conserved in several species with inefficiently-targeted or absent HIF, supporting the hypothesis of important HIF-independent functions for FIH. Overall, these data show that while oxygen-dependent HIF-α hydroxylation by FIH is highly conserved in many species, HIF-independent roles for FIH have evolved in others.
Highlights
In mammals, communication of oxygen availability within cells is achieved in part by enzymes which directly use dioxygen as a co-substrate [1]
Through functional testing of these enzymes, we provide the first evidence of active Factor Inhibiting Hypoxia-Inducible Factor (HIF) (FIH) enzymes outside of the Vertebrata, and demonstrate that ankyrin repeat domains (ARDs) hydroxylation is likely conserved among metazoan FIHs, while HIF-α C-terminal transactivation domain (CAD) modulation is less consistently conserved
Species which had undergone large-scale sequencing of their genome or transcriptome were included in the analysis, unless both FIH and HIF-α CAD homologs could be found amongst less comprehensive sequence submissions
Summary
Communication of oxygen availability within cells is achieved in part by enzymes which directly use dioxygen as a co-substrate [1]. One such enzyme is the oxygen and 2-oxoglutarate (2-OG)-dependent dioxygenase, Factor Inhibiting HIF (FIH), an asparaginyl hydroxylase first characterised through its role in modulation of the Hypoxia-Inducible Factor (HIF) transcription factors [2,3,4,5]. The HIFs, which are master regulators of the genomic response to hypoxia (oxygen insufficiency), are dimers of two basic helix-loop-helix-Per ARNT Sim homology (bHLH-PAS) transcription factors: an oxygen-responsive α-subunit (Fig 1A), which may be any of HIF-1α, -2α or -3α, and a common, constitutively active β-subunit, known as the aryl hydrocarbon. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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