Abstract
In diverse types of organisms, cellular hypoxic responses are mediated by prolyl 4-hydroxylases that use O(2) and α-ketoglutarate as substrates to hydroxylate conserved proline residues in target proteins. Whereas in metazoans these enzymes control the stability of the HIFα family of transcription factor subunits, the Dictyostelium enzyme (DdPhyA) contributes to O(2) regulation of development by a divergent mechanism involving hydroxylation and subsequent glycosylation of DdSkp1, an adaptor subunit in E3(SCF) ubiquitin ligases. Sequences related to DdPhyA, DdSkp1, and the glycosyltransferases that cap Skp1 hydroxyproline occur also in the genomes of Toxoplasma and other protists, suggesting that this O(2) sensing mechanism may be widespread. Here we show by disruption of the TgphyA locus that this enzyme is required for Skp1 glycosylation in Toxoplasma and that disrupted parasites grow slowly at physiological O(2) levels. Conservation of cellular function was tested by expression of TgPhyA in DdphyA-null cells. Simple gene replacement did not rescue Skp1 glycosylation, whereas overexpression not only corrected Skp1 modification but also restored the O(2) requirement to a level comparable to that of overexpressed DdPhyA. Bacterially expressed TgPhyA protein can prolyl hydroxylate both Toxoplasma and Dictyostelium Skp1s. Kinetic analyses showed that TgPhyA has similar properties to DdPhyA, including a superimposable dependence on the concentration of its co-substrate α-ketoglutarate. Remarkably, however, TgPhyA had a significantly higher apparent affinity for O(2). The findings suggest that Skp1 hydroxylation by PhyA is a conserved process among protists and that this biochemical pathway may indirectly sense O(2) by detecting the levels of O(2)-regulated metabolites such as α-ketoglutarate.
Highlights
A cytoplasmic prolyl 4-hydroxylase of Dictyostelium contributes to O2 sensing by modifying Skp[1]
Whereas in metazoans these enzymes control the stability of the HIF␣ family of transcription factor subunits, the Dictyostelium enzyme (DdPhyA) contributes to O2 regulation of development by a divergent mechanism involving hydroxylation and subsequent glycosylation of DdSkp[1], an adaptor subunit in E3SCF ubiquitin ligases
3 The abbreviations used are: prolyl 4-hydroxylases (P4Hs), prolyl 4-hydroxylase; HIF␣, hypoxia-inducible factor-␣; PHD, animal prolyl 4-hydroxylase domain protein (HIF␣-targeting); ␣KG, ␣-ketoglutarate (2-oxoglutarate); HFF, human foreskin fibroblast; Hyp, 4R,2S-hydroxyproline (or 4(trans)hydroxy-L-proline); Ub, ubiquitin; TgPhyA, Toxoplasma PhyA; rER, rough endoplasmic reticulum; SCF, protein complex consisting of SKP1, Cullin-1, RGX1, and an
Summary
A cytoplasmic prolyl 4-hydroxylase of Dictyostelium contributes to O2 sensing by modifying Skp[1]. In diverse types of organisms, cellular hypoxic responses are mediated by prolyl 4-hydroxylases that use O2 and ␣-ketoglutarate as substrates to hydroxylate conserved proline residues in target proteins. Whereas in metazoans these enzymes control the stability of the HIF␣ family of transcription factor subunits, the Dictyostelium enzyme (DdPhyA) contributes to O2 regulation of development by a divergent mechanism involving hydroxylation and subsequent glycosylation of DdSkp[1], an adaptor subunit in E3SCF ubiquitin ligases. Under conditions of normoxia and sufficient metabolic availability of ␣KG, cytoplasmic animal P4Hs (known as PHDs, for prolyl hydroxylase domain containing) hydroxylate the HIF␣ family of transcriptional factor subunits, resulting in recognition by the von Hippel-Lindau tumor suppressor protein (2). Oxidants like H2O2 or other metals may influence PHD activity via effects on the ascorbate pool, indirectly affect-
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