Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
J Biol Chem. 2012 Jul 20;287(30):25098-110. doi: 10.1074/jbc.M112.355446. Epub 2012 May 30.
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.
在不同类型的生物体中，细胞缺氧反应是由脯氨酰 4-羟化酶介导的，该酶利用 O(2)和α-酮戊二酸作为底物，使靶蛋白中的保守脯氨酸残基羟化。在后生动物中，这些酶控制着 HIFα 转录因子亚基家族的稳定性，而粘菌的酶（DdPhyA）通过一种不同的机制，通过羟化和随后的 DdSkp1 糖基化来参与 O(2)对发育的调节，DdSkp1 是 E3(SCF)泛素连接酶中的衔接子亚基。与 DdPhyA、DdSkp1 和糖基转移酶相关的序列也存在于弓形虫和其他原生动物的基因组中，这表明这种 O(2)感应机制可能很普遍。在这里，我们通过破坏 TgphyA 基因座表明，该酶是弓形虫 Skp1 糖基化所必需的，并且破坏的寄生虫在生理 O(2)水平下生长缓慢。通过在 DdphyA 缺失细胞中表达 TgPhyA 来测试细胞功能的保守性。简单的基因替换不能挽救 Skp1 糖基化，而过度表达不仅纠正了 Skp1 的修饰，而且还将 O(2)的需求恢复到与过度表达的 DdPhyA 相当的水平。细菌表达的 TgPhyA 蛋白可以使弓形虫和粘菌的 Skp1 发生脯氨酰羟化。动力学分析表明，TgPhyA 具有与 DdPhyA 相似的特性，包括对其共底物α-酮戊二酸浓度的依赖性相似。然而，值得注意的是，TgPhyA 对 O(2)的表观亲和力明显更高。这些发现表明，Skp1 的羟化作用是原生动物中普遍存在的过程，并且这种生化途径可能通过检测 O(2)调节的代谢物（如α-酮戊二酸）的水平间接感应 O(2)。
