Hypoxia up-regulates prolyl hydroxylase activity - A feedback mechansim that limits HIF-1 responses during reoxygenation
Hypoxia up-regulates prolyl hydroxylase activity - A feedback mechansim that limits HIF-1 responses during reoxygenation
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DOI：
10.1074/jbc.m302244200
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发表时间：
2003-10-03
影响因子：
4.8
通讯作者：
Frelin, C
中科院分区：
文献类型：
--
作者：
D'Angelo, G;Duplan, E;Frelin, C
低氧诱导基因转录的机制现在已经很好地建立起来。缺氧可降低脯氨酰羟化酶（PHD）的活性，该酶可羟化缺氧诱导因子-1 α（HIF-1 α）氧依赖性降解结构域（ODD）中的特定脯氨酸残基。因此，HIF-1 α积累并通过激活基因转录促进低氧耐受。本文鉴定了大鼠PHDs的三种形式，并表明一段时间的缺氧选择性地增加PHD-2 mRNA水平的表达。我们开发了PHD活性测定法，该测定法使用（i）标记的2-氧戊二酸肽特异性转化为琥珀酸，以及（ii）von Hippel-Lindau蛋白与谷胱甘肽S-转移酶-ODD融合蛋白的结合。这两项测定表明，常氧和缺氧细胞中的酶活性较低，而在复氧期间迅速增加。我们还开发了羟脯氨酸特异性抗体，该抗体识别融合蛋白（ODD-绿色荧光蛋白）的羟基化形式，该融合蛋白结合了HIF-1 α的ODD结构域和绿色荧光蛋白。使用这种抗体，我们证明了复氧诱导Pro-564的快速羟基化，随后是蛋白质的大量降解。结果表明，缺氧上调PHD（推测PHD-2）作为一种反馈机制，以停止缺氧反应在复氧细胞。我们认为脯氨酸羟化可能在低氧预适应中起作用。
The mechanism by which hypoxia induces gene transcription is now well established. Hypoxia reduces activity of prolyl hydroxylases (PHD) that hydroxylate specific proline residues in the oxygen-dependent degradation domain (ODD) of hypoxia-inducible factor-1alpha (HIF-1alpha). As a consequence, HIF-1alpha accumulates and promotes hypoxic tolerance by activating gene transcription. This paper identifies the three forms of PHDs in rats and shows that a period of hypoxia selectively increases expression of PHD-2 mRNAs levels. We developed assays for PHD activity that used (i) the peptide-specific conversion of labeled 2-oxoglutarate into succinate and (ii) the binding of the von Hippel-Lindau protein to a glutathione S-transferase-ODD fusion protein. The two assays indicated a low enzymatic activity in normoxic and hypoxic cells and a rapid increase during reoxygenation. We also developed hydroxyproline-specific antibodies that recognized hydroxylated forms of a fusion protein (ODD-green fluorescent protein) that combined the ODD domain of HIF-1alpha and the green fluorescent protein. Using this antibody, we demonstrated that reoxygenation induced a rapid hydroxylation of Pro-564, which was followed by a massive degradation of the proteins. The results suggest that a hypoxic up-regulation of PHD (presumably PHD-2) acts as a feedback mechanism to stop hypoxic responses in reoxygenated cells. We propose that proline hydroxylation might play a role in hypoxic preconditioning.