Leu-574 of human HIF-1a is a molecular determinant of prolyl hydroxylation
Leu-574 of human HIF-1a is a molecular determinant of prolyl hydroxylation
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DOI：
10.1096/fj.03-1233fje
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发表时间：
2004-04-01
期刊：
影响因子：
4.8
通讯作者：
Huang, LE
中科院分区：
文献类型：
--
作者：
Kageyama, Y;Koshiji, M;Huang, LE
缺氧诱导因子 (HIF)-1alpha 是氧稳态的主要调节因子，调节对细胞生长和存活至关重要的基因。在常氧条件下，HIF-1α 通过泛素-蛋白酶体途径不断降解。 von Hippel-Lindau (VHL) E3 泛素连接酶通过特异性识别羟基化 Pro-402 或 Pro-564 来结合 HIF-1α，这两者均由氧依赖性 HIF 脯氨酰羟化酶 (PHD/HPH) 修饰。尽管鉴定出保守的 Leu-X-X-Leu-Ala-Pro 基序，但 HIF-1α 对 PHD/HPH 结合的分子要求仍然难以捉摸。最近，我们证明 Pro-564 下游的人 HIF-1alpha-10 残基的 Leu-574 对于 VHL 识别至关重要。我们在此表明 Leu-574 的作用是招募 PHD2/HPH2 进行 Pro-564 羟基化。羟基化 Pro-564 特异性抗体已用于确定羟基化状态； Leu-574的突变或缺失导致羟基化HIF-1α占总量的比例显着下降。 Pro-564 和 Leu-574 之间的九个残基间隔对于脯氨酰羟基化不是必需的。此外，Leu-574 的突变会破坏 PHD2/HPH2 的结合，PHD2/HPH2 是 HIF-1α 氧依赖性蛋白水解的关键脯氨酰羟化酶。因此，我们的研究结果表明 Leu-574 对于招募 PHD2/HPH2 至关重要，从而为调节 HIF-1α 活性提供了分子基础。
Hypoxia-inducible factor (HIF)-1alpha, a master regulator of oxygen homeostasis, regulates genes crucial for cell growth and survival. In normoxia, HIF-1alpha is constantly degraded via the ubiquitin-proteasome pathway. The von Hippel-Lindau (VHL) E3 ubiquitin ligase binds HIF-1alpha through specific recognition of hydroxylated Pro-402 or Pro-564, both of which are modified by the oxygen-dependent HIF prolyl hydroxylases (PHDs/HPHs). Despite the identification of a conserved Leu-X-X-Leu-Ala-Pro motif, the molecular requirement of HIF-1alpha for PHDs/HPHs binding remains elusive. Recently, we demonstrated that Leu-574 of human HIF-1alpha-10 residues downstream of Pro-564-is essential for VHL recognition. We show here that the role of Leu-574 is to recruit PHD2/HPH2 for Pro-564 hydroxylation. An antibody specific for hydroxylated Pro-564 has been used to determine the hydroxylation status; mutation or deletion of Leu-574 results in a significant decrease in the ratio of the hydroxylated HIF-1alpha to the total amount. The nine-residue spacing between Pro-564 and Leu-574 is not obligatory for prolyl hydroxylation. Furthermore, mutation of Leu-574 disrupts the binding of PHD2/HPH2, a key prolyl hydroxylase for oxygen-dependent proteolysis of HIF-1alpha. Hence, our findings indicate that Leu-574 is essential for recruiting PHD2/HPH2, thereby providing a molecular basis for modulating HIF-1alpha activity.