Coordinate regulation of the oxygen-dependent degradation domains of hypoxia-inducible factor 1α
Coordinate regulation of the oxygen-dependent degradation domains of hypoxia-inducible factor 1α
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DOI：
10.1128/mcb.25.15.6415-6426.2005
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发表时间：
2005-08-01
影响因子：
5.3
通讯作者：
Giaccia, AJ
中科院分区：
文献类型：
--
作者：
Chan, DA;Sutphin, PD;Giaccia, AJ
氧依赖性蛋白水解是调节缺氧诱导因子（HIF）家族转录因子的主要手段。HIF-1的α亚基含有两个高度保守的氧依赖性降解结构域（402 ODD和564 ODD），每个结构域都包含一个在氧存在下被羟基化的脯氨酸，允许von Hippel-Lindau（VHL）E3泛素连接酶相互作用并将HIF-1 α靶向蛋白酶体进行降解。脯氨酸的突变足以在常氧条件下部分稳定HIF-1 α，但每个羟基化事件对HIF-1 α调节的具体贡献尚不清楚。在这里，我们表明，HIF-1 α的两个ODD在HIF-1 α蛋白质周转的调节中具有独立但相互作用的作用，每个ODD的相对参与取决于氧的水平。使用羟化特异性抗体，我们发现，在常氧条件下，脯氨酸564在脯氨酸402之前被羟化，并且脯氨酸564的突变导致脯氨酸402的羟化显著降低。然而，脯氨酸402的突变对脯氨酸564的羟基化几乎没有影响。为了确定脯氨酸564在常氧条件下更快的羟基化是否是由于对脯氨酸564周围的特定序列或蛋白质内的该位点的偏好，我们交换了全长HIF-1 α蛋白内的降解结构域。在这些结构域交换实验中，脯氨酰羟化酶结构域1（PHD 1）和PHD 2优先羟基化位于原始564 ODD的位点的脯氨酸，而PHD 3优选脯氨酸564序列，无论其位置如何。在限制氧张力，我们发现，脯氨酸402表现出氧依赖性降低羟化在较高的氧张力相对于脯氨酸564羟基化。这些结果表明，脯氨酸402的羟基化对氧的生理变化高度响应，因此，在缺氧条件下比在常氧条件下在HIF-1 α调节中起更重要的作用。总之，这些发现表明HIF-1 α的每个羟基化脯氨酸在响应于不同水平的氧合而控制HIF-1 α稳定性方面具有不同的活性。
Oxygen-dependent proteolysis is the primary means of regulating the hypoxia-inducible factor (HIF) family of transcription factors. The alpha-subunit of HIF factor 1 (HIF-1) contains two highly conserved oxygen-dependent degradation domains (402 ODD and 564 ODD), each of which includes a proline that is hydroxylated in the presence of oxygen, allowing the von Hippel-Lindau (VHL) E3 ubiquitin ligase to interact and target HIF-1 alpha to the proteasome for degradation. Mutation of either proline is sufficient to partially stabilize HIF-1 alpha under conditions of normoxia, but the specific contributions of each hydroxylation event to the regulation of HIF-1 alpha are unknown. Here we show that the two ODDs of HIF-1 alpha have independent yet interactive roles in the regulation of HIF-1 alpha protein turnover, with the relative involvement of each ODD depending on the levels of oxygen. Using hydroxylation-specific antibodies, we found that under conditions of normoxia proline 564 is hydroxylated prior to proline 402, and mutation of proline 564 results in a significant reduction in the hydroxylation of proline 402. Mutation of proline 402, however, has little effect on the hydroxylation of proline 564. To determine whether the more rapid hydroxylation of the proline 564 under conditions of normoxia is due to a preference for the particular sequence surrounding proline 564 or for that site within the protein, we exchanged the degradation domains within the full-length HIF-1 alpha protein. In these domain-swapping experiments, prolyl hydroxylase domain 1 (PHD1) and PHD2 preferentially hydroxylated the proline located in the site of the original 564 ODD, while PHD3 preferred the proline 564 sequence, regardless of its location. At limiting oxygen tensions, we found that proline 402 exhibits an oxygen-dependent decrease in hydroxylation at higher oxygen tensions relative to proline 564 hydroxylation. These results indicate that hydroxylation of proline 402 is highly responsive to physiologic changes in oxygen and, therefore, plays a more important role in HIF-1 alpha regulation under conditions of hypoxia than under conditions of normoxia. Together, these findings demonstrate that each hydroxylated proline of HIF-1 alpha has a distinct activity in controlling HIF-1 alpha stability in response to different levels of oxygenation.