Department of Biochemistry and Molecular Cell Biology, Key Laboratory of the Shanghai Science and Technology Commission for Cancer Microenvironment and Inflammation, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, China.
Acta Pharmacol Sin. 2012 Jul;33(7):953-63. doi: 10.1038/aps.2012.40. Epub 2012 Jun 11.
To investigate the mechanisms underlying the biphasic redox regulation of hypoxia-inducible factor-1 (HIF-1) transcriptional activity under different levels of oxidative stress caused by reactive oxidative species (ROS).
HeLa cells were exposed to different concentrations of H(2)O(2) as a simple model for mild and severe oxidative stress. Luciferase reporter assay and/or quantitative real-time PCR were used to investigate the transcriptional activity. Immunoblot was used to detect protein expression. Chromatin immunoprecipitation assay was used to detect HIF-1/DNA binding. The interaction of p300 with HIF-1α or with SENP3, and the SUMO2/3 conjugation states of p300 were examined by coimmunoprecipitation.
HIF-1 transcriptional activity in HeLa cells was enhanced by low doses (0.05-0.5 mmol/L) of H(2)O(2), but suppressed by high doses (0.75-8.0 mmol/L) of H(2)O(2). The amount of co-activator p300 bound to HIF-1α in HeLa cells was increased under mild oxidative stress, but decreased under severe oxidative stress. The ROS levels differentially modified cysteines 243 and 532 in the cysteine protease SENP3, regulating the interaction of SENP3 with p300 to cause different SUMOylation of p300, thus shifting HIF-1 transcriptional activity.
The shift of HIF-1 transactivation by ROS is correlated with and dependent on the biphasic redox sensing of SENP3 that leads to the differential SENP3/p300 interaction and the consequent fluctuation in the p300 SUMOylation status.
研究活性氧（ROS）引起的不同水平氧化应激下缺氧诱导因子-1（HIF-1）转录活性的双相氧化还原调节机制。
以 HeLa 细胞暴露于不同浓度 H₂O₂为例，模拟轻度和重度氧化应激。采用荧光素酶报告基因检测和/或实时定量 PCR 检测转录活性。免疫印迹法检测蛋白表达。染色质免疫沉淀法检测 HIF-1/DNA 结合。通过共免疫沉淀检测 p300 与 HIF-1α 或 SENP3 的相互作用，以及 p300 的 SUMO2/3 缀合状态。
HIF-1 转录活性在 HeLa 细胞中被低剂量（0.05-0.5 mmol/L）H₂O₂增强，但被高剂量（0.75-8.0 mmol/L）H₂O₂抑制。在轻度氧化应激下，HeLa 细胞中与 HIF-1α 结合的共激活剂 p300 数量增加，但在重度氧化应激下减少。ROS 水平差异修饰半胱氨酸蛋白酶 SENP3 中的半胱氨酸 243 和 532，调节 SENP3 与 p300 的相互作用，导致 p300 的不同 SUMO 化，从而改变 HIF-1 转录活性。
ROS 对 HIF-1 反式激活的转变与 SENP3 的双相氧化还原感应相关，并依赖于 SENP3，导致 SENP3/p300 相互作用的差异和 p300 SUMO 化状态的波动。
