Under hypoxic condition, copper (Cu) accumulates in cell nuclei, and regulates the activity of hypoxia-inducible factor-1 (HIF-1) through Cu-binding proteins (CuBPs). To understand the CuBPs in the nucleus, proteomic approach was undertaken to explore the dynamic changes of the CuBPs in response to hypoxia. Human umbilical vein endothelial cells (HUVECs) were treated with dimethyloxalylglycine in a final concentration of 100 μM for 4 h to induce hypoxia, resulting in the accumulation of HIF-1α and Cu in the nucleus. Cu immobilized metal affinity chromatography was applied to extract the CuBPs, followed by identification using nanoliter-liquid chromatograpy combined with quadrupole time of flight tandem mass spectrometry (nanoLC-Q-TOF-MS/MS). There were 278 nuclear proteins that were found as CuBPs in the induced hypoxic group in contrast to 218 CuBPs in the control group. Functional annotation of these proteins in gene ontology category revealed that proteins participating in negative regulation of transcription from RNA polymerase II promoter were dramatically enriched by induced hypoixc treatment. Label-free quantitative proteomic approach identified quantitative changes of nuclear proteome; of 17 differentially expressed proteins, 8 were downregulated and 9 were upregulated in the induced hypoxic nuclei. Four of the 17 proteins were CuBPs, including ILF2 and TRA2B, both were downregulated, and LMNA and HSPB1, both were upregulated. We confirmed the protein change of ALB, LMNA and HSPB1 (HSP27) in real hypoxia, and suggested that the identified CuBPs could be the target for further study of Cu regulation of HIF-1 activity in the nucleus.

中文翻译：

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低氧条件下核铜结合蛋白的分析。

在缺氧条件下，铜（Cu）积聚在细胞核中，并通过Cu结合蛋白（CuBPs）调节缺氧诱导因子1（HIF-1）的活性。为了了解细胞核中的CuBPs，采用蛋白质组学方法探索CuBPs对缺氧反应的动态变化。用终浓度为100μM的二甲基草酰甘氨酸处理人脐静脉内皮细胞（HUVEC）4小时，以诱导缺氧，导致HIF-1α和Cu在细胞核中积聚。应用固定化铜的金属亲和色谱法提取CuBP，然后使用纳升液相色谱结合四极杆飞行时间串联质谱（nanoLC-Q-TOF-MS / MS）进行鉴定。缺氧诱导组中有278种核蛋白被发现为CuBP，而对照组中则为218种CuBP。这些蛋白质在基因本体论类别中的功能注释显示，通过诱导的hypoixc处理，参与RNA聚合酶II启动子转录的负调控的蛋白质得到了极大的富集。无标记的定量蛋白质组学方法可确定核蛋白质组的定量变化；在诱导的低氧核中，有17种差异表达的蛋白质中有8种被下调，有9种被上调。17种蛋白质中有4种是CuBP，包括ILF2和TRA2B，均被下调，而LMNA和HSPB1，均被上调。我们确认了在实际缺氧状态下ALB，LMNA和HSPB1（HSP27）的蛋白质变化，