Profiling of nuclear copper-binding proteins under hypoxic condition
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.
KeywordsCu Hypoxia Copper binding proteins Proteomics
This study was funded by the National Natural Science Foundation of China (Grant Number 81230004 to Y. J. Kang and Grant Number 81400218 to X. Ding).
All authors participated in the design, analysis of the data, interpretation of the results, and review of the manuscript; HF and XD carried out the experiments; YJK, XD, HF and WZ wrote the manuscript. The authors like to thank Wen Yin, Lily Zheng and Liying Qiu for assistance in preparation of the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
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