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Purification and Characterization of Prolyl Hydroxylase 3/Pyruvate Kinase Isoform 2 Protein Complex

  • Sunil Kumar
  • Ashok Kumar PatelEmail author
Original Paper
  • 57 Downloads

Abstract

The prolyl hydroxylase 3 (PHD3) protein is less abundant in normal oxygen conditions (normoxia) but increases under deficient oxygen condition (hypoxia). Since cancerous cells often thrive in hypoxic conditions and predominantly express the Pyruvate kinase isoforms 2 (PKM2), the PHD3/PKM2 interaction might be particularly important in cancer development. In the present study, the PHD3/PKM2 complex was co-expressed and purified by size-exclusion chromatography. The interaction of PHD3 with PKM2 was confirmed in Native gel as well as western blot analysis. The PHD3/PKM2 complex formed discreet crystals under suitable conditions, and diffraction data revealed that crystal belonged to the P1 space group with 3.0 Å resolution. This is the first crystal report of PHD3/PKM2 complex as well as this study demonstrates a direct physical binding through protein–protein interaction. The structural analysis of complex will provide the information regarding the amino acid residues critical for the catalytic mechanism. Based on the structural information thus obtained, pharmacological interference with the PHD3/PKM2 interaction could be used as a novel strategy to reduce the cancer progression.

Keywords

PHD3/PKM2 complex Protein crystal Protein interaction Cancer proteins 

Notes

Acknowledgements

We are thankful to the beamline scientists at the European Synchrotron Radiation Facility, Grenoble, France for assisting us with the use of beamline ID30A-3. The authors acknowledge the infrastructural support from Indian Institute of Technology Delhi. SK acknowledges the research grant from SERB, Department of Science & Technology, and Govt. of India. Authors thank Dr. Dushyant Garg and Dr. Ruma Karmakar for continuous help in research experiments.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Hitosugi, T., Kang, S., Vander Heiden, M. G., Chung, T. W., Elf, S., Lythgoe, K., et al. (2009). Tyrosine phosphorylation inhibits PKM2 to promote the Warburg effect and tumor growth. Science Signaling,2, ra73–ra73.CrossRefGoogle Scholar
  2. 2.
    Gui, D. Y., Lewis, C. A., & Vander Heiden, M. G. (2013). Allosteric regulation of PKM2 allows cellular adaptation to different physiological states. Science Signaling,6, pe7–pe7.CrossRefGoogle Scholar
  3. 3.
    Srivastava, D., Razzaghi, M., Henzl, M. T., & Dey, M. (2017). Structural investigation of a dimeric variant of pyruvate kinase muscle isoform 2. Biochemistry,56, 6517–6520.CrossRefGoogle Scholar
  4. 4.
    Luo, W., Hu, H., Chang, R., Zhong, J., Knabel, M., O’Meally, R., et al. (2011). Pyruvate kinase M2 is a PHD3-stimulated coactivator for hypoxia-inducible factor 1. Cell,145, 732–744.CrossRefGoogle Scholar
  5. 5.
    Lee, J., Kim, H. K., Han, Y.-M., & Kim, J. (2008). Pyruvate kinase isozyme type M2 (PKM2) interacts and cooperates with Oct-4 in regulating transcription. The International Journal of Biochemistry and Cell Biology,40, 1043–1054.CrossRefGoogle Scholar
  6. 6.
    Yang, W., Xia, Y., Ji, H., Zheng, Y., Liang, J., Huang, W., et al. (2011). Nuclear PKM2 regulates β-catenin transactivation upon EGFR activation. Nature,480, 118.CrossRefGoogle Scholar
  7. 7.
    Azoitei, N., Becher, A., Steinestel, K., Rouhi, A., Diepold, K., Genze, F., et al. (2016). PKM2 promotes tumor angiogenesis by regulating HIF-1α through NF-κB activation. Molecular Cancer,15, 3.CrossRefGoogle Scholar
  8. 8.
    Chen, N., Rinner, O., Czernik, D., Nytko, K. J., Zheng, D., Stiehl, D. P., et al. (2011). The oxygen sensor PHD3 limits glycolysis under hypoxia via direct binding to pyruvate kinase. Cell Research,21, 983.CrossRefGoogle Scholar
  9. 9.
    Epstein, A. C., Gleadle, J. M., McNeill, L. A., Hewitson, K. S., O’Rourke, J., Mole, D. R., et al. (2001). C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell,107, 43–54.CrossRefGoogle Scholar
  10. 10.
    McNeill, L. A., Flashman, E., Buck, M. R., Hewitson, K. S., Clifton, I. J., Jeschke, G., et al. (2005). Hypoxia-inducible factor prolyl hydroxylase 2 has a high affinity for ferrous iron and 2-oxoglutarate. Molecular BioSystems,1, 321–324.CrossRefGoogle Scholar
  11. 11.
    Kaelin, W. G., Jr., & Ratcliffe, P. J. (2008). Oxygen sensing by metazoans: The central role of the HIF hydroxylase pathway. Molecular Cell,30, 393–402.CrossRefGoogle Scholar
  12. 12.
    Semenza, G. L. (2010). Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene,29, 625.CrossRefGoogle Scholar
  13. 13.
    Hasan, D., Gamen, E., Tarboush, N. A., Ismail, Y., Pak, O., & Azab, B. (2018). PKM2 and HIF-1α regulation in prostate cancer cell lines. PLoS ONE,13, e0203745.CrossRefGoogle Scholar
  14. 14.
    Metzen, E., Berchner-Pfannschmidt, U., Stengel, P., Marxsen, J. H., Stolze, I., Klinger, M., et al. (2003). Intracellular localisation of human HIF-1α hydroxylases: Implications for oxygen sensing. Journal of Cell Science,116, 1319–1326.CrossRefGoogle Scholar
  15. 15.
    Christofk, H. R., Vander Heiden, M. G., Harris, M. H., Ramanathan, A., Gerszten, R. E., Wei, R., et al. (2008). The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature,452, 230.CrossRefGoogle Scholar
  16. 16.
    Studier, F. W. (2014). Stable expression clones and auto-induction for protein production in E. coli, in structural genomics (pp. 17–32). Totowa: Humana Press.Google Scholar
  17. 17.
    Kumar, S., Dhembla, C., Hariprasad, P., Sundd, M., & Patel, A. K. (2019). Differential expression of structural and functional proteins during bean common mosaic virus-host plant interaction. Microbial Pathogenesis.  https://doi.org/10.1016/j.micpath.2019.103812.CrossRefPubMedGoogle Scholar
  18. 18.
    Kumar, S., Karmakar, R., Garg, D. K., Gupta, I., & Patel, A. K. (2019). Elucidating the functional aspects of different domains of bean common mosaic virus coat protein. Virus Research,273, 197755.CrossRefGoogle Scholar
  19. 19.
    Merril, C. R., Goldman, D., Sedman, S. A., & Ebert, M. H. (1981). Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science,211, 1437–1438.CrossRefGoogle Scholar
  20. 20.
    Kabsch, W. (2010). XDS. Acta Crystallographica Section D,66, 125–132.CrossRefGoogle Scholar
  21. 21.
    Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., et al. (2011). Overview of the CCP4 suite and current developments. Acta Crystallographica Section D,67, 235–242.CrossRefGoogle Scholar
  22. 22.
    Roy, A., Kucukural, A., & Zhang, Y. (2010). I-TASSER: A unified platform for automated protein structure and function prediction. Nature Protocols,5, 725.CrossRefGoogle Scholar
  23. 23.
    McDonough, M. A., Li, V., Flashman, E., Chowdhury, R., Mohr, C., Liénard, B. M., et al. (2006). Cellular oxygen sensing: CRYSTAL structure of hypoxia-inducible factor prolyl hydroxylase (PHD2). Proceedings of the National Academy of Sciences,103, 9814–9819.CrossRefGoogle Scholar
  24. 24.
    Laskowski, R. A., MacArthur, M. W., Moss, D. S., & Thornton, J. M. (1993). PROCHECK: A program to check the stereochemical quality of protein structures. Journal of Applied Crystallography,26, 283–291.CrossRefGoogle Scholar
  25. 25.
    Yan, Y., Zhang, D., Zhou, P., Li, B., & Huang, S.-Y. (2017). HDOCK: A web server for protein–protein and protein–DNA/RNA docking based on a hybrid strategy. Nucleic Acids Research,45, W365–W373.CrossRefGoogle Scholar
  26. 26.
    Matsui, Y., Yasumatsu, I., Asahi, T., Kitamura, T., Kanai, K., Ubukata, O., et al. (2017). Discovery and structure-guided fragment-linking of 4-(2, 3-dichlorobenzoyl)-1-methyl-pyrrole-2-carboxamide as a pyruvate kinase M2 activator. Bioorganic and Medicinal Chemistry,25, 3540–3546.CrossRefGoogle Scholar
  27. 27.
    Fedulova, N., Hanrieder, J., Bergquist, J., & Emrén, L. O. (2007). Expression and purification of catalytically active human PHD3 in Escherichia coli. Protein Expression and Purification,54, 1–10.CrossRefGoogle Scholar
  28. 28.
    Yang, W. (2015). Structural basis of PKM2 regulation. Protein and Cell,6, 238–240.CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Kusuma School of Biological SciencesIndian Institute of Technology DelhiNew DelhiIndia

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