Skip to main content
SpringerLink
Log in

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

  • Original Paper
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  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.

    Article  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  12. Semenza, G. L. (2010). Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene,29, 625.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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. 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.

    Article  PubMed  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  20. Kabsch, W. (2010). XDS. Acta Crystallographica Section D,66, 125–132.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  22. Roy, A., Kucukural, A., & Zhang, Y. (2010). I-TASSER: A unified platform for automated protein structure and function prediction. Nature Protocols,5, 725.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  28. Yang, W. (2015). Structural basis of PKM2 regulation. Protein and Cell,6, 238–240.

    Article  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India

    Sunil Kumar & Ashok Kumar Patel

Authors
  1. Sunil Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ashok Kumar Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ashok Kumar Patel.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kumar, S., Patel, A.K. Purification and Characterization of Prolyl Hydroxylase 3/Pyruvate Kinase Isoform 2 Protein Complex. Mol Biotechnol 62, 111–118 (2020). https://doi.org/10.1007/s12033-019-00228-9

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-019-00228-9

Keywords

Search

Navigation