Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Arginine refolds, stabilizes, and restores function of mutant pVHL proteins in animal model of the VHL cancer syndrome

Abstract

The von Hippel–Lindau (VHL) syndrome is a rare inherited cancer, caused by mutations in the VHL gene, many of which render the VHL protein (pVHL) unstable. pVHL is a tumor-suppressor protein implicated in a variety of cellular processes, most notably in response to changes in oxygen availability, due to its role as part of an E3-ligase complex which targets the hypoxia-inducible factor (HIF) for degradation. Previously we reported, using in silico and in vitro analyses, that common oncogenic VHL mutations render pVHL less stable than the wild-type protein, distort its core domain and as a result reduce the ability of the protein to bind its target HIF-1α. Among various chemical chaperones tested, arginine was the most effective in refolding mutant of pVHL. Here we examined the consequences of administering L- or D-arginine to a Drosophila VHL model and to human renal carcinoma cells, both expressing misfolded versions of human pVHL. Arginine treatment increased pVHL solubility in both models and increased the half-life of the mutant pVHL proteins in the cell culture. In both models, L- as well as D-arginine enhanced the ability of wild-type pVHL and certain misfolded mutant versions of pVHL to bind ODD, the HIF-derived target peptide, reflecting restoration of pVHL function. Moreover, continuous feeding of Drosophila expressing misfolded versions of pVHL either L- or D-arginine rich diet rescued their lethal phenotype. Collectively, these in vivo results suggest that arginine supplementation should be examined as a potential novel treatment for VHL cancer syndrome.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Varshney N, Kebede AA, Owusu-Dapaah H, Lather J, Kaushik M, Bhullar JS. A review of Von Hippel-Lindau syndrome. J Kidney Cancer VHL. 2017;4:20–9.

    Article  Google Scholar 

  2. Tarade D, Ohh M. The HIF and other quandaries in VHL disease. Oncogene. 2018;37:139–47.

    Article  CAS  Google Scholar 

  3. Ryan HE, Lo J, Johnson RS. HIF-1 alpha is required for solid tumor formation and embryonic vascularization. EMBO J. 1998;17:3005–15.

    Article  CAS  Google Scholar 

  4. Bai C, Sen P, Hofmann K, Ma L, Goebl M, Harper JW, et al. SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell. 1996;86:263–74.

    Article  CAS  Google Scholar 

  5. Ohh M, Takagi Y, Aso T, Stebbins CE, Pavletich NP, Zbar B, et al. Synthetic peptides define critical contacts between elongin C, elongin B, and the von Hippel-Lindau protein. J Clin Invest. 1999;104:1583–91.

    Article  CAS  Google Scholar 

  6. Gossage L, Eisen T, Maher ER. VHL, the story of a tumour suppressor gene. Nat Rev Cancer. 2015;15:55–64.

    Article  CAS  Google Scholar 

  7. Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME, et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 1999;399:271–5.

    Article  CAS  Google Scholar 

  8. Cockman ME, Masson N, Mole DR, Jaakkola P, Chang GW, Clifford SC, et al. Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein. J Biol Chem. 2000;275:25733–41.

    Article  CAS  Google Scholar 

  9. Kamura T, Sato S, Iwai K, Czyzyk-Krzeska M, Conaway RC, Conaway JW. Activation of HIF1alpha ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex. Proc Natl Acad Sci USA. 2000;97:10430–5.

    Article  CAS  Google Scholar 

  10. D’Angelo G, Duplan E, Boyer N, Vigne P, Frelin C. Hypoxia up-regulates prolyl hydroxylase activity: a feedback mechanism that limits HIF-1 responses during reoxygenation. J Biol Chem. 2003;278:38183–7.

    Article  Google Scholar 

  11. Ohh M, Park CW, Ivan M, Hoffman MA, Kim TY, Huang LE, et al. Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein. Nat Cell Biol. 2000;2:423–7.

    Article  CAS  Google Scholar 

  12. Miller F, Kentsis A, Osman R, Pan ZQ. Inactivation of VHL by tumorigenic mutations that disrupt dynamic coupling of the pVHL.hypoxia-inducible transcription factor-1alpha complex. J Biol Chem. 2005;280:7985–96.

    Article  CAS  Google Scholar 

  13. Berra E, Benizri E, Ginouves A, Volmat V, Roux D, Pouyssegur J. HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1alpha in normoxia. EMBO J. 2003;22:4082–90.

    Article  CAS  Google Scholar 

  14. Yu F, White SB, Zhao Q, Lee FS. HIF-1alpha binding to VHL is regulated by stimulus-sensitive proline hydroxylation. Proc Natl Acad Sci USA. 2001;98:9630–5.

    Article  CAS  Google Scholar 

  15. Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science. 2001;292:468–72.

    Article  CAS  Google Scholar 

  16. Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, et al. HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science. 2001;292:464–8.

    Article  CAS  Google Scholar 

  17. Min JH, Yang H, Ivan M, Gertler F, Kaelin WG Jr, Pavletich NP. Structure of an HIF-1alpha -pVHL complex: hydroxyproline recognition in signaling. Science. 2002;296:1886–9.

    Article  CAS  Google Scholar 

  18. Tabaro F, Minervini G, Sundus F, Quaglia F, Leonardi E, Piovesan D, et al. VHLdb: a database of von Hippel-Lindau protein interactors and mutations. Sci Rep. 2016;6:31128.

    Article  CAS  Google Scholar 

  19. Beroud C, Joly D, Gallou C, Staroz F, Orfanelli MT, Junien C. Software and database for the analysis of mutations in the VHL gene. Nucleic Acids Res. 1998;26:256–8.

    Article  CAS  Google Scholar 

  20. Stebbins CE, Kaelin WG Jr, Pavletich NP. Structure of the VHL-ElonginC-ElonginB complex: implications for VHL tumor suppressor function. Science. 1999;284:455–61.

    Article  CAS  Google Scholar 

  21. Knauth K, Bex C, Jemth P, Buchberger A. Renal cell carcinoma risk in type 2 von Hippel-Lindau disease correlates with defects in pVHL stability and HIF-1alpha interactions. Oncogene. 2006;25:370–7.

    Article  CAS  Google Scholar 

  22. Shmueli MD, Schnaider L, Rosenblum D, Herzog G, Gazit E, Segal D. Structural insights into the folding defects of oncogenic pVHL lead to correction of its function in vitro. PLoS ONE. 2013;8:e66333

    Article  CAS  Google Scholar 

  23. Bangiyeva V, Rosenbloom A, Alexander AE, Isanova B, Popko T, Schoenfeld AR. Differences in regulation of tight junctions and cell morphology between VHL mutations from disease subtypes. BMC Cancer. 2009;9:229.

    Article  Google Scholar 

  24. Shmueli MD, Schnaider L, Herzog G, Gazit E, Segal D. Computational and experimental characterization of dVHL establish a Drosophila model of VHL syndrome. PLoS ONE. 2014;9:e109864

    Article  Google Scholar 

  25. Duchi S, Fagnocchi L, Cavaliere V, Hsouna A, Gargiulo G, Hsu T. Drosophila VHL tumor-suppressor gene regulates epithelial morphogenesis by promoting microtubule and aPKC stability. Development. 2010;137:1493–503.

    Article  CAS  Google Scholar 

  26. Hsouna A, Nallamothu G, Kose N, Guinea M, Dammai V, Hsu T. Drosophila von Hippel-Lindau tumor suppressor gene function in epithelial tubule morphogenesis. Mol Cell Biol. 2010;30:3779–94.

    Article  CAS  Google Scholar 

  27. Khan SH, Ahmad N, Ahmad F, Kumar R. Naturally occurring organic osmolytes: from cell physiology to disease prevention. IUBMB Life. 2010;62:891–5.

    Article  CAS  Google Scholar 

  28. Li J, Garg M, Shah D, Rajagopalan R. Solubilization of aromatic and hydrophobic moieties by arginine in aqueous solutions. J Chem Phys. 2010;133:054902.

    Article  Google Scholar 

  29. Semenza GL. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol. 1999;15:551–78.

    Article  CAS  Google Scholar 

  30. Cho NK, Keyes L, Johnson E, Heller J, Ryner L, Karim F, et al. Developmental control of blood cell migration by the Drosophila VEGF pathway. Cell. 2002;108(6):865–76.

    Article  CAS  Google Scholar 

  31. Kurban G, Duplan E, Ramlal N, Hudon V, Sado Y, Ninomiya Y, et al. Collagen matrix assembly is driven by the interaction of von Hippel-Lindau tumor suppressor protein with hydroxylated collagen IV alpha 2. Oncogene. 2008;27(7):1004–12.

    Article  CAS  Google Scholar 

  32. Ohh M, Yauch RL, Lonergan KM, Whaley JM, Stemmer-Rachamimov AO, Louis DN, et al. The von Hippel-Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix. Mol Cell. 1998;1:959–68.

    Article  CAS  Google Scholar 

  33. Guo Y, Schoell MC, Freeman RS. The von Hippel-Lindau protein sensitizes renal carcinoma cells to apoptotic stimuli through stabilization of BIM(EL). Oncogene. 2009;28:1864–74.

    Article  CAS  Google Scholar 

  34. Yang H, Minamishima YA, Yan Q, Schlisio S, Ebert BL, Zhang X, et al. pVHL acts as an adaptor to promote the inhibitory phosphorylation of the NF-kappaB agonist Card9 by CK2. Mol Cell. 2007;28:15–27.

    Article  Google Scholar 

  35. Hergovich A, Lisztwan J, Barry R, Ballschmieter P, Krek W. Regulation of microtubule stability by the von Hippel-Lindau tumour suppressor protein pVHL. Nat Cell Biol. 2003;5:64–70.

    Article  CAS  Google Scholar 

  36. Chen CH, Terentjev EM. Aging and metastability of monoglycerides in hydrophobic solutions. Langmuir: the ACS journal of surfaces and colloids. 2009;25:6717–24.

    Article  CAS  Google Scholar 

  37. Arakawa T, Ejima D, Tsumoto K, Obeyama N, Tanaka Y, Kita Y, et al. Suppression of protein interactions by arginine: a proposed mechanism of the arginine effects. Biophys Chem. 2007;127:1–8.

    Article  CAS  Google Scholar 

  38. Ding Z, German P, Bai S, Feng Z, Gao M, Si W, et al. Agents that stabilize mutated von Hippel-Lindau (VHL) protein: results of a high-throughput screen to identify compounds that modulate VHL proteostasis. J Biomol Screen. 2012;17:572–80.

    Article  CAS  Google Scholar 

  39. Hon WC, Wilson MI, Harlos K, Claridge TD, Schofield CJ, Pugh CW, et al. Structural basis for the recognition of hydroxyproline in HIF-1 alpha by pVHL. Nature. 2002;417:975–8.

    Article  CAS  Google Scholar 

  40. McNeal CJ, Meininger CJ, Reddy D, Wilborn CD, Wu G. Safety and effectiveness of arginine in adults. J Nutr. 2016;146:2587S–93S.

    Article  CAS  Google Scholar 

  41. Navarro E, Alonso SJ, Martin FA, Castellano MA. Toxicological and pharmacological effects of D-arginine. Basic Clin Pharmacol Toxicol. 2005;97:149–54.

    Article  CAS  Google Scholar 

  42. Groth AC, Fish M, Nusse R, Calos MP. Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics. 2004;166:1775–82.

    Article  CAS  Google Scholar 

  43. Arquier N, Vigne P, Duplan E, Hsu T, Therond PP, Frelin C, et al. Analysis of the hypoxia-sensing pathway in Drosophila melanogaster. Biochem J. 2006;393:471–80.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to Sivan Peled and the Segal-Gazit research groups for fruitful discussions. This work was supported in part by the Israel Cancer Association, the Cancer Biology Research Center in Tel Aviv University, and the VHL Alliance (to DS).

Funding

This work was supported in part by the Israel Cancer Association, the Cancer Biology Research Center in Tel Aviv University, and the VHL Alliance (to DS).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Daniel Segal.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shmueli, M.D., Levy-Kanfo, L., Haj, E. et al. Arginine refolds, stabilizes, and restores function of mutant pVHL proteins in animal model of the VHL cancer syndrome. Oncogene 38, 1038–1049 (2019). https://doi.org/10.1038/s41388-018-0491-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41388-018-0491-x

Search

Quick links