Article | Published:

Perforin proteostasis is regulated through its C2 domain: supra-physiological cell death mediated by T431D-perforin

Cell Death & Differentiationvolume 25pages15171529 (2018) | Download Citation

Abstract

The pore forming, Ca2+-dependent protein, perforin, is essential for the function of cytotoxic lymphocytes, which are at the frontline of immune defence against pathogens and cancer. Perforin is a glycoprotein stored in the secretory granules prior to release into the immune synapse. Congenital perforin deficiency causes fatal immune dysregulation, and is associated with various haematological malignancies. At least 50% of pathological missense mutations in perforin result in protein misfolding and retention in the endoplasmic reticulum. However, the regulation of perforin proteostasis remains unexplored. Using a variety of biochemical assays that assess protein stability and acquisition of complex glycosylation, we demonstrated that the binding of Ca2+ to the C2 domain stabilises perforin and regulates its export from the endoplasmic reticulum to the secretory granules. As perforin is a thermo-labile protein, we hypothesised that by altering its C2 domain it may be possible to improve protein stability. On the basis of the X-ray crystal structure of the perforin C2 domain, we designed a mutation (T431D) in the Ca2+ binding loop. Mutant perforin displayed markedly enhanced thermal stability and lytic function, despite its trafficking from the endoplasmic reticulum remaining unchanged. Furthermore, by introducing the T431D mutation into A90V perforin, a pathogenic mutation, which results in protein misfolding, we corrected the A90V folding defect and completely restored perforin’s cytotoxic function. These results revealed an unexpected role for the Ca2+-dependent C2 domain in maintaining perforin proteostasis and demonstrated the possibility of designing perforin with supra-physiological cytotoxic function through stabilisation of the C2 domain.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Additional information

Edited by RA Knight

References

  1. 1.

    de Saint Basile G, Ménasché G, Fischer A. Molecular mechanisms of biogenesis and exocytosis of cytotoxic granules. Nat Rev Immunol. 2010;10:568–79.

  2. 2.

    Voskoboinik I, Whisstock JC, Trapani JA. Perforin and granzymes: function, dysfunction and human pathology. Nat Rev Immunol. 2015;15:388–400.

  3. 3.

    Yagi H, Conroy PJ, Leung EW, Law RH, Trapani JA, Voskoboinik I, et al. Structural Basis for Ca2 + -mediated Interaction of the Perforin C2 Domain with Lipid Membranes. J Biol Chem. 2015;290:25213–26.

  4. 4.

    Traore DA, Brennan AJ, Law RH, Dogovski C, Perugini MA, Lukoyanova N, et al. Defining the interaction of Prf1 with calcium and the phospholipid membrane. Biochem J. 2013;456:323–35.

  5. 5.

    Leung C, Hodel AW, Brennan AJ, Lukoyanova N, Tran S, House CM, et al. Real-time visualization of Prf1 nanopore assembly. Nat Nanotechnol. 2017;12:467–73.

  6. 6.

    Law RH, Lukoyanova N, Voskoboinik I, Caradoc-Davies TT, Baran K, Dunstone MA, et al. The structural basis for membrane binding and pore formation by lymphocyte Prf1. Nature. 2010;468:447–51.

  7. 7.

    Metkar SS, Marchioretto M, Antonini V, Lunelli L, Wang B, Gilbert RJ, et al. Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes. Cell Death Differ. 2015;22:74–85.

  8. 8.

    Lopez JA, Susanto O, Jenkins MR, Lukoyanova N, Sutton VR, Law RH, et al. Perforin forms transient pores on the target cell plasma membrane to facilitate rapid access of granzymes during killer cell attack. Blood. 2013;121:2659–68.

  9. 9.

    Janka GE. Familial and acquired hemophagocytic lymphohistiocytosis. Annu Rev Med. 2012;63:233–46.

  10. 10.

    Voskoboinik I, Smyth MJ, Trapani JA. Perforin-mediated target-cell death and immune homeostasis. Nat Rev Immunol. 2006;6:940–52.

  11. 11.

    Lofstedt A, Chiang SC, Onelov E, Bryceson YT, Meeths M, Henter JI. Cancer risk in relatives of patients with a primary disorder of lymphocyte cytotoxicity: a retrospective cohort study. Lancet Haematol. 2015;2:e536–42.

  12. 12.

    Chia J, Yeo KP, Whisstock JC, Dunstone MA, Trapani JA, Voskoboinik I. Temperature sensitivity of human Prf1 mutants unmasks subtotal loss of cytotoxicity, delayed FHL, and a predisposition to cancer. Proc Natl Acad Sci USA. 2009;106:9809–14.

  13. 13.

    Wiseman RL, Powers ET, Buxbaum JN, Kelly JW, Balch WE. An adaptable standard for protein export from the endoplasmic reticulum. Cell. 2007;131:809–21.

  14. 14.

    Brennan AJ, Chia J, Browne KA, Ciccone A, Ellis S, Lopez JA, et al. Protection from endogenous Prf1: glycans and the C terminus regulate exocytic trafficking in cytotoxic lymphocytes. Immunity. 2011;34:879–92.

  15. 15.

    House IG, House CM, Brennan AJ, Gilan O, Dawson MA, Whisstock JC, et al. Regulation of Prf1 activation and presynaptic toxicity through C-terminal glycosylation. EMBO Rep. 2017;18:1775–85.

  16. 16.

    Voskoboinik I, Thia MC, Fletcher J, Ciccone A, Browne K, Smyth MJ, et al. Calcium-dependent plasma membrane binding and cell lysis by Prf1 are mediated through its C2 domain: a critical role for aspartate residues 429, 435, 483, and 485 but not 491. J Biol Chem. 2005;280:8426–34.

  17. 17.

    Clementi R, Locatelli F, Dupre L, Garaventa A, Emmi L, Bregni M, et al. A proportion of patients with lymphoma may harbor mutations of the Prf1 gene. Blood. 2005;105:4424–8.

  18. 18.

    Trambas C, Gallo F, Pende D, Marcenaro S, Moretta L, De Fusco C, et al. A single amino acid change, A91V, leads to conformational changes that can impair processing to the active form of Prf1. Blood. 2005;106:932–7.

  19. 19.

    Voskoboinik I, Thia MC, Trapani JA. A functional analysis of the putative polymorphisms A91V and N252S and 22 missense Prf1 mutations associated with familial hemophagocytic lymphohistiocytosis. Blood. 2005;105:4700–6.

  20. 20.

    Voskoboinik I, Sutton VR, Ciccone A, House CM, Chia J, Darcy PK, et al. Perforin activity and immune homeostasis: the common A91V polymorphism in Prf1 results in both presynaptic and postsynaptic defects in function. Blood. 2007;110:1184–90.

  21. 21.

    Pankow S, Bamberger C, Calzolari D, Martinez-Bartolome S, Lavallee-Adam M, Balch WE, et al. F508 CFTR interactome remodelling promotes rescue of cystic fibrosis. Nature. 2015;528:510–6.

  22. 22.

    Veit G, Avramescu RG, Chiang AN, Houck SA, Cai Z, Peters KW, et al. From CFTR biology toward combinatorial pharmacotherapy: expanded classification of cystic fibrosis mutations. Mol Biol Cell. 2016;27:424–33.

  23. 23.

    Voskoboinik I, Thia MC, De Bono A, Browne K, Cretney E, Jackson JT, et al. The functional basis for hemophagocytic lymphohistiocytosis in a patient with co-inherited missense mutations in the Prf1 (PFN1) gene. J Exp Med. 2004;200:811–6.

  24. 24.

    Sutton VR, Waterhouse NJ, Baran K, Browne K, Voskoboinik I, Trapani JA. Measuring cell death mediated by cytotoxic lymphocytes or their granule effector molecules. Methods. 2008;44:241–9.

  25. 25.

    Kabsch W. Xds. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 2):125–32.

  26. 26.

    Evans PR. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr D Biol Crystallogr. 2011;67(Pt 4):282–92.

  27. 27.

    Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr. 2011;67(Pt 4):235–42.

  28. 28.

    Evans PR, Murshudov GN. How good are my data and what is the resolution? Acta Crystallogr D Biol Crystallogr. 2013;69(Pt 7):1204–14.

  29. 29.

    McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. J Appl Crystallogr. 2007;40(Pt 4):658–74.

  30. 30.

    Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 4):486–501.

  31. 31.

    Chen VB, Arendall WB 3rd, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 1):12–21.

Download references

Acknowledgements

This work was supported by project and programme grants from the National Health and Medical Research Council of Australia (to IV, JCW and JAT), and Wellcome Trust equipment grant 079605 to HS. IV and JCW are supported by a National Health and Medical Research Council of Australia Fellowships. We thank Colin House and Conor Kearney for critical reading of the manuscript, and Mr Samuel J Redmond for his technical assistance.

Author information

Affiliations

  1. Killer Cell Biology Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia

    • Amelia J. Brennan
    • , Tahereh Noori
    •  & Ilia Voskoboinik
  2. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, VIC, Australia

    • Ruby H. P. Law
    • , Paul J. Conroy
    •  & James C. Whisstock
  3. The ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, VIC, Australia

    • Ruby H. P. Law
    • , Paul J. Conroy
    •  & James C. Whisstock
  4. Department of Crystallography/Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, London, UK

    • Natalya Lukoyanova
    •  & Helen Saibil
  5. Department of Immunology, Juntendo University School of Medicine, Tokyo, 113-8421, Japan

    • Hideo Yagita
  6. Cancer Cell Death Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia

    • Annette Ciccone
    • , Sandra Verschoor
    •  & Joseph A. Trapani
  7. Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC, Australia

    • Joseph A. Trapani
    •  & Ilia Voskoboinik

Authors

  1. Search for Amelia J. Brennan in:

  2. Search for Ruby H. P. Law in:

  3. Search for Paul J. Conroy in:

  4. Search for Tahereh Noori in:

  5. Search for Natalya Lukoyanova in:

  6. Search for Helen Saibil in:

  7. Search for Hideo Yagita in:

  8. Search for Annette Ciccone in:

  9. Search for Sandra Verschoor in:

  10. Search for James C. Whisstock in:

  11. Search for Joseph A. Trapani in:

  12. Search for Ilia Voskoboinik in:

Conflict of interest

The authors declare that they have no conflict of interest.

Corresponding authors

Correspondence to Amelia J. Brennan or Ilia Voskoboinik.

Electronic supplementary material

About this article

Publication history

Received

Revised

Accepted

Published

DOI

https://doi.org/10.1038/s41418-018-0057-z