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.
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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.
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Brennan, A.J., Law, R.H.P., Conroy, P.J. et al. Perforin proteostasis is regulated through its C2 domain: supra-physiological cell death mediated by T431D-perforin. Cell Death Differ 25, 1517–1529 (2018). https://doi.org/10.1038/s41418-018-0057-z
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DOI: https://doi.org/10.1038/s41418-018-0057-z
