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Publication Abstract from PubMed

The pore forming, Ca(2+)-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 Ca(2+) 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 Ca(2+) 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 Ca(2+)-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.

Perforin proteostasis is regulated through its C2 domain: supra-physiological cell death mediated by T431D-perforin.,Brennan AJ, Law RHP, Conroy PJ, Noori T, Lukoyanova N, Saibil H, Yagita H, Ciccone A, Verschoor S, Whisstock JC, Trapani JA, Voskoboinik I Cell Death Differ. 2018 Feb 7. pii: 10.1038/s41418-018-0057-z. doi:, 10.1038/s41418-018-0057-z. PMID:29416110^[6]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.