The pore forming, Ca2+-dependent protein, perforin, is essential for the function of cytotoxic lymphocytes, which are at the frontline of immune defence1. Carriers of missense PRF1 mutations lack cytotoxic lymphocyte function due to perforin protein misfolding in the endoplasmic reticulum (ER), and consequently develop an aggressive immunoregulatory disorder, familial haemophagocytic lymphohistiocytosis (FHL2) or haematological malignancies.
Cytotoxic lymphocytes store cytotoxic effector molecules, including perforin and pro-apoptotic serine proteases granzymes, in secretory granules. Cytotoxic lymphocytes form a tight contact (immunological synapse) with target cells, which results in the release of perforin and granzymes into the synaptic cleft. Perforin remains inert at the acidic pH of the granules2, but when it reaches the neutral pH and high Ca2+ conditions of the synapse it binds to target cell membranes through its C2 domain3. Once bound, perforin oligomerises into large transmembrane pores that penetrate the lipid bilayer to allow the entry of granzymes, which trigger apoptotic cell death.
Recently, we discovered that Ca2+ binding to the C2 domain was necessary for the stabilisation, correct folding and trafficking of perforin from the ER. Remarkably, improving Ca2+ binding through the C2 domain stabilised misfolded and disease-causing perforin mutants. This was achieved by designing a “super-lytic” perforin mutant, which was >5-fold more cytotoxic than its wild-type counterpart and had a dramatically increased affinity for Ca2+. By combining “super-lytic” perforin with a disease-causing mutant, misfolded perforin was stabilised and its exocytic trafficking and function in cytotoxic lymphocytes restored.
Our studies demonstrate a novel role for Ca2+ in perforin proteostasis and provide an experimental rationale for the development of specific drug therapies for immunoregulatory disorders.
1. Brennan et al. (2010) Cell Death Differ. 17:607
2. Brennan et al. (2011) Immunity. 34:879
3. Traore*, Brennan* et al. (2013) Biochem J. 456:323 (*equal 1st author)