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

Abstract

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 stabilizes perforin and regulates its export from the endoplasmic reticulum to the secretory granules. Since perforin is a thermo-labile protein, we hypothesized that by altering its C2 domain it may be possible to improve protein stability. Based on the X-ray crystal structure of the perforin C2 domain, we designed a mutation (T431D) in the Ca 2+ binding loop. Mutant perforin displayed dramatically 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 stabilization of the C2 domain.3

Chemical priming of natural killer cells with branched polyethylenimine for cancer immunotherapy

“…Perforin is synthesized as a 70 kDa monomeric precursor in the ER, and binding of Ca 2+ to perforin is required for stable folding in the ER and subsequent trafficking to the cytoplasm, indicating that [Ca 2+ ] i is associated with perforin proteostasis in cytolytic lymphocytes. 26 Consistent with these reports, our data show that 25KbPEI-mediated calcium influx leads to accumulation of perforin.…”

“…Specifically, [Ca 2+ ] i is closely related to perforin proteostasis (eg, proper folding in the endoplasmic reticulum (ER), and trafficking from the Golgi to cytosolic vesicles) in cytolytic lymphocytes. 26 Since 25KbPEI induces perforin accumulation without transcriptional activation ( figure 2C ), we questioned whether this phenotype in NK cells was caused by 25KbPEI-mediated perturbation of [Ca 2+ ] i . As expected, a time-lapse experiment monitoring calcium concentrations in the cytoplasm of both NK92MI and primary NK cells clearly showed a rapid increase in [Ca 2+ ] i after treatment of 25KbPEI, but not after treatment with 25KfbPEI ( figure 5A , online supplemental figure 12 , online supplemental video 2 ).…”

Mechanisms of natural killer cell-mediated cellular cytotoxicity

“…However, recombinant perforin with and without the dodecapeptide shows the same pore‐forming activity and the X‐ray structure of perforin indicates that its C‐terminus is diverted away from Ca 2+ ‐binding region of the C2 domain, thereby challenging this hypothesis. Additionally, Ca 2+ binding to the C2 domain actually seems to be necessary for perforin protein stability and it regulates the export of perforin from the ER to the lytic granules . However, studies using endogenous instead of recombinant perforin showed that N‐linked glycosylation of the C‐terminal dodecapeptide inhibits its oligomerization and thereby its pore‐forming activity .…”

Degranulation enhances presynaptic membrane packing, which protects NK cells from perforin-mediated autolysis

“…Ultimately, this results in the death of the target and survival of the NK cell upon its degranulation. In considering this unidirectional attack of perforin, it is interesting that the membrane targeting of perforin to the target cell does not rely on any particular receiving ligand and is solely mediated by hydrophobic interaction [ 22 ]. Since these hydrophobic interactions are charge sensitive, we postulated that coalescence of high order lipid membrane domains, which can greatly affect the charge distribution on presynaptic membrane [ 23 ], might prevent perforin binding.…”

“…To more specifically investigate the mechanism of 7KC-induced NK cell death, especially its linkage with the activation induced by target cells and ultimately degranulation, we measured the effect of 7KC treatment under calcium-depleted conditions. Calcium is critical in the signaling required for NK cell activation and is also necessary for lytic granule release as well as perforin activation and membrane binding [ 22 ]. Thus, we depleted calcium from medium using the chelator EDTA at concentrations previously defined as effective to inactivate perforin [ 30 ].…”