Delivering the kiss of death: progress on understanding how perforin works

Abstract

Killer lymphocytes release perforin and granzymes from cytotoxic granules into the immunological synapse to destroy target cells as a critical mechanism in the defense against viruses and cancer. Perforin, a Ca(2+)-dependent pore-forming protein that multimerizes in membranes, delivers granzymes into the target cell cytosol. The original model for perforin (acting by forming a cell membrane channel through which granzymes pass) does not fit the experimental data. Recently, an alternative model has been proposed that involves active target cell collaboration with perforin to deliver granzymes and direct the target cell to an apoptotic, rather than necrotic, death.

Figure 1

Perforin sequence. Mature human perforin is produced by removal of the signal peptide (SP) and a poorly defined carboxy-terminal peptide (CTP). The C₂ domain is thought to be crucial for Ca²⁺-dependent membrane binding and the complement homology domain is needed for membrane insertion. Glycosylation sites are indicated by red stars. Numbering corresponds to that of the mature human protein.

Figure 2

Three models for how perforin delivers granzymes. (a) The original model for perforin delivery of granzymes was via multimerization in the cell membrane to form pores large enough for granzymes to pass through. (b) This model was revised by Froelich [71] to propose that granzymes are endocytosed independently of perforin and that perforin then acts as an endosomolysin. (c) We propose a hybrid model in which perforin forms small pores in the cell membrane that trigger a Ca²⁺ influx, which in turn activates a membrane repair response in which internal vesicles donate their membranes to patch the holes. The next step involves rapid co-endocytosis of granzymes and perforin into giant endosomes, followed by perforin-mediated release of granzymes to the cytosol. We do not know what triggers the rapid endocytosis or whether perforin pores in endosomal membranes destabilize the endosome (causing it to burst) or whether perforin forms endosomal membrane pores large enough to allow granzymes to escape. We think it is unlikely that granzymes enter the cell through plasma membrane pores, but that remains possible. In the figure the plasma membrane is black, endosome membranes are green and lysosomal membranes are blue.

Figure 3

Perforin triggers the rapid endocytosis of granzyme B into giant endosomes. (a) Target cells loaded with granzyme B (GzmB) and perforin (PFN) are rapidly taken up into gigantic early endosome antigen 1 (EEA-1)-staining endosomes. In the absence of perforin, some granyzme B is taken up into endosomes, but uptake is much less efficient. Images are obtained 2 min after incubating cells with granzyme B and perforin. (b) Large EEA-1-staining endosomes are also seen in target cells attacked by lymphokine-activated killer cells. Cells were preincubated in medium containing ethylene glycol tetraacetic acid (EGTA), and then granule exocytosis was triggered by adding Ca²⁺. Images reproduced with permission from [37^•].