Perforin Rapidly Induces Plasma Membrane Phospholipid Flip-Flop

Consejo Superior de Investigaciones Cientificas, Spain
PLoS ONE (Impact Factor: 3.23). 09/2011; 6(9):e24286. DOI: 10.1371/journal.pone.0024286
Source: PubMed


The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of intracellular protein delivery where granule proteases (granzymes) from cytotoxic lymphocytes are thought to diffuse through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN) and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm) when applied to liposomal membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidylserine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining highly correlated with apoptosis after Granzyme B (GzmB) treatment. We propose the structures that PFN oligomers form in the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB to translocate across the bilayer to the cytosolic leaflet of target cells.

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    • "During this time the target cell continues its death process, primarily by apoptosis (Figure 1q). Perforin induces membrane flipping on the target cell, resulting in exposure of phosphatidylserine.142 Phosphatidylserine on the surface of the cell may provide signaling to the NK cell to terminate the response, as NK cells express proteins that have been shown to bind to phosphatidylserine such as the ITIM-containing molecule CD300a.143 "
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    • "This indicates that the membrane repair response does not alter the intracellular signaling events (i.e., pyroptosis), even when membrane repair reduced direct lysis. Similar results have been observed with the immune PFT perforin, where decreasing direct lysis of cells by modulating calcium concentration did not alter the ability of cells to externalize phosphatidylserine or receive granzyme B [33]. We propose that membrane repair responses are protective, and bacterial toxins have evolved to provide rapid and effective lysis of target cells. "
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    • "This may be related to the absence of the signal peptide and inhibitory C-terminal. A recent study indicated that perforin pore structures can favor phosphatidylserine flip-flops, as measured by Annexin-V, at the concentrations that encourage granzyme-mediated apoptosis [31]–[32]. However, in our experiments, the apoptotic cell death induced by both full-length and truncated perforin was verified by TUNEL and caspase-3 activation assay. "
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