Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes

Abstract

Perforin-mediated cytotoxicity is an essential host defense, in which defects contribute to tumor development and pathogenic disorders including autoimmunity and autoinflammation. How perforin (PFN) facilitates intracellular delivery of pro-apoptotic and inflammatory granzymes across the bilayer of targets remains unresolved. Here we show that cellular susceptibility to granzyme B (GzmB) correlates with rapid PFN-induced phosphatidylserine externalization, suggesting that pores are formed at a protein-lipid interface by incomplete membrane oligomers (or arcs). Supporting a role for these oligomers in protease delivery, an anti-PFN antibody (pf-80) suppresses necrosis but increases phosphatidylserine flip-flop and GzmB-induced apoptosis. As shown by atomic force microscopy on planar bilayers and deep-etch electron microscopy on mammalian cells, pf-80 increases the proportion of arcs which correlates with the presence of smaller electrical conductances, while large cylindrical pores decline. PFN appears to form arc structures on target membranes that serve as minimally disrupting conduits for GzmB translocation. The role of these arcs in PFN-mediated pathology warrants evaluation where they may serve as novel therapeutic targets. Cell Death and Differentiation ( The cytotoxic cell granule-secretory pathway depends on perforin (PFN) to deliver granzyme (Gzm) proteases to the cytosol of target cells where they induce apoptosis and other biological effects, such as inflammation. 1 Ring-shaped transmembrane PFN pores hereafter called 'cylindrical pores', are presumed to act as the gateway for cytosolic entry, either at the plasma membrane or after endocytosis. [2][3][4] In either case the highly cationic Gzms are thought to diffuse through these cylindrical pores formed by poly-PFN. Nevertheless, a mechanistic understanding of the phenomenon (how the cationic globular protein exchanges from its carrier proteoglycan, serglycin, to the pore and crosses the plasma and/or vesicular membranes) has been lacking due to limitations in imaging technology and in our detailed understanding of the molecular forms that PFN may adopt following interaction with a target cell plasma membrane.Here we show under conditions where cylindrical pore formation is minimal, 5 that granzyme B (GzmB) translocation readily occurs. We previously demonstrated that a prelude to granzyme translocation is PFN-mediated, Ca-independent phosphatidylserine (PS) externalization (flip-flop) measured by annexin-V and lactadherin binding. 6 This rapid PS flip-flop also occurs when mouse CD8 cells contact antigen-pulsed target cells. Inasmuch as the proteinaceous cylinders offer a formidable barrier to lipid flow, we have speculated that the observed movement of anionic phospholipids to the external leaflet is due to the formation of proteo-lipidic structures, which consists of oligomerized PFN monomers bearing an arc morphology and plasma membrane lipids. [6][7][8] In the work reported here, the topology of PFN embedded into homogeneous planar bilayers and t...

Analysis of Perforin Assembly by Quartz Crystal Microbalance Reveals a Role for Cholesterol and Calcium-independent Membrane Binding

“…5G). It has recently been reported that atomic force microscopy images of perforin indeed include a mixture of arcs and fully formed rings (45). The full rings have a mean diameter of 20 nm, which is similar to previous studies using EM (8,9,(23)(24)(25)(26)(27)45).…”

More Than a Pore: The Interplay of Pore-Forming Proteins and Lipid Membranes

“…According to this, increased flip-flop of lipids was observed in vesicles in the presence of cytolytic peptides such as gramicidin (Classen et al 1987), melittin (Rapson et al 2011), and magainin (Nguyen et al 2009). Transbilayer movement of lipids has been also shown for proteins like colicin E1 (Sobko et al 2004), Bax (Epand et al 2002(Epand et al , 2003, actinoporins (Anderluh et al 2003;Valcarcel et al 2001), and perforin (Metkar et al 2015).…”

“…The pore presumably evolves via a toroidal structure (Color figure online) stable architecture in which amphipathic protein oligomers form an arc on one side of the pore and lipids located on the opposite side complete the toroidal structure. Pores formed by actinoporins are archetypal of ''matrix-type'' (Anderluh et al 2003;Valcarcel et al 2001), while MACPF/CDCs pores (like perforin and pneumolysin) represent the ''arc-type'' (Metkar et al 2015;Sonnen et al 2014). Direct evidences support the formation of pores by MACPF/CDCs oligomeric arcs (Fig.…”

“…Not only AMPs but also larger PFPs have been proposed to form protein-lipid pores. The list includes proteins from the Bcl-2/colicin family (Basanez 2002), actinoporins (Valcarcel et al 2001), and the membrane attack complex-perforin/cholesterol-dependent cytolysin (MACPF/CDC) superfamily Metkar et al 2015).…”

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

“…Once bound, the neutral pH of the synapse facilitates Prf1 oligomerisation 5 into ring and arc-shaped pores 6,7 , which are required for the diffusion of granzymes into the cytoplasm of the target cell to initiate apoptosis 8 . Prf1 therefore sits at the apex of the signaling cascade that triggers apoptotic target cell death and immune defense by cytotoxic lymphocytes.…”