title: Analysis of Perforin Assembly by Quartz Crystal Microbalance Reveals a Role for Cholesterol and Calcium-independent Membrane Binding.
abstract: Perforin is an essential component in the cytotoxic lymphocyte-mediated cell death pathway. The traditional view holds that perforin monomers assemble into pores in the target cell membrane via a calcium-dependent process and facilitate translocation of cytotoxic proteases into the cytoplasm to induce apoptosis. Although many studies have examined the structure and role of perforin, the mechanics of pore assembly and granzyme delivery remain unclear. Here we have employed quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate binding and assembly of perforin on lipid membranes, and show that perforin monomers bind to the membrane in a cooperative manner. We also found that cholesterol influences perforin binding and activity on intact cells and model membranes. Finally, contrary to current thinking, perforin efficiently binds membranes in the absence of calcium. When calcium is added to perforin already on the membrane, the QCM-D response changes significantly, indicating that perforin becomes membranolytic only after calcium binding.
authors.affiliation: From the Department of Biochemistry and Molecular Biology.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Stewart
authors.affiliation: From the Department of Biochemistry and Molecular Biology.
authors.forename: Catherina H
authors.initials: CH
authors.lastname: Bird
authors.affiliation: School of Chemistry, and.
authors.forename: Rico F
authors.initials: RF
authors.lastname: Tabor
authors.affiliation: From the Department of Biochemistry and Molecular Biology.
authors.forename: Michael E
authors.initials: ME
authors.lastname: D'Angelo
authors.affiliation: School of Chemistry, and.
authors.forename: Stefania
authors.initials: S
authors.lastname: Piantavigna
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Australian Research Council (ARC) Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria 3800 and.
authors.forename: James C
authors.initials: JC
authors.lastname: Whisstock
authors.affiliation: the Cancer Cell Death Laboratory, Cancer Immunology Program, Peter MacCallum Cancer Centre, St Andrew's Place, East Melbourne, Victoria 3002, Australia.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Trapani
authors.affiliation: School of Chemistry, and.
authors.forename: Lisandra L
authors.initials: LL
authors.lastname: Martin
authors.affiliation: From the Department of Biochemistry and Molecular Biology, phil.bird@monash.edu.
authors.forename: Phillip I
authors.initials: PI
authors.lastname: Bird
mesh_terms: D000818:Animals; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism; D008567:Membranes, Artificial; D051379:Mice; D054353:Perforin / Q000737:chemistry* / Q000378:metabolism; D058525:Quartz Crystal Microbalance Techniques / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D008567:Membranes, Artificial; D011994:Recombinant Proteins; D054353:Perforin; D002784:Cholesterol; D002118:Calcium
keywords: MACPF; Streptolysin O; calcium; cellular immune response; cholesterol; membrane bilayer; perforin; pore; protein self-assembly; quartz crystal microbalance with dissipation monitoring