Analysis of perforin assembly by quartz crystal microbalance reveals a role for cholesterol and calcium-independent membrane binding

Sarah E Stewart, Catherina H Bird, Rico F Tabor, Michael D'Angelo, Stefania Piantavigna, James C Whisstock, Joseph A Trapani, Lisandra L Martin, Phillip I Bird

Research output: Contribution to journalArticleResearchpeer-review

3 Citations (Scopus)

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. While 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.
Original languageEnglish
Pages (from-to)31101 - 31112
Number of pages12
JournalJournal of Biological Chemistry
Volume290
Issue number52
DOIs
Publication statusPublished - 2015

Cite this

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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. While 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.",
author = "Stewart, {Sarah E} and Bird, {Catherina H} and Tabor, {Rico F} and Michael D'Angelo and Stefania Piantavigna and Whisstock, {James C} and Trapani, {Joseph A} and Martin, {Lisandra L} and Bird, {Phillip I}",
year = "2015",
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language = "English",
volume = "290",
pages = "31101 -- 31112",
journal = "Journal of Biological Chemistry",
issn = "1083-351X",
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Analysis of perforin assembly by quartz crystal microbalance reveals a role for cholesterol and calcium-independent membrane binding. / Stewart, Sarah E; Bird, Catherina H; Tabor, Rico F; D'Angelo, Michael; Piantavigna, Stefania; Whisstock, James C; Trapani, Joseph A; Martin, Lisandra L; Bird, Phillip I.

In: Journal of Biological Chemistry, Vol. 290, No. 52, 2015, p. 31101 - 31112.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Analysis of perforin assembly by quartz crystal microbalance reveals a role for cholesterol and calcium-independent membrane binding

AU - Stewart, Sarah E

AU - Bird, Catherina H

AU - Tabor, Rico F

AU - D'Angelo, Michael

AU - Piantavigna, Stefania

AU - Whisstock, James C

AU - Trapani, Joseph A

AU - Martin, Lisandra L

AU - Bird, Phillip I

PY - 2015

Y1 - 2015

N2 - 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. While 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.

AB - 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. While 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.

UR - http://www.jbc.org/content/290/52/31101.full.pdf+html

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