Abstract
Perforin is a key protein of the vertebrate immune system. Secreted by cytotoxic lymphocytes as soluble monomers, perforin can self-assemble into oligomeric pores of 10–20 nm inner diameter in the membranes of virus-infected and cancerous cells. These large pores facilitate the entry of pro-apoptotic granzymes, thereby rapidly killing the target cell. To elucidate the pathways of perforin pore assembly, we carried out real-time atomic force microscopy and electron microscopy studies. Our experiments reveal that the pore assembly proceeds via a membrane-bound prepore intermediate state, typically consisting of up to approximately eight loosely but irreversibly assembled monomeric subunits. These short oligomers convert to more closely packed membrane nanopore assemblies, which can subsequently recruit additional prepore oligomers to grow the pore size.
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Acknowledgements
This work was funded by the BBSRC (BB/J005932/1, BB/J006254/1 and BB/N015487/1), the ERC (advanced grant no. 294408), the Wellcome Trust (no. 079605/2/06/02), NHMRC Fellowship (1059126), Project (1062706) and Program (1013667) grants, and the Sackler Foundation. The authors thank N. Nand Gosvami and J. Pegman for assistance with early experiments and analysis, and R. Thorogate, A. Ciccone and S. Verschoor for technical support.
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C.L. designed, performed and analysed AFM experiments and co-wrote the paper. A.W.H. designed, performed and analysed AFM and FRAP experiments and co-wrote the paper. A.J.B. expressed mutant perforin, developed the TMH1-lock mutant and carried out haemolysis experiments. N.L. carried out and analysed electron microscopy experiments and co-wrote the paper. S.T. performed and analysed haemolysis experiments. C.M.H. performed mass spectrometric analysis and co-wrote the paper. S.C.K., J.C.W. and M.A.D. contributed to the TMH1-lock mutant design. J.A.T. designed in vitro experiments. I.V., H.R.S. and B.W.H. analysed the data, led the research and co-wrote the paper. All authors read and commented on the manuscript.
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Leung, C., Hodel, A., Brennan, A. et al. Real-time visualization of perforin nanopore assembly. Nature Nanotech 12, 467–473 (2017). https://doi.org/10.1038/nnano.2016.303
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DOI: https://doi.org/10.1038/nnano.2016.303
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