Abstract
The NLRP3 inflammasome is a critical component of innate immunity that activates caspase-1 to induce inflammation in response to a wide spectrum of endogenous and exogenous stimuli. NLRP3 inflammasome activation has been shown by assays for the cleavage of caspase-1 and gasdermin D, the maturation of IL-1β and IL-18, and ASC speck formation in innate immune cells such as macrophages and monocytes. Recently, NEK7 has been revealed as an essential regulator for NLRP3 inflammasome activation by forming high-molecular-weight complexes with NLRP3. Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used to study multi-protein complexes in many experimental systems. Here, we provide a detailed protocol to detect NLRP3 inflammasome activation and NLRP3-NEK7 complex assembly in mouse macrophages by Western blot and BN-PAGE.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Schroder K, Tschopp J (2010) The Inflammasomes. Cell 140(6):821–832. https://doi.org/10.1016/j.cell.2010.01.040
Broz P, Dixit VM (2016) Inflammasomes: mechanism of assembly, regulation and signalling. Nat Rev Immunol 16(7):407–420. https://doi.org/10.1038/nri.2016.58
Jin T, Xiao TS (2015) Activation and assembly of the inflammasomes through conserved protein domain families. Apoptosis 20(2):151–156. https://doi.org/10.1007/s10495-014-1053-5
Srinivasula SM, Poyet JL, Razmara M, Datta P, Zhang ZJ, Alnemri ES (2002) The PYRIN-CARD protein ASC is an activating adaptor for caspase-1. J Biol Chem 277(24):21119–21122. https://doi.org/10.1074/jbc.C200179200
Shi J, Zhao Y, Wang K, Shi X, Wang Y, Huang H, Zhuang Y, Cai T, Wang F, Shao F (2015) Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526(7575):660–665. https://doi.org/10.1038/nature15514
Kayagaki N, Stowe IB, Lee BL, O’Rourke K, Anderson K, Warming S, Cuellar T, Haley B, Roose-Girma M, Phung QT, Liu PS, Lill JR, Li H, Wu J, Kummerfeld S, Zhang J, Lee WP, Snipas SJ, Salvesen GS, Morris LX, Fitzgerald L, Zhang Y, Bertram EM, Goodnow CC, Dixit VM (2015) Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526(7575):666–671. https://doi.org/10.1038/nature15541
Swanson KV, Deng M, Ting JP (2019) The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat Rev Immunol 19(8):477–489. https://doi.org/10.1038/s41577-019-0165-0
Sutterwala FS, Haasken S, Cassel SL (2014) Mechanism of NLRP3 inflammasome activation. Ann NY Acad Sci 1319:82–95. https://doi.org/10.1111/nyas.12458
Kelley N, Jeltema D, Duan Y, He Y (2019) The NLRP3 Inflammasome: an overview of mechanisms of activation and regulation. Int J Mol Sci 20(13). https://doi.org/10.3390/ijms20133328
He Y, Zeng MY, Yang D, Motro B, Nunez G (2016) NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux. Nature 530(7590):354–357. https://doi.org/10.1038/nature16959
Shi H, Wang Y, Li X, Zhan X, Tang M, Fina M, Su L, Pratt D, Bu CH, Hildebrand S, Lyon S, Scott L, Quan J, Sun Q, Russell J, Arnett S, Jurek P, Chen D, Kravchenko VV, Mathison JC, Moresco EM, Monson NL, Ulevitch RJ, Beutler B (2016) NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component. Nat Immunol 17(3):250–258. https://doi.org/10.1038/ni.3333
Schmid-Burgk JL, Chauhan D, Schmidt T, Ebert TS, Reinhardt J, Endl E, Hornung V (2015) A genome-wide CRISPR screen identifies NEK7 as an essential component of NLRP3 inflammasome activation. J Biol Chem. https://doi.org/10.1074/jbc.C115.700492
Swamy M, Kulathu Y, Ernst S, Reth M, Schamel WW (2006) Two dimensional blue native-/SDS-PAGE analysis of SLP family adaptor protein complexes. Immunol Lett 104(1-2):131–137. https://doi.org/10.1016/j.imlet.2005.11.004
Strecker V, Wumaier Z, Wittig I, Schagger H (2010) Large pore gels to separate mega protein complexes larger than 10 MDa by blue native electrophoresis: isolation of putative respiratory strings or patches. Proteomics 10(18):3379–3387. https://doi.org/10.1002/pmic.201000343
Wittig I, Braun HP, Schagger H (2006) Blue native PAGE. Nat Protoc 1(1):418–428. https://doi.org/10.1038/nprot.2006.62
Kofoed EM, Vance RE (2011) Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity. Nature 477(7366):592–595. https://doi.org/10.1038/nature10394
Duan Y, Zhang L, Angosto-Bazarra D, Pelegrin P, Nunez G, He Y (2020) RACK1 mediates NLRP3 Inflammasome activation by promoting NLRP3 active conformation and Inflammasome assembly. Cell Rep 33(7):108405. https://doi.org/10.1016/j.celrep.2020.108405
Acknowledgments
This work was supported by NIH grant AI148544 and Wayne State Startup funds to Y. H.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kelley, N., He, Y. (2023). Assessment of NLRP3 Inflammasome Activation and NLRP3-NEK7 Complex Assembly. In: Fink, S.L. (eds) Pyroptosis. Methods in Molecular Biology, vol 2641. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3040-2_2
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3040-2_2
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3039-6
Online ISBN: 978-1-0716-3040-2
eBook Packages: Springer Protocols