1、Mechanism and regulation of NLRP3 inflammasome activationOUTLINEIntroduction of NLRP3DefinitionStructureFunctionSignificanceMechanism of NLRP3 Inflammasome ActivationPriming the NLRP3 InflammasomeActivating the NLRP3 InflammasomeK+ EffluxCa2+ SignalingMitochondrial Dysfunction and ROSLysosomal Leaka

2、geNoncanonical Inflammasome PathwayAlternative NLRP3 Inflammasome PathwayRegulators of NLRP3 Inflammasome ActivationPKR and GBP5Nek7Conclusion and ProspectintroductionDefinition1Inflammasome is a pro-inflammatory protein complex that is formed after stimulation of the intracellular NOD-like receptor

3、s. It contains the enzyme caspase, which is activated in the complex and can then process cytokine proproteins into active cytokines.PRRPAMP and DAMPPyroptosisNLRP3 (NALP3) is an important sensor of cellular damage or stress. In stressed cells, such as those exposed to infection, it assembles with a

4、n adaptor protein and the protease caspase 1 to form a complex called an inflammasome. 1 Travers, Paul. Janeways immunobiology /M. Garland Science, 2012.introductionStructureNOD-like receptor (NLR)C terminal: Leucine-rich repeat (LRR)nucleotide-binding oligomerization domain (NOD)N terminal: pyrin d

5、omain (PYD)Apoptosis-associated speck-like protein containing a CARD (ASC)Caspase-12 Young Bae H H P J. Crystal structure of NALP3 protein pyrin domain (PYD) and its implications in inflammasome assembly.J. Journal of Biological Chemistry, 2011, 286(45):39528-36.Crystal Structure of Nalp3 PYD2introd

6、uctionFunctionStimuliConformational change of NLRP3 and oligomerizationRecruiting apoptosis-associated speck-like protein containing a CARD (ASC) and pro-caspase-1Activating caspase-1Cleavage of pro-IL-1 and pro-IL-18IL-1 and IL-18 for inflammatory reactionNALP proteins sense cellular damage and act

7、ivating1introductionSignificanceSystem diseasesCentral nervous systemAlzheimer s diseaseMultiplesclerosisCardiovascular systemAtherosclerosisRespiratory systemChronic obstructive pulmonary diseaseDigestive systemHepatocarcinomaAutoinflammatory diseasesFamilial cold inammatory syndromeMuckleWells syn

8、dromeMECHANISMC57BL / 6 macrophage3ASC-yellow fluorescent protein (YFP) Resting cells or cells treated with ATP or LPS alone3LPS-primed cells were treated with ATP, ASC-YFP formed large, irregularly shaped pyroptosomes.3 Bauernfeind F, Horvath G, Stutz A, et al. NF-kB activating pattern recognition

9、and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expressionJ. Journal of Immunology, 2009, 183(2):787-791.Caspase-1 activation and the formationof ASC pyroptosomes require priming3MECHANISMPriming step3Pretreatment (The first signal)Microbial ligandsThe protein amount

10、s of NLRP33InsufficientActivation of NF-BMacrophages4A retroviral vector Stimulation without priming High levels of NLRP34 Schroder K, Sagulenko V, Zamoshnikova A, et al. Acute lipopolysaccharide priming boosts inflammasome activation independently of inflammasome sensor inductionJ. Immunobiology, 2

11、012, 217(12):1325-1329.LPS pre-treatment4MECHANISMPost-transcriptional level5Priming macrophages for 10 minNot sufficient for the induction of NLRP3 expressionSignificantly enhancing inflammasome activationNF-kB-deficient mouse macrophage6The adaptor MyD88 and IL-1 receptor-associated kinase 1 (IRAK

12、1) and IRAK4The adaptor TRIF and IRAK15 Juliana C, Fernandesalnemri T, Kang S, et al. Non-transcriptional Priming and Deubiquitination Regulate NLRP3 Inflammasome ActivationJ. Journal of Biological Chemistry, 2012, 287(43):36617-22.6 Lin K M, Hu W, Troutman T D, et al. IRAK-1 bypasses priming and di

13、rectly links TLRs to rapid NLRP3 inflammasome activation.J. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(2):775-80.MECHANISMDeubiquitination7Lys-63-specific deubiquitinase BRCC36 (BRCC3)A JAMM domain-containing Zn2+ metalloproteasePromoting NLRP3 deubiqu

14、itination Regulating NLRP3 activation 7 Py B, Kim M S, Vakifahmetoglu-Norberg H, et al. Deubiquitination of NLRP3 by BRCC3 Critically Regulates Inflammasome ActivityJ. Molecular Cell, 2013, 49(2):331338. 8 Yuan H, Hideki H, Gabriel N. Mechanism and Regulation of NLRP3 Inflammasome ActivationJ. Trend

15、s in Biochemical Sciences, 2016, 41(12):10121021. NLRP3 priming8MECHANISMActivationK+ effluxCa2+ signalingMitochondrial dysfunction and reactive oxygen species (ROS)Lysosomal ruptureMECHANISMK+ efflux9,10,11A common denominatorPotassium ionophores such as nigericin Lowering cytosolic K+ concentratio

16、nNoncanonical inflammasome pathwayLittle about the mechanistic linkUpstream of NLRP3 activationNLRP3-activating mutation (NLRP3R258W)Conformational changesK+ efflux regulates NLRP3 inflammasome11 9 Muozplanillo R, Kuffa P, Martnezcoln G, et al. K+ efflux is the Common Trigger of NLRP3 inflammasome A

17、ctivation by Bacterial Toxins and Particulate MatterJ. Immunity, 2013, 38(6):1142-53.10 Yang D, He Y, Muoz-Planillo R, et al. Caspase-11 Requires the Pannexin-1 Channel and the Purinergic P2X7 Pore to Mediate Pyroptosis and Endotoxic ShockJ. Immunity, 2015, 43(5):1-10.11 Yaron J R, Gangaraju S, Rao

18、M Y, et al. K(+) regulates Ca(2+) to drive inflammasome signaling: dynamic visualization of ion flux in live cells.J. Cell Death & Disease, 2015, 6(e1954):420-425.MECHANISMCa2+ signaling12-14Ca2+ chelator BAPTA-AMpharmacological inhibition or small hairpin RNAKnockdown of the IP3REndoplasmic reticul

19、umCalcium-sensing receptor (CASR)G protein-coupled receptor (GPCR)Upstream of PLC Ca2+ mobilization and mitochondrial Ca2+ overloadMitochondrial dysfunctionControversy12 Lee G S, Subramanian N, Kim A I, et al. The calcium-sensing receptor regulates the NLRP3 inflammasome through Ca2+ and cAMP.J. Nat

20、ure, 2012, 492(7427):123-7.13 Murakami T, Ockinger J, Yu J, et al. Critical role for calcium mobilization in activation of the NLRP3 inflammasome.J. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(28):11282-7.14 Katsnelson M A, Rucker L G, Russo H M, et al.

21、 K+ Efflux Agonists Induce NLRP3 Inflammasome Activation Independently of Ca2+ Signaling.J. Journal of Immunology, 2015, 194(8):3937-52.Ca2+ signaling regulates NLRP3 inflammasome11 MECHANISMROS and Mitochondrial Dysfunction15-19Reactive oxygen species (ROS)NAPDH oxidaseHuman peripheral blood mononu

22、clear cellMitochondrial dysfunctionPerturbation of mitochondriaOxidized mitochondrial DNACardiolipinMitochondrial antiviral signaling protein (MAVS)15 Van B R, Kker M Y, Jansen M, et al. Human NLRP3 inflammasome activation is Nox1-4 independent.J. Blood, 2010, 115(26):5398-400.16 Zhou R, Yazdi A S,

23、Menu P, et al. A role for mitochondria in NLRP3 inflammasome activation.J. Nature, 2011, 469(7329):221-225.17 Nakahira K, Haspel J A, Rathinam V A, et al. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome.J. Nature I

24、mmunology, 2011, 12(3):222-230.18 Iyer S S, He Q, Janczy J R, et al. Mitochondrial cardiolipin is required for Nlrp3 inflammasome activationJ. Immunity, 2013, 39(2):311323. 19 Subramanian N, Natarajan K, Clatworthy M R, et al. The adaptor MAVS promotes NLRP3 mitochondrial localization and inflammaso

25、me activation.J. Cell, 2013, 153(2):348-61.MECHANISMLysosomal rupture9, 20Cathepsin BParticulate matterLysosome membraneCA-074-MeOff-target effectK+ efflux Lysosomotropic agent Leu-Leu-O-methyl ester 20 Dostert C, Guarda G, Romero J F, et al. Malarial hemozoin is a Nalp3 inflammasome activating dang

26、er signal.J. Plos One, 2009, 4(8):e6510. A Two-Signal Model for NLRP3 Inflammasome Activation 8MECHANISMNoncanonical Inflammasome Pathway21-24Caspase-11IL-1, IL-18pyroptosisGram-negative bacteriaDual-recognition mechanism of LPS Gasdermin D (GSDMD)Pannexin-1ATP channelP2X721 Kayagaki N, Warming S, L

27、amkanfi M. Non-canonical inflammasome activation targets caspase-11.J. Nature, 2011, 479(7371):117-21.22 Kayagaki N, Wong M T, Stowe I B, et al. Noncanonical inflammasome activation by intracellular LPS independent of TLR4.J. Science, 2013, 341(6151):1246-9.23 Kayagaki N, Stowe I B, Lee B L, et al.

28、Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.J. Nature, 2015, 526(7575):666-71.24 Ding J, Wang K, Wang L, et al. Pore-forming activity and structural autoinhibition of the gasdermin familyJ. Nature, 2016, 535(7610). Mechanisms of Activation for Noncanonical NLRP3 Inflamma

29、somes 8MECHANISMAlternative NLRP3 Inflammasome Pathway25Human monocyteLPSNo K+ efflux25 Gaidt M M, Ebert T S, Chauhan D, et al. Human Monocytes Engage an Alternative Inflammasome Pathway.J. Immunity, 2016, 44(4):833-846. Mechanisms of Activation for Alternative NLRP3 Inflammasomes8regulationRegulato

30、rs of NLRP3 inflammasome activationDouble-stranded RNA-dependent protein kinase (PKR)Guanylate-binding protein 5 (GBP5)Nek7regulationPKR & GBP526, 27Double-stranded RNA-dependent protein kinase (PKR)Regulating all known inflammasomesPromotionControversyGuanylate-binding protein 5 (GBP5)PromotionCont

31、roversy26 He Y, Franchi L, Nez G. The protein kinase PKR is critical for LPS-induced iNOS production but dispensable for inflammasome activation in macrophages.J. European Journal of Immunology, 2013, 43(5):1147-52.27 Meunier E, Dick M S, Dreier R F, et al. Caspase-11 activation requires lysis of pa

32、thogen-containing vacuoles by IFN-induced GTPases.J. Nature, 2014, 509(7500):366-370.regulationNek728, 29NIMA-related kinase familyMitotic progression and DNA damage responseEssentialNLRP3 oligomerizationASC speck formationCaspase-1 activationSpecificityNek6 & Nek9 PromotionMechanismK+ efflux28 He Y

33、, Zeng M Y, Yang D, et al. NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux.J. Nature, 2016, 530(7590):354-357.29 Shi H, Ying W, Li X, et al. NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome componentJ. Nature Immunolo

34、gy, 2016, 17(3):250-258.NLRP3 inflammasome8Conclusion and ProspectConclusion No unified mechanismA critical role for K+ effluxNek7 as an essential regulatorNoncanonical & alternative pathwayProspect Nek7 regulationSignals integrationPathobiologyPharmacologyreference1 Travers, Paul. Janeways immunobi

35、ology /M. Garland Science, 2012.2 Young Bae H H P J. Crystal structure of NALP3 protein pyrin domain (PYD) and its implications in inflammasome assembly.J. Journal of Biological Chemistry, 2011, 286(45):39528-36.3 Bauernfeind F, Horvath G, Stutz A, et al. NF-kB activating pattern recognition and cyt

36、okine receptors license NLRP3 inflammasome activation by regulating NLRP3 expressionJ. Journal of Immunology, 2009, 183(2):787-791.4 Schroder K, Sagulenko V, Zamoshnikova A, et al. Acute lipopolysaccharide priming boosts inflammasome activation independently of inflammasome sensor inductionJ. Immuno

37、biology, 2012, 217(12):1325-1329.5 Juliana C, Fernandesalnemri T, Kang S, et al. Non-transcriptional Priming and Deubiquitination Regulate NLRP3 Inflammasome ActivationJ. Journal of Biological Chemistry, 2012, 287(43):36617-22.6 Lin K M, Hu W, Troutman T D, et al. IRAK-1 bypasses priming and directl

38、y links TLRs to rapid NLRP3 inflammasome activation.J. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(2):775-80.7 Py B, Kim M S, Vakifahmetoglu-Norberg H, et al. Deubiquitination of NLRP3 by BRCC3 Critically Regulates Inflammasome ActivityJ. Molecular Cell

39、, 2013, 49(2):331338.8 Yuan H, Hideki H, Gabriel N. Mechanism and Regulation of NLRP3 Inflammasome ActivationJ. Trends in Biochemical Sciences, 2016, 41(12):10121021.9 Muozplanillo R, Kuffa P, Martnezcoln G, et al. K+ efflux is the Common Trigger of NLRP3 inflammasome Activation by Bacterial Toxins

40、and Particulate MatterJ. Immunity, 2013, 38(6):1142-53.10 Yang D, He Y, Muoz-Planillo R, et al. Caspase-11 Requires the Pannexin-1 Channel and the Purinergic P2X7 Pore to Mediate Pyroptosis and Endotoxic ShockJ. Immunity, 2015, 43(5):1-10.11 Yaron J R, Gangaraju S, Rao M Y, et al. K(+) regulates Ca(

41、2+) to drive inflammasome signaling: dynamic visualization of ion flux in live cells.J. Cell Death & Disease, 2015, 6(e1954):420-425.12 Lee G S, Subramanian N, Kim A I, et al. The calcium-sensing receptor regulates the NLRP3 inflammasome through Ca2+ and cAMP.J. Nature, 2012, 492(7427):123-7.13 Mura

42、kami T, Ockinger J, Yu J, et al. Critical role for calcium mobilization in activation of the NLRP3 inflammasome.J. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(28):11282-7.14 Katsnelson M A, Rucker L G, Russo H M, et al. K+ Efflux Agonists Induce NLRP3 I

43、nflammasome Activation Independently of Ca2+ Signaling.J. Journal of Immunology, 2015, 194(8):3937-52.15 Van B R, Kker M Y, Jansen M, et al. Human NLRP3 inflammasome activation is Nox1-4 independent.J. Blood, 2010, 115(26):5398-400.reference16 Zhou R, Yazdi A S, Menu P, et al. A role for mitochondri

44、a in NLRP3 inflammasome activation.J. Nature, 2011, 469(7329):221-225.17 Nakahira K, Haspel J A, Rathinam V A, et al. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome.J. Nature Immunology, 2011, 12(3):222-230.18 Iye

45、r S S, He Q, Janczy J R, et al. Mitochondrial cardiolipin is required for Nlrp3 inflammasome activationJ. Immunity, 2013, 39(2):311323. 19 Subramanian N, Natarajan K, Clatworthy M R, et al. The adaptor MAVS promotes NLRP3 mitochondrial localization and inflammasome activation.J. Cell, 2013, 153(2):3

46、48-61.20 Dostert C, Guarda G, Romero J F, et al. Malarial hemozoin is a Nalp3 inflammasome activating danger signal.J. Plos One, 2009, 4(8):e6510.21 Kayagaki N, Warming S, Lamkanfi M. Non-canonical inflammasome activation targets caspase-11.J. Nature, 2011, 479(7371):117-21.22 Kayagaki N, Wong M T, Stowe I B, et al. Noncanonical inflammasome activation by intracellular LPS independent of TLR4.J. Science, 2013, 341(6151):1