Inflammasome

Inflammasome

1. Discovery

   • "molecular platform triggering activation of immune caspases"
   • Apaf-1 (NBD) found in apoptosome ∴ looked for multiprotein complexes containing caspase-1
     • 2002 - first inflammasome detected (NALP1) but bound to caspase 5 & ASC
       • Took CARD containing ASC and saw binding with Caspase-1
         • NLRP3 found in 2004 in protein interaction experiements
           • Muckle Wells: ↑ activity leading to fever, neutrophillia, rashes ect - treated with IL-1R antagonists to IL-1RA (anakinra)
           • Agostini et al (2004)
       • Martinon 2002
   • KO animals
     • no difference in TNFa/IL-12 or pro-IL-1ß but active IL-1b was ↓. WB used to look at processing and subunits

2. structure

   • ASC
     • CARD - caspase activation and recruitment domain
       • 100 amino acids @ N-terminal of caspase-1 in apaf-1
     • activation nucleates the pion like polymerisation of ASC
       • Filiments form which function as a platform for C1 activation
     • PYD
       • Mutuations causes familial mediterrianian fever: ↑ IL-1ß (thought pyrin competes with C1 for binding to ASC
     • recruits C1 via CARD/CARD interactions
       • forms higher order signalosome
         • brings caspase domains into closer proximity for dimerisation and cleavage
     • sprial assembley shown but cryoelectron microscopy
   • sensor protein (NLRP3)
     • PYD, NBD, LRR
     • released from autoinhibition after activation so PYD can interact with ASC
   • Caspase

3. Activation

   • ATP @ P2X7
   • K+ efflux agents
     • cells in high K+ solutions inhibit inflammasome/IL-1b
       • due to either depolarising membrane or K+ thought P2X7
   • Inflammatory crystals
     • monosodium urate (gout)
     • cholesterol (atherosclerosis)
     • asbestoes
   • Thirodoxin signalling
     • involved in redox signalling in cells
       • Bound to TXNIP in reduced form in resting cells. Oxidised by ROS, realising TXNIP
         • TXNIP then binds to NLRP3 to faciltate inflammasome assembaly
   • Denitrosylation
     • aka NO addition to a protein (SNO group) occurs as cystines w/ SH groups
       • C1 has cysteine on active site
     • Nitrosylation inhibits inflammasome.
       • ie S-Nitrosylation: Thiol group addition on a cyseine ie C1 active site. S-O bond alters proteins function
         • prevents interaction between NLRP3 & ASC
   • ROS (mitochondrial dysfunction)
     • many NLRP3 activators couple w/ NADPH oxidase
     • inhibited by antioxidants
     • mitochondria stimulated with nigercin/alim, ↑ ROS and inflammasome seen to recruit to MAM
       • NLRP3 @ ER
       • ASC & C1 i cytoplasms
     • autophagy is stimulated when mitochondria dysfunction and prodcude ↑ ROS
       • mitochondria deficient cells have inactive inflammasome
     • misense mutations in TNFR1 have TRAPS which increases mito activity and ROS ∴ ↑ IL-1ß
       • Bulua 2011
   • lysosomal dysfunction
     • protease (cathepsin) release
       • serine proteases in neuts independently cleave pro-IL-1b
         • inflammation or mechanical stim triggers lysome destabalisation
           • Protease inhibitors result in inactive NLRP3
       • DAMPS/PAMPS able to move via pannexin-1 and activate
   • Deubiquitination (cAMP & Ca++)
   • Amyloid ß
     • first shown in vitro then in vivo. Hu data showed and lysates showed increases in cleaved C1
       • APP/PS1: hu/mu chimeric AB and hu presselin-1
         • chronic Aß deposition, neuroinflam, cognitive impairment
         • Crossed with NLRP3 KO
           • Effects reversed, reduced cleaved C1 and IL-1ß. Cognition better as shown in morris water maze.
           • Heneka 2013
           • Sanz 2009 showed P2X7 dependence
   • IFNy @ GBP5

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