J Biol Chem. 2012 Oct 12.

Multiple binding sites on the ASC pyrin domain allow self-association and interaction with NLRP3.External

Vajjhala, P. R., Mirams, R. E., Hill, J. M.,
--- - The University of Queensland, Australia.
A key process underlying an innate immune response to pathogens or cellular stress is activation of members of the NOD-like receptor (NLR) family, such as NLRP3, to assemble caspase-1 activating inflammasome complexes. Activated caspase-1 processes proinflammatory cytokines into active forms that mediate inflammation. Activation of the NLRP3 inflammasome is also associated with common diseases including cardiovascular disease, diabetes, chronic kidney disease and Alzheimer's disease. However, the molecular details of NLRP3 inflammasome assembly are not established. The adaptor protein ASC plays a key role in inflammasome assembly. It is composed of an N-terminal pyrin domain (PYD) and a C-terminal caspase recruitment domain (CARD), which are protein interaction domains of the death-fold superfamily. ASC interacts with NLRP3 via a homotypic PYD interaction and recruits procaspase-1 via a homotypic CARD interaction. Here we demonstrate that ASC PYD contains two distinct binding sites important for self-association and interaction with NLRP3 and the modulatory protein POP1. Modelling of the homodimeric ASC PYD complex formed via an asymmetric interaction using both sites resembles a type I interaction found in other death-fold domain complexes. This interaction mode also permits assembly of ASC PYDs into filaments. Furthermore, this binding mode is likely conserved in interactions with NLRP3 and POP1, as residues critical for interaction of ASC PYD are conserved in these PYDs. We also demonstrate that ASC PYD can simultaneously self-associate and interact with NLRP3, rationalising the model whereby ASC self-association upon recruitment to NLRP3 promotes clustering and activation of procaspase-1.
PMID: 23066025External
Arrow2 In vitro interaction Arrow2 In vivo interaction Arrow2 Characterization Arrow2 Functional role Arrow2 top
In vitro interaction
  DD1 DD2 Reference
Family DD1 DD2 Method Species Region Expression Species Region Expression
PYD ASC Link ASC GST fusion protein pull-down Not specified 1-91 E.coli Not specified 1-96 In vitro translation 23066025
PYD ASC Link NLRP3 GST fusion protein pull-down Not specified 1-91 E.coli Not specified Full length In vitro translation 23066025
PYD ASC Link POP1 GST fusion protein pull-down Not specified 1-91 E.coli Not specified 1-89 In vitro translation 23066025
(Link: click this icon to show interactions only between the two corresponding DDs)
Arrow2 In vitro interaction Arrow2 In vivo interaction Arrow2 Characterization Arrow2 Functional role Arrow2 top
In vivo interaction
  Endogenous
expression
Overexpression DD1 DD2 Reference
Family DD1 DD2 Method Species Region Species Region
PYD ASC Link ASC Co-immunoprecipitation HEK293 Not specified 1-91 Not specified Full length 23066025
PYD ASC Link ASC Co-immunoprecipitation HEK293 Not specified 1-91 Not specified 1-96 23066025
PYD ASC Link NLRP3 Co-immunoprecipitation HEK293 Not specified 1-91 Not specified Full length 23066025
PYD ASC Link POP1 Co-immunoprecipitation HEK293 Not specified 1-91 Not specified 1-89 23066025
(Link: click this icon to show interactions only between the two corresponding DDs)
Arrow2 In vitro interaction Arrow2 In vivo interaction Arrow2 Characterization Arrow2 Functional role Arrow2 top
Characterization
  Stoichiometry Affinity Protein-Protein interface Reference
Family DD1 DD2 Method Mutation Complex structure
PYD ASC Link ASC Co-immunoprecipitation E13A, K21A, R41A, D48A, D51A (ASC) 23066025
PYD ASC Link ASC GST fusion protein pull-down E13A, K21A, R41A, D48A, D51A (ASC) 23066025
PYD ASC Link NLRP3 Co-immunoprecipitation E13A, K21A, R41A, D48A, D51A (ASC) 23066025
PYD ASC Link NLRP3 GST fusion protein pull-down E13A, K21A, R41A, D48A, D51A (ASC) 23066025
PYD ASC Link POP1 Co-immunoprecipitation E13A, K21A, R41A, D48A, D51A (ASC) 23066025
PYD ASC Link POP1 GST fusion protein pull-down R3A, D10A, E13A, K21A, R38A, R41A, D48A, D51A, D54A, Q84A (ASC) 23066025
(Link: click this icon to show interactions only between the two corresponding DDs)