title: A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly.
abstract: Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro-caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface. VHHASC not only impairs ASC(CARD) interactions in vitro, but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHHASC leaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHHASC-EGFP into these structures allowed the visualization of endogenous ASC(PYD) filaments for the first time. These data revealed that cross-linking of ASC(PYD) filaments via ASC(CARD) mediates the assembly of ASC foci.
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142.
authors.forename: Florian I
authors.initials: FI
authors.lastname: Schmidt
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115.
authors.forename: Alvin
authors.initials: A
authors.lastname: Lu
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142.
authors.forename: Jeff W
authors.initials: JW
authors.lastname: Chen
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115.
authors.forename: Jianbin
authors.initials: J
authors.lastname: Ruan
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115.
authors.forename: Catherine
authors.initials: C
authors.lastname: Tang
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115 wu@crystal.harvard.edu ploegh@wi.mit.edu.
authors.forename: Hao
authors.initials: H
authors.lastname: Wu
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 wu@crystal.harvard.edu ploegh@wi.mit.edu.
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
mesh_terms: D000072283:A549 Cells; D053468:CARD Signaling Adaptor Proteins; D003598:Cytoskeletal Proteins / Q000276:immunology*; D004268:DNA-Binding Proteins / Q000276:immunology; D005260:Female; D006801:Humans; D058847:Inflammasomes / Q000276:immunology*; D008297:Male; D009000:Monocytes / Q000276:immunology*; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein / Q000276:immunology; D051616:Neuronal Apoptosis-Inhibitory Protein / Q000276:immunology; D057127:Single-Chain Antibodies / Q000276:immunology / Q000494:pharmacology*
chemical_list: C410167:AIM2 protein, human; D053468:CARD Signaling Adaptor Proteins; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D058847:Inflammasomes; C496800:NAIP protein, human; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; D051616:Neuronal Apoptosis-Inhibitory Protein; C402202:PYCARD protein, human; D057127:Single-Chain Antibodies