Functional characterization of apoptosis-associated speck-like protein (ASC) of the goldfish (Carassius auratus L.)

• ASC mRNA levels were highest in the spleen and macrophages.
• ASC expression was up-regulated in nigericin-treated macrophages.
• Goldfish ASC aggregated to form multimers and formed speck-like structures.
• ASC interacted with goldfish RIP2 and Caspase-1 in vitro.
• ASC did not induce activation of NF-κB, but down-regulated RIP2 ability to activated NF-κB.

Quantitative expression analysis of goldfish ASC indicated the highest and lowest mRNA levels in spleen and muscle, respectively. The ASC was differentially expressed in normal goldfish tissues and different immune cell populations. The highest ASC mRNA levels were observed in the spleen and macrophages. We generated a recombinant form of the molecule (rgfASC) and an anti-ASC IgG antibody, and report that treatment of goldfish macrophages with nigericin, an inducer of inflammasome pathway, up-regulated the expression of ASC at both mRNA and protein levels. rgfASC aggregated to form multimers in cross-linking assays, and formed speck-like structures visualized by confocal microscopy. Co-immunoprecipitation assays showed that rgfASC interacted with caspase-1 and receptor-interacting serine/threonine kinase 2 (RIP2). The dual luciferase reporter assay showed that ASC over-expression did not cause the activation of NF-κB directly, but down-regulated RIP2 ability to activate NF-κB. Goldfish ASC was found to interact with both Nod-like receptor and inflammasome signaling pathway molecules, suggesting multifunctional roles for ASC in regulation of different NLR signaling pathways and eventual proinflammatory cytokine production by activated macrophages.