The protease calpain2a limits innate immunity by targeting TRAF6 in teleost fish.

TNF receptor-associated factor 6 (TRAF6) plays a key signal transduction role in both antibacterial and antiviral signaling pathways. However, the regulatory mechanisms of TRAF6 in lower vertebrates are less reported. In this study, we identify calpain2a, is a member of the calcium-dependent proteases family with unique hydrolytic enzyme activity, functions as a key regulator for antibacterial and antiviral immunity in teleost fish. Upon lipopolysaccharide (LPS) stimulation, knockdown of calpain2a promotes the upregulation of inflammatory cytokines. Mechanistically, calpain2a interacts with TRAF6 and reduces the protein level of TRAF6 by hydrolyzing. After loss of enzymatic activity, mutant calpain2a competitively inhibits dimer formation and auto-ubiquitination of TRAF6. Knockdown of calpain2a also promotes cellular antiviral response. Mutant calpain2a lacking hydrolase activity represses ubiquitination of IFN regulatory factor (IRF) 3/7 from TRAF6. Taken together, these findings classify calpain2a is a negative regulator of innate immune responses by targeting TRAF6 in teleost fish.