A non-classical necroptosis pathway mediated by caspases

Necroptosis is a regulated form of cell death mediated by the RIPK1/RIPK3−MLKL pathway. In humans, caspase (CASP) 8 negatively regulates the necroptotic pathway by inactivating RIPK1 and RIPK3. In teleosts and other non-mammalian vertebrates, the role of caspase in necroptosis remains to be explored. By using large yellow croaker Larimichthys crocea as a representative teleost species, this work investigated caspase-mediated regulation on teleost necroptosis. Necroptosis was investigated with microscopy and biochemical assays. Caspase cleavages were analyzed via immunoblotting. Protein-protein interactions were examined by co-immunoprecipitation. Evolutionary conservations were assessed through sequence alignment and WebLogo analysis. The three core necroptotic machinery components (RIPK1, RIPK3, and MLKL) were identified from L. crocea and named LcRIPK1, LcRIPK3, and LcMLKL, respectively. Cellular transfection studies showed that LcRIPK3 complexed with LcRIPK1 and recruited LcMLKL. The recruited LcMLKL was subsequently activated by LcRIPK3. LcMLKL possessed two conserved phosphorylation sites essential to LcMLKL activation, and mimetic phosphorylation of these sites induced strong necroptosis. The activities of LcRIPK1, LcRIPK3, and LcMLKL were all subjected to caspase regulation. L. crocea CASP (LcCASP) 3 and 6 inactivated LcRIPK1 by preferentially cleaving at 366 VEVD 369 , while LcCASP1/3/6 inactivated LcRIPK3 by preferentially cleaving at 377 CDVD 380 . In contrast, LcCASP1/3/7 activated LcMLKL by cleaving LcMLKL at 133 DAVD 136 to generate a constitutively active necroptosis executor, thus bypassing the RIPK1/3 signaling. A survey across Actinopterygii, Amphibia, Aves, and Mammalia showed that the standard caspase cleavage site (p4-xxxD-p1) was broadly conserved in vertebrate RIPK1/3, but the critical P4 residue varied in teleosts and exhibited lineage-specific conservation in mammals and amphibians. The DxxD motif was universally present in teleost, mammalian, and avian MLKL, while the NxxD motif was highly conserved in amphibian MLKL. These results suggested that caspase-mediated necroptosis regulation may be a common feature shared by most vertebrates. This study revealed a non-canonical necroptotic pathway mediated by integrated caspase regulation networks in teleosts and provided a strong hypothesis for the existence of a conserved CASPs−MLKL pathway in other vertebrate lineages. These findings added new insights into the complex regulation mechanisms of necroptosis in Vertebrata.