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Published on: January 20, 2023
A non-classical necroptosis pathway mediated by caspases
Qingyue Wang1,2, Hang Xu3, Xin Ding1,2
1State Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture and Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China.
Background:
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.
Methods:
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.
Results:
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 366VEVD369, while LcCASP1/3/6 inactivated LcRIPK3 by preferentially cleaving at 377CDVD380. In contrast, LcCASP1/3/7 activated LcMLKL by cleaving LcMLKL at 133DAVD136 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.
Conclusions:
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.
Insights
This study reveals that caspases regulate necroptosis in teleosts by cleaving key pathway proteins. This caspase-mediated necroptosis regulation is likely conserved across vertebrates, offering new insights into cell death mechanisms.
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- Necroptosis is a regulated cell death pathway involving RIPK1, RIPK3, and MLKL.
- In humans, caspase-8 inhibits necroptosis by inactivating RIPK1 and RIPK3.
- The role of caspases in necroptosis regulation in teleosts is largely unexplored.
Purpose of the Study:
- Investigate caspase-mediated regulation of necroptosis in teleosts using the large yellow croaker (Larimichthys crocea).
- Identify and characterize the core necroptosis components in L. crocea.
- Determine how caspases interact with and modulate the RIPK1/RIPK3-MLKL pathway in teleosts.
Main Methods:
- Microscopy and biochemical assays to study necroptosis.
- Immunoblotting to analyze caspase cleavages.
- Co-immunoprecipitation to examine protein-protein interactions.
- Sequence alignment and WebLogo analysis for evolutionary conservation.
Main Results:
- Identified and characterized LcRIPK1, LcRIPK3, and LcMLKL in L. crocea.
- Demonstrated that LcRIPK3 complexes with LcRIPK1 and recruits/activates LcMLKL.
- Showed that L. crocea caspases (LcCASP) 3, 6, and 7 differentially regulate LcRIPK1, LcRIPK3, and LcMLKL.
- LcCASP3/6 inactivate LcRIPK1 and LcRIPK3, while LcCASP1/3/7 activate LcMLKL.
- Found conserved caspase cleavage sites in RIPK1/3 and MLKL across vertebrates, suggesting a common regulatory mechanism.
Conclusions:
- Revealed a teleost necroptosis pathway with integrated caspase regulation networks.
- Proposed a conserved CASPs-MLKL pathway across vertebrate lineages.
- Provided new insights into the complex regulation of necroptosis in Vertebrata.
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