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Published on: April 10, 2018
Molecular cloning and characterization of mouse caspase-8
K Sakamaki1, S Tsukumo, S Yonehara
1Department of Viral Oncology, Institute for Virus Research, Kyoto University, Japan.
Abstract:
Fas (APO-1/CD95) is a transmembrane receptor protein which induces apoptosis upon activation. In apoptosis triggered by Fas, a subset of cysteine proteases designated caspases is activated, playing a central role as effector molecules. Among these caspases, human caspase-8 (FLICE/MACH/Mch5) has been isolated and shown to be indispensable for Fas-mediated apoptotic signaling. In this study, we isolated the mouse homologue to human caspase-8 from a BaF3 cell cDNA library. This molecule conserved the death effector domain (DED) and protease domain as detected in human caspase-8, and was capable of inducing apoptosis in KB and Rat-1 cells when overexpressed. Expression of caspase-8 was detected in the various tissues of adult mouse and in embryos at 9.5 days and 17.5 days of development by Northern-blot analysis. Further, we isolated a chromosomal gene for caspase-8 from a mouse genomic library and analyzed the genomic structure of the isolated gene. This gene consisted of eight exons and seven introns spanning about 26 kb in the coding region.
Insights
Researchers identified the mouse caspase-8, a key protein in Fas-mediated apoptosis. This molecule is crucial for apoptosis signaling and its gene structure was analyzed.
Area of Science:
- Molecular Biology
- Cell Biology
- Immunology
Background:
- Fas receptor triggers apoptosis via caspase activation.
- Human caspase-8 is essential for Fas-mediated apoptosis.
- Caspases are cysteine proteases central to apoptosis.
Purpose of the Study:
- To isolate and characterize the mouse homologue of human caspase-8.
- To investigate the role and expression of mouse caspase-8.
- To analyze the genomic structure of the mouse caspase-8 gene.
Main Methods:
- Isolation of mouse caspase-8 from a BaF3 cell cDNA library.
- Overexpression studies in KB and Rat-1 cells to assess apoptosis induction.
- Northern-blot analysis for expression in adult mouse tissues and embryos.
- Isolation and analysis of the mouse caspase-8 gene from a genomic library.
Main Results:
- Mouse caspase-8 was isolated, sharing conserved death effector and protease domains with human caspase-8.
- Overexpression of mouse caspase-8 induced apoptosis in tested cell lines.
- Caspase-8 expression was detected in various adult mouse tissues and developing embryos.
- The mouse caspase-8 gene comprises eight exons and seven introns, spanning approximately 26 kb.
Conclusions:
- Mouse caspase-8 is a functional homologue of human caspase-8 and plays a role in apoptosis.
- The expression pattern suggests caspase-8 is important for both development and adult tissue function.
- The characterized genomic structure provides a basis for further genetic studies of caspase-8.
