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1.
Nat Commun ; 5: 3350, 2014 Feb 28.
Article in English | MEDLINE | ID: mdl-24577104

ABSTRACT

Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages the DEDs of procaspase 8 and its inhibitor FLIP to form death-inducing signalling complexes (DISCs). The DEDs of FADD, FLIP and procaspase 8 interact with one another using two binding surfaces defined by α1/α4 and α2/α5 helices, respectively. Here we report that FLIP has preferential affinity for the α1/α4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD's α2/α5 surface. These relative affinities contribute to FLIP being recruited to the DISC at comparable levels to procaspase 8 despite lower cellular expression. Additional studies, including assessment of DISC stoichiometry and functional assays, suggest that following death receptor recruitment, the FADD DED preferentially engages FLIP using its α1/α4 surface and procaspase 8 using its α2/α5 surface; these tripartite intermediates then interact via the α1/α4 surface of FLIP DED1 and the α2/α5 surface of procaspase 8 DED2.


Subject(s)
CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Caspase 8/metabolism , Fas-Associated Death Domain Protein/metabolism , Blotting, Western , Chromatography, Gel , HCT116 Cells , Humans , Immunoprecipitation , Protein Binding
2.
Cell Death Differ ; 19(8): 1317-27, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22322857

ABSTRACT

FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors. We report a novel interaction between FLIP and the DNA repair protein Ku70 that regulates FLIP protein stability by inhibiting its polyubiquitination. Furthermore, we found that the histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) enhances the acetylation of Ku70, thereby disrupting the FLIP/Ku70 complex and triggering FLIP polyubiquitination and degradation by the proteasome. Using in vitro and in vivo colorectal cancer models, we further demonstrated that SAHA-induced apoptosis is dependant on FLIP downregulation and caspase 8 activation. In addition, an HDAC6-specific inhibitor Tubacin recapitulated the effects of SAHA, suggesting that HDAC6 is a key regulator of Ku70 acetylation and FLIP protein stability. Thus, HDAC inhibitors with anti-HDAC6 activity act as efficient post-transcriptional suppressors of FLIP expression and may, therefore, effectively act as 'FLIP inhibitors'.


Subject(s)
Antigens, Nuclear/metabolism , Apoptosis/drug effects , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , DNA-Binding Proteins/metabolism , Histone Deacetylase Inhibitors/pharmacology , Acetylation , Amino Acid Sequence , Animals , Antigens, Nuclear/genetics , CASP8 and FADD-Like Apoptosis Regulating Protein/biosynthesis , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , Caspase 8/metabolism , DNA-Binding Proteins/genetics , Down-Regulation , Female , HCT116 Cells , HT29 Cells , Histone Deacetylase 6 , Histone Deacetylases/metabolism , Humans , Hydroxamic Acids/pharmacology , Ku Autoantigen , Mice , Mice, Inbred BALB C , Protein Processing, Post-Translational , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Transfection , Vorinostat
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