TRAF3 interacts with Smac/DIABLO and enhances the proapoptotic effect of Smac/DIABLO in cytoplasm.
Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI) is a 29 kDa mitochondrial precursor protein, which is proteolytically processed in mitochondria into a 23 kDa mature protein. It is released from the mitochondrial intermembrane space to cytosol after an apoptotic trigger. Smac/DIABLO acts as a dimer and it contributes to caspase activation by sequestering the inhibitor of apoptosis proteins (IAPs). In order to further investigate the mechanism of Smac/DIABLO action, we used the mature form of Smac/DIABLO as a bait and screened proteins that interact with mature Smac/DIABLO in human liver cDNA library using the yeast two-hybrid system. Forty-two colonies were obtained after 5.8x10(6) colonies were screened by nutrition limitation and X-galactosidase assay. After DNA sequence analysis and homology retrieval, one of the candidate proteins was identified as TRAF domain of the TNF receptor associated factor 3 (TRAF3). The interaction site between TRAF3 and Smac/DIABLO was identified by beta-galactosidase test. The interaction between TRAF3 and Smac/DIABLO via TRAF domain was identified in vivo by co-immunoprecipitation in HepG2 cells, and the direct interaction between TRAF3 and Smac/DIABLO in vitro was identified by GST-pull down assay. Co-expression of TRAF3 and mature Smac/DIABLO in 293 cells could enhance the Smac/DIABLO-mediated apoptosis. These results suggested that TRAF3 interacted with Smac/DIABLO via TRAF domain, leading to an increased proapoptotic effect of Smac/DIABLO in cytoplasm.
Zhang
Cuili
C
Clinical Laboratory Sichuan Corps Hospital of The People's Armed Police Forces, Leshan 614000, China.
Xie
Lei
L
Cheng
Hai'en
H
Wang
Yingxiong
Y
D000328:Adult; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D001483:Base Sequence; D017931:DNA Primers; D005784:Gene Amplification; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000502:physiology*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D048008:TNF Receptor-Associated Factor 3 / Q000235:genetics / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; C412403:DIABLO protein, human; D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D048008:TNF Receptor-Associated Factor 3; C090656:TRAF3 protein, human; D005982:Glutathione Transferase