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.