Studies Of The Functions Of SGT And Its Interaction Proteins

SGT (Small Glutamine-rich Teratricopeptiderepeat TPR) was cloned in 1998. It consists of 313 amino acids with a glutamine-rich sequence in its carboxyl terminal and three tandom TPR repeats in the center of the polypeptide chain. It's reported that SGT interacts with Hsp70 and Hsc70 and regulates their chaperon activities, which antagonize against heat shock, serum depletion and the apoptosis induced by drugs used in chemical therapies. Further more, many TPR domain-containig proteins such as CHIP, TPR2 and Strap play important roles in cell apoptosis with different mechanisms. To clarify whether SGT participates in the progress of cell apoptosis, we first constructed the SMMC-7721 cell lines stably expressing SGT. SMMC-7721 cells and the stable transfected cells were treated with CHX, followed by PI staining, Annexin V and PI co-staining, DNA fragmentation test, and Hoechest 33258 staining, apoptosis bodies assay. It was found in all the experiments that SGT promoted the apoptosis of 7721 cells induced by CHX, and its role in the regulation of apoptosis was caspase-dependent. SGT potentiated the activity of caspases and facilitated the cleavage of PARP, a substrate of caspase3. Overexpression of SGT led to the localization of more p53 on mitochondria than control group. To further investigate the function of SGT protein, we screened the cDNA library of human fetal liver using SGT as the bait in yeast two-hybrid system. PIAS1 was identified as a new interacting partner of SGT, and their interactions in vitro and in vivo were confirmed by GST-pull down, co-immunoprecipitation and confocal assays.β-1,4-galactosyltransferase 1 (GalT I) is the first cloned glycosyltransferase. It had been regarded as a house-keeping gene for a long time. In our previous investigation, it was found that GaT I was an intraction protein of SGT, and EIAF, one of the Ets family members could regulat its expression. In this paper, we investigated whether Ets-1, another member of Ets family could regulate its expression. Cotransfection of GalT I promoter /luciferase reporter and Ets-1 expression plasmid increased the luciferase reporter activity in a dose dependent manner. By deletion analysis, we identified a region between nucleotide -215—139 in GalT I promoter which was critical for responsiveness to Ets-1. Site specific mutagenesis revealed this region contained an Ets binding site (-205—200) which was necessary for GalT I activation. We confirmed that Ets-1 coud bind to and activate GalT I promoter by EMSA and chromatin-immunoprecipitation. These data suggest that Ets-1 is a transcriptional factor of GalT I.