ER stress induced autophagy plays an important role in main taining cellular

How ever, transfection of Flag PIAS1 at 1. 6 ug apparently decreased cell survival. PIAS1 plasmid transfection Amuvatinib 分子量 and expression was confirmed by immunoblot against Flag and PIAS1. Discussion In this study, we have identified the transcriptional re pressor Hes 1 as a novel SUMO substrate. In the cell, endogenous Hes 1 SUMOylation was not readily ob served due to low Hes 1 antibody efficiency and very few amount of IP product obtained. Thus, overexpres sion of Myc SUMO 1 was adopted which allows the detection of Hes 1 SUMOylation, suggesting that endogenous Hes 1 SUMOylation does take place in the cell. How ever, it is known that the SUMO molecule is attached to most substrates at the lysine residue of the ψ K X E consensus motif, which is directly bound by the E2 ligase UBC9.<br><br> This direct interaction explains why E1 and E2 only are sufficient to sumoylate many sub strates at the correct lysine residue in the absence AT-406 chemical 構造 of any E3. Actually, SUMO attach at non consensus sites has also been reported and it is suggested that the E3 ligase activity is particularly important for SUMOy lation at atypical consensus motif. Therefore, the association of E3 ligase and Hes 1 was further exam ined in our study. In support of this hypothesis, we have found that PIAS1 greatly enhanced the SUMOyla tion of Hes 1, and this effect was abolished by both PIAS1 siRNA and PIAS1W372A transfections. Further more, PIAS2 and PIAS3 also apparently increased the SUMOylation of Hes 1, but RanBP2 and Pc2 did not affect the SUMOylaiton of Hes 1. These results together reveal the important role of the PIAS family E3 ligase in Hes 1 SUMOylation in the cell.<br><br> There AG-490 分子量 are seven members of the Hes protein in the Hes protein family. Among these Hes proteins, Hes 1 and Hes 5 are important Notch ef fectors. Once activated by Delta, the NICD is cleaved by secretase and leads to the induction of Hes 1 and Hes 5 expression. In the absence of Hes 1 and Hes 5, NICD is unable to inhibit neurogenesis. Studies using knockout mice have shown that Hes 1 and Hes 5 oper ate in a common signaling pathway and they functionally compensate each other. However, in the present study we have found that Hes 1 could be SUMO modi fied by PIAS1, but Hes 5 could not. In another study, we have found that Hes 1 strongly regulates GluR1 expres sion in cultured cortical neurons but Hes 5 only moder ately does so.<br><br> These results together suggest that although both Hes 1 and Hes 5 are Notch effectors, their post translational modifications could be different and it is conceivable that they also participate in differ ent cellular functions. In the present study, we have identified Lys8, Lys27 and Lys39 as the major SUMO sites on Hes 1 and muta tion at these residues significantly decreased the DNA binding activity of Hes 1 to GADD45 promoter. Among these three lysine residues, Lys39 is located in the basic domain of GADD45 and Hes 1 can directly bind to DNA through its basic domain. Sequence alignment indi cated that Lys39 of Hes 1 is highly conserved in different species of vertebrates. Facilitation of DNA binding upon protein SUMOylation has also been demonstrated for other transcription factors.