Fig. 4
From: O-GlcNAcylation of SIRT1 enhances its deacetylase activity and promotes cytoprotection under stress
O-GlcNAcylation of SIRT1 at Ser549 increases its deacetylase activity. a NCI-H1299 cells were cotransfected with p53, shSIRT1 and either Flag-tagged wtSIRT1 or SIRT1S549A vectors. The cells were then treated by 1 μM TSA combining with or without 2 μM TMG for 3 h. The whole-cell extracts were immunoblotted with antibodies against p53 acetylated at Lys 382, p53, or SIRT1. Data represent three independent experiments. b NCI-H1299 cells were cotransfected with shSIRT1 and either Flag-tagged wtSIRT1 or SIRT1S549A vectors. The cells were then treated by 1 μM TSA combining with or without 2 μM TMG for 3 h. The whole-cell extracts were immunoblotted with antibodies against acetylated H3 (Lys 9/14), H3, or SIRT1. Data represent two independent experiments. c NCI-H1299 cells transfected with Flag-tagged wtSIRT1 or SIRT1S549A were treated with or without 2 μM TMG for 4 h. SIRT1 was immunopurified from cell extracts using anti-FLAG M2 Affinity Gel, and the relative deacetylase activity was determined in vitro at the concentration of 25 μM fluorogenic acetylated p53 peptide substrate and 500 μM NAD+ using a fluorometric assay system. Data represent two independent experiments. d The enzyme kinetic parameters were detected by varying the p53 (K382Ac) fluorometric peptide substrate concentration while keeping NAD+ at a saturating concentration for wtSIRT1 (black square), wtSIRT1 bearing O-GlcNAc (wtSIRT1-GlcNAc) (black triangle), SIRT1S549A (gray square), and SIRT1S549A bearing O-GlcNAc (SIRT1S549A-GlcNAc) (gray triangle). The Lineweaver–Burk plot and Km values were showed. Student’s t-test. Results are expressed as the mean ± s.d. (n = 3 biologic replicates). P-values: ns (not significant), ***P < 0.001
