Peptide Switch Is Essential for Sirt1 Deacetylase Activity Hyeog Kang, Jeong-Yong Suh, Young-Sang Jung, Jae-Won Jung, Myung K. Kim, Jay H. Chung  Molecular Cell  Volume 44, Issue 2, Pages 203-213 (October 2011) DOI: 10.1016/j.molcel.2011.07.038 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 44, 203-213DOI: (10.1016/j.molcel.2011.07.038) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Identification of a Region Essential for Sirt1 Deacetylase Activity (ESA) in the C-Terminal Domain (A) A summary of the deacetylase activity of recombinant Sirt1 containing the indicated C-terminal truncations, measured by using Ac-p53 as substrate. The conserved Sirtuin domain is shown as a black rectangle. See also Figures S1A and S1B. (B) A summary of the deacetylase activity of recombinant Sirt1 containing internal deletions (indicated by black bars) in the C-terminal domain. Original data for Sirt1 activity are shown in Figure S1B. The ESA region is overlined. (C) Evolutionary conservation of the ESA region. Gly 644 and Asp 650 are indicated with asterisks (∗). The locations of the Sirtuin domain and the ESA are shown below. See Figure S1C for a comparison of the C-terminal sequences of vertebrate Sirt1. (D) Mutations of the ESA region abolish Sirt1 activity. (Left) Deacetylation reactions were performed with recombinant Sirt1 containing either a single mutation of Gly 644→Pro (G644P) or Asp 650→Arg (D650R) or a double mutation (GPDR) of these two amino acid residues. Ac-p53 is visualized by immunoblotting with an antibody specific for Ac-K382 of p53. (Right) Deacetylation reactions were performed with WT and mutant Sirt1 (including ΔESA) using 3H-Ac-H4 as the substrate. Sirt1 activity was quantified by measuring the levels of O-[3H]acetyl-ADP-ribose that was liberated from deacetylase reaction by using scintillation counting (N = 4). Results are expressed as the mean ± SEM. (E) The ESA region is essential for Sirt1 activity in vivo. (Left) H1299 cells were transiently cotransfected with an expression vector for p53 and an expression vector for either WT Sirt1 or ΔESA Sirt1. Ac-p53 was visualized by immunoblotting with Ac-K328 (p53) antibody. (Right) HeLa cells were transiently cotransfected with an expression vector for FLAG-tagged NF-κB p65 (p65) and an expression vector for either V5-tagged WT Sirt1 or ΔESA Sirt1. Ac-p65 was visualized by immunoblotting with anti-Ac-Lys antibody after immunoprecipitating with FLAG antibody. Transfection with an empty vector (−) was used as a negative control. (F) Sirt1-mediated suppression of p53 activity requires the ESA region. The expression levels of p53 activated genes p21, PUMA, and Bim were visualized in H1299 cells cotransfected with an expression vector for p53 and an expression vector for either WT Sirt1 or ΔESA Sirt1. Transfection with an empty vector (−) was used as a control. (G) The ESA region is sufficient to confer activity to the deacetylase core. Deacetylation reactions were performed with recombinant deacetylase core (Core, aa 184–510), which includes additional sequences flanking the conserved Sirtuin domain (aa 236–490), as well as recombinant deacetylase core fused either to the ESA region (Core-ESA) or to the ESA region containing the GPDR double mutation (Core-GPDR). Molecular Cell 2011 44, 203-213DOI: (10.1016/j.molcel.2011.07.038) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 The ESA Interacts with the Deacetylase Core (A) The position of the C-terminal domain relative to the deacetylase core is not critical for Sirt1 deacetylase activity. Deacetylation reactions were performed with WT Sirt1 or Sirt1 in which the C-terminal domain (gray, aa 611–737) was placed on the N-terminal side of the deacetylase core (C-Core, aa 184–510). See Figure S2A–S2D for the disordered nature of the C-terminal domain of Sirt1. (B) The ESA binds to the Sirtuin domain. Pull-down assays were performed after incubating biotin-tagged ESA peptide immobilized on streptavidin beads with either F1a (aa 1–236) or F1b (aa 236–490) fragments. Streptavidin-immobilized biotin without the peptide is used as negative control. F1a or F1b bound to the ESA peptide is shown above, and the input levels of F1a and F1b are shown below. (C) The GPDR mutant ESA peptide can inhibit the deacetylase activity of Sirt1 (left panel), but not of Sirt6 (right panel), in trans. The deacetylase activity of recombinant Sirt1 and Sirt6 was measured by a fluorometric assay in the presence of 150 μM of either the WT ESA peptide, or the GPDR mutant ESA peptide (GPDR) or no peptide (−) (N = 4). Results are expressed as the mean ± SEM. See also Figure S2E–S2G. (D and E) The GPDR mutant ESA peptide inhibits Sirt1 in a noncompetitive manner. A Lineweaver-Burk plot derived from a fluorometric measurement of WT Sirt1 activity in the absence or presence of 0 (♦), 60 (□), 120 (▴) or 240 (×) μM of the GPDR mutant ESA peptide for the indicated concentration range of Ac-H4 (D) and NAD+ (E). Inhibition constants (Kii or Kis) for the GPDR peptide are shown for both Ac-H4 and for NAD+ substrates. For each inhibition analysis, the concentrations of the indicated substrate and the inhibitors were varied while the concentration of the other substrate was kept constant (500 μM NAD+ for D and 100 μM Ac H4 for E). Each experiment was performed in triplicate. To calculate the inhibition constants, nonlinear mixed-effects model fitting function in the programming language R was used for data fitting against double-reciprocal form of the noncompetitive model equation as described in the Experimental Procedures section. Molecular Cell 2011 44, 203-213DOI: (10.1016/j.molcel.2011.07.038) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 The ESA Increases the Sirt1-Substrate Interaction (A) Deletion of the ESA region decreases Sirt1-substrate interaction in vivo. HeLa cells were transiently cotransfected with an expression vector for FLAG-tagged NF-κB p65 (p65) and either an expression vector for V5-tagged full-length Sirt1 or Sirt1 with the ESA region deleted (ΔESA Sirt1). FLAG-tagged NF-κB p65 was immunoprecipitated with FLAG antibody and coimmunoprecipitated Sirt1 was visualized by immunoblotting with V5 antibody. The input levels of the proteins are shown on the right. (B) Deletion of the ESA region decreases Sirt1-substrate interaction in vitro. Recombinant FLAG-tagged full-length Sirt1 or ΔESA Sirt1 (aa 631–655 deleted) was incubated with either GST fused to Ac-p53 or GST alone. Pull-down assay was performed with anti-FLAG antibody and bound GST-Ac p53 was visualized by immunoblotting with GST antibody (top). Pulled-down FLAG-tagged Sirt1 (WT or ΔESA) was visualized by Coommassie staining (bottom). The light chain of the anti-FLAG antibody that was used for pull-down is visible and is indicated with an arrow. (C) The ESA region is sufficient to increase deacetylase core-substrate interaction. (Left) The ESA region increases the deacetylase core-Ac-p53 interaction. GST pull-down experiments were performed with recombinant deacetylase core fragment (Core, aa 184–510) and the deacetylase core fragment fused to the ESA (aa 631–655) (Core-ESA) and either GST-Ac p53 fusion protein or GST alone (negative control). Bound Core or Core-ESA was visualized by immunoblotting with Sirt1 antibody. Input levels of Core, Core-ESA, GST-Ac p53, and GST are also shown. (Right) The ESA region increases the deacetylase core-Ac-H4 interaction. Streptavidin-immobilized Ac-H4 peptide was incubated with either the deacetylase core (Core) or the deacetylase core fused to the ESA region (Core-ESA), and pull-down experiments were performed. Streptavidin bound to biotin without an attached peptide was used as a negative control. (D) The ESA alone does not bind to substrate. Pull-down experiments were performed by incubating biotin-tagged acetylated-histone H4 peptide (Ac-H4) immobilized on streptavidin beads with either full-length Sirt1 (GST-FL Sirt1), ESA fused to GST (GST-ESA), or GST alone. As a negative control, we used streptavidin beads bound to free biotin. Bound GST fusion proteins were detected by immunoblotting with GST antibody. Input levels of the GST fusion proteins are shown on the right. (E) GPDR ESA does not increase deacetylase core-substrate interaction. Pull-down experiments were performed by incubating biotin-tagged Ac-H4 that was immobilized on streptavidin beads with either the deacetylase core fragment, or the core fragment fused to the ESA (Core-ESA) or the core fragment fused to the GPDR mutant ESA (Core-GPDR). Bound core fusion proteins were visualized by immunoblotting with Sirt1 antibody. As a negative control, we used streptavidin beads bound to free biotin. Input levels of the core fusion proteins are also shown. A quantification of the levels of bound core fusion proteins is shown on the right (N = 8). Results are expressed as the mean ± SEM. (F) The leucine-zipper (LZ) domain of DBC1 inhibits the ESA-deacetylase core interaction. We incubated biotin-tagged ESA peptide immobilized on streptavidin beads with the deacetylase core fragment in the presence of competing 2 μg of either GST fragment or GST fused to the LZ (aa 243–264) domain of DBC1. The levels of bound deacetylase core and of input GST fusion proteins are shown. Streptavidin-immobilized biotin without the peptide is used as negative control. A quantification of the levels of bound deacetylase core fragment is shown on the right (N = 5). Results are expressed as the mean ± SEM. (G) The ESA peptide inhibits the LZ domain-deacetylase core interaction. GST pull-down experiments were performed by incubating either 2 μg GST fragment alone or GST-LZ fusion protein with the deacetylase core in the presence of either a control peptide or the ESA peptide. The levels of bound deacetylase core and that of the GST fusion proteins are shown. (H) The interaction of the deacetylase core with the LZ domain of DBC1 is stronger than with the ESA region. Pull-down experiments were performed by incubating GST, GST-ESA, or GST-LZ with the deacetylase core. The levels of bound deacetylase core and of the GST fusion proteins are shown. Molecular Cell 2011 44, 203-213DOI: (10.1016/j.molcel.2011.07.038) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 The GPDR Mutated ESA Peptide Can Inhibit Sirt1 Activity In Vivo (A) The GPDR mutated ESA peptide inhibits deacetylation of p53 by Sirt1 in H1299 cells. Expression vectors for p53 and V5-tagged Sirt1 were cotransfected with a 10-fold molar excess of an expression vector for HA-tagged DBC1, FLAG-tagged 3×GPDR (FLAG-3×GPDR), or FLAG-tagged 3×GPDR with a nuclear localization signal (FLAG-NLS-3×GPDR). Levels of acetylated p53 were visualized by antibody specific for acetylated K382 in p53. (B) The GPDR mutated ESA peptide inhibits deacetylation of Foxo1 by Sirt1 in the prostate cancer cell line DU145. An expression vector for HA-tagged Foxo1 was cotranfected with an expression vector for either V5-tagged-NLS-GPDR (3×) peptide or V5-tagged NLS without GPDR. Acetylation of Foxo1 was visualized by immunoblotting with an antibody specific for acetylated lysine after HA-Foxo1 was immunoprecipitated with anti-HA antibody. (C) The GPDR mutated ESA peptide restores sensitivity to the chemotherapeutic agent 5-FU. DU145 cells were cotransfected with an expression vector for V5-tagged-NLS-GPDR or control and treated with 20 μM 5-FU for two days before apoptosis levels were measured. Results are expressed as the mean ± SEM. ∗∗∗∗p < 0.0001 between empty and GPDR vectors. Molecular Cell 2011 44, 203-213DOI: (10.1016/j.molcel.2011.07.038) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 A Schematic Diagram of the ESA Peptide Switch The ESA-deacetylase core interaction turns “on” the deacetylase core and increases its affinity for substrates. The GPDR mutated ESA peptide or the LZ domain of DBC1 bind to the deacetylase core in a manner that is different from that of the ESA region and, therefore, cannot activate it or increase its affinity for substrates. DBC1 could also sterically hinder the deacetylase core-substrate interaction (not shown). Molecular Cell 2011 44, 203-213DOI: (10.1016/j.molcel.2011.07.038) Copyright © 2011 Elsevier Inc. Terms and Conditions