Journal: Nature Communications

Article Title: Crystallographic structure of a small molecule SIRT1 activator-enzyme complex

doi: 10.1038/ncomms8645

Figure Lengend Snippet: ( a ) Schematic diagram of human full-length hSIRT1 and Mini-hSIRT1 constructs. The N-terminal SBD, the central catalytic domain and the CTR are highlighted in red, green and orange. ( b ) Heat map of the HDX-MS perturbation of binding of 1 to hSIRT1 in the absence or presence of Ac-p53(W5) (Trp-5mer) at six different time points (15–5000, s). ( c ) Pivot plot of the activation by a chemically diverse STAC set using the Ac-p53(W5) substrate for mini-hSIRT1 versus full-length hSIRT1, as measured by OAcADPR assay. The red line represents y = x correlation. ( d ) Pivot plot of the STAC activation of mini-hSIRT1(ΔSBD) versus mini-hSIRT1. ( e ) Pivot plot the STAC activation of mini-hSIRT1(ΔCTR) versus mini-hSIRT1. ( f ) Pivot plot of the STAC activation of mini-hSIRT1(E230K) versus mini-hSIRT1.

Article Snippet: The p53-based Ac-p53(W5) (Ac-RHKKAcW-NH2) and FOXO-3a 21-mer (Ac-SADDSPSQLSKAcWPGSPTSRSS-NH2) peptides were obtained from Biopeptide.

Techniques: Construct, Binding Assay, Activation Assay

Journal: Nature Communications

Article Title: Crystallographic structure of a small molecule SIRT1 activator-enzyme complex

doi: 10.1038/ncomms8645

Figure Lengend Snippet: ( a ) Structure of mini-hSIRT1/ 1 /Ac-p53 7-mer/CarbaNAD quaternary complex. The STAC 1 and Ac-p53 7-mer are shown in green, red and blue for carbon, oxygen and nitrogen atoms. The CarbaNAD is shown in cyan, red, blue and orange for carbon, oxygen, nitrogen and phosphate atoms. The protein ribbon is rainbow-colored from blue at the N-terminus to red at the C-terminus. ( b ) Structure of mini-hSIRT1/ 1 / 2 complex. The mini-hSIRT1 shown in this complex is hSIRT1(183–516)-GS-CTR as the same complex containing hSIRT1(183–505)-(GGGS) 2 -CTR diffracts to 3.5 Å even though the structures are almost identical. The STAC 1 is shown in green, red and blue for carbon, oxygen and nitrogen atoms. The Inhibitor 2 is shown in cyan, red, blue and yellow for carbon, oxygen, nitrogen and sulfur atoms. The protein ribbon is rainbow-colored from blue at the N-terminus to red at the C-terminus. ( c ) Structural comparison of mini-hSIRT1/ 1 complex (green), mini-hSIRT1/ 1 /Ac-p53 7-mer/CarbaNAD quaternary complex (orange) and mini-hSIRT1/ 1 / 2 complex (magenta). ( d ) Superimposition of the SBD domains from mini-hSIRT1/ 1 complex (green), mini-hSIRT1/ 1 /Ac-p53 7-mer/CarbaNAD quaternary complex (orange) and mini-hSIRT1/ 1 / 2 complex (magenta). ( e ) Comparison of the STAC-mediated dimer interface of mini-hSIRT1/ 1 complex (green), mini-hSIRT1/ 1 /Ac-p53 7-mer/CarbaNAD quaternary complex (orange) and mini-hSIRT1/ 1 / 2 complex (magenta).

Article Snippet: The p53-based Ac-p53(W5) (Ac-RHKKAcW-NH2) and FOXO-3a 21-mer (Ac-SADDSPSQLSKAcWPGSPTSRSS-NH2) peptides were obtained from Biopeptide.

Techniques:

Journal: Nature Communications

Article Title: Crystallographic structure of a small molecule SIRT1 activator-enzyme complex

doi: 10.1038/ncomms8645

Figure Lengend Snippet: ( a ) Heat map of the ratio of wild-type/mutant fold-activation for hSIRT1 mutants for each compound from a structurally diverse collection of 246 STACs. Ratios from 0.80 to 1.16 are colored gray. This range covers one s.d. of the mean for V224A (0.98±0.18-fold, ) which does not affect activation. Activation impairment denoted as a red gradient (ratios of 1.17/6.78). Activation enhancement is shown as a blue gradient (ratios of 0.78/0.24). ( b ) Comparison of the fold-activation of I223R versus wild-type hSIRT1 with a structurally diverse collection of STACs. All of the data were generated with the OAADPr assay using the Ac-p53(W5) substrate. A different compound set (∼250 STACs) was used for the site-directed mutagenesis studies compared with that used for the initial characterization of mini-hSIRT1 (∼80 STACs).

Article Snippet: The p53-based Ac-p53(W5) (Ac-RHKKAcW-NH2) and FOXO-3a 21-mer (Ac-SADDSPSQLSKAcWPGSPTSRSS-NH2) peptides were obtained from Biopeptide.

Techniques: Mutagenesis, Activation Assay, Generated

Journal: Nature Communications

Article Title: Crystallographic structure of a small molecule SIRT1 activator-enzyme complex

doi: 10.1038/ncomms8645

Figure Lengend Snippet: ( a ) Heat map of the HDX-MS perturbation of binding of 1 to hSIRT1(E230K) in the absence or presence of Ac-p53(W5) at six different time points (15–5000, s). ( b ) Pivot plot of the STAC activation of mini-hSIRT1(R446E) versus mini-hSIRT1. ( c ) Pivot plot of the STAC activation of the double charge-reversal mutant mini-hSIRT1(R446E/E230K) versus mini-hSIRT1. ( d ) Speculative model of the activated conformation of SIRT1. Glu 230 and Arg 446 are shown in stick representation. The protein ribbon is rainbow-colored from blue at the N-terminus to red at the C-terminus. 1 and modeled Ac-p53(W5) are shown in cyan and green, respectively.

Article Snippet: The p53-based Ac-p53(W5) (Ac-RHKKAcW-NH2) and FOXO-3a 21-mer (Ac-SADDSPSQLSKAcWPGSPTSRSS-NH2) peptides were obtained from Biopeptide.

Techniques: Binding Assay, Activation Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: A Novel Sirtuin 2 (SIRT2) Inhibitor with p53-dependent Pro-apoptotic Activity in Non-small Cell Lung Cancer *

doi: 10.1074/jbc.M113.487736

Figure Lengend Snippet: Identification of a SIRT2-selective inhibitor, compound AEM1. A, chemical structure of the SIRT2 inhibitor AEM1 is shown. B, SIRT2-dependent MAL deacetylation was inhibited by compound AEM1. C, compound AEM1 showed weak inhibition of MAL deacetylation by SIRT1. D, compound AEM1 and AEM2 were potent inhibitors of p53 deacetylation by SIRT2, but not SIRT1. E, compound AEM1 showed weak inhibition of truncated SIRT1(Δ1–234) or the sirtuins SIRT3 and Sir2 from S. cerevisiae (ySir2). In all cases, deacetylation in a control reaction containing dimethyl sulfoxide was set to 100%. Results are given as the average ± S.D. (error bars) of three to six independent determinations.

Article Snippet: After preincubation of SIRT2 or SIRT1 with the inhibitors for 10 min, the reaction was started by adding the substrate, an acetylated p53 peptide (HLKSKKGQSTSRHKK(Ac)LMFK, synthesized by Biosyntan, Berlin, Germany), at 100 μ m in reaction buffer containing 1 m m NAD + .

Techniques: Inhibition, Control

Journal: The Journal of Biological Chemistry

Article Title: A Novel Sirtuin 2 (SIRT2) Inhibitor with p53-dependent Pro-apoptotic Activity in Non-small Cell Lung Cancer *

doi: 10.1074/jbc.M113.487736

Figure Lengend Snippet: SIRT2 inhibition sensitized NSCLC cells to etoposide-mediated apoptosis in a p53-dependent fashion. A and B, the cell lines A549 (p53+/+) (A) and H1299 (p53−/−) (B) were treated with increasing concentrations of compound AEM1 (left) or AEM2 (right) in the absence (dark bars) or presence of 1 μm etoposide (VP-16, light bars). After 48 h, the percentage of cells with subdiploid DNA content (sub-G1) was measured by flow cytometry as an indicator of apoptosis. Values of three independent determinations ± S.D. (error bars) are shown. C, p53−/− H1299 cells stably transduced with a tamoxifen-inducible p53-ERTM fusion (H1299 p53-ERTM) were treated as in B with compound AEM1 or AEM2 in the absence or presence of etoposide (VP-16), and apoptosis was measured as in B. D, p53 function in H1299 p53-ERTM cells was induced with 100 nm tamoxifen in parallel, and the effect of treatment with compounds AEM1 and AEM2 in combination with etoposide was measured as in A.

Article Snippet: After preincubation of SIRT2 or SIRT1 with the inhibitors for 10 min, the reaction was started by adding the substrate, an acetylated p53 peptide (HLKSKKGQSTSRHKK(Ac)LMFK, synthesized by Biosyntan, Berlin, Germany), at 100 μ m in reaction buffer containing 1 m m NAD + .

Techniques: Inhibition, Flow Cytometry, Stable Transfection, Transduction

Journal: The Journal of Biological Chemistry

Article Title: A Novel Sirtuin 2 (SIRT2) Inhibitor with p53-dependent Pro-apoptotic Activity in Non-small Cell Lung Cancer *

doi: 10.1074/jbc.M113.487736

Figure Lengend Snippet: Enhancement of p53 acetylation signal by pharmacological SIRT2 inhibition. A, the cell line A549 (p53+/+) was treated with 20 μm compound AEM1 or AEM2 in the absence or presence of 1 μm etoposide (VP-16). The acetylation level of p53 protein was determined by Western blotting using an α-p53-K382Ac antibody. Tubulin served as a loading control. The cell line H1299 (p53−/−) was used as a negative control. B, expression levels of SIRT2 and SIRT1 were unaffected by compounds AEM1 and AEM2. mRNA expression levels were measured by real-time PCR analysis. Expression levels of untreated cells (n. C.) was set to 1 and normalized to actin mRNA in individual experiments. Values of three independent determinations ± S.D. (error bars) are shown.

Article Snippet: After preincubation of SIRT2 or SIRT1 with the inhibitors for 10 min, the reaction was started by adding the substrate, an acetylated p53 peptide (HLKSKKGQSTSRHKK(Ac)LMFK, synthesized by Biosyntan, Berlin, Germany), at 100 μ m in reaction buffer containing 1 m m NAD + .

Techniques: Inhibition, Western Blot, Control, Negative Control, Expressing, Real-time Polymerase Chain Reaction