Over expression of wild type or a catalytically dead mutant of Sirtuin 6 does not influence NFκB responses

Abstract

SIRT6 is involved in inflammation, aging and metabolism potentially by modulating the functions of both NFκB and HIF1α. Since it is possible to make small molecule activators and inhibitors of Sirtuins we wished to establish biochemical and cellular assays both to assist in drug discovery efforts and to validate whether SIRT6 represents a valid drug target for these indications. We confirmed in cellular assays that SIRT6 can deacetylate acetylated-histone H3 lysine 9 (H3K9Ac), however this deacetylase activity is unusually low in biochemical assays. In an effort to develop alternative assay formats we observed that SIRT6 overexpression had no influence on TNFα induced nuclear translocation of NFκB, nor did it have an effect on nuclear mobility of RelA/p65. In an effort to identify a gene expression profile that could be used to identify a SIRT6 readout we conducted genome-wide expression studies. We observed that overexpression of SIRT6 had little influence on NFκB-dependent genes, but overexpression of the catalytically inactive mutant affected gene expression in developmental pathways.

Figure 1. Full length SIRT6 and mutant expression.

Human SIRT6 was amplified by PCR and cloned into a modified pET28a expression vector which incorporated a FLAG tag upstream of a 6xHIS tag and thrombin cleavage site (th). In addition to this Flag-6His-th-tag, the N-terminus of SIRT6 was modified with a Tev or Precission protease site. Wild type SIRT6 and a series of site directed mutants were expressed and purified as described. Samples were analysed by SDS-PAGE. Lanes 1 and 6: Molecular mass markers; Lane 2: wild type SIRT6; Lane 3: H133W SIRT6; Lane 4: H133Y SIRT6; Lane 5: N114A SIRT6; Lane 7: wild type SIRT6 with Precission tag; Lane 8: wild type SIRT6 with N-terminal tag removed.

Figure 2. Kinetic characteristics of SIRT6 deacetylase activity.

(A) Progress curves for purified, recombinant full-length wild-type (wt) and mutant SIRT6 proteins, expressed in E.coli. ▪ wt SIRT6 (2 µM), ▾H133W (10 µM), ▴ H133Y (10 µM), ○ N114A (10 µM), ◊ inhibition of wt (2 µM) with 20 mM NAM (this also generates an intrinsic level of background fluorescence). Error bars represent the standard error of mean duplicate values. (B) K _m,app determination for peptide gave a value of 14.7+/−1.5 µM. Error bars represent the standard error of mean duplicate values. (C) K _m,app determination for NAD⁺ gave a value of 52.4+/−18 µM. Error bars represent the standard error of mean duplicate values. (D) Deacetylation of purified H3/H4 histones with SIRT6. Deacetylation could be detected at a substrate:enzyme ratio of 1∶1.

Figure 3. SIRT6 activity in mammalian cells.

Panel A shows SIRT6 (anti-Flag stained in green) and H3K9Ac (red) double staining. Panel B shows SIRT6 staining alone. Panel C shows H3K9Ac staining alone and panel D shows Hoechst staining. Top row shows wild type SIRT6 transfection and lower row shows H133W transfection. Upward pointing arrowheads show the position of a transfected cell nucleus whereas a right pointing arrowhead shows an untransfected cell nucleus.

Figure 4. Influence of SIRT6 on nuclear translocation and nuclear mobility of RelA/p65.

(a) Medium magnification confocal microscopy images of cells immunostained for RelA/p65 (green) and flag tag-SIRT6 (red). The images show cells with low intensity of RelA/p65 staining in the nucleus in unstimulated cells (A and C) and with high intensity for RelA/p65 in the nucleus after stimulation with TNFα (B and D). A and B are cells that had been transfected with 5 µg of flag-tagged wild-type SIRT6 and C and D cells which had a control transfection with an empty vector. Transfected cells that were stimulated by TNFα show orange/yellow nuclei demonstrating co-localisation of immunofluorescence for RelA/p65 and flag tag. Figure 4b Histograms of measurements of RelA/p65 nuclear:cytoplasmic ratio. The data shows an approximate doubling of this ratio after stimulation by TNFα. There were no significant differences between the responses of of untransfected cells (column 8), cells transfected with an empty vector (column 7) or cells transfected with various concentrations of wild-type (WT) SIRT6 vector (column 1, 0.5 µg; column 2, 2 µg: column 3, 5 µg) or H133W mutant SIRT6 vector (column 4, 0.5 µg; column 5, 2 µg; column 6, 5 µg). The ratios are the mean results for 100 cells (+/− SEM). Figure 4c Histogram showing the intranuclear mobility of YFP-RelA/p65 measured as half-time to recovery after photo-bleaching. Mean data from 40 cells measured in 2 experiments. YFP-RelA/p65 was not significantly more mobile in cells following transient transfection and overexpression of wild type SIRT6 than in cells following a control transfection of H133W or empty plasmid.

Figure 5. Influence of SIRT6 over expression on TNFα induced MCP1 expression.

(a) HEK293 cells were transiently transfected with increasing amounts (0.5–20 µg) of expression plasmids for wild type SIRT6 or the H133W mutant. Control cells were transfected with an empty plasmid (100% Bluescript) or untransfected. Cells were stimulated with 10 ng/ml TNFα for 48 hours and MCP1 secretion into the culture supernatant was measured and normalised per live cell. Results represent means +/− SEM from triplicate experiments. (b) HEK293 cells were transiently transfected as above with increasing amounts (0.5–20 µg) of expression plasmids for wild type SIRT6 (lanes 1–6) or the H133W mutant (lanes 9–14). Control cells were transfected with an empty plasmid (lanes 7 and 15) or untransfected (lanes 8 and 16). Cells were stimulated with 10 ng/ml TNFα for 48 hours. Cell extracts were made and analysed by SDS-PAGE and Western blotting as described. Blots were probed with antibodies to FLAG (upper panel) and SIRT6 (lower panel).

Figure 6. Over expression of SIRT6 or H133W.

HEK293 cells were transfected with plasmid expressing Flag-tagged wild type SIRT6 (lanes 3 and 7) or deacetylase-dead (H133W) mutant of human SIRT6 in (lanes 4 and 8). Control cells were transfected with no DNA (lanes 1 and 5) or empty vector DNA pCDNA3 only (lanes 2 and 6). Each lane was loaded with 50 µg total protein of cell extract and analysed by SDS-PAGE followed by Western blotting with either anti-SIRT6 or anti-Flag. RNA was extracted from these cells was used for transcriptomic analysis without and with TNFα stimulation for 4 h.

Figure 7. Changes in gene expression of the TNFα profile.

Heat map of normalised expression levels (average intensity) of the 198 genes in the TNFα profile under the 8 treatment conditions.