SIRT6 upregulated AR expression under hyperosmotic stress.

<p>Control (C) indicates U937 cells treated with 5 mM glucose containing SFM for 16 hrs in (A) and (B). 100 mM N indicates U937 cells further treated with 100 mM NaCl (N) for 16 hrs in (A) and (B). U937 cells were pretreated either with solvent (DMSO) or indicated concentrations of TSA for 1 hr, followed by 16 hrs of 100 mM N treatment in (A) and (B). (A) (B) 200 nM TSA pretreatment increased nuclear NFAT5, nuclear SIRT6 and total AR expressions, without altering SIRT1 and PARP1 expressions in U937 cells under hyperosmotic stress. Control (C) indicates HeLa cells treated serum containing medium with 5 mM glucose in (C) and (D). 100 mM N indicates HeLa cells further treated with 100 mM NaCl (N) for 16 hrs in (C). (C) Overexpression of wildtype flag tagged SIRT6 enhanced AR expression both under normosmotic and hyperosmotic conditions in HeLa cells, compared with mock controls. (D) Overexpression of wildtype flag tagged SIRT6 and overexpression of myc-NFAT5 alone displayed similarly increased AR expression, compared with the mock control in HeLa cells. Expressions were evaluated using immunoblotting in cytoplasmic and nuclear extracts in (A) and in total extracts in (B), (C) & (D). Expression of SIRT6, flag and myc was used for confirmation of overexpression. Mock1 indicates the backbone plasmid of myc-NFAT5 and mock2 indicates backbone plasmid of flag-SIRT6. Results obtained from this figure was based on the densitometry based statistical analyses, given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161494#pone.0161494.s002" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161494#pone.0161494.s003" target="_blank">S3</a> Figs.</p

Hydrophobic pockets of SIRT1 and SIRT6.

<p>Complexes obtained by docking the inhibitor, Ex-527, to the close proximity of hydrophobic pocket of SIRT1 (A) and to the analogous hydrophobic pocket of SIRT6 (B), were used to simulate the permenance of the binding of the inhibitor to the hydrophobic cluster amino acids. Each amino acid was shown for clear positioning of the pockets. SIRT6 hydrophobic pocket amino acids (B) were predicted based on the corresponding amino acids of SIRT1 (A): V113, I183, I59, F62, V68 of SIRT6 were selected based on similarity to I347, I411, I270, F273, I279 of SIRT1. F297, a member of SIRT1 hydrophobic pocket shown in (A), does not have a corresponding hydrophobic residue in SIRT6 structure (B). (C)(D) Alanine and glycine mutations of SIRT1 hydrophobic pocket residues showed destabilizing characteristic (positive ΔΔG values) similar to the mutations of SIRT6, in part, validating the presence of a hydrophobic pocket in SIRT6 structure. FoldX was used to compute ΔΔG values to determine stability change. Protein structures obtained from PDB ID: 4I5I chain A and PDB ID: 3K35 chain A were used as SIRT1 and SIRT6, respectively.</p

The cofactor of SIRT6, NAD⁺ positively regulated nuclear SIRT6 accumulation and total AR expression under hyperosmotic stress.

<p>Control (C) indicates U937 cells treated with 5 mM glucose containing SFM for 16 hrs in (A) and (B). 100 mM N indicates U937 cells further treated with 100 mM NaCl (N) for 16 hrs in (A), (B) and (C). U937 cells were pretreated either with solvent (DMSO) or indicated concentrations of NAD⁺ or Ex-527 for 1 hr, followed by 16 hrs of 100 mM N treatment in (A), (B) and (C). (A)(C) The cofactor of SIRT6, NAD⁺, enhanced the nuclear accumulation of SIRT6 and total AR expression in dose-dependent manner under hyperosmotic stress. (B) A specific SIRT1 inhibitor and recently suggested SIRT6 inhibitor, Ex-527 did not altered nuclear SIRT6 expression under hyperosmotic stress, in part, justifying the data obtained in (A). Expressions were evaluated using immunoblotting in cytoplasmic and nuclear extracts in (A) & (B) and in total extracts in (C). Results obtained from this figure was based on the densitometry based statistical analyses, given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161494#pone.0161494.s004" target="_blank">S4 Fig</a>.</p

Ex-527 docked to SIRT6, displayed less flexibility and remained in closer proximity to the hydrophobic pocket compared with Ex-527 of SIRT1.

<p>(A) RMSF of Ex-527 atoms in SIRT6-Ex-527 complex under high salt (HS) conditions (blue) showed lower flexibility, compared with its positive control, Ex-527 of SIRT1-Ex-527 complex (red) during 20 ns of MD simulations. Under low salt, Ex-527 of SIRT1 (green) had shown lower flexibility than Ex-527 of SIRT6 (gray), for most of the residues. (B) Name of all atoms of Ex-527 were given for clear representation of the results in (A). (C) The distance between the center of mass of Ex-527 and the center of mass of the hydrophobic pocket residues of SIRT6 under high salt concentration (blue) persisted around 7 to 10 Å range, while its corresponding positive control (red) had a clear shift from 6 to 12 Å after ~7 ns of MD simulations. Under low salt, distance between Ex-527 to SIRT6 (gray) even got shortened, while that of SIRT1 (green) was lengthened, compared with Ex-527 of SIRT6 under high salt (blue). (D) Distance distribution vs. distance of Ex-527 to the hydrophobic pockets of all complexes in all simulations were given for clear representation of the distance shifts during the MD simulations in (C). a.u. refers to the arbitrary units, indicating the number of times that each distance was encountered during the simulations. Results obtained from this figure was based on the trends shown and statistical analyses given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161494#pone.0161494.s006" target="_blank">S6 Fig</a>. Å: Angstrom, C: Carbon, O: Oxygen, N: Nitrogen, Cl: Chloride</p

Ex-527 had more hydrophobic contacts and interacted mainly with SIRT6 active site residues compared with Ex-527 of SIRT1.

<p>Interaction schemes in (A) and (B) were obtained using Ligplot⁺ interaction analysis tool on the complex structures of SIRT1-Ex-527 and SIRT6-Ex-527 under high salt at the 20^th ns of MD simulations. Legend for the items in (A), (B), (C) and (D) were given as supplementary information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161494#pone.0161494.s007" target="_blank">S7 Fig</a>). (A) Ex-527 had only one hydrophobic contact remained with the previously established hydrophobic pocket (I279) and it was held close to the hydrophobic pocket with two other hydrophobic contacts (R282, D286) together with a hydrogen bond to E315 (Bond Length: 2.61 Å). (B) Ex-527 of SIRT6 had more hydrophobic contacts than that of SIRT1 and these contacts were made almost completely with SIRT6 active site residues (H131, W186, F62, Q111, T213). Distance vs. time analysis of the selected contacts observed in (A) and (B) for the whole simulations were given as in (C) and (D), respectively. (C) Side chain carbon (CG1) of I279 of SIRT1 was closer to N1 of Ex-527 for first ~7 ns (blue), while it was nearer to C2 of Ex-527 for the rest of the simulation (green). After ~14 ns, side chain oxygen (OE1) of E315 of SIRT1 was in close proximity of N1 of Ex-527, corresponding to the newly formed hydrogen bond (red). (D) For the first ~6 ns, side chain carbon (CE1) of F62 of SIRT6 was closer to the C8 of Ex-527, but this adjacency was fluctuated for the rest of the simulation (green). Yet, active site H131 of SIRT6 was in close proximity to Ex-527 throughout the simulation, presented with distance of its side chain nitrogen (ND1) to N1 of Ex527 (red). Naming for Ex-527 atoms were given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161494#pone.0161494.g004" target="_blank">Fig 4B</a>.</p

Proposed mechanism for SIRT6 based regulation of AR expression under hyperosmotic environment.

<p>Proposed mechanism for SIRT6 based regulation of AR expression under hyperosmotic environment.</p