Fig 2: Acetylation of human recombinant CKM disrupts dimer formation.(A) Nonacetylated and acetylated human rCKM was prepared under reducing and nonreducing conditions and run on native-PAGE prior to Western transfer and immunoblotting with anti-CKM antibodies. Representative immunoblot (left) to determine dimeric versus monomeric CKM. Statistical analysis (right) of densitomeric measurements of monomer and dimer CKM; n = 5 per group. (B) rCKM was incubated over a range of acetyl-CoA concentration (0–500 µM) prior to being run on native-PAGE. Dimeric versus monomeric CKM was determined; n = 5 per group. (C) Crosslinking mass spectrometry (XL-MS) revealed distance constraints between crosslinked lysine residues and obtained information on the structure of the rCKM dimer mapped to PDB 1U6R. XL-MS analysis of rCKM found the homodimer links K196-K196 and K11-K11 were heavily disrupted with acetylation of rCKM. Sirt2 deaceytlation restored the K196-K196 and K11-K11 homodimer crosslink back toward control (right). (D) MS analysis of in vitro acetylated rCKM compared with Sirt2 deacetylated rCKM. Data shown as mean ± SEM; n = 5 per group. P values calculated by 1-way ANOVA followed by Tukey post hoc analysis. *P < 0.05 versus nonacetylated rCKM dimer, #P < 0.05 versus nonacetylated Monomer rCKM (A and B).

Fig 3: Sirt2-dependent deacetylation of NQO1. Quantitative MS analysis was used to identify lysine residues on NQO1 that were deacetylated by SIRT2/NAD+ (A) For MS analysis Ac-NQO1(2 µg) was incubated in the absence and presence of SIRT2 (100 ng) and NAD+ (1 mM) for 5 and 30 min at 37°C. Samples (n = 4 per treatment) were then prepared for MS as shown above. Representative immunoblot showing the levels of lysine acetylation on NQO1 using the reaction conditions described above (B) Diagram showing the amino acid sequence of hNQO1 and the identity of lysine residues (kx red text) which were discovered to be acetylated by Ac-GSH. The location of redox-dependent regions of NQO1 (helix seven and C-terminus), identified in previous studies using redox-dependent anti-NQO1 antibodies (A180 epitope in blue text, C-term epitope in green text) (C) *Denotes lysine residues which showed a significant decrease (p <0.05) in the levels of acetylation when NQO1 was treated with SIRT2/NAD+.

Fig 4: Acetylation reduced enzymatic activity of human CKM.(A) Representative immunoblot (left) and quantification (middle) showing increased acetylation of human recombinant CKM (rCKM) by PCAF; n = 5 per group. Ponceau S staining demonstrates equal protein loading. Activity of rCKM (right) from aliquots of lane 1 and lane 2 and representative immunoblot (top) of rCKM acetylation level; n = 5 per group. (B) Acetylation level of rCKM was detected in the absence (lane 1) or presence of Sirt2 (lanes 2–5). In lanes 2–5, NAD+ was added at increasing concentrations from 0–1 mM and deacetylation of rCKM assessed by WB; n = 5 per group. (C) Activity of rCKM was determined in aliquots from non–in vitro acetylated rCKM (lane 1), unloaded lane (lane 2), no rCKM (lane 3), PCAF-mediated acetylated rCKM (lane 4), and Sirt2 deacetylated aliquots (lane 5); n = 5 per group (D and E) Concentration of acetyl-CoA (D) and NAD+, NADH, and NAD+/NADH ratio (E) were determined in TAC-stressed hearts; n = 5 for all groups. Data represent n = 5 for all groups and are shown as mean ± SEM. P values were calculated by 1-way ANOVA followed by Tukey post hoc analysis (B and C) or by Student’s t test (A, D, E and F). *P < 0.05 versus lane 1 (A, B, and C) or versus sham (D, E, and F). #P < 0.05 versus lane 2 (B) or lane 4 (C), or $P < 0.05 versus lane 3 (B).

Fig 5: Deacetylation of NQO1. The deacetylation of NQO1 was monitored by immunoblot analysis using anti-acetylated lysine antibodies. Ac-NQO1 was generated by incubation of NQO1 with Ac-GSH as described in the Materials and methods section (A) Ac-NQO1 was incubated with purified SIRT1 (470 ng), SIRT2 (100 ng) or SIRT3 (830 ng) in the absence and presence of 1 mM NAD+ for 15 min (B) Ac-NQO1 was incubated with SIRT2 (100 ng) in the absence and presence of 1 mM NAD+ and the sirtuin inhibitor AGK2 (100 µM) for 15min (C) Ac-NQO1 was incubated with HDAC6 (1 µg) or SIRT2 (100 ng) and NAD+ (1 mM) for the indicated times. Reaction conditions are described in the Materials and methods section.