An Artificial Reaction Promoter Modulates Mitochondrial Functions via Chemically Promoting Protein Acetylation

Acetylation, which modulates protein function, is an important process in intracellular signalling. In mitochondria, protein acetylation regulates a number of enzymatic activities and, therefore, modulates mitochondrial functions. Our previous report showed that tributylphosphine (PBu3), an artificial reaction promoter that promotes acetylransfer reactions in vitro, also promotes the reaction between acetyl-CoA and an exogenously introduced fluorescent probe in mitochondria. In this study, we demonstrate that PBu3 induces the acetylation of mitochondrial proteins and a decrease in acetyl-CoA concentration in PBu3-treated HeLa cells. This indicates that PBu3 can promote the acetyltransfer reaction between acetyl-CoA and mitochondrial proteins in living cells. PBu3-induced acetylation gradually reduced mitochondrial ATP concentrations in HeLa cells without changing the cytoplasmic ATP concentration, suggesting that PBu3 mainly affects mitochondrial functions. In addition, pyruvate, which is converted into acetyl-CoA in mitochondria and transiently increases ATP concentrations in the absence of PBu3, elicited a further decrease in mitochondrial ATP concentrations in the presence of PBu3. Moreover, the application and removal of PBu3 reversibly alternated mitochondrial fragmentation and elongation. These results indicate that PBu3 enhances acetyltransfer reactions in mitochondria and modulates mitochondrial functions in living cells.

biological molecules, mitochondrial functions might be controlled by the application of an exogenous artificial reaction promoter, since the activities of a number of mitochondrial proteins are regulated by acetylation [16][17][18] . In this study, we examined the ability of PBu 3 to promote the acetyltransfer reaction from acetyl-CoA to mitochondrial proteins and to modulate mitochondrial functions in living cells.

Results
PBu 3 promoted protein acetylation in the mitochondria. To verify our hypothesis that PBu 3 promotes the acetyltransfer reaction between acetyl-CoA and mitochondrial proteins, protein acetylation levels in mitochondria, cytoplasm, and nucleus were estimated by Western blotting using an anti-acetylated lysine antibody. The concentrations of PBu 3 used were the same as those used in our previous study measuring acetylation reactions using a fluorescent probe 15 . Exposure of HeLa cells to 5 mM and 10 mM PBu 3 for 10 min elicited acetylation of mitochondrial proteins, especially in the 30-55 kDa range (Fig. 1A). The acetylation signal increased significantly, depending on the PBu 3 concentration, in the indicated bands of mitochondrial proteins (Fig. 1B). No significant changes in protein acetylation level were observed in the cytoplasm or nucleus (Fig. 1A). Moreover, we observed that mitochondrial superoxide dismutase (SOD2), which is regulated by acetylation 19 , was also acetylated by the 10 min PBu 3 treatment (see Supplementary Fig. S1), indicating that PBu 3 induces acetylation of mitochondrial proteins.
To confirm that PBu 3 promotes acetyltransfer reaction between acetyl-CoA and mitochondrial proteins, we estimated cellular acetyl-CoA concentrations (Fig. 2). The concentration decreased in PBu 3 -treated cells, suggesting that acetyl-CoA is the substrate for protein acetylation. However, a high concentration of PBu 3 might inhibit protein deacetylase, instead of promoting the acetyltranfer reaction, because PBu 3 also acts as an ion chelator and Zn 2+ binds to SIRT3 20 . We therefore confirmed that application of PBu 3 (1-20 mM) has no effect on the activity of SIRT3 in vitro (see Supplementary Fig. S2), indicating that PBu 3 does not inhibit protein deacetylation but promotes protein acetylation. Based on these results, we concluded that PBu 3 successfully promotes the acetyltransfer reaction from acetyl-CoA to the neighbouring proteins in mitochondria, which probably occurs because of the high concentration of mitochondrial acetyl-CoA.
The toxicity of PBu 3 was evaluated by exposing HeLa cells to PBu 3 for 10 min. Concentrations of less than 10 mM had no toxic effect on cell viability after 24 h (Fig. 3). Although exposure to PBu 3 at concentrations higher than 2 mM for 24 h or 5 mM for longer than 2 h decreased cell viability (see Supplementary Fig. S3), brief treatment to promote protein acetylation in mitochondria (less than 10 mM for 10 min) did not exhibit any toxic Protein acetylation levels were compared using Western blotting probed with anti-acetylated lysine antibody in control and PBu 3 -treated cells (5 mM and 10 mM for 10 min). Mitochondrial, cytoplasmic and nuclear proteins were run under the same experimental conditions in separate runs. Cox4, β -actin and PARP1 were blotted as a loading control for mitochondrial, cytoplasmic and nuclear proteins, respectively. Data are representative of n = 3 experiments. (B) Relative acetylation levels in the arrowed bands of mitochondrial protein were calculated from integrated densitometry values relative to Cox4 levels. Data were normalized using control values. PBu 3 elicited significant protein acetylation in the mitochondria. Bars represent the mean ± SEM of three sets of data run in the same gel. * Indicates P < 0.05 (Dunnett's test).
Scientific RepoRts | 6:29224 | DOI: 10.1038/srep29224 effects. These results indicate that short-term exposure to PBu 3 at an appropriate concentration promotes the acetyltransfer reaction non-invasively in living cells.
Candidate proteins acetylated by PBu 3 . As shown in Fig. 2A, mitochondrial proteins in the 30-55 kDa range were strongly acetylated by PBu 3 . We therefore aimed to identify the proteins contained in these bands using LC-MS/MS, and succeeded in identifying the following proteins: Succinyl-CoA synthetase subunit β (SUCB1); E3 ubiquitin-protein ligase (MARCH5); monoamine oxidase type A (AOFA); and serine β -lactamase-like protein (LACTB). Among these candidate proteins, SUCB1 is a tricarboxylic acid (TCA) cycle enzyme that catalyses the reaction of succinyl-CoA to succinate, and is involved in ATP production in mitochondria 21 . MARCH5 is involved in mitochondrial quality control and Drp1-dependent mitochondrial fission 22 . These results suggest that PBu 3 -induced acetylation of mitochondrial proteins modifies mitochondrial functions. A number of other proteins are likely to be acetylated by PBu 3 in addition to the four candidates that we identified PBu 3 -induced protein acetylation affected ATP synthesis in mitochondria. It has been reported that the cellular ATP concentration is lower in SIRT3 knockout cells than that in normal cells 10 , indicating that protein acetylation inhibits ATP synthesis in mitochondria. We therefore examined the effect of PBu 3 -induced protein acetylation on ATP concentration using the ATP sensor protein, ATeam 23 . While 5 mM PBu 3 had no effect on the ATP concentration in cytoplasm (Fig. 4A), it elicited a gradual but significant decrease in the ATP concentration in mitochondria (Fig. 4B,C). The mitochondrial ATP concentration was lower than that in cytoplasm as reported before 23 (Fig. 4C). PBu 3 decreased the mitochondrial ATP concentration in a dose-dependent manner (0-10 mM; Fig. 4D).
To ascertain whether the PBu 3 -induced decrease in ATP concentration was the caused by protein acetylation in the mitochondria, we compared changes in ATP concentration resulting from PBu 3 application between normal cells and SIRT3-overexpressing cells (Fig. 4E). The decrease in ATP concentration was partially suppressed in the SIRT3-overexpressing cells, since it was attenuated by the protein deacetylase SIRT3 (Fig. 4F), indicating that PBu 3 modulates ATP concentration via mitochondrial protein acetylation.
The protein acetylation induced by PBu 3 thus resulted in a decrease in mitochondrial ATP concentration, probably due to the inhibition of enzymes involved in ATP production by acetylation: ATP synthase; the enzymes in the TCA cycle; and the electron transport chain [24][25][26][27] . Pharmacological inhibition of mitochondrial ATP synthesis by oligomycin induced a similar magnitude of decrease in ATP concentration, and the collapse of the  mitochondrial inner membrane potential induced by carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) elicited a greater decrease in ATP (see Supplementary Fig. S4). While the levels of decrease were comparable to those induced by PBu 3 , the rate of decrease induced by these inhibitors was faster, suggesting that the inhibition of mitochondrial ATP synthesis by PBu 3 is moderate by comparison. Moreover, we observed PBu 3 -induced modulation of mitochondrial ATP concentration in cells of the non-cancerous tissue-derived cell line HEK293 (see Supplementary Fig. S5). PBu 3 therefore promoted the acetyltransfer reaction from acetyl-CoA to mitochondrial proteins, which inhibited ATP production in mitochondria. Although acetyl-CoA is normally an essential substrate in mitochondrial energy production, mitochondrial ATP concentrations are decreased by PBu 3 -induced protein acetylation involving acetyl-CoA. We next assessed whether the dominant role of acetyl-CoA in the PBu 3 -treated cells was to act as a substrate for the TCA cycle or protein acetylation. To address this, changes in the mitochondrial ATP concentration in response to pyruvate were compared between control and PBu 3 -treated cells, because acetyl-CoA is produced through pyruvate decarboxylation. In the control cells, pyruvate (5 mM) induced a transient increase in mitochondrial ATP concentration (Fig. 5A blue line and B upper panels). In contrast, it decreased ATP concentrations in the PBu 3 -treated cells (Fig. 5A red line and B lower panels). These results indicate that acetyl-CoA contributes predominantly to protein acetylation in the presence of PBu 3 , which results in the further decrease in ATP concentrations.

PBu 3 -induced protein acetylation elicited alterations in mitochondrial morphology.
Recent studies have reported that the acetylation and deacetylation of mitochondrial proteins regulates mitochondrial fusion and fission, resulting in alterations in mitochondrial morphology 10,11 . These studies showed that mitochondria are fragmented in cells defective for SIRT3 or with a mutation in its downstream protein, and indicated that hyperacetylation elicits mitochondrial fragmentation, while deacetylation reverses this process. To demonstrate this process directly, we monitored mitochondrial shapes with mitochondria targeted TagCFP, before and after the application of PBu 3 . PBu 3 induced mitochondrial fragmentation within 10 min (Fig. 6A,B). Furthermore, the mitochondria returned to their normal shapes in 10 min after the PBu 3 was washed out (Fig. 6C). In SIRT3-overexpressing cells, the effect of PBu 3 appeared to be attenuated (see Supplementary Fig. S6). These results indicate that PBu 3 -induced protein acetylation reversibly regulates mitochondrial morphology, and that acetylation-induced mitochondrial morphological change are occurs quickly, within 10 min. Based on these results, we conclude that our method involving PBu 3 successfully modulates mitochondrial functions via mitochondrial protein acetylation, which enabled us to observe the time-course of the effects in mitochondria.

Discussion
In this study, we have shown that PBu 3 promotes mitochondrial protein acetylation and modulates mitochondrial functions. PBu 3 has been used as a catalyst in the acylation reaction 28 . It also catalyses the reaction in living cells, as shown in our previous study using a newly developed fluorescent probe 15 . The chemicals that promote the specific reaction intracellularly are referred to as "artificial reaction promoters". In our previous studies, these molecules were used to promote the reaction between specific biological molecules and fluorescent probes, which allowed us to measure biological molecules, such as acetyl-CoA and NAD(P)H, using the fluorescence imaging method 15,29 . In this study, the artificial reaction promoter, PBu 3 , was used to non-invasively promote the reaction between the biological molecules, acetyl-CoA and mitochondrial proteins (Figs 1-3), and to modulate mitochondrial functions in living cells (Figs 4-6) as summarized in Fig. 7. Our data show that PBu 3 promotes the reaction between acetyl-CoA and mitochondrial proteins, and modulates mitochondrial functions, at least in part, via the protein acetylation, although there might be other route. PBu 3 is therefore a useful tool for the control of cellular functions. To our knowledge, this is the first report to demonstrate the modulation of cellular function via the promotion of a specific chemical reaction in living cells.
While the protein acetylation process in mitochondria is not fully understood, the non-enzymatic chemical reaction between acetyl-CoA and lysine residues might be sufficient to explain mitochondrial protein  acetylation 30 . If there is a sufficient amount of acetyl-CoA to maintain the protein acetylation state in mitochondria in contrast to the other compartments in the cell, it makes sense that PBu 3 induces protein acetylation specifically in mitochondria. Acetylation of mitochondrial proteins negatively regulates mitochondrial functions in many cases 14 . In this study, we demonstrated that PBu 3 -induced acetylation down-regulates mitochondrial ATP production (Figs 4 and 5) and elicits mitochondrial fragmentation (Fig. 6) in HeLa cells. Although energy metabolism in cancer cell lines is different from that in non-cancerous cells, we observed this effect of PBu 3 on mitochondrial ATP concentration in both the cancerous HeLa cells and the non-cancerous HEK293 cells (see Supplementary Fig. S5). These results indicate that the effects of PBu 3 shown here are not unique to cancer cells. In addition to the processes observed in this study, mitochondrial protein acetylation is also related to oxidative damage 11,31 , fatty acid oxidation 2 , mitochondrial autophagy 32,33 , and apoptosis 17 . These mechanisms are important for maintaining the normal functions of cells and tissues; hence, abnormal acetylation of mitochondrial proteins has been implicated in a number of diseases, such as metabolic syndromes 34 , diabetes 35 , Parkinson's disease 36 , and Alzheimer's disease 37 . Using PBu 3 at suitable concentrations, protein acetylation levels in mitochondria can be reversibly controlled without the knockout or inhibition of acetyltransferase. Reversible regulation of this significant physiological process might therefore be a powerful tool for investigating the pathogenesis of these diseases.
Recent studies have reported methods referred to as "bioorthogonal chemistry", which allow artificial reactions to proceed in the cellular environments 38,39 . These methods enable the occurrence of specific reactions between artificially-induced compounds, or between endogenous molecules and artificially-induced compounds, for tagging and probing intracellular molecules in living cells 40,41 . In contrast, our method enhances a specific reaction between intrinsic biological molecules in living cells. Modulating biological reactions and functions without the knockout or inhibition of proteins is a novel approach to understanding intracellular events and physiological functions in living cells. We refer to this concept as "bioparallel chemistry" 29 . With the use of artificial reaction promoters, cellular functions other than protein acetylation might be controlled. These methods can reveal novel aspects of chemical reactions under physiological conditions, and allow for the control of cellular functions.

Cell Culture. HeLa cells and HEK293 cells were cultured in DMEM supplemented with 10% (v/v) FBS and 1%
(v/v) penicillin/ streptomycin in an incubator maintained at 37 °C and with a humidified atmosphere of 5% CO 2 . For the fluorescence measurements, the cells were seeded onto glass-based dishes.
Fluorescence measurements. Changes in cytoplasmic and mitochondrial ATP concentrations were measured using an ATP sensor protein, ATeam, localized to the cytoplasm and mitochondria, respectively 23 . Mitochondrial shapes in the HeLa cells were visualized using TagCFP-mito (Evrogen, Moscow, Russia). The plasmids coding for ATeam or TagCFP-mito were transfected into HeLa cells using Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) one day before the fluorescence measurements were conducted. DNA coding human SIRT3 was cloned from HeLa cell cDNA and inserted to the pmCherry-N1 vector using the BglII and EcoRI restriction enzyme sites, then transfected into HeLa cells following ATeam-transfection. The bath solution was changed to Fluorescence imaging was performed using a confocal laser scanning microscope system (FluoView FV1000; Olympus, Tokyo, Japan) mounted on an inverted microscope (IX81; Olympus) with 40 × and 60 × oil-immersion objective lenses. The temperature of the microscope stage was maintained at 37 °C during the experiments using a stage top incubator (IN-OIN-F2, Tokai hit, Shizuoka, Japan). TagCFP-mito was excited at 440 nm with a laser diode, and a signal was observed at 460-560 nm. ATeam was excited at 440 nm, and the fluorescence signals were separated using a 510 nm dichroic mirror and observed at 460-500 nm for CFP and 515-615 nm for YFP. Fluorescence images were acquired and analysed with the FluoView software package (Olympus). Fluorescence intensities were calculated as mean intensity over a defined region of interest (ROI) containing the entire cell body of each cell. Western Blotting. Control and PBu 3 -treated cells were harvested and the mitochondria, cytoplasm, and nucleus isolated using the Mitochondria Isolation Kit (BioChain Institute, Gibbstown, NJ, USA). The samples were lysed in RIPA buffer containing 25 mM HEPES, 1.5% TritonX-100 (v/v), 1.0% sodium-deoxycholate (w/v), 0.1% SDS (w/v), 500 mM NaCl, 5 mM EDTA, 50 mM NaF, 100 μ M Na 3 VO 4 , and 0.1 mg/mL leupeptin and protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). The protein lysates were diluted to the same protein concentrations, separated using SDS-PAGE, transferred onto a PVDF membrane (Millipore, Billerica, MA, USA), and probed with an acetylated lysine-specific antibody (Sigma-Aldrich, St. Louis, MO, USA). The secondary antibody used was a horseradish peroxidase (HRP)-conjugated anti mouse IgG (GE Healthcare, Little Chalfont, UK). The ECL Western blotting detection system (Millipore) was used for detection with imaging by LAS-1000 (Fuji Film, Tokyo, Japan). After detecting acetylated lysine signals, the HRP conjugated to the secondary antibody was inactivated by incubating the membrane in 15% H 2 O 2 for 30 min. Loading control proteins were then probed with a β -actin-specific antibody for the cytoplasmic protein sample, a Cox4-specific antibody for the mitochondrial protein sample, and a PARP1-specific antibody for the nuclear protein sample (GeneTex, Irvine, CA, USA). The secondary antibody was HRP-conjugated anti rabbit IgG (GE Healthcare) and the signals were detected as described above.
Quantification of Acetyl-CoA. Control and PBu 3 -treated (5 mM for 10 min) cells were harvested in ice-cold PBS and sonicated. The protein concentration of each sample was estimated using Coomassie Brilliant