Abstract
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.
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Introduction
Mitochondria are essential organelles for cellular energy metabolism and intracellular signalling for cell death and protein acetylation plays important roles in the modulation of mitochondrial functions1,2,3. Acetylation levels of mitochondrial proteins are regulated by the NAD+-dependent deacetylase activity of sirtuins. The mammalian genome encodes seven sirtuin isoforms, three of which are localized to mitochondria (SIRT3, 4 and 5)4. Among these, SIRT3 plays a major role in protein deacetylation and the resulting modification of enzymatic activities5. The knockout or inhibition of SIRT3 elicits hyperacetylation of mitochondrial proteins, resulting in a decrease in cellular ATP concentration via depolarization of the mitochondrial membrane potential6,7, an increase in the production of reactive oxygen species8,9 and alteration of mitochondrial morphology10,11. SIRT3 is thus the main regulator of mitochondrial protein acetylation levels. However, the acetylation processes of mitochondrial proteins remain unclear. While the mitochondrial protein GCN5L1 has been shown to be related to one of the mitochondrial protein acetylation mechanisms12, it is also possible that mitochondrial protein acetylation is caused by a reaction between lysine residues and acetyl-CoA in a non-enzymatic process13,14. Hence, mitochondrial protein acetylation is regulated by both enzymatic and non-enzymatic processes and controls mitochondrial functions.
“Artificial reaction promoters” are compounds that ptromte chemical reactions in vitro and also in cells. In our previous study, we demonstrated that the application of one such artificial reaction promoter, tributylphosphine (PBu3), elicited the acetylation of a fluorescent probe in the mitochondria of HeLa cells15. PBu3 probably enhanced the reactivity of acetyl-CoA in an acetyltransfer reaction in the cells, as well as in vitro and facilitated a chemical reaction between acetyl-CoA and the fluorescent probe in the mitochondria. Based on this result, we hypothesized that PBu3 non-enzymatically promotes the acetyltransfer reaction between acetyl-CoA and lysine residues of neighbouring proteins. If PBu3 enhances the intracellular non-enzymatic acetyltransfer reaction between those 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 acetylation16,17,18. In this study, we examined the ability of PBu3 to promote the acetyltransfer reaction from acetyl-CoA to mitochondrial proteins and to modulate mitochondrial functions in living cells.
Results
PBu3 promoted protein acetylation in the mitochondria
To verify our hypothesis that PBu3 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 PBu3 used were the same as those used in our previous study measuring acetylation reactions using a fluorescent probe15. Exposure of HeLa cells to 5 mM and 10 mM PBu3 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 PBu3 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 acetylation19, was also acetylated by the 10 min PBu3 treatment (see Supplementary Fig. S1), indicating that PBu3 induces acetylation of mitochondrial proteins.
To confirm that PBu3 promotes acetyltransfer reaction between acetyl-CoA and mitochondrial proteins, we estimated cellular acetyl-CoA concentrations (Fig. 2). The concentration decreased in PBu3-treated cells, suggesting that acetyl-CoA is the substrate for protein acetylation. However, a high concentration of PBu3 might inhibit protein deacetylase, instead of promoting the acetyltranfer reaction, because PBu3 also acts as an ion chelator and Zn2+ binds to SIRT320. We therefore confirmed that application of PBu3 (1–20 mM) has no effect on the activity of SIRT3 in vitro (see Supplementary Fig. S2), indicating that PBu3 does not inhibit protein deacetylation but promotes protein acetylation. Based on these results, we concluded that PBu3 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 PBu3 was evaluated by exposing HeLa cells to PBu3 for 10 min. Concentrations of less than 10 mM had no toxic effect on cell viability after 24 h (Fig. 3). Although exposure to PBu3 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 effects. These results indicate that short-term exposure to PBu3 at an appropriate concentration promotes the acetyltransfer reaction non-invasively in living cells.
Candidate proteins acetylated by PBu3
As shown in Fig. 2A, mitochondrial proteins in the 30–55 kDa range were strongly acetylated by PBu3. 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 mitochondria21. MARCH5 is involved in mitochondrial quality control and Drp1-dependent mitochondrial fission22. These results suggest that PBu3-induced acetylation of mitochondrial proteins modifies mitochondrial functions. A number of other proteins are likely to be acetylated by PBu3 in addition to the four candidates that we identified
PBu3-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 cells10, indicating that protein acetylation inhibits ATP synthesis in mitochondria. We therefore examined the effect of PBu3-induced protein acetylation on ATP concentration using the ATP sensor protein, ATeam23. While 5 mM PBu3 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 before23 (Fig. 4C). PBu3 decreased the mitochondrial ATP concentration in a dose-dependent manner (0–10 mM; Fig. 4D).
To ascertain whether the PBu3-induced decrease in ATP concentration was the caused by protein acetylation in the mitochondria, we compared changes in ATP concentration resulting from PBu3 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 PBu3 modulates ATP concentration via mitochondrial protein acetylation.
The protein acetylation induced by PBu3 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 chain24,25,26,27. 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 PBu3, the rate of decrease induced by these inhibitors was faster, suggesting that the inhibition of mitochondrial ATP synthesis by PBu3 is moderate by comparison. Moreover, we observed PBu3-induced modulation of mitochondrial ATP concentration in cells of the non-cancerous tissue-derived cell line HEK293 (see Supplementary Fig. S5).
PBu3 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 PBu3-induced protein acetylation involving acetyl-CoA. We next assessed whether the dominant role of acetyl-CoA in the PBu3-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 PBu3-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 PBu3-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 PBu3, which results in the further decrease in ATP concentrations.
PBu3-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 morphology10,11. 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 PBu3. PBu3 induced mitochondrial fragmentation within 10 min (Fig. 6A,B). Furthermore, the mitochondria returned to their normal shapes in 10 min after the PBu3 was washed out (Fig. 6C). In SIRT3-overexpressing cells, the effect of PBu3 appeared to be attenuated (see Supplementary Fig. S6). These results indicate that PBu3-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 PBu3 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 PBu3 promotes mitochondrial protein acetylation and modulates mitochondrial functions. PBu3 has been used as a catalyst in the acylation reaction28. It also catalyses the reaction in living cells, as shown in our previous study using a newly developed fluorescent probe15. 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 method15,29. In this study, the artificial reaction promoter, PBu3, was used to non-invasively promote the reaction between the biological molecules, acetyl-CoA and mitochondrial proteins (Figs 1, 2, 3) and to modulate mitochondrial functions in living cells (Figs 4, 5, 6) as summarized in Fig. 7. Our data show that PBu3 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. PBu3 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 acetylation30. 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 PBu3 induces protein acetylation specifically in mitochondria. Acetylation of mitochondrial proteins negatively regulates mitochondrial functions in many cases14. In this study, we demonstrated that PBu3-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 PBu3 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 PBu3 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 damage11,31, fatty acid oxidation2, mitochondrial autophagy32,33 and apoptosis17. 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 syndromes34, diabetes35, Parkinson’s disease36 and Alzheimer’s disease37. Using PBu3 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 environments38,39. 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 cells40,41. 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.
Methods
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% CO2. 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, respectively23. 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 Hanks’ balanced salt solutions (HBSS) containing (in mM): NaCl, 137; KCl, 5.4; CaCl2, 1.3; MgCl2, 0.5; MgSO4, 0.4; Na2HPO4, 0.3; KH2PO4, 0.4; NaHCO3, 4.2; D-Glucose, 5.6; HEPES, 5 (pH adjusted to 7.4 with NaOH) before the fluorescence measurements were conducted.
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 PBu3-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 μM Na3VO4 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% H2O2 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 PBu3-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 Blue (CBB) protein assay. The cell lysates were deproteinised using the Deproteinising Sample Preparation kit (BioVision, Milpitas, CA, USA). The acetyl-CoA concentration was then quantified using the PicoProbe Acetyl CoA Assay kit (BioVision). The probe was excited at 535 nm and the fluorescence measured at 589 nm using a microplate reader (Fluoroskan Ascent FL, Thermo Fisher Scientific, Waltham, MA, USA). The concentration was normalized using the protein concentration of each sample.
Identification of acetylated proteins
Mitochondrial protein samples were separated using SDS-PAGE and the gel was stained using the Silver Stain MS kit (Wako, Osaka, Japan). The gel was cut at an appropriate position and proteins contained in the gel fragment were digested using 20 ng/mL Trypsine. The digested proteins were eluted and resolved in elution buffer (50% acetonitrile and 5% trifluoroacetic acid). The sample was analysed using LC-MS/MS (Impact HD, Bruker Daltonics, Billerica, MA, USA).
Measurement of cell viability
Cell viability was measured using the MTT assay. After the cells had been exposed to PBu3-containing medium, this was replaced with a medium containing 0.5 mg/mL of MTT. The cells were incubated for 2 h at 37 °C. The medium was removed and 100 μL of DMSO was added to each well to dissolve the precipitate. Absorbance at 570 nm was measured using a microplate reader. Values from control cells were used to estimate the cell viability.
Statistical Analysis
Significant differences between two data-sets were determined using the Student’s t-test and the Dunnett’s test was used for multiple comparisons. P values lower than 0.05 were considered significant.
Additional Information
How to cite this article: Shindo, Y. et al. An Artificial Reaction Promoter Modulates Mitochondrial Functions via Chemically Promoting Protein Acetylation. Sci. Rep. 6, 29224; doi: 10.1038/srep29224 (2016).
References
Newman, J. C., He, W. & Verdin, E. Mitochondrial protein acylation and intermediary metabolism: regulation by sirtuins and implications for metabolic disease. J Biol Chem. 287, 42436–42443 (2012).
Hirschey, M. D. et al. SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation. Nature. 464, 121–125 (2010).
Rardin, M. J. et al. Label-free quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways. Proc Natl Acad Sci USA 110, 6601–6606 (2013).
Rauh, D. et al. An acetylome peptide microarray reveals specificities and deacetylation substrates for all human sirtuin isoforms. Nat Commun. 4, 2327 (2013).
Hebert, A. S. et al. Calorie restriction and SIRT3 trigger global reprogramming of the mitochondrial protein acetylome. Mol Cell. 49, 186–199 (2013).
Wu, Y. T., Lee, H. C., Liao, C. C. & Wei, Y. H. Regulation of mitochondrial FoF1ATPase activity by Sirt3-catalyzed deacetylation and its deficiency in human cells harboring 4977bp deletion of mitochondrial DNA. Biochim Biophys Acta. 1832, 216–227 (2013).
Cimen, H. et al. Regulation of succinate dehydrogenase activity by SIRT3 in mammalian mitochondria. Biochemistry. 49, 304–311 (2010).
Bause, A. S., Matsui, M. S. & Haigis, M. C. The Protein Deacetylase SIRT3 Prevents Oxidative Stress-induced Keratinocyte Differentiation. J Biol Chem. 288, 36484–36491 (2013).
Rato, L. et al. Pre-diabetes alters testicular PGC1-alpha/SIRT3 axis modulating mitochondrial bioenergetics and oxidative stress. Biochim Biophys Acta. 1837, 335–344 (2014).
Samant, S. A. et al. SIRT3 Deacetylates and Activates OPA1 To Regulate Mitochondrial Dynamics during Stress. Mol Cell Biol. 34, 807–819 (2014).
Tseng, A. H., Shieh, S. S. & Wang, D. L. SIRT3 deacetylates FOXO3 to protect mitochondria against oxidative damage. Free Radic Biol Med. 63, 222–234 (2013).
Scott, I., Webster, B. R., Li, J. H. & Sack, M. N. Identification of a molecular component of the mitochondrial acetyltransferase programme: a novel role for GCN5L1. Biochem J. 443, 655–661 (2012).
Wagner, G. R. & Payne, R. M. Widespread and enzyme-independent Nepsilon-acetylation and Nepsilon-succinylation of proteins in the chemical conditions of the mitochondrial matrix. J Biol Chem. 288, 29036–29045 (2013).
Baeza, J., Smallegan, M. J. & Denu, J. M. Mechanisms and Dynamics of Protein Acetylation in Mitochondria. Trends Biochem Sci (2016).
Komatsu, H. et al. Intracellular activation of acetyl-CoA by an artificial reaction promoter and its fluorescent detection. Chem Commun (Camb). 49, 2876–2878 (2013).
Hafner, A. V. et al. Regulation of the mPTP by SIRT3-mediated deacetylation of CypD at lysine 166 suppresses age-related cardiac hypertrophy. Aging (Albany NY). 2, 914–923 (2010).
Li, S. et al. p53-induced growth arrest is regulated by the mitochondrial SirT3 deacetylase. PLoS One. 5, e10486 (2010).
Yu, W., Dittenhafer-Reed, K. E. & Denu, J. M. SIRT3 protein deacetylates isocitrate dehydrogenase 2 (IDH2) and regulates mitochondrial redox status. J Biol Chem. 287, 14078–14086 (2012).
Cheng, A. et al. Mitochondrial SIRT3 Mediates Adaptive Responses of Neurons to Exercise and Metabolic and Excitatory Challenges. Cell Metab. 23, 128–142 (2016).
Jin, L. et al. Crystal structures of human SIRT3 displaying substrate-induced conformational changes. J Biol Chem. 284, 24394–24405 (2009).
Jaberi, E. et al. The novel mutation p.Asp251Asn in the beta-subunit of succinate-CoA ligase causes encephalomyopathy and elevated succinylcarnitine. J Hum Genet. 58, 526–530 (2013).
Fang, L. et al. Inactivation of MARCH5 prevents mitochondrial fragmentation and interferes with cell death in a neuronal cell model. PLoS One. 7, e52637 (2012).
Imamura, H. et al. Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators. Proc Natl Acad Sci USA 106, 15651–15656 (2009).
Fritz, K. S., Galligan, J. J., Hirschey, M. D., Verdin, E. & Petersen, D. R. Mitochondrial acetylome analysis in a mouse model of alcohol-induced liver injury utilizing SIRT3 knockout mice. J Proteome Res. 11, 1633–1643 (2012).
Schwer, B., Bunkenborg, J., Verdin, R. O., Andersen, J. S. & Verdin, E. Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2. Proc Natl Acad Sci USA 103, 10224–10229 (2006).
Ahn, B. H. et al. A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis. Proc Natl Acad Sci USA 105, 14447–14452 (2008).
Ozden, O. et al. SIRT3 deacetylates and increases pyruvate dehydrogenase activity in cancer cells. Free Radic Biol Med. 76, 163–172 (2014).
Vedejs, E. & Diver, S. T. Tributylphosphine: A remarkable acylation catalyst. J Am Chem Soc. 115, 3358–3359 (1993).
Komatsu, H., Shindo, Y., Oka, K., Hill, J. P. & Ariga, K. Ubiquinone-Rhodol (UQ-Rh) for Fluorescence Imaging of NAD(P)H through Intracellular Activation. Angew Chem Int Ed Engl (2014).
Baeza, J., Smallegan, M. J. & Denu, J. M. Site-specific reactivity of nonenzymatic lysine acetylation. ACS Chem Biol. 10, 122–128 (2015).
Someya, S. et al. Sirt3 mediates reduction of oxidative damage and prevention of age-related hearing loss under caloric restriction. Cell. 143, 802–812 (2010).
Guedes-Dias, P. & Oliveira, J. M. Lysine deacetylases and mitochondrial dynamics in neurodegeneration. Biochim Biophys Acta. 1832, 1345–1359 (2013).
Webster, B. R. et al. Restricted mitochondrial protein acetylation initiates mitochondrial autophagy. J Cell Sci. 126, 4843–4849 (2013).
Hirschey, M. D. et al. SIRT3 deficiency and mitochondrial protein hyperacetylation accelerate the development of the metabolic syndrome. Mol Cell. 44, 177–190 (2011).
Vazquez, E. J. et al. Mitochondrial complex I defect and increased fatty acid oxidation enhance protein lysine acetylation in the diabetic heart. Cardiovasc Res. 107, 453–465 (2015).
Hu, W., Guan, L. S., Dang, X. B., Ren, P. Y. & Zhang, Y. L. Small-molecule inhibitors at the PSD-95/nNOS interface attenuate MPP+-induced neuronal injury through Sirt3 mediated inhibition of mitochondrial dysfunction. Neurochem Int. 79, 57–64 (2014).
Yang, W. et al. Mitochondrial Sirt3 Expression is Decreased in APP/PS1 Double Transgenic Mouse Model of Alzheimer’s Disease. Neurochem Res. 40, 1576–1582 (2015).
Grammel, M. & Hang, H. C. Chemical reporters for biological discovery. Nat Chem Biol. 9, 475–484 (2013).
Patterson, D. M., Nazarova, L. A. & Prescher, J. A. Finding the right (bioorthogonal) chemistry. ACS Chem Biol. 9, 592–605 (2014).
Beatty, K. E., Xie, F., Wang, Q. & Tirrell, D. A. Selective dye-labeling of newly synthesized proteins in bacterial cells. J Am Chem Soc. 127, 14150–14151 (2005).
Yusop, R. M., Unciti-Broceta, A., Johansson, E. M., Sanchez-Martin, R. M. & Bradley, M. Palladium-mediated intracellular chemistry. Nat Chem. 3, 239–243 (2011).
Acknowledgements
We thank Dr. H. Imamura and Dr. H. Noji for providing the ATeam plasmid. This research was supported by the Grant-in-Aid for Scientific Research, KAKENHI (24240045 and 25750395).
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Y.S. designed the study, performed the experiments, analysed the data and wrote the paper. H.K. designed the study and edited the paper. K.H. and K.A. designed the study. K.O. designed the study and edited the paper. All authors reviewed the manuscript.
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Shindo, Y., Komatsu, H., Hotta, K. et al. An Artificial Reaction Promoter Modulates Mitochondrial Functions via Chemically Promoting Protein Acetylation. Sci Rep 6, 29224 (2016). https://doi.org/10.1038/srep29224
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DOI: https://doi.org/10.1038/srep29224
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