Cyclophilin D Modulates Mitochondrial AcetylomeNovelty and Significance
Rationale: Mice lacking cyclophilin D (CypD−/−), a mitochondrial chaperone protein, have altered cardiac metabolism. As acetylation has been shown to regulate metabolism, we tested whether changes in protein acetylation might play a role in these metabolic changes in CypD−/− hearts.
Objective: Our aim was to test the hypothesis that loss of CypD alters the cardiac mitochondrial acetylome.
Methods and Results: To identify changes in lysine-acetylated proteins and to map acetylation sites after ablation of CypD, we subjected tryptic digests of isolated cardiac mitochondria from wild-type and CypD−/− mice to immunoprecipitation using agarose beads coupled to antiacetyl lysine antibodies followed by mass spectrometry. We used label-free analysis for the relative quantification of the 875 common peptides that were acetylated in wild-type and CypD−/− samples and found 11 peptides (10 proteins) decreased and 96 peptides (48 proteins) increased in CypD−/− samples. We found increased acetylation of proteins in fatty acid oxidation and branched-chain amino acid metabolism. To evaluate whether this increase in acetylation might play a role in the inhibition of fatty acid oxidation that was previously reported in CypD−/− hearts, we measured the activity of l-3-hydroxyacyl-CoA dehydrogenase, which was acetylated in the CypD−/− hearts. Consistent with the hypothesis, l-3-hydroxyacyl-CoA dehydrogenase activity was inhibited by ≈50% compared with the wild-type mitochondria.
Conclusions: These results implicate a role for CypD in modulating protein acetylation. Taken together, these results suggest that ablation of CypD leads to changes in the mitochondrial acetylome, which may contribute to altered mitochondrial metabolism in CypD−/− mice.
Cyclophilin D (CypD) is a peptidyl prolyl cis-trans isomerase, which functions as a mitochondrial protein chaperone and is, therefore, likely to have multiple targets. CypD is also known to be an activator of the mitochondrial permeability transition pore (mPTP). mPTP is a nonselective pore in the inner mitochondrial membrane that is opened by high matrix calcium or reactive oxygen species.1–3 Sustained opening of the mPTP results in loss of membrane potential, uncoupling of oxidative phosphorylation, matrix swelling, ATP depletion, and increased production of reactive oxygen species, ultimately leading to cell death. mPTP plays a critical role in mediating cell death during ischemia/reperfusion injury, and inhibition of mPTP is proposed to be the end-effector of cardioprotective signaling cascades.4,5 In addition to sustained activation of mPTP, which leads to cell death, mPTP has been shown to open transiently. It has been proposed that transient opening of mPTP can serve as a mitochondrial calcium release mechanism.1,6
Editorial, see p 1268
Loss or inhibition of CypD has been shown to lead to an increase in mitochondrial calcium, which results in activation of mitochondrial NADH dehydrogenases, such as pyruvate dehydrogenase and alterations in the ratio of carbohydrate to fatty acid oxidation.6 Using both proteomic and metabolomic approaches, we previously found that mitochondria from CypD−/− hearts have alterations in pyruvate and branched-chain amino acid metabolism, as well as changes in levels of mitochondrial histone proteins.7 Because acetylation has been shown to regulate mitochondrial metabolism, we consider that the metabolic changes observed with loss of CypD might be because of alterations in the mitochondrial acetylome. Protein acetylation is a reversible post-translational modification, which adds an acetyl moiety to the ε-amino group of lysine residues.8–11 Protein acetylation has been shown to play a key role in histone modifications and is well known to influence changes in gene expression,12 but much less is known about its role in nonnuclear protein acetylation and cellular regulation. In particular, emerging data indicate that many proteins within the mitochondria are reversibly acetylated.13,14 The acetylation of many metabolic enzymes has been shown in liver and yeast, but there is little comprehensive data in heart. We, therefore, examined the effect of loss of CypD on cardiac mitochondrial protein acetylation. In the present study, we found that loss of CypD resulted in an increase in acetylation of many mitochondrial proteins in a profile consistent with cardiac metabolic remodeling in CypD−/− mice.
See Online Data Supplement for detailed materials and methods related to this study.
Adult male 12- to 16-week-old wild-type (WT) and CypD−/− mice, obtained from Dr Jeffery Molkentin (Cincinnati Children’s Hospital Medical Center), were studied. All animals were treated and cared for in accordance with the Guide for the Care and Use of Laboratory Animals of National Institutes of Health, Revised 2011, and protocols were approved by the Institutional Animal Care and Use Committee.
Mitochondria were isolated by differential centrifugation according to standard procedures.15
Isolated mitochondria (500 μg) were subjected to immunoprecipitation followed by Western blot analyses as previously described16 using anti-GRP75, anti-F1Fo ATP synthase subunit A, antipyruvate dehydrogenase E1 component subunit α antibodies.
Equivalent amounts of protein (20–40 μg) from each sample were separated on NuPAGE 4% to 12% Bis-Tris gels (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membranes. Gel transfer efficiency and equal loading were verified using reversible Ponceau S staining. The resulting blots were probed with antiacetylated lysine antibody (Cell Signaling, Danvers, MA), anti-GCN5L1, anti-sirtuin 3 (SIRT3), anti-VDAC-1 (Santa Cruz Biotechnology), anti-F1Fo ATP synthase subunit α, or anti-CypD antibody (Mitosciences, Eugene, OR).
Affinity Purification of Lysine-Acetylated Peptides for Mass Spectrometry
Isolated mitochondrial pellets (1 mg) were subjected to immunoprecipitation as previously described13 to identify lysine-acetylated peptides by mass spectrometry. The LCMS data were searched against the Swiss-Prot database (taxonomy Musculus [mouse]) using Mascot server (Matrix Science, London, United Kingdom; version 2.3). Relative quantification of acetylated peptides was performed using QUOIL (QUantification withOut Isotope Labeling), an in-house software program designed as a label-free approach to peptide quantification by LC-MS/MS.17
Mitochondria were isolated from WT and CypD−/− mouse hearts. SIRT3 activity was measured using SIRT3 Direct Fluorescent Screening Assay Kit (Cayman Chemical, Ann Arbor, MI) in the presence and absence of nicotinamide.
Langendorff Heart Perfusion and Protocol
Mouse hearts were subjected to Langendorff perfusion as previously described.18
Adult mouse ventricular myocytes were isolated by collagenase digestion as described previously19 and attached to matrigel-coated coverslips for 30 minutes in a 5% CO2 incubator at 37°C in medium 199 supplemented with 5 mmol/L creatine, 2 mmol/L l-carnitine, 5 mmol/L taurine, 2.5 mmol/L sodium pyruvate, 26 mmol/L NaHCO3, 100 U/mL penicillin, and 100 μg/mL streptomycin.
Mitochondrial NADH Measurement
Myocytes were mounted on the stage of a fluorescence microscope (Nikon Diaphot) with a 20× objective and superfused with Tyrode solution (25°C) containing (mmol/L): 140 NaCl, 4 KCl, 1 MgCl2, 5 HEPES, and 10 d-glucose (pH 7.4). The endogenous mitochondrial NADH autofluorescence was excited at λexc=340 nm (band pass filter) and its emission recorded at λem=415 nm (long pass filter) into a QuantEM 512 SC electron-multiplying charge-coupled device camera (Photometrics, Tucson, AZ). NADH levels were expressed as a percentage of the reduced NADH/NAD+ pool, which was calibrated by applying 4 mmol/L NaCN (100%) and 5 μmol/L carbonyl cyanide 3-chlorophenyl hydrazone (0%) for each experiment.
Mitochondrial Swelling and Calcium Retention Capacity Assays
mPTP opening in isolated heart mitochondria from WT and CypD−/− mouse hearts was assessed using the calcium retention capacity and Ca2+-induced swelling assays. Calcium retention capacity was assessed using 10 μmol/L fluorescent Ca2+ indicator (Calcium Green-5N; Molecular Probes, Eugene, OR) with the addition of 10 μmol/L Ca2+ pulses to induce mPTP opening. Ca2+-induced swelling assay was measured spectrophotometrically as a decrease in absorbance at 540 nm after pore opening that was induced by 250 μmol/L CaCl2. Both assays were assessed in the presence and absence of 200 nmol/L cyclosporine A, a known CypD and mPTP inhibitor.
Trifunctional Protein Enzyme Subunit α Activity Measurements
The activity of l-3-hydroxyacyl-CoA dehydrogenase (LCHAD) was measured in isolated mitochondria from WT and CypD−/− mouse hearts. After mitochondria isolation, the pellet was suspended in 25 mmol/L potassium phosphate, 50 mmol/L 3-(N-morpholino)propanesulfonic acid, 0.2 mmol/L EDTA, pH 8.0. The mitochondria suspensions were freeze-thawed 3× and sonicated on ice for 3×10 seconds with a 1-minute interval in between. Triton X-100 was then added to the suspensions to give a Triton X-100-to-protein ratio of 1:1. After incubation on ice for 30 minutes, the extracts were centrifuged at 11 600g for 10 minutes. Enzyme activities were then measured in the supernatant at 37oC in the presence of 100 μmol/L NADH and 100 μmol/L acetoacetyl-CoA. LCHAD activity was calculated as the rate of NADH oxidized per minute per milligram protein.
All data were expressed as means±SE. Student 2-sample t test or 1-way ANOVA with Bonferroni post hoc analyses were used for comparison of differences between groups, and a probability value ≤0.05 was considered significant.
Alterations in the Mitochondrial Acetylation Profile After Loss of CypD
We previously found that CypD−/− hearts have increased pyruvate dehydrogenase activity and a reduction in fatty acid oxidation relative to glucose oxidation.6 We also found changes in branched-chain amino acid metabolism in CypD−/− hearts.7 As mitochondrial acetylation has emerged as a key regulator of mitochondrial fatty acid and branched-chain amino acid metabolism, we tested whether CypD−/− hearts might have altered mitochondrial acetylation. Acetylation/deacetylation of protein lysine residues has emerged as an important post-translational modification for dynamic regulation of many proteins.8–11 We initially examined changes in lysine-acetylated proteins after the ablation of CypD. As shown in Figure 1A and 1B, Western blot analysis shows that acetylation levels were increased by 45±12% in CypD−/− mitochondria.
The acetylome has been compiled for yeast, mouse, and rat liver, but the cardiac mitochondrial acetylome has not been well defined. We, therefore, decided to define the cardiac mitochondrial acetylome, including sites of acetylation at baseline, in WT hearts. We accomplished this by subjecting tryptic digests of isolated cardiac mitochondria from WT mice to immunoprecipitation using antiacetyl lysine antibody coupled to agarose beads followed by mass spectrometry. In WT mitochondria, we found 198 acetylated proteins (with 864 peptides) at baseline (Figure 2A; Online Table I; n=3 biological replicates). As reported in Table 1 57 of these proteins were from the electron transport complexes I to V (258 acetylated peptides). More than 50% of the electron transport complexes were found to be acetylated at baseline. We also found that all of the TCA cycle proteins as well as a number of enzymes involved in fatty acid oxidation were acetylated. We also found 11 mitochondrial carrier proteins acetylated, including regulators (CCDC90A and B) of the recently described mitochondrial Ca2+ uniporter20 and mitochondrial pyruvate carrier 2 (Brain protein 44). Of interest, we found CypD was acetylated, which is consistent with previous data.21 Thus, most of the enzymes involved in cardiac mitochondrial metabolism and bioenergetics are acetylated at baseline.
As shown in Figure 1A, there is an increase in global acetylation in mitochondria isolated from mice lacking CypD. We, therefore, compared the acetylome of WT mitochondria to that of CypD−/−. We manually inspected all the spectra of identified acetylated peptides using Scaffold post-translational modification software in conjunction with the Proteome Discover software. As shown in Figure 2A, in the CypD−/− mitochondria, we identified 955 acetylated peptides (from 219 proteins), with 875 acetylated peptides common between WT and CypD−/− samples (see Online Table II for a complete list of acetylated proteins and peptides). Using the criteria defined in the caption to Figure 2A, we found 45 peptides that were acetylated only in WT (Online Table III) and 35 peptides that were acetylated only in CypD−/− mitochondria (Online Table IV). We concentrated our analysis on the 875 acetylated peptides that were detected in both WT and CypD−/− samples. A label-free analysis program for relative quantification of the common acetylated peptides between WT and CypD−/− samples was used and representative ion chromatographs are shown (Figure 2B–2D). We found 11 peptides (from 10 proteins) decreased in CypD−/− mitochondria by ≥20% with P<0.05 (Table 2). Using similar criteria, we found 96 peptides from 48 proteins including 19 proteins from the electron transport chain complexes (Table 3) that showed an increase in acetylation in CypD−/− mitochondria (Online Table V). We confirmed the increase in acetylation of several proteins by subsequent immunoprecipitation of acetylated proteins followed by immunoblot analysis with antibodies recognizing GRP75, F1F0 ATP synthase subunit α, and pyruvate dehydrogenase E1 component subunit α (Figure 1C). The increase or decrease in acetylation noted in Figure 2A could be because of a change in protein level in CypD−/− hearts. In a previous study, we examined changes in protein levels in CypD−/− hearts.7 We compared changes in protein levels in CypD−/− heart to changes in acetylation. Two (VDAC3 and DHSA) of the 10 proteins that showed a decrease in acetylation (Table 2) had reduced expression levels in CypD−/− hearts. Thus, the decrease in acetylation in these proteins is likely because of decreased protein level. Of the 48 proteins that showed an increase in acetylation (Online Table V), only 3 proteins (PRDX5, ODPA, and NDUA5) showed an elevated protein level. Therefore, the increase in protein acetylation for the vast majority of proteins cannot be attributed to an increase in protein expression. A change in acetylation has also been suggested to alter protein stability. Only 3 proteins increased in both acetylation and protein levels (PRDX5, ODPA, and NDUA5), and these proteins are potential candidates whereby acetylation may increase their stability.22 Of the 30 proteins that showed a decrease in protein level in CypD−/− hearts, only 2 (M2OM and NDUV1) showed an increase in acetylation. Thus, acetylation does not seem to have a dramatic effect on protein expression in adult myocardium.
Although acetylation does not have a large impact on protein stability, it could have a major impact on enzyme activity and metabolism. The reduction in fatty acid oxidation observed previously in CypD−/− hearts6 is entirely consistent with changes in acetylation observed in CypD−/− hearts. Increased acetylation has been shown previously to inhibit fatty acid oxidation.23,24 Consistent with the reduction in fatty acid oxidation in CypD−/− hearts, we saw an increase in acetylation of key enzymes involved in fatty acid oxidation. We found an increase in acetylation in both subunits of trifunctional enzyme (ECHA and ECHB), long-chain specific acyl-CoA dehydrogenase, 2,4-dienoyl-CoA reductase, 3-ketoacyl-CoA thiolase, and enoyl-CoA δ isomerase 1. To test the functional effect of acetylation on enzyme activity, we measured the LCHAD activity of trifunctional protein subunit α, an enzyme involved in fatty acid oxidation, which showed an increase in acetylation in CypD−/− mitochondria. We found that the LCHAD activity was inhibited by ≈50% in CypD−/− mitochondria as compared with WT mitochondria (Figure 3). To further evaluate whether an increase in acetylation results in a decrease in LCHAD activity, we preincubated WT sonicated mitochondria with 100 μmol/L acetyl-CoA (confirming an increase in acetylation of LCHAD by Western blot analysis). Consistent with the hypothesis, with acetyl-CoA treatment, LCHAD activity was inhibited similarly to the CypD−/− group. To identify the pathways affected by the increase in protein acetylation in CypD−/− hearts, we performed pathway analysis using Ingenuity software. The acetylated peptides comprise pathways involved in oxidative phosphorylation, mitochondrial dysfunction, butanoate metabolism, pyruvate metabolism, and branched-chain amino acid metabolism (Online Figure I).
We examined the mechanisms responsible for changes in the mitochondrial acetylome in CypD−/− mice. A recent study has shown in an in vitro assay that increasing NADH while NAD+ is held constant leads to a significant decrease in SIRT3 activity.25 As the NADH/NAD+ ratio can change during isolation of mitochondria, we measured the endogenous NADH fluorescence in WT and CypD−/− cardiomyocytes (Figure 4A). Baseline NADH levels were increased by ≈80% in CypD−/− hearts relative to WT (29.44±2.63% versus 16.20±2.86% of fully reduced NADH; Figure 4A). Western blot analysis showed no difference in either mitochondrial acetyl transferase (GCN5L1; Figure 4B) or deacetylases (SIRT3, Figure 4C; SIRT4, Figure 4D; or SIRT5, Figure 4E) between WT and CypD−/− mitochondria, which would support a primary role for an increase in NADH in the increase in acetylation. Finally, there was no difference in SIRT3 activity between WT and CypD−/− mitochondria (Figure 4F) measured under conditions where the NAD and NADH were fixed in the assay.
To gain additional insight into the mechanism by which loss of CypD leads to an increase in acetylation, we determined whether the peptides acetylated in CypD−/− hearts were SIRT3 substrates. To accomplish this, we compared the peptides showing an increase in acetylation after ablation of SIRT3 to the list of acetylated peptides increased in hearts lacking CypD. We found 151 peptides that exhibited an increase in acetylation in SIRT3−/− hearts compared with WT littermates; we defined these peptides as potential SIRT3 substrates (Online Table VI). Of interest, CypD itself is a SIRT3 substrate. If we compare these 151 peptides to the 96 peptides that exhibited an increase in acetylation in CypD−/− hearts, we found only 19 common peptides that are potential SIRT3 substrates (Online Table V, bolded).
CypD−/− hearts have reduced ischemia/reperfusion injury,4 and this raises the question as to whether the increase in mitochondrial acetylation plays a role in cardioprotection. Mitochondria isolated from CypD−/− mice have been shown to be more resistant to mPTP opening compared with WT mice.4 To address whether an increase in mitochondrial acetylation reduces mPTP opening, we used SIRT3−/− mice. These mice have been shown (and we confirmed) to have increased protein acetylation.26 Consistent with previous studies,27 we found no difference in mPTP opening between WT and SIRT3−/− mitochondria isolated from 3-month-old mice (Figure 5). mPTP opening was sensitive to cyclosporine A, a known mPTP inhibitor. These data suggest that an increase in mitochondrial acetylation per se is not sufficient to alter mPTP.
Several studies have shown that acetylation regulates metabolic enzymes by multiple mechanisms, including enzymatic activation,10 inhibition,28 or protein stability.22 A recent proteomics study by Kim et al13 revealed that >20% of liver mitochondrial proteins and enzymes are acetylated and that changes occur in acetylation status in response to acute fasting. Previous studies have identified several acetylated proteins in cardiac mitochondria29; however, this is the first study to evaluate large-scale acetylation in cardiac mitochondria. In this study, we identified >200 cardiac mitochondrial proteins that are acetylated under basal conditions. We found that >50% of electron transport complex proteins are acetylated at baseline. We also found baseline acetylation of TCA and fatty oxidation enzymes.
This study demonstrates that loss of CypD results in an increase in acetylation. We found an increase in acetylation of many enzymes involved in fatty acid oxidation, and previous studies have reported that increased acetylation of specific fatty acid oxidation enzymes leads to their inhibition.23,24 We provide new data showing that acetylation of LCHAD results in its inhibition.
What is the mechanism responsible for the increase in acetylation in CypD−/− hearts? Reversible lysine acetylation is regulated by opposing activities of protein acetyltransferases and deacetylases. We found no increase in the protein level of recently described mitochondrial acetyltransferase (GCN5L1).30 We also found no change in the levels of 3 mitochondrial deacytylases, SIRT3, SIRT4, and SIRT5. We also found no change in activity of SIRT3. These data are most consistent with the hypothesis that loss of CypD leads to an increase in NADH/NAD+, resulting in the inhibition of deacetylase(s) and an increase in protein acetylation (Online Figure II). Previous studies have suggested that a change in NADH/NAD+ ratio can alter the activity of sirtuins.25,31–33 A change in NADH/NAD+ would not carry over in an assay of SIRT from mitochondria, in which the NAD+/NADH level is set as part of the assay. It is likely that this increase in NADH/NAD+ activates all mitochondrial sirtuins. This would be consistent with our data showing that CypD−/− hearts exhibited acetylation of many proteins that are not SIRT3-dependent substrates, as determined either in this study (Online Table V) or previous studies.34–37 The current consensus (mostly obtained from data in liver) is that SIRT3 is the primary mitochondrial deacetylase.26 SIRT4 and SIRT5 have been shown to exhibit ADP-ribosyltransferase and succinyl deacetylase activity, respectively.26 However, there are data suggesting that SIRT438 and SIRT525 can also contribute to mitochondrial deacetylation.
It is well established that CypD−/− mitochondria have a decreased susceptibility to mPTP opening. As suggested in Online Figure II, we propose that inhibition of transient opening of mPTP leads to the increase in Ca2+, increased pyruvate dehydrogenase activity, and, ultimately, an increase in acetylation. The increase in acetylation per se does not seem to alter mPTP, although acetylation of a specific protein could be involved in regulation. Sinclair and colleagues27 reported that mitochondria from SIRT3−/− hearts have an increase in mPTP when measured at 18 months of age, but not at 3 or 6 months of age. They attributed the increase in mPTP at 18 months to an increase in acetylation of CypD. In SIRT3−/− heart mitochondria, we find a general increase in acetylation and a specific increase in acetylation of CypD at 3 months of age, but we find no change in mPTP, which is consistent with the study of Sinclair group.27 The increase in mPTP observed at 18 months in SIRT3−/− hearts could be because of metabolic changes that are known to occur in these mice rather than a direct effect of acetylation.
In summary, this study provides insights into changes in the cardiac mitochondrial acetylome occurring with loss of CypD and loss of SIRT3. Loss of CypD leads to an increase in protein acetylation, which could account for metabolic changes previously reported in these mice.6
We thank Drs Marcia Haigis and Gaëlle Laurent from Harvard Medical School for kindly providing the SIRT4 antibody.
Sources of Funding
This work was supported by the National Institutes of Health’s National Heart, Lung, and Blood Institute intramural program.
In August 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 12.8 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.113.301867/-/DC1.
- Nonstandard Abbreviations and Acronyms
- cyclophilin D
- mitochondrial permeability transition pore
- sirtuin 3
- Received May 28, 2013.
- Revision received September 16, 2013.
- Accepted September 20, 2013.
- © 2013 American Heart Association, Inc.
- Crompton M
- Elrod JW,
- Wong R,
- Mishra S,
- Vagnozzi RJ,
- Sakthievel B,
- Goonasekera SA,
- Karch J,
- Gabel S,
- Farber J,
- Force T,
- Brown JH,
- Murphy E,
- Molkentin JD
- Lu Z,
- Scott I,
- Webster BR,
- Sack MN
- Zhao S,
- Xu W,
- Jiang W,
- et al
- Choudhary C,
- Kumar C,
- Gnad F,
- Nielsen ML,
- Rehman M,
- Walther TC,
- Olsen JV,
- Mann M
- Sack MN
- Scholz TD,
- Balaban RS
- Sun J,
- Morgan M,
- Shen RF,
- Steenbergen C,
- Murphy E
- Sun J,
- Picht E,
- Ginsburg KS,
- Bers DM,
- Steenbergen C,
- Murphy E
- Mallilankaraman K,
- Cardenas C,
- Doonan PJ,
- et al
- Shulga N,
- Wilson-Smith R,
- Pastorino JG
- Lombard DB,
- Alt FW,
- Cheng HL,
- et al
- Shinmura K,
- Tamaki K,
- Sano M,
- Nakashima-Kamimura N,
- Wolf AM,
- Amo T,
- Ohta S,
- Katsumata Y,
- Fukuda K,
- Ishiwata K,
- Suematsu M,
- Adachi T
- Scott I,
- Webster BR,
- Li JH,
- Sack MN
- Lin SJ,
- Ford E,
- Haigis M,
- Liszt G,
- Guarente L
- Newman JC,
- He W,
- Verdin E
- Yu W,
- Dittenhafer-Reed KE,
- Denu JM
- Rardin MJ,
- Newman JC,
- Held JM,
- Cusack MP,
- Sorensen DJ,
- Li B,
- Schilling B,
- Mooney SD,
- Kahn CR,
- Verdin E,
- Gibson BW
Novelty and Significance
What Is Known?
Loss of cyclophilin D (CypD) leads to a decrease in the ratio of fatty acid oxidation relative to glucose oxidation.
Lysine acetylation has recently emerged as an important post-translational protein modification in the regulation of mitochondrial metabolism.
What New Information Does This Article Contribute?
Identification of the cardiac mitochondrial acetylome.
Loss of CypD results in changes to cardiac mitochondrial acetylome, with the majority of proteins showing an increase in acetylation.
Acetylation of mitochondrial trifunctional protein subunit α leads to the inhibition of its activity.
We examined whether CypD regulates mitochondrial metabolism by modulating changes in protein acetylation. We used a mass spectrometry–based approach and identified acetylated proteins in cardiac mitochondria under baseline conditions. We then compared differences in protein acetylation profiles in heart mitochondria from wild-type mice and mice lacking CypD. We found a general increase in mitochondrial protein acetylation in CypD−/− hearts, including several protein targets involved in fatty acid oxidation. A further examination of 1 of these protein targets, trifunctional protein subunit α, showed that acetylation leads to the inhibition of enzyme activity. This study provides an important link between acetylation and mitochondrial function and suggests that mitochondrial acetylome represents a new layer of protein regulation mediating adaptive changes in mitochondrial metabolism.