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Ethanol intoxication increases hepatic N-lysyl protein acetylation
Biochemical and Biophysical Research Communications 376 (2008) 615–619
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Ethanol intoxication increases hepatic N-lysyl protein acetylation Matthew J. Picklo Sr. Department of Pharmacology, Physiology, and Therapeutics, University of North Dakota, School of Medicine and Health Sciences, 501 N. Columbia Road, Grand Forks, ND 58203-9037, USA Department of Chemistry, University of North Dakota, Grand Forks, ND 58203-9037, USA
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Article history: Received 8 September 2008 Available online 18 September 2008
Keywords: Sirtuin Acetylation Mitochondria Ethanol CYP2E1
a b s t r a c t The acetylation of the e-amino group of lysine to form N-acetyl lysine (N-AcLys)-modified proteins regulates the activity of metabolic proteins. Because of the multiple effects of ethanol upon hepatic metabolism, it was hypothesized that ethanol exposure increases the hepatic content of N-AcLys-modified proteins. To test this hypothesis, rats or mice were exposed to ethanol using a liquid diet regimen. Content of N-AcLys-modified proteins was elevated more than 5-fold after 6 weeks of ethanol exposure and persisted after ethanol withdrawal. Use of CYP2E1-knockout mice demonstrated that ethanol-induced acetylation was not dependent solely on CYP2E1 expression. The mitochondrial content of N-AcLysmodified proteins was elevated almost 5-fold following 6 weeks of ethanol exposure. Mitochondrial content of the deacetylase Sirt3 was unchanged by 6 weeks of ethanol exposure. These data indicate ethanol intoxication changes the acetylation status of, and likely the activity of, multiple mitochondrial proteins. Ó 2008 Elsevier Inc. All rights reserved.
The N-lysyl acetylation of proteins is a post-translational modification that impacts multiple cellular functions [1–3]. The acetylation of the e-amino group of lysine to form N-acetyl lysine (N-AcLys)-modified proteins affects the function of multiple proteins including histones, p53, and a-tubulin (for review see [3]). Recent data indicate that over 20% of the mitochondrial proteome is subject to N-AcLys modification and that this process is regulated by diet [4,5]. Mitochondrial proteins such as acetyl CoA synthetase 2 (ACS2) and glutamate dehydrogenase-1 (GDH-1) undergo reversible acetylation that regulates enzymatic activity [5–7]. Recent research has focused upon cellular functions of the Silent Information Regulator 2 family of deacetylases, termed sirtuins (Sirt’s). Sirtuin deacetylases are NAD+ dependent [8]. Multiple classes of sirtuins (Sirt1–7) have been characterized that differ in subcellular localization, protein targets, and mechanisms of action (for reviews, see ([9,10]). Sirt1 is localized to the nucleus and Sirt2 is cytoplasmic. Sirt3, Sirt4, and Sirt5 reside in the mitochondria [5,11–13]. Sirt3 and Sirt5 have deacetylase activity whereas Sirt4 ADP-ribosylates proteins [12–15]. Ethanol exposure alters Sirt1 and Sirt5 pathways resulting in altered cellular signaling [16–18]. Ethanol intoxication decreases expression of Sirt5 and while increasing acetylation of p53 and PGC-1a [16]. Ethanol decreases hepatic Sirt1 content and increases the acetylation of PGC1a in animals fed a low fat diet [18]. In contrast, in animals fed diet containing high saturated fat content, ethanol increased Sirt1 levels [17].
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Because of the multiple biochemical effects of ethanol upon hepatic metabolism, it was hypothesized that ethanol exposure increases the hepatic content of multiple N-AcLys-modified proteins. To test this hypothesis, animals were exposed to ethanol. After 6 weeks of exposure, hepatic content of N-AcLys-modified proteins was elevated over 5-fold, particularly in the mitochondria. Multiple proteins were hyper-acetylated by ethanol exposure. On the other hand, mitochondrial content of Sirt3, the deacetylase known to regulate the N-lysyl acetylation of multiple mitochondrial proteins, was unchanged by 6 weeks of ethanol exposure. Methods Ethanol exposure. All experimental protocols were in accordance with the NIH guidelines for use of live animals and were approved by the University of North Dakota Institutional Animal Care and Use Committee. Lieber–DeCarli formula (18% protein, 35% fat, and 47% carbohydrate) liquid diet was purchased from BioServ (Frenchtown, NJ). Adult, male Sprague–Dawley rats (250 g) were purchased from Charles River Laboratories (Wilmington, MA). Rats were housed for 1 week on normal solid diet. Animals were then placed on liquid diet without ethanol for 1 week followed by liquid diet containing 36% of total calories from ethanol or control diet in which the ethanol calories were replaced by calories from maltose and dextrin. After 6 weeks, rats receiving the ethanol containing diet were given liquid diet without ethanol for zero (0) days, 2 days, or 7 days before killing. Control, non-ethanol-treated rats were pair-fed against the ethanol-treated animals killed on day zero of ethanol withdrawal. Another group of ethanol-treated rats and their pair-fed controls were treated for 7 days on the diet.
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Rats were deeply anesthetized by i.p. injection of ketamine (100 mg/kg) and xylazine (13 mg/kg). Livers and hearts were quickly removed. A portion of whole liver sample was frozen in liquid nitrogen and stored at 80 °C. In some studies, the remaining portions of liver were used for isolation of mitochondria (see below). Cyp2e1 / mice. Liver homogenates from female cyp2e1 / and female wild-type mice exposed to ethanol or control diet were provided as the kind gift of Professor Arthur Cederbaum, Mt. Sinai Medical Center (New York, NY) [19,20]. Cyp2e1 / and wild-type mice were on the SV/129 background and were generated as described previously [19]. Mice were bred at the Mt. Sinai Medical Center animal facility. At 8–10 weeks of age (16–20 g), mice were exposed to ethanol using a Lieber–DeCarli liquid diet with a gradient dosing of ethanol from 10% of caloric intake to a final of 35% of caloric over intake over 2 weeks followed by 1 week at 35% of caloric intake [20]. Pair-fed controls received dextrose instead of ethanol. Homogenates were prepared in ice-cold 0.15 M KCl as described previously and frozen at 80 °C [20]. Mitochondrial isolation. Mitochondria were isolated using differential centrifugation in a sucrose/mannitol buffer as described previously [21]. Mitochondria were aliquoted and frozen at 80 °C. Immunoblotting. Whole liver samples were homogenized in buffer consisting of sodium phosphate (20 mM, final pH 7.2 at 4 °C), 0.2 % Triton X-100, 1 mM DEPA, 250 lM BHT, and mammalian protease inhibitor cocktail (Sigma, St. Louis, MO) diluted according to manufacturer’s instructions. The homogenate was sonicated, centrifuged at 10,000g for 10 min at 4 °C to pellet any insoluble material, and the supernatant frozen at 80 °C. Proteins from purified mitochondria or whole liver homogenates were separated on gradient (4–20%) SDS–PAGE gels (Invitrogen, Carlsbad, CA). Proteins were transferred to an Immobilon Transfer Membrane (Millipore, Inc.) for 90 min at 90 V. Blots were blocked with 5% (w/v) nonfat dry milk in Tris-buffered saline for 2 h at 22 °C. All antibodies were diluted in 5% (w/v) nonfat dry milk in TBS containing 0.2% Tween 20. The following antibodies and dilutions were used: mouse anti-nicotinamide nucleotide transhydrogenase (NNT; 1:2000; Abnova; Taiwan), mouse anti-b-actin (1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit antib-actin (1:10,000; Lab Vision, Corp., Fremont, CA), rabbit CYP2E1 (1:1500; Chemicon, Temecula, CA), and rabbit anti-N-AcLys (1:2000; Chemicon, Temecula, CA). N-AcLys antibodies were prepared in rabbits using acetylated keyhole limpet hemocyanin as immunogen followed by purification against immobilized acetylated lysine on agarose. Anti-mouse Sirt3 was the kind gift of Dr. Eric Verdin (University of California, San Francisco) and anti-acetylated ACS2 was the kind gift of Dr. John Denu (University of Wisconsin, Madison) [5,6]. Blots were incubated with primary antibodies overnight at 4 °C, washed, and incubated with the appropriate secondary antibody conjugated to horse radish peroxidase for 2 h at 22 °C. Secondary antibody-HRP conjugates used were goat anti-mouse (1:6000; Promega, Madison, WI) and goat anti-rabbit (1:6000; Promega, Madison, WI). Blots were developed with chemiluminescent substrate (Pierce Detection Reagent, Pierce Biotechnology, Inc. Rockford, IL), imaged using a UVP gel documentation system (UVP, Inc., Upland, CA) and the immunoblot signals quantified. For homogenates of whole liver, b-actin was used as a loading control and all target antibody signals were normalized to the b-actin signal. For purified mitochondria, NNT was used a loading control and all target antibody signals were normalized to the NNT signal. Statistical analysis. Analysis of data was performed using Prism 4.0 software (GraphPad, San Diego, CA). One-way ANOVA with Dunnett’s posthoc test or Student’s t-test were used as appropriate. Significance was achieved with p < 0.05. In the immunoblot data presented, each lane is a sample from an individual animal.
Results The specificity of N-AcLys-modified protein detection by immunoblot blot analysis is shown in Fig. 1A. Levels of N-AcLys-modified proteins from control rats were compared to those on the ethanol diet for 6 weeks that underwent withdrawal for 0, 2, or 7 days. The level of N-AcLys signal was elevated over 5.5-fold in rats killed before withdrawal compared to their pair-fed controls (Fig. 1B and C). This level was maintained for at least 2 days following withdrawal. After 7 days, levels of acetylated proteins were not significantly different than controls. Rat exposed for 1 week to the same ethanol
Fig. 1. Multiple proteins are acetylated by ethanol intoxication. (A) Proteins (50 lg each lane) from liver homogenates from a rat exposed to ethanol for 6 weeks (ETOH) and its pair-fed control (C) were analyzed by immunoblot for the presence of N-AcLys-modified proteins b-actin was used as loading control. Specificity of immunoreactivity is demonstrated by loss of signal with preincubation of anti-NAcLys with acetylated BSA (Ac-BSA). (B) Rats were exposed to ethanol for 6 weeks followed by withdrawal for zero (0) days (EW01-3), 2 days (EW2), or 7 days (EW7) followed by removal of the liver. Control animals (C1–3) were pair-fed against their corresponding EW0 counterparts. Proteins (50 lg per lane) were analyzed by immunoblot for the presence of N-AcLys-modified proteins. Each lane is a sample from an individual rat. Blots were stripped and probed with anti-CYP2E1 followed by b-actin, as a loading control. Signal intensity was quantified and the samples compared (C). The effect of time on N-AcLys content and CYP2E1 content was analyzed by one-way ANOVA with Dunnett’s posttest. Ethanol-treated data were compared to the pair-fed control data. *p < 0.05. Data are means ± the standard deviation, n = 3 individual rats.
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diet demonstrated a doubling in protein acetylation (see Supplementary Data). CYP2E1 levels are elevated by ethanol intoxication owing to inhibition of CYP2E1 degradation by ethanol [22]. Thus, change in CYP2E1 content provides one measure against which resolution of ethanol-mediated effects could be compared. In contrast to the prolonged levels of N-AcLys-modified proteins, levels of the ethanol-metabolizing enzyme CYP2E1 were returned to baseline levels within 2 days following withdrawal of ethanol (Fig. 1B and C) consistent with data from other laboratories [22,23]. The data in Fig. 1 demonstrated that N-AcLys-modification did not return to basal levels in the same time frame as return of CYP2E1. CYP2E1 activity contributes to the metabolism of ethanol, hepatic oxidative damage, and ethanol-induced fatty liver [20,24]. Owing to the multiple effects of CYP2E1, it was hypothesized that elevated N-AcLys content was dependent upon CYP2E1. To test this hypothesis, liver homogenates were analyzed from transgenic mice lacking expression of CYP2E1 that were exposed to ethanol for 3 weeks [19]. Both wild-type mice and mice lacking CYP2E1 protein had increases of 3- to 5-fold in N-AcLys-modified protein when exposed to ethanol as compared to control, non-ethanolfed animals (Fig. 2). Mitochondrial protein acetylation has gained a large amount of attention owing to the fact that reversible N-AcLys-modification regulates the function of metabolic enzymes such as ACS2 and GDH-1 [6,7]. Analysis of mitochondrial fractions from control and
Fig. 2. CYP2E1 expression is not requisite for elevated ethanol-induced hepatic protein acetylation. CYP2E1 wild-type (W) and CYP2E1 null (null) animals were exposed to ethanol for 3 weeks prior to sacrifice. Hepatic proteins (50 lg per lane) from ethanol-treated (E) and pair-fed controls (C) were analyzed for N-AcLys content by immunoblot. Lack of CYP2E1 was confirmed by immunoblot. Immunoreactivity of b-actin was used as a loading control. Each lane is a sample from an individual mouse. Immunoreactive signal was quantified and the mean data are presented, n = 2 individual mice.
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ethanol-treated animals demonstrated that ethanol treatment caused a significant elevation (>4.5-fold) in N-AcLys-modified proteins that lasted for at least 2 days after cessation of ethanol (Fig. 3A and B). After 7 days, levels of acetylated proteins were not significantly different than controls. Using an antibody against acetylated form of ACS2, the effect of ethanol intoxication on the mitochondrial content of acetylated ACS2 was examined [6]. Mitochondrial levels of acetylated ACS2 were more than 4-fold higher in rats killed without withdrawal as compared to controls (Fig. 3C and D). Even after 7 days post-withdrawal, levels of acetylated ACS2 were significantly elevated. Decreases in SIRT3 protein may account for the increase in the content of N-AcLys-modified proteins as demonstrated in Sirt3 null mice [5]. Using an antibody raised against murine SIRT3, immunoblot analysis was performed comparing liver mitochondria from rats treated for 6 weeks with ethanol and their pair-fed controls. The data demonstrate that ethanol intoxication does not alter mitochondrial levels of Sirt3 protein (Fig. 4). The data contrast the elevated levels of acetylated ACS2, a substrate protein for Sirt3.
Discussion This current work demonstrates that ethanol intoxication increased the content of N-AcLys-modified proteins in the liver, especially in the mitochondrial compartment. The study by Kim and colleagues suggests that more than 20% of mitochondrial proteins can undergo the acetylation process [4]. Only in two instances so far, ACS2 and GDH-1, is it demonstrated that N-lysyl acetylation affect the activity of mitochondrial proteins [5–7]. In the case of ethanol intoxication, reducing acetyl CoA synthesis may be viewed as a compensatory mechanism of mitochondria. However, experiments studying Sirt3 suggest that unregulated mitochondrial acetylation is harmful. Si-RNA-mediated knockdown of Sirt3 in HEK293 cells renders the cells more vulnerable to the DNA alkylating agent methylmethane sulfonate [25]. In HIB1B brown adipocytes, inhibition of Sirt3 activity, through expression of a dominant negative, inactive Sirt3 enzyme, decreases the expression of the uncoupling protein UCP-1, an effect reversed by expression of PGC-1a [26]. These data suggest that Sirt3 (and by analogy mitochondrial protein acetylation) plays a role in nuclear-mitochondrial signaling. On the other hand, overexpression of Sirt3 (which presumably decreases mitochondrial protein acetylation) increases respiratory rates while reducing reactive oxygen species generation [26]. In the whole animal context, Sirt3 null mice possess elevated acetylation of mitochondrial proteins but do not have overt abnormalities [5]. It may be that hyper-acetylation is pathologic under stressed conditions with multiple factors like those that occur with chronic ethanol intoxication. The consequences of elevated mitochondrial acetylation in the context of chronic ethanol intoxication are not well studied, but the above in vitro evidence suggests that elevated acetylation plays a mechanistic role in the development of ethanol-induced hepatic pathology. The immunoblot data demonstrate that several mitochondrial proteins exhibited elevated N-lysyl acetylation. These same acetylated proteins are present in mitochondria isolated from control rats and suggest that these same proteins are predisposed to acetylation either enzymatically or non-enzymatically. The current immunoblot data using the anti-N-AcLys antibodies likely would not distinguish between different acetylated sequences on the same protein. Furthermore, we are cognizant that the labeled proteins may represent high abundance proteins of which only a few percent are acetylated and vice versa. Multiple pathways acting in concert may be responsible for the ethanol-mediated elevation in N-AcLys-modified proteins
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Fig. 3. Ethanol intoxication increases the content of acetylated proteins in hepatic mitochondria. Rats were exposed to ethanol for 6 weeks followed by withdrawal for zero (0) days (EW0), 2 days (EW2), or 7 days (EW7) followed by isolation of hepatic mitochondria. Control animals (C1–3) were pair-fed against their corresponding EW0 counterparts. Mitochondrial proteins (50 lg per lane for N-AcLys, 100 lg for acetylated ACS2 (Ac-ACS2)) were analyzed for N-AcLys content (A) or acetylated ACS2 (C) content by immunoblot. Each lane is a sample from an individual rat. Samples were normalized to content of NNT. Comparison of N-AcLys immunoreactive signals is shown in (B) and Ac-ACS2 signals shown in (D). The effect of ethanol exposure time on N-AcLys and Ac-ACS2 content was analyzed by one-way ANOVA with Dunnett’s posttest. Ethanoltreated data were compared to the pair-fed control data. *p < 0.05. Data are means ± the standard deviation, n = 3 individual rats.
Fig. 4. Ethanol intoxication does not alter mitochondria Sirt3 content. Rats were exposed to ethanol (ETOH 1–4) or the corresponding pair-fed control diet (C1–4) for 6 weeks followed by isolation of hepatic mitochondria. Mitochondrial protein (50 lg/lane) was analyzed by immunoblot for content of Sirt3 and signal normalized using NNT content. Each lane is a sample from an individual rat. Sirt3 content of ethanol and pair-fed rats was compared using a paired Student’s t-test. Data are means ± the standard deviation, n = 4 individual rats.
including (1) an increase in enzymatic lysyl acetylation activity, (2) an increase in non-enzymatic lysyl acetylation, (3) a decrease in NAD+-dependent sirtuin deacetylase activity, and (4) formation of
acetylated proteins that are not Sirt substrates. To date, a mitochondrial enzyme with protein N-lysyl acetyl transferase activity has not been reported, although non-mitochondrial N-lysyl acetylases exist in cells [27]. Potentially, non-enzymatic lysyl acetylation may occur. The thioester bond of acetyl CoA can potentially react non-enzymatically with the e-amino group of lysine leading to N-lysyl acetylation. Another mechanism of non-enzymatic acetylation involving hydrogen peroxide and acetaldehyde is proposed by Ishino and colleagues [28]. Unlike ethanol intoxication experiments examining Sirt1 and Sirt 5 levels, the current experiments demonstrate that Sirt3 protein levels were not altered by 6 weeks of ethanol exposure [16,18]. Potentially, Sirt3 activity may be diminished by chronic ethanol exposure. Sirts are NAD+-dependent enzymes with KM’s in the millimolar range for NAD+ [8]. Ethanol intoxication rapidly increases the hepatic NADH/NAD+ ratio 3-fold in the cytosol and mitochondria [29]. This is in part due to metabolic shuttling of NADH equivalents from the cytosol (elevated as a result of alcohol dehydrogenase activity) into the mitochondria [30], inhibition of respiration by ethanol exposure [31,32], and production of NADH through aldehyde dehydrogenase activity [33]. CYP2E1 has many roles in the progression of alcohol-mediated liver damage through production of acetaldehyde and generation of hydroxyl radical and ethyl radicals [34]. CYP2E1 contributes to the generation of alcohol-mediated hepatic steatosis [20]. The data presented in our work indicate that CYP2E1 expression is not requisite for elevated N-AcLys content. CYP2E1 levels returned to baseline while protein acetylation remained elevated for several days. Furthermore, CYP2E1 null mice demonstrated elevated levels of protein acetylation when exposed to ethanol. However, these data do not rule out that specific proteins may be affected by CYP2E1-dependent manner.
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In summary, there is a growing body of evidence indicating that ethanol intoxication greatly impacts the N-lysyl acetylation signaling pathway [16–18]. Our data indicate that ethanol intoxication causes an increase in mitochondrial protein acetylation that lasts for days after withdrawal and is independent of Sirt3 expression levels. Subsequent study is needed to determine the physiologic impact of this elevated acetylation and the extent to which it contributes to ethanol-induced toxicity. Acknowledgments This work was supported by a grant from the National Institute on Alcohol Abuse and Alcoholism, AA15145-01. The author thanks Tonya Murphy and Eric Long for their assistance with the animal husbandry and preparation of the samples. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2008.09.039. References [1] T. Yang, M. Fu, R. Pestell, A.A. Sauve, SIRT1 and endocrine signaling, Trends Endocrinol. Metab. 17 (2006) 186–191. [2] T. Yang, A.A. Sauve, NAD metabolism and sirtuins: metabolic regulation of protein deacetylation in stress and toxicity, AAPS J. 8 (2006) E632–E643. [3] X.J. Yang, S. Gregoire, Metabolism, cytoskeleton and cellular signalling in the grip of protein Nepsilon- and O-acetylation, EMBO Rep. 8 (2007) 556–562. [4] S.C. Kim, R. Sprung, Y. Chen, Y. Xu, H. Ball, J. Pei, T. Cheng, Y. Kho, H. Xiao, L. Xiao, N.V. Grishin, M. White, X.J. Yang, Y. Zhao, Substrate and functional diversity of lysine acetylation revealed by a proteomics survey, Mol. Cell 23 (2006) 607–618. [5] D.B. Lombard, F.W. Alt, H.L. Cheng, J. Bunkenborg, R.S. Streeper, R. Mostoslavsky, J. Kim, G. Yancopoulos, D. Valenzuela, A. Murphy, Y. Yang, Y. Chen, M.D. Hirschey, R.T. Bronson, M. Haigis, L.P. Guarente, R.V. Farese Jr., S. Weissman, E. Verdin, B. Schwer, Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation, Mol. Cell. Biol. 27 (2007) 8807–8814. [6] W.C. Hallows, S. Lee, J.M. Denu, Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases, Proc. Natl. Acad. Sci. USA 103 (2006) 10230–10235. [7] B. Schwer, J. Bunkenborg, R.O. Verdin, J.S. Andersen, E. Verdin, Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2, Proc. Natl. Acad. Sci. USA 103 (2006) 10224–10229. [8] M.T. Schmidt, B.C. Smith, M.D. Jackson, J.M. Denu, Coenzyme specificity of Sir2 protein deacetylases: implications for physiological regulation, J. Biol. Chem. 279 (2004) 40122–40129. [9] N. Dali-Youcef, M. Lagouge, S. Froelich, C. Koehl, K. Schoonjans, J. Auwerx, Sirtuins: the ’magnificent seven’, function, metabolism and longevity, Ann. Med. 39 (2007) 335–345. [10] E. Michishita, J.Y. Park, J.M. Burneskis, J.C. Barrett, I. Horikawa, Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins, Mol. Biol. Cell 16 (2005) 4623–4635. [11] H.M. Cooper, J.N. Spelbrink, The human Sirt3 protein deacetylase is exclusively mitochondrial, Biochem. J. (2008). [12] B. Schwer, B.J. North, R.A. Frye, M. Ott, E. Verdin, The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase, J. Cell Biol. 158 (2002) 647–657.
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Ethanol intoxication increases hepatic N-lysyl protein acetylation Matthew J. Picklo Sr. Department of Pharmacology, Physiology, and Therapeutics, University of North Dakota, School of Medicine and Health Sciences, 501 N. Columbia Road, Grand Forks, ND 58203-9037, USA Department of Chemistry, University of North Dakota, Grand Forks, ND 58203-9037, USA
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Article history: Received 8 September 2008 Available online 18 September 2008
Keywords: Sirtuin Acetylation Mitochondria Ethanol CYP2E1
a b s t r a c t The acetylation of the e-amino group of lysine to form N-acetyl lysine (N-AcLys)-modified proteins regulates the activity of metabolic proteins. Because of the multiple effects of ethanol upon hepatic metabolism, it was hypothesized that ethanol exposure increases the hepatic content of N-AcLys-modified proteins. To test this hypothesis, rats or mice were exposed to ethanol using a liquid diet regimen. Content of N-AcLys-modified proteins was elevated more than 5-fold after 6 weeks of ethanol exposure and persisted after ethanol withdrawal. Use of CYP2E1-knockout mice demonstrated that ethanol-induced acetylation was not dependent solely on CYP2E1 expression. The mitochondrial content of N-AcLysmodified proteins was elevated almost 5-fold following 6 weeks of ethanol exposure. Mitochondrial content of the deacetylase Sirt3 was unchanged by 6 weeks of ethanol exposure. These data indicate ethanol intoxication changes the acetylation status of, and likely the activity of, multiple mitochondrial proteins. Ó 2008 Elsevier Inc. All rights reserved.
The N-lysyl acetylation of proteins is a post-translational modification that impacts multiple cellular functions [1–3]. The acetylation of the e-amino group of lysine to form N-acetyl lysine (N-AcLys)-modified proteins affects the function of multiple proteins including histones, p53, and a-tubulin (for review see [3]). Recent data indicate that over 20% of the mitochondrial proteome is subject to N-AcLys modification and that this process is regulated by diet [4,5]. Mitochondrial proteins such as acetyl CoA synthetase 2 (ACS2) and glutamate dehydrogenase-1 (GDH-1) undergo reversible acetylation that regulates enzymatic activity [5–7]. Recent research has focused upon cellular functions of the Silent Information Regulator 2 family of deacetylases, termed sirtuins (Sirt’s). Sirtuin deacetylases are NAD+ dependent [8]. Multiple classes of sirtuins (Sirt1–7) have been characterized that differ in subcellular localization, protein targets, and mechanisms of action (for reviews, see ([9,10]). Sirt1 is localized to the nucleus and Sirt2 is cytoplasmic. Sirt3, Sirt4, and Sirt5 reside in the mitochondria [5,11–13]. Sirt3 and Sirt5 have deacetylase activity whereas Sirt4 ADP-ribosylates proteins [12–15]. Ethanol exposure alters Sirt1 and Sirt5 pathways resulting in altered cellular signaling [16–18]. Ethanol intoxication decreases expression of Sirt5 and while increasing acetylation of p53 and PGC-1a [16]. Ethanol decreases hepatic Sirt1 content and increases the acetylation of PGC1a in animals fed a low fat diet [18]. In contrast, in animals fed diet containing high saturated fat content, ethanol increased Sirt1 levels [17].
E-mail address: [email protected] 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.09.039
Because of the multiple biochemical effects of ethanol upon hepatic metabolism, it was hypothesized that ethanol exposure increases the hepatic content of multiple N-AcLys-modified proteins. To test this hypothesis, animals were exposed to ethanol. After 6 weeks of exposure, hepatic content of N-AcLys-modified proteins was elevated over 5-fold, particularly in the mitochondria. Multiple proteins were hyper-acetylated by ethanol exposure. On the other hand, mitochondrial content of Sirt3, the deacetylase known to regulate the N-lysyl acetylation of multiple mitochondrial proteins, was unchanged by 6 weeks of ethanol exposure. Methods Ethanol exposure. All experimental protocols were in accordance with the NIH guidelines for use of live animals and were approved by the University of North Dakota Institutional Animal Care and Use Committee. Lieber–DeCarli formula (18% protein, 35% fat, and 47% carbohydrate) liquid diet was purchased from BioServ (Frenchtown, NJ). Adult, male Sprague–Dawley rats (250 g) were purchased from Charles River Laboratories (Wilmington, MA). Rats were housed for 1 week on normal solid diet. Animals were then placed on liquid diet without ethanol for 1 week followed by liquid diet containing 36% of total calories from ethanol or control diet in which the ethanol calories were replaced by calories from maltose and dextrin. After 6 weeks, rats receiving the ethanol containing diet were given liquid diet without ethanol for zero (0) days, 2 days, or 7 days before killing. Control, non-ethanol-treated rats were pair-fed against the ethanol-treated animals killed on day zero of ethanol withdrawal. Another group of ethanol-treated rats and their pair-fed controls were treated for 7 days on the diet.
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Rats were deeply anesthetized by i.p. injection of ketamine (100 mg/kg) and xylazine (13 mg/kg). Livers and hearts were quickly removed. A portion of whole liver sample was frozen in liquid nitrogen and stored at 80 °C. In some studies, the remaining portions of liver were used for isolation of mitochondria (see below). Cyp2e1 / mice. Liver homogenates from female cyp2e1 / and female wild-type mice exposed to ethanol or control diet were provided as the kind gift of Professor Arthur Cederbaum, Mt. Sinai Medical Center (New York, NY) [19,20]. Cyp2e1 / and wild-type mice were on the SV/129 background and were generated as described previously [19]. Mice were bred at the Mt. Sinai Medical Center animal facility. At 8–10 weeks of age (16–20 g), mice were exposed to ethanol using a Lieber–DeCarli liquid diet with a gradient dosing of ethanol from 10% of caloric intake to a final of 35% of caloric over intake over 2 weeks followed by 1 week at 35% of caloric intake [20]. Pair-fed controls received dextrose instead of ethanol. Homogenates were prepared in ice-cold 0.15 M KCl as described previously and frozen at 80 °C [20]. Mitochondrial isolation. Mitochondria were isolated using differential centrifugation in a sucrose/mannitol buffer as described previously [21]. Mitochondria were aliquoted and frozen at 80 °C. Immunoblotting. Whole liver samples were homogenized in buffer consisting of sodium phosphate (20 mM, final pH 7.2 at 4 °C), 0.2 % Triton X-100, 1 mM DEPA, 250 lM BHT, and mammalian protease inhibitor cocktail (Sigma, St. Louis, MO) diluted according to manufacturer’s instructions. The homogenate was sonicated, centrifuged at 10,000g for 10 min at 4 °C to pellet any insoluble material, and the supernatant frozen at 80 °C. Proteins from purified mitochondria or whole liver homogenates were separated on gradient (4–20%) SDS–PAGE gels (Invitrogen, Carlsbad, CA). Proteins were transferred to an Immobilon Transfer Membrane (Millipore, Inc.) for 90 min at 90 V. Blots were blocked with 5% (w/v) nonfat dry milk in Tris-buffered saline for 2 h at 22 °C. All antibodies were diluted in 5% (w/v) nonfat dry milk in TBS containing 0.2% Tween 20. The following antibodies and dilutions were used: mouse anti-nicotinamide nucleotide transhydrogenase (NNT; 1:2000; Abnova; Taiwan), mouse anti-b-actin (1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit antib-actin (1:10,000; Lab Vision, Corp., Fremont, CA), rabbit CYP2E1 (1:1500; Chemicon, Temecula, CA), and rabbit anti-N-AcLys (1:2000; Chemicon, Temecula, CA). N-AcLys antibodies were prepared in rabbits using acetylated keyhole limpet hemocyanin as immunogen followed by purification against immobilized acetylated lysine on agarose. Anti-mouse Sirt3 was the kind gift of Dr. Eric Verdin (University of California, San Francisco) and anti-acetylated ACS2 was the kind gift of Dr. John Denu (University of Wisconsin, Madison) [5,6]. Blots were incubated with primary antibodies overnight at 4 °C, washed, and incubated with the appropriate secondary antibody conjugated to horse radish peroxidase for 2 h at 22 °C. Secondary antibody-HRP conjugates used were goat anti-mouse (1:6000; Promega, Madison, WI) and goat anti-rabbit (1:6000; Promega, Madison, WI). Blots were developed with chemiluminescent substrate (Pierce Detection Reagent, Pierce Biotechnology, Inc. Rockford, IL), imaged using a UVP gel documentation system (UVP, Inc., Upland, CA) and the immunoblot signals quantified. For homogenates of whole liver, b-actin was used as a loading control and all target antibody signals were normalized to the b-actin signal. For purified mitochondria, NNT was used a loading control and all target antibody signals were normalized to the NNT signal. Statistical analysis. Analysis of data was performed using Prism 4.0 software (GraphPad, San Diego, CA). One-way ANOVA with Dunnett’s posthoc test or Student’s t-test were used as appropriate. Significance was achieved with p < 0.05. In the immunoblot data presented, each lane is a sample from an individual animal.
Results The specificity of N-AcLys-modified protein detection by immunoblot blot analysis is shown in Fig. 1A. Levels of N-AcLys-modified proteins from control rats were compared to those on the ethanol diet for 6 weeks that underwent withdrawal for 0, 2, or 7 days. The level of N-AcLys signal was elevated over 5.5-fold in rats killed before withdrawal compared to their pair-fed controls (Fig. 1B and C). This level was maintained for at least 2 days following withdrawal. After 7 days, levels of acetylated proteins were not significantly different than controls. Rat exposed for 1 week to the same ethanol
Fig. 1. Multiple proteins are acetylated by ethanol intoxication. (A) Proteins (50 lg each lane) from liver homogenates from a rat exposed to ethanol for 6 weeks (ETOH) and its pair-fed control (C) were analyzed by immunoblot for the presence of N-AcLys-modified proteins b-actin was used as loading control. Specificity of immunoreactivity is demonstrated by loss of signal with preincubation of anti-NAcLys with acetylated BSA (Ac-BSA). (B) Rats were exposed to ethanol for 6 weeks followed by withdrawal for zero (0) days (EW01-3), 2 days (EW2), or 7 days (EW7) followed by removal of the liver. Control animals (C1–3) were pair-fed against their corresponding EW0 counterparts. Proteins (50 lg per lane) were analyzed by immunoblot for the presence of N-AcLys-modified proteins. Each lane is a sample from an individual rat. Blots were stripped and probed with anti-CYP2E1 followed by b-actin, as a loading control. Signal intensity was quantified and the samples compared (C). The effect of time on N-AcLys content and CYP2E1 content was analyzed by one-way ANOVA with Dunnett’s posttest. Ethanol-treated data were compared to the pair-fed control data. *p < 0.05. Data are means ± the standard deviation, n = 3 individual rats.
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diet demonstrated a doubling in protein acetylation (see Supplementary Data). CYP2E1 levels are elevated by ethanol intoxication owing to inhibition of CYP2E1 degradation by ethanol [22]. Thus, change in CYP2E1 content provides one measure against which resolution of ethanol-mediated effects could be compared. In contrast to the prolonged levels of N-AcLys-modified proteins, levels of the ethanol-metabolizing enzyme CYP2E1 were returned to baseline levels within 2 days following withdrawal of ethanol (Fig. 1B and C) consistent with data from other laboratories [22,23]. The data in Fig. 1 demonstrated that N-AcLys-modification did not return to basal levels in the same time frame as return of CYP2E1. CYP2E1 activity contributes to the metabolism of ethanol, hepatic oxidative damage, and ethanol-induced fatty liver [20,24]. Owing to the multiple effects of CYP2E1, it was hypothesized that elevated N-AcLys content was dependent upon CYP2E1. To test this hypothesis, liver homogenates were analyzed from transgenic mice lacking expression of CYP2E1 that were exposed to ethanol for 3 weeks [19]. Both wild-type mice and mice lacking CYP2E1 protein had increases of 3- to 5-fold in N-AcLys-modified protein when exposed to ethanol as compared to control, non-ethanolfed animals (Fig. 2). Mitochondrial protein acetylation has gained a large amount of attention owing to the fact that reversible N-AcLys-modification regulates the function of metabolic enzymes such as ACS2 and GDH-1 [6,7]. Analysis of mitochondrial fractions from control and
Fig. 2. CYP2E1 expression is not requisite for elevated ethanol-induced hepatic protein acetylation. CYP2E1 wild-type (W) and CYP2E1 null (null) animals were exposed to ethanol for 3 weeks prior to sacrifice. Hepatic proteins (50 lg per lane) from ethanol-treated (E) and pair-fed controls (C) were analyzed for N-AcLys content by immunoblot. Lack of CYP2E1 was confirmed by immunoblot. Immunoreactivity of b-actin was used as a loading control. Each lane is a sample from an individual mouse. Immunoreactive signal was quantified and the mean data are presented, n = 2 individual mice.
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ethanol-treated animals demonstrated that ethanol treatment caused a significant elevation (>4.5-fold) in N-AcLys-modified proteins that lasted for at least 2 days after cessation of ethanol (Fig. 3A and B). After 7 days, levels of acetylated proteins were not significantly different than controls. Using an antibody against acetylated form of ACS2, the effect of ethanol intoxication on the mitochondrial content of acetylated ACS2 was examined [6]. Mitochondrial levels of acetylated ACS2 were more than 4-fold higher in rats killed without withdrawal as compared to controls (Fig. 3C and D). Even after 7 days post-withdrawal, levels of acetylated ACS2 were significantly elevated. Decreases in SIRT3 protein may account for the increase in the content of N-AcLys-modified proteins as demonstrated in Sirt3 null mice [5]. Using an antibody raised against murine SIRT3, immunoblot analysis was performed comparing liver mitochondria from rats treated for 6 weeks with ethanol and their pair-fed controls. The data demonstrate that ethanol intoxication does not alter mitochondrial levels of Sirt3 protein (Fig. 4). The data contrast the elevated levels of acetylated ACS2, a substrate protein for Sirt3.
Discussion This current work demonstrates that ethanol intoxication increased the content of N-AcLys-modified proteins in the liver, especially in the mitochondrial compartment. The study by Kim and colleagues suggests that more than 20% of mitochondrial proteins can undergo the acetylation process [4]. Only in two instances so far, ACS2 and GDH-1, is it demonstrated that N-lysyl acetylation affect the activity of mitochondrial proteins [5–7]. In the case of ethanol intoxication, reducing acetyl CoA synthesis may be viewed as a compensatory mechanism of mitochondria. However, experiments studying Sirt3 suggest that unregulated mitochondrial acetylation is harmful. Si-RNA-mediated knockdown of Sirt3 in HEK293 cells renders the cells more vulnerable to the DNA alkylating agent methylmethane sulfonate [25]. In HIB1B brown adipocytes, inhibition of Sirt3 activity, through expression of a dominant negative, inactive Sirt3 enzyme, decreases the expression of the uncoupling protein UCP-1, an effect reversed by expression of PGC-1a [26]. These data suggest that Sirt3 (and by analogy mitochondrial protein acetylation) plays a role in nuclear-mitochondrial signaling. On the other hand, overexpression of Sirt3 (which presumably decreases mitochondrial protein acetylation) increases respiratory rates while reducing reactive oxygen species generation [26]. In the whole animal context, Sirt3 null mice possess elevated acetylation of mitochondrial proteins but do not have overt abnormalities [5]. It may be that hyper-acetylation is pathologic under stressed conditions with multiple factors like those that occur with chronic ethanol intoxication. The consequences of elevated mitochondrial acetylation in the context of chronic ethanol intoxication are not well studied, but the above in vitro evidence suggests that elevated acetylation plays a mechanistic role in the development of ethanol-induced hepatic pathology. The immunoblot data demonstrate that several mitochondrial proteins exhibited elevated N-lysyl acetylation. These same acetylated proteins are present in mitochondria isolated from control rats and suggest that these same proteins are predisposed to acetylation either enzymatically or non-enzymatically. The current immunoblot data using the anti-N-AcLys antibodies likely would not distinguish between different acetylated sequences on the same protein. Furthermore, we are cognizant that the labeled proteins may represent high abundance proteins of which only a few percent are acetylated and vice versa. Multiple pathways acting in concert may be responsible for the ethanol-mediated elevation in N-AcLys-modified proteins
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Fig. 3. Ethanol intoxication increases the content of acetylated proteins in hepatic mitochondria. Rats were exposed to ethanol for 6 weeks followed by withdrawal for zero (0) days (EW0), 2 days (EW2), or 7 days (EW7) followed by isolation of hepatic mitochondria. Control animals (C1–3) were pair-fed against their corresponding EW0 counterparts. Mitochondrial proteins (50 lg per lane for N-AcLys, 100 lg for acetylated ACS2 (Ac-ACS2)) were analyzed for N-AcLys content (A) or acetylated ACS2 (C) content by immunoblot. Each lane is a sample from an individual rat. Samples were normalized to content of NNT. Comparison of N-AcLys immunoreactive signals is shown in (B) and Ac-ACS2 signals shown in (D). The effect of ethanol exposure time on N-AcLys and Ac-ACS2 content was analyzed by one-way ANOVA with Dunnett’s posttest. Ethanoltreated data were compared to the pair-fed control data. *p < 0.05. Data are means ± the standard deviation, n = 3 individual rats.
Fig. 4. Ethanol intoxication does not alter mitochondria Sirt3 content. Rats were exposed to ethanol (ETOH 1–4) or the corresponding pair-fed control diet (C1–4) for 6 weeks followed by isolation of hepatic mitochondria. Mitochondrial protein (50 lg/lane) was analyzed by immunoblot for content of Sirt3 and signal normalized using NNT content. Each lane is a sample from an individual rat. Sirt3 content of ethanol and pair-fed rats was compared using a paired Student’s t-test. Data are means ± the standard deviation, n = 4 individual rats.
including (1) an increase in enzymatic lysyl acetylation activity, (2) an increase in non-enzymatic lysyl acetylation, (3) a decrease in NAD+-dependent sirtuin deacetylase activity, and (4) formation of
acetylated proteins that are not Sirt substrates. To date, a mitochondrial enzyme with protein N-lysyl acetyl transferase activity has not been reported, although non-mitochondrial N-lysyl acetylases exist in cells [27]. Potentially, non-enzymatic lysyl acetylation may occur. The thioester bond of acetyl CoA can potentially react non-enzymatically with the e-amino group of lysine leading to N-lysyl acetylation. Another mechanism of non-enzymatic acetylation involving hydrogen peroxide and acetaldehyde is proposed by Ishino and colleagues [28]. Unlike ethanol intoxication experiments examining Sirt1 and Sirt 5 levels, the current experiments demonstrate that Sirt3 protein levels were not altered by 6 weeks of ethanol exposure [16,18]. Potentially, Sirt3 activity may be diminished by chronic ethanol exposure. Sirts are NAD+-dependent enzymes with KM’s in the millimolar range for NAD+ [8]. Ethanol intoxication rapidly increases the hepatic NADH/NAD+ ratio 3-fold in the cytosol and mitochondria [29]. This is in part due to metabolic shuttling of NADH equivalents from the cytosol (elevated as a result of alcohol dehydrogenase activity) into the mitochondria [30], inhibition of respiration by ethanol exposure [31,32], and production of NADH through aldehyde dehydrogenase activity [33]. CYP2E1 has many roles in the progression of alcohol-mediated liver damage through production of acetaldehyde and generation of hydroxyl radical and ethyl radicals [34]. CYP2E1 contributes to the generation of alcohol-mediated hepatic steatosis [20]. The data presented in our work indicate that CYP2E1 expression is not requisite for elevated N-AcLys content. CYP2E1 levels returned to baseline while protein acetylation remained elevated for several days. Furthermore, CYP2E1 null mice demonstrated elevated levels of protein acetylation when exposed to ethanol. However, these data do not rule out that specific proteins may be affected by CYP2E1-dependent manner.
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In summary, there is a growing body of evidence indicating that ethanol intoxication greatly impacts the N-lysyl acetylation signaling pathway [16–18]. Our data indicate that ethanol intoxication causes an increase in mitochondrial protein acetylation that lasts for days after withdrawal and is independent of Sirt3 expression levels. Subsequent study is needed to determine the physiologic impact of this elevated acetylation and the extent to which it contributes to ethanol-induced toxicity. Acknowledgments This work was supported by a grant from the National Institute on Alcohol Abuse and Alcoholism, AA15145-01. The author thanks Tonya Murphy and Eric Long for their assistance with the animal husbandry and preparation of the samples. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2008.09.039. References [1] T. Yang, M. Fu, R. Pestell, A.A. Sauve, SIRT1 and endocrine signaling, Trends Endocrinol. Metab. 17 (2006) 186–191. [2] T. Yang, A.A. Sauve, NAD metabolism and sirtuins: metabolic regulation of protein deacetylation in stress and toxicity, AAPS J. 8 (2006) E632–E643. [3] X.J. Yang, S. Gregoire, Metabolism, cytoskeleton and cellular signalling in the grip of protein Nepsilon- and O-acetylation, EMBO Rep. 8 (2007) 556–562. [4] S.C. Kim, R. Sprung, Y. Chen, Y. Xu, H. Ball, J. Pei, T. Cheng, Y. Kho, H. Xiao, L. Xiao, N.V. Grishin, M. White, X.J. Yang, Y. Zhao, Substrate and functional diversity of lysine acetylation revealed by a proteomics survey, Mol. Cell 23 (2006) 607–618. [5] D.B. Lombard, F.W. Alt, H.L. Cheng, J. Bunkenborg, R.S. Streeper, R. Mostoslavsky, J. Kim, G. Yancopoulos, D. Valenzuela, A. Murphy, Y. Yang, Y. Chen, M.D. Hirschey, R.T. Bronson, M. Haigis, L.P. Guarente, R.V. Farese Jr., S. Weissman, E. Verdin, B. Schwer, Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation, Mol. Cell. Biol. 27 (2007) 8807–8814. [6] W.C. Hallows, S. Lee, J.M. Denu, Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases, Proc. Natl. Acad. Sci. USA 103 (2006) 10230–10235. [7] B. Schwer, J. Bunkenborg, R.O. Verdin, J.S. Andersen, E. Verdin, Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2, Proc. Natl. Acad. Sci. USA 103 (2006) 10224–10229. [8] M.T. Schmidt, B.C. Smith, M.D. Jackson, J.M. Denu, Coenzyme specificity of Sir2 protein deacetylases: implications for physiological regulation, J. Biol. Chem. 279 (2004) 40122–40129. [9] N. Dali-Youcef, M. Lagouge, S. Froelich, C. Koehl, K. Schoonjans, J. Auwerx, Sirtuins: the ’magnificent seven’, function, metabolism and longevity, Ann. Med. 39 (2007) 335–345. [10] E. Michishita, J.Y. Park, J.M. Burneskis, J.C. Barrett, I. Horikawa, Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins, Mol. Biol. Cell 16 (2005) 4623–4635. [11] H.M. Cooper, J.N. Spelbrink, The human Sirt3 protein deacetylase is exclusively mitochondrial, Biochem. J. (2008). [12] B. Schwer, B.J. North, R.A. Frye, M. Ott, E. Verdin, The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase, J. Cell Biol. 158 (2002) 647–657.
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