Original Article
Acta Cardiol Sin 2014;30:67-73
Basic
Rosuvastatin Modulates the Post-Translational Acetylome in Endothelial Cells Ming Chung Lin,1,2 Chung Hsi Hsing,3 Fu An Li,4 Chien Hsing Wu,5 Yaw Syan Fu,6 Jen Kun Cheng7 and Bin Huang6
Background: Statins are lipid-lowering drugs that can simultaneously evoke pleiotropic effects on cardioprotection, vasodilation, and diabetes prevention. Recently, statins have been reported to be able to activate the AMPactivated protein kinase, thereby up-regulating sirtuin (SIRT) that functions as non-histone deacetylases. Therefore, it is essential to investigate the post-translational acetylome that might explain the mechanism of statinmodulated pleiotropic effects. Method: Endothelial cells EAhy 926 treated with rosuvastatin were used to monitor the expression of SIRTs proteins. The protein lysates of both mock- and rosuvastatin-treated cells were further separated by twodimensional gel electrophoresis coupled with western blotting analysis. The significantly changed acetylcontaining proteins detected by using an anti-acetyl lysine antibody were collected from another preparative gel for mass spectrometric assay to identify the acetylated site in the proteins. Results: Rosuvastatin treatment was shown to increase the SIRT1 expression when compared with SIRT2. Among 100 detected proteins with acetylated signal, 12 showed an increased level of acetylation, whereas 6 showed a decreased level of acetylation (deacetylation). The acetylated lysine (K) sites of 3 heat shock proteins, i.e., HSP47/K165, HSP70/K380, and heat shock-inducible protein/K417, were determined. We also found that beta-filamin, elongation factor, galectin and hCG22067 have 2 acetylated lysine sites in their peptide sequences. These dynamic acetylations might alter the protein’s function and are thought to be important in regulating statin-mediated pleiotropic effect. Conclusions: Our study provided a feasible methodology for detecting acetylated proteins. This acetylome information may be utilized to explain, at least partially, the mechanisms of statin-derived pleiotropic effects.
Key Words:
Acetylation/deacetylation · Acetylome · Endothelial cell · Proteomics · Rosuvastatin · Sirtuin
INTRODUCTION Received: January 12, 2013 Accepted: February 6, 2013 1 Department of Anesthesiology, Chi Mei Medical Center; 2Department of Medical Laboratory Science and Biotechnology, Chung Hwa University of Medical Technology; 3Department of Anesthesiology, Chi Mei Medical Center, Tainan; 4Institute of Biomedical Science, Academia Sinica, Taipei; 5Division of Nephrology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine; 6Department of Biomedical Science and Environmental Biology, College of Life Science, Kaohsiung Medical University, Kaohsiung; 7Department of Anesthesiology, Mackay Memorial Hospital, Taipei, Taiwan. Address correspondence and reprint requests to: Dr. Bin Huang, Department of Biomedical Science and Environmental Biology, College of Life Science, Kaohsiung Medical University, No. 100, Shihchuan 1st Rd., San Ming District, Kaohsiung 80708, Taiwan. Tel: 886-7-312-1101 ext. 2704; Fax: 886-7-322-7508; E-mail: huangpin2 @yahoo.com.tw
Statins, or 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, were originally developed to reduce the level of total blood cholesterol. In the past decades, statin-derived pleiotropic effects have been reported that include improved endothelial functions, reduced platelet aggregation, and stabilized atherosclerotic plaques; however, the mechanism underlying these pleiotropic effects remains insufficiently investigated.1-3 Post-translational modifications (PTMs) of proteins are considered important processes in signal transduction and physiological responses.4 Statin-regulated PTMs such as S-nitrosylation and phosphorylation that 67
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lead to their pleiotropic effects have been previously described.5-7 Recently, the patterns of lysine acetylation and deacetylation in proteins, also known as the acetylome, have been regarded as a new means by which protein function is modulated.8 The acetylation of lysine residues was originally described to play a role in the histone protein modulation of gene expression. However, an increasing number of studies are focusing on non-histone proteins that have altered physiological functions following acetylation.9,10 To date, over 60 transcription factors and 133 mitochondrial proteins have been identified as being acetylated.11,12 The effects of acetylation on protein function have also been demonstrated. For example, acetylation on lysine 221 (K 221 ) and K 310 in NF-kB is associated with enhanced transcription of its target genes.13 The enzymatic activity of protein kinase ATM increases after protein acetylation, which results in decreased enzymatic activity of PTEM, Mdm2, and acetyl-CoA synthetase. 9 Concurrent with these findings, the protein acetylome can be considered to play an increasingly important role in the adaptation of cells to different physiological responses. The sirtuin (SIRT) protein family contains 7 members and shares a high level of homology with the yeast silent information regulator 2 (Sir2) protein, which functions in the NAD-dependent deacetylation of histones in the regulation of gene expression. These sirtuins occupy different subcellular compartments, such as the nucleus (SIRT1, 2, 6, and 7), cytoplasm (SIRT1 and 2), and the mitochondrion (SIRT3, 4, and 5). 14 Unlike the known deacetylases, SIRT-mediated deacetylation occurs only on the lysine residues of proteins. The deacetylation of non-histone proteins has gained increasing recognition, as these proteins play essential roles in regulating cellular signaling and homeostasis.15,16 The published studies found that activation of SIRT1 has a therapeutic effect during excessive ROS production. 17 SIRT2 has been suggested to increase resistance to oxidative stress through deacetylation of downstream proteins such as forkhead box O proteins (FOXOs).18,19 These findings revealed the possible critical involvement of SIRT1 and SIRT2 in the response to oxidative stress. Rosuvastatin is a newly designed statin that possesses the lowest inhibition coefficient (IC50 = 5.4 nM) when compared with other statins.20 Recently, studies were performed on statin-induced post-translational Acta Cardiol Sin 2014;30:67-73
modifications, such as phosphorylation and S-nitrosylation, that might be involved in pleiotropic effects.6,21 In terms of acetylation, statins increase cyclin-dependent kinase inhibitor p21 gene expression through inhibition of histone deacetylase activity and release of promoter-associated HDAC1/2.22 Rosuvastatin induces acetylation of histones H3 and H4 and C-C chemokine receptor type 7 gene expression, which promotes macrophage migration out of plaques and reduces atherosclerosis. 23 Clinical studies have shown that statins markedly decrease miR-34a levels and increase SIRT1 levels, which possibly contributes to the beneficial effects of statins on endothelial cell function.24 In the present study, ECs treated with rosuvastatin specifically increased the protein levels of SIRT1 as compared to SIRT2. Using two-dimensional gel electrophoresis (2-DE) analysis, we identified 16 proteins with differential levels of lysine acetylation and deacetylation. These proteins will be further studied to elucidate the effect of the acetylome on the function of each protein and subsequently the mechanism of statinregulated pleiotropic effects.
MATERIALS AND METHODS Cell culture The endothelial cell line EAhy 926 was kindly donated by Cora-Jean S. Edgell, University of North Carolina, Chapel Hill. ECs were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with fetal bovine serum (FBS, 10%), streptomycin (100 mg/mL), and penicillin (100 U/mL). The medium was then replaced with DMEM medium containing 2% FBS, and the ECs were incubated overnight prior to treatment with rosuvastatin (AstraZeneca, Taipei, Taiwan). The antibodies for SIRT1 and SIRT2 were purchased from Sigma-Aldrich (St. Louis, MO, USA), and the antibody against anti-lysine acetylation was obtained from Calbiochem (San Diego, CA, USA). Protein extraction and western blotting analysis After rosuvastatin treatment, the ECs were washed with cord buffer: NaCl (0.14 M), KCl (4 mM), glucose (11 mM), HEPES (10 mM, pH 7.4) and then lysed with 300 mL of lysis buffer: HEPES (250 mM, pH 7.7), EDTA (1 mM), neocuproine (0.1 mM), and CHAPS (0.4%, w/v). 68
Rosuvastatin Modulates Acetylome
After centrifugation at 10000 ´ g for 10 min at 4 °C, the protein supernatant was collected, and protein concentrations were determined using the BCA assay reagent (Thermo Fisher Scientific Inc., Rockford, IL, USA). Forty micrograms of cell lysate was mixed with SDS-PAGE sample buffer: Tris-HCl (62.5 mM, pH 6.8), SDS (3%, w/v), 2-mercaptoethanol (5%, v/v), and glycerol (10%, v/v), and then separated by SDS-PAGE. The blotted membranes were hybridized with either anti-SIRT1 (1:2,000) or anti-SIRT2 (1:3,000) antibodies to determine the level of protein expression.
films. The images on X-ray films were scanned using a digital scanner (Microtek International Inc.), and the results represent the level of post-translational acetylation. The acetylation levels were calculated using the Progenesis Samespots v2.0 software (NonLinear Dynamics, Newcastle, UK).
Mass spectrometric (MS) analysis The excised gel slices were digested with trypsin for 4 h at 37 °C (In-Gel Tryptic Digestion Kit, Thermo Fisher Scientific) to allow the subsequent identification of peptides by mass spectrometry (MS). The tryptic peptides were desalted on a Proteomics C18 Column (Mass Solution Ltd., Taipei, Taiwan) and then subjected to MS analysis using an nLC/Q-TOF (Micromass, Manchester, UK). MS data were used to search against entries in the National Center for Biotechnology Information (NCBI) database with a MASCOT in-house search program (Matrix Science, London, UK). In addition, peptides that contained acetylated lysine sites were determined. The search parameters were set as follows: mass values: monoisotopic; protein mass: unrestricted; peptide mass tolerance: ± 0.4 Da; fragment mass tolerance: ± 0.4 Da; max missed cleavages: 1; variable modification: acetylation in lysine, carbamidomethylation in cysteine, and oxidation in methionine.
Two-dimensional gel electrophoresis (2-DE) and lysine acetylome detection The protein lysates obtained from cells treated with either mock or rosuvastatin (10 mM) for 24 h were precipitated in ice-cold acetone at -20 °C. After centrifugation, the protein pellets were air-dried and then dissolved in sample buffer: urea (9 M), CHAPS (2%, w/v), DL-dithiothreitol (60 mM), and IPG solution (2%, v/v, pH 4-7; GE Healthcare, Piscataway, NJ, USA) at room temperature and shaken for 1 h to ensure that the proteins were thoroughly denatured. The lysates were mixed with rehydration buffer: urea (8 M), CHAPS (2%, w/v), IPG solution (0.5%, v/v, pH 4-7), and DL-dithiothreitol (30 mM) to reach a final volume of 340 mL. The samples were then soaked into an 18-cm DryStrip (pH 4-7, GE Healthcare) for up to 12 h on the Ettan IPGphor system (GE Healthcare), followed by isoelectric focusing (IEF) at an accumulated voltage of 60 kV/h. The strip gels were then incubated with equilibration buffer: SDS (2%, w/v), Tris-HCl (50 mM, pH 8.8), urea (6 M), glycerol (30%, v/v), and DL-dithiothreitol (60 mM) for 20 min, and then equilibrated with the same buffer containing iodoacetic acid (IAA, 135 mM) for an additional 20 min. The equilibrated IEF strip was laid on the top of a vertical SDSPAGE apparatus to separate the proteins. After 2-DE, the gels were stained using the VisPRO dye and scanned using a digital scanner (Microtek International Inc., Hsinchu, Taiwan). This obtained image represents the translational level of total proteins. After de-staining, the gels were blotted onto a nitrocellulose membrane and hybridized with an anti-acetyl lysine monoclonal antibody (1:500; Cell Signaling Tech., Boston, MA, USA). The membranes were developed with the SuperSignal West Femto reagent (Thermo Fisher Scientific) on X-ray
RESULTS Increased SIRT1 protein expression after rosuvastatin treatment As shown in Figure 1, the expression levels of SIRT1 and SIRT2 were investigated in dose- and time-dependent manners. Treatment of ECs with 10 mM rosuvastatin resulted in a significant (> 3 fold) increase in the SIRT1 protein level compared with that observed after mock treatment from 6 h to 24 h. However, the SIRT2 protein level did not change under similar conditions. The concentration of rosuvastatin applied in the present study is similar to previous studies.6 Rosuvastatin regulation of the acetylome According to the flowchart of Figure 2, the total protein lysates from mock- and rosuvastatin-treated cells were separated by 2-DE; the images obtained after 69
Acta Cardiol Sin 2014;30:67-73
Ming Chung Lin et al.
A
B
C
D
Figure 2. An experimental flowchart for the detection of rosuvastatin-modulated acetylome. After rosuvastatin treatment, the cell lysates were separated by 2-DE. The gels were stained with VisPRO dye to detect the levels of translational proteins. After reversible destaining using SDS-PAGE running buffer, the gels were subsequently subjected to western blotting analyses using an anti-acetyl-lysine antibody to obtain post-translational acetylation patterns. The proteins exhibited equal translational levels, but significantly changed acetylation levels were found in another 2-DE gel with rosuvastatin-treated samples, which were analyzed by mass spectrometry.
Figure 1. Sirtuin expression levels during treatment with rosuvastatin. (A) Endothelial cells were treated with a series dilution of rosuvastatin (1, 10 and 100 mM) to determine the optimal concentration applied in the experiment. Forty micrograms of protein lysates were separated by SDS-PAGE. Western blotting studies were performed using monoclonal antibodies against SIRT1 (1:2,000) and SIRT2 (1:3,000). (B) The relative fold-changes of protein levels were statistically calculated from 3 repeated experiments and the relative fold-changes are shown as the mean ± S.E. compared to control treatments. The changes were individually compared to the control by Fisher’s LSD for significant difference (p < 0.05). (C, D) For time-dependent protein expression, cells were treated with 10 mM rosuvastatin for 0.5, 6, and 24 h. The expression level of proteins was also statistically calculated.
with altered acetylation status, peptides containing acetyl lysine residues were also determined by tandem MS (MS/MS). Four proteins were found to contain 2 different acetylated lysine residues, and the other 14 proteins were identified to contain only 1 acetylated lysine residue (Table 1). Moreover, because trypsin can only digest those lysine residues that are not acetylated, the same peptides with or without acetylation can be specifically distinguished. These proteins are mostly cytoskeletal proteins and transcription factors.
VisPRO staining were used to determine the levels of translational proteins. Meanwhile, the post-translational acetylation levels were determined by subsequent western blotting using an anti-acetyl lysine antibody. Despite the detection of > 100 acetylated proteins from the control (126 ± 20) and rosuvastatin (142 ± 22) treatment groups, only approximately 15% of proteins showed a significant change in their acetylation status. We found 12 proteins with enhanced acetylation status (> 1.5-fold in protein spot density) and 6 proteins with decreased acetylation status (deacetylation, < 0.5-fold in protein spot density) (Figure 3). These proteins are annotated as heat shock response (EA3, EA5 and EA10), cell proliferation (EA1, EA4, EA6, EA11, DA1 and DA4) and protein expression (EA8 and DA3). In addition, these proteins showed no difference at the translational levels, indicating that the rosuvastatin-regulated acetylation/deacetylation process was actually a posttranslational event (Figure 3).
DISCUSSION Attempts have previously been made to explain the mechanisms underlying statin-derived pleiotropic effects in terms of post-translational modification. In addition to our previous study on the S-nitrosoproteome, in which we proposed a possible mechanism of nitric oxide generating pleiotropic effects, 6 we examined acetylome in the present study as an alternative mechanism for this pleiotropic effect. The most prominent benefit of 2-DE-based proteomics is that the translational and post-translational levels of proteins can be observed simultaneously. Therefore, post-translational evidence of these modifications can be clearly defined.
Determination of acetylated lysine residues In addition to the identification of the 18 proteins Acta Cardiol Sin 2014;30:67-73
70
Rosuvastatin Modulates Acetylome
A
B
Figure 3. Quantification of the rosuvastatin-modulated acetylome. (A) After incubation with 10 mM rosuvastatin for 24 h, 1 mg of protein lysates were separated by 2-DE. The gels were stained with VisPRO (VisPRO panel) prior to western blotting with a mouse anti-acetyl-lysine monoclonal antibody (1:500). The arrowheads indicate proteins whose expression levels are significantly increased/decreased (western blot panel). The corresponding protein spots on the VisPRO-stained gels were subjected to MS analysis. DA, decreased acetylation; EA, enhanced acetylation. (B) The normalized statistical data show the comparison of the translational (VisPRO) and post-translational levels (western blot), which demonstrated that rosuvastatin-regulated protein acetylation is a post-translational event. The data are shown as the mean ± S.E. values from 3 experiments, and the relative fold-changes are indicated under the statistic bar.
The levels of SIRT1, the most studied deacetylase in recent years, were significantly enhanced by rosuvastatin treatment from 6 h to 24 h (Figure 1). In contrast to the prediction that the amount of acetylated protein should decrease, a higher number of acetylated proteins was observed in the 2-DE assay. This implies that the rosuvastatin-regulated cellular acetylome might not act singly through SIRT1. Several of the identified proteins, when characterized, exhibited a close relationship with acetylomemediated physiologies. Heat shock proteins (HSPs), for example, are known to play important roles in cell homeostasis.25 The effects of lysine (Lys, K) acetylation on HSPs function have mostly been evaluated in HSP90. The deacetylation of HSP90 attenuates ATP-binding capacity, and thus involves a chaperone function, which regulates cancer cell invasion.26 In the present study, 3 HSPs (collagen (HSP47)/K 165 , HSP70/K 380 , and heat shock-inducible protein/K417) and their acetylated lysine sites were separately identified. Because HSPs have been shown to be important in modulating statinderived endothelial homeostasis, 27 post-translational
acetylation might play a role in the early regulatory functions of these HSPs. Therefore, more evidence in support of this hypothesis should be collected through future studies. The elongation factor is important in protein synthesis via promoting the binding of acryl-tRNA to ribosomes in a GTP-dependent manner. The activity of this elongation factor has recently been reported to be modulated by post-translational modifications such as phosphorylation, S-nitrosylation, and acetylation.6,28 In this study, elongation factor 1 alpha 1, also named elongation factor Tu (EF-Tu), was identified to be acetylated on residues K79 and K408 after rosuvastatin treatment. However, there is little information regarding the modulation of EF-Tu activity by acetylation. Therefore, whether acetylation/deacetylation of EF-Tu regulates downstream protein synthesis still needs to be elucidated. Despite the conflicting results that showed that fewer deacetylated proteins were detected under the condition of increased expression levels of SIRT1 deacetylase, the putative functions of these deacetylated proteins still deserve further discussion. EPB41L3 is an 71
Acta Cardiol Sin 2014;30:67-73
Ming Chung Lin et al. Table 1. Acetylated proteins and lysine residues identified by nLC-MS/MS Spot No(a)
Protein name(b)
Ac No(c)
EA1 EA2
Myosin-9 Annexin A2 isoform 1 HSP70 protein 5 Beta actin variant Colligin Beta-filamin
12667788 50845388
EA3 EA4 EA5 EA6
MW (kD) pI Theor/ Peptide Sequence Peptide Theor/Exp(d) Exp(e) match coverage % score(f)
Acetylated lysine residue(g)
146.4/139.5 40.4/47.2
6.5/6.8 8.5/6.3
48 48
25 64
49 25
MQLAK1080KEEELQAALAR MGRQLAGCGDAGK13K
119608027 51.1/52.6 62897625 41.7/24.9 30130 46.0/42.9 3298597 278.0/138.9
4.9/5.4 5.4/5.9 8.3/6.1 5.5/6.2
27 31 19 26
38 50 32 12
LGGK380LSSEDKETMEK CPEALFQPSFLGMESCGIHETTFNSIMK284RCDVDIR INFPDK165RSALQSINEWAAQTTDGK CVNK35RIGNLQTDLSDGLR VEVGK960DQEFTVDTR RDLAK166DITSDTSGDFR GITIDISLWK79FETSK SGDAAIVDMVPGK408PMCVESFSDYPPLGR LNLEAINYMAADGDFK127IK LNLEAINYMAADGDFKIK129HVAFD VQDLLLLDVAPLSLGLETVGGVMTALIK417ER
4502101 4503471
38.7/35.4 50.1/58.1
6.6/5.8 9.1/4.6
14 20
43 39
310942925
14.6/32.1
5.36.4
14
76
EA10 Heat shock188492 induced protein EA11 Protein S100-A11 5032057 EA12 Metallothionein-2, 157835360 chain A DA1 EPB41L3 protein 13544009 DA2 hCG22067 119572363
70.4/53.6
5.8/6.7
5
10
28 32 40 44 29 27 48 57 44 38 30
11.7/35.6 3.02/31.2
6.6/6.2 6.3/4.9
3 4
46 83
45 27
LDTNSDGQLDFSEFLNLIGGLAMACHDSFLK97AVPSQK SDPNCSCAAGDSCTCAGSCKCK22ECK
96.5/139.2 29.7/75.2
5.3/6.8 8.0/4.9
3 4
4 13
DA3
62420916
61.5/70.1
8.0/6.8
3
6
41 26 33 27
TDTAADGETTATEELEK542TQDDLMK NKPGVYTK255VCNYVK VCNYVK261WIQQTIAAN DTGK278PKGEATVSFDDPPSAK
157881403 22748615
41.5/68.2 11.7/23.3
5.3/5.7 9.3/6.7
2 4
7 31
20 39
DDDIAALVVDNGSGMCK17AGFAGDDAPR GGVSAVAGGVTAVGSAVVNK104VPLTGKK
62898329
10.0/53.2
11.7/6.2
3
23
27
K45NAGCGTR
EA7 EA8 EA9
DA4 DA5 DA6
Annexin A1 Elongation factor 1-alpha 1 Galectin-1
TATA-binding protein-associated factor Profilin-Beta-Actin Hypothetical protein LOC90488 Protein translocation complex beta variant
(a)
Spot numbers represent proteins that were enhanced (EA) or decreased (DA) in acetylation levels on the 2-DE gels. (b) Protein name was annotated in National Center for Biotechnology Information (NCBI) database. (c) Accession number was annotated in NCBI database. (d) Protein molecular weight was annotated in NCBI database (Theory, Theor) and was calculated from 2-DE gels (Experiment, Exp). (e) Protein pI was annotated in NCBI database (Theory, Theor) and was calculated from 2-DE gels (Experiment, Exp). (f) The MOWSE (MOlecular Weight SEarch) score of acetylated peptide. (g) Acetylated lysine residues were determined on peptide sequence.
actin-binding protein that is now identified as an inducer of membrane remodeling, and thus is also a tumor suppressor candidate.29 In addition to the EPB protein, a profilin-b-actin complex was detected that has functions similar to those of the EPB protein. In atherosclerosis, increased oxLDL levels are accompanied by elevated profilin protein levels.30
western blotting analysis due to the lack of available specific antibodies, we provided a feasible methodology as well as a putative physiological mechanism of the acetylome in statin-derived pleiotropic effects. Moreover, in addition to the acetylome on lysine residues, other modifications that interact with acetylation may also be involved in pleiotropic effects, and these should accordingly be investigated in future studies.
CONCLUSIONS ACKNOWLEDGEMENT In conclusion, although we were unable to conduct further studies on the acetylated proteins detected by Acta Cardiol Sin 2014;30:67-73
This work was supported in part by grants from the 72
Rosuvastatin Modulates Acetylome 14. Guarente L. Sirtuins, aging, and medicine. N Engl J Med 2011; 364:2235-44. 15. Verdin E, Hischey MD, Finley LWS, et al. Sirtuin regulation of mitochondria: energy production, apoptosis, and signaling. Trends Biochem Sci 2010;35:669-75. 16. He W, Newman JC, Wang MZ, et al. Mitochondrial sirtuins: regulators of protein acylation and metabolism. Trends Endocrin Met 2012;23:67-476. 17. Knutson MD, Leeuwenburgh C. Resveratrol and novel potent activators of SIRT1: effects on aging and age-related diseases. Nutr Rev 2008;66:591-6. 18. Wang F, Nguyen M, Qin FX, et al. SIRT2 deacetylates FOXO3a in response to oxidative stress and caloric restriction. Aging Cell 2007;6:505-14. 19. Oellerich MF, Potente M. FOXOs and sirtuins in vascular growth, maintenance, and aging. Circ Res 2012;110:1238-51. 20. McKenney JM. Pharmacologic characteristics of statins. Clin Cardiol 2003;26(Suppl III):32-8. 21. Enomoto S, Sata M, Fukuda D, et al. Rosuvastatin prevents endothelial cell death and reduces atherosclerotic lesion formation in ApoE-deficient mice. Biomed Pharmacother 2009;63:19-26. 22. Lin YC, Lin JH, Chou CW, et al. Statins increase p21 through inhibition of histone deacetylase activity and release of promoter-associated HDAC1/2. Cancer Res 2008;68:2375-83. 23. Feig JE, Shang Y, Rotllan N, et al. Statins promote the regression of atherosclerosis via activation of the CCR7-dependent emigration pathway in macrophages. PLoS ONE 2011;6:e28534. 24. Tabuchi T, Satoh M, Itoh T, et al. MicroRNA-34a regulates the longevity-associated protein SIRT1 in coronary artery disease: effect of statins on SIRT1 and microRNA-34a expression. Clin Sci 2012;123:161-71. 25. Madrigal-Matute J, Martin-Ventura JL, Blanco-Colio LM, et al. Heat-shock proteins in cardiovascular disease. Adv Clin Chem 2011;54:1-43. 26. Patel HJ, Modi S, Chiosis G, et al. Advances in the discovery and development of heat-shock protein 90 inhibitors for cancer treatment. Expert Opin Drug Discov 2011;6:559-87. 27. Uchiyama T, Atsuta H, Utsugi T, et al. HSF1 and constitutively active HSF1 improve vascular endothelial function (heat shock proteins improve vascular endothelial function). Atherosclerosis 2007;190:321-9. 28. Hu JL, Xu G, Lei L, et al. Etoposide phosphate enhances the acetylation level of translation elongation factor 1A in PLC5 cells. Z Naturforsch C 2012;67:327-30. 29. Dafou D, Grun B, Sinclair J, et al. Microcell-mediated chromosome transfer identifies EPB41L3 as a functional suppressor of epithelial ovarian cancers. Neoplasia 2010;12:579-89. 30. Romeo GR, Moulton KS, Kazlauskas A. Attenuated expression of profilin-1 confers protection from atherosclerosis in the LDL receptor-null mouse. Circ Res 2007;101:357-67.
National Science Council (NSC 101-2320-B-037-041) and projects from Chi-Mei Medical Center and Kaohsiung Medical University Research Foundation (100CM-KMU01 and 101CM-KMU-01). We are grateful to the core facility laboratory of the Institute of Biomedical Sciences, Academia Sinica, and the Center for Resources, Research & Development of Kaohsiung Medical School for the mass spectrometric analysis. We also acknowledge AstraZeneca, Taiwan, for providing the rosuvastatin powder.
REFERENCES 1. Ludman A, Venugopal V, Yellon DM, et al. Statins and cardioprotection-more than just lipid lowering? Pharmacol Ther 2009;122:30-43. 2. Zhou Q, Liao JK. Pleiotropic effects of statins. Circ J 2010;74: 818-26. 3. Kong W, Zhu Y. The pleiotropic effects of statins in the prevention of atherosclerosis. Cardiovasc Drugs Ther 2012;26:5-7. 4. Deribe YL, Pawson T, Dikic I. Post-translational modifications in signal integration. Nat Stru Mol Biol 2010;17:666-72. 5. Haendeler J, Hoffmann J, Zeiher AM, et al. Antioxidant effects of statins via S-nitrosylation and activation of thioredoxin in endothelial cells: a novel vasculoprotective function of statins. Circulation 2004;110:856-61. 6. Huang B, Li FA, Wu CH, et al. The role of nitric oxide on rosuvastatin-mediated S-nitrosylation and translational proteomes in human umbilical vein endothelial cells. Proteome Sci 2012; 10:43. 7. Paajarvi G, Roudier E, Crisby M, et al. HMG-CoA reductase inhibitors, statins, induce phosphorylation of Mdm2 and attenuate the p53 response to DNA damage. FASEB J 2005;19:476-8. 8. Sadoul K, Boyault C, Pabion M, et al. Regulation of protein turnover by acetyltransferases and deacetylases. Biochimie 2008; 90:306-12. 9. Glozak MA, Sengupta N, Zhang X, et al. Acetylation and deacetylation of non-histone proteins. Gene 2005;363:15-23. 10. Spange S, Wagner T, Heinzel T, et al. Acetylation of non-histone proteins modulates cellular signalling at multiple levels. Int J Biochem Cell Biol 2009;41:185-98. 11. Cohen T, Yao TP. AcK-knowledge reversible acetylation. Sci STKE 2004;2004:e42. 12. Kim SC, Sprung R, Chen Y, et al. Substrate and functional diversity of lysine acetylation revealed by a proteomic survey. Mol Cell 2006;23:607-18. 13. Chen LF, Greene WC. Shaping the nuclear action of NF-kappaB. Nat Rev Mol Cell Biol 2004;5:392-401.
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Acta Cardiol Sin 2014;30:67-73
Basic
Rosuvastatin Modulates the Post-Translational Acetylome in Endothelial Cells Ming Chung Lin,1,2 Chung Hsi Hsing,3 Fu An Li,4 Chien Hsing Wu,5 Yaw Syan Fu,6 Jen Kun Cheng7 and Bin Huang6
Background: Statins are lipid-lowering drugs that can simultaneously evoke pleiotropic effects on cardioprotection, vasodilation, and diabetes prevention. Recently, statins have been reported to be able to activate the AMPactivated protein kinase, thereby up-regulating sirtuin (SIRT) that functions as non-histone deacetylases. Therefore, it is essential to investigate the post-translational acetylome that might explain the mechanism of statinmodulated pleiotropic effects. Method: Endothelial cells EAhy 926 treated with rosuvastatin were used to monitor the expression of SIRTs proteins. The protein lysates of both mock- and rosuvastatin-treated cells were further separated by twodimensional gel electrophoresis coupled with western blotting analysis. The significantly changed acetylcontaining proteins detected by using an anti-acetyl lysine antibody were collected from another preparative gel for mass spectrometric assay to identify the acetylated site in the proteins. Results: Rosuvastatin treatment was shown to increase the SIRT1 expression when compared with SIRT2. Among 100 detected proteins with acetylated signal, 12 showed an increased level of acetylation, whereas 6 showed a decreased level of acetylation (deacetylation). The acetylated lysine (K) sites of 3 heat shock proteins, i.e., HSP47/K165, HSP70/K380, and heat shock-inducible protein/K417, were determined. We also found that beta-filamin, elongation factor, galectin and hCG22067 have 2 acetylated lysine sites in their peptide sequences. These dynamic acetylations might alter the protein’s function and are thought to be important in regulating statin-mediated pleiotropic effect. Conclusions: Our study provided a feasible methodology for detecting acetylated proteins. This acetylome information may be utilized to explain, at least partially, the mechanisms of statin-derived pleiotropic effects.
Key Words:
Acetylation/deacetylation · Acetylome · Endothelial cell · Proteomics · Rosuvastatin · Sirtuin
INTRODUCTION Received: January 12, 2013 Accepted: February 6, 2013 1 Department of Anesthesiology, Chi Mei Medical Center; 2Department of Medical Laboratory Science and Biotechnology, Chung Hwa University of Medical Technology; 3Department of Anesthesiology, Chi Mei Medical Center, Tainan; 4Institute of Biomedical Science, Academia Sinica, Taipei; 5Division of Nephrology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine; 6Department of Biomedical Science and Environmental Biology, College of Life Science, Kaohsiung Medical University, Kaohsiung; 7Department of Anesthesiology, Mackay Memorial Hospital, Taipei, Taiwan. Address correspondence and reprint requests to: Dr. Bin Huang, Department of Biomedical Science and Environmental Biology, College of Life Science, Kaohsiung Medical University, No. 100, Shihchuan 1st Rd., San Ming District, Kaohsiung 80708, Taiwan. Tel: 886-7-312-1101 ext. 2704; Fax: 886-7-322-7508; E-mail: huangpin2 @yahoo.com.tw
Statins, or 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, were originally developed to reduce the level of total blood cholesterol. In the past decades, statin-derived pleiotropic effects have been reported that include improved endothelial functions, reduced platelet aggregation, and stabilized atherosclerotic plaques; however, the mechanism underlying these pleiotropic effects remains insufficiently investigated.1-3 Post-translational modifications (PTMs) of proteins are considered important processes in signal transduction and physiological responses.4 Statin-regulated PTMs such as S-nitrosylation and phosphorylation that 67
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lead to their pleiotropic effects have been previously described.5-7 Recently, the patterns of lysine acetylation and deacetylation in proteins, also known as the acetylome, have been regarded as a new means by which protein function is modulated.8 The acetylation of lysine residues was originally described to play a role in the histone protein modulation of gene expression. However, an increasing number of studies are focusing on non-histone proteins that have altered physiological functions following acetylation.9,10 To date, over 60 transcription factors and 133 mitochondrial proteins have been identified as being acetylated.11,12 The effects of acetylation on protein function have also been demonstrated. For example, acetylation on lysine 221 (K 221 ) and K 310 in NF-kB is associated with enhanced transcription of its target genes.13 The enzymatic activity of protein kinase ATM increases after protein acetylation, which results in decreased enzymatic activity of PTEM, Mdm2, and acetyl-CoA synthetase. 9 Concurrent with these findings, the protein acetylome can be considered to play an increasingly important role in the adaptation of cells to different physiological responses. The sirtuin (SIRT) protein family contains 7 members and shares a high level of homology with the yeast silent information regulator 2 (Sir2) protein, which functions in the NAD-dependent deacetylation of histones in the regulation of gene expression. These sirtuins occupy different subcellular compartments, such as the nucleus (SIRT1, 2, 6, and 7), cytoplasm (SIRT1 and 2), and the mitochondrion (SIRT3, 4, and 5). 14 Unlike the known deacetylases, SIRT-mediated deacetylation occurs only on the lysine residues of proteins. The deacetylation of non-histone proteins has gained increasing recognition, as these proteins play essential roles in regulating cellular signaling and homeostasis.15,16 The published studies found that activation of SIRT1 has a therapeutic effect during excessive ROS production. 17 SIRT2 has been suggested to increase resistance to oxidative stress through deacetylation of downstream proteins such as forkhead box O proteins (FOXOs).18,19 These findings revealed the possible critical involvement of SIRT1 and SIRT2 in the response to oxidative stress. Rosuvastatin is a newly designed statin that possesses the lowest inhibition coefficient (IC50 = 5.4 nM) when compared with other statins.20 Recently, studies were performed on statin-induced post-translational Acta Cardiol Sin 2014;30:67-73
modifications, such as phosphorylation and S-nitrosylation, that might be involved in pleiotropic effects.6,21 In terms of acetylation, statins increase cyclin-dependent kinase inhibitor p21 gene expression through inhibition of histone deacetylase activity and release of promoter-associated HDAC1/2.22 Rosuvastatin induces acetylation of histones H3 and H4 and C-C chemokine receptor type 7 gene expression, which promotes macrophage migration out of plaques and reduces atherosclerosis. 23 Clinical studies have shown that statins markedly decrease miR-34a levels and increase SIRT1 levels, which possibly contributes to the beneficial effects of statins on endothelial cell function.24 In the present study, ECs treated with rosuvastatin specifically increased the protein levels of SIRT1 as compared to SIRT2. Using two-dimensional gel electrophoresis (2-DE) analysis, we identified 16 proteins with differential levels of lysine acetylation and deacetylation. These proteins will be further studied to elucidate the effect of the acetylome on the function of each protein and subsequently the mechanism of statinregulated pleiotropic effects.
MATERIALS AND METHODS Cell culture The endothelial cell line EAhy 926 was kindly donated by Cora-Jean S. Edgell, University of North Carolina, Chapel Hill. ECs were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with fetal bovine serum (FBS, 10%), streptomycin (100 mg/mL), and penicillin (100 U/mL). The medium was then replaced with DMEM medium containing 2% FBS, and the ECs were incubated overnight prior to treatment with rosuvastatin (AstraZeneca, Taipei, Taiwan). The antibodies for SIRT1 and SIRT2 were purchased from Sigma-Aldrich (St. Louis, MO, USA), and the antibody against anti-lysine acetylation was obtained from Calbiochem (San Diego, CA, USA). Protein extraction and western blotting analysis After rosuvastatin treatment, the ECs were washed with cord buffer: NaCl (0.14 M), KCl (4 mM), glucose (11 mM), HEPES (10 mM, pH 7.4) and then lysed with 300 mL of lysis buffer: HEPES (250 mM, pH 7.7), EDTA (1 mM), neocuproine (0.1 mM), and CHAPS (0.4%, w/v). 68
Rosuvastatin Modulates Acetylome
After centrifugation at 10000 ´ g for 10 min at 4 °C, the protein supernatant was collected, and protein concentrations were determined using the BCA assay reagent (Thermo Fisher Scientific Inc., Rockford, IL, USA). Forty micrograms of cell lysate was mixed with SDS-PAGE sample buffer: Tris-HCl (62.5 mM, pH 6.8), SDS (3%, w/v), 2-mercaptoethanol (5%, v/v), and glycerol (10%, v/v), and then separated by SDS-PAGE. The blotted membranes were hybridized with either anti-SIRT1 (1:2,000) or anti-SIRT2 (1:3,000) antibodies to determine the level of protein expression.
films. The images on X-ray films were scanned using a digital scanner (Microtek International Inc.), and the results represent the level of post-translational acetylation. The acetylation levels were calculated using the Progenesis Samespots v2.0 software (NonLinear Dynamics, Newcastle, UK).
Mass spectrometric (MS) analysis The excised gel slices were digested with trypsin for 4 h at 37 °C (In-Gel Tryptic Digestion Kit, Thermo Fisher Scientific) to allow the subsequent identification of peptides by mass spectrometry (MS). The tryptic peptides were desalted on a Proteomics C18 Column (Mass Solution Ltd., Taipei, Taiwan) and then subjected to MS analysis using an nLC/Q-TOF (Micromass, Manchester, UK). MS data were used to search against entries in the National Center for Biotechnology Information (NCBI) database with a MASCOT in-house search program (Matrix Science, London, UK). In addition, peptides that contained acetylated lysine sites were determined. The search parameters were set as follows: mass values: monoisotopic; protein mass: unrestricted; peptide mass tolerance: ± 0.4 Da; fragment mass tolerance: ± 0.4 Da; max missed cleavages: 1; variable modification: acetylation in lysine, carbamidomethylation in cysteine, and oxidation in methionine.
Two-dimensional gel electrophoresis (2-DE) and lysine acetylome detection The protein lysates obtained from cells treated with either mock or rosuvastatin (10 mM) for 24 h were precipitated in ice-cold acetone at -20 °C. After centrifugation, the protein pellets were air-dried and then dissolved in sample buffer: urea (9 M), CHAPS (2%, w/v), DL-dithiothreitol (60 mM), and IPG solution (2%, v/v, pH 4-7; GE Healthcare, Piscataway, NJ, USA) at room temperature and shaken for 1 h to ensure that the proteins were thoroughly denatured. The lysates were mixed with rehydration buffer: urea (8 M), CHAPS (2%, w/v), IPG solution (0.5%, v/v, pH 4-7), and DL-dithiothreitol (30 mM) to reach a final volume of 340 mL. The samples were then soaked into an 18-cm DryStrip (pH 4-7, GE Healthcare) for up to 12 h on the Ettan IPGphor system (GE Healthcare), followed by isoelectric focusing (IEF) at an accumulated voltage of 60 kV/h. The strip gels were then incubated with equilibration buffer: SDS (2%, w/v), Tris-HCl (50 mM, pH 8.8), urea (6 M), glycerol (30%, v/v), and DL-dithiothreitol (60 mM) for 20 min, and then equilibrated with the same buffer containing iodoacetic acid (IAA, 135 mM) for an additional 20 min. The equilibrated IEF strip was laid on the top of a vertical SDSPAGE apparatus to separate the proteins. After 2-DE, the gels were stained using the VisPRO dye and scanned using a digital scanner (Microtek International Inc., Hsinchu, Taiwan). This obtained image represents the translational level of total proteins. After de-staining, the gels were blotted onto a nitrocellulose membrane and hybridized with an anti-acetyl lysine monoclonal antibody (1:500; Cell Signaling Tech., Boston, MA, USA). The membranes were developed with the SuperSignal West Femto reagent (Thermo Fisher Scientific) on X-ray
RESULTS Increased SIRT1 protein expression after rosuvastatin treatment As shown in Figure 1, the expression levels of SIRT1 and SIRT2 were investigated in dose- and time-dependent manners. Treatment of ECs with 10 mM rosuvastatin resulted in a significant (> 3 fold) increase in the SIRT1 protein level compared with that observed after mock treatment from 6 h to 24 h. However, the SIRT2 protein level did not change under similar conditions. The concentration of rosuvastatin applied in the present study is similar to previous studies.6 Rosuvastatin regulation of the acetylome According to the flowchart of Figure 2, the total protein lysates from mock- and rosuvastatin-treated cells were separated by 2-DE; the images obtained after 69
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Ming Chung Lin et al.
A
B
C
D
Figure 2. An experimental flowchart for the detection of rosuvastatin-modulated acetylome. After rosuvastatin treatment, the cell lysates were separated by 2-DE. The gels were stained with VisPRO dye to detect the levels of translational proteins. After reversible destaining using SDS-PAGE running buffer, the gels were subsequently subjected to western blotting analyses using an anti-acetyl-lysine antibody to obtain post-translational acetylation patterns. The proteins exhibited equal translational levels, but significantly changed acetylation levels were found in another 2-DE gel with rosuvastatin-treated samples, which were analyzed by mass spectrometry.
Figure 1. Sirtuin expression levels during treatment with rosuvastatin. (A) Endothelial cells were treated with a series dilution of rosuvastatin (1, 10 and 100 mM) to determine the optimal concentration applied in the experiment. Forty micrograms of protein lysates were separated by SDS-PAGE. Western blotting studies were performed using monoclonal antibodies against SIRT1 (1:2,000) and SIRT2 (1:3,000). (B) The relative fold-changes of protein levels were statistically calculated from 3 repeated experiments and the relative fold-changes are shown as the mean ± S.E. compared to control treatments. The changes were individually compared to the control by Fisher’s LSD for significant difference (p < 0.05). (C, D) For time-dependent protein expression, cells were treated with 10 mM rosuvastatin for 0.5, 6, and 24 h. The expression level of proteins was also statistically calculated.
with altered acetylation status, peptides containing acetyl lysine residues were also determined by tandem MS (MS/MS). Four proteins were found to contain 2 different acetylated lysine residues, and the other 14 proteins were identified to contain only 1 acetylated lysine residue (Table 1). Moreover, because trypsin can only digest those lysine residues that are not acetylated, the same peptides with or without acetylation can be specifically distinguished. These proteins are mostly cytoskeletal proteins and transcription factors.
VisPRO staining were used to determine the levels of translational proteins. Meanwhile, the post-translational acetylation levels were determined by subsequent western blotting using an anti-acetyl lysine antibody. Despite the detection of > 100 acetylated proteins from the control (126 ± 20) and rosuvastatin (142 ± 22) treatment groups, only approximately 15% of proteins showed a significant change in their acetylation status. We found 12 proteins with enhanced acetylation status (> 1.5-fold in protein spot density) and 6 proteins with decreased acetylation status (deacetylation, < 0.5-fold in protein spot density) (Figure 3). These proteins are annotated as heat shock response (EA3, EA5 and EA10), cell proliferation (EA1, EA4, EA6, EA11, DA1 and DA4) and protein expression (EA8 and DA3). In addition, these proteins showed no difference at the translational levels, indicating that the rosuvastatin-regulated acetylation/deacetylation process was actually a posttranslational event (Figure 3).
DISCUSSION Attempts have previously been made to explain the mechanisms underlying statin-derived pleiotropic effects in terms of post-translational modification. In addition to our previous study on the S-nitrosoproteome, in which we proposed a possible mechanism of nitric oxide generating pleiotropic effects, 6 we examined acetylome in the present study as an alternative mechanism for this pleiotropic effect. The most prominent benefit of 2-DE-based proteomics is that the translational and post-translational levels of proteins can be observed simultaneously. Therefore, post-translational evidence of these modifications can be clearly defined.
Determination of acetylated lysine residues In addition to the identification of the 18 proteins Acta Cardiol Sin 2014;30:67-73
70
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A
B
Figure 3. Quantification of the rosuvastatin-modulated acetylome. (A) After incubation with 10 mM rosuvastatin for 24 h, 1 mg of protein lysates were separated by 2-DE. The gels were stained with VisPRO (VisPRO panel) prior to western blotting with a mouse anti-acetyl-lysine monoclonal antibody (1:500). The arrowheads indicate proteins whose expression levels are significantly increased/decreased (western blot panel). The corresponding protein spots on the VisPRO-stained gels were subjected to MS analysis. DA, decreased acetylation; EA, enhanced acetylation. (B) The normalized statistical data show the comparison of the translational (VisPRO) and post-translational levels (western blot), which demonstrated that rosuvastatin-regulated protein acetylation is a post-translational event. The data are shown as the mean ± S.E. values from 3 experiments, and the relative fold-changes are indicated under the statistic bar.
The levels of SIRT1, the most studied deacetylase in recent years, were significantly enhanced by rosuvastatin treatment from 6 h to 24 h (Figure 1). In contrast to the prediction that the amount of acetylated protein should decrease, a higher number of acetylated proteins was observed in the 2-DE assay. This implies that the rosuvastatin-regulated cellular acetylome might not act singly through SIRT1. Several of the identified proteins, when characterized, exhibited a close relationship with acetylomemediated physiologies. Heat shock proteins (HSPs), for example, are known to play important roles in cell homeostasis.25 The effects of lysine (Lys, K) acetylation on HSPs function have mostly been evaluated in HSP90. The deacetylation of HSP90 attenuates ATP-binding capacity, and thus involves a chaperone function, which regulates cancer cell invasion.26 In the present study, 3 HSPs (collagen (HSP47)/K 165 , HSP70/K 380 , and heat shock-inducible protein/K417) and their acetylated lysine sites were separately identified. Because HSPs have been shown to be important in modulating statinderived endothelial homeostasis, 27 post-translational
acetylation might play a role in the early regulatory functions of these HSPs. Therefore, more evidence in support of this hypothesis should be collected through future studies. The elongation factor is important in protein synthesis via promoting the binding of acryl-tRNA to ribosomes in a GTP-dependent manner. The activity of this elongation factor has recently been reported to be modulated by post-translational modifications such as phosphorylation, S-nitrosylation, and acetylation.6,28 In this study, elongation factor 1 alpha 1, also named elongation factor Tu (EF-Tu), was identified to be acetylated on residues K79 and K408 after rosuvastatin treatment. However, there is little information regarding the modulation of EF-Tu activity by acetylation. Therefore, whether acetylation/deacetylation of EF-Tu regulates downstream protein synthesis still needs to be elucidated. Despite the conflicting results that showed that fewer deacetylated proteins were detected under the condition of increased expression levels of SIRT1 deacetylase, the putative functions of these deacetylated proteins still deserve further discussion. EPB41L3 is an 71
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Ming Chung Lin et al. Table 1. Acetylated proteins and lysine residues identified by nLC-MS/MS Spot No(a)
Protein name(b)
Ac No(c)
EA1 EA2
Myosin-9 Annexin A2 isoform 1 HSP70 protein 5 Beta actin variant Colligin Beta-filamin
12667788 50845388
EA3 EA4 EA5 EA6
MW (kD) pI Theor/ Peptide Sequence Peptide Theor/Exp(d) Exp(e) match coverage % score(f)
Acetylated lysine residue(g)
146.4/139.5 40.4/47.2
6.5/6.8 8.5/6.3
48 48
25 64
49 25
MQLAK1080KEEELQAALAR MGRQLAGCGDAGK13K
119608027 51.1/52.6 62897625 41.7/24.9 30130 46.0/42.9 3298597 278.0/138.9
4.9/5.4 5.4/5.9 8.3/6.1 5.5/6.2
27 31 19 26
38 50 32 12
LGGK380LSSEDKETMEK CPEALFQPSFLGMESCGIHETTFNSIMK284RCDVDIR INFPDK165RSALQSINEWAAQTTDGK CVNK35RIGNLQTDLSDGLR VEVGK960DQEFTVDTR RDLAK166DITSDTSGDFR GITIDISLWK79FETSK SGDAAIVDMVPGK408PMCVESFSDYPPLGR LNLEAINYMAADGDFK127IK LNLEAINYMAADGDFKIK129HVAFD VQDLLLLDVAPLSLGLETVGGVMTALIK417ER
4502101 4503471
38.7/35.4 50.1/58.1
6.6/5.8 9.1/4.6
14 20
43 39
310942925
14.6/32.1
5.36.4
14
76
EA10 Heat shock188492 induced protein EA11 Protein S100-A11 5032057 EA12 Metallothionein-2, 157835360 chain A DA1 EPB41L3 protein 13544009 DA2 hCG22067 119572363
70.4/53.6
5.8/6.7
5
10
28 32 40 44 29 27 48 57 44 38 30
11.7/35.6 3.02/31.2
6.6/6.2 6.3/4.9
3 4
46 83
45 27
LDTNSDGQLDFSEFLNLIGGLAMACHDSFLK97AVPSQK SDPNCSCAAGDSCTCAGSCKCK22ECK
96.5/139.2 29.7/75.2
5.3/6.8 8.0/4.9
3 4
4 13
DA3
62420916
61.5/70.1
8.0/6.8
3
6
41 26 33 27
TDTAADGETTATEELEK542TQDDLMK NKPGVYTK255VCNYVK VCNYVK261WIQQTIAAN DTGK278PKGEATVSFDDPPSAK
157881403 22748615
41.5/68.2 11.7/23.3
5.3/5.7 9.3/6.7
2 4
7 31
20 39
DDDIAALVVDNGSGMCK17AGFAGDDAPR GGVSAVAGGVTAVGSAVVNK104VPLTGKK
62898329
10.0/53.2
11.7/6.2
3
23
27
K45NAGCGTR
EA7 EA8 EA9
DA4 DA5 DA6
Annexin A1 Elongation factor 1-alpha 1 Galectin-1
TATA-binding protein-associated factor Profilin-Beta-Actin Hypothetical protein LOC90488 Protein translocation complex beta variant
(a)
Spot numbers represent proteins that were enhanced (EA) or decreased (DA) in acetylation levels on the 2-DE gels. (b) Protein name was annotated in National Center for Biotechnology Information (NCBI) database. (c) Accession number was annotated in NCBI database. (d) Protein molecular weight was annotated in NCBI database (Theory, Theor) and was calculated from 2-DE gels (Experiment, Exp). (e) Protein pI was annotated in NCBI database (Theory, Theor) and was calculated from 2-DE gels (Experiment, Exp). (f) The MOWSE (MOlecular Weight SEarch) score of acetylated peptide. (g) Acetylated lysine residues were determined on peptide sequence.
actin-binding protein that is now identified as an inducer of membrane remodeling, and thus is also a tumor suppressor candidate.29 In addition to the EPB protein, a profilin-b-actin complex was detected that has functions similar to those of the EPB protein. In atherosclerosis, increased oxLDL levels are accompanied by elevated profilin protein levels.30
western blotting analysis due to the lack of available specific antibodies, we provided a feasible methodology as well as a putative physiological mechanism of the acetylome in statin-derived pleiotropic effects. Moreover, in addition to the acetylome on lysine residues, other modifications that interact with acetylation may also be involved in pleiotropic effects, and these should accordingly be investigated in future studies.
CONCLUSIONS ACKNOWLEDGEMENT In conclusion, although we were unable to conduct further studies on the acetylated proteins detected by Acta Cardiol Sin 2014;30:67-73
This work was supported in part by grants from the 72
Rosuvastatin Modulates Acetylome 14. Guarente L. Sirtuins, aging, and medicine. N Engl J Med 2011; 364:2235-44. 15. Verdin E, Hischey MD, Finley LWS, et al. Sirtuin regulation of mitochondria: energy production, apoptosis, and signaling. Trends Biochem Sci 2010;35:669-75. 16. He W, Newman JC, Wang MZ, et al. Mitochondrial sirtuins: regulators of protein acylation and metabolism. Trends Endocrin Met 2012;23:67-476. 17. Knutson MD, Leeuwenburgh C. Resveratrol and novel potent activators of SIRT1: effects on aging and age-related diseases. Nutr Rev 2008;66:591-6. 18. Wang F, Nguyen M, Qin FX, et al. SIRT2 deacetylates FOXO3a in response to oxidative stress and caloric restriction. Aging Cell 2007;6:505-14. 19. Oellerich MF, Potente M. FOXOs and sirtuins in vascular growth, maintenance, and aging. Circ Res 2012;110:1238-51. 20. McKenney JM. Pharmacologic characteristics of statins. Clin Cardiol 2003;26(Suppl III):32-8. 21. Enomoto S, Sata M, Fukuda D, et al. Rosuvastatin prevents endothelial cell death and reduces atherosclerotic lesion formation in ApoE-deficient mice. Biomed Pharmacother 2009;63:19-26. 22. Lin YC, Lin JH, Chou CW, et al. Statins increase p21 through inhibition of histone deacetylase activity and release of promoter-associated HDAC1/2. Cancer Res 2008;68:2375-83. 23. Feig JE, Shang Y, Rotllan N, et al. Statins promote the regression of atherosclerosis via activation of the CCR7-dependent emigration pathway in macrophages. PLoS ONE 2011;6:e28534. 24. Tabuchi T, Satoh M, Itoh T, et al. MicroRNA-34a regulates the longevity-associated protein SIRT1 in coronary artery disease: effect of statins on SIRT1 and microRNA-34a expression. Clin Sci 2012;123:161-71. 25. Madrigal-Matute J, Martin-Ventura JL, Blanco-Colio LM, et al. Heat-shock proteins in cardiovascular disease. Adv Clin Chem 2011;54:1-43. 26. Patel HJ, Modi S, Chiosis G, et al. Advances in the discovery and development of heat-shock protein 90 inhibitors for cancer treatment. Expert Opin Drug Discov 2011;6:559-87. 27. Uchiyama T, Atsuta H, Utsugi T, et al. HSF1 and constitutively active HSF1 improve vascular endothelial function (heat shock proteins improve vascular endothelial function). Atherosclerosis 2007;190:321-9. 28. Hu JL, Xu G, Lei L, et al. Etoposide phosphate enhances the acetylation level of translation elongation factor 1A in PLC5 cells. Z Naturforsch C 2012;67:327-30. 29. Dafou D, Grun B, Sinclair J, et al. Microcell-mediated chromosome transfer identifies EPB41L3 as a functional suppressor of epithelial ovarian cancers. Neoplasia 2010;12:579-89. 30. Romeo GR, Moulton KS, Kazlauskas A. Attenuated expression of profilin-1 confers protection from atherosclerosis in the LDL receptor-null mouse. Circ Res 2007;101:357-67.
National Science Council (NSC 101-2320-B-037-041) and projects from Chi-Mei Medical Center and Kaohsiung Medical University Research Foundation (100CM-KMU01 and 101CM-KMU-01). We are grateful to the core facility laboratory of the Institute of Biomedical Sciences, Academia Sinica, and the Center for Resources, Research & Development of Kaohsiung Medical School for the mass spectrometric analysis. We also acknowledge AstraZeneca, Taiwan, for providing the rosuvastatin powder.
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