Phosphoproteome Analysis Reveals Regulatory Sites in Major Pathways of Cardiac Mitochondria
2010; Elsevier BV; Volume: 10; Issue: 2 Linguagem: Inglês
10.1074/mcp.m110.000117
ISSN1535-9484
AutoresNing Deng, Jun Zhang, Chenggong Zong, Yueju Wang, Haojie Lu, Pengyuan Yang, Wenhai Wang, Glen Young, Yibin Wang, Paavo Kôrge, Christopher Lotz, Philip Doran, David A. Liem, Rolf Apweiler, James N. Weiss, Huilong Duan, Peipei Ping,
Tópico(s)ATP Synthase and ATPases Research
ResumoMitochondrial functions are dynamically regulated in the heart. In particular, protein phosphorylation has been shown to be a key mechanism modulating mitochondrial function in diverse cardiovascular phenotypes. However, site-specific phosphorylation information remains scarce for this organ. Accordingly, we performed a comprehensive characterization of murine cardiac mitochondrial phosphoproteome in the context of mitochondrial functional pathways. A platform using the complementary fragmentation technologies of collision-induced dissociation (CID) and electron transfer dissociation (ETD) demonstrated successful identification of a total of 236 phosphorylation sites in the murine heart; 210 of these sites were novel. These 236 sites were mapped to 181 phosphoproteins and 203 phosphopeptides. Among those identified, 45 phosphorylation sites were captured only by CID, whereas 185 phosphorylation sites, including a novel modification on ubiquinol-cytochrome c reductase protein 1 (Ser-212), were identified only by ETD, underscoring the advantage of a combined CID and ETD approach. The biological significance of the cardiac mitochondrial phosphoproteome was evaluated. Our investigations illustrated key regulatory sites in murine cardiac mitochondrial pathways as targets of phosphorylation regulation, including components of the electron transport chain (ETC) complexes and enzymes involved in metabolic pathways (e.g. tricarboxylic acid cycle). Furthermore, calcium overload injured cardiac mitochondrial ETC function, whereas enhanced phosphorylation of ETC via application of phosphatase inhibitors restored calcium-attenuated ETC complex I and complex III activities, demonstrating positive regulation of ETC function by phosphorylation. Moreover, in silico analyses of the identified phosphopeptide motifs illuminated the molecular nature of participating kinases, which included several known mitochondrial kinases (e.g. pyruvate dehydrogenase kinase) as well as kinases whose mitochondrial location was not previously appreciated (e.g. Src). In conclusion, the phosphorylation events defined herein advance our understanding of cardiac mitochondrial biology, facilitating the integration of the still fragmentary knowledge about mitochondrial signaling networks, metabolic pathways, and intrinsic mechanisms of functional regulation in the heart. Mitochondrial functions are dynamically regulated in the heart. In particular, protein phosphorylation has been shown to be a key mechanism modulating mitochondrial function in diverse cardiovascular phenotypes. However, site-specific phosphorylation information remains scarce for this organ. Accordingly, we performed a comprehensive characterization of murine cardiac mitochondrial phosphoproteome in the context of mitochondrial functional pathways. A platform using the complementary fragmentation technologies of collision-induced dissociation (CID) and electron transfer dissociation (ETD) demonstrated successful identification of a total of 236 phosphorylation sites in the murine heart; 210 of these sites were novel. These 236 sites were mapped to 181 phosphoproteins and 203 phosphopeptides. Among those identified, 45 phosphorylation sites were captured only by CID, whereas 185 phosphorylation sites, including a novel modification on ubiquinol-cytochrome c reductase protein 1 (Ser-212), were identified only by ETD, underscoring the advantage of a combined CID and ETD approach. The biological significance of the cardiac mitochondrial phosphoproteome was evaluated. Our investigations illustrated key regulatory sites in murine cardiac mitochondrial pathways as targets of phosphorylation regulation, including components of the electron transport chain (ETC) complexes and enzymes involved in metabolic pathways (e.g. tricarboxylic acid cycle). Furthermore, calcium overload injured cardiac mitochondrial ETC function, whereas enhanced phosphorylation of ETC via application of phosphatase inhibitors restored calcium-attenuated ETC complex I and complex III activities, demonstrating positive regulation of ETC function by phosphorylation. Moreover, in silico analyses of the identified phosphopeptide motifs illuminated the molecular nature of participating kinases, which included several known mitochondrial kinases (e.g. pyruvate dehydrogenase kinase) as well as kinases whose mitochondrial location was not previously appreciated (e.g. Src). In conclusion, the phosphorylation events defined herein advance our understanding of cardiac mitochondrial biology, facilitating the integration of the still fragmentary knowledge about mitochondrial signaling networks, metabolic pathways, and intrinsic mechanisms of functional regulation in the heart. Mitochondria are the source of energy to sustain life. In addition to their evolutionary origin as an energy-producing organelle, their functionality has integrated into every aspect of life, including the cell cycle, ROS 1The abbreviations used are:ROSreactive oxygen speciesAmBCammonium bicarbonateANTadenine nucleotide translocatorETCelectron transport chainETDelectron transfer dissociationQCR1ubiquinol-cytochrome c reductase core protein 1CIcomplex ICIIIcomplex IIIOMSSAOpen Mass Spectrometry Search AlgorithmGOgene ontologyFPRfalse positive rateGSK3glycogen synthase kinase 3. production, apoptosis, and ion balance (1.Johnson D.T. Harris R.A. Blair P.V. Balaban R.S. 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To meet tissue-specific functional demands, mitochondria acquire heterogeneous properties in individual organs, a first statement of their plasticity in function and proteome composition (1.Johnson D.T. Harris R.A. Blair P.V. Balaban R.S. Functional consequences of mitochondrial proteome heterogeneity.Am. J. Physiol. Cell Physiol. 2007; 292: C698-C707Crossref PubMed Scopus (94) Google Scholar, 6.Mootha V.K. Bunkenborg J. Olsen J.V. Hjerrild M. Wisniewski J.R. Stahl E. Bolouri M.S. Ray H.N. Sihag S. Kamal M. Patterson N. Lander E.S. Mann M. Integrated analysis of protein composition, tissue diversity, and gene regulation in mouse mitochondria.Cell. 2003; 115: 629-640Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar). The heterogeneity is evident even in an individual cardiomyocyte (7.Riva A. Tandler B. Loffredo F. Vazquez E. Hoppel C. Structural differences in two biochemically defined populations of cardiac mitochondria.Am. J. Physiol. Heart Circ. 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Systematic characterization of the murine mitochondrial proteome using functionally validated cardiac mitochondria.Proteomics. 2008; 8: 1564-1575Crossref PubMed Scopus (87) Google Scholar). The dynamics of the mitochondrial proteome manifest at multiple levels, including post-translational modifications, such as phosphorylation. Our investigative goal is to decode this organellar proteome and its post-translational modification in a biological and functional context. In cardiomyocytes, mitochondria are also constantly exposed to fluctuation in energy demands and in ionic conditions. The capacity of mitochondria to cope with such a dynamic environment is essential for the functional role of mitochondria in normal and disease phenotypes (8.Foster D.B. O'Rourke B. Van Eyk J.E. What can mitochondrial proteomics tell us about cardioprotection afforded by preconditioning?.Expert Rev. Proteomics. 2008; 5: 633-636Crossref PubMed Scopus (14) Google Scholar, 9.Weiss J.N. Korge P. Honda H.M. 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In mitochondria, ∼30 kinases and phosphatases have been identified thus far within the expected organellar proteome of a few thousand (3.Kislinger T. Cox B. Kannan A. Chung C. Hu P. Ignatchenko A. Scott M.S. Gramolini A.O. Morris Q. Hallett M.T. Rossant J. Hughes T.R. Frey B. Emili A. Global survey of organ and organelle protein expression in mouse: combined proteomic and transcriptomic profiling.Cell. 2006; 125: 173-186Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 4.Pagliarini D.J. Calvo S.E. Chang B. Sheth S.A. Vafai S.B. Ong S.E. Walford G.A. Sugiana C. Boneh A. Chen W.K. Hill D.E. Vidal M. Evans J.G. Thorburn D.R. Carr S.A. Mootha V.K. A mitochondrial protein compendium elucidates complex I disease biology.Cell. 2008; 134: 112-123Abstract Full Text Full Text PDF PubMed Scopus (1509) Google Scholar, 5.Zhang J. Li X. Mueller M. Wang Y. Zong C. Deng N. Vondriska T.M. Liem D.A. Yang J.I. Korge P. Honda H. Weiss J.N. Apweiler R. Ping P. 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Pedersen P.L. Robinson J. Murata M. Murphy A.M. Marbán E. Van Eyk J.E. Proteomic analysis of pharmacological preconditioning: novel protein targets converge to mitochondrial metabolism pathways.Circ. Res. 2006; 99: 706-714Crossref PubMed Scopus (118) Google Scholar, 13.Boja E.S. Phillips D. French S.A. Harris R.A. Balaban R.S. Quantitative mitochondrial phosphoproteomics using iTRAQ on an LTQ-Orbitrap with high energy collision dissociation.J. Proteome Res. 2009; 8: 4665-4675Crossref PubMed Scopus (87) Google Scholar, 14.Hopper R.K. Carroll S. Aponte A.M. Johnson D.T. French S. Shen R.F. Witzmann F.A. Harris R.A. Balaban R.S. Mitochondrial matrix phosphoproteome: effect of extra mitochondrial calcium.Biochemistry. 2006; 45: 2524-2536Crossref PubMed Scopus (215) Google Scholar, 16.Lee J. Xu Y. Chen Y. Sprung R. Kim S.C. Xie S. Zhao Y. Mitochondrial phosphoproteome revealed by an improved IMAC method and MS/MS/MS.Mol. Cell. Proteomics. 2007; 6: 669-676Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) demonstrated the prevalence as well as the dynamic nature of phosphorylation in cardiac mitochondria, suggesting that obtaining a comprehensive map of the mitochondrial phosphoproteome is feasible. In this study, we took a systematic approach to tackle the phosphorylation of murine cardiac mitochondrial pathways. We applied the unique strengths of both electron transfer dissociation (ETD) and collision-induced dissociation (CID) LC-MS/MS to screen phosphorylation events in a site-specific fashion. A total of 236 phosphorylation sites in 203 unique phosphopeptides were identified and mapped to 181 phosphoproteins. Novel phosphorylation modifications were discovered in diverse pathways of mitochondrial biology, including ion balance, proteolysis, and apoptosis. Consistent with the role of mitochondria as the major source of energy production under delicate control, metabolic pathways claimed one-third of phosphorylation sites captured in this analysis. To study molecular players steering mitochondrial phosphorylation, we probed the effects of calcium loading on phosphorylation. In addition, a number of kinases with previously unappreciated mitochondrial residence are suggested as potential players modulating mitochondrial pathways. Taken together, the cohort of novel phosphorylation events discovered in this study constitutes an essential step toward the full delineation of the cardiac mitochondrial phosphoproteome. All procedures were performed in accordance with the Animal Research Committee guidelines at UCLA and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. N-Dodecyl β-d-maltoside (Avanti Polar Lipids), protease inhibitor mixture (Roche Applied Science), fenvalerate and calyculin A (EMD Chemicals), sequencing grade modified trypsin (Promega), MonoTip TiO2 enrichment resin (GL Sciences) were obtained from the indicated sources. All other reagents were acquired from Sigma-Aldrich. Mitochondria were freshly isolated and purified from murine hearts as described previously (5.Zhang J. Li X. Mueller M. Wang Y. Zong C. Deng N. Vondriska T.M. Liem D.A. Yang J.I. Korge P. Honda H. Weiss J.N. Apweiler R. Ping P. Systematic characterization of the murine mitochondrial proteome using functionally validated cardiac mitochondria.Proteomics. 2008; 8: 1564-1575Crossref PubMed Scopus (87) Google Scholar). Briefly, 45 mouse hearts (8–10 weeks old, ICR strain, male) were pooled immediately after collection and homogenized as a biological replicate (isolation buffer: 250 mm sucrose, 1 mm EGTA, 20 mm HEPES, pH 7.5, protease inhibitor mixture, and phosphatase inhibitor mixtures). After removal of the nuclear fraction and tissue debris, the crude mitochondrial fraction was collected by centrifugation at 4000 × g for 20min. The resultant pellet was then resuspended with 19% Percoll solution in isolation buffer and slowly layered on top of a preformed discontinuous Percoll gradient, 30 and 60% (v/v), respectively. After 15-min centrifugation at 10,000 × g, purified mitochondria were retrieved at the interface of the two layers. All procedures were performed at 4 °C. A total of nine biological replicates were prepared and examined independently. Four independent biological replicates were analyzed by ETD with three technical replicates for each biological replicate. Five independent biological replicates were analyzed by CID with one technical replicate each. The purity as well as structural/functional integrity of each preparation was validated as described previously (10.Zhang J. Liem D.A. Mueller M. Wang Y. Zong C. Deng N. Vondriska T.M. Korge P. Drews O. Maclellan W.R. Honda H. Weiss J.N. Apweiler R. Ping P. Altered proteome biology of cardiac mitochondria under stress conditions.J. Proteome Res. 2008; 7: 2204-2214Crossref PubMed Scopus (48) Google Scholar). To examine the functional consequence of phosphorylation, we subjected cardiac mitochondria to calcium loading and characterized the impact of phosphorylation on ETC complex activities. Phosphorylation was modulated via the application of three distinct phosphatase inhibitors: fenvalerate (primarily targeting against PP2B family), calyculin A (primarily targeting against PP2A and PP1), and okadaic acid (primarily targeting against PP2A and PP1). Freshly isolated mitochondria (100 μg) were resuspended in reaction buffer (120 mm KCl, 10 mm HEPES, pH 7.4, 2.5 mm potassium phosphate, 5 mm glutamate, 5 mm malate, 0.1 mm ADP, 2.5 mm MgCl2, and 0.5 mg/ml BSA) and subsequently treated for 5 min with Ca2+ at a concentration of 40 μm at ambient temperature. In parallel groups, mitochondria were treated with Ca2+ in the presence of phosphatase inhibitor fenvalerate (100 nm), calyculin A (40 nm), or okadaic acid (300 nm). Subsequently, mitochondria were lysed with a hypotonic buffer (5 mm Tris-HCl, pH 7.4) and then subjected to biochemical assays. The specific activities of ETC complexes I (CI) and III (CIII) were measured in a 96-well microplate format (21.Wittig I. Braun H.P. Schägger H. Blue native PAGE.Nat. Protoc. 2006; 1: 418-428Crossref PubMed Scopus (1271) Google Scholar, 22.Zerbetto E. Vergani L. Dabbeni-Sala F. Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels.Electrophoresis. 1997; 18: 2059-2064Crossref PubMed Scopus (254) Google Scholar). Briefly, CI activity assays were initiated by mixing 100 μl of mitochondrial lysates (15 μg of protein) with 100 μl of CI assay buffer (5 mm Tris-HCl, pH 7.4, 0.2 mg/ml NADH, 2.0/ml nitro blue tetrazolium, 2 mm KCN, and 2 μm antimycin A). Assays were conducted at ambient temperature, and CI inhibitor (10 μm diphenyleneiodonium)-added groups served as negative controls to determine the inhibitor-insensitive background activity of the samples. The absorbance at 595 nm was monitored spectrometrically. Similarly, CIII activity assays were initiated by mixing mitochondrial lysates (20 μg of protein) with CIII assay buffer (50 mm sodium phosphate, pH 7.2, 50 μm cytochrome c, 0.1% BSA, 2 mm KCN, and 1 μg/ml rotenone) and 100 μm freshly reduced decylubiquinone (DBH2). CIII-specific inhibitor antimycin A (2 μm) was applied in negative control groups. CIII activity was monitored spectrometrically at 550 nm. Purified cardiac mitochondria were lysed with 0.5% N-dodecyl β-d-maltoside in the isolation buffer on ice. After centrifugation at 13,000 × g for 30 min, mitochondrial proteins were collected from the supernatant and then incubated with Laemmli sample buffer for 30 min. Denatured proteins were resolved by 12.5% SDS-PAGE (23.Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar). Subsequently, the gels were fixed and stained with colloidal Coomassie Blue. A total of 21 gel slices were excised from each lane. Gel slices were destained with 25% acetonitrile (ACN) in 50 mm ammonium bicarbonate (AmBC). Proteins were sequentially reduced with DTT and alkylated with iodoacetamide. Partial in-gel digestions (for ETD) were carried out with trypsin (1:100 ratio of trypsin versus protein in 50 mm AmBC, pH 8.0) at 37 °C for 3 h. For CID analysis, the proteins were digested overnight at 37 °C (1:50 ratio of trypsin versus protein). Peptides were extracted from gel slices with 50% ACN and 0.1% TFA and then with ACN only. Dried peptides were reconstituted with 100 μl of 50% ACN containing 0.1% TFA (v/v). Phosphopeptides were enriched using a TiO2 MonoTip as described previously (24.Pinkse M.W. Uitto P.M. Hilhorst M.J. Ooms B. Heck A.J. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns.Anal. Chem. 2004; 76: 3935-3943Crossref PubMed Scopus (829) Google Scholar). Briefly, the TiO2 MonoTip was sequentially equilibrated with three solutions: Solution 1, 50 mm AmBC containing 25% ACN; Solution 2, 50% ACN solution containing 5% TFA (v/v); and Solution 3, 50% ACN solution containing 0.1% TFA (v/v). Subsequently, peptides were loaded onto the tip with 50 repetitions and then rinsed for 10 cycles with 100 μl of 50% ACN containing 0.1% TFA (v/v). Finally, the enriched phosphopeptides were eluted sequentially with three buffers: Buffer 1, 100 μl of 50% ACN containing 5% TFA (v/v); Buffer 2, 50 mm AmBC supplemented with 25% ACN (pH 10.5); and Buffer 3, 1% aqueous ammonia solution. All eluted fractions were dried and subjected to mass spectrometric analyses. Enriched phosphopeptides were analyzed in parallel in both CID mode (LTQ-Orbitrap) and ETD mode (LTQ-XL-ETD). On-line peptide separation was achieved on a prepacked PicoFrit column (BioBasic C18, 75 μm × 10 cm, 5 μm, 300 Å; New Objective). The flow rate of chromatography was set to 5 μl/min for loading and 220 nl/min for separation (buffer A, 0.1% formic acid and 2% ACN; buffer B, 0.1% formic acid and 80% ACN). The resolving gradient was set as follows: 5–40% buffer B over 90 min to 100% buffer B over 10 min, maintained at 100% for 10 min, and then back to 0% buffer B. The mass spectrometer was operated in a data-dependent acquisition mode (25.Good D.M. Wirtala M. McAlister G.C. Coon J.J. Performance characteristics of electron transfer dissociation mass spectrometry.Mol. Cell. Proteomics. 2007; 6: 1942-1951Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar); one full MS scan was followed by five MS2 scans. Each biological sample was analyzed in three parallel technical replicates. In ETD mode, the default precursor charge state was set at 5+. The automatic gain control for fluoranthene was set to 4 × 105, and the ion/ion reaction time was set to a 100 ms. In CID mode, the LTQ-Orbitrap was operated in a data-dependent mode without using the FT module to assure the sensitivity and duty cycle of the analyses. An MS3 scan was triggered automatically when a neutral loss peak of 98, 49, or 32.7 m/z was detected within the top 10 most intense peaks in the MS2 spectrum. For both ETD and CID analyses, Bioworks version 3.3.1 was used for the .dta file generation. The minimal ion threshold was set to 10, intensity threshold was 1000, precursor tolerance was 1.4 amu, and mass range was 400–4500 Da. The ETD spectra were searched against the International Protein Index mouse database (version 3.34 with 51,424 entries) using the Open Mass Spectrometry Search Algorithm (OMSSA) (version 2.2.1) (26.Chi A. Huttenhower C. Geer L.Y. Coon J.J. Syka J.E. Bai D.L. Shabanowitz J. Burke D.J. Troyanskaya O.G. Hunt D.F. Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 2193-2198Crossref PubMed Scopus (498) Google Scholar). The following search parameters were set: partial enzymatic digestion (trypsin) permitting three missed cleavages; fixed modification of cysteine carboxyamidomethylation (+57 Da); and dynamic modifications of methionine oxidation (+16 Da) and serine, threonine, and tyrosine phosphorylation (+80 Da). The precursor charge states were set between 1+ and 6+. Precursor ion mass tolerance was set at ±1.5 Da; product ion tolerance was set at ±0.5 Da. The cutoff value of the filter threshold (E-value) was set to be better than 0.1, and then peptide spectra were subjected to manual inspection with the following criteria: Criterion 1, the charge state of the precursor was confirmed by characteristic precursor signals generated by charge stripping (27.Molina H. Horn D.M. Tang N. Mathivanan S. Pandey A. Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 2199-2204Crossref PubMed Scopus (465) Google Scholar); and Criterion 2, the mass spectrum was in agreement with the theoretical fragmentation profile obtained from ProteinProspector (28.Clauser K.R. Baker P. Burlingame A.L. Role of accurate mass measurement (+/−10 ppm) in protein identification strategies employing MS or MS/MS
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