Comparative Profiling of the Mammalian Mitochondrial Proteome: Multiple Aconitase-2 Isoforms Including N-formylkynurenine Modifications as Part of a Protein Biomarker Signature for Reactive Oxidative Species Christian Hunzinger,† Wojciech Wozny,† Gerhard P. Schwall,† Slobodan Poznanovic´ ,† Werner Stegmann,† Helmut Zengerling,† Rainer Schoepf,† Karlfried Groebe,† Michael A. Cahill,† Heinz D. Osiewacz,‡ Nora Ja1 gemann,§ Monika Bloch,§ Norbert A. Dencher,§ Frank Krause,§ and Andre´ Schrattenholz*,† ProteoSys AG, Carl Zeiss Strasse 51, 55129 Mainz, Germany, Institute of Molecular Biosciences, Molecular Developmental Biology, Johann Wolfgang Goethe-University, Marie-Curie-Str. 9, D-60439 Frankfurt, Germany. Physical Biochemistry, Department of Chemistry, Darmstadt University of Technology, Petersenstrasse 22, D-64287 Darmstadt, Germany Received November 4, 2005
The activity of mitochondria induces, as a byproduct, a variety of post-translational modifications in associated proteins, which have functional downstream consequences for processes such as apoptosis, autophagy, and plasticity; e.g., reactive oxygen species (ROS), which induce N-formyl-kynurenine from oxidized tryptophans in certain mitochondrial proteins which are localized in close spatial proximity to their source. This type of fast molecular changes has profound influence on cell death and survival with implications in a number of pathologies. The quantitative and differential analysis of bovine heart mitochondria by four 2D-PAGE methods, including 2D-PAGE with high-resolution IEF as first dimension, revealed that due to limited resolution, those methods employing blue native-, tricine-urea-, and 16BAC-PAGE as the first dimension are less applicable for the differential quantitative analysis of redundant protein spots which might give insight into post-translational modifications that are relevant in ageand stress-related changes. Moreover, 2D-PAGE with high resolution IEF was able to resolve a surprisingly large number of membrane proteins from mitochondrial preparations. For aconitase-2, an enzyme playing an important role in mitochondrial aging, a more thorough molecular analysis of all separable isoforms was performed, leading to the identification of two particular N-formylkynurenine modifications. Next to protein redundancy, native protein-protein interactions, with the potential of relating certain post-translational modification patterns to distinct oligomeric states, e.g., oxidative phosphorylation super complexes, might provide novel and (patho-) physiologically relevant information. Among proteins identified, 14 new proteins (GenBank entries), previously not associated with mitochondria, were found. Keywords: mitochondria • proteomics • two-dimensional polyacrylamide gel electrophoresis • IEF-SDS-PAGE • blue native-SDS-PAGE • tricine-urea-SDS-PAGE • 16-BAC-SDS-PAGE • quantitative differential protein expression • membrane proteins • reactive oxygen species • aconitase-2 • N-formylkynurenine • post-translational modifications
1. Introduction There have been a considerable number of studies using and comparing different proteomic methods for the mapping of mitochondrial proteins.1-9 The main focus of these studies and related database resources was essentially inventory: addressing how many mitochondrial proteins can be identified with a given method, and how mitochondrial proteins can be recognized as such. * To whom correspondence should be addressed. Fax: +49-6131-5019211. E-mail:
[email protected]. † ProteoSys AG. ‡ Johann Wolfgang Goethe-University, Frankfurt. § Darmstadt University of Technology. 10.1021/pr050377+ CCC: $33.50
2006 American Chemical Society
The mitochondrial proteome has the highest percentage of membrane proteins (nearly 80% of the inner membrane mass are made up of membrane proteins), and thus the controversial discussion of the efficiency of separation and mass spectrometrybased identification of membrane proteins has been intimately associated with these general mapping efforts.1-9 From a functional point of view, it is necessary to reach the optimal compromise between the resolution of a given method and the functional significance of fractionated proteins. For studies related to oxidative stress or aging, the mere identification of known intrinsic membrane proteins from the oxidative phosphorylation chain appears not to be sufficient. Instead the analysis of quantitative differences of post-translational isoforms of certain proteins may provide important information Journal of Proteome Research 2006, 5, 625-633
625
Published on Web 01/21/2006
research articles about underlying mechanisms. Here, we present data from a comparative analysis of four different SDS-2D-PAGE methods, comparing separations obtained by first dimension highresolution IEF,10 blue native-PAGE,11-14 tricine-urea-PAGE,15 and 16-BAC-PAGE,16 using a standard preparation of bovine heart mitochondria. Surprisingly, it was found that in terms of mitochondrial membrane proteins IEF is not particularly inferior to the other three methods, which moreover are shown to be less favorable when a differential quantification of multiple post-translationally modified spots is of interest. Although each of the methods provides unique protein identifications, the superior resolution of redundant protein spots by high-resolution IEF-SDS-PAGE proves to be highly advantageous, as we show for examples such as aconitase-2 and mitochondrial NADP+-dependent isocitrate dehydrogenase (IDPm), which both play important roles in mitochondrial aging17-20 and stress.21-25 Aconitase-2 is a fascinating case of an old, well-known Krebscycle protein, which has recently been shown to have completely unexpected functions in the integration of metabolic signals and maintenance of mtDNA,17,19 potentially mediated by ROS-induced post-translational modifications. The Nformylkynurenine modification detected in our study, a product of the dioxidation of tryptophan residues, has been suggested to occur at specific susceptible tryptophan residues, which are in close proximity to the mitochondrial source of reactive oxygen species.26
2. Materials and Methods 2.1. Preparation of Mitochondria from Bovine Heart. Bovine heart was excised from a freshly slaughtered animal and immediately stored on ice. About half of the total heart muscle tissue was processed shortly thereafter, the remaining tissue was cubed, shock frozen in liquid nitrogen, and stored at -80 °C for 6 days, before isolation of mitochondria. Bovine heart mitochondria were prepared using ice-cold buffers and equipments according to Smith27 by differential centrifugations without further purification, resuspended in 250 mM sucrose, 10 mM Tris-HCl, 1 mM Tris-succinate pH 7.8, 0.2 mM EDTA, 0.5 mM Pefabloc SC, frozen as aliquots in liquid nitrogen and stored at -80 °C until further usage (not later than 3-6 months after preparation). 2.2. Protein Separation. 2.2.1. High-Resolution IEF-SDSPAGE. High-Resolution IEF-SDS-PAGE was performed by 54 cm daisy chain serial IPG-IEF as described.10 Sample loading was by the rehydration to the pH 5-6 IPG overnight, using 300 µg protein per gel. Sample loading to the pH 4-5 and pH 6-9 IPGs was by electrophoretic migration from the pH 5-6 IPG. Briefly, shock-frozen samples were thawed at 25 °C and dissolved in 8 M Urea, 4% CHAPS, 0.1 M Tris pH 7.4. The volume was adjusted to 20 µL if necessary, followed by incubation of the sample at room temperature for 30 min with shaking at 1000 rpm in a Thermomixer comfort (Eppendorf). The samples were centrifuged for 5 min at 12 000 rcf and 25 °C, and the soluble extracts were collected by removing the supernatant. 2.2.2. Blue Native Electrophoresis/SDS-PAGE was performed as described13,14 with slight modifications to separate stable oxidative phosphorylation supercomplexes.13,28-30 100 µg aliquots (silver stain) or 400 µg aliquots (Coomassie R-250 stain) of mitochondrial protein suspension were pelleted and resuspended in 50 mM NaCl, 5 mM 6-aminocaproic acid, 10% glycerol, 50 mM imidazole pH 7.0, 1% digitonin (Calbiochem, 626
Journal of Proteome Research • Vol. 5, No. 3, 2006
Hunzinger et al.
high purity) to give a digitonin/protein ratio of 3 (w/w), followed by incubation on ice for 30 min. After centrifugation, the total supernatants were directly loaded on a 3-13% blue native-PAGE overlaid with a 3% stacking gel (0.15 × 1 cm gel wells).14 Running conditions: 100 V at 4 °C until the samples had completely entered the separating gel, subsequently electrophoresis was limited to 500 V, 15 mA until the dye front had reached the gel bottom (4-5 h). For second dimension tricineSDS-PAGE31 using 5% stacking gels and a 13% separating gel, blue native gel lanes were excised and incubated in a solution of 1% SDS and 1% mercaptoethanol at 20 °C for 1-2 h; 2Dgels were run overnight, at 80 V and eventually stained with Coomassie R-250 or silver nitrate.32,33 2.2.3. 16 BAC/SDS-PAGE. Two equilibration conditions were used: 5 min boiling in SDS buffer or equilibration by dipping the cut gel lane for 5 min in 3× SDS sample buffer. Other methods follow Hartinger et al.16 exactly; gels were run on a Hoefer SE600 gel electrophoresis system (18 × 16 cm) and were stained with silver nitrate.34 2.2.4. Tricine-Urea/Tricine SDS-PAGE. Protein separation of bovine heart mitochondrial proteins was performed by the tricine-urea/tricine method introduced by Scha¨gger and von Jagow31 and subsequently modified by Rais et al.15 Protein mixtures in loading buffer were applied to a 10% acrylamide, 6 M urea tricine gel (Hoefer). The gel was electrophoresed overnight at 70 V and 15 °C. For the second dimension, 2 lanes were cut and placed on top of 16% tricine gel (Hoefer). The gel pieces with lanes were covered with 2% SDS, 50 mM Tris pH 6.8, 12% glycerol, 0.02% Coomassie, and 20 mM DTT. The gels were run at 75 V overnight on a Hoefer SE600 gel electrophoresis system (18 × 16 cm). Silver staining was performed as described.34 2.3. Protein Identification. Analysis on the respective gel images was performed to detect protein spots using the Pic/ Greg software package of the Fraunhofer Gesellschaft in Sankt Augustin (http://www.fit.fraunhofer.de/projekte/greg/index_en.xml). Protein spots were included if spot volume was at least 1.e10 (in units used by Greg) and background intensity was at least four times less than spot intensity. All spots were checked manually using the Pic program and accepted or rejected depending on their actual appearance. In this way, artifacts were sorted out and obvious spots that did not quite match the automatic selection criteria could be manually added to the selection. Spots were robotically excised from the rehydrated silver-stained preparative gel and subjected to MALDI-TOF peptide mass fingerprinting, as described.35,36 Briefly, selected spots were excised from the gel by a picking robot (ProPick, Genomic Solutions Ltd, Huntingdon, UK) and proteins in gel pieces were trypsin digested using a ProGestrobot (Genomic Solutions Ltd, Huntington, UK). A ProMS-robot (Genomic Solutions Ltd, Huntingdon, UK) was used to apply samples for MALDI-TOF mass spectrometry onto an anchor target (Bruker, Bremen, Germany). Mass spectra of peptide ions were obtained using an Ultraflex MALDI time-of-flight (TOF) mass spectrometer (Bruker, Bremen, Germany) in reflector mode within a mass range from m/z 800 to 4000. The MS spectra were internally calibrated (trypsin autodigestion peptides m/z 842.50, 1045.56, 2211.10, and 2283.17) and annotated automatically using in-house software. The resulting peptide mass fingerprints were searched against the non redundant NCBI Protein Sequence Database using Mascot Server software v. 1.8. (Matrix Science, London, UK), with the following standard settings: taxonomy: mammalia, allowed missed
research articles
Profiling of the Mammalian Mitochondrial Proteome
Table 1. Protein Spot Numbers Detected and Identified via MALDI-TOF Peptide Mass Fingerprinting after Separation of the Same Bovine Heart Mitochondria Preparation by the Indicated Gel Electrophoresis Systemsa
spots detected spots picked proteins identified nonredundant proteins exclusive proteins a
IEF/ PAGE
blue native
tricineurea
16-BAC
1008 745 400 123 80
172 160 152 71 23
117 80 55 41 10
121 106 67 30 6
Spots were detected using the GREG software, as described above.
Table 2. Summary of Distribution of Nonredundant Protein Identifications Across the Four Methodsa Distribution of Identified Proteins over Methods IEF-PAGE
x x x x x x x
Figure 1. Separation of bovine heart mitochondria by four different SDS-2D-PAGE methods; Images of silver-stained representative gels are shown as follows: 54 cm IPG-IEF, covering pH 4-9 (A), tricine-urea/tricine (B), blue native/SDS-2D-PAGE of digitonin-solubilized mitochondria (C), and 16-BAC/SDS-2DPAGE (D). For details see ‘Materials and Methods’, sections 2.2.1-2.2.4.
cleavages: 1, variable modifications: oxidation (Met); mass tolerance: 75 ppm. All protein identifications included here showed significant Mascot Scores (>80). Protein modification analysis was performed employing a proprietary in-house software solution. Briefly, unmatched m/z values of every protein identified were searched against the theoretical masses of peptides comprising selected ROS-related modifications. 2.4. Protein Hydropathy. As an indicator for protein hydrophobicity and protein solubility the Grand Average of Hydropathy (GRAVY) score was calculated according to Kyte and Doolittle.37 Briefly, the GRAVY score was calculated as the sum of hydropathy values of all amino acids in a protein, divided by the total number of amino acids in the sequence.
3. Results For the comparative proteomic study presented here a superior preparation of bovine heart mitochondria was used with a particularly high yield of oxidative phosphorylation supercomplexes, as can be seen from Figure 1C in the context of previously published data.13,28 As shown in Figure 1, with representative gels for each method, the resolution of an identical sample from bovine heart mitochondria by high resolution 54 cm IEF-SDS-PAGE is far superior to all other methods. Proteins were separated by 2D-PAGE using the following separation systems as first dimension: high resolution 54 cm IPG-IEF10 (Figure 1A), tricine-urea/tricine15 (Figure 1B), blue native,12-14 (Figure 1C), and 16-BAC16 (Figure 1D). The number of spots detected from the same preparation of bovine heart mitochondria was 1008 with high-resolution IEF-SDS-PAGE, 117 with tricine-urea/ tricine-SDS-PAGE, 172 with blue native/SDS-PAGE and 121
blue native
tricine-urea
x x found in all 4 methods x x x x x x found in only 3 methods x x
16-BAC
joint ID’s
x
6 6 11 4 1 1 17 11 5 2 5 8 1 32 80 23 10 6 119 174
x x x
x x x
x
x found in only 2 methods
x x
x x x x found in only 1 method Total a
IEF/PAGE was performed with high resolution standard IPG’s covering the pI range 4-9; blue native, tricine-urea/tricine and 16-BAC electrophoretic methods were performed as described. The second dimension was a 12% polyacrylamide gel, using a standard SDS-PAGE according to Laemmli, or tricine-gels as described in the ‘Materials and Methods’ section.
with 16-BAC/SDS-PAGE, respectively. No comprehensive effort was undertaken to identify all visible spots, but only those spots with medium to high abundance, which where easily accessible to automated procedures of detection, subsequent enzymatic digestion and MALDI-TOF-based identification. The focus was on uniform conditions for all four methods, not on being comprehensive. In total, 674 from 1091 picked spots (out of 1418 detected spots) were identified using automated standard procedures. A total of 174 nonredundant proteins were found across all methods. A summary of the distribution of corresponding proteins is shown in Table 1. We are sure that many more proteins could have been identified from each one of the methods here with appropriate effort,15,38,39 but we wanted to compare yields of nonredundant and in particular redundant (i.e., post-translationally modified) proteins. Nonredundant proteins which were only detected in one of the methods were called “exclusive” proteins. The comparison of the four methods with regard to potential effects of ROS and aging to the complexity of protein patterns is on one level a matter of resolution. As shown and specified in Table 2, there are only 6 nonredundant proteins found by all four methods, 17 found in three methods, 32 found in only 2 methods and the remaining 119 found by only one method Journal of Proteome Research • Vol. 5, No. 3, 2006 627
research articles
Hunzinger et al.
Table 3. Redundancy Analysis of the Six Proteins Identified by All Methods and Their Respective Pattern of Isoformsa
theoretical
IEF-PAGE
blue native
tris/tricine urea
16-BAC
accession
description
MW
pI
MW
pI
MW
MW
MW
gi|27806769
aconitase 2, mitochondrial [Bos taurus]
87606
8.1
93000 93000 92000 92000 92000 92000 92000 92000 91000
6.5 6.55 6.6 6.7 6.8 6.9 7.0 7.1 7.2
93000 88000
66000
92000 83000
gi|27807143
ubiquinol-cytochrome c reductase core protein II [Bos taurus]
48473
9.1
45000 45000 45000 45000
6.6 6.8 7.1 7.3
53000 51000 50000
41000 41000
45000 43000 43000 41000
gi|27807355
NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75kDa (NADH-coenzyme Q reductase) precursor [Bos taurus]
79391
6.1
80000 80000 79000 79000 79000 78000 77000 76000
5.15 5.2 5.25 5.3 5.35 5.4 5.45 5.5
77000
61000 59000
83000 74000
gi|28461205
isocitrate dehydrogenase 2 (NADP+), mitochondrial [Bos taurus]
52209
9.1
44000 44000
8.1 8.3
50000
42000 41000
54000 41000
gi|27807237
ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit [Bos taurus]
59683
9.7
56000 56000 55000 55000
7.2 7.4 6.9 7.0
61000 60000 59000 54000
66000 50000 50000
55000 53000 55000 52000
gi|28461221
ATP synthase, H+ transporting, mitochondrial F1 complex, beta subunit [Bos taurus]
56249
4.8
54000 53000 53000 53000 50000 50000
4.95 5.0 5.05 5.1 4.9 4.95
57000 56000 46000 39000
49000 48000 46000 45000
52000 50000 49000 40000 39000 38000 27000 26000
a Which have been identified under conditions described: resolution of different isoforms, with potential underlying functional molecular differences is best in high resolution IEF-PAGE. Additional isoforms, some of them apparently truncated are revealed by the other methods, in particular 16-BAC. Theoretical molecular weights (MW) and isoelectric points (pI) as calculated from amino acid sequences of open reading frames according to the GeneBank accession numbers (accession) are compared to apparent experimental MW and pI found across the different methods. No systematic effort was undertaken to identify all isoforms potentially detectable by one of the methods, but rather an unbiased and uniform automated procedure of spot detection and identification was applied in a standardized fashion to obtain a comparison of said methods.
(the vast majority of these were found in IEF/PAGE). A table summarizing all 174 nonredundant identified proteins, and attributing them to the respective methods by which they were identified, is provided as Supporting Information (Table S1). As shown in Table 3, among the six nonredundant proteins identified by all four methods, there are two subunits of the ATP-synthase complex, two further proteins from the respiratory chain and in particular two citric acid cycle enzymes: aconitase-2 and NADP+-dependent isocitrate dehydrogenase (IDPm). The latter two have recently been implicated in oxidative stress regulation and related diseases.17-19,21-25,40 IDPm is crucial because it is one of the main sources of NADPH generation, a cofactor in many biosynthetic pathways and particularly in the regeneration of reduced glutathione that is critically important in cellular defense against oxidative damage.22-25,40 Significantly, IDPm has also been implicated in age-related mechanisms.25 For IDPm, we observed a variety of 628
Journal of Proteome Research • Vol. 5, No. 3, 2006
isoforms across all methods; however their distinct molecular features were not investigated further. Since a uniform test sample preparation of bovine heart mitochondria was employed in all cases, it is interesting to explore the number and nature of redundant spots and their respective potential relevance for ROS-mediated posttranslational modifications. In this regard, we chose to focus on a more detailed molecular analysis of the 14 isoforms of aconitase-2, which were found mainly by IEF/PAGE. This iron-sulfur protein plays a unique role in age-related maintenance of mtDNA and has a unique pattern of expression during aging as compared to other mitochondrial proteins.41 A thorough analysis of the sequence coverage of aconitase-2 peptides in MALDI-TOF spectra from peptide mass fingerprints (PMF) revealed no clear indications of the nature of differences observed in terms of sequence coverage or posttranslational modifications.
Profiling of the Mammalian Mitochondrial Proteome
Figure 2. Representative MALDI spectrum showing the unmodified and potentially N-formylkynurenine modified tryptic peptides 371-378 (A) and 657-671 (B) of aconitase-2 (gi27806769). The signal intensity the unmodified peptide 371-378 is lower than the modified one, which suggests that tryptophan 373 is predominantly oxidized. The opposite is true for peptide 657-671, leading to the conclusion that tryptophan 657 is predominantly nonmodified.
Two potential N-formylkynurenine modifications of two doubly oxidized tryptophan residues were identified. The peak (MH+) at 1017.501 can be assigned to peptide 371-378 (aconitase-2 - gi27806769) with formylkynurenine indicated as fW (sequence is EGfWPLDIR); a further peak at MH+ ) 1699.756 can be assigned to peptide 657-671 (fWVVIGDENYGEGSSR) from the same protein. Although significant MS/ MS data could not be obtained for unambiguous verification, these peaks most likely represent the N-formylkynurenine modified peptides, since both modifications have been identified previously,26 making them candidates for a functional and kinetic age-related screening. For both sequences, we also find the unmodified peptides (Figure 2): The ratios of unmodified versus modified peptides are always 0 for peptide 657-671 (tryptophan 657 is predominantly nonmodi-
research articles
Figure 3. Histogram of GRAVY scores and GRAVY score distributions of proteins for each of the four methods. In (A), the GRAVY score distribution of the four methods is shown, absolute counts are plotted versus GRAVY scores: light blue is the graph for 2D-IEF-SDS-PAGE; dark blue is for blue native PAGE as first dimension; the pink line is is for tricine-urea/tricine method according to Rais et al.; and the yellow line is for the 16-BAC method. In (B), the color coding is the same, but now the percentage of the respective total counts is plotted against GRAVY scores. Surprisingly, on the level of mere abundance of hydrophilic or hydrophobic amino acids in the proteins identified, there appears to be no particular bias of one of the four methods for hydrophobic proteins. The histogram distributions of the GRAVY scores, by sorting the individual GRAVY scores of the identified proteins of each method according to amplitude, are shown in (C-F): (C) is the histogram for blue native-PAGE as first dimension as described in the methods section, (D) is for the tricine-urea/tricine method, (E) is for 16-BAC and 3F is for 2 D-IEF-SDS-PAGE.
fied). A further possible aconitase modification, found predominantly for two IEF/PAGE isoforms (92 kD, pI 6.8 and 6.9), is a carbonylation (malondialdehyde) at lysine 700 (peptide MH+ ) 916.489, amino acids 694-700, IHETNLmK), which is in line with published data implicating aconitase as a target of malondialdehyde modification.18,41 Additionally to resolution, the question of which of the four methods would be most suitable for the analysis of ROS- or age-related protein changes, can be decided by the very nature of the identified proteins. Standard 2D IEF-SDS-PAGE is in many cases considered to be very problematic with regard to integral membrane proteins, which is essentially due to their generally poor solubilization in IEF-buffers.42 The 16-BAC16-PAGE, tricine-urea/tricineJournal of Proteome Research • Vol. 5, No. 3, 2006 629
research articles
Hunzinger et al.
Table 4. Identified Proteins with a Positive Grand Average Hydropathy (GRAVY) Score as an Indicator for Protein Hydrophobicity and Solubilitya GRAVY| score
accession
pI
MW
description
gi|27806831 gi|61866502
8.3 10.2
113780 11409
gi|54310686 gi|27805907
4.5 8.5
26004 27655
gi|61822295
10.1
15587
gi|61863144
5.8
16782
gi|61867324
8.8
35646
gi|27805917 gi|32189340 gi|27806561 gi|61842751
7.7 10.4 6.5 7.5
17490 32946 37487 18936
gi|61553497
8.8
32286
gi|28461221
4.8
56249
gi|448581 gi|27806307
5.3 10.6
36828 23335
gi|61831678
6.9
46913
gi|59858383 gi|30583661 gi|3660252
6.5 5.5 4.8
36674 29786 51397
gi|51247981
4.8
51673
gi|3660253
4.8
51449
gi|11514059
4.8
51655
gi|61858852
9.1
45768
nicotinamide nucleotide transhydrogenase predicted: similar to ATP synthase, H+ transporting, mitochondrial F0 complex, subunit g cytochrome c oxidase subunit II [Bos grunniens] hydroxyacyl-Coenzyme A dehydrogenase, type II hydroxyacyl-Coenzyme A [Bos taurus] predicted: similar to ES1 protein homolog, mitochondrial precursor (Protein KNP-I) (GT335 protein), partial [Bos taurus] predicted: similar to FabG-like protein, partial [Bos taurus] predicted: similar to malate dehydrogenase, mitochondrial precursor [Bos taurus] histidine triad nucleotide binding protein 2 solute carrier family 25 member 4 [Bos taurus] lactate dehydrogenase B [Bos taurus] predicted: similar to serine/threonine/tyrosine kinase 1, partial [Bos taurus] mitochondrial short-chain enoyl-coenzyme A hydratase 1 precursor [Bos taurus] ATP synthase, H+ transporting, mitochondrial F1 complex, beta subunit [Bos taurus] pyruvate dehydrogenase:SUBUNIT)beta mitochondrial ATP synthase, O subunit [Bos taurus] predicted: similar to dihydrolipoamide dehydrogenase, precursor, partial [Bos taurus] lactate dehydrogenase B [Bos taurus] prohibitin [Homo sapiens] chain E, the structure of bovine F1-Atpase covalently inhibited with 4-chloro-7nitrobenzofurazan chain F, beryllium fluoride inhibited bovine F1-atpase chain F, the structure of bovine F1-Atpase covalently inhibited with 4-chloro-7nitrobenzofurazan chain D, bovine F1-atpase inhibited by Dccd (dicyclohexylcarbodiimide) predicted: similar to mitochondrial acetoacetyl-CoA thiolase, partial [Bos taurus]
IEF‘PAGE
0.305 0.230
blue native
tricineurea
x x
16-BAC
x
0.225 0.183
x
x
0.124
x
0.115
x
0.108
x
0.082 0.076 0.067 0.065
x
0.061
x
x
0.042
x
x
x
0.031 0.026
x x
x
x
x
x
x
x
x x
0.026
x
0.026 0.024 0.011
x x
x
0.011
x
x
x
x
x
0.008
x x
x
x x
0.007
x
x
0.006
x
x
x
x
a The successful identification of a given protein from one or more of the investigated separation methods is indicated. The proteins are sorted according to their GRAVY score, starting with the more hydrophobic and less soluble proteins. All proteins identified are from Bos taurus or Bos sp.
PAGE15 and blue native-PAGE43 have received particular attention as successful alternatives in some cases. One method of assessing the hydrophobicity of protein employs GRAVY plots,37 which provide the average hydropathy score for all the amino acids in a protein. Highly hydrophobic integral membrane proteins typically have higher GRAVY scores (>0) however this alone cannot reliably predict the membrane localization without the help of hydropathy plots.37 As shown in Figure 3 and Table 4, under conditions described, without any other prior enrichment than preparation of mitochondria by differential centrifugation (to obtain a global view of mitochondrial proteins) there are, as expected, slight advantages for hydrophobic proteins when using blue native and 16BAC-PAGE (blue and yellow graphs in Figure 3A,B; 3C,E). Proteins with GRAVY scores above 0.2 were almost exclusively found in blue native gels, including subunit II of cytochrome oxidase. Among proteins with GRAVY scores between 0.1 and 0.2, the standard 2D-IEF-PAGE performs notably well, including hydroxyacyl-Coenzyme A dehydrogenase (0.183), which has been reported previously using 2D-PAGE.44 Notably, 2D-IEF630
Journal of Proteome Research • Vol. 5, No. 3, 2006
PAGE detected more protein species than any other method, and a comparable number of species with Gravy scores >0 as any other method. No further analysis of transmembrane spanning regions of any of the proteins found was performed, because this was beyond the focus of this study. An overview of all proteins with GRAVY scores higher than 0 and the methods where they were found is given in Table 4. Among the 174 nonredundant proteins (GenBank entries) found in this study, to our knowledge 14 have not been previously associated with mitchondria, which are the following proteins from Table S1, #56, 120, 139 (purine nucleoside phosphorylase, different subunits), #58 (sarcoplasmic reticulum 53K glycoprotein precursor), # 65 (cytosol aminopeptidase ) leucine aminopeptidase), # 75 (histidine triad nucleotide binding protein 2), #83 (anti-oxidant protein 2 ) non-selenium glutathione peroxidase ) acidic calcium-independent phospholipase A2), #108 (casein alpha-S1), #119 (CGI-105 protein), #135 (coiled-coil-helix-coiled-coil-helix domain containing 3), #140 (retinol binding protein 1), #155 (similar to serine/
research articles
Profiling of the Mammalian Mitochondrial Proteome
threonine/tyrosine kinase 1), #157 (similar to hypothetical protein, gi|61850233) and #169 (similar to QIL1 protein).
4. Discussion In summary, mammalian mitochondria are predicted to have approximately 1500-4000 different proteins7,45,46 in contrast to the 680 that have been found so far.7,8 In our study, 1008 spots have been detected, from the 54 cm IEF/2D-PAGE experiment. 745 of these have been picked, digested and analyzed by MALDI-TOF peptide mass fingerprint resulting in identification of 123 products of different genes. The corresponding numbers for blue native-PAGE are 172 detected and 71 identified, for tricine-urea/tricine-PAGE 117 detected and 41 identified, for 16-BAC-PAGE 121 detected and 30 identified non redundant proteins, respectively. The scope of our study was not to extend the current inventory of mitochondrial proteins, or to confirm existing data compilations, but to assess the best technical compromise in terms of defining an experimental window likely to reflect a maximum of information about corresponding post-translational modifications in relevant proteins, frequently occurring as ROS- and age-related changes. Therefore we considered and quantified not only resolution, and thus applicability to quantitative differential display and statistical analysis of a method, but also the bias toward detecting certain types of proteins. In a noncomprehensive fashion, and in a selected experimental window, the comparison of four 2D-PAGE methods demonstrated the superiority of 2D-IEF-SDS-PAGE over all other methods in terms of number of resolved spots and separation of protein isoforms. This finding is not surprising, but implies (because the same uniform preparation of standard bovine heart mitochondria was used in all experiments) that the other methods are poorly applicable for a differential gel-based study, that does not involve pre-electrophoretic isotopic labeling of the samples. Multiple nonresolved protein species in a single position would obscure isoform assignments and their quantification by isotopic labeling; a problem associated with all lower resolution methods, including most LC-MS approaches.42,47 However, 2D-blue native-PAGE allows the examination of protein redundancy in terms of native proteinprotein interactions, with the potential of relating certain posttranslational modification patterns to distinct oligomeric states of mitochondrial proteins, like individual oxidative phosphorylation complexes and their super complexes, which might provide novel and (patho-) physiologically relevant information. Beyond resolution, the biological relation of the proteins identified to ROS- and age-related processes and mitochondrial or intrinsic apoptotic mechanisms48,49 was of interest for this study. At one level, hydrophobic integral membrane proteins appear to be relevant in a variety of studies concerning mitochondrial proteomes, and recently GRAVY scores have been frequently used to address the coverage of these proteins by various proteomic methods.50-52 Under our given conditions, without using prior fractionations in order to obtain sufficient coverage of imported/exported proteins that potentially include signaling of age-related ROS-effects to the cytosol (especially components of the intrinsic or mitochondrial apoptotic pathway), essentially all methods were able to resolve MPTP proteins, like e.g., ANT-1 (solute carrier family 25, member 4, this study)53 whereas the AIF here could exclusively be identified in the 16-BAC method. Together with recent, more comprehensive reports concerning single methods, like e.g.,
blue native-PAGE, we can be confident to adequately address apoptosis-related molecular events.54 It is well-known, that some mitochondrial citric acid cycle enzymes are altered during the normal aging process. This is true for both NADP+-dependent isocitrate dehydrogenase (IDPm),25 which was found in several isoforms in this study, and in particular for aconitase-2, which has been implicated as playing a key role as an age-related marker in a variety of organisms and organs.17,20,55,56 In flight muscle mitochondria of house flies of different ages, aconitase was the only enzyme detected that exhibited altered activity during aging.41 It has moreover been suggested that post-translational oxidative modifications occur during the aging process and thus result in functional alterations. Specifically aconitase-2 appeared to be selectively carbonylated during aging.20 Likewise, adducts with malondialdehyde (MDA), a product of lipid peroxidation, were among those found for aconitase, very long chain acyl coenzyme A dehydrogenase, ATP synthase, and alphaketoglutarate dehydrogenase.57 The decreasing activities of these proteins have also been implicated in progress of agingrelated processes and all of them were identified in this study, indeed we observed candidate MDA adducts. The ferric iron of aconitase is moreover a target of signaling via nitric oxide and its metabolites peroxynitrite and the nitroxyl anion.58-60 Since mitochondria provide both a major source of oxidants and a target for their damaging effects, since mitochondrial oxidative stress is thought to be a major cause of cell aging, and since oxidative damage in aging is particularly high in mitochondrial DNA and aconitase,61-63 we rather paid special attention to the isoform pattern of this enzyme. The separation of different aconitase isoforms on IEF-SDS-PAGE now allows using this method for age-related investigations by employing mitochondrial preparations from individuals of different age, different genetic background or from those subjected to special dietary or exercise regimens.
Acknowledgment. This work was supported by EC FP6 Contract No. LSHM-CT-2004-512020; (http://www.mimage.unifrankfurt.de). This publication reflects only the authors’ views. The EC is not liable for any use that may be made of the information herein. Note Added after ASAP Publication: This paper was originally published on the Web (01/21/2006) with formatting errors in Table 3 and missing several decimal points in Table 4. The version posted to the Web 01/27/2006 and in print is correct.
Supporting Information Available: A table summarizing all 174 nonredundant identified proteins, and attributing them to the respective methods by which they were identified (Table S1). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Bailey, S. M.; Landar, A.; Darley-Usmar, V. Mitochondrial proteomics in free radical research. Free Radic Biol. Med. 2005, 38, 175-188. (2) Fukada, K.; Zhang, F.; Vien, A.; Cashman, N. R.; Zhu, H. Mitochondrial proteomic analysis of a cell line model of familial amyotrophic lateral sclerosis. Mol. Cell Proteomics 2004, 3, 12111223. (3) Gabaldon, T.; Huynen, M. A. Shaping the mitochondrial proteome. Biochim Biophys. Acta 2004, 1659, 212-220.
Journal of Proteome Research • Vol. 5, No. 3, 2006 631
research articles (4) Jin, J.; Meredith, G. E.; Chen, L.; Zhou, Y.; Xu, J.; Shie, F. S.; Lockhart, P.; Zhang, J. Quantitative proteomic analysis of mitochondrial proteins: relevance to Lewy body formation and Parkinson’s disease. Brain Res. Mol. Brain Res. 2005, 134, 119138. (5) Lopez, M. F.; Melov, S.; Johnson, F.; Nagulko, N.; Golenko, E.; Kuzdzal, S.; Ackloo, S.; Mikulskis, A. Proteomic analysis of mitochondrial proteins. Int. Rev. Neurobiol. 2004, 61, 31-48. (6) Sickmann, A.; Reinders, J.; Wagner, Y.; Joppich, C.; Zahedi, R.; Meyer, H. E.; Schonfisch, B.; Perschil, I.; Chacinska, A.; Guiard, B.; Rehling, P.; Pfanner, N.; Meisinger, C. The proteome of Saccharomyces cerevisiae mitochondria. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13207-13212. (7) Taylor, S. W.; Fahy, E.; Zhang, B.; Glenn, G. M.; Warnock, D. E.; Wiley, S.; Murphy, A. N.; Gaucher, S. P.; Capaldi, R. A.; Gibson, B. W.; Ghosh, S. S. Characterization of the human heart mitochondrial proteome. Nat. Biotechnol. 2003, 21, 281-286. (8) Gaucher, S. P.; Taylor, S. W.; Fahy, E.; Zhang, B.; Warnock, D. E.; Ghosh, S. S.; Gibson, B. W. Expanded coverage of the human heart mitochondrial proteome using multidimensional liquid chromatography coupled with tandem mass spectrometry. J. Proteome Res. 2004, 3, 495-505. (9) Alonso, J.; Rodriguez, J. M.; Baena-Lopez, L. A.; Santaren, J. F. Characterization of the Drosophila melanogaster Mitochondrial Proteome. J. Proteome Res. 2005, 4, 1636-1645. (10) Poznanovic, S.; Schwall, G.; Zengerling, H.; Cahill, M. A. Isoelectric focusing in serial immobilized pH gradient gels to improve protein separation in proteomic analysis. Electrophoresis 2005, 26, 3185-3190. (11) Scha¨gger, H.; von Jagow, G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem 1991, 199, 223-231. (12) Brookes, P. S.; Pinner, A.; Ramachandran, A.; Coward, L.; Barnes, S.; Kim, H.; Darley-Usmar, V. M. High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of mitochondria and signaling complexes. Proteomics 2002, 2, 969-977. (13) Krause, F.; Reifschneider, N. H.; Goto, S.; Dencher, N. A. Active oligomeric ATP synthases in mammalian mitochondria. Biochem. Biophys. Res. Commun. 2005, 329, 583-590. (14) Scha¨gger, H. Blue-native gels to isolate protein complexes from mitochondria. Methods Cell Biol. 2001, 65, 231-244. (15) Rais, I.; Karas, M.; Scha¨gger, H. Two-dimensional electrophoresis for the isolation of integral membrane proteins and mass spectrometric identification. Proteomics 2004, 4, 2567-2571. (16) Hartinger, J.; Stenius, K.; Hogemann, D.; Jahn, R. 16-BAC/SDSPAGE: a two-dimensional gel electrophoresis system suitable for the separation of integral membrane proteins. Anal. Biochem. 1996, 240, 126-133. (17) Shadel, G. S. Mitochondrial DNA, aconitase ‘wraps’ it up. Trends Biochem. Sci. 2005, 30, 294-296. (18) Yarian, C. S.; Rebrin, I.; Sohal, R. S. Aconitase and ATP synthase are targets of malondialdehyde modification and undergo an agerelated decrease in activity in mouse heart mitochondria. Biochem. Biophys. Res. Commun. 2005, 330, 151-156. (19) Chen, X. J.; Wang, X.; Kaufman, B. A.; Butow, R. A. Aconitase couples metabolic regulation to mitochondrial DNA maintenance. Science 2005, 307, 714-717. (20) Yan, L. J.; Levine, R. L.; Sohal, R. S. Oxidative damage during aging targets mitochondrial aconitase. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 11168-11172. (21) Yang, E. S.; Yang, J. H.; Park, J. E.; Park, J. W. Oxalomalate, a competitive inhibitor of NADP+ -dependent isocitrate dehydrogenase, regulates lipid peroxidation-mediated apoptosis in U937 cells. Free Radic Res. 2005, 39, 89-94. (22) Jo, S. H.; Son, M. K.; Koh, H. J.; Lee, S. M.; Song, I. H.; Kim, Y. O.; Lee, Y. S.; Jeong, K. S.; Kim, W. B.; Park, J. W.; Song, B. J.; Huh, T. L. Control of mitochondrial redox balance and cellular defense against oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase. J. Biol. Chem. 2001, 276, 16168-16176. (23) Kil, I. S.; Lee, J. H.; Shin, A. H.; Park, J. W. Glycation-induced inactivation of NADP(+)-dependent isocitrate dehydrogenase: implications for diabetes and aging. Free Radic Biol. Med. 2004, 37, 1765-1778. (24) Kil, I. S.; Park, J. W. Regulation of mitochondrial NADP+dependent isocitrate dehydrogenase activity by glutathionylation. J. Biol. Chem. 2005, 280, 10846-10854. (25) Kil, I. S.; Lee, Y. S.; Bae, Y. S.; Huh, T. L.; Park, J. W. Modulation of NADP(+)-dependent isocitrate dehydrogenase in aging. Redox Rep. 2004, 9, 271-277.
632
Journal of Proteome Research • Vol. 5, No. 3, 2006
Hunzinger et al. (26) Taylor, S. W.; Fahy, E.; Murray, J.; Capaldi, R. A.; Ghosh, S. S. Oxidative post-translational modification of tryptophan residues in cardiac mitochondrial proteins. J. Biol. Chem. 2003, 278, 19587-19590. (27) Smith, A. L. Preparations, properties and conditions for assay of mitochondria: slaughterhouse material, small scale. Methods Enzymol. 1967, 10, 81-86. (28) Scha¨gger, H.; Pfeiffer, K. Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. Embo J. 2000, 19, 17771783. (29) Krause, F.; Scheckhuber, C. Q.; Werner, A.; Rexroth, S.; Reifschneider, N. H.; Dencher, N. A.; Osiewacz, H. D. Supramolecular organization of cytochrome c oxidase- and alternative oxidasedependent respiratory chains in the filamentous fungus Podospora anserina. J. Biol. Chem. 2004, 279, 26453-26461. (30) Krause, F.; Reifschneider, N. H.; Vocke, D.; Seelert, H.; Rexroth, S.; Dencher, N. A. “Respirasome”-like supercomplexes in green leaf mitochondria of spinach. J. Biol. Chem. 2004, 279, 4836948375. (31) Scha¨gger, H.; von Jagow, G. Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 1987, 166, 368379. (32) Blum, H.; Beier, H.; Gross, H. J. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 1987, 8, 93-99. (33) Rabilloud, T.; Carpentier, G.; Tarroux, P. Improvement and simplification of low-background silver staining of proteins by using sodium dithionite. Electrophoresis 1988, 9, 288-291. (34) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 1996, 68, 850-858. (35) Cahill, M. A.; Wozny, W.; Schwall, G.; Schroer, K.; Holzer, K.; Poznanovic, S.; Hunzinger, C.; Vogt, J. A.; Stegmann, W.; Matthies, H.; Schrattenholz, A. Analysis of relative isotopologue abundances for quantitative profiling of complex protein mixtures labeled with the acrylamide/D3-acrylamide alkylation tag system. Rapid Commun. Mass Spectrom. 2003, 17, 1283-1290. (36) Sommer, S.; Hunzinger, C.; Schillo, S.; Klemm, M.; Biefang-Arndt, K.; Schwall, G.; Pu ¨ tter, S.; Hoelzer, K.; Schroer, K.; Stegmann, W.; Schrattenholz, A. Molecular analysis of homocysteic acid-induced neuronal stress. J. Proteome Res. 2004, 3, 572-581. (37) Kyte, J.; Doolittle, R. F. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 1982, 157, 105132. (38) Zahedi, R. P.; Meisinger, C.; Sickmann, A. Two-dimensional benzyldimethyl-n-hexadecylammonium chloride/SDS-PAGE for membrane proteomics. Proteomics 2005, 1, 1. (39) Devreese, B.; Vanrobaeys, F.; Smet, J.; Van Beeumen, J.; Van Coster, R. Mass spectrometric identification of mitochondrial oxidative phosphorylation subunits separated by two-dimensional blue-native polyacrylamide gel electrophoresis. Electrophoresis 2002, 23, 2525-2533. (40) Bulteau, A. L.; Lundberg, K. C.; Ikeda-Saito, M.; Isaya, G.; Szweda, L. I. Reversible redox-dependent modulation of mitochondrial aconitase and proteolytic activity during in vivo cardiac ischemia/ reperfusion. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5987-5991. (41) Yarian, C. S.; Sohal, R. S. In the aging housefly aconitase is the only citric acid cycle enzyme to decline significantly. J. Bioenerg. Biomembr. 2005, 37, 91-96. (42) Schrattenholz, A. Proteomics: how to control highly dynamic patterns of millions of molecules and interpret changes correctly? Drug Discov. Today Technol. 2004, 1, 1-8. (43) Rexroth, S.; Meyer zu Tittingdorf, J. M.; Krause, F.; Dencher, N. A.; Seelert, H. Thylakoid membrane at altered metabolic state: challenging the forgotten realms of the proteome. Electrophoresis 2003, 24, 2814-2823. (44) Sanchez, J. C.; Chiappe, D.; Converset, V.; Hoogland, C.; Binz, P. A.; Paesano, S.; Appel, R. D.; Wang, S.; Sennitt, M.; Nolan, A.; Cawthorne, M. A.; Hochstrasser, D. F. The mouse SWISS-2D PAGE database: a tool for proteomics study of diabetes and obesity. Proteomics 2001, 1, 136-163. (45) Richly, E.; Chinnery, P. F.; Leister, D. Evolutionary diversification of mitochondrial proteomes: implications for human disease. Trends Genet. 2003, 19, 356-362. (46) Heazlewood, J. L.; Millar, A. H.; Day, D. A.; Whelan, J. What makes a mitochondrion? Genome Biol. 2003, 4, 218. (47) Zolg, J. W.; Langen, H. How industry is approaching the search for new diagnostic markers and biomarkers. Mol. Cell Proteomics 2004, 3, 345-354.
research articles
Profiling of the Mammalian Mitochondrial Proteome (48) Eldadah, B. A.; Faden, A. I. Caspase pathways, neuronal apoptosis, and CNS injury. J. Neurotrauma 2000, 17, 811-829. (49) Saraste, A.; Pulkki, K. Morphologic and biochemical hallmarks of apoptosis. Cardiovasc. Res. 2000, 45, 528-537. (50) Blonder, J.; Goshe, M. B.; Xiao, W.; Camp, D. G., 2nd; Wingerd, M.; Davis, R. W.; Smith, R. D. Global analysis of the membrane subproteome of Pseudomonas aeruginosa using liquid chromatography-tandem mass spectrometry. J. Proteome Res. 2004, 3, 434-444. (51) Goshe, M. B.; Blonder, J.; Smith, R. D. Affinity labeling of highly hydrophobic integral membrane proteins for proteome-wide analysis. J. Proteome Res. 2003, 2, 153-161. (52) Jiang, X. S.; Zhou, H.; Zhang, L.; Sheng, Q. H.; Li, S. J.; Li, L.; Hao, P.; Li, Y. X.; Xia, Q. C.; Wu, J. R.; Zeng, R. A high-throughput approach for subcellular proteome: identification of rat liver proteins using subcellular fractionation coupled with twodimensional liquid chromatography tandem mass spectrometry and bioinformatic analysis. Mol. Cell Proteomics 2004, 3, 441555. (53) Faustin, B.; Rossignol, R.; Rocher, C.; Benard, G.; Malgat, M.; Letellier, T. Mobilization of adenine nucleotide translocators as molecular bases of the biochemical threshold effect observed in mitochondrial diseases. J. Biol. Chem. 2004, 279, 20411-20421. (54) Vahsen, N.; Cande, C.; Briere, J. J.; Benit, P.; Joza, N.; Larochette, N.; Mastroberardino, P. G.; Pequignot, M. O.; Casares, N.; Lazar, V.; Feraud, O.; Debili, N.; Wissing, S.; Engelhardt, S.; Madeo, F.; Piacentini, M.; Penninger, J. M.; Schagger, H.; Rustin, P.; Kroemer,
(55) (56) (57) (58) (59) (60) (61) (62) (63)
G. AIF deficiency compromises oxidative phosphorylation. Embo J. 2004, 23, 4679-4689. Epub 2004 Nov 4. Delaval, E.; Perichon, M.; Friguet, B. Age-related impairment of mitochondrial matrix aconitase and ATP-stimulated protease in rat liver and heart. Eur. J. Biochem. 2004, 271, 4559-4564. Das, N.; Levine, R. L.; Orr, W. C.; Sohal, R. S. Selectivity of protein oxidative damage during aging in Drosophila melanogaster. Biochem. J. 2001, 360, 209-216. Liang, L. P.; Patel, M. Iron-sulfur enzyme mediated mitochondrial superoxide toxicity in experimental Parkinson’s disease. J. Neurochem. 2004, 90, 1076-1084. Cooper, C. E. Nitric oxide and iron proteins. Biochim. Biophys. Acta 1999, 1411, 290-309. Gardner, P. R.; Costantino, G.; Szabo, C.; Salzman, A. L. Nitric oxide sensitivity of the aconitases. J. Biol. Chem. 1997, 272, 25071-25076. Castro, L.; Rodriguez, M.; Radi, R. Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide. J. Biol. Chem. 1994, 269, 29409-29415. Sastre, J.; Pallardo, F. V.; Vina, J. The role of mitochondrial oxidative stress in aging. Free Radic Biol. Med. 2003, 35, 1-8. Patel, M.; Li, Q. Y. Age dependence of seizure-induced oxidative stress. Neuroscience 2003, 118, 431-437. Bota, D. A.; Davies, K. J. Lon protease preferentially degrades oxidized mitochondrial aconitase by an ATP-stimulated mechanism. Nat. Cell Biol. 2002, 4, 674-680.
PR050377+
Journal of Proteome Research • Vol. 5, No. 3, 2006 633
