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VDAC Differential Interactome in Chicken Brain: Possible Hints to an Intrinsically Distinct Metabolism

Carla Rossini Crepaldi1, Helen Julie Laure2, Jose Cesar Rosa2 and Marcelo de Cerqueira Cesar1*

1Department of Basic Sciences, School of Animal Science and Food Engineering, University of Sao Paulo, Pirassununga, Brazil

2Protein Chemistry Center and Department of Molecular and Cellular Biology and Pathogenic Bio agents, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil

*Corresponding Author:
Marcelo de Cerqueira Cesar
Laboratory of Neuroscience and Proteomics
School of Animal Science and Food Engineering
University of Sao Paulo, Av. Duque de Caxias Norte 225, 13635-900, Pirassununga, SP, Brazil
Tel: 19 35654095
E-mail: [email protected]

Received date:February 18, 2016; Accepted date: March 23, 2016; Published date: March 30, 2016

Citation: Crepaldi CR, Laure HJ, Rosa JC, et al. VDAC Differential Interactome in Chicken Brain: Possible Hints to an Intrinsically Distinct Metabolism. J Transl Neurosci. 2016, 1:1.

Copyright: © 2016 Crepaldi CR, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

 
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Abstract

The kinetic assembly of protein complexes containing the VDAC (voltage dependent anion channel) follows a quite different pattern in bovine brain in comparison with rat neuronal cells. In order to investigate if the differential expression of VDACs between chicken brain when compared with bovine and rat brain mitochondria was linked to a differential VDAC interactome, we utilized BN-PAGE of mitochondria treated with dodecyl maltoside, followed by a seconddimensional SDS-PAGE. Unusually perhaps, several VDAC interactant proteins in chicken brain were not members of VDAC interactome in rat and bovine brain, which possibly suggests that these cells exhibit intrinsically differential responses to events such as neuronal cell death, bioenergetics, oxygen consumption and oxidative insults.

Keywords

Hexokinase; VDAC; Interactome; BN / SDS PAGE; Mitochondria; Chicken; Brain

Introduction

Hexokinase (HK, EC 2.7.1.1) mediates the first step of glucose catabolism, phosphorylating glucose to produce glucose 6-phosphate. The regulation of HK activity plays a major role in governing the rate of cerebral Glc utilization while avoiding production of neurotoxic lactate [1,2]. The Type I isozyme of hexokinase (HK 1) accounts for more than 90% of the total hexokinase activity in mammalian brain, where it exists predominantly in a mitochondrially bound form [2].

HK I attaches to the voltage dependent anion channel (VDAC) via their N-terminal region facilitating access to intra-mitochondrial ATP. It has been suggested that binding of HK modulates VDAC’s role in apoptosis [3]. VDAC proteins constitute the major pathway for metabolic exchange across the mitochondrial outer membrane (MOM). Two isoforms of VDAC, i.e., VDAC1 and VDAC2 are known to be expressed ubiquitously in chicken brain mitochondria [4].

VDAC1 regulates metabolic and energetic functions of mitochondria; its down-expression should affect cell metabolism and normal mitochondrial function [5]. The VDAC isoform 2 has anti-apoptotic roles as a specific inhibitor of BAK-dependent mitochondrial apoptosis [6]. Changes in the levels of VDAC1 and VDAC2 expression were observed under various pathological conditions [7]. It is interesting to note that cells with low levels of VDAC1 showed 4-fold-lower ATP-synthesis capacity and contained low ATP and ADP levels. In these cells there was a strong correlation between ATP levels and cell growth, suggesting limited metabolite exchange between mitochondria and cytosol [8].

It has been studied the possibility that differences in the relative expression of VDAC isoforms could be a factor in determining the differences in species-dependent ratio of hexokinase binding sites on bovine, avian and rat brain mitochondria. In this research, VDAC1 was the most abundantly expressed of the three isoforms. Moreover, chicken brain mitochondria showed the highest VDAC1 expression and the lowest VDAC2 levels between the three species [4]. The same phenomenon, increase of VDAC1 and decrease of VDAC2 has been detected in pharmaco-resistant epilepsy [9].

There are a variety of conventional (liquid chromatography, ultracentrifugation, and sucrose density gradient centrifugation) and nonconventional methods (co-immunoprecipitation, epitope-tagging, tandem affinity purification, and GST-pull-down) available for isolation of multiprotein complexes. However, most of these techniques often separate a population of such assemblies. To isolate individual complexes, further separation is required, which can be achieved by two-dimensional blue native SDS-PAGE [10]. In BN-PAGE, the Coomassie Brilliant Blue G-250 dye is added into the electrophoresis buffer. This anionic dye binds to the surface of the membrane proteins and facilitates their migration in the native polyacrylamide gel [11]. As a result, distinct protein complexes are separated due the sieving effect of the polyacrylamide gel, but protein – protein interactions among the multiprotein complex subunits are still retained. Although initially developed for the separation of mitochondrial and chloroplast membrane proteins, recent contributions of this approach have been made in studies of protein complexes in termophilic (Clostridium termocellum), antibiotic productive (Streptomyces coelicolor), sulfate-reducing bacteria and in pathogens associated with chronic periodontitis in humans [11-15]. BN / SDS-PAGE have also been used to study protein complexes in a variety of tissues, such as erythrocytes, rat muscle, colorectal cancer and bladder epithelial cells [10,12,16,17].

Recent studies indicate that the kinetic assembly of protein complexes containing the VDAC follows a pattern quite different in bovine and rat brain [18]. In order to investigate if the differential expression of VDACs between chicken brain when compared with bovine and rat brain mitochondria was linked to a differential VDAC interactome, we utilized BN-PAGE of mitochondria treated with dodecyl maltoside. After BN-PAGE, a second-dimensional SDS-PAGE was performed to separate polypeptides as components of VDAC complexes.

Materials and Method

Preparation of brain mitochondria

Chicken brains were obtained from the school slaughterhouse located on campus. Mitochondria were isolated from avian brain as described previously [2]. Mitochondrial protein concentrations were determined by the Bicinchoninic Acid Method, using the assay kit from Thermo Scientific (Rockford, IL, USA) with bovine serum albumin as standard.

Sample preparation

To determine the optimal conditions for the solubilization of VDAC protein complexes, a series of five different concentrations of dodecyl-maltoside (DDM) (0.25, 0.5, 0.75, 1.0 and 1.5%) were evaluated. The solubilization with 1.0% (w/v) was found to be the most effective. Mitochondrial membranes were solubilized as previously reported [18]. The detergent extracts of mitochondria containing approximately 200 μg of protein before solubilization, were loaded per lane.

2D – BN / Tricine SDS PAGE

BN PAGE was performed with linear 6% - 13% gradient gels, overlaid with a 4% stacking gel [19]. GE Healthcare HMW-Native protein markers (GE Healthcare, Buckinghamshire, UK) were used. For a second dimension, BN-PAGE gel lanes containing the proteins of interest were excised and incubated for 30 min. in equilibrating buffer A, containing 12,5 mM Tris (pH 6.8), 4% SDS, 20% Glycerol and 9% β-mercaptoethanol. The lanes were then dipped into equilibrating buffer B supplemented with 12,5 mM Tris HCl pH 6.8, 4% SDS, 20% Glycerol, and 2.5% Iodoacetamide for 15 min. at room temperature.

A 10% Tricine SDS-PAGE was utilized for the separation of proteins [20]. Proteins were subjected to Coomassie Blue R-250 staining or immunoblotting, to detect the complexes in which VDACs 1 and 2 were found.

Each stained gel was digitized and processed using the ImageQuantTL Capture software system (GE Healthcare). This software contains a graph that shows the intensity at each point along the length of the current lane. This is a threshold parameter, which discards peaks under a certain size (30) in relation to the highest peak on the gel. The higher the percentage value entered here the fewer the peaks likely to be detected in the profile. The sizes of the peaks were calculated after background subtraction.

Protein detection by western blotting

Proteins separated as described above were transferred to nitrocellulose membranes using TE62 Transfer Unit (GE Halthcare) and the buffer system of [21]. Membranes were blocked by incubation (overnight, 4°C) with 5% non-fat dried milk in TBS (20 mM Tris - 0.5 M NaCl, pH 7.5). Blots were probed with suitable dilutions of primary antibodies, and then washed extensively with TBS containing 0.1% (v/v) Tween 20, and incubated with 1:10,000-15,000 dilutions of horseradish peroxidase conjugated secondary antibodies; both primary and secondary antibodies were diluted in TBS containing 1% (w/v) gelatin. After further washing with TBS -Tween, chemi-luminescence was developed using the Super Signal West Pico system from Thermo Scientific (Rockford, IL, USA).

Mass spectrometry and protein identification

The second dimension gel bands were selected and proteins were identified by MALDI-TOF-TOF mass spectrometer after in gel trypsin digestion. Briefly, selected gel bands were excised and combined. SDS and CBB were removed by washing the gels three times with 50% ACN in 0.1 M ammonium bicarbonate, pH 7.8, followed by dehydration in neat ACN. Gel bands were dried in a Speed Vac instrument (Savant, New York, NY) and were swollen in 20 mL of 0.5 mg trypsin (Promega, Madison, USA) in 0.1 M ammonium bicarbonate, pH 7.8, followed by the addition of 50 mL of 0.1 M ammonium bicarbonate to cover the entire gel piece. Trypsin hydrolysis was carried out at 37°C for 24 h and the reaction was stopped by the addition of 5 mL of neat formic acid. Peptides were extracted from gel pieces and desalted in microtips filled with POROS R2 (PerSeptive Biosystems, Foster City, CA) previously equilibrated in 0.2% formic acid. After loading, the sample was desalted with two washes of 150 mL 0.2% formic acid. Peptides were eluted from the microtips with 30 mL of 60% methanol / 5% formic acid. The sample was concentrated, and samples were mixed with matrix solution (5 mg/mL α-cyano-4 hydroxycinnamic acid in 50% acetonitrile / 0.1% trifluoroacetic acid), applied on the MALDI target plate and air dried at room temperature. MALDI-TOF-TOF-MS instrument (Axima Performance, Kratos-Shimadzu, Manchester, UK) was calibrated with a mixture of bradykynin fragment, angiotensin II, renin and ACTH (mass accuracy < 50 ppm). The spectra of CID-MS / MS of each gel band were obtained in data dependent acquisition mode. The peak list was obtained from CID-MS / MS spectra using Launchpad v. 2.8 (Kratos-Shimadzu, Manchester, UK) and submitted to a database search using MASCOT version 2.2.04 against NCBI database version 52.9 selected for taxonomy filter of Gallus gallus. The database search parameters accept one missing trypsin cleavage, carbamidomethylation and methionine oxidation. Mass tolerance was 1.2 Da for precursor ions and 0.8 Da for product ions. Protein was considered to be identified by MASCOT for proteins corresponded to a level of significance of p < 0.05 and FDR less than 0.2%.

Results and Discussion

To analyze proteins interacting with VDAC, BN / SDS-PAGE was performed. The VDACs 1 and 2 associated protein complexes were revealed by antibody detection and confirmed by mass spectrometry. In the present paper, we utilized a well-characterized antibody against VDAC1, which was first raised by Poleti et al., which is highly specific for their desired targets [4]. However, the antibody against VDAC2 showed cross-immunoreactivity with VDAC1 from chick brain mitochondria. VDACs were detected in four pre-complexes (II, III, IV and V) and one complex (I) in avian brain mitochondria (Figure 1). The apparent masses of complex I and pre-complexes II, III, IV and V were 762, 707, 625, 571 and 464 kDa, respectively.

translational-neuroscience-Native-gel

Figure 1: (A) Blue Native gel gradient (6% - 13%) of DDM solubilized chicken brain mitochondria (B) Immunoblot to VDACs 1 and 2, showing the overlap proteins in the first complex (762 Kda), pre-complex II (707 KDa), pre-complex III (625 KDa), pre-complex IV (571 KDa) and pre-complex V (464 KDa) in chicken brain.

The general interpretative rule for the analysis of the BN / SDSPAGE is that all protein spots which are situated vertically in the gel are potential subunits of a same protein complex. A second rule is that if it is combined information about the subunit composition of the protein complexes (vertical) and the molecular mass of the complexes (horizontal) and is assumed that the molecular mass of a protein complex is increasing during assembly of the structural subunits of this complex, the analysis of the protein pattern after BN / SDS-PAGE resolves the direction of the stepwise subunit assembly from the lower towards the higher molecular mass of the protein complexes [22].

BN / SDS-PAGE were performed on five replicates of five different mitochondrial preparations (each preparation extracted from 3 adult chickens). The analysis of the representative gel from Figure 2 detected 85 spots of VDAC interacting proteins. Among these spots, 54 different proteins were identified with a MASCOT score above the threshold to validate MS data, i.e., 32. The identification of subunits of individual OXPHOs complexes, as VDAC interacting proteins in chicken brain, confirmed the validity of our MS approach. These interactants represent a large variety of functions such as components of TCA cycle, ketone body’s metabolism, scavengers of reactive oxygen species, members of mitochondrial permeability transition pore and cell proliferation between many others.

translational-neuroscience-protein-subunits

Figure 2: Identification of protein subunits in 2D BN / SDS gels of the pre-complexes II to V and complex I from dodecylmatoside-solubilized chicken brain mitochondria. Corresponding spots 1-20 from complex I, 1-20, 1-18, 1-16, 1-11 from pre-complexes II, III, IV and V, respectively, were excised from the gel and identified by MALDI-TOF / TOF MS. Results are summarized in Table 1.

The identified proteins and their corresponding MASCOT score, sequence coverage, and number of matched MS / MS are listed in Tables 1-6. The four pre-complexes and the major complex from chicken brain mitochondria can be observed in Figure 2 and Table 1.

I II III IV V
3CWB_D 3CWB_A 3CWB_A ANT  
ANTI ANT1 ANT1 ANT1 ANT1
ANT3 ANT3 ANT3 ANT3 ANT3
  AATM AATM AATM AATM
  ACON   ACON  
  ATPG ATPG ATPG  
AT5F1 AT5F1 AT5F1 AT5F1  
ATP5H ATP5H ATP5H ATP5H  
ATP5O ATP5O ATP5O ATP5O  
ATPB ATPA ATPA ATPA  
ATPA ATPB ATPB ATPB AL1I2
ARALAR2   ATPD ATPD  
avANT avANT avANT avANT avANT
  BDH1 BDH1   BDH1
CH60 CH60 CH60 CH60 CH60
CHPF2        
    CMBL CMBL  
COXII COXII COXII COXII COXII
    COX4I1 COX4I1 COX4I1
  DOCK2 DESM    
  E1C825 HADHA   HADHA
IMMT IMMT IMMT IMMT  
      QN1  
    MRP1 MRP1  
  MDH2 MDH2   MDH2
NDUA9     MYH11  
NDUAA NDUAA     MPCP
NDUFA8 NDUFA8 NDUFA8    
    NDUFB4    
NDUFA12 NDUFA12      
NDUFB8 NDUFB8      
NDUFB10 NDUFB10 NDUFB10    
NDUFS7 NDUFS7 NDUFS7    
NDUFS8 NDUFS8      
NDUFS1 NDUFS1 NDUFS1    
NDUFS3 NDUFS3      
NDUV2 NDUV2 NDUV2    
PHB PHB PRDX3 PRDX3 PRDX3
PHB2 PHB2 RBBP8 RBBP8  
QCRC1   QCRC1    
QCRC2 QCRC2 QCRC2 QCRC2 QCRC2
VDAC2 VDAC2 VDAC2 VDAC2 VDAC2
        XIRP1
        WWC2

Table 1: Identification results of VDAC-associated protein complex (I) and pre-complexes (II to V) in chicken brain mitochondria.

Complex I
Spot Accession NCBI Name of protein Function Mascot score Sequence coverage (%) Matches MS/MS Theoretical
pI Mr
1 gi|57530041 Mitochondrial inner membrane protein Other functions 578 13,2 14 5,72 79249
2 gi|57529753 NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial Oxidative phosphorylation 1024 18,7 16 6,5 79576
3 gi|61098440 Calcium-binding mitochondrial carrier protein Aralar2 Other functions 142 4 4 8,93 74102
4 gi|61098372 60 kDa heat shock protein, mitocondrial Chaperone;
Cellular defense
122 5,2 3 5,72 60972
5 gi|118109616 PREDICTED: similar to mitochondrial ATP synthase alpha subunit, partial Oxidative phosphorylation 77 29,2 2 5,8 5292
6 gi|71897237 ATP synthase subunit beta, mitochondrial precursor Oxidative phosphorylation 988 27,4 20 5,59 56627
7 gi|50754375 PREDICTED: cytochrome b-c1 complex subunit 1, mitochondrial Oxidative phosphorylation 145 4,6 4 6,58 52758
8 gi|118098350
PREDICTED: cytochrome b-c1 complex subunit 2, mitochondrial-like isoform X4
Oxidative phosphorylation 458 19 11 9,04 48579
10 gi|71895153 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10, mitochondrial Oxidative phosphorylation 350 14,4 8 6,15 41431
11 gi|57529307 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, mitochondrial Oxidative phosphorylation 474 20,1 12 9,4 43079
13 gi|46048903 Voltage-dependent anion-selective channel protein 2 Transport and carrier protein 334 19,1 6 8,61 30197
gi|124249322 Prohibitin-2 Regulates cell proliferation. May play a role in regulating mitochondrial respiration activity 302 14 8 9,89 33336
14 gi|295148230 Prohibitin Regulates cell proliferation. May play a role in regulating mitochondrial respiration activity 207 12,5 6 5,57 29892
gi|196049778 Chain D, Chicken Citocromo Bc1 complex Inhibited By An Iodinated Analogue Of The PolyketideCrocacin-d Oxidative phosphorylation 164 10,8 4 6,32 26939
gi|22775582 ATP/ADP antiporter Transport and carrier protein 105 6,7 3 9,78 32847
gi|118089692 PREDICTED: similar to ADP/ATP translocase Transport and carrier protein 105 7,6 3 9,72 29307
gi|54020693 ADP/ATP translocase 3 Transport and carrier protein 102 6,7 3 9,73 32748
15 gi|226437575 NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrial Oxidative phosphorylation 273 17,1 8 6,55 29232
16 gi|118086790 PREDICTED: similar to NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial precursor (NADH-ubiquinone oxidoreductase 24 kDa subunit) (NADH dehydrogenase subunit II) isoform 1 Oxidative phosphorylation 308 22,4 7 7,6 26893
gi|5834847 Cytochrome c oxidase subunit II (mitochondrion) Oxidative phosphorylation 127 9,3 3 4,57 25568
gi|50755667 PREDICTED: NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 Oxidative phosphorylation 62 6,3 2 5,98 20497
17 gi|118103240 PREDICTED: NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial-like Oxidative phosphorylation 120 8,6 4 10,02 20473
gi|118102465 PREDICTED: ATP synthase subunit b, mitochondrial Oxidative phosphorylation 107 8,5 2 9,34 31751
lgi|118090950 PREDICTED: NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, mitochondrial Oxidative phosphorylation 104 11 2 5,84 23824
gi|118085386 PREDICTED: chondroitin sulfate glucuronyltransferase-like Tissue development and morphogenesis 45 1,4 1 5,81 83804
18 gi|50745451 PREDICTED: similar to ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d isoform 3 (BLAST: PREDICTED: ATP synthase subunit d, mitochondrial isoform 1 ) Oxidative phosphorylation 313 30,4 8 8,73 18343
gi|118099484 PREDICTED: NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8 Oxidative phosphorylation 97 11,6 4 8,42 20125
19 gi|118099484 PREDICTED: NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8 Oxidative phosphorylation 91 11,6 4 8,42 20125
gi|118083809 PREDICTED: similar to LOC446923 protein isoform 1 (BLAST: ATP sinthasesubunidade O, isoformamitocondrial 2) Oxidative phosphorylation 66 6,7 2 9,88 22803
20 gi|296090732 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 Oxidative phosphorylation 165 27,4 6 9,57 16892
gi|57529832 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8, mitochondrial precursor Oxidative phosphorylation 151 18,3 6 7,9 21945

Table 2: Summary of proteins from 2D BN / SDS Gels Identified by MALDI-TOF-TOF in chicken brain mitochondria.

Pre-complex II
Spot Accession NCBI Name of protein Function Mascot score Sequence coverage (%) Matches MS/MS Theoretical
pI Mr
1 gi|57530041 Mitochondrial inner membrane protein Other functions 586 12,7 13 5,72 79249
gi|45383738 aconitatehydratase, mitochondrial Metabolic enzyme (TCA); mitochondrial DNA stability 52 1,9 1 8,05 85790
2 gi|57529753 NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial Oxidative phosphorylation 766 16,1 16 6,5 79576
3 gi|61098372 60 kDa heat shock protein, mitocondrial precursor Chaperone; Cellular defense 49 3,1 1 5,72 60972
gi|45383566 ATP synthase subunit alpha, mitochondrial Oxidative phosphorylation 40 2,4 2 9,29 60186
4 gi|118084029 PREDICTED: uncharacterized protein LOC418583   34 0,9 1 5,64 146912
gi|118097244 PREDICTED: dedicator of cytokinesis protein 2 Modulates microglia secretion, phagocytosis and paracrine neurotoxicity 34 0,7 1 8,3 219618
5 gi|71897237 ATP synthase subunit beta, mitochondrial precursor Oxidative phosphorylation 887 23,5 17 5,59 56627
6 gi|196049775 Chain A, Chicken Cytochrome Bc1 complexo Inhibited By An Iodinated Analogue Of The PolyketideCrocacin-D Oxidative phosphorylation 406 12,6 10 5,95 49441
7 gi|118098350 PREDICTED: cytochrome b-c1 complex subunit 2, mitochondrial-like isoform X4 Oxidative phosphorylation 577 19 11 9,04 48579
8 gi|45382953 Aspartate aminotransferase, mitochondrial precursor Amino acid metabolism 633 19,4 12 9,38 47241
9 gi|71895153 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10, mitochondrial  Oxidative phosphorylation 455 18,6 9 6,15 41431
10 gi|50758110  PREDICTED: malate dehydrogenase, mitochondrial Metabolic enzyme (TCA); 318 12,5 5 8,83 36968
gi|57529615 D-beta-hydroxybutyrate dehydrogenase, mitochondrial precursor Ketogenesis pathway 61 2,9 2 8,37 38237
gi|46048903 voltage-dependent anion-selective channel protein 2 Transport and carrier protein 46 3,9 1 8,61 30197
11 gi|46048903 voltage-dependent anion-selective channel protein 2 Transport and carrier protein 464 31,1 9 8,61 30197
  gi|124249322 Prohibitin-2 Regulates cell proliferation. May play a role in regulating mitochondrial respiration activity 140 13,6 6 9,89 33336
12 gi|118123062 PREDICTED: ATP synthase subunit gamma, mitochondrial isoform 1 Oxidative phosphorylation 269 11,3 6 9,39 32608
gi|295148230 Prohibitin Regulates cell proliferation. May play a role in regulating mitochondrial respiration activity 187 12,5 6 5,57 29892
13 gi|22775582 ATP/ADP antiporter Transport and carrier protein 206 10,7 4 9,78 32847
gi|54020693 ADP/ATP translocase 3 Transport and carrier protein 204 10,7 4 9,73 32748
gi|57530120 ADP/ATP translocase 1 Transport and carrier protein 97 8,4 2 9,74 32968
14 gi|226437575 NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrial Oxidative phosphorylation 561 27,2 14 6,55 29232
16 gi|5834847 cytochrome c oxidase subunit II (mitochondrion) Oxidative phosphorylation 287 25,1 8 4,57 25568
gi|50755667 PREDICTED: NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 Oxidative phosphorylation 155 7,4 4 5,98 20497
gi|118086790 PREDICTED: similar to NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial precursor (NADH-ubiquinone oxidoreductase 24 kDa subunit) (NADH dehydrogenase subunit II) isoform 1 Oxidative phosphorylation 121 9,8 3 7,6 26893
17 gi|118103240 PREDICTED: NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial-like Oxidative phosphorylation 125 8,6 4 10,02 20473
gi|118102465 PREDICTED: ATP synthase subunit b, mitochondrial Oxidative phosphorylation 71 4,2 1 9,34 31751
gi|118090950 PREDICTED: NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, mitochondrial Oxidative phosphorylation 56 5,7 2 5,84 23824
18 gi|50745451 PREDICTED: similar to ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d isoform 3 (BLAST: PREDICTED: ATP synthase subunit d, mitochondrial isoform 1 ) Oxidative phosphorylation 324 30,4 8 8,73 18343
gi|118099484 PREDICTED: NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8 Oxidative phosphorylation 59 7,6 2 8,42 20125
gi|118083809 PREDICTED: similar to LOC446923 protein isoform 1 (BLAST: ATP sinthasesubunidade O, isoformamitocondrial 2 ) Oxidative phosphorylation 46 6,7 2 9,88 22803
19 gi|118083809 PREDICTED: similar to LOC446923 protein isoform 1 (BLAST: ATP sinthase subunit O, isoformamitocondrial 2 ) Oxidative phosphorylation 144 11,9 3 9,88 22803
gi|118099484 PREDICTED: NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8 Oxidative phosphorylation 50 7,6 2 8,42 20125
20 gi|296090732 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 Oxidative phosphorylation 142 15,8 2 9,57 16892
gi|57529832 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8, mitochondrial precursor Oxidative phosphorylation 116 11 2 7,9 21945

Table 3: Summary of proteins from 2D BN / SDS Gels Identified by MALDI-TOF-TOF in chicken brain mitochondria (Pre-complex II).

Pre-complex III
Spot Accession NCBI Name of protein Function Mascot score Sequence coverage (%) Matches MS/MS Theoretical
pI Mr
1 gi|57530041 Mitochondrial inner membrane protein Other functions 344 8,2 10 5,72 79249
2 gi|57529753 NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial Oxidative phosphorylation 90 4 4 6,5 79576
gi|45384238 Trifunctional enzyme subunit alpha, mitochondrial Lipid metabolism; fatty acid beta-oxidation 39 1,4 2 9,19 83186
3 gi|61098372 60 kDa heat shock protein, mitocondrial precursor Chaperone; Cellular defense 184 5,2 3 5,72 60972
gi|45383566 ATP synthase subunit alpha, mitochondrial Oxidative phosphorylation 35 2,4 1 9,29 60186
4 gi|45383566 ATP synthase subunit alpha, mitochondrial Oxidative phosphorylation 658 17,5 12 9,29 60186
5 gi|71897237 ATP synthase subunit beta, mitochondrial precursor Oxidative phosphorylation 886 23,5 16 5,59 56627
6 gi|50754375 PREDICTED: cytochrome b-c1 complex subunit 1, mitochondrial Oxidative phosphorylation 273 9,2 9 6,58 52758
gi|196049775 Chain A, Chicken Cytochrome Bc1 complexo Inhibited By An Iodinated Analogue Of The PolyketideCrocacin-D Oxidative phosphorylation 273 9,9 9 5,95 49441
7 gi|118098350 PREDICTED: cytochrome b-c1 complex subunit 2, mitochondrial-like isoform X4 Oxidative phosphorylation 360 15,1 9 9,04 48579
gi|71897237 ATP synthase subunit beta, mitochondrial precursor Oxidative phosphorylation 72 2,6 1 5,59 56627
8 gi|45382953 Aspartate aminotransferase, mitochondrial precursor Aminoacid metabolism 337 15,4 11 9,38 47241
9 gi|50758110  PREDICTED: malate dehydrogenase, mitochondrial Metabolic enzyme (TCA) 233 12,8 6 8,83 36968
gi|46048903 voltage-dependent anion-selective channel protein 2 Transport and carrier protein 57 7,1 2 8,61 30197
gi|57529615 D-beta-hydroxybutyrate dehydrogenase, mitochondrial precursor Ketogenesis pathway 45 7,1 3 8,37 38237
10 gi|46048903 voltage-dependent anion-selective channel protein 2 Transport and carrier protein 584 24 9 8,61 30197
11 gi|118123062 PREDICTED: ATP synthase subunit gamma, mitochondrial isoform 1 Oxidative phosphorylation 300 11,3 8 9,39 32608
gi|50734923 PREDICTED: carboxymethylenebutenolidase homolog isoform 3 Prodrugbioactivation 40 4,9 2 6,45 28170
gi|22775582 ATP/ADP antiporter Transport and carrier protein 35 3 2 9,78 32847
gi|54020693 ADP/ATP translocase 3 Transport and carrier protein 35 3 2 9,73 32748
gi|118089692 PREDICTED: similar to ADP/ATP translocase Transport and carrier protein 35 3,4 2 9,72 29307
12 gi|54020693 ADP/ATP translocase 3 Transport and carrier protein 309 17,8 7 9,73 32748
14 gi|5834847 cytochrome c oxidase subunit II (mitochondrion) Oxidative phosphorylation 345 25,1 9 4,57 25568
gi|50755667 PREDICTED: NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 Oxidative phosphorylation 142 7,4 4 5,98 20497
gi|118086790 PREDICTED: similar to NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial precursor (NADH-ubiquinone oxidoreductase 24 kDa subunit) (NADH dehydrogenase subunit II) isoform 1 Oxidative phosphorylation 106 9,8 3 7,6 26893
gi|118093103 PREDICTED: thioredoxin-dependent peroxide reductase, mitochondrial isoform X4 Antioxidant protein 72 4,9 2 8,4 30992
15 gi|118102465 PREDICTED: ATP synthase subunit b, mitochondrial Oxidative phosphorylation 239 11,6 5 9,34 31751
gi|118103240 PREDICTED: NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial-like Oxidative phosphorylation 103 8,6 4 10,02 20473
gi|118085922 PREDICTED: similar to multidrug resistance protein 1a Transport protein,
limit access of drug to the central nervous system
42 1,3 1 8,87 148466
gi|50745451 PREDICTED: similar to ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d isoform 3 (BLAST: PREDICTED: ATP synthase subunit d, mitochondrial isoform 1 ) Oxidative phosphorylation 40 6,2 2 8,73 18343
16 gi|50745451 PREDICTED: similar to ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d isoform 3 (BLAST: PREDICTED: ATP synthase subunit d, mitochondrial isoform 1 ) Oxidative phosphorylation 354 30,4 7 8,73 18343
gi|118083809 PREDICTED: similar to LOC446923 protein isoform 1 (BLAST: ATP sinthasesubunidade O, isoformamitocondrial 2) Oxidative phosphorylation 58 6,7 2 9,88 22803
gi|118099484 PREDICTED: NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8 Oxidative phosphorylation 50 7,6 2 8,42 20125
gi|50737115 PREDICTED: DNA endonuclease RBBP8 Cell cycle progression, DNA repair and transcriptional regulation 42 1,1 1 6,14 103530
17 gi|118083809 PREDICTED: similar to LOC446923 protein isoform 1 (BLAST: ATP sinthasesubunidade O, isoformamitocondrial 2 ) Oxidative phosphorylation 154 11,9 4 9,88 22803
18 gi|118124369 PREDICTED: ATP synthase subunit delta, mitochondrial-like, partial Oxidative phosphorylation 100 35 2 4,75 4159
gi|71895513 citocromo c oxidase subunidade 4 isoforma 1, mitocondrial Oxidative phosphorylation 47 7 2 8,91 19631
gi|118083465 PREDICTED: NADH dehydrogenase [ubiquinone] 1 beta subcomplex       subunit 4 isoform X1   Oxidative phosphorylation 37 6,2 2 9,46 18701
gi|2959450 Desmin Cell morphology 36 2,9 1 5,3 51663

Table 4: Summary of proteins from 2D BN / SDS Gels Identified by MALDI-TOF-TOF in chicken brain mitochondria (Pre-complex III).

Pre-complex IV
Spot Accession NCBI Name of protein Function Mascot score Sequence average (%) Matches MS/MS Theoretical
pI Mr
1 gi|57530041 Mitochondrial inner membrane protein Other functions 314 8,2 9 5,72 79249
gi|45383738 Aconitatehydratase, mitochondrial Metabolic enzyme (TCA); mitochondrial DNA stability 106 3,8 3 8,05 85790
gi|45383566 ATP synthase subunit alpha, mitochondrial Oxidative phosphorylation 53 2,4 2 9,29 60186
2 gi|61098372 60 kDa heat shock protein, mitocondrial precursor Chaperone; Cellular defense 468 11,7 8 5,72 60972
gi|45383566 ATP synthase subunit alpha, mitochondrial Oxidative phosphorylation 107 2,4 2 9,29 60186
4 gi|71897237 ATP synthase subunit beta, mitochondrial precursor Oxidative phosphorylation 816 23,5 16 5,59 56627
5 gi|118088850 PREDICTED: similar to QN1 orf Cell division 53 0,6 2 5,86 170342
6 gi|118098350 PREDICTED: cytochrome b-c1 complex subunit 2, mitochondrial-like isoform X4 Oxidative phosphorylation 541 19 11 9,04 48579
7 gi|45382953 Aspartate aminotransferase, mitochondrial precursor Aminoacid metabolism 364 15,4 12 9,38 47241
gi|71897237 ATP synthase subunit beta, mitochondrial precursor Oxidative phosphorylation 76 2,6 2 5,59 56627
8 gi|46048903 Voltage-dependent anion-selective channel protein 2 Transport and carrier protein 603 30,4 8 8,61 30197
9 gi|118123062 PREDICTED: ATP synthase subunit gamma, mitochondrial isoform 1 Oxidative phosphorylation 296 11,3 8 9,39 32608
gi|50734923 PREDICTED: carboxymethylenebutenolidase homolog isoform 3 Prodrugbioactivation 40 4,9 2 6,45 28170
10 gi|22775582 ATP/ADP antiporter Transport and carrier protein 51 3 2 9,78 32847
gi|54020693 adenine nucleotide translocator 3 Transport and carrier protein 51 3 2 9,73 32748
gi|118089692 PREDICTED: similar to ADP/ATP translocase Transport and carrier protein 51 3,4 2 9,72 29307
12 gi|5834847 cytochrome c oxidase subunit II (mitochondrion) Oxidative phosphorylation 260 16,3 8 4,57 25568
gi|118093103 PREDICTED: thioredoxin-dependent peroxide reductase, mitochondrial isoform X4 Antioxidant protein 64 4,9 2 8,4 30992
13 gi|118102465 PREDICTED: similar to ATP synthase subunidade b Oxidative phosphorylation 428 15,8 12 9,34 31751
gi|50745451 PREDICTED: similar to ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d isoform 3 (BLAST: PREDICTED: ATP synthase subunit d, mitochondrial isoform 1 ) Oxidative phosphorylation 66 6,2 2 8,73 18343
gi|45384060 Myosin-11 Transport of vesicles 40 0,9 1 5,5 228891
gi|118085922 PREDICTED: similar to multidrug resistance protein 1a Transport protein, limit access of drug to the central nervous system 40 1,3 1 8,87 148466
14 gi|50745451 PREDICTED: similar to ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d isoform 3 (BLAST: PREDICTED: ATP synthase subunit d, mitochondrial isoform 1 )   Oxidative phosphorylation 345 30,4 8 8,73 18343
gi|118083809 PREDICTED: similar to LOC446923 protein isoform 1 (BLAST: ATP sinthasesubunidade O, isoformamitocondrial 2 )   Oxidative phosphorylation 56 6,7 2 9,88 22803
gi|50737115 DNA endonuclease RBBP8 Other functions 45 1,1 1 6,14 103530
15 gi|118083809 PREDICTED: similar to LOC446923 protein isoform 1 (BLAST: ATP sinthasesubunidade O, isoformamitocondrial 2 )   Oxidative phosphorylation 53 6,7 2 9,88 22803
16 gi|118124369 PREDICTED: ATP synthase subunit delta, mitochondrial-like, partial Oxidative phosphorylation 101 35 2 4,75 4159
gi|71895513 Cytochrome c oxidase subunit 4 isoform 1, mitochondrial Oxidative phosphorylation 37 7 2 8,91 19631

Table 5: Summary of proteins from 2D BN / SDS Gels Identified by MALDI-TOF-TOF in chicken brain mitochondria (Pre-complex IV).

Pre-complex V
Spot Accession NCBI Name of protein Function Mascot score Sequence coverage (%) Matches MS/MS Theoretical
pI Mr
1 gi|118082834 PREDICTED: mitochondrial 10-formyltetrahydrofolate dehydrogenase (Alternative name: Aldehyde dehydrogenase family 1 member L2 Detoxification, cell death 39 1,4 2 6,5 101470
2 gi|45384238 Trifunctional enzyme subunit alpha, mitochondrial Lipid metabolism; fatty acid beta-oxidation 181 5,5 7 9,19 83186
3 gi|61098372 60 kDa heat shock protein, mitocondrial precursor Chaperone; Cellular defense 539 16,4 11 5,72 60972
gi|4521320 unnamed protein product (BLAST: xin actin-binding repeat-containing protein 1) Protects actin filaments from depolymerization 39 1,6 1 7,3 87986
4 gi|118090114 PREDICTED: similar to WW, C2 and coiled-coil domain containing 2 Celular death 32 0,8 1 5,07 132717
5 gi|118098350 PREDICTED: cytochrome b-c1 complex subunit 2, mitochondrial-like isoform X4 Oxidative phosphorylation 207 10,5 7 9,04 48579
6 gi|45382953 Aspartate aminotransferase, mitochondrial precursor Aminoacid metabolism 207 13,5 10 9,38 47241
7 gi|50758110  PREDICTED: malate dehydrogenase, mitochondrial Metabolic enzyme (TCA) 195 17,4 7 8,83 36968
gi|46048903 voltage-dependent anion-selective channel protein 2 Transport and carrier protein 116 11 3 8,61 30197
gi|57529615 D-beta-hydroxybutyrate dehydrogenase, mitochondrial precursor Ketogenesis pathway 48 2,9 2 8,37 38237
8 gi|46048903 voltage-dependent anion-selective channel protein 2 Transport and carrier protein 492 31,1 10 8,61 30197
9 gi|22775582 ATP/ADP antiporter Transport and carrier protein 251 13,4 7 9,78 32847
gi|54020693 adenine nucleotide translocator 3 Transport and carrier protein 249 13,4 7 9,73 32748
gi|57530120 ADP/ATP translocase 1 Transport and carrier protein 185 11,1 5 9,74 32968
gi|57525378 Phosphate carrier protein, mitochondrial Transport and carrier protein 58 3,6 2 9,33 37421
10 gi|5834847 Cytochrome c oxidase subunit II (mitochondrion) Oxidative phosphorylation 248 16,3 8 4,57 25568
gi|118093103 PREDICTED: thioredoxin-dependent peroxide reductase, mitochondrial isoform X4 Antioxidant protein 148 8,7 4 8,4 30992
11 gi|71895513 Cytochrome c oxidase subunit 4 isoform 1, mitochondrial Oxidative phosphorylation 469 36,3 14 8,91 19631

Table 6: Summary of proteins from 2D BN / SDS Gels Identified by MALDI-TOF-TOF in chicken brain mitochondria (Pre-complex V).

Twenty eight identified non-OXPHOS proteins were interacting with VDAC in avian brain mitochondria. The identified components of VDAC interacting chicken brain proteins in precomplex V (Table 1 and Figure 2) are ADP / ATP Translocase 1 (ANT1); Adenine Nucleotide Translocator 3 (ANT3); Mitochondrial 10-formyltetrahydrofolate dehydrogenase (AL1L2); ATP / ADP Antiporter (avANT); Aspartate Aminotransferase (AATM); D-Beta-Hydroxybutyrate Dehydrogenase (BDH1); 60 kDa heat shock protein (CH60); Cytochrome C Oxidase subunit II (COXII); Cytochrome C Oxidase subunit 4 Isoform 1 (COX4I1); Trifunctional enzyme subunit alpha (HADHA); Malate Dehydrogenase Mitochondrial (MDHM); unnamed protein product (BLAST: xin actin-binding repeat-containing protein 1, XIRP1); Voltage-Dependent Anion-selective Channel Protein 2 (VDAC2); Thioredoxin-dependent Peroxide Reductase Prdx3 protein (PRDX3); Phosphate Carrier Protein Mitochondrial (MPCP); Cytochrome b-c1 complex subunit 2, mitochondrial-like isoform X4 (QCR2) and similar to WW, C2 and coiled-coil domain containing 2 (WWC2). The proteins AL1L2, avANT, BDH1, CH60, COXII, COX4I1, HADHA, MDHM, PRDX3, XIRP1 and WWC2 were not VDAC interactants in bovine and rat brain mitochondria [18].

D-Beta-Hydroxybutyrate Dehydrogenase (BDH1) catalyzes the interconversion of acetoacetate and (R)-3-hydroxybutyrate, the two major ketone bodies produced during fatty acid catabolism. The authors showed that ketolytic (BDH1) and glycolytic enzymatic (Hexokinase) profiles of malignant brain tumors were different from the normal non-neoplastic brain tissue. A decrease in the mitochondrial enzymes BDH1 and OXCT1( 3-Oxoacid CoA Transferase) was coupled with positive expression of the glycolytic enzymes HK2 and PKM2 (Pyruvate Kinase M2 isoform), supporting the notion that many high grade brain tumors in humans have aberrant metabolism of ketones, and may preferentially use glucose for their energy needs [23].

PRDX3 is a mitochondrial antioxidant protein and a member of the peroxiredoxin family that can scavenge not only hydrogen peroxide (H2O2) in co-operation with thiol, but also peroxynitrite (ONOO–). PRDX3 markedly reduced gliosis, a post- neuronal cell death event and seems to be neuroprotective against oxidative insults [24].

Another VDAC interactant protein just observed in chicken brain mitochondria was xin actin-binding repeat-containing protein 1 (XIRP1). It was found as one of the proteins with an altered level in serum from schizophrenic patients [25]. The phosphate carrier protein (MPCP) was also found as a VDAC interactant in bovine brain mitochondria [18], and is a key component of the mitochondrial permeability transition pore (mPTP) [26], in the same way as VDAC [27]. MPCP undergoes a calcium-induced conformational change to induce pore formation.

In addition to the proteins identified in pre-complex V, fifteen other proteins were identified in pre-complex IV (Table 1 and Figure 2). They are Aconitate hydratase (ACON); similar to ADP / ATP translocase (ANT); ATP synthase subunit alpha (ATPA); ATP synthase subunit beta (ATPB); similar to ATP synthase subunit b (ATP5F1); ATP synthase subunit gamma (ATPG); ATP synthase subunit delta (ATPD); similar to ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d isoform 3 (ATP5H); similar to LOC446923 protein isoform 1 (BLAST: ATP synthase subunit O, mitochondrial isoform 2) (ATP5O); Carboxymethylenebutenolidase homolog isoform 3 (CMBL); DNA endonuclease RBBP8 (RBBP8); Myosin-11 (MYH11); Mitochondrial inner membrane protein (IMMT); similar to Multidrug Resistance Protein 1a (MRP1) and similar to QN1 orf (QN1). CMBL, RBBP8, MYH11, MRP1 and QN1 do not interact with VDACs in bovine and rat brain mitochondria [18]. Schizophrenic patients showed differences in activities of some enzymes from TCA cycle, like Aconitase (ACON) which presented a decreased activity [28]. The proteins ACON, F1-ATPase chains α (ATPA) and β (ATPB) were found differentially decreased in response to an acute hypobaric hypoxic episode and the subsequent re-oxygenation in rat brain cortex [29]. The authors suggest that these results could be due to the loss of proteins coupled with the destabilization of the mitochondria found after a hypobaric hypoxic insult, which would alter both the structure and functionality of ATPase and more specifically its catalytic subunit F1.

CMBL serves as a key enzyme in the activation of olmesartan medoxomil a prodrug type angiotensin II type I receptor antagonist. It is distributed in 20 tissues, including whole brain, but the highest level is found in liver [30]. And the protein RBBP8 or CtIP (C-terminal binding protein Interacting Protein) is a multifunctional protein involved in transcription, DNA replication, DNA repair by homologous recombination and the G1 and the G2 checkpoints. Both its functions and interactions point to a putative oncogenic potential of RBBP8 loss [31].

Our results demonstrated in avian brain several VDAC interactant proteins associated with neurological diseases, like the Multidrug resistance-associated protein 1 (MRP1). Drug resistance is one of the most serious problems in treatment of epilepsy [32]. Accumulating experimental evidence suggests that increased expression of MRP1 has been determined in epileptogenic brain regions of patients with pharmacoresistant epilepsy [33]. Over expression of such transporters in epileptogenic tissue is thus likely to reduce the amount of drug that reaches the epileptic neurons, which would be a likely explanation for pharmacoresistance [32].

In addition to the proteins identified in pre-complex IV, nine other proteins were identified in pre-complex III (Table 1 and Figure 2). They are Chain A, Chicken Cytochrome Bc1 complex Inhibited By An Iodinated Analogue Of The Polyketide Crocacin-D (3 CWB_A); Cytochrome b-c1 complex subunit 1 (QCRC1); Desmin, partial (DESM); NADH dehydrogenase [ubiquinone] 1 alpha sub complex subunit 8 (NDUFA8); NADH dehydrogenase [ubiquinone] 1 beta sub complex subunit 4 (NDUFB4); NADH dehydrogenase [ubiquinone] 1 beta sub complex subunit 10 (NDUFB10); NADH dehydrogenase [ubiquinone] iron-sulfur protein 7 (NDUFS7);

NADH-ubiquinone oxidoreductase 75 kDa subunit (NDUS1) and similar to NADH dehydrogenase [ubiquinone] flavoprotein 2 (NDUV2). Between these 8 proteins, just DESM was not interacting with VDACs in rat and bovine brain mitochondria [18]. Regarding molecular evidence of abnormal mitochondrial function in psychiatric disorders, in studies of schizophrenic patients, the 24 kDa and 51 kDa subunits of complex I from the electron transport chain were significantly decreased in the pre frontal cortex [34]. Mitochondrial dysfunction and abnormal brain bioenergetics have also been implicated in autism, with reduced expression of eleven genes of electron transport chain complex I [35].

Desmin plays an essential role in maintaining cell cytoarchitecture, positioning and functioning of organelles, and the intercellular signaling pathway [36]. Desmin was shown to regulate mitochondria affinity to ADP and oxygen consumption supposedly through direct binding to VDAC [37].

The identified components of pre-complex II, not found in precomplex III (Table 2) are uncharacterized protein LOC418583 (E1C825); Dedicator of cytokinesis protein 2 (DOCK2); NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10 (NDUAA); NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 (NDUFA12); NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8 (NDUFB8); NADH dehydrogenase [ubiquinone] iron-sulfur protein 8 (NDUFS8); NADH dehydrogenase [ubiquinone] iron-sulfur protein 3 (NDUFS3); Prohibitin (PHB) and Prohibitin-2 (PHB2).

DOCK2 and E1C825 interact with VDACs just in chicken brain mitochondria [18]. DOCK2 is a guanyl nucleotide exchange factor expressed exclusively in brain microglia, and is regulated by PGE2 receptor EP2. It has been described that ablation of DOCK2 reduced amyloid burden in a model of Alzheimer´s disease [38].

PHBs have been functionally linked to diverse processes, such as transcriptional regulation, cell proliferation, development, and mitochondrial function [39]. Prohibitins exert a neuroprotective effect, by suppressing Reactive Oxygen Species (ROS) production and have a critical role in the activity of respiratory chain complex I [40]. It has been proposed that prohibitins promote longevity by moderating fat metabolism, mitochondrial proliferation and energy levels [41]. Its interaction with chicken brain VDAC, a gatekeeper in mitochondria mediated apoptosis is noteworthy.

In addition to proteins found in pre-complex II, four other proteins were identified in complex I. They are Calcium-binding mitochondrial carrier protein Aralar2 (Aralar2); Chondroitin sulfate glucuronyltransferase-like (CHPF2); Chain D Chicken Cytochrome Bc1 Complex Inhibited By An Iodinated Analogue Of The Polyketide Crocacin-D (3CWB_D) and NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (NDUA9).

Proteins Aralar2, CHPF2 and 3CWB_D interact with VDAC just in chicken mitochondria [18]. Aralar2 or Citrin is an aspartategluatamate carrier [42]. The presence of citrin in this study highlights a differential VDAC interactome in avian neuronal mitochondria in comparison with bovine and rat brains.

The protein CHPF2 is involved in glycosaminoglycan biosynthesis and plays a key role in tissue development and morphogenesis, and also contributes to tumor formation and development [43].

Analysis of the data reported above indicate that the kinetic assembly of protein complexes containing the VDAC follows a pattern quite different between chicken, bovine and rat brain [18]. The presence of ACON, Aralar2, CHPF2, CMBL, DOCK2, MRP1, PRDX3, RBBP8, Xirp1 and other proteins associated with VDAC only in chicken brain mitochondria, is in fact remarkable, and differentiate them from those of mammals, certainly in terms of developmental mechanisms of diseases, cell death and bioenergetics. Further studies are required to investigate if the differences in VDAC interactome reflect in differential metabolic and pathologic mechanisms between these species.

Acknowledgement

The work was supported by a grant from Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (2010/05560-6). Carla Rossini Crepaldi had a fellowship from Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (2009/06697-2). Biological samples were provided by Globoaves.

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