Proteomic approach to the identification of voltage‐dependent anion channel protein isoforms in guinea pig brain synaptosomes

Liberatori, Sabrina; Cañas, Benito; Tani, Chiara; Bini, Luca; Buonocore, Giuseppe; Godovac‐Zimmermann, Jasminka; Mishra, Om P.; Delivoria‐Papadopoulos, Maria; Bracci, Rodolfo; Pallini, Vitaliano

doi:10.1002/pmic.200300734

Cited by 49 publications

(43 citation statements)

References 36 publications

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“…The nature of the difference of the two forms of VDAC2, which may be related to the two VDAC2 species detected by 2-DE analysis of various rat tissues by Yamamoto et al (96), is still unclear. VDAC2 is known to be subject to tyrosine phosphorylation (97) and lysine acetylation (98), but the very small pI difference of the two observed spots of Ͻ0.1 pH unit suggests that the two forms do not differ by one of these PTMs but, presumably, some other modification.…”

Section: Discussionmentioning

confidence: 92%

Analysis of 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced Proteome Changes in 5L Rat Hepatoma Cells Reveals Novel Targets of Dioxin Action Including the Mitochondrial Apoptosis Regulator VDAC2

Sarioglu

Brandner

Haberger

et al. 2008

Molecular & Cellular Proteomics

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As part of a comprehensive survey of the impact of the environmental pollutant and hepatocarcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the proteome of hepatic cells, we have performed a high resolution twodimensional gel electrophoresis study on the rat hepatoma cell line 5L. 78 protein species corresponding to 73 different proteins were identified as up-or down-regulated following exposure of the cells to 1 nM TCDD for 8 h. There was an overlap of only nine proteins with those detected as altered by TCDD in our recent study using the non-gel-based isotope-coded protein label method (Sarioglu, H., Brandner, S., Jacobsen, C., Meindl, T., Schmidt, A., Kellermann, J., Lottspeich, F., and Andrae, U. The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 1 is the prototype of a class of compounds known as halogenated aromatic hydrocarbons that includes dibenzo-p-dioxins, dibenzofurans, and polychlorinated biphenyls. TCDD has been shown to cause a wide variety of toxic effects and is regarded as the most potent hepatocarcinogen in experimental animals (1). Epidemiological evidence suggests that TCDD is also a human carcinogen (2, 3). The molecular mechanisms underlying the tumorigenic activity of the compound are still largely unknown, which strongly hampers the estimation of the risk to humans associated with dioxin exposure and necessitates further studies aimed at the clarification of these mechanisms. Moreover the multifaceted toxicity of TCDD makes it a very attractive model compound for studies addressing the identification of basic principles associated with the disturbance of cellular homeostasis.

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Section: Discussionmentioning

confidence: 92%

Analysis of 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced Proteome Changes in 5L Rat Hepatoma Cells Reveals Novel Targets of Dioxin Action Including the Mitochondrial Apoptosis Regulator VDAC2

Sarioglu

Brandner

Haberger

et al. 2008

Molecular & Cellular Proteomics

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“…Using a functional proteomics approach involving MALDI-TOF mass spectrometry, Yan et al identified VDAC as phosphorylated, but neither the isoform or the site of phosphorylation was specified [59]. VDAC-1 and -2 were found to contain phosphorylated tyrosines by immunoblotting in guinea pig brain synaptosomes under hypoxic conditions [60]. The location of phosphotyrosine was not identified.…”

Section: Phosphorylationmentioning

confidence: 99%

Post-translational modifications of rat liver mitochondrial outer membrane proteins identified by mass spectrometry

Distler

Kerner

Hoppel

2007

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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SUMMARYThe identification of post-translational modifications is difficult especially for hydrophobic membrane proteins. Here we present the identification of several types of protein modifications on membrane proteins isolated from mitochondrial outer membranes. We show, in vivo, the mature rat liver mitochondrial carnitine palmitoyltransferase-I enzyme is N-terminally acetylated, phosphorylated on two threonine residues, and nitrated on two tyrosine residues. We show long chain acyl-CoA synthetase 1 is acetylated at both the N-terminal end and at a lysine residue and tyrosine residues are found to be phosphorylated and nitrated. For the three voltage-dependent anion channel isoforms present in the mitochondria, the N-terminal regions of the protein were determined and sites of phosphorylation were identified. These novel findings raise questions about regulatory aspects of carnitine palmitoyltransferase-I, long chain acyl-CoA synthetase and voltage dependent anion channel and further studies should advance our understanding about regulation of mitochondrial fatty acid oxidation in general and these three proteins in specific.

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“…8). Indeed, the peptide includes a threonine and a tyrosine residue, and VDAC phosphorylation has been previously reported (32), specifically tyrosine phosphorylation (33). In addition, the second proposed NBS partly overlaps a peptide that is a part of the 14-kDa V8-generated radiolabeled fragment (positions 98 -111, Fig.…”

Section: Characterization Of the Vdac Nbss-[␣-mentioning

confidence: 99%

Nucleotide-binding Sites in the Voltage-dependent Anion Channel

Yehezkel

Hadad

Zaid

et al. 2006

Journal of Biological Chemistry

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In this study, we addressed the presence and location of nucleotide-binding sites in the voltage-dependent anion channel (VDAC). VDAC bound to reactive red 120-agarose, from which it was eluted by ATP, less effectively by ADP and AMP, but not by NADH. The photoreactive ATP analog, benzoyl-benzoyl-ATP (BzATP), was used to identify and characterize the ATP-binding sites in VDAC.[␣-32 P]BzATP bound to purified VDAC at two or more binding sites with apparent high and low binding affinities. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis of BzATP-labeled VDAC confirmed the binding of at least two BzATP molecules to VDAC. The VDAC BzATP-binding sites showed higher specificity for purine than for pyrimidine nucleotides and higher affinity for negatively charged nucleotide species. VDAC treatment with the lysyl residue modifying reagent, fluorescein 5-isothiocyanate, markedly inhibited VDAC labeling with BzATP. The VDAC nucleotide-binding sites were localized using chemical and enzymatic cleavage. Digestion of [␣-32 P]BzATP-labeled VDAC with CNBr or V8 protease resulted in the appearance of ϳ17-and ϳ14-kDa labeled fragments. Further digestion, high performance liquid chromatography separation, and sequencing of the selected V8 peptides suggested that the labeled fragments originated from two different regions of the VDAC molecule. MALDI-TOF analysis of BzATP-labeled, tryptic VDAC fragments indicated and localized three nucleotide binding sites, two of which were at the N and C termini of VDAC. Thus, the presence of two or more nucleotidebinding sites in VDAC is suggested, and their possible function in the control of VDAC activity, and, thereby, of outer mitochondrial membrane permeability is discussed.It has recently been recognized that there exists a metabolic coupling between the cytosol and the mitochondria, with the outer mitochondrial membrane (OMM), 2 the boundary between these compartments, fulfilling an important function in this coupling (1-4). In the cross-talk between mitochondria and cytosol, mitochondrial ATP and Ca 2ϩ play major roles. As the primary transporter of nucleotides, Ca 2ϩ , and other ions and metabolites across the OMM (reviewed in Refs. 5 and 6), the voltage-dependent anion channel (VDAC) mediates a substantial portion of the OMM molecular traffic. It has been demonstrated that VDAC is permeable to Ca 2ϩ and possesses Ca 2ϩ -binding sites that control its activity (reviewed in Ref. 5). VDAC permeability is also regulated by associated protein(s) (reviewed in Refs. 5-7), and by different ligands that bind to VDAC, such as glutamate, ruthenium red, La 3ϩ , and NADH (reviewed in Refs. 5 and 6). Thus, by providing the pathway for anions, cations, ATP, and other metabolites into and out of the mitochondria, as well as possessing ligand-binding sites, VDAC plays an important role in the regulation of mitochondrial functions. Indeed, VDAC has been suggested to control OMM permeability, coupled respiration, and cell survival (3,4,8).It has been proposed that activati...

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Proteomic approach to the identification of voltage‐dependent anion channel protein isoforms in guinea pig brain synaptosomes

Cited by 49 publications

References 36 publications

Analysis of 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced Proteome Changes in 5L Rat Hepatoma Cells Reveals Novel Targets of Dioxin Action Including the Mitochondrial Apoptosis Regulator VDAC2

Analysis of 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced Proteome Changes in 5L Rat Hepatoma Cells Reveals Novel Targets of Dioxin Action Including the Mitochondrial Apoptosis Regulator VDAC2

Post-translational modifications of rat liver mitochondrial outer membrane proteins identified by mass spectrometry

Nucleotide-binding Sites in the Voltage-dependent Anion Channel

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