Cardiovascular Research Advance Access first published online on June 16, 2008
This version [Corrected Proof] published online on July 2, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn161
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Phosphoproteome analysis of isoflurane-protected heart mitochondria: phosphorylation of adenine nucleotide translocator-1 on Tyr194 regulates mitochondrial function
,*
1 Cardiovascular Anesthesia Research Laboratory, Institute of Anesthesiology, E-HOF, University Hospital Zurich, Switzerland, and Center of Integrative Human Physiology, University of Zurich, Rämistrasse 100, CH-8091 Zurich, Switzerland
2 Institute of Pharmacology and Toxicology, University of Zurich, Switzerland
3 Functional Genomics Center Zurich, Swiss Federal Institute of Technology Zurich and University of Zurich, Switzerland
* Corresponding author. Tel: +41 44 255 46 00; fax: +41 44 255 44 09. E-mail address: michael.zaugg{at}usz.ch
Aims: Reversible phosphorylation of mitochondrial proteins is essential in the regulation of respiratory function, energy metabolism, and mitochondrion-mediated cell death. We hypothesized that mitochondrial protein phosphorylation plays a critical role in cardioprotection during pre and postconditioning, two of the most efficient anti-ischaemic therapies.
Methods and results: Using phosphoproteomic approaches, we investigated the profiles of phosphorylated proteins in Wistar rat heart mitochondria protected by pharmacological pre and postconditioning elicited by isoflurane. Sixty-one spots were detected by two-dimensional blue-native gel electrophoresis-coupled Western blotting using a phospho-Ser/Thr/Tyr-specific antibody, and 45 of these spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Eleven protein spots related to oxidative phosphorylation, energy metabolism, chaperone, and carrier functions exhibited significant changes in their phosphorylation state when protected mitochondria were compared with unprotected. Using a phosphopeptide enrichment protocol followed by liquid chromatography-MS/MS, 26 potential phosphorylation sites were identified in 19 proteins. Among these, a novel phosphorylation site was detected in adenine nucleotide translocator-1 (ANT1) at residue Tyr194. Changes in ANT phosphorylation between protected and unprotected mitochondria were confirmed by immunoprecipitation. The biological significance of ANT phosphorylation at Tyr194 was further tested with site-directed mutagenesis in yeast. Substitution of Tyr194 with Phe, mimicking the non-phosphorylated state, resulted in the inhibition of yeast growth on non-fermentable carbon sources, implying a critical role of phosphorylation at this residue in regulating ANT function and cellular respiration.
Conclusions: Our analysis emphasizes the regulatory functions of the phosphoproteome in heart mitochondria and reveals a novel, potential link between bioenergetics and cardioprotection.
KEYWORDS Preconditioning; Ischaemia/reperfusion; Adenine nucleotide translocator; Protein phosphorylation
Time for primary review: 22 days
Both authors equally contributed to this work.
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Cardiovasc Res 2008 80: 1-2.
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  M. Tanno and T. Miura Adenine nucleotide translocator, a mitochondrial carrier protein, and fate of cardiomyocytes after ischaemia/reperfusion Cardiovasc Res, October 1, 2008; 80(1): 1 - 2. [Full Text] [PDF]
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