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Preventing Nitration of Specific Tyrosine Sites in Adenine Nucleotide Translocase Differentially Protects Against Cell Oxidative Stress Injury
Author(s) -
Yang Meiying,
Kwok WaiMeng,
Bienengraeber Martin,
Camara Amadou,
Heisner James,
Stowe David
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.635.5
Subject(s) - ant , chemistry , biochemistry , oxidative stress , mitochondrion , oxidative phosphorylation , inner mitochondrial membrane , mutant , adenine nucleotide translocator , nitration , microbiology and biotechnology , biology , ecology , organic chemistry , gene
The adenine nucleotide translocase (ANT) is the most abundant protein in the inner mitochondrial membrane and the main transporter for ATP and ADP exchange across the membrane. Ischemia reperfusion (IR) injury causes mitochondrial accumulation of reactive O 2 and N 2 species (ROS/RNS), which can induce irreversible and deleterious tyrosine nitration ( N Tyr) of protein. In this study, we investigated IR‐induced N Tyr of ANT and its impact on cell damage during oxidative stress. Isolated guinea pig hearts were subjected to 35 min ischemia and 20 min reperfusion or no‐IR (control). At the end of reperfusion, ANT was purified from IR or control heart mitochondria and subjected to Western blotting with antibodies to ANT and nitro‐Tyr. We found that IR induced N Tyr of ANT. Mass spectrometry analysis identified ANT tyrosines (Y) residues Y 81 , Y 191 and Y 195 were nitrated when mitochondria were treated with 100 µM peroxynitrite, an oxidant. To determine the contribution of nitration of ANT Y 81 , Y 191 and Y 195 on cellular injury induced by oxidative stress, a single mutant Y 81 F (phenyalanine) and a double mutant Y 192 F and Y 195 F were constructed. After transfection of these mutants into a cardiomyocyte cell line, HL‐1, we found that overexpression of both the single and double mutants of ANT displayed less lactic acid dehydrogenase release induced by hypoxia and reoxygeneration compared to the wild type ANT. Moreover, the association of Voltage‐dependent anion channels with ANT was reduced after IR. In summary, we have identified specific N Tyr residues in ANT following oxidative stress, and demonstrated that preventing nitration of these sites attenuated cell oxidative stress injury.