Acetylation and level of mitochondrial transcription factor A in several organs of young and old rats

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Abstract

To gain further information on the role of mitochondrial transcription factor A (TFAM) in mitochondrial biogenesis, we studied the post-translational modifications of the protein in 6- and 28-month-old rat liver. Mass spectrometry and immunoblot analysis revealed that TFAM was acetylated at a single lysine residue and that the level of acetylation did not change with age. The measurement of the content of TFAM and of mitochondrial DNA (mtDNA) in several organs (cerebellum, heart, kidney, and liver) of young and old rats showed an age-related increase of mtDNA and TFAM in all the organs analyzed, except in heart. These data are discussed in the light of the multiple roles of TFAM in mitochondrial biogenesis and of the age-related change of the mitochondrial transcription.

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Materials and methods

Animal samples. Male Wistar rats were maintained two per cage on a 12:12 h light–dark cycle at 25 °C and had access to standard laboratory chow and water ad libitum. All procedures were in accordance with the guiding principles in the care and use of laboratory animals of Bari University. Four rats for each of different ages (6 and 28 month) were killed and four of their own organs (cerebellum, heart, kidney, and liver) were quickly excised, immediately frozen in isopentane cooled by liquid

Acetylation of TFAM

In order to test whether TFAM was acetylated, the protein was purified from liver of 6-month old rat and subjected to MALDI-TOF mass spectrometry. As shown in Fig. 1A mass analysis, using bovine carbonic anhydrase as internal standard, showed a molecular species at m/z 23,721. Other internally calibrated spectra (not shown) resulted in molecular masses from 23,706 to 23,730 Da. The observed mass is significantly above the theoretical average molecular mass of 23,673, supporting the presence of a

Discussion

The results reported in this paper provide further information on qualitative and quantitative changes of TFAM during aging in different rat organs. Post-translational modification of a mitochondrial DNA-binding protein is reported here for the first time. TFAM is acetylated at a single lysine residue and the extent of acetylation does not change substantially with age. Protein acetylation is a regulatory mechanism for the expression of nuclear genes. In particular histone acetylation activates

Acknowledgements

We thank Dr. H. Hinagaki (Department of Chemistry, National Industrial Research Institute of Nagoya, Japan) for the gift of rat TFAM antiserum. This work was supported by Progetto di Ateneo ‘Regolazione della biogenesi mitocondriale mediata da proteine nucleari in diversi sistemi sperimentali,’ by Sigma-Tau Research Program “Effetti dell’acetil-L-carnitina sulla replicazione ed espressione del DNA mitocondriale nel ratto,” and by M.U.R.S.T. project Cluster C03 “Studio di geni di interesse

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