M.M. Dinardo, C. Musicco, V. Pesce, F. Fracasso, F. Milella, A.M.S. Lezza, G. Gadaleta, P. Cantatore, M.N. Gadaleta

Replication and transcription of the mitochondrial genome depend exclusively on nuclear DNA-encoded products. One of these products, mitochondrial transcription factor A (TFAM), plays a complex role in the regulation of both processes: it is required for mtDNA mainteinance and together with two other factors, TFB1 and TFB2, stimulates mitochondrial transcription [1]. Moreover homozygous disruption of TFAM gene and tissue-specific TFAM knockout cause severe respiratory chain deficiency and increased apoptosis in mice embryos [2]. Protein acetylation on the -amino groups of lysine is an important reversible modification that regulates gene expression. Although acetylation has been described for histone proteins, site-specific acetylation of a growing list of non-histone proteins has been shown to play an important role in transcriptional regulation and cell proliferation [3, 4]. The content of TFAM and mtDNA increased during aging in human skeletal muscle [5, 6].

Here we report results obtained by studying acetylation of TFAM as well as the relationship between mtDNA and TFAM levels in several rat tissues of young and aged rats. TFAM protein was purified from rat liver and subjected to MALDI-TOF. Mass analysis, using bovine carbonic anhydrase as internal standard, showed molecular species at m/z 23, 721. The observed mass is significantly above the theoretical average molecular mass of 23, 673, supporting the presence of a single acetylation (the mass of the acetyl-group is 42). The acetylation of TFAM was confirmed by western blot analysis. To test whether acetylation of TFAM changes during aging, we purified TFAM from 18- and 28-month-old rat liver. The level of acetylated TFAM did not appreciably change during aging [7].

To measure mtDNA content, total DNA was extracted from about 50-100 mg of frozen tissues (cerebellum, heart, kidney, liver and hindlimb skeletal muscles) of male Wistar 6-month-old and 28-month-old rats, digested with PvuII, run on a 0.35% agarose gel, blotted onto Hybond-N membrane and simultaneosly hybridized with a mitochondrial and a nuclear probe. Total proteins were extracted from the same rat tissues, separated in 12% SDS-polyacrylamide slab minigels and electroblotted onto PVDF membrane. The membrane was subjected to immunoblotting. Primary antibody for TFAM, a rabbit anti-rat TFAM antiserum, was a gift from Dr. H. Hinagaki. The hybridization signals were quantified by densitometry with LKB-Pharmacia Ultrascan-XL laser densitometer [7]. The quantitative estimate of the mtDNA copy number and TFAM content showed a similar age-related increase from 1.5 to 2.4-fold in cerebellum, liver and kidney whereas in the heart the TFAM and mtDNA levels were unchanged [7]. As far as hindlimb skeletal muscles, the TFAM and mtDNA levels were unchanged in gastrocnemius, extensorum digitorum longus and tibialis anterior muscles whereas mtDNA decreased and TFAM increased in the most oxidative hindlimb skeletal muscle that is the soleus. These data suggest the existence of tissue-specific differences in the regulation of mitochondrial gene expression probably related to different bioenergetic requirements and antioxidant defences.

1. Falkenberg M. et al. (2002) Nat. Genet. 31, 289-294.

2. Wang J. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 4038-4043.

3. Liu L. et al. (1999) Mol. Cell. Biol. 19, 1202-1209.

4. Martinez-Balbas M.A. et al. (2000) EMBO J. 19 (4), 662-671.

5. Pesce V. et al. (2001) Free Radic. Biol. Med. 30, 1223 -1233.

6. Lezza A.M.S. et al. (2001) FEBS Lett. 501, 74-78.

7. Dinardo M.M. et al. (2003) Biochem. Biophys. Res. Commun. 301, 187-191.

Keywords (Optional): 
aging rat
mtDNA content