Metabolic and gene expression changes induced by the naturally occurring Np53 isoform link mTOR pathway and mitochondrial alterations to the progeroid phenotype.

This study is to find the cellular and molecular mechanisms by which a naturally-occurring Np53 isoform causes accelerated aging in humans. The biological function of Np53, which lacks only 40 N-terminal amino acids, represents an example of p53 as a regulator of mammalian aging. When expressed together with WTp53 in mice, Np53 causes an aging phenotype such as shorter life span, reduced body mass, organ atrophy and osteoporosis. Because p53 must form a tetramer to regulate transcription, we generated p53 clones (based upon the structure of the native p53 tetramer) containing one Np53 linked with one WTp53 to form a functional Np53:WTp53 tetramer with 1:1 stoichiometry. Thus, our strategy ensured each p53 tetramer contained 2 Np53 and 2 WTp53 proteins. Importantly, Np53:WTp53 form stable tetramers, based upon gel filtration chromatography and structural analysis using electron microscopy. Furthermore, the Np53:WTp53 tetramer activates transcription equally well compared with WTp53 tetramers in an in vitro reconstituted transcription system. Having verified the stoichiometry, stability, structure, and activity of these Np53:WTp53 tetramers, here we used microarray analysis to compare global gene expression patterns in p53-null H1299 cells expressing either WTp53 or Np53:WTp53. As expected, global gene expression was largely similar, since the differences between Np53:WTp53 tetramers and WTp53 tetramers are slight: only 2 of 4 p53 proteins will be different in the Np53:WTp53 tetramer. Among only several dozen genes that were selectively up- or down-regulated 2-fold or greater, many genes known to regulate mammalian aging were altered in cells expressing Np53:WTp53, including insulin signaling pathway members (IRS1, INPP5D, PLK3, MAP3K1, FGF5) and regulators of glucose metabolism (SLC2A2, CRYAB, LRCH1). Expression of other key metabolic genes were also altered in cells expressing Np53:WTp53 tetramers, suggesting that global me tabolic changes might contribute to Np53:WTp53 pathology. In collaboration with Metabolon (Durham, NC), we identified approximately one hundred metabolites that were significantly up- or down-regulated in H1299 cells expressing Np53:WTp53. The metabolome analysis was a powerful complement to the gene expression data, and further suggested that the mTOR pathway (e.g. across-the-board up-regulation of amino acid levels) and mitochondrial function (e.g. up-regulation of carnitine, important for a-oxidation of fatty acids) was altered in cells expressing Np53:WTp53. These findings were subsequently validated using biochemical and cell-based approaches. Furthermore, whereas equal expression of Np53 and WTp53 cause accelerated aging in mammals, due to alternative splicing and translation initiation Np53 is a naturally-occurring isoform whose expression levels can change throughout the lifetime. Thus, the cellular and molecular mechanisms identified from this work will likely reflect changes common to normal, physiological aging.

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Lin SC, Karoly ED, Taatjes DJ