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The human ?Np53 isoform triggers metabolic and gene expression changes that activate mTOR and alter mitochondrial function.

ABSTRACT: A naturally occurring p53 isoform that lacks 39 residues at the N-terminus (denoted ?Np53), when expressed with wild-type p53 (WTp53), forms mixed ?Np53:WTp53 tetramers and causes accelerated aging in mice. Cellular alterations specific to ?Np53:WTp53 have been difficult to assess because ?Np53 and WTp53 coexpression results in tetramer heterogeneity, including formation of contaminating WTp53 tetramers. Based on the p53 tetramer structure, we expressed ?Np53 and WTp53 as a single transcript that maintained tetramer architecture, ensuring a 2:2 ?Np53:WTp53 stoichiometry. As expected, ?Np53:WTp53 tetramers were stable and transcriptionally active in vitro and in cells, largely mimicking the function of WTp53 tetramers. Microarray analyses, however, revealed about 80 genes whose expression was altered twofold or more in ?Np53:WTp53 cells. Moreover, global metabolomic profiling quantitated hundreds of biochemicals across different experiments (WTp53, ?Np53:WTp53, plus controls). When evaluated collectively, these data suggested altered mTOR signaling and mitochondrial function-each canonical regulators of longevity-in cells expressing ?Np53:WTp53 vs. WTp53. Increased levels of free amino acids, increased expression of IRS-1, and decreased expression of INPP5D/SHIP-1 suggested activated mTOR signaling in ?Np53:WTp53 cells; this was confirmed upon comparative analyses of several mTOR pathway intermediates. We also observed changes in mitochondrial function in ?Np53:WTp53 cells, which correlated with increased MARS2 expression and increased levels of carnitine, acetyl CoA, ATP, and Krebs cycle intermediates. Finally, increased levels of succinate and 2-hydroxyglutarate indicate potential epigenetic means to propagate ?Np53:WTp53-induced gene expression changes to cell progeny. This may be especially important for aging, as biological effects manifest over time.

SUBMITTER: Lin SC

PROVIDER: S-EPMC3773055 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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The human ΔNp53 isoform triggers metabolic and gene expression changes that activate mTOR and alter mitochondrial function.

Lin Shih-Chieh SC Karoly Edward D ED Taatjes Dylan J DJ

Aging cell 20130625 5

A naturally occurring p53 isoform that lacks 39 residues at the N-terminus (denoted ΔNp53), when expressed with wild-type p53 (WTp53), forms mixed ΔNp53:WTp53 tetramers and causes accelerated aging in mice. Cellular alterations specific to ΔNp53:WTp53 have been difficult to assess because ΔNp53 and WTp53 coexpression results in tetramer heterogeneity, including formation of contaminating WTp53 tetramers. Based on the p53 tetramer structure, we expressed ΔNp53 and WTp53 as a single transcript tha ...[more]

PMID: 23734707

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Project description:This study is to find the cellular and molecular mechanisms by which a naturally-occurring deltaNp53 isoform causes accelerated aging in humans. The biological function of deltaNp53, 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, ΔNp53 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 ΔNp53 linked with one WTp53 to form a functional ΔNp53:WTp53 tetramer with 1:1 stoichiometry. Thus, our strategy ensured each p53 tetramer contained 2 ΔNp53 and 2 WTp53 proteins. Importantly, ΔNp53:WTp53 form stable tetramers, based upon gel filtration chromatography and structural analysis using electron microscopy. Furthermore, the ΔNp53: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 ΔNp53:WTp53 tetramers, here we used microarray analysis to compare global gene expression patterns in p53-null H1299 cells expressing either WTp53 or ΔNp53:WTp53. As expected, global gene expression was largely similar, since the differences between ΔNp53:WTp53 tetramers and WTp53 tetramers are slight: only 2 of 4 p53 proteins will be different in the ΔNp53: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 ΔNp53: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 ΔNp53:WTp53 tetramers, suggesting that global me tabolic changes might contribute to ΔNp53: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 ΔNp53: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 ΔNp53:WTp53. These findings were subsequently validated using biochemical and cell-based approaches. Furthermore, whereas equal expression of ΔNp53 and WTp53 cause accelerated aging in mammals, due to alternative splicing and translation initiation ΔNp53 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. Comparison of global gene expression profiles in WTp53 or ΔNp53:WTp53 expressed H1299 RNA from FACS sorted GFP positive hybrid to Affymatrix Gene The exp 0, exp 4 and exp 5 are independent replicate/transfections/biological experiments. The exp d, exp e and exp f are control experiments.

Project description:This study is to find the cellular and molecular mechanisms by which a naturally-occurring deltaNp53 isoform causes accelerated aging in humans. The biological function of deltaNp53, 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, ÎNp53 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 ÎNp53 linked with one WTp53 to form a functional ÎNp53:WTp53 tetramer with 1:1 stoichiometry. Thus, our strategy ensured each p53 tetramer contained 2 ÎNp53 and 2 WTp53 proteins. Importantly, ÎNp53:WTp53 form stable tetramers, based upon gel filtration chromatography and structural analysis using electron microscopy. Furthermore, the ÎNp53: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 ÎNp53:WTp53 tetramers, here we used microarray analysis to compare global gene expression patterns in p53-null H1299 cells expressing either WTp53 or ÎNp53:WTp53. As expected, global gene expression was largely similar, since the differences between ÎNp53:WTp53 tetramers and WTp53 tetramers are slight: only 2 of 4 p53 proteins will be different in the ÎNp53: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 ÎNp53: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 ÎNp53:WTp53 tetramers, suggesting that global me tabolic changes might contribute to ÎNp53: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 ÎNp53: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 ÎNp53:WTp53. These findings were subsequently validated using biochemical and cell-based approaches. Furthermore, whereas equal expression of ÎNp53 and WTp53 cause accelerated aging in mammals, due to alternative splicing and translation initiation ÎNp53 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. Comparison of global gene expression profiles in WTp53 or ÎNp53:WTp53 expressed H1299 RNA from FACS sorted GFP positive hybrid to Affymatrix Gene The exp 0, exp 4 and exp 5 are independent replicate/transfections/biological experiments. The exp d, exp e and exp f are control experiments.

Project description:This study is to find the cellular and molecular mechanisms by which a naturally-occurring ΔNp53 isoform causes accelerated aging in humans. The biological function of ΔNp53, 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, ΔNp53 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 ΔNp53 linked with one WTp53 to form a functional ΔNp53:WTp53 tetramer with 1:1 stoichiometry. Thus, our strategy ensured each p53 tetramer contained 2 ΔNp53 and 2 WTp53 proteins. Importantly, ΔNp53:WTp53 form stable tetramers, based upon gel filtration chromatography and structural analysis using electron microscopy. Furthermore, the ΔNp53: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 ΔNp53:WTp53 tetramers, here we used microarray analysis to compare global gene expression patterns in p53-null H1299 cells expressing either WTp53 or ΔNp53:WTp53. As expected, global gene expression was largely similar, since the differences between ΔNp53:WTp53 tetramers and WTp53 tetramers are slight: only 2 of 4 p53 proteins will be different in the ΔNp53: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 ΔNp53: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 ΔNp53:WTp53 tetramers, suggesting that global me tabolic changes might contribute to ΔNp53: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 ΔNp53: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 ΔNp53:WTp53. These findings were subsequently validated using biochemical and cell-based approaches. Furthermore, whereas equal expression of ΔNp53 and WTp53 cause accelerated aging in mammals, due to alternative splicing and translation initiation ΔNp53 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.

Dataset Information

The human ?Np53 isoform triggers metabolic and gene expression changes that activate mTOR and alter mitochondrial function.

Publications

The human ΔNp53 isoform triggers metabolic and gene expression changes that activate mTOR and alter mitochondrial function.

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