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p53-induced growth arrest is regulated by the mitochondrial SirT3 deacetylase.

Li S, Banck M, Mujtaba S, Zhou MM, Sugrue MM, Walsh MJ - PLoS ONE (2010)

Bottom Line: Human SirT3 function appears coupled with p53 early during the initiation of p53 expression in the mitochondria by biochemical and cellular localization analysis.Additionally, we identified the chaperone protein BAG-2 in averting SirT3 targeting of p53 -mediated senescence.These studies identify a complex relationship between p53, SirT3, and chaperoning factor BAG-2 that may link the salvaging and quality assurance of the p53 protein for control of cellular fate independent of transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Mount Sinai School of Medicine, New York, New York, United States of America.

ABSTRACT
A hallmark of p53 function is to regulate a transcriptional program in response to extracellular and intracellular stress that directs cell cycle arrest, apoptosis, and cellular senescence. Independent of the role of p53 in the nucleus, some of the anti-proliferative functions of p53 reside within the mitochondria [1]. p53 can arrest cell growth in response to mitochondrial p53 in an EJ bladder carcinoma cell environment that is naïve of p53 function until induced to express p53 [2]. TP53 can independently partition with endogenous nuclear and mitochondrial proteins consistent with the ability of p53 to enact senescence. In order to address the role of p53 in navigating cellular senescence through the mitochondria, we identified SirT3 to rescue EJ/p53 cells from induced p53-mediated growth arrest. Human SirT3 function appears coupled with p53 early during the initiation of p53 expression in the mitochondria by biochemical and cellular localization analysis. Our evidence suggests that SirT3 partially abrogates p53 activity to enact growth arrest and senescence. Additionally, we identified the chaperone protein BAG-2 in averting SirT3 targeting of p53 -mediated senescence. These studies identify a complex relationship between p53, SirT3, and chaperoning factor BAG-2 that may link the salvaging and quality assurance of the p53 protein for control of cellular fate independent of transcriptional activity.

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Association of p53 with mitochondrial proteins contains NAD+ dependent protein deacetylase activity of human SirT3.(A). Total cell lysate from (+)tet, (-)tet 6 h and 24 h EJ-p53 cells were collected and solubilized in lysis buffer containing complete proteolysis inhibitor. Monoclonal antibody against human p53 (DO1) and then protein G-agarose beads were added to the sample. The immunoprecipitated proteins from the washed beads were separated on a 10% gel, then transfer to filters for immunoblotting. Blots were blocked and probed with anti-p53 (mouse monoclonal DO-1), polyclonal rabbit anti-mthsp 70 (HSPA9) and mouse monoclonal anti-Bcl 2 (clone 124, Dako USA) and anti- SirT3 followed by HRP-conjugated anti-goat, HRP-conjugated anti-mouse and HRP-conjugated anti-rabbit, and then developed using ECL. (B). Total cell, nuclear, and mitochondrial fractions were obtained from EJ-p53 cells after 12 hours of tetracycline withdrawal and p53 expression. Each fractionated lysate was analyzed by SDS-PAGE and immunoblotted with antibodies against p53, SirT1 and SirT3. (C) Protein deacetylase activity was measured against a synthetic peptide corresponding to the human p53 protein sequence (HLKSKKGQSTSRHKKLMFK-C*) radiolabeled with [14C]-acetylCoA (GE Healthcare) and purified acetyltransferases CBP and PCAF in vitro. Deacetylase activity was determined from mitochondrial and nuclear fractions taken from EJ-p53 cells following 12 hours after induction of p53 expression by tetracycline withdrawal. Deacetylase activity was determined from p53, Sirt1, and Sirt3 immunopreciptates taken from nuclear and mitochondrial fractions. (D) Co-localization of p53 and SirT3 was performed by transient expression of human Sirt3 tagged with the Green Fluorescent Protein (GFP) and visualized by laser confocal microscopy. Inducible expression of p53 was monitored after 6 and 24 hours post-induction through the withdrawal of tetracycline (Tet) from growth medium. Separate images at each wavelength were merged to determine the signal overlay.
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pone-0010486-g004: Association of p53 with mitochondrial proteins contains NAD+ dependent protein deacetylase activity of human SirT3.(A). Total cell lysate from (+)tet, (-)tet 6 h and 24 h EJ-p53 cells were collected and solubilized in lysis buffer containing complete proteolysis inhibitor. Monoclonal antibody against human p53 (DO1) and then protein G-agarose beads were added to the sample. The immunoprecipitated proteins from the washed beads were separated on a 10% gel, then transfer to filters for immunoblotting. Blots were blocked and probed with anti-p53 (mouse monoclonal DO-1), polyclonal rabbit anti-mthsp 70 (HSPA9) and mouse monoclonal anti-Bcl 2 (clone 124, Dako USA) and anti- SirT3 followed by HRP-conjugated anti-goat, HRP-conjugated anti-mouse and HRP-conjugated anti-rabbit, and then developed using ECL. (B). Total cell, nuclear, and mitochondrial fractions were obtained from EJ-p53 cells after 12 hours of tetracycline withdrawal and p53 expression. Each fractionated lysate was analyzed by SDS-PAGE and immunoblotted with antibodies against p53, SirT1 and SirT3. (C) Protein deacetylase activity was measured against a synthetic peptide corresponding to the human p53 protein sequence (HLKSKKGQSTSRHKKLMFK-C*) radiolabeled with [14C]-acetylCoA (GE Healthcare) and purified acetyltransferases CBP and PCAF in vitro. Deacetylase activity was determined from mitochondrial and nuclear fractions taken from EJ-p53 cells following 12 hours after induction of p53 expression by tetracycline withdrawal. Deacetylase activity was determined from p53, Sirt1, and Sirt3 immunopreciptates taken from nuclear and mitochondrial fractions. (D) Co-localization of p53 and SirT3 was performed by transient expression of human Sirt3 tagged with the Green Fluorescent Protein (GFP) and visualized by laser confocal microscopy. Inducible expression of p53 was monitored after 6 and 24 hours post-induction through the withdrawal of tetracycline (Tet) from growth medium. Separate images at each wavelength were merged to determine the signal overlay.

Mentions: Identification of the retroviral clone A478-34 as the SirT3 cDNA reveals that mammalian SirT3 may have a direct role in regulating p53 –induced cell growth arrest in the EJ-p53 cell line. Furthermore our studies indicate that SirT3 can direct cell fate prior to the inducible cell growth arrest by p53, as shown (Fig. 3). New evidence reveals that p53 may be acetylated at the post-translational level prior to entering the nucleus. This finding has important implications for p53 –mediated senescence since acetylation of p53 at the intracellular level plays a critical role in navigating senescence by p53. As a result a large amount of effort was needed to create the necessary reagents to appropriately investigate this new area of p53 function. Specifically, it was necessary to produce new antisera for the post-translational modifications of p53 not commercially available. This includes antisera for the acetylated form of p53 unique to the mitochondrial form of p53 in the cell model used. Our study indicates that p53 is a unique target for the anti-senescence protein deacetylase called SirT3 (Fig. 4). SirT3, like the Sirt1 protein (previously known to deacetylate p53 in nucleus) can also deacetylate a synthetic peptide corresponding to p53 (Fig. 4A). However, this deacetylation by SirT3 seems to occur in the mitochrondria (Fig. 4C). New reagents for these novel investigations now require the generation of new expression vectors for both SirT3 and p53 as well as other targets for p53 function in the mitochrondria. These results indicate post-translational acetylation of p53 is significant for understanding the role of p53 in mitochrondria in promoting growth arrest.


p53-induced growth arrest is regulated by the mitochondrial SirT3 deacetylase.

Li S, Banck M, Mujtaba S, Zhou MM, Sugrue MM, Walsh MJ - PLoS ONE (2010)

Association of p53 with mitochondrial proteins contains NAD+ dependent protein deacetylase activity of human SirT3.(A). Total cell lysate from (+)tet, (-)tet 6 h and 24 h EJ-p53 cells were collected and solubilized in lysis buffer containing complete proteolysis inhibitor. Monoclonal antibody against human p53 (DO1) and then protein G-agarose beads were added to the sample. The immunoprecipitated proteins from the washed beads were separated on a 10% gel, then transfer to filters for immunoblotting. Blots were blocked and probed with anti-p53 (mouse monoclonal DO-1), polyclonal rabbit anti-mthsp 70 (HSPA9) and mouse monoclonal anti-Bcl 2 (clone 124, Dako USA) and anti- SirT3 followed by HRP-conjugated anti-goat, HRP-conjugated anti-mouse and HRP-conjugated anti-rabbit, and then developed using ECL. (B). Total cell, nuclear, and mitochondrial fractions were obtained from EJ-p53 cells after 12 hours of tetracycline withdrawal and p53 expression. Each fractionated lysate was analyzed by SDS-PAGE and immunoblotted with antibodies against p53, SirT1 and SirT3. (C) Protein deacetylase activity was measured against a synthetic peptide corresponding to the human p53 protein sequence (HLKSKKGQSTSRHKKLMFK-C*) radiolabeled with [14C]-acetylCoA (GE Healthcare) and purified acetyltransferases CBP and PCAF in vitro. Deacetylase activity was determined from mitochondrial and nuclear fractions taken from EJ-p53 cells following 12 hours after induction of p53 expression by tetracycline withdrawal. Deacetylase activity was determined from p53, Sirt1, and Sirt3 immunopreciptates taken from nuclear and mitochondrial fractions. (D) Co-localization of p53 and SirT3 was performed by transient expression of human Sirt3 tagged with the Green Fluorescent Protein (GFP) and visualized by laser confocal microscopy. Inducible expression of p53 was monitored after 6 and 24 hours post-induction through the withdrawal of tetracycline (Tet) from growth medium. Separate images at each wavelength were merged to determine the signal overlay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2864751&req=5

pone-0010486-g004: Association of p53 with mitochondrial proteins contains NAD+ dependent protein deacetylase activity of human SirT3.(A). Total cell lysate from (+)tet, (-)tet 6 h and 24 h EJ-p53 cells were collected and solubilized in lysis buffer containing complete proteolysis inhibitor. Monoclonal antibody against human p53 (DO1) and then protein G-agarose beads were added to the sample. The immunoprecipitated proteins from the washed beads were separated on a 10% gel, then transfer to filters for immunoblotting. Blots were blocked and probed with anti-p53 (mouse monoclonal DO-1), polyclonal rabbit anti-mthsp 70 (HSPA9) and mouse monoclonal anti-Bcl 2 (clone 124, Dako USA) and anti- SirT3 followed by HRP-conjugated anti-goat, HRP-conjugated anti-mouse and HRP-conjugated anti-rabbit, and then developed using ECL. (B). Total cell, nuclear, and mitochondrial fractions were obtained from EJ-p53 cells after 12 hours of tetracycline withdrawal and p53 expression. Each fractionated lysate was analyzed by SDS-PAGE and immunoblotted with antibodies against p53, SirT1 and SirT3. (C) Protein deacetylase activity was measured against a synthetic peptide corresponding to the human p53 protein sequence (HLKSKKGQSTSRHKKLMFK-C*) radiolabeled with [14C]-acetylCoA (GE Healthcare) and purified acetyltransferases CBP and PCAF in vitro. Deacetylase activity was determined from mitochondrial and nuclear fractions taken from EJ-p53 cells following 12 hours after induction of p53 expression by tetracycline withdrawal. Deacetylase activity was determined from p53, Sirt1, and Sirt3 immunopreciptates taken from nuclear and mitochondrial fractions. (D) Co-localization of p53 and SirT3 was performed by transient expression of human Sirt3 tagged with the Green Fluorescent Protein (GFP) and visualized by laser confocal microscopy. Inducible expression of p53 was monitored after 6 and 24 hours post-induction through the withdrawal of tetracycline (Tet) from growth medium. Separate images at each wavelength were merged to determine the signal overlay.
Mentions: Identification of the retroviral clone A478-34 as the SirT3 cDNA reveals that mammalian SirT3 may have a direct role in regulating p53 –induced cell growth arrest in the EJ-p53 cell line. Furthermore our studies indicate that SirT3 can direct cell fate prior to the inducible cell growth arrest by p53, as shown (Fig. 3). New evidence reveals that p53 may be acetylated at the post-translational level prior to entering the nucleus. This finding has important implications for p53 –mediated senescence since acetylation of p53 at the intracellular level plays a critical role in navigating senescence by p53. As a result a large amount of effort was needed to create the necessary reagents to appropriately investigate this new area of p53 function. Specifically, it was necessary to produce new antisera for the post-translational modifications of p53 not commercially available. This includes antisera for the acetylated form of p53 unique to the mitochondrial form of p53 in the cell model used. Our study indicates that p53 is a unique target for the anti-senescence protein deacetylase called SirT3 (Fig. 4). SirT3, like the Sirt1 protein (previously known to deacetylate p53 in nucleus) can also deacetylate a synthetic peptide corresponding to p53 (Fig. 4A). However, this deacetylation by SirT3 seems to occur in the mitochrondria (Fig. 4C). New reagents for these novel investigations now require the generation of new expression vectors for both SirT3 and p53 as well as other targets for p53 function in the mitochrondria. These results indicate post-translational acetylation of p53 is significant for understanding the role of p53 in mitochrondria in promoting growth arrest.

Bottom Line: Human SirT3 function appears coupled with p53 early during the initiation of p53 expression in the mitochondria by biochemical and cellular localization analysis.Additionally, we identified the chaperone protein BAG-2 in averting SirT3 targeting of p53 -mediated senescence.These studies identify a complex relationship between p53, SirT3, and chaperoning factor BAG-2 that may link the salvaging and quality assurance of the p53 protein for control of cellular fate independent of transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Mount Sinai School of Medicine, New York, New York, United States of America.

ABSTRACT
A hallmark of p53 function is to regulate a transcriptional program in response to extracellular and intracellular stress that directs cell cycle arrest, apoptosis, and cellular senescence. Independent of the role of p53 in the nucleus, some of the anti-proliferative functions of p53 reside within the mitochondria [1]. p53 can arrest cell growth in response to mitochondrial p53 in an EJ bladder carcinoma cell environment that is naïve of p53 function until induced to express p53 [2]. TP53 can independently partition with endogenous nuclear and mitochondrial proteins consistent with the ability of p53 to enact senescence. In order to address the role of p53 in navigating cellular senescence through the mitochondria, we identified SirT3 to rescue EJ/p53 cells from induced p53-mediated growth arrest. Human SirT3 function appears coupled with p53 early during the initiation of p53 expression in the mitochondria by biochemical and cellular localization analysis. Our evidence suggests that SirT3 partially abrogates p53 activity to enact growth arrest and senescence. Additionally, we identified the chaperone protein BAG-2 in averting SirT3 targeting of p53 -mediated senescence. These studies identify a complex relationship between p53, SirT3, and chaperoning factor BAG-2 that may link the salvaging and quality assurance of the p53 protein for control of cellular fate independent of transcriptional activity.

Show MeSH
Related in: MedlinePlus