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The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide–dependent deacetylase

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      Abstract

      The yeast silent information regulator (Sir)2 protein links cellular metabolism and transcriptional silencing through its nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase activity. We report that mitochondria from mammalian cells contain intrinsic NAD-dependent deacetylase activity. This activity is inhibited by the NAD hydrolysis product nicotinamide, but not by trichostatin A, consistent with a class III deacetylase. We identify this deacetylase as the nuclear-encoded human Sir2 homologue hSIRT3, and show that hSIRT3 is located within the mitochondrial matrix. Mitochondrial import of hSIRT3 is dependent on an NH 2-terminal amphipathic α-helix rich in basic residues. hSIRT3 is proteolytically processed in the mitochondrial matrix to a 28-kD product. This processing can be reconstituted in vitro with recombinant mitochondrial matrix processing peptidase (MPP) and is inhibited by mutation of arginines 99 and 100 . The unprocessed form of hSIRT3 is enzymatically inactive and becomes fully activated in vitro after cleavage by MPP. These observations demonstrate the existence of a latent class III deacetylase that becomes catalytically activated upon import into the human mitochondria.

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      Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.

      Bcl-2 is an integral membrane protein located mainly on the outer membrane of mitochondria. Overexpression of Bcl-2 prevents cells from undergoing apoptosis in response to a variety of stimuli. Cytosolic cytochrome c is necessary for the initiation of the apoptotic program, suggesting a possible connection between Bcl-2 and cytochrome c, which is normally located in the mitochondrial intermembrane space. Cells undergoing apoptosis were found to have an elevation of cytochrome c in the cytosol and a corresponding decrease in the mitochondria. Overexpression of Bcl-2 prevented the efflux of cytochrome c from the mitochondria and the initiation of apoptosis. Thus, one possible role of Bcl-2 in prevention of apoptosis is to block cytochrome c release from mitochondria.
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        High-efficiency transformation of mammalian cells by plasmid DNA.

        We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells, and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus 40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.
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          The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms.

          The SIR genes are determinants of life span in yeast mother cells. Here we show that life span regulation by the Sir proteins is independent of their role in nonhomologous end joining. The short life span of a sir3 or sir4 mutant is due to the simultaneous expression of a and alpha mating-type information, which indirectly causes an increase in rDNA recombination and likely increases the production of extrachromosomal rDNA circles. The short life span of a sir2 mutant also reveals a direct failure to repress recombination generated by the Fob1p-mediated replication block in the rDNA. Sir2p is a limiting component in promoting yeast longevity, and increasing the gene dosage extends the life span in wild-type cells. A possible role of the conserved SIR2 in mammalian aging is discussed.
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            Author and article information

            Affiliations
            [1 ]Gladstone Institute of Virology and Immunology, University of California San Francisco, San Francisco, CA 94103
            [2 ]Applied Tumor Virology, Deutsches Krebsforschungszentrum, D–69120 Heidelberg, Germany
            [3 ]VA Medical Center, Pittsburgh, PA 15240
            Author notes

            Address correspondence to Eric Verdin, Gladstone Institute of Virology and Immunology, 365 Vermont St., San Francisco, CA 94103. Tel.: (415) 695-3815. Fax: (415) 695-1364. E-mail: everdin@ 123456gladstone.ucsf.edu

            Journal
            J Cell Biol
            The Journal of Cell Biology
            The Rockefeller University Press
            0021-9525
            1540-8140
            19 August 2002
            : 158
            : 4
            : 647-657
            2174009
            200205057
            10.1083/jcb.200205057
            12186850
            Copyright © 2002, The Rockefeller University Press
            Categories
            Article

            Cell biology

            hdac; chromatin; 11p15.5; apoptosis; acetylation

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