NAD-dependent Deacetylase Activity Research Articles

SIRT3, a Mitochondrial Sirtuin Deacetylase, Regulates Mitochondrial Function and Thermogenesis in Brown Adipocytes

SIRT3 is one of the seven mammalian sirtuin homologs of the yeast Sir2 gene, which mediates the effect of caloric restriction on life span extension in yeast and Caenorhabditis elegans. Because adipose tissue is essential in energy homeostasis and also plays a role in life span determination, we decided to investigate the function of sirtuin members in fat. We report here that murine SIRT3 is expressed in brown adipose tissue and is localized on the mitochondria inner membrane. Caloric restriction activates SIRT3 expression in both white and brown adipose. Additionally, cold exposure up-regulates SIRT3 expression in brown fat, whereas elevated climate temperature reduces the expression. Enforced expression of SIRT3 in the HIB1B brown adipocytes enhances the expression of the uncoupling protein PGC-1alpha, UCP1, and a series of mitochondria-related genes. Both ADP-ribosyltransferase and deacetylase activities of SIRT3 are required for this action. Furthermore, the SIRT3 deacetylase mutant exhibits a dominant negative effect by inhibiting UCP1 expression. This inhibitive effect can be abolished by the coexpression of PGC-1alpha, indicating a major role of PGC-1alpha in the SIRT3 action. In addition, SIRT3 stimulates CREB phosphorylation, which reportedly activates PGC-1alpha promoter directly. Functionally, sustained expression of SIRT3 decreases membrane potential and reactive oxygen species production while increasing cellular respiration. Finally, SIRT3, along with genes related to mitochondrial function, is down-regulated in the brown adipose tissue of several genetically obese mice. In summary, our results demonstrate that SIRT3 activates mitochondria functions and plays an important role in adaptive thermogenesis in brown adipose.

SIRT1 Deacetylation and Repression of p300 Involves Lysine Residues 1020/1024 within the Cell Cycle Regulatory Domain 1

The SIR2 family of nicotinamide adenosine dinucleotide (NAD)-dependent deacetylases modulates diverse biological functions in different species, including longevity, apoptosis, cell cycle exit, and cellular differentiation. SIRT1, the closest mammalian ortholog of the yeast SIR2 (silent information regulator 2) gene, represses several transcription factors, including p53, NFkappaB and forkhead proteins. The p300 protein serves as a rate-limiting transcriptional cointegrator of diverse transcription factors either to activate or to repress transcription through modular subdomains. Herein, SIRT1 physically interacted with and repressed p300 transactivation, requiring the NAD-dependent deacetylase activity of SIRT1. SIRT1 repression involved the CRD1 transcriptional repression domain of p300. Two residues within the CRD1 domain (Lys-1020 and Lys-1024) were required for SIRT1 repression and served as substrates for SIRT1 deacetylation. These residues also serve as acceptor lysines for modification by the ubiquitin-like SUMO protein. The SUMO-specific protease SSP3 relieved SIRT1 repression of p300. SSP3 antagonism of SIRT1 required the SUMO-deconjugating function of SSP3. Thus, p300 serves as a deacetylase substrate for SIRT1 through a conserved SUMO consensus motif. Because p300 is a limiting transcriptional cofactor, deacetylation and repression of p300 by SIRT1 may serve an important integration point during metabolism and cellular differentiation.

In Vitro Antileishmanial Activity of Nicotinamide

Our study represents the first report demonstrating the antileishmanial activity of nicotinamide (NAm), a form of vitamin B(3). A 5 mM concentration of NAm significantly inhibited the intracellular growth of Leishmania amastigotes and the NAD-dependent deacetylase activity carried by parasites overexpressing Leishmania major SIR2 (LmSIR2). However, the transgenic parasites were as susceptible as the wild-type parasites to NAm-induced cell growth arrest. Therefore, we conclude that NAm inhibits leishmanial growth and that overexpression of LmSIR2 does not overcome this inhibition. The mechanism of the inhibition is not defined but may include other in vivo targets. NAm may thus represent a new antileishmanial agent which could potentially be used in combination with other drugs during therapy.

A chromosomal SIR2 homologue with both histone NAD-dependent ADP-ribosyltransferase and deacetylase activities is involved in DNA repair in Trypanosoma brucei.

SIR2-like proteins have been implicated in a wide range of cellular events including chromosome silencing, chromosome segregation, DNA recombination and the determination of life span. We report here the molecular and functional characterization of a SIR2-related protein from the protozoan parasite Trypanosoma brucei, which we termed TbSIR2RP1. This protein is a chromosome-associated NAD-dependent enzyme which, in contrast to other known proteins of this family, catalyses both ADP-ribosylation and deacetylation of histones, particulary H2A and H2B. Under- or overexpression of TbSIR2RP1 decreased or increased, respectively, cellular resistance to DNA damage. Treatment of trypanosomal nuclei with a DNA alkylating agent resulted in a significant increase in the level of histone ADP-ribosylation and a concomitant increase in chromatin sensitivity to micrococcal nuclease. Both of these responses correlated with the level of TbSIR2RP1 expression. We propose that histone modification by TbSIR2RP1 is involved in DNA repair.

Enzymatic assays for NAD-dependent deacetylase activities

The NAD-dependent deacetylases are a new class of enzymes responsible for the removal of acetyl groups from lysines on proteins. Instead of water, the NAD-dependent deacetylases use a highly reactive ADP-ribose intermediate as a recipient for the acetyl group. The products of the reaction are nicotinamide, acetyl-ADP-ribose, and a deacetylated substrate. Many assays have been developed for the measurement of NAD-dependent deacetylase activity. In this review we present assays based on each of the two reactions catalyzed by these enzymes, deacetylation and NAD hydrolysis. First we describe methods for the production of acetylated protein and peptide substrates for use in deacetylation reactions. Then we describe four methods for assaying deacetylation, three of which directly measure the loss of acetyl groups from a protein or peptide substrate, and one that measures acetate production. We also describe two indirect methods for following enzyme activity, NAD hydrolysis and a novel NAD-nicotinamide exchange reaction. Finally, a quantitative method using a monoacetylated peptide as a substrate and HPLC to measure products is described.

Role for human SIRT2 NAD-dependent deacetylase activity in control of mitotic exit in the cell cycle.

Studies of yeast have shown that the SIR2 gene family is involved in chromatin structure, transcriptional silencing, DNA repair, and control of cellular life span. Our functional studies of human SIRT2, a homolog of the product of the yeast SIR2 gene, indicate that it plays a role in mitosis. The SIRT2 protein is a NAD-dependent deacetylase (NDAC), the abundance of which increases dramatically during mitosis and is multiply phosphorylated at the G(2)/M transition of the cell cycle. Cells stably overexpressing the wild-type SIRT2 but not missense mutants lacking NDAC activity show a marked prolongation of the mitotic phase of the cell cycle. Overexpression of the protein phosphatase CDC14B, but not its close homolog CDC14A, results in dephosphorylation of SIRT2 with a subsequent decrease in the abundance of SIRT2 protein. A CDC14B mutant defective in catalyzing dephosphorylation fails to change the phosphorylation status or abundance of SIRT2 protein. Addition of 26S proteasome inhibitors to human cells increases the abundance of SIRT2 protein, indicating that SIRT2 is targeted for degradation by the 26S proteasome. Our data suggest that human SIRT2 is part of a phosphorylation cascade in which SIRT2 is phosphorylated late in G(2), during M, and into the period of cytokinesis. CDC14B may provoke exit from mitosis coincident with the loss of SIRT2 via ubiquitination and subsequent degradation by the 26S proteasome.

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

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 NH2-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.

Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence.

The yeast Sir2 protein mediates chromatin silencing through an intrinsic NAD-dependent histone deacetylase activity. Sir2 is a conserved protein and was recently shown to regulate lifespan extension both in budding yeast and worms. Here, we show that SIRT1, the human Sir2 homolog, is recruited to the promyelocytic leukemia protein (PML) nuclear bodies of mammalian cells upon overexpression of either PML or oncogenic Ras (Ha-rasV12). SIRT1 binds and deacetylates p53, a component of PML nuclear bodies, and it can repress p53-mediated transactivation. Moreover, we show that SIRT1 and p53 co-localize in nuclear bodies upon PML upregulation. When overexpressed in primary mouse embryo fibroblasts (MEFs), SIRT1 antagonizes PML-induced acetylation of p53 and rescues PML-mediated premature cellular senescence. Taken together, our data establish the SIRT1 deacetylase as a novel negative regulator of p53 function capable of modulating cellular senescence.

The S. cerevisiae SET3 complex includes two histone deacetylases, Hos2 and Hst1, and is a meiotic-specific repressor of the sporulation gene program.

Set3 is one of two proteins in the yeast Saccharomyces cerevisiae that, like Drosophila Trithorax, contains both SET and PHD domains. We found that Set3 forms a single complex, Set3C, with Snt1, YIL112w, Sif2, Cpr1, and two putative histone deacetylases, Hos2 and NAD-dependent Hst1. Set3C includes NAD-dependent and independent deacetylase activities when assayed in vitro. Homology searches suggest that Set3C is the yeast analog of the mammalian HDAC3/SMRT complex. Set3C represses genes in early/middle of the yeast sporulation program, including the key meiotic regulators ime2 and ndt80. Whereas Hos2 is only found in Set3C, Hst1 is also present in a complex with Sum1, supporting previous characterizations of Hst1 and Sum1 as repressors of middle sporulation genes during vegetative growth. However, Hst1 is not required for meiotic repression by Set3C, thus implying that Set3C (-Hst1) and not Hst1-Sum1, is the meiotic-specific repressor of early/middle sporulation genes.

Sir2p exists in two nucleosome-binding complexes with distinct deacetylase activities.

The absolute requirement for the histone deacetylase activity of Sir2p in silencing coupled with the conservation of Sir2p-like proteins in larger eukaryotes suggests that this molecule plays an important role in gene regulation in all organisms. Here we report the purification and characterization of two Sir2p-containing protein complexes; one of which contains Sir4p and the other Net1p. The Sir4p-containing complex has an NAD-dependent histone deacetylase activity, while the Net1p-containing complex possesses deacetylase activity but only weak NAD-dependent histone deacetylase activity. Finally, we demonstrate that the Sir2p-containing complexes bind nucleosomes efficiently and partially restrict accessibility of the linker DNA to enzymatic probes.