Regulation of yeast sirtuins by NAD(+) metabolism and calorie restriction

Abstract

The Sir2 family proteins (sirtuins) are evolutionally conserved NAD(+) (nicotinamide adenine dinucleotide)-dependent protein deacetylases and ADP-ribosylases, which have been shown to play important roles in the regulation of stress response, gene transcription, cellular metabolism and longevity. Recent studies have also suggested that sirtuins are downstream targets of calorie restriction (CR), which mediate CR-induced beneficial effects including life span extension in an NAD(+)-dependent manner. CR extends life span in many species and has been shown to ameliorate many age-associated disorders such as diabetes and cancers. Understanding the mechanisms of CR as well as the regulation of sirtuins will therefore provide insights into the molecular basis of these age-associated metabolic diseases. This review focuses on discussing advances in studies of sirtuins and NAD(+) metabolism in genetically tractable model system, the budding yeast Saccharomyces cerevisiae. These studies have unraveled key metabolic longevity factors in the CR signaling and NAD(+) biosynthesis pathways, which may also contribute to the regulation of sirtuin activity. Many components of the NAD(+) biosynthesis pathway and CR signaling pathway are conserved in yeast and higher eukaryotes including humans. Therefore, these findings will help elucidate the mechanisms underlying age-associated metabolic disease and perhaps human aging.

Fig. 1

Sir2 functions in yeast. (A) The SIR complexes mediate silencing at the mating type loci, telomeres, and rDNA loci. Sir2 associates with Sir1/3/4 to repress the transcription of cryptic mating type loci (HML and HMR), while Sir2/3/4 are recruited to telomeres by telomere binding proteins to mediate telomere maintenance. In these two loci, Sir3 is suggested to mediate the spreading of SIR complex along the region to accomplish the assembly of silent chromatin. At the rDNA loci, Sir2 is recruited to the nucleolus to form RENT complexes with nucleolar protein Net1 and phosphatase Cdc14 to repress the recombination of rDNA repeats and to maintain genome integrity. (B) A model for ERC formation at the rDNA loci. Fob1-dependent replication fork block (STOP) induces double strain breaks (DBSs) within rDNA repeats during DNA replication. The repair of DBS could be initiated via recombination by using unequal sister chromatid (left) or equal sister chromatid (right) as template. The unequal sister-chromatid recombination results in the excision of rDNA repeats and the formation of extrachromosomal rDNA circle (ERC). ERCs are preferentially retained in the mother cell during asymmetric cytokinesis, and this accumulation of ERCs is suggested to be the primary cause of replicative aging in yeast. Sir2 functions in maintaining rDNA stability by promoting the cohesion of sister chromatids, which favors the proceeding of equal sister-chromatid recombination to fill up the double strand break. (C) Asymmetric segregation of oxidatively damaged proteins in yeast. In wild type cells (WT), oxidatively damaged proteins (shown as grey dots within cells) are asymmetrically retained in the mother cells, which is lost in old cells. In the sir2Δ deletion mutant, this age-dependent asymmetry is abolished, suggesting a role of Sir2 in protecting newborn cells by promoting an asymmetric segregation of damaged proteins.

Fig. 2

A model for CR-mediated life span extension in yeast. Multiple pathways mediate CR-induced life span extension. CR increases mitochondrial respiration and the concomitant elevation in NAD⁺/NADH ratio. The malate-aspartate NADH shuttle components (Mdh1/Aat1) function to balance redox equivalents between the mitochondrial and the cytosolic/nuclear pools, and are essential for relaying the metabolic signals in the mitochondria to downstream longevity factors, such as Sir2. CR also increases protein level of nicotinamidase Pnc1 in NAD⁺ salvage pathway. Up-regulation of Pnc1 facilitates the clearance of Nam and enhances Sir2 activity. Other Sir2-independent pathways also exist to mediate CR-induced beneficial effects including life span extension.

Fig. 3

Schematic presentations of the yeast mitochondrial respiratory chain and the NADH shuttle system. Mitochondrial NADH dehydrogenases (ND) oxidize NADH from both the cytosolic and mitochondrial pools to regenerate NAD⁺. The electrons acquired are then passed down the electron transport chain to the terminal acceptor oxygen (O₂). CR activates mitochondrial activity, which results in an increase in NAD⁺/NADH ratio. Since the mitochondrial inner membrane is impermeable to NAD⁺ and NADH, this increase in NAD⁺/NADH ratio is transmitted into the cytosolic/nuclear pools via the NADH shuttle system. The NADH shuttle system affects the NAD/NADH ratio indirectly by coupling the redox reaction of NAD⁺ and NADH with the redox reactions of permeable redox equivalents of NAD⁺ and NADH. In the example shown here, cytosolic malate dehydrogenase (Mdh2) couples the oxidation of NADH with the reduction of oxaloacetate (O). The resulting reduced product malate (M) enters the mitochondria via specific carriers and thereby shuttling redox equivalents into the mitochondrial matrix. In the mitochondrial matrix, mitochondrial Mdh1 catalyzes the generation of NADH from NAD⁺ and electrons carried by malate (M) are concurrently oxidized to oxaloacetate (O). Mitochondrial aspartate aminase (Aat1) then converts oxaloacetate to aspartate (A), which is subsequently transported to the cytosol via other carriers. Cytosolic Aat2 then regenerates oxaloacetate from aspartate, and another round of NADH shuttling is ready to resume. With the NADH shuttle system, alterations of the mitochondrial NAD⁺/NADH ratio resulted from the increase of mitochondrial respiration could be conveyed to the cytosolic/nuclear compartments.

Fig. 4

NAD⁺ biosynthesis and homeostasis in yeast. (A) The model of NAD⁺ biosynthesis pathways in yeast. NAD⁺ is produced via de novo synthesis from tryptophan or the salvage of NAD⁺ metabolites from NAD⁺-consuming reactions and from environment. See text for detail steps. (B) A schematic model of extended NAD⁺ pool. Yeast cells constitutively produce, release, and retrieve nicotinamide riboside (NmR). This extended cycle encompasses intracellular and extracellular compartments and might functions as a flexible pyridine nucleotide reservoir.