The common risk factors for atherosclerosis increase production of reactive oxygen species (ROS) by endothelial, vascular smooth muscle, and adventitial cells. These ROS initiate processes involved in atherogenesis through several important enzyme systems, including xanthine oxidase, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, and nitric oxide synthase. Physical forces also regulate vascular production of ROS. Oscillatory shear, which is present at sites where atherosclerosis develops, seems a particularly potent stimulus of superoxide production. The signaling cascade for activation of the NAD(P)H oxidase by angiotensin II has recently been elucidated and seems to involve a feed-forward mechanism that permits ongoing production of ROS for prolonged periods. Oxidative stress in humans with coronary artery disease is also exacerbated by a reduction of vascular extracellular superoxide dismutase, normally an important protective enzyme against the superoxide anion.

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2), are present in most gliomas and secondary glioblastomas, but are rare in other neoplasms. IDH1/2 mutations are heterozygous, and affect a single arginine residue. Recently, IDH1 mutations were identified in 8% of acute myelogenous leukemia (AML) patients. A glioma study revealed that IDH1 mutations cause a gain-of-function, resulting in the production and accumulation of 2-hydroxyglutarate (2-HG). Genotyping of 145 AML biopsies identified 11 IDH1 R132 mutant samples. Liquid chromatography-mass spectrometry metabolite screening revealed increased 2-HG levels in IDH1 R132 mutant cells and sera, and uncovered two IDH2 R172K mutations. IDH1/2 mutations were associated with normal karyotypes. Recombinant IDH1 R132C and IDH2 R172K proteins catalyze the novel nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of alpha-ketoglutarate (alpha-KG) to 2-HG. The IDH1 R132C mutation commonly found in AML reduces the affinity for isocitrate, and increases the affinity for NADPH and alpha-KG. This prevents the oxidative decarboxylation of isocitrate to alpha-KG, and facilitates the conversion of alpha-KG to 2-HG. IDH1/2 mutations confer an enzymatic gain of function that dramatically increases 2-HG in AML. This provides an explanation for the heterozygous acquisition of these mutations during tumorigenesis. 2-HG is a tractable metabolic biomarker of mutant IDH1/2 enzyme activity.

Clock:BMAL1 and NPAS2:BMAL1 are heterodimeric transcription factors that control gene expression as a function of the light-dark cycle. Although built to fluctuate at or near a 24-hour cycle, the clock can be entrained by light, activity, or food. Here we show that the DNA-binding activity of the Clock:BMAL1 and NPAS2:BMAL1 heterodimers is regulated by the redox state of nicotinamide adenine dinucleotide (NAD) cofactors in a purified system. The reduced forms of the redox cofactors, NAD(H) and NADP(H), strongly enhance DNA binding of the Clock:BMAL1 and NPAS2:BMAL1 heterodimers, whereas the oxidized forms inhibit. These observations raise the possibility that food, neuronal activity, or both may entrain the circadian clock by direct modulation of cellular redox state.

Nicotinamide adenine dinucleotide (NAD(+)) is both a coenzyme for hydride-transfer enzymes and a substrate for NAD(+)-consuming enzymes, which include ADP-ribose transferases, poly(ADP-ribose) polymerases, cADP-ribose synthases and sirtuins. Recent results establish protective roles for NAD(+) that might be applicable therapeutically to prevent neurodegenerative conditions and to fight Candida glabrata infection. In addition, the contribution that NAD(+) metabolism makes to lifespan extension in model systems indicates that therapies to boost NAD(+) might promote some of the beneficial effects of calorie restriction. Nicotinamide riboside, the recently discovered nucleoside precursor of NAD(+) in eukaryotic systems, might have advantages as a therapy to elevate NAD(+) without inhibiting sirtuins, which is associated with high-dose nicotinamide, or incurring the unpleasant side-effects of high-dose nicotinic acid.

The primary circulating form of vitamin D, 25-hydroxy-vitamin D [25(OH)D], is associated with multiple medical outcomes, including rickets, osteoporosis, multiple sclerosis and cancer. In a genome-wide association study (GWAS) of 4501 persons of European ancestry drawn from five cohorts, we identified single-nucleotide polymorphisms (SNPs) in the gene encoding group-specific component (vitamin D binding) protein, GC, on chromosome 4q12-13 that were associated with 25(OH)D concentrations: rs2282679 (P=2.0x10(-30)), in linkage disequilibrium (LD) with rs7041, a non-synonymous SNP (D432E; P=4.1x10(-22)) and rs1155563 (P=3.8x10(-25)). Suggestive signals for association with 25(OH)D were also observed for SNPs in or near three other genes involved in vitamin D synthesis or activation: rs3829251 on chromosome 11q13.4 in NADSYN1 [encoding nicotinamide adenine dinucleotide (NAD) synthetase; P=8.8x10(-7)], which was in high LD with rs1790349, located in DHCR7, the gene encoding 7-dehydrocholesterol reductase that synthesizes cholesterol from 7-dehydrocholesterol; rs6599638 in the region harboring the open-reading frame 88 (C10orf88) on chromosome 10q26.13 in the vicinity of ACADSB (acyl-Coenzyme A dehydrogenase), involved in cholesterol and vitamin D synthesis (P=3.3x10(-7)); and rs2060793 on chromosome 11p15.2 in CYP2R1 (cytochrome P450, family 2, subfamily R, polypeptide 1, encoding a key C-25 hydroxylase that converts vitamin D3 to an active vitamin D receptor ligand; P=1.4x10(-5)). We genotyped SNPs in these four regions in 2221 additional samples and confirmed strong genome-wide significant associations with 25(OH)D through meta-analysis with the GWAS data for GC (P=1.8x10(-49)), NADSYN1/DHCR7 (P=3.4x10(-9)) and CYP2R1 (P=2.9x10(-17)), but not C10orf88 (P=2.4x10(-5)).

Loss of mitochondrial complex I catalytic activity in the electron transport chain (ETC) is found in multiple tissues from individuals with sporadic Parkinson's disease (PD) and is a property of some PD model neurotoxins. Using special ETC subunit-specific and complex I immunocapture antibodies directed against the entire complex I macroassembly, we quantified ETC proteins and protein oxidation of complex I subunits in brain mitochondria from 10 PD and 12 age-matched control (CTL) samples. We measured nicotinamide adenine dinucleotide (NADH)-driven electron transfer rates through complex I and correlated these with complex I subunit oxidation levels and reductions of its 8 kDa subunit. PD brain complex I shows 11% increase in ND6, 34% decrease in its 8 kDa subunit and contains 47% more protein carbonyls localized to catalytic subunits coded for by mitochondrial and nuclear genomes We found no changes in levels of ETC proteins from complexes II-V. Oxidative damage patterns to PD complex I are reproduced by incubation of CTL brain mitochondria with NADH in the presence of rotenone but not by exogenous oxidant. NADH-driven electron transfer rates through complex I inversely correlate with complex I protein oxidation status and positively correlate with reduction in PD 8 kDa subunit. Reduced complex I function in PD brain mitochondria appears to arise from oxidation of its catalytic subunits from internal processes, not from external oxidative stress, and correlates with complex I misassembly. This complex I auto-oxidation may derive from abnormalities in mitochondrial or nuclear encoded subunits, complex I assembly factors, rotenone-like complex I toxins, or some combination.

Because the mucosal epithelia are in constant contact with large numbers of microorganisms, these surfaces must be armed with efficient microbial control systems. Here, we show that the Drosophila nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme, dual oxidase (dDuox), is indispensable for gut antimicrobial activities. Adult flies in which dDuox expression is silenced showed a marked increase in mortality rate even after a minor infection through ingestion of microbe-contaminated food. This could be restored by the specific reintroduction of dDuox, demonstrating that this oxidase generates a unique epithelial oxidative burst that limits microbial proliferation in the gut. Thus, oxidant-mediated antimicrobial responses are not restricted to the phagocytes, but rather are used more broadly, including in mucosal barrier epithelia.

Apoptotic and necrotic cell death are well characterized and are influenced by intracellular ATP levels. Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by DNA strand breaks, physiologically participates in DNA repair. Overactivation of PARP after cellular insults can lead to cell death caused by depletion of the enzyme's substrate beta-nicotinamide adenine dinucleotide and of ATP. In this study, we have differentially elicited apoptosis or necrosis in mouse fibroblasts. Fibroblasts from PARP-deficient (PARP(-/-)) mice are protected from necrotic cell death and ATP depletion but not from apoptotic death. These findings, together with cell death patterns in PARP(-/-) animals receiving other types of insults, indicate that PARP activation is an active trigger of necrosis, whereas other mechanisms mediate apoptosis.

Neutrophils constitute the dominant cell in the circulation that mediates the earliest innate immune human responses to infection. The morbidity and mortality from infection rise dramatically in patients with quantitative or qualitative neutrophil defects, providing clinical confirmation of the important role of normal neutrophils for human health. Neutrophil-dependent anti-microbial activity against ingested microbes represents the collaboration of multiple agents, including those prefabricated during granulocyte development in the bone marrow and those generated de novo following neutrophil activation. Furthermore, neutrophils cooperate with extracellular agents as well as other immune cells to optimally kill and degrade invading microbes. This brief review focuses attention on two examples of the integrated nature of neutrophil-mediated anti-microbial action within the phagosome. The importance and complexity of myeloperoxidase-mediated events illustrate a collaboration of anti-microbial responses that are endogenous to the neutrophil, whereas the synergy between the phagocyte NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and plasma-derived group IIA phospholipase A(2) exemplifies the collective effects of the neutrophil with an exogenous factor to achieve degradation of ingested staphylococci.

Redox status changes exert critical impacts on necrotic/apoptotic and normal cellular processes. We report here a widely expressed Ca2+-permeable cation channel, LTRPC2, activated by micromolar levels of H2O2 and agents that produce reactive oxygen/nitrogen species. This sensitivity of LTRPC2 to redox state modifiers was attributable to an agonistic binding of nicotinamide adenine dinucleotide (beta-NAD+) to the MutT motif. Arachidonic acid and Ca2+ were important positive regulators for LTRPC2. Heterologous LTRPC2 expression conferred susceptibility to death on HEK cells. Antisense oligonucleotide experiments revealed physiological involvement of "native" LTRPC2 in H2O2- and TNFalpha-induced Ca2+ influx and cell death. Thus, LTRPC2 represents an important intrinsic mechanism that mediates Ca2+ and Na+ overload in response to disturbance of redox state in cell death.

Abuse of the dissociative anesthetic ketamine can lead to a syndrome indistinguishable from schizophrenia. In animals, repetitive exposure to this N-methyl-d-aspartate-receptor antagonist induces the dysfunction of a subset of cortical fast-spiking inhibitory interneurons, with loss of expression of parvalbumin and the gamma-aminobutyric acid-producing enzyme GAD67. We show here that exposure of mice to ketamine induced a persistent increase in brain superoxide due to activation in neurons of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Decreasing superoxide production prevented the effects of ketamine on inhibitory interneurons in the prefrontal cortex. These results suggest that NADPH oxidase may represent a novel target for the treatment of ketamine-induced psychosis.

Spinal cord injury (SCI) provokes an inflammatory response that generates substantial secondary damage within the cord but also may contribute to its repair. Anti-inflammatory treatment of human SCI and its timing must be based on knowledge of the types of cells participating in the inflammatory response, the time after injury when they appear and then decrease in number, and the nature of their actions. Using post-mortem spinal cords, we evaluated the time course and distribution of pathological change, infiltrating neutrophils, monocytes/macrophages and lymphocytes, and microglial activation in injured spinal cords from patients who were 'dead at the scene' or who survived for intervals up to 1 year after SCI. SCI caused zones of pathological change, including areas of inflammation and necrosis in the acute cases, and cystic cavities with longer survival (Zone 1), mantles of less severe change, including axonal swellings, inflammation and Wallerian degeneration (Zone 2) and histologically intact areas (Zone 3). Zone 1 areas increased in size with time after injury whereas the overall injury (size of the Zones 1 and 2 combined) remained relatively constant from the time (1-3 days) when damage was first visible. The distribution of inflammatory cells correlated well with the location of Zone 1, and sometimes of Zone 2. Neutrophils, visualized by their expression of human neutrophil alpha-defensins (defensin), entered the spinal cord by haemorrhage or extravasation, were most numerous 1-3 days after SCI, and were detectable for up to 10 days after SCI. Significant numbers of activated CD68-immunoreactive ramified microglia and a few monocytes/macrophages were in injured tissue within 1-3 days of SCI. Activated microglia, a few monocytes/macrophages and numerous phagocytic macrophages were present for weeks to months after SCI. A few CD8(+) lymphocytes were in the injured cords throughout the sampling intervals. Expression by the inflammatory cells of the oxidative enzymes myeloperoxidase (MPO) and nicotinamide adenine dinucleotide phosphate oxidase (gp91(phox)), and of the pro-inflammatory matrix metalloproteinase (MMP)-9, was analysed to determine their potential to cause oxidative and proteolytic damage. Oxidative activity, inferred from MPO and gp91(phox) immunoreactivity, was primarily associated with neutrophils and activated microglia. Phagocytic macrophages had weak or no expression of MPO or gp91(phox). Only neutrophils expressed MMP-9. These data indicate that potentially destructive neutrophils and activated microglia, replete with oxidative and proteolytic enzymes, appear within the first few days of SCI, suggesting that anti-inflammatory 'neuroprotective' strategies should be directed at preventing early neutrophil influx and modifying microglial activation.

Necrosis has been considered a passive form of cell death in which the cell dies as a result of a bioenergetic catastrophe imposed by external conditions. However, in response to alkylating DNA damage, cells undergo necrosis as a self-determined cell fate. This form of death does not require the central apoptotic mediators p53, Bax/Bak, or caspases and actively induces an inflammatory response. Necrosis in response to DNA damage requires activation of the DNA repair protein poly(ADP-ribose) polymerase (PARP), but PARP activation is not sufficient to determine cell fate. Cell death is determined by the effect of PARP-mediated beta-nicotinamide adenine dinucleotide (NAD) consumption on cellular metabolism. Cells using aerobic glycolysis to support their bioenergetics undergo rapid ATP depletion and death in response to PARP activation. In contrast, cells catabolizing nonglucose substrates to maintain oxidative phosphorylation are resistant to ATP depletion and death in response to PARP activation. Because most cancer cells maintain their ATP production through aerobic glycolysis, these data may explain the molecular basis by which DNA-damaging agents can selectively induce tumor cell death independent of p53 or Bcl-2 family proteins.

We have found that two-photon fluorescence imaging of nicotinamide adenine dinucleotide (NADH) provides the sensitivity and spatial three-dimensional resolution to resolve metabolic signatures in processes of astrocytes and neurons deep in highly scattering brain tissue slices. This functional imaging reveals spatiotemporal partitioning of glycolytic and oxidative metabolism between astrocytes and neurons during focal neural activity that establishes a unifying hypothesis for neurometabolic coupling in which early oxidative metabolism in neurons is eventually sustained by late activation of the astrocyte-neuron lactate shuttle. Our model integrates existing views of brain energy metabolism and is in accord with known macroscopic physiological changes in vivo.

11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) interconverts inactive cortisone and active cortisol. Although bidirectional, in vivo it is believed to function as a reductase generating active glucocorticoid at a prereceptor level, enhancing glucocorticoid receptor activation. In this review, we discuss both the genetic and enzymatic characterization of 11beta-HSD1, as well as describing its role in physiology and pathology in a tissue-specific manner. The molecular basis of cortisone reductase deficiency, the putative "11beta-HSD1 knockout state" in humans, has been defined and is caused by intronic mutations in HSD11B1 that decrease gene transcription together with mutations in hexose-6-phosphate dehydrogenase, an endoluminal enzyme that provides reduced nicotinamide-adenine dinucleotide phosphate as cofactor to 11beta-HSD1 to permit reductase activity. We speculate that hexose-6-phosphate dehydrogenase activity and therefore reduced nicotinamide-adenine dinucleotide phosphate supply may be crucial in determining the directionality of 11beta-HSD1 activity. Therapeutic inhibition of 11beta-HSD1 reductase activity in patients with obesity and the metabolic syndrome, as well as in glaucoma and osteoporosis, remains an exciting prospect.

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 alpha-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.

The voltage-dependent anion channel (VDAC) mediates trafficking of small molecules and ions across the eukaryotic outer mitochondrial membrane. VDAC also interacts with antiapoptotic proteins from the Bcl-2 family, and this interaction inhibits release of apoptogenic proteins from the mitochondrion. We present the nuclear magnetic resonance (NMR) solution structure of recombinant human VDAC-1 reconstituted in detergent micelles. It forms a 19-stranded beta barrel with the first and last strand parallel. The hydrophobic outside perimeter of the barrel is covered by detergent molecules in a beltlike fashion. In the presence of cholesterol, recombinant VDAC-1 can form voltage-gated channels in phospholipid bilayers similar to those of the native protein. NMR measurements revealed the binding sites of VDAC-1 for the Bcl-2 protein Bcl-x(L), for reduced beta-nicotinamide adenine dinucleotide, and for cholesterol. Bcl-x(L) interacts with the VDAC barrel laterally at strands 17 and 18.

Pseudomonas aeruginosa toxin (PA toxin) inhibits protein synthesis in a reticulocyte cell-free system. The inhibition requires NAD and results in a block at an elongation step of polypeptide assembly. PA toxin was found to act like diphtheria toxin fragment A. Both toxins catalyze the transfer of radioactivity from nicotinamide(U-14-C)adenine dinucleotide ((14-C)NAD) into covalent linkage with the 100,000 dalton elongation (EF-2) protein. Furthermore, in the presence of a limiting amount of EF-2, excess toxin, and (14-C)NAD, the two toxins were non-additive in the amount of label transferred to EF-3. Unlike free fragment A of diphtheria toxin, the enzymatic activity of PA toxin is heat labile and neutralizable with antibody to PA toxin but not with antibody to fragment A. Although PA and diphtheria toxins have different cellular specificities and molecular properties and produce different clinical symptoms, their intracellular mechanisms of action appear to be identical.

Signaling by receptor activator of NF-kappaB (nuclear factor-kappaB) ligand (RANKL) is essential for differentiation of bone marrow monocyte-macrophage lineage (BMM) cells into osteoclasts. Here, we show RANKL stimulation of BMM cells transiently increased the intracellular level of reactive oxygen species (ROS) through a signaling cascade involving TNF (tumor necrosis factor) receptor-associated factor (TRAF) 6, Rac1, and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (Nox) 1. A deficiency in TRAF6 or expression of a dominant-interfering mutant of TRAF6 blocks RANKL-mediated ROS production. Application of N-acetylcysteine (NAC) or blocking the activity of Nox, a protein leading to the formation of ROS, with diphenylene iodonium (DPI) inhibits the responses of BMM cells to RANKL, including ROS production, activation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, and extracellular signal-regulated kinase (ERK), and osteoclast differentiation. Moreover, both RANKL-mediated ROS production and osteoclast differentiation were completely blocked in precursors depleted of Nox1 activity by RNA interference or by expressing a dominant-negative mutant of Rac1. Together, these results indicate that ROSs act as an intracellular signal mediator for osteoclast differentiation.

Phagocytes such as neutrophils and macrophages produce reactive oxygen species (ROS) during phagocytosis or stimulation with a wide variety of agents through activation of nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase that is assembled at the plasma membrane from resident plasma membrane and cytosolic protein components. One of the subunits of the phagocyte NADPH oxidase is now recognized as a member of a family of NADPH oxidases, or NOX, present in cells other than phagocytes. Physiologic generation of ROS has been implicated in a variety of physiologic responses from transcriptional activation to cell proliferation and apoptosis. The increase in superoxide and hydrogen peroxide (H2O2) that results from stimulation of the NADPH oxidase is transient, in part due to the presence of the antioxidant enzymes, which return their concentrations to the prestimulation steady state level. Thus, the antioxidant enzymes may function in the "turn-off" phase of signal transduction by ROS. During its transient elevation, H2O2 may act as a modifier of key signaling enzymes through reversible oxidation of critical thiols. The rapid reaction of thiols with H2O2 when in their unprotonated state would provide a potential mechanism for the specificity that is necessary for physiologic cell signaling.

Nicotinamide adenine dinucleotide (NAD)+-dependent sirtuins have been identified to be key regulators in the lifespan extending effects of calorie restriction (CR) in a number of species. In this study we report for the first time that promotion of the NAD+-dependent sirtuin, SIRT1-mediated deacetylase activity, may be a mechanism by which CR influences Alzheimer disease (AD)-type amyloid neuropathology. Most importantly, we report that the predicted attenuation of beta-amyloid content in the brain during CR can be reproduced in mouse neurons in vitro by manipulating cellular SIRT1 expression/activity through mechanisms involving the regulation of the serine/threonine Rho kinase ROCK1, known in part for its role in the inhibition of the non-amyloidogenic alpha-secretase processing of the amyloid precursor protein. Conversely, we found that the expression of constitutively active ROCK1 in vitro cultures significantly prevented SIRT1-mediated response, suggesting that alpha-secretase activity is required for SIRT1-mediated prevention of AD-type amyloid neuropathology. Consistently we found that the expression of exogenous human (h) SIRT1 in the brain of hSIRT1 transgenics also resulted in decreased ROCK1 expression and elevated alpha-secretase activity in vivo. These results demonstrate for the first time a role for SIRT1 activation in the brain as a novel mechanism through which CR may influence AD amyloid neuropathology. The study provides a potentially novel pharmacological strategy for AD prevention and/or treatment.

The corepressor CtBP (carboxyl-terminal binding protein) is involved in transcriptional pathways important for development, cell cycle regulation, and transformation. We demonstrate that CtBP binding to cellular and viral transcriptional repressors is regulated by the nicotinamide adenine dinucleotides NAD+ and NADH, with NADH being two to three orders of magnitude more effective. Levels of free nuclear nicotinamide adenine dinucleotides, determined using two-photon microscopy, correspond to the levels required for half-maximal CtBP binding and are considerably lower than those previously reported. Agents capable of increasing NADH levels stimulate CtBP binding to its partners in vivo and potentiate CtBP-mediated repression. We propose that this ability to detect changes in nuclear NAD+/NADH ratio allows CtBP to serve as a redox sensor for transcription.

Acetyl-coenzyme A (CoA) synthetase (Acs) is an enzyme central to metabolism in prokaryotes and eukaryotes. Acs synthesizes acetyl CoA from acetate, adenosine triphosphate, and CoA through an acetyl-adenosine monophosphate (AMP) intermediate. Immunoblotting and mass spectrometry analysis showed that Salmonella enterica Acs enzyme activity is posttranslationally regulated by acetylation of lysine-609. Acetylation blocks synthesis of the adenylate intermediate but does not affect the thioester-forming activity of the enzyme. Activation of the acetylated enzyme requires the nicotinamide adenine dinucleotide-dependent protein deacetylase activity of the CobB Sir2 protein from S. enterica. We propose that acetylation modulates the activity of all the AMP-forming family of enzymes, including nonribosomal peptide synthetases, luciferase, and aryl- and acyl-CoA synthetases. These findings extend our knowledge of the roles of Sir2 proteins in gene silencing, chromosome stability, and cell aging and imply that lysine acetylation is a common regulatory mechanism in eukaryotes and prokaryotes.

The surface of streptococci presents an array of different proteins, each designed to perform a specific function. In an attempt to understand the early events in group A streptococci infection, we have identified and purified a major surface protein from group A type 6 streptococci that has both an enzymatic activity and a binding capacity for a variety of proteins. Mass spectrometric analysis of the purified molecule revealed a monomer of 35.8 kD. Molecular sieve chromatography and sodium dodecyl sulfate (SDS)-gel electrophoresis suggest that the native conformation of the protein is likely to be a tetramer of 156 kD. NH2-terminal amino acid sequence analysis revealed 83% homology in the first 18 residues and about 56% in the first 39 residues with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of eukaryotic or bacterial origin. This streptococcal surface GAPDH (SDH) exhibits a dose-dependent dehydrogenase activity on glyceraldehyde-3-phosphate in the presence of beta-nicotinamide adenine dinucleotide both in its pure form and on the streptococcal surface. Its sensitivity to trypsin on whole organism and its inability to be removed with 2 M NaCl or 2% SDS support its surface location and tight attachment to the streptococcal cell. Affinity-purified antibodies to SDH detected the presence of this protein on the surface of all M serotypes of group A streptococcal tested. Purified SDH was found to bind to fibronectin, lysozyme, as well as the cytoskeletal proteins myosin and actin. The binding activity to myosin was found to be localized to the globular heavy meromyosin domain. SDH did not bind to streptococcal M protein, tropomyosin, or the coiled-coil domain of myosin. The multiple binding capacity of the SDH in conjunction with its GAPDH activity may play a role in the colonization, internalization, and the subsequent proliferation of group A streptococci.