hMOF Acetylation of DBC1/CCAR2 Prevents Binding and Inhibition of SirT1

The NAD⁺-dependent deacetylase SirT1 regulates gene silencing and genomic stability in response to nutrient deprivation and DNA damage. An important regulator of SirT1 in mammalian cells is DBC1 (deleted in breast cancer 1; KIAA1967 or CCAR2), which binds to SirT1 and inhibits the deacetylation of substrates. Recent studies have revealed that ATM/ATR-mediated phosphorylation of DBC1 promotes binding to SirT1. Here we show that DBC1 is modified by acetylation on two N-terminal lysine residues (K112 and K215). The MYST family histone acetyltransferase hMOF (human MOF) is responsible for DBC1 acetylation. Acetylation of K112 and K215 inhibits DBC1-SirT1 binding and increases SirT1 deacetylase activity. SirT1 also promotes DBC1 deacetylation, suggesting the presence of a negative-feedback mechanism that stabilizes the SirT1-DBC1 complex and limits SirT1 activity. hMOF binding and acetylation of DBC1 are inhibited after DNA damage in an ATM-dependent fashion, contributing to increased SirT1-DBC1 binding after DNA damage. Furthermore, a DBC1 mutant that mimics the acetylated state fails to promote apoptosis after DNA damage. These results suggest that acetylation of DBC1 inhibits binding to SirT1 and serves as a mechanism that connects DNA damage signaling to SirT1 and cell fate determination.     

A Redox-resistant Sirtuin-1 Mutant Protects against Hepatic Metabolic and Oxidant Stress

Sirtuin-1 (SirT1), a member of the NAD⁺-dependent class III histone deacetylase family, is inactivated in vitro by oxidation of critical cysteine thiols. In a model of metabolic syndrome, SirT1 activation attenuated apoptosis of hepatocytes and improved liver function including lipid metabolism. We show in SirT1-overexpressing HepG2 cells that oxidants (nitrosocysteine and hydrogen peroxide) or metabolic stress (high palmitate and high glucose) inactivated SirT1 by reversible oxidative post-translational modifications (OPTMs) on three cysteines. Mutating these oxidation-sensitive cysteines to serine preserved SirT1 activity and abolished reversible OPTMs. Overexpressed mutant SirT1 maintained deacetylase activity and attenuated proapoptotic signaling, whereas overexpressed wild type SirT1 was less protective in metabolically or oxidant-stressed cells. To prove that OPTMs of SirT1 are glutathione (GSH) adducts, glutaredoxin-1 was overexpressed to remove this modification. Glutaredoxin-1 overexpression maintained endogenous SirT1 activity and prevented proapoptotic signaling in metabolically stressed HepG2 cells. The in vivo significance of oxidative inactivation of SirT1 was investigated in livers of high fat diet-fed C57/B6J mice. SirT1 deacetylase activity was decreased in the absence of changes in SirT1 expression and associated with a marked increase in OPTMs. These results indicate that glutathione adducts on specific SirT1 thiols may be responsible for dysfunctional SirT1 associated with liver disease in metabolic syndrome.     

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD⁺ biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD⁺ in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD⁺ content, we have expressed plant and yeast mitochondrial NAD⁺ carriers in human cells and observed a profound increase in mitochondrial NAD⁺. None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD⁺ content. Surprisingly, constitutive redistribution of NAD⁺ from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD⁺ transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD⁺ levels. These results suggest that a mitochondrial NAD⁺ transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD⁺ synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells.     

The chemical biology of NAD+ regulation in axon degeneration

During axon degeneration, NAD⁺ levels are largely controlled by two enzymes: nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) and sterile alpha and toll interleukin motif containing protein 1 (SARM1). NMNAT2, which catalyzes the formation of NAD⁺ from NMN and ATP, is actively degraded leading to decreased NAD⁺ levels. SARM1 activity further decreases the concentration of NAD⁺ by catalyzing its hydrolysis to form nicotinamide and a mixture of ADPR and cADPR. Notably, SARM1 knockout mice show decreased neurodegeneration in animal models of axon degeneration, highlighting the therapeutic potential of targeting this novel NAD⁺ hydrolase. This review discusses recent advances in the SARM1 field, including SARM1 structure, regulation, and catalysis as well as the identification of the first SARM1 inhibitors.     

Enhanced radiosensitivity and radiation-induced apoptosis in glioma CD133-positive cells by knockdown of SirT1 expression

CD133-expressing glioma cells play a critical role in tumor recovery after treatment and are resistant to radiotherapy. Herein, we demonstrated that glioblastoma-derived CD133-positive cells (GBM-CD133⁺) are capable of self-renewal and express high levels of embryonic stem cell genes and SirT1 compared to GBM-CD133⁻ cells. To evaluate the role of SirT1 in GBM-CD133⁺, we used a lentiviral vector expressing shRNA to knock-down SirT1 expression (sh-SirT1) in GBM-CD133⁺. Silencing of SirT1 significantly enhanced the sensitivity of GBM-CD133⁺ to radiation and increased the level of radiation-mediated apoptosis. Importantly, knock-down of SirT1 increased the effectiveness of radiotherapy in the inhibition of tumor growth in nude mice transplanted with GBM-CD133⁺. Kaplan-Meier survival analysis indicated that the mean survival rate of GBM-CD133⁺ mice treated with radiotherapy was significantly improved by Sh-SirT1 as well. In sum, these results suggest that SirT1 is a potential target for increasing the sensitivity of GBM and glioblastoma-associated cancer stem cells to radiotherapy.     

The NAD+ synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) is a p53 downstream target

NAD⁺ metabolism plays key roles not only in energy production but also in diverse cellular physiology. Aberrant NAD⁺ metabolism is considered a hallmark of cancer. Recently, the tumor suppressor p53, a major player in cancer signaling pathways, has been implicated as an important regulator of cellular metabolism. This notion led us to examine whether p53 can regulate NAD⁺ biosynthesis in the cell. Our search resulted in the identification of nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2), a NAD⁺ synthetase, as a novel downstream target gene of p53. We show that NMNAT-2 expression is induced upon DNA damage in a p53-dependent manner. Two putative p53 binding sites were identified within the human NMNAT-2 gene, and both were found to be functional in a p53-dependent manner. Furthermore, knockdown of NMNAT-2 significantly reduces cellular NAD⁺ levels and protects cells from p53-dependent cell death upon DNA damage, suggesting an important functional role of NMNAT-2 in p53-mediated signaling. Our demonstration that p53 modulates cellular NAD⁺ synthesis is congruent with p53’s emerging role as a key regulator of metabolism and related cell fate.     

NMNAT2‐mediated NAD+ generation is essential for quality control of aged oocytes

Advanced maternal age has been reported to impair oocyte quality; however, the underlying mechanisms remain to be explored. In the present study, we identified the lowered NAD⁺ content and decreased expression of NMNAT2 protein in oocytes from old mice. Specific depletion of NMNAT2 in mouse oocytes disturbs the meiotic apparatus assembly and metabolic activity. Of note, nicotinic acid supplementation during in vitro culture or forced expression of NMNAT2 in aged oocytes was capable of reducing the reactive oxygen species (ROS) production and incidence of spindle/chromosome defects. Moreover, we revealed that activation or overexpression of SIRT1 not only partly prevents the deficient phenotypes of aged oocytes but also ameliorates the meiotic anomalies and oxidative stress in NMNAT2‐depleted oocytes. To sum up, our data indicate a role for NMNAT2 in controlling redox homeostasis during oocyte maturation and uncover that NMNAT2‐ NAD⁺‐SIRT1 is an important pathway mediating the effects of maternal age on oocyte developmental competence.     

Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants

Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD⁺ biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD⁺ synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome.     

NAMPT maintains mitochondria content via NRF2-PPARα/AMPKα pathway to promote cell survival under oxidative stress

Mitochondria plays a key role in regulating cell death process under stress conditions and it has been indicated that NAMPT overexpression promotes cell survival under genotoxic stress by maintaining mitochondrial NAD⁺ level. NAMPT is a rate-limiting enzyme for NAD⁺ production in mammalian cells and it was suggested that NAMPT and NMNAT3 are responsible for mitochondrial NAD⁺ production to maintain mitochondrial NAD⁺ pool. However, subsequent studies suggested mitochondrial may lack the NAMPT-NMANT3 pathway to maintain NAD⁺ level. Therefore, how NAMPT overexpression rescues mitochondrial NAD⁺ content to promote cell survival in response to genotoxic stress remains elusive. Here, we show that NAMPT promotes cell survival under oxidative stress via both SIRT1 dependent p53-CD38 pathway and SIRT1 independent NRF2-PPARα/AMPKα pathway, and the NRF2-PPARα/AMPKα pathway plays a more profound role in facilitating cell survival than the SIRT1-p53-CD38 pathway does. Mitochondrial content and membrane potential were significantly reduced in response to H2O2 treatment, whereas activated NRF2-PPARα/AMPKα pathway by NAMPT overexpression rescued the mitochondrial membrane potential and content, suggesting that maintained mitochondrial content and integrity by NAMPT overexpression might be one of the key mechanisms to maintain mitochondrial NAD⁺ level and subsequently dictate cell survival under oxidative stress. Our results indicated that NRF2 is a novel down-stream target of NAMPT, which mediates anti-apoptosis function of NAMPT via maintaining mitochondrial content and membrane potential.     

Fueling genome maintenance: On the versatile roles of NAD+ in preserving DNA integrity

NAD⁺ is a versatile biomolecule acting as a master regulator and substrate in various cellular processes, including redox regulation, metabolism, and various signaling pathways. In this article, we concisely and critically review the role of NAD⁺ in mechanisms promoting genome maintenance. Numerous NAD⁺-dependent reactions are involved in the preservation of genome stability, the cellular DNA damage response, and other pathways regulating nucleic acid metabolism, such as gene expression and cell proliferation pathways. Of note, NAD⁺ serves as a substrate to ADP-ribosyltransferases, sirtuins, and potentially also eukaryotic DNA ligases, all of which regulate various aspects of DNA integrity, damage repair, and gene expression. Finally, we critically analyze recent developments in the field as well as discuss challenges associated with therapeutic actions intended to raise NAD⁺ levels.     

Interaction of Sirt3 with OGG1 contributes to repair of mitochondrial DNA and protects from apoptotic cell death under oxidative stress

Sirtuin 3 (Sirt3), a major mitochondrial NAD⁺-dependent deacetylase, targets various mitochondrial proteins for lysine deacetylation and regulates important cellular functions such as energy metabolism, aging, and stress response. In this study, we identified the human 8-oxoguanine-DNA glycosylase 1 (OGG1), a DNA repair enzyme that excises 7,8-dihydro-8-oxoguanine (8-oxoG) from damaged genome, as a new target protein for Sirt3. We found that Sirt3 physically associated with OGG1 and deacetylated this DNA glycosylase and that deacetylation by Sirt3 prevented the degradation of the OGG1 protein and controlled its incision activity. We further showed that regulation of the acetylation and turnover of OGG1 by Sirt3 played a critical role in repairing mitochondrial DNA (mtDNA) damage, protecting mitochondrial integrity, and preventing apoptotic cell death under oxidative stress. We observed that following ionizing radiation, human tumor cells with silencing of Sirt3 expression exhibited deteriorated oxidative damage of mtDNA, as measured by the accumulation of 8-oxoG and 4977 common deletion, and showed more severe mitochondrial dysfunction and underwent greater apoptosis in comparison with the cells without silencing of Sirt3 expression. The results reported here not only reveal a new function and mechanism for Sirt3 in defending the mitochondrial genome against oxidative damage and protecting from the genotoxic stress-induced apoptotic cell death but also provide evidence supporting a new mtDNA repair pathway.     

Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi

The intracellular parasite Trypanosoma cruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasite''s drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD⁺). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzi throughout its life cycle. NAD⁺ biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number: 2.7.7.1). The identification of the NMNAT of T. cruzi is important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi. The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD⁺ metabolism in these parasites.     

Tankyrase-1 overexpression reduces genotoxin-induced cell death by inhibiting PARP1

Poly(ADP-ribose) polymerases or PARPs are a family of NAD⁺-dependent enzymes that modify themselves and other substrate proteins with ADP-ribose polymers. The founding member PARP1 is localized predominantly in the nucleus and is activated by binding to DNA lesions. Excessive PARP1 activation following genotoxin treatment causes NAD⁺ depletion and cell death, whereas pharmacological PARP1 inhibition protects cells from genotoxicity. This study investigates whether cellular viability and NAD⁺ metabolism are regulated by tankyrase-1, a PARP member localized predominantly in the cytosol. Using a tetracycline-sensitive promoter to regulate tankyrase-1 expression in Madin–Darby canine kidney (MDCK) cells, we found that a 40-fold induction of tankyrase-1 (from 1500 to 60,000 copies per cell) lowers steady-state NAD⁺ levels but does not affect basal cellular viability. Moreover, the induction confers protection against the oxidative agent H₂O₂ and the alkylating agent MNNG, genotoxins that kill cells by activating PARP1. The cytoprotective effect of tankyrase-1 is not due to enhanced scavenging of oxidants or altered expression of Mcl-1, an anti-apoptotic molecule previously shown to be down-regulated by tankyrase-1 in CHO cells. Instead, tankyrase-1 appears to protect cells by preventing genotoxins from activating PARP1-mediated reactions such as PARP1 automodification and NAD⁺ consumption. Our findings therefore indicate a cytoprotective function of tankyrase-1 mediated through altered NAD⁺ homeostasis and inhibition of PARP1 function.