Sirt1 is the most evolutionarily conserved mammalian sirtuin. It plays a vital role in the regulation of metabolism, stress responses, genome stability, and ultimately aging. Although much attention ...has focused on the identification of the cellular targets and functional networks controlled by Sirt1, the mechanisms that regulate Sirt1 activity by biological stimuli have only recently begun to emerge. As an enzyme, the activity of Sirt1 can be controlled by the availability of its substrates, post-translational modifications, interactions with other proteins, or changes in its expression levels. In this review, we briefly discuss the ways and means by which the activity of Sirt1 is fine-tuned under different conditions.
Glucocorticoids regulate diverse biological processes throughout the body via the glucocorticoid receptor (GR). Ligand‐bound GR translocates into the nucleus and can elicit changes in gene expression ...by direct contact with the DNA or by protein–protein interactions with other transcription factors. The GR can also mediate rapid nongenomic signaling events initiated in the cytoplasm. In this chapter, we review the biological and physiological implications of glucocorticoids, the GR, and many of the signal transduction mechanisms that mediate their action.
Recent studies have revealed new roles for NAD and its derivatives in transcriptional regulation. The evolutionarily conserved Sir2 protein family requires NAD for its deacetylase activity and ...regulates a variety of biological processes, such as stress response, differentiation, metabolism, and aging. Despite its absolute requirement for NAD, the regulation of Sir2 function by NAD biosynthesis pathways is poorly understood in mammals. In this study, we determined the kinetics of the NAD biosynthesis mediated by nicotinamide phosphoribosyltransferase (Nampt) and nicotinamide/nicotinic acid mononucleotide adenylyltransferase (Nmnat), and we examined its effects on the transcriptional regulatory function of the mouse Sir2 ortholog, Sir2α, in mouse fibroblasts. We found that Nampt was the ratelimiting component in this mammalian NAD biosynthesis pathway. Increased dosage of Nampt, but not Nmnat, increased the total cellular NAD level and enhanced the transcriptional regulatory activity of the catalytic domain of Sir2α recruited onto a reporter gene in mouse fibroblasts. Gene expression profiling with oligonucleotide microarrays also demonstrated a significant correlation between the expression profiles of Nampt- and Sir2α-overexpressing cells. These findings suggest that NAD biosynthesis mediated by Nampt regulates the function of Sir2α and thereby plays an important role in controlling various biological events in mammals.
Direct DNA sequencing can be used for characterizing mutagenicity in simple and complex biological models. Recently we described a method of whole-genome sequencing for detecting mutations in simple ...models of cultured bacteria, mammalian cells, and nematode. In the current proof-of-concept study, we expand and improve our method for evaluating a more complex mammalian biological model in outbred mice. We detail the method by applying it to a small set of animals treated with a mutagen with known mutagenicity profiles, N-ethyl-N-nitrosourea (ENU), for consistency with the known data. Whole-genome high-fidelity sequencing (HiFi Sequencing) showed frequencies and spectra of background mutations in tissues of untreated mice that were consistent with normal ageing and characterized by spontaneous or enzymatic deamination of 5-methylcytosine. In mice treated with a single 40 mg/kg dose of ENU, the frequency of mutations in the genomic DNA of solid tissues increased up to 7-fold, with the greatest increase observed in the spleen and the smallest increase in the liver. The most common mutations detected in ENU-treated mice were T > A transitions and T > C transversions, consistent with the types of mutations caused by alkylating agents. The data suggest that HiFi Sequencing may be useful for characterizing mutagenicity of novel compounds in various biological models.
•Mutations are involved in cancer onset and progression.•High-Fidelity Sequencing detects mutations induced by prototypical mutagens.•HiFi Sequencing may be useful for identification of potential in vivo carcinogens.
Molnupiravir (MOV) is used to treat COVID‐19. In cells, MOV is converted to the ribonucleoside analog N4‐hydroxycytidine (NHC) and incorporated into the SARS‐CoV‐2 RNA genome during its replication, ...resulting in RNA mutations. The widespread accumulation of such mutations inhibits SARS‐CoV‐2 propagation. Although safety assessments by many regulatory agencies across the world have concluded that the genotoxic risks associated with the clinical use of MOV are low, concerns remain that it could induce DNA mutations in patients, particularly because numerous in vitro studies have shown that NHC is a DNA mutagen. In this study, we used HiFi sequencing, a technique that can detect ultralow‐frequency substitution mutations in whole genomes, to evaluate the mutagenic effects of MOV in E. coli and of MOV and NHC in mouse lymphoma L5178Y cells and human lymphoblastoid TK6 cells. In all models, exposure to these compounds increased genome‐wide mutation frequencies in a dose‐dependent manner, and these increases were mainly composed of A:T → G:C transitions. The NHC exposure concentrations used for mammalian cells were comparable to those observed in the plasma of humans who received clinical doses of MOV.
DNA base editors have enabled genome editing without generating DNA double strand breaks. The applications of this technology have been reported in a variety of animal and plant systems, however, ...their editing specificity in human stem cells has not been studied by unbiased genome-wide analysis. Here we investigate the fidelity of cytidine deaminase-mediated base editing in human induced pluripotent stem cells (iPSCs) by whole genome sequencing after sustained or transient base editor expression. While base-edited iPSC clones without significant off-target modifications are identified, this study also reveals the potential of APOBEC-based base editors in inducing unintended point mutations outside of likely in silico-predicted CRISPR-Cas9 off-targets. The majority of the off-target mutations are C:G->T:A transitions or C:G->G:C transversions enriched for the APOBEC mutagenesis signature. These results demonstrate that cytosine base editor-mediated editing may result in unintended genetic modifications with distinct patterns from that of the conventional CRISPR-Cas nucleases.
Nicotinamide adenine dinucleotide (NAD) is a classic coenzyme in cellular redox reactions. Recently, NAD biochemistry has also been implicated in a broader range of biological functions in mammals, ...but the regulation of NAD biosynthesis has been poorly investigated. Recent progress in the field of NAD biochemistry has fueled new interest in the NAD biosynthetic pathways from its precursors and their physiological roles in metabolism. This review summarizes the latest knowledge on the NAD biosynthetic pathways and focuses on one of the key NAD biosynthetic enzymes, namely, nicotinamide phosphoribosyltransferase.
Mammals predominantly use nicotinamide rather than nicotinic acid as a precursor for NAD biosynthesis. Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme that converts nicotinamide to nicotinamide mononucleotide in the NAD biosynthetic pathway from nicotinamide in mammals. The same protein has also been identified as a cytokine (pre-B-cell colony-enhancing factor or PBEF) or an insulin-mimetic hormone (visfatin).
We propose that the presumed multiple effects of Nampt/PBEF/visfatin may be entirely explained by its role as an intra and extracellular NAD biosynthetic enzyme. We also propose a new model of Namp/PBEF/visfatin-mediated systemic NAD biosynthesis and its possible physiological significance. Our model provides an important insight into developing preventive/therapeutic interventions for metabolic complications, such as obesity and diabetes.
Genetic toxicology uses several assays to identity mutagens and protects the public. Most of these assays, however, rely on reporter genes, can only measure mutation indirectly based on phenotype, ...and often require specific cell lines or animal models—features that impede their integration with existing and emerging toxicological models, such as organoids. In this study, we show that PacBio Single‐Molecule, Real‐Time (PB SMRT) sequencing identified substitution mutations caused by chemical mutagens in Escherichia coli by generating nearly error‐free consensus reads after repeatedly inspecting both strands of circular DNA molecules. Using DNA from E. coli exposed to ethyl methanosulfonate (EMS) or N‐ethyl‐N‐nitrosourea (ENU), PB SMRT sequencing detected mutation frequencies (MFs) and spectra comparable to those obtained by clone‐sequencing from the same exposures. The optimized background MF of PB SMRT sequencing was ≤ 1 × 10−7 mutations per base pair (mut/bp).
Nicotinamide phosphoribosyltransferase (Nampt) synthesizes nicotinamide mononucleotide (NMN) from nicotinamide in a mammalian NAD+ biosynthetic pathway and is required for SirT1 activity in vivo. ...Nampt has also been presumed to be a cytokine (PBEF) or a hormone (visfatin). The crystal structure of Nampt in the presence and absence of NMN shows that Nampt is a dimeric type II phosphoribosyltransferase and provides insights into the enzymatic mechanism.
Intracellular nicotinamide phosphoribosyltransferase (iNampt) is an essential enzyme in the NAD biosynthetic pathway. An extracellular form of this protein (eNampt) has been reported to act as a ...cytokine named PBEF or an insulin-mimetic hormone named visfatin, but its physiological relevance remains controversial. Here we show that eNampt does not exert insulin-mimetic effects in vitro or in vivo but rather exhibits robust NAD biosynthetic activity. Haplodeficiency and chemical inhibition of Nampt cause defects in NAD biosynthesis and glucose-stimulated insulin secretion in pancreatic islets in vivo and in vitro. These defects are corrected by administration of nicotinamide mononucleotide (NMN), a product of the Nampt reaction. A high concentration of NMN is present in mouse plasma, and plasma eNampt and NMN levels are reduced in Nampt heterozygous females. Our results demonstrate that Nampt-mediated systemic NAD biosynthesis is critical for β cell function, suggesting a vital framework for the regulation of glucose homeostasis.
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