Project description:Nutrient-driven O-GlcNAcylation of key components of the transcription machinery may epigenetically modulate gene expression in metazoans. The global effects of GlcNAcylation on transcription can be addressed directly in C. elegans because knockouts of the O-GlcNAc cycling enzymes are viable and fertile. Using anti-O-GlcNAc ChIP-on-chip whole-genome tiling arrays on wild-type and mutant strains, we detected over 800 promoters where O-GlcNAc cycling occurs, including microRNA loci and multigene operons. Intriguingly, O-GlcNAc-marked promoters are biased toward genes associated with PIP3 signaling, hexosamine biosynthesis, and lipid/carbohydrate metabolism. These marked genes are linked to insulin-like signaling, metabolism, aging, stress, and pathogen-response pathways in C. elegans. Whole-genome transcriptional profiling of the O-GlcNAc cycling mutants confirmed dramatic deregulation of genes in these key pathways. As predicted, the O-GlcNAc cycling mutants show altered lifespan and UV stress susceptibility phenotypes. We propose that O-GlcNAc cycling at promoters participates in a molecular program impacting nutrient-responsive pathways in C. elegans, including stress, pathogen response, and adult lifespan. The observed impact of O-GlcNAc cycling on both signaling and transcription in C. elegans has important implications for human diseases of aging, including diabetes and neurodegeneration.
Project description:The modification of nuclear, mitochondrial, and cytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is a dynamic and essential post-translational modification of metazoans. Numerous forms of cellular injury lead to elevated levels of O-GlcNAc in both in vivo and in vitro models, and elevation of O-GlcNAc levels before, or immediately after, the induction of cellular injury is protective in models of heat stress, oxidative stress, endoplasmic reticulum (ER) stress, hypoxia, ischemia reperfusion injury, and trauma hemorrhage. Together, these data suggest that O-GlcNAc is a regulator of the cellular stress response. However, the molecular mechanism(s) by which O-GlcNAc regulates protein function leading to enhanced cell survival have not been identified. In order to determine how O-GlcNAc modulates stress tolerance in these models we have used stable isotope labeling with amino acids in cell culture to determine the identity of proteins that undergo O-GlcNAcylation in response to heat shock. Numerous proteins with diverse functions were identified, including NF-90, RuvB-like 1 (Tip49α), RuvB-like 2 (Tip49β), and several COPII vesicle transport proteins. Many of these proteins bind double-stranded DNA-dependent protein kinase (PK), or double-stranded DNA breaks, suggesting a role for O-GlcNAc in regulating DNA damage signaling or repair. Supporting this hypothesis, we have shown that DNA-PK is O-GlcNAc modified in response to numerous forms of cellular stress.
Project description:O-GlcNAcylation is an abundant posttranslational modification in the brain implicated in human neurodegenerative diseases. We have exploited viable null alleles of the enzymes of O-GlcNAc cycling to examine the role of O-GlcNAcylation in well-characterized Caenorhabditis elegans models of neurodegenerative proteotoxicity. O-GlcNAc cycling dramatically modulated the severity of the phenotype in transgenic models of tauopathy, amyloid β-peptide, and polyglutamine expansion. Intriguingly, loss of function of O-GlcNAc transferase alleviated, whereas loss of O-GlcNAcase enhanced, the phenotype of multiple neurodegenerative disease models. The O-GlcNAc cycling mutants act in part by altering DAF-16-dependent transcription and modulating the protein degradation machinery. These findings suggest that O-GlcNAc levels may directly influence neurodegenerative disease progression, thus making the enzymes of O-GlcNAc cycling attractive targets for neurodegenerative disease therapies.
Project description:Arbutin (p-hydroxyphenyl-β-D-glucopyranoside), a well-known tyrosinase inhibitor, has been widely used as a cosmetic whitening agent. Although its natural role is to scavenge free radicals within cells, it has also exhibited useful activities for the treatment of diuresis, bacterial infections and cancer, as well as anti-inflammatory and anti-tussive activities. Because function of free radical scavenging is also related to antioxidant and the effects of arbutin on longevity and stress resistance in animals have not yet been confirmed, here the effects of arbutin on Caenorhabditis elegans were investigated. The results demonstrated that optimal concentrations of arbutin could extend lifespan and enhance resistance to oxidative stress. The underlying molecular mechanism for these effects involves decreased levels of reactive oxygen species (ROS), improvement of daf-16 nuclear localization, and up-regulated expression of daf-16 and its downstream targets, including sod-3 and hsp16.2. In this work the roles of arbutin in lifespan and health are studied and the results support that arbutin is an antioxidant for maintaining overall health.
Project description:Intricate and interconnected pathways modulate longevity, but screens to identify the components of these pathways have not been saturating. Because biological processes are often executed by protein complexes and fine-tuned by regulatory factors, the first-order protein-protein interactors of known longevity genes are likely to participate in the regulation of longevity. Data-rich maps of protein interactions have been established for many cardinal organisms such as yeast, worms, and humans. We propose that these interaction maps could be mined for the identification of new putative regulators of longevity. For this purpose, we have constructed longevity networks in both humans and worms. We reasoned that the essential first-order interactors of known longevity-associated genes in these networks are more likely to have longevity phenotypes than randomly chosen genes. We have used C. elegans to determine whether post-developmental inactivation of these essential genes modulates lifespan. Our results suggest that the worm and human longevity networks are functionally relevant and possess a high predictive power for identifying new longevity regulators.
Project description:Removal of the reproductive system of many animals including fish, flies, nematodes, mice and humans can increase lifespan through mechanisms largely unknown. The abrogation of the germline in Caenorhabditis elegans increases longevity by 60% due to a signal emitted from the somatic gonad. Apart from increased longevity, germline-less C. elegans is also resistant to other environmental stressors such as feeding on bacterial pathogens. However, the evolutionary conservation of this pathogen resistance, its genetic basis and an understanding of genes involved in producing this extraordinary survival phenotype are currently unknown. To study these evolutionary aspects we used the necromenic nematode Pristionchus pacificus, which is a genetic model system used in comparison to C. elegans. By ablation of germline precursor cells and subsequent feeding on the pathogen Serratia marcescens we discovered that P. pacificus shows remarkable resistance to bacterial pathogens and that this response is evolutionarily conserved across the Genus Pristionchus. To gain a mechanistic understanding of the increased resistance to bacterial pathogens and longevity in germline-ablated P. pacificus we used whole genome microarrays to profile the transcriptional response comparing germline ablated versus un-ablated animals when fed S. marcescens. We show that lipid metabolism, maintenance of the proteasome, insulin signaling and nuclear pore complexes are essential for germline deficient phenotypes with more than 3,300 genes being differentially expressed. In contrast, gene expression of germline-less P. pacificus on E. coli (longevity) and S. marcescens (immunity) is very similar with only 244 genes differentially expressed indicating that longevity is due to abundant gene expression also involved in immunity. By testing existing mutants of Ppa-DAF-16/FOXO and the nuclear hormone receptor Ppa-DAF-12 we show a conserved function of both genes in resistance to bacterial pathogens and longevity. This is the first study to show that the influence of the reproductive system on extending lifespan and innate immunity is conserved in evolution.
Project description:Thousands of nuclear and cytosolic proteins are modified with a single β-N-acetylglucosamine on serine and threonine residues in mammals, a modification termed O-GlcNAc. This modification is essential for normal development and plays important roles in virtually all intracellular processes. Additionally, O-GlcNAc is involved in many disease states, including cancer, diabetes, and X-linked intellectual disability. Given the myriad of functions of the O-GlcNAc modification, it is therefore somewhat surprising that O-GlcNAc cycling is mediated by only two enzymes: the O-GlcNAc transferase (OGT), which adds O-GlcNAc, and the O-GlcNAcase (OGA), which removes it. A significant outstanding question in the O-GlcNAc field is how do only two enzymes mediate such an abundant and dynamic modification. In this review, we explore the current understanding of mechanisms for substrate selection for the O-GlcNAc cycling enzymes. These mechanisms include direct substrate interaction with specific domains of OGT or OGA, selection of interactors via partner proteins, posttranslational modification of OGT or OGA, nutrient sensing, and localization alteration. Altogether, current research paints a picture of an exquisitely regulated and complex system by which OGT and OGA select substrates. We also make recommendations for future work, toward the goal of identifying interaction mechanisms for specific substrates that may be able to be exploited for various research and medical treatment goals.
Project description:Discriminating pathogenic bacteria from bacteria used as a food source is key to Caenorhabidits elegans immunity. Using mutants defective in the enzymes of O-linked N-acetylglucosamine (O-GlcNAc) cycling, we examined the role of this nutrient-sensing pathway in the C. elegans innate immune response. Genetic analysis showed that deletion of O-GlcNAc transferase (ogt-1) yielded animals hypersensitive to the human pathogen S. aureus but not to P. aeruginosa. Genetic interaction studies revealed that nutrient-responsive OGT-1 acts through the conserved β-catenin (BAR-1) pathway and in concert with p38 MAPK (PMK-1) to modulate the immune response to S. aureus. Moreover, whole genome transcriptional profiling revealed that O-GlcNAc cycling mutants exhibited deregulation of unique stress- and immune-responsive genes. The participation of nutrient sensor OGT-1 in an immunity module evolutionarily conserved from C. elegans to humans reveals an unexplored nexus between nutrient availability and a pathogen-specific immune response.
Project description:The antagonistic pleiotropy theory of aging proposes that aging takes place because natural selection favors genes that confer benefit early on life at the cost of deterioration later in life. This theory predicts that genes that impact development would play a key role in shaping adult lifespan. To better understand the link between development and adult lifespan, we examined the genes previously known to be essential for development. From a pool of 57 genes that cause developmental arrest after inhibition using RNA interference, we have identified 24 genes that extend lifespan in Caenorhabditis elegans when inactivated during adulthood. Many of these genes are involved in regulation of mRNA translation and mitochondrial functions. Genetic epistasis experiments indicate that the mechanisms of lifespan extension by inactivating the identified genes may be different from those of the insulin/insulin-like growth factor 1 (IGF-1) and dietary restriction pathways. Inhibition of many of these genes also results in increased stress resistance and decreased fecundity, suggesting that they may mediate the trade-offs between somatic maintenance and reproduction. We have isolated novel lifespan-extension genes, which may help understand the intrinsic link between organism development and adult lifespan.