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David Gems - One of the best experts on this subject based on the ideXlab platform.
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Clustering of genetically defined allele classes in the Caenorhabditis elegans Daf-2 insulin/IGF-1 receptor.
Genetics, 2008Co-Authors: David GemsAbstract:The Daf-2 insulin/IGF-1 receptor regulates development, metabolism, and aging in the nematode Caenorhabditis elegans. However, complex differences among Daf-2 alleles complicate analysis of this gene. We have employed epistasis analysis, transcript profile analysis, mutant sequence analysis, and homology modeling of mutant receptors to understand this complexity. We define an allelic series of nonconditional Daf-2 mutants, including nonsense and deletion alleles, and a putative null allele, m65. The most severe Daf-2 alleles show incomplete suppression by daf-18(0) and daf-16(0) and have a range of effects on early development. Among weaker Daf-2 alleles there exist distinct mutant classes that differ in epistatic interactions with mutations in other genes. Mutant sequence analysis (including 11 newly sequenced alleles) reveals that class 1 mutant lesions lie only in certain extracellular regions of the receptor, while class 2 (pleiotropic) and nonconditional missense mutants have lesions only in the ligand-binding pocket of the receptor ectodomain or the tyrosine kinase domain. Effects of equivalent mutations on the human insulin receptor suggest an altered balance of intracellular signaling in class 2 alleles. These studies consolidate and extend our understanding of the complex genetics of Daf-2 and its underlying molecular biology.
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Sex‐specific Effects of the DAF‐12 Steroid Receptor on Aging in Caenorhabditis elegans
Annals of the New York Academy of Sciences, 2007Co-Authors: Diana Mcculloch, David GemsAbstract:Sex differences in longevity and aging are seen through- out the animal kingdom. These are likely to result, in part, from sex differences in endocrinology. In the nematode Caenorhabditis elegans, males are the longer-lived sex. Here we explore the possibility that sex differences in insulin/insulin-like growth factor 1 (IGF-1) and steroid endocrinology contribute to this sex difference in aging by studying C. elegans populations in liquid culture. We report that in hermaphrodite populations, mutational loss of the DAF-12 steroid receptor affected life span as in previous plate-culture studies: mutant longevity is suppressed in a weak Daf-2 insulin/IGF-1 receptor mutant but enhanced in a stronger Daf-2 mutant. However, in males, mutation of daf-12 had little effect on aging in either weak or strong Daf-2 mutants. Moreover, while mutation of daf-12 marginally reduced life span in Daf-2(+) hermaphrodites, as in plate-cultured populations, it did not in Daf-2(+) males. These results could imply that in C. elegans, as in mammals, sex differences in steroid endocrinology contribute to sex differences in aging.
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Sex-specific effects of the DAF-12 steroid receptor on aging in Caenorhabditis elegans.
Ann N Y Acad Sci, 2007Co-Authors: David GemsAbstract:Sex differences in longevity and aging are seen throughout the animal kingdom. These are likely to result, in part, from sex differences in endocrinology. In the nematode Caenorhabditis elegans, males are the longer-lived sex. Here we explore the possibility that sex differences in insulin/insulin-like growth factor 1 (IGF-1) and steroid endocrinology contribute to this sex difference in aging by studying C. elegans populations in liquid culture. We report that in hermaphrodite populations, mutational loss of the DAF-12 steroid receptor affected life span as in previous plate-culture studies: mutant longevity is suppressed in a weak Daf-2 insulin/IGF-1 receptor mutant but enhanced in a stronger Daf-2 mutant. However, in males, mutation of daf-12 had little effect on aging in either weak or strong Daf-2 mutants. Moreover, while mutation of daf-12 marginally reduced life span in Daf-2(+) hermaphrodites, as in plate-cultured populations, it did not in Daf-2(+) males. These results could imply that in C. elegans, as in mammals, sex differences in steroid endocrinology contribute to sex differences in aging.
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Erratum to "Diapause-associated metabolic traits reiterated in long-lived Daf-2 mutants in the nematode Caenorhabditis elegans" [Mech. Ageing Dev. 127 (5) (2006) 458-472].
Mechanisms of Ageing and Development, 2006Co-Authors: Joshua J. Mcelwee, Eugene Schuster, Eric Blanc, Janet M. Thornton, David GemsAbstract:The longevity of the Caenorhabditis elegans diapausal dauer larva greatly exceeds that of the adult. Dauer formation and adult ageing are both regulated by insulin/IGF-1 signalling (IIS). Reduced IIS, e.g. by mutation of the Daf-2 insulin/IGF-1 receptor gene, increases adult lifespan. This may reflect mis-expression in the adult of dauer longevity-assurance processes. Since IIS plays a central role in the regulation of metabolism, metabolic alterations shared by dauer larvae and Daf-2 adults represent candidate mechanisms for lifespan determination. We have conducted a detailed comparison of transcript profile data from dauers and Daf-2 mutant adults, focusing on expression of metabolic pathway genes. Our results imply up-regulation in both dauers and Daf-2 mutant adults of gluconeogenesis, glyoxylate pathway activity, and trehalose biosynthesis. Down-regulation of the citric acid cycle and mitochondrial respiratory chain occurs in dauers, but not Daf-2 adults. However, the F1 ATPase inhibitor was up-regulated in both, implying enhanced homeostasis in conditions where mitochondria are stressed. Overall, the data implies increased conversion of fat to carbohydrate, and conservation of ATP stocks in Daf-2 mutant adults, suggesting a state of increased energy availability. We postulate that this fuels increased somatic maintenance activity, as suggested by the disposable soma theory.
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Erratum to "Diapause-associated metabolic traits reiterated in long-lived Daf-2 mutants in the nematode Caenorhabditis elegans" [Mech. Ageing Dev. 127 (5) (2006) 458-472] (DOI:10.1016/j.mad.2006.01.006)
2006Co-Authors: Joshua J. Mcelwee, Eugene Schuster, Eric Blanc, Janet M. Thornton, David GemsAbstract:The longevity of the Caenorhabditis elegans diapausal dauer larva greatly exceeds that of the adult. Dauer formation and adult ageing are both regulated by insulin/IGF-1 signalling (IIS). Reduced IIS, e.g. by mutation of the Daf-2 insulin/IGF-1 receptor gene, increases adult lifespan. This may reflect mis-expression in the adult of dauer longevity-assurance processes. Since IIS plays a central role in the regulation of metabolism, metabolic alterations shared by dauer larvae and Daf-2 adults represent candidate mechanisms for lifespan determination. We have conducted a detailed comparison of transcript profile data from dauers and Daf-2 mutant adults, focusing on expression of metabolic pathway genes. Our results imply up-regulation in both dauers and Daf-2 mutant adults of gluconeogenesis, glyoxylate pathway activity, and trehalose biosynthesis. Down-regulation of the citric acid cycle and mitochondrial respiratory chain occurs in dauers, but not Daf-2 adults. However, the F1 ATPase inhibitor was up-regulated in both, implying enhanced homeostasis in conditions where mitochondria are stressed. Overall, the data implies increased conversion of fat to carbohydrate, and conservation of ATP stocks in Daf-2 mutant adults, suggesting a state of increased energy availability. We postulate that this fuels increased somatic maintenance activity, as suggested by the disposable soma theory. © 2006 Elsevier Ireland Ltd. All rights reserved.
Gary Ruvkun - One of the best experts on this subject based on the ideXlab platform.
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The C. elegans Daf-2 Insulin-Like Receptor is Abundantly Expressed in the Nervous System and Regulated by Nutritional Status
Cold Spring Harbor Symposia on Quantitative Biology, 2011Co-Authors: Koutarou D. Kimura, Donald L Riddle, Gary RuvkunAbstract:A Caenorhabditis elegans insulin-like signaling pathway regulates development, metabolism, and longevity. We detected abundant Daf-2 insulin-like receptor protein mainly in the nervous system, consistent with the assignment of Daf-2 pathway regulation of longevity to the nervous system. Daf-2 abundance in the nervous system is dependent on food intake, showing environmental modulation of pathway signaling. Daf-2 abundance is not dependent on downstream PI-3 kinase to DAF-16 transcription factor signaling. The modulation of Daf-2 protein level by nutritional status may constitute an important component in the irreversible commitment to dauer arrest.
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gene activities that mediate increased life span of c elegans insulin like signaling mutants
Genes & Development, 2007Co-Authors: Andrew V Samuelson, Christopher E Carr, Gary RuvkunAbstract:Genetic and RNA interference (RNAi) screens for life span regulatory genes have revealed that the Daf-2 insulin-like signaling pathway plays a major role in Caenorhabditis elegans longevity. This pathway converges on the DAF-16 transcription factor and may regulate life span by controlling the expression of a large number of genes, including free-radical detoxifying genes, stress resistance genes, and pathogen resistance genes. We conducted a genome-wide RNAi screen to identify genes necessary for the extended life span of Daf-2 mutants and identified ∼200 gene inactivations that shorten Daf-2 life span. Some of these gene inactivations dramatically shorten Daf-2 mutant life span but less dramatically shorten Daf-2; daf-16 mutant or wild-type life span. Molecular and behavioral markers for normal aging and for extended life span in low insulin/IGF1 (insulin-like growth factor 1) signaling were assayed to distinguish accelerated aging from general sickness and to examine age-related phenotypes. Detailed demographic analysis, molecular markers of aging, and insulin signaling mutant test strains were used to filter progeric gene inactivations for specific acceleration of aging. Highly represented in the genes that mediate life span extension in the Daf-2 mutant are components of endocytotic trafficking of membrane proteins to lysosomes. These gene inactivations disrupt the increased expression of the DAF-16 downstream gene superoxide dismutase sod-3 in a Daf-2 mutant, suggesting trafficking between the insulin-like receptor and DAF-16. The activities of these genes may normally decline during aging.
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daf 16 target genes that control c elegans life span and metabolism
Science, 2003Co-Authors: Siu Sylvia Lee, Scott Kennedy, Andrew C Tolonen, Gary RuvkunAbstract:Signaling from the Daf-2/insulin receptor to the DAF-16/FOXO transcription factor controls longevity, metabolism, and development in disparate phyla. To identify genes that mediate the conserved biological outputs of Daf-2 /insulin-like signaling, we usedcomparative genomics to identify 17 orthologous genes from Caenorhabditis and Drosophila , each of which bears a DAF-16 binding site in the promoter region. One-third of these DAF-16 downstream candidate genes were regulatedby Daf-2 /insulin-like signaling in C. elegans , and RNA interference inactivation of the candidates showed that many of these genes mediate distinct aspects of daf-16 function, including longevity, metabolism, anddevelopment.
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Daf-28 encodes a C. elegans insulin superfamily member that is regulated by environmental cues and acts in the Daf-2 signaling pathway
Genes & Development, 2003Co-Authors: Weiqing Li, Scott Kennedy, Gary RuvkunAbstract:Environmental conditions determine whether Caenorhabditis elegans grows directly into adulthood or arrests at an alternative L3 larval stage to form a dauer larva (Cassada and Russell 1975). Dauer larvae are induced by harsh environmental conditions such as starvation, high population density, or high temperature. Dauers can survive in harsh environments because of their distinctive adaptive features in morphology, behavior, metabolism, and life span. When environmental conditions improve, a dauer undergoes a series of developmental changes and reenters the reproductive cycle by molting into an L4 larva and subsequently into an adult. Molecular genetic analysis of dauer arrest has revealed that the dauer versus reproductive growth decision is controlled by at least two signaling cascades: the Daf-2 (insulin/IGF-1 receptor-like; Kimura et al. 1997) and the DAF-7 (TGFβ-like) pathways (Ren et al. 1996; Schackwitz et al. 1996). A decrease in either of the signals causes dauer arrest, indicating that both pathways are required for reproductive growth. Downstream components of the Daf-2 pathway include the AGE-1/PI3 kinase (Morris et al. 1996), PDK-1/PDK1 (Paradis et al. 1999), AKT-1 and AKT-2/Akt/PKB (Paradis and Ruvkun 1998), DAF-18/PTEN (Ogg and Ruvkun 1998), and the DAF-16/forkhead transcription factor (Lin et al. 1997; Ogg et al. 1997). Down-regulation of Daf-2 results in nuclear localization and thus activation of the DAF-16 transcription factor (Henderson and Johnson 2001; Lee et al. 2001; Lin et al. 2001). The Daf-2 signaling pathway also regulates metabolism and aging, analogously and probably homologously to the regulation of metabolism by insulin signaling in mammals. When Daf-2 signaling is decreased, life span is greatly extended (Kenyon et al. 1993; Larsen et al. 1995). Insulin-like signaling has recently been shown to regulate longevity in mice as well (Bluher et al. 2003; Holzenberger et al. 2003). Because the dauer arrest, longevity, and metabolic-shift phenotypes caused by the Daf-2(lf) (lf: loss-of-function) or age-1(lf) mutation are fully suppressed by a daf-16(lf) mutation (Gottlieb and Ruvkun 1994), daf-16 is the major output of Daf-2/IR signaling. daf-7 encodes a divergent member of the TGFβ superfamily that is a ligand for the parallel neuroendocrine pathway (Ren et al. 1996; Schackwitz et al. 1996). Downstream components of the DAF-7 pathway include the DAF-1/TGFβ type I receptor (Georgi et al. 1990; Gunther et al. 2000), DAF-4/TGFβ type II receptor (Estevez et al. 1993), DAF-8, DAF-14 and DAF-3/Smads (Patterson et al. 1997; Inoue and Thomas 2000), and DAF-5 (Riddle et al. 1981; Thomas et al. 1993). A nearly congruent mammalian TGFβ signaling pathway has also been detected in mammals, and although its regulation of metabolism and convergence with insulin signaling have not yet been noted, there are indications that these pathways do intersect (McPherron et al. 1997; McPherron and Lee 2002). Similarly to daf-16 in the insulin-like pathway, daf-3 and daf-5 are the major outputs of the DAF-7/TGFβ-like pathway. Sensory neurons mediate initiation of dauer arrest in response to environmental input of population density (Albert et al. 1981; Perkins et al. 1986; Vowels and Thomas 1994). A pheromone reflecting this population density is the normal physiological trigger of dauer arrest. Although its biochemical identity remains unknown, dauer pheromone has been surmised to be a small fatty acid, based on its partitioning pattern. Addition of a crude pheromone preparation to wild-type C. elegans causes dauer arrest (Golden and Riddle 1982, 1984). Of all the sensory inputs to dauer arrest, although food and temperature are modulatory, dauer pheromone is the most potent regulator and can overcome, for example, low temperature and high food concentrations, which favor reproductive development. Proper morphology and function of sensory neurons are necessary for pheromone-induced dauer arrest (Albert et al. 1981; Perkins et al. 1986; Vowels and Thomas 1994). Mutant animals defective in ciliated sensory neurons or the glial-like sheath cells that facilitate exposure of sensory neurons to the environment do not arrest as dauers when exposed to dauer pheromone, but do form dauers if the animals also carry a mutation in the insulin-like or TGFβ-like neuroendocrine signaling pathways. This indicates that sensory neurons are involved in responding to dauer pheromone to, in turn, regulate the production of neuroendocrine insulin-like and TGFβ-like signals. A set of 12 sensory neurons in the head amphid sensilla has ciliated endings, and 8 of them are directly exposed to the environment (Perkins et al. 1986). Laser ablation of three of these sensory neurons, ADF, ASG, and ASI, causes dauer arrest, indicating that these neurons signal reproductive growth and lack of such signals causes dauer arrest (Bargmann and Horvitz 1991). Similarly, ablation of the sensory neuron ASJ inhibits dauer recovery (Bargmann and Horvitz 1991). The DAF-7 TGFβ-like protein is clearly one of those neuroendocrine signals: a daf-7∷GFP transcriptional fusion gene is expressed in the ASI neuron, and its expression is down-regulated by dauer pheromone (Ren et al. 1996; Schackwitz et al. 1996). The dauer pheromone is likely to be detected by an as yet unidentified pheromone receptor that couples to a cyclic GMP signaling pathway that includes daf-11. Mutations in DAF-11, a transmembrane guanylyl cyclase that is expressed in ciliated sensory neurons, including ASI and ASJ, cause dauer arrest (Birnby et al. 2000). This observation suggests that the signaling pathway downstream of the pheromone receptor uses a cGMP second messenger, as do G-protein-coupled receptors involved in olfaction and vision (Birnby et al. 2000). Moreover, cGMP-gated channels have also been implicated in regulation of dauer arrest (Coburn and Bargmann 1996; Komatsu et al. 1996; Ailion and Thomas 2000). Because dauer arrest caused by daf-11(lf) can be partially suppressed either by daf-3(lf) or daf-16(lf), DAF-11 is likely to regulate both the DAF-7/TGFβ and the Daf-2/IR pathways (Thomas et al. 1993; Gottlieb and Ruvkun 1994; Riddle 1997). Many of the 37 C. elegans ins (insulin-like) genes identified by genome analysis are expressed in sensory neurons (Pierce et al. 2001), consistent with the expected sensory input to the Daf-2 pathway. Sensory neurons have been implicated in control of life span (Apfeld and Kenyon 1998), perhaps by regulating the production and/or secretion of insulin-like ligands, which, in turn, engage the Daf-2 pathway. However, there is not yet direct evidence linking sensory neuron function to ins gene activities. Although multiple components of the Daf-2/IR pathway have been isolated from various genetic screens, no loss-of-function (lf) mutation has been found in an ins gene that might encode a ligand for Daf-2/IR. Failure to isolate such an lf mutation may be attributed to the possible redundant functions of the many ins genes in dauer arrest. Among the ins genes, ins-1 and ins-18 have been shown to regulate dauer arrest and life span, respectively. Increased ins-1 gene dosage promotes dauer arrest, most likely by antagonizing Daf-2 activity (Pierce et al. 2001). INS-18 has been suggested to regulate life span by activating Daf-2, based on RNAi (RNA interference) experiments (Kawano et al. 2000). Daf-28(sa191) has been isolated from a screen for mutants that arrest at the dauer stage (Malone and Thomas 1994). Daf-28(sa191) causes transient dauer arrest and a 10% extension in life span. Daf-28(sa191) dauer arrest is partially suppressed by a daf-16(lf) mutation . Because Daf-28(sa191) shares the extended life span and suppression by daf-16 features with mutations in the Daf-2 insulin-like pathway, Daf-28 was predicted to function in the Daf-2 pathway (Malone et al. 1996). Genetic analysis also showed that Daf-28(sa191) is a semidominant allele that poisons Daf-28 wild-type function (Malone and Thomas 1994). We show here that Daf-28 encodes an insulin-like protein that regulates DAF-16 nuclear localization. Therefore, we propose that Daf-28 functions as a ligand of Daf-2. A Daf-28∷GFP transcriptional fusion is expressed in two pairs of sensory neurons, ASI and ASJ, and this expression is down-regulated in response to dauer-inducing environmental and sensory signals. We propose that Daf-28 is expressed in sensory neurons to inhibit dauer arrest as a Daf-2 agonist. The Daf-28(sa191) mutation R37C disrupts a probable proteolytic cleavage site that is expected to be necessary for processing the Daf-28 propeptide into a mature insulin. We propose that the Daf-28R37C mutant protein acts as a competitive inhibitor to down-regulate the activities of Daf-28+ and other INS proteins. Consistent with this hypothesis, increased gene dosage of Daf-28+ or its closest relatives INS-4 or INS-6 suppresses the dauer arrest caused by Daf-28R37C. Daf-28 has a broadened expression pattern in aged animals and increased expression levels in animals defective in sensory functions, suggesting that the production of insulin-like neuroendocrine signals is influenced by aging-related factors and sensory neuron functions.
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Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the Daf-2 insulin-like signaling pathway.
Current Biology, 2001Co-Authors: Jürgen Hench, Gary RuvkunAbstract:Abstract C. elegans insulin-like signaling regulates metabolism, development, and life span. This signaling pathway negatively regulates the activity of the forkhead transcription factor DAF-16. daf-16 encodes multiple isoforms that are expressed in distinct tissue types and are probable orthologs of human FKHRL1, FKHR, and AFX. We show that human FKHRL1 can partially replace DAF-16, proving the orthology. In mammalian cells, insulin and insulin-like growth factor signaling activate AKT/PKB kinase to negatively regulate the nuclear localization of DAF-16 homologs (reviewed in [1]). We show that the absence of AKT consensus sites on DAF-16 is sufficient to cause dauer arrest in Daf-2(+) animals, proving that daf-16 is the major output of insulin signaling in C. elegans . FKHR, FKRHL1, and AFX may similarly be the major outputs of mammalian insulin signaling. Daf-2 insulin signaling, via AKT kinases, negatively regulates DAF-16 by controlling its nuclear localization. Surprisingly, we find that daf-7 TGF-β signaling also regulates DAF-16 nuclear localization specifically at the time when the animal makes the commitment between diapause and reproductive development. daf-16 function is supported by the combined action of two distinct promoter/enhancer elements, whereas the coding sequences of two major DAF-16 isoforms are interchangeable. Together, these observations suggest that the combined effects of transcriptional and posttranslational regulation of daf-16 transduce insulin-like signals in C. elegans and perhaps more generally.
Donald L Riddle - One of the best experts on this subject based on the ideXlab platform.
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interaction of structure specific and promiscuous g protein coupled receptors mediates small molecule signaling in caenorhabditis elegans
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Donha Park, Inish Odoherty, Rishi K Somvanshi, Axel Bethke, Frank C Schroeder, Ujendra Kumar, Donald L RiddleAbstract:Abstract A chemically diverse family of small-molecule signals, the ascarosides, control developmental diapause (dauer), olfactory learning, and social behaviors of the nematode model organism, Caenorhabditis elegans. The ascarosides act upstream of conserved signaling pathways, including the insulin, TGF-β, serotonin, and guanylyl cyclase pathways; however, the sensory processes underlying ascaroside function are poorly understood. Because ascarosides often are multifunctional and show strongly synergistic effects, characterization of their receptors will be essential for understanding ascaroside biology and may provide insight into molecular mechanisms that produce synergistic outcomes in small-molecule sensing. Based on DAF-8 immunoprecipitation, we here identify two G-protein–coupled receptors, DAF-37 and DAF-38, which cooperatively mediate ascaroside perception. daf-37 mutants are defective in all responses to ascr#2, one of the most potent dauer-inducing ascarosides, although this mutant responds normally to other ascarosides. In contrast, daf-38 mutants are partially defective in responses to several different ascarosides. Through cell-specific overexpression, we show that DAF-37 regulates dauer when expressed in ASI neurons and adult behavior when expressed in ASK neurons. Using a photoaffinity-labeled ascr#2 probe and amplified luminescence assays (AlphaScreen), we demonstrate that ascr#2 binds to DAF-37. Photobleaching fluorescent energy transfer assays revealed that DAF-37 and DAF-38 form heterodimers, and we show that heterodimerization strongly increases cAMP inhibition in response to ascr#2. These results suggest that that the ascarosides' intricate signaling properties result in part from the interaction of highly structure-specific G-protein–coupled receptors such as DAF-37 with more promiscuous G-protein–coupled receptors such as DAF-38.
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The C. elegans Daf-2 Insulin-Like Receptor is Abundantly Expressed in the Nervous System and Regulated by Nutritional Status
Cold Spring Harbor Symposia on Quantitative Biology, 2011Co-Authors: Koutarou D. Kimura, Donald L Riddle, Gary RuvkunAbstract:A Caenorhabditis elegans insulin-like signaling pathway regulates development, metabolism, and longevity. We detected abundant Daf-2 insulin-like receptor protein mainly in the nervous system, consistent with the assignment of Daf-2 pathway regulation of longevity to the nervous system. Daf-2 abundance in the nervous system is dependent on food intake, showing environmental modulation of pathway signaling. Daf-2 abundance is not dependent on downstream PI-3 kinase to DAF-16 transcription factor signaling. The modulation of Daf-2 protein level by nutritional status may constitute an important component in the irreversible commitment to dauer arrest.
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Caenorhabditis elegans SDF-9 enhances insulin/insulin-like signaling through interaction with Daf-2.
Genetics, 2007Co-Authors: Victor L. Jensen, Patrice S Albert, Donald L RiddleAbstract:SDF-9 is a modulator of Caenorhabditis elegans insulin/IGF-1 signaling that may interact directly with the Daf-2 receptor. SDF-9 is a tyrosine phosphatase-like protein that, when mutated, enhances many partial loss-of-function mutants in the dauer pathway except for the temperature-sensitive mutant Daf-2(m41). We propose that SDF-9 stabilizes the active phosphorylated state of Daf-2 or acts as an adaptor protein to enhance insulin-like signaling.
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the tor pathway interacts with the insulin signaling pathway to regulate c elegans larval development metabolism and life span
Development, 2004Co-Authors: Kailiang Jia, Di Chen, Donald L RiddleAbstract:The highly conserved target-of-rapamycin (TOR) protein kinases control cell growth in response to nutrients and growth factors. In mammals, TOR has been shown to interact with raptor to relay nutrient signals to downstream translation machinery. We report that in C. elegans , mutations in the genes encoding CeTOR and raptor result in dauer-like larval arrest, implying that CeTOR regulates dauer diapause. The daf-15 (raptor) and let-363 (CeTOR) mutants shift metabolism to accumulate fat, and raptor mutations extend adult life span. daf-15 transcription is regulated by DAF-16, a FOXO transcription factor that is in turn regulated by Daf-2 insulin/IGF signaling. This is a new mechanism that regulates the TOR pathway. Thus, Daf-2 insulin/IGF signaling and nutrient signaling converge on DAF-15 (raptor) to regulate C. elegans larval development, metabolism and life span.
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two pleiotropic classes of daf 2 mutation affect larval arrest adult behavior reproduction and longevity in caenorhabditis elegans
Genetics, 1998Co-Authors: David Gems, Pamela L. Larsen, Amy J Sutton, Mark L Sundermeyer, Patrice S Albert, Kevin V King, Mark L Edgley, Donald L RiddleAbstract:The nematode Caenorhabditis elegans responds to overcrowding and scarcity of food by arresting development as a dauer larva, a nonfeeding, long-lived, stress-resistant, alternative third-larval stage. Previous work has shown that mutations in the genes Daf-2 (encoding a member of the insulin receptor family) and age-1 (encoding a PI 3-kinase) result in constitutive formation of dauer larvae (Daf-c), increased adult longevity (Age), and increased intrinsic thermotolerance (Itt). Some Daf-2 mutants have additional developmental, behavioral, and reproductive defects. We have characterized in detail 15 temperature-sensitive and 1 nonconditional Daf-2 allele to investigate the extent of Daf-2 mutant defects and to examine whether specific mutant traits correlate with each other. The greatest longevity seen in Daf-2 mutant adults was approximately three times that of wild type. The temperature-sensitive Daf-2 mutants fell into two overlapping classes, including eight class 1 mutants, which are Daf-c, Age, and Itt, and exhibit low levels of L1 arrest at 25.5 degrees. Seven class 2 mutants also exhibit the class 1 defects as well as some or all of the following: reduced adult motility, abnormal adult body and gonad morphology, high levels of embryonic and L1 arrest, production of progeny late in life, and reduced brood size. The strengths of the Daf-c, Age, and Itt phenotypes largely correlated with each other but not with the strength of class 2-specific defects. This suggests that the Daf-2 receptor is bifunctional. Examination of the null phenotype revealed a maternally rescued egg, L1 lethal component, and a nonconditional Daf-c component. With respect to the Daf-c phenotype, the dauer-defective (Daf-d) mutation daf-12(m20) was epistatic to Daf-2 class 1 alleles but not the severe class 2 alleles tested. All Daf-2 mutant defects were suppressed by the daf-d mutation daf-16(m26). Our findings suggest a new model for Daf-2, age-1, daf-12, and daf-16 interactions.
Jeffrey M Bergelson - One of the best experts on this subject based on the ideXlab platform.
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specificity of coxsackievirus b3 interaction with human but not murine decay accelerating factor replacement of a single residue within short consensus repeat 2 prevents virus attachment
Journal of Virology, 2015Co-Authors: Lili Zhang, Jeffrey M Bergelson, Susan Hafenstein, Lindsey J OrgantiniAbstract:Many coxsackievirus B (CVB) isolates bind to human decay-accelerating factor (DAF) as well as to the coxsackievirus and adenovirus receptor (CAR). However, the virus does not interact with murine DAF. To understand why CVB3 binds specifically to human DAF, we constructed a series of chimeric molecules in which specific regions of the human DAF molecule were replaced by the corresponding murine sequences. We found that replacement of human short consensus repeat 2 (SCR2) with murine SCR2 ablated virus binding to human DAF, as did deletion of human SCR2. Although replacement of human SCR4 had a partial inhibitory effect, deletion of SCR4 had no effect. Within human SCR2, replacement of serine 104 (S104) with the proline residue found in murine DAF eliminated virus binding. On the basis of the structure of the CVB3-DAF complex determined by cryo-electron microscopy, DAF S104 is in close contact with a viral capsid residue, a threonine at VP1 position 271. Replacement of this capsid residue with larger amino acids specifically eliminated virus attachment to human DAF but had no effect on attachment to CAR or replication in HeLa cells. Taken together, these results support the current model of virus-DAF interaction and point to a specific role for VP1 T271 and DAF S104 at the virus-DAF interface. IMPORTANCE The results of the present study point to a specific role for VP1 T271 and DAF S104 at the interface between CVB3 and DAF, and they demonstrate how subtle structural changes can dramatically influence virus-receptor interactions. In addition, the results support a recent pseudoatomic model of the CVB3-DAF interaction obtained by cryo-electron microscopy.
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the crystal structure of a coxsackievirus b3 rd variant and a refined 9 angstrom cryo electron microscopy reconstruction of the virus complexed with decay accelerating factor daf provide a new footprint of daf on the virus surface
Journal of Virology, 2012Co-Authors: Joshua D Yoder, Javier O Cifuente, Jeffrey M Bergelson, Susan HafensteinAbstract:The coxsackievirus-adenovirus receptor (CAR) and decay-accelerating factor (DAF) have been identified as cellular receptors for coxsackievirus B3 (CVB3). The first described DAF-binding isolate was obtained during passage of the prototype strain, Nancy, on rhabdomyosarcoma (RD) cells, which express DAF but very little CAR. Here, the structure of the resulting variant, CVB3-RD, has been solved by X-ray crystallography to 2.74 Å, and a cryo-electron microscopy reconstruction of CVB3-RD complexed with DAF has been refined to 9.0 Å. This new high-resolution structure permits us to correct an error in our previous view of DAF-virus interactions, providing a new footprint of DAF that bridges two adjacent protomers. The contact sites between the virus and DAF clearly encompass CVB3-RD residues recently shown to be required for binding to DAF; these residues interact with DAF short consensus repeat 2 (SCR2), which is known to be essential for virus binding. Based on the new structure, the mode of the DAF interaction with CVB3 differs significantly from the mode reported previously for DAF binding to echoviruses.
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dynamin and lipid raft dependent entry of decay accelerating factor daf binding and non daf binding coxsackieviruses into nonpolarized cells
Journal of Virology, 2009Co-Authors: Jeffrey M Bergelson, Kunal P Patel, Carolyn B CoyneAbstract:Group B coxsackieviruses (CVB) use the CVB and adenovirus receptor (CAR) to enter and infect cells. Some CVB also bind to decay-accelerating factor (DAF), but that interaction alone is insufficient for infection. We previously found that CVB3 entry into polarized human intestinal cells (Caco-2) occurs by a caveolin-dependent but dynamin-independent mechanism that requires DAF-mediated tyrosine kinase signals. In this study, we examined how CVB enter and infect nonpolarized HeLa cells and how DAF binding affects these processes. Using immunofluorescence microscopy and a combination of dominant-negative proteins, small interfering RNAs, and drugs targeting specific endocytic pathways, we found that both DAF-binding and non-DAF-binding virus isolates require dynamin and lipid rafts to enter and infect cells. Unlike what we observed in Caco-2 cells, CVB3 entered HeLa cells with CAR. We found no role for clathrin, endosomal acidification, or caveolin. Inhibition of tyrosine kinases blocked an early event in infection but did not prevent entry of virus into the cell. These results indicate that CVB3 entry into nonpolarized HeLa cells differs significantly from entry into polarized Caco-2 cells and is not influenced by virus binding to DAF.
Ralf Baumeister - One of the best experts on this subject based on the ideXlab platform.
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C. elegans SGK-1 Is the Critical Component in the Akt/PKB Kinase Complex to Control Stress Response and Life Span
Developmental Cell, 2004Co-Authors: Maren Hertweck, Christine Göbel, Ralf BaumeisterAbstract:The Daf-2 insulin receptor-like signaling pathway controls metabolism, development, longevity, and stress response in C. elegans. Here we show that SGK-1, the C. elegans homolog of the serum- and glucocorticoid-inducible kinase SGK, acts in parallel to the AKT kinases to mediate Daf-2 signaling. Loss of sgk-1 results in defective egg-laying, extended generation time, increased stress resistance, and an extension of life span. SGK-1 forms a protein complex with the AKT kinases, and is activated by and strictly depends on PDK-1. All three kinases of this complex are able to directly phosphorylate DAF-16/FKHRL1, yet have different functions in Daf-2 signaling. Whereas AKT-1 and AKT-2 are more important for regulating dauer formation, SGK-1 is the crucial factor for the control of development, stress response, and longevity. Our data also suggest the existence of a second pathway from Daf-2 to DAF-16 that does not depend on AKT-1, AKT-2, and SGK-1.
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c elegans sgk 1 is the critical component in the akt pkb kinase complex to control stress response and life span
Developmental Cell, 2004Co-Authors: Maren Hertweck, Christine Göbel, Ralf BaumeisterAbstract:Abstract The Daf-2 insulin receptor-like signaling pathway controls metabolism, development, longevity, and stress response in C. elegans . Here we show that SGK-1, the C. elegans homolog of the serum- and glucocorticoid-inducible kinase SGK, acts in parallel to the AKT kinases to mediate Daf-2 signaling. Loss of sgk-1 results in defective egg-laying, extended generation time, increased stress resistance, and an extension of life span. SGK-1 forms a protein complex with the AKT kinases, and is activated by and strictly depends on PDK-1. All three kinases of this complex are able to directly phosphorylate DAF-16/FKHRL1, yet have different functions in Daf-2 signaling. Whereas AKT-1 and AKT-2 are more important for regulating dauer formation, SGK-1 is the crucial factor for the control of development, stress response, and longevity. Our data also suggest the existence of a second pathway from Daf-2 to DAF-16 that does not depend on AKT-1, AKT-2, and SGK-1.
