Rasmussen, N. et al. (2015) International Worm Meeting "Putative RAP-1 function in a positive feedback loop to reinforce Ras/LET-60-dependent 1deg VPC commitment."

Rasmussen, N., Reiner, D.

[International Worm Meeting, 2015]

Patterning of the vulval epithelial tube has helped elucidate many well conserved signaling networks at the level of individual cells. The six equipotent vulval precursor cells (VPCs) are induced by EGF/LIN-3 to form a distinctive pattern of 3deg-3deg-2deg-1deg-2deg-3deg, where 1deg and 2deg cells proliferate to form the vulva while 3deg cells divide once and join the surrounding quiescent hypodermis. Initial cell fate specification is thought to be promptly followed by a reinforcement phase, where re-wired, focused signals ensure fate commitment and exclusion of contradictory signals. A central player in the patterning process is the small GTPase Ras/LET-60. Ras is the most mutated oncoprotein, yet therapies directly targeting it and its effectors have proven largely ineffectual. Ras/LET-60 serves as a key signaling node downstream of EGFR/LET-23 in initiating vulval patterning in presumptive 1deg cells by utilizing its canonical effector, the Raf/LIN-45-MEK-ERK cascade. Of particular interest is the small GTPase RAP-1/Rap1, which shares high sequence identity with LET-60/Ras, including 100% identity in the effector-binding region. As C. elegans encodes a single RAP-1 isoform that is not required for viability or fertility, we are using the vulval system to define the role of RAP-1 signaling relative to LET-60/Ras. We found that targeted knockdown or deletion of RAP-1 resulted in a significant decrease in LET-60-dependent vulval hyper-induction We observed a similar effect from vulval-specific RNAi depletion of both the RapGEF PXF-1 and RAP-1. To test if RAP-1 functions cell autonomously, we transgenically expressed mutationally activated RAP-1 using a vulval-specific promoter and observed a proliferative increase relative to non-transgenic control siblings. We examined the spatiotemporal expression of transgenic GFP driven by the PXF-1 promoter and found it to be expressed at low levels in all six of the VPCs prior to reception of the EGF signal, but that subsequent GFP expression was restricted to proliferative cells after the advent of EGF signal. We hypothesize that as a key mechanism of fate reinforcement, the Ras/LET-60-Raf-MEK-ERK signaling cascade functions to magnify PXF-1 expression in presumptive 1deg cells, thus focusing RAP-1 activation specifically to presumptive 1deg cells and reinforcing the Ras/LET-60 1deg induction signal in a positive feedback loop. We plan to further investigate this hypothesis and define how PXF-1/RapGEF and RAP-1 signals are focused exclusively to presumptive 1deg cells, as well as the downstream signaling partners for RAP-1. Our results may point to general signaling mechanisms of ensuring developmental fidelity, and identify druggable targets for Ras-dependent tumors.

Sakoguchi H et al. (2016) Biosci Biotechnol Biochem "Screening of biologically active monosaccharides: growth inhibitory effects of d-allose, d-talose, and l-idose against the nematode Caenorhabditis elegans."

Izumori K, Yoshihara A, Sakoguchi H, Sato M

[Biosci Biotechnol Biochem, 2016]

We compared the growth inhibitory effects of all aldohexose stereoisomers against the model animal Caenorhabditis elegans. Among the tested compounds, the rare sugars d-allose (d-All), d-talose (d-Tal), and l-idose (l-Ido) showed considerable growth inhibition under both monoxenic and axenic culture conditions. 6-Deoxy-d-All had no effect on growth, which suggests that C6-phosphorylation by hexokinase is essential for inhibition by d-All.

Zhang, Q. et al. (2019) International Worm Meeting "Multiple regulatory mechanisms control expression of the LIN-31/FOXB transcription factor."

Zhang, Q., Reiner, D

[International Worm Meeting, 2019]

The equipotent vulval precursor cells (VPCs) are induced by the anchor cell to assume 1?, 2?, or 3? cell fates. LIN-31, an ortholog of the FOXB transcription factor, is the terminal selector for VPC fates. By antibody staining, LIN-31 was previously reported to be expressed in P1.p-P11.p (including the VPCs, P3.p-P8.p). The regulatory sequences in the pB255 vector (P. Tan and S. Kim), drive GFP expression only in P3.p-P8.p. This "promoter" is used for heterologous gene expression in the VPCs, but is not truly specific. This reporter additionally expresses GFP in a small number of cells in the head and tail, probably neurons (N. Dudley, and M. Sundaram, our unpublished results). We set out to generate a VPC-specific promoter for analysis of gene expression in the VPCs, and so dissected the regulatory sequences present in pB255. To improve cell identification, we added 2xNLS sequences to GFP-encoding sequences. To counter variable somatic silencing in VPCs, we also used complex arrays to obtain consistent expression. Expression from resulting extrachromosomal arrays was not silenced. Sequences of lin-31exons and introns are present in inverted orientation upstream of the 4.5 kb "promoter" sequence in pB255. Deletion of this region caused GFP expression in VC neurons.Keeping this repressive sequence in place, we deleted promoter sequences, leaving 3.5 kb, 2.5 kb, 1.5 kb, 500 bp, 220 bp and 100 bpadjacent to the ATG. None of these constructs abolished VPC expression, though shorter sequences conferred variable expression. Additionally, control of head and tail neuronal expression may not be genetically separable from control of VPC expression. We used CRISPR to tag the 5' end of the endogenous lin-31gene. We observed expression in P1.p-P11.p from mid-L1 to L2 stage; after, expression in vulval lineages gradually decreased until it disappeared at the mid-L4 stage, while expression in non-vulval Pn.p cells persisted. Expression in head and tail neurons was present in the mid-L1 until L3, when it began to get progressively weaker. We will continue toward our goal of a VPC-specific promoter. However, we unexpectedly discovered that LIN-31 expression is excluded from VPCs after their induction.

Sakoguchi H et al. (2016) Bioorg Med Chem Lett "Growth inhibitory effect of d-arabinose against the nematode Caenorhabditis elegans: Discovery of a novel bioactive monosaccharide."

Shintani T, Izumori K, Sato M, Sakoguchi H, Okuma K, Yoshihara A

[Bioorg Med Chem Lett, 2016]

Biological activities of unusual monosaccharides (rare sugars) have largely remained unstudied until recently. We compared the growth inhibitory effects of aldohexose stereoisomers against the animal model Caenorhabditis elegans cultured in monoxenic conditions with Escherichia coli as food. Among these stereoisomers, the rare sugar d-arabinose (d-Ara) showed particularly strong growth inhibition. The IC50 value for d-Ara was estimated to be 7.5mM, which surpassed that of the potent glycolytic inhibitor 2-deoxy-d-glucose (19.5mM) used as a positive control. The inhibitory effect of d-Ara was also observed in animals cultured in axenic conditions using a chemically defined medium; this excluded the possible influence of E. coli. To our knowledge, this is the first report of biological activity of d-Ara. The d-Ara-induced inhibition was recovered by adding either d-ribose or d-fructose, but not d-glucose. These findings suggest that the inhibition could be induced by multiple mechanisms, for example, disturbance of d-ribose and d-fructose metabolism.

Yoshihara A et al. (2019) Bioorg Med Chem Lett "Growth inhibition by 1-deoxy-d-allulose, a novel bioactive deoxy sugar, screened using Caenorhabditis elegans assay."

Sakoguchi H, Fleet GWJ, Izumori K, Shintani T, Sato M, Yoshihara A

[Bioorg Med Chem Lett, 2019]

The biological activities of deoxy sugars (deoxy monosaccharides) have remained largely unstudied until recently. We compared the growth inhibition by all 1-deoxyketohexoses using the animal model Caenorhabditis elegans. Among the eight stereoisomers, 1-deoxy-d-allulose (1d-d-Alu) showed particularly strong growth inhibition. The 50% inhibition of growth (GI₅₀) concentration by 1d-d-Alu was estimated to be 5.4mM, which is approximately 10 times lower than that of d-allulose (52.7mM), and even lower than that of the potent glycolytic inhibitor, 2-deoxy-d-glucose (19.5mM), implying that 1d-d-Alu has a strong growth inhibition. In contrast, 5-deoxy- and 6-deoxy-d-allulose showed no growth inhibition of C. elegans. The inhibition by 1d-d-Alu was alleviated by the addition of d-ribose or d-fructose. Our findings suggest that 1d-d-Alu-mediated growth inhibition could be induced by the imbalance in d-ribose metabolism. To our knowledge, this is the first report of biological activity of 1d-d-Alu which may be considered as an antimetabolite drug candidate.

Katane M et al. (2020) Biochim Biophys Acta Proteins Proteom "Biochemical characterization of d-aspartate oxidase from Caenorhabditis elegans: Its potential use in the determination of free D-glutamate in biological samples."

Katane M, Sekine M, Nakayama K, Homma H, Kuwabara H, Saitoh Y, Miyamoto T

[Biochim Biophys Acta Proteins Proteom, 2020]

d-Aspartate oxidase (DDO) is a flavin adenine dinucleotide (FAD)-containing flavoprotein that stereospecifically acts on acidic D-amino acids (i.e., free d-aspartate and D-glutamate). Mammalian DDO, which exhibits higher activity toward d-aspartate than D-glutamate, is presumed to regulate levels of d-aspartate in the body and is not thought to degrade D-glutamate in vivo. By contrast, three DDO isoforms are present in the nematode Caenorhabditis elegans, DDO-1, DDO-2, and DDO-3, all of which exhibit substantial activity toward D-glutamate as well as d-aspartate. In this study, we optimized the Escherichia coli culture conditions for production of recombinant C. elegans DDO-1, purified the protein, and showed that it is a flavoprotein with a noncovalently but tightly attached FAD. Furthermore, C. elegans DDO-1, but not mammalian (rat) DDO, efficiently and selectively degraded D-glutamate in addition to d-aspartate, even in the presence of various other amino acids. Thus, C. elegans DDO-1 might be a useful tool for determining these acidic D-amino acids in biological samples.

Shintani T et al. (2019) J Appl Glycosci (1999) "D-Allose, a Stereoisomer of D-Glucose, Extends the Lifespan of Caenorhabditis elegans via Sirtuin and Insulin Signaling."

Izumori K, Sakoguchi H, Yoshihara A, Shintani T, Sato M

[J Appl Glycosci (1999), 2019]

D-Allose (D-All), C-3 epimer of D-glucose, is a rare sugar known to suppress reactive oxygen species generation and prevent hypertension. We previously reported that D-allulose, a structural isomer of D-All, prolongs the lifespan of the nematode Caenorhabditis elegans. Thus, D-All was predicted to affect longevity. In this study, we provide the first empirical evidence that D-All extends the lifespan of C. elegans. Lifespan assays revealed that a lifespan extension was induced by 28 mM D-All. In particular, a lifespan extension of 23.8 % was achieved (p< 0.0001). We further revealed that the effects of D-All on lifespan were dependent on the insulin gene daf-16 and the longevity gene sir-2.1, indicating a distinct mechanism from those of other hexoses, such as D-allulose, with previously reported antiaging effects.

Sato M et al. (2008) J Nat Med "Potential anthelmintic: D-psicose inhibits motility, growth and reproductive maturity of L1 larvae of Caenorhabditis elegans."

Izumori K, Yamasaki T, Kurose H, Sato M

[J Nat Med, 2008]

No anthelmintic sugars have yet been identified. Eight ketohexose stereoisomers (D- and L-forms of psicose, fructose, tagatose and sorbose), along with D-galactose and D-glucose, were examined for potency against L1 stage Caenorhabditis elegans fed Escherichia coli. Of the sugars, D-psicose specifically inhibited the motility, growth and reproductive maturity of the L1 stage. D-Psicose probably interferes with the nematode nutrition. The present results suggest that D-psicose, one of the rare sugars, is a potential anthelmintic.

Yasuaki Saitoh et al. (2010) East Asia Worm Meeting "Study on physiological functions of D-amino acid degradative enzymes in nematode Caenorhabditis elegans"

Yousuke Seida, Kazuhiro Maeda, Tomonori Kawata, Masumi Katane, Hiroyuki Kobuna, Takao Inoue, Yasuaki Saitoh, Hiroyuki Arai, Yasuhito Nakagawa, Masae Sekine, Taro Sakamoto, Hiroshi Homma, Takemitsu Furuchi

[East Asia Worm Meeting, 2010]

Among free D-amino acids existing in living organisms, D-serine (D-Ser) and D-aspartate (D-Asp) are the most actively studied. D-Ser has been proposed as a neuromodulator that regulates L-glutamate-mediated activation of the N-methyl-D-Asp (NMDA) receptor by acting as a co-agonist. On the other hand, several lines of evidence suggest that D-Asp plays important roles in regulating developmental processes, hormone secretion and steroidogenesis. D-Amino acid oxidase (DAO) and D-Asp oxidase (DDO) are known as stereospecific degradative enzymes that catalyze the oxidative deamination of D-amino acids. DAO displays broad substrate specificity and acts on several neutral and basic D-amino acids, while DDO is highly specific for acidic D-amino acids. DAO and DDO are presumed to regulate endogenous D-Ser and D-Asp levels, respectively, as well as mediate the elimination of accumulated exogenous D-amino acids in various organs. Previously, we demonstrated that nematode Caenorhabditis elegans, a multicellular model animal has at least one active DAO gene and three active DDO genes, while it had been thought that most organisms bear only one copy of each DAO and DDO gene. In addition, our previous study revealed that the spatiotemporal distributions of these enzymes in the body of C. elegans are different from one another. In this study, to elucidate the physiological roles of the C. elegans DAO and DDOs, we characterized several phenotypes of four C. elegans mutants in which each gene is partially deleted and inactivated. We also determined free D-amino acid contents in several worm samples using high-performance liquid chromatography (HPLC) techniques. We will report the phenotypes of the C. elegans mutants in comparison with those of wild-type C. elegans, as well as alterations in D-amino acid levels within the body.

Saitoh, Yasuaki et al. (2013) International Worm Meeting "D-Aspartate oxidase is involved in the caloric restriction-induced lifespan extension in C. elegans."

Inoue, Takao, Sekine, Masae, Saitoh, Yasuaki, Arai, Hiroyuki, Furuchi, Takemitsu, Sakamoto, Taro, Okutsu, Mari, Homma, Hiroshi, Katane, Masumi

[International Worm Meeting, 2013]

Among free D-amino acids existing in living organisms, D-serine (D-Ser) and D-aspartate (D-Asp) are the most intensively studied. In mammals, D-Ser has been proposed as a neuromodulator that regulates L-glutamate (L-Glu)-mediated activation of the N-methyl-D-Asp (NMDA) receptor by acting as a co-agonist. On the other hand, several lines of evidence suggest that D-Asp plays important roles in regulating hormone secretion and steroidogenesis. D-Amino acid oxidase (DAO) and D-Asp oxidase (DDO) are known as stereospecific degradative enzymes that catalyze the oxidative deamination of D-amino acids. Mammalian DAO and DDO are presumed to regulate endogenous D-Ser and D-Asp levels, respectively. Previously, we demonstrated that D-Ser, D-Asp, D-Glu and D-alanine (D-Ala) are present in nematode Caenorhabditis elegans, a multicellular model animal. We also found that C. elegans has at least one active DAO gene and three active DDO genes (DDO-1, DDO-2 and DDO-3), and that the spatiotemporal distributions of these enzymes in the body of C. elegans differ from one another. Furthermore, our previous study showed that alterations of brood size and hatching rate are observed in four C. elegans mutants lacking each gene for the DAO and DDOs. Interestingly, lifespan extension was observed in the DDO-3 mutant. To characterize the mechanism of lifespan extension in the DDO-3 mutant, we performed genetic epistasis experiments to test interactions between the DDO-3 gene and other known longevity pathways. The results suggest that DDO-3 is involved in caloric restriction-induced lifespan extension but not in insulin/IGF signaling pathway, NAD/sir2 pathway nor mitochondrial electron transport system. We also found that D-Glu and L-tryptophan (L-Trp) accumulate throughout life in the DDO-3 mutant. Now we are investigating the relationship between aging and the accumulations of D-Glu and L-Trp.