Cecchetelli, Alyssa D et al. (2015) International Worm Meeting "RHO-1 is required for normal oocyte transit through the spermatheca."

Cecchetelli, Alyssa D, Cram, Erin J

[International Worm Meeting, 2015]

Mechanotransduction, converting a mechanical signal into a biochemical response, is vital for proper development, tissue function and homeostasis of biological tubes. Defects in a cell's ability to sense and respond to external stimuli can lead to complications and diseases such as asthma, atherosclerosis and cancer. The C. elegans spermatheca, the site of fertilization within the worm, provides an ideal in vivo model to study this biological process. Stretch of the incoming oocyte is converted into waves of calcium potentiated by PLC-1 that culminates in acto-myosin contractility and expulsion of fertilized embryos into the uterus. Loss of functional PLC-1 results in trapping of embryos within the spermatheca due to a lack of calcium signaling within the tissue. What remains unknown however, is what proteins act up or downstream of PLC-1 in the mechanotransduction pathway. One possible regulator is the small GTPase RHO-1. RHO-1 overexpression using a gain of function (gf) allele results in a highly constricted spermathecae and rapid transit, similar to loss of SPV-1, a recently identified GTPase-activating protein (GAP). Conversely, RHO-1 depletion via RNAi leads to embryos that become trapped in the spermatheca and/or worms that exhibit abnormal ovulations, where incoming oocytes are pinched in half upon entry. Preliminary evidence shows that RHO-1 has an affect on proper calcium oscillations, providing evidence that RHO-1 may be acting upstream of PLC-1. PLC-1 interestingly contains a unique RHO-1 interaction domain in addition to a CDC25 RAS guanine nucleotide exchange factor (GEF), EF hand, and RAS association domains. A structure function analysis of PLC-1 is in progress to determine the role of each PLC-1 domain in oocyte transit through the spermatheca. Although the presence of a unique RHO-1 interaction site suggests RHO-1 as a direct regulator, preliminary evidence does not support this hypothesis. It is possible that RHO-1 is indirectly regulating PLC-1 activity and subsequently calcium release. Given the conservation of PLC-1, RHO-1 and the identified mechanotransduction pathway that controls C. elegans ovulation, this work will provide insights into the biological mechanisms required to convert a mechanical stimulus into a biochemical response.

Cecchetelli, Alyssa D. et al. (2017) International Worm Meeting "A novel role for heterotrimeric G protein alpha subunits in C. elegans ovulation."

Cecchetelli, Alyssa D., Cram, Erin J.

[International Worm Meeting, 2017]

Mechanotransduction, converting a mechanical signal into a biochemical response, is vital for proper development, tissue function and homeostasis. Defects in a cell's ability to sense and respond to external stimuli can lead to complications in development and diseases such as asthma. The C. elegans spermatheca, the site of fertilization, provides an ideal in vivo model to study this biological process. Stretch of the incoming oocyte is converted into waves of calcium potentiated by PLC-1 that culminates in acto-myosin contractility and expulsion of fertilized embryos into the uterus. What remains unknown however, is what activates PLC-1. Loss of functional PLC-1 results in trapping of embryos within the spermatheca due to a lack of calcium signaling within the tissue. To identify potential activators of PLC-1, we conducted a candidate RNAi screen and identified two heterotrimeric G protein alpha subunits, GOA-1 (Gi/Go class) and GSA-1 (Gs class) as essential for proper oocyte transit through the spermatheca. Depletion of goa-1 via RNAi and a loss of function allele result in a significant increase in the number of embryos occupying spermathecae compared to wild type animals. Further analysis revealed that this increase in occupied spermathecae coincides with abnormal calcium signaling and extremely long embryo transits through the spermatheca, resulting in a significant reduction in broodsize. Depletion of gsa-1 via RNAi results in trapping of embryos in the spermatheca due to a lack of calcium signaling in the spermathecal bag comparable to the loss of plc-1. These results provide novel evidence that heterotrimeric G protein signaling may play an essential role in the stimulation of calcium signaling perhaps through the regulation of phospholipases like PLC-1. Given the conservation of PLC-1, GOA-1, GSA-1 and the pathway that regulates C. elegans ovulation, this work should provide insights into stretch activation of phospholipase signaling in vivo.

Pettit, Hannah N et al. (2019) International Worm Meeting "G-protein alpha subunit, GOA-1, is a regulator of contraction and relaxation in the C. elegans spermatheca."

Cram, Erin J, Pettit, Hannah N, Cecchetelli, Alyssa D, Castaneda, Perla

[International Worm Meeting, 2019]

Caenorbabditis elegans is a transparent roundworm used to study a multitude of cellular processes in an in vivo system. The C. elegans reproductive system is an ideal model to study mechanotransduction and contractility. Fertilization occurs in hermaphroditic worms in the spermatheca, when oocytes get pumped in to the spermatheca, are fertilized, and then pumped out as mature embryos. This process is highly regulated and requires cellular communication and coordination between the cells of the reproductive system. In a genetic screen for regulators of this process, we identified the small G protein GOA-1 as an important regulator of calcium signaling in the spermatheca. GOA-1 is a heterotrimeric G-protein a-subunit that can inhibit the production of second messengers such as cAMP. Normally, oocyte entry into the spermatheca triggers a characteristic set of calcium transients that increase in intensity, peaking concomitantly with the strong contractions that push the fertilized embryo into the uterus. GOA-1 deficient animals have a delayed and uncoordinated calcium response, which results in defects in tissue contractility and delayed embryo exit. In order to better understand the role of GOA-1 in the proposed pathway, we created a gain of function GOA-1 strain to determine how hyperactivation of GOA-1 would affect calcium signaling in the spermatheca. Additionally, in order to understand the role of GOA-1 in cellular communication, GOA-1 was locally rescued in specific cells of the spermatheca in a GOA-1 deficient animal. Data collected from these strains will help elucidate the role of GOA-1 in modulating Ca2+ signaling in vivo, allowing us to better understand the signaling networks that drive actomyosin contractility in a conserved, intact biological system.

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.

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.