Ward, Jordan D. et al. (2011) International Worm Meeting "Sumoylation of C. elegans nuclear receptor NHR-25 promotes proper organogenesis."

Jindra, Marek, Asahina, Masako, Ward, Jordan D., Bojanala, Nagagireesh, Ashrafi, Kaveh, Yamamoto, Keith R., Bernal, Teresita

[International Worm Meeting, 2011]

Tissue-specific regulatory programs governed by nuclear receptors (NRs) are a critical component of metazoan development and homeostasis with aberrations in these programs leading to pathophysiology. We are using the simplicity and powerful genetics of C. elegans to deconvolute how regulatory inputs differentially converge on NRs in specific cell and tissue-types. The single C. elegans NR5A family receptor, NHR-25, coordinates diverse tissue-specific developmental events such as molting, seam cell differentiation, fat metabolism, cell fate decisions and vulva formation. To uncover novel co-regulators of NHR-25 activity we performed a genome-wide yeast two-hybrid (Y2H) screen that uncovered the C. elegans SUMO1 homolog (SMO-1) as an NHR-25 interacting protein. SUMO Y2H interactions can reflect both covalent sumoylation and non-covalent interaction; to distinguish between these possibilities we performed mutational analysis. Both deletion of the SMO-1 C-terminal di-glycine repeat and mutations in the SMO-1 beta sheet, which abrogate covalent sumoylation and non-covalent binding, respectively, prevented NHR-25 binding in Y2H assays. in vitro biochemical assays confirmed the sumoylation of NHR-25. We have identified three lysines in NHR-25 responsible for the interaction, as well as non-covalent Sumo interacting motifs (SIM) located in the ligand binding domain of NHR-25. These data argue that NHR-25 initially binds SMO-1 non-covalently in order to promote its sumoylation, similar to thymine DNA glycosylase sumoylation. The C. elegans vulva is a paradigm of organogenesis since the progression from 22 epithelial precursor cells to the seven toroidal disks in the completed organ involve a combination of reproducible cell divisions, migrations, remodeling of adherens junctions, cell fusions and muscle attachments. Deletion of the smo-1 gene causes severe protruding vulva (Pvl) and weak multivulva (Muv) phenotypes. When NHR-25 activity was reduced in a smo-1 mutant, Muv induction in P3.p, P4.p and P8.p was enhanced. Interestingly, daughters of P(5-7).p vulval precursor cells (VPCs) were also affected and could not complete the full program of vulval cell divisions, thereby suppressing the smo-1 Pvl phenotype. Our work suggests that SMO-1 and NHR-25 function together during VPC cell division in a cell context-dependent manner. These data highlight how the sumoylation of nuclear receptors fine-tunes target gene regulation and thus ensures proper organ development. Supported by GACR 204/09/H058, 204/07/0948, NPVII 2B06129, TFF postdoctoral fellowship 700046, NIH grant CA020535.

Ward, Jordan D. et al. (2011) International Worm Meeting "Vulval organogenesis involves an interaction between SMO-1 and the nuclear receptor NHR-25"

Asahina, Masako, Ashrafi, Kaveh, Ward, Jordan D., Jindra, Marek, Bernal, Teresita, Yamamoto, Keith R., Bojanala, Nagagireesh

[International Worm Meeting, 2011]

Organogenesis at the cellular level involves a complex interplay of cell fate acquisition, cell cycle control, remodeling of adherens junctions, cell migrations and cell fusions. Thus, this process is tightly regulated temporarily as well as spatially. Tissue-specific regulatory programs governed by nuclear receptors (NRs) are a critical component of metazoan development and homeostasis with aberrations in these programs leading to pathophysiology. We are using the simplicity and powerful genetics of C. elegans to deconvolute how regulatory inputs differentially converge on NRs in specific cell and tissue-types. The single C. elegans NR5A family receptor, NHR-25, coordinates diverse tissue-specific developmental events such as molting, seam cell differentiation, fat metabolism, cell fate decisions and vulva formation. To uncover novel co-regulators of NHR-25 activity we performed a genome-wide yeast two-hybrid (Y2H) screen that uncovered the C. elegans SUMO homolog (SMO-1) as an NHR-25 interacting protein. Both deletion of the SMO-1 C-terminal di-glycine repeat and mutations in the SMO-1 beta sheet, which abrogate covalent sumoylation and non-covalent binding, respectively, prevented NHR-25 binding in Y2H assays. in vitro biochemical assays confirmed the sumoylation of NHR- 25. We have identified three lysines in NHR-25 responsible for the interaction, as well as non-covalent SUMO interacting motifs (SIM) located in the ligand binding domain of NHR-25. These data argue that NHR-25 initially binds SMO-1 non-covalently in order to promote its sumoylation, similar to thymine DNA glycosylase sumoylation. Deletion of the smo-1 gene in C. elegans causes severe protruding vulva (Pvl) and weak multivulva (Muv) phenotypes. When NHR-25 activity was reduced in a smo-1 mutant, Muv induction in P3.p, P4.p and P8.p was enhanced. Interestingly, daughters of P(5-7).p vulval precursor cells (VPCs) were also affected and could not complete the full program of vulval cell divisions, thereby suppressing the smo-1 Pvl phenotype. Our work suggests that SMO-1 and NHR-25 function together during VPC cell division in a cell context-dependent manner. The C. elegans vulva is a paradigm of organogenesis and these data highlight how the sumoylation of nuclear receptors fine-tunes target gene regulation and thus ensures proper organ development. Supported by GACR 204/09/H058, 204/07/0948, NPVII 2B06129, TFF postdoctoral fellowship 700046, NIH grant CA020535.

Ward, Jordan D. et al. (2013) International Worm Meeting "Sumoylated NHR-25/NR5A regulates cell fate during C. elegans vulval development."

Asahina, Masako, Bernal, Teresita, Bojanala, Nagagireesh, Ashrafi, Kaveh, Ward, Jordan D., Yamamoto, Keith

[International Worm Meeting, 2013]

Individual metazoan transcription factors (TFs) regulate distinct sets of genes depending on cell type and developmental or physiological context. The exquisite cell and tissue specificity of gene regulation by a given TF appear to be achieved combinatorially, within multifactor regulatory complexes with distinct compositions and activities. The precise mechanisms by which regulatory information from ligands, genomic sequence elements, co-factors, and post-translational modifications are integrated by TFs remain challenging questions. Here we examine how a single regulatory input (sumoylation) differentially modulates the activity of the conserved C. elegans nuclear hormone receptor, NHR-25, in different cell types. Through a combination of yeast two-hybrid analysis and in vitro biochemistry we identified the single C. elegans SUMO (SMO-1) as an NHR-25 interacting protein, and showed that NHR-25 is sumoylated on three lysines. Genetic studies revealed that loss of smo-1 phenocopied NHR-25 overexpression, with respect to maintenance of the 3o cell fate in vulval precursor cells (VPCs) during development. Furthermore, in vivo overexpression using NHR-25 alleles that could not be sumoylated and SUMO-NHR-25 fusions indicated that NHR-25 sumoylation is critical for maintaining 3o cell fate. SUMO also regulated formation of an NHR-25 accumulation gradient in VPCs. Using an NHR-25::GFP translational reporter, we discovered that NHR-25 levels were uniform across VPCs at the beginning of development, but as cells began dividing a smo-1-dependent NHR-25 gradient formed with highest levels in 1o fated VPCs, lower levels in 2o fated VPCs, and the lowest levels in 3o fated VPCs. Our findings support a model where the ratio of unsumoylated to sumoylated NHR-25 regulates 3o cell fate determination and maintenance during vulval development. Supported by GAAV IAA500960906, MODBIOLIN 7FP-REGPOT-2012-2013-1, Terry Fox Foundation (#700046), CIHR (#234765) and the NIH (CA20535).

Ward, Jordan D. et al. (2013) International Worm Meeting "Acyl-CoA synthase-3 and the nuclear receptor NHR-25 regulate innate immunity genes and promote pathogen resistance."

Petnic, Sarah, Schiller, Benjamin, Ewbank, Jonathan, Bernal, Teresita, Yamamoto, Keith, Couillault, Carol, Mullaney, Brendan, Ashrafi, Kaveh, van Gilst, Marc, Ward, Jordan D.

[International Worm Meeting, 2013]

We have previously demonstrated that the acyl-CoA synthetase , acs-3, negatively regulates the nuclear hormone receptor, nhr-25, to promote an endocrine program of lipid uptake and synthesis. Given that NHR-25 is a transcription factor, we performed microarray analysis to identify regulated genes promoting this metabolic program. Much to our surprise, despite the observation that nhr-25(lf) suppresses all known acs-3 mutant phenotypes, loss of function of either acs-3 or nhr-25 had a similar effect at the transcriptional level. In both cases, EASE and gene ontology analysis revealed a significant upregulation of genes involved in cuticle formation and pathogen response. qPCR verification of the microarray data suggested that acs-3 and nhr-25 are epistatic with respect to pathogen response gene induction. Elevated pathogen response gene expression can be caused by osmotic stress resistance phenotypes and cuticular damage. acs-3 mutants have: i) a subtle resistance to acute hyperosmotic stress; ii) a partially penetrant sensitivity to hypo-osmotic stress; and iii) an intact cuticular barrier, as exhibited by elevated staining with the cuticle impermeable Hoechst 33358 dye in tail nuclei. nhr-25 mutants showed essentially wild-type responses in all of these assays. We then performed survival assays with the pathogens Drechmeria coniospora and Pseudomonas aeruginosa to test whether loss of acs-3 or nhr-25 activity affected resistance to pathogens. Loss of acs-3 activity caused hypersensitivity to Drechmeria, while nhr-25 mutants were sensitive to Pseudomonas. Intriguingly, acs-3 mutation suppressed the sensitivity of nhr-25 mutants to Pseudomonas. Together, these results highlight a link between the ACS-3-NHR-25 endocrine program and resistance to pathogens.

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.