Divergence in enzyme regulation between Caenorhabditis elegans and human tyrosine hydroxylase, the key enzyme in the synthesis of dopamine

2011 ◽  
Vol 434 (1) ◽  
pp. 133-141 ◽  
Author(s):  
Ana C. Calvo ◽  
Angel L. Pey ◽  
Antonio Miranda-Vizuete ◽  
Anne P. Døskeland ◽  
Aurora Martinez
Keyword(s):  
Caenorhabditis Elegans ◽  
Tyrosine Hydroxylase ◽  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Feedback Inhibition ◽  
Substrate Inhibition ◽  
Enzyme Regulation ◽  
C Elegans ◽  
Key Enzyme ◽  
Human Orthologue

TH (tyrosine hydroxylase) is the rate-limiting enzyme in the synthesis of catecholamines. The cat-2 gene of the nematode Caenorhabditis elegans is expressed in mechanosensory dopaminergic neurons and has been proposed to encode a putative TH. In the present paper, we report the cloning of C. elegans full-length cat-2 cDNA and a detailed biochemical characterization of the encoded CAT-2 protein. Similar to other THs, C. elegans CAT-2 is composed of an N-terminal regulatory domain followed by a catalytic domain and a C-terminal oligomerization domain and shows high substrate specificity for L-tyrosine. Like hTH (human TH), CAT-2 is tetrameric and is phosphorylated at Ser35 (equivalent to Ser40 in hTH) by PKA (cAMP-dependent protein kinase). However, CAT-2 is devoid of characteristic regulatory mechanisms present in hTH, such as negative co-operativity for the cofactor, substrate inhibition or feedback inhibition exerted by catecholamines, end-products of the pathway. Thus TH activity in C. elegans displays a weaker regulation in comparison with the human orthologue, resembling a constitutively active enzyme. Overall, our data suggest that the intricate regulation characteristic of mammalian TH might have evolved from more simple models to adjust to the increasing complexity of the higher eukaryotes neuroendocrine systems.

scholarly journals The Caenorhabditis elegans unc-13 gene product is a phospholipid-dependent high-affinity phorbol ester receptor

1992 ◽  
Vol 287 (3) ◽  
pp. 995-999 ◽  
Author(s):  
S Ahmed ◽  
I N Maruyama ◽  
R Kozma ◽  
J Lee ◽  
S Brenner ◽  
...  
Keyword(s):  
Amino Acid ◽  
Caenorhabditis Elegans ◽  
Phorbol Ester ◽  
Catalytic Domain ◽  
Amino Acid Residues ◽  
C Elegans ◽  
High Affinity ◽  
Cell Extracts ◽  
Neuronal Connections ◽  
Phorbol Ester Receptor

The Caenorhabditis elegans unc-13 mutant is a member of a class of mutants that have un-coordinated movement. Mutations of the unc-13 gene cause diverse defects in C. elegans, including abnormal neuronal connections and modified synaptic transmission in the nervous system. unc-13 cDNA encodes a protein (UNC-13) of 1734 amino acid residues with a predicted molecular mass of 198 kDa and sequence identity to the C1/C2 regions but not to the catalytic domain of the ubiquitously expressed protein kinase C family [Maruyama & Brenner (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5729-5733]. To characterize the phorbol ester binding site of the UNC-13 protein, cDNA encoding the C1/C2-like regions (amino acid residues 546-940) was expressed in Escherichia coli and the 43 kDa recombinant protein was purified. Phorbol ester binding to the 43 kDa protein was zinc- and phospholipid-dependent, stereospecific and of high affinity (Kd 67 nM). UNC-13 specific antisera detected a protein of approx. 190 kDa in wild-type (N2) but not in mutant (e1019) C. elegans cell extracts. We conclude that UNC-13 represents a novel class of phorbol ester receptor.

scholarly journals Molecular and biochemical characterization of S-adenosylmethionine decarboxylase from the free-living nematode Caenorhabditis elegans

1998 ◽  
Vol 336 (3) ◽  
pp. 545-550 ◽  
Author(s):  
Akram A. DA'DARA ◽  
Rolf D. WALTER
Keyword(s):  
Caenorhabditis Elegans ◽  
Biochemical Characterization ◽  
Active Form ◽  
Nematode Caenorhabditis Elegans ◽  
Free Living ◽  
Polyamine Biosynthesis ◽  
Calculated Molecular Mass ◽  
C Elegans ◽  
Adenosylmethionine Decarboxylase ◽  
Free Living Nematode

S-Adenosylmethionine decarboxylase (SAMDC) is a major regulatory enzyme in the polyamine biosynthesis and is considered a potentially important drug target for the chemotherapy of proliferative and parasitic diseases. To study regulatory mechanisms which are involved in the expression of SAMDC of the free-living nematode Caenorhabditis elegans, we have isolated the SAMDC gene and cDNA. Genomic Southern-blot analysis suggests that the C. elegans SAMDC is encoded by a single-copy gene which spans 3.9 kb and consists of six exons and five introns. The first two introns are located in the 5´-untranslated region (UTR). Analyses of the 5´-flanking region of the gene revealed several consensus sequences for the binding of different transcription factors such as CBP, AP2, cMyb, VPE2 and others. The C. elegans SAMDC mRNA possesses an open reading frame (ORF) which encodes a polypeptide of 368 amino acids, corresponding to a SAMDC proenzyme with a calculated molecular mass of 42141 Da. The active form of the C. elegans SAMDC is a heterotetramer, consisting of two subunits of 32 and 10 kDa derived from cleavage of the pro-enzyme. The SAMDC mRNA has an unusually long 5´-UTR of 477 nucleotides. This region has a small ORF which could encode a putative peptide of 17 residues. Moreover, the C. elegans SAMDC mRNA is trans-spliced with the 22 nucleotides spliced leader sequence at the 5´-end.

scholarly journals Requirement of the Caenorhabditis elegans RapGEF pxf-1 and rap-1 for Epithelial Integrity

2005 ◽  
Vol 16 (1) ◽  
pp. 106-116 ◽  
Author(s):  
W. Pellis-van Berkel ◽  
M.H.G. Verheijen ◽  
E. Cuppen ◽  
M. Asahina ◽  
J. de Rooij ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Nucleotide Exchange ◽  
Epithelial Integrity ◽  
C Elegans ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Polarized Secretion ◽  
Green Fluorescent

The Rap-pathway has been implicated in various cellular processes but its exact physiological function remains poorly defined. Here we show that the Caenorhabditis elegans homologue of the mammalian guanine nucleotide exchange factors PDZ-GEFs, PXF-1, specifically activates Rap1 and Rap2. Green fluorescent protein (GFP) reporter constructs demonstrate that sites of pxf-1 expression include the hypodermis and gut. Particularly striking is the oscillating expression of pxf-1 in the pharynx during the four larval molts. Deletion of the catalytic domain from pxf-1 leads to hypodermal defects, resulting in lethality. The cuticle secreted by pxf-1 mutants is disorganized and can often not be shed during molting. At later stages, hypodermal degeneration is seen and animals that reach adulthood frequently die with a burst vulva phenotype. Importantly, disruption of rap-1 leads to a similar, but less severe phenotype, which is enhanced by the simultaneous removal of rap-2. In addition, the lethal phenotype of pxf-1 can be rescued by expression of an activated version of rap-1. Together these results demonstrate that the pxf-1/rap pathway in C. elegans is required for maintenance of epithelial integrity, in which it probably functions in polarized secretion.

scholarly journals The genes pme-1 and pme-2 encode two poly(ADP-ribose) polymerases in Caenorhabditis elegans

2002 ◽  
Vol 368 (1) ◽  
pp. 263-271 ◽  
Author(s):  
Steve N. GAGNON ◽  
Michael O. HENGARTNER ◽  
Serge DESNOYERS
Keyword(s):  
Caenorhabditis Elegans ◽  
Messenger Rna ◽  
Catalytic Domain ◽  
Post Translational Modification ◽  
Developmentally Regulated ◽  
Splice Leader ◽  
C Elegans ◽  
Zinc Finger Motifs ◽  
Multicellular Organisms ◽  
Parp 1

Poly(ADP-ribose) polymerases (PARPs) are an expanding, well-conserved family of enzymes found in many metazoan species, including plants. The enzyme catalyses poly(ADP-ribosyl)ation, a post-translational modification that is important in DNA repair and programmed cell death. In the present study, we report the finding of an endogenous source of poly(ADP-ribosyl)ation in total extracts of the nematode Caenorhabditis elegans. Two cDNAs encoding highly similar proteins to human PARP-1 (huPARP-1) and huPARP-2 are described, and we propose to name the corresponding enzymes poly(ADP-ribose) metabolism enzyme 1 (PME-1) and PME-2 respectively. PME-1 (108kDa) shares 31% identity with huPARP-1 and has an overall structure similar to other PARP-1 subfamily members. It contains sequences having considerable similarity to zinc-finger motifs I and II, as well as with the catalytic domain of huPARP-1. PME-2 (61kDa) has structural similarities with the catalytic domain of PARPs in general and shares 24% identity with huPARP-2. Recombinant PME-1 and PME-2 display PARP activity, which may partially account for the similar activity found in the worm. A partial duplication of the pme-1 gene with pseudogene-like features was found in the nematode genome. Messenger RNA for pme-1 are 5′-tagged with splice leader 1, whereas those for pme-2 are tagged with splice leader 2, suggesting an operon-like expression for pme-2. The expression pattern of pme-1 and pme-2 is also developmentally regulated. Together, these results show that PARP-1 and −2 are conserved in evolution and must have important functions in multicellular organisms. We propose using C. elegans as a model to understand better the functions of these enzymes.

scholarly journals Molecular genetic and biochemical characterization of a putative family of zinc metalloproteins in Caenorhabditis elegans

Metallomics ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 1814-1823 ◽  
Author(s):  
Poulami Chaudhuri ◽  
Hasan Tanvir Imam ◽  
Yona Essig ◽  
Jovaras Krasauskas ◽  
Samuel M. Webb ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Fluorescence Microscopy ◽  
Phase Contrast ◽  
Biochemical Characterization ◽  
Molecular Genetic ◽  
Phase Contrast Microscopy ◽  
C Elegans ◽  
Contrast Microscopy

The first characterization of W08E12.2, W08E12.3, W08E12.4 and W08E12.5, four putative metalloproteins in C. elegans. (A) phase contrast microscopy, (B) fluorescence microscopy of PW08E12.3;W08E12.4::GFP.

scholarly journals Metabolic shift from glycogen to trehalose promotes lifespan and healthspan in Caenorhabditis elegans

2018 ◽  
Vol 115 (12) ◽  
pp. E2791-E2800 ◽  
Author(s):  
Yonghak Seo ◽  
Samuel Kingsley ◽  
Griffin Walker ◽  
Michelle A. Mondoux ◽  
Heidi A. Tissenbaum
Keyword(s):  
Caenorhabditis Elegans ◽  
Metabolic Diseases ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Glycogen Storage ◽  
The Body ◽  
Metabolic Shift ◽  
C Elegans ◽  
High Sugar ◽  
Key Enzyme

As Western diets continue to include an ever-increasing amount of sugar, there has been a rise in obesity and type 2 diabetes. To avoid metabolic diseases, the body must maintain proper metabolism, even on a high-sugar diet. In both humans and Caenorhabditis elegans, excess sugar (glucose) is stored as glycogen. Here, we find that animals increased stored glycogen as they aged, whereas even young adult animals had increased stored glycogen on a high-sugar diet. Decreasing the amount of glycogen storage by modulating the C. elegans glycogen synthase, gsy-1, a key enzyme in glycogen synthesis, can extend lifespan, prolong healthspan, and limit the detrimental effects of a high-sugar diet. Importantly, limiting glycogen storage leads to a metabolic shift whereby glucose is now stored as trehalose. Two additional means to increase trehalose show similar longevity extension. Increased trehalose is entirely dependent on a functional FOXO transcription factor DAF-16 and autophagy to promote lifespan and healthspan extension. Our results reveal that when glucose is stored as glycogen, it is detrimental, whereas, when stored as trehalose, animals live a longer, healthier life if DAF-16 is functional. Taken together, these results demonstrate that trehalose modulation may be an avenue for combatting high-sugar-diet pathology.

scholarly journals Biochemical characterization of Caenorhabditis elegans UBC-1: self-association and auto-ubiquitination of a RAD6-like ubiquitin-conjugating enzyme in vitro

1997 ◽  
Vol 327 (2) ◽  
pp. 357-361 ◽  
Author(s):  
S. David LEGGETT ◽  
M. E. Peter CANDIDO
Keyword(s):  
Caenorhabditis Elegans ◽  
Biochemical Characterization ◽  
Quaternary Structure ◽  
Catalytic Domain ◽  
Site Directed Mutagenesis ◽  
Post Translational Modification ◽  
Ubiquitin Conjugating Enzyme ◽  
Self Association ◽  
Conjugating Enzyme

The Caenorhabditis elegans ubiquitin-conjugating enzyme UBC-1 is distinct from other RAD6 homologues in possessing a C-terminal tail 40 amino acid residues long [Leggett, Jones and Candido (1995) DNA Cell Biol. 14, 883-891]. Such extensions from the core catalytic domain have been found in a subset of known conjugating enzymes, where they have been shown to have diverse roles including target recognition, membrane attachment and sporulation. In the present study we used mutagenesis in vitro to examine the role of the tail in specific aspects of UBC-1 structure and activity. Cross-linking experiments with purified recombinant UBC-1 reveal that it forms dimers and probably tetramers. The acidic tail of UBC-1 has an important role in this interaction because deletions of the tail significantly decrease, but do not abolish, this self-association. Ubiquitin conjugation assays show that, in addition to accepting a thiol-bound ubiquitin at its active site, UBC-1 is stably mono-ubiquitinated. Deletion analysis and site-directed mutagenesis localize the site of ubiquitination to Lys-162 in the tail. These findings demonstrate that the C-terminal tail of UBC-1 is important both for its quaternary structure and post-translational modification in vitro.

The glycomes of Caenorhabditis elegans and other model organisms

2002 ◽  
Vol 69 ◽  
pp. 117-134 ◽  
Author(s):  
Stuart M. Haslam ◽  
David Gems ◽  
Howard R. Morris ◽  
Anne Dell
Keyword(s):  
Caenorhabditis Elegans ◽  
Current Knowledge ◽  
Structural Data ◽  
Model Organisms ◽  
Entire Genome ◽  
C Elegans ◽  
The Face ◽  
Area Of Concern ◽  
Nematode Worm

There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research. However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited. This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced. To date, only limited structural data on the glycosylated molecules of this organism have been reported. Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C. elegans; high-mannose structures dominate, with only minor amounts of complex-type structures. Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fucα1–2Gal1–2Man as peripheral structures. The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter. Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.

The Effect of Mianserin on Lifespan of Caenorhabditis elegans is Abolished by Glucose

2021 ◽  
Vol 13 ◽  
Author(s):  
Abdullah Almotayri ◽  
Jency Thomas ◽  
Mihiri Munasinghe ◽  
Markandeya Jois
Keyword(s):  
Caenorhabditis Elegans ◽  
Scaffolding Protein ◽  
Lifespan Extension ◽  
Wild Type ◽  
E Coli ◽  
C Elegans ◽  
Risk Of Death ◽  
Increased Risk ◽  
Antidepressant Use ◽  
Extension Effect

Background: The antidepressant mianserin has been shown to extend the lifespan of Caenorhabditis elegans (C. elegans), a well-established model organism used in aging research. The extension of lifespan in C. elegans was shown to be dependent on increased expression of the scaffolding protein (ANK3/unc-44). In contrast, antidepressant use in humans is associated with an increased risk of death. The C. elegans in the laboratory are fed Escherichia coli (E. coli), a diet high in protein and low in carbohydrate, whereas a typical human diet is high in carbohydrates. We hypothesized that dietary carbohydrates might mitigate the lifespan-extension effect of mianserin. Objective: To investigate the effect of glucose added to the diet of C. elegans on the lifespan-extension effect of mianserin. Methods: Wild-type Bristol N2 and ANK3/unc-44 inactivating mutants were cultured on agar plates containing nematode growth medium and fed E. coli. Treatment groups included (C) control, (M50) 50 μM mianserin, (G) 73 mM glucose, and (M50G) 50 μM mianserin and 73 mM glucose. Lifespan was determined by monitoring the worms until they died. Statistical analysis was performed using the Kaplan-Meier version of the log-rank test. Results: Mianserin treatment resulted in a 12% increase in lifespan (P<0.05) of wild-type Bristol N2 worms but reduced lifespan by 6% in ANK3/unc-44 mutants, consistent with previous research. The addition of glucose to the diet reduced the lifespan of both strains of worms and abolished the lifespan-extension by mianserin. Conclusion: The addition of glucose to the diet of C. elegans abolishes the lifespan-extension effects of mianserin.

scholarly journals Functional Redundancy of the B9 Proteins and Nephrocystins in Caenorhabditis elegans Ciliogenesis

2008 ◽  
Vol 19 (5) ◽  
pp. 2154-2168 ◽  
Author(s):  
Corey L. Williams ◽  
Marlene E. Winkelbauer ◽  
Jenny C. Schafer ◽  
Edward J. Michaud ◽  
Bradley K. Yoder
Keyword(s):  
Caenorhabditis Elegans ◽  
Joubert Syndrome ◽  
Cystic Kidney ◽  
Basal Bodies ◽  
The Novel ◽  
C Elegans ◽  
Human Genes ◽  
Cilia Formation ◽  
Strong Candidate ◽  
Candidate Loci

Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and Joubert syndrome (JBTS) are a group of heterogeneous cystic kidney disorders with partially overlapping loci. Many of the proteins associated with these diseases interact and localize to cilia and/or basal bodies. One of these proteins is MKS1, which is disrupted in some MKS patients and contains a B9 motif of unknown function that is found in two other mammalian proteins, B9D2 and B9D1. Caenorhabditis elegans also has three B9 proteins: XBX-7 (MKS1), TZA-1 (B9D2), and TZA-2 (B9D1). Herein, we report that the C. elegans B9 proteins form a complex that localizes to the base of cilia. Mutations in the B9 genes do not overtly affect cilia formation unless they are in combination with a mutation in nph-1 or nph-4, the homologues of human genes (NPHP1 and NPHP4, respectively) that are mutated in some NPHP patients. Our data indicate that the B9 proteins function redundantly with the nephrocystins to regulate the formation and/or maintenance of cilia and dendrites in the amphid and phasmid ciliated sensory neurons. Together, these data suggest that the human homologues of the novel B9 genes B9D2 and B9D1 will be strong candidate loci for pathologies in human MKS, NPHP, and JBTS.

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