Characterization of gana-1, a Caenorhabditis elegans gene encoding a single ortholog of vertebrate α-galactosidase and α-N-acetylgalactosaminidase

BACKGROUND: Human α-galactosidase A (α-GAL) and α-N-acetylgalactosaminidase (α-NAGA) are presumed to share a common ancestor. Deficiencies of these enzymes cause two well-characterized human lysosomal storage disorders (LSD) – Fabry (α-GAL deficiency) and Schindler (α-NAGA deficiency) diseases. Caenorhabditis elegans was previously shown to be a relevant model organism for several late endosomal/lysosomal membrane proteins associated with LSDs. The aim of this study was to identify and characterize C. elegans orthologs to both human lysosomal luminal proteins α-GAL and α-NAGA. RESULTS: BlastP searches for orthologs of human α-GAL and α-NAGA revealed a single C. elegans gene (R07B7.11) with homology to both human genes (α-galactosidase and α-N-acetylgalactosaminidase) – gana-1. We cloned and sequenced the complete gana-1 cDNA and elucidated the gene organization. Phylogenetic analyses and homology modeling of GANA-1 based on the 3D structure of chicken α-NAGA, rice α-GAL and human α-GAL suggest a close evolutionary relationship of GANA-1 to both human α-GAL and α-NAGA. Both α-GAL and α-NAGA enzymatic activities were detected in C. elegans mixed culture homogenates. However, α-GAL activity on an artificial substrate was completely inhibited by the α-NAGA inhibitor, N-acetyl-D-galactosamine. A GANA-1::GFP fusion protein expressed from a transgene, containing the complete gana-1 coding region and 3 kb of its hypothetical promoter, was not detectable under the standard laboratory conditions. The GFP signal was observed solely in a vesicular compartment of coelomocytes of the animals treated with Concanamycin A (CON A) or NH(4)Cl, agents that increase the pH of the cellular acidic compartment. Immunofluorescence detection of the fusion protein using polyclonal anti-GFP antibody showed a broader and coarsely granular cytoplasmic expression pattern in body wall muscle cells, intestinal cells, and a vesicular compartment of coelomocytes. Inhibition of gana-1 by RNA interference resulted in a decrease of both α-GAL and α-NAGA activities measured in mixed stage culture homogenates but did not cause any obvious phenotype. CONCLUSIONS: GANA-1 is a single C. elegans ortholog of both human α-GAL and α-NAGA proteins. Phylogenetic, homology modeling, biochemical and GFP expression analyses support the hypothesis that GANA-1 has dual enzymatic activity and is localized in an acidic cellular compartment

Novel structural arrangement of nematode cystathionine β-synthases: characterization of Caenorhabditis elegans CBS-1

CBSs (cystathionine β-synthases) are eukaryotic PLP (pyridoxal 5 *-phosphate)-dependent proteins that maintain cellular homocysteine homoeostasis and produce cystathionine and hydrogen sulfide. In the present study, we describe a novel structural arrangement of the CBS enzyme encoded by the cbs-1 gene of the nematode Caenorhabditis elegans. The CBS-1 protein contains a unique tandem repeat of two evolutionarily conserved catalytic regions in a single polypeptide chain. These repeats include a catalytically active C-terminal module containing a PLP-binding site and a less conserved N-terminal module that is unable to bind the PLP cofactor and cannot catalyse CBS reactions, as demonstrated by analysis of truncated variants and active-site mutant proteins. In contrast with other metazoan enzymes, CBS-1 lacks the haem and regulatory Bateman domain essential for activation by AdoMet (S-adenosylmethionine) and only forms monomers. We determined the tissue and subcellular distribution of CBS-1 and showed that cbs-1 knockdown by RNA interference leads to delayed development and to an approximately 10-fold elevation of homocysteine concentrations in nematode extracts. The present study provides the first insight into the metabolism of sulfur amino acids and hydrogen sulfide in C. elegans and shows that nematode CBSs possess a structural feature that is unique among CBS proteins

Folia2.qxp

Abstract. Valproic acid (2-propyl pentanoic acid) is a drug used for the treatment of epilepsy and bipolar disorder. Although very rare, side effects such as spina bifida and other defects of neural tube closure indicate that valproic acid interferes with developmental regulatory pathways. Recently obtained data show that valproic acid affects cell growth, differentiation, apoptosis and immunogenicity of cultured cancer cells and tumours. Focused studies uncovered the potential of valproic acid to interfere with multiple regulatory mechanisms including histone deacetylases, GSK3 α α and β β, Akt, the ERK pathway, the phosphoinositol pathway, the tricarboxylic acid cycle, GABA, and the OXPHOS system. Valproic acid is emerging as a potential anticancer drug and may also serve as a molecular lead that can help design drugs with more specific and more potent effects on the one side and drugs with wide additive but weaker effects on the other. Valproic acid is thus a powerful molecular tool for better understanding and therapeutic targeting of pathways that regulate the behaviour of cancer cells

Trichoplax adhaerens reveals a network of nuclear receptors sensitive to 9-cis-retinoic acid at the base of metazoan evolution

Trichoplax adhaerens, the only known species of Placozoa is likely to be closely related to an early metazoan that preceded branching of Cnidaria and Bilateria. This animal species is surprisingly well adapted to free life in the World Ocean inhabiting tidal costal zones of oceans and seas with warm to moderate temperatures and shallow waters. The genome of T. adhaerens (sp. Grell) includes four nuclear receptors, namely orthologue of RXR (NR2B), HNF4 (NR2A), COUP-TF (NR2F) and ERR (NR3B) that show a high degree of similarity with human orthologues. In the case of RXR, the sequence identity to human RXR alpha reaches 81% in the DNA binding domain and 70% in the ligand binding domain. We show that T. adhaerens RXR (TaRXR) binds 9-cis retinoic acid (9-cis-RA) with high affinity, as well as high specificity and that exposure of T. adhaerens to 9-cis-RA regulates the expression of the putative T. adhaerens orthologue of vertebrate L-malate-NADP+ oxidoreductase (EC 1.1.1.40) which in vertebrates is regulated by a heterodimer of RXR and thyroid hormone receptor. Treatment by 9-cis-RA alters the relative expression profile of T. adhaerens nuclear receptors, suggesting the existence of natural ligands. Keeping with this, algal food composition has a profound effect on T. adhaerens growth and appearance. We show that nanomolar concentrations of 9-cis-RA interfere with T. adhaerens growth response to specific algal food and causes growth arrest. Our results uncover an endocrine-like network of nuclear receptors sensitive to 9-cis-RA in T. adhaerens and support the existence of a ligand-sensitive network of nuclear receptors at the base of metazoan evolution

Perilipin-related protein regulates lipid metabolism in C. elegans

Perilipins are lipid droplet surface proteins that contribute to fat metabolism by controlling the access of lipids to lipolytic enzymes. Perilipins have been identified in organisms as diverse as metazoa, fungi, and amoebas but strikingly not in nematodes. Here we identify the protein encoded by the W01A8.1 gene in Caenorhabditis elegans as the closest homologue and likely orthologue of metazoan perilipin. We demonstrate that nematode W01A8.1 is a cytoplasmic protein residing on lipid droplets similarly as human perilipins 1 and 2. Downregulation or elimination of W01A8.1 affects the appearance of lipid droplets resulting in the formation of large lipid droplets localized around the dividing nucleus during the early zygotic divisions. Visualization of lipid containing structures by CARS microscopy in vivo showed that lipid-containing structures become gradually enlarged during oogenesis and relocate during the first zygotic division around the dividing nucleus. In mutant embryos, the lipid containing structures show defective intracellular distribution in subsequent embryonic divisions and become gradually smaller during further development. In contrast to embryos, lipid-containing structures in enterocytes and in epidermal cells of adult animals are smaller in mutants than in wild type animals. Our results demonstrate the existence of a perilipin-related regulation of fat metabolism in nematodes and provide new possibilities for functional studies of lipid metabolism

Rescue experiment of <i>gei-8(ok1671)</i> with overlapping amplified regions of genomic DNA injected into the gonads of parents.

<p>Rescue experiment of <i>gei-8(ok1671)</i> with overlapping amplified regions of genomic DNA injected into the gonads of parents.</p

Comparison of N-terminal regions of GEI-8-related proteins to NCoR/SMRT.

<p>Sequence alignment of GEI-8 nematode orthologues with their nearest Metazoa/Fungi homologues, both human orthologues NCoR1 and SMRT (NCoR2) are shown. Green bars indicate the position of the alpha-helices in the structure of the upstream DAD domain of human SMRT and homology predicted positions in the second SANT domain. Residues indispensable for regulating HDAC interactions and function are highlighted in blue (needed for the structural integrity), magenta (interaction with HDAC) and red (activation of HDAC). Only the N-terminal part of the sequences is shown. The identical and similar residues are highlighted by different intensity of shading. Sequence identifiers: <i>C. elegans</i>: GEI8_CAEEL, <i>C. brenneri</i>: CN15693, <i>C. briggsae</i>: A8X8F0_CAEBR, <i>C. remanei</i>: RP40355, <i>C. japonica</i>: JA23925 ABLE03010463.1 ABLE03032768.1 ABLE03032771.1 ABLE03032769.1 ABLE03032772.1, <i>Loa loa</i>: E1FVE0_LOALO, <i>Brugia malayi</i>: A8NSC3_BRUMA, <i>Ixodes scapularis</i>: B7PZ26_IXOSC, <i>Saccharomyces cerevisiae</i>: SNT1_YEAST, <i>Drosophila melanogaster</i>: Q9VYK0_DROME, <i>Trichoplax adhaerens</i>: B3SAN1_TRIAD, <i>Branchiostoma floridae</i>: C3XV35_BRAFL, <i>Danio rerio</i>: A8B6H7_DANRE, <i>Xenopus tropicalis</i>: NCOR1_XENTR AAMC01044136.1, <i>Anolis carolinensis</i>: ANOCA15679 2 ENSACAP00000014806; ENSACAT00000015107, <i>Gallus gallus</i>: UPI0000E813A6, <i>Homo sapiens</i>: NCOR2_HUMAN (NCoR2), <i>Homo sapiens</i>: NCOR1_HUMAN (NCoR1). PDB structure 1XC5 was used to determine the position of the helices.</p

GEI-8, a Homologue of Vertebrate Nuclear Receptor Corepressor NCoR/SMRT, Regulates Gonad Development and Neuronal Functions in <em>Caenorhabditis elegans</em>

<div><p>NCoR and SMRT are two paralogous vertebrate proteins that function as corepressors with unliganded nuclear receptors. Although <i>C. elegans</i> has a large number of nuclear receptors, orthologues of the corepressors NCoR and SMRT have not unambiguously been identified in <i>Drosophila</i> or <i>C. elegans</i>. Here, we identify GEI-8 as the closest homologue of NCoR and SMRT in <i>C. elegans</i> and demonstrate that GEI-8 is expressed as at least two isoforms throughout development in multiple tissues, including neurons, muscle and intestinal cells. We demonstrate that a homozygous deletion within the <i>gei-8</i> coding region, which is predicted to encode a truncated protein lacking the predicted NR domain, results in severe mutant phenotypes with developmental defects, slow movement and growth, arrested gonadogenesis and defects in cholinergic neurotransmission. Whole genome expression analysis by microarrays identified sets of de-regulated genes consistent with both the observed mutant phenotypes and a role of GEI-8 in regulating transcription. Interestingly, the upregulated transcripts included a predicted mitochondrial sulfide:quinine reductase encoded by Y9C9A.16. This locus also contains non-coding, 21-U RNAs of the piRNA class. Inhibition of the expression of the region coding for 21-U RNAs leads to irregular gonadogenesis in the homozygous <i>gei-8</i> mutants, but not in an otherwise wild-type background, suggesting that GEI-8 may function in concert with the 21-U RNAs to regulate gonadogenesis. Our results confirm that GEI-8 is the orthologue of the vertebrate NCoR/SMRT corepressors and demonstrate important roles for this putative transcriptional corepressor in development and neuronal function.</p> </div