Characterization of the caspase family in zebrafish.
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ABSTRACT:
SUBMITTER: Spead O
PROVIDER: S-EPMC5965869 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Calcium Binding Proteins (CaBPs), part of the vast family of EF-Hand-domain containing proteins, modulate intracellular calcium levels. They thereby contribute to a broad spectrum of biological processes - amongst others cell migration, gene expression and neural activity. In this study we identified twelve members of this protein family in zebrafish including one gene (cabp4b) currently not present in the zebrafish genome assembly. To gain insight into their biological functions, we carried out a detailed analysis of the expression patterns of these genes during zebrafish late embryonic and early larval development. We detected specific transcription for most of them in different neuronal cell populations including the neuroretina, the inner ear and the notochord. Our data supports potential roles for CaBPs during neuronal development and function and provides a starting point for genetic studies to examine CaBPs' function in these tissues and organs.
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Project description:Caspase-3 was cloned from zebrafish embryos and its properties were characterized to identify the biological implications of caspase in embryogenesis and apoptosis in zebrafish, which is a model organism in vertebrate developmental biology and genetics. The predicted amino acid sequence, totalling 282 amino acid residues, consisted of the prodomain and large and small subunits. Phylogenetic analysis showed that the cloned zebrafish caspase was a member of the caspase-3 subfamily with approx. 60% identity with caspase-3 from Xenopus, chicken and mammals. In addition, recombinant zebrafish caspase hydrolysed acetyl-Asp-Glu-Val-Asp-4-methyl-coumaryl-7-amide, and exhibited similar substrate specificity to the mammalian caspase-3 subfamily. Therefore this caspase was designated zebrafish caspase-3. Overexpression of zebrafish caspase-3 induced apoptosis and increased ceramide levels in fish fathead minnow tailbud cells and zebrafish embryos. Both ceramide generation and apoptosis induction were inhibited by treatment with a caspase inhibitor, benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone. Moreover, zebrafish caspase-3 mRNA was present in early embryos up to the 1000-cell stage as a maternal factor, and was then expressed throughout the body after the gastrula stage by zygotic expression. These findings indicate that the isolated caspase-3 plays an important role in the induction of ceramide generation as well as apoptosis in fish cells and the zebrafish embryo, and suggest that caspase-3 functions as a modulator of the pro-apoptotic signal in development.
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Project description:BackgroundThe cell suicide pathway of apoptosis is a necessary event in the life of multicellular organisms. It is involved in many biological processes ranging from development to the immune response. Evolutionarily conserved proteases, called caspases, play a central role in regulating apoptosis. Reception of death stimuli triggers the activation of initiator caspases, which in turn activate the effector caspases. In Lepidoptera, apoptosis is crucial in processes such as metamorphosis or defending against baculovirus infection. The discovery of p35, a baculovirus protein inhibiting caspase activity, has led to the characterization of the first lepidopteran caspase, Sf-Caspase-1. Studies on Sf-Caspase-1 mode of activation suggested that apoptosis in Lepidoptera requires a cascade of caspase activation, as demonstrated in many other species.ResultsIn order to get insights into this gene family in Lepidoptera, we performed an extensive survey of lepidopteran-derived EST datasets. We identified 66 sequences distributed among 27 species encoding putative caspases. Phylogenetic analyses showed that Lepidoptera possess at least 5 caspases, for which we propose a unified nomenclature. According to homology to their Drosophila counterparts and their primary structure, we determined that Lep-Caspase-1, -2 and -3 are putative effector caspases, whereas Lep-Caspase-5 and -6 are putative initiators. The likely function of Lep-Caspase-4 remains unclear. Lep-Caspase-2 is absent from the silkworm genome and appears to be noctuid-specific, and to have arisen from a tandem duplication of the Caspase-1 gene. In the tobacco hawkmoth, 3 distinct transcripts encoding putative Caspase-4 were identified, suggesting at least 2 duplication events in this species.ConclusionsThe basic repertoire of five major types of caspases shared among Lepidoptera seems to be smaller than for most other groups studied to date, but gene duplication still plays a role in lineage-specific increases in diversity, just as in Diptera and mammals.
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Project description:Zebrafish express five cytochrome P450 1 genes: CYP1A, CYP1B1, CYP1C1, CYP1C2, inducible by aryl hydrocarbon receptor agonists, and CYP1D1, a constitutively expressed CYP1A-like gene. We examined substrate selectivity of CYP1s expressed in yeast.CYP1s were expressed in W(R) yeast, engineered to over-express P450 reductase, via pYES/DEST52 and via pYeDP60. Microsomal fractions from transformed yeast were examined for activity with fluorogenic substrates, benzo[a]pyrene and testosterone. Modeling and docking approaches were used to further evaluate sites of oxidation on benzo[a]pyrene and testosterone.CYP1s expressed in yeast dealkylated ethoxy-, methoxy-, pentoxy- and benzoxy-resorufin (EROD, MROD, PROD, BROD). CYP1A and CYP1C2 had the highest rates of EROD activity, while PROD and BROD activities were low for all five CYP1s. The relative rates of resorufin dealkylation by CYP1C1, CYP1C2 and CYP1D1 expressed via pYeDP60 were highly similar to relative rates obtained with pYES/DEST52-expressed enzymes. CYP1C1 and CYP1C2 dealkylated substituted coumarins and ethoxy-fluorescein-ethylester, while CYP1D1 did not. The CYP1Cs and CYP1D1 co-expressed with epoxide hydrolase oxidized BaP with different rates and product profiles, and all three produced BaP-7,8,9,10-tetrol. The CYP1Cs but not CYP1D1 metabolized testosterone to 6?-OH-testosterone. However, CYP1D1 formed an unidentified testosterone metabolite better than the CYP1Cs. Testosterone and BaP docked to CYP homology models with poses consistent with differing product profiles.Yeast-expressed zebrafish CYP1s will be useful in determining further functionality with endogenous and xenobiotic compounds.Determining the roles of zebrafish CYP1s in physiology and toxicology depends on knowing the substrate selectivity of these enzymes.
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Project description:Although it is known that tumor necrosis factor receptor (TNFR) signaling plays a crucial role in vascular integrity and homeostasis, the contribution of each receptor to these processes and the signaling pathway involved are still largely unknown. Here, we show that targeted gene knockdown of TNFRSF1B in zebrafish embryos results in the induction of a caspase-8, caspase-2 and P53-dependent apoptotic program in endothelial cells that bypasses caspase-3. Furthermore, the simultaneous depletion of TNFRSF1A or the activation of NF-?B rescue endothelial cell apoptosis, indicating that a signaling balance between both TNFRs is required for endothelial cell integrity. In endothelial cells, TNFRSF1A signals apoptosis through caspase-8, whereas TNFRSF1B signals survival via NF-?B. Similarly, TNF? promotes the apoptosis of human endothelial cells through TNFRSF1A and triggers caspase-2 and P53 activation. We have identified an evolutionarily conserved apoptotic pathway involved in vascular homeostasis that provides new therapeutic targets for the control of inflammation- and tumor-driven angiogenesis.
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Project description:The Annexins (ANXs) are a family of calcium- and phospholipid-binding proteins that have been implicated in many cellular processes, including channel formation, membrane fusion, vesicle transport, and regulation of phospholipase A2 activity. As a first step toward understanding in vivo function, we have cloned 11 zebrafish anx genes. Four genes (anx1a, anx2a, anx5,and anx11a) were identified by screening a zebrafish cDNA library with a Xenopus anx2 fragment. For these genes, full-length cDNA sequences were used to cluster 212 EST sequences generated by the Zebrafish Genome Resources Project. The EST analysis revealed seven additional anx genes that were subsequently cloned. The genetic map positions of all 11 genes were determined by using a zebrafish radiation hybrid panel. Sequence and syntenic relationships between zebrafish and human genes indicate that the 11 genes represent orthologs of human anx1,2,4,5,6,11,13,and suggest that several zebrafish anx genes resulted from duplications that arose after divergence of the zebrafish and mammalian genomes. Zebrafish anx genes are expressed in a wide range of tissues during embryonic and larval stages. Analysis of the expression patterns of duplicated genes revealed both redundancy and divergence, with the most similar genes having almost identical tissue-specific patterns of expression and with less similar duplicates showing no overlap. The differences in gene expression of recently duplicated anx genes could explain why highly related paralogs were maintained in the genome and did not rapidly become pseudogenes.
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Project description:To study characterization of zebrafish glycine receptors (zGlyRs), we assessed expression and function of five ?- and two ß-subunit encoding GlyR in zebrafish. Our qPCR analysis revealed variable expression during development, while in situ hybridizations uncovered expression in the hindbrain and spinal cord; a finding consistent with the reported expression of GlyR subunits in these tissues from other organisms. Electrophysiological recordings using Xenopus oocytes revealed that all five ? subunits form homomeric receptors activated by glycine, and inhibited by strychnine and picrotoxin. In contrast, ß subunits only formed functional heteromeric receptors when co-expressed with ? subunits. Curiously, the second transmembranes of both ß subunits were found to lack a phenylalanine at the sixth position that is commonly associated with conferring picrotoxin resistance to heteromeric receptors. Consistent with the absence of phenylalanines at the sixth position, heteromeric zGlyRs often lacked significant picrotoxin resistance. Subsequent efforts revealed that resistance to picrotoxin in both zebrafish and human heteromeric GlyRs involves known residues within transmembrane 2, as well as previously unknown residues within transmembrane 3. We also found that a dominant mutation in human GlyR?1 that gives rise to hyperekplexia, and recessive mutations in zebrafish GlyRßb that underlie the bandoneon family of motor mutants, result in reduced receptor function. Lastly, through the use of a concatenated construct we demonstrate that zebrafish heteromeric receptors assemble with a stoichiometry of 3?:2ß. Collectively, our findings have furthered our knowledge regarding the assembly of heteromeric receptors, and the molecular basis of ß subunit-conferred picrotoxin resistance. These results should aid in future investigations of glycinergic signaling in zebrafish and mammals.
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Project description:This SuperSeries is composed of the SubSeries listed below.
2024-04-01 | GSE218958 | GEO
Project description:The caspase-recruitment domain (CARD) mediates homotypic protein-protein interactions that assemble large oligomeric signaling complexes such as the inflammasomes during innate immune responses. Structural studies of the mammalian CARDs demonstrate that their six-helix bundle folds belong to the death-domain superfamily, whereas such studies have not been reported for other organisms. Here, the zebrafish interferon-induced guanylate-binding protein 1 (zIGBP1) was identified that contains an N-terminal GTPase domain and a helical domain typical of the mammalian guanylate-binding proteins, followed by a FIIND domain and a C-terminal CARD similar to the mammalian inflammasome proteins NLRP1 and CARD8. The structure of the zIGBP1 CARD as a fusion with maltose-binding protein was determined at 1.47 Å resolution. This revealed a six-helix bundle fold similar to the NLRP1 CARD structure with the bent ?1 helix typical of all known CARD structures. The zIGBP1 CARD surface contains a positively charged patch near its ?1 and ?4 helices and a negatively charged patch near its ?2, ?3 and ?5 helices, which may mediate its interaction with partner domains. Further studies using binding assays and other analyses will be required in order to address the physiological function(s) of this zebrafish protein.
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Project description:We have characterized the protein cross-linking enzyme transglutaminase (TGs) genes in zebrafish, Danio rerio, based on the analysis of their genomic organization and phylogenetics. Thirteen zebrafish TG genes (zTGs) have been identified, of which 11 show high homology to only 3 mammalian enzymes: TG1, TG2 and FXIIIa. No zebrafish homologues were identified for mammalian TGs 3-7. Real-time PCR analysis demonstrated distinct temporal expression profiles for zTGs in larvae and adult fish. Analysis by in situ hybridization revealed restricted expression of zTG2b and zFXIIIa in skeletal elements, resembling expression of their mammalian homologues in osteo-chondrogenic cells. Mammalian TG2 and FXIIIa have been implicated in promoting osteoblast differentiation and bone mineralization in vitro, however, mouse models lacking either gene have no skeletal phenotype likely due to a compensation effect. We show in this study that mineralization of the newly formed vertebrae is significantly reduced in fish grown for 5 days in the presence of TG inhibitor KCC-009 added at 3-5 days post fertilization. This treatment reduces average vertebrae mineralization by 30%, with complete inhibition in some fish, and no effect on the overall growth and vertebrae number. This is the first in vivo demonstration of the crucial requirement for the TG-catalyzed cross-linking activity in bone mineralization.
| S-EPMC3266987 | biostudies-literature