Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A neutral ceramidase homologue from Dictyostelium discoideum exhibits an acidic pH optimum.

ABSTRACT: The nucleotide sequence reported for the Dictyostelium discoideum ceramidase is available on the DNA Data Bank of Japan (DDBJ). Ceramidases (CDases) are currently classified into three categories (acid, neutral and alkaline) based on their optimal pHs and primary structures. Here, we report the first exception to this rule. We cloned the CDase cDNA, consisting of 2142 nucleotides encoding 714 amino-acid residues, from the slime mould, Dictyostelium discoideum. The putative amino-acid sequence indicates 32-42% identity with various neutral CDases, but does not show any similarity to the acid and alkaline CDases, indicating the enzyme should be classified as a neutral CDase. However, overexpression of the cDNA in D. discoideum resulted in increased CDase activity at an acidic, but not a neutral pH range. Knockout of the gene in slime mould eliminated CDase activity at acidic pH. The recombinant enzyme expressed in the slime mould was purified and then characterized. Consequently, the purified CDase was found to exhibit the maximal activity at approx. pH 3.0. The singular pH dependency of slime mould CDase is not derived from the specific post-translational modification in the slime mould, because the enzyme showed an acidic pH optimum even when expressed in Chinese hamster ovary cells, whereas rat neutral-CDase exhibited a neutral pH optimum when expressed in slime mould.

SUBMITTER: Monjusho H

PROVIDER: S-EPMC1223776 | biostudies-other | 2003 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Disruption of a dynamin homologue affects endocytosis, organelle morphology, and cytokinesis in Dictyostelium discoideum.

Project description:The identification and functional characterization of Dictyostelium discoideum dynamin A, a protein composed of 853 amino acids that shares up to 44% sequence identity with other dynamin-related proteins, is described. Dynamin A is present during all stages of D. discoideum development and is found predominantly in the cytosolic fraction and in association with endosomal and postlysosomal vacuoles. Overexpression of the protein has no adverse effect on the cells, whereas depletion of dynamin A by gene-targeting techniques leads to multiple and complex phenotypic changes. Cells lacking a functional copy of dymA show alterations of mitochondrial, nuclear, and endosomal morphology and a defect in fluid-phase uptake. They also become multinucleated due to a failure to complete normal cytokinesis. These pleiotropic effects of dynamin A depletion can be rescued by complementation with the cloned gene. Morphological studies using cells producing green fluorescent protein-dynamin A revealed that dynamin A associates with punctate cytoplasmic vesicles. Double labeling with vacuolin, a marker of a postlysosomal compartment in D. discoideum, showed an almost complete colocalization of vacuolin and dynamin A. Our results suggest that that dynamin A is likely to function in membrane trafficking processes along the endo-lysosomal pathway of D. discoideum but not at the plasma membrane.

| S-EPMC25165 | biostudies-literature

Mef2A, a homologue of animal Mef2 transcription factors, regulates cell differentiation in Dictyostelium discoideum.

Project description:BackgroundTranscription factors from the MADS-box family play a relevant role in cell differentiation and development and include the animal SRF (serum response factor) and MEF2 (myocyte enhancer factor 2) proteins. The social amoeba Dictyostelium discoideum contains four genes coding for MADS-box transcription factors, two of these genes code for proteins that are more similar to SRF, and the other two code for proteins that are more similar to MEF2 animal factors.ResultsThe biological function of one of the two genes that codes for MEF2-related proteins, a gene known as mef2A, is described in this article. This gene is expressed under the transcriptional control of two alternative promoters in growing cells, and its expression is induced during development in prespore cells. Mutant strains where the mef2A gene has been partially deleted were generated to study its biological function. The mutant strains showed reduced growth when feeding on bacteria and were able to develop and form fruiting bodies, but spore production was significantly reduced. A study of developmental markers showed that prespore cells differentiation was impaired in the mutant strains. When mutant and wild-type cells were set to develop in chimeras, mutant spores were underrepresented in the fruiting bodies. The mutant cells were also unable to form spores in vitro. In addition, mutant cells also showed a poor contribution to the formation of the tip-organizer and the upper region of slugs and culminant structures. In agreement with these observations, a comparison of the genes transcribed by mutant and wild-type strains during development indicated that prestalk gene expression was enhanced, while prespore gene expression decreased in the mef2A- strain.ConclusionsOur data shows that mef2A plays a role in cell differentiation in D. discoideum and modulates the expression of prespore and prestalk genes.

| S-EPMC3640940 | biostudies-literature

The human homologue of Dictyostelium discoideum phg1A is expressed by human metastatic melanoma cells.

Project description:Tumour cannibalism is a characteristic of malignancy and metastatic behaviour. This atypical phagocytic activity is a crucial survival option for tumours in conditions of low nutrient supply, and has some similarities to the phagocytic activity of unicellular microorganisms. In fact, Dictyostelium discoideum has been used widely as a model to study phagocytosis. Recently, phg1A has been described as a protein that is primarily involved in the phagocytic process of this microorganism. The closest human homologue to phg1A is transmembrane 9 superfamily protein member 4 (TM9SF4). Here, we report that TM9SF4 is highly expressed in human malignant melanoma cells deriving from metastatic lesions, whereas it is undetectable in healthy human tissues and cells. TM9SF4 is predominantly expressed in acidic vesicles of melanoma cells, in which it co-localizes with the early endosome antigens Rab5 and early endosome antigen 1. TM9SF4 silencing induced marked inhibition of cannibal activity, which is consistent with a derangement of intracellular pH gradients, with alkalinization of acidic vesicles and acidification of the cell cytosol. We propose TM9SF4 as a new marker of malignancy, representing a potential new target for anti-tumour strategies with a specific role in tumour cannibalism and in the establishment of a metastatic phenotype.

| S-EPMC2799214 | biostudies-literature

Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA).

Project description:Although the DNA methyltransferase 2 family is highly conserved during evolution and recent reports suggested a dual specificity with stronger activity on transfer RNA (tRNA) than DNA substrates, the biological function is still obscure. We show that the Dictyostelium discoideum Dnmt2-homologue DnmA is an active tRNA methyltransferase that modifies C38 in tRNA(Asp(GUC)) in vitro and in vivo. By an ultraviolet-crosslinking and immunoprecipitation approach, we identified further DnmA targets. This revealed specific tRNA fragments bound by the enzyme and identified tRNA(Glu(CUC/UUC)) and tRNA(Gly(GCC)) as new but weaker substrates for both human Dnmt2 and DnmA in vitro but apparently not in vivo. Dnmt2 enzymes form transient covalent complexes with their substrates. The dynamics of complex formation and complex resolution reflect methylation efficiency in vitro. Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress. However, dnmA expression only partially correlated with tRNA methylation in vivo. Strikingly, dnmA expression in the laboratory strain AX2 was significantly lower than in the NC4 parent strain. As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation.

| S-EPMC3794594 | biostudies-literature

Ocular Effects of Glycyrrhizin at Acidic and Neutral pH.

Project description:PurposeTo test the effects of acidic vs. neutral pH glycyrrhizin (GLY) on the unwounded and wounded normal mouse cornea and after infection with Pseudomonas aeruginosa isolates KEI 1025 and multidrug-resistant MDR9.MethodsAcidic or neutral GLY vs. phosphate-buffered saline (PBS) was topically applied to normal or wounded corneas of C57BL/6 mice. In unwounded corneas, goblet cells and corneal nerves were stained and quantitated. After wounding, corneas were fluorescein stained and photographed using a slit lamp. Mice also were infected with KEI 1025 or MDR9 and the protective effects of GLY pH evaluated comparatively.ResultsIn the unwounded cornea, application of acidic or neutral GLY vs. PBS reduced the number of bulbar conjunctival goblet cells but did not alter corneal nerve density. Similar application of GLY to scarified corneas delayed wound closure. After KEI 1025 infection, none of the GLY vs. PBS-treated corneas perforated; GLY treatment also decreased plate count (neutral pH more effective) and reduced MPO and several cytokines. Similarly, for MDR9, GLY at either pH was protective and also enhanced the effects of moxifloxacin to which MDR9 is resistant.ConclusionAcidic or neutral pH GLY decreased goblet cell number but had no effect on nerve density. After corneal wounding, GLY at either pH (1) delayed wound closure and, (2) after infection, decreased keratitis when used alone or in combination with moxifloxacin. Neutral pH did not alter the therapeutic effect of GLY and would be preferred if used clinically.

| S-EPMC8814321 | biostudies-literature

Dictyostelium discoideum protein phosphatase-1 catalytic subunit exhibits distinct biochemical properties.

Project description:Protein phosphatase-1 (PP1) is expressed ubiquitously and is involved in many eukaryotic cellular functions, although PP1 enzyme activity could not be detected in the social amoeba Dictyostelium discoideum cell extracts. In the present paper, we show that D. discoideum has a single copy gene that codes for the catalytic subunit of PP1 (DdPP1c). DdPP1c is expressed throughout the D. discoideum life cycle with constant levels of mRNA, and its protein and amino acid sequence show a mean identity of 80% with other PP1c enzymes. However, it has a distinctive difference: the substitution of a phenylalanine residue (Phe(269) in the DdPP1c) for a highly conserved cysteine residue (Cys(273) in rabbit PP1c) in a region that was shown to have a critical role in the interaction of rabbit PP1c with toxin inhibitors. Wild-type DdPP1c and an engineered mutant form in which Phe(269) was replaced by a cysteine residue were expressed in Escherichia coli. Both recombinant activities were similarly inhibited by okadaic acid, tautomycin and microcystin. However, the Phe(269)-->Cys mutation resulted in a large increase in enzyme activity towards phosphorylase a and a higher sensitivity to calyculin A. These results, together with the molecular modelling of DdPP1c structure, indicate that the Phe(269) residue, which occurs naturally in D. discoideum, confers distinct biochemical properties on this enzyme.

| S-EPMC1223547 | biostudies-other

Developmental commitment in Dictyostelium discoideum.

Project description:Upon starvation, Dictyostelium discoideum cells halt cell proliferation, aggregate into multicellular organisms, form migrating slugs, and undergo morphogenesis into fruiting bodies while differentiating into dormant spores and dead stalk cells. At almost any developmental stage cells can be forced to dedifferentiate when they are dispersed and diluted into nutrient broth. However, migrating slugs can traverse lawns of bacteria for days without dedifferentiating, ignoring abundant nutrients and continuing development. We now show that developing Dictyostelium cells revert to the growth phase only when bacteria are supplied during the first 4 to 6 h of development but that after this time, cells continue to develop regardless of the presence of food. We postulate that the cells' inability to revert to the growth phase after 6 h represents a commitment to development. We show that the onset of commitment correlates with the cells' loss of phagocytic function. By examining mutant strains, we also show that commitment requires extracellular cyclic AMP (cAMP) signaling. Moreover, cAMP pulses are sufficient to induce both commitment and the loss of phagocytosis in starving cells, whereas starvation alone is insufficient. Finally, we show that the inhibition of development by food prior to commitment is independent of contact between the cells and the bacteria and that small soluble molecules, probably amino acids, inhibit development during the first few hours and subsequently the cells become unable to react to the molecules and commit to development. We propose that commitment serves as a checkpoint that ensures the completion of cooperative aggregation of developing Dictyostelium cells once it has begun, dampening the response to nutritional cues that might inappropriately block development.

| S-EPMC2168402 | biostudies-literature