Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Our previous studies identified two iron-regulated cytoplasmic membrane proteins of 32 and 36 kDa expressed by both Staphylococcus epidermidis and Staphylococcus aureus. In this study we show by Triton X-114 phase partitioning and tritiated palmitic acid labelling that these proteins are lipoproteins which are anchored into the cytoplasmic membrane by their lipid-modified N termini. In common with those of some other gram-positive bacteria, these highly immunogenic lipoproteins were released from the bacterial cell into the culture supernatants, with release being promoted by growth of the bacteria under iron-restricted conditions. Immunoelectron microscopy with a monospecific rabbit antiserum to the 32-kDa S. epidermidis lipoprotein showed that the majority of the antigen was distributed throughout the staphylococcal cell wall. Only minor quantities were detected in the cytoplasmic membrane, and exposure of the lipoprotein on the bacterial surface was minimal. A monoclonal antibody raised to the 32-kDa lipoprotein of S. aureus was used in immunoblotting studies to investigate the conservation of this antigen among a variety of staphylococci. The monoclonal antibody reacted with polypeptides of 32 kDa in S. epidermidis and S. aureus and of 40 kDa in Staphylococcus hominis. No reactivity was detected with Staphylococcus lugdunensis, Staphylococcus cohni, or Staphylococcus haemolyticus. The gene encoding the 32-kDa lipoprotein from S. epidermidis has been isolated from a Lambda Zap II genomic DNA library and found to be a component of an iron-regulated operon encoding a novel ABC-type transporter. The operon contains three genes, designated sitA, -B, and -C, encoding an ATPase, a cytoplasmic membrane protein, and the 32-kDa lipoprotein, respectively. SitC shows significant homology both with a number of bacterial adhesins, including FimA of Streptococcus parasanguis and ScaA of Streptococcus gordonii, and with lipoproteins of a recently described family of ABC transporters with proven or putative metal ion transport functions. Although the solute specificity of this novel transporter has not yet been determined, we speculate that it may be involved in either siderophore- or transferrin-mediated iron uptake in S. epidermidis.
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PMID:Molecular cloning of a 32-kilodalton lipoprotein component of a novel iron-regulated Staphylococcus epidermidis ABC transporter. 967 60

The pH and trafficking of recycling endosomes have previously been studied using transferrin. We have used another approach, one in which the vesicle transport protein cellubrevin was appended with a luminal IgG epitope to allow targeting of fluorescein-5'-isothiocyanate (FITC)-labeled anti-IgG F(ab) antibodies to the recycling endosomes in living cells. FITC-F(ab) was specifically internalized by COS cells transfected with cellubrevin-Ig, which at steady state accumulated in a pericentriolar region similar to rhodamine-transferrin. Confocal microscopic analysis showed that endosome labeling by these two markers was heterogeneous. This differential distribution was not induced by the IgG tag, since endogenous Cb and Tf were also partitioned into separate endosomal populations. We used fluorescence ratio imaging of internalized FITC-F(ab) to measure the pH of cellubrevin-enriched recycling endosomes (pHCb) and FITC-transferrin to measure the pH of transferrin-enriched recycling endosomes (pHTf). In COS cells, cellubrevin endosomes (mean pHCb 6.1 +/- 0.05; range, 5.2-6.6) were more acidic than transferrin endosomes (mean pHTf 6.5 +/- 0.05; range, 5.6-7.2). Similar results were obtained in Chinese hamster ovary cells. Treatment with the vacuolar H+-ATPase inhibitor bafilomycin A1 caused pHTf to increase (DeltapHTf = 1.2 pH units) to a greater extent than pHCb (DeltapHCb = 0.5 pH units). Furthermore, inhibition of the Na+/K+-ATPase by ouabain or acetylstrophanthidin caused pHTf to decrease by 0.6 pH units but had no effect on pHCb. Based on the combination of these morphological and functional data, we suggest that the recycling endosomes are heterogeneous in their biochemical compositions, ion transport properties, and pH values.
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PMID:Cellubrevin-targeted fluorescence uncovers heterogeneity in the recycling endosomes. 967 89

Amyloidogenic processing of the beta-amyloid precursor protein (APP) has been implicated in the pathology of Alzheimer's disease. Because it has been suggested that catabolic processing of the APP holoprotein occurs in acidic intracellular compartments, we studied the effects of the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) and the H+-ATPase inhibitor bafilomycin A1 on APP catabolism in human embryonic kidney 293 cells expressing either wild-type or "Swedish" mutant APP. Unlike bafilomycin A1, which inhibits beta-amyloid production in cells expressing mutant but not wild-type APP, FCCP inhibited beta-amyloid production in both cell types. Moreover, the effects of FCCP were independent of alterations in total cellular APP levels or APP maturation, and the concentrations used did not alter either cellular ATP levels or cell viability. Bafilomycin A1, which had no effect on beta-amyloid production in wild-type cells, inhibited endocytosis of fluorescent transferrin, whereas concentrations of FCCP that inhibited beta-amyloid production in these cells had no effect on endosomal function. Thus, in wild-type-expressing cells it appears that the beta-amyloid peptide is not produced in the classically defined endosome. Although bafilomycin A1 decreased beta-amyloid release from cells expressing mutant APP but not wild-type APP, it altered lysosomal function in both cell types, suggesting that in normal cells beta-amyloid is not produced in the lysosome. Although inhibition of beta-amyloid production by bafilomycin A1 in mutant cells may occur via changes in endosomal/lysosomal pH, our data suggest that FCCP inhibits wild-type beta-amyloid production by acting on a bafilomycin A1-insensitive acidic compartment that is distinct from either the endosome or the lysosome.
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PMID:Novel effects of FCCP [carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone] on amyloid precursor protein processing. 1009 49

Histoplasma capsulatum (Hc) maintains a phagosomal pH of about 6.5. This strategy allows Hc to obtain iron from transferrin, and minimize the activity of macrophage (Mo) lysosomal hydrolases. To determine the mechanism of pH regulation, we evaluated the function of the vacuolar ATPase (V-ATPase) in RAW264.7 Mo infected with Hc yeast or the nonpathogenic yeast Saccharomyces cerevisae (Sc). Incubation of Hc-infected Mo with bafilomycin, an inhibitor of the V-ATPase, did not affect the intracellular growth of Hc, nor did it affect the intraphagosomal pH. In contrast, upon addition of bafilomycin, phagosomes containing Sc rapidly changed their pH from 5 to 7. Hc-containing phagosomes had 5-fold less V-ATPase than Sc-containing phagosomes as quantified by immunoelectron microscopy. Furthermore, Hc-containing phagosomes inhibited phagolysosomal fusion as quantified by the presence of acid phosphatase, accumulation of LAMP2, and fusion with rhodamine B-isothiocyanate-labeled dextran-loaded lysosomes. Finally, in Hc-containing phagosomes, uptake of ferritin was equivalent to phagosomes containing Sc, indicating that Hc-containing phagosomes have full access to the early "bulk flow" endocytic pathway. Thus, Hc yeasts inhibit phagolysosomal fusion, inhibit accumulation of the V-ATPase in the phagosome, and actively acidify the phagosomal pH to 6.5 as part of their strategy to survive in Mo phagosomes.
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PMID:Regulation of the macrophage vacuolar ATPase and phagosome-lysosome fusion by Histoplasma capsulatum. 1022 58

The mouse SKD1 is an AAA-type ATPase homologous to the yeast Vps4p implicated in transport from endosomes to the vacuole. To elucidate a possible role of SKD1 in mammalian endocytosis, we generated a mutant SKD1, harboring a mutation (E235Q) that is equivalent to the dominant negative mutation (E233Q) in Vps4p. Overexpression of the mutant SKD1 in cultured mammalian cells caused defect in uptake of transferrin and low-density lipoprotein. This was due to loss of their receptors from the cell surface. The decrease of the surface transferrin receptor (TfR) was correlated with expression levels of the mutant protein. The mutant protein displayed a perinuclear punctate distribution in contrast to a diffuse pattern of the wild-type SKD1. TfR, the lysosomal protein lamp-1, endocytosed dextran, and epidermal growth factor but not markers for the secretory pathway were accumulated in the mutant SKD1-localized compartments. Degradation of epidermal growth factor was inhibited. Electron microscopy revealed that the compartments were exaggerated multivesicular vacuoles with numerous tubulo-vesicular extensions containing TfR and endocytosed horseradish peroxidase. The early endosome antigen EEA1 was also redistributed to these aberrant membranes. Taken together, our findings suggest that SKD1 regulates morphology of endosomes and membrane traffic through them.
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PMID:The mouse SKD1, a homologue of yeast Vps4p, is required for normal endosomal trafficking and morphology in mammalian cells. 1067 28

Within the clathrin-coated vesicle (CCV) cycle, coat assembly drives the internalization of receptors from the cell surface and disassembly allows for the processing of internalized ligands. The heat shock cognate protein, hsc70, has been implicated in regulating coat disassembly. We find that in cells overexpressing ATPase-deficient hsc70 mutants, uncoating of CCVs is inhibited in vivo, and the majority of unassembled cytosolic clathrin shifts to an assembled pool that cofractionates with AP1 and AP2. Surprisingly, this assembled pool of coat proteins accumulates in the absence of cargo receptors, suggesting that disruption of hsc70 activity may cause misassembly of empty clathrin cages. The strongest effect of overexpression of hsc70 mutants is a block in transferrin receptor (TfnR) recycling, which cannot be accounted for by the degree of inhibition of uncoating of endocytic CCVs. These results suggest that hsc70 participates in multiple transport and/or sorting events between endosomal compartments. Additionally, the mutant-expressing cells are defective at internalizing transferrin. In the most potent case, the initial rate of uptake is inhibited 10-fold, and TfnR levels double at the cell surface. Our findings demonstrate that hsc70 indeed regulates coat disassembly and also suggest that this chaperone broadly modulates clathrin dynamics throughout the CCV cycle.
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PMID:Dominant-interfering Hsc70 mutants disrupt multiple stages of the clathrin-coated vesicle cycle in vivo. 1115 86

1. Receptor-mediated endocytosis in epithelial cells is a crucial mechanism for transport of macromolecules and regulation of cell-surface protein expression. Na+-H+ exchanger type 3 (NHE3) has been shown to cycle between the apical plasma membrane and the early endosomal compartment and to interfere with endocytosis. 2. In the present study we investigated in detail the NHE3-dependent step of apical endocytosis in an epithelial cell line (opossum kidney cells). 3. Inhibition of NHE3 led to a rapid dose-dependent inhibition of apical albumin endocytosis but did not affect basolateral transferrin endocytosis. Re-exocytosis of albumin was not increased by NHE3 inhibition. 4. NHE3 dependency of albumin endocytosis was still observed at 20 degrees C or when microtubules had been disrupted. This was not the case for inhibition of vacuolar H+-ATPase. 5. NHE3 inhibition rapidly blocked internalisation of pre-bound albumin and attenuated degradation of internalised albumin without changing general protein degradation. 6. Furthermore, NHE3 inhibition reduced the rate of endocytic vesicle fusion significantly. 7. In summary, our data indicate that NHE3 is important for the early phase of the apical endocytic pathway, located between the plasma membrane and early endosomes, at least in part due to its involvement in endocytic vesicle fusion.
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PMID:Inhibition of Na+-H+ exchanger-3 interferes with apical receptor-mediated endocytosis via vesicle fusion. 1125 Oct 45

Iron is an essential mineral for normal cellular physiology, but an excess can result in cell injury. Iron in low-molecular-weight forms may play a catalytic role in the initiation of free radical reactions. The resulting oxyradicals have the potential to damage cellular lipids, nucleic acids, proteins, and carbohydrates; the result is wide-ranging impairment in cellular function and integrity. The rate of free radical production must overwhelm the cytoprotective defenses of cells before injury occurs. There is substantial evidence that iron overload in experimental animals can result in oxidative damage to lipids in vivo, once the concentration of iron exceeds a threshold level. In the liver, this lipid peroxidation is associated with impairment of membrane-dependent functions of mitochondria and lysosomes. Iron overload impairs hepatic mitochondrial respiration primarily through a decrease in cytochrome C oxidase activity, and hepatocellular calcium homeostasis may be compromised through damage to mitochondrial and microsomal calcium sequestration. DNA has also been reported to be a target of iron-induced damage, and this may have consequences in regard to malignant transformation. Mitochondrial respiratory enzymes and plasma membrane enzymes such as sodium-potassium-adenosine triphosphatase (Na(+) + K(+)-ATPase) may be key targets of damage by non-transferrin-bound iron in cardiac myocytes. Levels of some antioxidants are decreased during iron overload, a finding suggestive of ongoing oxidative stress. Reduced cellular levels of ATP, lysosomal fragility, impaired cellular calcium homeostasis, and damage to DNA all may contribute to cellular injury in iron overload. Evidence is accumulating that free-radical production is increased in patients with iron overload. Iron-loaded patients have elevated plasma levels of thiobarbituric acid reactants and increased hepatic levels of aldehyde-protein adducts, indicating lipid peroxidation. Hepatic DNA of iron-loaded patients shows evidence of damage, including mutations of the tumor suppressor gene p53. Although phlebotomy therapy is effective in removing excess iron in hereditary hemochromatosis, chelation therapy is required in the treatment of many patients who have combined secondary and transfusional iron overload due to disorders in erythropoiesis. In patients with beta-thalassemia who undergo regular transfusions, deferoxamine treatment has been shown to be effective in preventing iron-induced tissue injury and in prolonging life expectancy. The use of the oral chelator deferiprone remains controversial, and work is continuing on the development of new orally effective iron chelators.
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PMID:Iron toxicity and chelation therapy. 1241 32

Fusion of enhanced green fluorescent protein (EGFP) to the C-terminal of rat Na,K-ATPase a1-subunit is introduced as a novel procedure for visualizing trafficking of Na,K-pumps in living COS-1 renal cells in response to PKA or PKC stimulation. Stable, functional expression of the fluorescent chimera (Na,K-EGFP) was achieved in COS-1 cells using combined puromycin and ouabain selection procedures. Na,K-pump activities were unchanged after fusion with EGFP, both in basal and regulated states. In confocal laser scanning and fluorescence microscopes, the Na,K-EGFP chimera was distributed mainly along the plasma membrane of COS cells. In unstimulated COS cells, Na,K-EGFP was also present in lysosomes and in vesicles en route from the endoplasmic reticulum to the plasma membrane, but it was almost absent from recycling endosomes labelled with fluorescent transferrin. After activation of protein kinase A or C, the density of co-localizing Na,K-EGFP and transferrin vesicles was increased 3-4-fold, while the ouabain-sensitive 86Rb uptake was reduced by 22%. Simultaneous activation of PKA and PKC had additive effects with a 6-fold increase of co-localization and a 38% reduction of 86Rb uptake. Responses of similar magnitude were seen after inhibition of protein phosphatase by okadaic acid. Reduction of the amount of Na,K-ATPase in surface plasma membranes through internalization in recycling endosomes may thus in part explain a decrease in Na,K-pump activity following protein kinase activation or protein phosphatase inhibition.
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PMID:Trafficking of Na,K-ATPase fused to enhanced green fluorescent protein is mediated by protein kinase A or C. 1253 74

Subosteoclastic bone resorption is a result of HCl and proteinase secretion through a late endosome-like bone facing membrane domain called ruffled border. As bone matrix is degraded, it enters osteoclasts' transcytotic vesicles for further processing and is then finally exocytosed to the intercellular space. The present study clarifies the spatial relationship between these vesicle fusion and matrix uptake processes at the ruffled border. Our results show the presence of vacuolar H+-ATPase, small GTPase rab7 as well as dense aggregates of F-actin at the peripheral ruffled border, where basolaterally endocytosed transferrin and cathepsin K are delivered. On the contrary, rhodamine-labeled bone matrix enters transcytotic vesicles at the central ruffled border, where the vesicle budding proteins such as clathrin, AP-2 and dynamin II are also localized. We present a model for the mechanism of ruffled border turnover and suggest that, due to its late endosomal characteristics, the ruffled border serves as a valuable model for studying the dynamic organization of other endosomal compartments as well.
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PMID:Osteoclast ruffled border has distinct subdomains for secretion and degraded matrix uptake. 1255 37


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