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)

Cystinosis is an inherited metabolic disease characterized by accumulation of lysosomal cystine and renal impairment. In an attempt to better understand the link between cystine accumulation and renal functions, we studied the effects of cystine loading on the Na(+)-H+ antiporter and the sodium pump in renal epithelial cells (LLC-PK1) in culture. Incubation of LLC-PK1 with 1 mM cystine dimethyl ester (CDME) for 48 h caused lysosomal cystine loading and reduced by 22 +/- 2% the maximal velocity of sodium-hydrogen antiport with no significant change in the affinity of sodium for the transporter. Rubidium influx decreased to 46 +/- 5% of control. Ouabain binding experiments revealed a 10% reduction in the number of Na(+)-K(+)-ATPase units in the intact cells. Na(+)-K(+)-ATPase activity in the particulate fraction of the cells homogenate declined to 50 +/- 7.5% of controls. No significant change was observed in the activity of ouabain-insensitive phosphatases. The intracellular concentration of sodium increased from 20.6 +/- 3.7 to 64.8 +/- 10 mM, and potassium concentration decreased from 103 +/- 6 to 80 +/- 13 mM. In addition to the observed reduction in the sodium gradient and in agreement with the reduction in the intracellular potassium concentration, the membrane potential changed from -80.8 +/- 7.5 to -69.9 +/- 7.0 mV. The results suggest that intracellular accumulation of cystine is associated with reduction in the number and the activity of membrane transporters. The consequence of the changes in the activity of Na(+)-K(+)-ATPase is a reduction in the electrochemical forces that drive transport in the renal cells tested.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cystine dimethyl ester reduces the forces driving sodium-dependent transport in LLC-PK1 cells. 132 21

Cellular cystine loading with cystine dimethyl ester inhibits volume absorption, transepithelial potential difference, glucose transport, and bicarbonate transport in proximal convoluted tubules perfused in vitro. This study examined the roles of ATP and NaK ATPase in this in vitro model of the Fanconi syndrome of cystinosis. Intracellular ATP was measured using the luciferin-luciferase assay. Intracellular ATP was reduced by 60% in proximal convoluted tubules incubated with 0.5 mM cystine dimethyl ester for 15 min at 37 degrees C (P less than 0.001). Incubation of cystine loaded tubules with 1 mM exogenous ATP increased intracellular ATP to levels not significantly different than that of controls. On the other hand, Vmax NaK ATPase activity was unchanged even though the incubation times and the concentration of cystine dimethyl ester were doubled to 30 min and 1 mM, respectively. In proximal convoluted tubules perfused in vitro, 0.5 mM cystine dimethyl ester resulted in an 89% inhibition in volume absorption (0.81 +/- 0.14 to 0.09 +/- 0.09 nl/mm.min), while there was only a 45% inhibition in volume absorption (P less than 0.01) due to cellular cystine loading in the presence of 1 mM lumen and bath ATP (0.94 +/- 0.05 to 0.52 +/- 0.11 nl/mm.min). These data demonstrate that proximal tubule cellular cystine loading decreases cellular ATP concentration, but does not directly inhibit NaK ATPase activity. The inhibition in transport and decrease in intracellular ATP due to cellular cystine loading was ameliorated by exogenous ATP. These data are consistent with cellular ATP depletion playing a major role in the inhibition of proximal tubule transport due to intracellular cystine loading.
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PMID:Role of adenosine triphosphate (ATP) and NaK ATPase in the inhibition of proximal tubule transport with intracellular cystine loading. 184 41

Lysosomes containing large amounts of the amino acid, cystine, were obtained from transformed, cultured, human lymphoblasts which had been exposed to cystine dimethyl ester. Lysosomal cystine efflux was greatly enhanced by exogenous ATP in cell lines from normal individuals. Cystine efflux was unresponsive to ATP in lysosomes from individuals with the disorder, cystinosis. Efflux of cystine from normal cell lysosomes was inhibited by both the ATP analog, 5-adenylylimidodiphosphate, and the proton translocator, carbonyl cyanide m-chlorophenylhydrazone. Efflux was not affected by ouabain or oligomycin. Thus, lysosomal cystine efflux is dependent upon the functioning of a proton-pump ATPase. ATPase-dependent cystine efflux appears to be aberrant in cystinotic cell lysosomes.
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PMID:ATP-dependent lysosomal cystine efflux is defective in cystinosis. 629 78

The cause of Fanconi syndrome in cystinosis is enigmatic. It has previously been shown that renal tubules could be loaded with cystine by incubating them with cystine dimethylester (CDE), mimicking the biochemical hallmark of cystinosis. Such tubules have impaired transport, decreased whole-cell O2 consumption, and substrate utilization. In this study, the metabolic disturbances in cystine-loaded renal tubule cells were further characterized. Isolated rat renal tubules were loaded with cystine by incubating them with 2 mM CDE for 10 min. This had no significant effect on total ATPase, Na(+)-K(+)-ATPase, or the ouabain-insensitive ATPase activity of renal tissue homogenates from these cystine-loaded tubules. Intracellular K was significantly lower in the cystine-loaded tubules (37 +/- 2 versus 47 +/- 3 nEq/mg; P < 0.008). Intracellular ATP was reduced by 39% in the cystine-loaded tubules (23.7 +/- 2.4 versus 38.1 +/- 3.3 nmol/mg of protein; P < 0.0025). CDE (2 mM) reduced isolated mitochondrial O2 consumption with glutamate as the substrate by 66% (4.7 +/- 0.7 versus 13.9 +/- 0.8 nm/min per mg of protein, P < 0.001) but had no effect on mitochondrial O2 consumption with succinate as the substrate. It was speculated that the impaired transport from cystine loading with CDE is secondary to a decrease in energy generation.
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PMID:Metabolic studies of rat renal tubule cells loaded with cystine: the cystine dimethylester model of cystinosis. 757 95

To better understand the link between lysosomal cystine accumulation and the renal impairment seen in cystinosis, we have studied the effect of cystine loading in vivo, on renal function of rats, and in brush-border membrane vesicles (BBMV) prepared from the kidney cortex of the treated rats. Intraperitoneal injection of cystine dimethyl ester (CDME) (400 mumol, twice a day, for 5 days) led to an increased urine volume and excretion of glucose, phosphate, and protein. Kinetic analysis of alpha-methylglucoside initial flux in BBMV showed reduction in maximal transport capacity (Vmax, from 10.1 +/- 1.3 to 8.5 +/- 0.7 nmol.min-1.mg protein-1; P < 0.01) with no change in Michaelis constant (Km, 4.80 +/- 0.08 and 4.90 +/- 0.05 mM). The number of phlorizin binding sites declined (from 6.5 +/- 0.7 to 4.1 +/- 0.4 pmol/mg protein; P < 0.01) with no significant change in the affinity for phlorizin (0.64 +/- 0.08 and 0.59 +/- 0.06 microM). In the cortex homogenate, cystine concentration, which was undetectable in controls, increased to 0.97 +/- 0.09 nmol 1/2 cystine/mg protein. Two hours after CDME administration, ATP content declined to approximately 50% of control values. This decline was transient, and ATP content was recovered to control values 5 h after CDME administration. The treatment did not affect ouabain-sensitive adenosinetriphosphatase activity (40.0 +/- 3.9 and 38.6 +/- 4.7 nmol Pi.mg protein-1.min-1) or the number and affinity of ouabain binding sites (Bmax = 1.48 +/- 0.25 and 1.44 +/- 0.18 pmol/mg, and Kd = 0.68 +/- 0.09 and 0.72 +/- 0.12 microM, respectively). (ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cystine loading induces Fanconi's syndrome in rats: in vivo and vesicle studies. 828 17

Cystinosis is a lysosomal storage disease which is the most-common inherited cause of the Fanconi syndrome. Insights into the pathophysiology of the proximal tubular defect have come from in vitro studies of the cystine-loaded tubule. Proximal tubules loaded with cystine have a generalized proximal tubule transport defect characteristic of the Fanconi syndrome. The decrease in proximal tubular transport with cystine loading is not due to an increase in paracellular permeability with backflux of solute transport from the blood to the tubular lumen, but due to a decrease in active transport. The Na-K-ATPase activity is intact under Vmax conditions in cystine-loaded tubules; however, the production of ATP is severely compromised. The cystine-loaded tubule has a lower intracellular phosphate concentration than that of control tubules. This low intracellular phosphate concentration in cystine-loaded tubules likely plays a critical role in the decrease in intracellular ATP. Preservation of intracellular phosphate at control levels prevents the decrease in intracellular ATP and the proximal tubule respiratory dysfunction with cystine loading. The clinical significance and future directions for investigation are discussed.
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PMID:The Fanconi syndrome of cystinosis: insights into the pathophysiology. 974 76

Cystinosis is an autosomal recessive lysosomal storage disorder caused by a defect in the lysosomal cystine carrier cystinosin. Cystinosis is the most common cause of inherited Fanconi syndrome leading to renal failure, in which the pathogenesis is still enigmatic. Based on studies of proximal tubules loaded with cystine dimethyl ester (CDME), altered mitochondrial adenosine triphosphate (ATP) production was proposed to be an underlying pathologic mechanism. Thus far, however, experimental evidence supporting this hypothesis in humans is lacking. In this study, energy metabolism was extensively investigated in primary fibroblasts derived from eight healthy subjects and eight patients with cystinosis. Patient's fibroblasts accumulated marked amounts of cystine and displayed a significant decrease in intracellular ATP content. Remarkably, overall energy-generating capacity, activity of respiratory chain complexes, ouabain-dependent rubidium uptake reflecting Na,K-ATPase activity, and bradykinin-stimulated mitochondrial ATP production were all normal in these cells. In conclusion, the data presented demonstrate that mitochondrial energy-generating capacity and Na,K-ATPase activity are intact in cultured cystinotic fibroblasts, thus questioning the idea of altered mitochondrial ATP synthesis as a keystone for the pathogenesis of cystinosis.
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PMID:Decreased intracellular ATP content and intact mitochondrial energy generating capacity in human cystinotic fibroblasts. 1643 94

Alterations in ATP metabolism have been proposed to be involved in the pathogenesis of cystinosis, the most common form of inherited Fanconi syndrome. A recent study showed normal activity of respiratory chain complexes I-IV with decreased ATP levels in cystinotic fibroblasts. Here, we show normal complex V expression and activity in mitochondria of cystinotic fibroblasts. This indicates that alterations in mitochondrial oxidative phosphorylation enzymes are not responsible for ATP decrease in cystinotic fibroblasts.
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PMID:Mitochondrial complex V expression and activity in cystinotic fibroblasts. 1859 76

Lysosomal membrane proteins act at several crucial steps of the lysosome life cycle, including lumen acidification, metabolite export, molecular motor recruitment and fusion with other organelles. This review summarizes the molecular mechanisms of lysosomal storage diseases caused by defective transport of small molecules or ions across the lysosomal membrane, as well as Danon disease. In cystinosis and free sialic acid storage diseases, transporters for cystine and acidic monosaccharides, respectively, are blocked or retarded. A putative cobalamin transporter and a hybrid transporter/transferase of acetyl groups are defective in cobalamin F type disease and mucopolysaccharidosis type IIIC, respectively. In neurodegenerative forms of osteopetrosis, mutations of a proton/chloride exchanger impair the charge balance required for sustained proton pumping by the V-type ATPase, thus resulting in bone-resorption lacuna neutralization. However, the mechanism leading to lysosomal storage and neurodegeneration remains unclear. Mucolipidosis type IV is caused by mutations of a lysosomal cation channel named TRPML1; its gating properties are still poorly understood and the ion species linking this channel to lipid storage and membrane traffic defects is debated. Finally, the autophagy defect of Danon disease apparently arises from a role of LAMP2 in lysosome/autophagosome fusion, possibly secondary to a role in dynein-based centripetal motility.
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PMID:Molecular and cellular basis of lysosomal transmembrane protein dysfunction. 1914 88

Cystinosis is a rare autosomal recessive storage disorder characterized by defective lysosomal efflux of cystine due to mutations in the CTNS gene encoding the lysosomal cystine transporter, cystinosin. Lysosomal cystine accumulation leads to crystal formation and functional impairment of multiple organs. Moreover, cystinosis is the most common inherited cause of renal Fanconi syndrome in children. Oral cysteamine therapy delays disease progression by reducing intracellular cystine levels. However, because cysteamine does not correct all complications of cystinosis, including Fanconi syndrome, we hypothesized that cystinosin could have novel roles in addition to transporting cystine out of the lysosome. By coimmunoprecipitation experiments and mass spectrometry, we found cystinosin interacts with almost all components of vacuolar H(+)-ATPase and the Ragulator complex and with the small GTPases Ras-related GTP-binding protein A (RagA) and RagC. Furthermore, the mammalian target of rapamycin complex 1 (mTORC1) pathway was downregulated in proximal tubular cell lines derived from Ctns(-/-) mice. Decrease of lysosomal cystine levels by cysteamine did not rescue mTORC1 activation in these cells, suggesting that the downregulation of mTORC1 is due to the absence of cystinosin rather than to the accumulation of cystine. Our results show a dual role for cystinosin as a cystine transporter and as a component of the mTORC1 pathway, and provide an explanation for the appearance of Fanconi syndrome in cystinosis. Furthermore, this study highlights the need to develop new treatments not dependent on lysosomal cystine depletion alone for this devastating disease.
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PMID:Cystinosin is a Component of the Vacuolar H+-ATPase-Ragulator-Rag Complex Controlling Mammalian Target of Rapamycin Complex 1 Signaling. 2644 7


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