Cystine: Compartmentalization within Lysosomes in Cystinotic Leukocytes

Schulman, J D; Bradley, Kathryn H.; Seegmiller, J. Edwin

doi:10.1126/science.166.3909.1152

Cited by 102 publications

(37 citation statements)

References 12 publications

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“…6,7 Like cystinotic fibroblasts, which actively generate but poorly retain (approximately 1%) free soluble cysteine continuously discharged by exocytosis, [10][11][12] PTCs in Ctns 2/2 kidneys incompletely retain cystine generated in lysosomes from recaptured albumin (calculations not shown). In various noncrystalline lysosome storage diseases, lysosomal expansion impacts gene expression by the transcription factor, transcription factor EB, 37 which triggers lysosomal discharge.…”

Section: Ctnsmentioning

confidence: 99%

“…6 Furthermore, by electron microscopy, enlarged acidphosphatase-labeled structures enclose an amorphous matrix without systematic association with crystals. 7 Cystinotic fibroblasts show enlarged lysosomes without acidification defect 8 and accumulate cystine on degradation of endocytosed disulfiderich proteins, such as albumin, in proportion to extracellular concentration.…”

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confidence: 99%

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Time Course of Pathogenic and Adaptation Mechanisms in Cystinotic Mouse Kidneys

Chevronnay

Janssens

Smissen

et al. 2014

Journal of the American Society of Nephrology

126

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Cystinosis, a main cause of Fanconi syndrome, is reproduced in congenic C57BL/6 cystinosin knockout (KO) mice. To identify the sequence of pathogenic and adaptation mechanisms of nephropathic cystinosis, we defined the onset of Fanconi syndrome in KO mice between 3 and 6 months of age and analyzed the correlation with structural and functional changes in proximal tubular cells (PTCs), with focus on endocytosis of ultrafiltrated disulfide-rich proteins as a key source of cystine. Despite considerable variation between mice at the same age, typical event sequences were delineated. At the cellular level, amorphous lysosomal inclusions preceded cystine crystals and eventual atrophy without crystals. At the nephron level, lesions started at the glomerulotubular junction and then extended distally. In situ hybridization and immunofluorescence revealed progressive loss of expression of megalin, cubilin, sodiumglucose cotransporter 2, and type IIa sodium-dependent phosphate cotransporter, suggesting apical dedifferentiation accounting for Fanconi syndrome before atrophy. Injection of labeled proteins revealed that defective endocytosis in S1 PTCs led to partial compensatory uptake by S3 PTCs, suggesting displacement of endocytic load and injury by disulfide-rich cargo. Increased PTC apoptosis allowed luminal shedding of cystine crystals and was partially compensated for by tubular proliferation. We conclude that lysosomal storage triggered by soluble cystine accumulation induces apical PTC dedifferentiation, which causes transfer of the harmful load of disulfide-rich proteins to more distal cells, possibly explaining longitudinal progression of swan-neck lesions. Furthermore, our results suggest that subsequent adaptation mechanisms include lysosomal clearance of free and crystalline cystine into urine and ongoing tissue repair.

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Section: Ctnsmentioning

confidence: 99%

mentioning

confidence: 99%

Time Course of Pathogenic and Adaptation Mechanisms in Cystinotic Mouse Kidneys

Chevronnay

Janssens

Smissen

et al. 2014

Journal of the American Society of Nephrology

126

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“…Although lysosomal accumulation of cystine in cystinosis was demonstrated already in the 1960s (5), studies on the pathogenesis of this disease were initially hampered by the absence of a proper in vitro model of lysosomal cystine accumulation. This issue was solved with the introduction of dimethyl esters of amino acids, which readily passed the lysosomal membrane and, once inside the organelle, were rapidly degraded by lysosomal hydrolases to yield free amino acid and methanol thus allowing amino acid loading of the lysosomes (6,7).…”

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Decreased Intracellular ATP Content and Intact Mitochondrial Energy Generating Capacity in Human Cystinotic Fibroblasts

et al. 2006

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ABSTRACT: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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“…Cystinotic cells have normal reduced glutathione content (27,34). Because cysteamine supplied exogenously can reduce cystinotic intracellular cystine levels to near normal (42), the possibility that cystinosis might be due to a defect in endogenous MEA generation needed critical evaluation.…”

Section: Discussionmentioning

confidence: 99%

Pantetheinase Activity and Cysteamine Content in Cystinotic and Normal Fibroblasts and Leukocytes

Orloff¹,

Butler²,

Towne³

et al. 1981

Pediatr Res

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Summaryas a hydrogen carrier between cytoplasmic-reduced glutathione (which appears to penetrate membranes poorly) and &tralysoso-Cysteamine is the most effective agent known for the reduction mal cystine. A genetic deficiency in endogenous cystearnine genof the elevated cystine content of cells from patients with cysti-eration might therefore lead to diminished capacity for intralysonosis. A defect in endogenous cysteamine generation could ac-somal reduction of cysthe to cysteke, a molecule to which the count for many of the metabolic features of this disorder. To test lysosomal membrane should be permeable (33) and hence could this we have for measur-be the fundamental etiology of abnormal cysthe accumulation in ing pantetheinase (cysteamine-generating) activity and intracellu-cystinosis. lar cysteamine levels and used these methods to measure such Cystearnine ( (4,6,8,9,(14)(15)(16)(17). 9.4 f 1.5; cystinotic, 7.7 2 1.71. Cysteamine levels were normal in There is some evidence that pantetheinase is present in both leukocytes from c~stinotics receiving no 'ysteamine Or doses of soluble and lysosomal-microsomal cell fractions. This enzyme has oral cysteamine too low to reduce leukocyte cystine content. The not previously been described in human cells or tissues. results indicate that the cause of cystinosis is unlikely to be related Pantetheinase activity has been determined by several methods. to a failure to generate or sustain normal intracellular cysteamine levels.One involves quantitation of pantothenic acid (PoA), an end product of PTSH cleavage, using a microbiological assay in which PoA is a necessary growth factor (e.g., using Lactobacillus arabiSpeculation nosus) (2, 5, 9, 10, 39). The radiochemical assay of Dupre et al. (6, cystearnine is an extremely effective cystine depleting agent for 14-17) utilizes labeled PTSH as substrate and measures radioaccystinotic fibroblasts in vitro and can greatly reduce cystinotic tive product after PTSH. A pHleukocyte cystine content in vivo. Its pharmacologic properties Stat has been proposed (l l). The first is time suggest that it might prove to be of value in the therapy of consuming and proved erratic in our hands, whereas the radicystinosis. However, we do not believe that a defect in endogenous Ochemical assay requires an radiolabeled substrate cysteamine generation is a characteristic of cystinotic cells. The which must be We therefore a eventual elucidation of the cystinotic defect may require analysis system for low levels pantetheinase of the permeability characteristics of cystinotic ~ysosomes or the activity in small cell samples; our assay involves rapid quantitation discovery of presently unidentified pathways for lysosomal metab-MEA On an acid after generation PTSH olism of cystine.and reaction with N-ethylmaleimide (NEM). The present report describes our use of this new method to determine panthetheinase activity and define certain properties of Cystinosis, a metabolic disease inherited as an autosomal Feces-this enzyme in extracts of cultured skin ...

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Cystine: Compartmentalization within Lysosomes in Cystinotic Leukocytes

Abstract: The large amount of cystine compartmentalized in cystinotic leukocytes cosediments in isopycnic sucrose density gradients with dense lysosomal particles, within which it is presumably contained. Such cystine appears to be primarily noncrystalline in these organelles.

Cited by 102 publications

References 12 publications

Time Course of Pathogenic and Adaptation Mechanisms in Cystinotic Mouse Kidneys

Time Course of Pathogenic and Adaptation Mechanisms in Cystinotic Mouse Kidneys

Decreased Intracellular ATP Content and Intact Mitochondrial Energy Generating Capacity in Human Cystinotic Fibroblasts

Pantetheinase Activity and Cysteamine Content in Cystinotic and Normal Fibroblasts and Leukocytes

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