Endolysosomal transport of newly-synthesized cathepsin D in a sucrose model of lysosomal storage

Hamer, Isabelle; Jadot, Michel

doi:10.1016/j.yexcr.2005.06.006

Cited by 8 publications

(9 citation statements)

References 34 publications

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“…This is a strong argument in favor of its lysosomal localization. The full‐length Hyal‐1 form remained enriched in fraction 3 (Figure D, closed arrow) as in control cells (Figure B, closed arrow); its increase of abundance likely the consequence of a retarded maturation in the presence of sucrose .…”

Section: Resultsmentioning

confidence: 84%

“…Nevertheless, because co‐distribution does not always equal co‐localization, we decided to test the lysosomal localization of Hyal‐1 by inducing a specific decrease of the lysosome density with sucrose, prior to fractionation of the cells on a Percoll TM density gradient [i.e. endocytosed sucrose induces the osmotic swelling of lysosomes, resulting in a decrease of their density ]. Interestingly, the activity of the cleaved Hyal‐1 form that was detected in fraction 7 of the untreated cells (Figure B, open arrow) shifted to fractions 2–3 in the sucrose treated cells, similarly to β ‐galactosidase (Figure C and D, open arrow).…”

Section: Resultsmentioning

confidence: 99%

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Subcellular Trafficking and Activity of Hyal‐1 and Its Processed Forms in Murine Macrophages

Puissant

Gilis

Dogné

et al. 2014

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The hyaluronidase Hyal-1 is an acid hydrolase that degrades hyaluronic acid (HA), a component of the extracellular matrix. It is often designated as a lysosomal protein. Yet few data are available on its intracellular localization and trafficking. We demonstrate here that in RAW264.7 murine macrophages, Hyal-1 is synthesized as a glycosylated precursor that is only weakly mannose 6-phosphorylated. Nevertheless, this precursor traffics to endosomes, via a mannose 6-phosphate-independent secretion/recapture mechanism that involves the mannose receptor.Once in endosomes, it is processed into a lower molecular mass form that is transported to lysosomes, where its activity could be detected using native gel zymography. Indeed, this activity co-distributed with lysosomal hydrolases in the densest fraction of a self-forming Percoll TM density gradient. Moreover, it shifted toward the lower density region, in parallel with those hydrolases, when a decrease of lysosomal density was induced by the endocytosis of sucrose. Interestingly, the activity of the processed form of Hyal-1 was largely underestimated when assayed by zymography after SDS-PAGE and subsequent renaturation of the proteins, by contrast to the full-length protein that could efficiently degrade HA in those conditions. These results suggest that noncovalent associations support the lysosomal activity of Hyal-1.

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Section: Resultsmentioning

confidence: 84%

Section: Resultsmentioning

confidence: 99%

Subcellular Trafficking and Activity of Hyal‐1 and Its Processed Forms in Murine Macrophages

Puissant

Gilis

Dogné

et al. 2014

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“…To this purpose, we used a simple cellular model of lysosomal accumulation represented by human fibroblasts subjected to sucrose loading for 14 d (43)(44)(45)(46)(47)(48). Sucrose is not degraded in fibroblasts because of the lack of invertase and does not induce osmotic intracellular stress at 88 mM, the concentration we used (44,48).…”

Section: Discussionmentioning

confidence: 99%

A lysosome‐plasma membrane‐sphingolipid axis linking lysosomal storage to cell growth arrest

Samarani

Loberto²,

Soldà

et al. 2018

FASEB j.

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Lysosomal accumulation of undegraded materials is a common feature of lysosomal storage diseases, neurodegenerative disorders, and the aging process. To better understand the role of lysosomal storage in the onset of cell damage, we used human fibroblasts loaded with sucrose as a model of lysosomal accumulation. Sucrose-loaded fibroblasts displayed increased lysosomal biogenesis followed by arrested cell proliferation. Notably, we found that reduced lysosomal catabolism and autophagy impairment led to an increase in sphingolipids ( i.e., sphingomyelin, glucosylceramide, ceramide, and the gangliosides GM3 and GD3), at both intracellular and plasma membrane (PM) levels. In addition, we observed an increase in the lysosomal membrane protein Lamp-1 on the PM of sucrose-loaded fibroblasts and a greater release of the soluble lysosomal protein cathepsin D in their extracellular medium compared with controls. These results indicate increased fusion between lysosomes and the PM, as also suggested by the increased activity of lysosomal glycosphingolipid hydrolases on the PM of sucrose-loaded fibroblasts. The inhibition of β-glucocerebrosidase and nonlysosomal glucosylceramidase, both involved in ceramide production resulting from glycosphingolipid catabolism on the PM, partially restored cell proliferation. Our findings indicate the existence of a new molecular mechanism underlying cell damage triggered by lysosomal impairment.-Samarani, M., Loberto, N., Soldà, G., Straniero, L., Asselta, R., Duga, S., Lunghi, G., Zucca, F. A., Mauri, L., Ciampa, M. G., Schiumarini, D., Bassi, R., Giussani, P., Chiricozzi, E., Prinetti, A., Aureli, M., Sonnino, S. A lysosome-plasma membrane-sphingolipid axis linking lysosomal storage to cell growth arrest.

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“…Mitochondria were isolated by subcellular fractionation (differential centrifugations) from 3T3-L1 adipocytes treated or not with 10 ng/ml TNF␣ or 0.5 M FCCP for 6 days, followed by a Nycodenz gradient to prepare highly purified mitochondrial fractions. Subcellular fractionation was performed at 4°C in HEPES-EDTA-sucrose buffer (255 mM sucrose, 1 mM EDTA, 20 mM N-2-hydroxyethylpiperazine-N=-2-ethanesulfonic acid, pH 7.4) according to a slightly modified, previously described protocol (28), with cell homogenate preparations from 3T3-L1 adipocytes requiring 40 changeovers through the Dounce. Mitochondrial fractions were resuspended in 1.24 Nycodenz (Gentaur) density before being added on top of a 1.05-1.24 Nycodenz density gradient and submitted to 144,203 g at 4°C for 150 min (OptimaTM LE-80K Ultracentrifuge; Beckman Coulter, rotor Sw 55Ti from Beckman).…”

Section: Methodsmentioning

confidence: 99%

Mild mitochondrial uncoupling does not affect mitochondrial biogenesis but downregulates pyruvate carboxylase in adipocytes: role for triglyceride content reduction

Pauw

Demine

Tejerina

et al. 2012

American Journal of Physiology-Endocrinology and Metabolism

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De Pauw A, Demine S, Tejerina S, Dieu M, Delaive E, Kel A, Renard P, Raes M, Arnould T. Mild mitochondrial uncoupling does not affect mitochondrial biogenesis but downregulates pyruvate carboxylase in adipocytes: role for triglyceride content reduction. Am J Physiol Endocrinol Metab 302: E1123-E1141, 2012. First published February 21, 2012 doi:10.1152/ajpendo.00117.2011In adipocytes, mitochondrial uncoupling is known to trigger a triglyceride loss comparable with the one induced by TNF␣, a proinflammatory cytokine. However, the impact of a mitochondrial uncoupling on the abundance/composition of mitochondria and its connection with triglyceride content in adipocytes is largely unknown. In this work, the effects of a mild mitochondrial uncoupling triggered by FCCP were investigated on the mitochondrial population of 3T3-L1 adipocytes by both quantitative and qualitative approaches. We found that mild mitochondrial uncoupling does not stimulate mitochondrial biogenesis in adipocytes but induces an adaptive cell response characterized by quantitative modifications of mitochondrial protein content. Superoxide anion radical level was increased in mitochondria of both TNF␣-and FCCP-treated adipocytes, whereas mitochondrial DNA copy number was significantly higher only in TNF␣-treated cells. Subproteomic analysis revealed that the abundance of pyruvate carboxylase was reduced significantly in mitochondria of TNF␣-and FCCP-treated adipocytes. Functional study showed that overexpression of this major enzyme of lipid metabolism is able to prevent the triglyceride content reduction in adipocytes exposed to mitochondrial uncoupling or TNF␣. These results suggest a new mechanism by which the effects of mitochondrial uncoupling might limit triglyceride accumulation in adipocytes.adipocytes; carbonyl cyanide p-trifluoromethoxyphenylhydrazone; tumor necrosis factor-␣; mitoproteome OBESITY RESULTS LARGELY FROM A CHRONICALLY POSITIVE energy balance. Therefore, reducing body energy intake and increasing energy expenditure are two approaches that have been translated into strategies to limit fat accumulation. Because mitochondria play a critical role in the process of energy expenditure, the alteration of oxidative phosphorylation (OX-PHOS) efficiency through OXPHOS uncoupling from ATP production in white adipocytes has been considered as an attractive approach to fight obesity (7, 69). In other words, uncoupling increases cellular metabolic demand since uncoupling OXPHOS lowers ATP production and increases the demand for reducing equivalents to restore/maintain mitochondrial membrane potential, thereby increasing substrate catabolism, which would theoretically decrease triglyceride (TG) stores in adipocytes. Indeed, although mitochondrial dysfunction could represent a major cause of lipid metabolism disorders and pathological triglyceride accumulation in several cell lines (50, 75), we and others have shown that mitochondrial uncoupling in adipocytes triggers lipolysis, limits fatty acid synthesis, and leads to a reduction in TG ...

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Endolysosomal transport of newly-synthesized cathepsin D in a sucrose model of lysosomal storage

Cited by 8 publications

References 34 publications

Subcellular Trafficking and Activity of Hyal‐1 and Its Processed Forms in Murine Macrophages

Subcellular Trafficking and Activity of Hyal‐1 and Its Processed Forms in Murine Macrophages

A lysosome‐plasma membrane‐sphingolipid axis linking lysosomal storage to cell growth arrest

Mild mitochondrial uncoupling does not affect mitochondrial biogenesis but downregulates pyruvate carboxylase in adipocytes: role for triglyceride content reduction

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