Disturbed Cholesterol Traffic but Normal Proteolytic Function in LAMP-1/LAMP-2 Double-deficient Fibroblasts

Eskelinen, Eeva‐Liisa; Schmidt, Christine K.; Neu, Silja; Willenborg, Marion; Fuertes, Graciela; Salvador, Natalia; Tanaka, Yoshitaka; Lüllmann‐Rauch, Renate; Hartmann, Dieter; Heeren, J.; Figura, Kurt Von; Knecht, Erwin; Säftig, Paul

doi:10.1091/mbc.e04-02-0103

Cited by 241 publications

(279 citation statements)

References 36 publications

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“…Similar findings were obtained upon analysis of LAMP1/LAMP2 double-deficient fibroblasts (29). Lysosomes in double-deficient cells also accumulated significant amounts of lipid.…”

supporting

confidence: 82%

Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells

Deffieu

Lee

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Lysosomes are lined with a glycocalyx that protects the limiting membrane from the action of degradative enzymes. We tested the hypothesis that Niemann-Pick type C 1 (NPC1) protein aids the transfer of low density lipoprotein-derived cholesterol across this glycocalyx. A prediction of this model is that cells will be less dependent upon NPC1 if their glycocalyx is decreased in density. Lysosome cholesterol content was significantly lower after treatment of NPC1-deficient human fibroblasts with benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside, an inhibitor of O-linked glycosylation. Direct biochemical measurement of cholesterol showed that lysosomes purified from NPC1-deficient fibroblasts contained at least 30% less cholesterol when O-linked glycosylation was blocked. As an independent means to modify protein glycosylation, we used Chinese hamster ovary ldl-D cells defective in UDP-Gal/UDP-GalNAc 4-epimerase in which N-and O-linked glycosylation can be controlled. CRISPR generated, NPC1-deficient ldl-D cells supplemented with galactose accumulated more cholesterol than those in which sugar addition was blocked. In the absence of galactose supplementation, NPC1-deficient ldl-D cells also transported more cholesterol from lysosomes to the endoplasmic reticulum, as monitored by an increase in cholesteryl [ 14 C]-oleate levels. These experiments support a model in which NPC1 protein functions to transfer cholesterol past a lysosomal glycocalyx.L ow-density lipoprotein-derived cholesterol is delivered to cells by receptor-mediated endocytosis and transport to lysosomes. Within lysosomes, cholesterol esters are hydrolyzed and cholesterol is exported to the cytoplasm for cellular use (1). Cholesterol export requires two lysosomal glycoproteins: NPC1 and NPC2 (2, 3). Patients carrying homozygous mutations in either of these proteins present with Niemann-Pick type C (NPC) disease, a neurological disorder that is associated with massive accumulation of unesterified cholesterol and glycosphingolipids in lysosomes (2-4).NPC2 is a small, soluble, cholesterol binding protein that is thought to pick up cholesterol both from lipoprotein lipase and from the abundant, intralumenal membranes present within lysosomes (5, 6). NPC1 is a much larger glycoprotein that contains 13 transmembrane domains and three, relatively large, lumenally oriented domains (2). NPC1's first (N-terminal), luminal domain binds cholesterol directly (7,8); the second domain can bind NPC2 in a cholesterol-dependent manner and has been proposed to facilitate transfer of cholesterol from NPC2 onto NPC1's N-terminal domain (9).The requirement for NPC1 and NPC2 proteins for cholesterol export from lysosomes is somewhat enigmatic because cholesterol can generally partition freely into membrane bilayers (10). The limiting membrane of lysosomes is lined predominantly by densely packed, highly glycosylated, lysosomal membrane proteins; their oligosaccharides are thought to protect the phospholipid bilayer from hydrolytic destruction (11,12). This glycoprotein coat ...

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“…Similar findings were obtained upon analysis of LAMP1/LAMP2 double-deficient fibroblasts (29). Lysosomes in double-deficient cells also accumulated significant amounts of lipid.…”

supporting

confidence: 82%

Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells

Deffieu

Lee

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…In contrast, we observed accumulation of abnormal mitochondria, which are substrates for macroautophagy and increased oxidative stress in LAMP2 deficient mice on a fed day, paralleling impairment in macroautophagy, which is the major pathway for removal of damaged mitochondria. 60 LAMP2, together with LAMP1, plays an important role in cholesterol efflux, 61 which also may be mechanistically important in the observed benefits of intermittent fasting, Figure 10. Intermittent fasting induces autophagy without depletion of myocardial autophagy-lysosome machinery proteins.…”

Section: Discussionmentioning

confidence: 99%

Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury

et al. 2015

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(2015) Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemiareperfusion injury, Autophagy, 11:9, 1537-1560, DOI: 10.1080/15548627.2015 Keywords: autophagy, fasting, ischemia-reperfusion, lysosome, myocardial infarctionAbbreviations: CMA, chaperone-mediated autophagy; CQ, chloroquine; GFP, green fluorescent protein; LAD, left anterior descending; LAMP2, lysosomal-associated membrane protein 2; MOI, multiplicity of infection; NRCMs, neonatal rat ventricular cardiac myocytes; q4D, quaque qutra die/every fourth day; qOD, quaque otra die/every other day; TFEB, transcription factor EB; MAP1LC3B (also abbreviated as LC3), microtubule-associated protein 1 light chain 3, isoform B; TTC, triphenyl tetrazolium chloride; LV, left ventricle; AAR, area at risk; WT, wild type.Autophagy, a lysosomal degradative pathway, is potently stimulated in the myocardium by fasting and is essential for maintaining cardiac function during prolonged starvation. We tested the hypothesis that intermittent fasting protects against myocardial ischemia-reperfusion injury via transcriptional stimulation of the autophagy-lysosome machinery. Adult C57BL/6 mice subjected to 24-h periods of fasting, every other day, for 6 wk were protected from invivo ischemia-reperfusion injury on a fed day, with marked reduction in infarct size in both sexes as compared with nonfasted controls. This protection was lost in mice heterozygous null for Lamp2 (coding for lysosomal-associated membrane protein 2), which demonstrate impaired autophagy in response to fasting with accumulation of autophagosomes and SQSTM1, an autophagy substrate, in the heart. In lamp2 null mice, intermittent fasting provoked progressive left ventricular dilation, systolic dysfunction and hypertrophy; worsening cardiomyocyte autophagosome accumulation and lack of protection to ischemia-reperfusion injury, suggesting that intact autophagy-lysosome machinery is essential for myocardial homeostasis during intermittent fasting and consequent ischemic cardioprotection. Fasting and refeeding cycles resulted in transcriptional induction followed by downregulation of autophagy-lysosome genes in the myocardium. This was coupled with fasting-induced nuclear translocation of TFEB (transcription factor EB), a master regulator of autophagy-lysosome machinery; followed by rapid decline in nuclear TFEB levels with refeeding. Endogenous TFEB was essential for attenuation of hypoxia-reoxygenation-induced cell death by repetitive starvation, in neonatal rat cardiomyocytes, in-vitro. Taken together, these data suggest that TFEBmediated transcriptional priming of the autophagy-lysosome machinery mediates the beneficial effects of fastinginduced autophagy in myocardial ischemia-reperfusion injury.

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“…Autophagic degradation has been shown to be impaired in hepatocytes isolated from LAMP-2-deficient mice [71]. However, no defect in autophagy was observed in LAMP-2-deficient mouse fibroblasts [72]. A blockade in the later stage of autophagy only occurs in fibroblasts that are deficient for both LAMP-1 and LAMP-2.…”

Section: Escrt and Hrsmentioning

confidence: 99%

Overview of macroautophagy regulation in mammalian cells

et al. 2010

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Macroautophagy is a multistep, vacuolar, degradation pathway terminating in the lysosomal compartment, and it is of fundamental importance in tissue homeostasis. In this review, we consider macroautophagy in the light of recent advances in our understanding of the formation of autophagosomes, which are double-membrane-bound vacuoles that sequester cytoplasmic cargos and deliver them to lysosomes. In most cases, this final step is preceded by a maturation step during which autophagosomes interact with the endocytic pathway. The discovery of AuTophaGyrelated genes has greatly increased our knowledge about the mechanism responsible for autophagosome formation, and there has also been progress in the understanding of molecular aspects of autophagosome maturation. Finally, the regulation of autophagy is now better understood because of the discovery that the activity of Atg complexes is targeted by protein kinases, and owing to the importance of nuclear regulation via transcription factors in regulating the expression of autophagy genes.

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Disturbed Cholesterol Traffic but Normal Proteolytic Function in LAMP-1/LAMP-2 Double-deficient Fibroblasts

Cited by 241 publications

References 36 publications

Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells

Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells

Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury

Overview of macroautophagy regulation in mammalian cells

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