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1.
Methods Enzymol ; 588: 31-40, 2017.
Article in English | MEDLINE | ID: mdl-28237108

ABSTRACT

Macroautophagy, a major lysosomal degradative pathway for cytoplasmic components, is a process that can be stimulated in response to many stressful situations including cancer treatment. The central autophagic organelle is the autophagosome, a double-membrane-bound vacuole that sequesters cytoplasmic material. The ultimate destiny of the autophagosome is fusion with the lysosomal compartment, where cargo, including proteins, is degraded. Here, we report a method to measure the lysosomal degradation of long-lived proteins along the autophagic pathway.


Subject(s)
Autophagy , Lysosomes/metabolism , Proteins/metabolism , Proteolysis , Animals , Biochemistry/methods , Humans , Neoplasms/metabolism
2.
Int Rev Cell Mol Biol ; 328: 1-23, 2017.
Article in English | MEDLINE | ID: mdl-28069131

ABSTRACT

Macroautophagy is a lysosomal catabolic process that maintains the homeostasis of eukaryotic cells, tissues, and organisms. Macroautophagy plays important physiological roles during development and aging processes and also contributes to immune responses. The process of macroautophagy is compromised in diseases, such as cancer, neurodegenerative disorders, and diabetes. The autophagosome, the double-membrane-bound organelle that sequesters cytoplasmic material to initiate macroautophagy, is formed by the hierarchical recruitment of about 15 autophagy-related (ATG) proteins and associated proteins, such as DFCP1, AMBRA1, the class III phosphatidyl-inositol 3-kinase VPS34, and p150/VPS15. Evidence suggests that in addition to the canonical pathway, noncanonical pathways that do not require the entire repertoire of ATGs can also result in formation of autophagosomes. Here we will discuss recent discoveries concerning the molecular regulation of these noncanonical forms of macroautophagy and their potential roles in cellular responses to stressful situations.


Subject(s)
Autophagy/genetics , Animals , Autophagosomes/metabolism , Humans , Models, Biological , Signal Transduction , Ubiquitins/metabolism
3.
Cell Death Differ ; 22(3): 389-97, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25361082

ABSTRACT

Motile and primary cilia (PC) are microtubule-based structures located at the cell surface of many cell types. Cilia govern cellular functions ranging from motility to integration of mechanical and chemical signaling from the environment. Recent studies highlight the interplay between cilia and autophagy, a conserved cellular process responsible for intracellular degradation. Signaling from the PC recruits the autophagic machinery to trigger autophagosome formation. Conversely, autophagy regulates ciliogenesis by controlling the levels of ciliary proteins. The cross talk between autophagy and ciliated structures is a novel aspect of cell biology with major implications in development, physiology and human pathologies related to defects in cilium function.


Subject(s)
Autophagy/physiology , Cilia/physiology , Animals , Cell Movement/physiology , Humans , Signal Transduction
4.
Oncogene ; 32(18): 2261-72, 2272e.1-11, 2013 May 02.
Article in English | MEDLINE | ID: mdl-22733132

ABSTRACT

Malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew; these cells are known as cancer stem-like cells (CSCs) or tumor-initiating cells. Primitive mammary CSCs/progenitor cells can be propagated in culture as floating spherical colonies termed 'mammospheres'. We show here that the expression of the autophagy protein Beclin 1 is higher in mammospheres established from human breast cancers or breast cancer cell lines (MCF-7 and BT474) than in the parental adherent cells. As a result, autophagic flux is more robust in mammospheres. We observed that basal and starvation-induced autophagy flux is also higher in aldehyde dehydrogenase 1-positive (ALDH1(+)) population derived from mammospheres than in the bulk population. Beclin 1 is critical for CSC maintenance and tumor development in nude mice, whereas its expression limits the development of tumors not enriched with breast CSCs/progenitor cells. We found that decreased survival in autophagy-deficient cells (MCF-7 Atg7 knockdown cells) during detachment does not contribute to an ultimate deficiency in mammosphere formation. This study demonstrates that a prosurvival autophagic pathway is critical for CSC maintenance, and that Beclin 1 plays a dual role in tumor development.


Subject(s)
Apoptosis Regulatory Proteins/genetics , Autophagy/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Membrane Proteins/genetics , Neoplastic Stem Cells/pathology , Adult , Aldehyde Dehydrogenase 1 Family , Animals , Apoptosis Regulatory Proteins/metabolism , Beclin-1 , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Survival/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Isoenzymes/metabolism , Membrane Proteins/metabolism , Mice , Mice, Nude , Middle Aged , Neoplastic Stem Cells/metabolism , Retinal Dehydrogenase/metabolism , Tumor Cells, Cultured
5.
Cell Death Dis ; 1: e87, 2010 Oct 21.
Article in English | MEDLINE | ID: mdl-21368860

ABSTRACT

Proton pump inhibitors (PPI) target tumour acidic pH and have an antineoplastic effect in melanoma. The PPI esomeprazole (ESOM) kills melanoma cells through a caspase-dependent pathway involving cytosolic acidification and alkalinization of tumour pH. In this paper, we further investigated the mechanisms of ESOM-induced cell death in melanoma. ESOM rapidly induced accumulation of reactive oxygen species (ROS) through mitochondrial dysfunctions and involvement of NADPH oxidase. The ROS scavenger N-acetyl-L-cysteine (NAC) and inhibition of NADPH oxidase significantly reduced ESOM-induced cell death, consistent with inhibition of cytosolic acidification. Autophagy, a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, represents a defence mechanism in cancer cells under metabolic stress. ESOM induced the early accumulation of autophagosomes, at the same time reducing the autophagic flux, as observed by WB analysis of LC3-II accumulation and by fluorescence microscopy. Moreover, ESOM treatment decreased mammalian target of rapamycin signalling, as reduced phosphorylation of p70-S6K and 4-EBP1 was observed. Inhibition of autophagy by knockdown of Atg5 and Beclin-1 expression significantly increased ESOM cytotoxicity, suggesting a protective role for autophagy in ESOM-treated cells. The data presented suggest that autophagy represents an adaptive survival mechanism to overcome drug-induced cellular stress and cytotoxicity, including alteration of pH homeostasis mediated by proton pump inhibition.


Subject(s)
Antineoplastic Agents/therapeutic use , Autophagy , Esomeprazole/therapeutic use , Melanoma/drug therapy , Oxidative Stress , Proton Pump Inhibitors/therapeutic use , Acetylcysteine/pharmacology , Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Autophagy-Related Protein 5 , Beclin-1 , Cell Cycle Proteins , Cell Line, Tumor , Humans , Hydrogen-Ion Concentration , Melanoma/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , NADPH Oxidases/metabolism , Phosphoproteins/metabolism , Phosphorylation , Reactive Oxygen Species/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism
6.
Cell Death Differ ; 16(1): 12-20, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18600232

ABSTRACT

Macroautophagy is an evolutionarily conserved vacuolar, self-digesting mechanism for cellular components, which end up in the lysosomal compartment. In mammalian cells, macroautophagy is cytoprotective, and protects the cells against the accumulation of damaged organelles or protein aggregates, the loss of interaction with the extracellular matrix, and the toxicity of cancer therapies. During periods of nutrient starvation, stimulating macroautophagy provides the fuel required to maintain an active metabolism and the production of ATP. Macroautophagy can inhibit the induction of several forms of cell death, such as apoptosis and necrosis. However, it can also be part of the cascades of events that lead to cell death, either by collaborating with other cell death mechanisms or by causing cell death on its own. Loss of the regulation of bulk macroautophagy can prime self-destruction by cells, and some forms of selective autophagy and non-canonical forms of macroautophagy have been shown to be associated with cell demise. There is now mounting evidence that autophagy and apoptosis share several common regulatory elements that are crucial in any attempt to understand the dual role of autophagy in cell survival and cell death.


Subject(s)
Autophagy/physiology , Adenosine Triphosphate/metabolism , Animals , Apoptosis/physiology , Cell Survival/physiology , Energy Metabolism/physiology , Extracellular Matrix/metabolism , Humans , Multiprotein Complexes/metabolism , Necrosis/metabolism , Organelles/metabolism
7.
Cell Death Differ ; 15(8): 1318-29, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18421301

ABSTRACT

Resveratrol, a polyphenol found in grapes and other fruit and vegetables, is a powerful chemopreventive and chemotherapeutic molecule potentially of interest for the treatment of breast cancer. The human breast cancer cell line MCF-7, which is devoid of caspase-3 activity, is refractory to apoptotic cell death after incubation with resveratrol. Here we show that resveratrol arrests cell proliferation, triggers death and decreases the number of colonies of cells that are sensitive to caspase-3-dependent apoptosis (MCF-7 casp-3) and also those that are unresponsive to it (MCF-7vc). We demonstrate that resveratrol (i) acts via multiple pathways to trigger cell death, (ii) induces caspase-dependent and caspase-independent cell death in MCF-7 casp-3 cells, (iii) induces only caspase-independent cell death in MCF-7vc cells and (iv) stimulates macroautophagy. Using BECN1 and hVPS34 (human vacuolar protein sorting 34) small interfering RNAs, we demonstrate that resveratrol activates Beclin 1-independent autophagy in both cell lines, whereas cell death via this uncommon form of autophagy occurs only in MCF-7vc cells. We also show that this variant form of autophagic cell death is blocked by the expression of caspase-3, but not by its enzymatic activity. In conclusion, this study reveals that non-canonical autophagy induced by resveratrol can act as a caspase-independent cell death mechanism in breast cancer cells.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Autophagy , Breast Neoplasms/pathology , Membrane Proteins/metabolism , Stilbenes/pharmacology , Vesicular Transport Proteins/metabolism , Apoptosis/drug effects , Autophagy-Related Protein 7 , Beclin-1 , Breast Neoplasms/metabolism , Caspase 3/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , RNA, Small Interfering/metabolism , Resveratrol , Signal Transduction , Ubiquitin-Activating Enzymes/metabolism
8.
Cell Death Differ ; 12 Suppl 2: 1509-18, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16247498

ABSTRACT

Macroautophagy is a vacuolar, self-digesting mechanism responsible for the removal of long-lived proteins and damaged organelles by the lysosome. The discovery of the ATG genes has provided key information about the formation of the autophagosome, and about the role of macroautophagy in allowing cells to survive during nutrient depletion and/or in the absence of growth factors. Two connected signaling pathways encompassing class-I phosphatidylinositol 3-kinase and (mammalian) target of rapamycin play a central role in controlling macroautophagy in response to starvation. However, a considerable body of literature reports that macroautophagy is also a cell death mechanism that can occur either in the absence of detectable signs of apoptosis (via autophagic cell death) or concomitantly with apoptosis. Macroautophagy is activated by signaling pathways that also control apoptosis. The aim of this review is to discuss the signaling pathways that control macroautophagy during cell survival and cell death.


Subject(s)
Autophagy/physiology , Cell Death/physiology , Cell Survival/physiology , Animals , Humans , Signal Transduction
9.
Biochimie ; 85(1-2): 33-45, 2003.
Article in English | MEDLINE | ID: mdl-12765773

ABSTRACT

Prion diseases form a group of neurodegenerative disorders with the unique feature of being transmissible. These diseases involve a pathogenic protein, called PrP(Sc) for the scrapie isoform of the cellular prion protein (PrP(C)) which is an abnormally-folded counterpart of PrP(C). Many questions remain unresolved concerning the function of PrP(C) and the mechanisms underlying prion replication, transmission and neurodegeneration. PrP(C) is a glycosyl-phosphatidylinositol-anchored glycoprotein expressed at the cell surface of neurons and other cell types. PrP(C) may be present as distinct isoforms depending on proteolytic processing (full length and truncated), topology(GPI-anchored, transmembrane or soluble) and glycosylation (non- mono- and di-glycosylated). The present review focuses on the implications of PrP(C) glycosylation as to the function of the normal protein, the cellular pathways of conversion into PrP(Sc), the diversity of prion strains and the related selective neuronal targeting.


Subject(s)
Prion Diseases/metabolism , Prions/metabolism , Animals , Glycosylation , Humans , PrPC Proteins/chemistry , PrPC Proteins/metabolism , PrPSc Proteins/chemistry , PrPSc Proteins/metabolism , Prions/chemistry , Prions/pathogenicity , Protein Processing, Post-Translational
10.
Exp Cell Res ; 268(2): 139-49, 2001 Aug 15.
Article in English | MEDLINE | ID: mdl-11478840

ABSTRACT

Autophagy is a major catabolic process allowing the renewal of intracellular organelles by which cells maintain their homeostasis. We have previously shown that autophagy is controlled by two transduction pathways mediated by a heterotrimeric Gi3 protein and phosphatidylinositol 3-kinase activities in the human colon cancer cell line HT-29. Here, we show that 3-methyladenine, an inhibitor of autophagy, increases the sensitivity of HT-29 cells to apoptosis induced by sulindac sulfide, a nonsteroidal anti-inflammatory drug which inhibits the cyclooxygenases. Similarly, HT-29 cells overexpressing a GTPase-deficient mutant of the G(alpha i3) protein (Q204L), which have a low rate of autophagy, were more sensitive to sulindac sulfide-induced apoptosis than parental HT-29 cells. In both cell populations we did not observe differences in the expression patterns of COX-2, Bcl-2, Bcl(XL), Bax, and Akt/PKB activity. However, the rate of cytochrome c release was higher in Q204L-overexpressing cells than in HT-29 cells. These results suggest that autophagy could retard apoptosis in colon cancer cells by sequestering mitochondrial death-promoting factors such as cytochrome c.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Apoptosis/physiology , Autophagy/drug effects , Colonic Neoplasms/metabolism , Protein Serine-Threonine Kinases , Sulindac/pharmacology , Adenine/analogs & derivatives , Adenine/pharmacology , Caspases/metabolism , Cyclooxygenase 2 , Cytochrome c Group/metabolism , Dose-Response Relationship, Drug , Drug Antagonism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Humans , Isoenzymes/biosynthesis , Membrane Proteins , Prostaglandin-Endoperoxide Synthases/biosynthesis , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins c-akt , Sulindac/analogs & derivatives , Tumor Cells, Cultured
11.
J Biol Chem ; 276(38): 35243-6, 2001 Sep 21.
Article in English | MEDLINE | ID: mdl-11477064

ABSTRACT

The tumor suppressor PTEN is a dual protein and phosphoinositide phosphatase that negatively controls the phosphatidylinositol (PI) 3-kinase/protein kinase B (Akt/PKB) signaling pathway. Interleukin-13 via the activation of the class I PI 3-kinase has been shown to inhibit the macroautophagic pathway in the human colon cancer HT-29 cells. Here we demonstrate that the wild-type PTEN is expressed in this cell line. Its overexpression directed by an inducible promoter counteracts the interleukin-13 down-regulation of macroautophagy. This effect was dependent upon the phosphoinositide phosphatase activity of PTEN as determined by using the mutant G129E, which has only protein phosphatase activity. The role of Akt/PKB in the signaling control of interleukin-13-dependent macroautophagy was investigated by expressing a constitutively active form of the kinase ((Myr)PKB). Under these conditions a dramatic inhibition of macroautophagy was observed. By contrast a high rate of autophagy was observed in cells expressing a dominant negative form of PKB. These data demonstrate that the signaling control of macroautophagy overlaps with the well known PI 3-kinase/PKB survival pathway and that the loss of PTEN function in cancer cells inhibits a major catabolic pathway.


Subject(s)
Autophagy , Genes, Tumor Suppressor , Phosphoinositide-3 Kinase Inhibitors , Phosphoric Monoester Hydrolases/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/antagonists & inhibitors , Tumor Suppressor Proteins , Base Sequence , Cell Division , DNA Primers , Enzyme Activation , HT29 Cells , Humans , Interleukin-13/physiology , PTEN Phosphohydrolase , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Up-Regulation
12.
Glycobiology ; 10(11): 1217-24, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11087714

ABSTRACT

Using bioinformatics methods, we have previously identified Glu235 and Glu340 as the putative acid/base catalyst and nucleophile, respectively, in the active site of human glucocerebrosidase. Thus, we undertook site-directed mutagenesis studies to obtain experimental evidence supporting these predictions. Recombinant retroviruses were used to express wild-type and E235A and E340A mutant proteins in glucocerebrosidase-deficient murine cells. In contrast to wild-type enzyme, the mutants were found to be catalytically inactive. We also report the results of various studies (Western blotting, glycosylation analysis, subcellular fractionation, and confocal microscopy) indicating that the wild-type and mutant enzymes are identically processed and sorted to the lysosomes. Thus, enzymatic inactivity of the mutant proteins is not the result of incorrect folding/processing. These findings indicate that Glu235 plays a key role in the catalytic machinery of human glucocerebrosidase and may indeed be the acid/base catalyst. As concerns Glu340, the results both support our computer-based predictions and confirm, at the biological level, previous identification of Glu340 as the nucleophile by use of active site labeling techniques. Finally, our findings may help to better understand the molecular basis of Gaucher disease, the human lysosomal disease resulting from deficiency in glucocerebrosidase.


Subject(s)
Glucosylceramidase/genetics , Animals , Base Sequence , Catalytic Domain/genetics , Cell Line , DNA, Complementary/genetics , Gene Expression , Glucosylceramidase/chemistry , Glucosylceramidase/metabolism , Glycosylation , Humans , Mice , Mice, Knockout , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Subcellular Fractions/enzymology , Transduction, Genetic
13.
J Biol Chem ; 275(50): 39090-5, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-10993892

ABSTRACT

Galpha-interacting protein (GAIP) is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by the alpha-subunit of the trimeric G(i3) protein. Both proteins are part of a signaling pathway that controls lysosomal-autophagic catabolism in human colon cancer HT-29 cells. Here we show that GAIP is phosphorylated by an extracellular signal-regulated (Erk1/2) MAP kinase-dependent pathway sensitive to amino acids, MEK1/2 (PD098059), and protein kinase C (GF109203X) inhibitors. An in vitro phosphorylation assay demonstrates that Erk2-dependent phosphorylation of GAIP stimulates its GTPase-activating protein activity toward the Galpha(i3) protein (k = 0.187 +/- 0.001 s(-)(1), EC(50) = 1.12 +/- 0.10 microm) when compared with unphosphorylated recombinant GAIP (k = 0.145 +/- 0.003 s(-)(1), EC(50) = 3.16 +/- 0. 12 microm) or to GAIP phosphorylated by other Ser/Thr protein kinases (protein kinase C, casein kinase II). This stimulation and the phosphorylation of GAIP by Erk2 were abrogated when serine at position 151 in the RGS domain was substituted by an alanine residue using site-directed mutagenesis. Furthermore, the lysosomal-autophagic pathway was not stimulated in S151A-GAIP mutant-expressing cells when compared with wild-type GAIP-expressing cells. These results demonstrate that the GTPase-activating protein activity of GAIP is stimulated by Erk2 phosphorylation. They also suggested that Erk1/2 and GAIP are engaged in the signaling control of a major catabolic pathway in intestinal derived cells.


Subject(s)
Colonic Neoplasms/metabolism , GTP Phosphohydrolases/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phosphoproteins/metabolism , Alanine/chemistry , Casein Kinase II , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Humans , Immunoblotting , Indoles/pharmacology , Kinetics , MAP Kinase Signaling System , Maleimides/pharmacology , Mitogen-Activated Protein Kinase 3 , Mutagenesis, Site-Directed , Phosphoproteins/genetics , Phosphorylation , Precipitin Tests , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , RGS Proteins/metabolism , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Serine/chemistry , Signal Transduction , Transfection , Tumor Cells, Cultured
14.
J Biol Chem ; 275(2): 992-8, 2000 Jan 14.
Article in English | MEDLINE | ID: mdl-10625637

ABSTRACT

3-Methyladenine which stops macroautophagy at the sequestration step in mammalian cells also inhibits the phosphoinositide 3-kinase (PI3K) activity raising the possibility that PI3K signaling controls the macroautophagic pathway (Blommaart, E. F. C., Krause, U., Schellens, J. P. M., Vreeling-Sindelárová, H., and Meijer, A. J. (1997) Eur. J. Biochem. 243, 240-246). The aim of this study was to identify PI3Ks involved in the control of macroautophagic sequestration in human colon cancer HT-29 cells. An increase of class I PI3K products (phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate) caused by either feeding cells with synthetic lipids (dipalmitoyl phosphatidylinositol 3, 4-bisphosphate and dipalmitoyl phosphatidylinositol 3,4, 5-triphosphate) or by stimulating the enzymatic activity by interleukin-13 reduced macroautophagy. In contrast, an increase in the class III PI3K product (phosphatidylinositol 3-phosphate), either by feeding cells with a synthetic lipid or by overexpressing the p150 adaptor, stimulates macroautophagy. Transfection of a specific class III PI3K antisense oligonucleotide greatly inhibited the rate of macroautophagy. In accordance with a role of class III PI3K, wortmannin (an inhibitor of PI3Ks) inhibits macroautophagic sequestration and protein degradation in the low nanomolar range (IC(50) 5-15 nM). Further in vitro enzymatic assay showed that 3-methyladenine inhibits the class III PI3K activity. Dipalmitoyl phosphatidylinositol 3-phosphate supplementation or p150 overexpression rescued the macroautophagic pathway in HT-29 cells overexpressing a GTPase-deficient mutant of the Galpha(i3) protein suggesting that both class III PI3K and trimeric G(i3) protein signaling are required in the control macroautophagy in HT-29 cells. In conclusion, our results demonstrate that distinct classes of PI3K control the macroautophagic pathway in opposite directions. The roles of PI3Ks in macroautophagy are discussed in the context of membrane recycling.


Subject(s)
Autophagy/physiology , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol Phosphates/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Signal Transduction/physiology , Adenocarcinoma , Androstadienes/pharmacology , Autophagy/drug effects , Chromones/pharmacology , Colonic Neoplasms , Enzyme Inhibitors/pharmacology , Homeostasis , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Kinetics , L-Lactate Dehydrogenase/analysis , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol Phosphates/pharmacology , Proto-Oncogene Proteins c-akt , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Wortmannin
15.
Biochem J ; 345 Pt 3: 459-66, 2000 Feb 01.
Article in English | MEDLINE | ID: mdl-10642502

ABSTRACT

The macroautophagic-lysosomal pathway is a bulk degradative process for cytosolic proteins and organelles including the endoplasmic reticulum (ER). We have previously shown that the human colonic carcinoma HT-29 cell population is characterized by a high rate of autophagic degradation of N-linked glycoproteins substituted with ER-type glycans. In the present work we demonstrate that glucosidase inhibitors [castanospermine (CST) and deoxynojirimycin] have a stabilizing effect on newly synthesized glucosylated N-linked glycoproteins and impaired their lysosomal delivery as shown by subcellular fractionation on Percoll gradients. The inhibition of macroautophagy was restricted to N-linked glycoproteins because macroautophagic parameters such as the rate of sequestration of cytosolic markers and the fractional volume occupied by autophagic vacuoles were not affected in CST-treated cells. The protection of glucosylated glycoproteins from autophagic sequestration was also observed in inhibitor-treated Chinese hamster ovary (CHO) cells and in Lec23 cells (a CHO mutant deficient in glucosidase I activity). The interaction of glucosylated glycoproteins with the ER chaperone binding protein (BiP) was prolonged in inhibitor-treated cells in comparison with untreated CHO cells. These results show that the removal of glucose from N-glycans of glycoproteins is a key event for their delivery to the autophagic pathway and that interaction with BiP could prevent or delay newly synthesized glucosylated N-linked glycoproteins from being sequestered by the autophagic pathway.


Subject(s)
Autophagy/physiology , Glucose/metabolism , Glycoproteins/metabolism , Heat-Shock Proteins , Oligosaccharides/metabolism , Animals , Autophagy/drug effects , CHO Cells/drug effects , Carbohydrate Conformation , Carcinoma/metabolism , Carrier Proteins/metabolism , Colonic Neoplasms/metabolism , Cricetinae , Endoplasmic Reticulum Chaperone BiP , Enzyme Inhibitors/pharmacology , Glucosidases/antagonists & inhibitors , Glycoproteins/chemistry , Glycoproteins/drug effects , Humans , Indolizines/pharmacology , Lysosomes/drug effects , Lysosomes/metabolism , Mannose/chemistry , Mannose/metabolism , Molecular Chaperones/metabolism , Tumor Cells, Cultured
16.
Glycobiology ; 10(12): 1277-81, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11159919

ABSTRACT

The biochemical hallmark of Congenital Disorders of Glycosylation (CDG) including type Ia is a defective N-glycosylation of serum glycoproteins. Hypoglycosylated forms of alpha1-antitrypsin have been detected by Western blot in serum from CDG Ia patients. In contrast we were not able to detect hypoglycosylation in alpha1-antitrypsin synthesized by fibroblasts, keratinocytes, enterocytes, and leukocytes. Similarly no hypoglycosylation was detectable in a membrane-associated N-linked glycoprotein, the facilitative glucose transporter GLUT-1 and also in serum immunoglobulin G isolated from sera of CDG Ia patients. We conclude that the phenotypic expression of CDG Ia is tissue-dependent.


Subject(s)
Carbohydrate Metabolism, Inborn Errors/metabolism , Blotting, Western , Glycosylation , Humans , Immunoglobulin G/metabolism , Isoelectric Focusing
17.
J Biol Chem ; 274(45): 31903-8, 1999 Nov 05.
Article in English | MEDLINE | ID: mdl-10542217

ABSTRACT

Lu and Lu(v13) are two glycoprotein (gp) isoforms that belong to the immunoglobulin superfamily and carry both the Lutheran (Lu) blood group antigens and the basal cell adhesion molecule epithelial cancer antigen. Lu (85 kDa) and Lu(v13) (78 kDa) gps, which differ only in the length of their cytoplasmic domain, are adhesion molecules that bind laminin. In nonerythroid tissues, the Lu/basal cell adhesion molecule antigens are predominantly expressed in the endothelium of blood vessel walls and in the basement membrane region of normal epithelial cells, whereas they exhibit a nonpolarized expression in some epithelial cancers. Here, we analyzed the polarization of Lu and Lu(v13) gps in epithelial cells by confocal microscopy and domain-selective biotinylation assays. Differentiated human colon carcinoma Caco-2 cells exhibited a polarized expression of endogenous Lu antigens associated with a predominant expression of the Lu isoform at the basolateral domain of the plasma membrane and a very low expression of the Lu(v13) isoform at both the apical and basolateral domains. Analysis of transfected Madin-Darby canine kidney cells revealed a basolateral expression of Lu gp and a nonpolarized expression of Lu(v13) gp. Delivery of Lu(v13) to both apical and basolateral surfaces showed similar kinetics, indicating that this isoform is directly transported to each surface domain. A dileucine motif at position 608-609, specific to the Lu isoform, was characterized as a dominant basolateral sorting signal that prevents Lu gp from taking the apical delivery pathway.


Subject(s)
Cell Adhesion Molecules/physiology , Cell Polarity , Epithelial Cells/metabolism , Lutheran Blood-Group System , Neoplasm Proteins/physiology , Amino Acid Sequence , Animals , Caco-2 Cells , Dogs , Humans , Leucine/metabolism , Molecular Sequence Data , Peptide Mapping , Signal Transduction , Structure-Activity Relationship
18.
J Biol Chem ; 274(19): 13547-55, 1999 May 07.
Article in English | MEDLINE | ID: mdl-10224124

ABSTRACT

In hepatocellular carcinoma HepG2 cells, free polymannose-type oligosaccharides appearing in the cytosol during the biosynthesis and quality control of glycoproteins are rapidly translocated into lysosomes by an as yet poorly defined process (Saint-Pol, A., Bauvy, C., Codogno, P., and Moore, S. E. H. (1997) J. Cell Biol. 136, 45-59). Here, we demonstrate an ATP-dependent association of [2-3H]mannose-labeled Man5GlcNAc with isolated rat liver lysosomes. This association was only observed in the presence of swainsonine, a mannosidase inhibitor, which was required for the protection of sedimentable, but not nonsedimentable, Man5GlcNAc from degradation, indicating that oligosaccharides were transported into lysosomes. Saturable high affinity transport (Kuptake, 22.3 microM, Vmax, 7.1 fmol/min/unit of beta-hexosaminidase) was dependent upon the hydrolysis of ATP but independent of vacuolar H+/ATPase activity. Transport was inhibited strongly by NEM and weakly by vanadate but not by sodium azide, and, in addition, the sugar transport inhibitors phloretin, phloridzin, and cytochalasin B were without effect on transport. Oligosaccharide import did not show absolute specificity but was selective toward partially demannosylated and dephosphorylated oligosaccharides, and, furthermore, inhibition studies revealed that the free reducing GlcNAc residue of the oligosaccharide was of critical importance for its interaction with the transporter. These results demonstrate the presence of a novel lysosomal free oligosaccharide transporter that must work in concert with cytosolic hydrolases in order to clear the cytosol of endoplasmic reticulum-generated free oligosaccharides.


Subject(s)
Cytosol/metabolism , Liver/metabolism , Lysosomes/metabolism , Mannans/metabolism , Acetylglucosamine/metabolism , Animals , Biological Transport , Intracellular Membranes/metabolism , Inulin/metabolism , Kinetics , Liver/ultrastructure , Male , Phosphorylation , Rats , Rats, Wistar , Tritium , Tumor Cells, Cultured
19.
Biochim Biophys Acta ; 1453(3): 369-77, 1999 Mar 30.
Article in English | MEDLINE | ID: mdl-10101255

ABSTRACT

The aim of the present study was to explore how mannose enters fibroblasts derived from a panel of children suffering from different subtypes of type I carbohydrate deficient glycoprotein syndrome: seven carbohydrate deficient glycoprotein syndrome subtype Ia (phosphomannomutase deficiency), two carbohydrate deficient glycoprotein syndrome subtype Ib (phosphomannose isomerase deficiency) and two carbohydrate deficient glycoprotein syndrome subtype Ix (not identified deficiency). We showed that a specific mannose transport system exists in all the cells tested but has different characteristics with respect to carbohydrate deficient glycoprotein syndrome subtypes. Subtype Ia fibroblasts presented a mannose uptake equivalent or higher (maximum 1.6-fold) than control cells with a D-[2-3H]-mannose incorporation in nascent N-glycoproteins decreased up to 7-fold. Compared to control cells, the mannose uptake was greatly stimulated in subtype Ib (4.0-fold), due to lower Kuptake and higher Vmax values. Subtype Ib cells showed an increased incorporation of D-[2-3H]-mannose into nascent N-glycoproteins. Subtype Ix fibroblasts presented an intermediary status with mannose uptake equivalent to the control but with an increased incorporation of D-[2-3H]-mannose in nascent N-glycoproteins. All together, our results demonstrate quantitative and/or qualitative modifications in mannose transport of all carbohydrate deficient glycoprotein syndrome fibroblasts in comparison to control cells, with a relative homogeneity within a considered subtype of carbohydrate deficient glycoprotein syndrome. These results are consistent with the possible use of mannose as a therapeutic agent in carbohydrate deficient glycoprotein syndrome Ib and Ix.


Subject(s)
Congenital Disorders of Glycosylation/metabolism , Mannose/metabolism , Biological Transport/drug effects , Cells, Cultured , Congenital Disorders of Glycosylation/drug therapy , Fibroblasts/metabolism , Glycoproteins/metabolism , Humans , Mannose/pharmacology , Skin/metabolism
20.
Biochem J ; 337 ( Pt 2): 289-95, 1999 Jan 15.
Article in English | MEDLINE | ID: mdl-9882627

ABSTRACT

Autophagic sequestration is controlled by the Galphai3 protein in human colon cancer HT-29 cells. Immunofluorescence and subcellular fractionation studies showed that the Galphai3 protein is preferentially associated with Golgi membranes but co-localization was also observed with the endoplasmic reticulum (ER) membrane. The Galphai2 protein, which is not involved in the control of autophagic sequestration, is associated with the plasma membrane. Transfection of chimaeric Galphai proteins (Galphai3/2, Galphai2/3) containing the N- and C-terminal parts of the relevant Galphai demonstrated that the C-terminal part of the Galphai3 protein, by governing its membrane localization [de Almeida, Holtzman, Peters, Ercolani, Ausiello and Stow (1994) J. Cell Sci. 107, 507-515], is important in the control of macroautophagic sequestration. G alpha interacting protein (GAIP),which stimulates the GTPase activity of the Galphai3 protein and favours macroautophagic sequestration in HT-29 cells,was shown, by immunofluorescence studies using confocal microscopy, to be confined to the cytoplasm. The cytoplasmic distribution of GAIP only partially overlaps with that of the Galphai3 protein. However, the presence of the two proteins on Golgi and ER membranes was confirmed by subcellular fractionation. These results point to the importance of the cytoplasmic localization of the Galphai3 protein and GAIP in controlling autophagic sequestration in HT-29 cells.


Subject(s)
Autophagy , Colonic Neoplasms/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , Phosphoproteins/isolation & purification , Cell Compartmentation , Cell Fractionation , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/ultrastructure , Fluorescent Antibody Technique , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Golgi Apparatus/chemistry , Golgi Apparatus/ultrastructure , HT29 Cells , Humans , Phosphoproteins/genetics , Phosphoproteins/immunology , RGS Proteins , Recombinant Fusion Proteins/isolation & purification
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