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:3.4.23.5 (cathepsin D)
4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the present work, we analyzed the variations in the expression and trafficking of cathepsin D (CD), a lysosomal endopeptidase, associated with the enterocytic differentiation of the human colon carcinoma HT-29 cell line. In spite of the fact that the abundance of CD mRNA was severalfold higher in undifferentiated HT-29 cells than in their enterocyte-like differentiated counterparts, the intracellular levels of CD activity and protein were found to be much higher in the latter. The kinetic of transport of newly synthesized proCD was different in the two cell populations: (a) full conversion of proCD into the lysosomal mature form required more than 24 h in differentiated cells, whereas it was almost complete within 8 h in undifferentiated HT-29 cells; and (b) the extracellular release of proCD was shown to occur more rapidly and to a higher degree in undifferentiated than in differentiated cells. Most of the secreted proCD contained phosphomannoses. Secretion of beta-hexosaminidase activity doubled, whereas that of CD activity was unchanged, upon vacuolar alkalinization with ammonium chloride or chloroquine. Inhibition of the lysosomal-autophagic degradative pathway resulted in the accumulation of proCD molecules in undifferentiated HT-29 cells. Altogether these data suggest that: (a) the expression and the posttranslational fate of CD in HT-29 colon cancer cells are largely affected by the state of their enterocytic differentiation; and (b) in this cell line the acid-dependent mannose 6-phosphate receptor pathway is, at best, little involved in the trafficking of CD.
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PMID:Expression and posttranslational fate of cathepsin D in HT-29 tumor cells depend on their enterocytic differentiation state. 930 Jan 84

The transport of pro-cathepsin D from the trans-Golgi network (TGN) to the endosomal pathway is dependent on binding to the calcium-independent mannose 6-phosphate receptor (ci-M6PR), which is incorporated into TGN-derived clathrin-coated transport vesicles (CCVs). Inhibition of this transport step by wortmannin has led to the proposal that it is dependent upon a phosphoinositide 3-kinase activity necessary for ci-M6PR recruitment into TGN-derived CCVs or in the formation of those vesicles (Brown, W. J., DeWald, D. B., Emr, S. D., Plutner, H., and Balch, W. E. (1995) J. Cell Biol. 130, 781-796; Davidson, H. W. (1995) J. Cell Biol. 130, 797-806). In this study we have addressed the effect of wortmannin on the TGN step of the ci-M6PR cycle. CCVs from K562 cells, pretreated or not with 250 nM wortmannin, were purified on equilibrium density gradients. Quantification of TGN-derived CCVs, assessed by gamma-adaptin content in purified vesicle fractions, showed that the formation of the vesicles was only marginally decreased after 20 min of treatment with the drug, while for the same wortmannin treatment, the amount of ci-M6PR recruited into those vesicles was decreased by 70% compared with control. At a later time point (2 h), a reduction in the amount of gamma-adaptin in CCV fractions was also observed. These findings demonstrate that inhibition of ci-M6PR recruitment into CCVs but not of vesicle formation is the primary reason for the observed defect in cathepsin D transport following wortmannin treatment.
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PMID:Inhibition of calcium-independent mannose 6-phosphate receptor incorporation into trans-Golgi network-derived clathrin-coated vesicles by wortmannin. 930 67

Stable BHK cell lines inducibly expressing wild-type or dominant negative mutant forms of the rab7 GTPase were isolated and used to analyze the role of a rab7-regulated pathway in lysosome biogenesis. Expression of mutant rab7N125I protein induced a dramatic redistribution of cation-independent mannose 6-phosphate receptor (CI-MPR) from its normal perinuclear localization to large peripheral endosomes. Under these circumstances approximately 50% of the total receptor and several lysosomal hydrolases cofractionated with light membranes containing early endosome and Golgi markers. Late endosomes and lysosomes were contained exclusively in well-separated, denser gradient fractions. Newly synthesized CI-MPR and cathepsin D were shown to traverse through an early endocytic compartment, and functional rab7 was crucial for delivery to later compartments. This observation was evidenced by the fact that 2 h after synthesis, both markers were more prevalent in fractions containing light membranes. In addition, both were sensitive to HRP-DAB- mediated cross-linking of early endosomal proteins, and the late endosomal processing of cathepsin D was impaired. Using similar criteria, the lysosomal membrane glycoprotein 120 was not found accumulated in an early endocytic compartment. The data are indicative of a post-Golgi divergence in the routes followed by different lysosome-directed molecules.
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PMID:Mutant Rab7 causes the accumulation of cathepsin D and cation-independent mannose 6-phosphate receptor in an early endocytic compartment. 949 Jul 21

Rab7 is a small molecular weight GTPase that is known to be associated with late endocytic compartments. Studies in which wild-type or mutant forms of this protein have been overexpressed in mammalian cells have indicated that rab7 plays a role in controlling membrane transport between late endocytic compartments. However, both the precise site(s) of action and localization of rab7 remain unclear. In the present study, we have used density-gradient centrifugation in combination with a new epitope-specific flow cytometric sorting method to isolate rab7-containing vesicles from baby hamster kidney (BHK) cells. Electron-micrographs of sorted elements showed a homogeneous population of vesicles that resembles late endosomes. The polypeptide composition of rab7-containing vesicles was then analyzed by two-dimensional (2-D) gel electrophoresis. Rab7-containing vesicles were enriched in the cation-independent mannose 6-phosphate receptor and especially in the precursor forms of cathepsin D. Taken together, these results show that the rab7-containing vesicles are a component of the endocytic pathway that connects late endosomes and lysosomes and in which precursor forms of lysosomal hydrolases, segregated from their receptor, might be included.
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PMID:Flow cytometric sorting and biochemical characterization of the late endosomal rab7-containing compartment. 952 98

We have previously shown that in HEp-2 cells, multivesicular bodies (MVBs) processing internalized epidermal growth factor-epidermal growth factor receptor complexes mature and fuse directly with lysosomes in which the complexes are degraded. The MVBs do not fuse with a prelysosomal compartment enriched in mannose 6-phosphate receptor (M6PR) as has been described in other cell types. Here we show that the cation-independent M6PR does not become enriched in the endocytic pathway en route to the lysosome, but if a pulse of M6PR or an M6PR ligand, cathepsin D, is followed, a significant fraction of these proteins are routed from the trans-Golgi to MVBs. Accumulation of M6PR does not occur because when the ligand dissociates, the receptor rapidly leaves the MVB. At steady state, most M6PR are distributed within the trans-Golgi and trans-Golgi network and in vacuolar structures distributed in the peripheral cytoplasm. We suggest that these M6PR-rich vacuoles are on the return route from MVBs to the trans-Golgi network and that a separate stable M6PR-rich compartment equivalent to the late endosome/prelysosome stage does not exist on the endosome-lysosome pathway in these cells.
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PMID:The kinetics of mannose 6-phosphate receptor trafficking in the endocytic pathway in HEp-2 cells: the receptor enters and rapidly leaves multivesicular endosomes without accumulating in a prelysosomal compartment. 952 79

The two mannose 6-phosphate (Man-6-P) binding sites of the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) have been localized to domains 1-3 and 7-9, and studies have shown that Arg435 in domain 3 and Arg 1334 in domain 9 are essential for Man-6-P binding. To determine whether the IGF-II/MPR containing a single Man-6-P binding site is functional, clonal mouse L cell lines stably transfected with either mutant bovine IGF-II/MPR cDNA, containing substitutions at position 435 and/or 1334, or the wild type receptor cDNA were assayed for their ability to sort lysosomal enzymes to the lysosome. Mutant receptors containing a single Man-6-P binding site were approximately 50% less efficient than the wild type receptor in the overall targeting of lysosomal enzymes to the lysosome. Mutant receptors containing a substitution at Arg1334 (Dom9(Ala)), in contrast to those containing a substitution at Arg435 (Dom3(Ala)), were unable to target cathepsin D and beta-hexosaminidase to the lysosome. Equilibrium binding assays using 125I-labeled beta-glucuronidase demonstrated that Dom3(Ala) and Dom9(Ala) had a Kd of 2.0 and 4.3 nM, respectively. In addition, Dom3(Ala), unlike Dom9(Ala), was unable to completely dissociate from ligand under acidic pH conditions. These data indicate that the two Man-6-P binding sites of the IGF-II/MPR are not functionally equivalent.
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PMID:The two mannose 6-phosphate binding sites of the insulin-like growth factor-II/mannose 6-phosphate receptor display different ligand binding properties. 971 56

Doc2 has one Munc13-interacting domain at the N-terminal region and two C2-like domains interacting with Ca2+ and phospholipid at the C-terminal region. Doc2 consists of two isoforms, Doc2alpha and -beta. Doc2alpha is specifically expressed in neuronal cells and implicated in Ca2+-dependent neurotransmitter release, whereas Doc2beta is ubiquitously expressed and its function is unknown. We show here that both Doc2alpha and -beta interact with rat tctex-1, a light chain of cytoplasmic dynein, in both cell-free and intact cell systems. Overexpression of the N-terminal fragment of Doc2 containing the tctex-1-interacting domain induces changes in the intracellular localization of cation-independent mannose 6-phosphate receptor and its ligand, cathepsin D, which are transported from trans-Golgi network to late endosomes. Overexpression of the C-terminal fragment containing two C2-like domains shows the similar effect, but to a lesser extent, whereas overexpression of full-length Doc2 or the C-terminal fragment of rabphilin3 containing two C2-like domains does not show this effect. Because dynein is a minus-end-directed microtubule-based motor protein, these results suggest that Doc2, especially Doc2beta, plays a role in dynein-dependent intracellular vesicle transport.
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PMID:Interaction of Doc2 with tctex-1, a light chain of cytoplasmic dynein. Implication in dynein-dependent vesicle transport. 980 56

The interaction of Salmonella and Yersinia with macrophages is critical to the pathogenesis of these organisms. After internalization into macrophages, these bacteria reside in membrane-enclosed vacuoles. In this report, we present an approach to isolate and characterize bacteria-containing vacuoles (BCVs) to study intracellular trafficking of pathogenic bacteria within the membrane system of host cells. Using the mouse monocyte-macrophage cell line J774A.1, we found that Salmonella typhimurium replicated intracellularly to approximately 5 times its original numbers over a 9 hour infection course, while Yersinia pseudotuberculosis and Escherichia coli did not replicate inside these cells. Analysis of isolated latex bead-containing vacuoles confirmed that they trafficked normally from endosomes to lysosomes within the endocytic pathway of J774A.1 cells. We isolated BCVs free of contaminating endosomes and lysosomes using sucrose step gradients, and used quantitative immunoblotting to characterize the contents of these vacuoles at different time points after internalization. We found that the isolated BCVs contained endosomal and lysosomal marker proteins including lamp-1, mannose 6-phosphate receptor (M 6-PR), cathepsin D and cathepsin L. Further, we report on differential processing of lysosomal hydrolases (such as cathepsin D and cathepsin L) associated with the isolated BCVs. Although there was some contamination of the S. typhimurium-containing vacuoles with endoplasmic reticulum (ER) marker protein calnexin, the Y. pseudotuberculosis-containing vacuoles were predominately free of ER contamination. The Y. pseudotuberculosis-containing vacuoles displayed properties of lysosomes, containing the M 6-PR-dependent lysosomal hydrolases cathepsin D and cathepsin L, which were shown to be processed to their mature forms incrementally over time. These results, coupled with intracellular growth and microscopic examination of infected cells over time, indicated that Y. pseudotuberculosis traffics to lysosomes where they are degraded. The described method for isolation and characterization of BCVs proved to be a valuable tool to characterize the vacuolar compartment occupied by Y. pseudotuberculosis, and has potential to be applied to other vacuole resident pathogens whose trafficking is thought to play a role in pathogenesis.
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PMID:Isolation and characterization of Salmonella typhimurium and Yersinia pseudotuberculosis-containing phagosomes from infected mouse macrophages: Y. pseudotuberculosis traffics to terminal lysosomes where they are degraded. 980 87

Chinese hamster ovary cell mutants defective in the post-uptake degradation of low-density lipoprotein (LDL) in lysosomes were selected from mutagenized cells by novel three-step screening. First, in the presence of LDL, clones sensitive to an inhibitor of the rate-limiting enzyme of the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl-CoA reductase, were isolated. Second, from the selected clones, those lacking in the degradation of a constituent of a fluorescent LDL were qualitatively screened by microscopy. Third, the clones were further screened by previously established quantitative analytical flow cytometry that detects the early-phase disintegration of LDL by lysosomal acid hydrolases. One of the isolated mutant clones, LEX1 (Lysosome-Endosome X 1), was a recessive mutant, and exhibited a specific disorder in the late endocytic pathway. LEX1 cells showed an unusual perinuclear aggregate of vesicles, heterogeneously positive for lysosomal glycoprotein-B/cathepsin D and rab7, yet negative for the cation-independent mannose 6-phosphate receptor. The aggregate was formed around the microtubule organizing center, and was disrupted by nocodazole treatment. Internalized octadecyl rhodamine B-labeled LDL (R18-LDL) was accumulated in the perinuclear rab7-positive vesicles. In a Percoll density gradient, neither internalized R18-LDL nor internalized horseradish peroxidase was efficiently chased into heavy lysosomal fractions positive for beta-hexosaminidase. LEX1 cells showed differences in the activity and subcellular distribution of lysosomal enzymes. These characteristics of LEX1 cells are consistent with the ideas that the perinuclear vesicle aggregate is an arrested intermediate of direct fusion or divergence between lysosomes and rab7-positive, cation-independent mannose 6-phosphate receptor-negative late endosomes, and that equilibrium between the lysosomes and the late endosomes is shifted towards the late endosomes in LEX1 cells. Such fusion or divergence between the late endosomes and the lysosomes would determine an appropriate equilibrium between them, and might thereby play an important role for proper lysosomal digestive functions. LEX1 mutant cells would be helpful for the dissection of the as yet unrevealed details of the late endocytic membrane dynamics and for the identification of factors involved in the process arrested by the mutation.
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PMID:An arrested late endosome-lysosome intermediate aggregate observed in a Chinese hamster ovary cell mutant isolated by novel three-step screening. 1008 48

The molecular machinery behind lysosome biogenesis and the maintenance of the perinuclear aggregate of late endocytic structures is not well understood. A likely candidate for being part of this machinery is the small GTPase Rab7, but it is unclear whether this protein is associated with lysosomes or plays any role in the regulation of the perinuclear lysosome compartment. Previously, Rab7 has mainly been implicated in transport from early to late endosomes. We have now used a new approach to analyze the role of Rab7: transient expression of Enhanced Green Fluorescent Protein (EGFP)-tagged Rab7 wt and mutant proteins in HeLa cells. EGFP-Rab7 wt was associated with late endocytic structures, mainly lysosomes, which aggregated and fused in the perinuclear region. The size of the individual lysosomes as well as the degree of perinuclear aggregation increased with the expression levels of EGFP-Rab7 wt and, more dramatically, the active EGFP-Rab7Q67L mutant. In contrast, upon expression of the dominant-negative mutants EGFP-Rab7T22N and EGFP-Rab7N125I, which localized mainly to the cytosol, the perinuclear lysosome aggregate disappeared and lysosomes, identified by colocalization of cathepsin D and lysosome-associated membrane protein-1, became dispersed throughout the cytoplasm, they were inaccessible to endocytosed molecules such as low-density lipoprotein, and their acidity was strongly reduced, as determined by decreased accumulation of the acidotropic probe LysoTracker Red. In contrast, early endosomes associated with Rab5 and the transferrin receptor, late endosomes enriched in the cation-independent mannose 6-phosphate receptor, and the trans-Golgi network, identified by its enrichment in TGN-38, were unchanged. These data demonstrate for the first time that Rab7, controlling aggregation and fusion of late endocytic structures/lysosomes, is essential for maintenance of the perinuclear lysosome compartment.
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PMID:Rab7: a key to lysosome biogenesis. 1067 7


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