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)

Dynamin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracycline-inducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgi-derived coated vesicles, were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.
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PMID:Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. 796 76

The transport of proteins from the secretory to the endocytic pathway is mediated by carrier vesicles coated with the AP-1 Golgi assembly proteins and clathrin. The mannose 6-phosphate receptors (MPHs) are two major transmembrane proteins segregated into these transport vesicles. Together with the GTPase ARF-1, these cargo proteins are essential components for the efficient translocation of the cytosolic AP-1 onto membranes of the trans-Golgi network, the first step of clathrin coat assembly, MPR-negative fibroblasts have a low capacity of recruiting AP-1 which can be restored by re-expressing the MPRs in these cells. This property was used to identify the protein motif of the cation-dependent mannose 6-phosphate receptor (CD-MPR) cytoplasmic domain that is essential for these interactions. Thus, the affinity of AP-1 for membranes and in vivo transport of cathepsin D were measured for MPR-negative cells re-expressing various CD-MPR mutants. The results indicate that the targeting of lysosomal enzymes requires the CD-PDR cytoplasmic domain that are different from tyrosine-based endocytosis motifs. The first is a casein kinase II phosphorylation site (ESEER) that is essential for high affinity binding of AP-1 and therefore probably acts as a dominant determinant controlling CD-MPR sorting in the trans-Golgi network. The second is the adjacent di-leucine motif (HLLPM), which, by itself, is not critical for AP-1 binding, but is absolutely required for a downstream sorting event.
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PMID:A casein kinase II phosphorylation site in the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor determines the high affinity interaction of the AP-1 Golgi assembly proteins with membranes. 856 75

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

The expression, processing, and intracellular localization of cathepsin D (CD), an endosomal-lysosomal protease involved in malignancy, were studied in rat embryo fibroblasts transformed with an active mutant of c-Ha-ras oncogene. The pattern of the processed molecular forms of CD, comprising two single-chain mature forms of 45 and 43 kDa and two double-chain mature forms of 34 + 9 kDa and 30 + 14 kDa, expressed by the parental cell line was similar to that found in normal rat liver cells. By contrast, in the ras-transfected counterpart this pattern was profoundly altered in that the 45 kDa species was much less represented and the 30 + 14 kDa species virtually absent. In both untransformed and ras-transformed cells the conversion of proCD into mature forms was not inhibited by ammonium chloride, which is known to increase the intravacuolar pH of post-Golgi compartments. Yet, this drug induced the accumulation of the 43 and 45 kDa molecular forms of mature CD in ras-transformed cells and of the 34 kDa molecule in untransformed cells. As compared to controls, in ras-transformed fibroblasts vacuolar compartments containing CD were reduced in number and mostly located toward the periphery of the cell. This contrasted with the perinuclear distribution of CD-positive granules in untransformed cells. Serum deprivation did not affect the growth, nor the intra- and extracellular accumulation of CD activity in ras-transformed cultures, while it blocked the growth and strongly stimulated the accumulation of CD in the medium in cultures of control fibroblasts. Altogether these data are indicative for a crucial role of ras GTPase in the regulation of the transport between post-Golgi organelles.
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PMID:Transformation by oncogenic ras-p21 alters the processing and subcellular localization of the lysosomal protease cathepsin D. 1032 36

Rab5 regulates endocytic membrane traffic by specifically recruiting cytosolic effector proteins to their site of action on early endosomal membranes. We have characterized a new Rab5 effector complex involved in endosomal fusion events. This complex includes a novel protein, Rabenosyn-5, which, like the previously characterized Rab5 effector early endosome antigen 1 (EEA1), contains an FYVE finger domain and is recruited in a phosphatidylinositol-3-kinase-dependent fashion to early endosomes. Rabenosyn-5 is complexed to the Sec1-like protein hVPS45. hVPS45 does not interact directly with Rab5, therefore Rabenosyn-5 serves as a molecular link between hVPS45 and the Rab5 GTPase. This property suggests that Rabenosyn-5 is a closer mammalian functional homologue of yeast Vac1p than EEA1. Furthermore, although both EEA1 and Rabenosyn-5 are required for early endosomal fusion, only overexpression of Rabenosyn-5 inhibits cathepsin D processing, suggesting that the two proteins play distinct roles in endosomal trafficking. We propose that Rab5-dependent formation of membrane domains enriched in phosphatidylinositol-3-phosphate has evolved as a mechanism for the recruitment of multiple effector proteins to mammalian early endosomes, and that these domains are multifunctional, depending on the differing activities of the effector proteins recruited.
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PMID:Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain. 1106 61

Salmonella typhimurium is a facultative intracellular pathogen that colonizes host cells throughout the course of infection. A unique feature of this pathogen is its ability to enter into (invade) epithelial cells and elongate the vacuole within which it resides into tubular structures called Salmonella-induced filaments (Sifs). In this study we sought to characterize the mechanism of Sif formation by immunofluorescence analysis using subcellular markers. The late endosomal lipid lysobisphosphatidic acid associated in a punctate pattern with the Salmonella-containing vacuole, starting 90 min after infection and increasing thereafter. Lysobisphosphatidic acid-rich vesicles were also found to interact with Sifs, at numerous sites along the tubules. Similarly, cholesterol-rich vesicles were also found in association with intracellular bacteria and Sifs. The lysosomal hydrolase cathepsin D was present in Sifs, both in a punctate pattern and, at later times, predominantly in an uninterrupted linear pattern. Rab7 associated with Sifs and expression of the N125I dominant negative mutant of this GTPase inhibited Sif formation. Transfection of HeLa cells with a vector encoding SifA fused to the green fluorescent protein caused swelling and aggregation of lysobisphosphatidic acid-containing compartments, suggesting that this virulence factor directs membrane fusion events involving late endosomes. Our findings demonstrate that Sif formation involves fusion of late endocytic compartments with the Salmonella-containing vacuole, and suggest that SifA modulates this event.
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PMID:Characterization of Salmonella-induced filaments (Sifs) reveals a delayed interaction between Salmonella-containing vacuoles and late endocytic compartments. 1155 18

The functioning of the endocytic pathway is influenced by a distinct set of rab GTPases, including rab5a, which regulates homotypic fusion of early endosomes. Expression of a dominant active, GTPase-defective rab5a accelerates endosome fusion, causing the formation of a greatly enlarged endocytic compartment. Here we present evidence that rab5a also regulates trafficking between endosomes and lysosomes and may play a role in lysosome biogenesis. The GTPase defective rab5aQ79L mutant was inducibly expressed as an EGFP fusion in HEK293 cells, and the distribution of lysosome proteins and endocytic markers then assessed by deconvolution fluorescence microscopy. During expression of EGFP-rab5aQ79L, the lysosome proteins LAMP-1, LAMP-2 and cathepsin D were found in dilated EGFP-rab5aQ79L-positive vesicles, which also rapidly labeled with transferrin Texas Red. Exogenous tracers that normally traffic to lysosomes after prolonged chase (dextran Texas Red and DiI-LDL) also accumulated in these vesicles. Dextran Texas Red preloaded into lysosomes localized with subsequently expressed EGFP-rab5a Q79L, suggesting the existence of lysosome to endosome traffic. Cells expressing EGFP-rab5a wt or the dominant negative EGFP-rab5aS34N did not exhibit these abnormalities. Despite the dramatic alterations in lysosome protein distribution caused by expression of EGFP-rab5a Q79L, there was little change in the endocytosis or recycling of a cell-surface receptor (beta2-adrenergic receptor). However, there was a deficiency of dense beta-hexosaminidase-containing lysosomes in cells expressing EGFP-rab5aQ79L, as assessed by Percoll gradient fractionation. These results suggest that expression of a GTPase-defective rab5a affects lysosome biogenesis by alteration of traffic between lysosomes and endosomes.
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PMID:Lysosome proteins are redistributed during expression of a GTP-hydrolysis-defective rab5a. 1179 15

Small GTPase RhoA regulates signal transduction from receptors in the membrane to a variety of cellular events related to cell morphology, motility, cytoskeletal dynamics, cytokinesis, and tumour progression, but it is unclear how RhoA regulates intracellular membrane dynamics of lysosomes. We showed previously by confocal immunofluorescence microscopy that the transfection of dominant active RhoA in MM1 cells causes the dispersal translocation of lysosomes stained for cathepsin D throughout the cytoplasm. Y-27632, a selective inhibitor of p160ROCK, impeded the cellular redistribution of lysosomes and promoted reclustering of lysosomes toward the perinuclear region. Here we have further investigated whether the acidic lysosomal vesicles dispersed throughout the cytoplasm are applied to the early endosomes in the endocytic pathway, and we demonstrate that the dispersed lysosomes were accessible to endocytosed molecule such as dextran, and their acidity was not changed, as determined by increased accumulation of the acidotropic probe LysoTracker Red. Brefeldin A did not induce the tabulation of these dispersed lysosomes, but it caused early endosomes to form an extensive tubular network. The dispersed lysosomes associated with cathepsin D and LIMPII were not colocalized with early endosomes, and these vesicles were not inaccessible to the endocytosed anti-transferrin receptor antibody. Moreover, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, induced a dramatic change in LIMPII-containing structures in which LIMPII-positive swollen large vacuoles were increased and small punctate structures disappeared in the cytoplasm. These swollen vacuoles were not doubly positive for LIMPII and transferrin receptor, and were not inaccessible to the internalized anti-transferrin receptor antibody. Therefore, our novel findings presented in this paper indicate that RhoA activity causes a selective translocation of lysosomes without perturbing the machinery of endocytic pathway.
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PMID:A role for small GTPase RhoA in regulating intracellular membrane traffic of lysosomes in invasive rat hepatoma cells. 1258 97

Small GTPase Rho and its downstream effectors, ROCK family of Rho-associated serine-threonine kinases, are thought to participate in cell morphology, motility, and tumor progression through regulating the rearrangement of actin cytoskeleton. Here we present evidence that transfection of human breast cancer cells with cDNA encoding a dominant active mutant of ROCK causes dispersal of lysosomal vesicles throughout the cytoplasm without perturbing the machinery of the endocytic pathway. The intracellular distribution of lysosomes and endocytosed transferrin, an early endosomal marker, were further assessed by confocal immunofluorescence microscopy. In the active ROCK transfected cells the lysosomal proteins, cathepsin D, LIMPII, and LAMP1, were found throughout the cytoplasm in dispersed small vesicles, which were accessible to the endocytosed Texas Red-labeled transferrin. 3D-image analysis of lysosomal distribution in the active ROCK transfectants revealed abundant punctate signals in the peripheral region of the basal plasma membrane. Cells expressing vector alone did not exhibit these alterations. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, induced LIMPII-positive/ transferrin negative large vacuoles in the perinuclear region, and disappearence of the dispersed small vesicular structures. To our knowledge, this is the first evidence that increasing ROCK expression contributes to selective cellular dispersion of lysosomes in invasive breast cancer cells.
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PMID:Overexpression of ROCK in human breast cancer cells: evidence that ROCK activity mediates intracellular membrane traffic of lysosomes. 1285 12


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