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)

Antigen presentation requires intracellular processing of native antigens to produce immunogenic peptides that bind to major histocompatibility complex class II (MHC-II) molecules. In functional studies of antigen processing by elicited peritoneal macrophages, MHC-II-peptide complexes were formed intracellularly. Immunogenic peptides were not released to bind surface MHC-II molecules. Ultrastructural studies employing immunogold staining in ultrathin cryosections of these macrophages showed large amounts of MHC-II molecules in intracellular sac-like vacuoles in the peripheral cytoplasm; most of these were negative for the lamp 1 lysosomal/endosomal membrane protein and cathepsin D. MHC-II molecules were also present in endosomes containing cathepsin D and lamp 1 as well as previously internalized gold-transferrin. The intracellular pool of MHC-II molecules was only slightly decreased by treatment with cycloheximide for 3 hr, indicating that it consisted mainly of endocytosed, recycling molecules, as opposed to nascent ones. These ultrastructural studies support the notion that there is endocytosis of MHC-II molecules into endocytic compartments, consistent with our earlier biochemical data. Furthermore, we have defined the distinct endocytic compartments that must mediate important functions in antigen processing, including the formation of MHC-II-peptide complexes.
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PMID:Functional and ultrastructural evidence for intracellular formation of major histocompatibility complex class II-peptide complexes during antigen processing. 237 Dec 88

The polarized delivery of membrane proteins to the cell surface and the initial secretion of lysosomal proteins into the culture medium were studied in the polarized human intestinal adenocarcinoma cell line Caco-2 in the presence or absence of the microtubule-active drug nocodazole. The appearance of newly synthesized proteins at the plasma membrane was measured by their sensitivity to proteases added either to the apical or the basolateral surface of cells grown on nitrocellulose filters. Nocodazole was found to reduce the delivery to the cell surface of an apical membrane protein, aminopeptidase N, and to lead to its partial missorting to the basolateral surface, whereas the drug had no influence on the delivery of a basolateral 120-kD membrane protein defined by a monoclonal antibody. Furthermore, nocodazole selectively blocked the apical secretion of two lysosomal proteins, cathepsin D and acid alpha-glucosidase, whereas the drug had no influence on their basolateral secretion. These results suggest that in Caco-2 cells an intact microtubular network is important for the transport of newly synthesized proteins to the apical cell surface.
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PMID:Nocodazole, a microtubule-active drug, interferes with apical protein delivery in cultured intestinal epithelial cells (Caco-2). 264 10

Myelin basic protein (MBP), an extrinsic membrane protein from the myelin sheath, binds dicyanohemin. The binding generates absorption bands in the Soret region and quenches the fluorescence emitted by the sole tryptophan residue. The absorption titration curves in the Soret demonstrate that the binding is stoichiometric, one heme per protein, with a large value of the extinction coefficient (8 X 10(4) M-1 cm-1 at 420 nm). Fluorescence quenching titration curves indicate an identical stoichiometry and a low quenching efficiency of 20%. From the heme titration curve the association constant between dicyanohemin and MBP is estimated to be greater than or equal to 10 nM-1 in 50 mM 4-morpholinepropanesulfonic acid buffer, pH 7.0, at 20 degrees C. Digestion of MBP by Staphylococcus aureus V8 protease yields a peptide (38-118) whose heme binding properties are identical to those of MBP. In contrast, peptides obtained by digestion of MBP with cathepsin D do not exhibit any specific binding of dicyanohemin. The cleavage of the Phe-Phe (42-43) bond appears to be critical in this respect. A comparison of the sequence immediately preceding, including these residues with a probable heme binding site of a mitochondrial cytochrome b, reveals a high degree of homology. The possible significance of heme binding is discussed.
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PMID:A heme binding site on myelin basic protein: characterization, location, and significance. 620 38

We have previously demonstrated that human peripheral blood low density mononuclear cells cultured in granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 develop into dendritic cells (DCs) that are extremely efficient in presenting soluble antigens to T cells. To identify the mechanisms responsible for efficient antigen capture, we studied the endocytic capacity of DCs using fluorescein isothiocyanate-dextran, horseradish peroxidase, and lucifer yellow. We found that DCs use two distinct mechanisms for antigen capture. The first is a high level of fluid phase uptake via macropinocytosis. In contrast to what has been found with other cell types, macropinocytosis in DCs is constitutive and allows continuous internalization of large volumes of fluid. The second mechanism of capture is mediated via the mannose receptor (MR), which is expressed at high levels on DCs. At low ligand concentrations, the MR can deliver a large number of ligands to the cell in successive rounds. Thus, while macropinocytosis endows DCs with a high capacity, nonsaturable mechanism for capture of any soluble antigen, the MR gives an extra capacity for antigen capture with some degree of selectivity for non-self molecules. In addition to their high endocytic capacity, DCs from GM-CSF + IL-4-dependent cultures are characterized by the presence of a large intracellular compartment that contains high levels of class II molecules, cathepsin D, and lysosomal-associated membrane protein-1, and is rapidly accessible to endocytic markers. We investigated whether the capacity of DCs to capture and process antigen could be modulated by exogenous stimuli. We found that DCs respond to tumor necrosis factor alpha, CD40 ligand, IL-1, and lipopolysaccharide with a coordinate series of changes that include downregulation of macropinocytosis and Fc receptors, disappearance of the class II compartment, and upregulation of adhesion and costimulatory molecules. These changes occur within 1-2 d and are irreversible, since neither pinocytosis nor the class II compartment are recovered when the maturation-inducing stimulus is removed. The specificity of the MR and the capacity to respond to inflammatory stimuli maximize the capacity of DCs to present infectious non-self antigens to T cells.
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PMID:Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. 762 94

Chediak-Higashi Syndrome (CHS) is an autosomal recessive disease affecting secretory granules and lysosomes-like organelles. In CHS fibroblasts, acidic organelles are abnormally large and clustered in the perinuclear area. We have analyzed fibroblast cell lines from a CHS patient and from the murine model for CHS, the beige mouse, to determine which lysosome-like compartments are affected. Uptake of neutral red showed that in both beige and CHS cell lines, the acidic organelles were markedly clustered in the perinuclear region of the cells. Giant organelles (> 4 microns) were observed in a fraction of the cells, and these were more dramatic in the beige fibroblasts than in the CHS fibroblasts. The total dye uptake of both mutant cell lines was similar to their respective wild type fibroblasts, suggesting that the overall volume of acidic compartments is unaffected by the disorder. Histochemistry and immunofluorescence showed that the giant organelles in both beige and CHS fibroblasts were positive for cathepsin D, lysosome-associated membrane protein (LAMP) 1, LAMP 2, and a 120-kD lysosomal glycoprotein, all marker proteins for late endosomes and lysosomes. The giant organelles were also negative for transferrin receptor and mannose-6-phosphate receptor, and most of them were also negative for rab 7. This distribution of marker proteins shows that the giant organelles in both beige and CHS are derived from late compartments of the endocytic pathway. This conclusion was confirmed using endocytic tracers. BSA was transported to the giant organelles, but only after long incubation times, and only at 37 degrees C. alpha 2-Macroglobulin was taken up and degraded at similar rates by CHS or beige cells and their respective wild type control cells. Taken together, our results indicate that the mutation in CHS specifically affects late endosomes and lysosomes, with little or no effect on early endosomes. Although the mutation clearly causes mislocalization of these organelles, it appears to have little effect on their endocytic and degradative functions.
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PMID:The giant organelles in beige and Chediak-Higashi fibroblasts are derived from late endosomes and mature lysosomes. 790 7

Epithelial cells in situ can internalize their desmosomes. This can be induced in cell cultures after removal of calcium ions from the cell medium. To study this endocytic process, a nontumorigenic human breast epithelial cell line, HMT-3522, was used. HMT-3522 cells were grown in serum-free, chemically defined medium, containing epidermal growth factor (EGF). Removal of EGF from the medium led to growth arrest and a kind of epithelial differentiation process in which adjacent cells interdigitated and formed more desmosomes than in the proliferating state. Growth-inhibited HMT-3522 cells dissociated following EGTA treatment, the desmosomes divided in a symmetrical fashion, and the desmosomal plaques (half-desmosomes) on the cell surface became internalized. The internalization was independent of clathrin, since immunogold labeling of ultracryosections never showed clathrin on desmosomal plaque-associated membrane domains. Moreover, cytosol acidification, which selectively inhibits endocytosis from clathrin-coated pits, practically blocked the uptake of transferrin, whereas internalization of desmosomal plaques continued. In contrast, actin filaments appeared to be involved in the desmosomal internalization. Thus, depolymerization of actin filaments by cytochalasin D significantly reduced endocytosis of half-desmosomes. Immunogold labeling showed that the vesicles with desmosomal plaques were not enriched in MPR (cation-independent mannose-6-phosphate receptor), cathepsin D or the lysosome-associated membrane protein lamp-1. In addition, the morphology was different. Thus, the endocytic vesicles with desmosomal plaques represent a special compartment, distinct from typical endosomes and lysosomes.
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PMID:Endocytosis of desmosomal plaques depends on intact actin filaments and leads to a nondegradative compartment. 792 92

We have compared the intracellular transport and subcellular distribution of MHC class II-invariant chain complexes in a wild-type HLA-DR3 homozygous cell line and a mutant cell line, T2.DR3. The latter has a defect in antigen processing and accumulates HLA-DR3 molecules associated with an invariant chain-derived peptide (CLIP) rather than the normal complement of peptides derived from endocytosed proteins. We find that in the wild-type cells, CLIP is transiently associated with HLA-DR3 molecules, suggesting that the peptide is a normal class II-associated intermediate generated during proteolysis of the invariant chain. In the mutant cell line proteolysis of the invariant chain is less efficient, and HLA-DR3/CLIP complexes are generated much more slowly. Examination of the mutant cell line by immunoelectronmicroscopy shows that class II-invariant chain complexes accumulate intracellularly in large acidic vesicles which contain lysosomal markers, including beta-hexosaminidase, cathepsin D, and the lysosomal membrane protein CD63. The markers in these vesicles are identical to those seen in the class II-containing vesicles (MIICs) seen in the wild-type cells but the morphology is drastically different. The vesicles in the mutant cells are endocytic, as measured by the internalization of BSA-gold conjugates. The implication of these findings for antigen processing in general and the nature of the mutation in particular are discussed.
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PMID:Transport and intracellular distribution of MHC class II molecules and associated invariant chain in normal and antigen-processing mutant cell lines. 820 55

The membrane-association of early biosynthetic form of cathepsin D has been demonstrated in hepatoma cells, and this membrane-association is not mediated by mannose 6-phosphate residues, implying that a mannose 6-phosphate receptor-independent mechanism operates in the sorting of cathepsin D. In this paper, to demonstrate whether cathepsin D is associated with the lysosomal membranes, an in vitro binding experiment was carried out employing lysosomal cathepsin D or microsomal procathepsin D isolated from rat liver. Immunoblotting analysis revealed that an intermediate form of cathepsin D was associated with the lysosomal membranes; this lysosomal membrane-associated cathepsin D was released from the membranes by washing with Na2CO3 (pH 10.6) but not with solutions containing mannose 6-phosphate. This suggested that cathepsin D associates with the membranes by ionic-interaction, and that the membrane-associated cathepsin D resides as a peripheral membrane protein in the lysosomal membrane fraction. To confirm that the intermediate form of cathepsin D specifically interacts with the lysosomal integral membrane proteins, the lysosomal membrane fraction was treated with trypsin and the binding experiment was conducted. The result showed that the binding capacity of cathepsin D to the lysosomal membranes was apparently abolished and cathepsin D did not rebind to the membranes. These data suggest that the intermediate form of cathepsin D is preferentially recognized by the lysosomal membranous protein which complements the mannose 6-phosphate receptor-dependent intracellular sorting mechanism.
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PMID:Cathepsin D associates with lysosomal membranous protein. 859 33

We studied the biogenesis of the acrosome in sperm cells in immunogold-labeled ultrathin cryosections of rat testis, using a variety of antibodies against endosomal/lysosomal marker protein and acrosin, the major secretory protein of sperm cells. As expected, acrosomes and proacrosomal vesicles in the trans-Golgi region contained abundant acrosin. Rat lysosomal membrane glycoprotein (lgp) 120 and mouse lysosome-associated membrane protein-1 were not detectable in the acrosomal membrane. Similarly, the late endosomal markers cation-dependent and -independent mannose 6-phosphate receptors were absent from the acrosome and proacrosomal vesicles. Therefore, acrosomes do not exhibit these endosomal/lysosomal features. Apart from (pro) acrosomal vesicles, both spermatocytes and spermatids contained classical lysosomes (positive for rat lgp 120, mouse lysosome-associated membrane protein-1, and cathepsin D) that were negative for acrosin. Quantitative analysis of the immunogold labeling showed that spermatocytes express more mannose 6-phosphate receptors and lgp 120 than spermatids, whereas the opposite situation existed for acrosin. These data indicate differential synthetic activity of lysosomal and acrosomal constituents in different states of sperm differentiation. Together, our observations argue against a lysosomal /endosomal origin of the acrosome.
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PMID:Evidence for a nonlysosomal origin of the acrosome. 860 90

Melanomas exhibiting mutated ras genes are frequently invasive and amelanotic. Transfecting melanocytes with ras oncogenes causes transformation and a loss of visible pigmentation. We analyzed murine melanocytes rendered amelanotic by transfection with the v-rasHa oncogene. Consistent with previous reports, tyrosinase and tyrosinase-related protein-1 (TRP-1) were not expressed by transformed cells. In addition, lack of expression of TRP-2 and the product of the silver locus was documented. Levels of melanosomal matrix antigens, the pink-eyed dilution locus protein and lysosome-associated membrane protein-1 were markedly reduced. Residual matrix antigens were localized by immunofluorescence to large vacuoles distributed peri-nuclearly in transfected cells. Electron microscopy demonstrated the absence of typical melanosomes and the presence of large vacuolar structures, also in a peri-nuclear distribution. Although levels of lysosomal hydrolases, such as beta-glucuronidase and cathepsin D, were diminished, marked elevations were observed in the expression of cathepsins B and L, 2 thiol proteases implicated in the acquisition of invasiveness. Our data demonstrate that transfection of melanocytes with v-rasHa is sufficient to disrupt the biogenesis of melanosomes and to up-regulate thiol protease synthesis, providing insights into the amelanotic and invasive nature of melanomas exhibiting mutations in ras genes.
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PMID:Melanosomal and lysosomal alterations in murine melanocytes following transfection with the v-rasHa oncogene. 863 74


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