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)

We used a combination of subcellular fractionation and lactoperoxidase-mediated iodination to examine the polypeptide compositions of three hepatocyte endocytic compartments: early endosomes, late endosomes, and lysosomes. A chemical conjugate of asialoorosomucoid and lactoperoxidase which binds specifically to asialoglycoprotein receptors was perfused through isolated rat livers at 37 degrees C. Subcellular fractions enriched in various endocytic compartments were then isolated by differential and isopycnic centrifugation, and the lactoperoxidase moiety of the internalized conjugate was used to catalyze the iodination of lumenal-facing proteins. The 125I profiles of early and late endosomes were strikingly similar after gel electrophoresis. Using immunoprecipitation, we directly identified and compared the relative amounts of the Na+,K(+)-ATPase and several different acid hydrolases and membrane receptors in all three fractions. The asialoglycoprotein receptor and the low density lipoprotein related protein were approximately nine times more abundant in early endosomes than late endosomes, suggesting that they recycle from early endosomes. In addition, cathepsin D, but not cathepsin L, beta-glucuronidase, and lgp 120, was detected in early endosomes; however, all of these molecules were detected in lysosomes. Our findings provide strong evidence that early endosomes mature into late endosomes and that there is either selective delivery or selective retention of hydrolases at discrete points in the endocytic pathway.
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PMID:Lumenal labeling of rat hepatocyte endocytic compartments. Distribution of several acid hydrolases and membrane receptors. 131 3

Effects of bafilomycin A1, an inhibitor of vacuolar H(+)-ATPase, on the synthesis and processing of cathepsin D and cathepsin H were investigated in primary cultured rat hepatocytes. Pulse-chase experiments showed that after being synthesized as procathepsin D and procathepsin H the precursors were converted into mature forms in the control cells as the chase time elapsed. However, in the presence of 5 x 10(-7) M of bafilomycin A1, both precursors were largely secreted into the medium and no mature forms were found within the cells. Thus bafilomycin A1 mimics lysosomotropic amines with regard to perturbation of the targeting of lysosomal acid hydrolases. In contrast, bafilomycin A1 was found not to inhibit processings of proalbumin and procomplement component 3, which are thought to occur at the acidic trans-Golgi, implying that the proteolytic event of the proproteins is not sensitive to an increase of intra-Golgi pH. The results suggest that bafilomycin A1 is useful as a pH-perturbant to study the role of acidity in living cells.
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PMID:Bafilomycin A1 inhibits the targeting of lysosomal acid hydrolases in cultured hepatocytes. 206 75

Homogenates of the posterior latissimus dorsi muscle, a phasic muscle, were fractionated by a one-step zonal centrifugation technique into four major organelle populations and cytoplasmic constituents. These were: (1) Plasma membrane fragments with a modal equilibrium density of 1.10 and containing 5'-nucleotidase, alkaline phosphodiesterase, p-nitrophenylphosphatase and acid phosphatase (beta-glycerophosphate was used as the substrate). (2) Sarcoplasmic reticular fragments which could be further subdivided into calcium transport vesicles, with a model equilibrium density of 1.16, that exhibited calcium uptake; K+-ATPase; leucyl-bet-naphthylamidase; acid phosphodiesterase; acid phosphatase (using cytidine monophosphate as the substrate); and sarcoplasmic reticular lysosomes, with a model equilibrium density of 1.18, possessing dipeptidyl-aminopeptidase II, cathepsin D, alpha-glucosidase, N-acetyl-beta-glucosaminidase, and NADH oxidase activity. (3) Mitochondria with a modal equilibrium density of 1.21. (4) Catalase-containing vesicles with a modal equilibrium density of 1.22; and cytoplasmic constituents (modal density of 1.25) with phosphorylase, pyruvate kinase, myosin-ATPase, aldolase, and protein and RNA content. The purity of these organelles was equal to or better than previous efforts, with a 30-fold purification achieved for 5'-nucleotidase and alkaline phosphodiesterase. These results lend support to the hypothesis that the sarcoplasmic reticulum of phasic muscle, in addition to its specialized role in excitation-contraction coupling, represents a multifunctional membrane system, and that, similar to the smooth endoplasmic reticulum of other cells, it includes some membrane-bound lysosomal enzymes and NADH oxidase.
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PMID:Isopycnic-zonal centrifugation of plasma membrane, sarcoplasmic reticular fragments, lysosomes, and cytoplasmic proteins from phasic skeletal muscle. 721 87

Human metastatic breast cancer cells in culture contain large acidic vesicles (diameter 5-10 microns) in which endocytosed extracellular matrix can be digested by activated lysosomal proteinases such as cathepsin D (P. Montcourrier et al. (1990). Cancer Res. 50, 6045-6054). We examined these large compartments by transmission electron microscopy, measured their pH by video-enhanced epifluorescence using FITC-dextran, and studied their functional significance. Their presence in metastatic MDA-MB231 cells was found to be correlated with an increased ability of cells to migrate through Matrigel and a high cathepsin D concentration. These cells were able to phagocytose 1.24 microns diameter latex beads and fluorescence Matrigel and incorporate this extracellular material into large acidic vesicles. This indicated that large acidic vesicles were associated with both phagocytosis and invasion, and are heterophagolysosomes rather than autophagosomes. Large acidic vesicles were actively acidified with a H(+)-ATPase vacuolar pump specifically inhibited by bafilomycin A1, and reached pH values (< 4), lower than the lysosomal value (pH approximately 5) in the same cells and in specialized phagocytotic cells such as macrophages. We conclude that the phagocytotic activity of breast cancer cells, associated with high cathepsin D expression, and high acidification potential, characterize cancer cells that have migrated through Matrigel.
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PMID:Characterization of very acidic phagosomes in breast cancer cells and their association with invasion. 784 58

Inactivation of Na+/K(+)-ATPase by partially reduced oxygen metabolites has been implicated in ischemia-reperfusion injury to heart and other organs. Because oxidation of many proteins makes them more susceptible to degradation by intracellular proteinases, we studied the effects of several such proteinases on native and H2O2-oxidized preparations of Na+/K(+)-ATPase from canine kidney (containing alpha 1 isoform of the catalytic subunit) and rat axolemma (containing alpha 2 and alpha 3 isoforms). Lysosomal cathepsin D degraded the native and the oxidized preparations at acid pH, but it was significantly more effective against the oxidized forms. m-Calpain had little or no effect on the native Na+/K(+)-ATPase preparations, but it digested the oxidized alpha-subunits of the axolemma and the kidney enzymes. mu-Calpain's effects were similar to those of m-calpain. Multi-catalytic proteinase which is known to degrade a large number of oxidized proteins, did not affect the native or the oxidized forms of Na+/K(+)-ATPase. The findings suggest that (a) during oxidative stress there may be accelerated degradation of the oxidatively damaged Na+/K(+)-ATPase, either through internalization and transport to lysosomes, or by the action of calpains at the membrane; and (b) those isoforms of the enzyme that are more sensitive to oxidants are more susceptible to degradation by the above processes.
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PMID:Different sensitivities of native and oxidized forms of Na+/K(+)-ATPase to intracellular proteinases. 820 42

Coxiella burnetii and Chlamydia trachomatis are bacterial obligate intracellular parasites that occupy distinct vacuolar niches within eucaryotic host cells. We have employed immunofluorescence, cytochemistry, fluorescent vital stains, and fluid-phase markers in conjunction with electron, confocal, and conventional microscopy to characterize the vacuolar environments of these pathogens. The acidic nature of the C. burnetii-containing vacuole was confirmed by its acquisition of the acidotropic base acridine orange (AO). The presence of the vacuolar-type (H+) ATPase (V-ATPase) within the Coxiella vacuolar membrane was demonstrated by indirect immunofluorescence, and growth of C. burnetii was inhibited by bafilomycin A1 (Baf A), a specific inhibitor of the V-ATPase. In contrast, AO did not accumulate in C. trachomatis inclusions nor was the V-ATPase found in the inclusion membrane. Moreover, chlamydial growth was not inhibited by Baf A or the lysosomotropic amines methylamine, ammonium chloride, and chloroquine. Vacuoles harboring C. burnetii incorporated the fluorescent fluid- phase markers, fluorescein isothiocyanate-dextran (FITC-dex) and Lucifer yellow (LY), indicating trafficking between that vacuole and the endocytic pathway. Neither FITC-dex nor LY was sequestered by chlamydial inclusions. The late endosomal-prelysosomal marker cation-independent mannose 6-phosphate receptor was not detectable in the vacuolar membranes encompassing either parasite. However, the lysosomal enzymes acid phosphatase and cathepsin D and the lysosomal glycoproteins LAMP-1 and LAMP-2 localized to the C. burnetii vacuole but not the chlamydial vacuole. Interaction of C. trachomatis inclusions with the Golgi-derived vesicles was demonstrated by the transport of sphingomyelin, endogenously synthesized from C6-NBD-ceramide, to the chlamydial inclusion and incorporation into the bacterial cell wall. Similar trafficking of C-NBD-ceramide was not evident in C. burnetii-infected cells. Collectively, the data indicate that C. trachomatis replicates within a nonacidified vacuole that is disconnected from endosome-lysosome trafficking but may receive lipid from exocytic vesicles derived from the trans-Golgi network. These observations are in sharp contrast to those for C. burnetii, which by all criteria resides in a typical phagolysosome.
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PMID:Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis. 864 84

In the human carcinoma cell line HEp-2, endosomes are multivesicular bodies (MVBs) which gradually mature and eventually fuse with other mature endosomes and/or with preexisting lysosomes. Selective inhibition of the vacuolar H(+)-ATPase with bafilomycin A1 (Baf) did not influence endocytic uptake and recycling of the protein toxin ricin. Moreover, quantification of immunogold labeling on ultracryosections revealed that the frequency by which MVBs containing internalized cationized gold (Cat.Au) also labeled for the cation-independent mannose-phosphate receptor (MPR) was the same with and without Baf, suggesting that formation and maturation of MVBs were unchanged in the presence of Baf. However, degradation of ricin was strongly reduced by bafilomycin, and this reduction was not only due to increased pH in lysosomes. Thus, quantitative lmmunogold labeling showed that the Baf-induced alkalinization reduced the transfer of internalized Cat.Au to lysosomes, as defined by their content of human lysosome-associated membrane protein 1 (h-lamp-1) and cathepsin D. Accordingly, although low vacuolar pH does not seem to be required for transport to MPR-containing endosomes, it seems to be important for a late fusion step along the endocytic pathway.
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PMID:Inhibition of the vacuolar H(+)-ATPase with bafilomycin reduces delivery of internalized molecules from mature multivesicular endosomes to lysosomes in HEp-2 cells. 874 Dec 16

We characterized the Mycobacterium marinum phagosome by using a variety of endocytic markers to follow the path of the bacteria through a mouse macrophage cell line. Using a laser confocal microscope, we found that the majority of viable M. marinum cells were in nonacidic vacuoles that did not colocalize with the vacuolar proton ATPase (V-ATPase), the calcium-independent mannose-6-phosphate receptor (CI-M6PR), or cathepsin D. In contrast, heat-killed organisms and latex beads were in acidic vacuoles which contained the V-ATPase, the CI-M6PR, and cathepsin D. A population of vesicles that contained live M. marinum labeled with the lysosomal glycoprotein LAMP-1, but the percentage of vacuoles that labeled was lower than for heat-killed organisms or latex beads. When testing live and heat-killed Mycobacterium tuberculosis, we found levels of colocalization with LAMP- and cathepsin D comparable to those for the M. marinum isolate. We conclude that M. marinum, like M. tuberculosis, can circumvent the host endocytic pathway and reside in an intracellular compartment which is not acidic and does not fuse with lysosomes. In addition, we describe a system for sampling a large population of intracellular organisms by using a laser confocal microscope.
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PMID:Differential trafficking of live and dead Mycobacterium marinum organisms in macrophages. 911 92

Little is known about the development of the central nervous system (CNS) in humans. Ethical considerations preclude experimental studies in this field, and as a result most available data on human ontogenesis are descriptive. Comparative anatomic and embryologic studies have demonstrated that the main developmental milestones are conserved across species, and their results can be used to suggest a likely scenario for human development. The development of the ventricles, meninges, and choroid plexuses are discussed in this article. The central cavity of the neural tube is formed during neurulation, which occurs during the fourth gestational week. The first milestone is occlusion of the spinal neurocele (the central canal in the neural tube) shortly after neurulation. This prevents free communication between the ventricular system and the amniotic cavity. The second milestone is development of the meninges, which separate the central nervous system from the rest of the body. The embryonic origin of the meninges varies across species. In birds (and probably in mammals), the spinal meninges are derived from the somitic mesoderm, the brainstem meninges from the cephalic mesoderm, and the telencephalic meninges from the neural crest. Differentiation of the meninges, which involves formation of the subarachnoid space, occurs early, before the cerebrospinal fluid (CSF) begins to flow around the CNS. During ontogenesis, the meninges play a key role in regulating the growth of underlying nervous structures. They induce the formation of the superficial glial limiting layer and stimulate the growth of precursors located in the superficial blastemas of the cerebellum and hippocampus. The choroid plexuses are complex specialized structures that produce most of the CSF. Their epithelium derives from the neural tube epithelium and their mesenchyma from the meninges. Of the many enzymes produced in the choroid plexuses, some reflect the pivotal metabolic role of these structures (alkaline and acid phosphatases, magnesium-dependent ATPase, glucose-6-phosphatase, thiamine pyrophosphatase, adenylate cyclase, oxidoreductase, esterases, hydrolases, cathepsin D, and glutathion S-transferase). The two enzymes that are crucial to the production of CSF are Na+/K+ ATPase and carbonic anhydrase. Inactivation of catecholamines is mediated by catechol-O-methyltransferase and by the monoamine oxidases A and B. The morphology and synthesis profile of the choroid plexuses changes during development, although little is known about these changes in humans.
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PMID:Embryonic and fetal development of structures associated with the cerebro-spinal fluid in man and other species. Part I: The ventricular system, meninges and choroid plexuses. 975 71

Bordetella pertussis is readily killed after uptake by professional phagocytes, whereas its close relative Bordetella bronchiseptica is not and can persist intracellularly for days. Phagocytosis of members of either species by a mouse macrophage cell line results in transport of the bacteria to a phagosomal compartment positive for the lysosome-associated membrane protein 1, the protease cathepsin D, and the late endosomal vacuolar proton-pumping ATPase but negative for the early endosome antigen 1 and the early endosomal transferrin receptor. In addition, we demonstrate that Bordetella-containing phagosomes rapidly acidify to pH 4.5 to 5.0. Taken together, these data demonstrate that Bordetella-containing phagosomes rapidly mature to an acidic late endosomal/lysosomal compartment. Following up on this observation, we determined that B. pertussis does not survive in bacterial growth media adjusted to a pH of 4.5, whereas this pH has only minor effects on the growth of B. bronchiseptica. Raising the intracellular pH in infected macrophages by the addition of bafilomycin A(1), ammonium chloride, or monensin increases the survival of acid-sensitive B. pertussis but, surprisingly, decreases that of acid-tolerant B. bronchiseptica. In summary, we hypothesize that the differential survival of B. pertussis and B. bronchiseptica in macrophages is, at least in part, due to the differences in their acid tolerance.
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PMID:Phagosome acidification has opposite effects on intracellular survival of Bordetella pertussis and B. bronchiseptica. 1108 29


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