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)

To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed (24 h) to media containing leupeptin (0-10 mM), E 64 (0-10 mM), or chloroquine (0-50 microM). Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D (Mr 53,000) within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D (Mr 48,000). Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate (not detectable in control fibroblasts). Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation.
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PMID:Effects of cysteine protease inhibitors on rabbit cathepsin D maturation. 261 Feb 47

After isolating monoclonal antibodies specific for the 52-kDa precursor of cathepsin D (cath-D), which is secreted in excess in both hormone-dependent and hormone-independent breast cancer, we developed a two-step double-determinant immunoenzymometric assay that is specific for this pro-enzyme. The assay combines the use of a monoclonal antibody specific for the precursor and bound to microtiter plates, and a second antibody directed against a smaller processed form of the mature enzyme, coupled to alkaline phosphatase. The specificity of the assay relies on separate and sequential additions of the antigen and the conjugated second antibody. It allows rapid measurement of the analyte in plasma and cytosols of normal and neoplastic mammary tissues, with a detection limit of 5 fmol and a maximal interassay coefficient of variation of 9%. This assay is particularly useful for tissue cytosol samples where the pro-enzyme form co-exists with large quantities of the mature processed forms of the enzyme. Comparative assays of 52-kDa pro-cath-D and total cath-D in cytosols of breast cancers and benign mastopathies indicate that the present assay better discriminates between benign and cancerous mammary tumors.
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PMID:A two-site immunoenzymometric assay of 52-kDa pro-cathepsin D, and its use in human breast diseases. 264 58

Cathepsin D from normal (Hs578Bst) and malignant (MCF7, MDA-MB-231) breast cell lines has been characterized with regard to its kinetic properties, activity levels, precursor and processed M(r) forms, and isoform composition. Normal cell cathepsin D appears to have a more neutral pH optimum (pH 3.5) than the cancer cell line (pH 3.0-3.2) and greater activity between pH values of 4.0 to 4.5. The two cancer cell lines have approximately 1.5 to 2.0-fold increased total acid protease activity and 2 to 3-fold increased pepstatin-inhibitable protease activity (i.e. cathepsin D) when compared to the normal breast cell line. Western blotting indicates that a major processed form of cathepsin D for all three cell lines occurs at 31 kDa. The cancer cell lines contain significant amounts of cathepsin D precursors of 47 and 42 kDa whereas the normal cell line contains little if any of these precursors. Isoelectric focusing indicates that the normal cell line contains approximately 50% of its total acid protease activity at pIs above 4 whereas the cancer cell lines contain 70-80% of their protease activity at such pIs. In addition, the cancer cell lines contain two to three major isoforms between pIs of 5.5 and 6.3 which were not present in the normal cell line. The isoforms from pI values of 5.5 to 7.3 for all three cell lines are 100% pepstatin-inhibitable. In addition, Western blot analysis indicates that these isoforms contain the processed 31 kDa form of cathepsin D. The combined results indicate that the two breast cancer cell lines are similar to biopsied malignant breast tissue in exhibiting altered acid protease isoform profiles with increased relative amounts of pepstatin-inhibitable and immunoreactive acid protease activity (cathepsin D) compared to normal breast tissue or cells.
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PMID:Western blotting and isoform analysis of cathepsin D from normal and malignant human breast cell lines. 764 43

The structure of rabbit procathepsin E was determined by molecular cloning of its cDNA. The proenzyme consisted of 379 amino acids and had structural features common to human and guinea-pig procathepsin E species. The highly conserved tripeptide sequence at the active site of aspartic proteinases, Asp-Thr(Ser)-Gly, is, however, replaced by Asp-Thr-Val in rabbit procathepsin E. To our knowledge, this is the first case of such a variation in aspartic proteinases. The processed form, cathepsin E, hydrolyzed various biologically active peptides maximally at around pH5. Tachykinins, such as substance P and neurokinin A, were hydrolyzed most rapidly, with specific cleavage of sequences essential for their activity. The rates of hydrolysis were several hundred-fold higher than those of cathepsin D. Furthermore, cathepsin E was able to inactivate a functional-domain peptide of fibroblast growth factor, the sequence of which resembles those of tachykinins, and it was active in the generation of functional peptides, such as endothelin and angiotensin I, from their respective precursors. Procathepsin E was detected at high levels in various fetal tissues, such as the liver, stomach and blood cells. At the adult stage, the proenzyme was detectable only in specific tissues, such as the urinary bladder, duodenum and colon. Northern-blot analysis showed similar stage-specific and tissue-specific expression of the mRNA for procathepsin E. Since tachykinins and other suited peptide substrates of cathepsin E have been shown to have mitogenic activity, (pro)cathepsin E may regulate the growth and differentiation of embryonic and fetal tissues by degrading or processing these peptides. The enzyme may also regulate the physiological activities of adult tissues which are mediated by substance P and related tachykinins.
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PMID:Rabbit procathepsin E and cathepsin E. Nucleotide sequence of cDNA, hydrolytic specificity for biologically active peptides and gene expression during development. 840 90

Cathepsin D carries a mannose 6-phosphate sorting signal which is recognized by a specific mannose 6-phosphate receptor, presumably at the site of the trans Golgi network, which segregates cathepsin D from the secretory proteins, and results in targeting of the enzyme to the acidic prelysosomal compartments and lysosomes in mammalian cells. Recent evidence implies that another sorting signal resides within the polypeptide backbone of the precursor cathepsin D. To evaluate the role of the propeptide region of cathepsin D in mannose 6-phosphate receptor-independent targeting to lysosomes, we prepared a deletion mutant of rat cathepsin D lacking the propeptide portion and analyzed its intracellular targeting mechanism after transfection of the mutant cDNA as well as the wild-type cDNA into COS cells. The glycosylated mutant protein was retained intracellularly, and extracellular release of mutant protein was not observed after a 48 h chase. A cell fractionation experiment demonstrated that in the cells expressing the wild-type cathepsin D, the processed form of 44 kDa cathepsin D was recovered in the dense lysosomal fraction. In contrast, in the cells expressing the mutant protein, virtually all of the cell-associated cathepsin D was present in the light fraction which was enriched in the marker enzyme NADPH cytochrome c reductase, and this molecular form of cathepsin D was not observed in the dense lysosomal fraction. An immunofluorescence study revealed that the deletion mutant protein was accumulated within the endoplasmic reticulum, unlike the wild-type protein. These results suggest that the mutant cathepsin D is not correctly recognized by the intracellular sorting system in the endoplasmic reticulum, implying that the propeptide region of cathepsin D is essential for the export of cathepsin D from the endoplasmic reticulum.
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PMID:Intracellular targeting of lysosomal cathepsin D in COS cells. 874 16

Lysosomes are considered to be a terminal degradative compartment of the endocytic pathway, into which transport is mostly unidirectional. However, specialized secretory vesicles regulated by Ca2+, such as neutrophil azurophil granules, mast cell-specific granules, and cytotoxic lymphocyte lytic granules, share characteristics with lysosomes that may reflect a common biogenesis. In addition, the involvement of Ca2+ transients in the invasion mechanism of the parasite Trypanosoma cruzi, which occurs by fusion of lysosomes with the plasma membrane, suggested that lysosome exocytosis might be a generalized process present in most cell types. Here we demonstrate that elevation in the intracellular free Ca2+ concentration of normal rat kidney (NRK) fibroblasts induces fusion of lysosomes with the plasma membrane. This was verified by measuring the release of the lysosomal enzyme beta-hexosaminidase, the appearance on the plasma membrane of the lysosomal glycoprotein lgp120, the release of fluid-phase tracers previously loaded into lysosomes, and the release of the lysosomally processed form of cathepsin D. Exposure to the Ca2+ ionophore ionomycin or addition of Ca2+-containing buffers to streptolysin O-permeabilized cells induced exocytosis of approximately 10% of the total lysosomes of NRK cells. The process was also detected in other cell types such as epithelial cells and myoblasts. Lysosomal exocytosis was found to require micromolar levels of Ca2+ and to be temperature and ATP dependent, similar to Ca2+-regulated secretory mechanisms in specialized cells. These findings highlight a novel role for lysosomes in cellular membrane traffic and suggest that fusion of lysosomes with the plasma membrane may be an ubiquitous form of Ca2+-regulated exocytosis.
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PMID:Lysosomes behave as Ca2+-regulated exocytic vesicles in fibroblasts and epithelial cells. 910 39

Synaptotagmins (Syts) I and II are believed to act as Ca2+ sensors in the control of neurotransmission. Here we demonstrate that mast cells express Syt II in their lysosomal fraction. We further show that activation of mast cells by either aggregation of FcepsilonRI or by Ca2+ ionophores results in exocytosis of lysosomes, in addition to the well documented exocytosis of their secretory granules. Syt II directly regulates lysosomal exocytosis, whereby overexpression of Syt II inhibited Ca2+-triggered release of the lysosomal processed form of cathepsin D, whereas suppression of Syt II expression markedly potentiated this release. These findings provide evidence for a novel function of Syt II in negatively regulating Ca2+-triggered exocytosis of lysosomes, and suggest that Syt II-regulated secretion from lysosomes may play an important role in mast cell biology.
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PMID:Synaptotagmin II negatively regulates Ca2+-triggered exocytosis of lysosomes in mast cells. 1033 Apr 44

We tested the hypothesis that chronically ischemic (IS) myocardium induces autophagy, a cellular degradation process responsible for the turnover of unnecessary or dysfunctional organelles and cytoplasmic proteins, which could protect against the consequences of further ischemia. Chronically instrumented pigs were studied with repetitive myocardial ischemia produced by one, three, or six episodes of 90 min of coronary stenosis (30% reduction in baseline coronary flow followed by reperfusion every 12 h) with the non-IS region as control. In this model, wall thickening in the IS region was chronically depressed by approximately 37%. Using a nonbiased proteomic approach combining 2D gel electrophoresis with in-gel proteolysis, peptide mapping by MS, and sequence database searches for protein identification, we demonstrated increased expression of cathepsin D, a protein known to mediate autophagy. Additional autophagic proteins, cathepsin B, heat shock cognate protein Hsc73 (a key protein marker for chaperone-mediated autophagy), beclin 1 (a mammalian autophagy gene), and the processed form of microtubule-associated protein 1 light chain 3 (a marker for autophagosomes), were also increased. These changes, not evident after one episode, began to appear after two or three episodes and were most marked after six episodes of ischemia, when EM demonstrated autophagic vacuoles in chronically IS myocytes. Conversely, apoptosis, which was most marked after three episodes, decreased strikingly after six episodes, when autophagy had increased. Immunohistochemistry staining for cathepsin B was more intense in areas where apoptosis was absent. Thus, autophagy, triggered by ischemia, could be a homeostatic mechanism, by which apoptosis is inhibited and the deleterious effects of chronic ischemia are limited.
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PMID:Autophagy in chronically ischemic myocardium. 1617 25

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