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)

Newly synthesized enzymes destined for lysosomal localization contain mannose 6-phosphate (Man6-P) residues, allowing interaction with Man6-P receptors (MPRs) and subsequent intracellular targeting to the lysosome. In most cultured cells, lysosomal enzymes are rapidly dephosphorylated after targeting, but in some transformed cell lines, these proteins retain the Man6-P marker. To investigate the significance of this in human malignancy, we examined the persistence of the Man6-P marker in human breast biopsy specimens using MPR derivatives as affinity probes. In one approach, extracts of frozen tissue were standardized to protein content, fractionated by SDS-PAGE, immobilized on nitrocellulose, and probed with iodinated MPR. On average, carcinomas contained 4-fold higher levels of Man6-P glycoproteins than did benign tumors or normal breast samples. In about 15% of the carcinomas, levels of Man6-P glycoproteins were highly elevated (7-10-fold). Multiple Man6-P glycoproteins were detected, suggesting a general alteration in the synthesis or processing of many lysosomal enzymes in carcinomas. In a second approach, sections of formalin-fixed breast biopsy specimens were probed with biotinylated MPR. Malignant cells in 25 of 75 carcinomas exhibited granular cytoplasmic staining in what appears to be intracellular vesicles. Staining was specifically inhibited by Man6-P and was not observed in stromal components or lymphocytes. In addition, Man6-phosphorylated proteins were not detected in the 14 normal or benign biopsy samples examined. Staining appeared to be independent of most prognostic factors examined, including p53, cathepsin D, DNA ploidy, and hormone (estrogen and progesterone) receptor status. However, positive staining was significantly associated with high histological and nuclear grades (P < 0.05) and potentially with c-erbB-2 (P < 0.10), suggesting that elevated levels of Man6-P glycoproteins are associated with the more aggressive tumors.
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PMID:Increased levels of glycoproteins containing mannose 6-phosphate in human breast carcinomas. 761 83

An acid protease activity from human brain was found to cleave a fluorogenic peptide substrate encompassing the amino terminus of Alzheimer's amyloid-beta peptide (A beta). The protease was isolated and determined to be cathepsin D based on chromatographic, immunological, and enzymatic data. Analysis of the cleavage sites indicated that cathepsin D hydrolyzed the methionine--aspartate bond generating the in vivo amino terminus of A beta. These data suggested that cathepsin D could be involved in amyloidogenic processing of the amyloid precursor protein. Consequently, cathepsin D from both Alzheimer's-diseased and control brains was compared to determine whether there were any differences which could account for an increase in A beta production in Alzheimer's disease. No differences were detected in isoform composition or tissue content of cathepsin D as measured by 2-D IEF-SDS-PAGE. Enzymological characterization of brain cathepsin D demonstrated that it could undergo a previously undescribed pH-dependent reversible activation. However, that activation appeared identical for both AD and normal brain enzymes. These data demonstrate that concentration, isoform distribution, and several enzymological characteristics of cathepsin D are not distinguishable between AD and normal brain. The pH dependence of cathepsin D activity suggests, however, that its intracellular localization may be important in considering the potential role of cathepsin D in Alzheimer's disease.
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PMID:Cathepsin D from Alzheimer's-diseased and normal brains. 764 17

Plasminogen activator inhibitor-1 (PAI-1) is the most important inhibitor of tissue-type plasminogen activator (t-PA) in plasma and plays a major role in the regulation of fibrinolysis. Plasma t-PA/PAI-1 complexes are cleared via a receptor-dependent mechanism in hepatocytes, while the fate of complexes formed in the extracellular matrix and in thrombi is less well understood. In this study, the degradation of t-PA/PAI-1 complexes by monocytes was examined. THP-1 monocytoid cells and freshly isolated human monocytes internalize and degrade [125I]t-PA/PAI-1 complexes at rates of 11.4 +/- 5.9 (mean +/- S.D.) and 44.6 +/- 6.3 ng/10(6) cells/h, respectively. Degradation is blocked by receptor-associated protein (RAP), indicating a member of the low density lipoprotein (LDL) receptor family is involved in the uptake/degradation of t-PA/PAI-1 complexes by monocytes. Degradation of t-PA/PAI-1 complexes is also inhibited by chloroquine and by pepstatin A, suggesting that a lysosomal aspartyl protease is likely involved. SDS-PAGE and Western blotting demonstrated that the purified lysosomal aspartyl protease, cathepsin D, is capable of digesting t-PA (t1/2 15 min), active PAI-1 (t1/2 2 h), and t-PA/PAI-1 complex (t1/2 30 min). Cathepsin D sequentially cleaves PAI-1 after hydrophobic amino acids, yielding lower molecular weight fragments. PAI-1 conformation influences the degradative efficiency of cathepsin D, with vitronectin-bound PAI-1 and latent PAI-1 exhibiting resistance to proteolysis and > 10-fold prolongation in t1/2. These data provide evidence that t-PA/PAI-1 complexes are internalized by human monocytes via a member of the low density lipoprotein (LDL) receptor family, and identifies cathepsin D-like aspartyl protease activity as largely responsible for the degradation of these complexes. Furthermore, vitronectin-bound PAI-1 and latent PAI-1 are relatively resistant to degradation by cathepsin D, which may be of importance in complex physiological environments.
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PMID:Cathepsin D-like aspartyl protease activity mediates the degradation of tissue-type plasminogen activator/plasminogen activator inhibitor-1 complexes in human monocytes. 766 1

Ligands such as complement fragments (C3, C4), IgG or alpha 2-macroglobulin, which bind antigen (Ag) before their uptake by antigen-presenting cells (APC), are likely to modulate the different steps of Ag processing and presentation. These ligands contribute to internalization and endosomal targeting of Ag; they also influence its processing and, consequently, the binding of resulting peptides to major histocompatibility complex (MHC) class II molecules before presentation to T cells. Complement protein C3 contains, like other members of the alpha 2-macroglobulin family, an intrachain thiolester bond. Conformational alteration or limited proteolysis of C3 into C3b leads to breaking of the thiolester with transient capacity of the revealed carbonyl group to esterify hydroxyl groups of Ag. Ester-linked complexes including tetanus toxin (TT) and C3b were prepared to analyse the influence of bound C3b on TT processing and presentation by APC. Covalent binding of C3b to TT resulted in increased and prolonged stimulation of specific T-cell proliferation. This effect was observed with non-specific B cells, as well as with a TT-specific B-cell clone, as APC. On the other hand, SDS-PAGE analysis of proteolysates of TT or C3b-TT, obtained with endosome/lysosome-enriched subcellular fractions prepared from human Epstein-Barr virus (EBV)-transformed B cells, indicated a delay of TT proteolysis when TT was associated to C3b. Treatment of APC with protease inhibitors, before and during exposure of the cells to Ag, resulted in differences in the inhibition of TT and C3b-TT proteolysis. Using purified cathepsins B and D, we demonstrated that covalent binding of C3b to TT totally abolished TT proteolysis by cathepsin D, while proteolysis by cathepsin B was preserved. This finding and the absence of cathepsin B in endosomes may explain a delay in TT processing when it is associated to C3b. Confirming these data, presentation by formaldehyde-fixed cells of C3b-TT proteolysates showed higher stimulation of specific T-cell clones than formaldehyde-fixed TT proteolysates.
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PMID:Modulation of antigen processing and presentation by covalently linked complement C3b fragment. 789 Mar 1

To study the mechanism involved in mannose-6-phosphate (Man-6-P) independent lysosomal proenzyme membrane association, we used a reversible cross-linker to probe radiolabeled human HepG2 cells permeabilized with saponin in the presence of Man-6-P. After immunoprecipitation of the extracted and cross-linked cells with anti-cathepsin D antibody, followed by complete reduction of the immunoprecipitates and SDS-polyacrylamide gel electrophoresis analysis, we found that procathepsin D was specifically and transiently associated, independent of Man-6-P, with two co-synthesized glycoproteins having molecular masses of 68 and 72 kDa. Pulse-chase and cell fractionation experiments showed that the Man-6-P independent association of procathepsin D with the 68-kDa protein started in the rough endoplasmic reticulum, continued in the Golgi, but had no association with either membrane. The Man-6-P independent association of procathepsin D with the 72-kDa protein and the membrane was found in compartments all the way from the Golgi to the dense lysosome, where processing of procathepsin D is believed to occur and where procathepsin D dissociated from the 72-kDa protein and the membrane. Endo H digestion of the 72-kDa protein showed that this protein was partially resistant to Endo H, suggesting that membrane association of the procathepsin D-72-kDa protein complex probably began in a late Golgi compartment. Endo F digestion of the proteins showed both have the same molecular mass around 58 kDa. Using antiserum against human saposin C, we identified the two glycoproteins as forms of prosaposin with different glycosylation. The transient, Man-6-P independent, membrane association of the procathepsin D-prosaposin complex and the presence of this complex in heavy lysosomes indicated that the proteins were transported to the lysosome as a complex. The association of two lysosomal proteins in the endoplasmic reticulum early after synthesis suggested that preassembly of some lysosomal components occurs before the earliest previously identified steps in the sorting pathway.
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PMID:Intermolecular association of lysosomal protein precursors during biosynthesis. 810 29

Murine peritoneal macrophages (PMO) and veiled cells (VC) isolated from the thoracic duct of irradiated lymphadenectomized (MNLX) mice presented intact human serum albumin (HSA) to stimulated T lymphocytes, but VC were not as effective as PMO in presenting the antigen. Pepstatin A significantly inhibited the presentation of HSA by VC. Lysates prepared from PMO degraded [125I]HSA at pH 4.0 to peptides as demonstrated by SDS-polyacrylamide-gel electrophoresis and autoradiography. Degradation was inhibited by pepstatin A, suggesting that cathepsin D might be responsible for processing the antigen. In contrast, lysates prepared from VC did not degrade [125I]HSA. The localization of cathepsin D, by light microscopy, was examined on cytospins of PMO and VC by means of a peroxidase antiperoxidase technique (PAP). Cathepsin D was found in vacuoles in the cytoplasm of PMO and, in some cases, appeared to be bound to some areas of the cell surface, but the enzyme could not be detected in VC.
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PMID:Role of cathepsin D in the degradation of human serum albumin by peritoneal macrophages and veiled cells in antigen presentation. 825 54

The in vitro degradation of dystrophin protein by endogenous proteases in human skeletal muscle has been investigated using a tissue homogenate assay system with subsequent protein analysis via SDS polyacrylamide electrophoresis and immunoblotting (using a monoclonal antibody to the central rod region of dystrophin). The rate of dystrophin degradation and nature of the proteolytic fragments formed at pH 5.5 and pH 7.5 (corresponding to the two major protease groups of relevance to intracellular protein catabolism) were broadly similar; incorporation of protease inhibitors in the above system suggested that Ca2+ activated proteinase and cathepsin D are principally responsible for the degradation of dystrophin at pH 7.5 and pH 5.5 respectively. The rate of dystrophin degradation at pH 7.5 was reduced by approximately 20% in the presence of 10(-5) M clenbuterol, a beta-adrenoceptor agonist with therapeutic potential in the treatment of human muscle wasting diseases.
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PMID:Effect of protease inhibitors and clenbuterol on the in vitro degradation of dystrophin by endogenous proteases in human skeletal muscle. 826 23

The amino acid sequence of the propart of bovine procathepsin D was determined at the protein level. Incubation of the isolated procathepsin D at pH 3.5-5.0 for 30-120 min leads to a 2 kDa reduction in its molecular mass, as seen by SDS-PAGE. The activation product is pseudocathepsin D and is the result of a proteolytic cleavage between LeuP26 and IleP27 in the propart. Incubation at pH 5.0 for 20 h of either procathepsin D or pseudocathepsin D results in both cases in approximately equal amounts of pseudocathepsin D and a further processed intermediate, nine amino acids shorter than pseudocathepsin D. No reaction products corresponding to cathepsin D with a mature amino terminus were observed, showing that autoproteolysis alone cannot generate the mature form found in the lysosomes.
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PMID:Procathepsin D cannot autoactivate to cathepsin D at acid pH. 845 61

In order to characterize the intracellular processing event of lysosomal cathepsin B, the proenzyme was purified from the rat liver microsomal contents using a Con A-Sepharose column, a Sepharose-Gly-Phe-GlySc column, and an anti-cathepsin B IgG column. The purified proenzyme gave a single protein band of 39 kDa on SDS/polyacrylamide gel electrophoresis. The proenzyme showed no appreciable enzymatic activity. When the purified proenzyme was incubated with the cathepsin B-free tritosomal contents, prepared by treatment of the tritosomal contents with anti-cathepsin B IgG Sepharose, at pH 3.0, 30 degrees C, a remarkable increase of enzymatic activity was observed. Immunoblot analysis showed that the proenzyme was completely converted to the active intermediate form of 31 kDa after 1 h incubation. These processing and activation events were blocked in the presence of pepstatin. When the proenzyme was incubated with the cathepsins B- and D-free tritosomal contents, prepared by treatment of the cathepsin B-free tritosomal contents with anti-cathepsin D IgG Sepharose, the processing and activation did not occur. These results indicate that cathepsin D is involved in the processing and activation of procathepsin B in rat liver lysosome. In the NH2-terminal sequence analysis of the 31 kDa form, the terminal was assigned as proline (66th residue). Since the NH2-terminus of the mature single-chain form of cathepsin B (29 kDa) ends at leucine (80th residue), the NH2-terminus of the 31 kDa form is 14 amino acid residues longer than that of the single-chain form.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and processing of rat liver procathepsin B. 848 12

A proteinase accumulated in breast secretions from women with breast cancer has been characterised. Inhibition of the proteolytic activity of breast secretions by pepstatin A showed that the main enzyme involved was an aspartyl proteinase. Determination of its cleavage specificity by SDS-PAGE and amino acid sequence analysis revealed that it was identical to that of cathepsin D, an aspartyl proteinase suggested to be involved in breast cancer development. The identity between both proteins was further confirmed by immunological analysis with monoclonal antibodies against cathepsin D. Quantification of cathepsin D in nipple fluids from 41 women with benign or malignant breast diseases and from 19 control women without breast pathology revealed the presence of variable amounts of this proteinase. The average concentration of cathepsin D in breast secretions from cancer-bearing breasts was 7.2 +/- 2.2 fmol micrograms of protein, which was significantly higher than those of nipple fluids from control women (2.9 +/- 0.6 fmol micrograms-1) (P = 0.04) or from patients with benign breast diseases (2.1 +/- 0.3 fmol micrograms-1) (P = 0.004). Though the number of cancer patients studied was small (n = 21), no correlations were found with cytosolic concentrations of cathepsin D or oestrogen receptors, neither with other parameters such as tumour size, histological grade, axillary node involvement or menopausal status.
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PMID:Cathepsin D in breast secretions from women with breast cancer. 849 1


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