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)

The degradation of 135I-apoprotein B of human low-density lipoprotein by cell extracts of cultured bovine aortic smooth muscle cells was determined by measuring the formation of acid-soluble products and by analyzing the electrophoretic patterns of digested apoprotein in gels containing sodium dodecyl sulfate. Degradation resulted in an initial rapid accumulation of a limited number of distinct smaller fragments. Two products with apparent molecular weights of 220,000 and 200,000 predominated. Pepstatin inhibited proteolysis almost completely, as measured by either assay. Leupeptin decreased hydrolysis to acid-soluble products by approximately 50%, but had no effect on the initial cleavage of intact apoprotein B. Similar results were found in the case of extracts from cultured human skin fibroblasts and from adult bovine arterial smooth muscle. Leupeptin inhibited intracellular degradation of 125I-apoprotein B in cultured cells by approximately 50%. It is concluded that the intralysosomal degradation of apoprotein B involves an initial limited endoproteolytic attack at susceptible sites by cathepsin D. This and other enzymes, including cathepsin B, then act synergistically to bring degradation to completion.
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PMID:Cathepsin-D-dependent initiation of the hydrolysis by lysosomal enzymes of apoprotein B from low-density lipoproteins. 744 60

We previously reported that a substantial amount of newly synthesized apoE in mouse macrophages is degraded prior to secretion; a portion of this pool of apoE can be rescued by the addition of HDL3 to the incubation medium. In the present studies, the location and nature of the intracellular degradation of apoE were more closely examined. Inhibitors of protein trafficking (brefeldin A) as well as a number of protease inhibitors were used. The experiments using brefeldin A (5 micrograms/ml) clearly established that neither the endoplasmic reticulum nor the Golgi complex are the sites of apoE degradation. Using a pulse-chase design, [35S]apoE cannot be chased out in the presence of brefeldin A and remains undegraded within the cell. The accumulated apoE lacks the sialic acid residues, indicating that this final stage of processing must occur in the trans-Golgi network or later. Lysosomotropic agents, ammonium chloride and chloroquine, on the other hand, inhibit apoE degradation by over 70 and 80%, respectively, while total cell protein degradation remains unaffected. Similarly, a cocktail consisting of four lysosomal protease inhibitors (pepstatin, E-64, chymostatin, and antipain), inhibits specifically apoE degradation by over 60%. In contrast, ALLN, an inhibitor of Ca(2+)-dependent cysteine proteases, has a moderate effect on apoE degradation (30% inhibition) and a more pronounced effect on total protein degradation. These data suggest that the site of intracellular apoE degradation in the macrophage is the lysosome. These conclusions are supported by light and electron microscopy of macrophages, clearly showing the presence of immunoreactive apoE (along with cathepsin D) in the endosomal/lysosomal compartment of control and lysosomotropic agent-treated cells. In contrast, little or no labeling is seen in this compartment in brefeldin A-treated cells. At lower concentrations of the lysosomotropic agents, the extent of inhibition of apoE degradation is compensated for by its increased secretion, in a manner analogous to the effect of these agents on lysosomal enzymes. Higher concentrations of these agents, which lead to a profound inhibition of apoE degradation, also specifically block apoE secretion. The block in apoE secretion in the presence of high concentrations of chloroquine leads to undiminished or higher concentrations of immunoreactive apoE in the endosomal/lysosomal compartment, suggesting that apoE is targeted for lysosomal degradation directly, without prior secretion or surface association. These data strongly suggest pH-dependent sorting of apoE in macrophages to the degradative and secretory pathways and imply a protein-protein interaction in the process.
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PMID:Lysosomal degradation and sorting of apolipoprotein E in macrophages. 857 39

Quantitative proteomics can be used as a screening tool for identification of differentially expressed proteins as potential biomarkers for cancers. Candidate biomarkers from such studies can subsequently be tested using other techniques for use in early detection of cancers. Here we demonstrate the use of stable isotope labeling with amino acids in cell culture (SILAC) method to compare the secreted proteins (secretome) from pancreatic cancer-derived cells with that from non-neoplastic pancreatic ductal cells. We identified 145 differentially secreted proteins (>1.5-fold change), several of which were previously reported as either up-regulated (e.g. cathepsin D, macrophage colony stimulation factor, and fibronectin receptor) or down-regulated (e.g. profilin 1 and IGFBP-7) proteins in pancreatic cancer, confirming the validity of our approach. In addition, we identified several proteins that have not been correlated previously with pancreatic cancer including perlecan (HSPG2), CD9 antigen, fibronectin receptor (integrin beta1), and a novel cytokine designated as predicted osteoblast protein (FAM3C). The differential expression of a subset of these novel proteins was validated by Western blot analysis. In addition, overexpression of several proteins not described previously to be elevated in human pancreatic cancer (CD9, perlecan, SDF4, apoE, and fibronectin receptor) was confirmed by immunohistochemical labeling using pancreatic cancer tissue microarrays suggesting that these could be further pursued as potential biomarkers. Lastly the protein expression data from SILAC were compared with mRNA expression data obtained using gene expression microarrays for the two cell lines (Panc1 and human pancreatic duct epithelial), and a correlation coefficient (r) of 0.28 was obtained, confirming previously reported poor associations between RNA and protein expression studies.
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PMID:Biomarker discovery from pancreatic cancer secretome using a differential proteomic approach. 1621 74