Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.23.5 (cathepsin D)
 4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Saposins A, B, C, and D, which are required for the enzymatic hydrolysis of sphingolipids by specific lysosomal hydrolases, are produced by proteolytic processing of their common precursor protein, prosaposin. Our previous observation suggested that lysosomal cathepsin D may be involved in the proteolysis of prosaposin. Herein we report the involvement of cathepsin D in the proteolytic processing of prosaposin. An antibody against human placental cathepsin D blocked the proteolytic activity toward prosaposin in a human testicular lysosomal protease mixture (glycoprotein fraction). On immunoblot analysis using a monoclonal antibody against human saposin C, cathepsin D showed a similar proteolytic pattern as that of a human testicular glycoprotein fraction and hydrolyzed prosaposin into products of 48 and 29 kDa. The Km and Vmax values were 0.9 microM and 167 nmol/h/mg, respectively. N-Terminal sequence analysis indicated that the 48-kDa band was a mixture of two trisaposins, including domains for saposins A, B, and C and saposins B, C, and D, respectively. A similar study also showed that the 29-kDa band contained two disaposins, including domains for saposins A and B and saposins C and D, respectively. By longer treatment with cathepsin D, disaposins were further processed into mature saposin A and small fragments (14.5-17.5 kDa) containing individual saposins and portions of interdomain sequences. These small fragments were no longer processed by cathepsin D, but trimmed to fragments having similar molecular sizes (10.5-11.5 kDa) to those of mature saposins by a rat lysosome preparation. These findings indicated that cathepsin D is involved in the maturation of saposins but that, in addition to cathepsin D, other proteases appear to be involved in the maturation of saposin B, C, and D in lysosomes. Gangliosides, which specifically form complexes with prosaposin and saposins, inhibit proteolysis of prosaposin by cathepsin D. This finding indicates that prosaposin may be protected from lysosomal proteolysis by forming a complex with gangliosides in vivo.
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PMID:Lysosomal proteolysis of prosaposin, the precursor of saposins (sphingolipid activator proteins): its mechanism and inhibition by ganglioside. 914 48

Before delivery to endosomes, portions of proCD (procathepsin D) and proSAP (prosaposin) are assembled into complexes. We demonstrate that such complexes are also present in secretions of cultured cells. To study the formation and properties of the complexes, we purified proCD and proSAP from culture media of Spodoptera frugiperda cells that were infected with baculoviruses bearing the respective cDNAs. The biological activity of proCD was demonstrated by its pH-dependent autoactivation to pseudocathepsin D and that of proSAP was demonstrated by feeding to saposin-deficient cultured cells that corrected the storage of radioactive glycolipids. In gel filtration, proSAP behaved as an oligomer and proCD as a monomer. ProSAP altered the elution of proCD such that the latter was shifted into proSAP-containing fractions. ProSAP did not change the elution of mature cathepsin D. Using surface plasmon resonance and an immobilized biotinylated proCD, binding of proSAP was demonstrated under neutral and weakly acidic conditions. At pH 6.8, specific binding appeared to involve more than one binding site on a proSAP oligomer. The dissociation of the first site was characterized by a K(D1) of 5.8+/-2.9x10(-8) M(-1) (calculated for the monomer). ProSAP stimulated the autoactivation of proCD and also the activity of pseudocathepsin D. Concomitant with the activation, proSAP behaved as a substrate yielding tri- and disaposins and smaller fragments. Our results demonstrate that proSAP forms oligomers that are capable of binding proCD spontaneously and independent of the mammalian type N-glycosylation but not capable of binding mature cathepsin D. In addition to binding proSAP, proCD behaves as an autoactivable and processing enzyme and its binding partner as an activator and substrate.
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PMID:Purified recombinant human prosaposin forms oligomers that bind procathepsin D and affect its autoactivation. 1525 80

To identify genes involved in the regulation of plasma high density lipoprotein (HDL) cholesterol (HDL-C) levels, patients with low HDL-C and age- and sex-matched controls (normal HDL-C) were extensively characterized. Comparative transcriptome analysis was carried out in cholesterol-loaded monocyte-derived macrophages from low HDL subjects segregated into groups with or without cholesterol efflux defects or ABCA1 mutations. Clusters of differentially regulated genes were evident in the low HDL groups as compared with controls. Of particular note, expression of cathepsin D (CTSD), a lysosomal proteinase, was reduced by approximately 50% in monocyte-derived macrophages of low HDL-C subjects, most significantly those with cholesterol efflux defects but without mutations in ABCA1 (p < 0.01). These results were verified by reverse transcription-PCR and replicated in a second cohort. We show here that blocking the activity or expression of CTSD, by pepstatin or CTSD small interfering RNA, respectively, reduced ABCA1 expression and protein abundance in both macrophages and CHO cells and apolipoprotein A-I-mediated lipid efflux by more than 70%. Conversely, expression of CTSD increased both ABCA1 mRNA expression and cellular ABCA1 protein. Consistent with its role in the proteolytic processing of prosaposin, inactivation of CTSD function resulted in the accumulation of glycosphingo-lipid and free cholesterol in late endosomes/lysosomes, a phenotype similar to NPC1 deficiency. Inhibition of CTSD also caused retention of ABCA1 in lysosomal compartments, reducing its trafficking to the plasma membrane. These studies demonstrate a novel and potentially important role for CTSD in intracellular cholesterol trafficking and ABCA1-mediated efflux. Therefore, decreased CTSD expression may contribute to low plasma HDL-C levels.
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PMID:Cathepsin D, a lysosomal protease, regulates ABCA1-mediated lipid efflux. 1703 48

Prosaposin, the precursor of the sphingolipid hydrolase activator proteins called saposins A, B, C, and D, is abundant in the nervous system and muscles. Besides its role as the precursor of saposins, prosaposin is reported to function as a neurotrophic factor, initiating neural differentiation and preventing neuronal cell death in vivo and in vitro. In this study, we examined the localization and synthesis of prosaposin in the rat cochlea. Intense prosaposin immunoreactivity was observed in the organ of Corti, stria vascularis, and spiral ganglion. In an immuno-electron microscopic study, prosaposin immunoreactivity was found mainly in lysosomal granules of the cells in these regions. In the lysosome, prosaposin does not always colocalize with cathepsin D, but was localized mainly in the dark area of the lysosome. Prosaposin mRNA was observed in these same regions. Our results suggest that prosaposin plays a role in homeostasis in the peripheral auditory system.
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PMID:Localization of prosaposin in rat cochlea. 1715 77

Most mammalian cells contain two types of mannose 6-phosphate (Man-6-P) receptors (MPRs): the 300 kDa cation-independent (CI) MPR and 46 kDa cation-dependent (CD) MPR. The two MPRs have overlapping function in intracellular targeting of newly synthesized lysosomal proteins, but both are required for efficient targeting. Despite extensive investigation, the relative roles and specialized functions of each MPR in targeting of specific proteins remain questions of fundamental interest. One possibility is that most Man-6-P glycoproteins are transported by both MPRs, but there may be subsets that are preferentially transported by each. To investigate this, we have conducted a proteomics analysis of serum from mice lacking either MPR with the reasoning that lysosomal proteins that are selectively transported by a given MPR should be preferentially secreted into the bloodstream in its absence. We purified and identified Man-6-P glycoproteins and glycopeptides from wild-type, CDMPR-deficient, and CIMPR-deficient mouse serum and found both lysosomal proteins and proteins not currently thought to have lysosomal function. Different mass spectrometric approaches (spectral count analysis of nanospray LC-MS/MS experiments on unlabeled samples and LC-MALDI/TOF/TOF experiments on iTRAQ-labeled samples) revealed a number of proteins that appear specifically elevated in serum from each MPR-deficient mouse. Man-6-P glycoforms of cellular repressor of E1A-stimulated genes 1, tripeptidyl peptidase I, and heparanase were elevated in absence of the CDMPR and Man-6-P glycoforms of alpha-mannosidase B1, cathepsin D, and prosaposin were elevated in the absence of the CIMPR. Results were confirmed by Western blot analyses for select proteins. This study provides a comparison of different quantitative mass spectrometric approaches and provides the first report of proteins whose cellular targeting appears to be MPR-selective under physiological conditions.
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PMID:Proteomics analysis of serum from mutant mice reveals lysosomal proteins selectively transported by each of the two mannose 6-phosphate receptors. 1784 85

DNA diagnostics are useful but are hampered by difficult ethical issues. Moreover, it cannot provide enough information on the environmental factors that are important for pathogenesis of certain diseases. However, this is not a problem for RNA diagnostics, which evaluate the expression of the gene in question. We here report a novel RNA diagnostics tool that can be employed with peripheral blood mononuclear cells (PBMCs). To establish this tool, we identified 290 genes that are highly expressed in normal PBMCs but not in TIG-1, a normal human fibroblast cell. These genes were entitled PREP after predominantly expressed in PBMC and included 50 uncharacterized genes. We then conducted PREP gene-focused microarray analysis on PBMCs from seven cases of Churg-Strauss syndrome (CSS), which is a small-vessel necrotizing vasculitis. We found that PREP135 (coactosin-like protein), PREP77 (prosaposin), PREP191 (cathepsin D), PREP234 (c-fgr), and PREP136 (lysozyme) were very highly up-regulated in all seven CSS patients. Another 28 genes were also up-regulated, albeit more moderately, and three were down-regulated in all CSS patients. The nature of these up- and down-regulated genes suggest that the immune systems of the patients are activated in response to invading microorganisms. These observations indicate that focused microarray analysis of PBMCs may be a practical, useful, and low-cost bedside diagnostics tool.
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PMID:Focused microarray analysis of peripheral mononuclear blood cells from Churg-Strauss syndrome patients. 1826 71

The notion that prosaposin (Prosap) is likely involved in brain development and regeneration led us to explore its expression in stem/progenitor neural cells and its fate after cell differentiation. The expression of procathepsin-cathepsin D (proCath-Cath D), an endoprotease that plays an important role in the processing and sorting of Prosap, has been concomitantly examined. Our data evidenced that in embryonic human neural progenitor cells (eHNPCs) intact and high molecular weight intermediate forms of Prosap and intermediate forms of Cath D accumulated inside the cells, while the formation of saposins and mature Cath D was impaired. Furthermore, neither Prosap nor proCath D were secreted from eHNPCs. The block of the processing and secretion shared by Prosap and proCath D was overcome during the course of differentiation of eHNPCs into a mixed population of astrocytes and neuronal cells. Upon differentiation, large amounts of Prosap and proCath D were secreted from the cells, while saposins and mature Cath D were produced inside the cells. The dramatic accumulation of Prosap (an antiapoptotic factor) and reduction of mature Cath D (a proapoptotic factor) in the undifferentiated eHNPCs most likely play a role in the molecular mechanisms regulating the resistance to apoptotic signals of these cells and might represent a critically important issue in HNPCs biology.
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PMID:The secretion and maturation of prosaposin and procathepsin D are blocked in embryonic neural progenitor cells. 1834 66

Most soluble lysosomal hydrolases are sorted in the trans-Golgi network (TGN) and delivered to the lysosomes by the mannose 6-phosphate receptor (M6PR). However, the non-enzymic sphingolipid activator protein (SAP), prosaposin, as well as certain soluble lysosomal hydrolases, is sorted and trafficked to the lysosomes by sortilin. Based on previous results demonstrating that prosaposin requires sphingomyelin to be targeted to the lysosomes, we hypothesized that sortilin and its ligands are found in detergent-resistant membranes (DRMs). To test this hypothesis we have analyzed DRM fractions and demonstrated the presence of sortilin and its ligand, prosaposin. Our results showed that both the M6PR and its cargo, cathepsin B, were also present in DRMs. Cathepsin H has previously been demonstrated to interact with sortilin, while cathepsin D interacts with both sortilin and the M6PR. Both of these soluble lysosomal proteins were also found in DRM fractions. Using sortilin shRNA we have showed that prosaposin is localized to DRM fractions only in the presence of sortilin. These observations suggest that in addition to interacting with the same adaptor proteins, such as GGAs, AP-1 and retromer, both sortilin and the M6PR localize to similar membrane platforms, and that prosaposin must interact with sortilin to be recruited to DRMs.
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PMID:Sortilin and prosaposin localize to detergent-resistant membrane microdomains. 1899 38

Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, prosaposin and GM(2)AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin-deficient mice.
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PMID:The inactivation of the sortilin gene leads to a partial disruption of prosaposin trafficking to the lysosomes. 1973 68


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