Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Up to 4% of the human 46-kDa mannose 6-phosphate receptor (MPR46) expressed in Madin-Darby canine kidney (MDCK) cells are localized at the cell surface. At steady state, the expression of MPR46 on the apical surface of filter-grown MDCK cells is about sixfold lower than on the basolateral surface. The cytoplasmic domain of the MPR46 is phosphorylated on serine 56 at low stoichiometry. By expressing mutant MPR46 we have shown that the MPR46 phosphorylation site is required for delivery to the plasma membrane. In addition, mutant MPR46 expressed in MPR-deficient mouse embryonic fibroblasts were not detected at the cell surface and their ability to sort newly synthesized cathepsin D was not altered. Since the loss of MPR46 phosphorylation correlates with the lack of cell surface expression, phosphorylation of serine 56 may either function as a direct plasma membrane targeting signal or inhibit MPR46 recycling from endosomes to Golgi, resulting in trafficking to the cell surface.
Mol Biol Cell 1997 Apr
PMID:Serine phosphorylation site of the 46-kDa mannose 6-phosphate receptor is required for transport to the plasma membrane in Madin-Darby canine kidney and mouse fibroblast cells. 924 38

Mannose 6-phosphate receptors carry newly synthesized lysosomal hydrolases from the trans-Golgi network to endosomes, then return to the trans-Golgi network for another round of enzyme delivery. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, interferes with the delivery of newly synthesized lysosomal enzymes to lysosomes. We used two independent assays of mannose 6-phosphate receptor trafficking to determine the precise step that is blocked by wortmannin. Using an assay that monitors resialylation of desialylated cell surface 300-kDa mannose 6-phosphate receptors, we found that receptor endocytosis and transport to the trans-Golgi network were not inhibited by 2 microM wortmannin. In addition, this concentration of drug had no effect on the transport of the mannose 6-phosphate receptor from late endosomes to the trans-Golgi network using a system that reconstitutes this transport process in cell extracts. Under the same conditions, wortmannin significantly inhibited the generation of mature cathepsin D. In addition, the structurally unrelated phosphatidylinositol 3-kinase inhibitor, LY294002, was also without effect when added to in vitro endosome-trans-Golgi network transport reactions. These experiments demonstrate that the interruption in lysosomal enzyme targeting is most likely due to a wortmannin-sensitive process required for the export of these receptors from the trans-Golgi network, consistent with the established role of phosphatidylinositol 3-kinase in the equivalent transport process in Saccharomyces cerevisiae.
Mol Biol Cell 1997 Apr
PMID:Phosphatidylinositol 3-kinase is not required for recycling of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network. 924 39

Human beta-secretase candidates, MP78 (h-MP78, EC 3.4.24.15) and cathepsin D (Cat D, EC 3.4.23.5), were evaluated for their ability to enhance amyloid-beta-protein (A beta) secretion when overexpressed in beta APP-containing cells. HEK-293 cells stably co-expressing h-MP78 or Cat D and h-beta APP695 were metabolically labeled with [35S]methionine and A beta secretion was quantified in the conditioned media by immunoprecipitation and ELISA without showing any significant increase in A beta production. Because Cat D is known to have a higher affinity for APP-substrate containing the Swedish familial Alzheimer's disease double mutation (SFAD, K595N and M596L substitutions in beta APP695) than for the wild type substrate [Dreyer et al., Eur. J. Biochem., 224 (1994) 265-271], the effect of Cat D overexpression was tested in a HEK293/beta APPSFAD stable cell line. ELISA analysis of the conditioned media from these cells did also not reveal any increase in A beta generation. In addition, recombinant h-MP78 purified from E. coli cleaved an APP-derived substrate spanning the beta-secretase site (ISEVKMD1AEFRHDS) at multiple sites, but the beta-site cleavage was only a minor one; cleavage occurred predominantly at K-M and E-F bonds. Human liver Cat D also cleaved the same substrate at multiple sites, yet the major cleavage at pH 4.0 occurred at the amyloidogenic D1 site. These findings indicate that h-MP78 does not have the cleavage specificity required for a beta-secretase protease and although Cat D fulfilled the amyloidogenic cleavage specificity, the results of the co-expression experiments make both enzymes less likely candidates as relevant beta-secretases.
Brain Res Mol Brain Res 1997 Sep
PMID:Expression and characterization of human beta-secretase candidates metalloendopeptidase MP78 and cathepsin D in beta APP-overexpressing cells. 933 17

Degradation of yolk protein is essential for the early development of the avian embryo. In Japanese quail (Coturnix coturnix japonica), proteolysis in the surrounding tissue of the yolk, the yolk-sac membrane, can be inhibited by class-specific inhibitors of cysteine proteinases as well as of aspartic proteinases. Purification of the enzymes leads to one cysteine proteinase and one aspartic proteinase with an apparent molecular mass of 29 kD and 44 kD, respectively. Both enzymes were purified in a two-chain form, although a single-chain form is also present in the homogenate of yolk-sac membrane. The cysteine proteinase was identified by NH2-terminal sequence analysis as well as by kinetic studies as a new cathepsin B from quail. Like mammalian cathepsin B, this avian cathepsin B exhibits two different kinds of proteolytic activity, an endopeptidase activity and a dipeptidyl carboxypeptidase activity. Chicken egg white cystatin, a protein-aceous cysteine proteinase inhibitor, inhibits quail cathepsin B with an equilibrium dissociation constant (Ki) of 3.3 nM. Likewise the aspartic proteinase was identified as a new cathepsin D from quail. This avian cathepsin D has a different processing site to all known mammalian cathepsins D. In quail cathepsin D one NH2-termini is homologous to amino acids 211-230 in mammalian cathepsin D. This is more than 100 amino acids downstream of the mammalian processing site. Comparison of the enzymatic properties of quail and bovine cathepsin D indicate that the different processing site has no influence on the enzymatic properties.
Comp Biochem Physiol B Biochem Mol Biol 1997 Sep
PMID:Proteolytic enzymes in yolk-sac membrane of quail egg. Purification and enzymatic characterisation. 941 5

The subfraction of low density lipoprotein (LDL) with low sialic acid content that caused accumulation of cholesterol esters in human aortic smooth muscle cells has been found in the blood of coronary atherosclerosis patients. It was demonstrated that this subfraction consists of LDL with small size, high electronegative charge, reduced lipid content, altered tertiary structure of apolipoprotein B, etc. LDL of this subfraction is naturally occurring multiple-modified LDL (nomLDL). In this study we compared the binding, uptake and proteolytic degradation of native LDL and nomLDL by smooth muscle cells cultured from human grossly normal intima, fatty streaks, and atherosclerotic plaques. Uptake of nomLDL by normal and atherosclerotic cells was 3.5- and 6-fold, respectively, higher than uptake of native LDL. Increased uptake of nomLDL was due to increased binding of this LDL by intimal smooth muscle cells. The enhanced binding is explained by the interaction of nomLDL with cellular receptors other than LDL-receptor. Modified LDL interacted with the scavenger receptor, asialoglycoprotein receptor, and also with cell surface proteoglycans. Rates of degradation of nomLDL were 1.5- and 5-fold lower than degradation of native LDL by normal and atherosclerotic cells, respectively. A low rate of nomLDL degradation was also demonstrated in homogenates of intimal cells. Activities of lysosomal proteinases of atherosclerotic cells were decreased compared with normal cells. Pepstatin A, a cathepsin D inhibitor, completely inhibited lipoprotein degradation, while serine, thiol, or metallo-proteinase inhibitors had partial effect. This fact reveals that cathepsin D is involved in initial stages of apoB degradation by intimal smooth muscle cells. Obtained data show that increased uptake and decreased lysosomal degradation of nomLDL may be the main cause of LDL accumulation in human aortic smooth muscle cells, leading to foam cell formation.
Exp Mol Pathol 1997
PMID:Metabolism of native and naturally occurring multiple modified low density lipoprotein in smooth muscle cells of human aortic intima. 943 79

We have previously shown that in HEp-2 cells, multivesicular bodies (MVBs) processing internalized epidermal growth factor-epidermal growth factor receptor complexes mature and fuse directly with lysosomes in which the complexes are degraded. The MVBs do not fuse with a prelysosomal compartment enriched in mannose 6-phosphate receptor (M6PR) as has been described in other cell types. Here we show that the cation-independent M6PR does not become enriched in the endocytic pathway en route to the lysosome, but if a pulse of M6PR or an M6PR ligand, cathepsin D, is followed, a significant fraction of these proteins are routed from the trans-Golgi to MVBs. Accumulation of M6PR does not occur because when the ligand dissociates, the receptor rapidly leaves the MVB. At steady state, most M6PR are distributed within the trans-Golgi and trans-Golgi network and in vacuolar structures distributed in the peripheral cytoplasm. We suggest that these M6PR-rich vacuoles are on the return route from MVBs to the trans-Golgi network and that a separate stable M6PR-rich compartment equivalent to the late endosome/prelysosome stage does not exist on the endosome-lysosome pathway in these cells.
Mol Biol Cell 1998 Apr
PMID:The kinetics of mannose 6-phosphate receptor trafficking in the endocytic pathway in HEp-2 cells: the receptor enters and rapidly leaves multivesicular endosomes without accumulating in a prelysosomal compartment. 952 79

HEC1A endometrial cancer cells express the wild-type form of the estrogen receptor (ER) and 17beta-estradiol (E2) induces proliferation of these cells. In contrast, tamoxifen only causes a minimal increase (<20%) in cell proliferation. In HEC1A cells transiently transfected with the C3-Luc plasmid derived from the complement C3 gene, both E2 and tamoxifen exhibited ER agonist activity and tamoxifen was also a partial antagonist for this response. The relative ER agonist/antagonist activities of E2, tamoxifen and ICI 182,780 were also investigated in HEC1A1 cells transiently transfected with two E2-responsive plasmids, pCATHD-CAT and pCKB-CAT which contain 5'-promoter inserts from the cathepsin D and creatine kinase B genes, respectively. The results showed that E2 and tamoxifen induced reporter gene activity in cells transiently transfected with both constructs. ICI 182,780 exhibited partial ER agonist activity only in cells transiently transfected with pCKB-CAT and antagonized E2-induced reporter gene activity using both the CKB- and CATHD-derived constructs. These results demonstrate that HEC1A endometrial cancer cells are E2-responsive and represent a useful cell culture model for understanding hormone/antihormone-induced endometrial cell responses.
J Steroid Biochem Mol Biol 1998 Mar
PMID:Estrogen- and antiestrogen-responsiveness of HEC1A endometrial adenocarcinoma cells in culture. 961 30

IIB-BR-G is an undifferentiated, highly heterogeneous, hormone receptor negative human breast cancer cell line previously established in our laboratory from a patient's primary tumor. An in vitro growing cell line (IIB-BR-G) and a xenotransplanted tumor growing in nude mice (IIB-BR-G(NUDE)) were derived. To further characterize these systems, immunocytochemical analysis was performed for differentiation antigens (PEM 200 kDa, CEA, NCA 90 kDa), blood-group related antigens (Le(x), sTn), oncogenes and tumor suppressor gene products (Her-2/neu protein, p53), metastasis-related cathepsin D and CD63/5.01 Ag, and the chemokine monocyte chemotactic protein 1 (MCP-1). Expression of markers was heterogeneous in these different systems. Previously reported karyotypic analysis has shown extensive chromosomal alterations including double min. Searching for oncogene amplification, we detected augmented copy number of c-myc and c-fos, the last one with two rearranged fragments. No amplification was found for c-erbB-2 in the cell line or in IIB-BR-G(NUDE), although this oncogene was amplified in the patient's primary tumor DNA. The differences observed between the patient's tumor, the cell line and the IIB-BR-G(NUDE) tumors are probably due to clonal expansion of cell variants not present in the original tumor. Electron microscopy of IIB-BR-G growing cells revealed epithelial characteristics with abundant dense granules, presumably secretory, distributed all over the cytoplasm and great nuclear pleomorphism. In vitro, IIB-BR-G cells showed a significant number of invading cells by Matrigel assay. After nearly 40 sequential subcutaneous passages of the original xenograft through nude mice, 80% of recipients developed spontaneous metastases, primarily to the lung and lymph nodes. Since this experimental model allowed to analyze changes produced in cancer cells from the primary tumor during adaptation to in vitro and in vivo growth, our results provide novel insights on the behaviour of hormone independent metastatic breast cancer.
Cell Mol Biol (Noisy-le-grand) 1998 May
PMID:The human breast cancer cell line IIB-BR-G has amplified c-myc and c-fos oncogenes in vitro and is spontaneously metastatic in vivo. 962 Apr 46

A lysozyme (pI 5.5) was purified to homogeneity from heated acid extracts of Drosophila melanogaster larvae, using gel filtration in a Superose column and ion-exchange chromatography in a Mono Q column. The final yield was 67%. The purified lysozyme with Mr 13,700 (determined by SDS-polyacrylamide gel electrophoresis) decreases in activity and has its pH optimum displaced towards acidic values and Km increases as the ionic strength of the medium becomes higher. The lysozyme is resistant to a cathepsin D-like proteinase present in cyclorrhaphous Diptera and displays a chitinase activity which is 11-fold higher than that of chicken lysozyme. Microsequencing of an internal peptide of the purified lysozyme showed that this enzyme is the product of the previously sequenced Lys D gene. The results suggest that the product of the Lys P gene has pI 7.2, a pH optimum around 5 and is not a true digestive enzyme. The most remarkable sequence convergence of D. melanogaster lysozyme D and lysozymes from vertebrate foregut fermenters are serine 104 and a decrease in the number of basic amino acids, suggesting that these features are necessary for digestive function in an acid environment. Adaptive residues putatively conferring stability in an acid proteolytic environment differ between insects and vertebrates, probably because they depend on the overall three-dimensional structure of the lysozymes. A maximum likelihood phylogeny and inferences from insect lysozyme sequences showed that the recruitment of lysozymes as digestive enzymes is an ancestral condition of the flies (Diptera: Cyclorrhapha).
Insect Biochem Mol Biol
PMID:Molecular adaptation of Drosophila melanogaster lysozymes to a digestive function. 969 34

Cathepsin D, a lysosomal aspartic protease, has been purified from porcine liver using a combination of pepstatin-A agarose and Affi-Gel Blue affinity chromatography, followed by size-exclusion chromatography. The purified protein consists of two polypeptide chains of 15 and 30 kDa, and has an isoelectric point of 6.8. Porcine liver cathepsin D has maximum activity at pH 2.5-3.0 as determined by its activity against hemoglobin, with a Kcat of 14.3 s-1 and a kcat/KM of 2.70 x 10(6) s-1M-1 as determined by the hydrolysis of a fluorogenic peptide substrate.
Biochem Mol Biol Int 1998 Jul
PMID:Purification and partial characterization of cathepsin D from porcine (Sus scrofa) liver using affinity chromatography. 971 4


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