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)

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

The mature form of cathepsin D (Cat D), purified to homogeneity from postmortem human brain or mouse brain, behaved as a 42-kDa protein in its native state but revealed additional proteolytic processing under denaturing conditions. Human brain Cat D was composed of a 30-32 kDa heavy chain and a protein doublet consisting of 14 and 15 kDa light chains. Mouse Cat D, which closely resembled the human enzyme in amino acid composition, existed mainly as the uncleaved 42-kDa protein, but up to 40% existed as a complex of 30-32 kDa and 12-14 kDa chains. The 3:1 ratio of light to heavy (30-32 kDa) chains suggested processing of some 30-kDa chains. Cleavage of the 42-kDa chain could not be induced autolytically. Human brain Cat D had a 2-3-fold higher specific activity than the mouse enzyme but shared other properties, including similar biphasic pH optima (peaks at pH 3.30 and 4.2), Km values for methemoglobin and inhibitor profiles. Human Cat D displayed the same polypeptide chain composition when purified from brains differing in postmortem interval (3-28 h). Fresh SH-SY5Y human neuroblastoma cells analyzed on Western blots with anti-Cat D antibodies also displayed only cleaved forms of mature Cat D. Furthermore, brain Cat D isolated from mice stored after death for 5, 15 or 30 h at 25 degrees C contained the same molar ratios of cleaved and uncleaved enzyme found in fresh mouse brain . Cat D activity was stable in human brains with postmortem intervals up to 27 h and stored frozen for up to 3 years. Similarly, total Cat D activity was essentially unchanged in brains of mice subjected to stimulated postmortem conditions for 0.5-4.2 h, although 20% of the total soluble brain protein became insoluble during this postmortem interval. These results demonstrate a remarkable postmortem stability of Cat D and strongly suggest that limited proteolytic cleavage of mature brain Cat D is an in vivo event, the extent of which varies markedly in different species.
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PMID:Limited proteolytic processing of the mature form of cathepsin D in human and mouse brain: postmortem stability of enzyme structure and activity. 884 39

By use of the signal sequence trap method, we isolated a cDNA encoding a novel aspartic protease-like protein from the mouse kidney, and termed it 'kidney-derived aspartic protease-like protein (KAP).' The protein, a 419-amino-acid polypeptide with a 16-amino-acid signal sequence, had 47% identity with mouse cathepsin D, and its overall structure was closely related to known aspartic proteases. Northern blot analysis revealed that KAP mRNA is expressed at the highest level in the kidney, at a moderate level in the lung, and at low levels in the spleen and adipose tissue. In situ hybridization analysis demonstrated that the mRNA is expressed abundantly in the proximal straight tubule and slightly, but significantly, in the proximal convoluted tubule in the kidney. This intra-renal distribution differs distinctly from those of previously reported proteases such as cathepsins B, D, and H.
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PMID:Molecular cloning of a novel mouse aspartic protease-like protein that is expressed abundantly in the kidney. 901 90

Three protein inhibitors of proteolytic enzymes with molecular weights 21, 22, and 23 kD were isolated from potato tubers (Solanum tuberosum L.) by ammonium sulfate precipitation followed by gel and ion-exchange chromatography. The 21- and 22-kD proteins were shown to be serine proteinase inhibitors with different specificities. The 21-kD protein inhibits human leucocyte elastase and trypsin effectively, but it is less effective towards chymotrypsin. The 22-kD protein is an inhibitor of cysteine proteinases and suppresses the activities of papain, ficin, and bromelain with the same affinities. None of the isolated proteins inhibit subtilisin, pepsin, or cathepsin D. The 21-kD protein consists of two disulfide-linked polypeptide chains with molecular weights of 16.5 +/- 1 kD and 4.5 +/- 1 kD. The 22-kD and 23-kD proteins have a single polypeptide chain. The N-terminal 22-25 amino acid sequences of these three proteins were determined. These sequences have significant homology to other plant inhibitors from the Kunitz soybean inhibitor superfamily.
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PMID:Potato tuber protein proteinase inhibitors belonging to the Kunitz soybean inhibitor family. 948 70

Rab7 is a small molecular weight GTPase that is known to be associated with late endocytic compartments. Studies in which wild-type or mutant forms of this protein have been overexpressed in mammalian cells have indicated that rab7 plays a role in controlling membrane transport between late endocytic compartments. However, both the precise site(s) of action and localization of rab7 remain unclear. In the present study, we have used density-gradient centrifugation in combination with a new epitope-specific flow cytometric sorting method to isolate rab7-containing vesicles from baby hamster kidney (BHK) cells. Electron-micrographs of sorted elements showed a homogeneous population of vesicles that resembles late endosomes. The polypeptide composition of rab7-containing vesicles was then analyzed by two-dimensional (2-D) gel electrophoresis. Rab7-containing vesicles were enriched in the cation-independent mannose 6-phosphate receptor and especially in the precursor forms of cathepsin D. Taken together, these results show that the rab7-containing vesicles are a component of the endocytic pathway that connects late endosomes and lysosomes and in which precursor forms of lysosomal hydrolases, segregated from their receptor, might be included.
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PMID:Flow cytometric sorting and biochemical characterization of the late endosomal rab7-containing compartment. 952 98

Thyrocytes are known for their ability to iodinate thyroglobulin from which the thyroid hormones are generated. In the intact thyroid gland the iodination process is almost exclusively executed at the apical plasma membrane of thyroid epithelial cells. Here, we show that freshly isolated thyrocytes iodinated polypeptides other than thyroglobulin and that one of the major iodinated polypeptides was the mature form of the lysosomal protease cathepsin D (CD). The detection of mature CD as an iodinated polypeptide suggested that a fraction of the lysosomally maturated enzyme was delivered to the apical plasma membrane where it became available for iodination. After labeling of thyrocytes with [35S]methionine/cysteine overnight part of the mature CD was released into the culture medium. This was abolished by inhibiting maturation of CD with NH4Cl, indicating that mature CD appeared in the medium after its proteolytic maturation in an acidic compartment. Besides CD other soluble lysosomal polypeptides like the beta-N-acetylhexosaminidase and the sphingolipid-activating protein D (Sap D) were iodinated and partially secreted as mature polypeptides. In contrast, the membrane-associated lysosomal ceramidase was iodinated and partially secreted as immature single-chain enzyme and not as fully maturated two-chain enzyme. These data indicate that a portion of mature CD and other soluble lysosomal enzymes is delivered from lysosomes to the cell surface whereas some membrane-associated enzymes from the terminal lysosomal compartment are efficiently excluded from this process.
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PMID:Iodination of mature cathepsin D in thyrocytes as an indicator for its transport to the cell surface. 965 Jul 83

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.
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PMID:Purification and partial characterization of cathepsin D from porcine (Sus scrofa) liver using affinity chromatography. 971 4

The crystal structure of a pepsin from the gastric mucosa of Atlantic cod has been determined to 2.16 A resolution. Data were collected on orthorhombic crystals with cell dimensions a = 35.98, b = 75.40 and c = 108.10 A, on a FAST area-detector system. The phase problem was solved by the molecular-replacement method using porcine pepsin (PDB entry 5PEP) as a search model. The structure has been refined to a crystallographic R factor of 20.8% using all reflections between 8.0 and 2.16 A, without prior knowledge of the primary sequence. The resulting crystal structure is very similar to the porcine enzyme, consisting of two domains with predominantly beta-sheet structure in the same sequential positions as the enzyme from pig. In the course of the model building, 122 residues were substituted and two residues deleted from the starting model to give a polypeptide chain of 324 amino acids and a sequence identity of 57.7% with the pig pepsin. No carbohydrate residues were located. Sequence alignment with available aspartic proteinases, indicates that the fish enzyme seems to be more related to mammalian gastric pepsins than to the mammalian gastricsins and chymosins, lysosomal cathepsin D's and a pepsin from tuna fish. The amino-acid composition of the cod enzyme, however, is more in accordance with the cathepsin D's.
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PMID:Structure and proposed amino-acid sequence of a pepsin from atlantic cod (Gadus morhua). 976 15

Gaucher's disease (GD) is caused by an inherited deficiency of acid beta-glucosidase with storage of glucosylceramides in the lysosomes of macrophages. This study identifies a G202R mutation in the acid beta-glucosidase gene in an infant with severe neuronopathic (type 2) GD and only slightly reduced acid beta-glucosidase activity. Western blot analysis, pulse chase experiments, and the thin frozen section immunogold method were used to analyse the implications of this mutation on the pathogenesis, clinical heterogeneity and diagnostic evaluation of GD. The results show that acid beta-glucosidase persists in the patient's fibroblasts as a mannose-rich polypeptide in the endoplasmic reticulum and is not transported to the lysosomes. By contrast, high expression of the lysosome-associated membrane proteins LAMP-1 and LAMP-2, saposin C, and cathepsin D was observed in the patient's lysosomes. Immunogold labelling of the integral membrane proteins LAMP-1 and LAMP-2 increases significantly at the cell surface of Kupffer cells and fibroblasts as well as at the apical membrane of hepatocytes. In addition, LAMP-1 and LAMP-2 associate with the bilayer of stored glucosylceramide. It is concluded that defective intracellular transport of mutant acid beta-glucosidase from the endoplasmic reticulum to lysosomes leads to a more severe clinical phenotype than the residual enzyme activity may indicate. Furthermore, the detection of LAMP in the tubular bundles of undigested glucosylceramides, as well as their increased concentration at the surfaces of the affected cells, suggests that these proteins play a role in the storage or removal of substrate in GD. Intracellular targeting of acid beta-glucosidase and LAMP contributes to the broad phenotypic heterogeneity of GD.
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PMID:Intracellular transport of acid beta-glucosidase and lysosome-associated membrane proteins is affected in Gaucher's disease (G202R mutation). 1044 Jul 52

Full-length zebrafish cDNAs encoding two aspartic proteinases were cloned and sequenced. One of the two cDNAs was a 1708 bp product with an open reading frame of 398 amino acid residues corresponding to a cathepsin D. The other was a 1383 bp product encoding a polypeptide chain of 416 amino acids homologous to nothepsin, an aspartic proteinase first identified by us in the liver of Antarctic Notothenioidei. Gene expression assessed by RT-PCR and northern blot hybridization of RNA from different tissues showed that the expression was tissue- and sex-specific. Whereas the cathepsin D gene was expressed in all the tissues examined independently of the sex, the nothepsin gene was expressed exclusively in female livers.
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PMID:Sex- and tissue-specific expression of aspartic proteinases in Danio rerio (zebrafish). 1113 92


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