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)

Recombinant mouse (Mus musculus) and rat (Rattus norvegicus) cystatin C were produced by expression in Escherichia coli, isolated and functionally characterized. The mouse and rat inhibitors were both fully active in titrations of papain. Determination of equilibrium constants for dissociation (Ki) for their complexes with the target proteinase, cathepsin B, produced values not largely different from that for human cystatin C (Ki 0.07-0.13 nM). Rabbit antisera against mouse and rat cystatin C were produced and used for improved affinity purification of the recombinant inhibitors. Affinity purified immunoglobulins isolated from the antiserum against mouse cystatin C were used for construction of a sensitive enzyme-linked immunosorbent assay. The assay was used to demonstrate a high degree of immunological cross-reactivity between mouse and rat cystatin C and could be used for cystatin C quantification in mouse and rat tissue homogenates. All tissues analyzed contained cystatin C, with a relative content very similar to that of human tissues. For all species, brain tissue contained the highest cystatin C amounts and liver the lowest, whereas kidney, spleen and muscle tissues were intermediate in content. In the mouse, a notable high cystatin C content in parotid gland tissue was observed. The high degree of similarity in distribution pattern and functional properties for mouse, rat and human cystatin C indicates that a murine model should be relevant for studies of the human disease, hereditary cystatin C amyloid angiopathy.
Comp Biochem Physiol B Biochem Mol Biol 1996 Jul
PMID:Mouse and rat cystatin C: Escherichia coli production, characterization and tissue distribution. 876 Nov 77

1,25-Dihydroxyvitamin D3 [1,25(OH)2(D)3], the active metabolite of vitamin D, is a potent inhibitor of breast cancer cell growth both in vivo and in vitro. To complement data which documents the anti-proliferative effects of 1,25(OH)2(D)3, we assessed the role of apoptosis in vitamin D-mediated growth arrest of MCF-7 cells. Time course studies indicated that 100 nM 1,25(OH)2(D)3 significantly reduces MCF-7 cell numbers within 48 h of treatment. Morphological assessment demonstrated that MCF-7 cells treated with 1,25(OH)2(D)3 for 48 h exhibit characteristic apoptotic features, including cytoplasmic condensation, pyknotic nuclei, condensed chromatin and nuclear matrix re-organization. In situ end labelling with terminal transferase indicated that cells exhibiting apoptotic morphology in 1,25(OH)2(D)3-treated cultures were positive for DNA strand breaks. These morphological features of apoptosis were accompanied by an increase in the cell death rate assessed as soluble DNA-histone complexes indicative of DNA fragmentation. To complement the morphological data, we assessed the temporal expression of two proteins which have been associated with apoptosis in mammary cells and tumors. The steady state mRNA levels for TRPM-2/clusterin and cathepsin B mRNA were significantly up-regulated in MCF-7 cells treated with 1,25(OH)2(D)3 compared to control cells. Time-dependent increases in the expression of TRPM-2/clusterin and cathepsin B proteins were detected by Western blotting in 1,25(OH)2(D)3-treated cells. These findings indicate that, in addition to its anti-proliferative effects, 1,25(OH)2(D)3 activates the apoptotic cell death pathway in MCF-7 breast cancer cells.
J Steroid Biochem Mol Biol 1996 Jul
PMID:1,25-Dihydroxyvitamin D3 induces morphological and biochemical markers of apoptosis in MCF-7 breast cancer cells. 890 20

The phenotype of autosomal dominant polycystic kidney disease (ADPKD) is characterized by basement membrane abnormalities, hyperproliferation, and alterations in epithelial cell polarity. Since proteinases have been implicated in matrix degradation and growth factor activation, lysosomal enzymes were compared in normal and ADPKD tissues and cell cultures. Acidic proteolytic activity (azocasein) was reduced in ADPKD, and specific enzymatic assays detected disease-dependent decreases in the specific activities of beta-galactosidase, beta-hexosaminidase, and cathepsins, B, L, and H. Cathepsin D-specific activities were unchanged. Lucifer yellow fluorescence in ADPKD cells was consistent with an alteration in heterogeneity of lysosomal enzyme content in ADPKD rather than a decrease in total lysosomal number. Western analysis, metabolic labeling, and immunoprecipitation analysis confirmed decreases in the expression and synthesis of the major normal molecular immunoreactive species of beta-galactosidase and cathepsins B and H in ADPKD tissue and cells but no changes in cathepsin D. In addition, ADPKD-specific high-molecular-weight species of cathepsin H were seen and abnormal forms of cathepsin B and beta-galactosidase were common in ADPKD, suggesting abnormal molecular processing and posttranslational modifications. In addition, immunolocalization studies showed abnormal apical plasma-membrane localization of cathepsins B and H in ADPKD cyst epithelial cells, consistent with a protein sorting defect in ADPKD. Increased extracellular secretion of lysosomal enzymes was also measured in ADPKD cultured cells and in filter-grown epithelia shown to be predominantly directed to the basal compartment. These results demonstrate that lysosomal enzyme alterations in ADPKD may play a role in aberrant processing of the basement membrane. Alterations in the polarized secretion of lysosomal enzymes by ADPKD epithelia in vitro were also detected. Whereas all normal epithelia cells secreted lysosomal enzymes predominantly to the apical medium compartments, basally directed secretion was increased in all ADPKD epithelia and attained an overall reversal of polarity for cathepsins B + L. It is concluded that alterations in lysosomal enzyme function in ADPKD are the result of alterations in synthesis, molecular processing, and polarized secretion of specific enzymes and may have impact on proliferative and basement membrane abnormalities in this genetic disease. These results are consistent with a fundamental defect in protein processing sorting, and trafficking in ADPKD.
Biochem Mol Med 1997 Feb
PMID:Functional defects in lysosomal enzymes in autosomal dominant polycystic kidney disease (ADPKD): abnormalities in synthesis, molecular processing, polarity, and secretion. 906 78

The interleukin-1 beta converting enzyme (ICE) family of cysteine proteases has been implicated in apoptosis. This study tested the effects of a novel pan-ICE family inhibitor, bocaspartyl(OMe)-fluoromethylketone (boc-Asp-CH2F), against low potassium-induced apoptosis of cultured rat cerebellar granule neurons (CGN). A single application of this cell-permeant compound (20 microM) inhibited apoptotic cell death up to 48 h. Classical apoptotic changes were monitored by fluorescence microscopy, DNA fragmentation and scanning electron microscopy (SEM). A control peptidic fluoromethylketone (boc-Thr-CH2F), and inhibitors to calpain (Ac-Leu-Leu-norleucinal), cathepsin B (Z-Phe-Ala-CH2F), and CPP32-like proteases (Z-DEVD-CH2F), failed to prevent apoptotic death. An 35S-methionine incorporation assay verified that, unlike cycloheximide, boc-Asp-CH2F did not inhibit protein synthesis, hence excluding this as a rescuing mechanism. Although ICE was not detected by northern blot analysis, both CPP32 and Nedd2 expression were found to increase during apoptosis. Kinetic assays with cell extracts from boc-Asp-CH2F-treated neurons measured reduced rates of cleavage for DEVD-pNA and LEVD-pNA. At present, ICE-like proteases remain viable candidates for mediating neuronal death.
Mol Psychiatry 1997 May
PMID:Inhibition of the interleukin-1 beta converting enzyme family rescues neurons from apoptotic death. 915 87

In the seminiferous epithelium, germ cell development behind the blood-testis barrier involves continual degradation and renewal of inter-testicular cell junctions. This allows: (i) the translocation of developing germ cells from the basal lamina to the adluminal compartment during spermatogenesis, and (ii) the eventual release of mature spermatids into the tubular lumen during spermiation. Throughout spermatogenesis, cellular debris must also be removed from the epithelium Thus, it is conceivable that proteases, protease inhibitors, and cell junctional components are involved in these events. The present study sought to examine whether testicular cells can express multiple cathepsin mRNAs given that these proteases are involved in the degradation and processing of proteins as well as in tissue regeneration. By using total RNA isolated from primary cultures of Sertoli, Leydig, and germ cells for reverse-transcription and polymerase chain reaction (RT-PCR), the mRNAs of cathepsin B, C, D, H, L, and S were shown to be expressed by Sertoli and Leydig cells, whereas germ cells isolated from adult rats expressed all of the above cathepsin mRNAs except cathepsin D. Throughout postnatal development and maturation, the testicular steady-state mRNA levels of cathepsin B, C, D, L, and S remain relatively unchanged with the exception of cathepsin H whose mRNA level increased during maturation and peaked at 45-60 days of age. Using lonidamine, an anti-spermatogenic drug which is known to induce premature release of germ cells without affecting Leydig cell function by disrupting the inter-Sertoli-germ cell junctions, we have examined the differential expression of these cathepsin mRNAs in the testis at the time of extensive tissue restructuring. It was noted that the expression of cathepsin L and S in the testis increased significantly concomitant with the disappearance of elongate spermatids whereas the expression of cathepsin B, C, D, and H increased significantly when most of the round spermatids and spermatocytes were depleted. These results illustrate the intricate inter-relationship between these proteases in the testis during maturation and tissue restructuring.
Biochem Mol Biol Int 1997 Jun
PMID:Differential expression of multiple cathepsin mRNAs in the rat testis during maturation and following lonidamine induced tissue restructuring. 923 20

Mutants null for the cathepsin B-like cysteine proteinase gene (cpc) of Leishmania mexicana have been generated by targeted gene disruption. The gene deletion was confirmed using a polymerase chain reaction (PCR) method with cpc-specific primers and genomic DNA isolated from the mutants. cpc was re-expressed in the null mutants from an episomal vector. Re-expression of the enzyme (CPC) was detected by Western blotting with a specific anti-peptide antiserum. The cpc null mutants grew apparently normally as promastigotes and amastigotes in axenic cultures, but they showed greatly reduced infectivity to macrophages in vitro with only a low percentage of the cells being infected. Re-expression of cpc in the null mutant increased the parasite's infectivity in vitro. The null mutant parasites formed lesions in mice at a similar rate as wild type parasites, although somewhat smaller lesions were produced. The results suggest that although the cysteine proteinase encoded by cpc plays a role in the parasite's interaction with macrophages it alone is not crucial for infectivity or virulence.
Mol Biochem Parasitol 1997 Sep
PMID:Cathepsin B-like cysteine proteinase-deficient mutants of Leishmania mexicana. 927 67

The structure of the wild-type human procathepsin B has been refined to a crystallographic R-value of 0.18 and R-free of 0.23 exploiting the data obtained from new crystals that diffract beyond 2.5 A resolution. The structure confirms two previously presented, lower-resolution structures. The structure of the propeptide chain folds on the surface of the enzyme domains and blocks access of substrate to the already formed active site. Abundant solvent molecules fill the cavities between the propeptide and the enzyme part of the molecule. The propeptide structure is compared with a substrate model in the S2, S1, S1' and S2' binding sites. In this crystal form the cathepsin B occluding loop residues adopt yet another conformation. The structures show that the occluding loop region between the residues Cys108 and Cys119 behaves quite independently from the rest of the structure and easily adapts to changes in environment. The variety of the observed conformations of the occluding loop is in agreement with other data showing that the loop is responsible for limiting cathepsin B activity to that of a carboxydipeptidase. The region before Cys108 is essentially the same as in the mature structure, whereas the region from Cys119 to Thr125 is raised compared to the mature form by the propeptide squeezed between it and the enzyme domains, surface. The structure strongly suggests that processing of procathepsin B during its autoactivation is not unimolecular.
J Mol Biol 1997 Sep 05
PMID:Crystal structure of the wild-type human procathepsin B at 2.5 A resolution reveals the native active site of a papain-like cysteine protease zymogen. 929 26

To improve the level of purity of cathepsin B, we have modified the published procedure [Agarwal, S.K. and Khan, M.Y. (1987) Biochem. Int. 15,785-792] by incorporating CM-Sephadex ion exchange chromatography and chromatofocusing. The enzyme thus isolated could be resolved into one 26 kDa major and a minor 27 kDa protein bands on SDS-PAGE. The two components, however, could not be separated by gel filtration and they eluted, in a single peak corresponding to a molecular mass of 28.1 kDa. Among the various substrates tested, Z-Phe-Arg-MCA with a Km of 0.058 mM and hemoglobin with a Km of 1.449 microM were the most preferred synthetic and protein substrates respectively. It was found to be a glycoprotein with an acidic pI of 4.8. The enzyme was activated by various thiol-reducing reagents and inhibited by cysteine proteinase inhibitors, divalent cations, lysyl group modifiers, anti-inflammatory drug and denaturing agents. The hydrodynamic behaviour of cathepsin B suggested a compact and globular conformation. Immunodiffusion studies with anti-goat cathepsin B indicated a tissue/ species dependence.
Biochem Mol Biol Int 1997 Sep
PMID:Catalytic and physico-chemical characteristics of goat spleen cathepsin B. 930 39

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

Characterization of cathepsin B from buffalo kidney and goat spleen showed the presence of isozymes in case of the goat spleen (GSCB-I and GSCB-II) whereas cathepsin B from buffalo kidney exhibited only one form (BKCB). The molecular weights determined by SDS-PAGE for GSCB-I, GSCB-II, and BKCB were 25.7, 26.6 and 25.5 kDa respectively. The kinetic parameters (Km and Vmax) of GSCB-I showed close similarities with BKCB against alpha-N-benzoyl-DL-arginine-2-napthylamide whereas GSCB-II was closer to the buffalo enzyme with regards to its activity against Z-Arg-Arg-MCA and Z-Phe-Arg-MCA. All the three enzymes had similar sensitivities towards urea, antipain and leupeptin. However, clear differences were observed in the inhibition patterns of the enzyme with iodoacetic acid and iodoacetamide. Differences in the kinetic, immunogenic and some catalytic properties of GSCB-I and II, which had similarities with regard to most of their physico-chemical properties, were considered to be due to the existence of two isozyme forms in goat spleen cathepsin B preparations. Absence of such a multiplicity in forms of the enzyme from buffalo kidney was accordingly attributed to the absence of cathepsin B isozymes in this species. These observations taken together therefore, indicate a probable species/tissue dependence of cathepsin B.
Mol Cell Biochem 1997 Dec
PMID:On the tissue/species dependence of cathepsin B isozymes. 945 Jun 49


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