Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Earlier work has suggested that calcium-containing lysosomes are involved in 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-stimulated intestinal absorption of the divalent cation. In the present report immunofluorescent labelling studies on fixed frozen sections of chick intestine were undertaken to determine whether lysosomes could respond to calcium transport conditions in less than 5 min. Tissue prepared from vitamin D-deficient chicks dosed with vehicle or 1.3 nmol of 1,25(OH)2D3 15 h prior to use was immunofluorescently labelled for cathepsin B, a lysosomal protease. In the absence of calcium absorption, punctate staining was found in the region below the terminal web, and more diffusely in the cytoplasm. The intensity of staining was noticeably greater in sections from 1,25(OH)2D3-treated than control chicks. In sections prepared after 3 min of calcium absorption, cathepsin B staining was localized near the basal and lateral membranes of the epithelial cells. After 30 min of transport, the protease was found in the villus core regardless of vitamin D status; however, immunoreactivity within the epithelial cells of 1,25(OH)2D3-treated chick intestine had returned to pretransport intensity, whereas that of controls had not. To further investigate the specificity of the cathepsin B antibody, the intracellular compartmentalization of the protease was determined by biochemical methods. Using dosing procedures and calcium transport times equivalent to those for the immunofluorescent studies mucosae were collected by scraping, homogenized, and subcellular fractions prepared by a combination of differential and Percoll gradient centrifugation. In the absence of calcium transport, cathepsin B-specific activity was enhanced in whole homogenates, endocytic vesicles, and a lysosomal fraction prepared from intestinal epithelium of 1,25(OH)2D3-treated chicks, relative to vitamin D-deficient controls. After 3 min of calcium absorption, a profound (approximately 4-fold) decrease in endocytic vesicle cathepsin B activity was found regardless of vitamin D status, as well as a similar marked decrease in lysosomes prepared from vitamin D-deficient, but not -treated, chicks. After 30 min of calcium transport, endocytic vesicles prepared from either vitamin D-deficient or 1,25(OH)2D3-treated birds had recovered cathepsin B activity to pretransport levels. However, lysosomes prepared from rachitic chicks remained low in protease levels relative to equivalent fractions from dosed chicks. Thus, biochemical analysis of cathepsin B activity in putative endocytic vesicles and lysosomes supports the intracellular redistribution of protease visualized with immunofluorescence microscopy.
Mol Cell Endocrinol 1991 Jun
PMID:Redistribution of cathepsin B activity from the endosomal-lysosomal pathway in chick intestine within 3 min of calcium absorption. 193 26

When coupled with separation of alveolar macrophages (AM) into four different density fractions (I, II, III and IV) by discontinuous Percoll gradient centrifugation, ultrastructural heterogeneity was evident in secreting process of lysosomal enzymes. Lower dense AM (I and II) released high levels of acid phosphatase and cathepsin B, whereas higher dense ones (III and IV) did not. Ultrastructurally, there were multiple ruffling and active extension of long cytoplasmic processes from one pole or around the cell surface of AM obtained from the higher density fractions. In contrast, AM from lower dense fractions had much less cytoplasmic processes and contained more cytoplasmic vacuoles showing positive reactions of acid phosphatase. These cells featured more frequently round or ovoid knobs with acid phosphatase activity along and from the tips of the cytoplasmic processes, suggestive of exocytosis. It was suggested that these ultrastructural changes linked to the maturation process and release of lysosomal enzymes from differentiated AM.
Cell Mol Biol 1991
PMID:Morphological heterogeneity among fractionated alveolar macrophages in their release of lysosomal enzymes. 205 88

The nucleotide sequence of a gene encoding a 35-kDa thiol protease of the parasitic nematode Haemonchus contortus has been determined. The gene, designated AC-2, shares 97% nucleotide sequence identity and 98% amino acid identity with previously characterized AC-1 cDNAs encoding the thiol protease. The AC-2 gene spans 8 kb and appears to contain 11 introns, ranging in size from 57 bp to over 5.2 kb. One of the introns interrupts the proposed active site region that is conserved between the H. contortus protease and the related thiol proteases cathepsin B and papain. Southern blot hybridization experiments indicate that the protease is encoded by a small gene family in H. contortus. Rabbit antisera prepared against the recombinant protein react on Western blots with 35 and 37-kDa proteins of adult worms. These proteins were not detectable by Western blot analysis in three larval parasitic developmental stages of H. contortus. Northern blot hybridizations indicate that mRNA transcripts for the gene family are present at low levels in a mixed population of third- and fourth-stage larvae but highly abundant in adult worms. Expression of the protease correlates with blood-feeding and suggests a role for the protease in blood digestion.
Mol Biochem Parasitol 1990 Dec
PMID:A developmentally regulated cysteine protease gene family in Haemonchus contortus. 209 Sep 40

We have cloned cDNAs encoding a 35-kilodalton cysteine protease that is a major component of protective extracts isolated from blood-feeding Haemonchus contortus adult worms. Near full-length cDNAs for the protease were isolated by immunoscreening an adult worm cDNA expression library with a rabbit antiserum prepared against the protein eluted from preparative SDS gels and by rescreening the library with oligonucleotide probes. The protein predicted from the nucleotide sequence of the cDNAs and of a genomic DNA clone comprises 342 amino acids and contains an N-terminal signal sequence, 16 cysteine residues and four potential N-linked glycosylation sites. The enzyme appears to be glycosylated in vivo. The H. contortus protease, called AC-1, displays an overall 42% sequence identity with the human lysosomal thiol protease cathepsin B. The similarities between cathepsin B and AC-1 are localized primarily to regions of cathepsin B that comprise the mature, active form of the enzyme. A stretch of six amino acids that includes the active site cysteine of cathepsin B is conserved, and is present in the same relative location in AC-1, suggesting that this region comprises the active site of the H. contortus enzyme.
Mol Biochem Parasitol 1990 Jun
PMID:Molecular cloning and primary sequence of a cysteine protease expressed by Haemonchus contortus adult worms. 238 65

Cathepsin L activity was quantitated in alveolar macrophages (AMs) and bronchoalveolar lavage (BAL) fluid from Sprague-Dawley rats exposed through the nose only to fresh mainstream smoke from University of Kentucky reference cigarettes (2Rl), and in AMs and BAL fluid of room control (RC) and sham control (SH) animals. Activity was determined with the methylcoumarylamide substrate, benzyloxycarbonyl-Phe-Arg-4-methyl-7-coumarylamide (Z-Phe-Arg-NMec). Total activity of cysteine proteinases in AM homogenates towards Z-Phe-Arg-NMec was measured by determining inhibition with E-64 (0.5 microM), a specific inhibitor of this class of enzymes. The activities of cathepsin B and L towards the substrate were differentiated by use of the diazomethane inhibitor, Z-Phe-Phe-CHN2 (0.01 microM), which selectively inhibits cathepsin L at this concentration. We found that cathepsin L activity was significantly elevated in AMs of animals exposed to 10 puffs of cigarette smoke, twice a day, 7 days/wk for 22 wk (1,620 +/- 870 units/mg protein) as compared with cells from RC (420 +/- 150) and SH (550 +/- 160) animals. Cathepsin B activity was also increased in AMs from animals exposed to cigarette smoke (2,360 +/- 890) as compared to RC (930 +/- 170) and SH (1,020 +/- 70) animals. Cathepsin L and B activity was also present in unconcentrated BAL fluid, but levels did not differ significantly among the three groups. The results demonstrate that AMs contain significant levels of cathepsin L activity as measured with methylcoumarylamide substrates, and that activity increases in cells from rats exposed to cigarette smoke in amounts comparable to those inhaled by a healthy 70-kg human smoker using 20 cigarettes a day.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1989 Nov
PMID:Cathepsin L activity in alveolar macrophages of rats: response to cigarette smoke. 263 53

Trophozoites of Plasmodium falciparum obtain free amino acids for protein synthesis by degrading host erythrocyte hemoglobin in an acidic food vacuole. We previously reported that leupeptin and L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64), two inhibitors of the cysteine class of proteinases, blocked hemoglobin degradation in the trophozoite food vacuole, and we identified a 28-kDa trophozoite cysteine proteinase as a potential food vacuole hemoglobinase. We now report that the biochemical properties of the trophozoite cysteine proteinase closely resembled those of the lysosomal cysteine proteinases cathepsin B and cathepsin L. The trophozoite proteinase had a pH optimum of 5.5-6.0, near that of both lysosomal proteinases, and it was efficiently inhibited by highly specific diazomethylketone and fluoromethylketone inhibitors of cathepsin B and cathepsin L. The trophozoite proteinase preferred peptide substrates with arginine adjacent to hydrophobic amino acids, as does cathepsin L. Micromolar concentrations of the fluoromethylketone inhibitor Z-Phe-Ala-Ch2F blocked the degradation of hemoglobin in the trophozoite food vacuole and prevented parasite multiplication. In previous studies much higher concentrations of the inhibitor were not toxic for mice. Our results provide additional evidence that the 28-kDa trophozoite proteinase is a food vacuole hemoglobinase and suggest that specific inhibitors of the enzyme may have potential as antimalarial drugs.
Mol Biochem Parasitol 1989 Jun 15
PMID:Plasmodium falciparum: inhibitors of lysosomal cysteine proteinases inhibit a trophozoite proteinase and block parasite development. 267 22

We have constructed cDNA clones containing the complete nucleotide sequences coding for two highly antigenic Schistosoma mansoni adult worm proteins, Sm31 and Sm32. The predicted amino acid sequence of Sm31 shows significant homology to mouse, rat and human cathepsin B. The nucleotide sequence of Sm32 is identical to that reported by others for S. mansoni "haemoglobinase'. The different nucleotide sequences demonstrate the existence of two different proteolytic enzymes, both of which are synthesised in the form of precursor molecules. Structural homology of the schistosome cathepsin B to the mammalian ones indicates that the mature protein is processed from a propeptide. The calculated molecular weight of haemoglobinase of 47,000 suggests that post-translational processing is also involved in generating an active protease.
Mol Biochem Parasitol 1989 Mar 01
PMID:Primary structures of Sm31/32 diagnostic proteins of Schistosoma mansoni and their identification as proteases. 272 81

In order to study the regulation of cathepsin B expression in the thyroid, cathepsin B mRNA concentrations were measured in rat thyroid cells (FRTL5) in culture. Northern blot analysis demonstrated that cathepsin B mRNA concentrations were increased in FRTL5 cells cultured for up to 6 days in TSH. The effect of TSH on cathepsin B mRNA concentrations was dose dependent over the range 25-150 mu units/ml. Cytoplasmic dot-blot analysis was used to characterize this effect further. The TSH-induced increase in cathepsin B mRNA concentrations (approximately fivefold over that in untreated cells) was partially mimicked by forskolin (approximately threefold) and ionomycin, while phorbol ester decreased cathepsin B mRNA concentrations. Similar changes were observed for thyroglobulin and actin mRNA concentrations. TSH had no effect on cathepsin B enzymatic activity or immunoreactive protein concentration. These results demonstrate (1) that cathepsin B expression in the thyroid is regulated in parallel with that of thyroglobulin and actin, and (2) that cyclic AMP- and Ca2+-dependent processes stimulate gene expression, while phorbol ester treatment inhibits gene expression in FRTL5 cells.
J Mol Endocrinol 1989 May
PMID:Thyrotrophin, forskolin and ionomycin increase cathepsin B mRNA concentrations in rat thyroid cells in culture. 275 29

We have cloned and analyzed a developmentally and spatially regulated prestalk cell-specific gene from Dictyostelium discoideum. The gene encodes a protein highly homologous to the lysosomal cysteine proteinases cathepsin H and cathepsin B. Amino acid comparisons between these enzymes showed that the active-site amino acids were conserved, as were amino acids known to be important for catalysis and residues which form the intramolecular cysteine bridges. We have constructed a series of internal deletions, duplications, and linker scanner mutations within the region 300 base pairs 5' to the cap site. Analysis of expression of the mutations in transformants identified a approximately 35-base pair GC-rich region containing a dAdC/dGdT palindromic repeat and a G-rich box which is homologous to the 3' GT half of the palindromic repeat. Deletion or disruption of the G box resulted in a approximately 50-fold drop in the level of expression of the gene fusion in transformants in response to cyclic AMP in single-cell culture but did not affect the temporal pattern of regulation or control by cyclic AMP. The expression of such constructs during normal multicellular differentiation paralleled that of the endogenous gene; however, the level of RNA from the constructs was only approximately 10-fold lower than that of constructs containing the G box. Deletion of the 3' half of the palindromic sequence and the G box region resulted in a dramatic decrease in the level of transcription, although the constructs still showed proper temporal expression. These results suggest that this 35-base-pair region acts as an important part of the regulatory region for cell type and cyclic AMP regulation.
Mol Cell Biol 1987 Jan
PMID:Identification of the sequences controlling cyclic AMP regulation and cell-type-specific expression of a prestalk-specific gene in Dictyostelium discoideum. 303 53

The comparison of the amino acid sequences of 5 cysteine proteinases: papain, actinidin, rat cathepsins B and H and chicken cathepsin L, demonstrates a striking homology among their sequences. The N-terminal region (residues 1-70 in papain) and C-terminal region (residues 118-212 in papain) display the highest sequence homologies, whereas the lowest sequence homologies are observed in the middle region (residues 71-117 in papain); a segment where most insertions/deletions are observed. The highest sequence homology is observed between rat cathepsin H and chicken cathepsin L. As shown by X-ray studies, papain and actinidin have a clearly defined double domain structure. Each domain contains a core of non-polar side chains, which are retained in cathepsins B, H and L, except for the non-polar residue 203 of the core which is replaced by glutamic acid in cathepsin B. The percentage and the location of alpha-helix and beta-sheets of cathepsins B, H and L, assessed using the methods of Garnier et al. (1978, J. Mol. Biol. 120, 97-120) and Chou and Fasman (1974, Biochemistry 13, 222-245), show that the main ordered structures in papain and actinidin are probably retained in cathepsins B, H and L. The differences observed occur essentially in the middle region, a place where sequences display the lowest homologies and which is far removed from the active site.
 ...
PMID:Sequence homologies, hydrophobic profiles and secondary structures of cathepsins B, H and L: comparison with papain and actinidin. 314 20


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