In vitro and in vivo inhibition of rat liver cathepsin L by epidermal proteinase inhibitor

A monoclonal antibody against chick type II collagen has been produced by lymphocyte-myeloma cell hybridization. The antibody, harvested either from spent medium of hybridoma cultures or from ascites fluid of hybridoma-containing mice, has an extremely high titer against type II collagen but shows no activity against type I. The antigenic site of the collagen seems to be located within the helical portion of native molecules. Using fluorescence histochemical procedures, the antibody can be used to localize type II collagen in sectioned material.     

Participation of lysosomes in basal proteolysis in perfused rat liver. Discrepancy between leupeptin-induced lysosomal enlargement and inhibition of proteolysis

Livers of non-starved rats were perfused cyclically for 2 h in the presence of either 4 x the normal concentration of amino acids (known to suppress proteolysis to basal level), or the same medium together with leupeptin (an inhibitor of cathepsin B, H and L). Stereologic analysis revealed that the drug elicited a linear increase in the fractional cytoplasmic volume of the hepatocytic autophagic vacuolar compartment amounting to 1.34%/h. Measurements of proteolysis under the same experimental conditions showed that addition of leupeptin to the perfusate reduced proteolysis from 1.74%/h to 1.34%/h, i.e. an inhibition of 21.6% was observed. Thus, although proteolysis was only little inhibited by leupeptin, cytoplasm was sequestered at a rate that almost fully accounted for overall protein breakdown during basal state. The reasons for this discrepancy, such as subtotal inhibition of proteinases, increase of lysosomal contents and compensatory increased non-lysosomal degradation are discussed.     

Rat liver thiol proteinases: cathepsin B, cathepsin H and cathepsin L

Data on following points of lysosomal thiol proteinases (cathepsins B, H and L) from rat liver are described in this paper: Partial amino acid sequence of cathepsin B, substrate specificity of cathepsin L, immunological studies of cathepsin B and H and effectiveness of E-64, specific thiol proteinase inhibitor in vivo.     

Bombyx cysteine proteinase inhibitor (BCPI) homologous to propeptide regions of cysteine proteinases is a strong, selective inhibitor of cathepsin L-like cysteine proteinases.

Bombyx cysteine proteinase inhibitor (BCPI) is a novel cysteine proteinase inhibitor. The protein sequence is homologous to the proregions of certain cysteine proteinases. Here we report the mechanism of its inhibition of several cysteine proteinases. BCPI strongly inhibited Bombyx cysteine proteinase (BCP) activity with a K(i) = 5.9 pM, and human cathepsin L with a K(i) = 36 pM. The inhibition obeyed slow-binding kinetics. The inhibition of cathepsin H was much weaker (K(i) = 82 nM), while inhibition of papain (K(i) > 1 microM) and cathepsin B (K(i) > 4 microM) was negligible. Following incubation with BCP, BCPI was first truncated at the C-terminal end, and then gradually degraded over time. The truncation mainly involved two C-terminal amino acid residues. Recombinant BCPI lacking the two C-terminal amino acid residues still retained substantial inhibitory activity. Our results indicate that BCPI is a stable and highly selective inhibitor of cathepsin L-like cysteine proteinases.     

The genes of the lysosomal cysteine proteinases cathepsin B, H, L, and S map to different mouse chromosomes

The mouse genes for the lysosomal cysteine proteinases cathepsin B, H, L, and S were mapped to Chromosomes (Chrs) 14, 9, 13, and 3, respectively. Two of the DNA probes used in this study detected an additional, independently segregating locus. The cathepsin B-specific probe hybridized to a locus on Chr 2, and the cathepsin H probe to a locus on the X Chr. These loci either correspond to pseudogenes or to cathepsin B- and cathepsin H-related genes. The four cysteine proteinases mapped in this study lie within known regions of conserved synteny between mouse and human chromosomes, when compared with the corresponding positions of their human homologs. Assuming that the genes of the cysteine proteinase gene family arose from a common ancestral gene, our results suggest that these four cysteine proteinases had been dispersed over different chromosomes before separation of mouse and human in evolution. Received: 22 August 1996 / Accepted: 20 November 1996     

The design of peptidyldiazomethane inhibitors to distinguish between the cysteine proteinases calpain II, cathepsin L and cathepsin B.

A series of peptidyldiazomethanes was synthesized and tested as inactivators of the cysteine proteinases calpain II, cathepsin L and cathepsin B. Inactivators that react rapidly and that show a degree of selectivity between the enzymes were identified. Z-Tyr(I)-Ala-CHN2 (where Z represents benzyloxycarbonyl) reacts rapidly with cathepsin L and more slowly with cathepsin B, but does not inhibit calpain II. Z-Leu-Leu-Tyr-CHN2 reacts rapidly with cathepsin L and calpain II but very slowly with cathepsin B. Boc-Val-Lys(epsilon-Z)Leu-Tyr-CHN2 (where Boc represents t-butyloxycarbonyl) reacts more rapidly with calpain II than with cathepsin L or cathepsin B. The discriminating inhibitory effects of these compounds make them potentially useful for investigation of enzyme functions in vivo. The data presented also provide insights into the subsite specificity of calpain.     

Long-term ingestion of ammonium inhibits lysosomal proteolysis in rat liver

A standard diet was supplemented with ammonium acetate (20%, w/w). The effect on liver protein degradation of oral administration of the ammonium diet to rats for 6 weeks has been studied. It is shown that lysosomal proteolysis is markedly decreased (by 62%) while non-lysosomal proteolysis is inhibited by 11%. This is the first report showing that ammonium ingestion inhibits liver proteolysis.     

Inactivation and degradation of rat liver catalase by mitochondrial and lysosomal proteinases

The initial phases of catalase degradation in rat hepatocytes were studied. Preparations of highly purified fractions of lysosomes and mitochondria from rat liver were obtained. The proteinase activity was measured by the radio-isotope method by the increase of the free amino groups or by the decrease of the catalase activity, using labelled catalase as a substrate. It was found that the initial step of catalase degradation occurs in the enzyme localized in the inner membrane as well as in the mitochondrial matrix and that the total degradation of catalase is completed in the lysosomal fraction of rat liver.     

Towards specific functions of lysosomal cysteine peptidases: phenotypes of mice deficient for cathepsin B or cathepsin L

The lysosomal cysteine peptidases cathepsin B and cathepsin L are abundant and ubiquitously expressed members of the papain family, and both enzymes contribute to the terminal degradation of proteins in the lysosome. However, there is accumulating evidence for specific functions of lysosomal proteases in health and disease. The generation of 'knock out' mouse strains that are deficient in lysosomal proteases provides a valuable tool for evaluation of existing hypotheses and gaining new insights into the in vivo functions of these proteases. In this minireview, we summarise and discuss the findings obtained by analysis of mice that are devoid of cathepsin B or cathepsin L. In brief, cathepsin L appears to be critically involved in epidermal homeostasis, regulation of the hair cycle, and MHC class II-mediated antigen presentation in cortical epithelial cells of the thymus. Cathepsin B plays a major role in pathological trypsinogen activation in the early course of experimental pancreatitis and contributes significantly to TNF-alpha induced hepatocyte apoptosis.     

Crystallization and properties of cathepsin B from rat liver.

Cathepsin B from rat liver was purified to apparent homogeneity by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, DEAE-Sephadex and CM-Sephadex column chromatography, and was crystallized. The purified enzyme formed spindle-shaped crystals and its homogeneity was proved by disc gel electrophoresis in the presence of sodium dodecyl sulfate and by ultracentrifugal analysis. Its s20,w value was 2.8 S and its relative molecular mass was calculated to be 22,500 (+/- 900) by sedimentation equilibrium analysis. Crystalline cathepsin B was shown to consist of four isozymes with isoelectric points between pH 4.9 and 5.3, the main isozyme having an isoelectric point of pH 5.0. The enzyme was irreversibly inactivated by exposure to weak alkali. The pH optimum was 6.0 with alpha-N-benzoyl-DL-arginine-4-nitroanilide as substrate. Amino acid analysis showed that the enzyme contained hexosamine, glucosamine and galactosamine. Cathepsin B inactivated aldolase, glucokinase, apo-ornithine aminotransferase, and apo-cystathionase, but the rates of inactivation of glucokinase, apo-ornithine aminotransferase, and apocystathionase were lower than that of aldolase. Studies by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate showed that cathepsin B degraded apo-ornithine aminotransferase to two polypeptide chains differing in relative molecular mass and electrophoretic mobility.     

Effects of lysosomal proteinase inhibition on the development of the rat embryo in vitro

Altered lysosomal function in the visceral yolk sac can result in abnormal development. As proteolysis is an important function of the rodent visceral yolk sac during early and mid-gestation, we characterized the lysosomal proteolytic enzyme activity of this extraembryonic membrane and determined the effects of inhibitors of protein degradation on embryonic development. Constituent activities of cysteine and aspartic acid proteinases were measured in rat visceral yolk sac on gestation day 12, and the effects of the cysteine proteinase inhibitors leupeptin, E-64 [trans-epoxysuccinyl-l-leucylamido(4-guanido)butane] and N-ethylmaleimide and the aspartic acid proteinase inhibitor pepstatin were determined in Sprague-Dawley rat embryos cultured in vitro from gestation days 10-12. It was determined that only cysteine proteinases, primarily cathepsins B and L, are active in the mid-gestation visceral yolk sac. The cysteine proteinase inhibitors leupeptin and E-64 both produced a concentration-related decrease in embryonic growth, as measured by crown-rump length, somite number, and embryonic protein content, and a concentration-related increase in incidence of abnormalities. A characteristic pattern of abnormalities was produced which involved a decrease in neural tube volume and the formation of a subectodermal blister opposite the point of attachment of the vitelline vessels. At high concentrations, anophthalmia was also observed. The decreased neural tube volume was associated with increased osmolality of the exocoelomic fluid, the major extraembryonic fluid compartment. It is possible that the osmotic change decreased neural tube volume by causing water to move to the compartment with a higher solute concentration, out of the embryo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potent and selective inactivation of cysteine proteinases with N-peptidyl-O-acyl hydroxylamines.

In all of eight tested human cells and cell lines with inducible indoleamine 2,3-dioxygenase (EC 1.13.11.17) tetrahydrobiopterin biosynthesis was activated by interferon-gamma. This was demonstrated by GTP cyclohydrolase I (EC 3.5.4.16) activities and intracellular neopterin and biopterin concentrations. Pteridine synthesis was influenced by extracellular tryptophan. In T 24-cell extracts, submillimolar concentrations of tetrahydrobiopterin stimulated the indoleamine 2,3-dioxygenase reaction.     

Studies on activation and inhibition of cathepsin B from buffalo liver

Cathepsin B (EC 3.4.22.1) was purified from buffalo liver. The enzyme activity against-benzoyl-dl-arginine-naphthylamme (BANA) was substantially reduced by heat (above 37C) and by nondenaturing concentrations of urea (3 M) and guanidine hydrochloride (1 M). Cathepsin B was significantly activated by 1.5 mM EDTA alone. The activation of the enzyme was further enhanced in the presence of thiol compounds, e.g., cysteine thioglycolic acid, 2,3-dimercapto-1-propenol, and dithioerythritol (DTE). The minimum concentration of the thiol compound required for optimal activation of cathepsin B was found to be lowest (0.2 mM) for DTE. The BANA hydrolyzing activity of cathepsin B was substantially reduced by Cu²⁺ (20–200M) and Ca²⁺ (30–250 mM) as well as by thiol blocking reagents, e.g., iodoacetate, 5,5-dithiobis(2-nitro-benzoic acid) (DTNB), andp-hydroxymercuribenzoate (pHMB). The enzyme activity was completely abolished when the molar ratio of the reagent: cathepsin B was close to 1. The number of free sulfhydryl groups in cathepsin B was determined to be 2 by titration against DTNB and pHMB. Modification of one free thiol group of cathepsin B resulted in complete loss of BANA hydrolyzing activity.     

Evidence for the selective release of lysosomal proteinases in fasted rabbits.

The enzyme responsible for the conversion of "neutral" to "alkaline" fructose 1,6-bisphosphatase (EC 3.1.3.11) by removal of a 7000 dalton peptide (converting enzyme, Proteinase I) has been shown to be localized in rat liverlysosomes. Lysosomes also contain a specific proteinase (Proteinase II) that catalyzes the release of a small peptide from the NH2-terminus of the native subunits. In fasted rabbits Proteinase II is released into the cytoplasm, together with Cathepsin A, but Proteinase I remains associated with the lysosomal fraction. Increased osmotic fragility of liver lysosomes in fasted rabbits has also been observed, but this increased fragility does not result in the release of Proteinase I. The appearance of Proteinase II in the cytoplasm may be due either to its selective release from the lysosomes, without release of Proteinase I, or its localization in a different lysosomal fraction. Changes in lysosomal structure induced by fasting may play a dual role in : 1) the mobilization of amino acids for gluconeogenesis and 2) the modulation of activity of gluconeogenic enzymes.     

Participation of intracellular cysteine proteinases, in particular cathepsin B, in degradation of collagen in periosteal tissue explants

The involvement of cysteine proteinases in the degradation of soft connective tissue collagen was studied in cultured periosteal explants. Using cysteine proteinase inhibitors that were active intracellularly or extracellularly (Ep453 and Ep475, respectively), it was shown that over-all collagen degradation, as measured by the release of hydroxyproline, decreased significantly on inhibition of the intracellular pool of cysteine proteinases by Ep453. This inhibitor also induced an accumulation of intracellular fibrillar collagen in fibroblasts, indicating a decreased degradation of phagocytosed collagen. The extracellular inhibitor, Ep475, had minor or no effects.Histochemical analysis using a substrate for the cysteine proteinases cathepsins B and L revealed a high level of enzyme activity, which was completely blocked in explants preincubated with a selective intracellular inhibitor of cathepsin B, Ca074-Mc. Moreover, the cathepsin B inhibitor strongly affected collagen degradation, decreasing the release of hydroxyproline and increasing the accumulation of phagocytosed collagen. These effects were comparable or slightly stronger than those found with the general intracellular inhibitor (Ep453). Taken together, these data strongly suggest that intracellular cysteine proteinases, in particular cathepsin B, play an important role in the digestion of soft connective tissue collagen.