Purification and properties of a new cathepsin from rat liver.

1) A lysosomal protease, a new cathepsin that inactivates glucose-6-phosphate dehydrogenase [EC 1.1.1.49] and some other enzymes and differs from cathepsin B [EC 3.4.22.1] was purified about 2,200-fold from crude extracts of rat liver by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, and DEAE Sephadex and CM-Sephadex column chromatographies. 2) The new cathepsin was markedly activated by the thiol-reagent, 2-mercaptoethanol and inhibited by monoiodoacetate. 3) The molecular weight of the new cathepsin was found by Sephadex G-75 column chromatography to be 22,000, which is smaller than that of cathepsin B. 4) The optimum pH of the enzyme for inactivation of glucose-6-phosphate dehydrogenase was pH 5.0--5.5. The enzyme was unstable in alkali and on heat treatment. 5) The rates of inactivation of glucose-6-phosphate dehydrogenase, apo-ornithine aminotransferase [EC 2.6.1.13], apo-tyrosine aminotransferase [EC 2.6.1.5], apo-cystathionase [EC 4.4.1.1], glucokinase [EC 2.7.1.2], glyceraldehyde-3-phosphate dehydrogenase [EC 1.2.1.12], and malate dehydrogenase [EC 1.1.1.37] by the new cathepsin were higher than those by cathepsin B. However aldolase [EC 4.1.2.13] was inactivated more rapidly by cathepsin B than by the new cathepsin. Lactate dehydrogenase [EC 1.1.1.27], glutamate dehydrogenase [EC 1.4.1.2] and alcohol dehydrogenase [EC 1.1.1.1] were not inactivated by either cathepsin. Unlike cathepsin B, the new cathepsin scarcely hydrolyzes N-substituted derivatives of arginine.     

An immunocytochemical study on co-localization of cathepsin B and atrial natriuretic peptides in secretory granules of atrial myoendocrine cells of rat heart

To examine localization of cathepsin B, a representative lysosomal cysteine protease, in atrial myoendocrine cells of the rat heart, immunohistochemistry at the light and electron microscopic level was applied to the atrial tissue, using a monospecific antibody for rat liver cathepsin B. In serial semi-thin sections, immunoreactivity for cathepsin B and atrial natriuretic peptides (ANP) was detected in the para-nuclear region of atrial myoendocrine cells. Several large granules and many fine granules in the region of the cells were positively stained by the cathepsin B antibody. Gold particles indicating cathepsin B antigenicity labeled secretory granules in the cells, which were also labeled by those indicating ANP, using thin sections of the Lowicryl K4M-embedded material. Moreover, some granules labeled densely by immunogold particles for cathepsin B seemed to be lysosomes. By double immunostaining using thin sections of the Epon-embedded material, gold particles indicating cathepsin B and ANP antigenicities were co-localized in secretory granules of the cells. By enzyme assay, activity of cathepsin B was three times higher in atrial tissue than ventricular tissue. The results suggest that co-localization of cathepsin B and ANP in secretory granules is compatible with the possibility that cathepsin B participates in the maturation process of ANP.     

Binding specificity of papain and cathepsin B

In order to obtain useful information for the design of inhibitors of cathepsin B, an important enzyme in both physiological and pathological processes, the modes of interaction between ligands and thiol proteases (papain and cathepsin B) were analyzed. A new and powerful method was developed to quantitatively understand geometrical features of the interaction. Fairly good electrostatic complementarities were found for the oxygen atoms of P1 and P2 amide, and for the hydrogen atom of P2 amide. Electrostatic correlation potentials on the side chains of P1 and P2 were nearly neutral, so that interacting conformations in these regions are anticipated to be stabilized by hydrophobic forces rather than electrostatic ones. A three-dimensional structure of rat liver cathepsin B was deduced based on the assumption that the tertiary structure of cathepsin B is similar to that of actinidin. Furthermore, an inhibitor of the thiol proteases, benzyloxycarbonyl-L-phenylalanyl-L-alanine chloromethyl ketone was model fitted to cathepsin B, in order to investigate its mode of interaction with the enzyme. It was possible to account for some structure-activity relationships in the enzyme-ligand interactions.     

A catalytically active high-Mr form of human cathepsin B from sputum.

A cysteine proteinase from purulent sputum was partially purified by a method involving affinity chromatography on Sepharose-aminohexanoylphenylalanylglycinaldehyde semicarbazone. It was immunologically related to lysosomal cathepsin B from human liver and was similar in many, but not all, other aspects. It was catalytically active, as demonstrated by active-site-directed radioiodination, and hydrolysed three cathepsin B substrates, two with Km values similar to those of lysosomal cathepsin B. In addition, the rates of inactivation of the sputum and lysosomal forms of the enzyme by L-3-carboxy-2,3-transepoxypropionyl-leucylamido(4-guanidino) butane (Compound E-64) were very similar. However, the sputum enzyme differed from lysosomal cathepsin B in the following respects. Inhibition by chicken cystatin was much weaker for sputum cathepsin B than for the lysosomal enzyme. Sputum cathepsin B had greater stability at pH 7.5 and a higher apparent Mr, even after deglycosylation, than lysosomal cathepsin B. We conclude that the form of cathepsin B found in sputum is probably a truncated form of human procathepsin B, with some differences in properties that could be of physiological importance.     

Fluorescent microplate assay for cancer cell-associated cathepsin B.

Cathepsin B and in particular cell-surface and secreted cathepsin B has been implicated in the invasive and metastatic phenotype of numerous types of cancer. We describe here a method to easily survey cancer cell lines for cathepsin B activity using the highly selective substrate Z-Arg-Arg-AMC. Intact human U87 glioma cells hydrolyze Z-Arg-Arg-AMC with a Km of 460 microM at pH 7.0 and 37 degrees C. This is nearly the same as the Km of 430 microM obtained with purified cathepsin B assayed under the same conditions. The pericellular (i.e. both cell-surface and released) cathepsin B activity was inhibited by the cysteine protease inhibitors E-64, leupeptin, Mu-Np2-HphVS-2Np, Mu-Leu-HpHVSPh and the cathepsin B selective inhibitor Mu-Tyr(3,5 I2)-HphVSPh with IC50 values similar to those observed for the inhibition of purified human liver cathepsin B. Other human cancer cell lines with measurable pericellular cathepsin B activity included HT-1080 fibrosarcoma, MiaPaCa pancreatic, PC-3 prostate and HCT-116 colon. Cathepsin B activity correlated with protein levels of cathepsin B as determined by immunoblot analysis. Pericellular cathepsin B activity was also detected in the rat cell lines MatLyLu prostate and Mat B III adenocarcinoma and in the murine lines B16a melanoma and Lewis lung carcinoma. The ability to determine pericellular cathepsin B activity will be useful in selecting appropriate cell lines for use in vivo when analyzing the effects of inhibiting cathepsin B activity on tumor growth and metastasis.     

Bovine spleen cathepsin B1 and collagenolytic cathepsin. A comparative study of the properties of the two enzymes in the degradation of native collagen.

Bovine spleen cathepsin B1 and collagenolytic cathepsin were separated by chromatography on Amberlite IRC-50 and collagenolytic cathepsin was partially purified by chromatography on DEAE-Sephadex (A-50). 2. Collagenolytic cathepsin degraded insoluble tendon collagen maximally at pH 3.5 and 28 degrees C; mainly alpha-chain components were released into solution. At 28 degrees C the telopeptides in soluble skin collagen were also cleaved to yield alpha-chain components. Collagenolytic cathepsin was thus similar to cathepsin B1 in its action against native collagen, but mixtures of these two enzymes exhibited a synergistic effect. 3. The addition of thiol-blocking compounds produced similar inhibition of collagenolytic cathepsin and cathepsin B1. The enzyme responded similarly to all other compounds tested except to 6-aminohexanoic acid, when collagenolytic cathepsin was slightly activated and cathepsin B1 was almost unaffected. 4. Leupeptin, which is a structural analogue of arginine-containing synthetic substrates, inhibited collagenolytic cathepsin as effectively as cathepsin B1. Collagenolytic cathepsin was shown to retain a low residual activity against alpha-N-benzoyl-DL-arginine p-nitroanilide during purification which was equivalent to 0.2% of the activity of cathepsin B1. 5. Cathepsin B1 and collagenolytic cathepsin could not be separated by affinity chromatography on organomercurial-Sepharose 4B. The two enzymes could be resolved on DEAE-Sephadex (A-50) and by isoelectric focusing in an Ampholine pH gradient. The pI of the major cathepsin B1 isoenzyme was 4.9 and the pI of collagenolytic cathepsin was 6.4. 6. From chromatography on Sephadex G-75 (superfine grade) the molecular weights were calculated to be 26000 for cathepsin B1 and 20000 for collagenolytic cathepsin. The difference in molecular weight was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.     

High molecular weight kininogen as substrate for cathepsin B

We investigated the influence of pH and divalent cations (Zn2+, Mg2+ and Ca2+) on high molecular weight kininogen processing by cathepsin B. At pH 6.3, high molecular weight kininogen is hydrolyzed by cathepsin B at three sites generating fragments of 80, 60 and 40 kDa. Cathepsin B has kininogenase activity at this pH which is improved in the absence of divalent cations. At pH 7.35, high molecular weight kininogen is slightly cleaved by cathepsin B into fragments of 60 kDa, and cathepsin B kininogenase activity is impaired. Our results suggest that high molecular weight kininogen is a substrate for cathepsin B under pathophysiological conditions.     

Downregulation of uPAR and cathepsin B induces apoptosis via regulation of Bcl-2 and Bax and inhibition of the PI3K/Akt pathway in gliomas

Background

Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes.

Methodology/Principal Findings

In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results.

Conclusions/Significance

In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.     

Human cathepsin B. Application of the substrate N-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide to a study of the inhibition by leupeptin.

1. The kinetic parameters Kcat. and Km were determined for the hydrolysis of some arginine naphthylamides by human cathepsin B. 2. A new and efficient synthesis of Z-Arg-Arg-NNap (benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide) was developed. 3. Z-Arg-Arg-NNap was a specific and sensitive substrate for cathepsin B, and was used for kinetic studies. 4. Values of kcat. were maximal in the pH range 5.4--6.2, and depended on a single ionizing group of pKa 4.4. 5. Leupeptin was a purely competitive inhibitor of human cathepsin B. 6. The effect of pH on the apparent inhibitor constant, Ki (app.), was determined. Ki (app.) was pH-independent in the range pH 4.3--6.0, with the mean value 7 x 10(-9) M.     

Membrane permeable fluorogenic rhodamine substrates for selective determination of cathepsin L.

The dipeptidyl rhodamine diamide substrates (Z-Phe-Arg)2-R110 and (Z-Arg-Arg)2-R110 are 820- and 360-fold more selective for cathepsin L than for cathepsin B allowing a sensitive determination of cathepsin L activity in the presence of high activity of cathepsin B. The results obtained with cell lysates suggest that the cysteine proteinase activity of vital macrophages detected by flow cytometry with these substrates is mainly due to cathepsin L.     

Characterization of cathepsin B proteinase (AcCP-2) in eggs and larvae stages of hookworm Ancylostoma caninum

Cathepsin B proteinase constitutes a large multigenes family in parasitic and non-parasitic nematodes. The localization of cathepsin B proteinases (AcCP-1 and AcCP-2) in adult worm of Ancylostoma caninum has been characterized (Harrop et al., 1995), but the localization and function in eggs and larval stages remained undiscovered. Here we described the expressing of cathepsin B proteinase (AcCP-2) in Escherichia coli, and immuno-localization of cathepsin B proteinase in eggs and larvae stages of A. caninum. A cDNA fragment encoding a cathepsin B proteinase (AcCP-2) was cloned from A. caninum and expressed in E. coli. Gelatin digestion showed that recombinant cathepsin B proteinase (AcCP-2) has protease activity. The protein level of cathepsin B proteinase in larval and adult worm was detected by western blot. The immuno-localization of cathepsin B proteinase in eggs and larval stages was characterized. The expression of cathepsin B proteinase was more abundant in eggs and larvae stages of A. caninum. It implied that cathepsin B proteinase might play roles in the early development of A. caninum.     

Cathepsin B localizes to plasma membrane caveolae of differentiating myoblasts and is secreted in an active form at physiological pH

Our in vitro studies support a functional link between the induction of cathepsin B gene expression and the catabolic restructuring associated with myotube formation during myogenesis in vivo. We have tested two predictions that are basic to this hypothesis: (1) that active cathepsin B is localized to plasma membrane caveolae of fusing myoblasts; and (2) that active cathepsin B is secreted from fusing myoblasts at physiological pH. During differentiation, L6 rat myoblasts demonstrated a fusion-related increase in activity associated with the 25/26-kDa, fully processed, active form of cathepsin B. Immunocytochemical studies demonstrated a redistribution of lysosomal cathepsin B protein toward the membrane of fusing myoblasts, and a colocalization of cathepsin B with caveolin-3, the muscle-specific structural protein of membrane caveolae. Sucrose density fractionation and Western blot analysis demonstrated that an active form of cathepsin B localizes to caveolar fractions along with caveolin-3, annexin-VII, beta-dystroglycan and dystrophin. Finally, 'real-time' activity assays and Western blot analysis demonstrated that active cathepsin B is secreted from fusing myoblasts at physiological pH. Collectively, these studies support an association of active cathepsin B with plasma membrane caveolae and the secretion of active cathepsin B from differentiating myoblasts during myoblast fusion.     

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.     

Exploring the role of 5' alternative splicing and of the 3'-untranslated region of cathepsin B mRNA

The cysteine peptidase cathepsin B is responsible for connective tissue breakdown in several diseases. The pathological expression of cathepsin B may depend on the structure of its mRNA. We investigated the translational efficiency of the cathepsin B mRNA untranslated regions (UTRs) using fusion constructs to green fluorescent protein (GFP) and luciferase. Transfection of fusion constructs with GFP and luciferase containing the full-length 5'-UTR, the variant lacking exon 2, and that lacking exons 2 and 3 into mammalian cells, resulted in modulation of the biosynthetic rate of cathepsin B in a cell-specific manner. Constructs missing these exons were biosynthetically more efficient than the full-length counterpart. Luciferase was cloned upstream of the 3'-UTR, downstream of the 5'-UTR, or sandwiched between the 5'- and the 3'-UTR. The UTRs of cathepsin B downregulated luciferase biosynthesis moderately when present individually, with the 3'-UTR being more efficient than the 5'-UTR, and downregulated it even more when present simultaneously. A truncated cathepsin B-GFP chimeric product derived from the 5'-UTR missing exons 2 and 3 induced cell death. The increased biosynthetic rate and abnormal trafficking of cathepsin B observed in pathologies such as cancer and osteoarthritis may depend on alternative splicing of pre-mRNA.     

Roles of cathepsins in reperfusion-induced apoptosis in cultured astrocytes

Astrocytic apoptosis may play a role in the central nervous system injury. We previously showed that reperfusion of cultured astrocytes with normal medium after exposure to hydrogen peroxide (H(2)O(2))-containing medium causes apoptosis. This study examines the involvement of the lysosomal enzymes cathepsins B and D in the astrocytic apoptosis. Reperfusion after exposure to H(2)O(2) caused a marked increase in caspase-3 and cathepsin D activities and a marked decrease in cathepsin B activity. Pepstatin A, an inhibitor of cathepsin D, and acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspart-1-aldehyde (Ac-DMQD-CHO), a specific inhibitor of caspase-3, blocked the H(2)O(2)-induced decrease in cell viability and DNA ladder formation in cultured rat astrocytes. The (L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester (CA074 Me), a specific inhibitor of cathepsin B, did not affect the H(2)O(2)-induced cell injury. On the other hand, CA074 Me decreased cell viability with DNA ladder formation when cultured in the presence of Ac-DMQD-CHO. This caspase-independent apoptosis was attenuated by the addition of the cathepsin D inhibitor pepstatin A. Caspase-3 like activity was markedly inhibited by Ac-DMQD-CHO and partially by pepstatin A. Pepstatin A and CA074 Me inhibited cathepsin B and cathepsin D activities, respectively, in the presence and absence of Ac-DMQD-CHO. These results suggest that cathepsins B and D are involved in astrocytic apoptosis: cathepsin D acts as a death-inducing factor upstream of caspase-3 and the caspase-independent apoptosis is regulated antagonistically by cathepsins B and D.     

Effect of cysteine proteinase inhibitors on murine B16 melanoma cell invasion in vitro

Various types of proteinases are implicated in the malignant progression of human and animal tumors. Proteinase inhibitors may therefore be useful as therapeutic agents in anti-invasive and anti-metastatic treatment. The aims of this study were (1) to estimate the relative importance of proteinases in B16 cell invasion in vitro using synthetic, class-specific proteinase inhibitors and (2) to assess the inhibitory effect of some naturally occurring cysteine proteinase inhibitors. Serine proteinase inhibitor reduced invasiveness by up to 24%, whereas inhibition of aspartic proteinases reduced invasion by 11%. Synthetic inhibitors of cysteine proteinases markedly impaired invasion: cathepsin B inhibitors, particularly Ca-074Me, inhibited invasion from 20-40%, whereas cathepsin L inhibitor Clik 148 reduced invasion by 11%. The potato cysteine proteinase inhibitor PCPI 8.7 inhibited invasion by 21%, whereas another potato inhibitor, PCPI 6.6, and the mushroom cysteine proteinase inhibitor clitocypin had no effects. As the inhibitors that inhibited cathepsin B were in general more efficient at impairing the invasiveness, we conclude that of the two cysteine proteinases, cathepsin B plays a more important role than cathepsin L in murine melanoma cell invasion.