Cathepsin D inactivates cysteine proteinase inhibitors, cystatins

The formation of inactive complexes in excess molar amounts of human cathepsins H and L with their protein inhibitors human stefin A, human stefin B and chicken cystatin at pH 5.6 has been shown by measurement of enzyme activity coupled with reverse-phase HPLC not to involve covalent cleavage of the inhibitors. Inhibition must be the direct result of binding. On the contrary the interaction of cystatins with aspartic proteinase cathepsin D at pH 3.5 for 60 min followed by HPLC resulted in their inactivation accompanied by peptide bond cleavage at several sites, preferentially those involving hydrophobic amino acid residues. The released peptides do not inhibit papain and cathepsin L. These results explain reported elevated levels of cysteine proteinases and lead to the proposal that cathepsin D exerts an important function, through inactivation of cystatins, in the increased activities of cysteine proteinases in human diseases including muscular distrophy.     

Further characterization of cysteine proteinase inhibitors purified from rat and human epidermis

Cysteine proteinase inhibitors isolated from rat and human epidermis were purified to homogeneity and had isoelectric points of pH 4.31 and pH 5.10, respectively, Both inhibitors caused noncompetitive inhibition to the same degree against papain (EC 3.4.22.2), but the activity of human inhibitor against rat liver cathepsins B (EC 3.4.22.1), H (EC 3.4.22.16), and L (EC 3.422.-) was more effective than that of rat inhibitor. Dependency on pH was observed with rat inhibitor for cathepsins B and H, and with human inhibitor for cathepsin L. The reaction of the inhibitors with papain and cathepsins H and L occurred immediately, while the inhibition reaction of cathepsin B increased progressively during a preincubation time up to 40 min. Incubation at pH 7.0 maximized the progressive inhibitory activity. These findings demonstrate that cysteine proteinase inhibitors from rat and human epidermis inhibited a variety of cysteine proteinases. However, the inhibitor and enzyme interaction depends upon the enzyme, inhibitor source, and experimental conditions such as pH and preincubation time.     

Inhibitory effect of the sugarcane cystatin CaneCPI-4 on cathepsins B and L and human breast cancer cell invasion

The lysosomal cysteine proteases cathepsin B and L play important roles in tumor cell invasion. An imbalance between these cathepsins and their endogenous inhibitors, the cystatins, has been associated with development of the metastatic phenotype. Accordingly, many studies have indicated potential use of cystatins in therapeutic approaches. We report a novel cystatin from sugarcane (Saccharum officinarum), CaneCPI-4, with strong inhibitory activity against cathepsins B (K(i) = 0.83 nM) and L (K(i) = 0.021 nM). The invasive ability of MDA-MB-231 human breast cancer cells expressing CaneCPI-4 was only slightly decreased. In contrast, addition of low, non-toxic concentrations of recombinant His-tagged CaneCPI-4 significantly reduced invasion through a Matrigel matrix. Immunoblot analyses failed to detect the recombinant protein inside cells, indicating that the cystatin was not internalized by endocytosis, but exerted its anti-invasive effect mainly through inhibition of extracellular cathepsins. Our findings open the possibility of considering phytocystatins for anti-cancer strategies.     

Conserved cystatin segments as models for designing specific substrates and inhibitors of cysteine proteinases

Peptide segments derived from consensus sequences of the inhibitory site of cystatins, the natural inhibitors of cysteine proteinases, were used to develop new substrates and inhibitors of papain and rat liver cathepsins B, H, and L. Papain hydrolyzedAbz-QVVAGA-EDDnp andAbz-LVGGA-EDDnp at about the same rate, with specificity constants in the 10⁷M^–1 sec^–1 range; cathepsin L also hydrolyzes both substrates with specificity constants in the 10⁵ M^–1 sec^–1 range due to lowerk _cat values, with theK _m 's being identical to those with papain. OnlyAbz-LVGGA-EDDnp was rapidly hydrolyzed by cathepsin B, and to a lesser extent by cathepsin H. Peptide substrates that alternate these two building blocks (LVGGQVVAGAPWK and QVVAGALVGGAPWK) discriminate the activities of cathepsins B and L and papain. Cathepsin L was highly selective for cleavage at the G-G bond of the LVGG fragment in both peptides. Papain and cathepsin B cleaved either the LVGG fragment or the QVVAG fragment, depending on their position within the peptide. While papain was more specific for the segment located C-terminally, cathepsin B was specific for that in N-terminal position. Peptidyl diazomethylketone inhibitors based on these two sequences also reacted differently with papain and cathepsins. GlcA-QVVA-CHN₂ was a potent inhibitor of papain and reacted with papain 60 times more rapidly (k ₊₀= 1,100,000 M^–1 sec^–1) than with cathepsin L, and 220 times more rapidly than with cathepsin B. Cathepsins B and L were preferentially inhibited by Z-RLVG-CHN₂. Thus cystatin-derived peptides provide a valuable framework for designing sensitive, selective substrates and inhibitors of cysteine proteinases.     

Cystatin S: a cysteine proteinase inhibitor of human saliva

An acidic protein of human saliva, which we named SAP-1 previously, is now shown to be an inhibitor of several cysteine proteinases. The protein inhibited papain and ficin strongly, and stem bromelain and bovine cathepsin C partially. However, it did not inhibit either porcine cathepsin B or clostripain. The mode of the inhibition of papain was found to be non-competitive. The name cystatin S has been proposed for this salivary protein in view of the similarities in activity and structure to other cysteine proteinase inhibitors such as chicken egg-white cystatin and human cystatins A, B, and C. The cystatin S antigen was detected immunohistochemically in the serous cells of human parotid and submaxillary glands.     

Role of the single cysteine residue, Cys 3, of human and bovine cystatin B (stefin B) in the inhibition of cysteine proteinases

Cystatin B is unique among cysteine proteinase inhibitors of the cystatin superfamily in having a free Cys in the N-terminal segment of the proteinase binding region. The importance of this residue for inhibition of target proteinases was assessed by studies of the affinity and kinetics of interaction of human and bovine wild-type cystatin B and the Cys 3-to-Ser mutants of the inhibitors with papain and cathepsins L, H, and B. The wild-type forms from the two species had about the same affinity for each proteinase, binding tightly to papain and cathepsin L and more weakly to cathepsins H and B. In general, these affinities were appreciably higher than those reported earlier, perhaps because of irreversible oxidation of Cys 3 in previous work. The Cys-to-Ser mutation resulted in weaker binding of cystatin B to all four proteinases examined, the effect varying with both the proteinase and the species variant of the inhibitor. The affinities of the human inhibitor for papain and cathepsin H were decreased by threefold to fourfold and that for cathepsin B by approximately 20-fold, whereas the reductions in the affinities of the bovine inhibitor for papain and cathepsins H and B were approximately 14-fold, approximately 10-fold and approximately 300-fold, respectively. The decreases in affinity for cathepsin L could not be properly quantified but were greater than threefold. Increased dissociation rate constants were responsible for the weaker binding of both mutants to papain. By contrast, the reduced affinities for cathepsins H and B were due to decreased association rate constants. Cys 3 of both human and bovine cystatin B is thus of appreciable importance for inhibition of cysteine proteinases, in particular cathepsin B.     

The place of human gamma-trace (cystatin C) amongst the cysteine proteinase inhibitors

Native gamma-trace, a small basic protein present in high concentration in cerebrospinal fluid, semen and neuroendocrine cells, but of unknown biological function, is shown to be a potent inhibitor of the cysteine proteinases papain, ficin, and human cathepsins B, H and L. It proves to be the tightest -binding protein inhibitor of cathepsin B so far discovered. The name cystatin C is proposed for gamma-trace to reflect the many similarities in activity and structure to chicken egg-white cystatin and mammalian cystatins A and B. The inhibition constants of cystatin C, taken together with its widespread distribution in human tissues and extracellular fluids, suggest that a physiological function could well be the regulation of cysteine proteinase activity.     

Decreased activity of cathepsins L+B and decreased invasive ability of PC3 prostate cancer cells

Cancer metastasis involves multiple factors, one of which is the production and secretion of matrix degrading proteases by the cancer cells. Many metastasizing cancer cells secrete the lysosomal proteases, cathepsins L and B, which implicates them in the metastatic process. Cathepsins L and B are regulated by endogenous cysteine proteinase inhibitors (CPI) known as cystatins. An imbalance between cathepsin L and/or B and cystatin expression/activity may be a characteristic of the metastatic phenotype. To determine whether cystatins can attenuate the invasive ability of PC3 prostate cancer cells, cells were transfected with a cDNA coding for chicken cystatin. Expression of chicken cystatin mRNA was determined by PCR analysis. Total cysteine proteinase inhibitory activity, cathepsins L+B activity, and invasion through a Matrigel® matrix were assessed. Stably transfected cells expressed the chicken cystatin mRNA and exhibited a significant decrease in secreted cathepsin L+B activity and a small increase in secreted cysteine proteinase inhibitor activity. The ability of cystatin transfected cells to invade the reconstituted basement membrane, Matrigel®, was attenuated compared to nontransfected cells or cells transfected with vector alone. We have demonstrated that the cysteine proteinases cathepsins L and B participate in the invasive ability of the PC3 prostate cancer cell line, and we discuss here the potential of using cysteine proteinase inhibitors such as the cystatins as anti-metastatic agents.     

Human plasma alpha-cysteine proteinase inhibitor. Purification by affinity chromatography, characterization and isolation of an active fragment.

Human plasma alpha-cysteine proteinase inhibitor (alpha CPI) was purified by a two-stage method: affinity chromatography on S-carboxymethyl-papain-Sepharose, and high-resolution anion-exchange chromatography. The protein was obtained as a form of Mr about 64 000 and material of higher Mr (about 100 000). In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with reduction, both forms showed a major component of Mr 64 000. An antiserum was raised against alpha CPI, and 'rocket' immunoassays showed the mean concentration in sera from 19 individuals to be 35.9 mg/dl. Both low-Mr and high-Mr forms of alpha CPI were confirmed to be sialoglycoproteins by the decrease in electrophoretic mobility after treatment with neuraminidase. alpha CPI was shown immunologically to be distinct from antithrombin III and alpha 1-antichymotrypsin, two serine proteinase inhibitors from plasma with somewhat similar Mr values. alpha CPI was also distinct from cystatins A and B, the two intracellular low-Mr cysteine proteinase inhibitors from human liver. Complexes of alpha CPI with papain were detectable in immunoelectrophoresis, but dissociated to free enzyme and intact inhibitor in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The stoichiometry of binding of papain was close to 1:1 for both low-Mr and high-Mr forms. alpha CPI was found to be a tight-binding inhibitor of papain and human cathepsins H and L (Ki 34 pM, 1.1 nM and 62 pM respectively). By contrast, inhibition of cathepsin B was much weaker, Ki being about 35 microM. Dipeptidyl peptidase I also was weakly inhibited. Digestion of alpha CPI with bromelain gave rise to an inhibitory fragment of Mr about 22 000, which was isolated.     

Synthetic domains of cystatins linked to enkephalins are novel inhibitors of brain cathepsins L/B

Cystatin domains or homologous sequences were synthesized and tested as inhibitors of papain, and rat brain cathepsins B and L. These domains included: I, an enzyme substrate binding site containing a -GG- cleavage site (YGGFL); II, known cystatin consensus sequences (-QVVAG- or -QLVSG-); and III, the proposed ancillary site for binding of chicken cystatin to papain (-IPWLN-). A Domain II analog QVVAG(K-NH2) inhibited cathepsin L and papain with Ki 1-4 X 10(-4) M but was inactive towards cathepsin B. A peptide containing Domains I and II, YGGFL-QVVAG(K-NH2), inhibited papain and cathepsin B with Ki 10(-4)-10(-5) M, and cathepsin L with Ki 10(-6) M. The presence of Domain III in the analog YGGFL-QVVAG-IPWLN(K-NH2) resulted in a 10-fold increase in potency towards papain. These data demonstrated that putative cystatin domains are: 1) probably proximal in the intact cystatins; 2) can be linked directly to each other to yield smaller peptides active as inhibitors; 3) showed some specificity towards the three cysteine proteinases.     

Decreased activity of cathepsins L+B and decreased invasive ability of PC3 prostate cancer cells

Cancer metastasis involves multiple factors, one of which is the production and secretion of matrix degrading proteases by the cancer cells. Many metastasizing cancer cells secrete the lysosomal proteases, cathepsins L and B, which implicates them in the metastatic process. Cathepsins L and B are regulated by endogenous cysteine proteinase inhibitors (CPI) known as cystatins. An imbalance between cathepsin L and/or B and cystatin expression/activity may be a characteristic of the metastatic phenotype. To determine whether cystatins can attenuate the invasive ability of PC3 prostate cancer cells, cells were transfected with a cDNA coding for chicken cystatin. Expression of chicken cystatin mRNA was determined by PCR analysis. Total cysteine proteinase inhibitory activity, cathepsins L+B activity, and invasion through a Matrigel® matrix were assessed. Stably transfected cells expressed the chicken cystatin mRNA and exhibited a significant decrease in secreted cathepsin L+B activity and a small increase in secreted cysteine proteinase inhibitor activity. The ability of cystatin transfected cells to invade the reconstituted basement membrane, Matrigel®, was attenuated compared to nontransfected cells or cells transfected with vector alone. We have demonstrated that the cysteine proteinases cathepsins L and B participate in the invasive ability of the PC3 prostate cancer cell line, and we discuss here the potential of using cysteine proteinase inhibitors such as the cystatins as anti-metastatic agents.     

The emerging role of cystatins in Alzheimer's disease

Recently opposing effects of cysteine protease inhibitors, the human cystatins, on neurodegeneration have been reported. Human cystatin C is a risk factor for late‐onset Alzheimer's disease (AD), whereas human stefin B (cystatin B) has no direct involvement in AD. Conflicting data show that their target protease, cathepsin B, might be anti‐amyloidogenic, helping in amyloid‐beta (Aβ) clearance or, instead, might be involved in Aβ production. Some reports claim that cystatin C binds soluble Aβ, making transgenic animals healthier, others, in contrast, that deleting cystatins genes may contribute to amyloid pathology in animal models of AD.     

Sensitization of stefin B-deficient thymocytes towards staurosporin-induced apoptosis is independent of cysteine cathepsins

Stefin B (cystatin B) is an inhibitor of lysosomal cysteine cathepsins and does not inhibit cathepsin D, E (aspartic) or cathepsin G (serine) proteinases. In this study, we have investigated apoptosis triggered by camptothecin, staurosporin (STS), and anti-CD95 monoclonal antibody in the thymocytes from the stefin B-deficient mice and wild-type mice. We have observed increased sensibility to STS-induced apoptosis in the thymocytes of stefin B-deficient mice. Pretreatment of cells with pan-caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone completely inhibited phosphatidylserine externalization and caspase activation, while treatment with inhibitor of calpains- and papain-like cathepsins (2S,3S)-trans-epoxysuccinyl-leucylamido-3-methyl-butane ethyl ester did not prevent caspase activation nor phosphatidylserine exposure. We conclude that sensitization to apoptosis induced by STS in thymocytes of stefin B-deficient and wild-type mice is not dependent on cathepsin inhibition by stefin B.     

Inhibition of rat liver cathepsins B and L by the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal and its analogues

Cathepsins B and L belong to the papain superfamily of cysteine proteases and play important roles in various physiological and pathological processes. In the course of studies on their inhibitors, we examined the inhibitory effects of the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLal) and its analogues. As a result, rat liver cathepsins B and L were shown to be strongly inhibited by them. The concentration required for 50% inhibition (IC(50)) by ZLLLal was 88 nM for cathepsin B and 163 nM for cathepsin L. Moreover, various analogues of ZLLLal, including 2-furancarbonyl-, nicotinyl-, isonicotinyl- and 4-morpholinylsuccinyl-LLLals, and some acetyl-Pro (AcP)-containing analogues, AcPLLLal and AcPPLLLal, were shown to inhibit both enzymes more strongly than ZLLLal. Among them, isonicotinyl-LLLal was most inhibitory against both cathepsins B (IC(50), 12 nM) and L (IC(50), 20 nM). Several of these inhibitors were indicated to be somewhat more soluble in aqueous media than ZLLLal.     

Identification of cysteine protease inhibitors that belong to cystatin family 1 in the cellular slime mold Dictyostelium discoideum

Family 1 cystatins are cytosolic inhibitors of cysteine proteases, and they are conserved in higher eukaryotes. We characterized two newly identified family 1 cystatins of the cellular slime mold Dictyostelium discoideum, cystatin A1 and A2. Their recombinant proteins showed specific inhibitory activity against papain and cathepsin B, respectively. Using specific polyclonal antibodies, we found that cystatin A1 is stably expressed throughout the life cycle of Dictyostelium, whereas cystatin A2 expression is up-regulated during the course of development.     

A novel type of bifunctional inhibitor directed against proteolytic activity and receptor/ligand interaction. Cystatin with a urokinase receptor binding site

Cancer invasion and metastasis is a process requiring a coordinated series of (anti-)adhesive, migratory, and pericellular proteolytic events involving various proteases such as urokinase-type plasminogen activator (uPA)/plasmin, cathepsins B and L, and matrix metalloproteases. Novel types of double-headed inhibitors directed to different tumor-associated proteolytic systems were generated by substitution of a loop in chicken cystatin, which is nonessential for cysteine protease inhibition, with uPA-derived peptides covering the human uPA receptor binding sequence uPA-(19-31). The inhibition constants of these hybrids toward cysteine proteases are similar to those of wild-type cystatin (K(i), papain (pm), 1.9-2.4; K(i), cathepsin B (nm), 1.0-1.7; K(i), cathepsin L (pm), 0.12-0.61). FACS analyses revealed that the hybrids compete for binding of uPA to the cell surface-associated uPA receptor (uPAR) expressed on human U937 cells. The simultaneous interaction of the hybrid molecules with papain and uPAR was analyzed by surface plasmon resonance. The measured K(D) value of a papain-bound cystatin variant harboring the uPAR binding sequence of uPA (chCys-uPA-(19-31)) and soluble uPAR was 17 nm (K(D) value for uPA/uPAR interaction, 5 nm). These results indicate that cystatins with a uPAR binding site are efficient inhibitors of cysteine proteases and uPA/uPAR interaction at the same time. Therefore, these compact and small bifunctional inhibitors may represent promising agents for the therapy of solid tumors.     

Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides.

The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S-transferase-fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S-transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time-dependent and occurred by an apparent one-step mechanism. The cathepsin K propeptide had a Ki of 3.6-6.3 nM for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240-fold selective inhibitor of cathepsin K (Ki = 0.27 nM) and cathepsin L (Ki = 0.12 nM) compared with cathepsin S (Ki = 65 nM). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nM) than cathepsin S (Ki = 7.6 nM) itself or cathepsin K (Ki = 7.0 nM). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J. E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745-750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained.     

Inhibition of rat liver cathepsins B and L by the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal and its analogues

Cathepsins B and L belong to the papain superfamily of cysteine proteases and play important roles in various physiological and pathological processes. In the course of studies on their inhibitors, we examined the inhibitory effects of the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLal) and its analogues. As a result, rat liver cathepsins B and L were shown to be strongly inhibited by them. The concentration required for 50% inhibition (IC₅₀) by ZLLLal was 88 nM for cathepsin B and 163 nM for cathepsin L. Moreover, various analogues of ZLLLal, including 2-furancarbonyl-, nicotinyl-, isonicotinyl- and 4-morpholinylsuccinyl-LLLals, and some acetyl-Pro (AcP)-containing analogues, AcPLLLal and AcPPLLLal, were shown to inhibit both enzymes more strongly than ZLLLal. Among them, isonicotinyl-LLLal was most inhibitory against both cathepsins B (IC₅₀, 12 nM) and L (IC₅₀, 20 nM). Several of these inhibitors were indicated to be somewhat more soluble in aqueous media than ZLLLal.     

Identification and discrimination of extracellularly active cathepsins B and L in high-invasive melanoma cells

We established a novel protocol for lithium dodecyl sulfate (LDS) gelatin zymography, which operates under reducing conditions and at a slightly acidic pH value (6.5). This zymographic assay is based on polyacrylamide gel electrophoresis and facilitates the electrophoretic separation of human cathepsins in an active state. By this technique, activity of purified human liver cathepsin B was detected at a concentration as low as 50 ng and was blocked only in the presence of the cysteine protease inhibitor E-64 and the specific cathepsin B inhibitor CA-074 but not by aspartate, serine, or matrix metalloprotease inhibitors. The method was applied to analyze cathepsin activities in cell culture supernatants of the high-invasive melanoma cell line MV3. Interestingly, LDS zymography of MV3 cell supernatants in combination with specific inhibitors of cathepsins B and L identified three forms of extracellularly active cathepsin B and two forms of proteolytically active cathepsin L. We herein describe the generation and biochemical significance of acidic LDS zymography. This novel method permits not only the enzymatic analysis of purified cysteine proteases but also the identification and discrimination of different cathepsin activities in biological fluids, cell lysates, or supernatants, especially of cathepsins B and L, which are closely linked to major inflammatory and malignant processes.