Breast cancer cells express cathepsins B and L but not cathepsins K or H

Lysosomal cysteine proteinases (cathepsins) are considered to play a role in bone degradation mediated by metastatic breast cancers. To evaluate which cathepsin contributes to the osteolysis, we quantitatively determined the expression levels of four cathepsins in two breast cancer cell lines, MCF-7 and MDA-MB-231, by competitive RT-PCR. Cathepsin K, which is the most abundant cathepsin in osteoclasts, was not detected in either cell lines. We also failed to detect cathepsin H mRNA. By contrast, we found significant expression of cathepsins B and L in both cell lines. By Northern blot analysis cathepsin B mRNA was detected in a single form in these cells, whereas osteoclasts contained multiple forms of the mRNA. Cathepsin B protein was also detected by Western blotting as a single immunoreactive band corresponding to its mature enzyme. These findings suggest that osteolysis associated with metastatic breast cancers takes place in a different way from osteoclast-mediated bone resorption.     

Amino acid sequences of the human kidney cathepsins H and L

The complete amino acid sequences of human kidney cathepsin H (EC 3.4.22.16) and human kidney cathepsin L (EC 3.4.22.15) were determined. Cathepsin H contains 230 residues and has an Mr of 25116. The sequence was obtained by sequencing the light, heavy and mini chain and the peptides produced by cyanogen bromide cleavage of the single-chain form of the enzyme. The glycosylated mini chain is a proteolytic fragment of the propeptide of cathepsin H. Human cathepsin L has 217 amino acid residues and an Mr of 23720. Its amino acid sequence was deduced from N-terminal sequences of the heavy and light chains and from the sequences of cyanogen bromide fragments of the heavy chain. The fragments were aligned by comparison with known sequences of cathepsins H and L from other species. Cathepsins H and L exhibit a high degree of sequence homology to cathepsin B (EC 3.4.22.1) and other cysteine proteinases of the papain superfamily.     

Optimization of detergents for the assay of cathepsins B, L, S, and K

The differential effects of representative, commonly available ionic (SDS), nonionic (Brij 35, Tween 20, and Triton X-100), and zwitterionic (Chaps) detergents on the catalytic activity and properties of human cathepsins B, L, S, and K were examined. The presence of detergents in the assay buffer affected the activity of cathepsins to variable extents; Chaps enhanced the activity of all the enzymes while SDS was most detrimental. Tween 20 lowered cathepsin S activity, while it slightly enhanced that of all other cathepsins studied. The presence of detergents in the activation buffer was clearly beneficial to both cathepsins L and K, possibly by favoring the release of the enzyme from the walls of the incubation vessel. Overall, the results indicate that Chaps is the optimal detergent for use with this family of enzymes.     

Active cathepsins B, H, K, L and S in human inflammatory bronchoalveolar lavage fluids

BACKGROUND INFORMATION: Chronic inflammation and tissue remodelling result from an imbalance between proteolytic enzymes and their inhibitors in the lungs in favour of proteolysis. While many studies have examined serine proteases (e.g. cathepsin G and neutrophil elastase) and matrix metalloproteases, little is known about the role of papain-like CPs (cysteine proteases). The present study focuses on the thiol-dependent cathepsins (CPs) and their specific cystatin-like inhibitors [CPIs (CP inhibitors)] in human inflammatory BALFs (BAL fluids, where BAL stands for broncho-alveolar lavage). RESULTS: Cathepsins B, K and S found were mostly zymogens, whereas cathepsins H and L were predominantly in their mature forms. Little immunoreactive cystatin C was found and the high- and low-molecular-mass ('weight') kininogens were extensively degraded. The BALF procathepsins B and L could be activated autocatalytically, indicating that alveolar fluid pro-CPs are reservoirs of mature enzymes. Hydrolysis patterns of 7-amino-4-methylcoumarin-derived peptide substrates showed that extracellular alveolar CPs remain proteolytically active, and that cathepsins B and L are the most abundant thiol-dependent endoproteases. The CP/CPI balance was significantly tipped in favour of cathepsins (3- or 5-fold), as confirmed by the extensive CP-dependent degradation of exogenous kininogens by BALFs. CONCLUSIONS: Although their importance for inflammation remains to be clarified, the presence of active cathepsins L, K and S suggests that they contribute to the extracellular breakdown of the extracellular matrix.     

Cysteine cathepsins S and L modulate anti-angiogenic activities of human endostatin

Human endostatin, a potent anti-angiogenic protein, is generated by release of the C terminus of collagen XVIII. Here, we propose that cysteine cathepsins are involved in both the liberation and activation of bioactive endostatin fragments, thus regulating their anti-angiogenic properties. Cathepsins B, S, and L efficiently cleaved in vitro FRET peptides that encompass the hinge region corresponding to the N terminus of endostatin. However, in human umbilical vein endothelial cell-based assays, silencing of cathepsins S and L, but not cathepsin B, impaired the generation of the ～22-kDa endostatin species. Moreover, cathepsins L and S released two peptides from endostatin with increased angiostatic properties and both encompassing the NGR sequence, a vasculature homing motif. The G10T peptide (residues 1455-1464: collagen XVIII numbering) displayed compelling anti-proliferative (EC(50) = 0.23 nm) and proapoptotic properties. G10T inhibited aminopeptidase N (APN/CD13) and reduced tube formation of endothelial cells in a manner similar to bestatin. Combination of G10T with bestatin resulted in no further increase in anti-angiogenic activity. Taken together, these data suggest that endostatin-derived peptides may represent novel molecular links between cathepsins and APN/CD13 in the regulation of angiogenesis.     

Antifibrotic effects of curcumin are associated with overexpression of cathepsins K and L in bleomycin treated mice and human fibroblasts

Background

Lung fibrosis is characterized by fibroblast proliferation and the deposition of collagens. Curcumin, a polyphenol antioxidant from the spice tumeric, has been shown to effectively counteract fibroblast proliferation and reducing inflammation and fibrotic progression in animal models of bleomycin-induced lung injury. However, there is little mechanistic insight in the biological activity of curcumin. Here, we study the effects of curcumin on the expression and activity of cathepsins which have been implicated in the development of fibrotic lung diseases.

Methods

We investigated the effects of curcumin administration to bleomycin stimulated C57BL/6 mice and human fetal lung fibroblasts (HFL-1) on the expression of cathepsins K and L which have been implicated in matrix degradation, TGF-β1 modulation, and apoptosis. Lung tissues were evaluated for their contents of cathepsins K and L, collagen, and TGF-β1. HFL-1 cells were used to investigate the effects of curcumin and cathepsin inhibition on cell proliferation, migration, apoptosis, and the expression of cathepsins K and L and TGF-β1.

Results

Collagen deposition in lungs was decreased by 17-28% after curcumin treatment which was accompanied by increased expression levels of cathepsins L (25%-39%) and K (41%-76%) and a 30% decrease in TGF-β1 expression. Moreover, Tunel staining of lung tissue revealed a 33-41% increase in apoptotic cells after curcumin treatment. These in vivo data correlated well with data obtained from the human fibroblast line, HFL-1. Here, cathepsin K and L expression increased 190% and 240%, respectively, in the presence of curcumin and the expression of TGF-β1 decreased by 34%. Furthermore, curcumin significantly decreased cell proliferation and migration and increased the expression of surrogate markers of apoptosis. In contrast, these curcumin effects were partly reversed by a potent cathepsin inhibitor.

Conclusion

This study demonstrates that curcumin increases the expression of cathepsins K and L in lung which an effect on lung fibroblast cell behavior such as proliferation, migration and apoptosis rates and on the expression of TGF-β1 in mouse lung and HFL-1 cells. These results suggest that cathepsin-inducing drugs such as curcumin may be beneficial in the treatment of lung fibrosis.     

Potent vasoactive properties of endothelin 1 in human skin.

The effect of the endothelial cell-derived peptide endothelin 1 was investigated in human skin. Intradermal injection of endothelin 1 (1-100 pmol) caused a dose-dependent area of pallor that was associated with a significant reduction in basal skin blood flow, measured by laser-Doppler blood flowmeter (with 1 pmol endothelin, P = 0.012, analysis of variance). The coadministration of endothelin 1 (1-100 pmol) with the neuropeptide vasodilator calcitonin gene-related peptide (CGRP) inhibited the vasodilator response to CGRP (10 pmol) by up to 82.7 +/- 9.2% (with 100 pmol endothelin, P less than 0.001). The response of the prostanoid vasodilator prostaglandin E2 (10 pmol) was inhibited by endothelin in a similar manner. In addition to the vasoconstrictor effects, endothelin 1 produced a dose-dependent flare that surrounded the area of pallor, and this was associated with a significant increase in blood flow (P less than 0.05) within the flare area. The H1 antagonist terfenadine (120 mg po) significantly reduced the flare area associated with endothelin 1: flare 5 min after intradermal endothelin (10 pmol, placebo treated), 668 +/- 405 mm2; terfenadine treated, 201 +/- 257 mm2 (P less than 0.05). The flare was also significantly attenuated when endothelin (10 pmol) was injected into local anesthetic-treated skin. Thus intradermal injection of endothelin in humans causes long-lasting vasoconstriction at the site of injection and a surrounding flare. Results suggest that the flare component is partially histamine dependent and the result of an axon reflex. This study demonstrates the potent activity of endothelin in human skin. It is possible that endothelin could be relevant to the local response of skin to injury.     

Interaction between human cathepsins K, L, and S and elastins: mechanism of elastinolysis and inhibition by macromolecular inhibitors

Proteolytic degradation of elastic fibers is associated with a broad spectrum of pathological conditions such as atherosclerosis and pulmonary emphysema. We have studied the interaction between elastins and human cysteine cathepsins K, L, and S, which are known to participate in elastinolytic activity in vivo. The enzymes showed distinctive preferences in degrading elastins from bovine neck ligament, aorta, and lung. Different susceptibility of these elastins to proteolysis was attributed to morphological differences observed by scanning electron microscopy. Kinetics of cathepsin binding to the insoluble substrate showed that the process occurs in two steps. The enzyme is initially adsorbed on the elastin surface in a nonproductive manner and then rearranges to form a catalytically competent complex. In contrast, soluble elastin is bound directly in a catalytically productive manner. Studies of enzyme partitioning between the phases showed that cathepsin K favors adsorption on elastin; cathepsin L prefers the aqueous environment, and cathepsin S is equally distributed among both phases. Our results suggest that elastinolysis by cysteine cathepsins proceeds in cycles of enzyme adsorption, binding of a susceptible peptide moiety, hydrolysis, and desorption. Alternatively, the enzyme may also form a new catalytic complex without prior desorption and re-adsorption. In both cases the active center of the enzymes remains at least partly accessible to inhibitors. Elastinolytic activity was readily abolished by cystatins, indicating that, unlike enzymes such as leukocyte elastase, pathological elastinolytic cysteine cathepsins might represent less problematic drug targets. In contrast, thyropins were relatively inefficient in preventing elastinolysis by cysteine cathepsins.     

The S2 subsites of cathepsins K and L and their contribution to collagen degradation

The exchange of residues 67 and 205 of the S2 pocket of human cysteine cathepsins K and L induces a permutation of their substrate specificity toward fluorogenic peptide substrates. While the cathepsin L-like cathepsin K (Tyr67Leu/Leu205Ala) mutant has a marked preference for Phe, the Leu67Tyr/Ala205Leu cathepsin L variant shows an effective cathepsin K-like preference for Leu and Pro. A similar turnaround of inhibition was observed by using specific inhibitors of cathepsin K [1-(N-Benzyloxycarbonyl-leucyl)-5-(N-Boc-phenylalanyl-leucyl)carbohydrazide] and cathepsin L [N-(4-biphenylacetyl)-S-methylcysteine-(D)-Arg-Phe-beta-phenethylamide]. Molecular modeling studies indicated that mutations alter the character of both S2 and S3 subsites, while docking calculations were consistent with kinetics data. The cathepsin K-like cathepsin L was unable to mimic the collagen-degrading activity of cathepsin K against collagens I and II, DQ-collagens I and IV, and elastin-Congo Red. In summary, double mutations of the S2 pocket of cathepsins K (Y67L/L205A) and L (L67Y/A205L) induce a switch of their enzymatic specificity toward small selective inhibitors and peptidyl substrates, confirming the key role of residues 67 and 205. However, mutations in the S2 subsite pocket of cathepsin L alone without engineering of binding sites to chondroitin sulfate are not sufficient to generate a cathepsin K-like collagenase, emphasizing the pivotal role of the complex formation between glycosaminoglycans and cathepsin K for its unique collagenolytic activity.     

Promiscuous processing of human alphabeta-protryptases by cathepsins L, B, and C

Human α- and β-protryptase zymogens are abundantly and selectively produced by mast cells, but the mechanism(s) by which they are processed is uncertain. β-Protryptase is sequentially processed in vitro by autocatalysis at R(-3) followed by cathepsin (CTS) C proteolysis to the mature enzyme. However, mast cells from CTSC-deficient mice successfully convert protryptase (pro-murine mast cell protease-6) to mature murine mast cell protease-6. α-Protryptase processing cannot occur by trypsin-like enzymes due to an R(-3)Q substitution. Thus, biological mechanisms for processing these zymogens are uncertain. β-Tryptase processing activity(ies) distinct from CTSC were partially purified from human HMC-1 cells and identified by mass spectroscopy to include CTSB and CTSL. Importantly, CTSB and CTSL also directly process α-protryptase (Q(-3)) and mutated β-protryptase (R(-3)Q) as well as wild-type β-protryptase to maturity, indicating no need for autocatalysis, unlike the CTSC pathway. Heparin promoted tryptase tetramer formation and protected tryptase from degradation by CTSB and CTSL. Thus, CTSL and CTSB are capable of directly processing both α- and β-protryptases from human mast cells to their mature enzymatically active products.     

Cytokines suppress the shear stress-stimulated release of vasoactive peptides from human endothelial cells

Endothelial cells from human umbilical vein perfused at 0.5 ml/min released vasopressin, endothelin, and substance P. Upon perfusion of the cells at 3.0 ml/min, the release of endothelia and vasopressin was significantly increased whereas the release of substance P was significantly decreased. Endothelial cells precultured for 24 h with interleukin-I (IL-1) and interferon-γ (IFN-γ) released more endothelin and less substance P at low flow and there was no further increase in release at high flow rate. These results suggest that cytokines suppress the normal responses of endothelial cells to increased fluid shear stress.     

Some properties of cathepsins chemically fixed to carriers.

An insoluble preparation of rat liver cathepsin D was obtained by coupling the enzyme to Enzacryl Polyacetal (EPA-cathepsin) and to CNBr-activated Sepharose 4B. EPA-cathepsin was active toward the synthetic hexapeptides (Gly-Phe-Leu)2 and did not split hemoglobin. The optimum pH of splitting was displaced upward by 1.5 units to pH 5.0. The enzyme exhibited maximum activity at 60 degrees C. No appreciable loss of activity was seen on storage of the enzyme for 4 months or after repeated use of the preparations. Coupling of rat liver cathepsin D to activated Sepharose gave preparations active towards both protein and synthetic substrates. The preparations were totally inactive in acid media and exhibited maximum activity at pH 7.0, that is, under physiological conditions. Optimum temperature was 65 degrees. The specific activity of the preparations (pH 7.0, 65 degrees) was 60-110 percent that of the free enzyme in acid media. Proteolytic activity of the Sepharose-coupled cathepsin D was not inhibited by pepstatin, whereas that of the free enzyme was fully inhibited by this reagent. A sarcoma cathepsin, similar in some of its properties to the rat liver enzyme, was also coupled to CNBr-activated Sepharose 4B. The preparation split protein substrates at pH 7.0 and possessed enhanced thermostability. The enzymes fixed on Sepharose showed increased stability.     

Comprehensive search for cysteine cathepsins in the human genome

Our study was aimed at examinating whether or not the human genome encodes for previously unreported cysteine cathepsins. To this end, we used analyses of the genome sequence and mRNA expression levels. The program TBLASTN was employed to scan the draft sequence of the human genome for the 11 known cysteine cathepsins. The cathepsin-like segments in the genome were inspected, filtered, and annotated. In addition to the known cysteine cathepsins, the scan identified three pseudogenes, closely related to cathepsin L, on chromosome 10, as well as two remote homologs, tubulointerstitial protein antigen and tubulointerstitial protein antigen-related protein. No new members of the family were identified. mRNA expression profiles for 10 known human cysteine cathepsins showed varying expression levels in 46 different human tissues and cell lines. No expression of any of the three cathepsin L-like pseudogenes was found. Based on these results, it is likely that to date all human cysteine cathepsins are known.     

Enzyme-substrate interactions in the hydrolysis of peptides by cathepsins B and H from rat liver.

Free Ca2+ in the cytosol ([Ca2+]i) of individual rat ventricle cells injected with aequorin was measured under anoxia. In glucose-free medium myocytes spontaneously shortened after about 60 min, although [Ca2+]i was still at or near resting levels. However, within minutes a net inward movement of Ca2+ across the sarcolemma developed and [Ca2+]i began to rise. Provided oxygen was readmitted before [Ca2+]i exceeded 2-3 microM, cells were able to restore [Ca2+]i to resting levels through caffeine-sensitive sequestration of Ca2+ in the sarcoplasmic reticulum. We suggest that Ca2+-independent shortening of anoxic cardiomyocytes reflects onset of rigor which triggers loss of [Ca2+]i homoeostasis.     

6-Acylamino-penam derivatives: synthesis and inhibition of cathepsins B,L, K,and S

The synthesis of a new series of 6-acylamino penam derivatives and their inhibition of cysteine proteases cathepsins B, L, K, and S is described. The 6-acylamino-penam sulfone compounds showed excellent cathepsin L, K, and S inhibition activity with IC(50) values in the nanomolar and subnanomolar range.     

Control of the renal microvasculature by vasoactive peptides

In recent years, numerous techniques have been developed to study renal microcirculation. These technical advances have provided new insight regarding the specificity of action of vasoconstrictor peptides (angiotensin II, arginine vasopressin, endothelin) and vasodilator peptides (bradykinin, atrial natriuretic peptide) at discrete sites within the renal vascular bed. Differential signal transduction mechanisms, particularly those related to calcium regulation, appear to mediate the renal vascular actions of these compounds, both in a segment-specific and agonist-specific manner. These observations substantiate the concept that regulation of intrarenal and intraglomerular dynamics is accomplished by selective changes in pre- and postglomerular resistance induced by different endogenous peptides. This microvascular selectivity allows precise regulation of glomerular and peritubular capillary function, and ultimately exerts great influence on the volume and composition of the final urine.     

Human cathepsins K,L, and S: Related proteases,but unique fibrinolytic activity

Background

Fibrin formation and dissolution are attributed to cascades of protease activation concluding with thrombin activation, and plasmin proteolysis for fibrin breakdown. Cysteine cathepsins are powerful proteases secreted by endothelial cells and others during cardiovascular disease and diabetes. Their fibrinolytic activity and putative role in hemostasis has not been well described.

Bovine cathepsins S and L: isolation and amino acid sequences.

The purification procedure of cathepsin S includes acid activation of spleen homogenate, incubation at 37 degrees C, precipitation with (NH4)2SO4 in H2O/tert-butanol medium, gel chromatography, chromatofocusing, covalent chromatography and cation chromatography of FPLC system. Cathepsin S has a M(r) of about 24,000 Da with pI of 6.5 and 6.8. The mixture of both forms gave a single sequence. Cathepsin L was purified from bovine kidney by acid treatment and incubation of 37 degrees C, precipitation by (NH4)2SO4, two ion exchange chromatographies on CM-Sephadex, gel chromatography and ion exchange chromatography on FPLC system. Cathepsin L exists in multiple forms with pI 5.3-5.7 and M(r) of about 29,000 Da. N-terminal amino acid sequence confirms that cathepsin L and cathepsin S are different enzymes.