Evolution of placentally expressed cathepsins

Species and strain variants of a family of placentally expressed cathepsins (PECs) were cloned and sequenced in order to identify evolutionary conserved structural characteristics of this large family of cysteine proteases. Cathepsins M, P, Q, and R, are conserved in mice and rats but homologs of these genes are not found in human or rabbit placenta, showing that this family of proteases are probably restricted to rodents. Species-specific gene duplications have given rise to variants of cathepsin M in mice, and cathepsin Q in rats. Although the PECs have diverged at a greater rate than the other lysosomal cathepsins, residues around the specificity sub-sites of the individual enzymes are conserved. Strain-specific polymorphisms show that the evolutionary rate of divergence of cathepsins M and 3, the most recently duplicated pair of mouse genes, is even higher than the other PECs. In human placenta, critical functions of the PECs are probably performed by broader specificity proteases such as cathepsins B and L.     

The human cathepsin F gene--a fusion product between an ancestral cathepsin and cystatin gene

Human cathepsin F is a novel papain-like cysteine protease of unknown function. Here, we describe the complete human cathepsin F (CTSF) gene which is composed of 13 exons. In addition to a previous report, two novel upstream located exons whose splice sites interrupted the propeptide of cathepsin F within the 'cystatin-like' domain, recently described by Nagler et al. (Biochem. Biophys. Res. Comm. 257, 313-318, 1999) were identified. A comparison of the genomic structures between this novel part of the cathepsin F gene and those of several cystatin genes revealed striking similarities, supporting the hypothesis that the cathepsin F gene resulted from a gene fusion between an ancestral cystatin and cathepsin gene.     

Cysteine cathepsins in human cancer

Proteases play causal roles in the malignant progression of human tumors. This review centers on the roles in this process of cysteine cathepsins, i.e., peptidases belonging to the papain family (C1) of the CA clan of cysteine proteases. Cysteine cathepsins, most likely along with matrix metalloproteases (MMPs) and serine proteases, degrade the extracellular matrix, thereby facilitating growth and invasion into surrounding tissue and vasculature. Studies on tumor tissues and cell lines have shown changes in expression, activity and distribution of cysteine cathepsins in numerous human cancers. Molecular, immunologic and pharmacological strategies to modulate expression and activity of cysteine cathepsins have provided evidence for a causal role for these enzymes in tumor progression and invasion. Clinically, the levels, activities and localization of cysteine cathepsins and their endogenous inhibitors have been shown to be of diagnostic and prognostic value. Understanding the roles that cysteine proteases play in cancer could lead to the development of more efficacious therapies.     

Regulation of collagenase activities of human cathepsins by glycosaminoglycans

Cathepsin K, a lysosomal papain-like cysteine protease, forms collagenolytically highly active complexes with chondroitin sulfate and represents the most potent mammalian collagenase. Here we demonstrate that complex formation with glycosaminoglycans (GAGs) is unique for cathepsin K among human papain-like cysteine proteases and that different GAGs compete for the binding to cathepsin K. GAGs predominantly expressed in bone and cartilage, such as chondroitin and keratan sulfates, enhance the collagenolytic activity of cathepsin K, whereas dermatan, heparan sulfate, and heparin selectively inhibit this activity. Moreover, GAGs potently inhibit the collagenase activity of other cysteine proteases such as cathepsins L and S at 37 degrees C. Along this line MMP1-generated collagen fragments in the presence of GAGs are stable against further degradation at 28 degrees C by all cathepsins but cathepsin K, whereas thermal destabilization at 37 degrees C renders the fragments accessible to all cathepsins. These results suggest a novel mechanism for the regulation of matrix protein degradation by GAGs. It further implies that cathepsin K represents the only lysosomal collagenolytic activity under physiologically relevant conditions.     

Cysteine cathepsins B,X and K expression in peri-arteriolar glioblastoma stem cell niches

Glioblastoma (GBM) is the most lethal brain tumor also due to malignant and therapy-resistant GBM stem cells (GSCs) that are localized in protecting hypoxic GSC niches. Some members of the cysteine cathepsin family of proteases have been found to be upregulated in GBM. Cathepsin K gene expression is highly elevated in GBM tissue versus normal brain and it has been suggested to regulate GSC migration out of the niches. Here, we investigated the cellular distribution of cathepsins B, X and K in GBM tissue and whether these cathepsins are co-localized in GSC niches. Therefore, we determined expression of these cathepsins in serial paraffin sections of 14 human GBM samples and serial cryostat sections of two samples using immunohistochemistry and metabolic mapping of cathepsin activity using selective fluorogenic substrates. We detected cathepsins B, X and K in peri-arteriolar GSC niches in 9 out of 16 GBM samples, which were defined by co-expression of the GSC marker CD133, the niche marker stromal-derived factor-1α (SDF-1α) and smooth muscle actin as a marker for arterioles. The expression of cathepsin B and X was detected in stromal cells and cancer cells throughout the GBM sections, whereas cathepsin K expression was more restricted to arteriole-rich regions in the GBM sections. Metabolic mapping showed that cathepsin B, but not cathepsin K is active in GSC niches. On the basis of these findings, it is concluded that cathepsins B, X and K have distinct functions in GBM and that cathepsin K is the most likely GSC niche-related cathepsin of the three cathepsins investigated.     

Human cathepsin V functional expression, tissue distribution, electrostatic surface potential, enzymatic characterization, and chromosomal localization

Cathepsin V, a thymus and testis-specific human cysteine protease, was expressed in Pichia pastoris, and its physicokinetic properties were determined. Recombinant procathepsin V is autocatalytically activated at acidic pH and is effectively inhibited by various cysteine protease class-specific inhibitors. The S2P2 subsite specificity of cathepsin V was found to be intermediate between those of cathepsins S and L. The substrate binding pocket, S2, accepted both aromatic and nonaromatic hydrophobic residues, whereas cathepsins L and S preferred either an aromatic or nonaromatic hydrophobic residue, respectively. In contrast to cathepsin L, but similar to cathepsin S, cathepsin V exhibited only a very weak collagenolytic activity. Furthermore, cathepsin V was determined to be significantly more stable at mildly acidic and neutral pH than cathepsin L, but distinctly less stable than cathepsin S. A homology structure model of cathepsin V revealed completely different electrostatic potentials on the molecular surface when compared with human cathepsin L. The model-based electrostatic potential of human cathepsin V was neutral to weakly positive at and in the vicinity of the active site cleft, whereas that of cathepsin L was negative over extended regions of the surface. Surprisingly, the electrostatic potential of the human cathepsin V model structure resembled that of the model structure of mouse cathepsin L. These differences in the electrostatic potential at the molecular surfaces provide a reactivity determinant that may be the source of differences in substrate selectivity and pH stability. Cathepsin V was mapped to the chromosomal region 9q22.2, a site adjacent to the cathepsin L locus. The high sequence identity and the overlapping chromosomal gene loci suggest that both proteases evolved from an ancestral cathepsin L-like precursor by gene duplication.     

Mouse cathepsin M, a placenta-specific lysosomal cysteine protease related to cathepsins L and P

The complete nucleotide sequence of a novel cathepsin cDNA derived from mouse placenta was determined and is termed cathepsin M. The predicted protein of 333 amino acid is a member of the family C1A proteases and is related to mouse cathepsins L and P. Mouse cathepsin M is highly expressed in placenta, whereas no detectable levels were found in lung, spleen, heart, brain, kidney, thymus, testicle, liver, or embryo. Phylogenic analyses of the sequences of human and mouse cathepsins show that cathepsin M is most closely related to cathepsins P and L. However, the differences are sufficiently large to indicate that the enzymes will be found in other species. This is in contrast to human cathepsins L and V, which probably resulted from a gene duplication after divergence of mammalian species.     

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.     

Extracellular catalase activity protects cysteine cathepsins from inactivation by hydrogen peroxide

The resistance of secreted cysteine cathepsins to peroxide inactivation was evaluated using as model THP-1 cells. Differentiated cells released mostly cathepsin B, but also cathepsins H, K, and L, with a maximum of endopeptidase activity at day 6. Addition of non-cytotoxic concentrations of H(2)O(2) did not affect mRNA expression levels and activity of cathepsins, while the catalase activity remained also unchanged, consistently with RT-PCR analysis. Conversely inhibition of extracellular catalase led to a striking inactivation of secreted cysteine cathepsins by H(2)O(2). This report suggests that catalase may participate in the protection of extracellular cysteine proteases against peroxidation.     

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.     

Tumor cell- and microenvironment-specific roles of cysteine cathepsins in mouse models of human cancers

The human genome encodes for 11 papain-like endolysosomal cysteine peptidases, collectively known as the cysteine cathepsins. Based on their biochemical properties and with the help of experiments in cell culture, the cysteine cathepsins have acquired a reputation as promotors of progression and metastasis of various cancer entities. However, tumors are known to be complex tissues in which non-cancerous cells are also critical for tumorigenesis. Here we discuss the results of the intense investigation of cathepsins in mouse models of human cancers. We focus on models in immunocompetent mice, because only such models allow for analysis of cathepsins in a fully functional tumor microenvironment. An important outcome of those studies was the identification of cancer-promoting cathepsins in tumor-associated macrophages. Another interesting outcome of these animal studies was the identification of a homeostatic tumor-suppressive role for cathepsin L in skin and intestinal cancers. Taken together, these in vivo findings provide a basis for the use of cysteine cathepsins as therapeutic targets, prodrug activators, or as proteases for imaging tumors.     

Kininogen-derived peptides for investigating the putative vasoactive properties of human cathepsins K and L.

Macrophages at an inflammatory site release massive amounts of proteolytic enzymes, including lysosomal cysteine proteases, which colocalize with their circulating, tight-binding inhibitors (cystatins, kininogens), so modifying the protease/antiprotease equilibrium in favor of enhanced proteolysis. We have explored the ability of human cathepsins B, K and L to participate in the production of kinins, using kininogens and synthetic peptides that mimic the insertion sites of bradykinin on human kininogens. Although both cathepsins processed high-molecular weight kininogen under stoichiometric conditions, only cathepsin L generated significant amounts of immunoreactive kinins. Cathepsin L exhibited higher specificity constants (kcat/Km) than tissue kallikrein (hK1), and similar Michaelis constants towards kininogen-derived synthetic substrates. A 20-mer peptide, whose sequence encompassed kininogen residues Ile376 to Ile393, released bradykinin (BK; 80%) and Lys-bradykinin (20%) when incubated with cathepsin L. By contrast, cathepsin K did not release any kinin, but a truncated kinin metabolite BK(5-9) [FSPFR(385-389)]. Accordingly cathepsin K rapidly produced BK(5-9) from bradykinin and Lys-bradykinin, and BK(5-8) from des-Arg9-bradykinin, by cleaving the Gly384-Phe385 bond. Data suggest that extracellular cysteine proteases may participate in the regulation of kinin levels at inflammatory sites, and clearly support that cathepsin K may act as a potent kininase.     

Insights into the roles of cathepsins in antigen processing and presentation revealed by specific inhibitors

Eleven human cathepsins have been identified, however, the in vivo roles of individual cathepsins are still largely unknown. In this brief review we will summarize the functions of individual cathepsins in antigen processing and presentation, which are the initial steps of the immune response. Two general inhibitors of papain-like cysteine proteases, E-64 and pyridoxal phosphate, can completely suppress antigen presentation in vivo. To evaluate the contribution of individual cathepsins, specific inhibitors have been developed based on cathepsin tertiary structures: CA-074 for cathepsin B, CLIK-148 and -195 for cathepsin L, CLIK-60 for cathepsin S. Administration of CA-074, a cathepsin B inhibitor, suppresses the response to exogenous antigens, such as hepatitis B virus antigen, ovalbumin and Leishmania major antigen, and induces switching of the helper T cell responses from Th-2 to Th-1 of CD4+ T cells, thereby downregulating the production of IgE and IgG1. Administration of the cathepsin S inhibitor CLIK-60 impairs presentation of an autoantigen, alpha-fodrin, in Sjogren's syndrome and suppresses the Th-1 response and autoantibody production.     

Cysteine cathepsins and extracellular matrix degradation

Background

Cysteine cathepsins are normally found in the lysosomes where they are involved in intracellular protein turnover. Their ability to degrade the components of the extracellular matrix in vitro was first reported more than 25 years ago. However, cathepsins were for a long time not considered to be among the major players in ECM degradation in vivo. During the last decade it has, however, become evident that abundant secretion of cysteine cathepsins into extracellular milieu is accompanying numerous physiological and disease conditions, enabling the cathepsins to degrade extracellular proteins.

Scope of view

In this review we will focus on cysteine cathepsins and their extracellular functions linked with ECM degradation, including regulation of their activity, which is often enhanced by acidification of the extracellular microenvironment, such as found in the bone resorption lacunae or tumor microenvironment. We will further discuss the ECM substrates of cathepsins with a focus on collagen and elastin, including the importance of that for pathologies. Finally, we will overview the current status of cathepsin inhibitors in clinical development for treatment of ECM-linked diseases, in particular osteoporosis.

Major conclusions

Cysteine cathepsins are among the major proteases involved in ECM remodeling, and their role is not limited to degradation only. Deregulation of their activity is linked with numerous ECM-linked diseases and they are now validated targets in a number of them. Cathepsins S and K are the most attractive targets, especially cathepsin K as a major therapeutic target for osteoporosis with drugs targeting it in advanced clinical trials.

General significance

Due to their major role in ECM remodeling cysteine cathepsins have emerged as an important group of therapeutic targets for a number of ECM-related diseases, including, osteoporosis, cancer and cardiovascular diseases. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Cysteine protease cathepsin F is expressed in human atherosclerotic lesions, is secreted by cultured macrophages, and modifies low density lipoprotein particles in vitro

During atherogenesis, low density lipoprotein (LDL) particles in the arterial intima become modified and fuse to form extracellular lipid droplets. Proteolytic modification of apolipoprotein (apo) B-100 may be one mechanism of droplet formation from LDL. Here we studied whether the newly described acid protease cathepsin F can generate LDL-derived lipid droplets in vitro. Treatment of LDL particles with human recombinant cathepsin F led to extensive degradation of apoB-100, which, as determined by rate zonal flotation, electron microscopy, and NMR spectroscopy, triggered both aggregation and fusion of the LDL particles. Two other acid cysteine proteases, cathepsins S and K, which have been shown to be present in the arterial intima, were also capable of degrading apoB-100, albeit less efficiently. Cathepsin F treatment resulted also in enhanced retention of LDL to human arterial proteoglycans in vitro. Cultured monocyte-derived macrophages were found to secrete active cathepsin F. In addition, similarly with cathepsins S and K, cathepsin F was found to be localized mainly within the macrophage-rich areas of the human coronary atherosclerotic plaques. These results suggest that proteolytic modification of LDL by cathepsin F may be one mechanism leading to the extracellular accumulation of LDL-derived lipid droplets within the proteoglycan-rich extracellular matrix of the arterial intima during atherogenesis.     

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.     

Lysosomal cathepsins B, L, and D in the development of murine experimental leukemias

Lysosomal proteases are actively involved into pathogenesis of malignant tumors. Impairments in the interaction between proteases and their inhibitors are implicated in the processes of tumor invasion and metastasis. Among proteases associated with malignant growth, cysteine cathepsins B and L and aspartic cathepsin D are considered to play the major role in the tumor development. The present study was designed to investigate the activity of cathepsins B, L, and D during the development and treatment of murine experimental leukemias and to determine correlation between these proteases and course of pathological process as well as efficiency of the chemotherapeutic treatment. P-388 leukemia was characterized by a more aggressive development and unfavorable prognosis than L1210/1 leukemia. In mice with P-388 leukemia the activity of lysosomal cathepsins B, D, and L in the tumor tissue, liver and spleen, as well as the activity of cathepsins B and L in serum were lower than activities of these enzymes in mice with L1210/1 leukemia. Changes in the activity of cathepsins in liver and spleen of leukemic mice reflected a level of aggressiveness of the tumor development and invasion of these organs with tumor cells. Treatment of these experimental leukemias resulted in the increase of cathepsin B, L and D activity in the tumor tissue, liver, spleen and the increase in cathepsin B and L activity in serum. The highest protease activity was detected in the groups of mice characterized by the highest inhibition of the tumor growth. These data demonstrate that lysosomal proteases are involved in the progression of murine experimental leukemias and elimination of tumor cells in the result of treatment. Thus, determination of the activity of cysteine and aspartic proteases can be used for evaluation of cancer malignancy, tumor sensitivity for chemotherapy and efficiency of treatment.