Cysteine cathepsins: cellular roadmap to different functions

Cysteine cathepsins belong to the papain-like family C1 of clan CA cysteine peptidases. These enzymes are ubiquitously expressed and exert their proteolytic activity mainly, but not exclusively within the compartments along the endocytic pathway. Moreover, cysteine cathepsins are active in pericellular environments as soluble enzymes or bound to cell surface receptors at the plasma membrane, and possibly even within secretory vesicles, the cytosol, mitochondria, and within the nuclei of eukaryotic cells. Proteolytic actions performed by cysteine cathepsins are essential in the maintenance of homeostasis and depend heavily upon their correct sorting and trafficking within cells. As a consequence, the numerous and diverse approaches to identification, qualitative and quantitative determination, and visualization of cysteine cathepsin functions in vitro, in situ, and in vivo cover the entire spectrum of biochemistry, molecular and cell biology. This review focuses upon the transport pathways directing cysteine cathepsins to their points of action and thus emphasizes the broader role and functionality of cysteine cathepsins in a number of specific cellular locales. Such understanding will provide a foundation for future research investigating the involvement of these peptidases with their substrates, inhibitors, and the intertwined proteolytic networks at the hubs of complex biological systems.     

γ-Interferon-inducible Lysosomal Thiol Reductase (GILT) Maintains Phagosomal Proteolysis in Alternatively Activated Macrophages

Although it is known that lysosomal cysteine cathepsins require a reducing environment for optimal activity, it is not firmly established how these enzymes are maintained in their reduced-active state in the acidic and occasionally oxidative environment within phagosomes and lysosomes. γ-Interferon-inducible lysosomal thiol reductase (GILT) has been the only enzyme described in the endosomes, lysosomes, and phagosomes with the potential to catalyze the reduction of cysteine cathepsins. Our goal in the current study was to assess the effect of GILT on major phagosomal functions with an emphasis on proteolytic efficiency in murine bone marrow-derived macrophages. Assessment of phagosomal disulfide reduction upon internalization of IgG-opsonized experimental particles confirmed a major role for GILT in phagosomal disulfide reduction in both resting and interferon-γ-activated macrophages. Furthermore we observed a decrease in early phagosomal proteolytic efficiency in GILT-deficient macrophages, specifically in the absence of an NADPH oxidase-mediated respiratory burst. This deficiency was more prominent in IL-4-activated macrophages that inherently possess lower levels of NADPH oxidase activity. Finally, we provide evidence that GILT is required for optimal activity of the lysosomal cysteine protease, cathepsin S. In summary, our results suggest a role for GILT in maintaining cysteine cathepsin proteolytic efficiency in phagosomes, particularly in the absence of high NADPH oxidase activity, which is characteristic of alternatively activated macrophages.     

Thiol peptide hydrolases from animal tissues, their structure and function

Data on properties, structure and biological functions of a variety of thiol (cysteine) peptide hydrolases from animal tissues have been summarized. This large group of diverse intracellular enzymes involves both endo- and exopeptidases. Best studied are lysosomal thiol peptide hydrolases: cathepsins B, H and L, the primary structure of which is deciphered. They present a family of homologous proteins, structurally similar to papain. Ca2+-dependent neutral proteinases is another family of related proteins. The biological functions of various thiol peptide hydrolases are considered: their participation in protein turnover, post-translational processing, regulation of unidirectional biological processes and metabolic refolding. Data on endogenous inhibitors of thiol peptide hydrolases and on regulation of enzymic activity are presented.     

Lysosomal cysteine proteases: more than scavengers

Lysosomal cysteine proteases were believed to be mainly involved in intracellular protein degradation. Under special conditions they have been found outside lysosomes resulting in pathological conditions. With the discovery of a series of new cathepsins with restricted tissue distributions, it has become evident that these enzymes must be involved in a range of specific cellular tasks much broader than as simple housekeeping enzymes. It is therefore timely to review and discuss the various physiological roles of mammalian lysosomal papain-like cysteine proteases as well as their mechanisms of action and the regulation of their activity.     

Cathepsins L and Z are critical in degrading polyglutamine-containing proteins within lysosomes

In neurodegenerative diseases caused by extended polyglutamine (polyQ) sequences in proteins, aggregation-prone polyQ proteins accumulate in intraneuronal inclusions. PolyQ proteins can be degraded by lysosomes or proteasomes. Proteasomes are unable to hydrolyze polyQ repeat sequences, and during breakdown of polyQ proteins, they release polyQ repeat fragments for degradation by other cellular enzymes. This study was undertaken to identify the responsible proteases. Lysosomal extracts (unlike cytosolic enzymes) were found to rapidly hydrolyze polyQ sequences in peptides, proteins, or insoluble aggregates. Using specific inhibitors against lysosomal proteases, enzyme-deficient extracts, and pure cathepsins, we identified cathepsins L and Z as the lysosomal cysteine proteases that digest polyQ proteins and peptides. RNAi for cathepsins L and Z in different cell lines and adult mouse muscles confirmed that they are critical in degrading polyQ proteins (expanded huntingtin exon 1) but not other types of aggregation-prone proteins (e.g. mutant SOD1). Therefore, the activities of these two lysosomal cysteine proteases are important in host defense against toxic accumulation of polyQ proteins.     

Cysteine Cathepsins in Neurological Disorders

Increased proteolytic activity is a hallmark of several pathological processes, including neurodegeneration. Increased expression and activity of cathepsins, lysosomal cysteine proteases, during degeneration of the central nervous system is frequently reported. Recent studies reveal that a disturbed balance of their enzymatic activities is the first insult in brain aging and age-related diseases. Leakage of cathepsins from lysosomes, due to their membrane permeability, and activation of pro-apoptotic factors additionally contribute to neurodegeneration. Furthermore, in inflammation-induced neurodegeneration the cathepsins expressed in activated microglia play a pivotal role in neuronal death. The proteolytic activity of cysteine cathepsins is controlled by endogenous protein inhibitors—the cystatins—which evidently fail to perform their function in neurodegenerative processes. Exogenous synthetic inhibitors, which may augment their inhibitory potential, are considered as possible therapeutic tools for the treatment of neurological disorders.     

Cysteine proteases: destruction ability versus immunomodulation capacity in immune cells

Cysteine proteases (cathepsins) play a pivotal role in various physiological processes, as well as in several diseases. In the immune response, maturation of major histocompatibility class II (MHC II) molecules and processing of antigens for further presentation by MHC II is tightly linked to the enzymes of the endosomal/lysosomal system, of which cysteine proteases constitute a major proportion. Furthermore, the process of autophagy provides access for cytosolic antigens to proteolysis by lysosomal cathepsins and subsequent MHC II presentation. Other specific functions of proteolytic enzymes associated with the immune response, such as activation of granzymes by cathepsin C in T-lymphocytes, are introduced and covered in this review.     

Effect of proteinase inhibitors on intracellular processing of cathepsin B, H and L in rat macrophages

The effects of various proteinase inhibitors on the processing of lysosomal cathepsins B, H and L were investigated in cultured rat peritoneal macrophages. The processing of newly synthesized pro-cathepsins B, H and L to the mature single-chain enzymes was sensitive to a metal chelator,1,10-phenanthroline, and a synthetic metalloendopeptidase substrate, Z-Gly-Leu-NH2, and insensitive to inhibitors of serine proteinases, aspartic proteinases and cysteine proteinases. Inhibitors of cysteine proteinases, E-64-d and leupeptin, inhibited the processing of the single-chain forms of cathepsins B, H and L to the two-chain forms. These results suggest that (a) metal endopeptidase(s) is (are) involved in the propeptide processing of cathepsin B, H and L, and that proteolytic cleavages of the mature single-chain cathepsins are accomplished by cysteine proteinases in lysosomes.     

Cathepsin Inhibition-Induced Lysosomal Dysfunction Enhances Pancreatic Beta-Cell Apoptosis in High Glucose

Autophagy is a lysosomal degradative pathway that plays an important role in maintaining cellular homeostasis. We previously showed that the inhibition of autophagy causes pancreatic β-cell apoptosis, suggesting that autophagy is a protective mechanism for the survival of pancreatic β-cells. The current study demonstrates that treatment with inhibitors and knockdown of the lysosomal cysteine proteases such as cathepsins B and L impair autophagy, enhancing the caspase-dependent apoptosis of INS-1 cells and islets upon exposure to high concentration of glucose. Interestingly, treatment with cathepsin B and L inhibitors prevented the proteolytic processing of cathepsins B, D and L, as evidenced by gradual accumulation of the respective pro-forms. Of note, inhibition of aspartic cathepsins had no effect on autophagy and cell viability, suggesting the selective role of cathepsins B and L in the regulation of β-cell autophagy and apoptosis. Lysosomal localization of accumulated pro-cathepsins in the presence of cathepsin B and L inhibitors was verified via immunocytochemistry and lysosomal fractionation. Lysotracker staining indicated that cathepsin B and L inhibitors led to the formation of severely enlarged lysosomes in a time-dependent manner. The abnormal accumulation of pro-cathepsins following treatment with inhibitors of cathepsins B and L suppressed normal lysosomal degradation and the processing of lysosomal enzymes, leading to lysosomal dysfunction. Collectively, our findings suggest that cathepsin defects following the inhibition of cathepsin B and L result in lysosomal dysfunction and consequent cell death in pancreatic β-cells.     

Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues

As a model for defining the role of lysosomal cathepsins in apoptosis, we characterized the action of the lysosomotropic agent LeuLeuOMe using distinct cellular models. LeuLeuOMe induces lysosomal membrane permeabilization, resulting in release of lysosomal cathepsins that cleave the proapoptotic Bcl-2 family member Bid and degrade the antiapoptotic member Bcl-2, Bcl-xL, or Mcl-1. The papain-like cysteine protease inhibitor E-64d largely prevented apoptosis, Bid cleavage, and Bcl-2/Bcl-xL/Mcl-1 degradation. The pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone failed to prevent Bid cleavage and degradation of anti-apoptotic Bcl-2 homologues but substantially decreased cell death, suggesting that cathepsin-mediated apoptosis in these cellular models mostly follows a caspase-dependent pathway. Moreover, in vitro experiments showed that one or more of the cysteine cathepsins B, L, S, K, and H could cleave Bcl-2, Bcl-xL, Mcl-1, Bak, and BimEL, whereas no Bax cleavage was observed. On the basis of inhibitor studies, we demonstrate that lysosomal disruption triggered by LeuLeuOMe occurs before mitochondrial damage. We propose that degradation of anti-apoptotic Bcl-2 family members by lysosomal cathepsins synergizes with cathepsin-mediated activation of Bid to trigger a mitochondrial pathway to apoptosis. Moreover, XIAP (X-chromosome-linked inhibitor of apoptosis) was also found to be a target of cysteine cathepsins, suggesting that cathepsins can mediate caspase-dependent apoptosis also downstream of mitochondria.     

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.     

Inhibitory properties of cystatin F and its localization in U937 promonocyte cells

Cystatin F is a recently discovered type II cystatin expressed almost exclusively in immune cells. It is present intracellularly in lysosome-like vesicles, which suggests a potential role in regulating papain-like cathepsins involved in antigen presentation. Therefore, interactions of cystatin F with several of its potential targets, cathepsins F, K, V, S, H, X and C, were studied in vitro. Cystatin F tightly inhibited cathepsins F, K and V with Ki values ranging from 0.17 nM to 0.35 nM, whereas cathepsins S and H were inhibited with 100-fold lower affinities (Ki approximately 30 nM). The exopeptidases, cathepsins C and X were not inhibited by cystatin F. In order to investigate the biological significance of the inhibition data, the intracellular localization of cystatin F and its potential targets, cathepsins B, H, L, S, C and K, were studied by confocal microscopy in U937 promonocyte cells. Although vesicular staining was observed for all the enzymes, only cathepsins H and X were found to be colocalized with the inhibitor. This suggests that cystatin F in U937 cells may function as a regulatory inhibitor of proteolytic activity of cathepsin H or, more likely, as a protection against cathepsins misdirected to specific cystatin F containing endosomal/lysosomal vesicles. The finding that cystatin F was not colocalized with cystatin C suggests distinct functions for these two cysteine protease inhibitors in U937 cells.     

Effective extraction of lysosomal enzymes with digitonin

A method for rapid and effective extraction of rat liver lysosomal enzymes has been elaborated. It includes isolation of lysosomal-mitochondrial fraction by means of differential centrifugation, selective destruction of the lysosomal membrane by digitonin and centrifugal obtaining of the lysosomal matrix. Total labilization of the lysosomal membrane is achieved at 0.3 mM of the detergent. The maximal enrichment of an extract by lysosomal enzymes is observed in the range of 0.3-0.4 mM of digitonin. The level of lysosomal enzyme purification is 30.7 for cysteine cathepsins B, L, H, 24.9- for beta-galactosidase, 14.1- for acid phosphatase. The method gives high yield of lysosomal enzymes (40-80%).     

Cysteine cathepsins: from structure, function and regulation to new frontiers

It is more than 50 years since the lysosome was discovered. Since then its hydrolytic machinery, including proteases and other hydrolases, has been fairly well identified and characterized. Among these are the cysteine cathepsins, members of the family of papain-like cysteine proteases. They have unique reactive-site properties and an uneven tissue-specific expression pattern. In living organisms their activity is a delicate balance of expression, targeting, zymogen activation, inhibition by protein inhibitors and degradation. The specificity of their substrate binding sites, small-molecule inhibitor repertoire and crystal structures are providing new tools for research and development. Their unique reactive-site properties have made it possible to confine the targets simply by the use of appropriate reactive groups. The epoxysuccinyls still dominate the field, but now nitriles seem to be the most appropriate "warhead". The view of cysteine cathepsins as lysosomal proteases is changing as there is now clear evidence of their localization in other cellular compartments. Besides being involved in protein turnover, they build an important part of the endosomal antigen presentation. Together with the growing number of non-endosomal roles of cysteine cathepsins is growing also the knowledge of their involvement in diseases such as cancer and rheumatoid arthritis, among others. Finally, cysteine cathepsins are important regulators and signaling molecules of an unimaginable number of biological processes. The current challenge is to identify their endogenous substrates, in order to gain an insight into the mechanisms of substrate degradation and processing. In this review, some of the remarkable advances that have taken place in the past decade are presented. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Interaction of lysosomal cysteine proteinases with alpha 2-macroglobulin: conclusive evidence for the endopeptidase activities of cathepsins B and H

The lysosomal cysteine proteinases, cathepsins B, H, and L, were all shown to bind to alpha 2-macroglobulin. The bound enzymes remained active against low-molecular-weight synthetic substrates and bound the active-site-directed inhibitor, benzyloxycarbonyl-125I-Tyr-Ala-diazomethane. Binding of the radiolabeled inhibitor to high-molecular-weight protein on sodium dodecyl sulfate polyacrylamide gels indicated that a proportion of the enzymes was covalently bound to alpha 2-macroglobulin. Cleavage fragments of alpha 2-macroglobulin of Mr 92,000 and 86,000 were seen for cathepsins B, H, and L, indicating cleavage in the bait region. Binding and cleavage were observed for both single-chain and two-chain forms of cathepsin B from human, ox, and pig livers, showing that all active forms of cathepsins B, H, and L are endopeptidases.     

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.     

Protein kinase C-alpha and -delta are required for NADPH oxidase activation in WKYMVm-stimulated IMR90 human fibroblasts

Gene duplications in rodents have given rise to a family of proteases that are expressed exclusively in placenta. To define the biological role of these enzymes specific inhibitors are needed to differentiate their activities from other more ubiquitously expressed proteases, such as cathepsins B and L. Libraries of peptidyl inhibitors based upon a 4-cyclohexanone pharmacophore were screened for inhibition of cathepsins P, L, and B. The tightest binding dipeptidyl inhibitor for cathepsin P contained Tyr in P(2) and Trp in P(2)('), consistent with the specificity of this enzyme for hydrophobic amino acids at these sites in synthetic substrates. An inhibitor containing Trp in both P(2) and P(2)(') provided better discrimination between cathepsin P and cathepsins B and L. Extension of the inhibitors to include P(3), and P(3)(') amino acids identified an inhibitor with Trp in P(2), P(2)('), and P(3), and Phe in P(3)(') that bound to cathepsin P with a K(i) of 32 nM. This specificity for inhibitors with hydrophobic aromatic amino acids in these four positions is unique among the lysosomal cysteine proteases. This inhibitor bound to cathepsin P an order of magnitude tighter than to mouse and human cathepsin L and two orders of magnitude tighter than to human cathepsin B. Cbz-Trp-Trp-4-cyclohexanone-Trp-Phe-OMe can discriminate cathepsin P from cathepsins B and L and consequently can be used to specifically inhibit and identify cathepsin P in cellular systems.     

Cysteine cathepsins: From structure, function and regulation to new frontiers

It is more than 50 years since the lysosome was discovered. Since then its hydrolytic machinery, including proteases and other hydrolases, has been fairly well identified and characterized. Among these are the cysteine cathepsins, members of the family of papain-like cysteine proteases. They have unique reactive-site properties and an uneven tissue-specific expression pattern. In living organisms their activity is a delicate balance of expression, targeting, zymogen activation, inhibition by protein inhibitors and degradation. The specificity of their substrate binding sites, small-molecule inhibitor repertoire and crystal structures are providing new tools for research and development. Their unique reactive-site properties have made it possible to confine the targets simply by the use of appropriate reactive groups. The epoxysuccinyls still dominate the field, but now nitriles seem to be the most appropriate “warhead”. The view of cysteine cathepsins as lysosomal proteases is changing as there is now clear evidence of their localization in other cellular compartments. Besides being involved in protein turnover, they build an important part of the endosomal antigen presentation. Together with the growing number of non-endosomal roles of cysteine cathepsins is growing also the knowledge of their involvement in diseases such as cancer and rheumatoid arthritis, among others. Finally, cysteine cathepsins are important regulators and signaling molecules of an unimaginable number of biological processes. The current challenge is to identify their endogenous substrates, in order to gain an insight into the mechanisms of substrate degradation and processing. In this review, some of the remarkable advances that have taken place in the past decade are presented. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Inhibition of cathepsins and related proteases by amino acid, peptide, and protein hydroperoxides

Reaction of radicals in the presence of O2, and singlet oxygen, with some amino acids, peptides, and proteins yields hydroperoxides. These species are key intermediates in chain reactions and protein damage. Previously we have shown that peptide and protein hydroperoxides react rapidly with thiols, and that this can result in inactivation of thiol-dependent enzymes. The major route for the cellular removal of damaged proteins is via catabolism mediated by proteosomal and lysosomal pathways; cysteine proteases (cathepsins) play a key role in the latter system. We hypothesized that inactivation of cysteine proteases by hydroperoxide-containing oxidised proteins may contribute to the accumulation of modified proteins within cells. We show here that thiol-dependent cathepsins, either isolated or in cell lysates, are rapidly and efficiently inactivated by amino acid, peptide, and protein hydroperoxides in a time- and concentration-dependent manner; this occurs with similar efficacy to equimolar H2O2. Inactivation involves reaction of the hydroperoxide with Cys residues as evidenced by thiol loss and formation of sulfenic acid intermediates. Structurally related, non-thiol-dependent cathepsins are less readily inactivated by these hydroperoxides. This inhibition, by oxidized proteins, of the system designed to remove modified proteins, may contribute to the accumulation of damaged proteins in cells subject to oxidative stress.     

Exploring the role of cathepsin in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease which is marked by leukocytes infiltration inside synovial tissue, joints and also inside synovial fluid which causes progressive destruction of joint cartilage. There are numerous genetical and lifestyle factors, responsible for rheumatoid arthritis. One such factor can be cysteine cathepsins, which act as proteolytic enzymes. These proteolytic enzyme gets activated at acidic pH and are found in lysosomes and are also termed as cysteine proteases. These proteases belong to papain family and have their elucidated role in musculoskeletal disorders. Numerous cathepsins have their targeted role in rheumatoid arthritis. These proteases are secreted through various cell types which includes matrix metalloproteases and papain like cysteine proteases. These proteases can potentially lead to bone and cartilage destruction which causes an immune response in case of inflammatory arthritis.