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Cysteine cathepsins are a fascinating group of proteolytic enzymes that play diverse and crucial roles in numerous biological processes, both in health and disease. Understanding these proteases is essential for uncovering novel insights into the underlying mechanisms of a wide range of disorders, such as cancer. Cysteine cathepsins influence cancer biology by participating in processes such as extracellular matrix degradation, angiogenesis, immune evasion, and apoptosis. In this comprehensive review, we explore foundational research that illuminates the diverse and intricate roles of cysteine cathepsins as diagnostic markers and therapeutic targets for cancer. This review aims to provide valuable insights into the clinical relevance of cysteine cathepsins and explore their capacity to advance personalised and targeted medical interventions in oncology., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Biomedical research often focuses on properties that differentiate between diseased and healthy tissue; one of the current focuses is elevated expression and altered localisation of proteases. Among these proteases, dysregulation of cysteine cathepsins can frequently be observed in inflammation-associated diseases, which tips the functional balance from normal physiological to pathological manifestations. Their overexpression and secretion regularly exhibit a strong correlation with the development and progression of such diseases, making them attractive pharmacological targets. But beyond their mostly detrimental role in inflammation-associated diseases, cysteine cathepsins are physiologically highly important enzymes involved in various biological processes crucial for maintaining homeostasis and responding to different stimuli. Consequently, several challenges have emerged during the efforts made to translate basic research data into clinical applications. In this review, we present both physiological and pathological roles of cysteine cathepsins and discuss the clinical potential of cysteine cathepsin-targeting strategies for disease management and diagnosis., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cysteine cathepsin proteases are found under normal conditions in the lysosomal compartments of cells, where they play pivotal roles in a variety of cellular processes such as protein and lipid metabolism, autophagy, antigen presentation, and cell growth and proliferation. As a consequence, aberrant localization and activity contribute to several pathologic conditions such as a variety of malignancies, cardiovascular diseases, osteoporosis, and other diseases. Hence, there is a resurgence of interest to expand the toolkit to monitor intracellular cathepsin activity and better ascertain their functions under these circumstances. Previous fluorescent activity-based probes (ABPs) that target cathepsins B, L, and S enabled detection of their activity in intact cells as well as non-invasive detection in animal disease models. However, their binding potency is suboptimal compared to the cathepsin inhibitor on which they were based, as the P1 positive charge was capped by a reporter tag. Here, we show the development of an improved cathepsin ABP that has a P1 positive charge by linking the tag on an additional amino acid at the end of the probe. While enhancing potency towards recombinant cathepsins, the new probe had reduced cell permeability due to additional peptide bonds. At a second phase, the probe was trimmed; the fluorophore was linked to an extended carbobenzoxy moiety, leading to enhanced cell permeability and superb detection of cathepsin activity in intact cells. In conclusion, this work introduces a prototype design for the next generation of highly sensitive ABPs that have excellent detection of cellular cathepsin activity.

New therapeutic targets that could improve current antitumor therapy and overcome cancer resistance are urgently needed. Promising candidates are lysosomal cysteine cathepsins, proteolytical enzymes involved in various critical steps during cancer progression. Among them, cathepsin X, which acts solely as a carboxypeptidase, has received much attention. Our results indicate that the triazole-based selective reversible inhibitor of cathepsin X named Z9 (1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-((4-isopropyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one) significantly reduces tumor progression, both in vitro in cell-based functional assays and in vivo in two independent tumor mouse models: the FVB/PyMT transgenic and MMTV-PyMT orthotopic breast cancer mouse models. One of the mechanisms by which cathepsin X contributes to cancer progression is the compensation of cathepsin-B activity loss. Our results confirm that cathepsin-B inhibition is compensated by an increase in cathepsin X activity and protein levels. Furthermore, the simultaneous inhibition of both cathepsins B and X with potent, selective, reversible inhibitors exerted a synergistic effect in impairing processes of tumor progression in in vitro cell-based assays of tumor cell migration and spheroid growth. Taken together, our data demonstrate that Z9 impairs tumor progression both in vitro and in vivo and can be used in combination with other peptidase inhibitors as an innovative approach to overcome resistance to antipeptidase therapy., (© 2022. The Author(s).)

The interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction. To gain insight into this dynamic interface, we combined Click chemistry with pulsed stable isotope labelling of amino acids in cell culture to quantify the host proteome response during macrophage infection with the intracellular bacterial pathogen Salmonella enterica Typhimurium. We monitored newly synthesized proteins across different host cell compartments and infection stages. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus. We verified that active cathepsins re-traffic to the nucleus and that these are linked to cell death. Pharmacological cathepsin inhibition and nuclear targeting of a cellular cathepsin inhibitor (stefin B) suppressed S. enterica Typhimurium-induced cell death. We demonstrate that cathepsin activity is required for pyroptotic cell death via the non-canonical inflammasome, and that lipopolysaccharide transfection into the host cytoplasm is sufficient to trigger active cathepsin accumulation in the host nucleus and cathepsin-dependent cell death. Finally, cathepsin inhibition reduced gasdermin D expression, thus revealing an unexpected role for cathepsin activity in non-canonical inflammasome regulation. Overall, our study illustrates how resolution of host proteome dynamics during infection can drive the discovery of biological mechanisms at the host-microbe interface.

Introduction : Cysteine cathepsins are involved in the development and progression of numerous inflammation-associated diseases such as cancer, arthritis, bone and immune disorders. Consequently, there is a drive to progress research efforts focused on cathepsin use in diagnostics and as therapeutic targets in disease. Areas covered : This review discusses the potential of cysteine cathepsins as therapeutic targets in inflammation-associated diseases and recent advances in preclinical and clinical research. We describe direct targeting of cathepsins for treatment purposes and their indirect use in diagnostics. Expert opinion : The targeting of cysteine cathepsins has not translated into the clinic; this failure is attributed to off- and on-target side effects and/or the lack of companion biomarkers. This field now embraces developments in diagnostic imaging, the activation of prodrugs and antibody-drug conjugates for targeted drug delivery. The future lies in improved molecular tools and therapeutic concepts that will find a wide spectrum of uses in diagnostic and therapeutic applications.

Proteolytic activity in the tumor microenvironment is one of the key elements supporting tumor development and metastasis. One of the key families of proteases that are overexpressed in various types of cancer and implicated in different stages of tumor progression are cysteine cathepsins. Among them, cathepsins S and L can be secreted into the tumor microenvironment by tumor and/or immune cells, making them promising drug delivery targets. Here we present a new system for cathepsin S/L targeting using a liposomal drug carrier system functionalized with the endogenous cysteine cathepsin inhibitor, stefin A. The selective targeting of cathepsins by stefin A-conjugated liposomes was confirmed in vitro and in vivo, demonstrating the potential of this approach for cancer diagnosis and treatment., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

: For a long time, cysteine cathepsins were considered primarily as proteases crucial for nonspecific bulk proteolysis in the endolysosomal system. However, this view has dramatically changed, and cathepsins are now considered key players in many important physiological processes, including in diseases like cancer, rheumatoid arthritis, and various inflammatory diseases. Cathepsins are emerging as important players in the extracellular space, and the paradigm is shifting from the degrading enzymes to the enzymes that can also specifically modify extracellular proteins. In pathological conditions, the activity of cathepsins is often dysregulated, resulting in their overexpression and secretion into the extracellular space. This is typically observed in cancer and inflammation, and cathepsins are therefore considered valuable diagnostic and therapeutic targets. In particular, the investigation of limited proteolysis by cathepsins in the extracellular space is opening numerous possibilities for future break-through discoveries. In this review, we highlight the most important findings that establish cysteine cathepsins as important players in the extracellular space and discuss their roles that reach beyond processing and degradation of extracellular matrix (ECM) components. In addition, we discuss the recent developments in cathepsin research and the new possibilities that are opening in translational medicine., Competing Interests: The authors declare no conflict of interest.

Cysteine cathepsins have been for a long time considered to execute mainly nonspecific bulk proteolysis in the endolysosomal system. However, this view has been changing profoundly over the last decade as cathepsins were found in the cytoplasm, nucleus and in the extracellular milieu. Cathepsins are currently gaining increased attention largely because of their extracellular roles associated with disease development and progression. While kept under tight control under physiological conditions, their dysregulated and elevated activity in the extracellular milieu are distinctive hallmarks of numerous diseases such as various cancers, inflammatory disorders, rheumatoid arthritis, bone disorders and heart diseases. In this review, we discuss cysteine cathepsins with a major focus on their extracellular roles and extracellular proteolytic targets beyond degradation of the extracellular matrix. We further highlight the perspectives of cathepsin research and novel avenues in cathepsin-based diagnostic and therapeutic applications., (Copyright © 2018. Published by Elsevier B.V.)

Since the discovery of the key role of cathepsin K in bone resorption, cysteine cathepsins have been investigated by pharmaceutical companies as drug targets. The first clinical results from targeting cathepsins by activity-based probes and substrates are paving the way for the next generation of molecular diagnostic imaging, whereas the majority of antibody-drug conjugates currently in clinical trials depend on activation by cathepsins. Finally, cathepsins have emerged as suitable vehicles for targeted drug delivery. It is therefore timely to review the future of cathepsins in drug discovery. We focus here on inflammation-associated diseases because dysregulation of the immune system accompanied by elevated cathepsin activity is a common feature of these conditions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The development of reactive drug carriers that could actively respond to biological signals is a challenging task. Different peptides can self-assemble into biocompatible nanostructures of various functionalities, including drugs carriers. Minimal building blocks, such as diphenylalanine, readily form ordered nanostructures. Here we present the development of self-assembled tetra-peptides that include the diphenylalanine motif, serving as substrates of the cathepsin proteases. This is of great clinical importance as cathepsins, whose activity and expression are highly elevated in cancer and other pathologies, have been shown to serve as efficient enzymes for therapeutic release. Based on the cathepsins affinity around the active site, we generated a library of Phe-Phe-Lys-Phe (FFKF) tetra-peptide substrates (TPSs). We inserted various N-termini capping groups with different chemical properties to investigate the effect on protease affinity and self-assembly. All nine TPSs were cleaved by their targets, cathepsins B and L. However, solvent switching led to nanofibers self-assembly of only seven of them. Due to its rapid self-assembly and complete degradation by cathepsin B, we focused on TPS4, Cbz-FFKF-OH. Degradation of TPS4 nanofibers by cathepsin B led to the release of 91.8±0.3% of the incorporated anti-cancerous drug Doxorubicin from the nanofibers within 8h while only 55±0.2% was released without enzyme treatment. Finally, we demonstrated that tumor lysates fully degraded TPS4 nanofibers. Collectively, these results suggest that tetra-peptide substrates that form nanostructures could serve as a promising platform for targeted drug delivery to pathologies in which protease activity is highly elevated., (Copyright © 2016 Elsevier B.V. All rights reserved.)

To ensure successful feeding tick saliva contains a number of inhibitory proteins that interfere with the host immune response and help to create a permissive environment for pathogen transmission. Among the potential targets of the salivary cystatins are two host cysteine proteases, cathepsin S, which is essential for antigen- and invariant chain-processing, and cathepsin C (dipeptidyl peptidase 1, DPP1), which plays a critical role in processing and activation of the granule serine proteases. Here, the effect of salivary cystatin OmC2 from Ornithodoros moubata was studied using differentiated MUTZ-3 cells as a model of immature dendritic cells of the host skin. Following internalization, cystatin OmC2 was initially found to inhibit the activity of several cysteine cathepsins, as indicated by the decreased rates of degradation of fluorogenic peptide substrates. To identify targets, affinity chromatography was used to isolate His-tagged cystatin OmC2 together with the bound proteins from MUTZ-3 cells. Cathepsins S and C were identified in these complexes by mass spectrometry and confirmed by immunoblotting. Furthermore, reduced increase in the surface expression of MHC II and CD86, which are associated with the maturation of dendritic cells, was observed. In contrast, human inhibitor cystatin C, which is normally expressed and secreted by dendritic cells, did not affect the expression of CD86. It is proposed that internalization of salivary cystatin OmC2 by the host dendritic cells targets cathepsins S and C, thereby affecting their maturation.

Lysosomes and lysosomal hydrolases, including the cathepsins, have been shown to change their properties with aging brain a long time ago, although their function was not really understood. The first biochemical and clinical studies were followed by a major expansion in the last 20 years with the development of animal disease models and new approaches leading to a major advancement of understanding of the role of physiological and degenerative processes in the brain at the molecular level. This includes the understanding of the major role of autophagy and the cathepsins in a number of diseases, including its critical role in the neuronal ceroid lipofuscinosis. Similarly, cathepsins and some other lysosomal proteases were shown to have important roles in processing and/or degradation of several important neuronal proteins, thereby having either neuroprotective or harmful roles. In this review, we discuss lysosomal cathepsins and their regulation with the focus on cysteine cathepsins and their endogenous inhibitors, as well as their role in several neurodegenerative diseases., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Copper is an essential metal ion for embryonic development, iron acquisition, cardiac function, neuropeptide biogenesis, and other critical physiological processes. Ctr1 is a high affinity Cu(+) transporter on the plasma membrane and endosomes that exists as a full-length protein and a truncated form of Ctr1 lacking the methionine- and histidine-rich metal-binding ectodomain, and it exhibits reduced Cu(+) transport activity. Here, we identify the cathepsin L/B endolysosomal proteases functioning in a direct and rate-limiting step in the Ctr1 ectodomain cleavage. Cells and mice lacking cathepsin L accumulate full-length Ctr1 and hyper-accumulate copper. As Ctr1 also transports the chemotherapeutic drug cisplatin via direct binding to the ectodomain, we demonstrate that the combination of cisplatin with a cathepsin L/B inhibitor enhances cisplatin uptake and cell killing. These studies identify a new processing event and the key protease that cleaves the Ctr1 metal-binding ectodomain, which functions to regulate cellular Cu(+) and cisplatin acquisition., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

During lysosomal membrane permeabilization (LMP), lysosomal lumenal contents can be released into the cytosol. Small molecules are more likely to be released, and cysteine cathepsins, with mature forms possessing a mass of 25-30 kDa, are among the smallest lumenal lysosomal enzymes. In addition, specific substrates for cysteine cathepsins are available to investigators, and therefore the measurement of the cathepsin activity as a hallmark of LMP works well. Here, we present a protocol for measuring the activity of these enzymes after selective plasma membrane permeabilization with a low concentration of digitonin and after total cell membrane lysis with a high concentration of digitonin. A fluorogenic substrate can be added either directly to the well with lysed cells to show LMP or to the cell-free extract to show that the lysosomal membrane has been sufficiently destabilized to allow the translocation of lysosomal enzymes. Although the content of lysosomal cysteine cathepsins differs between cell lines, this method has general applicability, is sensitive, and has high throughput. The presented protocol shows how to measure cysteine cathepsin activity in the presence of lysed cells and also in cell-free extracts. Depending on the aim of the study, one or both types of measurements can be performed., (© 2016 Cold Spring Harbor Laboratory Press.)

Extracellular cysteine cathepsins are known to drive cancer progression, but besides degradation of extracellular matrix proteins little is known about their physiological substrates and thus the molecular mechanisms they deploy. One of the major mechanisms used by other extracellular proteases to facilitate cancer progression is proteolytic release of the extracellular domains of transmembrane proteins or ectodomain shedding. Here we show using a mass spectrometry-based approach that cathepsins L and S act as sheddases and cleave extracellular domains of CAM adhesion proteins and transmembrane receptors from the surface of cancer cells. In cathepsin S-deficient mouse pancreatic cancers, processing of these cathepsin substrates is highly reduced, pointing to an essential role of cathepsins in extracellular shedding. In addition to influencing cell migration and invasion, shedding of surface proteins by extracellular cathepsins impacts intracellular signaling as demonstrated for regulation of Ras GTPase activity, thereby providing a putative mechanistic link between extracellular cathepsin activity and cancer progression. The MS data is available via ProteomeXchange with identifier PXD002192., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Proteases are important effectors of numerous physiological and pathological processes. Reliable determination of a protease's specificity is crucial to understand protease function and to develop activity-based probes and inhibitors. During the last decade, various proteomic approaches for profiling protease substrate specificities were reported. Although most of these approaches can identify up to thousands of substrate cleavage events in a single experiment, they are often time consuming and methodologically challenging as some of these approaches require rather complex sample preparation procedures. For such reasons their application is often limited to those labs that initially introduced them. Here, we report on a fast and simple approach for proteomic profiling of protease specificities (fast profiling of protease specificity (FPPS)), which can be applied to complex protein mixtures. FPPS is based on trideutero-acetylation of novel N-termini generated by the action of proteases and subsequent peptide fractionation on Stage Tips containing ion-exchange and reverse phase chromatographic resins. FPPS can be performed in 2 days and does not require extensive fractionation steps. Using this approach, we have determined the specificity profiles of the cysteine cathepsins K, L and S. We further validated our method by comparing the results with the specificity profiles obtained by the N-terminal combined fractional diagonal chromatography method. This comparison pointed to almost identical substrate specificities for all three cathepsins and confirmed the reliability of the FPPS approach. All MS data have been deposited in the ProteomeXchange with identifiers PXD001536 and PXD001553 (http://proteomecentral.proteomexchange.org/dataset/PXD001536; http://proteomecentral.proteomexchange.org/dataset/PXD001553)., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cysteine cathepsins are primarily lysosomal proteases involved in general protein turnover, but they also have specific proteolytic functions in antigen presentation and bone remodeling. Cathepsins are most stable at acidic pH, although growing evidence indicates that they have physiologically relevant activity also at neutral pH. Post-translational proteolytic processing of mature chemokines is a key, yet underappreciated, level of chemokine regulation. Although the role of selected serine proteases and matrix metalloproteases in chemokine processing has long been known, little has been reported about the role of cysteine cathepsins. Here we evaluated cleavage of CXC ELR (CXCL1, -2, -3, -5, and -8) and non-ELR (CXCL9-12) chemokines by cysteine cathepsins B, K, L, and S at neutral pH by high resolution Tris-Tricine SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Whereas cathepsin B cleaved chemokines especially in the C-terminal region, cathepsins K, L, and S cleaved chemokines at the N terminus with glycosaminoglycans modulating cathepsin processing of chemokines. The functional consequences of the cleavages were determined by Ca(2+) mobilization and chemotaxis assays. We show that cysteine cathepsins inactivate and in some cases degrade non-ELR CXC chemokines CXCL9-12. In contrast, cathepsins specifically process ELR CXC chemokines CXCL1, -2, -3, -5, and -8 N-terminally to the ELR motif, thereby generating agonist forms. This study suggests that cysteine cathepsins regulate chemokine activity and thereby leukocyte recruitment during protective or pathological inflammation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Elevated cathepsins levels and activities are found in several types of human cancer, making them valuable biomarkers for detection and targeting therapeutics. We designed small molecule quenched activity-based probes (qABPs) that fluoresce upon activity-dependent covalent modification, yielding cell killing by Photodynamic Therapy (PDT). These novel molecules are highly selective theranostic probes that enable both detection and treatment of cancer with minimal side effects. Our qABPs carry a photosensitizer (PS), which is activated by light, resulting in oxidative stress and subsequent cell ablation, and a quencher that when removed by active cathepsins allow the PS to fluoresce and demonstrate PD properties. Our most powerful and stable PS-qABP, YBN14, consists of a selective cathepsin recognition sequence, a QC-1 quencher and a new bacteriochlorin derivative as a PS. YBN14 allowed rapid and selective non-invasive in vivo imaging of subcutaneous tumors and induced specific tumor macrophage apoptosis by light treatment, resulting in a substantial tumor shrinkage in an aggressive breast cancer mouse model. These results demonstrate for the first time that the PS-qABPs technology offers a functional theranostic tool, which can be applied to numerous tumor types and other inflammation-associated diseases.

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 25years 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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The synthesis and evaluation of two cathepsin S-specific probes is described. For long-term retention of the probe at the target site and a high signal-to-noise ratio, we introduced a lipidation approach via the simple attachment of palmitoic acid to the reporter. After cathepsin S-specific cleavage in cultured cells and in a grafted tumor mouse model, fluorescence increased owing to dequenching and we observed an intracellular accumulation of the fluorescence in the target tissue. The lipidated probe provided a prolonged and strongly fluorescent signal in tumors when compared to the very similar non-lipidated probe, demonstrating that non-invasive tumor identification is feasable. The homing principle by probe lipidation might also work for selective administration of cytotoxic compounds to specifically reduce tumor mass., (© 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.)

Since their discovery, cysteine cathepsins were generally considered to be involved mainly in the nonspecific bulk protein degradation that takes place within the lysosomes. However, it has become clear that their proteolytical activity can also influence various specific pathological processes such as cancer, arthritis, and atherosclerosis. Furthermore, their localization was found not to be limited strictly to the lysosomes. In the light of those findings, it is not surprising that cysteine cathepsins are currently considered as highly relevant clinical targets. Moreover, recent development of proteomic-based methods for identification of novel physiological substrates of proteases provides a major opportunity also in the field of cysteine cathepsins. In this review, we will therefore present cysteine cathepsin roles in disease progression and discuss their potential relevance as prognostic and diagnostic biomarkers., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Osteoarthritis and rheumatoid arthritis are destructive joint diseases that involve the loss of articular cartilage. Degradation of cartilage extracellular matrix is believed to occur due to imbalance between the catabolic and anabolic processes of resident chondrocytes. Previous work has suggested that various lysosomal cysteine cathepsins participate in cartilage degeneration; however, their exact roles in disease development and progression have not been elucidated. In order to study degradation processes under conditions resembling the in vivo milieu of the cartilage, we cultivated chondrocytes on a type II collagen-containing matrix. Stimulation of the cultivated chondrocytes with interleukin-1α and/or tumor necrosis factor α resulted in a time-dependent increase in cathepsin S expression and induced its secretion into the conditioned media. Using a novel bioluminescent activity-based probe, we were able to demonstrate a significant increase in proteolytic activity of cathepsin S in the conditioned media of proinflammatory cytokine-stimulated chondrocytes. For the first time, cathepsin S was demonstrated to be secreted from chondrocytes upon stimulation with the proinflammatory cytokines, and displayed proteolytic activity in culture supernatants. Its stability at neutral pH and potent proteolytic activity on extracellular matrix components mean that cathepsin S may contribute significantly to cartilage degradation and may thus be considered a potential drug target in joint diseases.

Stress-induced premature senescence (SIPS) of endothelial cells (ECs) has emerged as a contributor to global EC dysfunction. One of the cellular abnormalities mechanistically linked to SIPS is lysosomal dysfunction. In this study, we examined the impact of a range of cardiovascular risk factors on the expression of sirtuin 1 (SIRT1), SIPS, and apoptosis, and we documented the role of SIRT1 in reduced EC and endothelial progenitor cell (EPC) viability. These findings were confirmed in mice with selective endothelial SIRT1 knockout. The effects of stressors could be partially mimicked by inducing lysosomal membrane permeabilization or inhibiting autophagy, and were reversed by a cathepsin inhibitor. We provide evidence that SIRT1 is an important substrate of cysteine cathepsins B, S, and L. An antioxidant/peroxynitrite scavenger, ebselen, prevented stress-induced SIRT1 depletion and subversion of autophagy by mitigating lysosomal dysfunction. In conclusion, our data advance the concept of "stem cell aging" by establishing the critical role of lysosomal dysfunction in the development of SIPS through the cathepsin-induced proteolytic cleavage of SIRT1, a mechanism linking cell stress to apoptosis and SIPS. Ebselen potently protects lysosomal membrane integrity, preventing cathepsin-induced cleavage of SIRT 1 in EPCs and blunting SIPS and apoptotic cell death induced by relevant cardiovascular stressors. The proposed mechanism of SIRT1 depletion in stress has all of the attributes of being a paradigm of SIPS of EPCs., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Lysosomes are the key degradative compartments of the cell. Lysosomal cathepsins, which are enclosed in the lysosomes, help to maintain the homeostasis of the cell's metabolism by participating in the degradation of heterophagic and autophagic material. Following the targeted lysosomal membrane's destabilization, the cathepsins can be released into the cytosol and initiate the lysosomal pathway of apoptosis through the cleavage of Bid and the degradation of the anti-apoptotic Bcl-2 homologues. Cathepsins can also amplify the apoptotic signaling, when the lysosomal membranes are destabilized at a later stage of apoptosis, initiated by other stimuli. However, the functional integrity of the lysosomal compartment during apoptosis enables efficient autophagy, which can counteract apoptosis by providing the energy source and by disposing the damaged mitochondria, which generate the ROS. Impairing autophagy by disabling the lysosome function is being investigated as an adjuvant therapeutic approach to sensitize cells to apoptosis-inducing agents. Destabilization of the lysosomal membranes by the lysosomotropic detergents seems to be a promising strategy in this context as it would not only disable autophagy, but also promote apoptosis through the initiation of the lysosomal pathway. In contrast, the impaired autophagy and lysosomal degradation linked with the increased oxidative stress underlie degenerative changes in the aging neurons. This further suggests that lysosomes and lysosomal cathepsins have a dual role in cell death. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Near-infrared fluorophore (NIRF)-labeled imaging probes are becoming increasingly important in bio-molecular imaging applications, that is, in animal models for tumor imaging or inflammation studies. In this study we showed that the previously introduced chemical concept of 'Reverse Design' represents an efficient strategy for the generation of selective probes for cysteine proteases from chemically optimized protease inhibitors for investigations in proteomic lysates as well as for in vivo molecular imaging studies. The newly developed activity-based probe AW-091 was demonstrated to be highly selective for cathepsin S in vitro and proved useful in monitoring cysteine cathepsin activity in vivo, that is, in zymosan-induced mouse model of inflammation. AW-091 showed higher signal-to-background ratios at earlier time points than the commercially available polymer-based ProSense680 (VisEn Medical) and thus represents an efficient new tool for studying early proteolytic processes leading to various diseases, including inflammation, cancer, and rheumatoid arthritis. In addition, the fluorescent signal originating from the cleaved AW-091 was shown to be reduced by the administration of an anti-inflammatory drug, dexamethasone and by the cathepsin inhibitor E-64, providing a valuable system for the evaluation of small-molecule inhibitors of cathepsins., (Copyright © 2010. Published by Elsevier Ltd.)

Cysteine cathepsins and matrix metalloproteases are considered to play important roles in the development of arthritic diseases. Their accumulation in synovial fluid of primarily rheumatoid arthritis patients is also well documented. However, a detailed comparison between the protease levels and activities between rheumatoid arthritis samples and osteoarthritis samples has never been made. Here, we report that both cysteine cathepsins B and S and matrix metalloproteases-1, -3 and -13 are detected in patient synovial fluid samples with significantly higher levels detected in rheumatoid arthritis patients. Among the proteases, cathepsin S was found to be significantly elevated, consistent with its critical role in the immune response. These results suggest that cysteine cathepsins have a major role in inflammation at least in rheumatoid arthritis. In addition to proteases, interleukin-6 was detected at significant levels in most samples, suggesting that proinflammatory cytokines might be in-volved in the stimulation of expression of these proteases during inflammation.

The tumor necrosis factor (TNF) is a cytokine known to be an important mediator of apoptosis and inflammation. It has been implicated in the pathogenesis of a number of diseases, including cancer and rheumatoid arthritis. TNF apoptosis has been known for a number of years to be critically dependent on caspases; however, recently it has been suggested that cysteine cathepsins might also be involved in the pathway. In the present work the hypothesis that cathepsins can act as an essential downstream mediator of TNF-alpha-triggered apoptosis was tested. The TNF-alpha apoptosis was investigated in two tumor-cell lines: U937 and T98G. Based on the use of pharmacological caspase inhibitors, the TNF-alpha induced caspase-dependent apoptotic cell death in both cell lines, which was accompanied by lysosomal destabilization and the release of cathepsins in the cytosol. However, blocking cysteine cathepsins with a broad-spectrum inhibitor, E64d, or a more specific cathepsin B inhibitor, CA-074Me, had no effect on the progression of the apoptosis in both cell lines, suggesting that the TNF-alpha apoptosis is not critically dependent on the cathepsins in these two cellular models.

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.

Cysteine cathepsins have been known for a long time to play an important role in cancer progression and metastasis. Several studies have proposed the concept of anti-cathepsin therapy in cancer treatment. On the other hand, cysteine cathepsins have been recently found to play a role in tumour cell death through mediation of apoptosis. The purpose of this mini-review is therefore to provide an insight into the mechanisms by which cysteine cathepsins modulate apoptosis and/or participate in tumour invasion, and to evaluate the impact of these enzymes on both tumour progression and development of potential strategies for cancer treatment.

A balance between proteolytic activity and protease inhibition is crucial to the appropriate function of many biological processes. There is mounting evidence for the presence of both papain-like cysteine proteases and serpins with a corresponding inhibitory activity in the nucleus. Well characterized examples of cofactors fine tuning serpin activity in the extracellular milieu are known, but such modulation has not been studied for protease-serpin interactions within the cell. Accordingly, we present an investigation into the effect of a DNA-rich environment on the interaction between model serpins (MENT and SCCA-1), cysteine proteases (human cathepsin V and human cathepsin L), and cystatin A. DNA was indeed found to accelerate the rate at which MENT inhibited cathepsin V, a human orthologue of mammalian cathepsin L, up to 50-fold, but unexpectedly this effect was primarily effected via the protease and secondarily by the recruitment of the DNA as a "template" onto which cathepsin V and MENT are bound. Notably, the protease-mediated effect was found to correspond both with an altered substrate turnover and a conformational change within the protease. Consistent with this, cystatin inhibition, which relies on occlusion of the active site rather than the substrate-like behavior of serpins, was unaltered by DNA. This represents the first example of modulation of serpin inhibition of cysteine proteases by a co-factor and reveals a mechanism for differential regulation of cathepsin proteolytic activity in a DNA-rich environment.

The myristoylated alanine-rich C kinase substrate (MARCKS) is a substrate of protein kinase C (PKC). Besides regulation at the level of gene transcription, MARCKS concentrations within the cell are also regulated by proteolytic cleavage by cathepsins and calpains, which are cysteine proteinases. Stefin B (cystatin B) is an endogenous inhibitor of lysosomal cysteine cathepsins, but not calpains. We have observed increased cleavage of MARCKS in brain and macrophages, but not in liver and kidney extracts of stefin B-deficient mice compared to wild-type mice. Processing of cathepsin B was unaltered in the brain of stefin B-deficient mice and we conclude that increased cleavage of MARCKS could be attributed to the lack of inhibitor.

Apoptosis is the major mechanism by which eukaryotic organisms eliminate potentially dangerous, superfluous and damaged cells. Initially, nuclei and mitochondria were found to be the key organelles involved in the process. However, recent data suggest that lysosomes and the endoplasmic reticulum also play important roles in the process. A number of different stimuli were found to directly or indirectly target the lysosomal membrane, thereby inducing lysosomal permeabilization and the release of cysteine cathepsins and the aspartic protease cathepsin D into the cytosol. Once in the cytosol, cathepsins can trigger cell death by different mechanisms. Here we discuss the different signaling pathways used by lysosomal proteases to trigger apoptosis and their potential role in physiological processes.

The general view on cysteine cathepsins, which were long believed to be primarily involved in intracellular protein turnover, has dramatically changed in last 10 to 15 years. The discovery of new cathepsins, such as cathepsins K, V, X, F and O, and their tissue distribution suggested that at least some of them are involved in very specific cellular processes. Moreover, gene ablation experiments revealed that cathepsins play a vital role in numerous physiological processes, such as antigen processing and presentation, bone remodelling, prohormone processing and wound healing. Their involvement in several pathologies, including osteoporosis, rheumatoid arthritis, osteoarthritis, bronchial asthma and cancer have also been confirmed and today several of them have been validated as relevant targets for therapies. Compounds targeting cathepsins S and K are already in clinical evaluation, whereas others are in experimental phases. The cathepsin K inhibitor AAE-581 (balicatib) as the most advanced of them passed Phase II clinical trials in 2005. In this review, we discuss the current view on cathepsins as an emerging group of targets for several diseases and the development of cathepsin K and S inhibitors for treatment of osteoporosis and various immune disorders.

Cathepsin X is a lysosomal carboxypeptidase with a potential role in processes of inflammation and immune response. The integrin-binding motifs RGD and ECD, present in the pro- and in mature forms of cathepsin X, respectively, suggest that this enzyme might have a function in cell signaling and adhesion. In this study, we report that cysteine protease inhibitors E-64 and CA-074 and 2F12 monoclonal antibody, all of which inhibit cathepsin X activity, significantly reduced adhesion of differentiated U-937 cells to polystyrene- and fibrinogen-coated surfaces via Mac-1 integrin receptor, whereas their binding to vitronectin, fibronectin or Matrigel was not affected. On the other hand, cathepsin X, added to differentiating U-937 cells, stimulated their adhesion. Using confocal microscopy, we demonstrated that the pro-form of cathepsin X was co-localized with beta(2) and beta(3) integrin subunits and its mature form solely with the beta(2) integrin subunit with the most intense signal in cell-cell junctions in differentiated U-937 cells and in co-cultures with endothelial cells. Our results indicate that active cathepsin X mediates the function of beta(2) integrin receptors during cell adhesion and that it could also be involved in other processes associated with beta(2) integrin receptors such as phagocytosis and T cell activation.

Antigen (Ag) processing by major histocompatibility complex class II (MHC) class II molecules is tightly linked with the proteases of the endosomal/lysosomal system. Cysteine (Cys) cathepsins, which constitute a major portion of this proteolytic system, have been found to have essential roles in both Ag processing and maturation of the MHC class II molecules. In this review, we will cover some specific functions of individual Cys cathepsins and particularly those most relevant to the immune system.

The S1 and S2 subsite specificity of recombinant human cathepsins X was studied using fluorescence resonance energy transfer (FRET) peptides with the general sequences Abz-Phe-Xaa-Lys(Dnp)-OH and Abz-Xaa-Arg-Lys(Dnp)-OH, respectively (Abz=ortho-aminobenzoic acid and Dnp=2,4-dinitrophenyl; Xaa=various amino acids). Cathepsin X cleaved all substrates exclusively as a carboxymonopeptidase and exhibited broad specificity. For comparison, these peptides were also assayed with cathepsins B and L. Cathepsin L hydrolyzed the majority of them with similar or higher catalytic efficiency than cathepsin X, acting as an endopeptidase mimicking a carboxymonopeptidase (pseudo-carboxymonopeptidase). In contrast, cathepsin B exhibited poor catalytic efficiency with these substrates, acting as a carboxydipeptidase or an endopeptidase. The S1' subsite of cathepsin X was mapped with the peptide series Abz-Phe-Arg-Xaa-OH and the enzyme preferentially hydrolyzed substrates with hydrophobic residues in the P1' position.

Free propeptides are known to function as inhibitors of the parental mature cysteine cathepsins. This general rule, however, does not apply to the aminopeptidase cathepsin H. Screening of propeptide fragments for their inhibitory potency revealed no significant effect on the native mature cathepsin H. On the other hand, inhibitory interaction was established with recombinant cathepsin H that displays endopeptidase activity due to a lack of the mini-chain. This finding suggests that the propeptide-binding region is structurally rearranged during maturation processing and mini-chain formation, which impairs the effective recognition of mature cathepsin H by its own propeptide.

Endogenous cysteine proteases were given much attention lately, as their role in a variety of pathophysiological disorders became evident. Amongst them cathepsins, which are thought to be implicated in mediation of osteoporosis, cancer progression, atherosclerosis, and many other conditions, are of considerable interest as drug targets. In the presented work, papain was chosen as a model cysteine protease and panning protocol was optimized for selection of papain-binding phage-displayed peptides from a commercially available combinatorial peptide library. Different selection strategies were applied in order to select high-affinity binders. Ultimately, five cyclic peptides (CNWAAGYNCGGGS-NH2, CWSMMGFQCGGGS-NH2, CWEWGGWHCGGSS-OH, CNWTLGGYKCGGGS-NH2 (all cyclized through formation of intramolecular disulphide bond), and GNWTLGGYKGG (cyclized head-to-tail)) were synthesized and tested for inhibitory activity towards papain and human cathepsins L, B, H, and K. The peptides possess inhibitory constants in the low micromolar to mid-nanomolar range and exhibit certain selectivity for different lysosomal cysteine proteases included in this study.

Cathepsin X, a recently discovered lysosomal cysteine protease, shares common structural features and activity properties with cysteine protease cathepsin B. Based on its widespread mRNA distribution in primary tumors and tumor cell lines, a redundant function in tumor progression has been proposed. In this study, we have shown that these two related proteases exhibit different profiles with respect to their protein distribution in cells and tissues and to their possible roles in malignancy. Protein level of cathepsin X did not differ significantly between matched pairs of lung tumor and adjacent lung tissue obtained from patients with lung cancer whereas that of cathepsin B was 9.6-fold higher in tumor compared to adjacent lung tissue. Immunohistochemical analysis of lung tumor cathepsin X revealed very faint staining in tumor cells but positive staining in infiltrated histiocytes, alveolar macrophages, bronchial epithelial cells, and alveolar type II cells. Cathepsin X stained positive also in CD68+ cells in germinal centers of secondary follicles in lymph nodes, corresponding to tingible body macrophages. Two cell lines with proven invasive behavior, MCF-10A neoT and MDA-MB 231, showed positive staining for cathepsin B, but negative for cathepsin X. We showed that the invasive potential of MCF-10A neoT cells can be impaired by specific inhibitor of cathepsin B but not by that of cathepsin X. Cathepsin X was found in large amounts in the pro-monocytic U-937 cell line, in monocytes and in dendritic cells, generated from monocytes in vitro. Our results show that cathepsin X is not involved in degradation of extracellular matrix, a proteolytic event leading to tumor cell invasion and metastasis. Its expression, restricted to immune cells suggests a role in phagocytosis and the regulation of immune response.

Glycosaminoglycans have been shown to be important regulators of activity of several papain-like cathepsins. Binding of glycosaminoglycans to cathepsins thus directly affects catalytic activity, stability or the rate of autocatalytic activation of cathepsins. The interaction between cathepsin X and heparin has been revealed by affinity chromatography using heparin-Sepharose. Conformational changes were observed to accompany heparin-cathepsin X interaction by far UV-circular dichroism at both acidic (4.5) and neutral (7.4) pH. These conformational changes promoted a 4-fold increase in the dissociation constant of the enzyme-substrate interaction and increased 2.6-fold the kcat value also. The interaction between cathepsin X and heparin or heparan sulfate is specific since dermatan sulfate, chondroitin sulfate, and hyaluronic acid had no effect on the cathepsin X activity. Using flow cytometry cathepsin X was shown to bind cell surface heparan sulfate proteoglycans in wild-type CHO cells but not in CHO-745 cells, which are deficient in glycosaminoglycan synthesis. Moreover, fluorescently labeled cathepsin X was shown by confocal microscopy to be endocytosed by wild-type CHO cells, but not by CHO-745 cells. These results demonstrate the existence of an endocytosis mechanism of cathepsin X by the CHO cells dependent on heparan sulfate proteoglycans present at the cell surface, thus strongly suggesting that heparan sulfate proteoglycans can regulate the cellular trafficking and the enzymatic activity of cathepsin X.

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.

Cathepsin S is unique among mammalian cysteine cathepsins in being active and stable at neutral pH. We show that autocatalytic activation of procathepsin S at low pH is a bimolecular process that is considerably accelerated (approximately 20-fold) by glycosaminoglycans and polysaccharides such as dextran sulfate, chondroitin sulfates A and E, and dermatan sulfate through electrostatic interaction with the proenzyme. Procathepsin S is also shown to undergo autoactivation at neutral pH in the presence of dextran sulfate with t1/2 of approximately 20 min at pH 7.5. This novel property of procathepsin S may have implications in pathological conditions associated with the appearance of active cathepsins outside lysosomes.

Recombinant full-length human procathepsin F, produced in the baculovirus expression system, was partially processed during the purification procedure to a form lacking the N-terminal cystatin-like domain and activated with pepsin. Active cathepsin F efficiently hydrolyzed Z-FR-MCA (kcat/Km=106 mM(-1) s(-1)) and Bz-FVR-MCA (kcat/Km=8 mM(-1) s(-1)), whereas hydrolysis of Z-RR-MCA was very slow (kcat/Km<0.2 mM(-1) s(-1)). Cathepsin F was rapidly and tightly inhibited by cystatin C, chicken cystatin and equistatin with Ki values in the subnanomolar range (0.03-0.47 nM), whereas L-kininogen was a less strong inhibitor of the enzyme (Ki=4.7 nM). Stefin A inhibited cathepsin F slowly (kass=1.6 x 10(5) M(-1) s(-1)) and with a lower affinity (Ki=25 nM). These data suggest that cathepsin F differs from other related endopeptidases by considerably weaker inhibition by stefins.

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.

Increasing evidence suggests that lysosomal proteases are actively involved in apoptosis. Using HeLa cells as the model system, we show that selective lysosome disruption with L-leucyl-L-leucine methyl ester results in apoptosis, characterized by translocation of lysosomal proteases into the cytosol and by the cleavage of a proapoptotic Bcl-2-family member Bid. Apoptosis and Bid cleavage, but not translocation of lysosomal proteases to the cytosol, could be prevented by 15 microM L-trans-epoxysuccinyl(OEt)-Leu-3-methylbutylamide, an inhibitor of papain-like cysteine proteases. Incubation of cells with 15 microM N-benzoyloxycarbonyl-VAD-fluoromethyl ketone prevented apoptosis but not Bid cleavage, suggesting that cathepsin-mediated apoptosis in this system is caspase-dependent. In vitro experiments performed at neutral pH showed that papain-like cathepsins B, H, L, S, and K cleave Bid predominantly at Arg(65) or Arg(71). No Bid cleavage was observed with cathepsins C and X or the aspartic protease cathepsin D. Incubation of full-length Bid treated with cathepsins B, H, L, and S resulted in rapid cytochrome c release from isolated mitochondria. Thus, Bid may be an important mediator of apoptosis induced by lysosomal disruption.

Apoptosis can be mediated by mechanisms other than the traditional caspase-mediated cleavage cascade. There is growing recognition that alternative proteolytic enzymes such as the lysosomal cathepsin proteases can initiate or propagate proapoptotic signals, but it is currently unclear how cathepsins achieve these actions. Recent in vitro evidence suggests that cathepsins cleave the proapoptotic Bcl-2 family member Bid, thereby activating it and allowing it to induce the mitochondrial release of cytochrome c and subsequent apoptosis. We have tested this hypothesis in vivo by breeding mice that lack cathepsin inhibition (cystatin B-deficient mice) to Bid-deficient mice, to determine whether the apoptosis caused by cathepsins is dependent on Bid signaling. We found that cathepsins are still able to promote apoptosis even in the absence of Bid, indicating that these proteases mediate apoptosis via a different pathway, or that some other molecule can functionally substitute for Bid in this system.