Dušan Turk

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.

Lysosomal cysteine proteases: facts and opportunities

From their discovery in the first half of the 20th century, lysosomal cysteine proteases have come a long way: from being the enzymes non‐selectively degrading proteins in lysosomes to being those responsible for a number of important cellular processes. Some of the features and roles of their structures, specificity, regulation and physiology are discussed.

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

Abstract It is more than 50years 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 50years after the discovery of lysosome.

Emerging roles of cysteine cathepsins in disease and their potential as drug targets.

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.

Crystal structure of the complex formed by the membrane type 1-matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor

The proteolytic activity of matrix metalloproteinases (MMPs) towards extracellular matrix components is held in check by the tissue inhibitors of metalloproteinases (TIMPs). The binary complex of TIMP‐2 and membrane‐type‐1 MMP (MT1‐MMP) forms a cell surface located ‘receptor’ involved in pro‐MMP‐2 activation. We have solved the 2.75 Å crystal structure of the complex between the catalytic domain of human MT1‐MMP (cdMT1‐MMP) and bovine TIMP‐2. In comparison with our previously determined MMP‐3–TIMP‐1 complex, both proteins are considerably tilted to one another and show new features. CdMT1‐MMP, apart from exhibiting the classical MMP fold, displays two large insertions remote from the active‐site cleft that might be important for interaction with macromolecular substrates. The TIMP‐2 polypeptide chain, as in TIMP‐1, folds into a continuous wedge; the A‐B edge loop is much more elongated and tilted, however, wrapping around the S‐loop and the β‐sheet rim of the MT1‐MMP. In addition, both C‐terminal edge loops make more interactions with the target enzyme. The C‐terminal acidic tail of TIMP‐2 is disordered but might adopt a defined structure upon binding to pro‐MMP‐2; the Ser2 side‐chain of TIMP‐2 extends into the voluminous S1′ specificity pocket of cdMT1‐MMP, with its Oγ pointing towards the carboxylate of the catalytic Glu240. The lower affinity of TIMP‐1 for MT1‐MMP compared with TIMP‐2 might be explained by a reduced number of favourable interactions.

Cystatins: Biochemical and structural properties, and medical relevance

The cystatin superfamily comprises a large group of the cystatin domain containing proteins, present in a wide variety of organisms, including humans. Cystatin inhibitory activity is vital for the delicate regulation of normal physiological processes by limiting the potentially highly destructive activity of their target proteases such as the papain (C1) family, including cysteine cathepsins. Some of the cystatins also inhibit the legumain (C13) family of enzymes. Failures in biological mechanisms controlling protease activities result in many diseases such as neurodegeneration, cardiovascular diseases, osteoporosis, arthritis, and cancer. Cystatins have been classified into three types: the stefins, the cystatins and the kininogens, although other cystatin-related proteins, such as CRES proteins, are emerging. The stefins are mainly intracellular proteins, whereas the cystatins and the kininogens are extracellular. The cystatins are tight binding and reversible inhibitors. The basic mechanism of interaction between cystatins and their target proteases has been established, based mainly on the crystal structures of various cathepsins, stefins and cystatins and their enzyme-inhibitor complexes. Cystatins, as rather non-selective inhibitors, discriminate only slightly between endo- and exopeptidases. They are also prone to form amyloids. The levels of some stefins and cystatins in tissue and body fluids can serve as relatively reliable markers for a variety of diseases. In this review we summarize present knowledge about cystatins and their role in some diseases.

Regulating Cysteine Protease Activity: Essential Role of Protease Inhibitors As Guardians and Regulators

Cysteine proteases are widespread in nature. Their implication in numerous vital processes and pathologies make them highly attractive targets for drug design. The proper functioning and regulation of activity of cysteine proteases is a delicate balance of many factors, one of the most crucial being the protease inhibitors. In this review the basic principles of physiological protease inhibition by protein inhibitors are discussed with the focus on papain-like lysosomal cysteine proteases and the caspases, and their inhibitors.

Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity

AbstractWe report here that a number of commonly used small peptide caspase inhibitors consisting of a caspase recognition sequence linked to chloromethylketone, fluoromethylketone or aldehyde reactive group efficiently inhibit other cysteine proteases than caspases. The in vitro studies included cathepsins B, H, L, S, K, F, V, X and C, papain and legumain. Z-DEVD-cmk was shown to be the preferred irreversible inhibitor of most of the cathepsins in vitro, followed by Z-DEVD-fmk, Ac-YVAD-cmk, Z-YVAD-fmk and Z-VAD-fmk. Inactivation of legumain by all the inhibitors investigated was moderate, whereas cathepsins H and C were poorly inhibited or not inhibited at all. Inhibition by aldehydes was not very potent. All the three fluoromethylketones efficiently inhibited cathepsins in Jurkat and human embryonic kidney 293 cells at concentrations of 100 μM. Furthermore, they completely inhibited cathepsins B and X activity in tissue extracts at concentrations as low as 1 μM. These results suggest that data based on the use of these inhibitors should be taken with caution and that other proteases may be implicated in the processes previously ascribed solely to caspases.

Structure of human dipeptidyl peptidase I (cathepsin C): exclusion domain added to an endopeptidase framework creates the machine for activation of granular serine proteases

Dipeptidyl peptidase I (DPPI) or cathepsin C is the physiological activator of groups of serine proteases from immune and inflammatory cells vital for defense of an organism. The structure presented shows how an additional domain transforms the framework of a papain‐like endopeptidase into a robust oligomeric protease‐processing enzyme. The tetrahedral arrangement of the active sites exposed to solvent allows approach of proteins in their native state; the massive body of the exclusion domain fastened within the tetrahedral framework excludes approach of a polypeptide chain apart from its termini; and the carboxylic group of Asp1 positions the N‐terminal amino group of the substrate. Based on a structural comparison and interactions within the active site cleft, it is suggested that the exclusion domain originates from a metallo‐protease inhibitor. The location of missense mutations, characterized in people suffering from Haim–Munk and Papillon–Lefevre syndromes, suggests how they disrupt the fold and function of the enzyme.

Crystal structure of MHC class II‐associated p41 Ii fragment bound to cathepsin L reveals the structural basis for differentiation between cathepsins L and S

The lysosomal cysteine proteases cathepsins S and L play crucial roles in the degradation of the invariant chain during maturation of MHC class II molecules and antigen processing. The p41 form of the invariant chain includes a fragment which specifically inhibits cathepsin L but not S. The crystal structure of the p41 fragment, a homologue of the thyroglobulin type‐1 domains, has been determined at 2.0 Å resolution in complex with cathepsin L. The structure of the p41 fragment demonstrates a novel fold, consisting of two subdomains, each stabilized by disulfide bridges. The first subdomain is an α‐helix–β‐strand arrangement, whereas the second subdomain has a predominantly β‐strand arrangement. The wedge shape and three‐loop arrangement of the p41 fragment bound to the active site cleft of cathepsin L are reminiscent of the inhibitory edge of cystatins, thus demonstrating the first example of convergent evolution observed in cysteine protease inhibitors. However, the different fold of the p41 fragment results in additional contacts with the top of the R‐domain of the enzymes, which defines the specificity‐determining S2 and S1′ substrate‐binding sites. This enables inhibitors based on the thyroglobulin type‐1 domain fold, in contrast to the rather non‐selective cystatins, to exhibit specificity for their target enzymes.