Boris Turk

Targeting proteases: successes, failures and future prospects

Until fairly recently, proteases were considered primarily to be protein-degrading enzymes. However, this view has dramatically changed and proteases are now seen as extremely important signalling molecules that are involved in numerous vital processes. Protease signalling pathways are strictly regulated, and the dysregulation of protease activity can lead to pathologies such as cardiovascular and inflammatory diseases, cancer, osteoporosis and neurological disorders. Several small-molecule drugs targeting proteases are already on the market and many more are in development. The status of human protease research and prospects for future protease-targeted drugs are reviewed here, with reference to some key examples where protease drugs have succeeded or failed.

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.

Oligomeric Structure and Substrate Induced Inhibition of Human Cathepsin C

Cathepsin C has been purified from human kidney by a modified procedure. Human cathepsin C was isolated as pure protein with a pI close to 6.0. The enzyme was shown to have a molecular mass of 200 kDa and to consist of four identical subunits, each composed of three different polypeptide chains, two of them disulfidebound. Their NH-terminal amino acid sequences were determined. Two chains showed pronounced similarity with the heavy and light chains of other papain-like cysteine proteinases, whereas the third one corresponded to the prosequence of the enzyme, thus showing that a substantial part of the proregion remains bound in the mature enzyme. The kinetics of substrate hydrolysis deviated substantially from standard Michaelis-Menten kinetics, demonstrating substrate inhibition at higher substrate concentrations. These data are explained by a sequential cooperative interaction model, where an enzyme molecule can bind up to four substrate molecules but where only the binary enzyme-substrate complex is catalytically active. Substrate inhibition was observed over the whole range of pH activity. From the pH activity profile it can be concluded that at least three ionizable groups with pK values 4.2, 6.8, and 7.7 are involved in substrate hydrolysis. Human cathepsin C thus appears to differ qualitatively from other cysteine proteinases of different origin.

Lysosomal cysteine proteases: structural features and their role in apoptosis.

Among the variety of proteolytic enzymes enormous progress has been seen recently in our understanding of lysosomal cysteine proteases, also known as cysteine cathepsins. These enzymes play a crucial role in diverse biological processes in physiological and pathological states, including genetic diseases. In the present review, their properties and structural features that are important to an understanding of their biological function are presented. Special emphasis is given to the newly discovered role of lysosomal cathepsins in apoptotic pathways. IUBMB Life, 57: 347‐353, 2005

Recombinant human procathepsin S is capable of autocatalytic processing at neutral pH in the presence of glycosaminoglycans

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 (∼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 t 1/2 of ∼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.

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 0.17u2003nm to 0.35u2003nm, whereas cathepsins S and H were inhibited with 100‐fold lower affinities (Kiu2003≈u200330u2003nm). 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.

Identification of bovine stefin A, a novel protein inhibitor of cysteine proteinases

For the first time, three different stefins, A, B and C, have been isolated from a single species. The complete amino acid sequence of bovine stefin A was determined. The inhibitor, with a calculated M r of 11,123, consists of 98 amino acid residues. Although it exhibits considerable similarity to human and rat stefin A, some significant differences in inhibition kinetics were found. Bovine stefin A bound tightly and rapidly to cathepsin L (k ass = 9.6·106 M −1·s −1, K i = 29 pM). The binding to cathepsin H was also rapid (k ass = 2.1·106 M −1·s −1), but weaker (K i = 0.4 nM) due to a higher dissociation rate constant. In contrast, the binding to cathepsin B was much slower (k ass = 1.4·105 M −1·s −1), but still tight (K i = 1.9 nM).

High-molecular-weight kininogen binds two molecules of cysteine proteinases with different rate constants

Fluorescence titrations showed that high‐molecular‐weight kininogen binds two molecules of papain, cruzipain and cathepsin S with high affinity. The 2:1 binding stoichiometry was confirmed by stopped‐flow kinetic measurements of papain binding, which also revealed that the two sites bind the enzyme with different association rate constants (k ass,1 = 23.0 × 106 M− s−1 and k ass2, = 3.4 × 106 M−1s−1. As for low‐molecular‐weight kininogen, comparison of these kinetic constants with previous data for intact low‐ and high‐molecular‐weight kininogen and the separated domains indicated that the faster‐binding site is also the tighter‐binding site and is that of domain 3, whereas the slower‐binding, lower‐affinity site is on domain 2. The results further demonstrate that there is no appreciable steric interference between the two domains or by the kininogen light chain in the binding of proteinases. Similarly, the binding of kininogen via its light chain to a surface, as indicated by the binding to the model surface, heparin, did not affect the inhibitory properties of kininogen. The M r of high‐molecular‐weight kininogen was determined to be 83 500 by sedimentation equilibrium measurements, in agreement with the value calculated from amino acid sequence and carbohydrate analysis.

Sensitization of stefin B-deficient thymocytes towards staurosporin-induced apoptosis is independent of cysteine cathepsins

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.

Recombinant human cathepsin X is a carboxymonopeptidase only: a comparison with cathepsins B and L.

Abstract 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.