Human Cysteine Cathepsins Research Articles

N-terminally truncated forms of human cathepsin F accumulate in aggresome-like inclusions

The contribution of individual cysteine cathepsins as positive mediators of programmed cell death is dependent on several factors, such as the type of stimuli, intensity and duration of the stimulus, and cell type involved. Of the eleven human cysteine cathepsins, cathepsin F is the only cathepsin that exhibits an extended N-terminal proregion, which contains a cystatin-like domain. We predicted that the wild-type human cathepsin F contains three natively disordered regions within the enzyme's propeptide and various amino acid stretches with high fibrillation propensity. Wild-type human cathepsin F and its N-terminally truncated forms, Ala20–Asp484 (Δ19CatF), Pro126–Asp484 (Δ125CatF), and Met147–Asp484 (Δ146CatF) were cloned into the pcDNA3 vector and overexpressed in HEK 293T cells. Wild-type human cathepsin F displayed a clear vesicular labeling and colocalized with the LAMP2 protein, a lysosomal marker. However, all three N-terminally truncated forms of human cathepsin F were recovered as insoluble proteins, suggesting that the deletion of at least the signal peptides (Δ19CatF), results in protein aggregation. Noteworthy, they concentrated large perinuclear–juxtanuclear aggregates that accumulated within aggresome-like inclusions. These inclusions showed p62-positive immunoreactivity and were colocalized with the autophagy marker LC3B, but not with the LAMP2 protein. In addition, an approximately 2–3 fold increase in DEVDase activity was not sufficient to induce apoptotic cell death. These results suggested the clearance of the N-terminally truncated forms of human cathepsin F via the autophagy pathway, underlying its protective and prosurvival mechanisms.

Cysteine cathepsin S processes leptin, inactivating its biological activity

Leptin is a 16 kDa hormone mainly produced by adipocytes that plays an important role in many biological events including the regulation of appetite and energy balance, atherosclerosis, osteogenesis, angiogenesis, the immune response, and inflammation. The search for proteolytic enzymes capable of processing leptin prompted us to investigate the action of cysteine cathepsins on human leptin degradation. In this study, we observed high cysteine peptidase expression and hydrolytic activity in white adipose tissue (WAT), which was capable of degrading leptin. Considering these results, we investigated whether recombinant human cysteine cathepsins B, K, L, and S were able to degrade human leptin. Mass spectrometry analysis revealed that among the tested enzymes, cathepsin S exhibited the highest catalytic activity on leptin. Furthermore, using a Matrigel assay, we observed that the leptin fragments generated by cathepsin S digestion did not exhibit angiogenic action on endothelial cells and were unable to inhibit food intake in Wistar rats after intracerebroventricular administration. Taken together, these results suggest that cysteine cathepsins may be putative leptin activity regulators in WAT.

Gene Targeting of the Cysteine Peptidase Cathepsin H Impairs Lung Surfactant in Mice

BackgroundThe 11 human cysteine cathepsins are proteases mainly located in the endolysosomal compartment of all cells and within the exocytosis pathways of some secretory cell types. Cathepsin H (Ctsh) has amino- and endopeptidase activities. In vitro studies have demonstrated Ctsh involvement in the processing and secretion of the pulmonary surfactant protein B (SP-B). Furthermore, Ctsh is highly expressed in the secretory organelles of alveolar type II pneumocytes where the surfactant proteins are processed.Methodology/Principal FindingsHence, we generated Ctsh null mice by gene targeting in embryonic stem cells to investigate the role of this protease in surfactant processing in vivo. The targeting construct contains a ß-galactosidase (lacZ) reporter enabling the visualisation of Ctsh expression sites. Ctsh-deficiency was verified by northern blot, western blot, and measurement of the Ctsh aminopeptidase activity. Ctsh −/− mice show no gross phenotype and their development is normal without growth retardation. Broncho-alveolar lavage (BAL) from Ctsh −/− mice contained lower levels of SP-B indicating reduced SP-B secretion. The BAL phospholipid concentration was not different in Ctsh+/+ and Ctsh −/− mice, but measurement of surface tension by pulsating bubble surfactometry revealed an impairment of the tension reducing function of lung surfactant of Ctsh −/− mice.Conclusions/SignificanceWe conclude that cathepsin H is involved in the SP-B production and reduced SP-B levels impair the physical properties of the lung surfactant. However, Ctsh defiency does not reproduce the severe phenotype of SP-B deficient mice. Hence, other proteases of the secretory pathway of type II pneumocytes, i.e. cathepsins C or E, are still able to produce surfactant of sufficient quality in absence of Ctsh.

Human Cysteine Cathepsins Are Not Reliable Markers of Infection by Pseudomonas aeruginosa in Cystic Fibrosis

Cysteine cathepsins have emerged as new players in inflammatory lung disorders. Their activities are dramatically increased in the sputum of cystic fibrosis (CF) patients, suggesting that they are involved in the pathophysiology of CF. We have characterized the cathepsins in CF expectorations and evaluated their use as markers of colonization by Pseudomonas aeruginosa. The concentrations of active cathepsins B, H, K, L and S were the same in P. aeruginosa-positive (19 Ps+) and P. aeruginosa-negative (6 Ps−) samples, unlike those of human neutrophil elastase. Also the cathepsin inhibitory potential and the cathepsins/cathepsin inhibitors imbalance remained unchanged and similar (∼2-fold) in the Ps+ and Ps− groups (p<0.001), which correlated with the breakdown of their circulating cystatin-like inhibitors (kininogens). Procathepsins, which may be activated autocatalytically, are a potential proteolytic reservoir. Immunoblotting and active-site labeling identified the double-chain cathepsin B, the major cathepsin in CF sputum, as the main molecular form in both Ps+ and Ps− samples, despite the possible release of the ∼31 kDa single-chain form from procathepsin B by sputum elastase. Thus, the hydrolytic activity of cysteine cathepsins was not correlated with bacterial colonization, indicating that cathepsins, unlike human neutrophil elastase, are not suitable markers of P. aeruginosa infection.

Regulation of Chemerin Chemoattractant and Antibacterial Activity by Human Cysteine Cathepsins

Chemerin, a ligand for the G-protein coupled receptor chemokine-like receptor 1, requires C-terminal proteolytic processing to unleash its chemoattractant activity. Proteolytically processed chemerin selectively attracts specific subsets of immunoregulatory APCs, including chemokine-like receptor 1-positive immature plasmacytoid dendritic cells (pDC). Chemerin is predicted to belong to the structural cathelicidin/cystatin family of proteins composed of antibacterial polypeptide cathelicidins and inhibitors of cysteine proteinases (cystatins). We therefore hypothesized that chemerin may interact directly with cysteine proteases, and that it might also function as an antibacterial agent. In this article, we show that chemerin does not inhibit human cysteine proteases, but rather is a new substrate for cathepsin (cat) K and L. cat K- and L-cleaved chemerin triggered robust migration of human blood-derived pDC ex vivo. Furthermore, cat K- and L-truncated chemerin also displayed antibacterial activity against Enterobacteriaceae. Cathepsins may therefore contribute to host defense by activating chemerin to directly inhibit bacterial growth and to recruit pDC to sites of infection.

Stefin A displaces the occluding loop of cathepsin B only by as much as required to bind to the active site cleft

Cathepsin B (EC 3.4.22.1) is one of the most versatile human cysteine cathepsins. It is important for intracellular protein degradation under normal conditions and is involved in a number of pathological processes. The occluding loop makes cathepsin B unique among cysteine cathepsins. This ∼ 20 residue long insertion imbedded into the papain-like protease scaffold restricts access to the active site cleft and endows cathepsin B with its carboxydipeptidase activity. Nevertheless, the enzyme also exhibits endopeptidase activity and is inhibited by stefins and cystatins. To clarify the structural properties of the occluding loop upon the binding of stefins, we determined the crystal structure of the complex between wild-type human stefin A and wild-type human cathepsin B at 2.6 Å resolution. The papain-like part of cathepsin B structure remains unmodified, whereas the occluding loop residues are displaced. The part enclosed by the disulfide bridge containing histidines 110 and 111 (i.e. the 'lasso' part) is rotated by ∼ 45° away from its original position. A comparison of the structure of the unliganded cathepsin B with the structure of the proenzyme, its complexes with chagasin and stefin A shows that the magnitude of the shift of the occluding loop is related to the size of the binding region. It is smallest in the procathepsin structures and increases in the series of complexes with stefin A and chagasin, although it has no impact on the binding constant. Hence, cathepsin B can dock inhibitors and certain substrates regardless of the size of the binding region.

Irreversible inhibition of human cathepsins B, L, S and K by hypervalent tellurium compounds

The inhibition of human cysteine cathepsins B, L, S and K was evaluated by a set of hypervalent tellurium compounds (telluranes) comprising both organic and inorganic derivatives. All telluranes studied showed a time- and concentration-dependent irreversible inhibition of the cathepsins, and their second-order inactivation rate constants were determined. The organic derivatives were potent inhibitors of the cathepsins and clear specificities were detected, which were parallel to their known substrate specificities. In all cases, the activity of the tellurane-inhibited cathepsins was recovered by treatment of the inactivated enzymes with reducing agents. The maximum stoichiometry of the reaction between cysteine residues and telluranes were also determined. The presented data indicate that it is possible to design organic compounds with a tellurium(IV) moiety as a novel warhead that covalently modifies the catalytic cysteine, and which also form strong interactions with subsites of cathepsins B, L, S and K, resulting in more specific inhibition.

Pharmacological inhibitors to identify roles of cathepsin K in cell-based studies: a comparison of available tools

Cathepsin K (Cat K) degrades bone type I collagen and is a target for the pharmacological treatment of osteoporosis. Further roles for Cat K have been recently described, some of which are supported by the use of purportedly selective Cat K inhibitors in human and rodent cell-based assays. Twelve commercial and non-commercial Cat K inhibitors were profiled against a panel of purified human, rat, and mouse cysteine cathepsins and in two cell-based enzyme occupancy assays for activity against Cat K, B, and L. Ten inhibitors, including the carbohydrazide Cat K inhibitor II (Boc-Phe-Leu-NHNH-CO-NHNH-Leu-Z), the non-covalent K4b, and the epoxide NC-2300, have either little Cat K selectivity, or appear poorly cell penetrant. The amino-acetonitrile-containing inhibitors L-873724 and odanacatib show greater than 100-fold human Cat K enzyme selectivity and have similar IC(50) values against each cathepsin in cell-based and enzyme assays. The basic inhibitor balicatib has greater cellular potencies than expected on the basis of purified enzyme assays. The accumulation of [(14)C]-balicatib in fibroblasts is blocked by prior treatment of the cells with NH(4)Cl, consistent with balicatib having lysosomotropic properties. These results support the use of L-873724 and odanacatib as tools to identify novel roles for Cat K using human cell-based systems, but suggest using caution in the interpretation of studies employing the other compounds.

The S2 subsites of cathepsins K and L and their contribution to collagen degradation

The exchange of residues 67 and 205 of the S2 pocket of human cysteine cathepsins K and L induces a permutation of their substrate specificity toward fluorogenic peptide substrates. While the cathepsin L-like cathepsin K (Tyr67Leu/Leu205Ala) mutant has a marked preference for Phe, the Leu67Tyr/Ala205Leu cathepsin L variant shows an effective cathepsin K-like preference for Leu and Pro. A similar turnaround of inhibition was observed by using specific inhibitors of cathepsin K [1-(N-Benzyloxycarbonyl-leucyl)-5-(N-Boc-phenylalanyl-leucyl)carbohydrazide] and cathepsin L [N-(4-biphenylacetyl)-S-methylcysteine-(D)-Arg-Phe-beta-phenethylamide]. Molecular modeling studies indicated that mutations alter the character of both S2 and S3 subsites, while docking calculations were consistent with kinetics data. The cathepsin K-like cathepsin L was unable to mimic the collagen-degrading activity of cathepsin K against collagens I and II, DQ-collagens I and IV, and elastin-Congo Red. In summary, double mutations of the S2 pocket of cathepsins K (Y67L/L205A) and L (L67Y/A205L) induce a switch of their enzymatic specificity toward small selective inhibitors and peptidyl substrates, confirming the key role of residues 67 and 205. However, mutations in the S2 subsite pocket of cathepsin L alone without engineering of binding sites to chondroitin sulfate are not sufficient to generate a cathepsin K-like collagenase, emphasizing the pivotal role of the complex formation between glycosaminoglycans and cathepsin K for its unique collagenolytic activity.

Interaction between Human Cathepsins K, L, and S and Elastins: MECHANISM OF ELASTINOLYSIS AND INHIBITION BY MACROMOLECULAR INHIBITORS

Proteolytic degradation of elastic fibers is associated with a broad spectrum of pathological conditions such as atherosclerosis and pulmonary emphysema. We have studied the interaction between elastins and human cysteine cathepsins K, L, and S, which are known to participate in elastinolytic activity in vivo. The enzymes showed distinctive preferences in degrading elastins from bovine neck ligament, aorta, and lung. Different susceptibility of these elastins to proteolysis was attributed to morphological differences observed by scanning electron microscopy. Kinetics of cathepsin binding to the insoluble substrate showed that the process occurs in two steps. The enzyme is initially adsorbed on the elastin surface in a nonproductive manner and then rearranges to form a catalytically competent complex. In contrast, soluble elastin is bound directly in a catalytically productive manner. Studies of enzyme partitioning between the phases showed that cathepsin K favors adsorption on elastin; cathepsin L prefers the aqueous environment, and cathepsin S is equally distributed among both phases. Our results suggest that elastinolysis by cysteine cathepsins proceeds in cycles of enzyme adsorption, binding of a susceptible peptide moiety, hydrolysis, and desorption. Alternatively, the enzyme may also form a new catalytic complex without prior desorption and re-adsorption. In both cases the active center of the enzymes remains at least partly accessible to inhibitors. Elastinolytic activity was readily abolished by cystatins, indicating that, unlike enzymes such as leukocyte elastase, pathological elastinolytic cysteine cathepsins might represent less problematic drug targets. In contrast, thyropins were relatively inefficient in preventing elastinolysis by cysteine cathepsins.

(2S,3S)-Oxirane-2,3-dicarboxylic acid: A privileged platform for probing human cysteine cathepsins

The notion that human cysteine cathepsins contribute only to general protein turnover within the lysosomes has changed in the last decade in a substantial manner. A continuously growing number of data accumulated in different fields of life sciences revealed that these enzymes are involved in a variety of pivotal physiological processes. To investigate these particular fraction of proteolytical activity of the human degradome even in a complex cellular environment, chemical probes that covalently label the corresponding proteases proved to be versatile tools. (2S,3S)-Oxirane-2,3-dicarboxylic acid provides an ideal platform for the design of such probing systems. Depending on the complexity of the attached recognition elements, either the activity of the entire group of human cysteine cathepsins or individual members can be detected.

Substrate Profiling of Cysteine Proteases Using a Combinatorial Peptide Library Identifies Functionally Unique Specificities

The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. A microtiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathepsins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially match the cleavage site sequences in their physiological substrates. Capitalizing on its unique preference for proline and glycine at the P2 and P3 positions, respectively, selective substrates and a substrate-based inhibitor were developed for cathepsin K. A cluster analysis of the proteases based on the complete specificity profile provided a functional characterization distinct from standard sequence analysis. This approach provides useful information for developing selective chemical probes to study protease-related pathologies and physiologies.

Comprehensive search for cysteine cathepsins in the human genome.

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.

Cathepsin expression during skeletal development

Cysteine proteinases, cathepsins B, H, K, L and S, have been implicated in several proteolytic processes during development, growth, remodeling and aging, as well as in a variety of pathological processes. For systematic analysis of cathepsin gene expression we have produced cDNA clones for mouse and human cysteine cathepsins. Northern analysis of a panel of total RNAs isolated from 16–19 different human and mouse tissues revealed the presence of mRNAs for cathepsin B, H, K, L and S in most tissues, but each with a distinct profile. Of the different cathepsin mRNAs, those for cathepsin K were clearly the highest in bone and cartilage. However, relatively high mRNA levels for the other cathepsins were also present in these tissues. To better understand the roles of different cathepsins during endochondral ossification in mouse long bones, cathepsin mRNAs were localized by in situ hybridization. Cathepsin K mRNAs were predominantly seen in multinucleated chondroclastic and osteoclastic cells at the osteochondral junction and on the surface of bone spicules. The other cathepsin mRNAs were also seen in osteoclasts, and in hypertrophic and proliferating chondrocytes. These observations were confirmed by immunohistochemistry and suggest that all cysteine cathepsins are involved in matrix degradation during endochondral ossification.