Computational predictions of cysteine cathepsin‐mediated fibrinogen proteolysis

Fibrin clot formation is a proteolytic cascade of events with thrombin and plasmin identified as the main proteases cleaving fibrinogen precursor, and the fibrin polymer, respectively. Other proteases may be involved directly in fibrin(ogen) cleavage, clot formation, and resolution, or in the degradation of fibrin‐based scaffolds emerging as useful tools for tissue engineered constructs. Here, cysteine cathepsins are investigated for their putative ability to hydrolyze fibrinogen, since they are potent proteases, first identified in lysosomal protein degradation and known to participate in extracellular proteolysis. To further explore this, we used two independent computational technqiues, molecular docking and bioinformatics sequence analysis (PACMANS), to predict potential binding interactions and sites of hydrolysis between cathepsins K, L, and S and fibrinogen. By comparing the results from these two objective, computational methods, it was determined that cathepsins K, L, and S do bind and cleave fibrinogen α, β, and γ chains at similar and unique sites. These differences were visualized experimentally by the unique cleaved fibrinogen banding patterns after incubation with each of the cathepsins, separately. In conclusion, human cysteine cathepsins K, L, and S are a new class of proteases that should be considered during fibrin(ogen) degradation studies both for disease processes where coagulation is a concern, and also in the implementation and design of bioengineered systems.     

Multiple sites on SARS‐CoV‐2 spike protein are susceptible to proteolysis by cathepsins B,K, L,S, and V

SARS‐CoV‐2 is the coronavirus responsible for the COVID‐19 pandemic. Proteases are central to the infection process of SARS‐CoV‐2. Cleavage of the spike protein on the virus''s capsid causes the conformational change that leads to membrane fusion and viral entry into the target cell. Since inhibition of one protease, even the dominant protease like TMPRSS2, may not be sufficient to block SARS‐CoV‐2 entry into cells, other proteases that may play an activating role and hydrolyze the spike protein must be identified. We identified amino acid sequences in all regions of spike protein, including the S1/S2 region critical for activation and viral entry, that are susceptible to cleavage by furin and cathepsins B, K, L, S, and V using PACMANS, a computational platform that identifies and ranks preferred sites of proteolytic cleavage on substrates, and verified with molecular docking analysis and immunoblotting to determine if binding of these proteases can occur on the spike protein that were identified as possible cleavage sites. Together, this study highlights cathepsins B, K, L, S, and V for consideration in SARS‐CoV‐2 infection and presents methodologies by which other proteases can be screened to determine a role in viral entry. This highlights additional proteases to be considered in COVID‐19 studies, particularly regarding exacerbated damage in inflammatory preconditions where these proteases are generally upregulated.     

A lead discovery strategy driven by a comprehensive analysis of proteases in the peptide substrate space

We present here a comprehensive analysis of proteases in the peptide substrate space and demonstrate its applicability for lead discovery. Aligned octapeptide substrates of 498 proteases taken from the MEROPS peptidase database were used for the in silico analysis. A multiple‐category naïve Bayes model, trained on the two‐dimensional chemical features of the substrates, was able to classify the substrates of 365 (73%) proteases and elucidate statistically significant chemical features for each of their specific substrate positions. The positional awareness of the method allows us to identify the most similar substrate positions between proteases. Our analysis reveals that proteases from different families, based on the traditional classification (aspartic, cysteine, serine, and metallo), could have substrates that differ at the cleavage site (P1–P1′) but are similar away from it. Caspase‐3 (cysteine protease) and granzyme B (serine protease) are previously known examples of cross‐family neighbors identified by this method. To assess whether peptide substrate similarity between unrelated proteases could reliably translate into the discovery of low molecular weight synthetic inhibitors, a lead discovery strategy was tested on two other cross‐family neighbors—namely cathepsin L2 and matrix metallo proteinase 9, and calpain 1 and pepsin A. For both these pairs, a naïve Bayes classifier model trained on inhibitors of one protease could successfully enrich those of its neighbor from a different family and vice versa, indicating that this approach could be prospectively applied to lead discovery for a novel protease target with no known synthetic inhibitors.     

Mechanism of inhibition of cathepsin K by potent, selective 1, 5-diacylcarbohydrazides: a new class of mechanism-based inhibitors of thiol proteases

The nature of the inhibition of thiol proteases by a new class of mechanism-based inhibitors, 1,5-diacylcarbohydrazides, is described. These potent, time-dependent, active-site spanning inhibitors include compounds that are selective for cathepsin K, a cysteine protease unique to osteoclasts. The 1,5-diacylcarbohydrazides are slow substrates for members of the papain superfamily with inhibition resulting from slow enzyme decarbamylation. Enzyme-catalyzed hydrolysis of 2,2'-N, N'-bis(benzyloxycarbonyl)-L- leucinylcarbohydrazide is accompanied by formation of a hydrazide-containing product and a carbamyl-enzyme intermediate that is sufficiently stable to be observed by mass spectrometry and NMR. Stopped-flow studies yield a saturation limited value of 43 s(-)(1) for the rate of cathepsin K acylation by 2,2'N, N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide. Inhibition potency varies among proteases tested as reflected by 2-3 orders of magnitude differences in K(i) and K(obs)/I, but all eventually form the same stable covalent intermediate. Reactivation rates are equivalent for all enzymes tested (1 x 10(-)(4) s(-)(1)), indicating hydrolysis of a common carbamyl-enzyme form. NMR spectroscopic studies with cathepsin K and 2,2'-N,N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide provide evidence of inhibitor cleavage to generate a covalent carbamyl-enzyme intermediate rather than a tetrahedral complex. The product Cbz-leu-hydrazide does not appear enzyme-bound after cleavage in the NMR spectra, suggesting that the stable inhibited form of the enzyme is the thioester complex. 1, 5-diacylcarbohydrazides represent a new class of unreactive cysteine protease inhibitors that share a common mechanism of action across members of the papain superfamily. Both S and S' subsite interactions are exploited in achieving high selectivity and potency.     

Selective inhibition of the collagenolytic activity of human cathepsin K by altering its S2 subsite specificity

The primary specificity of papain-like cysteine proteases (family C1, clan CA) is determined by S2-P2 interactions. Despite the high amino acid sequence identities and structural similarities between cathepsins K and L, only cathepsin K is capable of cleaving interstitial collagens in their triple helical domains. To investigate this specificity, we have engineered the S2 pocket of human cathepsin K into a cathepsin L-like subsite. Using combinatorial fluorogenic substrate libraries, the P1-P4 substrate specificity of the cathepsin K variant, Tyr67Leu/Leu205Ala, was determined and compared with those of cathepsins K and L. The introduction of the double mutation into the S2 subsite of cathepsin K rendered the unique S2 binding preference of the protease for proline and leucine residues into a cathepsin L-like preference for bulky aromatic residues. Homology modeling and docking calculations supported the experimental findings. The cathepsin L-like S2 specificity of the mutant protein and the integrity of its catalytic site were confirmed by kinetic analysis of synthetic di- and tripeptide substrates as well as pH stability and pH activity profile studies. The loss of the ability to accept proline in the S2 binding pocket by the mutant protease completely abolished the collagenolytic activity of cathepsin K whereas its overall gelatinolytic activity remained unaffected. These results indicate that Tyr67 and Leu205 play a key role in the binding of proline residues in the S2 pocket of cathepsin K and are required for its unique collagenase activity.     

Contribution of cathepsin L to secretome composition and cleavage pattern of mouse embryonic fibroblasts

The endolysosomal cysteine endoprotease cathepsin L is secreted from cells in a variety of pathological conditions such as cancer and arthritis. We compared the secretome composition and extracellular proteolytic cleavage events in cell supernatants of cathepsin L-deficient and wild-type mouse embryonic fibroblasts (MEFs). Quantitative proteomic comparison of cell conditioned media indicated that cathepsin L deficiency affects, albeit in a limited manner, the abundances of extracellular matrix (ECM) components, signaling proteins, and further proteases as well as endogenous protease inhibitors. Immunodetection corroborated that cathepsin L deficiency results in decreased abundance of the ECM protein periostin and elevated abundance of matrix metalloprotease (MMP)-2. While mRNA levels of MMP-2 were not affected by cathepsin L ablation, periostin mRNA levels were reduced, potentially indicating a downstream effect. To characterize cathepsin L contribution to extracellular proteolysis, we performed terminal amine isotopic labeling of substrates (TAILS), an N-terminomic technique for the identification and quantification of native and proteolytically generated protein N-termini. TAILS identified >1500 protein N-termini. Cathepsin L deficiency predominantly reduced the magnitude of collagenous cleavage sites C-terminal to a proline residue. This contradicts cathepsin L active site specificity and indicates altered activity of further proteases as a result of cathepsin L ablation.     

High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays

Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized (X=all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXabeta, factor XIa and factor alpha XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates x 24 different proteases x 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P2 preference for Leu, Val, Phe, and Tyr in both the P1=Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage.     

Cleavage site specificity of cathepsin K toward cartilage proteoglycans and protease complex formation

Cathepsin K is a potent extracellular matrix-degrading protease that requires interactions with soluble glycosaminolycans for its collagenolytic activity in bone and cartilage. The major sources of glycosaminoglycans in cartilage are aggrecan aggregates. Therefore, we investigated whether cathepsin K activity is capable to hydrolyze aggrecan into fragments allowing the formation of glycosaminoglycan-cathepsin K complexes and determined the cleavage site specificity of cathepsin K toward the cartilage-resident link protein and aggrecan. The cleavage site specificity was compared with those of cathepsins S and L. All three cathepsins released glycosaminoglycans from native bovine cartilage at lysosomal pH and to a lesser degree at neutral extracellular pH. Cathepsin-predigested aggrecan complexes and cartilage provided suitable glycosaminoglycan fragments that allowed the formation of collagenolytically active cathepsin K complexes. A detailed analysis of the degradation of aggrecan aggregates revealed two cathepsin K cleavage sites in the link protein and several sites in aggrecan, including one site within the interglobular domain E1. In summary, these results demonstrate that cathepsin K is capable to degrade aggrecan complexes at specific cleavage sites and that cathepsin K activity alone is sufficient to self-provide the glycosaminoglycan fragments required for the formation of its collagenolytically active complex.     

Regulation of collagenase activities of human cathepsins by glycosaminoglycans

Cathepsin K, a lysosomal papain-like cysteine protease, forms collagenolytically highly active complexes with chondroitin sulfate and represents the most potent mammalian collagenase. Here we demonstrate that complex formation with glycosaminoglycans (GAGs) is unique for cathepsin K among human papain-like cysteine proteases and that different GAGs compete for the binding to cathepsin K. GAGs predominantly expressed in bone and cartilage, such as chondroitin and keratan sulfates, enhance the collagenolytic activity of cathepsin K, whereas dermatan, heparan sulfate, and heparin selectively inhibit this activity. Moreover, GAGs potently inhibit the collagenase activity of other cysteine proteases such as cathepsins L and S at 37 degrees C. Along this line MMP1-generated collagen fragments in the presence of GAGs are stable against further degradation at 28 degrees C by all cathepsins but cathepsin K, whereas thermal destabilization at 37 degrees C renders the fragments accessible to all cathepsins. These results suggest a novel mechanism for the regulation of matrix protein degradation by GAGs. It further implies that cathepsin K represents the only lysosomal collagenolytic activity under physiologically relevant conditions.     

Localization of rat cathepsin K in osteoclasts and resorption pits: inhibition of bone resorption and cathepsin K-activity by peptidyl vinyl sulfones.

We have localized cathepsin K in rat osteoclasts and within exposed resorption pits by immuno-fluorescence microscopy. Intracellular staining using an antibody raised against recombinant mouse cathepsin K was vesicular and uniformly distributed throughout the cell. Confocal microscopy analysis did not reveal an accumulation of cathepsin K containing vesicles opposing the ruffled border and the resorption lacuna. Exposed resorption pits exhibited a uniform distribution of cathepsin K, and no differences were observed between the edges and the centers of the pits. The immunostaining of resorption pits with anti-cathepsin K antibodies demonstrates that the protease is secreted into the sub-osteoclastic compartment. Cathepsin K-specific inhibition using peptidyl vinyl sulfones as selective cysteine protease inactivators reduced bone resorption by 80% in a dose-dependent manner at sub-micromolar concentrations. No reduction of bone resorption was observed at those low concentrations using a potent cathepsin L, S, B-specific inhibitor. That the inhibition of bone resorption can be attributed to cathepsin K-like protease inhibition was corroborated by the selective inhibition of the osteoclastic Z-Gly-Pro-Arg-MbetaNA hydrolyzing activity by the cathepsin K, L, S, B-inhibitor, but not by the cathepsin L, B, and S inhibitor. Z-Gly-Pro-Arg-MbetaNA is efficiently hydrolyzed by cathepsin K but only poorly by cathepsins L, S, and B. On the contrary, the intracellular hydrolysis of the cathepsin B-specific substrate, Z-Arg-Arg-MbetaNA, was prevented by both types of inhibitors. The identification of cathepsin K in resorption pits and the inhibition of bone resorption and intracellular cathepsin K activity by selective vinyl sulfone inhibitors indicate the critical role of the protease in osteoclastic bone resorption.     

Toward computer-based cleavage site prediction of cysteine endopeptidases

Identification of relevant substrates is essential for elucidation of in vivo functions of peptidases. The recent availability of the complete genome sequences of many eukaryotic organisms holds the promise of identifying specific peptidase substrates by systematic proteome analyses in combination with computer-based screening of genome databases. Currently available proteomics and bioinformatics tools are not sufficient for reliable endopeptidase substrate predictions. To address these shortcomings the bioinformatics tool 'PEPS' (Prediction of Endopeptidase Substrates) has been developed and is presented here. PEPS uses individual rule-based endopeptidase cleavage site scoring matrices (CSSM). The efficiency of PEPS in predicting putative caspase 3, cathepsin B and cathepsin L cleavage sites is demonstrated in comparison to established algorithms. Mortalin, a member of the heat shock protein family HSP70, was identified by PEPS as a putative cathepsin L substrate. Comparative proteome analyses of cathepsin L-deficient and wild-type mouse fibroblasts showed that mortalin is enriched in the absence of cathepsin L. These results indicate that CSSM/PEPS can correctly predict relevant peptidase substrates.     

Quantifying cathepsin S activity in antigen presenting cells using a novel specific substrate

Cathepsin S (CatS) is a lysosomal cysteine protease belonging to the papain superfamily. Because of the relatively broad substrate specificity of this family, a specific substrate for CatS is not yet known. Based on a detailed study of the CatS endopeptidase specificity, using six series of internally quenched fluorescent peptides, we were able to design a specific substrate for CatS. The peptide series was based on the sequence GRWHTVGLRWE-Lys(Dnp)-DArg-NH2, which shows only one single cleavage site between Gly and Leu and where every substrate position between P-3 and P-3' was substituted with up to 15 different amino acids. The endopeptidase specificity of CatS was mainly determined by the P-2, P-1', and the P-3' substrate positions. Based on this result, systematically modified substrates were synthesized. Two of these modified substrates, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2 and Mca-GRWHPMGAPWE-Lys(Dnp)-DArg-NH2, did not react with the purified cysteine proteases cathepsin B (CatB) and cathepsin L (CatL). Using a specific CatS inhibitor, we could further show that these two peptides were not cleaved by endosomal fractions of antigen presenting cells (APCs), when CatS was inhibited and related cysteine proteases cathepsin B, H, L and X were still active. Although aspartic proteases like cathepsin E and cathepsin D were also present, our substrates were suitable to quantify cathepsin S activity specifically in APCs, including B cells, macrophages, and dendritic cells without the use of any protease inhibitor. We find that CatS activity differs significantly not only between the three types of professional APCs but also between endosomal and lysosomal compartments.     

Protease cleavage site fingerprinting by label‐free in‐gel degradomics reveals pH‐dependent specificity switch of legumain

Determination of protease specificity is of crucial importance for understanding protease function. We have developed the first gel‐based label‐free proteomic approach (DIPPS—direct in‐gel profiling of protease specificity) that enables quick and reliable determination of protease cleavage specificities under large variety of experimental conditions. The methodology is based on in‐gel digestion of the gel‐separated proteome with the studied protease, enrichment of cleaved peptides by gel extraction, and subsequent mass spectrometry analysis combined with a length‐limited unspecific database search. We applied the methodology to profile ten proteases ranging from highly specific (trypsin, endoproteinase GluC, caspase‐7, and legumain) to broadly specific (matrix‐metalloproteinase‐3, thermolysin, and cathepsins K, L, S, and V). Using DIPPS, we were able to perform specificity profiling of thermolysin at its optimal temperature of 75°C, which confirmed the applicability of the method to extreme experimental conditions. Moreover, DIPPS enabled the first global specificity profiling of legumain at pH as low as 4.0, which revealed a pH‐dependent change in the specificity of this protease, further supporting its broad applicability.     

Activity,Specificity, and Probe Design for the Smallpox Virus Protease K7L

The K7L gene product of the smallpox virus is a protease implicated in the maturation of viral proteins. K7L belongs to protease Clan CE, which includes distantly related cysteine proteases from eukaryotes, pathogenic bacteria, and viruses. Here, we describe its recombinant high level expression, biochemical mechanism, substrate preference, and regulation. Earlier studies inferred that the orthologous I7L vaccinia protease cleaves at an AG-X motif in six viral proteins. Our data for K7L suggest that the AG-X motif is necessary but not sufficient for optimal cleavage activity. Thus, K7L requires peptides extended into the P7 and P8 positions for efficient substrate cleavage. Catalytic activity of K7L is substantially enhanced by homodimerization, by the substrate protein P25K as well as by glycerol. RNA and DNA also enhance cleavage of the P25K protein but not of synthetic peptides, suggesting that nucleic acids augment the interaction of K7L with its protein substrate. Library-based peptide preference analyses enabled us to design an activity-based probe that covalently and selectively labels K7L in lysates of transfected and infected cells. Our study thus provides proof-of-concept for the design of inhibitors and probes that may contribute both to a better understanding of the role of K7L in the virus life cycle and the design of novel anti-virals.     

Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides.

The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S-transferase-fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S-transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time-dependent and occurred by an apparent one-step mechanism. The cathepsin K propeptide had a Ki of 3.6-6.3 nM for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240-fold selective inhibitor of cathepsin K (Ki = 0.27 nM) and cathepsin L (Ki = 0.12 nM) compared with cathepsin S (Ki = 65 nM). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nM) than cathepsin S (Ki = 7.6 nM) itself or cathepsin K (Ki = 7.0 nM). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J. E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745-750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained.     

Identification of tubulins as substrates of serine protease HtrA1 by mixture‐based oriented peptide library screening

Serine protease HtrA1 belongs to a family of chymotrypsin‐like proteases that were first identified in bacteria and later in mammalian systems. These proteases were identified as components of protein quality control in prokaryotic systems and as regulators of diverse signaling pathways in mammalian systems. In particular, HtrA1 is implicated in trophoblast cell migration and invasion, tumor progression, chemotherapy‐induced cytotoxicity, osteoarthritis, age‐related macular degeneration, and pathogenesis of Alzheimer's disease. However, systematic analysis of its potential substrates in biological system is still lacking. Therefore, we performed a mixture‐based oriented peptide library screening to identify putative substrates of HtrA1. We identified [AEGR]‐[LAGR]‐[IAMLR]‐[TVIAL] as consensus residues for P1 to P4 sites. We identified several putative substrates of HtrA1 involved in the pathogenesis of various diseases. In this study, we report on the identification of tubulins as potential substrates of HtrA1, and validated tubulins as in vitro and intracellular substrates of HtrA1. These results provide initial insights into substrate identification and functional characterization of HtrA1 in pathogenesis of various diseases. J. Cell. Biochem. 107: 253–263, 2009. © 2009 Wiley‐Liss, Inc.     

Why specific anti‐integrase antibodies from HIV‐infected patients can efficiently hydrolyze 21‐mer oligopeptide corresponding to antigenic determinant of human myelin basic protein

Human immunodeficiency virus‐infected patients possess anti‐integrase (IN) catalytic IgGs and IgMs (abzymes), which, unlike canonical proteases, specifically hydrolyze only intact globular IN. Anti‐myelin MBP abzymes from patients with multiple sclerosis and systemic lupus erythematosus efficiently hydrolyze only intact MBP. Anti‐MBP and anti‐IN abzymes do not hydrolyze several other tested control globular proteins. Here, we show that anti‐IN abzymes efficiently hydrolyze a 21‐mer oligopeptide (OP21) corresponding to one antigenic determinant (AGD) of MBP, whereas anti‐MBP abzymes extremely poorly cleave oligopeptides corresponding to AGDs of IN. All sites of IgG‐mediated and IgM‐mediated proteolysis of OP21 by anti‐IN abzymes were found for the first time by a combination of reverse phase and thin layer chromatography and mass spectrometry. Several clustered sites of OP21 cleavage were revealed and compared with the cleavage sites within the complete IN. Several fragments of OP21 had good homology with many fragments of the IN sequence. The active sites of anti‐IN abzymes are known to be located on their light chains, whereas heavy chains are responsible for the affinity for protein substrates. Interactions of intact IN with both light and heavy chains of the abzymes provide high affinity for IN and the specificity of its hydrolysis. Our data suggest that OP21 interacts mainly with the light chains of polyclonal anti‐IN abzymes, which possess lower affinity and specificity for substrate. The hydrolysis of the non‐cognate OP21 oligopeptide may be also less specific than the hydrolysis of the globular IN because in contrast to previously described serine protease‐like abzymes against different proteins, anti‐IN abzymes possess serine, thiol, acidic, and metal‐dependent protease activities. Copyright © 2013 John Wiley & Sons, Ltd.