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L- trans -Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L
Abstract
1. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) at a concentration of 0.5 mM had no effect on the serine proteinases plasma kallikrein and leucocyte elastase or the metalloproteinases thermolysin and clostridial collagenase. In contrast, 10 muM-E-64 rapidly inactivated the cysteine proteinases cathepsins B, H and L and papain (t0.5 = 0.1-17.3s). The streptococcal cysteine proteinase reacted much more slowly, and there was no irreversible inactivation of clostripain. The cysteine-dependent exopeptidase dipeptidyl peptidase I was very slowly inactivated by E-64. 2. the active-site-directed nature of the interaction of cathepsin B and papain with E-64 was established by protection of the enzyme in the presence of the reversible competitive inhibitor leupeptin and by the stereospecificity for inhibition by the L as opposed to the D compound. 3. It was shown that the rapid stoichiometric reaction of the cysteine proteinases related to papain can be used to determine the operational molarity of solutions of the enzymes and thus to calibrate rate assays. 4. The apparent second-order rate constants for the inactivation of human cathepsins B and H and rat cathepsin L by a series of structural analogues of E-64 are reported, and compared with those for some other active-site-directed inhibitors of cysteine proteinases. 5. L-trans-Epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475) was found to inhibit cathepsins B and L more rapidly than E-64. 6. Fumaryl-leucylamido(3-methyl)butane (Dc-11) was 100-fold less reactive than the corresponding epoxide, but was nevertheless about as effective as iodoacetate.
... [31] One of the most encountered reactive groups of mechanism based ABP targeting papain family of the cysteine proteases is the epoxide which has been shown to label this type of enzyme efficiently and selectively. [32][33][34][35][36][37][38][39][40][41][42] The epoxide forms a thioether bond, irreversibly modifying the active site ( Figure 1). [26,38,39,43,44] The origin of the epoxide "warhead" in the form of epoxysuccinates stems from the natural product E-64 (l-trans-epoxysuccinyl-leucylamido(4-guanidino)butane), which was initially isolated in 1978, from Aspergillus japonicus TPR-46 by Hanada and coworkers. ...
... Over the years, [48] various groups have demonstrated the ability of E-64 to inhibit a large number of cysteine proteases such as cathepsins B, [49] L, [33,50] H [33] S, [51] K, [52] F, [53] O, [54] V, [55] and X, [56] calpain I, [57,58] calpain II [59] and cruzain. [60] However, not all cysteine proteases are inhibited by E-64, and a very good example is represented by caspases. ...
... Over the years, [48] various groups have demonstrated the ability of E-64 to inhibit a large number of cysteine proteases such as cathepsins B, [49] L, [33,50] H [33] S, [51] K, [52] F, [53] O, [54] V, [55] and X, [56] calpain I, [57,58] calpain II [59] and cruzain. [60] However, not all cysteine proteases are inhibited by E-64, and a very good example is represented by caspases. ...
Synthetic chemical probes are powerful tools for investigating biological processes. They are particularly useful for proteomic studies such as activity‐based protein profiling (ABPP). These chemical methods initially used mimics of natural substrates. As the techniques gained prominence, more and more elaborate chemical probes with increased specificity towards given enzyme/protein families and amenability to various reaction conditions were used. Among the chemical probes, peptidyl‐epoxysuccinates represent one of the first types of compounds used to investigate the activity of the cysteine protease papain‐like family of enzymes. Structurally derived from the natural substrate, a wide body of inhibitors and activity‐ or affinity‐based probes bearing the electrophilic oxirane unit for covalent labeling of active enzymes now exists. Herein, we review the literature regarding the synthetic approaches to epoxysuccinate‐based chemical probes together with their reported applications, from biological chemistry and inhibition studies to supramolecular chemistry and the formation of protein arrays.
... Cruzipain was purified from Dm28c clone epimastigotes of T. cruzi [12]. Cysteine proteinases were titrated with E64 as described previously [13]. The activation buffers for enzyme assays were: 0.1 m phosphate buffer pH 6.0, containing 1 mm EDTA, 2 mm dl-dithiothreitol for cathepsins B and L, and 0.1 m phosphate buffer pH 6.0, containing 10 mm dl-dithiothreitol for cruzipain. ...
... Even though most of them, including cruzipain, preferentially accommodate Arg or Gly residues at position P1, the S1 subsite of papain-like proteinases may accommodate a variety of residues and therefore is of little interest in the development of specific substrates for individual enzymes. Cysteine proteinases differ more by their P2 specificity, and this feature has been exploited to develop substrates of improved specificity that differentiate between the activities of mammalian lysosomal cathepsins B and L [13]. Although recent data on the structures of papain and rat cathepsin B complex with irreversible inhibitors suggest that the active site of papain-like proteinases may extend up to the S3 subsite [28,31], little information is available about the P3 specificity of these enzymes or parasite proteinases. ...
The substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, was investigated using a series of dansyl-peptides based on the putative autoproteolytic sequence of the proteinase (VVG-GP) located at the hinge region between the catalytic domain and the C-terminal extension. Replacing Val with Pro at P2 in this sequence greatly improved the rate of cleavage by cruzipain. Tyr and Val residues are preferred at P3 by all cysteine proteinases whatever their origin, whereas only cruzipain and cathepsin L cleaved substrate with a His at that position. The combination of a Pro at P2 and His at P3 abolished cleavage by cathepsin L, so that only cruzipain was able to cleave the HPGGP peptide at the GG bond. A substrate with intramolecularly quenched fluorescence was raised on this sequence (Abz-HPGGPQ-EDDnp) which was also specifically cleaved by cruzipain (k cat /K m of 157 000 m ±1´s±1) and by a homologous proteinase from Trypanosoma congolense. The pH activity profile of cruzipain on Abz-HPGGPQ-EDDnp showed a narrow peak with a maximum at pH 5.5 and no cleavage above pH 6.8, although trypanosomal cysteine proteinases remain active at basic pH. The lack of activity at neutral and basic pH was due to a decrease in k cat , while the K m remained essentially unchanged, demonstrating that the substrate still binds to the enzyme and therefore behaves as an inhibitor. Changing the substrate into an inhibitor depended on the deprotonation of the His residue in the substrate, as deduced from a comparison of the pH activity profile with that of a related, but uncharged, substrate. Abz-HPGGPQ-EDDnp also inhibited mammalian cathepsins B and L but was not cleaved by these proteinases at any pH. The importance of the His residue at P3 for cleavage by cruzipain was confirmed by substituting Lys for His at that position. The resulting peptide was not cleaved by cruzipain in spite of the presence of a positively charged group at P3, but still interacted with the enzyme. It was concluded that the presence of an imidazolium group at P3 was essential to endow the HPGGPQ sequence with the properties of a cruzipain substrate. Keywords: cysteine proteinase; cruzipain; enzyme specificity. Targeting parasite enzymes that are involved in the infection of host cells, is a new and promising chemotherapeutic approach, that differs from classical pathways in drug discovery [1]. Protozoan and helminth parasites contain cysteine proteinases that participate in many aspects of parasite development and infection, and are thus appropriate models for use in the development of this strategy [2,3]. Parasite cysteine proteinases have properties similar to those of their mammalian homologues from the papain family [3,4], and their activity is generally measured using synthetic substrates developed for lysosomal cathepsins B and L [5,6]. So far, no specific substrate has been developed for parasite proteinases. The subsite specificities of parasite proteinases need to be determined so as to develop peptidyl susbtrates and inhibitors that specifically target their active sites. These probes could ultimately be used to develop antiparasitic drugs, provided that the activity of the cysteine proteinase is shown to be essential for parasite development and spreading. We recently demonstrated the important contribution of the S2 H subsite of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, in the design of substrates that are 50-fold more specific for cruzipain than for cathepsin L, and 200-fold more specific for the parasite enzyme than for cathepsin B [7]. These fluorogenic substrates were derived from the sequence of natural cysteine proteinase inhibitors belonging to the cystatin family. It has also been shown recently that positively charged residues are preferred by cruzipain at positions P5±P3 and P3 H ± P5 H , whereas a hydrophobic residue is required at the P2 position [8]. It has been suggested that positions P2 and P3 are of major importance in the design of selective inhibitors of cysteine proteinases, due to their critical positions at the enzyme surface [9]. This work investigated the unprime specificities of cruzipain and its related mammalian proteinases so as to identify the residues at positions P2 and P3 that are selective for the parasite proteinase. Pentapeptide sequences based on the putative autoproteolytic sequence of cruzipain (VVGGP), located at the hinge region between the catalytic domain and the C-terminal extension [10], were synthesized and labelled N-terminally using a dansyl group. They were then assayed for their suitability as substrates for cruzipain and its mammalian homologues.
... Compound E− 64 (Sigma-Aldrich), a classic inhibitor of cysteine peptidase, was tested against AsCathL following the assays shown in item 2.9. AsCathL molar concentration was determined by the titration of the enzyme active site by the E− 64 compound [26], (Supplementary Data Fig. 3). ...
... E− 64 is a potent and irreversible inhibitor of papain-like cysteine peptidase and has been used to identify and classify enzymes as cysteine peptidase [26,32]. This compound inhibited approximately 90% of AsCathL activity against Z-Phe-Arg-AMC substrate, thus confirming that AsCathL is a cysteine peptidase belonging to the papain family. ...
Cysteine peptidases are involved in physiological processes of insect development and have been considered as potential targets for the development of insect control strategies. In this study, we obtained a recombinant cysteine cathepsin L (AsCathL) from leaf-cutting ant (Atta sexdens), a species from the order Hymenoptera who causes enormous damage to crops, natural forests and reforested areas. RT-qPCR showed AsCathL expression throughout insect development and in all body parts of the adult insect analysed, suggesting its role as a lysosomal cathepsin. AsCathL encodes a protein of 320 amino acid residues consisting of a pro-peptide and the mature with amino acids sequence over 67% similarity with lysosomal cathepsin L of species from Lepidoptera and Diptera. Phylogenetic tree revealed that AsCathL is very similar to predicted cathepsins found in other ants. Recombinant AsCathL was expressed in insoluble form by Escherichia coli Arctic Express (DE3) RIL, purified under denaturing conditions and refolded. The enzyme showed hydrolytic activity in vitro towards synthetic substrate Z-Phe-Arg-AMC at acidic pH. Synthetic inhibitor E−64 acted against peptidase activity and a study regarding the interaction between E−64 and AsCathL using nuclear magnetic resonance (NMR) revealed that 83.18% from all E−64 molecules are irreversibly bound to AsCathL. In addition, the proteolytic activity of AsCathL was strongly inhibited by recombinant sugarcane cystatins with Ki ranging from 0.6 nM to 2.95 nM. To the best of our knowledge this is the first report characterizing a cysteine peptidase from leaf-cutting ants, which may contribute to future studies of ants’ cathepsins.
... Therefore, we sought to examine the effect of pharmacological inhibition of lysosomal cathepsins on the mannose-mediated upregulation of tight junction proteins. Among the cathepsin inhibitors, aloxistatin 34,35 , pepstatin A 36 , and nafamostat mesylate 37 have inhibitory effects on cysteine proteases (e.g., cathepsins B, K, and L), aspartic proteases (e.g., cathepsins D and E), and serine proteases (e.g., cathepsins A and G), respectively. Interestingly, only aloxistatin treatment cooperated with the mannose-mediated upregulation of tight junction protein expression in DSS-treated cells, whereas pepstatin A and nafamostat mesylate treatment did not (Supplementary Fig. 12a-d). ...
... The primary colonic epithelial cells of mice were purified as previously described 34 . Briefly, the colonic tissues were cut into pieces and trained at 37°C in Dulbecco's modified Eagle medium (DMEM) containing 5% FBS and 1 mM dithiothreitol (DTT) for 30 min. ...
Metabolite alteration has been associated with the pathogenesis of inflammatory bowel disease (IBD), including colitis. Mannose, a natural bioactive monosaccharide that is involved in metabolism and synthesis of glycoproteins, exhibits anti-inflammatory and anti-oxidative activities. We show here that the circulating level of mannose is increased in patients with IBD and mice with experimental colitis. Mannose treatment attenuates intestinal barrier damage in two mouse colitis models, dextran sodium sulfate (DSS)-induced colitis and spontaneous colitis in IL-10 -deficient mice. We demonstrate that mannose treatment enhanced lysosomal integrity and limited the release of cathepsin B, preventing mitochondrial dysfunction and myosin light chain kinase (MLCK)-induced tight junction disruption in the context of intestinal epithelial damage. Mannose exerts a synergistic therapeutic effect with mesalamine on mouse colitis. Cumulatively, the results indicate that mannose supplementation may be an optional approach to the treatment of colitis and other diseases associated with intestinal barrier dysfunction.
... Over 200 approved recombinant proteins including albumin, growth hormone and insulin among others, are on the FDA list [8]. The recombinant DNA technology had boosted enzyme industries in several means: (i) the use of fermentation to produce enzymes of animals and plants origin; (ii) the use of industrial organisms to produce enzymes from microorganisms that are hard to propagate; (iii) enhancing productivity of enzymes with the help of manifold copies of genes, competent signal sequences and very strong promoters; (iv) using a safe host to produce functional enzymes from [9][10][11][12][13][14]. Using recombinant DNA technology, currently, enzymes can be custom-made to meet the requirements of the users or of the process. ...
Papain is a proteolytic enzyme obtained from the fruits of carica papaya with abundant therapeutic, food, industrial and analytical applications. The challenges related to the development of papain technology on industrial scale include cost of production and downstream processing of the Original Research Article Makeri et al.; Asian J. 80 enzyme. In the present research, the responsible for gene encoding papain enzyme was obtained from the carica papaya plant, sequenced and amplified using polymerase chain reaction. This papain gene was initially converted to cDNA and then the papain gene was sequence to identify the region of similarity that may be a classify base on functional, structure, and phylogenic relationships between the known papain DNA sequence and the sample Papain gene. The papain gene was then clone into the pBR322 cloning vector to create recombinant protein molecules. This showed that the DNA sequencing of the extracted genes exhibited an acceptable level of similarity to the corresponding gene (MEROO647) with identity of 98% and E-value 0.246 X10-9 from the public database NCBI (National Centre for Biotechnology information) In contrast, the similar papain genes sequence (P00784, MEROO647, and AT3G5470) in NCBI extracted from other sources in this study, the papaya gene sequences was obtained from carica papaya (fruit) and in the sequence similarity analysis because the product was amplified from mRNA that was extracted from carica papaya fruit.
... Two assays were used to measure the proteolytic activities of CCP1 in the presence of NIa-Pro. In all these assays, a specific chemical inhibitor of PLCPs, E-64 was used as a positive control [62,68]. ...
Papain-like cysteine proteases (PLCPs) play pivotal roles in plant defense against pathogen invasions. While pathogens can secrete effectors to target and inhibit PLCP activities, the roles of PLCPs in plant-virus interactions and the mechanisms through which viruses neutralize PLCP activities remain largely uncharted. Here, we demonstrate that the expression and activity of a maize PLCP CCP1 (Corn Cysteine Protease), is upregulated following sugarcane mosaic virus (SCMV) infection. Transient silencing of CCP1 led to a reduction in PLCP activities, thereby promoting SCMV infection in maize. Furthermore, the knockdown of CCP1 resulted in diminished salicylic acid (SA) levels and suppressed expression of SA-responsive pathogenesis-related genes. This suggests that CCP1 plays a role in modulating the SA signaling pathway. Interestingly, NIa-Pro, the primary protease of SCMV, was found to interact with CCP1, subsequently inhibiting its protease activity. A specific motif within NIa-Pro termed the inhibitor motif was identified as essential for its interaction with CCP1 and the suppression of its activity. We have also discovered that the key amino acids responsible for the interaction between NIa-Pro and CCP1 are crucial for the virulence of SCMV. In conclusion, our findings offer compelling evidence that SCMV undermines maize defense mechanisms through the interaction of NIa-Pro with CCP1. Together, these findings shed a new light on the mechanism(s) controlling the arms races between virus and plant.
... Inhibition of another lysosomal cysteine protease, Cathepsin S has been reported to reverse TGFβ -induced epithelial-mesenchymal transition, restore the turnover of TJ proteins and prevent invasive growth in glioblastoma cells (Wei, et al., 2021). In retinal pigment epithelial cells, To investigate if the depletion of claudin-1, claudin-4 and occludin in cells expressing Map was mediated by the lysosomal cathepsins, we treated the cells with E-64, an irreversible inhibitor of cathepsin B, H, and L (Barrett, et al., 1982) or CA-074 methyl ester (CA-074Me), a cellpermeable analog of CA-074 which irreversibly inhibits intracellular cathepsin B (Buttle, et al., 1992). Stable cell lines expressing either the AcGFP vector alone, AcGFP-Tir or AcGFP-Map were grown in individual wells of a 6 well plate until fully confluent and treated with E-64 (30µM) or CA-074Me (20µM) for 18 hours after which the total cell lysates were prepared and analyzed by western blotting (Figure 1). ...
Infections by Enteropathogenic E. coli (EPEC) cause acute diarrheal disease in infants accounting for severe morbidity and mortality. One of the underlying causes of the disease is the break-down of the intestinal barrier maintained by the tight junctions (TJs). EPEC uses a type 3 secretion system to translocate more than twenty effectors into infected cells which disrupt several functions of the host cells. The effectors EspF, Map, EspG1/G2 and NleA have been reported to disrupt the TJs and cause the leakage of charged ions and uncharged molecules through the barrier. We have reported earlier that EspF and Map cause the depletion of TJ proteins claudin-1, claudin-4 and occludin through both transcriptional and post-transcriptional mechanisms. Here, we show that the EPEC effector Map modulates the lysosomal protease, cathepsin B to deplete claudins and occludin. Expression of mutant Map proteins that lacked the mitochondrial targeting sequence (MTS) completely restored the total levels of occludin and its localization at the TJs and partially restored claudin-4 levels and its junctional localization. We also identified a novel interaction of Map with the GTPase Rab13. As Rab13 has been reported to mediate the recycling of occludin to the plasma membrane, its interaction with Map has important implications for the loss of TJ integrity in EPEC pathogenesis. Occludin regulates the passage of water and uncharged solutes through TJs and Map may block its recycling to compromise the TJs thus causing excessive leakage through the barrier.
... Beside CatL inhibition, E-64 is known to have a broad specificity for inhibiting other cysteine proteases. 21 In addition to a different specificity of E-64 and related succinyl-epoxide inhibitors, the IC50 values previously reported might be one obvious reason for a much higher anti-viral activity of CI-XII compared to E-64 or CLIK148 in the fluorescence-based assay. ...
Emerging RNA viruses including SARS-CoV-2 continue to be a major threat around the globe. The cell entry of SARS-CoV-2 particles via the endosomal pathway involves the cysteine protease cathepsin L (CatL) among other proteases. CatL is rendered as a promising drug target in the context of different viral and lysosome-related diseases. Hence, drug discovery and structure-based optimization of inhibitors is of high pharmaceutical interest. We herein verified and compared the anti-SARS-CoV-2 activity of a set of carbonyl and succinyl-epoxide-based inhibitors, which have previously been identified as cathepsin inhibitors. Calpain inhibitor XII (CI-XII), MG-101 and CatL inhibitor IV (CLI-IV) possess antiviral activity in the very low nanomolar IC50 range in Vero E6 cells. Experimental structural data on how these and related compounds bind to CatL are however notably lacking, despite their therapeutic potential. Consequently, we present and compare crystal structures of CatL in complex with 14 compounds, namely BOCA (N-BOC-2-aminoacetaldehyde), CLI-IV, CI-III, CI-VI, CI-XII, the main protease alpha-ketoamide inhibitor 13b, MG-101, MG-132 as well as E-64d (aloxistatin), E-64, CLIK148, CAA0225, TC-I (CID 16725315) and TPCK at resolutions better than 2 Angstrom. Overall, the presented data comprise a broad and solid basis for structure-guided understanding and optimization of CatL inhibitors towards protease drug development.
... Proteasome inhibition rescues TNF-induced degradation of BIRC2 and BIRC3. To further interrogate the role of protein degradation in regulating BIRC3 and 2 expression, A549 cells were incubated with MG-132, MG-262 or PR-171, which all inhibit the activity of the proteolytic 20S core of the 26S proteasome [52][53][54], and the cysteine proteinase inhibitor, E-64 [55]. Western blotting of whole A549 cell extracts for K48 ubiquitin revealed striking increases in signal following IL1B or TNF treatments in the presence of the three proteasome inhibitors, but not E-64 (S8A Fig in S1 File). ...
Roles for the baculoviral inhibitor of apoptosis repeat-containing (BIRC) genes, BIRC2 and BIRC3, may include signaling to the inflammatory transcription factor, nuclear factor-κB (NF-κB) and protection from cell death. However, distinct functions for each BIRC are not well-delineated. Given roles for the epithelium in barrier function and host defence, BIRC2 and BIRC3 expression was characterized in pulmonary epithelial cell lines and primary human bronchial epithelial cells (pHBECs) grown as undifferentiated cells in submersion culture (SC) or as highly differentiated cells at air-liquid interface (ALI). In A549 cells, interleukin-1β (IL1B) and tumor necrosis factor α (TNF) induced BIRC3 mRNA (~20-50-fold), with maximal protein expression from 6-24 h. Similar effects occurred in BEAS-2B and Calu-3 cells, as well as SC and ALI pHBECs. BIRC2 protein was readily detected in unstimulated cells, but was not markedly modulated by IL1B or TNF. Glucocorticoids (dexamethasone, budesonide) modestly increased BIRC3 mRNA and protein, but showed little effect on BIRC2 expression. In A549 cells, BIRC3 mRNA induced by IL1B was unchanged by glucocorticoids and showed supra-additivity with TNF-plus-glucocorticoid. Supra-additivity was also evident for IL1B-plus-budesonide induced-BIRC3 in SC and ALI pHBECs. Using A549 cells, IL1B- and TNF-induced BIRC3 expression, and to a lesser extent, BIRC2, was prevented by NF-κB inhibition. Glucocorticoid-induced BIRC3 expression was prevented by silencing and antagonism of the glucocorticoid receptor. Whereas TNF, but not IL1B, induced degradation of basal BIRC2 and BIRC3 protein, IL1B- and TNF-induced BIRC3 protein remained stable. Differential regulation by cytokines and glucocorticoids shows BIRC2 protein expression to be consistent with roles in rapid signaling events, whereas cytokine-induced BIRC3 may be more important in later effects. While TNF-induced degradation of both BIRCs may restrict their activity, cytokine-enhanced BIRC3 expression could prime for its function. Finally, shielding from glucocorticoid repression, or further enhancement by glucocorticoid, may indicate a key protective role for BIRC3.
... 44 Activation of the enzymes was optimized following the protocols established earlier in our laboratory. 45 The total concentration of the active enzymes was subsequently measured by active-site titration with the irreversible inhibitor, E-64, which binds in a 1:1 molecular ratio at the active sites of most cysteine proteases, 46 while Bradford assay was used for finding out the concentration of the inhibitors. 43 An initial set of protease−substrate reactions yielded the K m (Michaelis constant) and V max values of each enzyme, which were calculated by nonlinear fitting of the progress curves, following Michaelis−Menten kinetics, on Graphpad Prism 6.0 (http://www.graphpad.com/prism). ...
The emergence of resistance in Plasmodium falciparum to frontline artemisinin-based combination therapies has raised global concerns and emphasized the identification of new drug targets for malaria. Cysteine protease falcipain-2 (FP2), involved in host hemoglobin degradation and instrumental in parasite survival, has long been proposed as a promising malarial drug target. However, designing active-site-targeted small-molecule inhibitors of FP2 becomes challenging due to their off-target specificity toward highly homologous human cysteine cathepsins. The use of proteinaceous inhibitors, which have nonconserved exosite interactions and larger interface area, can effectively circumvent this problem. In this study, we report for the first time that human stefin-A (STFA) efficiently inhibits FP2 with Ki values in the nanomolar range. The FP2-STFA complex crystal structure, determined in this study, and sequence analyses identify a unique nonconserved exosite interaction, compared to human cathepsins. Designing a mutation Lys68 > Arg in STFA amplifies its selectivity garnering a 3.3-fold lower Ki value against FP2, and the crystal structure of the FP2-STFAK68R complex shows stronger electrostatic interaction between side-chains of Arg68 (STFAK68R) and Asp109 (FP2). Comparative structural analyses and molecular dynamics (MD) simulation studies of the complexes further confirm higher buried surface areas, better interaction energies for FP2-STFAK68R, and consistency of the newly developed electrostatic interaction (STFA-R68-FP2-D109) in the MD trajectory. The STFA-K68R mutant also shows higher Ki values against human cathepsin-L and stefin, a step toward eliminating off-target specificity. Hence, this work underlines the design of host-based proteinaceous inhibitors against FP2, with further optimization to render them more potent and selective.
... Details of the materials and methods employed are provided in the Supplemental Material online (see references [17,36,[44][45][46][47][48][49][50][51][52][53]). ...
Background:
Chronic obstructive pulmonary disease (COPD) is an irreversible disease mainly caused by smoking. COPD is characterized by emphysema and chronic bronchitis associated with enhanced epithelial permeability.
Hypothesis:
Lung biopsies from smokers revealed a decreased expression level of occludin, which is a protein involved in the cohesion of epithelial tight junctions. Moreover, the occludin level correlated negatively with smoking history (pack-years), COPD grades, and cathepsin S (CatS) activity. Thus, we examined whether CatS could participate in the modulation of the integrity of human lung epithelial barriers.
Methods and results:
Cigarette smoke extract (CSE) triggered the upregulation of CatS by THP-1 macrophages through the mTOR/TFEB signaling pathway. In a co-culture model, following the exposure of macrophages to CSE, an enhanced level of permeability of lung epithelial (16HBE and NHBE) cells towards FITC-Dextran was observed, which was associated with a decrease in occludin level. Similar results were obtained using 16HBE and NHBE cells cultured at the air-liquid interface. The treatment of THP-1 macrophages by CatS siRNAs or by a pharmacological inhibitor restored the barrier function of epithelial cells, suggesting that cigarette smoke-elicited CatS induced an alteration of epithelial integrity via the proteolytic injury of occludin.
Conclusions:
Alongside its noteworthy resistance to oxidative stress induced by cigarette smoke oxidants and its deleterious elastin-degrading potency, CatS may also have a detrimental effect on the barrier function of epithelial cells through the cleavage of occludin. The obtained data emphasize the emerging role of CatS in smoking-related lung diseases and strengthen the relevance of targeting CatS in the treatment of emphysema and COPD.
... Fluorescence release was monitored using a Spectramax Gemini spectrofluorometer (Molecular Devices, Saint Grégoire, France). The active concentration of CatS was determined by titration with E-64, a broad-spectrum inhibitor of cysteine cathepsins [2]. ...
Supplementary files (Bigot et al, 2022, antioxidants)
... CTSZ/X contains the domain of peptidase-C1, also belongs to cysteine peptidase family C1, subfamily C1A, exhibits carboxymonopeptidase and carboxydipeptidase activities, and is involved in maintaining homeostasis and immune cell function [56,57] (https://smart.embl.de/smart/, accessed on 30 December 2021). ...
Treatment of glioblastoma (GBM) remains very challenging, and it is particularly important to find sensitive and specific molecular targets. In this work, we reveal the relationship between the expression of cathepsins and radioresistance in GBM. We analyzed cathepsins (cathepsin B, cathepsin D, cathepsin L, and cathepsin Z/X), which are highly associated with the radioresistance of GBM by regulating different types of cell death. Cathepsins could be potential targets for GBM treatment.
... The antibiotic E-64 is a thiol protease inhibitor, and its producer is Aspergillus japonicus TPR-64, which contains (2R,3R)-oxirane-2,3-dicarboxylic acid (282) [437,438] and a lipopeptide called cystargamide has been identified in the fermentation broth of the actinomycete Kitasatospora cystarginea and contains 3-heptyloxirane-2-carboxylic acid (283) [439]. ...
The study of lipopeptides and their related compounds produced by various living organisms from bacteria to marine invertebrates is of fundamental interest for medicinal chemistry, pharmacology, and practical clinical medicine. Using the principles of retrosynthetic analysis of linear and cyclic peptides, the pharmacological activity of unique, unusual, and rare fatty acids (FA) that are part of natural lipopeptides was investigated. To search for new biologically active natural metabolites from natural sources, more than 350 FA incorporated into linear and cyclic peptides isolated from bacteria, cyanobacteria, microalgae, marine invertebrates, fungal endophytes, and microorganisms isolated from sediments are presented. Biological activities have been studied experimentally in various laboratories, as well as data obtained using QSAR (Quantitative Structure-Activity Relationships) algorithms. According to the data obtained, several FA were identified that demonstrated strong antibacterial, antimicrobial, antifungal, or antitumor activity. Along with this, FA have been found that have shown rare properties such as antiviral, antidiabetic, anti-helmintic, anti-inflammatory, anti-psoriatic, anti-ischemic, and anti-infective activities. In addition, FA have been found as potential regulators of lipid metabolism, as well as agents for the treatment of acute neurological disorders, as well as in the treatment of atherosclerosis and multiple sclerosis. For 36 FA, 3D graphs are presented, which demonstrate their predicted and calculated activities.
... The protease activity of CTSH could be effectively inhibited by E64, a compound first isolated from the fungus Aspergillus violaceus (Barrett et al., 1981(Barrett et al., , 1982. We treated the chemosensitive (F) Bar graph shows the relative expression levels of Ctsh in the sensitive, chemo32, chemo34, and resistant samples, measured by bulk RNA-seq analysis (n = 3 mice). ...
Cisplatin-based chemotherapy remains the primary treatment for unresectable and metastatic muscle-invasive bladder cancers (MIBCs). However, tumors frequently develop chemoresistance. Here, we established a primary and orthotopic MIBC mouse model with gene-edited organoids to recapitulate the full course of chemotherapy in patients. We found that partial squamous differentiation, called semi-squamatization, is associated with acquired chemoresistance in both mice and human MIBCs. Multi-omics analyses showed that cathepsin H (CTSH) is correlated with chemoresistance and semi-squamatization. Cathepsin inhibition by E64 treatment induces full squamous differentiation and pyroptosis, and thus specifically restrains chemoresistant MIBCs. Mechanistically, E64 treatment activates the tumor necrosis factor pathway, which is required for the terminal differentiation and pyroptosis of chemoresistant MIBC cells. Our study revealed that semi-squamatization is a type of lineage plasticity associated with chemoresistance, suggesting that differentiation via targeting of CTSH is a potential therapeutic strategy for the treatment of chemoresistant MIBCs.
... As the S1 0 pocket of both cathepsins V and L is nonspecific, compound 25 hardly exerts selectivity via its structural features. Moreover, data obtained from the dilution assay suggest irreversible binding of compound 25 to cathepsin V, in accordance with its epoxy warhead, similar to E-64, which forms a covalent bond with the thiol group of the active site of cysteine [61]. ...
Cathepsin V is a human lysosomal cysteine peptidase with specific functions during pathological processes and is as such a promising therapeutic target. Peptidase inhibitors represent powerful pharmacological tools for regulating excessive proteolytic activity in various diseases. Cathepsin V is highly related to cathepsin L but differs in tissue distribution, binding site morphology, substrate specificity, and function. To validate its therapeutic potential and extend the number of potent and selective cathepsin V inhibitors, we used virtual high-throughput screening of commercially available compound libraries followed by an evaluation of kinetic properties to identify novel potent and selective cathepsin V inhibitors. We identified the ureido methylpiperidine carboxylate derivative, compound 7, as a reversible, selective, and potent inhibitor of cathepsin V. It also exhibited the most preferable characteristics for further evaluation with in vitro functional assays that simulate the processes in which cathepsin V is known to play an important role. Compound 7 exerted significant effects on cell proliferation, elastin degradation, and immune cell cytotoxicity. The latter was increased because compound 7 impaired conversion of immunosuppressive factor cystatin F to its active monomeric form. Taken together, our results present novel potent inhibitors of cathepsin V and provide new hit compounds for detailed development and optimization. Further, we demonstrate that cathepsin V is a potential target for new approaches to cancer therapy.
... As a positive inhibitor control, each screening plate included CA-074 (1 μM final concentration), a known selective inhibitor of cathepsin B; 21,22 CA-074 is a natural product originally isolated from Aspergillus japonicus. 25 It is noted that the Z-Glu-Lys-AMC substrate at pH 4.6 and the Z-Arg-Lys-AMC substrate at pH 7.2 had similar K m values of 460 and 429 μM, respectively. These similar K m values of both substrates at a concentration of 60 μM in the screening assays represent equivalent substrate conditions. ...
Dysregulation of cathepsin B, which involves the translocation of the enzyme from acidic pH lysosomes to the neutral pH cytosol, followed by the initiation of cell death and inflammation, occurs in numerous brain disorders. The wide difference in the acidic pH (4.6) of lysosomes compared to the neutral pH (7.2) of the cytosol suggests that screening at different pH conditions may identify pH-selective modulators of cathepsin B. Therefore, a collection of pure marine and plant natural product (NP) compounds, with synthetic compounds, was screened at pH 4.6 and pH 7.2 in cathepsin B assays, which led to the identification of GER-12 (Crossbyanol B) and GER-24 ((7Z,9Z,12Z)-octadeca-7,9,12-trien-5-ynoic acid) marine NP inhibitors at acidic pH but not at neutral pH. GER-12 was effective for the reversible inhibition of cathepsin B, with an IC50 of 3 μM. GER-24 had an IC50 of 16 μM and was found to be an irreversible inhibitor. These results show that NP screening at distinct biological pH conditions can lead to the identification of pH-selective cathepsin B modulators. These findings suggest that screening efforts for molecular probes and drug discovery may consider the biological pH environment of the target in the disease process.
... Nevertheless, further studies are required to analyse the influence of metal ions on the enzymatic catalysis capacity of cathepsin L. The enzymatic activity of rTsCatL2 was significantly inhibited by E64 and PMSF and enhanced by EDTA, L-cysteine and DTT. E64 is a specific inhibitor of cysteine proteinases, PMSF inhibits serine protease and sulfhydryl protease activities, DTT is a reducing agent that prevents cross-linking of disulfide bonds, and L-cysteine is a sulfhydryl activator, which further confirms that rTsCatL2 is a sulfhydryl-containing cysteine protease [70]. Since pH is important in promoting the denaturation of various protein substrates to unfold their structures and make them more readily hydrolysed, we analysed the degradation of natural substrate proteins by rTsCatL2 at different pH values [67,71]. ...
Cathepsin L is an important cysteine protease, but its function in T. spiralis remains unclear. The aim of this research was to explore the biological characteristics of T. spiralis cathepsin L (TsCatL) and its role in T. spiralis -host interactions. Bioinformatic analysis revealed the presence of the cysteine protease active site residues Gln, Cys, His and Asn in mature TsCatL, as well as specific motifs of cathepsin L similar to ERFNIN and GYLND in the prepeptide of TsCatL. Molecular docking of mature TsCatL and E64 revealed hydrophobic effects and hydrogen bonding interactions. Two domains of TsCatL (TsCatL2) were cloned and expressed, and recombinant TsCatL2 (rTsCatL2) was autocatalytically cleaved under acidic conditions to form mature TsCatL. TsCatL was transcribed and expressed in larvae and adults and located in the stichosome, gut and embryo. Enzyme kinetic tests showed that rTsCatL2 degraded the substrate Z-Phe-Arg-AMC under acidic conditions, which was inhibited by E64 and PMSF and enhanced by EDTA, L-cysteine and DTT. The kinetic parameters of rTsCatL2 were a Km value of 48.82 μM and Vmax of 374.4 nM/min at pH 4.5, 37 °C and 5 mM DTT. In addition, it was shown that rTsCatL2 degraded haemoglobin, serum albumin, immunoglobulins (mouse IgG, human IgG and IgM) and extracellular matrix components (fibronectin, collagen I and laminin). The proteolytic activity of rTsCatL2 was host specific and significantly inhibited by E64. rTsCatL2 possesses the natural activity of a sulfhydryl-containing cysteine protease, and TsCatL is an important digestive enzyme that seems to be important for the nutrient acquisition, immune evasion and invasion of Trichinella in the host.
... E-64d是一种cathepsin非选择性共价抑制剂, 对cathepsin L/B均有抑制作用且毒副作用较低 [190] . 研究表 明, cathepsin抑制剂E-64d可与TMPRSS-2抑制剂卡莫 ...
Since coronavirus disease 2019 (COVID-19) was reported in late 2019, its causative agent, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been spreading worldwide for more than two years, and the trend continues unabated. SARS-CoV-2 is a single positive-stranded RNA virus from the same β-coronavirus family as SARS-CoV, which caused a severe acute respiratory syndrome (SARS) epidemic in 2003. Entry into the cell is mediated by the binding of the spike glycoprotein on the envelope to angiotensin converting enzyme 2 (ACE2) on the host cell, possibly with the help of co-receptors, adhesion factors, or alternative receptors. During the process of virus assembly, release, cell adhesion, and entry into the host cell, the SARS-CoV-2 spike protein must undergo a series of structural changes, including cleavage and conformational changes, to achieve membrane fusion function. After binding to cell surface receptors, SARS-CoV-2 has two invasion pathways: (i) The direct fusion pathway at the cell membrane interface, and (ii) the clathrin-mediated endocytosis pathway. In both cases, the SARS-CoV-2 spike protein must be cleaved by furin-like proteins at the S1/S2 site. Host proteases, mainly TMPRSS-2 or cathepsin, are involved in cleavage at the S2′ site, corresponding to pathway (i) or (ii). The entire entry process ends when the viral genome enters the cytosol. During the replication cycle of SARS-CoV-2, genomic RNA can be mutated. Accumulation of mutations, particularly in the spike protein, may result in new variants that differ in infectivity, transmissibility, virulence, and/or immune escape capability. These changes can alter the prevailing lineage in circulation, trigger a new wave of public health events, and threaten scientific research, clinical prevention, and treatment. The World Health Organization (WHO) has identified five mutant strains as variants of concern (VOCs) by assessing their public health significance worldwide: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529). The Omicron variant is currently in circulation. The entry-related characteristics of VOCs may indicate global epidemiological trends and shed light on future pandemic dynamics. The entry mechanism of SARS-CoV-2, the first and crucial step in the establishment of a viral infection, has been extensively studied over the past two years. As the molecular mechanisms involved in entry are gradually being elucidated, interventions in key events may prevent viral entry and provide information for drug development and clinical treatment. Because the cellular entry mechanism of coronaviruses is relatively conserved, the first step in interrupting viral infection —blocking the entry process—is a promising target for the development of new preventive and therapeutic drugs against COVID-19 and even pan-coronavirus inhibitors, including small molecules, peptides, and monoclonal antibodies. Researchers have identified potential drug targets involved in the SARS-CoV-2 entry process, such as spike proteins, ACE2, and host proteases. Inhibition of the viral entry process is expected to reduce viral load in patients, thereby alleviating symptoms and improving prognosis. Recently, computer-aided drug design has emerged as a powerful and productive tool for drug discovery, in addition to experimental methods in the wet lab. In future anti-SARS-CoV-2 drug discovery, the flexible combination and optimized design of drug discovery strategies to improve the efficiency of drug development will be the trend. In this review, we outline the mechanism of SARS-CoV-2 entry into cells, describe the entry properties of VOCs currently defined by the WHO, and present the progress in the discovery of SARS-CoV-2 entry inhibitors.
... These findings indicate changes in the composition of peptidases during egg maturation. In order to identify which peptidase classes contribute to the global proteolytic activity in F. hepatica eggs, the protein extracts were treated with five class-specific peptidase inhibitors which selectively target metallopeptidases (EDTA) 54 , cysteine (l-trans-epoxysuccinyl-leucylamido(4-guanidino)butane, E-64) 55 , serine 56 , aspartic peptidases (pepstatin) 57 , and threonine catalytic subunits of proteasome (benzyloxycarbonyl-l-leucyl-l-leucyl-leucinal, MG132) 58 . Reactions were performed at three different pH levels (3.5, 6, 8.5), which were selected according to the previously established pH optima of proteolytic activities of the egg extracts. ...
Fasciola hepatica is a global parasite of livestock which also causes a neglected zoonosis in humans. The parasite’s communication with the host during its complicated lifecycle is based on an ingenious enzymatic apparatus which includes a variety of peptidases. These enzymes are implicated in parasite migration, pathogenesis of the disease, and modification of host immune response. Although the dynamics of proteolytic machinery produced by intra-mammalian F. hepatica life stages has been previously investigated in great detail, peptidases of the eggs so far received little scientific attention. In this study, we performed a comparative RNA-seq analysis aimed at identification of peptidases expressed in F. hepatica eggs, cultured at 37 °C to represent gall bladder retained eggs, for different time periods and employed mass spectrometry in order to identify and quantify peptidases translated in F. hepatica egg lysates. We demonstrated that F. hepatica eggs undergo significant molecular changes when cultured at the physiological temperature of the definitive host. Egg transcriptome is subject to numerous subtle changes while their proteome is even more variable. The peptidase profile is considerably modified on both transcriptome and proteome level. Finally, we measured and classified proteolytic activities in extracts from F. hepatica eggs using a library of fluorogenic substrates and peptidase class-selective inhibitors. Activities of threonine peptidases were detected constantly, while the cysteine peptidases prevailing in freshly laid eggs are substituted by aspartic peptidase and metallopeptidase activities in the later stages of egg development.
... Recombinant human cathepsin L and benzyloxycarbonyl-Phe-Arg-7amino-4-methyl coumarin (Z-Phe-Arg-AMC) were from R&D Systems (Minneapolis, USA). Active site titration of cathepsin L was performed using the titrating agent, trans-epoxysuccinyl-L-leucylamido(4guanidino)butane (E-64), an irreversible inhibitor of cysteine proteases (Sigma-Aldrich, Saint-Quentin Fallavier, France) according to the method described elsewhere (Barrett et al., 1982). Heparinases I, II, III, Δ-4,5-glycuronidase, and chondroitinase B, all from Flavobacterium heparinum were purchased from Grampenz (Upper Ardoe, Aberdeen, Scotland). ...
Mucopolysaccharidoses (MPS) are a group of rare lysosomal storage diseases characterized by glycosaminoglycan (GAG) accumulation causing progressive multi-organs dysfunction and ultimately severe cardio-respiratory damages. Human cystatin C (hCC), a potent inhibitor of cysteine cathepsins, plays an important role in respiratory diseases. However, its regulation remained unknown in MPS. Herein, elevated hCC levels were measured in respiratory specimens from MPS-I, -II, and -III patients and were significantly correlated with severe respiratory symptoms (rs = 0.7173). Heparan sulfate (HS), a prominent GAG, dampened its inhibitory activity toward cathepsin L in a dose-dependent manner. HS and HS-oligosaccharides bound tightly hCC, in combination with a secondary structure rearrangement. Molecular modeling studies identified three HS binding regions in hCC, including the N-terminus, which is crucial in the inhibition of cathepsins. Impairment of inhibitory potential of hCC may reflect abnormal regulation of proteolytic activity of cathepsin L in lung, ultimately contributing to the severity of MPS.
... E-64 is a potent chemical inhibitor of papain-like proteases (Barrett et al. 1982) and prevents AtSerpin1 complex formation with RD21 in vivo (Lampl et al. 2010). The structural features of the protease active site are consistent with the striking and exclusive affinity for E-64. ...
Protease inhibitors of the serpin family are ubiquitous in the plant kingdom but relatively little is known about their biological functions in comparison with their counterparts in animals. X-ray crystal structures have provided crucial insights into animal serpin functions. The recently solved structure of AtSerpin1 from Arabidopsis thaliana, which has the highly conserved reactive center P2-P1 Leu-Arg-Xaa (Xaa = small residue), displays both conserved and plant-specific serpin features. Sequence homology suggests that AtSerpin1 belongs to serpin Clade B, composed of intracellular mammalian serpins, which is consistent with the lack of strong evidence for secretion of serpins from plant cells. The major in vivo target protease for AtSerpin1 is the papain-like cysteine RD21 protease, a match reminiscent of the inhibition of cathepsins K, L and S by the Clade-B mammalian serpin, SCCA-1 (SERPINB3). The function of AtSerpin1 and other serpins that contain P2-P1 Leu-Arg-Xaa (the 'LR' serpins) in plants remains unknown. However, based on its homol-ogy and interactive partners, AtSerpin1 and perhaps other serpins are likely to be involved in regulating programmed cell death or associated processes such as senescence. Abundant accumulation of serpins in seeds and their presence in phloem sap suggest additional functions in plant defense by irreversible inhibition of digestive proteases from pests or pathogens. Here we review the most recent findings in plant serpin biology, focusing on advances in describing the structure and inhibitory specificity of the LR serpins.
... Z-VAD-FMK or caspase inhibitor I or E64 or Q-VD-OPhH at two different concentrations (25 or 50 µM) for 1 h. These compounds were selected as they shown to inhibit caspase-3, other caspases and cysteine proteases.[32][33][34][35] Cells were treated with the Rh-VAD-FMK probe for 1 h, followed by the addition of coelenterazine h. ...
Non-invasive, real-time, longitudinal imaging of protein functions in living systems with unprecedented specificity is one of the critical challenges of modern biomedical research. Despite several advancements, it is estimated that nearly 35% of the human proteome is not completely characterized. Therefore, the development of new technologies is imperative for shining more light on so-called “dark proteomes”. Towards that goal, here we report a platform fusion technology called activity-based protein profiling-bioluminescence resonance energy transfer (ABPP-BRET). This method provides an opportunity to study the post-translational modification of a target protein in real-time in living systems in a longitudinal manner with a high spatio-temporal resolution. This semi-synthetic BRET biosensor method is used for target engagement studies and further for inhibitor profiling in live cells. The simplicity of this method coupled with the critical physical distance dependent BRET read-out turned out to be a powerful method, thus pushing the activity-based protein profiling technology to the next level.
... E-64, an epoxysuccinyl derivative is known to irreversibly inhibit the cysteine proteases (structure shown in Figure S4). [56] Pro-Cathepsin C was also activated by incubating with CatL. However, a muchreduced amount (1 : 100) of CatL was used to avoid CatL interference. ...
Defects in autophagy are implicated in many age‐related diseases that cause neurodegeneration including both Alzheimer's and Parkinson's. Within autophagy, the lysosome plays a crucial role by enabling the breakdown and recycling of a wide range of biomolecular species. Herein, the effects of isomerization of aspartic acid (Asp) on substrate recognition and degradation are investigated for a collection of lysosomal cathepsins using liquid chromatography coupled to mass spectrometry. By examining a series of synthetic peptides with sequences derived from long‐lived proteins known to undergo Asp isomerization, we demonstrate that isomerized forms of Asp significantly perturb cathepsin activity by impeding digestion and shifting preferential sites of proteolysis. Although the sensitivity to isomerization varies for each cathepsin, none of the cathepsins were capable of digesting sites within several residues of the C‐terminal side of the isomerized Asp. Under physiological conditions, the peptide fragments left behind after such incomplete digestion would not be suitable substrates for transporter recognition and could precipitate autophagic malfunction in the form of lysosomal storage.
... These male mice were also tested with a dose of 2.5 lg/5 lL of ficin combined with 50 lM of E-64, a highly selective cysteine protease inhibitor. 21,22 Behavioural measurements After spicule application or injection, we measured two site-directed behaviours: (i) the number of itch-like scratching bouts with the hind paw, and (ii) the number of pain-like wipes with the forepaw as described. 20 No mice were excluded from the study after testing. ...
Background:
Ficin, a cysteine protease derived from fig-tree latex, has been reported to elicit itch and nociceptive sensations, though quantitative sensory studies are lacking. Cowhage containing the pruritic cysteine Mucunain, on the other hand, has been widely studied as activating polymodal nociceptors and eliciting a histamine-independent itch.
Objectives:
We tested whether ficin in heat-inactivated cowhage spicules would elicit itch and nociceptive sensations in humans, and analogous behaviors in mice, that are similar to those evoked by native cowhage and, whether these behaviors in mice were dose-dependent when ficin was injected intradermally.
Methods:
Human volunteers rated the magnitude of itch and nociceptive sensations evoked by either native cowhage spicules or heat-inactivated spicules soaked in 1, 10 or 100 mg/ml ficin (0.03, 0.3, and 3 ng of ficin in spicule tip), applied to forearm. In mice, itch-like scratching and nociceptive-like wiping were recorded in response to either native cowhage, to heat-inactivated spicules that were either inactive or contained 100 mg/ml ficin, or to intradermal injections of 1.25, 2.5, or 5 μg/ 5 μl, each treatment applied to the cheek.
Results:
The dose of 100 mg/ml ficin in spicules evoked comparable magnitudes of itch, nociceptive sensations and areas of cutaneous dysesthesia as native cowhage in humans and comparable itch-like scratching and pain-like wiping behaviors in mice. But to elicit similar behaviors when injected intradermally in mice a greater amount of ficin (1.25 μg) was required.
Conclusion:
Spicule-delivery or intradermal injection of ficin elicits behaviors in mice that model itch and nociceptive sensations in humans, suggesting that ficin may be useful in translating mechanistic research on the neural mechanisms of pruritic and nociceptive effects of cysteine proteases between the two species.
... Auto-lysosomal degradation is another important pathway for removal of CFTR after targeting by ER-associated autophagy or endocytic removal CFTR from the plasma membrane (Okiyoneda et al., 2010;He et al., 2021). We used the cathepsin protease inhibitor E-64 to broadly block lysosomal protein degradation (Barrett et al., 1982). E-64 treatment did not result in any accumulation of F508del or P67L band B or band C (Figure 6E-F, Supplemental Figure S9A-D), confirming that autolysosomal degradation is not critical for the clearance of these variants under corrector treatment. ...
Pharmacological chaperones represent a class of therapeutic compounds for treating protein misfolding diseases. One of the most prominent examples is the FDA-approved pharmacological chaperone lumacaftor (VX-809), which has transformed cystic fibrosis (CF) therapy. CF is a fatal disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). VX-809 corrects folding of F508del CFTR, the most common patient mutation, yet F508del exhibits only mild VX-809 response. In contrast, rarer mutations P67L and L206W are hyper-responsive to VX-809, while G85E is non-responsive. Despite the clinical success of VX-809, the mechanistic origin for the distinct susceptibility of mutants remains unclear. Here, we use interactomics to characterize the impact of VX-809 on proteostasis interactions of P67L and L206W and compare these to F508del and G85E. We determine hyper-responsive mutations P67L and L206W exhibit decreased interactions with proteasomal, and autophagy degradation machinery compared to F508del and G85E. We then show inhibiting the proteasome attenuates P67L and L206W VX-809 response. Our data suggests a previously unidentified but required role for protein degradation in VX-809 correction. Furthermore, we present an approach for identifying proteostasis characteristics of mutant-specific therapeutic response to pharmacological chaperones.
... The fast-binding inhibitor G€ u2602 showed linear progress curves, and the apparent inhibition constant K i 0 was determined by non-linear regression using the Morrison equation for tight binding inhibition 58 with GraphPad Prism software. The concentration of mCatK was determined by active site titration as described previously 59 with E-64 used as the titrant 60 . The final concentration of DMSO in the assay systems did not exceed 1.5%. ...
Cathepsin K (CatK) is a target for the treatment of osteoporosis, arthritis, and bone metastasis. Peptidomimetics with a cyanohydrazide warhead represent a new class of highly potent CatK inhibitors; however, their binding mechanism is unknown. We investigated two model cyanohydrazide inhibitors with differently positioned warheads: an azadipeptide nitrile Gü1303 and a 3-cyano-3-aza-β-amino acid Gü2602. Crystal structures of their covalent complexes were determined with mature CatK as well as a zymogen-like activation intermediate of CatK. Binding mode analysis, together with quantum chemical calculations, revealed that the extraordinary picomolar potency of Gü2602 is entropically favoured by its conformational flexibility at the nonprimed-primed subsites boundary. Furthermore, we demonstrated by live cell imaging that cyanohydrazides effectively target mature CatK in osteosarcoma cells. Cyanohydrazides also suppressed the maturation of CatK by inhibiting the autoactivation of the CatK zymogen. Our results provide structural insights for the rational design of cyanohydrazide inhibitors of CatK as potential drugs.
... inhibit cathepsin B with nanomolar potency (20,21) and selectivity for inhibition of cathepsin B compared to the related cysteine cathepsins L, H, and S (20)(21)(22). These inhibitory properties of CA-074 used purified rat cathepsin B activity monitored at pH 5.5 with the fluorogenic substrate Z-Arg-Arg-AMC (21). ...
CA-074 is a selective inhibitor of cathepsin B, a lysosomal cysteine protease. CA-074 has been utilized in numerous studies to demonstrate the role of this protease in cellular and physiological functions. Cathepsin B in numerous human disease mechanisms involves its translocation from acidic lysosomes of pH 4.6 to neutral pH 7.2 of cellular locations, including the cytosol and extracellular environment. To gain in-depth knowledge of CA-074 inhibition under these different pH conditions, this study evaluated the molecular features, potency, and selectivity of CA-074 for cathepsin B inhibition under acidic and neutral pH conditions. This study demonstrated that CA-074 is most effective at inhibiting cathepsin B at an acidic pH of 4.6 with nM potency, which was more than 100-fold more potent than its inhibition at a neutral pH of 7.2. The pH-dependent inhibition of CA-074 was abolished by methylation of its C-terminal proline, indicating the requirement for the free C-terminal carboxyl group for pH-dependent inhibition. Under these acidic and neutral pH conditions, CA-074 maintained its specificity for cathepsin B over other cysteine cathepsins, displayed irreversible inhibition, and inhibited diverse cleavages of peptide substrates of cathepsin B assessed by profiling mass spectrometry. Molecular docking suggested that pH-dependent ionic interactions of the C-terminal carboxylate of CA-074 occur with His110 and His111 residues in the S2' subsite of the enzyme at pH 4.6, but these interactions differ at pH 7.2. While high levels of CA-074 or CA-074Me (converted by cellular esterases to CA-074) are used in biological studies to inhibit cathepsin B at both acidic and neutral pH locations, it is possible that adjusted levels of CA-074 or CA-074Me may be explored to differentially affect cathepsin B activity at these different pH values. Overall, the results of this study demonstrate the molecular, kinetic, and protease specificity features of CA-074 pH-dependent inhibition of cathepsin B.
... These results indicate a good interaction between NatProDB and SARS-CoV-2 Mpro residues, mainly on the active site. Additionally, the "top 10" NatProDB compounds presented better energy when compared to a positive control (E-64), which was a well-known cysteine protease inhibitor (562th better energy, Table 1) [65,66]. ...
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has received global attention due to the serious threat it poses to public health. Since the outbreak in December 2019, millions of people have been affected and its rapid global spread has led to an upsurge in the search for treatment. To discover hit compounds that can be used alone or in combination with repositioned drugs, we first analyzed the pharmacokinetic and toxicological properties of natural products from Brazil’s semiarid region. After, we analyzed the site prediction and druggability of the SARS-CoV-2 main protease (Mpro), followed by docking and molecular dynamics simulation. The best SARS-CoV-2 Mpro complexes revealed that other sites were accessed, confirming that our approach could be employed as a suitable starting protocol for ligand prioritization, reinforcing the importance of catalytic cysteine-histidine residues and providing new structural data that could increase the antiviral development mainly against SARS-CoV-2. Here, we selected 10 molecules that could be in vitro assayed in response to COVID-19. Two compounds (b01 and b02) suggest a better potential for interaction with SARS-CoV-2 Mpro and could be further studied.
... These results indicate that Sl-CathL-mutSC shows the expected hydrolytic activity of cysteine cathepsin L [37]. To confirm cysteine protease activity, we tested Sl-CathL-mutSC with E-64 and PMSF, specific cysteine and serine proteases inhibitors, respectively [38,39]. As expected, E-64 was able to inhibit Sl-CathL-mutSC ( Figure 3D), while the addition of PMSF caused no inhibition. ...
The Sphenophorus levis (Coleoptera, Curculionidae) is one of the main pests of sugarcane in Brazil. Although its major digestive proteases are known, its complex digestive process still needs to be further understood. We constructed a transcriptome from the midgut of 30-day-old larvae and identified sequences similar to its major digestive protease (cysteine cathepsin Sl-CathL), however, they presented a different amino acid than cysteine in the active cleft. We identified, recombinantly produced, and characterized Sl-CathL-CS, a pseudo cysteine protease, and verified that higher gene expression levels of Sl-CathL-CS occur in the midgut of 30-day old larvae. We reverted the serine residue to cysteine and compared the activity of the mutant (Sl-CathL-mutSC) with Sl-CathL-CS. Sl-CathL-CS presented no protease activity, but Sl-CathL-mutSC hydrolyzed Z-Phe-Arg-AMC (Vmax = 1017.60 ± 135.55, Km = 10.77 mM) and was inhibited by a cysteine protease inhibitor E-64 (Ki = 38.52 ± 1.20 μM), but not by the serine protease inhibitor PMSF. Additionally, Sl-CathL-CS interacted with a sugarcane cystatin, while Sl-CathL-mutSC presented weaker interaction. Finally, protein ligand docking reinforced the differences in the catalytic sites of native and mutant proteins. These results indicate that Sl-CathL-CS is a pseudo-cysteine protease that assists protein digestion possibly by interacting with canecystatins, allowing the true proteases to work.
The oxygen-labile transcription factor called hypoxia-inducible factor (HIF) is responsible for the cellular and organismal adaptive response to reduced oxygen availability. Deregulation of HIF is associated with the pathogenesis of major human diseases including cardiovascular disease and cancer. Under normoxia, the HIFα subunit is hydroxylated on conserved proline residues within the oxygen-dependent degradation domain (ODD) that labels HIFα for proteasome-mediated degradation. Despite similar oxygen-dependent degradation machinery acting on HIF1α and HIF2α, these two paralogs have been shown to exhibit unique kinetics under hypoxia, which suggests that other regulatory processes may be at play. Here, we characterize the protease activity found in rabbit reticulocytes that specifically cleaves the ODD of HIF1α but not HIF2α. Notably, the cleavage product is observed irrespective of the oxygen-dependent prolyl-hydroxylation potential of HIF1α, suggesting independence from oxygen. HIF1α M561T substitution, which mimics an evolutionary substitution that occurred during the duplication and divergence of HIF1α and HIF2α, diminished the cleavage of HIF1α. Protease inhibitor screening suggests that cysteine proteases cathepsins L and B preferentially cleave HIF1αODD, thereby revealing an additional layer of differential HIF regulation.
Emerging RNA viruses, including SARS-CoV-2, continue to be a major threat. Cell entry of SARS-CoV-2 particles via the endosomal pathway involves cysteine cathepsins. Due to ubiquitous expression, cathepsin L (CatL) is considered a promising drug target in the context of different viral and lysosome-related diseases. We characterized the anti-SARS-CoV-2 activity of a set of carbonyl- and succinyl epoxide-based inhibitors, which were previously identified as inhibitors of cathepsins or related cysteine proteases. Calpain inhibitor XII, MG-101, and CatL inhibitor IV possess antiviral activity in the very low nanomolar EC50 range in Vero E6 cells and inhibit CatL in the picomolar Ki range. We show a relevant off-target effect of CatL inhibition by the coronavirus main protease α-ketoamide inhibitor 13b. Crystal structures of CatL in complex with 14 compounds at resolutions better than 2 Å present a solid basis for structure-guided understanding and optimization of CatL inhibitors toward protease drug development.
Tetrahymena thermophila is a promising host for recombinant protein production, but its utilization in biotechnology is mostly limited due to the presence of intracellular and extracellular papain-family cysteine proteases (PFCPs). In this study, we employed bioinformatics approaches to investigate the T. thermophila PFCP genes and their encoded proteases (TtPFCPs), the most prominent protease family in the genome. Results from the multiple sequence alignment, protein modeling, and conserved motif analyses revealed that all TtPFCPs showed considerably high homology with mammalian cysteine cathepsins and contained conserved amino acid motifs. The total of 121 TtPFCP-encoding genes, 14 of which were classified as non-peptidase homologs, were found. Remaining 107 true TtPFCPs were divided into four distinct subgroups depending on their homology with mammalian lysosomal cathepsins: cathepsin L-like (TtCATLs), cathepsin B-like (TtCATBs), cathepsin C-like (TtCATCs), and cathepsin X-like (TtCATXs) PFCPs. The majority of true TtPFCPs (96 out of the total) were in TtCATL-like peptidase subgroup. Both phylogenetic and chromosomal localization analyses of TtPFCPs supported the hypothesis that TtPFCPs likely evolved through tandem gene duplication events and predominantly accumulated on micronuclear chromosome 5. Additionally, more than half of the identified TtPFCP genes are expressed in considerably low quantities compared to the rest of the TtPFCP genes, which are expressed at a higher level. However, their expression patterns fluctuate based on the stage of the life cycle. In conclusion, this study provides the first comprehensive in-silico analysis of TtPFCP genes and encoded proteases. The results would help designing an effective strategy for protease knockout mutant cell lines to discover biological function and to improve the recombinant protein production in T. thermophila.
Cathepsin L plays physiological and pathological roles in immune responses, cancer, metamorphosis, and oogenesis in several species. However, the function of Cathepsin L in medaka ovaries remains unclear. Therefore, here, we examined the physiological functions of Cathepsin L in the medaka ovaries. Cathepsin L mRNA transcripts and proteins were found to be constitutively expressed in the ovaries of Oryzias latipes over a 24-h spawning cycle. Expression was localized within the oocyte cytoplasm of growing follicles and the follicle layer of preovulatory and postovulatory follicles. Moreover, the active form of Cathepsin L was highly expressed in the follicle layer of periovulatory follicles and the ovaries 2–6 h after ovulation. Recombinant Cathepsin L was activated under acidic conditions and exhibited enzymatic activity in acidic and neutral pH conditions. However, extracellular matrix proteins were degraded by recombinant Cathepsin L under acidic, not neutral pH conditions. Cathepsin L was secreted from preovulatory follicles, while active recombinant Cathepsin L was detected in the conditioned medium of a medaka cell line, OLHNI-2. Mechanistically, recombinant Cathepsin L activates recombinant urokinase-type plasminogen activator-1, which is expressed within the follicle layers post-ovulation. Meanwhile, the treatment of medakas with an E-64 or anti-Cathepsin L antibody effectively blocked follicular layer degeneration and degradation after ovulation, whereas in vitro ovulation was not inhibited by either. Collectively, the findings of this study indicate that although Cathepsin L does not impact ovulation in medakas, it contributes to the degeneration and degradation of the follicle layers following ovulation via activation of urokinase-type plasminogen activator-1, and not via the degradation of extracellular matrix proteins.
Identification of novel drugs for anti-African swine fever (ASF) applications is of utmost urgency, as it negatively affects pig farming and no effective vaccine or treatment is currently available. African swine fever virus (ASFV) encoded pS273R is a cysteine protease that plays an important role in virus replication. E64, acting as an inhibitor of cysteine protease, has been established as exerting an inhibitory effect on pS273R. In order to obtain a better understanding of the interaction between E64 and pS273R, common docking, restriction docking, and covalent docking were employed to analyze the optimal bonding position between pS273R-E64 and its bonding strength. Additionally, three sets of 100 ns molecular dynamics simulations were conducted to examine the conformational dynamics of pS273R and the dynamic interaction of pS273R-E64, based on a variety of analytical methods including root mean square deviation (RMSD), root mean square fluctuation (RMSF), free energy of ligand (FEL), principal component analysis (PCA), and molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) analysis. The results show that E64 and pS273R exhibited close binding degrees at the activity center of ASFV pS273R protease. The data of these simulations indicate that binding of E64 to pS273R results in a reduction in flexibility, particularly in the ARM region, and a change in the conformational space of pS273R. Additionally, the ability of E64 to interact with polar amino acids such as ASN158, SER192, and GLN229, as well as charged amino acids such as LYS167 and HIS168, seems to be an important factor in its inhibitory effect. Finally, Octet biostratigraphy confirmed the binding of E64 and pS273R with a KD value of 903 uM. Overall, these findings could potentially be utilized in the development of novel inhibitors of pS273R to address the challenges posed by ASFV.
Prostate-specific Ag (PSA), which is expressed in a majority of prostate cancers, is a potential target for specific immunotherapy. Previous studies have shown that two 10-mer PSA peptides (designated PSA-1 and PSA-3) selected to conform to human HLA class I-A2 motifs can elicit CTL responses in vitro. A longer PSA peptide (30-mer) designated PSA-OP (oligoepitope peptide), which contains both the PSA-1 and PSA-3 HLA-A2 epitopes and an additional potential CTL epitope (designated PSA-9) for the HLA-class I-A3 allele, was investigated for the ability to induce cytotoxic T cell activity. T cell lines from different HLA-A2 and HLA-A3 donors were established by in vitro stimulation with PSA-OP; the CTL lines lysed PSA-OP as well as PSA-1- or PSA-3-pulsed C1R-A2 cells, and PSA-OP and PSA-9-pulsed C1R-A3 cells, respectively. The CTL lines derived from the PSA-OP peptide also lysed PSA-positive prostate cancer cells. PSA-OP-derived T cell lines also lysed recombinant vaccinia-PSA-infected targets but not targets infected with wild-type vaccinia. PSA-OP did not bind HLA-A2 and HLA-A3 molecules. The decrease in cytotoxicity in the presence of protease inhibitors suggests that the PSA-OP is cleaved into shorter peptides, which in turn can interact with HLA-class I molecules and, as a consequence, induce CTL-mediated lysis. We have also demonstrated that it is possible to induce CTL responses in HLA-A2.1/Kb transgenic mice by immunization with PSA-OP with adjuvant. These studies thus provide evidence that oligopeptides such as PSA-OP may be useful candidates for peptide-based cancer vaccines.
The complex interplay between immune cells and tumor cells within the tumor microenvironment (TME) can lead to disease progression. Specifically, signals generated in the TME can cause immunosuppression, promoting angiogenesis and immune evasion, which leads to tumor development. The interplay of M1 and M2 macrophage populations that coincide with these tumor markers is particularly important in the TME. Triple-negative breast cancer (TNBC) often presents as advanced disease, and these tumors are also often bereft of recognized molecular targets that can be found in other subtypes, limiting their therapeutic options. However, tumor-associated macrophages (TAMs) infiltration in TNBC is frequently observed. Moreover, a high density of TAMs, particularly M2 macrophages, is associated with poorer outcomes in various cancers, including TNBC. This provides a strong basis for exploiting TAMs as potential therapeutic targets. Specifically, efforts to increase M2 to M1 repolarization are promising therapeutic approaches in TNBC, and four recent studies wherein divergent approaches to target the M2-rich macrophage population and reverse immune subversion are described. These and similar efforts may yield promising diagnostic or therapeutic options for TNBC, a great clinical need.
p>To facilitate the safety assessment process, a predictive model of flavouring ester hydrolysis has been constructed through an evaluation of the hydrolysis of 44 esters of monoterpene alcohols (citronellol, geraniol, nerol, linalool, and α-terpineol), cinnamyl alcohol, cinnamic acid and furfuryl alcohol. This was achieved by investigating the rates of hydrolysis in artificial gastrointestinal fluids, rat and human tissue homogenates and following single dose oral and intraperitoneal administration to rats. Substrate specificity was found to be similar in each system investigated and was due to defined structural criteria (which are numbered in order of decreasing importance): 1. Steric hindrance of nucleophilic attack by esterases on the ester bond, by hydrocarbon groups present on carbon atoms directly adjacent to the ester bond; 2. The presence of bulky and rigid groups; 3. Substrate specificity as a result of the linear length of the alcohol and carboxylic acid carbon chains.
The serine esterases of rat liver which hydrolysed selected flavouring esters were found to consist of 2 isoenzymes: one has low affinity but high stereoselectivity for (S)-linalyl esters; the other has high affinity but is not stereoselective for linalyl esters. The low affinity but not the high affinity isoenzymes was found to be highly sensitive to inhibition by phenylmethylsulfonyl fluoride.
In vivo studies with rats demonstrated the rapid sequestration of lipophilic esters into adipose tissue and confirmed the glucuronic acid conjugation of citronellol, linalool and α-terpineol following the hydrolysis of their esters.
Overall these studies add increased confidence to the metabolic predictions necessary during the safety assessment of food flavouring esters.</p
Phage-related ribosomal proteases (Prps) are essential for the assembly and maturation of the ribosome in Firmicutes, including the human pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Clostridium difficile. These bacterial proteases cleave off an N-terminal extension of a precursor of ribosomal protein L27, a processing step that is essential for the formation of functional ribosomes. This essential role of Prp in these pathogens has identified this protease as a potential antibiotic target. In this work, we determine the X-ray crystal structure of a covalent inhibition complex at 2.35 Å resolution, giving the first complete picture of the active site of a functional Prp. We also characterize the kinetic activity and screen for potential inhibitors of Prp. This work gives the most complete characterization of the structure and specificity of this novel class of proteases to date.
The acidity of small alkylthiols depends mainly on the inductive effect of substituents, and the ionization state of neighbor functional groups. Protein thiols have a wide range of pKas, that depend, qualitatively on the electrostatic environment, due to charged neighboring groups and α-helix macrodipoles, hydrogen bonds. The desolvation of the thiol/thiolate group also causes major alterations of the pKa.
Nucleophilicity is the most important kinetic property of the thiolate catalytic cysteines in proteins, it is not related to pKa and depends on the regulation of the solvation as well as hydrogen bonding of protein thiolates. In general, aqueous environments greatly diminish the nucleophilicity of thiolates but the protein environment can delicately modulate it by tweaking its nonbonding and solvation interactions, and importantly enhancing its reactivity when the right substrate is bound and ready to react, this explains why protein thiolate reactivity is exquisitely selective.
This research was to investigate protein changes (protein molecular weight distribution, free amino acid (FAA), cathepsin activity) in silver carp (Hypophthalmichthys molitrix) muscle during the process of brine curing (10 g/L salt, 4 °C, 3 h) and air-drying (45 °C, 24 h). The results showed that after air-drying for 4 h, the proportion of protein aggregates with molecular weight >1000 kDa increased by 7.47% compared to fresh fish. Meantime, activities of cathepsin B and B + L increased from 1.52 ± 0.35 and 2.05 ± 0.45 U/g to 2.33 ± 0.22 and 3.27 ± 0.18 U/g, respectively. However, total FAA content had not significantly different (P > 0.05) in this drying period. Thus, although protein aggregation and protein degradation occurred simultaneously, protein aggregation was dominant in the early stage of drying processing. And the protein aggregation decreased, and total FAA content increased, activities of cathepsin decreased but remained relatively high with continuous drying. Thus, protein changes in silver carp muscle were mainly due to degradation. Protein analysis in this study favored to understand the silver carp muscle changes during drying processing and supported to improve freshwater fish drying processes.
Our laboratory has previously reported a strategy of employing cysteine cathepsin (CC) inhibitors as adduct forming, trapping agents to extend the tumor residence time of neurotensin receptor subtype 1 (NTSR1)-targeted radiopharmaceuticals. As a follow-up, we herein report a small library of CC trapping agent (CCTA)-incorporated, NTSR1-targeted conjugates with structural modifications that reduce the number of charged functional groups for both the CCTA and the peptide targeting sequence. These modifications were pursued to reduce the renal uptake and increase the translational potential of the CCTA-incorporated, NTSR1-targeted agents as radiotherapeutics. The biological performance of these constructs was examined using a battery of in vitro and in vivo studies employing the NTSR1-positive HT-29 human colon cancer cell line as our model. In vitro studies confirmed the ability of these constructs to target the NTSR1 and efficiently form intracellular adducts with cysteine proteases. Biodistribution studies using an HT-29 xenograft mouse model revealed that truncation (removal of Lys6-Pro7) of the NTSR1-targeted peptide (¹⁷⁷Lu-NE2a) had the greatest (3.7-fold) effect at lowering renal recognition/uptake relative to our previously reported construct. Other charge-reducing modifications to the CCTA resulted in unexpected increases in renal uptake. All of the constructs demonstrated similar levels of in vivo NTSR1-positive tumor targeting with the highest tumor residualization resulting from the construct containing the zwitterionic CCTA (¹⁷⁷Lu-NE2a). In vivo adduct formation of the conjugates was confirmed using autoradiographic SDS-PAGE analysis.
Cysteine proteases belonging to the falcipain (FP) family play a pivotal role in the biology of malaria parasites and have been extensively investigated as potential antimalarial drug targets. Three paralogous FP-family cysteine proteases of Plasmodium malariae, termed malapains 2–4 (MP2–4), were identified in PlasmoDB. The three MPs share similar structural properties with the FP-2/FP-3 subfamily enzymes and exhibit a close phylogenetic lineage with vivapains (VXs) and knowpains (KPs), FP orthologues of P. vivax and P. knowlesi. Recombinant MP-2 and MP-4 were produced in a bacterial expression system, and their biochemical properties were characterized. Both recombinant MP-2 and MP-4 showed enzyme activity across a broad range of pH values with an optimum activity at pH 5.0 and relative stability at neutral pHs. Similar to the FP-2/FP-3 subfamily enzymes in other Plasmodium species, recombinant MP-2 and MP-4 effectively hydrolyzed hemoglobin at acidic pHs. They also degraded erythrocyte cytoskeletal proteins, such as spectrin and band 3, at a neutral pH. These results imply that MP-2 and MP-4 are redundant hemoglobinases of P. malariae and may also participate in merozoite egression by degrading erythrocyte cytoskeletal proteins. However, compared with other FP-2/FP-3 enzymes, MP-2 showed a strong preference for arginine at the P2 position. Meanwhile, MP-4 showed a primary preference for leucine at the P2 position but a partial preference for phenylalanine. These different substrate preferences of MPs underscore careful consideration in the design of optimized inhibitors targeting the FP-family cysteine proteases of human malaria parasites.
The housekeeping sortase A (SrtA), a membrane-associated cysteine transpeptidase, is responsible for anchoring surface proteins to the cell wall peptidoglycan in Gram-positive bacteria. This process is essential for the regulation of bacterial virulence and pathogenicity. Therefore, SrtA is considered to be an ideal target for antivirulence therapy. In this study, we report that ML346, a compound with a barbituric acid and cinnamaldehyde scaffold, functions as an irreversible inhibitor of Staphylococcus aureus SrtA (SaSrtA) and Streptococcus pyogenes SrtA (SpSrtA) in vitro at low micromolar concentrations. According to our X-ray crystal structure of the SpSrtAΔN81/ML346 complex (Protein Data Bank ID: 7V6K), ML346 covalently modifies the thiol group of Cys208 in the active site of SpSrtA. Importantly, ML346 significantly attenuated the virulence phenotypes of S. aureus and exhibited inhibitory effects on Galleria mellonella larva infection caused by S. aureus. Collectively, our results indicate that ML346 has potential for development as a covalent antivirulence agent for treating S. aureus infections, including methicillin-resistant S. aureus.
Sickle cell disease (SCD) is the most common hereditary blood disorder in the United States. SCD is frequently associated with osteonecrosis, osteoporosis and osteopenia and other bone related complications such as vaso-occlusive pain, ischemic damage, osteomyelitis, and bone marrow hyperplasia known as sickle bone disease (SBD)1,2. Previous SBD models have failed to distinguish the age- and sex-specific characteristics of bone morphometry. In this study, we use the Townes mouse model of SCD to study the pathophysiological complications of SBD in both SCD and sickle cell trait. Changes in bone microarchitecture and bone development were assessed by high-resolution quantitative micro-computed tomography (microCT) and the 3D reconstruction of femurs from male and female mice. Our results indicate that SCD causes bone loss and sex-dependent anatomical changes in bone. Particularly, SCD female mice are prone to trabecular bone loss while cortical bone degradation occurs in both sexes. Additionally, we describe the impact of genetic knockdown of cathepsin K and E-64 mediated cathepsin inhibition on SBD.
Fasciola hepatica is a global parasite of livestock which also causes a neglected zoonosis in humans. The parasite’s communication with the host during its complicated lifecycle is based on an ingenious enzymatic apparatus which includes a variety of peptidases. These enzymes are implicated in parasite migration, pathogenesis of the disease, and modification of host immune response. Although the dynamics of proteolytic machinery produced by intra-mammalian F. hepatica life stages has been previously investigated in great detail, peptidases of the eggs so far received little scientific attention. In this study, we performed a comparative RNA-seq analysis aimed at identification of peptidases expressed in F. hepatica eggs at different ages and employed mass spectrometry in order to identify and quantify peptidases translated in F. hepatica egg lysates. Finally, we measured and classified proteolytic activities in extracts from F. hepatica eggs using a library of fluorogenic substrates and peptidase class-selective inhibitors. Using these methods, we observed significant changes in the composition of peptidases during egg maturation.
Cathepsin C, an important lysosomal cysteine protease, mediates the maturation process of neutrophil serine proteases, and participates in the inflammation and immune regulation process associated with polymorphonuclear neutrophils. Therefore, cathepsin C is considered to be an attractive target for treating inflammatory diseases. With INS1007 (trade name: brensocatib) being granted a breakthrough drug designation by FDA for the treatment of Adult Non-cystic Fibrosis Bronchiectasis and Coronavirus Disease 2019, the development of cathepsin C inhibitor will attract attentions from medicinal chemists in the future soon. Here, we summarized the research results of cathepsin C as a therapeutic target, focusing on the development of cathepsin C inhibitor, and provided guidance and reference opinions for the upcoming development boom of cathepsin C inhibitor.
Bromelain (Bro) is a multiprotein complex extracted from the pineapple plant Ananas comosus, composed of at least eight cysteine proteases. Bro has a wide range of applications in medicine and industry, where the stability of its active proteases is always a major concern. The present study describes the improvement of stability and gain of specific activity in the enzymatic content of Bro immobilized on gold nanoparticles (GNPs). GNPs were synthesized in situ using Bro as the reducing and stabilizing agents and characterized by surface plasmon resonance and transmission electron microscopy. Consistent with the structural changes observed by circular dichroism analysis, the association with GNPs affected enzyme activity. The active Bro immobilized on GNPs (NanoBro) remained stable under storage and gained thermal stability consistent with a thermophilic enzyme. Proteolytic assays were performed on type I collagen membranes using fluorescence spectroscopy of O-phthaldialdehyde (OPA), changes in the membrane superficial structure, and topography by scanning electron microscopy, FTIR, and scanning laser confocal microscopy. Another characteristic of the NanoBro observed was the significant increase in susceptibility to the inhibitory effect of E-64, indicating a gain in cysteine protease activity. The higher stability and specific activity of NanoBro contributed to the broadening and improvement of Bro applications.
The following sequence has been derived for streptococcal proteinase. (See article). The sequence permits the assignment of the single cysteine residue essential for catalytic action at position 47 from the NH2 terminus of the protein. The tryptophan residue at the binding site of the enzyme is at position 214. A histidine residue at position 195 has been assigned as the catalytically important entity in the molecule. Streptococcal proteinase and papain, an enzyme with similar properties, are compared with respect to structure and function.
In order to investigate the specificity of streptococcal proteinase, the peptides formed during proteolysis of the reduced, carboxymethylated phenylalanyl chain of insulin have been isolated and identified. One of the bonds most rapidly hydrolyzed was the Phe-Tyr linkage in the -Phe-Phe-Tyr-sequence. With this result as a lead, a series of synthetic substrates was tested. The results of experiments with acyl dipeptides and with the insulin chain showed that the chief requirement for hydrolysis is the presence of a bulky side chain on the ammo acid adjacent, on the amino-terminal side, to the residue contributing the carbonyl to the susceptible peptide bond. This side chain appears to be important for the binding of substrate to the active center of the enzyme. The residues actually linked by the bond are not so critical, although the results with the acyl dipeptides indicate that there may be a secondary hydrophobic binding of the side chain of the amino acid donating the nitrogen to the susceptible peptide bond. Studies of Vmax and Km for the hydrolysis of several benzyloxycarbonyl-L-phenylalanyl peptides showed that, in these cases, deacylation of an acyl enzyme is not rate-limiting. The variations of Vmax and Km with pH were studied. A graph of log Vmax with respect to pH showed one inflection at pH 6.4 whereas a graph of pKm with respect to pH had two inflections, one at pH 6.4 and the other at pH 8.4. These results are compatible with the conclusion that an unprotonated imidazole ring and the protonated form of the single sulfhydryl group are essential for activity.
Peptidyl diazomethyl ketones are specific inactivators of thiol proteinases being unreactive toward other classes of proteinases. Variation of the peptide portion of the reagents has provided affinity labels for the selective inactivation of the thiol endopeptidase cathepsin B, streptococcal proteinase, and clostripain and for the aminodipeptidase cathepsin C. Comparison of rates of inactivation of cathepsin B and streptococcal proteinase revealed a similarity in specificity, both enzymes showing a preference for a phenylalanine residue in the penultimate position of the peptide portion of the reagent. Reagents not satisfying the specificity of a particular thiol proteinase either did not inactivate or did so only very slowly. In some cases, the reagents bound to the enzyme in a manner unproductive for alkylation but productive for substrate behavior leading to destruction of the inhibitor by cleavage of the peptide portion. Thus, benzyloxycarbonyl-Phe-Gly-Phe diazomethyl ketone was rapidly cleaved by cathepsin B into benzyloxycarbonyl-Phe-Gly and phenylalanine diazomethyl ketone. Clostripain, an enzyme of trypsin-like specificity, was selectively inactivated by benzyloxycarbpnyl-Lys diazomethyl ketone at micromolar concentrations, but was inactivated extremely slowly by 2.5 x 10â»â´ M benzyloxycarbonyl-Phe-Ala diazomethyl ketone, a very effective inactivated by Gly-Phe diazomethyl ketone in the range of 10â»â· to 10â»â¸ M. By comparison, peptidyl diazomethyl ketones with blocked NHâ terminals were considerably less effective. Peptidyl diazomethyl ketones are unreactive with such thiols as mercaptoethanol and glutathione.
The use of rate assays is contrasted with the use of titrants to determine the absolute concentration of active sites of hydrolytic enzymes. Arguments are given which lead to the preference of a stoichiometric titration for the determination of the concentration of enzyme active sites. The absolute standard in this method is the small organic titrant rather than the enzyme which must be the absolute standard in a rate assay procedure. The mathematical basis of four methods of determining absolute enzyme concentration is presented. Three of these four methods were tested using p-nitrophenyl acetate and 2,4-dinitrophenyl acetate as titrants for α-chymotrypsin, showing good agreement. Of five titrants investigated for the determination of the concentration of trypsin solutions, p-nitrophenyl N2-benzyloxycarbonyl-L-lysinate hydrochloride was the most precise, and the one most closely resembling a specific substrate of trypsin. p-Nitrophenyl N-benzyloxycarbonyl-L-tyrosinate served as a titrant for papain solutions. N-trans-Cinnamoylimidazole was a titrant for the enzyme subtilisin. For the titration of elastase, no suitable specific substrate was found and diethyl p-nitrophenyl phosphate was used. o-Nitrophenyl dimethylcarbamate was used for the titration of acetylcholinesterase. For each titration, it was demonstrated that the criteria necessary for a rigorous titration were met. For most enzymes, a rate assay, based on a Kcat calculated from the titration data, was developed as a secondary standard for the determination of enzyme concentration. A discussion of the purity of each enzyme preparation, possible impurities in the preparations, and the possible effect of these impurities on titrations and kinetics is given.
Cathepsin L was purified from rat liver lysosomes by cell fractionation, osmotic disruption of the lysosomes in the lysosomal mitochondrial pellet, gel filtration of the lysosomal extract and chromatography on CM‐Sephadex.
Cathepsin L is a thiol proteinase and exists in several multiple forms visible on the disc electropherogram. By polyacrylamide‐gel electrophoresis in the presence of sodium dodecyl sulphate, its molecular weight was found to be 23000 – 24000. The isoelectric points of the multiple forms of cathepsin L extended from pH 5.8 – 6.1 ascertained by analytical isoelectric focusing.
Using various protein substrates, cathepsin L was found to be the most active endopeptidase from rat liver lysosomes acting at pH 6 – 7. In contrast to cathepsin B 1 , its capability of hydrolyzing N‐substituted derivatives of arginine is low and it does not split esters.
Greatest activity is obtained close to pH 5.0 with 70 – 90% of maximal activity at pH 4.0 and pH 6.0 and 30–40% at pH 7.0.
The enzyme is strongly inhibited by leupeptin and the chloromethyl ketone of tosyl‐lysine. Leupeptin acts as a pseudo‐irreversible inhibitor.
The enzyme is stable for several months at slightly acid pH values in the presence of thiol compounds in a deep‐frozen state.
1) A lysosomal protease, a new cathepsin that inactivates glucose-6-phosphate dehydrogenase [EC 1.1.1.49] and some other enzymes and differs from cathepsin B [EC 3.4.22.1] was purified about 2,200-fold from crude extracts of rat liver by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, and DEAE Sephadex and CM-Sephadex column chromatog-raphies.
2) The new cathepsin was markedly activated by the thiol-reagent, 2-mercaptoethanol and inhibited by monoiodoacetate.
3) The molecular weight of the new cathepsin was found by Sephadex G-75 column chro-matography to be 22,000, which is smaller than that of cathepsin B.
4) The optimum pH of the enzyme for inactivation of glucose-6-phosphate dehydrogenase was pH 5.0–5.5. The enzyme was unstable in alkali and on heat treatment.
5) The rates of inactivation of glucose-6-phosphate dehydrogenase, apo-ornithine amino-transferase [EC 2.6.1.13], apo-tyrosine aminotransferase [EC 2.6.1.5], apo-cystathionase [EC 4.4.1.1], glucokinase [EC 2.7.1.2], glyceraldehyde-3-phosphate dehydrogenase [EC 1.2.1.12], and malate dehydrogenase [EC 1.1.1.37] by the new cathepsin were higher than those by cathepsin B. However aldolase [EC 4.1.2.13] was inactivated more rapidly by cathepsin B than by the new cathepsin. Lactate dehydrogenase [EC 1.1.1.27], glutamate dehydrogenase [EC 1.4.1.2] and alcohol dehydrogenase [EC 1.1.1.1] were not inactivated by either cathepsin. Unlike cathepsin B, the new cathepsin scarcely hydrolyzes N-substituted derivatives of arginine.
Lysosomal cathepsins B and N complete the depolymerization of native fibrillar collagen in the phagolysosome after prior extracellular fragmentation by collagenase and other neutral proteinases. In vitro studies have confirmed that cathepsins B and N cleave native collagen only at the short non-helical telopeptides, which generate the intermolecular cross-links. This action occurs maximally at pH 3.5 and at 37 degrees C the released monomers denature spontaneously and are susceptible to further breakdown. In the phagolysosome the collagenous debris is already weakened and probably therefore, more easily disrupted by these cathepsins. Complete digestion would then be undertaken by the whole complement of proteases. The lysosomal glycosidases may assist this breakdown by degrading ground substance components which are normally tightly bound to collagen. In certain situations cells may instead generate an acidic pericellular environment that could permit the direct action of secreted lysosomal enzymes. This extracellular action may supersede the action of collagenase and the activity of these different enzymes would thus be regulated by changes in the nature of this microenvironment.
1. Cathepsin B1 was purified from human liver by a method involving autolysis, fractional precipitation with acetone, adsorption on, and stepwise elution from, CM-cellulose and an organomercurial adsorbent, gel chromatography and finally equilibrium chromatography on CM-cellulose. 2. The early stages of the procedure, including the use of the organomercurial adsorbent, were suitable for the simultaneous isolation of cathepsin D. The two cathepsins were sharply separated on the organomercurial column, and particular attention was given to the method for the preparation and use of this adsorbent. 3. A method is described for the staining of analytical isoelectric-focusing gels for cathepsin B1 activity, as well as protein. By this method it was shown that cathepsin B1 was represented by at least six isoenzymes during the greater part of the purification procedure. After the gel-chromatography step this group of isoenzymes was obtained essentially free of other proteins, in good yield. The isoenzymes were resolved from this mixture by chromatography on CM-cellulose. The purified enzyme was stable for several weeks at slightly acid pH values in the absence of thiol compounds; it was unstable above pH7. 4. The pI values of the isoenzymes of cathepsin B1 extended from pH4.5 to 5.5, that of the major isoenzyme tending to increase from 5.0 to 5.2 during the purification procedure. Gel chromatography indicated a molecular weight of 27500 for all of the isoenzymes, whereas polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate gave a value of 24000. 5. An antiserum raised in sheep against the purified enzyme reacted specifically with the alkali-denatured molecule. Purified cathepsin B1 contained no material precipitable by an anti-(human cathepsin D) serum. 6. The enzyme hydrolysed several N-substituted derivatives of l-arginine 2-naphthylamide, as well as haemoglobin, azo-haemoglobin, azo-globin and azo-casein. Greatest activity was obtained near pH6.0. 7. The sensitivity of human cathepsin B1 to chemical inhibitors was generally similar to that of other thiol proteinases. The enzyme was inactivated by the chloromethyl ketones derived from tosylphenylalanine, tosyl-lysine, acetyltetra-alanine and acetyldialanylprolylalanine. 8. The hydrolysis of alpha-N-benzoyl-dl-arginine 2-naphthylamide by extracts of human liver at pH6 was attributable entirely to cathepsin B1.
The active site of an enzyme performs the twofold function of binding a substrate and catalysing a reaction. The efficiency of these actions determines the overall activity of the enzyme towards the particular substrate, i.e. determines the specificity of the enzyme. It is therefore possible to obtain information on the active site by the kinetics of the enzyme’s reactions with different substrates and inhibitors. An important feature of the active site is its size. It should be possible to 'measure’ this by using substrates or inhibitors large enough to show up the interactions of the furthermost parts of the binding site. In the present series of investigations on proteolytic enzymes, our approach is to compare the activity of the enzyme towards ( a ) peptides of increasing length, ( b ) diastereoisomeric pairs of peptides in which a particular amino acid residue has been replaced by its antipode, and ( c ) pairs of substrates in which a particular side chain (say a methyl group) has been replaced by another (say an aromatic group). The influence of these changes on reaction rates as a function of distance from the point of cleavage indicates the extent of the active site (Schechter, Abramowitz & Berger 1965; Abramowitz, Schechter & Berger 1967).
Kinetic evidence consistent with the formation of an acyl-enzyme intermediate in the proteinase-catalyzed hydrolyses of N-Z-L-alaninates and α-N-Z-L-lysinates has been obtained. The effect of adding a simple nucleophile, such as methanol, was consistent with the partitioning of a common acyl-enzyme intermediate between water and the alcohol. However, for the hydrolysis of o-nitrophenyl N-Z-L-alaninate and for the effect of methanol, the kinetics were more complex. The kcat for o-nitrophenyl N-Z-L-alaninate was about 3.4-fold lower than the kcat values for p- or m-nitrophenyl N-Z-L-alaninates. The rate of increase of kcat with methanol concentration was dependent upon the nature of the leaving group of the N-Z-L-alanine ester. The kcat/Km ratio was also dependent upon the nucleophile concentration. These results are interpreted in terms of the binding of the leaving group and the methanol at the active site of proteinase, and indicate the existence of a kinetically important ternary enzyme-substrate-leaving group (nucleophile) complex. Proteinase showed specificity toward an unbranched carbon chain or an aromatic residue as the amino acid side-chain group of N-acylamino acid ester substrates; substitutions in the β-carbon position resulted in a marked decrease in activity. The presence of a charged group in the side-chain group, positive or negative, had no significant effect on the rate of proteinase hydrolysis. The most striking feature is the specificity of proteinase toward the N-acyl group of the substrate. The N-acyl group, benzyloxycarbonyl, was found to yield optimal activity with ester substrates and even small changes in this structure resulted in a dramatic decrease in activity. Evidence is presented indicating that the binding of a hydrophobic group in the N-acyl moiety, in a precise manner and at a precise distance with respect to the ester bond, is essential for optimal enzymatic activity and suggesting that the binding site for the N-acyl group in proteinase may be a relatively small inflexible hydrophobic cleft. The kinetic properties of proteinase are compared with those of papain and ficin.
1. The u.v.-spectral characteristics of 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs(2)), 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py), 4,4'-dipyridyl disulphide (4-Py-S-S-4-Py), 5-mercapto-2-nitrobenzoic acid (Nbs), 2-thiopyridone (Py-2-SH) and 4-thiopyridone (Py-4-SH) were determined over a wide range of pH and used to calculate their acid dissociation constants. 2. The reactions of l-cysteine, 2-mercaptoethanol and papain with the above-mentioned disulphides were investigated spectrophotometrically in the pH range 2.5-8.5. 3. Under the conditions of concentration used in this study the reactions of both low-molecular-weight thiols with all three disulphides resulted in the stoicheiometric release of the thiol or thione fragments Nbs, Py-2-SH and Py-4-SH at all pH values. The rates of these reactions are considerably faster at pH8 than at pH4, which suggests that the predominant reaction pathway in approximately neutral media is nucleophilic attack of the thiolate ion on the unprotonated disulphide. 4. The reaction of papain with Nbs(2) is markedly reversible in the acid region, and the pH-dependence of the equilibrium constant for this system in the pH range 5-8 at 25 degrees C and I=0.1 is described by: [Formula: see text] 5. Papain reacts with both 2-Py-S-S-2-Py and 4-Py-S-S-4-Py in the pH range 2.5-8.5 to provide release of the thione fragments, stoicheiometric with the thiol content of the enzyme. 6. Whereas the ratios of the second-order rate constant for the reaction at pH4 to that at pH8 for the cysteine-2-Py-S-S-2-Py reaction (k(pH4)/k(pH8)=0.015) and for the papain-4-Py-S-S-4-Py reaction (k(pH4)/k(pH8)=0.06) are less than 1, that for the papain-2-Py-S-S-2-Py reaction is greater than 1 (k(pH4)/k(pH8)=15). 7. This high reactivity of papain has been shown to involve reaction of the thiol group of cysteine-25, the enzyme's only cysteine residue, which is part of its catalytic site. 8. That this rapid and stoicheiometric reaction of the thiol group of native papain is not shown either by low-molecular-weight thiols or by the thiol group of papain after its active conformation has been destroyed by acid or heat denaturation, strongly commends 2-Py-S-S-2-Py as one of the most useful papain active-site titrants discovered to date. This reagent has been shown to allow accurate titration of papain active sites in the presence of up to 10-fold molar excess of l-cysteine and up to 100-fold molar excess of 2-mercaptoethanol.
Three peptide chloromethyl ketones have been prepared which irreversibly inhibit elastase. Amino acid analyses of the inactivated enzyme show that inhibition results from the alkylation of a histidine residue of the enzyme. The three peptide chloromethyl ketones differ markedly in their ability to inactivate the enzyme with the longer peptides reacting more rapidly. A parallel dependence of the ease of substrate hydrolysis on peptide chain length has been observed previously with amide substrates of elastase.
Extracts of rheumatoid synovial tissue obtained at surgical synovectomy contained neutral proteinases as well as cathepsin D. The neutral proteinase activity was particle-bound but could be solubilized by 1 M MgCl2. About half of the solubilized activity adsorbed to aproptinin-Sepharose at pH 7.5 and was desorbed at pH 3.3. This activity was shown to be due to leukocyte elastase and cathepsin G by enzymological and immunological criteria. The neutral proteinase activity that did not adsorb to aprotinin-Sepharose was not due to elastase or cathepsin G. It was able to hydrolyse proteoglycan and was inhibited by diisopropylfluorophosphate, soybean and lima bean trypsin inhibitors. It was, therefore, a serine proteinase. Its inhibition characteristics were different from those of plasmin, kallikrein or thrombin. All of the neutral proteinase activity of synovial extracts was attributable to serine proteinases, no evidence of metallo-proteinases was found. The possible role of the neutral proteinases in the degradation of the matrix of cartilage is discussed. A simple procedure for purifying leukocyte elastase and cathepsin G is described as well as the raising of specific antisera to these enzymes.
Cathepsin H was purified from human liver by a method involving autolysis and acetone fractionation, and chromatography on DEAE-cellulose, Ultrogel AcA 54, hydroxyapatite and concanavalin A-Sepharose. The procedure allowed for the simultaneous isolation of cathepsin B and cathepsin D. Cathepsin H was shown to consist of a single polypeptide chain of 28 000 mol.wt., and affinity for concanavalin A-Sepharose indicated that it was a glycoprotein. The enzyme existed in multiple isoelectric forms, the two major forms having pI values of 6.0 and 6.4; it hydrolysed azocasein (pH optimum 5.5), benzoylarginine 2-naphthylamide (Ba-Arg-NNap), leucyl 2-naphthylamide (Arg-NNap), (pH optimum 6.8). Arg-NNap and Arg-NMec, unlike Bz-Arg-NNap-, were not hydrolysed by human cathepsin B. Cathepsin H was similar to cathepsin B in being irreversibly inactivated by exposure to alkaline pH. Sensitivity to chemical inhibitors by 1 microM-leupeptin, which gave essentially complete inhibition of the other lysosomal cysteine proteinases, cathepsins B and L.
Ubiquitous distribution of Ca2+-dependent neutral protease and its specific inhibitor in various tissues and cells is described. A proposal is made to call the protease calpain and the inhibitor calpastatin, both as generic names.Calpain, which implies a Ca2+-dependent protease belonging to the thiol protease group, has been knoqn to be widely distributed under different names: e.g., phosphorylase kinase activating factor (KAF), mayofibrllar Z-line-removing protease, Ca2+-activated neutral protease (CANP), recpetor transforming factor (RTF), etc. Some of the common features of these enzymes are discussed with special reference to the effect on them of a common, endogenous inhibitor, calpastatin.The existence of two types of calpain was discovered from rat liver and kidney and human erythrocytes. Calpain I requires only 40 μm Ca2+ for 50% activation, while calpain II responds only to a Ca2+ concentration of mm order. Capain I is the minor component in rat liver, but it is the only type of calpain that can be found in human erythrocytes.Calpastatin was found to exist in the soluble fraction of rat liver, brain, kidney, spleen cells and thymocytes, human liver, erythrocytes and lymphocytes, and monkey and guinea pig erythrocytes. Calpastatin was partially purified by chromatography on DEAE-cellulose and Ultrogen AcA 34. It has a molecular weight of 280,000–300,000 and is extremely heat stable but readily digetible by trypsin. None of the proteases tested, except calpains I and II, were inhibited by calpastatin. Inhibition is not based on sequestering of Ca2+ from the medium by the inhibitor, while the binding of calpain to calpastatin does need Ca2+. Calpain frm one tissue can be inhibited by calpastatin not only from the same tissue but also from the other tissues of the same animal or even of a different species.The relative abundance of calpain to calpastatin found in one tissue differs greatly from that found in the other tissue. Teven in the same kind of tissue, the ratio seems to vary depending upon the sex, age and stag eof differentiation, possibly including neoplastic transformation.
Benzyloxycarbonyl-phenylalanyl-arginine 4-methyl-7-coumarylamide was found to be an excellent substrate for the fluorimetric assay of cathepsin B, and arginine 4-methyl-7-coumarylamide for cathepsin H. Procedures were developed that are very convenient, and avoid the hazards associated with the use of naphthylamides.
A simple method for isolation of kallikrein from human plasma is described. Before activation of the enzyme with acetone, the plasma was treated with 0.2 M-methylamine at pH 8.2 to inactivate alpha 2-macroglobulin and thus prevent the irreversible binding of the active enzyme to the inhibitor. The enzyme was adsorbed on soya-bean trypsin inhibitor-Sepharose 4B and eluted with 5 mM-NaOH, pH 11.3. It was further purified by immunoadsorption of contaminating proteins, and gel chromatography on Ultrogel AcA 44. About 3 mg of kallikrein was obtained from 400 ml of plasma (35% yield). The purified enzyme was shown to be homogeneous by electrophoretic and immunological criteria. The specific activities against benzyloxycarbonylphenylalanylarginine methylcoumarylamide, prolylphenylalanylarginine methylcoumarylamide and tosylarginine methyl ester were higher than any previously reported. The purified enzyme was resolved into two forms of mol.wts. 88 000 and 86 000 in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis without reduction. Each consisted of three chains linked by disulphide bonds, one containing the reactive serine residue (mol.wt. 36 000 or 34 000), and two additional chains (mol.wt. 28 000 and 22 000).
It has previously been demonstrated (Poole, A.R., Tiltman, K.J., Recklies, A.D. and Stoker, T.A.M. (1978) Nature 273, 545-547) that malignant human breast tumours maintained in organ culture secrete elevated amounts of a thiol proteinase. This enzyme has been shown to possess enzymic properties similar to those of cathepsin B (EC 3.4.22.1) with respect to specificity, affinity and pH optima for synthetic substrates. However, the tumour enzyme is much more stable than human liver cathepsin B to inactivation above neutral pH, and it also has a large molecular size and a more acidic isoenzyme pattern. The stability of this enzyme under physiological conditions may allow it to play a role in tumour invasion and metastasis.
1. The kinetic parameters Kcat. and Km were determined for the hydrolysis of some arginine naphthylamides by human cathepsin B. 2. A new and efficient synthesis of Z-Arg-Arg-NNap (benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide) was developed. 3. Z-Arg-Arg-NNap was a specific and sensitive substrate for cathepsin B, and was used for kinetic studies. 4. Values of kcat. were maximal in the pH range 5.4--6.2, and depended on a single ionizing group of pKa 4.4. 5. Leupeptin was a purely competitive inhibitor of human cathepsin B. 6. The effect of pH on the apparent inhibitor constant, Ki (app.), was determined. Ki (app.) was pH-independent in the range pH 4.3--6.0, with the mean value 7 x 10(-9) M.
Modification of E-64 focused on the terminal agmatine for practical use led to some potent analogs which were successfully obtained by a stereoselective synthesis using D-tartaric acid as a starting material. Ep-475 (id. E-64-c), in which the agmatine was replaced by 3-methyl-butylamine, was found to react with the essential SH of papain in accord with the decrease of activity. [3H]Ep-475 was irreversibly incorporated into papain in an equimolar ratio. None of the analogs showed any effect on thiol enzymes other than proteolytic ones.
Crystalline rat liver cathepsin B, a lysosomal thiol protease, is composed of two polypeptide chains with molecular weights of ca. 25,000 and 5,000, linked by disulfide bridge(s). The chains were separated by gel filtration after reduction and carboxymethylation. Sequence analyses of the two chains revealed that the enzyme has an extremely high sequence homology to papain, a plant thiol protease, especially in the active site related areas.
The mechanism of inhibition of cathepsin B [EC 3.4.22.1] and cathepsin L [EC 3.4.22.-] by E-64 was investigated. Kinetic studies indicated that E-64 was an irreversible inhibitor of these enzymes. [3H]E-64 is incorporated into cathepsin B in a one/one molar ratio in parallel with inactivation of the enzyme. Titration of one of the 10 SH groups of native cathepsin B with 2,2'-dithiodipyridine resulted in complete loss of enzyme activity. Decrease of titratable SH groups and activity of cathepsin B was proportional to the concentration of E-64 added, indicating that E-64 binds to an equimolar amount of active SH residues of cathepsin B. The effects of E-64 and its derivatives on lysosomal cathepsin B and cathepsin L in rat liver were studied in vitro and in vivo. The D form of E-64 inhibited the cathepsin both in vitro and in vivo, although its inhibitory effects were less than those of E-64-(L). E-64-b(RR), in which the terminal agmatine of E-64 is replaced by leucine, was as active as E-64-(L) in vitro, but was completely inactive in vivo. Among the E-64 derivatives tested, E-64-c(SS), in which the terminal agmatine of E-64 is replaced by isoarylamide, showed strong inhibitory activity in vivo, like E-64-(L).
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
Protein Research Foundation
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