scholarly journals Rational design of human cathepsin L with efficient collagenolytic activity

2020 ◽  
Author(s):  
Debi Choudhury ◽  
Sampa Biswas
Keyword(s):  
Cysteine Protease ◽  
Type I Collagen ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Rational Approach ◽  
Collagenolytic Activity ◽  
Type I ◽  
Cysteine Cathepsins ◽  
Binding Surface ◽  
Human Cathepsin

AbstractCollagens are the main structural components of ECM and collagenolysis is vital for ECM remodelling, which is essential for normal organ homeostasis. Any dysregulation of this process can result in various pathological conditions. The degradation of the interstitial collagens is generally mediated by specific collagenases which are capable of cleaving at specific loci in the collagen triple helix. Lysosomal cysteine cathepsins have general housekeeping as well as some highly specialized functions. Amongst them, human cathepsin K is the only lysosomal cysteine protease which has potent collagenolytic activity against type I collagen. In this study, we have imparted collagenolytic property to another human cysteine protease, cathepsin L, by systematically engineering proline-specificity and GAG-binding surface in the protease. The designed proline-specific mutant shows high specificity for peptidyl substrate containing proline at P2 position but is incapable of cleaving collagen. However, when the proline-specific mutant is further engineered for GAG-binding surface, it can degrade type I collagen in presence of Chondroitin 4-Sulfate (C4-S). We also present high resolution crystal structures of these proline-specific (1.4 Å) and collagen-specific (1.8 Å) mutants. Finally docking studies with prolyl-peptidic substrate (Ala-Gly-Pro-Arg-Ala) at the active site and a GAG molecule (C4-S) at the GAG binding site enables us to identify key structural features responsible for collagenolytic activity of papain-like cysteine proteases. This study provides a rational approach to engineer an efficient collagenase from a human template enzyme which may have various noninvasive therapeutic implications with an expected additional advantage of immuno-compatiblity.

scholarly journals Cysteine Protease Inhibitor (AcStefin) Is Required for Complete Cyst Formation of Acanthamoeba

2013 ◽  
Vol 12 (4) ◽  
pp. 567-574 ◽  
Author(s):  
Jung-Yub Lee ◽  
Su-Min Song ◽  
Eun-Kyung Moon ◽  
Yu-Ran Lee ◽  
Bijay Kumar Jha ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Protease Activity ◽  
Cathepsin L ◽  
Cyst Formation ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Content Type ◽  
Human Cathepsin ◽  
Interfering Rna

ABSTRACTThe encystation ofAcanthamoebaleads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions, such as those associated with starvation, low temperatures, and biocides. Furthermore, cysteine proteases have been implicated in the massive turnover of intracellular components required for encystation. Thus, strict modulation of the activities of cysteine proteases is required to protectAcanthamoebafrom intracellular damage. However, mechanisms underlying the control of protease activity during encystation have not been established inAcanthamoeba. In the present study, we identified and characterizedAcanthamoebacysteine protease inhibitor (AcStefin), which was found to be highly expressed during encystation and to be associated with lysosomes by fluorescence microscopy. Recombinant AcStefin inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, and papain. Transfection with small interfering RNA against AcStefin increased cysteine protease activity during encystation and resulted in incomplete cyst formation, reduced excystation efficiency, and a significant reduction in cytoplasmic area. Taken together, these results indicate that AcStefin is involved in the modulation of cysteine proteases and that it plays an essential role during the encystation ofAcanthamoeba.

Cryptostatin, a chagasin-family cysteine protease inhibitor of Cryptosporidium parvum

Parasitology ◽  
2012 ◽  
Vol 139 (8) ◽  
pp. 1029-1037 ◽  
Author(s):  
J.-M. KANG ◽  
H.-L. JU ◽  
J.-R. YU ◽  
W.-M. SOHN ◽  
B.-K. NA
Keyword(s):  
Cryptosporidium Parvum ◽  
Cysteine Protease ◽  
Cathepsin L ◽  
Sodium Dodecyl ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Immunogold Electron Microscopy ◽  
Electron Microscopy Analysis ◽  
Wide Ph Range ◽  
Human Cathepsin

SUMMARYCysteine proteases of pathogenic protozoan parasites play pivotal roles in the life cycle of parasites, but strict regulation of their activities is also essential for maintenance of parasite physiology and interaction with hosts. In this study, we identified and characterized cryptostatin, a novel inhibitor of cysteine protease (ICP) of Cryptosporidium parvum. Cryptostatin showed low sequence identity to other chagasin-family ICPs, but 3 motifs (NPTTG, GXGG, and RPW/F motifs), which are evolutionarily conserved in chagasin-family ICPs, were found in the sequence. The overall structure of cryptostatin consisted of 8 β-strands that progressed in parallel and closely resembled the immunoglobulin fold. Recombinant cryptostatin inhibited various cysteine proteases, including papain, human cathepsin B, human cathepsin L, and cryptopain-1, with Ki's in the picomolar range. Cryptostatin was active over a wide pH range and was highly stable under physiological conditions. The protein was thermostable and retained its inhibitory activity even after incubation at 95°C. Cryptostatin formed tight complexes with cysteine proteases, so the complexes remained intact in the presence of sodium dodecyl sulfate and β-mercaptoethanol, but they were disassembled by boiling. An immunogold electron microscopy analysis demonstrated diffused localization of cryptostatin within oocystes and meronts, but not within trophozoites, which suggests a possible role for cryptostatin in host cell invasion by C. parvum.

scholarly journals Challenges for targeting SARS-CoV-2 proteases as a therapeutic strategy for COVID-19

2020 ◽  
Author(s):  
Kas Steuten ◽  
Heeyoung Kim ◽  
John C. Widen ◽  
Brett M. Babin ◽  
Ouma Onguka ◽  
...  
Keyword(s):  
Viral Entry ◽  
Therapeutic Strategy ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Development Programs ◽  
Entry Pathway ◽  
Cysteine Cathepsins ◽  
Human Cathepsin ◽  
Transmembrane Serine Protease ◽  
Substantial Drop

ABSTRACTTwo proteases produced by the SARS-CoV-2 virus, Mpro and PLpro, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both Mpro and PLpro proteases. These efforts identified a small number of hits for the Mpro protease and no viable hits for the PLpro protease. Of the Mpro hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead Mpro inhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of Mpro inhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsin L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting Mpro and PLpro proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.

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

2009 ◽  
Vol 390 (9) ◽  
Author(s):  
Sylvie Desmarais ◽  
Frédéric Massé ◽  
M. David Percival
Keyword(s):  
Human Cell ◽  
Type I Collagen ◽  
Cathepsin K ◽  
The Other ◽  
Enzyme Assays ◽  
Type I ◽  
Treatment Of Osteoporosis ◽  
Cysteine Cathepsins ◽  
Rodent Cell ◽  
Enzyme Selectivity

Abstract Cathepsin K (Cat K) degrades bone type I collagen and is a target for the pharmacological treatment of osteoporosis. Further roles for Cat K have been recently described, some of which are supported by the use of purportedly selective Cat K inhibitors in human and rodent cell-based assays. Twelve commercial and non-commercial Cat K inhibitors were profiled against a panel of purified human, rat, and mouse cysteine cathepsins and in two cell-based enzyme occupancy assays for activity against Cat K, B, and L. Ten inhibitors, including the carbohydrazide Cat K inhibitor II (Boc-Phe-Leu-NHNH-CO-NHNH-Leu-Z), the non-covalent K4b, and the epoxide NC-2300, have either little Cat K selectivity, or appear poorly cell penetrant. The amino-acetonitrile-containing inhibitors L-873724 and odanacatib show greater than 100-fold human Cat K enzyme selectivity and have similar IC50 values against each cathepsin in cell-based and enzyme assays. The basic inhibitor balicatib has greater cellular potencies than expected on the basis of purified enzyme assays. The accumulation of [14C]-balicatib in fibroblasts is blocked by prior treatment of the cells with NH4Cl, consistent with balicatib having lysosomotropic properties. These results support the use of L-873724 and odanacatib as tools to identify novel roles for Cat K using human cell-based systems, but suggest using caution in the interpretation of studies employing the other compounds.

scholarly journals Presence of Anti-cystatin C–positive Dendritic Cells or Macrophages and Localization of Cysteine Proteases in the Apical Bud of the Enamel Organ in the Rat Incisor

2005 ◽  
Vol 53 (5) ◽  
pp. 643-651 ◽  
Author(s):  
Sumio Nishikawa
Keyword(s):  
Dendritic Cells ◽  
Cystatin C ◽  
Cysteine Protease ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Enamel Organ ◽  
Apical Bud ◽  
Apical Buds ◽  
Proliferation And Differentiation

Cystatin C, a cysteine protease inhibitor, was examined in the apical buds of rat incisors by immunohistochemistry, because in transition and maturation zones most of the dendritic cells in the papillary layer are anti-cystatin C–positive. Anti-cystatin C–labeled cells were sparse and localized to the proliferation and differentiation zones, constituting the apical bud of 5-week-old rat incisors. These cells were considered macrophages or dendritic cells, based on their reactivity with OX6 and ED1, as well as their ultrastructure. Basement membrane at the periphery of apical bud was also labeled by anti-cystatin C antibody. The apical buds included a few apoptotic fragments and weak reactivity with antibody to cathepsin L, a cysteine protease. Reactivity to anti-cystatin C and anti-cathepsin ∗∗∗L antibodies was also detected in the apical bud of newborn rat incisors. These results suggest that the cystatin C–positive macrophages or dendritic cells are involved in normal incisor formation. They may be related to the clearance of apoptotic cells or protection from putative cysteine protease activity.

scholarly journals MT1-MMP–dependent neovessel formation within the confines of the three-dimensional extracellular matrix

2004 ◽  
Vol 167 (4) ◽  
pp. 757-767 ◽  
Author(s):  
Tae-Hwa Chun ◽  
Farideh Sabeh ◽  
Ichiro Ota ◽  
Hedwig Murphy ◽  
Kevin T. McDonagh ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Cell Surface ◽  
Type I Collagen ◽  
Ex Vivo ◽  
Three Dimensional ◽  
Collagenolytic Activity ◽  
Type I ◽  
Ex Vivo Model

During angiogenesis, endothelial cells initiate a tissue-invasive program within an interstitial matrix comprised largely of type I collagen. Extracellular matrix–degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP–dependent processes. To identify proteinases critical to neovessel formation, an ex vivo model of angiogenesis has been established wherein tissue explants from gene-targeted mice are embedded within a three-dimensional, type I collagen matrix. Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., β3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation. Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.

Cysteine proteinase activities in the fish pathogenPhilasterides dicentrarchi(Ciliophora: Scuticociliatida)

Parasitology ◽  
2004 ◽  
Vol 128 (5) ◽  
pp. 541-548 ◽  
Author(s):  
A. PARAMÁ ◽  
R. IGLESIAS ◽  
M. F. ÁLVAREZ ◽  
J. LEIRO ◽  
F. M. UBEIRA ◽  
...  
Keyword(s):  
Crude Extract ◽  
Type I Collagen ◽  
Cysteine Proteinase ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Type I ◽  
Molecular Weights ◽  
Reducing Conditions ◽  
Sds Page

This study investigated protease activities in a crude extract andin vitroexcretion/secretion (E/S) products ofPhilasterides dicentrarchi, a ciliate fish parasite causing economically significant losses in aquaculture. Gelatin/SDS–PAGE analysis (pH 4, reducing conditions) detected 7 bands with gelatinolytic activity (approximate molecular weights 30–63 kDa) in the crude extract. The banding pattern observed in analysis of E/S products was practically identical, except for 1 low-molecular-weight band detected in the crude extract but not in the E/S products. In assays with synthetic peptidep-nitroanilide substrates, the crude extract hydrolysed substrates characteristic of cysteine proteases, namely Z-Arg-Arg pNA, Bz-Phe-Val-Arg pNA and Z-Phe-Arg pNA. These activities were strongly inhibited by the cysteine protease inhibitor E-64 and by Ac-Leu-Val-Lys aldehyde, a potent inhibitor of cysteine proteases of the cathepsin B protease subfamily. The proteases present in the crude extract degraded both type-I collagen and haemoglobinin vitro, consistent with roles in tissue invasion and nutrition respectively. Again, E-64 completely (collagen) or markedly (haemoglobin) inhibited this degradation. Finally, the histolytic activity of the ciliate in turbot fibroblast monolayers was strongly reduced in the presence of E-64, confirming the importance of secreted cysteine proteinases in the biology ofPhilasterides dicentrarchi.

scholarly journals Extracellular-matrix degradation at acid pH. Avian osteoclast acid collagenase isolation and characterization

1993 ◽  
Vol 290 (3) ◽  
pp. 873-884 ◽  
Author(s):  
H C Blair ◽  
S L Teitelbaum ◽  
L E Grosso ◽  
D L Lacey ◽  
H L Tan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Specificity ◽  
Cathepsin B ◽  
Type I Collagen ◽  
Sequence Similarity ◽  
Bone Matrix ◽  
Collagenolytic Activity ◽  
Type I ◽  
Isolation And Characterization

Osteoclasts degrade bone matrix, which is mainly type I collagen and hydroxyapatite, in an acidic extracellular compartment. Thus we reasoned that osteoclasts must produce an acid collagenase. We purified this enzyme, a 31 kDa protein, from avian osteoclast lysates (in 100 mM acetate/1 mM CHAPS/1 mM dithiothreitol, pH 4.4), fractionated by (NH2)2SO4 precipitation, gelatin-affinity, cation exchange, and gel filtration. Fraction activity was measured using diazotized collagen or 3H-labelled cross-linked collagen (decalcified and trypsin-treated metabolically L-[4,5-3H]proline-labelled bone) as substrates. Iodoacetate, leupeptin, antipain, pepstatin and mercurials inhibited collagenolysis by the isolated proteinase; mercurial derivatives could not be re-activated by dithiothreitol. Collagen degradation was maximal at pH 4.4; purified proteinase reproduced the collagenolytic activity of cell lysates. The N-terminal amino acid sequence from the isolated protein and its CNBr degradation fragments showed sequence similarity to mammalian cathepsin Bs, and near-identity with avian liver cathepsin B. Peptide substrate specificity of the osteoclastic enzyme resembled those of mammalian cathepsin B and its avian liver counterpart, but degradation of low-molecular-mass substrates by the osteoclastic enzyme was slower, reflecting generally lower kcat. values. Further, kcat/Km varied less between arginine-containing substrates than for previously reported cathepsin Bs, indicating different substrate specificity of the osteoclast enzyme. Polyclonal antibody raised to a 25 kDa fragment of the enzyme recognized a single 31 kDa band in SDS/PAGE of osteoclast lysates blotted to poly(vinylidene difluoride), adsorbed collagenolytic activity of osteoclast lysates, and stained avian osteoclasts in tissue sections. Degenerate sense- and antisense-oligonucleotide primers, predicted from segments of primary amino acid sequence, amplified a 486 bp DNA fragment; this was cloned and sequenced. Of 162 amino acids encoded, 77% are identical with those of human cathepsin B; hybridization identified a 2.4 kb RNA in osteoclast lysates. We conclude that the major avian osteoclast collagenolytic enzyme is a cathepsin B, whose activity varies from other enzymes of its class.

Zinc Inhibits Collagenolysis by Cathepsin K and Matrix Metalloproteinases in Demineralized Dentin Matrix

2017 ◽  
Vol 51 (6) ◽  
pp. 576-581 ◽  
Author(s):  
Pinar Altinci ◽  
Roda Seseogullari-Dirihan ◽  
Gulsen Can ◽  
David Pashley ◽  
Arzu Tezvergil-Mutluay
Keyword(s):  
Matrix Metalloproteinases ◽  
Mass Loss ◽  
Type I Collagen ◽  
Cathepsin K ◽  
Collagen Degradation ◽  
Type I ◽  
Level Control ◽  
Physiological Level ◽  
Cysteine Cathepsins ◽  
Demineralized Dentin

The enzymatic degradation of dentin organic matrix occurs via both the action of matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs). Zinc can prevent collagen hydrolysis by MMPs. However, its effect on the activity of dentin-bound CCs is not known. The aim of this study was to investigate the effect of zinc on matrix-bound cathepsin K and MMP activity in dentin. Completely demineralized dentin beams were divided into test groups (n = 9) and incubated at 37°C in an incubation media (1 mL) containing ZnCl2 of 0.02 (physiological level, control), 0.2, 0.5, 1, 5, 10, 20, 30, or 40 mM. The dry mass changes of the beams were determined, and incubation media were analyzed for cathepsin K- and MMP-specific collagen degradation end products - CTX (C-terminal cross-linked telopeptide of type I collagen) and ICTP (cross-linked carboxy-terminal telopeptide of type I collagen) - at 1, 3, and 7 days of incubation. The mass loss of the beams decreased when the zinc level in the incubation media was ≥5 mM (p < 0.05). The release of liberated collagen degradation telopeptides decreased in accordance with the decrease in the mass loss rates of the beams. Cathepsin K-induced dentin collagen degradation can be strongly inhibited by zinc. Zinc levels of ≥5 mM can be considered as a reliable threshold for the stabilization of dentin matrices.

scholarly journals Characterisation of a cysteine protease from poultry red mites and its potential use as a vaccine for chickens

Parasite ◽  
2021 ◽  
Vol 28 ◽  
pp. 9
Author(s):  
Shiro Murata ◽  
Ayaka Taniguchi ◽  
Masayoshi Isezaki ◽  
Sotaro Fujisawa ◽  
Eishi Sakai ◽  
...  
Keyword(s):  
Cysteine Protease ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Mite Species ◽  
Economic Losses ◽  
Dermanyssus Gallinae ◽  
Physiological Processes ◽  
Poultry Houses ◽  
Poultry Red Mites ◽  
Potential Use

Poultry red mites (PRMs, Dermanyssus gallinae) are ectoparasites that negatively affect farmed chickens, leading to serious economic losses worldwide. Acaricides have been used to control PRMs in poultry houses. However, some PRMs have developed resistance to acaricides, and therefore different approaches are required to manage the problems caused by PRMs. Vaccination of chickens is one of the methods being considered to reduce the number of PRMs in poultry houses. In a previous study, a cysteine protease, Deg-CPR-1, was identified as a candidate vaccine against PRMs distributed in Europe. In this study, we investigated the characteristics of Deg-CPR-1. A phylogenetic analysis revealed that Deg-CPR-1 is closely related to the digestive cysteine proteases of other mite species, and it was classified into a cluster different from that of chicken cathepsins. Deg-CPR-1 of PRMs in Japan has an amino acid substitution compared with that of PRMs in Europe, but it showed efficacy as a vaccine, consistent with previous findings. Deg-CPR-1 exhibited cathepsin L-like enzyme activity. In addition, the Deg-CPR-1 mRNA was expressed in the midgut and in all stages of PRMs that feed on blood. These results imply that Deg-CPR-1 in the midgut may have important functions in physiological processes, and the inhibition of its expression may contribute to the efficacy of a Deg-CPR-1-based vaccine. Further research is required to fully understand the mechanisms of vaccine efficacy.

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