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 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.

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.

A cysteine protease inhibitor blocks SARS-CoV-2 infection of human and monkey cells

2020 ◽  
Author(s):  
Drake M. Mellott ◽  
Chien-Te Tseng ◽  
Aleksandra Drelich ◽  
Pavla Fajtová ◽  
Bala C. Chenna ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Host Cell ◽  
Cysteine Protease ◽  
Cleavage Site ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Protein Processing ◽  
Spike Protein ◽  
Cysteine Protease Inhibitor ◽  
Viral Infectivity

ABSTRACTK777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 μM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 μM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2, differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.SIGNIFICANCEThe virus causing COVID-19 is highly infectious and has resulted in a global pandemic. We confirm that a cysteine protease inhibitor, approved by the FDA as a clinical-stage compound, inhibits SARS-CoV-2 infection of several human and monkey cell lines with notable(nanomolar) efficacy. The mechanism of action of this inhibitor is identified as a specific inhibition of host cell cathepsin L. This in turn inhibits host cell processing of the coronaviral spike protein, a step required for cell entry. Neither of the coronaviral proteases are inhibited, and the cleavage site of spike protein processing is different from that reported in other coronaviruses. Hypotheses to explain the differential activity of the inhibitor with different cell types are discussed.

scholarly journals Identification and design of vinyl sulfone inhibitors against Cryptopain-1 – a cysteine protease from cryptosporidiosis-causing Cryptosporidium parvum

2018 ◽  
Author(s):  
Arpita Banerjee
Keyword(s):  
Cryptosporidium Parvum ◽  
Cysteine Protease ◽  
Drug Targets ◽  
Computational Study ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Vinyl Sulfone ◽  
Docking Simulations ◽  
Stunted Growth ◽  
Ligand Interactions

AbstractCryptosporidiosis, a disease marked by diarrhea in adults and stunted growth in children, is associated with the unicellular protozoan pathogen Cryptosporidium; often the species parvum. Cryptopain-1, a cysteine protease characterized in the genome of Cryptosporidium parvum, had been earlier shown to be inhibited by a vinyl sulfone compound called K11777 (or K-777). Cysteine proteases have long been established as valid drug targets, which can be covalently and selectively inhibited by vinyl sulfones. This computational study was initiated to identify purchasable vinyl sulfone compounds, which could possibly inhibit cryptopain-1 with higher efficacy than K11777. Docking simulations screened a number of such possibly better inhibitors. The work was furthered to probe the enzymatic pocket of cryptopain-1, through in-silico mutations, to derive a map of receptor-ligand interactions in the docked complexes. The idea was to provide crucial clues to aid the design of inhibitors, which would be able to bind the protease well by making favorable interactions with important residues of the enzyme. The analyses dictated placement of ligands towards the front of the enzymatic cleft, and disfavored interactions deep within. The S1’ and S2 subsites of the enzyme preferred to remain occupied by polar ligand subgroups. Reasonably distanced ring systems and polar backbones of ligands were desired across the cleft. Large as well as inflexible subgroups were not tolerated. Double ringed systems such as substituted napthalene, especially in S1, were exceptions though. The S2 subsite, which is typically a specificity determinant in papain (C1) family cysteine proteases such as cathepsin L-like cryptopain-1, can possibly accommodate polar and hydrophobic ligand subgroups alike.

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 A Novel Cysteine Protease Inhibitor of Naegleria fowleri That Is Specifically Expressed during Encystation and at Mature Cysts

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 388
Author(s):  
Hương Giang Lê ◽  
A-Jeong Ham ◽  
Jung-Mi Kang ◽  
Tuấn Cường Võ ◽  
Haung Naw ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Expression Profiles ◽  
Critical Role ◽  
Cyst Formation ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Naegleria Fowleri ◽  
Nægleria Fowleri ◽  
Cystatin Family

Naegleria fowleri is a free-living amoeba that is ubiquitous in diverse natural environments. It causes a fatal brain infection in humans known as primary amoebic meningoencephalitis. Despite the medical importance of the parasitic disease, there is a great lack of knowledge about the biology and pathogenicity of N. fowleri. In this study, we identified and characterized a novel cysteine protease inhibitor of N. fowleri (NfCPI). NfCPI is a typical cysteine protease inhibitor belonging to the cystatin family with a Gln-Val-Val-Ala-Gly (QVVAG) motif, a characteristic motif conserved in the cystatin family of proteins. Bacterially expressed recombinant NfCPI has a dimeric structure and exhibits inhibitory activity against several cysteine proteases including cathespin Bs of N. fowleri at a broad range of pH values. Expression profiles of nfcpi revealed that the gene was highly expressed during encystation and cyst of the amoeba. Western blot and immunofluorescence assays also support its high level of expression in cysts. These findings collectively suggest that NfCPI may play a critical role in encystation or cyst formation of N. fowleri by regulating cysteine proteases that may mediate encystation or mature cyst formation of the amoeba. More comprehensive studies to investigate the roles of NfCPI in encystation and its target proteases are necessary to elucidate the regulatory mechanism and the biological significance of NfCPI.

scholarly journals Studies of Inhibitory Mechanisms of Propeptide-Like Cysteine Protease Inhibitors

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Bui T. T. Nga ◽  
Yuki Takeshita ◽  
Misa Yamamoto ◽  
Yoshimi Yamamoto
Keyword(s):  
Protease Inhibitors ◽  
Cysteine Protease ◽  
Cathepsin L ◽  
Cysteine Residue ◽  
Cysteine Protease Inhibitor ◽  
Inhibition Mechanism ◽  
Cysteine Protease Inhibitors ◽  
Inhibitory Potency ◽  
Inhibitory Mechanisms

Mouse cytotoxic T-lymphocyte antigen-2α (CTLA-2α), Drosophila CTLA-2-like protein (crammer), and Bombyx cysteine protease inhibitor (BCPI) belong to a novel family of cysteine protease inhibitors (I29). Their inhibitory mechanisms were studied comparatively. CTLA-2α contains a cysteine residue (C75), which is essential for its inhibitory potency. The CTLA-2α monomer was converted to a disulfide-bonded dimer in vitro and in vivo. The dimer was fully inhibitory, but the monomer, which possessed a free thiol residue, was not. A disulfide-bonded CTLA-2α/cathepsin L complex was isolated, and a cathepsin L subunit with a molecular weight of 24,000 was identified as the interactive enzyme protein. Crammer also contains a cysteine residue (C72). Both dimeric and monomeric forms of crammer were inhibitory. A crammer mutant with Cys72 to alanine (C72A) was fully inhibitory, while the replacement of Gly73 with alanine (G73A) caused a significant loss in inhibitory potency, which suggests a different inhibition mechanism from CTLA-2α. BCPI does not contain cysteine residue. C-terminal region (L77-R80) of BCPI was essential for its inhibitory potency. CTLA-2α was inhibitory in the acidic pH condition but stabilized cathepsin L under neutral pH conditions. The different inhibition mechanisms and functional considerations of these inhibitors are discussed.

Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor inTrypanosoma cruzi

2001 ◽  
Vol 114 (21) ◽  
pp. 3933-3942 ◽  
Author(s):  
Ana C. S. Monteiro ◽  
Magnus Abrahamson ◽  
Ana P. C. A. Lima ◽  
Marcos A. Vannier-Santos ◽  
Julio Scharfstein
Keyword(s):  
Cell Surface ◽  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Mammalian Cells ◽  
Tight Binding ◽  
Cysteine Proteases ◽  
Host Cells ◽  
Cysteine Protease Inhibitor ◽  
Reversible Inhibitor ◽  
Mammalian Host

Lysosomal cysteine proteases from mammalian cells and plants are regulated by endogenous tight-binding inhibitors from the cystatin superfamily. The presence of cystatin-like inhibitors in lower eukaryotes such as protozoan parasites has not yet been demonstrated, although these cells express large quantities of cysteine proteases and may also count on endogenous inhibitors to regulate cellular proteolysis. Trypanosoma cruzi, the causative agent of Chagas’ heart disease, is a relevant model to explore this possibility because these intracellular parasites rely on their major lysosomal cysteine protease (cruzipain) to invade and multiply in mammalian host cells. Here we report the isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cysteine protease inhibitor in T. cruzi. We used high temperature induced denaturation to isolate a heat-stable cruzipain-binding protein (apparent molecular mass, 12 kDa) from epimastigote lysates. This protein was subsequently characterized as a tight-binding and reversible inhibitor of papain-like cysteine proteases. Immunoblotting indicated that the expression of chagasin is developmentally regulated and inversely correlated with that of cruzipain. Gold-labeled antibodies localized chagasin to the flagellar pocket and cytoplasmic vesicles of trypomastigotes and to the cell surface of amastigotes. Binding assays performed by probing living parasites with fluorescein (FITC)-cruzipain or FITC-chagasin revealed the presence of both inhibitor and protease at the cell surface of amastigotes. The intersection of chagasin and cruzipain trafficking pathways may represent a checkpoint for downstream regulation of proteolysis in trypanosomatid protozoa.

scholarly journals Interaction of cysteine protease inhibitor mutants with cysteine proteases

2010 ◽  
Vol 76 (2) ◽  
pp. 406
Author(s):  
S.G. Van Wyk ◽  
K.J. Kunert ◽  
B.J. Vorster ◽  
U. Schluter
Keyword(s):  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor

Cryptopain-1, a cysteine protease of Cryptosporidium parvum, does not require the pro-domain for folding

Parasitology ◽  
2008 ◽  
Vol 136 (2) ◽  
pp. 149-157 ◽  
Author(s):  
B.-K. NA ◽  
J.-M. KANG ◽  
H.-I. CHEUN ◽  
S.-H. CHO ◽  
S.-U. MOON ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Cysteine Protease ◽  
Transmembrane Domain ◽  
Cathepsin L ◽  
Active Form ◽  
Protozoan Parasite ◽  
Biochemical Properties ◽  
Peptide Sequence ◽  
Signal Peptide Sequence ◽  
Mature Domain

SUMMARYCryptosporidium parvum is an intracellular protozoan parasite that causes cryptosporidiosis in mammals including humans. In the current study, the gene encoding the cysteine protease of C. parvum (cryptopain-1) was identified and the biochemical properties of the recombinant enzyme were characterized. Cryptopain-1 shared common structural properties with cathepsin L-like papain family enzymes, but lacked a typical signal peptide sequence and contained a possible transmembrane domain near the amino terminus and a unique insert in the front of the mature domain. The recombinant cryptopain-1 expressed in Escherichia coli and refolded to the active form showed typical biochemical properties of cathepsin L-like enzymes. The folding determinant of cryptopain-1 was characterized through multiple constructs with or without different lengths of the pro-domain of the enzyme expressed in E. coli and assessment of their refolding abilities. All constructs, except one that did not contain the full-length mature domain, successfully refolded into the active enzymes, suggesting that cryptopain-1 did not require the pro-domain for folding. Western blot analysis showed that cryptopain-1 was expressed in the sporozoites and the enzyme preferentially degraded proteins, including collagen and fibronectin, but not globular proteins. This suggested a probable role for cryptopain-1 in host cell invasion and/or egression by the parasite.

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