Biotechnological Potential of Araucaria angustifolia Pine Nuts Extract and the Cysteine Protease Inhibitor AaCI-2S

Protease inhibitors are involved in the regulation of endogenous cysteine proteases during seed development and play a defensive role because of their ability to inhibit exogenous proteases such as those present in the digestive tracts of insects. Araucaria angustifolia seeds, which can be used in human and animal feed, were investigated for their potential for the development of agricultural biotechnology and in the field of human health. In the pine nuts extract, which blocked the activities of cysteine proteases, it was detected potent insecticidal activity against termites (Nasutitermes corniger) belonging to the most abundant termite genus in tropical regions. The cysteine inhibitor (AaCI-2S) was purified by ion-exchange, size exclusion, and reversed-phase chromatography. Its functional and structural stability was confirmed by spectroscopic and circular dichroism studies, and by detection of inhibitory activity at different temperatures and pH values. Besides having activity on cysteine proteases from C. maculatus digestive tract, AaCI-2S inhibited papain, bromelain, ficin, and cathepsin L and impaired cell proliferation in gastric and prostate cancer cell lines. These properties qualify A. angustifolia seeds as a protein source with value properties of natural insecticide and to contain a protease inhibitor with the potential to be a bioactive molecule on different cancer cells.

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

10.1101/2020.10.23.347534 ◽

2020 ◽

Cited By ~ 1

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.

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

Eukaryotic Cell ◽

10.1128/ec.00308-12 ◽

2013 ◽

Vol 12 (4) ◽

pp. 567-574 ◽

Cited By ~ 17

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.

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

Pathogens ◽

10.3390/pathogens10040388 ◽

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.

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

Journal of Cell Science ◽

10.1242/jcs.114.21.3933 ◽

2001 ◽

Vol 114 (21) ◽

pp. 3933-3942 ◽

Cited By ~ 4

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.

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Interaction of cysteine protease inhibitor mutants with cysteine proteases

South African Journal of Botany ◽

10.1016/j.sajb.2010.02.053 ◽

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

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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

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.4a6533.2005 ◽

2005 ◽

Vol 53 (5) ◽

pp. 643-651 ◽

Cited By ~ 1

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.

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CHARACTERIZATION OF PARTIALLY PURIFIED CYSTEINE PROTEASE INHIBITOR FROM THE FRUITS AND SEEDS OF SOURSOP (ANNONA MURICATA)

JOURNAL OF RESEARCH AND REVIEW IN SCIENCE ◽

10.36108/jrrslasu/0202.70.0140 ◽

2020 ◽

Vol 7 (1) ◽

Author(s):

Segun Adeola ◽

Habeeb Bankole ◽

Rahmon Kanmodi

Keyword(s):

Cysteine Protease ◽

Anticancer Agent ◽

Therapeutic Potential ◽

Cysteine Proteases ◽

Exchange Chromatography ◽

Size Exclusion ◽

Cysteine Protease Inhibitor ◽

Tropical Fruit ◽

Isolation And Purification ◽

Wide Range

Introduction: Soursop (Annona muricataLinn) is an edible lowland tropical fruit-bearing tree that is widely cultivated across regions of the world. It has been extensively researched as a result of its store of acetogenin; a potent anticancer agent. However, there is a dearth of information on the precise mechanism of action of acetogenin; thereby subjecting it to rigorous scrutiny. It is therefore imperative to investigate this plant in the hope of discovering a different class of anticancer agent inherent in it. Various studies have demonstrated that cysteine protease inhibitors (CPIs) have considerable therapeutic potential which can be utilized in a variety of disease states including cancer. Aim: Study was designed to isolate, purify and characterize CPI from the fruits and seeds of Soursop. Method: Isolation and purification of CPI was achieved by simple methods consisting of ammonium sulphate precipitation, anion exchange chromatography and size exclusion chromatography. Mode of inhibition, optimum pH and temperature, as well as the effect of metals on the enzyme activity were determined using spectrophotometry. Result: The purified CPI from seeds and fruits exhibited competitive and noncompetitive inhibition against papain respectively. However, maximal inhibitory activities for both fruit and seed samples were observed at similar optimal pH and temperature of 8 and 40°C respectively. Although, metal cations such as cobalt (Co2+), copper (Cu2+) and zinc (Zn2+) did not effect a considerable decrease on the inhibitory activity of the CPI; Lead (Pb2+), Magnesium (Mg2+) and manganese (Mn2+) significantly inhibited CPI at a very low concentration (1mM). Conclusion: The antagonistic properties exhibited by the purified CPI certainly indicate its likely suitability for pharmaceutical application in the treatment of some pathological conditions such as cancer in which uncontrolled proteolytic activities of cysteine proteases are implicated. There is an ample scope for further research on structure elucidation and protein engineering to facilitate its usage in wide range of application.

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Predicting Diagnostic Potential of Cathepsin in Epithelial Ovarian Cancer: A Design Validated by Computational, Biophysical and Electrochemical Data

Biomolecules ◽

10.3390/biom12010053 ◽

2021 ◽

Vol 12 (1) ◽

pp. 53

Author(s):

Hemangi Ranade ◽

Priya Paliwal ◽

Anis Ahmad Chaudhary ◽

Sakshi Piplani ◽

Hassan Ahmed Rudayni ◽

...

Keyword(s):

Ovarian Cancer ◽

Protease Inhibitor ◽

Epithelial Ovarian Cancer ◽

Cystatin C ◽

Gynecological Cancer ◽

Cathepsin L ◽

Cathepsin K ◽

Binding Energies ◽

Cysteine Protease Inhibitor ◽

Diagnostic Potential

Background: Epithelial ovarian cancer remains one of the leading variants of gynecological cancer with a high mortality rate. Feasibility and technical competence for screening and detection of epithelial ovarian cancer remain a major obstacle and the development of point of care diagnostics (POCD) may offer a simple solution for monitoring its progression. Cathepsins have been implicated as biomarkers for cancer progression and metastasis; being a protease, it has an inherent tendency to interact with Cystatin C, a cysteine protease inhibitor. This interaction was assessed for designing a POCD module. Methods: A combinatorial approach encompassing computational, biophysical and electron-transfer kinetics has been used to assess this protease-inhibitor interaction. Results: Calculations predicted two cathepsin candidates, Cathepsin K and Cathepsin L based on their binding energies and structural alignment and both predictions were confirmed experimentally. Differential pulse voltammetry was used to verify the potency of Cathepsin K and Cathepsin L interaction with Cystatin C and assess the selectivity and sensitivity of their electrochemical interactions. Electrochemical measurements indicated selectivity for both the ligands, but with increasing concentrations, there was a marked difference in the sensitivity of the detection. Conclusions: This work validated the utility of dry-lab integration in the wet-lab technique to generate leads for the design of electrochemical diagnostics for epithelial ovarian cancer.

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Proteases as Therapeutic Targets Against the Parasitic Cnidarian Ceratonova shasta: Characterization of Molecules Key to Parasite Virulence In Salmonid Hosts

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.804864 ◽

2022 ◽

Vol 11 ◽

Author(s):

Gema Alama-Bermejo ◽

Pavla Bartošová-Sojková ◽

Stephen D. Atkinson ◽

Astrid S. Holzer ◽

Jerri L. Bartholomew

Keyword(s):

Pacific Northwest ◽

Cathepsin D ◽

Cell Metabolism ◽

Cathepsin L ◽

Cysteine Proteases ◽

Aminopeptidase N ◽

Cysteine Protease Inhibitor ◽

Parasite Development ◽

Future Research ◽

3D Protein Structure

Proteases and their inhibitors play critical roles in host-parasite interactions and in the outcomes of infections. Ceratonova shasta is a myxozoan pathogen that causes enteronecrosis in economically important salmonids from the Pacific Northwest of North America. This cnidarian parasite has host-specific genotypes with varying virulence, making it a powerful system to decipher virulence mechanisms in myxozoans. Using C. shasta genome and transcriptome, we identified four proteases of different catalytic types: cathepsin D (aspartic), cathepsin L and Z-like (cysteine) and aminopeptidase-N (metallo); and a stefin (cysteine protease inhibitor), which implied involvement in virulence and hence represent target molecules for the development of therapeutic strategies. We characterized, annotated and modelled their 3D protein structure using bioinformatics and computational tools. We quantified their expression in C. shasta genotype 0 (low virulence, no mortality) and IIR (high virulence and mortality) in rainbow trout Oncorhynchus mykiss, to demonstrate that there are major differences between the genotypes during infection and parasite development. High proliferation of genotype IIR was associated with high expression of the cathepsin D and the stefin, likely correlated with high nutrient demands and to regulate cell metabolism, with upregulation preceding massive proliferation and systemic dispersion. In contrast, upregulation of the cathepsin L and Z-like cysteine proteases may have roles in host immune evasion in genotype 0 infections, which are associated with low proliferation, low inflammation and non-destructive development. In contrast to the other proteases, C. shasta aminopeptidase-N appears to have a prominent role in nematocyst formation in both genotypes, but only during sporogenesis. Homology searches of C. shasta proteases against other myxozoan transcriptomes revealed a high abundance of cathepsin L and aminopeptidase homologs suggesting common gene requirements across species. Our study identified molecules of potential therapeutic significance for aquaculture and serves as a baseline for future research aimed at functional characterisation of these targets.

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