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

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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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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Using serpins cysteine protease cross-specificity to possibly trap SARS-CoV-2 Mpro with reactive center loop chimera

Clinical Science ◽

10.1042/cs20200767 ◽

2020 ◽

Vol 134 (17) ◽

pp. 2235-2241

Author(s):

Mohamad Aman Jairajpuri ◽

Shoyab Ansari

Keyword(s):

Cysteine Protease ◽

Active Site ◽

Cleavage Site ◽

Serine Proteases ◽

Therapeutic Potential ◽

Cysteine Proteases ◽

Protease Inhibition ◽

Reactive Center Loop ◽

Main Protease ◽

Reactive Center

Abstract Human serine protease inhibitors (serpins) are the main inhibitors of serine proteases, but some of them also have the capability to effectively inhibit cysteine proteases. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) main protease (Mpro) is a chymotrypsin-type cysteine protease that is needed to produce functional proteins essential for virus replication and transcription. Serpin traps its target proteases by presenting a reactive center loop (RCL) as protease-specific cleavage site, resulting in protease inactivation. Mpro target sites with its active site serine and other flanking residues can possibly interact with serpins. Alternatively, RCL cleavage site of serpins with known evidence of inhibition of cysteine proteases can be replaced by Mpro target site to make chimeric proteins. Purified chimeric serpin can possibly inhibit Mpro that can be assessed indirectly by observing the decrease in ability of Mpro to cleave its chromogenic substrate. Chimeric serpins with best interaction and active site binding and with ability to form 1:1 serpin–Mpro complex in human plasma can be assessed by using SDS/PAGE and Western blot analysis with serpin antibody. Trapping SARS-CoV-2 Mpro cysteine protease using cross-class serpin cysteine protease inhibition activity is a novel idea with significant therapeutic potential.

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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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Trypanosomatid cysteine protease activity may be enhanced by a kininogen-like moiety from host serum

Biochemical Journal ◽

10.1042/bj3050549 ◽

1995 ◽

Vol 305 (2) ◽

pp. 549-556 ◽

Cited By ~ 15

Author(s):

J D Lonsdale-Eccles ◽

G W N Mpimbaza ◽

Z R M Nkhungulu ◽

J Olobo ◽

L Smith ◽

...

Keyword(s):

Molecular Mass ◽

Protease Inhibitors ◽

Cysteine Protease ◽

Leishmania Donovani ◽

Leishmania Major ◽

Cysteine Proteases ◽

Cysteine Protease Inhibitor ◽

Cysteine Protease Inhibitors ◽

African Trypanosomes

African trypanosomes contain cysteine proteases (trypanopains) the activity of which can be measured by in vitro digestion of fibrinogen, after electrophoresis in fibrinogen-containing SDS/polyacrylamide gels. When assessed by this procedure, trypanopain from Trypanosoma brucei (trypanopain-Tb) is estimated to have a molecular mass of 28 kDa. However, two additional bands of trypanopain activity (87 kDa and 105 kDa) are observed if serum is added to the trypanopain before electrophoresis. Formation of the 87 and 105 kDa bands is frequently accompanied by a reduction in the intensity of the 28 kDa activity which suggests that the extra bands are complexes of the 28 kDa trypanopain-Tb and a molecule from rat serum called rat trypanopain moledulator (rTM). The rTM-induced activation of cysteine proteases is not restricted to T. brucei as it is also observed with proteases from other protozoan parasites such as bloodstream forms of Trypanosoma congolense and the mammalian-infective in vitro-derived promastigote forms of Leishmania donovani and Leishmania major. The physical properties of rTM resemble those of the kininogen family of cysteine protease inhibitors. rTM is an acidic (pI 4.7) heat-stable 68 kDa glycoprotein with 15 kDa protease-susceptible domains. This resemblance between rTM and kininogens was confirmed by the positive, albeit weak, immunoreactivity between anti-(human low-molecular-mass kininogen) antibody and rTM as well as anti-rTM antibody and human low-molecular-mass kininogen. Furthermore, commercial preparations of human-low-molecular-mass kininogen and chicken egg white cystatin mimicked rTM by forming extra bands of proteolytic activity in the presence of trypanopain-Tb. In some instances, low-molecular-mass kininogen was also observed to increase the rate of hydrolysis of 7-(benzyloxycarbonyl-phenylalanyl-arginyl-amido)-4- methylcoumarin by live T. brucei. Although this effect was rather erratic, in no instance was significant inhibition observed when this putative cysteine protease inhibitor was used under these conditions. The activation of parasite cysteine proteases by commonly accepted cysteine protease inhibitors is unexpected and may have important pathological repercussions.

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A Tick Cysteine Protease Inhibitor RHcyst-1 Exhibits Antitumor Potential

Cellular Physiology and Biochemistry ◽

10.1159/000489645 ◽

2018 ◽

Vol 46 (6) ◽

pp. 2385-2400 ◽

Cited By ~ 3

Author(s):

Nana Wei ◽

Zhibing Lin ◽

Zhengmao Xu ◽

Jie Cao ◽

Yongzhi Zhou ◽

...

Keyword(s):

Tumor Cells ◽

Cysteine Protease ◽

Tumor Therapy ◽

Cysteine Proteases ◽

Cysteine Protease Inhibitor ◽

Cell Counting ◽

Migration And Invasion ◽

Mouse Tumor ◽

Antitumor Effects

Background/Aims: We previously identified a potent and tight-binding inhibitor of cysteine proteases from Rhipicephalus haemaphysaloides, RHcyst-1, which belongs to the cystatin type 1 family. Cathepsins, which are members of the cysteine protease family, participate in various pathological processes, including the initiation and development of cancers. The present study aimed to investigate the antitumor effects of RHcyst-1 and to explore the underlying mechanism of these effects. Methods: Different tumor cells were treated with RHcyst-1 in vitro. Proliferation activity was evaluated using Cell Counting Kit-8, and migration and invasion were determined by wound healing and Transwell® invasion assays. In addition, a mouse tumor therapy model was established by inoculating the left forelimb of mice with B16-F10 cells, and tumor progression was evaluated by assessing tumor volume and survival. Flow cytometry was conducted to evaluate myeloid-derived suppressor cells (MDSCs), CD4+, and CD8+ T cell levels in PBMCs and spleens. Immunohistochemistry was performed to analyze immune cell infiltration and angiogenesis in the tumors. Results: RHcyst-1 significantly inhibited the proliferation, migration, and invasion of all four different tumor cells in vitro. Additionally, it inhibited tumor growth and improved survival in vivo. A decrease and an increase in MDSCs levels were observed in PBMCs and in the spleen, respectively, after RHcyst-1 application. Conclusions: Tick RHcyst-1 has potential antitumor efficacy, and the observed antitumor activities may be partly attributable to changes in cathepsin expression and MDSCs levels in the PBMCs and spleens. The findings of the present study suggest that RHcyst-1 may have the potential to be utilized in cancer treatment.

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Inhibition of Plasmodium falciparum Oocyst Production by Membrane-Permeant Cysteine Protease Inhibitor E64d

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01012-06 ◽

2006 ◽

Vol 51 (3) ◽

pp. 1064-1070 ◽

Cited By ~ 13

Author(s):

S. Eksi ◽

B. Czesny ◽

G.-J. van Gemert ◽

R. W. Sauerwein ◽

W. Eling ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Blood Cell ◽

Protease Inhibitor ◽

Red Blood Cell ◽

Cysteine Protease ◽

Malaria Parasite ◽

Cysteine Proteases ◽

Food Vacuole ◽

Cysteine Protease Inhibitor ◽

Parasite Transmission

ABSTRACT During asexual intraerythrocytic growth, Plasmodium falciparum utilizes hemoglobin obtained from the host red blood cell (RBC) as a nutrient source. Papain-like cysteine proteases, falcipains 2 and 3, have been reported to be involved in hemoglobin digestion and are targets of current antimalarial drug development efforts. However, their expression during gametocytogenesis, which is required for malaria parasite transmission, has not been studied. Many of the available antimalarials do not inhibit development of sexual stage parasites, and therefore, the persistence of gametocytes after drug treatment allows continued transmission of the disease. In the work reported here, incubation of stage V gametocytes with membrane-permeant cysteine protease inhibitor E64d significantly inhibited oocyst production (80 to 100%). The same conditions inhibited processing of gametocyte-surface antigen Pfs230 during gametogenesis but did not alter the morphology of the food vacuole in gametocytes, inhibit emergence, or block male exflagellation. E64d reduced the level of oocyst production more effectively than that reported previously for falcipain 1-knockout parasites, suggesting that falcipains 2 and 3 may also be involved in malaria parasite transmission. However, in this study only falcipain 3 and not falcipain 2 was found to be expressed in stage V gametocytes. Interestingly, during gametocytogenesis falcipain 3 was transported into the red blood cell and by stage V was localized in vesicles along the RBC surface, consistent with a role during gamete emergence. The ability of a membrane-permeant cysteine protease inhibitor to significantly reduce malaria parasite transmission suggests that future drug design should include evaluation of gametogenesis and sporogonic development.

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