Cystatin A protease inhibitor and cysteine proteases in human cervicovaginal fluid in term pregnancy and labor

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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Increased levels of the megakaryocyte and platelet expressed cysteine proteases stefin A and cystatin A prevent thrombosis

Scientific Reports ◽

10.1038/s41598-019-45805-9 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Anna Mezzapesa ◽

Delphine Bastelica ◽

Lydie Crescence ◽

Marjorie Poggi ◽

Michel Grino ◽

...

Keyword(s):

Cysteine Proteases ◽

Stefin A ◽

Cystatin A

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Extraction and Characterization of Cathepsin Inhibitor from Milkfish

Jurnal Pengolahan Hasil Perikanan Indonesia ◽

10.17844/jphpi.v18i1.9562 ◽

2015 ◽

Vol 18 (1) ◽

Author(s):

Tati Nurhayati ◽

Ruddy Suwandi ◽

Syefri Rusyadi

Keyword(s):

Protease Inhibitor ◽

Metal Ion ◽

Proteolytic Enzyme ◽

Cysteine Proteases ◽

Partial Purification ◽

Optimum Temperature ◽

Endogenous Inhibitors ◽

Protease Enzyme ◽

Myofibril Protein

Proteolytic enzyme is distributed acros all organism including fish. Cysteine proteases are the largest group of proteolytic enzyme. Lysosomal cathepsin, one of cysteine protease enzyme, cause softening and degradation of myofibril protein and it’s activity is regulated by endogenous inhibitors. The purposes of this study were to optimize the extraction cathepsin inhibitors from the skin, muscles, and viscera of fish, to partially purify the cathepsin inhibitors of selected sources, and to study the characteristics of the cathepsin inhibitor. The cathepsin inhibitor could be extracted from muscle fish and partially purified using ammonium sulfate of 70%. The purified cathepsin inhibitor had optimum temperature at 40°C and the optimum at pH 8. Metal ions decreased the activity of the protease inhibitor, except 1 mM of metal ion Mn2+ and Na+. Keywords: Cathepsin, characterization, partial purification, protease inhibitor

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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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The role of the second binding loop of the cysteine protease inhibitor, cystatin A (stefin A), in stabilizing complexes with target proteases is exerted predominantly by Leu73

European Journal of Biochemistry ◽

10.1046/j.1432-1033.2002.03273.x ◽

2002 ◽

Vol 269 (22) ◽

pp. 5649-5658 ◽

Cited By ~ 7

Author(s):

Alona Pavlova ◽

Sergio Estrada ◽

Ingemar Björk

Keyword(s):

Protease Inhibitor ◽

Cysteine Protease ◽

Cysteine Protease Inhibitor ◽

Stefin A ◽

Cystatin A ◽

Binding Loop

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Amblyomma americanumtick saliva serine protease inhibitor 6 is a cross-class inhibitor of serine proteases and papain-like cysteine proteases that delays plasma clotting and inhibits platelet aggregation

Insect Molecular Biology ◽

10.1111/imb.12024 ◽

2013 ◽

Vol 22 (3) ◽

pp. 306-319 ◽

Cited By ~ 41

Author(s):

A. Mulenga ◽

T. Kim ◽

A. M. G. Ibelli

Keyword(s):

Platelet Aggregation ◽

Protease Inhibitor ◽

Serine Protease ◽

Serine Proteases ◽

Cysteine Proteases ◽

Serine Protease Inhibitor

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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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