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

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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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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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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Repurposing the McoTI-II Rigid Molecular Scaffold in to Inhibitor of ‘Papain Superfamily’ Cysteine Proteases

Pharmaceuticals ◽

10.3390/ph14010007 ◽

2020 ◽

Vol 14 (1) ◽

pp. 7

Author(s):

Manasi Mishra ◽

Vigyasa Singh ◽

Meenakshi B. Tellis ◽

Rakesh S. Joshi ◽

Shailja Singh

Keyword(s):

Cyclic Peptide ◽

Homology Modelling ◽

Cathepsin L ◽

Cysteine Proteases ◽

Protein Docking ◽

Dynamics Simulation ◽

Cysteine Protease Inhibitor ◽

Full Potential ◽

Molecular Scaffold ◽

Mechanism Of Inhibition

Clan C1A or ‘papain superfamily’ cysteine proteases are key players in many important physiological processes and diseases in most living systems. Novel approaches towards the development of their inhibitors can open new avenues in translational medicine. Here, we report a novel design of a re-engineered chimera inhibitor Mco-cysteine protease inhibitor (CPI) to inhibit the activity of C1A cysteine proteases. This was accomplished by grafting the cystatin first hairpin loop conserved motif (QVVAG) onto loop 1 of the ultrastable cyclic peptide scaffold McoTI-II. The recombinantly expressed Mco-CPI protein was able to bind with micromolar affinity to papain and showed remarkable thermostability owing to the formation of multi-disulphide bonds. Using an in silico approach based on homology modelling, protein–protein docking, the calculation of the free-energy of binding, the mechanism of inhibition of Mco-CPI against representative C1A cysteine proteases (papain and cathepsin L) was validated. Furthermore, molecular dynamics simulation of the Mco-CPI–papain complex validated the interaction as stable. To conclude, in this McoTI-II analogue, the specificity had been successfully redirected towards C1A cysteine proteases while retaining the moderate affinity. The outcomes of this study pave the way for further modifications of the Mco-CPI design for realizing its full potential in therapeutics. This study also demonstrates the relevance of ultrastable peptide-based scaffolds for the development of novel inhibitors via grafting.

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

Plants ◽

10.3390/plants9121676 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1676

Author(s):

Roberto Carlos Sallai ◽

Bruno Ramos Salu ◽

Rosemeire Aparecida Silva-Lucca ◽

Flávio Lopes Alves ◽

Thiago Henrique Napoleão ◽

...

Keyword(s):

Protease Inhibitor ◽

Animal Feed ◽

Cathepsin L ◽

Cysteine Proteases ◽

Reversed Phase ◽

Size Exclusion ◽

Cysteine Protease Inhibitor ◽

Araucaria Angustifolia ◽

Tropical Regions ◽

Defensive Role

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

Parasitology ◽

10.1017/s0031182012000297 ◽

2012 ◽

Vol 139 (8) ◽

pp. 1029-1037 ◽

Cited By ~ 13

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.

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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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Optimization of Plasmodium vivax sporozoite production from Anopheles stephensi in South West India

Malaria Journal ◽

10.1186/s12936-021-03767-2 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Ajeet Kumar Mohanty ◽

Charles de Souza ◽

Deepika Harjai ◽

Prathamesh Ghavanalkar ◽

Mezia Fernandes ◽

...

Keyword(s):

South Asia ◽

Plasmodium Vivax ◽

Anopheles Stephensi ◽

Parasite Development ◽

Future Research ◽

High Quality ◽

Gametocyte Density ◽

Feeding Experiments ◽

South West ◽

Membrane Feeding

Abstract Background Efforts to study the biology of Plasmodium vivax liver stages, particularly the latent hypnozoites, have been hampered by the limited availability of P. vivax sporozoites. Anopheles stephensi is a major urban malaria vector in Goa and elsewhere in South Asia. Using P. vivax patient blood samples, a series of standard membrane-feeding experiments were performed with An. stephensi under the US NIH International Center of Excellence for Malaria Research (ICEMR) for Malaria Evolution in South Asia (MESA). The goal was to understand the dynamics of parasite development in mosquitoes as well as the production of P. vivax sporozoites. To obtain a robust supply of P. vivax sporozoites, mosquito-rearing and mosquito membrane-feeding techniques were optimized, which are described here. Methods Membrane-feeding experiments were conducted using both wild and laboratory-colonized An. stephensi mosquitoes and patient-derived P. vivax collected at the Goa Medical College and Hospital. Parasite development to midgut oocysts and salivary gland sporozoites was assessed on days 7 and 14 post-feeding, respectively. The optimal conditions for mosquito rearing and feeding were evaluated to produce high-quality mosquitoes and to yield a high sporozoite rate, respectively. Results Laboratory-colonized mosquitoes could be starved for a shorter time before successful blood feeding compared with wild-caught mosquitoes. Optimizing the mosquito-rearing methods significantly increased mosquito survival. For mosquito feeding, replacing patient plasma with naïve serum increased sporozoite production > two-fold. With these changes, the sporozoite infection rate was high (> 85%) and resulted in an average of ~ 22,000 sporozoites per mosquito. Some mosquitoes reached up to 73,000 sporozoites. Sporozoite production could not be predicted from gametocyte density but could be predicted by measuring oocyst infection and oocyst load. Conclusions Optimized conditions for the production of high-quality P. vivax sporozoite-infected An. stephensi were established at a field site in South West India. This report describes techniques for producing a ready resource of P. vivax sporozoites. The improved protocols can help in future research on the biology of P. vivax liver stages, including hypnozoites, in India, as well as the development of anti-relapse interventions for vivax malaria.

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Perceptions of Shared Leadership within Academic Libraries Suggest Room for Improvement

Evidence Based Library and Information Practice ◽

10.18438/b82d0h ◽

2011 ◽

Vol 6 (3) ◽

pp. 82

Author(s):

Genevieve C. Gore

Keyword(s):

Pacific Northwest ◽

Academic Libraries ◽

Shared Leadership ◽

Middle Managers ◽

The United States ◽

Future Research ◽

Cross Sectional Survey ◽

Cross Sectional ◽

The Third ◽

Level Of Agreement

Objective – To survey middle managers’ beliefs regarding their participation in shared leadership and their libraries’ practices of shared leadership. Design – Cross-sectional survey. Setting – Twenty-two academic libraries within four-year public master’s level institutions in the Pacific Northwest of the United States. Subjects – A total of 115 middle managers were invited to participate; 77 completed the survey for a response rate of 66.9%. Methods – Selected middle managers were contacted by email a total of five times and were invited to complete a Web-based survey composed of three sections. The first section contained 10 statements for rating shared leadership within their own institutions, which they were asked to rate on an eight-point Likert scale from 1 (strongly agree) to 7 (strongly disagree), with 8 as an option for no opinion. The second section used the same scale to rate their levels of agreement with conceptual definitions of shared leadership from Jackson’s Framework. Jackson’s Framework consists of four components for ascertaining levels of shared leadership from both managerial and staff perspectives: accountability, equity, partnership and ownership. The third section invited subjects to provide their own definition of shared leadership. A three-part pretest served to validate the survey instrument. Mean scores were calculated for each statement. Main Results – In the first section, there was the highest overall level of agreement (1.52) with the statement “I am accountable for the decisions within the scope of my responsibility” followed by “I share information with the senior library administration” (1.71). There was the lowest overall level of agreement (3.65) with the statement that “Ideas presented at all levels of staff in the library are equally considered.” In the second section, respondents’ mean scores for Jackson’s definitions of four concepts of shared leadership were 2.62 for ownership, 2.68 for both partnership and equity, and 2.77 for accountability. In the third section, respondents most often linked their definitions of shared leadership with communication, learning and collaboration. Conclusion – Examining middle managers’ perceptions of shared leadership may help us understand organizational trends and capacity for leadership within libraries. Future research could examine shared leadership at levels below middle management as well as the relationship between accountability and shared leadership throughout the organization.

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Molecular Characterization of a Novel Cathepsin L in Macrobrachium nipponense and Its Function in Ovary Maturation

Frontiers in Endocrinology ◽

10.3389/fendo.2021.816813 ◽

2022 ◽

Vol 12 ◽

Author(s):

Sufei Jiang ◽

Yiwei Xiong ◽

Wenyi Zhang ◽

Junpeng Zhu ◽

Dan Cheng ◽

...

Keyword(s):

Cathepsin L ◽

Cysteine Proteases ◽

Macrobrachium Nipponense ◽

Reading Frame ◽

Physiological Processes ◽

Gonad Somatic Index ◽

Oriental River Prawn ◽

Ovary Maturation

Cathepsin L genes, which belonged to cysteine proteases, were a series of multifunctional protease and played important roles in a lot of pathological and physiological processes. In this study, we analyzed the characteristics a cathepsin L (named Mn-CL2) in the female oriental river prawn, Macrobrachium nipponense which was involved in ovary maturation. The Mn-CL2 was1,582 bp in length, including a 978 bp open reading frame that encoded 326 amino acids. The Mn-CL2 was classified into the cathepsin L group by phylogenetic analysis. Real-time PCR (qPCR) analysis indicated that Mn-CL2 was highly expressed in the hepatopancreas and ovaries of female prawns. During the different ovarian stages, Mn-CL2 expression in the hepatopancreas and ovaries peaked before ovarian maturation. In situ hybridization studies revealed that Mn-CL2 was localized in the oocyte of the ovary. Injection of Mn-CL2 dsRNA significantly reduced the expression of vitellogenin. Changes in the gonad somatic index also confirmed the inhibitory effects of Mn-CL2 dsRNA on ovary maturation. These results suggest that Mn-CL2 has a key role in promoting ovary maturation.

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