scholarly journals Use of Recombinant Entamoeba histolytica Cysteine Proteinase 1 To Identify a Potent Inhibitor of Amebic Invasion in a Human Colonic Model

2007 ◽  
Vol 6 (7) ◽  
pp. 1130-1136 ◽  
Author(s):  
Samuel G. Meléndez-López ◽  
Scott Herdman ◽  
Ken Hirata ◽  
Min-Ho Choi ◽  
Youngchool Choe ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Combinatorial Libraries ◽  
Host Immune System ◽  
Dependent Manner ◽  
Vinyl Sulfone ◽  
Cysteine Proteinases ◽  
Ph Optimum

ABSTRACT Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.

scholarly journals Cystatins — Inhibitors of Cysteine Proteinases

1992 ◽  
Vol 3 (4) ◽  
pp. 307-332 ◽  
Author(s):  
Libuse A. Bobek ◽  
Michael J. Levine
Keyword(s):  
Common Ancestor ◽  
Molecular Level ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Diverse Group ◽  
Cysteine Proteinases ◽  
Future Directions ◽  
Cysteine Proteinase Inhibitors ◽  
Physicochemical And Functional Properties

The cystatin superfamily of proteins, derived from a common ancestor, is comprised of a diverse group of potent cysteine proteinase inhibitors and antibacterial/viral agents grouped into several families. This review concentrates on family 2 cystatins, namely, the human salivary cystatins and cystatin C. Emphasis is given to their physicochemical and functional properties at both the protein and the molecular level. The role of cystatins in disease processes, including those in the oral cavity, is also discussed. Finally, future directions for cystatin research in oral biology are presented.

scholarly journals Entamoeba histolytica Cysteine Proteinases Disrupt the Polymeric Structure of Colonic Mucin and Alter Its Protective Function

2003 ◽  
Vol 71 (2) ◽  
pp. 838-844 ◽  
Author(s):  
Darcy Moncada ◽  
Kathy Keller ◽  
Kris Chadee
Keyword(s):  
Entamoeba Histolytica ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Colonic Epithelium ◽  
Dependent Manner ◽  
Cysteine Proteinases ◽  
Protective Function ◽  
Mucous Layer ◽  
Colonic Mucin ◽  
Cscl Density Gradient

ABSTRACT The adherent mucous gel layer lining the colonic epithelium is the first line of host defense against invasive pathogens, such as Entamoeba histolytica. The mucous layer prevents the attachment of amoeba to the colonic epithelium by trapping and aiding in the expulsion of the parasite. Disruption of the mucous layer is thought to occur in invasive amebiasis, and the mechanism by which the parasite overcomes this barrier is not known. The aim of this study was to characterize the specific interactions occurring between E. histolytica secreted cysteine proteinases and colonic mucin as a model to examine the initial events of invasive amebiasis. E. histolytica secreted products were examined for mucinase activity utilizing mucin metabolically labeled with [35S]cysteine as a substrate. Cysteine proteinases degraded mucin in a time- and dose-dependent manner. A significant reduction (>50%) in high-molecular-weight mucin with altered buoyant density was observed when degraded mucin was analyzed by Sepharose 4B column chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and CsCl density gradient centrifugation. Mucinase activity was eliminated by the specific cysteine protease inhibitor trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane and was independent of glycosidase activity. Moreover, the degraded mucin was 38% less effective than native mucin at inhibiting amebic adherence to target epithelial cells. These results are the first to show that E. histolytica cysteine proteinases alter the protective function of the mucous barrier by disrupting the structure of the MUC2 polymer. Mechanistically, the parasite achieves this via proteolytic degradation of the terminal cysteine-rich domains.

Involvement of cysteine proteinases in excystment of Paragonimus ohirai metacercariae induced by sodium cholate and A23187

2003 ◽  
Vol 77 (1) ◽  
pp. 21-26 ◽  
Author(s):  
T. Ikeda
Keyword(s):  
Proteinase Inhibitor ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Serine Proteinase ◽  
Sodium Cholate ◽  
Cysteine Proteinase Inhibitor ◽  
Cysteine Proteinases ◽  
Serine Proteinase Inhibitor ◽  
Similar Activity

AbstractThe involvement of intrinsic proteinases in the excystment of Paragonimus ohirai metacercariae was studied in in vitro excystment induced by sodium (Na) cholate, a bile salt and A23187, a Ca2+ ionophore. The effects of various proteinase inhibitors on the in vitro excystment were examined and similar inhibitory profiles were obtained. Benzyloxycarbonyl-L-leucyl-L-leucinal (Z-Leu-Leu-H), a cysteine proteinase inhibitor and 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc SC), a serine proteinase inhibitor completely inhibited excystment, while L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido (4-guanidino)-butane (E-64), a cysteine proteinase inhibitor and leupeptin, a cysteine/serine proteinase inhibitor permitted partial excystment at a lower rate, but inhibited it from proceeding from the partial excystment stage. In secretions released from metacercariae during excystment, proteinase activities detected towards various fluorogenic peptidyl substrates were almost completely inhibited by Z-Leu-Leu-H and E-64, but not by Pefabloc SC. Sodium cholate induced a higher secretion of cysteine proteinases and a higher rate of excystment than A23187. Profiles of cysteine proteinase activities towards five peptidyl substrates detected were markedly different among the two secretions and the lysate of newly excysted juveniles. Newly excysted juveniles released cysteine proteinases with similar activity profiles and levels to metacercariae induced by Na cholate-incubation, whereas the release of cysteine proteinases was reduced compared with metacercariae induced by A23187-incubation. These results provide valuable information about the involvement of intrinsic proteinases in metacercarial excystment.

scholarly journals Candida albicans Secreted Aspartyl Proteinases in Virulence and Pathogenesis

2003 ◽  
Vol 67 (3) ◽  
pp. 400-428 ◽  
Author(s):  
Julian R. Naglik ◽  
Stephen J. Challacombe ◽  
Bernhard Hube
Keyword(s):  
Candida Albicans ◽  
Proteinase Inhibitors ◽  
Host Immune System ◽  
Human Proteins ◽  
Candida Infections ◽  
Human Infections ◽  
Host Cell Membranes ◽  
Aspartyl Proteinases

SUMMARY Candida albicans is the most common fungal pathogen of humans and has developed an extensive repertoire of putative virulence mechanisms that allows successful colonization and infection of the host under suitable predisposing conditions. Extracellular proteolytic activity plays a central role in Candida pathogenicity and is produced by a family of 10 secreted aspartyl proteinases (Sap proteins). Although the consequences of proteinase secretion during human infections is not precisely known, in vitro, animal, and human studies have implicated the proteinases in C. albicans virulence in one of the following seven ways: (i) correlation between Sap production in vitro and Candida virulence, (ii) degradation of human proteins and structural analysis in determining Sap substrate specificity, (iii) association of Sap production with other virulence processes of C. albicans, (iv) Sap protein production and Sap immune responses in animal and human infections, (v) SAP gene expression during Candida infections, (vi) modulation of C. albicans virulence by aspartyl proteinase inhibitors, and (vii) the use of SAP-disrupted mutants to analyze C. albicans virulence. Sap proteins fulfill a number of specialized functions during the infective process, which include the simple role of digesting molecules for nutrient acquisition, digesting or distorting host cell membranes to facilitate adhesion and tissue invasion, and digesting cells and molecules of the host immune system to avoid or resist antimicrobial attack by the host. We have critically discussed the data relevant to each of these seven criteria, with specific emphasis on how this proteinase family could contribute to Candida virulence and pathogenesis.

scholarly journals Changes in Proteolytic Activity and Cysteine Proteinase Gene Expression during the Senescence of etr1-1 Transgenic Petunias

HortScience ◽  
2005 ◽  
Vol 40 (4) ◽  
pp. 1105B-1105
Author(s):  
Michelle Jones ◽  
Gunching Chaffin ◽  
David Clark
Keyword(s):  
Proteolytic Activity ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Transcript Abundance ◽  
Flower Senescence ◽  
Cysteine Proteinases ◽  
University Of Florida ◽  
Nitrogen Levels ◽  
Protein Levels ◽  
The University

Corolla senescence in petunias was accompanied by a decrease in total proteins and a corresponding increase in proteolytic activity. Transgenic petunias that contain the mutated ethylene receptor (35S:etr1-1) have reduced sensitivity to ethylene and delayed flower senescence. Declines in total protein levels and increases in proteolytic activity were also delayed in etr1-1 flowers and corresponded with corolla wilting. Experiments using class-specific proteinase inhibitors indicated that proteolytic activity in petunia corollas was largely due to cysteine proteinases. Total nitrogen levels within the corollas of both wild type and etr1-1 flowers also decreased during senescence. Nine cDNAs encoding putative cysteine proteinases (CPs) were identified from a petunia EST database developed at the University of Florida. Six of these cysteine proteinases showed increased transcript abundance during corolla senescence (senescence-associated CPs) while three decreased in abundance. Of the six senescence-associated cysteine proteinases, only five showed delayed up regulation in etr1-1 flowers that corresponded with corolla wilting. The role of ethylene in the regulation of protein degradation during flower senescence will be discussed.

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