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Title: Cloning and partial characterization of Entamoeba histolytica PTPases

Abstract

Reversible protein tyrosine phosphorylation is an essential signal transduction mechanism that regulates cell growth, differentiation, mobility, metabolism, and survival. Two genes coding for protein tyrosine phophatases, designed EhPTPA and EhPTPB, were cloned from Entamoeba histolytica. EhPTPA and EhPTPB proteins showed amino acid sequence identity of 37%, both EhPTPases showed similarity with Dictyostelium discoideum and vertebrate trasmembranal PTPases. mRNA levels of EhPTPA gene are up-regulated in trophozoites recovered after 96 h of liver abscess development in the hamster model. EhPTPA protein expressed as a glutathione S-transferase fusion protein (GST::EhPTPA) showed enzymatic activity with p-nitrophenylphosphate as a substrate and was inhibited by PTPase inhibitors vanadate and molybdate. GST::EhPTPA protein selectively dephosphorylates a 130 kDa phosphotyrosine-containing protein in trophozoite cell lysates. EhPTPA gene codifies for a 43 kDa native protein. Up-regulation of EhPTPA expression suggests that EhPTPA may play an important role in the adaptive response of trophozoites during amoebic liver abscess development.

Authors:
 [1];  [1];  [1];  [2]
  1. Departamento de Patologia Experimental, Centro de Investigacion y de Estudios Avanzados del I.P.N., Mexico D.F, 07360 (Mexico)
  2. Departamento de Patologia Experimental, Centro de Investigacion y de Estudios Avanzados del I.P.N., Mexico D.F, 07360 (Mexico). E-mail: rosales@cinvestav.mx
Publication Date:
OSTI Identifier:
20798902
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 342; Journal Issue: 4; Other Information: DOI: 10.1016/j.bbrc.2006.02.055; PII: S0006-291X(06)00350-0; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ABSCESSES; AMINO ACID SEQUENCE; AMOEBA; CLONING; GENE REGULATION; GENES; GLUTATHIONE; HAMSTERS; LIVER; METABOLISM; MOLYBDATES; PHOSPHORYLATION; TYROSINE

Citation Formats

Herrera-Rodriguez, Sara Elisa, Baylon-Pacheco, Lidia, Talamas-Rohana, Patricia, and Rosales-Encina, Jose Luis. Cloning and partial characterization of Entamoeba histolytica PTPases. United States: N. p., 2006. Web. doi:10.1016/j.bbrc.2006.02.055.
Herrera-Rodriguez, Sara Elisa, Baylon-Pacheco, Lidia, Talamas-Rohana, Patricia, & Rosales-Encina, Jose Luis. Cloning and partial characterization of Entamoeba histolytica PTPases. United States. doi:10.1016/j.bbrc.2006.02.055.
Herrera-Rodriguez, Sara Elisa, Baylon-Pacheco, Lidia, Talamas-Rohana, Patricia, and Rosales-Encina, Jose Luis. Fri . "Cloning and partial characterization of Entamoeba histolytica PTPases". United States. doi:10.1016/j.bbrc.2006.02.055.
@article{osti_20798902,
title = {Cloning and partial characterization of Entamoeba histolytica PTPases},
author = {Herrera-Rodriguez, Sara Elisa and Baylon-Pacheco, Lidia and Talamas-Rohana, Patricia and Rosales-Encina, Jose Luis},
abstractNote = {Reversible protein tyrosine phosphorylation is an essential signal transduction mechanism that regulates cell growth, differentiation, mobility, metabolism, and survival. Two genes coding for protein tyrosine phophatases, designed EhPTPA and EhPTPB, were cloned from Entamoeba histolytica. EhPTPA and EhPTPB proteins showed amino acid sequence identity of 37%, both EhPTPases showed similarity with Dictyostelium discoideum and vertebrate trasmembranal PTPases. mRNA levels of EhPTPA gene are up-regulated in trophozoites recovered after 96 h of liver abscess development in the hamster model. EhPTPA protein expressed as a glutathione S-transferase fusion protein (GST::EhPTPA) showed enzymatic activity with p-nitrophenylphosphate as a substrate and was inhibited by PTPase inhibitors vanadate and molybdate. GST::EhPTPA protein selectively dephosphorylates a 130 kDa phosphotyrosine-containing protein in trophozoite cell lysates. EhPTPA gene codifies for a 43 kDa native protein. Up-regulation of EhPTPA expression suggests that EhPTPA may play an important role in the adaptive response of trophozoites during amoebic liver abscess development.},
doi = {10.1016/j.bbrc.2006.02.055},
journal = {Biochemical and Biophysical Research Communications},
number = 4,
volume = 342,
place = {United States},
year = {Fri Apr 21 00:00:00 EDT 2006},
month = {Fri Apr 21 00:00:00 EDT 2006}
}
  • The growth of Entamoeba histolytica in microtiter plates in vitro in a variety of environments with reduced oxygen tensions is reported. With 3% O/sub 2/, 3% CO/sub 2/, and 94% N/sub 2/, the parasite growth in microtiter plates was identical to that in screw-capped culture tubes, as measured by (/sup 3/H)thymidine incorporation and by quantitative parasite counts. There were no significant differences between the drug concentrations necessary to inhibit parasite growth by 50% based on (/sup 3/H)thymidine incorporation vs those defined by quantitative parasite counts for the 15 antimicrobial agents tested (including seven drugs used for the treatment of amebiasis).more » This technique provides a reproducible method to quantitate the activity of potential antiamebic agents in vitro. The isotopic method should be of particular value in defining the metabolism of the parasite and effects of antimicrobial agents on it, whereas the morphologic method may be more valuable for workers with limited resources available to them.« less
  • No abstract prepared.
  • The sequencing of the genome of Entamoeba histolytica has allowed a reconstruction of its metabolic pathways, many of which are unusual for a eukaryote. Based on the genome sequence, it appears that amino acids may play a larger role than previously thought in energy metabolism, with roles in both ATP synthesis and NAD regeneration. Arginine decarboxylase may be involved in survival of E. histolytica during its passage through the stomach. The usual pyrimidine synthesis pathway is absent, but a partial pyrimidine degradation pathway could be part of a novel pyrimidine synthesis pathway. Ribonucleotide reductase was not found in the E.more » histolytica genome, but it was found in the close relatives Entamoeba invadens and Entamoeba moshkovskii, suggesting a recent loss from E. histolytica. The usual eukaryotic glucose transporters are not present, but members of a prokaryotic monosaccharide transporter family are present.« less
  • No abstract prepared.
  • Cysteine proteases (CP) are key pathogenesis and virulence determinants of protozoan parasites. Entamoeba histolytica contains at least 50 cysteine proteases; however, only three (EhCP1, EhCP2 and EhCP5) are responsible for approximately 90% of the cysteine protease activity in this parasite. CPs are expressed as inactive zymogens. Because the processed proteases are potentially cytotoxic, protozoan parasites have developed mechanisms to regulate their activity. Inhibitors of cysteine proteases (ICP) of the chagasin-like inhibitor family (MEROPS family I42) were recently identified in bacteria and protozoan parasites. E. histolytica contains two ICP-encoding genes of the chagasin-like inhibitor family. EhICP1 localizes to the cytosol, whereasmore » EhICP2 is targeted to phagosomes. Herein, we report two crystal structures of EhICP2. The overall structure of EhICP2 consists of eight {beta}-strands and closely resembles the immunoglobulin fold. A comparison between the two crystal forms of EhICP2 indicates that the conserved BC, DE and FG loops form a flexible wedge that may block the active site of CPs. The positively charged surface of the wedge-forming loops in EhICP2 contrasts with the neutral surface of the wedge-forming loops in chagasin. We postulate that the flexibility and positive charge observed in the DE and FG loops of EhICP2 may be important to facilitate the initial binding of this inhibitor to the battery of CPs present in E. histolytica.« less