Two novel calcium‐binding proteins from cytoplasmic granules of the protozoan parasite <i>Entamoeba histolytica</i>

Nickel, Rosa; Jacobs, Thomas; Urban, Britta C.; Scholze, Henning; Bruhn, Heike; Leippe, Matthias

doi:10.1016/s0014-5793(00)02245-6

Cited by 33 publications

(22 citation statements)

References 24 publications

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“…The cytoplasm of E. histolytica has numerous granules. Calcium binding proteins have been identified in these granules that may have important role in granule discharge during cell killing (Nickel et al, 2000). Therefore it is probable that EhCaBP1 modulates actin-mediated processes through the participation of other proteins in a complex network.…”

Section: Discussionmentioning

confidence: 99%

“…A number of other calcium binding proteins have been identified in E. histolytica. Two EF-hand calcium binding proteins, grainin 1 and grainin 2 were purified from the granules of E. histolytica and are thought to be involved in endocytosis (Nickel et al, 2000). E. histolytica also has a functionally diverse calmodulin-like calcium binding protein (EhCaBP1) with no known homologue in the database (Prasad et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

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Calcium binding protein 1 of the protozoan parasiteEntamoeba histolyticainteracts with actin and is involved in cytoskeleton dynamics

Sahoo

Labruyère

Bhattacharya

et al. 2004

Journal of Cell Science

153

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demonstrated by a co-sedimentation assay. A variant of EhCaBP1 did not bind F-actin showing the specificity of the interaction between EhCaBP1 and actin. There is no significant change in the kinetics of in vitro polymerization of actin in presence of EhCaBP1, indicating that EhCaBP1 does not affect filament treadmilling. In addition, using atomic force microscopy; it was found that filaments of Factin, polymerized in presence of EhCaBP1, were thinner. These results indicate that EhCaBP1 may be involved in dynamic membrane restructuring at the time of cell pseudopod formation, phagocytosis and endocytosis in a process mediated by direct binding of EhCaBP1 to actin, affecting the bundling of actin filaments.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Calcium binding protein 1 of the protozoan parasiteEntamoeba histolyticainteracts with actin and is involved in cytoskeleton dynamics

Sahoo

Labruyère

Bhattacharya

et al. 2004

Journal of Cell Science

153

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“…Among these are two related EF-hand-containing proteins, grainin 1 and 2, which are localized in intracellular granules (16). They may be involved in phagocytosis, control of endocytotic pathways and Ca 2ϩ -dependent granular discharge.…”

Section: Camentioning

confidence: 99%

Identification and Characterization of EhCaBP2

Chakrabarty

Sethi

Padhan

et al. 2004

Journal of Biological Chemistry

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Entamoeba histolytica, an early branching eukaryote, is the etiologic agent of amebiasis. Calcium plays a pivotal role in the pathogenesis of amebiasis by modulating the cytopathic properties of the parasite. However, the mechanistic role of Ca 2؉ and calcium-binding proteins in the pathogenesis of E. histolytica remains poorly understood. We had previously characterized a novel calcium-binding protein (EhCaBP1) from E. histolytica. Here, we report the identification and partial characterization of an isoform of this protein, EhCaBP2. Both EhCaBPs have four canonical EF-hand Ca 2؉ binding domains. The two isoforms are encoded by genes of the same size (402 bp). Comparison between the two genes showed an overall identity of 79% at the nucleotide sequence level. This identity dropped to 40% in the 75-nucleotide central linker region between the second and third Ca 2؉ binding domains. Both of these genes are single copy, as revealed by Southern hybridization. Analysis of the available E. histolytica genome sequence data suggested that the two genes are non-allelic. Homology-based structural modeling showed that the major differences between the two EhCaBPs lie in the central linker region, normally involved in binding target molecules. A number of studies indicated that EhCaBP1 and EhCaBP2 are functionally different. They bind different sets of E. histolytica proteins in a Ca 2؉ -dependent manner. Activation of endogenous kinase was also found to be unique for the two proteins and the Ca 2؉ concentration required for their optimal functionality was also different. In addition, a 12-mer peptide was identified from a random peptide library that could differentially bind the two proteins. Our data suggest that EhCaBP2 is a new member of a class of E. histolytica calcium-binding proteins involved in a novel calcium signal transduction pathway.

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“…Internal changes in [Ca 2ϩ ] i have been observed in response to various stimuli (25,34,35), and several important calciumbinding proteins have been discovered in E. histolytica including a protein similar to calmodulin, EhCaBP (36,37). In addition, the multidrug resistance phenotype mediated by the up-regulation of the EhPgp1 and EhPgp5 genes of E. histolytica has been shown to be reversed by calcium-channel blocker verapamil (38 -41).…”

Section: Changes In Ure3-bp Occupancy Of the Hgl5 And Fdx Promoters Imentioning

confidence: 99%

Calcium Modulates Promoter Occupancy by the Entamoeba histolytica Ca2+-binding Transcription Factor URE3-BP

Gilchrist

Leo

Line

et al. 2003

Journal of Biological Chemistry

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The Entamoeba histolytica upstream regulatory element 3-binding protein (URE3-BP) binds to the URE3 sequence of the Gal/GalNAc-inhibitable lectin hgl5 and ferredoxin 1 (fdx) gene promoters. This binding can be inhibited in vitro by addition of calcium. Two EF-hand motifs, which are associated with the ability to bind calcium, are present in the amino acid sequence of URE3-BP. Mutation of the second EF-hand motif in URE3-BP resulted in the loss of calcium inhibition of DNA binding as monitored by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays revealed that URE3-BP was physically bound to the hgl5 and fdx promoters in vivo. Parasite intracellular calcium concentrations were altered by changes in extracellular calcium. Promoter occupancy was lost when intracellular calcium levels were increased by coordinate increases in extracellular calcium. Increased intracellular calcium also resulted in decreased levels of URE3-BP mRNA. Together these results demonstrate that changes in extracellular calcium result in changes in URE3-BP mRNA and in the ability of URE3-BP to bind to URE3-containing promoters. Modulation of URE3-BP by calcium may represent an important mechanism of control of gene expression in E. histolytica.The early branching eukaryote Entamoeba histolytica is a human parasite that is the etiologic agent of amebic dysentery and liver abscess. Only one of every 10 infections leads to disease (1), and the parasite and host factors that control the outcome of infection are not well understood. Alteration in transcriptional control of certain crucial genes may contribute to the expression of a virulence phenotype. Padilla-Vaca et al. There has been considerable divergence in the mechanisms of transcription of the early branching E. histolytica from later branching eukaryotes such as Homo sapiens and Saccharomyces cerevisiae. For instance the E. histolytica core promoter for protein encoding genes consists of a novel GAAC element, in addition to a TATA and INR (4 -6) and contains short regulatory 5Ј and 3Ј sequences (7). The RNA polymerase II is also unusual for a eukaryote in that it is resistant to ␣-amanitin (8). To increase our understanding of transcriptional regulation in this organism we investigated the mechanisms of transcriptional control of a well characterized virulence protein, the galactose-and N-acetyl-D-galactosamine-inhibitable lectin (Gal/GalNAc-inhibitable lectin), which is essential for parasite adherence and contact-mediated cytolysis. The promoter for one of the genes encoding the lectin heavy subunit (hgl5) contains five major regulatory regions (upstream regulatory elements 1-5 (URE1-5)) 1 upstream of the core promoter (4) and a GAAC sequence motif that predominantly influences the rate and site of transcription initiation (4 -6). The hgl5 UREs differ in whether they regulate transcription via the TATA or the GAAC elements, with the URE3 exerting its effect on mRNA transcription via GAAC (9). Interestingly, mutation of the URE3 motif in the hgl5 promoter leads to...

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Two novel calcium‐binding proteins from cytoplasmic granules of the protozoan parasite Entamoeba histolytica

Cited by 33 publications

References 24 publications

Calcium binding protein 1 of the protozoan parasiteEntamoeba histolyticainteracts with actin and is involved in cytoskeleton dynamics

Calcium binding protein 1 of the protozoan parasiteEntamoeba histolyticainteracts with actin and is involved in cytoskeleton dynamics

Identification and Characterization of EhCaBP2

Calcium Modulates Promoter Occupancy by the Entamoeba histolytica Ca2+-binding Transcription Factor URE3-BP

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