Calcium modulates promoter occupancy by the Entamoeba histolytica Ca2+-binding transcription factor URE3-BP

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.     

Identification and cellular localization of the actin-binding protein ABP-120 from Entamoeba histolytica

Several actin-binding proteins participate in the morphological changes that occur during amoeboid movement. The gene encoding one of these proteins, the gelation factor ABP-120, was identified and characterized from trophozoites of Entamoeba histolytica . The sequence contains 2574 nucleotides, with an open reading frame of 858 amino acids, giving a protein of 93 kDa belonging to the spectrin family. The N-terminal domain of ABP-120 from E. histolytica revealed a consensus site for actin binding homologous to the actin-binding sites of ABP-120 of Dictyostelium discoideum , α-actinin and spectrin. Analysis of the central domain revealed the presence of four repeats of a 73-amino-acid motif constituting 31% of the protein. In addition, a stretch of 105 amino acids was highly divergent when compared with the C-terminal domain of D. discoideum ABP-120. This sequence showed short motifs that are homologous to microtubule-binding domains. We found that ABP-120 from E. histolytica binds to F-actin. In addition, upon motility of the parasite, this protein localized in the pseudopod and the uroid region, implying a role for ABP-120 in movement and capping of surface receptors in E. histolytica .     

A Genome-Wide Over-Expression Screen Identifies Genes Involved in Phagocytosis in the Human Protozoan Parasite, Entamoeba histolytica

Functional genomics and forward genetics seek to assign function to all known genes in a genome. Entamoeba histolytica is a protozoan parasite for which forward genetics approaches have not been extensively applied. It is the causative agent of amoebic dysentery and liver abscess, and infection is prevalent in developing countries that cannot prevent its fecal-oral spread. It is responsible for considerable global morbidity and mortality. Given that the E. histolytica genome has been sequenced, it should be possible to apply genomic approaches to discover gene function. We used a genome-wide over-expression screen to uncover genes regulating an important virulence function of E. histolytica, namely phagocytosis. We developed an episomal E. histolytica cDNA over-expression library, transfected the collection of plasmids into trophozoites, and applied a high-throughput screen to identify phagocytosis mutants in the population of over-expressing cells. The screen was based on the phagocytic uptake of human red blood cells loaded with the metabolic toxin, tubercidin. Expression plasmids were isolated from trophozoites that survived exposure to tubercidin-charged erythrocytes (phagocytosis mutants), and the cDNAs were sequenced. We isolated the gene encoding profilin, a well-characterized cytoskeleton-regulating protein with a known role in phagocytosis. This supports the validity of our approach. Furthermore, we assigned a phagocytic role to several genes not previously known to function in this manner. To our knowledge, this is the first genome-wide forward genetics screen to be applied to this pathogen. The study demonstrates the power of forward genetics in revealing genes regulating virulence in E. histolytica. In addition, the study validates an E. histolytica cDNA over-expression library as a valuable tool for functional genomics.     

glp-1 and lin-12, genes implicated in distinct cell-cell interactions in C. elegans, encode similar transmembrane proteins

Genomic DNA closely related in sequence to lin-12, a gene that specifies certain cell fates during C. elegans development, was isolated from a C. elegans library by low stringency hybridization. DNA sequencing of genomic and cDNA clones predicts the new sequence to encode an integral membrane protein that shares three repeated amino acid sequence motifs with the lin-12 product and the Drosophila Notch product: an epidermal growth factor-like motif, the "lin-12/Notch Repeat," and a motif present in two yeast gene products that have cell cycle dependent functions. Austin and Kimble (see accompanying paper) present evidence that this sequence corresponds to glp-1, a gene implicated in cell-cell interactions distinct from those involving lin-12. Possible implications of the predicted structure of the glp-1 product with respect to these cell-cell interactions are discussed.     

The fragment transformation method to detect the protein structural motifs

To identify functional structural motifs from protein structures of unknown function becomes increasingly important in recent years due to the progress of the structural genomics initiatives. Although certain structural patterns such as the Asp-His-Ser catalytic triad are easy to detect because of their conserved residues and stringently constrained geometry, it is usually more challenging to detect a general structural motifs like, for example, the betabetaalpha-metal binding motif, which has a much more variable conformation and sequence. At present, the identification of these motifs usually relies on manual procedures based on different structure and sequence analysis tools. In this study, we develop a structural alignment algorithm combining both structural and sequence information to identify the local structure motifs. We applied our method to the following examples: the betabetaalpha-metal binding motif and the treble clef motif. The betabetaalpha-metal binding motif plays an important role in nonspecific DNA interactions and cleavage in host defense and apoptosis. The treble clef motif is a zinc-binding motif adaptable to diverse functions such as the binding of nucleic acid and hydrolysis of phosphodiester bonds. Our results are encouraging, indicating that we can effectively identify these structural motifs in an automatic fashion. Our method may provide a useful means for automatic functional annotation through detecting structural motifs associated with particular functions.     

A full-length cDNA of hREV3 is predicted to encode DNA polymerase zeta for damage-induced mutagenesis in humans

DNA damage can cause mutations which in turn may lead to carcinogenesis. In the yeast Saccharomyces cerevisiae, DNA damage-induced mutagenesis pathway requires the REV3 gene. It encodes the catalytic subunit of DNA polymerase zeta that specifically functions in translesion DNA synthesis. We have cloned a cDNA of the human homologue of REV3 (hREV3), which consists of 10,716 bp and codes for a protein of 3130 amino acid residues (352,737 Da). Its C-terminal 755 amino acids show extensive homology with the yeast protein at the C-terminus: 43% identity and 74% similarity. This region contains the six highly conserved DNA polymerase motifs. Furthermore, we have identified four sequence motifs in the N-terminal region outside the polymerase domain that are conserved in DNA polymerase delta from various sources. Three of which are present in DNA polymerase zeta encoded by human, yeast, and plant REV3 genes, indicating that this protein is a member of the DNA polymerase delta family. DNA polymerases delta and zeta are structurally distinguished by the presence of a specific delta IV motif in the former and motifs zeta I and zeta II in the latter, respectively. Human DNA polymerase zeta is ubiquitously expressed in various tissues, consistent with the notion that the hREV3 pathway may be a fundamental mechanism of damage-induced mutagenesis in humans.