Research Article: Structure of Ca2+-binding protein-6 from Entamoeba histolytica and its involvement in trophozoite proliferation regulation

Date Published: May 15, 2017

Publisher: Public Library of Science

Author(s): Deepshikha Verma, Aruna Murmu, Samudrala Gourinath, Alok Bhattacharya, Kandala V. R. Chary, William A. Petri.


Cell cycle of Entamoeba histolytica, the etiological agent of amoebiasis, follows a novel pathway, which includes nuclear division without the nuclear membrane disassembly. We report a nuclear localized Ca2+-binding protein from E. histolytica (abbreviated hereafter as EhCaBP6), which is associated with microtubules. We determined the 3D solution NMR structure of EhCaBP6, and identified one unusual, one canonical and two non-canonical cryptic EF-hand motifs. The cryptic EF-II and EF-IV pair with the Ca2+-binding EF-I and EF-III, respectively, to form a two-domain structure similar to Calmodulin and Centrin proteins. Downregulation of EhCaBP6 affects cell proliferation by causing delays in transition from G1 to S phase, and inhibition of DNA synthesis and cytokinesis. We also demonstrate that EhCaBP6 modulates microtubule dynamics by increasing the rate of tubulin polymerization. Our results, including structural inferences, suggest that EhCaBP6 is an unusual CaBP involved in regulating cell proliferation in E. histolytica similar to nuclear Calmodulin.

Partial Text

Ca2+ and Ca2+-binding proteins (CaBPs) such as Calmodulin (CaM), Centrins and Annexins have been implicated in cell cycle regulation and progression in many eukaryotes [1,2]. The in-vivo and in-vitro cell culture studies have revealed spikes in the levels of Ca2+ at the G1 and G1/S boundaries [3,4]. The nuclear localized CaM plays the role of a major signal-transducing factor during the cell cycle [2,5]. CaM associates with the microtubule organizing centre (MTOC) and acts as the key molecule that couples cell cycle with Ca2+ signaling [6,7]. The levels of intracellular CaM were found to increase during the G1/S transition in normal human fibroblasts [8]. Centrins, also known as Caltractins, are a family of EF-hand Ca2+-binding phosphoproteins found in the centrosomes of eukaryotes. Centrins are present in the centrioles and they are required for the centriole duplication [9,10]. They are also considered to play a role in severing of microtubules by causing calcium-mediated contraction. Similar function has been established for the Centrin homologue in yeast, CDC31 [11]. Cells in the mutant strain of Saccharomyces cerevisiae, lacking this protein, do not divide despite having grown abnormally large and having accumulated double the amount of DNA. Centrins undergo phosphorylation during G2/M phase boundary, thus assisting in the chromosome separation and mitotic spindle assembly [12]. Moreover, CaBP Annexin II was shown to have an increased expression in HeLa cells at the G1/S and S/G2 boundaries [13]. Taken together, a significant role of Ca2+ and CaBPs emerges in the regulation of cell cycle progression.

Ca2+ signaling has been demonstrated as one of the most pivotal pathways in amoebic pathogenesis. However, as pointed out earlier, the influence of Ca2+ in amoebic cells has not been extensively investigated till date. The occurrence of twenty-seven distinct CaBPs in E. histolytica suggests their massive contribution in the regulation of various Ca2+-signaling pathways. The involvement of a few CaBPs in phagocytosis and gene expression underlines the need for further detailed studies with other CaBPs [31].




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