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  • br Materials and methods br Results Heterologous expression


    Materials and methods
    Results Heterologous expression of P. locustae Hxk in methylotrophic yeast P. pastoris was not accompanied by nuclear localization of parasite protein (Fig. 1). The addition of leptomycin B, a specific inhibitor of nuclear export in fission yeast Schizosaccharomyces pombe (Nishi et al., 1994) and mammalian cells (Kudo et al., 1998), into cultivation medium also did not result in accumulation of parasite Hxk in the nuclei. Immunolabeling of Sf9 cells expressing P. locustae Hxk showed specific accumulation of the heterologous protein in insect nuclei even without Leptomycin B blocking of nuclear export (Fig. 2a–d). The clear co-localization of hexokinase with the nuclei of Sf9 cells was confirmed by DAPI staining. No specific labeling of nuclei was observed in the control preparations (Fig. 2d and e).
    Discussion In this study, microsporidian Hxk without N-terminal 12 aa signal peptide was expressed in yeasts and in insect cells in order to simulate the process of its secretion from microsporidia into the host cytoplasm. Despite the conservatism of the nuclear import apparatus (Macara, 2001), yeast cells did not recognize P. locustae Hxk as a nuclear protein. In contrast, the enzyme clearly accumulated in the nuclei of insect Sf9 cells. This result is in agreement with our previous data illustrating nuclear localization of P. locustae Hxk in infected host cells (Senderskiy et al., 2014) and demonstrates that cell lines derived from animals related to the natural hosts are the most suitable models to investigate molecular interactions between intracellular pathogens and their hosts. The physiological role of microsporidian Hxk in host nuclei may be associated with upregulation of glucose (hexoses) uptake by infected cells. This would intensify the replication and sporulation of parasites due to increased ATP production in host mitochondria (Hacker et al., 2014). In addition, during sporogenesis microsporidia require ursolic acid synthesis for trehalose storage, as well as for formation of the chitin-rich spore wall and the O-mannosylated polar tube (Xu et al., 2004). There is some evidence that yeast Hxk2 regulates transcriptional activity of genes encoding hexose carriers, diminishing expression of high-affinity transporters and elevating expression of low-affinity transporters in the presence of high glucose concentrations (Petit et al., 2000). Further analysis of the expression of hexose transporters and glucose uptake in host cells infected by microsporidia or in Hxk expressing cells should verify this hypothesis.
    Introduction Ovarian cancer is the fifth leading cancer type for the estimated new cancer deaths among women and the leading cause of deaths owing to gynecologic malignancy in United States [1]. Despite the use of standard therapeutic regimens including cytoreductive surgery followed by chemotherapies based on platinum and paclitaxel, the prognosis of ovarian cancer patients remains poor mainly because of the difficulty in early diagnosis and the ineffective control of advanced cancer growth, metastasis and recurrence [2]. Hence, there is an urgent need to elucidate the mechanism of ovarian cancer progression and identify new targets for ovarian cancer treatment. In the 1920s, Otto Warburg and colleagues discovered that tumors used aerobic glycolysis to ferment glucose to lactate even in the presence of oxygen and completely functioning mitochondria [3,4]; the phenomenon of this inefficient metabolic pathway to produce energy was later dubbed the Warburg effect, a metabolic hallmark of most cancers. The Warburg effect allows cancer cells to rapidly proliferate by satisfying the demand for rapid production of ATP, biosynthetic precursors of macromolecules and reducing equivalents in the form of NADPH [5,6]. Cancer cells benefit as well from an acidic environment caused by lactate secretion during the process of the Warburg effect. Estrella V et al. [7] and Vander Heiden et al. [6,8] demonstrated that the acidic environment promotes local invasion and proliferation of cancer cells. Glucose is the substrate for glycolysis, and the increase of glucose consumption is used as an indicator of aerobic glycolysis [9,10]. Lactate is the end product of aerobic glycolysis, and the increase of lactate production is considered as the index of the Warburg effect [11]. Hexokinases catalyze the first rate-limiting glycolytic reaction, converting glucose to glucose-6-phosphate (G6P). The hexokinase family is composed of four isoenzymes, HK1, HK2, HK3, and glucokinase. HK2 is highly expressed in various cancers but not in normal tissues, and its high expression is associated with poor prognosis [[12], [13], [14]]. In view of the crucial role of the Warburg effect in tumor growth and progression, strategies aiming to inhibit the expression of HK2 may aid the development of novel therapeutics for cancer.