The heat shock protein 90 plays a pivotal role in the life cycle control of Leishmania donovani promoting the fast-growing insect stage of this parasite. Equally important for insect stage growth is the co-chaperone Sti1. We show that replacement of Sti1 is only feasible in the presence of additional Sti1 transgenes indicating an essential role. To better understand the impact of Sti1 and its interaction with Hsp90, we performed a mutational analysis of Hsp90. We established that a single amino acid exchange in the Leishmania Hsp90 renders that protein resistant to the inhibitor radicicol (RAD), yet does not interfere with its functionality. Based on this RAD-resistant Hsp90, we established a combined chemical knockout/gene complementation (CKC) approach. We can show that Hsp90 function is required in both insect and mammalian life stages and that the Sti1-binding motif of Hsp90 is crucial for proliferation of insect and mammalian stages of the parasite. The Sti1-binding motif in Leishmania Hsp90 is suboptimal – optimizing the motif increased initial intracellular proliferation underscoring the importance of the Hsp90–Sti1 interaction for this important parasitic protozoan. The CKC strategy we developed will allow the future analysis of more Hsp90 domains and motifs in parasite viability and infectivity.
Molecular chaperone proteins play a pivotal role in the protozoan parasite Leishmania donovani, controlling cell fate and ensuring intracellular survival. In higher eukaryotes, the so-called co-chaperone proteins are required for client protein recognition and proper function of chaperones, among them the small glutamine-rich tetratricopeptide repeat proteins (SGT) which interact with both HSP70 and HSP90 chaperones. An atypical SGT homolog is found in the L. donovani genome, encoding a protein lacking the C-terminal glutamine-rich region, normally typical for SGT family members. The gene is expressed constitutively during the life cycle and is essential for survival and/or growth of the parasites. LdSGT forms large, stable complexes that also include another putative cochaperone, HSC70 interacting protein (HIP). The gene product forms cytoplasmic clusters, matching the subcellular distribution of HIP and partly that of the major cytoplasmic chaperones, HSP70 and HSP90, reflecting a direct molecular interaction with both chaperones.
Phytoestrogens have a controversial effect on hormone-dependent tumours. Herein, we investigated the effect of the pumpkin seed extract (PSE) on estradiol production and estrogen receptor (ER)-α/ER-β/progesterone receptor (PR) status on MCF7, Jeg3, and BeWo cells. The PSE was prepared and analyzed by mass spectrometry. MCF7, Jeg3, and BeWo cells were incubated with various concentrations of PSE. Untreated cells served as controls. Supernatants were tested for estradiol production with an ELISA method. Furthermore, the effect of the PSE on ER-α/ER-β/PR expression was assessed by immunocytochemistry. The PSE was found to contain both lignans and flavones. Estradiol production was elevated in MCF7, BeWo, and Jeg3 cells in a concentration-dependent manner. In MCF7 cells, a significant ER-α downregulation and a significant PR upregulation were observed. The above results after properly designed animal studies could highlight a potential role of pumpkin seed's lignans in breast cancer prevention and/or treatment.
We identified a Leishmania major-specific gene that can partly compensate for the loss of virulence observed for L. major HSP100 null mutants. The gene, encoding a 46 kD protein of unknown function and lineage, also enhances the virulence of wild type L. major upon overexpression. Surprisingly, the approximately sixfold overexpression of this protein also extends the host range of L. major to normally resistant C57BL/6 mice, causing persisting lesions in this strain, even while eliciting a strong cellular immune response. This enhanced virulence in vivo is mirrored in vitro by increased parasite burden inside bone marrow-derived macrophages. The localization of the protein in the macrophage cytoplasm suggests that it may modulate the macrophage effector mechanisms. In summary, our data show that even minor changes of gene expression in L. major may alter the outcome of an infection, regardless of the host's genetic predisposition.
Herein we investigated the effect of elderflower extracts (EFE) and of enterolactone/enterodiol on hormone production and proliferation of trophoblast tumor cell lines JEG-3 and BeWo, as well as MCF7 breast cancer cells. The EFE was analyzed by mass spectrometry. Cells were incubated with various concentrations of EFE. Untreated cells served as controls. Supernatants were tested for estradiol production with an ELISA method. Furthermore, the effect of the EFE on ERα/ERβ/PR expression was assessed by immunocytochemistry. EFE contains a substantial amount of lignans. Estradiol production was inhibited in all cells in a concentration-dependent manner. EFE upregulated ERα in JEG-3 cell lines. In MCF7 cells, a significant ERα downregulation and PR upregulation were observed. The control substances enterolactone and enterodiol in contrast inhibited the expression of both ER and of PR in MCF7 cells. In addition, the production of estradiol was upregulated in BeWo and MCF7 cells in a concentration dependent manner. The downregulating effect of EFE on ERα expression and the upregulation of the PR expression in MFC-7 cells are promising results. Therefore, additional unknown substances might be responsible for ERα downregulation and PR upregulation. These findings suggest potential use of EFE in breast cancer prevention and/or treatment and warrant further investigation.
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