8 research outputs found
Studies on the optimization of expression and purification & Functional characterization of Class C – GPCRs
G-protein coupled receptor (GPCRs) is a multigene family consisting of more than 1000 genes. They are the most abundant membrane proteins found on a cell surface and are involved in several signaling pathways. In a cell, the signal is transduced by diverse activating endogenous ligands binding on the extracellular surface. This results in the uncoupling of G-proteins from the cytoplasmic loops, leading to the activation of the second messengers. GPCRs have enormous therapeutic importance due to their involvement in basic physiological processes including sensory perception, neurotransmission, metabolism, hormonal balance, etc. Structural and biochemical data are the pre-requisites for designing the drugs involving GPCRs. The current study was focused on the optimization of expression, purification and functional characterization of class-C GPCRs involved in neurotransmission. The first part of the study was aimed at optimizing the expression and purification of the ligand binding extracellular domain (ECD) of rat metabotropic GABAB1b receptor (GBR1bNT) in the recombinant baculovirus (RBV) and E.coli expression systems. GBR1bNT was modeled based on the crystal structure coordinates of ECD of metabotropic glutamate receptor (mGluR). Depending on this GBR1bNT model, molecular cloning strategies were developed for the expression of GBR1bNT. In both systems, GBR1bNT was well expressed and purified. The second part of the study was aimed at biochemical characterization of a putative cholesterol binding motif (pCBM) in Drosophila metabotropic glutamate receptor (DmGluRA). This pCBM might be involved in regulating the binding of DmGluRA to glutamate with high affinity. During the study, it was inferred that a 12- amino-acid amphipathic peptide containing the pCBM might be crucial for the activity of the receptor. Upon conducting [3H]-glutamate binding and detergent-resistant-membrane (DRM) association studies on different truncation constructs of DmGluRA (1-910 amino acids), the N-terminal construct (1-624 amino acids) containing the ECD with one single pCBM was found to be capable of binding glutamate in high affinity state as observed with the full length DmGluRA. Together this study shows that 1) Using an interdisciplinary approach (computational and experimental strategies) GBR1bNT was efficiently expressed and purified in both E.coli and RBV expression systems and 2) the role of pCBM was studied in DmGluRA receptor regulation
Role of Transferrin Receptor and the ABC Transporters ABCB6 and ABCB7 for Resistance and Differentiation of Tumor Cells towards Artesunate
The anti-malarial artesunate also exerts profound anti-cancer activity. The susceptibility of tumor cells to artesunate can be enhanced by ferrous iron. The transferrin receptor (TfR) is involved in iron uptake by internalization of transferrin and is over-expressed in rapidly growing tumors. The ATP-binding cassette (ABC) transporters ABCB6 and ABCB7 are also involved in iron homeostasis. To investigate whether these proteins play a role for sensitivity towards artesunate, Oncotest's 36 cell line panel was treated with artesunate or artesunate plus iron(II) glycine sulfate (Ferrosanol®). The majority of cell lines showed increased inhibition rates, for the combination of artesunate plus iron(II) glycine sulfate compared to artesunate alone. However, in 11 out of the 36 cell lines the combination treatment was not superior. Cell lines with high TfR expression significantly correlated with high degrees of modulation indicating that high TfR expressing tumor cells would be more efficiently inhibited by this combination treatment than low TfR expressing ones. Furthermore, we found a significant relationship between cellular response to artesunate and TfR expression in 55 cell lines of the National Cancer Institute (NCI), USA. A significant correlation was also found for ABCB6, but not for ABCB7 in the NCI panel. Artesunate treatment of human CCRF-CEM leukemia and MCF7 breast cancer cells induced ABCB6 expression but repressed ABCB7 expression. Finally, artesunate inhibited proliferation and differentiation of mouse erythroleukemia (MEL) cells. Down-regulation of ABCB6 by antisense oligonucleotides inhibited differentiation of MEL cells indicating that artesunate and ABCB6 may cooperate. In conclusion, our results indicate that ferrous iron improves the activity of artesunate in some but not all tumor cell lines. Several factors involved in iron homeostasis such as TfR and ABCB6 may contribute to this effect
Effect of artesunate on MEL cell proliferation, differentiation, and ABCB6/ABCB7 expression.
<p>A. Effect on cell growth. MEL cells were cultured without or with 1.0 µg/ml artesunate for the indicated time. The cell numbers were measured by trypan blue exclusion. B. Protein expression of ABCB6 and ABCB7 after treatment with 2% DMSO for different time points. Actin expression served as loading control. C. Effect on differentiation. MEL cells (100,000 cells/ml) were cultured in the presence of various concentrations of artesunate for 48 h. The content of heme was estimated, by the method with oxalic acid. D. The cells transfected with sense- or antisense oligonucleotides for ABCB6 were induced with 2% DMSO without or with 0.18 µg/ml artesunate for 48 h.</p
Expression of ABCB6 and ABCB7 in MCF7 (A.) and CCRF-CEM cells (B.) after treatment with artesunate.
<p>The expression after 24 hours is given in relation to the expression before treatment.</p
Relationship between transferrin receptor (TfR) protein expression and degree of modulation to a combination therapy of artesunate plus iron(II) glycine sulfate (see Table I) in tumor cell lines of the Oncotest panel.
*<p>Relative expression. For details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000798#pone.0000798-Wirth1" target="_blank">[36]</a>.</p
TfR expression and in vitro anti-tumor activity of artesunate alone and in combination with iron(II) glycine sulfate (Ferrosanol®, 10 µg/ml) in Oncotest's 36 cell line panel.
*<p>TfR expression by a cell micro-array technique has been described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000798#pone.0000798-Wirth1" target="_blank">[36]</a>.</p
Relationship between transferrin receptor (<i>TFR</i>), <i>ABCB6</i>, and <i>ABCB7</i> mRNA expression and response to artesunate (ART) in tumor cell lines of the NCI drug screening panel.
*<p>The median log<sub>10</sub>IC<sub>50</sub> value for artesunate was used as a cut-off to separate tumor cell lines as being “sensitive” or “resistant”</p>**<p>Relative mRNA expression as reported (<a href="http://dtp.nci.nih.gov" target="_blank">http://dtp.nci.nih.gov</a>)</p