Ligand Binding in the Conserved Interhelical Loop of CorA, a Magnesium Transporter from Mycobacterium tuberculosis

CorA is a constitutively expressed magnesium transporter in many bacteria. The crystal structures of Thermotoga maritima CorA provide an excellent structural framework for continuing studies. Here, the ligand binding properties of the conserved interhelical loop, the only portion of the protein exposed to the periplasmic space, are characterized by solution nuclear magnetic resonance spectroscopy. Through titration experiments performed on the isolated transmembrane domain of Mycobacterium tuberculosis CorA, it was found that two CorA substrates (Mg²⁺ and Co²⁺) and the CorA-specific inhibitor (Co(III) hexamine chloride) bind in the loop at the same binding site. This site includes the glutamic acid residue from the conserved “MPEL” motif. The relatively large dissociation constants indicate that such interactions are weak but not atypical for channels. The present data support the hypothesis that the negatively charged loop could act as an electrostatic ring, increasing local substrate concentrations before transport across the membrane.The heterogeneous membrane environment is very challenging to mimic for structural, dynamic, and functional studies of membrane proteins. It is not surprising, therefore, that different aspects of the structure can be brought to light under different conditions. Recently, an excellent set of crystal structures of the CorA Mg²⁺ transporter have been published (1–3), and whereas many CorA mysteries were solved, the highly conserved periplasmic interhelical loops in the pentameric structure were not well resolved. Here, solution nuclear magnetic resonance (NMR) spectroscopy of the transmembrane domain resolves these loops, and the secondary structure and ion binding in this domain are characterized.The 2TM-GxN family of transporters is a large group of integral membrane proteins responsible for metal ion transport (especially for magnesium) across membranes (4, 5). CorA is a prototypical member in this family responsible for magnesium influx as well as efflux in some cases (5, 6). In the extensive phylogenetic analysis, it was shown that CorA is characterized by a universally conserved “GMN” motif in an interhelical loop connecting two conserved transmembrane helices at the C terminus of the full-length protein (4). As the only constitutively expressed magnesium transporter, CorA can play an important role in the viability of pathogens, such as Helicobacter pylori (7).Pentameric CorA from Thermotoga maritima forms two distinguishable domains; that is, a large cytoplasmic domain and a small transmembrane domain (1–3). In this latter domain there are two transmembrane helices connected by an interhelical periplasmic loop. The first transmembrane helix (TM1)2 lines the pore, whereas the second transmembrane helix (TM2) forms an outer ring of helices, which appears to have only weak interactions with the TM1 helices.Different mechanisms of substrate transport for CorA have been proposed (1–3, 6, 8), whereas the structure and function of the conserved loop is still an open issue. Based on phylogenetic analyses, it has been shown that there were two conserved sequences in the interhelical loop. One is the GMN motif that is universally conserved, and the other is the MPEL motif that is conserved throughout most bacterial genomes. The glutamic acid residue in the MPEL sequence is almost universally conserved in CorA and CorA homologs in eukaryotic cells, including yeast and humans (4). However, it is not conserved in Methanococcus jannaschii for which CorA has been functionally characterized (9). Although the Mycobacterium tuberculosis protein, studied here, has not been functionally characterized in detail, it has been shown to transport magnesium ions across the membrane.³ Because the interhelical loop is the only portion of the protein exposed to the periplasmic side and because there is a highly conserved negatively charged residue, it has been suggested that the loop could act as an initial magnesium binding site (1). This could result in enhancing Mg²⁺ concentration at the mouth of the pore, enhancing substrate selection and generating partial cation dehydration (1). Recent functional studies of CorA homologs from yeast have shown that this negatively charged residue in the loop plays an important role in function (10–12). Substitution of Glu by Lys results in a dramatic reduction in transport activity in yeast (10), and substitution of a positively charged residue (Arg in A1r2p, a CorA homolog in yeast) by a Glu increases channel activity (11). However, the CorA from M. jannaschii does not contain either this residue or a negatively charged loop. Based on these results, the negatively charged loop appears to be functionally important and may act as an initial substrate binding site for increasing the local substrate concentration to facilitate ion transport (10–12), whereas it may not be functionally essential at least for some CorA members. However, it has also been argued, based on limited crystallographic data, that the loop may not form a binding site for substrate selection and dehydration (3). Instead, it was speculated that the loop could mediate the relative movement of the two transmembrane helices (3). Accordingly, further investigation of this functionally important loop is warranted.In the present work we characterize the interaction between the isolated transmembrane domain of CorA (CorA-TMD) and its substrates as well as an inhibitor. Such a “divide and conquer” strategy has been successfully applied to several membrane proteins, such as the M2 protein (a proton channel from influenza A) (13–15), Vpu (a membrane protein encoded by human immunodeficiency virus involved in the budding of new viral particles from the host cell) (16, 17), GlpG (a rhomboid intra-membrane protease) (18, 19), and S2P (a intra-membrane metalloprotease) (20). In addition, the electron density map from the crystal structure of the isolated CorA soluble domain was used to solve the structure of full-length CorA by molecular replacement (1). This suggests that the structural influence of the transmembrane domain on the soluble domain is limited, at least for the conformational state that was crystallized (1). Hence, a divide and conquer strategy for CorA appears to be justified and provides an opportunity to characterize this domain by solution NMR.It has been shown that NMR has a unique ability of characterize weak interactions that are not readily characterized by other methods (21, 22). In the present work we characterize the binding of two substrates (Mg²⁺ and Co²⁺) to the loop as well as an inhibitor (Cobalt(III) hexamine chloride, HexCo³⁺) by NMR. Our data indicate that the ligands of CorA can weakly but specifically bind to the interhelical loop of CorA-TMD through their interaction with the conserved negatively charged glutamic acid residue in the MPEL motif, supporting the hypothesis that the loop acts as an initial binding site.     

Biofouling and stability of synthetic polymers in sea water

Commercial synthetic polymers namely Polycarbonate (PC), Low density polyethylene (LDPE), High density polyethylene (HDPE), and Polypropylene (PP) coupons were immersed for a period of 12 months (Feb 2006 – Feb 2007) in Bay of Bengal, East coast, India. Samples were retrieved every month and the extent of biofouling and biodegradation were monitored. Biofouling was found to depend not only on the season but also on the chemical nature of the polymer. Surface energy of all the four polymers is positively correlated with fouling only at the initial stages (three months) while surface roughness had a negative correlation. The later increased during the study period. Total suspended solids and organic matter were more abundant on HDPE, followed by PP and LDPE, indicating that among polyolefins hydrophobic surfaces (lower surface energy) favor biofouling over one year. Maximum fouling was observed on polycarbonate during initial three months. Chlorophyll a showed a decreasing trend during the study, as secondary foulers such as Balanus amphitrite, were dominant after the monsoon (6th month in the present study). Maximum weight loss was seen in LDPE (1.9%), followed by that in HDPE (1.6%), PC (0.69%) and finally in PP (0.65%) samples in the 12 months time period. FTIR spectra of PC displayed a decrease in carbonate carbonyl index, while an initial increase and a decrease in carbonyl index of polyolefins as a function of time indicated biodegradation.     

Combining selected reaction monitoring with discovery proteomics in limited biological samples

Simultaneous quantification of multiple proteins by selected reaction monitoring (SRM) has several applications in cell signaling studies including embryo proteomics. However, concerns have recently been raised over the specificity of SRM assays due to possible ion redundancy and/or sequence similarity of selected peptide with multiple non‐related proteins. In this Viewpoint article, we discuss some simple measures that can increase our confidence in the accuracy of SRM scans used in proteomic experiments. At least in embryonic samples from porcine species, these measures were found to be useful in validating MS‐identified differentially expressed proteins. Among the nine proteins analyzed by SRM assay, all the proteins that were found to be up‐ or down‐regulated in MS experiment were also faithfully up‐ or down‐regulated in SRM assay.     

Synthesis of functionalized amino acid derivatives as new pharmacophores for designing anticancer agents

A new series of functionalized amino acid derivatives N-substituted 1-N-(tert-butoxycarbonyl)-2,2-dimethyl-4-phenyl-5-oxazolidine carboxamide (1-17) and 1-N-substituted-3-amino-2-hydroxy-3-phenylpropane-1-carboxamide (18-34) were synthesized and evaluated for their in vitro cytotoxicity against human cancer cell lines. Compound 6 has shown interesting cytotoxicity (IC(50) = 5.67 microm) in ovarian cancer, while compound 10 exhibited promising cytotoxicity in ovarian (IC(50) = 6.1 microm) and oral (IC(50) = 4.17 microm) cancers. These compounds could be of use in designing new anti-cancer agents.     

Chitosan-Stearic Acid Based Polymeric Micelles for the Effective Delivery of Tamoxifen: Cytotoxic and Pharmacokinetic Evaluation

Chitosan is a widely employed polysaccharide with positive zeta-potential and better tissue/cell adhesion. Its hydrophilicity, high viscosity, and insolubility at physiological pH are major hurdles in proper utilization of this macromolecule. Therefore, it was conjugated with biocompatible stearic acid and the conjugate was employed to develop polymeric micelles for delivery of tamoxifen to breast cancer cells. The conjugate was characterized by FT-IR and NMR, and the nanocarrier was characterized for micromeritics, surface charge, drug loading, and morphological attributes. The efficacy was evaluated by in vitro MTT studies, safety by erythrocyte compatibility, and biodistribution by in vivo pharmacokinetic studies. Despite better drug loading and sustained drug release, cytotoxicity on MCF-7 breast cancer cells was substantially enhanced and the pharmacokinetic profile was significantly modified. The AUC was enhanced manifolds along with reduced clearance. The findings are unique and provide an alternative to the conventional lipid-based nanocarriers for better dose delivery, tissue adhesion, and desired pharmacokinetic modulation.     

LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy

Long non‐coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here, we investigated the role of a novel vascular endothelial‐associated lncRNA (VEAL2) in regulating endothelial permeability. Precise editing of veal2 loci in zebrafish (veal2 ^gib005Δ8/+) induced cranial hemorrhage. In vitro and in vivo studies revealed that veal2 competes with diacylglycerol for interaction with protein kinase C beta‐b (Prkcbb) and regulates its kinase activity. Using PRKCB2 as bait, we identified functional ortholog of veal2 in humans from HUVECs and named it as VEAL2. Overexpression and knockdown of VEAL2 affected tubulogenesis and permeability in HUVECs. VEAL2 was differentially expressed in choroid tissue in eye and blood from patients with diabetic retinopathy, a disease where PRKCB2 is known to be hyperactivated. Further, VEAL2 could rescue the effects of PRKCB2‐mediated turnover of endothelial junctional proteins thus reducing hyperpermeability in hyperglycemic HUVEC model of diabetic retinopathy. Based on evidence from zebrafish and hyperglycemic HUVEC models and diabetic retinopathy patients, we report a hitherto unknown VEAL2 lncRNA‐mediated regulation of PRKCB2, for modulating junctional dynamics and maintenance of endothelial permeability.     

In-vitro culture of Plasmodium falciparum: Utility of modified (RPNI) medium for drug-sensitivity studies using SYBR Green I assay

Studies were carried out to establish the potential of RPNI medium for drug-sensitivity studies using the MSF assay. The drug sensitivity of standard anti-malarials was compared using both the (³H) Hypoxanthine incorporation assay and the MSF assay. The media supplements used during the study have been human serum, FBS and ALBUMAX-II. Drug sensitivity of two parasite lines, adapted to grow separately in conventional as well as in RPNI medium was compared to observe the effect of RPNI medium on functional characteristics of the parasite. The results revealed identical IC₅₀ values of standard anti-malarials obtained by both the (³H) Hypoxanthine incorporation assay and the MSF assay and no untoward effect of FBS and ALBUMAX-II could be noticed on the chemo-sensitivity of standard anti-malarials. Apart from this the chemo-sensitive response of parasite line adapted to grow in RPNI medium was observed to be intact. These findings showed that RPNI medium has potential to be used for chemo-sensitivity studies and the MSF assay being more convenient was observed to be most suitable assay for bio evaluation of new molecules.     

Enhancing effects of 24-epibrassinolide and Putrescine on the antioxidant capacity and free radical scavenging activity of <Emphasis Type="Italic">Raphanus sativus</Emphasis> seedlings under Cu ion stress

The present investigation recorded significant restoration of seedling growth (root length, shoot length and fresh weight) upon application of 24-epibrassinolide (EBL) and putrescine (Put) to 7-day-old seedlings of Raphanus sativus L. cv. Pusa chetki grown under copper (Cu) ion stress. EBL and Put with/or without Cu ion treated seedlings showed increased titers of ascorbic acid, total phenols and proline when compared with Cu-stressed seedlings. Differential responses in the activities of guaiacol peroxidase (GPOX) and catalase (CAT) were noted for EBL and Put alone or with/or without Cu ion treatment. Decreased activities of glutathione reductase (GR) and superoxide dismutase (SOD) noted for EBL and Put alone were observed to enhance significantly when applied in combination with Cu ion solution. A remarkable decrease in malondialdehyde contents was observed in seedlings treated with EBL and Put alone and with/or without Cu ion stress. Enhanced free radical scavenging activities were also recorded for seedlings given EBL and Put alone or in combination over Cu ion stressed seedlings. Maximum DPPH activity was observed in seedlings treated with Put and EBL 10⁻⁹ M + Put. Significant enhancements in deoxyribose and reducing power activities were too recorded for Put, EBL and Put + 10⁻⁹ M EBL treatments. Improved seedling growth, antioxidant levels (ascorbic acid, total phenols and proline) and enzymic (GPOX, CAT, SOD and GR) activities and free radical scavenging capacities along with reduced membrane damage in seedlings given EBL and Put with/or without Cu stress suggests significant and positive interactions of EBL and Put in alleviating the Cu ion induced oxidative damage in radish seedlings.     

Spatial and temporal profiles of cytokinin biosynthesis and accumulation in developing caryopses of maize

Background and Aims

Cytokinins are a major group of plant hormones and are associated with various developmental processes. Developing caryopses of maize have high levels of cytokinins, but little is known about their spatial and temporal distribution. The localization and quantification of cytokinins was investigated in maize (Zea mays) caryopsis from 0 to 28 d after pollination together with the expression and localization of isopentenyltransferase ZmIPT1 involved in cytokinin biosynthesis and ZmCNGT, the gene putatively involved in N9-glucosylation.

Methods

Biochemical, cellular and molecular approaches resolved the overall cytokinin profiles, and several gene expression assays were used for two critical genes to assess cytokinin cell-specific biosynthesis and conversion to the biologically inactive form. Cytokinins were immunolocalized for the first time in maize caryopses.

Key Results

During the period 0–28 d after pollination (DAP): (1) large quantities of cytokinins were detected in the maternal pedicel region relative to the filial tissues during the early stages after fertilization; (2) unpollinated ovules did not accumulate cytokinins; (3) the maternal nucellar region showed little or no cytokinin signal; (4) the highest cytokinin concentrations in filial endosperm and embryo were detected at 12 DAP, predominantly zeatin riboside and zeatin-9-glucoside, respectively; and (5) a strong cytokinin immuno-signal was detected in specific cell types in the pedicel, endosperm and embryo.

Conclusions

The cytokinins of developing maize caryopsis may originate from both local syntheses as well as by transport. High levels of fertilization-dependent cytokinins in the pedicel suggest filial control on metabolism in the maternal tissue; they may also trigger developmental programmed cell death in the pedicel.