- Role of Dimers in the cAMP-Dependent Activation of Hyperpolarization-Activated Cyclic-Nucleotide-Modulated (HCN) Ion Channels. [Journal Article]
- JPJ Phys Chem B 2018 Feb 20
- Hyperpolarization-activated cyclic-nucleotide-modulated (HCN) ion channels control rhythmicity in neurons and cardiomyocytes. Cyclic AMP (cAMP) modulates HCN activity through the cAMP-induced formati...
Hyperpolarization-activated cyclic-nucleotide-modulated (HCN) ion channels control rhythmicity in neurons and cardiomyocytes. Cyclic AMP (cAMP) modulates HCN activity through the cAMP-induced formation of a tetrameric gating ring spanning the intracellular region (IR) of HCN. Although evidence from confocal patch-clamp fluorometry indicates that the cAMP-dependent gating of HCN occurs through a dimer of dimers, the structural and dynamical basis of cAMP allostery in HCN dimers has so far remained elusive. Thus, here we examine how dimers influence IR structural dynamics, and the role that such structural dynamics play in HCN allostery. To this end, we performed molecular dynamics (MD) simulations of HCN4 IR dimers in their fully apo, fully holo, and partially cAMP-bound states, resulting in a total simulated time of 1.2 μs. Comparative analyses of these MD trajectories, as well as previous monomer and tetramer simulations utilized as benchmarks for comparison, reveal that dimers markedly sensitize the HCN IR to cAMP-modulated allostery. Our results indicate that dimerization fine-tunes the IR dynamics to enhance, relative to both monomers and tetramers, the allosteric intra- and interprotomer coupling between the cAMP-binding domain and tetramerization domain components of the IR. The resulting allosteric model provides a viable rationalization of electrophysiological data on the role of IR dimers in HCN activation.
- Characterization of metabolic network of oxalic acid biosynthesis through RNA seq data analysis of developing spikes of finger millet (Eleusine coracana): Deciphering the role of key genes involved in oxalate formation in relation to grain calcium accumulation. [Journal Article]
- GENEGene 2018 Apr 05; 649:40-49
- In the present study, we identified seven major genes of oxalic acid biosynthesis pathway (SGAT, GGAT, ICL, GLO, MHAR, APO and OXO) from developing spike transcriptome of finger millet using rice as ...
In the present study, we identified seven major genes of oxalic acid biosynthesis pathway (SGAT, GGAT, ICL, GLO, MHAR, APO and OXO) from developing spike transcriptome of finger millet using rice as a reference. Sequence alignment of identified genes showed high similarity with their respective homolog in rice except for OXO and GLO. Transcript abundance (FPKM) reflects the higher accumulation of identified genes in GP-1 (low calcium genotype) as compared to GP-45 (high calcium genotype) which was further confirmed by qRT-PCR analysis, indicating differential oxalate formation in both genotypes. Determination of oxalic acid and tartaric acid content in developing spikes explain that higher oxalic acid content in GP-1 however, tartaric acid content was more in GP-45. Higher calcium content in GP-45 and lower oxalate accumulation may be due to the diversion of more ascorbic acid into tartaric acid and may correspond to less formation of calcium oxalate. Our results suggest that more than one pathway for oxalic acid biosynthesis might be present in finger millet with probable predominance of ascorbate-tartarate pathway rather than glyoxalate-oxalate conversion. Thus, finger millet can be use as an excellent model system for understanding more specific role of nutrients-antinutrients interactions, as evident from the present study.
- The Molecular Determinants of Small-Molecule Ligand Binding at P2X Receptors. [Journal Article]
- FPFront Pharmacol 2018; 9:58
- P2X receptors are trimeric eukaryotic ATP-gated cation channels. Extracellular ATP-their physiological ligand-is released as a neurotransmitter and in conditions of cell damage such as inflammation, ...
P2X receptors are trimeric eukaryotic ATP-gated cation channels. Extracellular ATP-their physiological ligand-is released as a neurotransmitter and in conditions of cell damage such as inflammation, and substantial evidence implicates P2X receptors in diseases including neuropathic pain, cancer, and arthritis. In 2009, the first P2X crystal structure,Danio rerioP2X4 in theapo- state, was published, and this was followed in 2012 by the ATP-bound structure. These structures transformed our understanding of the conformational changes induced by ATP binding and the mechanism of ligand specificity, and enabled homology modeling of mammalian P2X receptors for ligand docking and rational design of receptor modulators. P2X receptors are attractive drug targets, and a wide array of potent, subtype-selective modulators (mostly antagonists) have been developed. In 2016, crystal structures of human P2X3 in complex with the competitive antagonists TNP-ATP and A-317491, andAiluropoda melanoleucaP2X7 in complex with a series of allosteric antagonists were published, giving fascinating insights into the mechanism of channel antagonism. In this article we not only summarize current understanding of small-molecule modulator binding at P2X receptors, but also use this information in combination with previously published structure-function data and molecular docking experiments, to hypothesize a role for the dorsal fin loop region in differential ATP potency, and describe novel, testable binding conformations for both the semi-selective synthetic P2X7 agonist 2'-(3')-O-(4-benzoyl)benzoyl ATP (BzATP), and the P2X4-selective positive allosteric modulator ivermectin. We find that the distal benzoyl group of BzATP lies in close proximity to Lys-127, a residue previously implicated in BzATP binding to P2X7, potentially explaining the increased potency of BzATP at rat P2X7 receptors. We also present molecular docking of ivermectin to rat P2X4 receptors, illustrating a plausible binding conformation between the first and second transmembrane domains which not only tallies with previous mutagenesis studies, but would also likely have the effect of stabilizing the open channel structure, consistent with the mode of action of this positive allosteric modulator. From our docking simulations and analysis of sequence homology we propose a series of mutations likely to confer ivermectin sensitivity to human P2X1.
- Characteristics of suicidal attempts among farmers in rural South India. [Journal Article]
- IPInd Psychiatry J 2017 Jan-Jun; 26(1):28-33
- CONCLUSIONS: Pesticide poisoning was the most common mode for attempting suicide among the farmers. Contrary to public perception and other studies, relationship, and marital issues, not financial reasons were found to be the most common immediate precipitant for the attempters in our study.
- Structure Based Optimization Strategies for G Protein-Coupled Receptor (GPCR) Allosteric Modulators: A Case Study from Analyses of New Metabotropic Glutamate Receptor 5 (mGlu5) X-ray structures. [Journal Article]
- JMJ Med Chem 2018 Feb 18
- Two interesting new X-ray structures of negative allosteric modulator (NAM) ligands for the mGlu5receptor, M-MPEP (3) and fenobam (4), are reported. The new structures show how the binding of the lig...
Two interesting new X-ray structures of negative allosteric modulator (NAM) ligands for the mGlu5receptor, M-MPEP (3) and fenobam (4), are reported. The new structures show how the binding of the ligands induces different receptor water channel conformations to previously published structures. The structure of fenobam, where a urea replaces the acetylenic linker in M-MPEP and mavoglurant, reveals a binding mode where the ligand is rotated by 180º compared to a previously proposed docking model. The need for multiple ligand structures for accurate GPCR structure-based drug design is demonstrated by the different growing vectors identified for the head groups of M-MPEP and mavoglurant and by the unexpected water mediated receptor interactions of a new chemotype represented by fenobam. The implications of the new structures for ligand design are discussed, with extensive analysis of the energetics of the water networks of both pseudo-apo and bound structures providing a new design strategy for allosteric modulators.
- Apo-10'-lycopenoic acid inhibits cancer cell migration and angiogenesis and induces peroxisome proliferator-activated receptor γ. [Journal Article]
- JNJ Nutr Biochem 2018 Jan 31; 56:26-34
- CONCLUSIONS: ALA inhibits cancer cell motility and angiogenesis and induces PPARγ expression, which could be one of the potential mechanisms for ALA protecting against tumor progression.
- Study of manganese binding to the ferroxidase centre of human H-type ferritin. [Journal Article]
- JIJ Inorg Biochem 2018 Feb 09; 182:103-112
- Ferritins are ubiquitous and conserved proteins endowed with enzymatic ferroxidase activity, that oxidize Fe(II) ions at the dimetal ferroxidase centre to form a mineralized Fe(III) oxide core deposi...
Ferritins are ubiquitous and conserved proteins endowed with enzymatic ferroxidase activity, that oxidize Fe(II) ions at the dimetal ferroxidase centre to form a mineralized Fe(III) oxide core deposited within the apo-protein shell. Herein, the in vitro formation of a heterodimetal cofactor constituted by Fe and Mn ions has been investigated in human H ferritin (hHFt). Namely, Mn and Fe binding at the hHFt ferroxidase centre and its effects on Fe(II) oxidation have been investigated by UV-Vis ferroxidation kinetics, fluorimetric titrations, multifrequency EPR, and preliminary Mössbauer spectroscopy. Our results show that in hHFt, both Fe(II) and Mn(II) bind the ferroxidase centre forming a Fe-Mn cofactor. Moreover, molecular oxygen seems to favour Mn(II) binding and increases the ferroxidation activity of the Mn-loaded protein. The data suggest that Mn influences the Fe binding and the efficiency of the ferroxidation reaction. The higher efficiency of the Mn-Fe heterometallic centre may have a physiological relevance in specific cell types (i.e. glia cells), where the concentration of Mn is the same order of magnitude as iron.
- Inhibition of CD95/CD95L Signaling with APG101 Prevents Invasion and Enhances Radiation Therapy for Glioblastoma. [Journal Article]
- MCMol Cancer Res 2018 Feb 16
- CONCLUSIONS: APG101 (asunercept) successfully used in a controlled phase II glioblastoma trial (NCT01071837) acts anti-invasively by inhibiting matrix metalloproteinase signaling resulting in additive effects together with radiotherapy and helping to further develop a treatment for this devastating disease.
- Structural basis for the glycosyltransferase activity of the Salmonella effector SseK3. [Journal Article]
- JBJ Biol Chem 2018 Feb 15
- The Salmonella secreted effector SseK3 translocates into host cells, targeting innate immune responses including NF-κB activation. SseK3 is a glycosyltransferase that transfers an N-acetylglucosamine...
The Salmonella secreted effector SseK3 translocates into host cells, targeting innate immune responses including NF-κB activation. SseK3 is a glycosyltransferase that transfers an N-acetylglucosamine (GlcNAc) moiety onto the guanidino group of a target arginine, modulating host cell function. However, a lack of structural information has precluded elucidation of the molecular mechanisms in arginine and GlcNAc selection. We report here the crystal structure of SseK3 in its apo form and in complex with hydrolysed UDP-GlcNAc. SseK3 possesses the typical glycosyltransferase type-A (GT-A)-family fold and the metal-coordinating DXD motif essential for ligand binding and enzymatic activity. Several conserved residues were essential for arginine-GlcNAcylation and SseK3-mediated inhibition of NF-κB activation. Isothermal titration calorimetry revealed SseK3's preference for manganese coordination. The pattern of interactions in the substrate-bound SseK3 structure explained the selection of the primary ligand. Structural re-arrangement of the C-terminal residues upon ligand binding was crucial for SseK3's catalytic activity and NMR analysis indicated that SseK3 has limited UDP-GlcNAc hydrolysis activity. The release of free N-acetyl α-D-glucosamine, and the presence of the same molecule in the SseK3 active site, classified it as a retaining glycosyltransferase. A glutamate residue in the active site suggested a double-inversion mechanism for the arginine N-glycosylation reaction. Homology models of SseK1, SseK2, and the Escherichia coli orthologue NleB1, reveal differences in the surface electrostatic charge distribution possibly accounting for their diverse activities. This first structure of a retaining GT-A arginine N-glycosyltransferase provides an important step towards a better understanding of this enzyme class and their roles as bacterial effectors.
New Search Next
- Low apolipoprotein A-I levels in Friedreich's ataxia and in frataxin-deficient cells: Implications for therapy. [Journal Article]
- PlosPLoS One 2018; 13(2):e0192779
- Friedreich's ataxia (FA) is an autosomal recessive neurodegenerative disorder, which results primarily from reduced expression of the mitochondrial protein frataxin. FA has an estimated prevalence of...
Friedreich's ataxia (FA) is an autosomal recessive neurodegenerative disorder, which results primarily from reduced expression of the mitochondrial protein frataxin. FA has an estimated prevalence of one in 50,000 in the population, making it the most common hereditary ataxia. Paradoxically, mortality arises most frequently from cardiomyopathy and cardiac failure rather than from neurological effects. Decreased high-density lipoprotein (HDL) and apolipoprotein A-I (ApoA-l) levels in the general population are associated with an increased risk of mortality from cardiomyopathy and heart failure. However, the pathophysiology of heart disease in FA is non-vascular and there are conflicting data on HDL-cholesterol in FA. Two studies have shown a decrease in HDL-cholesterol compared with controls and two have shown there was no difference between FA and controls. One also showed that there was no difference in serum Apo-A-I levels in FA when compared with controls. Using a highly specific stable isotope dilution mass spectrometry-based assay, we demonstrated a 21.6% decrease in serum ApoA-I in FA patients (134.8 mg/dL, n = 95) compared with non-affected controls (172.1 mg/dL, n = 95). This is similar to the difference in serum ApoA-I levels between non-smokers and tobacco smokers. Knockdown of frataxin by > 70% in human hepatoma HepG2 cells caused a 20% reduction in secreted ApoA-I. Simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor caused a 200% increase in HMG-CoA in the control HepG2 cells with a similar increase in the frataxin knockdown HepG2 cells, back to levels found in the control cells. There was a concomitant 20% increase in secreted ApoA-I to levels found in the control cells that were treated with simvastatin. This study provides compelling evidence that ApoA-I levels are reduced in FA patients compared with controls and suggest that statin treatment would normalize the ApoA-I levels.