Metabolic coupling between glutamate and N-acetylaspartate in the human brain.

A metabolic coupling between glutamate and N-acetylaspartate measured by in vivo magnetic resonance spectroscopy has been recently reported in the literature with inconsistent findings. In this study, confounders originating from Pearson's spurious correlation of ratios and spectral correlation due to overlapping magnetic resonance spectroscopy signals of glutamate and N-acetylaspartate were practically eliminated to facilitate the determination of any metabolic link between glutamate and N-acetylaspartate in the human brain using in vivo magnetic resonance spectroscopy. In both occipital and medial prefrontal cortices of healthy individuals, correlations between glutamate and N-acetylaspartate were found to be insignificant. Our results do not lend support to a recent hypothesis that N-acetylaspartate serves as a significant reservoir for the rapid replenishment of glutamate during signaling or stress.

Elevated Brain Glutamate Levels in Bipolar Disorder and Pyruvate Carboxylase-Mediated Anaplerosis.

In vivo 1H magnetic resonance spectroscopy studies have found elevated brain glutamate or glutamate + glutamine levels in bipolar disorder with surprisingly high reproducibility. We propose that the elevated glutamate levels in bipolar disorder can be explained by increased pyruvate carboxylase-mediated anaplerosis in brain. Multiple independent lines of evidence supporting increased pyruvate carboxylase-mediated anaplerosis as a common mechanism underlying glutamatergic hyperactivity in bipolar disorder and the positive association between bipolar disorder and obesity are also described.

Cerebral phosphoester signals measured by 31P magnetic resonance spectroscopy at 3 and 7 Tesla.

Abnormal cell membrane metabolism is associated with many neuropsychiatric disorders. Free phosphomonoesters and phosphodiesters, which can be detected by in vivo 31P magnetic resonance spectroscopy (MRS), are important cell membrane building blocks. However, the quantification of phosphoesters has been highly controversial even in healthy individuals due to overlapping signals from macromolecule membrane phospholipids (MP). In this study, high signal-to-noise ratio (SNR) cerebral 31P MRS spectra were acquired from healthy volunteers at both 3 and 7 Tesla. Our results indicated that, with minimal spectral interference from MP, the [phosphocreatine (PCr)]/[phosphocholine (PC) + glycerophosphocholine (GPC)] ratio measured at 7 Tesla agreed with its value expected from biochemical constraints. In contrast, the 3 Tesla [PCr]/[PC+GPC] ratio obtained using standard spectral fitting procedures was markedly smaller than the 7 Tesla ratio and than the expected value. The analysis suggests that the commonly used spectral model for MP may fail to capture its complex spectral features at 3 Tesla, and that additional prior knowledge is necessary to reliably quantify the phosphoester signals at low magnetic field strengths when spectral overlapping is significant.

Local and Interregional Neurochemical Associations Measured by Magnetic Resonance Spectroscopy for Studying Brain Functions and Psychiatric Disorders.

Magnetic resonance spectroscopy (MRS) studies have found significant correlations among neurometabolites (e.g., between glutamate and GABA) across individual subjects and altered correlations in neuropsychiatric disorders. In this article, we discuss neurochemical associations among several major neurometabolites which underpin these observations by MRS. We also illustrate the role of spectral editing in eliminating unwanted correlations caused by spectral overlapping. Finally, we describe the prospects of mapping macroscopic neurochemical associations across the brain and characterizing excitation-inhibition balance of neural networks using glutamate- and GABA-editing MRS imaging.

Characterization of Carbonic Anhydrase In Vivo Using Magnetic Resonance Spectroscopy.

Carbonic anhydrase is a ubiquitous metalloenzyme that catalyzes the reversible interconversion of CO2/HCO3-. Equilibrium of these species is maintained by the action of carbonic anhydrase. Recent advances in magnetic resonance spectroscopy have allowed, for the first time, in vivo characterization of carbonic anhydrase in the human brain. In this article, we review the theories and techniques of in vivo 13C magnetization (saturation) transfer magnetic resonance spectroscopy as they are applied to measuring the rate of exchange between CO2 and HCO3- catalyzed by carbonic anhydrase. Inhibitors of carbonic anhydrase have a wide range of therapeutic applications. Role of carbonic anhydrases and their inhibitors in many diseases are also reviewed to illustrate future applications of in vivo carbonic anhydrase assessment by magnetic resonance spectroscopy.

Polyamine acetylation and substrate-induced oligomeric states in histone acetyltransferase of multiple drug resistant Acinetobacter baumannii.

Histone acetyltransferase (Hpa2) is an unusual acetyltransferase, with a wide range of substrates; including histones, polyamines and aminoglycosides antibiotic. Hpa2 belongs to GNAT superfamily and GNATs are well known for the formation of homo-oligomers. However, the reason behind their oligomerization remained unexplored. Here, oligomeric states of Hpa2 were explored, to understand the functional significance of oligomerization. Biochemical analysis suggests that Hpa2 exists as dimer in solution and self-assembles into tetramer in the spermine, spermidine and kanamycin bound form. Stability analysis with denaturants concludes that homo-oligomerization of Hpa2 relies on bound substrate and not on experimental conditions. Homo-oligomerization in Hpa2 depicts direct correlation with its polyamine acetylating capacity. This correlation and in silico model structures suggest that oligomerization of Hpa2 is associated with the hastening of acetylation process. Interestingly, polyamine acetylation down regulates biofilms formation in E. coli BL21/Hpa2-transformants cells. Therefore, we propose that Hpa2 manipulates survival strategies of the bacterium via polyamines and antibiotics acetylation.

Aminoglycoside antibiotic resistance conferred by Hpa2 of MDR Acinetobacter baumannii: an unusual adaptation of a common histone acetyltransferase.

Antibiotic-resistant bacteria pose the greatest threat to human health. Among the list of such bacteria released by WHO, carbapenem-resistant Acinetobacter baumannii, for which almost no treatment exists, tops the list. A. baumannii is one of the most troublesome ESKAPE pathogens and mechanisms that have facilitated its rise as a successful pathogen are not well studied. Efforts in this direction have resulted in the identification of Hpa2-Ab, an uncharacterized histone acetyltransferase enzyme of GNAT superfamily. Here, we show that Hpa2-Ab confers resistance against aminoglycoside antibiotics using Escherichia coli DH5α strains in which Hpa2 gene is expressed. Resistivity for aminoglycoside antibiotics is demonstrated with the help of CLSI-2010 and KB tests. Isothermal titration calorimetry, MALDI and acetylation assays indicate that conferred resistance is an outcome of evolved antibiotic acetylation capacity in this. Hpa2 is known to acetylate nuclear molecules; however, here it is found to cross its boundary and participate in other functions. An array of biochemical and biophysical techniques were also used to study this protein, which demonstrates that Hpa2-Ab is intrinsically oligomeric in nature, exists primarily as a dimer and its interface is mainly stabilized by hydrophobic interactions. Our work demonstrates an evolved survival strategy by A. baumannii and provides insights into the mechanism that facilitates it to rise as a successful pathogen.

A. baumannii histone acetyl transferase Hpa2: optimization of homology modeling, analysis of protein-protein interaction and virtual screening.

In the current scenario, widespread multidrug resistivity in ESKAPE pathogens demands identification of novel drug targets to keep their infections at bay. For this purpose, we have identified a novel target Hpa2 of A. baumannii, a member of GNAT superfamily of HATs. But due to sequence identity of equal or less than 35%, the correct sequence alignment and construction of 3D monomeric and dimeric models of Hpa2 having optimal structural parameters is a troublesome task. To circumvent these problems, we have designed an easy and optimized protocol for Hpa2 monomer modeling, and for generation of dimeric Hpa2 model using data-driven protein-protein docking experiment. Improvement in the structural features of generated model is an onerous process and generally achieved by paying time and computational cost. Herein, it is achieved by reconciliation of FoldX commands which takes less time in execution. Evaluations performed to validate structural parameters and stability of monomeric and dimeric Hpa2 attests to its quality. Analysis of interfacial residues, energy terms and RMSD values indicated a clear correlation between experimental and theoretical interface properties of the dimers, corroborating to the regime used for Hpa2 dimer generation. Structural information from the refined models was used for virtual screening of substrate-derived library and polyamines to achieve a new platform for developing A. baumannii inhibitory molecules. Molecules showing preferential binding at the dimer interface could be used as allosteric inhibitors. Binding of polyamines with model illustrated the same binding pattern as described experimentally in case of yeast Hpa2.

Characterization of substrate binding and enzymatic removal of a 3-methyladenine lesion from genomic DNA with TAG of MDR A. baumannii.

The rise of multiple-drug resistance in bacterial pathogens imposes a serious public health concern and has led to increased interest in studying various pathways as well as enzymes. Different DNA glycosylases collaborate during bacterial infection and disease by overcoming the effects of ROS- and RNS-mediated host innate immunity response. 3-Methyladenine DNA glycosylase I, an essential DNA repair enzyme, was chosen for the present study from the MDR species of A. baumannii. The enzyme was especially chosen because of its functional significance in A. baumannii and due to its structural variation from its human homologue. MDR strains such as A. baumannii are interesting targets owing to their evolved mechanisms of evading a host defence. In the absence of any structural information, the enzyme was characterized biophysically and biochemically. Binding studies with 3mA and Zn2+ indicated that the activity of TAG-Ab is an enthalpy-driven process. Fluorescence thermal denaturation studies described that the denaturation of TAG-Ab is a two-step process. Modified RP-HPLC-based glycosylase assay attested that the heterologously expressed and purified TAG-Ab enzyme is active and catalyses the removal of 3mA. Other binding parameters and the effect of adenine on substrate binding are also discussed in detail.

Optimized method for TAG protein homology modeling: In silico and experimental structural characterization.

The DNA glycosylases cleave CN glycosyl bond to release a free base and generate abasic sites concurrently. Function and structure of these enzymes in the pathogenic bacterium Acinetobacter baumannii and its closely related species are not well characterized. Inhibition of TAG enzyme is a promising drug design strategy against A. baumannii. Here optimized molecular modeling approaches were used to provide a structural scaffold of TAG. The recombinant TAG protein was expressed and purified to determine oligomeric state using size exclusion chromatography, which showed the existence of TAG protein as monomer (mwt ∼21kDa). Secondary structure and substrate binding were analyzed using CD are in good agreement with the in silico predictions. Near UV-CD spectrum shows the involvement of Tyr residues in substrate recognition. Molecular docking studies were performed to understand the molecular recognition interactions and this knowledge was used to identify the potent inhibitors using virtual screening. Residues crucial for DNA holding and enzyme catalysis are reconfirmed by the in silico mutational studies.

In-silico modeling studies of G-quadruplex with soy isoflavones having anticancerous activity.

Telomere forms t-loop and G-quadruplex as the protective structure and the formation of these structures hinder the telomerase enzyme action. The binding affinities of ligand which stabilize the G-quadruplex represent good correlation with telomerase inhibition depicted in the anti-cancerous action. Most of the potent G-quadruplex stabilizing compounds suffer from the poor drug like properties. Herein, natural dietary compounds isoflavones were taken for the theoretical study to examine their stabilizing effect on G-quadruplex structure. The experimental G-quadruplex complexes were reproduced to obtain and validate the theoretical parameters. The obtained theoretical binding energies are in significant correlation with the experimental data. Analysis of binding shows isoflavones to be groove binders, and differential nature of quadruplex grooves might be beneficial in the selectivity aspects. Among all, derrubone was found to have better selectivity as well as affinity for the G-quadruplex comparable to well known ligand TMPyP4. The GBSA rescoring result enlightens the various interaction terms involved in the binding process. Cumulative stabilizing effects coming from VDW, ES, and GB energy terms attest to optimal binding of derrubone molecule which can be considered as a lead for the higher phases of drug designing. These findings are of great value in terms of unexplored groove binding modes and the studied natural compounds might be helpful to direct the focus of synthetic chemists in designing of new generation of antitumor agents.