Terminal Regions Confer Plasticity to the Tetrameric Assembly of Human HspB2 and HspB3

Heterogeneity in small heat shock proteins (sHsps) spans multiple spatiotemporal regimes—from fast fluctuations of part of the protein, to conformational variability of tertiary structure, plasticity of the interfaces, and polydispersity of the inter-converting, and co-assembling oligomers. This heterogeneity and dynamic nature of sHsps has significantly hindered their structural characterization. Atomic coordinates are particularly lacking for vertebrate sHsps, where most available structures are of extensively truncated homomers. sHsps play important roles in maintaining protein levels in the cell and therefore in organismal health and disease. HspB2 and HspB3 are vertebrate sHsps that are found co-assembled in neuromuscular cells, and variants thereof are associated with disease. Here, we present the structure of human HspB2/B3, which crystallized as a hetero-tetramer in a 3:1 ratio. In the HspB2/B3 tetramer, the four α-crystallin domains (ACDs) assemble into a flattened tetrahedron which is pierced by two non-intersecting approximate dyads. Assembly is mediated by flexible “nuts and bolts” involving IXI/V motifs from terminal regions filling ACD pockets. Parts of the N-terminal region bind in an unfolded conformation into the anti-parallel shared ACD dimer grooves. Tracts of the terminal regions are not resolved, most likely due to their disorder in the crystal lattice. This first structure of a full-length human sHsp heteromer reveals the heterogeneous interactions of the terminal regions and suggests a plasticity that is important for the cytoprotective functions of sHsps.     

Longitudinal investigation of neuroinflammation and metabolite profiles in the APPswe×PS1Δe9 transgenic mouse model of Alzheimer's disease

There is increasing evidence linking neuroinflammation to many neurological disorders including Alzheimer's disease (AD); however, its exact contribution to disease manifestation and/or progression is poorly understood. Therefore, there is a need to investigate neuroinflammation in both health and disease. Here, we investigate cognitive decline, neuroinflammatory and other pathophysiological changes in the APP_swe×PS1_Δe9 transgenic mouse model of AD. Transgenic (TG) mice were compared to C57BL/6 wild type (WT) mice at 6, 12 and 18 months of age. Neuroinflammation was investigated by [¹⁸F]DPA‐714 positron emission tomography and myo‐inositol levels using ¹H magnetic resonance spectroscopy (MRS) in vivo. Neuronal and cellular dysfunction was investigated by looking at N‐acetylaspartate (NAA), choline‐containing compounds, taurine and glutamate also using MRS. Cognitive decline was first observed at 12 m of age in the TG mice as assessed by working memory tests . A significant increase in [¹⁸F]DPA‐714 uptake was seen in the hippocampus and cortex of 18 m‐old TG mice when compared to age‐matched WT mice and 6 m‐old TG mice. No overall effect of gene was seen on metabolite levels; however, a significant reduction in NAA was observed in 18 m‐old TG mice when compared to WT. In addition, age resulted in a decrease in glutamate and an increase in choline levels. Therefore, we can conclude that increased neuroinflammation and cognitive decline are observed in TG animals, whereas NAA alterations occurring with age are exacerbated in the TG mice. These results support the role of neuroinflammation and metabolite alteration in AD and in ageing.

     

Design,synthesis and molecular docking of thiazolidinedione based benzene sulphonamide derivatives containing pyrazole core as potential anti-diabetic agents

We herein report the design, synthesis and molecular docking studies of 2,4-thiazolidinedione derivatives containing benzene sulphonyl group which are docked against the Peroxisome Proliferator Activated Receptor (PPARγ) target. Compound 7p was most effective in lowering the blood glucose level as compared to standard drugs pioglitazone and rosiglitazone. Compound 7p exhibited potent PPAR-γ transactivation of 61.2% with 1.9 folds increase in gene expression. In molecular docking studies 7p showed excellent interactions with amino acids TYR 473, SER 289, HIE 449, TYR 327, ARG 288, MET 329 and LEU 228. Compound 7p did not cause any damage to the liver without any noteworthy weight gain and may be considered as promising candidates for the development of new antidiabetic agents.     

Multispectroscopic DNA-binding studies of a terbium(III) complex containing 2,2′-bipyridine ligand

Agarose gel electrophoresis, absorption, fluorescence, viscosity, and circular dichroism (CD) have been used in exploring the interaction of terbium(III) complex, [Tb(bpy)₂Cl₃(OH₂)] where bipy is 2,2′-bipyridine, with Fish salmon DNA. Agarose gel electrophoresis assay, along with absorption and fluorescence studies, reveal interaction between the corresponding complex and FS-DNA. Also, the binding constants (K_b) and the Stern–Volmer quenching constants (K_sv) of Tb(III) complex with FS-DNA were determined. The calculated thermodynamic parameters suggested that the binding of mentioned complex to FS-DNA was driven mainly by hydrophobic interactions. A comparative study of this complex with respect to the effect of iodide-induced quenching, ionic strength effect, and ethidium bromide exclusion assay reflects binding of explicit to the FS-DNA primarily in a groove fashion. CD and viscosity data also support the groove binding mode. Furthermore, Tb(III) complex have been simultaneously screened for their antibacterial and antifungal activities.     

Inclusion complexation between baicalein and β-cyclodextrin and the influence of β-cyclodextrin on the binding of baicalein with DNA: a spectroscopic approach

This work deals with the commonly studied cyclic oligosaccharide and gains importance as it is entered on a drug delivering carbohydrate and provides insight into the oligosaccharide complex–biomolecular interaction. The binding of a flavone, baicalein, to β-cyclodextrin and calf thymus DNA is studied. The binding of baicalein to calf thymus DNA in the presence of β-cyclodextrin is analysed using the UV–vis absorption and fluorescence spectroscopy. The mode of binding and structure of the baicalein–β-cyclodextrin complex are reported. The role of the structure and the stoichiometry of the inclusion complex of baicalein–β-cyclodextrin in its influence on DNA binding are analysed.

Highlights

? This paper deals with the binding of a flavone, baicalein to β-cyclodextrin and/or DNA.

? The inclusion complexation between baicalein and β-cyclodextrin is analysed.

? The stoichiometry and the binding strength of the inclusion complex is reported.

? The role of β-cyclodextrin in tuning the binding of baicalein to DNA is emphasized.

? Spectroscopic and docking analysis are used to articulate the results.     

Unravelling the mechanisms of a protein refolding process based on the association of detergents and co‐solvents

A new technique associating the detergent Sodium Dodecyl Sulphate (SDS) and an alcohol‐type co‐solvent has been set up, showing an unexpected efficiency to refold several types of soluble or membrane proteins. The present contribution deepens the fundamental knowledge on the phenomena underlying this process, considering the refolding of two model peptides featuring the main protein secondary structures: α‐helix and β‐sheet. Their refolding was monitored by fluorescence and circular dichroism, and it turns out that: (i) 100% recovery of the folded structure is observed for both peptides, (ii) the highest the SDS concentration, the more co‐solvent to be added to recover the peptides' native structures, (iii) a high alcohol concentration is required to alter the SDS denaturing properties, (iv) the co‐solvent performance relies on its specific lipophilic–hydrophilic balanced character, (v) the size of the micelle formed by the detergent does not enter the process critical parameters, and (vi) increasing the salt concentration up to 1 M NaCl has a beneficial impact on the process efficiency. These mechanistic aspects will help us to improve the method and extend its application. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Biogenic synthesis of gold nanoparticle using fractioned cellular components from eukaryotic algae and cyanobacteria

In the present investigation fractioned cellular components like intact pigment bearing thylakoids/chloroplasts, carotenoids, protein, polysaccharides were extracted from the cyanobacterium Anabaena sphaerica and green alga Chlorococcum infusionum. Each of these extracts was used separately in search for efficient reducing agents during gold nanoparticle (GNP) production in pro‐ and eukaryotic algal cell systems. The whole biomass and extracted compounds or cellular structures were exposed in 25 mg L^?1 aqueous hydrogen tetrachloroaurate solutions separately at room temperature. Isolated viable chloroplasts from C. infusionum and thylakoids from A. sphaerica were found to be able to reduce gold ions. The protein extracts of both strains were also able to synthesize GNP at 4°C. Extracted polysaccharides of the two strains responded differently. Polysaccharides from A. sphaerica showed positive response in GNP synthesis, whereas no change was observed for C. infusionum. The carotenoids extracts from both strains acted like an efficient reducing agent. Initially the reducing efficiency of these extracted components was confirmed by the appearance of purple color in biomass or in experimental media. The GNPs, synthesized within the biomass were extracted by sonication with sodium citrate. The UV–vis spectroscopy of extracted purple colored suspensions and media showed the absorption bands at approximately 530–540 nm indicating a strong positive signal of GNP synthesis. Transmission electro n microscopy determined the size and shapes of the particles. The X‐ray diffraction study of the synthesized GNP revealed that the 2θ values appeared at 38.2°, 44.5°, 64.8° and 77.8°. Amongst all, isolated thylakoids and chloroplast showed only spherical GNP production with variable size range at pH 4. Monodisperse GNPs were also synthesized by isolated thylakoids and chloroplast at pH 9. A detailed morphological change of gold treated biomass was revealed employing scanning electron microscopy. The fluorescent property of gold loaded cells was studied by fluorescence microscopy.     

Specific Inter-residue Interactions as Determinants of Human Monoacylglycerol Lipase Catalytic Competency: A ROLE FOR GLOBAL CONFORMATIONAL CHANGES*

The serine hydrolase monoacylglycerol lipase (MGL) functions as the main metabolizing enzyme of 2-arachidonoyl glycerol, an endocannabinoid signaling lipid whose elevation through genetic or pharmacological MGL ablation exerts therapeutic effects in various preclinical disease models. To inform structure-based MGL inhibitor design, we report the direct NMR detection of a reversible equilibrium between active and inactive states of human MGL (hMGL) that is slow on the NMR time scale and can be modulated in a controlled manner by pH, temperature, and select point mutations. Kinetic measurements revealed that hMGL substrate turnover is rate-limited across this equilibrium. We identify a network of aromatic interactions and hydrogen bonds that regulates hMGL active-inactive state interconversion. The data highlight specific inter-residue interactions within hMGL modulating the enzymes function and implicate transitions between active (open) and inactive (closed) states of the hMGL lid domain in controlling substrate access to the enzymes active site.     

Lipid-Protein Interactions in the Regulated Betaine Symporter BetP Probed by Infrared Spectroscopy

The Na⁺-coupled betaine symporter BetP senses changes in the membrane state and increasing levels of cytoplasmic K⁺ during hyperosmotic stress latter via its C-terminal domain and regulates transport activity according to both stimuli. This intriguing sensing and regulation behavior of BetP was intensively studied in the past. It was shown by several biochemical studies that activation and regulation depends crucially on the lipid composition of the surrounding membrane. In fact, BetP is active and regulated only when negatively charged lipids are present. Recent structural studies have revealed binding of phosphatidylglycerol lipids to functional important parts of BetP, suggesting a functional role of lipid interactions. However, a regulatory role of lipid interactions could only be speculated from the snapshot provided by the crystal structure. Here, we investigate the nature of lipid-protein interactions of BetP reconstituted in closely packed two-dimensional crystals of negatively charged lipids and probed at the molecular level with Fourier transform infrared (FTIR) spectroscopy. The FTIR data indicate that K⁺ binding weakens the interaction of BetP especially with the anionic lipid head groups. We suggest a regulation mechanism in which lipid-protein interactions, especially with the C-terminal domain and the functional important gating helices transmembrane helice 3 (TMH3) and TMH12, confine BetP to its down-regulated transport state. As BetP is also activated by changes in the physical state of the membrane, our results point toward a more general mechanism of how active transport can be modified by dynamic lipid-protein interactions.