Membrane potential accelerates sugar uptake by stabilizing the outward facing conformation of the Na/glucose symporter vSGLT.

Sodium-dependent glucose transporters (SGLTs) couple a downhill Na+ ion gradient to actively transport sugars. Here, we investigate the impact of the membrane potential on vSGLT structure and function using sugar uptake assays, double electron-electron resonance (DEER), electrostatic calculations, and kinetic modeling. Negative membrane potentials, as present in all cell types, shift the conformational equilibrium of vSGLT towards an outward-facing conformation, leading to increased sugar transport rates. Electrostatic calculations identify gating charge residues responsible for this conformational shift that when mutated reduce galactose transport and eliminate the response of vSGLT to potential. Based on these findings, we propose a comprehensive framework for sugar transport via vSGLT, where the cellular membrane potential facilitates resetting of the transporter after cargo release. This framework holds significance not only for SGLTs but also for other transporters and channels.

Crystal structure of an L-type lectin domain from archaea.

The crystal structures of an L-type lectin domain from Methanocaldococcus jannaschii in apo and mannose-bound forms have been determined. A thorough investigation of L-type lectin domains from several organisms provides insight into the differences in these domains from different kingdoms of life. While the overall fold of the L-type lectin domain is conserved, differences in the lengths of the carbohydrate-binding loops and significant variations in the Mn2+ -binding site compared to the Ca2+ -binding site are observed. Furthermore, the sequence and phylogenetic analyses suggest that the archaeal L-type lectin domain is evolutionarily closer to the plant legume lectins than to its bacterial or animal counterparts. This is the first report of the biochemical, structural, sequence, and phylogenetic analyses of an L-type lectin domain from archaea and serves to enhance our understanding of the species-specific differences and evolution of L-type lectin domains.

The Single Residue K12 Governs the Exceptional Voltage Sensitivity of Mitochondrial Voltage-Dependent Anion Channel Gating.

The voltage-dependent anion channel (VDAC) is a ß-barrel channel of the mitochondrial outer membrane (MOM) that passively transports ions, metabolites, polypeptides, and single-stranded DNA. VDAC responds to a transmembrane potential by "gating," i.e. transitioning to one of a variety of low-conducting states of unknown structure. The gated state results in nearly complete suppression of multivalent mitochondrial metabolite (such as ATP and ADP) transport, while enhancing calcium transport. Voltage gating is a universal property of ß-barrel channels, but VDAC gating is anomalously sensitive to transmembrane potential. Here, we show that a single residue in the pore interior, K12, is responsible for most of VDAC's voltage sensitivity. Using the analysis of over 40 µs of atomistic molecular dynamics (MD) simulations, we explore correlations between motions of charged residues inside the VDAC pore and geometric deformations of the ß-barrel. Residue K12 is bistable; its motions between two widely separated positions along the pore axis enhance the fluctuations of the ß-barrel and augment the likelihood of gating. Single channel electrophysiology of various K12 mutants reveals a dramatic reduction of the voltage-induced gating transitions. The crystal structure of the K12E mutant at a resolution of 2.6 Å indicates a similar architecture of the K12E mutant to the wild type; however, 60 µs of atomistic MD simulations using the K12E mutant show restricted motion of residue 12, due to enhanced connectivity with neighboring residues, and diminished amplitude of barrel motions. We conclude that ß-barrel fluctuations, governed particularly by residue K12, drive VDAC gating transitions.

Purification, characterization, and crystal structure of YhdA-type azoreductase from Bacillus velezensis.

Azoreductases are being extensively investigated for their ability to initiate degradation of recalcitrant azo dyes through reduction of azo bonds. There is great interest in studying their diversity, structure, and function to facilitate better understanding and effective application. Current study reports azoreductase enzyme from Bacillus velezensis, which showed 69.5% identity to the Bacillus subtilis azoreductase YhdA. The enzyme was homotetrameric and molecular weight of each subunit was 20 kDa. It decolorized azo dyes with different structures. The Vmax for decolorization of congo red, methyl orange and methyl red was 14.7, 28.6, and 77.9 nmol/min/mg, respectively. The enzyme contained FMN as cofactor and used NADPH as the favored co-substrate. It was oxygen-insensitive, but the presence of reducing agents enhanced its activity, which is a new finding. The azoreductase expression in B. velezensis was found to be unaffected by addition of azo dyes, although azo dyes are known to induce azoreductase expression in few organisms. The enzyme was thermostable with melting temperature of 89.5°C and functioned in wide temperature range. Further, the enzyme was crystallized and its structure was solved. The structural basis of its functional attributes is discussed. In our knowledge, this is the first report on characterization of azoreductase enzyme from B. velezensis.

MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel.

A structure of the murine voltage-dependent anion channel (VDAC) was determined by microcrystal electron diffraction (MicroED). Microcrystals of an essential mutant of VDAC grew in a viscous bicelle suspension, making it unsuitable for conventional X-ray crystallography. Thin, plate-like crystals were identified using scanning-electron microscopy (SEM). Crystals were milled into thin lamellae using a focused-ion beam (FIB). MicroED data were collected from three crystal lamellae and merged for completeness. The refined structure revealed unmodeled densities between protein monomers, indicative of lipids that likely mediate contacts between the proteins in the crystal. This body of work demonstrates the effectiveness of milling membrane protein microcrystals grown in viscous media using a focused ion beam for subsequent structure determination by MicroED. This approach is well suited for samples that are intractable by X-ray crystallography. To our knowledge, the presented structure is a previously undescribed mutant of the membrane protein VDAC, crystallized in a lipid bicelle matrix and solved by MicroED.

Charge Reduction of Membrane Proteins in Native Mass Spectrometry Using Alkali Metal Acetate Salts.

Native mass spectrometry (MS) provides the capacity to monitor membrane protein complexes and noncovalent binding of ligands and lipids to membrane proteins. The charge states produced by native MS of membrane proteins often result in gas-phase protein unfolding or loss of noncovalent interactions. In an effort to reduce the charge of membrane proteins, we examined the utility of alkali metal salts as a charge-reducing agent. Low concentrations of alkali metal salts caused marked charge reduction in the membrane protein, Erwinia ligand-gated ion channel (ELIC). The charge-reducing effect only occurred for membrane proteins and was detergent-dependent, being most pronounced in long polyethylene glycol (PEG)-based detergents such as C10E5 and C12E8. On the basis of these results, we propose a mechanism for alkali metal charge reduction of membrane proteins. Addition of low concentrations of alkali metals may provide an advantageous approach for charge reduction of detergent-solubilized membrane proteins by native MS.

Enhancement of the cancer inhibitory effect of the bioactive food component resveratrol by nanoparticle based delivery.

Naturally occurring bioactive food components such as dietary polyphenols have shown many beneficial biological activities due to their good antioxidant properties. Among them significant attention has been given to resveratrol (RV) in recent years as it plays a promising role in cancer prevention. It has demonstrated anti-proliferative effects, as well as the ability to inhibit the initiation and progression of induced cancer in a wide variety of tumor models. However, the benefits of its therapeutic effects were found to be limited due to its poor pharmacokinetic properties such as poor aqueous solubility, instability and extensive first pass metabolism. To overcome these limitations, the present study aimed to synthesize thermosensitive copolymeric nanoparticle encapsulated formulations of resveratrol-nanoresveratrol (NRV) and evaluate their in vitro anticancer activity and inhibitory effect on 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin inflammation and tumorigenesis in Swiss albino mice. For this purpose PNIPAAM-PEG based thermosensitive copolymeric nanoparticles were synthesized followed by the encapsulation of RV in their hydrophobic core. This enhanced the therapeutic bioavailability of resveratrol. Nanoresveratrol demonstrated stronger antioxidant activity and comparable anticancer efficacy to free resveratrol. Nanoparticles were characterized by IR, NMR, DLS and TEM. The best results were obtained with NRV at significantly lower doses. NRV demonstrated better in vitro anticancer activity against melanoma cell line B16. It showed comparable reduction of TPA induced skin edema, hyperplasia and oxidative stress response. In the promotion phase, a significant reduction was found in tumor incidence and tumor burden in mice pre-treated with NRV. Moreover, at all doses NRV altered Bax and Bcl2 expressions which lead to the induction of apoptosis.

Crystal structures of a ß-trefoil lectin from Entamoeba histolytica in monomeric and a novel disulfide bond-mediated dimeric forms.

ß-Trefoil lectins are galactose/N-acetyl galactosamine specific lectins, which are widely distributed across all kingdoms of life and are known to perform several important functions. However, there is no report available on the characterization of these lectins from protozoans. We have performed structural and biophysical studies on a ß-trefoil lectin from Entamoeba histolytica (EntTref), which exists as a mixture of monomers and dimers in solution. Further, we have determined the affinities of EntTref for rhamnose, galactose and different galactose-linked sugars. We obtained the crystal structure of EntTref in a sugar-free form (EntTref_apo) and a rhamnose-bound form (EntTref_rham). A novel Cys residue-mediated dimerization was revealed in the crystal structure of EntTref_apo while the structure of EntTref_rham provided the structural basis for the recognition of rhamnose by a ß-trefoil lectin for the first time. To the best of our knowledge, this is the only report of the structural, functional and biophysical characterization of a ß-trefoil lectin from a protozoan source and the first report of Cys-mediated dimerization in this class of lectins.

Targeted delivery of thermoresponsive polymeric nanoparticle-encapsulated lycopene: in vitro anticancer activity and chemopreventive effect on murine skin inflammation and tumorigenesis.

Naturally occurring lycopene has been reported for its chemopreventive and chemotherapeutic efficiency in various cancers, but its exceptional lipophilicity, poor aqueous solubility, instability, and consequently poor bioavailability limit its usage as a chemopreventive and chemotherapeutic agent. The present study aimed to synthesize co-polymeric nanoparticle-encapsulated formulations of commercial lycopene (NLY) and extracted lycopene (NLX) and evaluate their in vitro anticancer activity and inhibitory effect on 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin inflammation and tumorigenesis in Swiss albino mice. To prepare the nanoparticle-encapsulated formulations of lycopene, thermosensitive PNIPAAM-PEG-based co-polymeric nanoparticles were synthesized and characterized by FTIR spectroscopy, NMR spectroscopy, DLS, and TEM. Nanolycopene, unlike free lycopene, could be readily dispersed in aqueous media. Nanolycopene demonstrated stronger antioxidant activity and comparable in vitro anticancer efficacy to free lycopene against the melanoma cell line B16. Furthermore, nanolycopene showed comparable reduction of TPA-induced skin edema, expression of COX-2, and oxidative stress response. Additionally, it showed significant inhibition of tumor promotion. It also altered Bax and Bcl2 expressions, which led to the induction of apoptosis. The results also supported that the extracted lycopene-encapsulated nanoparticles may be a good alternative to the expensive commercial lycopene for cancer treatment.

Crystal structure of the legume lectin-like domain of an ERGIC-53-like protein from Entamoeba histolytica.

ERGIC-53-like proteins are type I membrane proteins that belong to the class of intracellular cargo receptors and are known to be indispensable for the intracellular transport of glycoproteins. They are implicated in transporting glycoproteins between the endoplasmic reticulum and the Golgi body. The crystal structure of the legume lectin-like domain of an ERGIC-53-like protein from Entamoeba histolytica has been determined at 2.4 Šresolution. Although the overall structure of the domain resembles those of its mammalian and yeast orthologs (ERGIC-53 and Emp46, respectively), there are significant changes in the carbohydrate-binding site. A sequence-based search revealed the presence of several homologs of ERGIC-53 in different species of Entamoeba. This is the first report of the structural characterization of a member of this class of proteins from a protozoan and serves to further knowledge and understanding regarding the species-specific differences.

Structural and functional characterization of mercuric reductase from Lysinibacillus sphaericus strain G1.

In response to the widespread presence of inorganic Hg in the environment, bacteria have evolved resistance systems with mercuric reductase (MerA) as the key enzyme. MerA enzymes have still not been well characterized from gram positive bacteria. Current study reports physico-chemical, kinetic and structural characterization of MerA from a multiple heavy metal resistant strain of Lysinibacillus sphaericus, and discusses its implications in bioremediation application. The enzyme was homodimeric with subunit molecular weight of about 60 kDa. The Km and Vmax were found to be 32 µM of HgCl2 and 18 units/mg respectively. The enzyme activity was enhanced by ß-mercaptoethanol and NaCl up to concentrations of 500 µM and 100 mM respectively, followed by inhibition at higher concentrations. The enzyme showed maximum activity in the pH range of 7-7.5 and temperature range of 25-50 °C, with melting temperature of 67 °C. Cu2+ exhibited pronounced inhibition of the enzyme with mixed inhibition pattern. The enzyme contained FAD as the prosthetic group and used NADPH as the preferred electron donor, but it showed slight activity with NADH as well. Structural characterization was carried out by circular dichroism spectrophotometry and X-ray crystallography. X-ray confirmed the homodimeric structure of enzyme and gave an insight on the residues involved in catalytic binding. In conclusion, the investigated enzyme showed higher catalytic efficiency, temperature stability and salt tolerance as compared to MerA enzymes from other mesophiles. Therefore, it is proposed to be a promising candidate for Hg2+ bioremediation.

Validation of PCR based detection system for aflatoxin producing molds.

Aflatoxins are polyketide secondary metabolites that are produced by certain fungal species in the Aspergillus section Flavi, particularly Aspergillus flavus and Aspergillus parasiticus which contaminate human food as well as animal feed. These are among the most carcinogenic substances known. Due to the toxic and carcinogenic properties of aflatoxins, there is a need to develop reliable methods to detect the presence of aflatoxigenic Aspergilli in contaminated food and feed. Not all Aspergillus strains are able to produce aflatoxins. It requires a detection methodology which can specifically distinguish between the aflatoxin producing and nonproducing strains of Aspergillus. Present communication reports validation of a PCR based detection system based on three genes viz., nor-1, apa-2 and omt-1 involved in aflatoxin biosynthesis, that can specifically distinguish the two aflatoxin producing species viz. Aspergillus flavus ,and Aspergillus parasiticus from non-producers i.e., A. niger, A. fumigates and A. oryzae.