Biodegradation of bisphenol A using psychrotolerant bacterial strain Pseudomonas palleroniana GBPI_508.

A psychrotolerant bacterial strain of Pseudomonas sp. (P. palleroniana GBPI_508), isolated from the Indian Himalayan region, is studied for analyzing its potential for degrading bisphenol A (BPA). Response surface methodology using Box-Behnken design was used to statistically optimize the environmental factors during BPA degradation and the maximum degradation (97%) was obtained at optimum conditions of mineral salt media pH 9, experimental temperature 25 °C, an inoculum volume of 10% (v/v), and agitation speed 130 rpm at the BPA concentration 270 mg L-1. The Monod model was used for understanding bacterial degradation kinetics, and 37.5 mg-1 half saturation coefficient (KS) and 0.989 regression coefficient (R2) were obtained. Besides, the utmost specific growth rate µmax was witnessed as 0.080 h-1 with the GBPI_508 during BPA degradation. Metabolic intermediates detected in this study by GC-MS were identified as valeric acid, propionic acid, diglycolic acid, and phenol. The psychrotolerant bacterial strain of Pseudomonas sp. (P. palleroniana GBPI_508), isolated from the Indian Himalayan region has shown good potential for remediation of BPA at variable conditions.

Characterization of APX and APX-R gene family in Brassica juncea and B. rapa for tolerance against abiotic stresses.

KEY MESSAGE: APX and APX-R gene families were identified and characterized in two important oilseed species of Brassica. Gene expression under abiotic stress conditions, recombinant protein expression, and analysis further divulged their drought, heat, and salt-responsive behavior. Ascorbate peroxidases (APX) are heme-dependent enzymes that rid the cells of H2O2 and regulate diverse biological processes. In the present study, we performed APX gene family characterization in two Brassica sp. (B. juncea and B. rapa) as these are commercially important oilseed crops and affected severely by abiotic stresses. We identified 16 BjuAPX and 9 BraAPX genes and 2 APX-R genes each in B. juncea and B. rapa genomes, respectively. Phylogenetic analysis divided the APX genes into five distinct clades, which exhibited conservation in the gene structure, motif organization, and sub-cellular location within the clade. Structural analysis of APX and APX-R proteins revealed the amino acid substitutions in conserved domains of APX-R proteins. The expression profiling of BjuAPX and BraAPX genes showed that 3 BjuAPX, 7BraAPX, and 2 BraAPX-R genes were drought and heat responsive. Notably, BjuAAPX1a, BjuAPX1d, BjuAAPX6, BraAAPX1a, BraAAPX2, and BraAAPX3b showed high expression levels in RT-qPCR. Cis-regulatory elements in APX and APX-R gene promoters supported the differential behavior of these genes. Further, two stress-responsive genes BjuAPX1d and BraAAPX2 were cloned, characterized, and their roles were validated under heat, drought, salt, and cold stress in bacterial expression system. This study for the first time reports the presence of APX activity in dimeric and LMW form of purified BraAAPX2 protein. The study may help pave way for developing abiotic stress-tolerant Brassica crops.

Exploration of glutathione reductase for abiotic stress response in bread wheat (Triticum aestivum L.).

KEY MESSAGE: A total of seven glutathione reductase (GR) genes were identified in Triticum aestivum, which were used for comparative structural characterization, phylogenetic analysis and expression profiling with the GR genes of other cereal plants. The modulated gene expression and enzyme activity revealed the role of GRs in abiotic stress response in T. aestivum. Glutathione reductase (GR) is an enzymatic antioxidant that converts oxidized glutathione (GSSG) into reduced glutathione (GSH) through the ascorbate-glutathione cycle. In this study, a total of seven GR genes forming two homeologous groups were identified in the allohexaploid genome of bread wheat (Triticum aestivum). Besides, we identified three GR genes in each Aegilops tauschii, Brachypodium distachyon, Triticum urartu and Sorghum bicolor, which were used for comparative characterization. Phylogenetic analysis revealed the clustering of GR proteins into two groups; class I and class II, which were predicted to be localized in cytoplasm and chloroplast, respectively. The exon-intron and conserved motif patterns were almost conserved in each group, in which a maximum of 10 and 17 exons were present in chloroplastic and cytoplasmic GRs, respectively. The protein structure analysis confirmed the occurrence of conserved pyridine nucleotide disulfide oxidoreductase (Pyr_redox) and pyridine nucleotide disulfide oxidoreductase dimerization (Pyr_redox_dim) domains in each GR. The active site of GR proteins consisted of two conserved cysteine residues separated by four amino acid residues. Promoter analysis revealed the occurrence of growth and stress-related cis-active elements. Tissue-specific expression profiling suggested the involvement of GRs in both vegetative and reproductive tissue development in various plants. The differential expression of TaGR genes and enhanced GR enzyme activity suggested their roles under drought, heat, salt and arsenic stress. Interaction of GRs with other proteins and chemical compounds of the ascorbate-glutathione cycle revealed their coordinated functioning. The current study will provide a foundation for the validation of the precise role of each GR gene in future studies.

Structural basis of UCUU RNA motif recognition by splicing factor RBM20.

The vertebrate splicing factor RBM20 (RNA binding motif protein 20) regulates protein isoforms important for heart development and function, with mutations in the gene linked to cardiomyopathy. Previous studies have identified the four nucleotide RNA motif UCUU as a common element in pre-mRNA targeted by RBM20. Here, we have determined the structure of the RNA Recognition Motif (RRM) domain from mouse RBM20 bound to RNA containing a UCUU sequence. The atomic details show that the RRM domain spans a larger region than initially proposed in order to interact with the complete UCUU motif, with a well-folded C-terminal helix encoded by exon 8 critical for high affinity binding. This helix only forms upon binding RNA with the final uracil, and removing the helix reduces affinity as well as specificity. We therefore find that RBM20 uses a coupled folding-binding mechanism by the C-terminal helix to specifically recognize the UCUU RNA motif.

OSCA Genes in Bread Wheat: Molecular Characterization, Expression Profiling, and Interaction Analyses Indicated Their Diverse Roles during Development and Stress Response.

The hyperosmolality-gated calcium-permeable channels (OSCA) are pore-forming transmembrane proteins that function as osmosensors during various plant developmental processes and stress responses. In our analysis, through in silico approaches, a total of 42 OSCA genes are identified in the Triticum aestivum genome. A phylogenetic analysis reveals the close clustering of the OSCA proteins of Arabidopsis thaliana, Oryza sativa, and T. aestivum in all the clades, suggesting their origin before the divergence of dicots and monocots. Furthermore, evolutionary analyses suggest the role of segmental and tandem duplication events (Des) and purifying selection pressure in the expansion of the OSCA gene family in T. aestivum. Expression profiling in various tissue developmental stages and under abiotic and biotic stress treatments reveals the probable functioning of OSCA genes in plant development and the stress response in T. aestivum. In addition, protein-protein and protein-chemical interactions reveal that OSCA proteins might play a putative role in Ca2+-mediated developmental processes and adaptive responses. The miRNA interaction analysis strengthens the evidence for their functioning in various biological processes and stress-induced signaling cascades. The current study could provide a foundation for the functional characterization of TaOSCA genes in future studies.

Identification, characterization and expression profiling of cation-proton antiporter superfamily in Triticum aestivum L. and functional analysis of TaNHX4-B.

The monovalent cation proton antiporter (CPA) superfamily comprises Na+/H+ exchanger (NHX), K+ efflux antiporter (KEA), and cation/H+ exchanger (CHX) family proteins, which play vital functions in plants. A total of 107 TaCPA proteins were identified in Triticum aestivum, and phylogenetically classified into 35 TaNHX, 24 TaKEA and 48 TaCHX proteins. These families had representatives derived from all three sub-genomes. TaKEA genes consisted of higher number of exons, followed by TaNHXs and TaCHXs. The occurrence of about 10 transmembrane regions and higher composition of helices and coils support their membrane-bound and hydrophobic nature. Diverse expression in various tissues and modulated expression under stress conditions suggested their role in development and in response to stress. Co-expression analyses revealed their complex interaction networks. Expression of TaNHX4-B.1 and TaNHX4-B.4 facilitated differential abiotic stress tolerance to Escherichia coli. Our study provides comprehensive information about CPA genes, which would be useful in their future functional characterization.

Molecular characterization of ascorbate peroxidase (APX) and APX-related (APX-R) genes in Triticum aestivum L.

Ascorbate peroxidases (APXs) are heme-dependent H2O2 scavenging enzymes involved in myriad biological processes. Herein, a total of 21 TaAPX and six TaAPX-R genes were identified from the A, B and D sub-genomes of Triticum aestivum L. The occurrence of three paralogous gene pairs with unequal evolutionary rate suggested functional divergence. The phylogenetic analysis formed four distinct clades having conserved gene and protein architecture, and sub-cellular localization. The tertiary structure analysis revealed the presence of helices and coils and residues involved in ligand binding. Transcriptional profiling of each TaAPX and TaAPX-R gene suggested their specific role during development and stress response. Modulated transcript expression and APX enzyme activity during various stress conditions indicated their role in stress response. Interaction analyses suggested their association with other genes, miRNAs and various legends. The present study reported numerous features of these genes, and may provide a platform for their detailed functional characterization in future studies.

Genome-wide characterization revealed role of NBS-LRR genes during powdery mildew infection in Vitis vinifera.

NBS-LRR comprises a large class of disease resistance (R) proteins that play a widespread role in plant protection against pathogens. In grapevine, powdery mildew cause significant losses in its productivity and efforts are being directed towards finding of resistance loci or genes imparting resistance/tolerance against such fungal diseases. In the present study, we performed genome-wide analysis of NBS-LRR genes during PM infection in grapevine. We identified 18, 23, 12, 16, 10, 10, 9, 20 and 14 differentially expressed NBS-LRR genes in response to PM infection in seven partially PM-resistant (DVIT3351.27, Husseine, Karadzhandal, Khalchili, Late vavilov, O34-16, Sochal) and 2 PM-susceptible (Carignan and Thompson seedless) V. vinifera accessions. Further, the identified sequences were characterized based on chromosomal locations, physicochemical properties, gene structure and motif analysis, and functional annotation by Gene Ontology (GO) mapping. The NBS-LRR genes responsive to powdery mildew could potentially be exploited to improve resistance in grapes.

Molecular characterization and differential expression suggested diverse functions of P-type II Ca2+ATPases in Triticum aestivum L.

BACKGROUND: Plant P-type II Ca2+ATPases are formed by two distinct groups of proteins (ACAs and ECAs) that perform pumping of Ca2+ outside the cytoplasm during homeostasis, and play vital functions during development and stress management. In the present study, we have performed identification and characterisation of P-type II Ca 2+ ATPase gene family in an important crop plant Triticum aestivum. RESULTS: Herein, a total of 33 TaACA and 9 TaECA proteins were identified from the various chromosomes and sub-genomes of Triticum aestivum. Phylogenetic analysis revealed clustering of the homoeologous TaACA and TaECA proteins into 11 and 3 distinct groups that exhibited high sequence homology and comparable structural organization as well. Both TaACA and TaECA group proteins consisted of eight to ten transmembrane regions, and their respective domains and motifs. Prediction of sub-cellular localization was found variable for most of the proteins; moreover, it was consistent with the evolutionarily related proteins from rice and Arabidopsis in certain cases. The occurrence of assorted sets of cis-regulatory elements indicated their diverse functions. The differential expression of various TaACA and TaECA genes during developmental stages suggested their roles in growth and development. The modulated expression during heat, drought, salt and biotic stresses along with the occurrence of various stress specific cis-regulatory elements suggested their association with stress response. Interaction of these genes with numerous development and stress related genes indicated their decisive role in various biological processes and signaling. CONCLUSION: T. aestivum genome consisted of a maximum of 42 P-type II Ca 2+ ATPase genes, derived from each A, B and D sub-genome. These genes may play diverse functions during plant growth and development. They may also be involved in signalling during abiotic and biotic stresses. The present study provides a comprehensive insight into the role of P-type II Ca 2+ ATPase genes in T. aestivum. However, the specific function of each gene needs to be established, which could be utilized in future crop improvement programs.

Genome-wide identification and characterization of LRR-RLKs reveal functional conservation of the SIF subfamily in cotton (Gossypium hirsutum).

BACKGROUND: As one of the largest subfamilies of the receptor-like protein kinases (RLKs) in plants, Leucine Rich Repeats-RLKs (LRR-RLKs) are involved in many critical biological processes including growth, development and stress responses in addition to various physiological roles. Arabidopsis contains 234 LRR-RLKs, and four members of Stress Induced Factor (SIF) subfamily (AtSIF1-AtSIF4) which are involved in abiotic and biotic stress responses. Herein, we aimed at identification and functional characterization of SIF subfamily in cultivated tetraploid cotton Gossypium hirsutum. RESULTS: Genome-wide analysis of cotton LRR-RLK gene family identified 543 members and phylogenetic analysis led to the identification of 6 cotton LRR-RLKs with high homology to Arabidopsis SIFs. Of the six SIF homologs, GhSIF1 is highly conserved exhibiting 46-47% of homology with AtSIF subfamily in amino acid sequence. The GhSIF1 was transiently silenced using Virus-Induced Gene Silencing system specifically targeting the 3' Untranslated Region. The transiently silenced cotton seedlings showed enhanced salt tolerance compared to the control plants. Further, the transiently silenced plants showed better growth, lower electrolyte leakage, and higher chlorophyll and biomass contents. CONCLUSIONS: Overall, 543 LRR-RLK genes were identified using genome-wide analysis in cultivated tetraploid cotton G. hirsutum. The present investigation also demonstrated the conserved salt tolerance function of SIF family member in cotton. The GhSIF1 gene can be knocked out using genome editing technologies to improve salt tolerance in cotton.

Binding and thermodynamics of REV peptide-ctDNA interaction.

The thermodynamics of DNA-ligand binding is important as it provides useful information to understand the details of binding processes. HIV-1 REV response element (RRE) located in the env coding region of the viral genome is reported to be well conserved across different HIV-1 isolates. In this study, the binding characteristics of Calf thymus DNA (ctDNA) and REV peptide from HIV-1 were investigated using spectroscopic (UV-visible, fluorescence, and circular dichroism (CD)) and isothermal titration calorimetric (ITC) techniques. Thermal stability and ligand binding properties of the ctDNA revealed that native ctDNA had a Tm of 75.5 °C, whereas the ctDNA-REV peptide complex exhibited an incremental shift in the Tm by 8 °C, indicating thermal stability of the complex. CD data indicated increased ellipticity due to large conformational changes in ctDNA molecule upon binding with REV peptide and two binding stoichiometric modes are apparent. The ctDNA experienced condensation due to large conformational changes in the presence of REV peptide and positive Bâ†’Ψ transition was observed at higher molar charge ratios. Fluorescence studies performed at several ligand concentrations revealed a gradual decrease in the fluorescence intensity of EtBr-bound ctDNA in response to increasing ligand concentrations. The fluorescence data further confirmed two stoichiometric modes of binding for ctDNA-REV peptide complex as previously observed with CD studies. The binding enthalpies were determined using ITC in the temperature range of 293 K-308 K. The ITC binding isotherm was exothermic at all temperatures examined, with low ΔH values indicating that the ctDNA-REV peptide interaction is driven largely by entropy. The heat capacity change (ΔCp ) was insignificant, an unusual finding in the area of DNA-peptide interaction studies. The variation in the values obtained for ΔH, ΔS, and ΔG with temperature further suggests that ctDNA-REV peptide interaction is entropically driven. ITC based analysis of salt dependence of binding constant gave a charge value (Z) = +4.01, as determined for the δlnK/δln[Na+ ] parameter, suggesting the participation of only 3-4 Arg out of 11 Arg charge from REV peptide. The stoichiometry observed for the complex was three molar charge of REV peptide binding per molar charge of ctDNA. ITC based analysis further confirmed that the binding between ctDNA and REV peptide is governed by electrostatic interaction. Molecular interactions including H-bonding, van der Waals forces, and solvent molecules rearrangement, underlie the binding of REV peptide to ctDNA.

Structural insights into the inclusion complexes between clomiphene citrate and ß-cyclodextrin: The mechanism of preferential isomeric selection.

A major challenge in pharmaceuticals for clinical applications is to alter the solubility, stability, and toxicity of drug molecules in living systems. Cyclodextrins (CDs) have the ability to form host-guest inclusion complexes with pharmaceuticals for further development of new drug formulations. The inclusion complex of clomiphene citrate (CL), a poorly water-soluble drug, with native ß-cyclodextrin (ß-CD) was characterized by a one and two-dimensional nuclear magnetic resonance (NMR) spectroscopic approach and also by molecular docking techniques. Here we report NMR and a computational approach in preferential isomeric selection of CL, which exists in two stereochemical isomers, enclomiphene citrate (ENC; E isomer) and zuclomiphene citrate (ZNC; Z isomer) with ß-CD. ß-CD cavity protons, namely, H-3' and H-5', experienced shielding in the presence of CL. The aromatic ring protons of the CL molecule were observed to be deshielded in the presence of ß-CD. The stoichiometric ratio of the ß-CD:CL inclusion complex was observed by NMR and found to be 1:1. The overall binding constant of ß-CD:CL inclusion complexes was based on NMR chemical shifts and was calculated to be 50.21 M-1 . The change in Gibb's free energy (∆G) was calculated to be -9.80 KJ mol-1 . The orientation and structure of the ß-CD:CL inclusion complexes are proposed on the basis of NMR and molecular docking studies. 2D 1 H-1 H ROESY confirmed the involvement of all three aromatic rings of CL in the inclusion complexation with ß-CD in the solution, confirming the multiple equilibria between ß-CD and CL. Molecular docking and 2D 1 H-1 H ROESY provide insight into the inclusion complexation of two isomers of CL into the ß-CD cavity. A molecular docking technique further provided the different binding affinities of the E and Z isomers of CL with ß-CD and confirmed the preference of the Z isomer binding for ß-CD:CL inclusion complexes. The study indicates that the formation of a hydrogen bond between -O- of CL and the hydrogen atom of the hydroxyl group of ß-CD was the main factor for noncovalent ß-CD:CL inclusion complex formation and stabilization in the aqueous phase.

In-depth 2-DE reference map of Aspergillus fumigatus and its proteomic profiling on exposure to itraconazole.

Aspergillus fumigatus (A. fumigatus) is a medically important opportunistic fungus that may lead to invasive aspergillosis in humans with weak immune system. Proteomic profiling of this fungus on exposure to itraconazole (ITC), an azole antifungal drug, may lead to identification of its molecular targets and better understanding on the development of drug resistance against ITC in A. fumigatus. Here, proteome analysis was performed using 2-DE followed by mass spectrometric analysis which resulted in identification of a total of 259 unique proteins. Further, proteome profiling of A. fumigatus was carried out on exposure to ITC, 0.154 µg/ml, the minimum inhibitory concentration (MIC50). Image analysis showed altered levels of 175 proteins (66 upregulated and 109 downregulated) of A. fumigatus treated with ITC as compared to the untreated control. Peptide mass fingerprinting led to the identification of 54 proteins (12 up-regulated and 42 down-regulated). The differentially expressed proteins include proteins related to cell stress, carbohydrate metabolism and amino acid metabolism. We also observed four proteins, including nucleotide phosphate kinase (NDK), that are reported to interact with calcineurin, a protein involved in regulation of cell morphology and fungal virulence. Comparison of differentially expressed proteins on exposure to ITC with artemisinin (ART), an antimalarial drug with antifungal activity(1), revealed a total of 26 proteins to be common among them suggesting that common proteins and pathways are targeted by these two antifungal agents. The proteins targeted by ITC may serve as important leads for development of new antifungal drugs.

H2AX phosphorylation is important for LANA-mediated Kaposi's sarcoma-associated herpesvirus episome persistence.

The DNA damage response (DDR) of host cells is utilized by a number of viruses to establish and propagate their genomes in the infected cells. We examined the expression of the DDR genes during Kaposi's sarcoma-associated herpesvirus (KSHV) infection of human peripheral blood mononuclear cells (PBMCs). The genes were mostly downregulated, except H2AX, which was upregulated during infection. H2AX is important for gammaherpesvirus infectivity, and its phosphorylation at serine 139 is crucial for maintenance of latency during mouse gamma-herpesvirus 68 (MHV-68) infection. We now also observed phosphorylation of H2AX at serine 139 during KSHV infection. H2AX is a histone H2A isoform shown to interact with the latency-associated nuclear antigen (LANA) encoded by KSHV. Here, we show that LANA directly interacted with H2AX through domains at both its N and C termini. The phosphorylated form of H2AX (γH2AX) was shown to colocalize with LANA. Chromatin immunoprecipitation (ChIP) assays showed that a reduction in H2AX levels resulted in reduced binding of LANA with KSHV terminal repeats (TRs). Binding preferences of H2AX and γH2AX along the KSHV episome were examined by whole-episome ChIP analysis. We showed that γH2AX had a higher relative binding activity along the TR regions than that of the long unique region (LUR), which highlighted the importance of H2AX phosphorylation during KSHV infection. Furthermore, knockdown of H2AX resulted in decreased KSHV episome copy number. Notably, the C terminus of LANA contributed to phosphorylation of H2AX. However, phosphorylation was not dependent on the ability of LANA to drive KSHV-infected cells into S-phase. Thus, H2AX contributes to association of LANA with the TRs, and phosphorylation of H2AX is likely important for its increased density at the TRs.

DPY1 as an osmosensor for drought signaling.

Drought stress has been extensively studied for its effect on the downstream signaling cascade and stress-responsive gene expression, but understanding the process has remained elusive. Recently, Zhao et al. identified DROOPY LEAF1 (DPY1) as an osmosensor and revealed a novel mechanism of DPY1-STRESS ACTIVATED PROTEIN KINASE6 (SAPK6)-mediated drought stress signaling in higher plants.

Investigation of two-pore K+ (TPK) channels in Triticum aestivum L. suggests their role in stress response.

Two-pore K+ (TPK) channels are voltage-independent and involved in stress response in plants. Herein, we identified 12 TaTPK genes located on nine chromosomes in the Triticum aestivum genome. The majority of TaTPK genes comprised two exons. Each TaTPK channel comprised four transmembrane (TM) helices, N- and C-terminal ion-channel domains, two EF-hand domains and one 14-3-3 binding site. Additionally, highly conserved 'GYGD' motif responsible for K+ ion specificity, was found in between the TMs in both the ion-channel domains. Nine TaTPK channels were predicted to be localised at the plasma membrane, while three were vacuolar. The protein-protein and protein-chemical interactions indicated the coordinated functioning of the TaTPK channels with the other K+ transporters and their possible interaction with the Ca2+-signaling pathway. Expression studies suggested their importance in both vegetative and reproductive tissues development. Significantly modulated expression of various TaTPK genes during heat, drought, combined heat and drought and salt stresses, and after fungal infestation, depicted their function in stress responses. The miRNAs and transcription factors interaction analyses suggested their role in the hormone, light, growth and development-related, and stress-responsive signaling cascades. The current study suggested vital functions of various TaTPK genes, especially in stress response, and would provide an opportunity for their detailed characterization in future studies.

Modulation in gene expression and enzyme activity suggested the roles of monodehydroascorbate reductase in development and stress response in bread wheat.

Monodehydroascorbate reductase (MDHAR) is a crucial enzymatic antioxidant of the ascorbate-glutathione pathway involved in reactive oxygen species scavenging. Herein, we identified 15 TaMDHAR genes in bread wheat. Phylogenetic analysis revealed their clustering into three groups, which are also related to the subcellular localization in the peroxisome matrix, peroxisome membrane, and chloroplast. Each TaMDHAR protein consisted of two conserved domains; Pyr_redox and Pyr_redox_2 of the pyridine nucleotide disulfide oxidoreductase family. The occurrence of diverse groups of cis-regulatory elements in the promoter region and their interaction with numerous transcription factors suggest assorted functions of TaMDHARs in growth and development and in light, phytohormones, and stress responses. Expression analysis in various tissues further revealed their importance in vegetative and reproductive development. In addition, the differential gene expression and enhanced enzyme activity during drought, heat, and salt treatments exposed their role in abiotic stress response. Interaction of MDHARs with various antioxidant enzymes and biochemicals related to the ascorbate-glutathione cycle exposed their synchronized functioning. Interaction with auxin indicated the probability of cross-talk between antioxidants and auxin signaling. The miR168a, miR169, miR172 and others interaction with various TaMDHARs further directed their association with developmental processes and stress responses. The current study provides extensive information about the importance of TaMDHARs, moreover, the precise role of each gene needs to be established in future studies.

Electrostatic interactions mediated defibrillation of ß-lactoglobulin fibrils using Keggin Polyoxometalates.

The whey protein ß-lactoglobulin (ßLG) forms fibrils similar to the amyloid fibrils in the neurodegenerative diseases due to its higher predisposition of ß-sheets. This study shed light on the understanding different inorganic Keggin polyoxometalates (POMs) interaction with the protein ßLG fibrils. POMs such as Phosphomolybdic acid (PMA), silicomolybdic acid (SMA), tungstosilicic acid (TSA), and phosphotungstic acid (PTA) were used due to their inherent higher anionic charges. The interaction studies were monitored with fluorescence spectra and Thioflavin T assay for both the ßLG monomers and the fibrils initially to elucidate the binding ability of the POMs. The binding of POMs and ßLG is also demonstrated by molecular docking studies. Zeta potential studies showed the electrostatic mediated higher interactions of the POMs with the protein fibrils. Isothermal titration calorimetry (ITC) studies showed that the molybdenum containing POMs have higher affinity to the protein fibrils than the tungsten. This study could help understanding formation of food grade protein fibrils which have profound importance in food industries.

Resolving the functions of peptidylprolyl isomerases: insights from the mutagenesis of the nuclear FKBP25 enzyme.

Peptidylprolyl isomerases have been implicated in chromatin regulation through their association with histones, chromatin-modifying enzymes and DNA-binding transcription factors. As with other post-translational modifications to proteins, a mechanistic understanding of the regulation of biological processes is fostered by loss-of-function studies both in vitro and in vivo. For peptidylprolyl isomerases, this can be accomplished with small-molecule inhibitors with high affinity for the isomerase active site or by mutation of amino acid residues that contribute to catalysis. In the present article, we review caveats to each of these approaches, and place emphasis on the thorough characterization of loss-of-function mutations in FKBPs (FK506-binding proteins). Using a case study of mutagenesis of the nuclear FKBP25 peptidylprolyl isomerase enzyme, we demonstrate that certain mutations generate a loss-of-function phenotype because they induce a complete loss of the FKBP domain fold, whereas other mutations are 'surgical' in that they ablate catalytic isomerase activity, while maintaining domain structure. Peptidylprolyl isomerases are thought to have both catalytic and non-catalytic functions, but differentiating between these mechanisms has proved to be challenging. The domain-destabilizing and surgical mutants described will facilitate the characterization of these two reported functions of peptidylprolyl isomerases.

Phosphate Deficiency: A Tale from the End of PILNCR2.

A deficiency in inorganic phosphate (Pi) induces the expression of miRNA399 and the accumulation of its target Pi transporters (PHT1s) mRNA, which is contrary to the goal of miRNA-mediated gene regulation. Recently, a novel mechanism of RNA/RNA-duplex formation between the transcripts of a Pi deficiency-induced long non-coding RNA (PILNCR2) and PHT1s has been reported, which prevents the binding and cleavage of miRNA399 to PHT1 mRNAs, thereby providing tolerance of Pi-deficient conditions. Moreover, the way in which ribosomes move through the RNA/RNA-duplex for the translation of PHT1 transporter proteins remains elusive.