TIA-1 RRM23 binding and recognition of target oligonucleotides

TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expressio

Biochemical characterization and molecular docking of cloned xylanase gene from Bacillus subtilis RTS expressed in E. coli

This study employed mesophilic Bacillus subtilis RTS strain isolated from soil with high xylanolytic activity. A 642 bp (xyn) xylanase gene (GenBank accession number MT677937) was extracted from Bacillus subtilis RTS and cloned in Escherichia coli BL21 cells using pET21c expression system. The cloned gene belongs to glycoside hydrolase family 11 with protein size of approximately 23 KDa. The recombinant xylanase showed optimal enzymeactivity at 60 degrees C and at pH6.5. Thermostability of recombinant xylanase was observed between the temperature range of 30-60 degrees C. Xylanase also remained stable in different concentration of various organic solvents (ethanol, butanol). This might be due to the formation of protein/organic solvent interface which prevents stripping of essential water molecules from enzyme, thus enzyme conformation and activity remained stable. Finally, the molecular docking analysis through AutoDock Vina showed the involvement of Tyr 108, Arg140 and Pro144 in protein-ligand interaction, which stabilizes this complex. The observed stability of recombinant xylanase at higher temperature and in the presence of organic solvent (ethanol, butanol) suggested possible application of this enzyme in biofuel and other industrial applications. (C) 2020 Elsevier B.V. All rights reserved

RNA Recognition and Stress Granule Formation by TIA Proteins

Stress granule (SG) formation is a primary mechanism through which gene expression is rapidly modulated when the eukaryotic cell undergoes cellular stresses (including heat, oxidative, viral infection, starvation). In particular, the sequestration of specifically targeted translationally stalled mRNAs into SGs limits the expression of a subset of genes, but allows the expression of heatshock proteins that have a protective effect in the cell. The importance of SGs is seen in several disease states in which SG function is disrupted. Fundamental to SG formation are the T cell restricted intracellular antigen (TIA) proteins (TIA-1 and TIA-1 related protein (TIAR)), that both directly bind to target RNA and self-associate to seed the formation of SGs. Here a summary is provided of the current understanding of the way in which TIA proteins target specific mRNA, and how TIA self-association is triggered under conditions of cellular stress

TIA-1 RRM23 binding and recognition of target oligonucleotides

TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression.Peer reviewe

Synthesis and Characterization of Silver and Graphene Nanocomposites and Their Antimicrobial and Photocatalytic Potentials

Microbial pathogens and bulk amounts of industrial toxic wastes in water are an alarming situation to humans and a continuous threat to aquatic life. In this study, multifunctional silver and graphene nanocomposites (Ag)1−x(GNPs)x [25% (x = 0.25), 50% (x = 0.50) and 75% (x = 0.75) of GNPs] were synthesized via ex situ approach. Further, the synthesized nanocomposites were explored for their physicochemical characteristics, such as vibrational modes (Raman spectroscopic analysis), optical properties (UV visible spectroscopic analysis), antibacterial and photocatalytic applications. We investigated the antimicrobial activity of silver and graphene nanocomposites (Ag-GNPs), and the results showed that Ag-GNPs nanocomposites exhibit remarkably improved antimicrobial activity (28.78% (E. coli), 31.34% (S. aureus) and 30.31% (P. aeruginosa) growth inhibition, which might be due to increase in surface area of silver nanoparticles (AgNPs)). Furthermore, we investigated the photocatalytic activity of silver (AgNPs) and graphene (GNPs) nanocomposites in varying ratios. Interestingly, the Ag-GNPs nanocomposites show improved photocatalytic activity (78.55% degradation) as compared to AgNPs (54.35%), which can be an effective candidate for removing the toxicity of dyes. Hence, it is emphatically concluded that Ag-GNPs hold very active behavior towards the decolorization of dyes and could be a potential candidate for the treatment of wastewater and possible pathogenic control over microbes. In the future, we also recommend different other in vitro biological and environmental applications of silver and graphene nanocomposites

Huntingtin Inclusions Trigger Cellular Quiescence, Deactivate Apoptosis, and Lead to Delayed Necrosis

Competing models exist in the literature for the relationship between mutant Huntingtin exon 1 (Httex1) inclusion formation and toxicity. In one, inclusions are adaptive by sequestering the proteotoxicity of soluble Httex1. In the other, inclusions compromise cellular activity as a result of proteome co-aggregation. Using a biosensor of Httex1 conformation in mammalian cell models, we discovered a mechanism that reconciles these competing models. Newly formed inclusions were composed of disordered Httex1 and ribonucleoproteins. As inclusions matured, Httex1 reconfigured into amyloid, and other glutamine-rich and prion domain-containing proteins were recruited. Soluble Httex1 caused a hyperpolarized mitochondrial membrane potential, increased reactive oxygen species, and promoted apoptosis. Inclusion formation triggered a collapsed mitochondrial potential, cellular quiescence, and deactivated apoptosis. We propose a revised model where sequestration of soluble Httex1 inclusions can remove the trigger for apoptosis but also co-aggregate other proteins, which curtails cellular metabolism and leads to a slow death by necrosis