Souvik Maiti

Zinc regulates the activity of kinase-phosphatase pair (BasPrkC/BasPrpC) in Bacillus anthracis

Bacillus anthracis Ser/Thr protein kinase PrkC (BasPrkC) is important for virulence of the bacterium within the host. Homologs of PrkC and its cognate phosphatase PrpC (BasPrpC) are the most conserved mediators of signaling events in diverse bacteria. BasPrkC homolog in Bacillus subtilis regulates critical processes like spore germination and BasPrpC modulates the activity of BasPrkC by dephosphorylation. So far, biochemical and genetic studies have provided important insights into the roles of BasPrkC and BasPrpC; however, regulation of their activities is not known. We studied the regulation of BasPrkC/BasPrpC pair and observed that Zn2+ metal ions can alter their activities. Zn2+ promotes BasPrkC kinase activity while inhibits the BasPrpC phosphatase activity. Concentration of Zn2+ in growing B. anthracis cells was found to vary with growth phase. Zn2+ was found to be lowest in log phase cells while it was highest in spores. This variation in Zn2+ concentration is significant for understanding the antagonistic activities of BasPrkC/BasPrpC pair. Our results also show that BasPrkC activity is modulated by temperature changes and kinase inhibitors. Additionally, we identified Elongation Factor Tu (BasEf-Tu) as a substrate of BasPrkC/BasPrpC pair and assessed the impact of their regulation on BasEf-Tu phosphorylation. Based on these results, we propose Zn2+ as an important regulator of BasPrkC/BasPrpC mediated phosphorylation cascades. Thus, this study reveals additional means by which BasPrkC can be activated leading to autophosphorylation and substrate phosphorylation.

Information Encoded in Non-Native States Drives Substrate-Chaperone Pairing

Many proteins refold inxa0vitro through kinetic folding intermediates that are believed to be by-products of native-state centric evolution. These intermediates are postulated to play only minor roles, if any, inxa0vivo because they lack any information related to translation-associated vectorial folding. We demonstrate that refolding intermediate of a test protein, generated inxa0vitro, is able to find its cognate chaperone, from the whole complement of Escherichia coli soluble chaperones. Cognate chaperone-binding uniquely alters the conformation of non-native substrate. Importantly, precise chaperone targeting of substrates are maintained as long as physiological molar ratios of chaperones remain unaltered. Using a library of different chaperone substrates, we demonstrate that kinetically trapped refolding intermediates contain sufficient structural features for precise targeting to cognate chaperones. We posit that evolution favors sequences that, in addition to coding for a functional native state, encode folding intermediates with higher affinity for cognate chaperones than noncognate ones.

CRISPR/Cas9: a historical and chemical biology perspective of targeted genome engineering

The CRISPR-Cas9 system has revolutionized the process of making changes to the DNA sequence of organisms. Relying on a simplistic model of RNA guided DNA binding and cleavage, this molecular toolbox has found application in nearly every branch of biological sciences. The story of CRISPR-Cas9 is one of discovery and development where a component of bacterial adaptive immunity has been harnessed to address important biological questions using significant inputs from physicochemical structure-function studies. In this review, we trace the evolution of CRISPR-Cas9 from its predecessor genome editing tools and document its current status with an emphasis on chemical biology aspects of modulating its activity to generate a potent tool for gene therapy applications.

A pH sensitive high-throughput assay for miRNA binding of a Peptide-Aminoglycoside (PA) library

MicroRNAs (miRNA) are small RNAs that have a regulatory role in gene expression. Because of this regulatory role, miRNAs have become a new target for therapeutic compounds. Here, we outline an approach to target specific miRNAs using a high throughput capable assay and a 215 compound peptidic-aminosugar (PA) library. Aminosugars have been shown in a number of recent reports as important lead compounds that bind miRNA. In order to screen for compounds that bind miRNA, we have developed a high throughput displacement assay using a fluorescein-neomycin conjugated molecule (F-neo) as a probe for competitive miRNA binding compounds. We have applied the F-neo assay to four different miRNA constructs and the assay is applicable to most miRNAs, at various stages of processing. The results of the screen were validated by the determination of the IC50 for a select group of compounds from the library. For example, we identified eight compounds that bind to hsa-miR 504 with higher affinity than the parent neomycin. From the F-neo displacement assay we found that the number of binding sites differs for each miRNA, and the binding sites appear to differ both physically and chemically, with different affinity of the compounds resulting from the size of the molecule as well as the chemical structure. Additionally, the affinity of the compounds was dependent on the identity and position of the amino acid position of conjugation and the affinity of the compounds relative to other compounds in the library was miRNA dependent with the introduction of a second amino acid.

Zinc Oxide Nanoparticles Dispersed in Ionic Liquids Show High Antimicrobial Efficacy to Skin-Specific Bacteria

Zinc oxide (ZnO) nanoparticles have been shown in the literature to have antibacterial properties and have been widely used in antibacterial formulations. However, one of the problems with ZnO nanoparticles is their tendency to aggregate, thereby causing damage to normal cells and lowering their antibacterial efficacy during application. In this work, we have attempted to avoid this by using a combination of ZnO nanoparticles and ionic liquids, a class of low melting salts containing organic cations and organic/inorganic anions that show antibacterial property as well, and tested the antibacterial activity of this dispersion. ZnO nanoparticles of 60 nm were dispersed in two different ionic liquids-choline acetate (IL1) and 1-butyl-3-methylimidazolium chloride (IL2)-to achieve high dispersibility, whereas ZnO dispersed in phosphate-buffered saline was taken as a control. These dispersions were tested on four strains- Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae, and Staphylococcus epidermidis. Maximum efficiency was obtained for ZnO nanoparticles dispersed in imidazolium-based ionic liquids against skin-specific S. epidermidis. Skin infections induced by S. epidermidis are prevalent in hospital-acquired diseases. In most cases, traditional antibiotic-based therapies fail to combat such infections. Our strategy of developing a dispersion of ZnO nanoparticles in ionic liquids shows superior antibacterial efficacy in comparison to that shown individually by ZnO nanoparticles or ionic liquids. We have also established that the mechanism of killing this skin-specific bacterium is possibly through the production of reactive oxygen species leading to bacterial cell lysis. Further, we showed that this formulation is biocompatible and nontoxic to normal keratinocyte cells even under coculture conditions.

Genome-wide computational analysis of potential long noncoding RNA mediated DNA:DNA:RNA triplexes in the human genome

BackgroundOnly a handful of long noncoding RNAs have been functionally characterized. They are known to modulate regulation through interacting with other biomolecules in the cell: DNA, RNA and protein. Though there have been detailed investigations on lncRNA-miRNA and lncRNA-protein interactions, the interaction of lncRNAs with DNA have not been studied extensively. In the present study, we explore whether lncRNAs could modulate genomic regulation by interacting with DNA through the formation of highly stable DNA:DNA:RNA triplexes.MethodsWe computationally screened 23,898 lncRNA transcripts as annotated by GENCODE, across the human genome for potential triplex forming sequence stretches (PTS). The PTS frequencies were compared across 5′UTR, CDS, 3′UTR, introns, promoter and 1000 bases downstream of the transcription termination sites. These regions were annotated by mapping to experimental regulatory regions, classes of repeat regions and transcription factors. We validated few putative triplex mediated interactions where lncRNA-gene pair interaction is via pyrimidine triplex motif using biophysical methods.ResultsWe identified 20,04,034 PTS sites to be enriched in promoter and intronic regions across human genome. Additional analysis of the association of PTS with core promoter elements revealed a systematic paucity of PTS in all regulatory regions, except TF binding sites. A total of 25 transcription factors were found to be associated with PTS. Using an interaction network, we showed that a subset of the triplex forming lncRNAs, have a positive association with gene promoters. We also demonstrated an in vitro interaction of one lncRNA candidate with its predicted gene target promoter regions.ConclusionsOur analysis shows that PTS are enriched in gene promoter and largely associated with simple repeats. The current study suggests a major role of a subset of lncRNAs in mediating chromatin organization modulation through CTCF and NSRF proteins.

Teratogenic, cardiotoxic and hepatotoxic properties of related ionic liquids reveal the biological importance of anionic components

Ionic liquids are fast becoming the solvent of choice for a number of diverse chemical, industrial and biological processes. Although they are considered a ‘greener’ alternative to conventional solvents, their effects on biological systems remain poorly understood. The zebrafish, a small fresh-water fish, is an ideal model for testing the effects of small molecules on whole organism assays. Because of conservation of molecular pathways between human and zebrafish, studies on zebrafish can lead to direct insights into human biology. We have evaluated the toxic effects of five different but related ionic liquids on zebrafish embryonic assays. We have discovered two related ionic liquids that show strong cardiotoxic and hepatotoxic effects suggesting previously unanticipated effects on health. Comparisons of ionic liquids with different cationic but the same anionic components suggest that both the anionic and the cationic components play important roles in determining the biological effects of an ionic liquid.

Identification of novel circadian transcripts in the zebrafish retina

High fecundity, transparent embryos for monitoring the rapid development of organs and the availability of a well-annotated genome has made zebrafish a model organism of choice for developmental biology and neurobiology. This vertebrate model, a favourite in chronobiology studies, shows striking circadian rhythmicity in behaviour. Here, we identify novel genes in the zebrafish genome, which shows their expression in the zebrafish retina. We further resolve the expression pattern over time and assign specific novel transcripts to the retinal cell type, predominantly in the inner nuclear layer. Using chemical ablation and free run experiments we segregate the transcripts that are rhythmic when entrained by light from those that show sustained oscillations in the absence of external cues. The transcripts reported here with rigorous annotation and specific functions in circadian biology provide the groundwork for functional characterisation of novel players in the zebrafish retinal clock.

Specific Cholesterol Binding Drives Drastic Structural Alterations in Apolipoprotein A1

Proteins typically adopt a multitude of flexible and rapidly interconverting conformers, many of which are governed by specific protein interaction domains. Whereas disc-shaped oligomeric HDL and its major protein component ApoA1 have been the focus of several investigations, the structural properties of monomeric ApoA1 remain poorly understood. Using tens of independent molecular simulations (>50 μs), we reveal that ApoA1 adopts a compact conformation. Upon the addition of a physiological concentration of cholesterol to ApoA1, the monomeric protein spontaneously formed a circular conformation. Remarkably, these drastic structural perturbations are driven by a specific cholesterol binding site at the C-terminal and a novel cholesterol binding site at the N-terminal. We propose a mechanism whereby ApoA1 opens in a stagewise manner and mutating the N-terminal binding site destroys the open belt-shaped topology. Complementary experiments confirm that the structural changes are induced by specific association of cholesterol with ApoA1, not by the nonspecific hydrophobic effect.

Superior HIV-1 TAR Binders with Conformationally Constrained R52 Arginine Mimics in the Tat(48-57) Peptide

We report a 100‐fold increase in binding affinity of the Tat(48–57) peptide to HIV‐1 transcriptional activator‐responsive element (TAR) RNA by replacing Arg52, an essential and critical residue for Tats specific binding, with (2S,4S)‐4‐guanidinoproline. The resulting αTat1M peptide is a far superior binder than γTat1M, a peptide containing another conformationally constrained arginine mimic, (2S,4S)‐4‐amino‐N‐(3‐guanidinopropyl)proline, or even the control Tat peptide (CtrlTat) itself. Our observations are supported by circular dichroism (CD), isothermal titration calorimetry (ITC), gel electrophoresis and UV spectroscopy studies. Molecular dynamics simulations suggest increased interactions between the more compact αTat1M and TAR RNA, relative to CtrlTat. The CD signature of the RNA itself remains largely unchanged upon binding of the peptides. The Tat mimetics further have better cell uptake properties than the control Tat peptide, thus increasing their potential application as specific TAR‐binding molecules.