Ni+2 permease system of Helicobacter pylori contains highly conserved G-quadruplex motifs.

The genome of a micro-organism contains all the information required for its survival inside its host cells. The guanine rich regions of the genome can form stable G-quadruplex structures that act as the regulators of gene expression. Herein, the completely sequenced genomes of Helicobacter pylori were explored for the identification and characterization of the conserved G-quadruplex motifs in this gastrointestinal pathogen. Initial in silico analysis revealed the presence of ~8241 GQ motifs in the H. pylori genome. Metal binding proteins of H. pylori are significantly enriched in the GQ motifs. Our study emphasizes the identification and characterization of four highly conserved G-quadruplex forming motifs (HPGQs) in the nickel transporter genes (nixA, niuB1, niuB2, and niuD) of the H. pylori. Nickel is a virulence determinant in H. pylori and is required as a co-factor for the urease and [NiFe] hydrogenase enzymes that are crucial for its survival in the stomach lining of humans. The presence of GQ motifs in these nickel transporter genes can affect their expression and may alter the functioning of Urease and [NiFe] hydrogenase. Similar to human and virus G-quadruplexes, targeting these conserved PGQs with bioactive molecules may represent a novel therapeutic avenue for combating infection of H. pylori. The identified HPGQs were characterized in-vitro by using CD spectroscopy, electrophoresis technique, and NMR spectroscopy at both acidic (4.5) and neutral pH (7.0). ITC revealed the specific interaction of these HPGQs with high affinity to the known G-quadruplex binding ligand, TMPyP4. The mTFP based reporter assay showed decrease in the gene expression of mTFP in the TMPyP4 treated cells as compared to the untreated and further affirmed the formation of stable G-quadruplex structures in the HPGQ motifs in vivo. This is the first report for characterizing G-quadruplex motifs in nickel transport-associated genes in the H. pylori bacterium.

Structures of Type III Secretion System Needle Filaments.

Among the Gram-negative bacterial secretion systems, type III secretion systems (T3SS) possess a unique extracellular molecular apparatus called the needle. This macromolecular protein assembly is a nanometre-size filament formed by the helical arrangement of hundreds of copies of a single, small protein, which is highly conserved between T3SSs from animal to plant bacterial pathogens. The needle filament forms a hollow tube with a channel ~20 Å in diameter that serves as a conduit for proteins secreted into the targeted host cell. In the past ten years, technical breakthroughs in biophysical techniques such as cryo-electron microscopy (cryo-EM) and solid-state NMR (SSNMR) spectroscopy have uncovered atomic resolution details about the T3SS needle assembly. Several high-resolution structures of Salmonella typhimurium and Shigella flexneri T3SS needles have been reported demonstrating a common structural fold. These structural models have been used to explain the active role of the needle in transmitting the host-cell contact signal from the tip to the base of the T3SS through conformational changes as well as during the injection of effector proteins. In this chapter, we summarize the current knowledge about the structure and the role of the T3SS needle during T3SS assembly and effector secretion.

Nanodomain Clustering of the Plant Protein Remorin by Solid-State NMR.

Nanodomains are dynamic membrane subcompartments, enriched in specific lipid, and protein components that act as functional platforms to manage an abundance of cellular processes. The remorin protein of plants is a well-established nanodomain marker and widely serves as a paradigm to study nanodomain clustering. Located at the inner leaflet of the plasma membrane, remorins perform essential functions during signaling. Using deuterium and phosphorus solid-state NMR, we inquire on the molecular determinants of the lipid-protein and protein-protein interactions driving nanodomain clustering. By monitoring thermotropism properties, lipid acyl chain order and membrane thickness, we report the effects of phosphoinositides and sterols on the interaction of various remorin peptides and protein constructs with the membrane. We probed several critical residues involved in this interaction and the involvement of the coiled-coil homo-oligomerisation domain into the formation of remorin nanodomains. We trace the essential role of the pH in nanodomain clustering based on anionic lipids such as phosphoinositides. Our results reveal a complex interplay between specific remorin residues and domains, the environmental pH and their resulting effects on the lipid dynamics for phosphoinositide-enriched membranes.

Rationally designed small molecules targeting toxic CAG repeat RNA that causes Huntington's disease (HD) and spinocerebellar ataxia (SCAs).

Huntington's diseases (HD) is a very devastating disease caused by r(CAG) expansion in HTT gene, encoding the huntingtin protein. r(CAG) expansion causes disease via multiple pathways including, 1) loss of normal protein function like sequestration of RNA binding protein such as Muscleblind-like (MBNL) and nucleolin, 2) Gain of function for mutant proteins and 3) repeat-associated non-ATG (RAN) translation; in which expanded r(CAG) translates into toxic poly glu, poly ser, or poly ala without the use of any canonical start codon. Herein, we have rationally designed and synthesized a unique class of pyridocoumarin derivatives that target the r(CAG)exp involved in HD and spinocerebellar ataxia (SCA) pathogenesis. Notably, compounds 3 and 15 showed higher affinity (nanomolar Kd) and selectivity for diseased r(CAG)exp RNA compared to regular duplex AU-paired RNA. Interestingly, both scaffolds are cell permeable, exhibit low toxicity to healthy fibroblast cells and are also capable of reducing the level of poly Q aggregation in cellular models. Indeed, our current study offers promising facet for selectively targeting repeats containing RNAs that cause severe diseases like HD and SCAs.

Characterization of highly conserved G-quadruplex motifs as potential drug targets in Streptococcus pneumoniae.

Several G-quadruplex forming motifs have been reported to be highly conserved in the regulatory regions of the genome of different organisms and influence various biological processes like DNA replication, recombination and gene expression. Here, we report the highly conserved and three potentially G-quadruplex forming motifs (SP-PGQs) in the essential genes (hsdS, recD, and pmrA) of the Streptococcus pneumoniae genome. These genes were previously observed to play a vital role in providing the virulence to the bacteria, by participating in the host-pathogen interaction, drug-efflux system and recombination- repair system. However, the presence and importance of highly conserved G-quadruplex motifs in these genes have not been previously recognized. We employed the CD spectroscopy, NMR spectroscopy, and electrophoretic mobility shift assay to confirm the adaptation of the G-quadruplex structure by the SP-PGQs. Further, ITC and CD melting analysis revealed the energetically favorable and thermodynamically stable interaction between a candidate G4 binding small molecule TMPyP4 and SP-PGQs. Next, TFP reporter based assay confirmed the regulatory role of SP-PGQs in the expression of PGQ harboring genes. All these experiments together characterized the SP-PGQs as a promising drug target site for combating the Streptococcus pneumoniae infection.

Restoration of miRNA-149 Expression by TmPyP4 Induced Unfolding of Quadruplex within Its Precursor.

Noncoding RNAs are functional RNA molecules that get transcribed from DNA but are not translated into proteins; yet, they can regulate gene expression at transcriptional and post-transcriptional levels. Secondary structures present within these RNAs play a major role in determining their nature of function. In the case of miRNAs, the precursor miRNA have a hairpin stem loop structure which is required for Dicer recognition and further maturation. Alternately, it might assume a G-quadruplex structure. The transition from hairpin to G-quadruplex depends upon the nucleotide sequence as well as the cellular microenvironment, and this might affect the miRNA maturation and other downstream activity. Formation of the G-quadruplex within precursor miRNA-149 has been shown to inhibit Dicer processing activity followed by suppression of miRNA-149 maturation in cancer cells. In this report, we show that suppression of cell proliferation by the upregulated miRNA-149 could be rescued by unfolding the G-quadruplex present in pre-miRNA-149 by TmPyP4 (Porphyrin) treatment. Using UV-visible spectroscopy, circular dichroism, and isothermal titration calorimetry, we observed that TmPyP4 binds strongly to G-quadruplex and unfolds it, which was further verified by NMR spectroscopy. In cellulo, qRT-PCR measurements of miRNA-149 in MCF-7 breast cancer cells showed concentration dependent enhancement of mature miRNA-149 upon treatment of TmPyP4. As a consequence of enhanced miRNA-149 activity, we also observe the reduction in miRNA-149 target protein ZBTB2 that eventually leads to reduced cell proliferation.

SMMDB: a web-accessible database for small molecule modulators and their targets involved in neurological diseases.

High-throughput screening and better understanding of small molecule's structure-activity relationship (SAR) using computational biology techniques have greatly expanded the face of drug discovery process in better discovery of therapeutics for various disease. Small Molecule Modulators Database (SMMDB) includes >1100 small molecules that have been either approved by US Food and Drug Administration, are under investigation or were rejected in clinical trial for any kind of neurological diseases. The comprehensive information about small molecules includes the details about their molecular targets (such as protein or enzyme, DNA, RNA, antisense RNA etc.), pharmacokinetic and pharmacodynamic properties such as binding affinity to their targets (Kd, Ki, IC50 and EC50 if available), mode of action, log P-value, number of hydrogen bond donor and acceptors, their clinical trial status, their 2D and three-dimensional structures etc. To enrich the basic annotation of every small molecule entry present in SMMDB, it is hyperlinked to their description present in PubChem, DrugBank, PubMed and KEGG database. The annotation about their molecular targets was enriched by linking it with UniProt and GenBank and STRING database that can be utilized to study the interaction and relation between various targets involved in single neurological disease. All molecules present in the SMMDB are made available to download in single file and can be further used in establishing the SAR, structure-based drug designing as well as shape-based virtual screening for developing the novel therapeutics against neurological diseases. The scope of this database majorly covers the interest of scientific community and researchers who are engaged in putting their endeavor toward therapeutic development and investigating the pathogenic mechanism of various neurological diseases. The graphical user interface of the SMMDB is accessible on http://bsbe.iiti.ac.in/bsbe/smmdb.

Myricetin Reduces Toxic Level of CAG Repeats RNA in Huntington's Disease (HD) and Spino Cerebellar Ataxia (SCAs).

Huntington's disease (HD) is a neurodegenerative disorder that is caused by abnormal expansion of CAG repeats in the HTT gene. The transcribed mutant RNA contains expanded CAG repeats that translate into a mutant huntingtin protein. This expanded CAG repeat also causes mis-splicing of pre-mRNA due to sequestration of muscle blind like-1 splicing factor (MBNL1), and thus both of these elicit the pathogenesis of HD. Targeting the onset as well as progression of HD by small molecules could be a potent therapeutic approach. We have screened a set of small molecules to target this transcript and found Myricetin, a flavonoid, as a lead molecule that interacts with the CAG motif and thus prevents the translation of mutant huntingtin protein as well as sequestration of MBNL1. Here, we report the first solution structure of the complex formed between Myricetin and RNA containing the 5'CAG/3'GAC motif. Myricetin interacts with this RNA via base stacking at the AA mismatch. Moreover, Myricetin was also found reducing the proteo-toxicity generated due to the aggregation of polyglutamine, and further, its supplementation also improves neurobehavioral deficits in the HD mouse model. Our study provides the structural and mechanistic basis of Myricetin as an effective therapeutic candidate for HD and other polyQ related disorders.

Structural insight for the recognition of G-quadruplex structure at human c-myc promoter sequence by flavonoid Quercetin.

Small molecule ligands that could stabilize G-quadruplex structure formed at the promoter region of human c-myc oncogene will regulate its expression in cancer cells. Flavonoids, a group of naturally available small molecule, have been known for their various promising effects on human health. In present study, we have performed detailed biophysical studies for the interaction of human c-myc G-quadruplex DNA with nine representative flavonoids: Luteolin, Quercetin, Rutin, Genistein, Kaempferol, Puerarin, Hesperidin, Myricetin and Daidzein. We found by using fluorescence titration that Quercetin interacts with c-myc G-quadruplex DNA sequence Pu24T with highest affinity. This interaction was further explored by using NMR spectroscopy and we have derived the first solution structure for the complex formed between Quercetin and biologically significant c-myc promoter DNA sequence forming G-quadruplex structure. In present solution structure, Quercetin stacks at 5' and 3' G-tetrads of Pu24T G-quadruplex structure and stabilize it via π-π stacking interactions. Furthermore, in vitro studies on HeLa cells suggested that Quercetin induces apoptosis-mediated cell death and down-regulated c-myc gene expression. This study emphasizes the potential of flavonoids as a promising candidate for targeting c-myc promoter region and thus, could act as a potential anti-cancer agent.

Evidences for Piperine inhibiting cancer by targeting human G-quadruplex DNA sequences.

Piperine, a naturally occurring alkaloid, is well known as anti-oxidant, anti-mutagenic, anti-tumor and anti-proliferative agent. Piperine exerts such pharmacological activities by binding or interacting with various cellular targets. Recently, the first report for Piperine interaction with duplex DNA has been published last year but its interaction with G-quadruplex structures has not been studied yet. Herein, we report for the first time the interaction of Piperine with various DNA G-quadruplex structures. Comprehensive biophysical techniques were employed to determine the basis of interaction for the complex formed between Piperine and G-quadruplex DNA sequences. Piperine showed specificity for G-quadruplex DNA over double stranded DNA, with highest affinity for G-quadruplex structure formed at c-myc promoter region. Further, in-vitro studies show that Piperine causes apoptosis-mediated cell death that further emphasizes the potential of this natural product, Piperine, as a promising candidate for targeting G-quadruplex structure and thus, acts as a potent anti-cancer agent.

G4IPDB: A database for G-quadruplex structure forming nucleic acid interacting proteins.

Nucleic acid G-quadruplex structure (G4) Interacting Proteins DataBase (G4IPDB) is an important database that contains detailed information about proteins interacting with nucleic acids that forms G-quadruplex structures. G4IPDB is the first database that provides comprehensive information about this interaction at a single platform. This database contains more than 200 entries with details of interaction such as interacting protein name and their synonyms, their UniProt-ID, source organism, target name and its sequences, ∆Tm, binding/dissociation constants, protein gene name, protein FASTA sequence, interacting residue in protein, related PDB entries, interaction ID, graphical view, PMID, author's name and techniques that were used to detect their interactions. G4IPDB also provides an efficient web-based "G-quadruplex predictor tool" that searches putative G-quadruplex forming sequences simultaneously in both sense and anti-sense strands of the query nucleotide sequence and provides the predicted G score. Studying the interaction between proteins and nucleic acids forming G-quadruplex structures could be of therapeutic significance for various diseases including cancer and neurological disease, therefore, having detail information about their interactions on a single platform would be helpful for the discovery and development of novel therapeutics. G4IPDB can be routinely updated (twice in year) and freely available on http://bsbe.iiti.ac.in/bsbe/ipdb/index.php.

Structural Insight into the interaction of Flavonoids with Human Telomeric Sequence.

Flavonoids are a group of naturally available compounds that are an attractive source for drug discovery. Their potential to act as anti-tumourigenic and anti-proliferative agents has been reported previously but is not yet fully understood. Targeting human telomeric G-quadruplex DNA could be one of the mechanisms by which these flavonoids exert anticancer activity. We have performed detailed biophysical studies for the interaction of four representative flavonoids, Luteolin, Quercetin, Rutin and Genistein, with the human telomeric G-quadruplex sequence tetramolecular d-(T2AG3T) (Tel7). In addition, we used NMR spectroscopy to derive the first model for the complex formed between Quercetin and G-quadruplex sequence. The model showed that Quercetin stabilises the G-quadruplex structure and does not open the G-tetrad. It interacts with the telomeric sequence through π-stacking at two sites: between T1pT2 and between G6pT7. Based on our findings, we suggest that Quercetin could be a potent candidate for targeting the telomere and thus, act as a potent anti-cancer agent.

Structural Insights Reveal the Dynamics of the Repeating r(CAG) Transcript Found in Huntington's Disease (HD) and Spinocerebellar Ataxias (SCAs).

In humans, neurodegenerative disorders such as Huntington's disease (HD) and many spinocerebellar ataxias (SCAs) have been found to be associated with CAG trinucleotide repeat expansion. An important RNA-mediated mechanism that causes these diseases involves the binding of the splicing regulator protein MBNL1 (Muscleblind-like 1 protein) to expanded r(CAG) repeats. Moreover, mutant huntingtin protein translated from expanded r(CAG) also yields toxic effects. To discern the role of mutant RNA in these diseases, it is essential to gather information about its structure. Detailed insight into the different structures and conformations adopted by these mutant transcripts is vital for developing therapeutics targeting them. Here, we report the crystal structure of an RNA model with a r(CAG) motif, which is complemented by an NMR-based solution structure obtained from restrained Molecular Dynamics (rMD) simulation studies. Crystal structure data of the RNA model resolved at 2.3 Å reveals non-canonical pairing of adenine in 5´-CAG/3´-GAC motif samples in different syn and anti conformations. The overall RNA structure has helical parameters intermediate to the A- and B-forms of nucleic acids due to the global widening of major grooves and base-pair preferences near internal AA loops. The comprehension of structural behaviour by studying the spectral features and the dynamics also supports the flexible nature of the r(CAG) motif.

DPP-IV inhibitory potential of naringin: an in silico, in vitro and in vivo study.

The incretin based therapies are an emerging class of antidiabetic drugs, with two categories: one is glucagone like peptide-1 (GLP-1) agonists and the other one is dipeptidyl peptidase (CD26; DPP-IV) inhibitors. However, in the DPP-IV inhibitors category only few compounds are commercially available and also have some undesirable effects. Therefore, in the present work we tried to explore a naturally occurring compound naringin for its potential DPP-IV inhibition and antidiabetic potential. It is noteworthy that this compound is abundantly present in orange peel and thus may provide cost effective treatment for diabetes, especially type 2 diabetes mellitus. In the present study, we have conducted virtual docking study and observed tight binding of naringin, as shown by higher negative values of H bond lengths, while in vitro DPP-IV inhibition assay has also shown better inhibition by naringin. In vivo study, in response to 10 days administration of 40 mg/kg of naringin twice daily to Wistar albino rats, inhibited the serum levels of DPP-IV activity, random glucose concentration with concomitant increase in insulin levels. All the comparisons were made with the standard commercially available drug sitagliptin.