Subhrangsu Chatterjee

Applications of saturation transfer difference NMR in biological systems.

The method of saturation transfer difference (STD) nuclear magnetic resonance (NMR) is an indispensable NMR tool in drug discovery. It identifies binding epitope(s) at the atomic resolution of small molecule ligands (e.g. organic drugs, peptides and oligosaccharides), while interacting with their receptors, such as proteins and/or nucleic acids. The method is widely used to screen active drug molecules, simultaneously ranking them in a qualitative way. STD NMR is highly successful for a variety of high molecular weight systems, such as whole viruses, platelets, intact cells, lipopolysaccharide micelles, membrane proteins, recombinant proteins and dispersion pigments. Modifications of STD pulse programs using (13)C and (15)N nuclei are now used to overcome the signal overlapping that occurs with more complex structures.

FoxP3 Acts as a Cotranscription Factor with STAT3 in Tumor-Induced Regulatory T Cells

FoxP3, a lineage-specification factor, executes its multiple activities mostly through transcriptional regulation of target genes. We identified an interleukin-10 (IL-10)-producing FoxP3(+) T regulatory cell population that contributes to IL-10-dependent type 2 cytokine bias in breast-cancer patients. Although genetic ablation of FOXP3 inhibited IL10 transcription, genome-wide analysis ruled out its role as a transcription factor for IL10. In-depth analysis revealed that histone acetyl transterase-1, in association with FoxP3, modified the IL10 promoter epigenetically, making a space for docking STAT3-FoxP3 complexes. A predictive docking module with target-receptor specificity, along with exon-deletion and site-directed mutagenesis studies, showed that STAT3 binds through its N-terminal floppy domain to the exon 2 β sheet region of FoxP3 to form STAT3-FoxP3 complexes. Such cotranscriptional activity of FoxP3 extended to other STAT3-target genes that lack FoxP3-binding sites. These results suggest a function of FoxP3, where, failing to achieve direct promoter occupancy, FoxP3 promotes transcription in association with the locus-specific transcription factor STAT3.

Indolicidin Targets Duplex DNA: Structural and Mechanistic Insight through a Combination of Spectroscopy and Microscopy

Indolicidin (IR13), a 13‐residue antimicrobial peptide from the cathelicidin family, is known to exhibit a broad spectrum of antimicrobial activity against various microorganisms. This peptide inhibits bacterial DNA synthesis resulting in cell filamentation. However, the precise mechanism remains unclear and requires further investigation. The central PWWP motif of IR13 provides a unique structural element that can wrap around, and thus stabilize, duplex B‐type DNA structures. Replacements of the central Trp‐Trp pair with Ala‐Ala, His‐His, or Phe‐Phe residues in the PxxP motif significantly affects the ability of the peptide to stabilize duplex DNA. Results of microscopy studies in conjunction with spectroscopic data confirm that the DNA duplex is stabilized by IR13, thereby inhibiting DNA replication and transcription. In this study we provide high‐resolution structural information on the interaction between indolicidin and DNA, which will be beneficial for the design of novel therapeutic antibiotics based on peptide scaffolds.

Use of a small peptide fragment as an inhibitor of insulin fibrillation process: a study by high and low resolution spectroscopy.

A non-toxic, nine residue peptide, NIVNVSLVK is shown to interfere with insulin fibrillation by various biophysical methods. Insulin undergoes conformational changes under certain stress conditions leading to amyloid fibrils. Fibrillation of insulin poses a problem in its long-term storage, reducing its efficacy in treating type II diabetes. The dissociation of insulin oligomer to monomer is the key step for the onset of fibrillation. The time course of insulin fibrillation at 62°C using Thioflavin T fluorescence shows an increase in the lag time from 120 min without peptide to 236 min with peptide. Transmission electron micrographs show branched insulin fibrils in its absence and less inter-fibril association in its presence. Upon incubation at 62°C and pH 2.6, insulin lost some α-helical structure as seen by Fourier transformed infra-red spectroscopy (FT-IR), but if the peptide is added, secondary structure is almost fully maintained for 3 h, though lost partially at 4 h. FT-IR spectroscopy also shows that insulin forms the cross beta structure indicative of fibrils beyond 2 h, but in the presence of the peptide, α-helix retention is seen till 4 h. Both size exclusion chromatography and dynamic light scattering show that insulin primarily exists as trimer, whose conversion to a monomer is resisted by the peptide. Saturation transfer difference nuclear magnetic resonance confirms that the hydrophobic residues in the peptide are in close contact with an insulin hydrophobic groove. Molecular dynamics simulations in conjunction with principal component analyses reveal how the peptide interrupts insulin fibrillation. In vitro hemolytic activity of the peptide showed insignificant cytotoxicity against HT1080 cells. The insulin aggregation is probed due to the inter play of two key residues, PheB24 and TyrB26 monitored from molecular dynamics simulations studies. Further new peptide based leads may be developed from this nine residue peptide.

Plant alkaloid chelerythrine induced aggregation of human telomere sequence--a unique mode of association between a small molecule and a quadruplex.

Small molecules that interact with G-quadruplex structures formed by the human telomeric region and stabilize them have the potential to evolve as anticancer therapeutic agents. Herein we report the interaction of a putative anticancer agent from a plant source, chelerythrine, with the human telomeric DNA sequence. It has telomerase inhibitory potential as demonstrated from telomerase repeat amplification assay in cancer cell line extract. We have attributed this to the quadruplex binding potential of the molecule and characterized the molecular details of the interaction by means of optical spectroscopy such as absorbance and circular dichroism and calorimetric techniques such as isothermal titration calorimetry and differential scanning calorimetry. The results show that chelerythrine binds with micromolar dissociation constant and 2:1 binding stoichiometry to the human telomeric DNA sequence. Chelerythrine association stabilizes the G-quadruplex. Nuclear magnetic resonance spectroscopy ((1)H and (31)P) shows that chelerythrine binds to both G-quartet and phosphate backbone of the quadruplex leading to quadruplex aggregation. Molecular dynamics simulation studies support the above inferences and provide further insight into the mechanism of ligand binding. The specificity toward quartet binding for chelerythrine is higher compared to that of groove binding. MM-PBSA calculation mines out the energy penalty for quartet binding to be -4.7 kcal/mol, whereas that of the groove binding is -1.7 kcal/mol. We propose that the first chelerythrine molecule binds to the quartet followed by a second molecule which binds to the groove. This second molecule might bring about aggregation of the quadruplex structure which is evident from the results of nuclear magnetic resonance.

Nuclear matrix-associated protein SMAR1 regulates alternative splicing via HDAC6-mediated deacetylation of Sam68

Significance Multiple studies highlight the role of various proteins in regulation of alternative splicing; however, the regulatory role of distinct posttranslational modifications during alternative splicing that contribute to tumorigenesis is enigmatic. Here we report a previously unidentified noncanonical mechanism of regulation of alternative splicing modulated by deacetylation of RNA-binding protein Sam68 (Src-associated substrate during mitosis of 68 kDa) via Scaffold/matrix-associated region-binding protein 1 (SMAR1)–histone deacetylase 6 (HDAC6) complex. SMAR1 in complex with HDAC6 maintains Sam68 in a deacetylated state. We observed that ERK-1/2–dependent phosphorylation of SMAR1, knockdown of SMAR1, or loss of heterozygosity facilitates CD44 variant exon inclusion via Sam68 acetylation and thus confers invasive and metastatic potential in breast tumor cells. Our findings provide key insights into regulation of alternative splicing and the potential for therapeutic intervention during tumor metastasis. Pre-mRNA splicing is a complex regulatory nexus modulated by various trans-factors and their posttranslational modifications to create a dynamic transcriptome through alternative splicing. Signal-induced phosphorylation and dephosphorylation of trans-factors are known to regulate alternative splicing. However, the role of other posttranslational modifications, such as deacetylation/acetylation, methylation, and ubiquitination, that could modulate alternative splicing in either a signal-dependent or -independent manner remain enigmatic. Here, we demonstrate that Scaffold/matrix-associated region-binding protein 1 (SMAR1) negatively regulates alternative splicing through histone deacetylase 6 (HDAC6)-mediated deacetylation of RNA-binding protein Sam68 (Src-associated substrate during mitosis of 68 kDa). SMAR1 is enriched in nuclear splicing speckles and associates with the snRNAs that are involved in splice site recognition. ERK–MAPK pathway that regulates alternative splicing facilitates ERK-1/2–mediated phosphorylation of SMAR1 at threonines 345 and 360 and localizes SMAR1 to the cytoplasm, preventing its interaction with Sam68. We showed that endogenously, SMAR1 through HDAC6 maintains Sam68 in a deacetylated state. However, knockdown or ERK-mediated phosphorylation of SMAR1 releases the inhibitory SMAR1–HDAC6–Sam68 complex, facilitating Sam68 acetylation and alternative splicing. Furthermore, loss of heterozygosity at the Chr.16q24.3 locus in breast cancer cells, wherein the human homolog of SMAR1 (BANP) has been mapped, enhances Sam68 acetylation and CD44 variant exon inclusion. In addition, tail-vein injections in mice with human breast cancer MCF-7 cells depleted for SMAR1 showed increased CD44 variant exon inclusion and concomitant metastatic propensity, confirming the functional role of SMAR1 in regulation of alternative splicing. Thus, our results reveal the complex molecular mechanism underlying SMAR1-mediated signal-dependent and -independent regulation of alternative splicing via Sam68 deacetylation.

Novel G-quadruplex stabilizing agents: in-silico approach and dynamics.

The stabilization of overhang G-rich repetitive DNA units at the 3′-end of telomeres, which are well known to form functionally important G-quadruplex structures, is a current goal in designing novel anticancer drugs. In the present study, we have undertaken an in silico approach by molecular docking using a small molecule library to find potential G-quadruplex stabilizing agents. Two molecules, A, [N′1-imino(2-pyridyl)methyl-3,4,5-trimethoxybenzene-1-carbohydrazide] and B, [(3-[4-({[3-({4-[(2cyanoethyl)(methyl)amino]benzylidene}amino)propyl]imino}methyl)(methyl) anilino]propanenitrile)], that had good docking scores have been investigated for interaction with G-quadruplexes in a Molecular Dynamics simulation study. Fluorescence spectroscopy of G-quadruplexes bound to the screened molecules A and B was used to experimentally validate the theoretical results. The binding of ligands A and B to G-quadruplexes resulted in blue shifts of 10–18 nm, respectively, in the fluorescence emission spectra of the G-quadruplexes, demonstrating that both molecules bind to the G-face of the quadruplex. The same experiment was performed for the complexation of these small molecules with a G-rich DNA duplex, . Interestingly, no blue shift was observed in the fluorescence emission spectra of the DNA duplex in the presence of these small molecules. Thus, these findings indicated that these ligands very selectively bind to G-quadruplexes instead of the duplex DNA. In addition, a one-dimensional water ligand observed via a gradient spectroscopy Nuclear Magnetic Resonance (NMR) experiment showed that both molecules bound to the 23-mer G-quadruplex DNA. The molecular properties of the ligand–quadruplex complex have been analyzed with the help of the Adaptive Poisson-Boltzmann Solver, revealing that electrostatics govern the binding of the small molecules to G-quadruplexes. Both molecules were investigated in detail using solvation free energy calculations and Absorption, Distribution, Metabolism, Elimination and Toxicity (ADMET) predictions, which provide insight into lead optimization for designing G-quadruplex stabilizing agents; therefore, these molecules have potential as new therapeutic agents.

Solution Structures, Dynamics, and Ice Growth Inhibitory Activity of Peptide Fragments Derived from an Antarctic Yeast Protein

Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.

Double GC:GC mismatch in dsDNA enhances local dynamics retaining the DNA footprint: a high-resolution NMR study.

Mutations in the genome are responsible for several fatal genetic disorders. The default DNA repair mechanism restores the malfunction of the gene caused by mutation to maintain functional regularity and sequential integrity of the cell. Here, we have elucidated the NMR structure and the dynamics of GC mismatched DNA (PDB code: 2MJX) and found that the mismatched DNA still retains the typical B‐type helical form, but in the process introduces backbone distortion resulting from frame‐shifted base pairs.

Discovery and Structural Characterization of G-quadruplex DNA in Human Acetyl-CoA Carboxylase Gene Promoters: Its Role in Transcriptional Regulation and as a Therapeutic Target for Human Disease

Accumulating evidence suggests that G-quadruplexes play vital roles in gene expression, DNA replication, and recombination. Three distinct promoters (PI, PII, and PIII) regulate human acetyl-CoA carboxylase 1 (ACC1) gene expression. In this study, we asked whether the G-rich sequences within the human ACC1 (PI and PII) promoters can form G-quadruplex structures and regulate normal DNA transactions. Using multiple complementary methods, we show that G-rich sequences of PI and PII promoters form intramolecular G-quadruplex structures and then establish unambiguously the topologies of these structures. Importantly, G-quadruplex formation in ACC1 gene promoter region blocks DNA replication and suppresses transcription, and this effect was further augmented by G-quadruplex stabilizing ligands. Altogether, these results are consistent with the notion that G-quadruplex structures exist within the human ACC1 gene promoter region, whose activity can be suppressed by G-quadruplex stabilizing ligands, thereby revealing a novel regulatory mechanism of ACC1 gene expression and as a possible therapeutic target.