Ansuman Lahiri

Genome wide gene expression regulation by HIP1 Protein Interactor, HIPPI: Prediction and validation

BackgroundHIP1 Protein Interactor (HIPPI) is a pro-apoptotic protein that induces Caspase8 mediated apoptosis in cell. We have shown earlier that HIPPI could interact with a specific 9 bp sequence motif, defined as the HIPPI binding site (HBS), present in the upstream promoter of Caspase1 gene and regulate its expression. We also have shown that HIPPI, without any known nuclear localization signal, could be transported to the nucleus by HIP1, a NLS containing nucleo-cytoplasmic shuttling protein. Thus our present work aims at the investigation of the role of HIPPI as a global transcription regulator.ResultsWe carried out genome wide search for the presence of HBS in the upstream sequences of genes. Our result suggests that HBS was predominantly located within 2 Kb upstream from transcription start site. Transcription factors like CREBP1, TBP, OCT1, EVI1 and P53 half site were significantly enriched in the 100 bp vicinity of HBS indicating that they might co-operate with HIPPI for transcription regulation. To illustrate the role of HIPPI on transcriptome, we performed gene expression profiling by microarray. Exogenous expression of HIPPI in HeLa cells resulted in up-regulation of 580 genes (p < 0.05) while 457 genes were down-regulated. Several transcription factors including CBP, REST, C/EBP beta were altered by HIPPI in this study. HIPPI also interacted with P53 in the protein level. This interaction occurred exclusively in the nuclear compartment and was absent in cells where HIP1 was knocked down. HIPPI-P53 interaction was necessary for HIPPI mediated up-regulation of Caspase1 gene. Finally, we analyzed published microarray data obtained with post mortem brains of Huntingtons disease (HD) patients to investigate the possible involvement of HIPPI in HD pathogenesis. We observed that along with the transcription factors like CREB, P300, SREBP1, Sp1 etc. which are already known to be involved in HD, HIPPI binding site was also significantly over-represented in the upstream sequences of genes altered in HD.ConclusionsTaken together, the results suggest that HIPPI could act as an important transcription regulator in cell regulating a vast array of genes, particularly transcription factors and at least, in part, play a role in transcription deregulation observed in HD.

Molecular Dynamics of the Anticodon Domain of Yeast tRNAPhe:Codon-Anticodon Interaction

We have studied the effect of codon-anticodon interaction on the structure and dynamics of transfer RNAs using molecular dynamics simulations over a nanosecond time scale. From our molecular dynamical investigations of the solvated anticodon domain of yeast tRNA(Phe) in the presence and absence of the codon trinucleotides UUC and UUU, we find that, although at a gross level the structures are quite similar for the free and the bound domains, there are small but distinct differences in certain parts of the molecule, notably near the Y37 base. Comparison of the dynamics in terms of interatomic or inter-residual distance fluctuation for the free and the bound domains showed regions of enhanced rigidity in the loop region in the presence of codons. Because fluorescence experiments suggested the existence of multiple conformers of the anticodon domain, which interconvert on a much larger time scale than our simulations, we probed the conformational space using five independent trajectories of 500 ps duration. A generalized ergodic measure analysis of the trajectories revealed that at least for this time scale, all the trajectories populated separate parts of the conformational space, indicating a need for even longer simulations or enhanced sampling of the conformational space to give an unequivocal answer to this question.

Conformational preferences of modified uridines: comparison of AMBER derived force fields.

The widespread occurrence of modified residues in RNA sequences necessitates development of accurate parameters for these modifications for reliable modeling of RNA structure and dynamics. A comprehensive set of parameters for the 107 naturally occurring RNA modifications was proposed by Aduri et al. (J. Chem. Theory Comput. 2007, 3, 1464-1475) for the AMBER FF99 force field. In this work, we tested these parameters on a set of modified uridine residues, namely, dihydrouridine, 2-thiouridine, 4-thiouridine, pseudouridine, and uridine-5-oxyacetic acid, by performing molecular dynamics and replica exchange molecular dynamics simulations of these nucleosides. Although our simulations using the FF99 force field did not, in general, reproduce the experimentally observed conformational characteristics well, combination of the parameter set with recent revisions of the FF99 force field for RNA showed noticeable improvement for some of the nucleosides.

Interactions of HIPPI, a molecular partner of Huntingtin interacting protein HIP1, with the specific motif present at the putative promoter sequence of the caspase-1, caspase-8 and caspase-10 genes

To investigate the mechanism of increased expression of caspase‐1 caused by exogenous Hippi, observed earlier in HeLa and Neuro2A cells, in this work we identified a specific motif AAAGACATG (− 101 to − 93) at the caspase‐1 gene upstream sequence where HIPPI could bind. Various mutations in this specific sequence compromised the interaction, showing the specificity of the interactions. In the luciferase reporter assay, when the reporter gene was driven by caspase‐1 gene upstream sequences (− 151 to − 92) with the mutation G to T at position − 98, luciferase activity was decreased significantly in green fluorescent protein–Hippi‐expressing HeLa cells in comparison to that obtained with the wild‐type caspase‐1 gene 60 bp upstream sequence, indicating the biological significance of such binding. It was observed that the C‐terminal ‘pseudo’ death effector domain of HIPPI interacted with the 60 bp (− 151 to − 92) upstream sequence of the caspase‐1 gene containing the motif. We further observed that expression of caspase‐8 and caspase‐10 was increased in green fluorescent protein–Hippi‐expressing HeLa cells. In addition, HIPPI interacted in vitro with putative promoter sequences of these genes, containing a similar motif. In summary, we identified a novel function of HIPPI; it binds to specific upstream sequences of the caspase‐1, caspase‐8 and caspase‐10 genes and alters the expression of the genes. This result showed the motif‐specific interaction of HIPPI with DNA, and indicates that it could act as transcription regulator.

Exploring the idea of self-guided dynamics

We have examined the recently proposed concept of self-guiding in the context of both stochastic and deterministic dynamics of a test particle in a double-well potential. In contrast to the original suggestion, results from our stochastic dynamics simulations indicate that enhancement of the systematic part of the force by introduction of a self-guiding term actually decreases the sampling efficiency of the system. It was found that only by substantially reducing the correlation between the actual force and the self-guiding force an enhancement in sampling could be achieved. In a deterministic analog of the same problem, constructed by coupling the test particle to a bath of a number of harmonic oscillators, an analogous reduction in the barrier crossing rate could be observed for the case when the actual force and the biasing force were strongly correlated. However, even a moderate decorrelation resulted in appreciable enhancement in barrier crossings. For the deterministic dynamics of the test particle uncoupled to the bath, no decrease in sampling was observed. We suggest that depending on the inertial memory of the system, the degree of correlation between the actual and the biasing force determines whether sampling will increase or not. This provides a unified picture and gives us insight about the applicability of the method under different simulation conditions.

Specificity determinants for the abscisic acid response element

Abscisic acid (ABA) response elements (ABREs) are a group of cis‐acting DNA elements that have been identified from promoter analysis of many ABA‐regulated genes in plants. We are interested in understanding the mechanism of binding specificity between ABREs and a class of bZIP transcription factors known as ABRE binding factors (ABFs). In this work, we have modeled the homodimeric structure of the bZIP domain of ABRE binding factor 1 from Arabidopsis thaliana (AtABF1) and studied its interaction with ACGT core motif‐containing ABRE sequences. We have also examined the variation in the stability of the protein–DNA complex upon mutating ABRE sequences using the protein design algorithm FoldX. The high throughput free energy calculations successfully predicted the ability of ABF1 to bind to alternative core motifs like GCGT or AAGT and also rationalized the role of the flanking sequences in determining the specificity of the protein‐DNA interaction.

PROPERTIES OF DIANIONIC OXYPHOSPHORANE INTERMEDIATES FROM HYBRID QM/MM SIMULATION : IMPLICATIONS FOR RIBOZYME REACTIONS

Abstract Dianionic oxyphosphorane intermediates have been suggested as good models for studying the mechanisms of self-cleavage reactions undergone by some RNA molecules (the so-called RNA enzymes or ribozymes). We report here the results of a hybrid quantum and molecular mechanical (QM/MM) free energy calculation for obtaining the reaction free energy profiles for such a system. The reactions are simulated in the gas phase as well as within a water sphere in the presence and absence of Mg2+ ion. The semiempirical quantum mechanical AM1 hamiltonian describes the oxyphosphorane species and the molecular mechanical CHARMM force field describes the solvent and Mg2+. We find that, when compared with accurate ab initio calculations, the present method shows a tendency to overestimate the stability of the intermediates. On the other hand, it correctly reproduces the qualitative features, namely, the stabilization of the intermediate with solvation and destabilization in the presence of Mg2+. It also shows a drastic lowering of the barrier for bond cleavage in the presence of Mg2+, in agreement with ab initio and experimental results. We discuss possible means of increasing the accuracy of the method for subsequent application to modelling the reactions within ribozymes.

Arabidopsis thaliana regulatory element analyzer

UNLABELLED In the Arabidopsis thaliana regulatory element analyzer (AtREA) server, we have integrated sequence data, genome-wide expression data and functional annotation data in three application modules which will be useful to identify major regulatory targets of a user-provided cis-regulatory element (CRE), study different features of CRE distribution and evaluate the role of a set of CREs in the regulation of gene expression--independently as well as in combination with other user-provided CREs. AVAILABILITY AtREA is freely available at http://www.bioinformatics.org/grn/atrea.html.

DYNAMICS OF LEUCINE-RICH REPEAT PROTEINS

We have studied the collective dynamics of leucine-rich repeat (LRR) proteins using an elastic network approximation. The slowest mode of the porcine ribonuclease inhibitor (pRI) protein could be visualized as bending fluctuations of a curved elastic strip leading to a planar opening–closing motion of the horseshoe which largely corresponded to the deformation of the protein on ligand binding. The second slowest mode however exhibited a significant out of plane splaying. The distribution of the lowest eigenvalues of different LRR proteins as a function of their repeat number was found to be close to the dispersion curve obtained from pRI whereas that of the leucine-rich variant (LRV) protein showed considerable deviation. The differing mechanical properties of these structurally similar solenoid proteins may have relevance to their function.

Effect of Inactivating Mutations on Peptide Conformational Ensembles: The Plant Polypeptide Hormone Systemin

As part of their basal immune mechanism against insect/herbivore attacks, plants have evolved systemic response mechanisms. Such a systemic wound response in tomato was found to involve an 18 amino acid polypeptide called systemin, the first polypeptide hormone to be discovered in plants. Systematic alanine scanning and deletion studies showed differential modulation in its activity, particularly a major loss of function due to alanine substitution at positions 13 and 17 and less extentive loss of function due to substitution at position 12. We have studied the conformational ensembles of wild-type systemin along with its 17 variants by carrying out a total of 5.76 μs of replica-exchange molecular dynamics simulation in an implicit solvent environment. In our simulations, wild-type systemin showed a lack of α-helical and β-sheet structures, in conformity with earlier circular dichroism and NMR data. On the other hand, two regions containing diproline segments showed a tendency to adopt polyproline II structures. Examination of conformational ensembles of the 17 variants revealed a change in the population distributions, suggesting a less flexible structure for alanine substitutions at positions 12 and 13 but not for position 17. Combined with the experimental observations that positions 1-14 of systemin are important for the formation of the peptide-receptor complex, this leads to the hypothesis that loss of conformational flexibility may play a role in the loss of activity of systemin due to the P12A and P13A substitutions, while T17A deactivation probably occurs for a different reason, most likely the loss of the threonine phosphorylation site. We also indicate possible structural reasons why the substitution of the prolines at positions 12 and 13 leads to a loss of conformational freedom in the peptide.