Pawan K. Dhar

Looking for a sequence based allostery definition: a statistical journey at different resolution scales.

The aim of this work was to detect allosteric hotspots signatures characterizing protein regions acting as the key drivers of global allosteric conformational change. We computationally estimated the relative strength of intra-molecular interaction in allosteric proteins between two putative allostery-susceptible sites using a co-evolution model based upon the optimization of the cross-correlation in terms of free-energy-transfer hydrophobicity scale (Tanford scale) distribution along the chain. Cross-Recurrence Quantification Analysis (Cross-RQA) applied on the sequences of allostery susceptible sites showed evidence of strong interaction amongst allosteric susceptible sites. This could be due to transient weak molecular bonds between allostery susceptible patches enabling regions far-apart to come together. Further, using a large protein dataset, by comparing allosteric protein set with a randomly generated sequence population as well as a generic protein set, we reconfirmed our earlier findings that hydrophobicity patterning (as formalized by Recurrence Quantification Analysis (RQA) descriptors) may serve as determinant of allostery and its relevance in the transmission of allosteric conformational change. We applied RQA to free-energy-transfer hydrophobicity-transformed amino acid sequences of the allostery dataset to extract allostery specific global sequence features. These free-energy-transfer hydrophobicity-based RQA markers proved to be representative of allosteric signatures and not related to the differences between randomly generated and real proteins. These free-energy-transfer hydrophobicity-based RQA markers when evaluated by pattern recognition tools could distinguish allosteric proteins with 92% accuracy.

Sequence signatures of allosteric proteins towards rational design

Allostery is the phenomenon of changes in the structure and activity of proteins that appear as a consequence of ligand binding at sites other than the active site. Studying mechanistic basis of allostery leading to protein design with predetermined functional endpoints is an important unmet need of synthetic biology. Here, we screened the amino acid sequence landscape in search of sequence-signatures of allostery using Recurrence Quantitative Analysis (RQA) method. A characteristic vector, comprised of 10 features extracted from RQA was defined for amino acid sequences. Using Principal Component Analysis, four factors were found to be important determinants of allosteric behavior. Our sequence–based predictor method shows 82.6% accuracy, 85.7% sensitivity and 77.9% specificity with the current dataset. Further, we show that Laminarity-Mean-hydrophobicity representing repeated hydrophobic patches is the most crucial indicator of allostery. To our best knowledge this is the first report that describes sequence determinants of allostery based on hydrophobicity. As an outcome of these findings, we plan to explore possibility of inducing allostery in proteins.

Computational identification of novel microRNAs and their targets in the malarial vector, Anopheles stephensi

AbstractMicroRNAs are a ~22 nucleotide small non-coding RNAs found in animals, plants and viruses. They regulate key cellular processes by enhancing, degrading or silencing protein coding targets. Currently most of the data on miRNA is available from Drosophilan. Given their important post-transcriptional role in several organisms, there is a need to understand the miRNA mediated processes in normal and abnormal conditions. Here we report four novel microRNAs ast-mir-2502, ast-mir-2559, ast-mir-3868 and ast-mir-9891 in Anopheles stephensi identified from a set of 3,052 transcriptome sequences, showing average minimum free energy of −31.8xa0kcal/mol of duplex formation with mRNA indicating their functional relevance. Phylogenetic study shows conservation of sequence signatures within the Class Insecta. Furthermore, 26 potential targets of these four miRNAs have been predicted that play an important role in the mosquito life-cycle. This work leads to novel leads and experimental possibilities for improved understanding of gene regulatory processes in mosquito.

Heat Stress-Induced Cup9-Dependent Transcriptional Regulation of SIR2

ABSTRACT The epigenetic writer Sir2 maintains the heterochromatin state of chromosome in three chromosomal regions, namely, the silent mating type loci, telomeres, and the ribosomal DNA (rDNA). In this study, we demonstrated the mechanism by which Sir2 is regulated under heat stress. Our study reveals that a transient heat shock causes a drastic reduction in the SIR2 transcript which results in sustained failure to initiate silencing for as long as 90 generations. Hsp82 overexpression, which is the usual outcome of heat shock treatment, leads to a similar downregulation of SIR2 transcription. Using a series of genetic experiments, we have established that heat shock or Hsp82 overexpression causes upregulation of CUP9 that, in turn, represses SIR2 transcription by binding to its upstream activator sequence. We have mapped the cis regulatory element of SIR2. Our study shows that the deletion of cup9 causes reversal of the Hsp82 overexpression phenotype and upregulation of SIR2 expression in heat-induced Hsp82-overexpressing cells. On the other hand, we found that Cup9 overexpression represses SIR2 transcription and leads to a failure in the establishment of heterochromatin. The results of our study highlight the mechanism by which environmental factors amend the epigenetic configuration of chromatin.

Discovering Novel Anti-Malarial Peptides from the Not-coding Genome - A Working Hypothesis

The not-coding regions of the genome describe sequences that do not have a history of transcription. They are also called ‘dark matter’ of the genome. Here we present a working hypothesis for finding novel anti-malarial peptides from such regions of the yeast genome that encode neither RNA nor protein. This is based on our previous experimental work where not-coding DNA sequences were artificially expressed leading to protein expression and phenotypic outcome. In this study, we explored the vast not-coding DNA space of Saccharomyces cerevisiae in search of novel antimalarial peptides. Given the lack of effective therapeutic solutions against malaria, there is an urgent unmet requirement to find novel antimalarial drugs and targets. Our initial efforts to find novel anti-malarial peptides have led to unexpected and interesting results. However, our work is preliminary and is based on computational studies only. In future, more computational and experimental work is needed to establish therapeutic potential of synthetic peptides that have origins in the not-coding genome space.

Application of Recurrence Quantification Analysis (RQA) in Biosequence Pattern Recognition

Recurrence Quantitative Analysis is a relatively new pattern recognition tool well suited for short, non-linear and non stationary systems. It is designed to detect recurrence patterns that are expressed as a set of Recurrence Quantification variables. In our work we made use of this tool on allosteric protein system to identify residues involved in the transmission of the structural rearrangements as an upshot of allostery. Allostery is the phenomenon of changes in the structure and activity of proteins that appear as a consequence of ligand binding at sites other than the active site. Here, we scrutinized the sequence landscape of ‘ras’ protein by partitioning its residues into windows of equal size. An 11 element characteristic vector, comprising of 10 features extracted from the Recurrence Quantification Analysis along with a feature relating to allosteric involvement, was defined for each windowed sequence set. By applying multivariate statistical analysis tools including Principal Component Analysis and Multiple Regression Analysis upon the characteristic feature vectors extracted from all the windowed sequence set, we could develop a significant linear model to identify the residues that are critical to allostery of ‘ras’ protein.

Building momentum for systems and synthetic biology in India.

Biological systems are inherently noisy. Predicting the outcome of a perturbation is extremely challenging. Traditional reductionist approach of describing properties of parts, vis-a-vis higher level behaviour has led to enormous understanding of fundamental molecular level biology. This approach typically consists of converting genes into junk (knock-down) and garbage (knock-out) and observe how a system responds. To enable broader understanding of biological dynamics, an integrated computational and experimental strategy was formally proposed in mid 1990s leading to the re-emergence of Systems Biology. However, soon it became clear that natural systems were far more complex than expected. A new strategy to address biological complexity was proposed at MIT (Massachusetts Institute of Technology) in June 2004, when the first meeting of synthetic biology was held. Though the term ‘synthetic biology’ was proposed during 1970s (Szybalski in Control of gene expression, Plenum Press, New York, 1974), the usage of the original concept found an experimental proof in 2000 with the demonstration of a three-gene circuit called repressilator (Elowitz and Leibler in Nature, 403:335–338, 2000). This encouraged people to think of forward engineering biology from a set of well described parts.

Decahydroquinolines from the venom of a formicinae ant, Oecophylla smaragdina

Ecologically significant species in controlling pests, Oecophylla smaragdina uses its venom to paralyze their prey and to communicate with their colony mates. But no significant analysis of the ants venom gland secretions has been carried out hitherto. This study describes the identification of venom constituents of Oecophylla smaragdina using coupled gas chromatography and mass spectroscopy (GC-MS) analysis. The results indicate the anticipated presence of a neurotoxin i.e., 2, 5 dipropyl decahydroquinoline and phenol, 2, 4-bis (1, 1 dimethylethyl). This is the first report on presence of decahydroquinolines in the venom of formicinae ant species of genera Oecophylla.

Making Synthetic Proteins From Non-coding DNA

Non-coding DNA describes regions of the genome for which no appar- ent function has been identified. The term Junk DNA, was introduced in 1972 by Susumu Ohno for such non-expressed regions of genome. A number of bioinfor- matics studies have been organized to understand the function of non-coding DNA. However, a clear understanding is lacking. To address this issue, we invented a method to make novel genes from non-coding DNA and study its expression. This chapter describes the general composition of non-coding DNA, describes a novel approach of studying these sequences and provides a first glimpse of some of the interesting results.

Engineering Biological Systems: A Brief Overview

Ever since synthetic biology as a formal discipline was launched, several parts, devices and circuits have been designed. The story of engineering-inspired approach to biology probably started from a publication in 2000 that described three gene circuit called repressilator and another one describing toggle switch. While repressilator is given a more detailed chapter in another chapter, this chapter provides a brief overview of key technical innovations in synthetic biology.