CDH1 gene as biomarker towards breast cancer prediction.

Breast cancer is considered to be happened due to genetic aberration. Out of several genes expressed, it is found that cadherin 1, type 1 (CDH1) is responsible in several ways to control the metabolic order in human. Deregulation of the function of protein E-cadherin, expressed from CDH1 plays an important role in lobular breast cancer. In order to understand the root cause of this recent claim, we focus on CDH1 gene: whether the genetic information translated due to any deviation/alteration/modification in its sequence is related to the occurrence of the different types breast cancer. Towards this end, quantitative analysis of different biophysical and bio-chemical properties of CDH1 gene in genomic and proteomic levels from the available genomic (cDNA) sequences of CDH1 gene (obtained from the COSMIC Database for 78 patients, suffering from various types of breast cancer) clearly emphasizes that alternation/modification in the sequence of the CDH1 gene can be detrimental. Furthermore, Random forest, K-nearest neighbour and stochastic gradient descent (SGD) algorithms are applied on the derived dataset to classify the types of breast cancer, and to validate our hypothesis regarding the acute role of CDH1 as potential bio marker for breast cancer. Analysis of the mutated CDH1 gene sequences, and their related parameters using aforesaid machine learning techniques clearly establish that CDH1 gene can take the deterministic role in predicting the chances of occurrences of different types of breast cancer with an accuracy of >90%. Such an observation opens a new paradigm in diagnostic approach of breast cancer.Communicated by Ramaswamy H. Sarma.

Intrinsic proclivity of left-handed conformation in large Nest motif peptides inferred from molecular dynamics.

The 'Nest' motif plays a functional role in protein owing to its ligand binding potential aided by geometric concavity. The presence of less favored left-handed conformation (L-state) in its structure makes this concavity possible and in shaping the native chemical environment amenable to stable binding interactions. To understand the persistent appearance of L-state torsion in the Nest motif, we analyzed 0.5µs Molecular Dynamics (MD) simulation trajectories of 35 six-residue peptides (out of a total of 50 large Nest sequences of ≥6 residues) identified in our previous study. Analysis of the MD trajectories of the individual peptides reveals initial L-state in 60% of the peptides persists for >40% of the trajectory. Further, Nests with different sequences appear to adopt a specific conformational state driven by the neighboring L-state residues. The sequences also possess short secondary structures and amino acid repeats, suggesting evolutionary conservation and the specific role of amino acids in locally predisposing the torsion angle to the L-state. These findings help us to understand how L-state conformation is an essential prerequisite in stabilizing the Nest motif and shed light on the sequence-structure-function paradigm in the rational design of peptides and peptidomimetics for therapeutics.Communicated by Ramaswamy H. Sarma.

Analysing the genetic code degeneracy: a consequence towards bacterial staining.

As 20 naturally occurring amino acids are coded by 61 mRNA codons out of 64, it is obvious that 61→20 cannot have one-to-one mapping which generates the problem of codon degeneracy. Despite several efforts there is no specific outcome which can describe this well-known enigmatic degeneracy of the codon table. Since, every biological behaviour is regulated by protein which in turn consists of amino acids bearing the inherent characteristics of degeneracy among mRNA codons (Crick F.H.C. The Origin of the Genetic Code. J. Mol. Biol.1968; 38: 367-379), it is worthy to analyse the impact of such degeneracy on biological behaviours. Here, based on mathematical models using the concept of b-type of the nucleotide bases and hamming distances, an effort has been initiated to understand the impact of biasness of genetic code degeneracy on biological behaviours. The proposed models have been utilized to understand the characteristic features of bacterial genes of gram-positive and gram-negative bacteria. To the best of our knowledge, this is the first mathematical model to capture the effect of genetic code degeneracy, showing a paradigm towards understanding the behavioural difference between gram-positive and gram-negative bacteria, and thereby opening a new avenue for revealing differential biological properties.Communicated by Ramaswamy H. Sarma.

Effect of biphosphate salt on dipalmitoylphosphatidylcholine bilayer deformation by Tat polypeptide.

Translocation of positively charged cell penetrating peptides (CPP) through cell membrane is important in drug delivery. Here we report all-atom molecular dynamics simulations to investigate how a biphosphate salt in a solvent affects the interaction of a CPP, HIV-1 Tat peptide with model dipalmitoylphosphatidylcholine (DPPC) lipid bilayer. Tat peptide has a large number of basic arginines and a couple of polar glutamines. We observe that in absence of salt, the basic residues of the polypeptide get localized in the vicinity of the membrane without altering the bilayer properties much; polypeptide induce local thinning of the bilayer membrane at the area of localization. In presence of biphosphate salt, the basic residues, dressed by the biphosphate ions, are repelled by the phosphate head groups of the lipid molecules. However, polar glutamine prefers to stay in the vicinity of the bilayer. This leads to larger local bilayer thickness at the contact point by the polar residue and non-uniform bilayer thickness profile. The thickness deformation of bilayer structure disappears upon mutating the polar residue, suggesting importance of the polar residue in bilayer deformation. Our studies point to control bilayer deformation by appropriate peptide sequence and solvent conditions.

Cα-Methyl-l-valine: A Preferential Choice over α-Aminoisobutyric Acid for Designing Right-Handed α-Helical Scaffolds.

In synthetic peptides containing Gly and coded α-amino acids, one of the most common practices to enhance their helical extent is to incorporate a large number of l-Ala residues along with noncoded, strongly foldameric α-aminoisobutyric acid (Aib) units. Earlier studies have established that Aib-based peptides, with propensity for both the 310- and α-helices, have a tendency to form ordered three-dimensional structure that is much stronger than that exhibited by their l-Ala rich counterparts. However, the achiral nature of Aib induces an inherent, equal preference for the right- and left-handed helical conformations as found in Aib homopeptide stretches. This property poses challenges in the analysis of a model peptide helical conformation based on chirospectroscopic techniques like electronic circular dichroism (ECD), a very important tool for assigning secondary structures. To overcome such ambiguity, we have synthesized and investigated a thermally stable 14-mer peptide in which each of the Aib residues of our previously designed and reported analogue ABGY (where B stands for Aib) is replaced by Cα-methyl-l-valine (L-AMV). Analysis of the results described here from complementary ECD and 1H nuclear magnetic resonance spectroscopic techniques in a variety of environments firmly establishes that the L-AMV-containing peptide exhibits a significantly stronger preference compared to that of its Aib parent in terms of conferring α-helical character. Furthermore, being a chiral α-amino acid, L-AMV shows an intrinsic, extremely strong bias for a quite specific (right-handed) screw sense. These findings emphasize the relevance of L-AMV as a more appropriate unit for the design of right-handed α-helical peptide models that may be utilized as conformationally constrained scaffolds.

A graph theoretic model to understand the behavioral difference of PPCA among its paralogs towards recognition of DXCA.

Among all the proteins of Periplasmic C type Cytochrome family obtained from cytochrome C7 found in Geobacter sulfurreducens, only the Periplasmic C type Cytochrome A (PPCA) protein can recognize the deoxycholate (DXCA), while its other paralogs do not, as observed from the crystal structures. Though some existing works have used graph-theoretic approaches to realize the 3-D structural properties of proteins, its usage in the rationalisation of the physiochemical behavior of proteins has been very limited. To understand the driving force towards the recognition of DXCA exclusively by PPCA among its paralogs, in this work, we propose two graph theoretic models based on the combinatorial properties, namely, base-pair-type and impact, of the nucleotide bases and the amino acid residues, respectively. Combinatorial analysis of the binding sequences using the proposed base-pair type based graph theoretic model reveals the differential behaviour of PPCA among its other paralogs. Further, to investigate the underlying chemical phenomenon, another graph theoretic model has been developed based on impact. Analysis of the results obtained from impact-based model clearly indicates towards the helix formation of PPCA which is essential for the recognition of DXCA, making PPCA a completely different entity from its paralogs.

Insight into the sequence-structure relationship of TLR cytoplasm's Toll/Interleukin-1 receptor domain towards understanding the conserved functionality of TLR 2 heterodimer in mammals.

The signaling response of TLR2 to ligands has always been as a homodimer or in heterodimerization with TLR1/TLR6. The Toll/Interleukin-1 Receptor (TIR) domain of the TLR cytoplasmic region regulates the dimerization and interactions with adaptor molecules to build an active signaling complex. To understand the conservation of functionality of the TLR2-heterodimers between the distantly related species human(h) and mice(m), the pattern of TIR-TIR interaction in heterodimers has been studied through the sequence-structural point of view. Comparative analysis of primary sequence and structural pattern of TLRs(1/2/6) corroborates higher sequence homology between TLR1 and TLR6. Molecular docking analysis of TLR2-TLR1 and TLR2-TLR6 cytoplasmic dimers in both mouse and human have identified that for interaction the BB loop/near-BB loop residues of TLR2 are involved with the near-DD loop of TLR1 and DD loop residues of TLR6 within the TIR domains, which may cause to differential signaling. Molecular dynamics simulation of dimers for both human and mice species recognize stable interface between near-BB/BB loop region of TLR2 and discrete near-DD and DD loop region of TLR1 and TLR6 respectively. The observed dimerization pattern in both the species is further supported by Alanine scanning mutation study. However, Solvent Accessible Surface Area (SASA) of BB and DD loop regions of the cytoplasmic monomers and the heterodimers suggests that while TLR2 BB loop is actively associated as the dimer interface with its heterodimer partners in both the species, the DD loop acts as the active interfacing region in hTLR1 and mTLR6. Communicated by Ramaswamy H. Sarma.

New facets of larger Nest motifs in proteins.

The Nest is a concave-shaped structural motif in proteins formed by consecutive enantiomeric left-handed (L) and right-handed (R) helical conformation of the backbone. This important motif subsumes many turn and helix capping structures and binds electron-rich ligands. Simple Nests are either RL or LR. Larger Nests (>2 residues long) may be RLR, LRL, RLRL, and so forth, being considered as composed of overlapping simple Nests. The larger Nests remain under-explored despite their widely known contributions to protein function. In our study, we address whether the recurrence of enantiomeric geometry in the larger Nests constrains the peptide backbone such that distinct compositional and conformational preferences are seen compared to simple Nests. Our analysis reveals the critical role of the L helical torsion angle in the formation of larger Nests. This can be observed through the higher propensity of residue or secondary structure combinations in LR and LRL backbone conformation in comparison to RL or RLR, although LR/LRL is considerably lower by occurrence. We also find that the most abundant doublets and triplets in Nests have a propensity for particular secondary structures, suggesting a strong sequence-structure relationship in the larger Nest. Overall, our analysis corroborates distinct features of simple and the larger Nests. Such insights would be helpful towards in-vitro design of peptides and peptidomimetic studies.

Design of a Peptide-Based Model Leads for Scavenging Anions.

Among several peptide-based anion recognition motifs, the "CαNN" motif containing C-1 α, N0, and N+1 of three consecutive residues is unique in its mode of interaction. Having a spatial geometry of ßαα or ßαß, this motif occurs in the N terminus of a helix and often found at the functional interface of a protein, mediating crucial biological significance upon interaction with anion(s). The interaction of anion(s) with chimeric peptide sequences containing the naturally occurring "CαNN" motif (CPS224Ac, CPS226, and CPS228) reported in our previous attempts strongly confirms that the information regarding the interaction is embedded within the local sequences of the motif segment. At these prevailing circumstances, an effort has been pursued to design novel scaffolds based on the "CαNN" motif for achieving better recognition of anion(s). Exploring the existing data set of the "CαNN" motif available in the FSSP database, four novel peptide-based scaffolds have been designed (DS1, DS2, DS3, and DS4), and preliminary screenings have been performed using computational approaches. Our initial work suggests that two (DS1 and DS3) out of the four scaffolds are potential candidates for better anion recognition. By employing biophysical characterization using both qualitative and quantitative measures, in this present study, we report the interaction of sulfate and phosphate ions with these two designed scaffolds, in which there is much better recognition of anions by these scaffolds than the natural sequences, justifying their logical engineering. Our observation strongly suggests that these designed scaffolds are better potential candidates than those of the naturally occurring "CαNN" motif in terms of anion recognition and could be utilized for the scavenging of anion(s) for different purposes.

Interaction landscape of a 'CαNN' motif with arsenate and arsenite: a potential peptide-based scavenger of arsenic.

Arsenic (As) is a toxic metalloid that has drawn immense attention from the scientific community recently due to its fatal effects through its unwanted occurrence in ground water around the globe. The presence of an excess amount of water soluble arsenate and/or arsenite salt (permissible limit 10 µg L-1 as recommended by the WHO) in water has been correlated with several human diseases. Although arsenate (HAsO4 2-) is a molecular analogue of phosphate (HPO4 2-), phosphate is indispensable for life, while arsenic and its salts are toxic. Therefore, it is worthwhile to focus on the removal of arsenic from water. Towards this end, the design of peptide-based scaffolds for the recognition of arsenate and arsenite would add a new dimension. Utilizing the stereochemical similarity between arsenate (HAsO4 2-) and phosphate (HPO4 2-), we successfully investigated the recognition of arsenate and arsenite with a naturally occurring novel phosphate binding 'CαNN' motif and its related designed analogues. Using computational as well as biophysical approaches, for the first time, we report here that a designed peptide-based scaffold based on the 'CαNN' motif can recognize anions of arsenic in a thermodynamically favorable manner in a context-free system. This peptide-based arsenic binding agent has the potential for future development as a scavenger of arsenic anions to obtain arsenic free water.

Quantum chemical studies on anion specificity of CαNN motif in functional proteins.

Anion binding CαNN motif is found in functionally important regions of protein structures. This motif based only on backbone atoms from three adjacent residues, recognizes free sulphate or phosphate ion as well as phosphate groups in nucleotides and in a variety of cofactors. The mode of anion recognition and microscopic picture of binding interaction remains unclear. Here we perform self-consistent quantum chemical calculations considering sulphate and phosphate bound CαNN motif fragments from crystal structures of functional proteins in order to figure out microscopic basis of anion recognition. Our calculations indicate that stability and preference of the anion in the motif depends on the sequence of the motif. The stabilization energy is larger in case of polar residue containing motif fragment. Nitrogen atom of the polar residue of motif mainly participates in the coordination at the lowest energy levels. Anion replacement decreases stabilization energy along with coordination between motif atoms and oxygen atoms of anion shifted to higher energies, suggesting preference of the motif residues to specific anion. Our analysis may be helpful to understand microscopic basis of interaction between proteins and ionic species.

A machine learning approach towards the prediction of protein-ligand binding affinity based on fundamental molecular properties.

There is an exigency of transformation of the enormous amount of biological data available in various forms into some significant knowledge. We have tried to implement Machine Learning (ML) algorithm models on the protein-ligand binding affinity data already available to predict the binding affinity of the unknown. ML methods are appreciably faster and cheaper as compared to traditional experimental methods or computational scoring approaches. The prerequisites of this prediction are sufficient and unbiased features of training data and a prediction model which can fit the data well. In our study, we have applied Random forest and Gaussian process regression algorithms from the Weka package on protein-ligand binding affinity, which encompasses protein and ligand binding information from PdbBind database. The models are trained on the basis of selective fundamental information of both proteins and ligand, which can be effortlessly fetched from online databases or can be calculated with the availability of structure. The assessment of the models was made on the basis of correlation coefficient (R 2) and root mean square error (RMSE). The Random forest model gave R 2 and RMSE of 0.76 and 1.31 respectively. We have also used our features and prediction models on the dataset used by others and found that our model with our features outperformed the existing ones.

TLR11 or TLR12 silencing reduces Leishmania major infection.

Toll-like receptors (TLRs) recognize the pathogen-associated molecular patterns (PAMPs) and induce host-protective immune response. The role of the profilin-recognizing TLR11/TLR12 in Leishmania infection is unknown. Herein, we report that TLR11/ TLR12 expression increases in virulent L. major-infected macrophages but is prevented by miltefosine, an anti-leishmanial drug. While lipohosphoglycan (LPG) increases, LPG or TLR2 blockade prevents, the heightened TLR11/TLR12 expression. LPG-TLR2 interaction triggers MyD88- and TIRAP-mediated signaling enhancing ERK-1/2 activation and increased production of IL-10 that promotes TLR11/TLR12 expression. Profilin expression was higher in the virulent L. major and L. donovani parasites than that observed in the avirulent parasites. TLR11 or TLR12 silencing reduces parasite burden and increases IFN-γ, but reduces IL-4, production indicating that TLR11 and TLR12 play a pro-leishmanial role.

Anion induced conformational preference of Cα NN motif residues in functional proteins.

Among different ligand binding motifs, anion binding Cα NN motif consisting of peptide backbone atoms of three consecutive residues are observed to be important for recognition of free anions, like sulphate or biphosphate and participate in different key functions. Here we study the interaction of sulphate and biphosphate with Cα NN motif present in different proteins. Instead of total protein, a peptide fragment has been studied keeping Cα NN motif flanked in between other residues. We use classical force field based molecular dynamics simulations to understand the stability of this motif. Our data indicate fluctuations in conformational preferences of the motif residues in absence of the anion. The anion gives stability to one of these conformations. However, the anion induced conformational preferences are highly sequence dependent and specific to the type of anion. In particular, the polar residues are more favourable compared to the other residues for recognising the anion.

Ratio of ellipticities between 192 and 208 nm (R1 ): An effective electronic circular dichroism parameter for characterization of the helical components of proteins and peptides.

Circular dichroism (CD) spectroscopy represents an important tool for characterization of the peptide and protein secondary structures that mainly arise from the conformational disposition of the peptide backbone in solution. In 1991 Manning and Woody proposed that, in addition to the signal intensity, the ratio between [θ]nπ* and [θ]ππ*ǁ ((R2 ) ≅ [θ]222 /[θ]208 ), along with [θ]ππ*⊥ and [θ]ππ*ǁ ((R1 ) ≅ [θ]192 /[θ]208 ), may be utilized towards identifying the peptide/protein conformation (especially 310 - and α-helices). However, till date the use of the ratiometric ellipticity component for helical structure analysis of peptides and proteins has not been reported. We studied a series of temperature dependent CD spectra of a thermally stable, model helical peptide and its related analogs in water as a function of added 2,2,2-trifluoroethanol (TFE) in order to explore their landscape of helicity. For the first time, we have experimentally shown here that the R1 parameter can characterize better the individual helices, while the other parameter R2 and the signal intensity do not always converge. We emphasize the use of the R1 ratio of ellipticities for helical characterization because of the common origin of these two bands (exciton splitting of the amide π→ π* transition in a helical polypeptide). This approach may become worthwhile and timely with the increasing accessibility of CD synchrotron sources.

PPIMpred: a web server for high-throughput screening of small molecules targeting protein-protein interaction.

PPIMpred is a web server that allows high-throughput screening of small molecules for targeting specific protein-protein interactions, namely Mdm2/P53, Bcl2/Bak and c-Myc/Max. Three different kernels of support vector machine (SVM), namely, linear, polynomial and radial basis function (RBF), and two other machine learning techniques including Naive Bayes and Random Forest were used to train the models. A fivefold cross-validation technique was used to measure the performance of these classifiers. The RBF kernel of SVM outperformed and/or was comparable with all other methods with accuracy values of 83%, 79% and 90% for Mdm2/P53, Bcl2/Bak and c-Myc/Max, respectively. About 80% of the predicted SVM scores of training/testing datasets from Mdm2/P53 and Bcl2/Bak have significant IC50 values and docking scores. The proposed models achieved an accuracy of 66-90% with blind sets. The three mentioned (Mdm2/P53, Bcl2/Bak and c-Myc/Max) proposed models were screened in a large dataset of 265 242 small chemicals from National Cancer Institute open database. To further realize the robustness of this approach, hits with high and random SVM scores were used for molecular docking in AutoDock Vina wherein the molecules with high and random predicted SVM scores yielded moderately significant docking scores (p-values < 0.1). In addition to the above-mentioned classification scheme, this web server also allows users to get the structural and chemical similarities with known chemical modulators or drug-like molecules based on Tanimoto coefficient similarity search algorithm. PPIMpred is freely available at http://bicresources.jcbose.ac.in/ssaha4/PPIMpred/.

Computational design of model scaffold for anion recognition based on the 'Cα NN' motif.

The 'novel phosphate binding 'Cα NN' motif', consisting of three consecutive amino acid residues, usually occurs in the protein loop regions preceding a helix. Recent computational and complementary biophysical experiments on a series of chimeric peptides containing the naturally occurring 'Cα NN' motif at the N-terminus of a designed helix establishes that the motif segment recognizes the anion (sulfate and phosphate ions) through local interaction along with extension of the helical conformation which is thermodynamically favored even in a context-free, nonproteinaceous isolated system. However, the strength of the interaction depends on the amino acid sequence/conformation of the motif. Such a locally-mediated recognition of anions validates its intrinsic affinity towards anions and confirms that the affinity for recognition of anions is embedded within the 'local sequence' of the motif. Based on the knowledge gathered on the sequence/structural aspects of the naturally occurring 'Cα NN' segment, which provides the guideline for rationally engineering model scaffolds, we have modeled a series of templates and investigated their interactions with anions using computational approach. Two of these designed scaffolds show more efficient anion recognition than those of the naturally occurring 'Cα NN' motif which have been studied. This may provide an avenue in designing better anion receptors suitable for various biochemical applications.

Genome-wide mitochondrial DNA sequence variations and lower expression of OXPHOS genes predict mitochondrial dysfunction in oral cancer tissue.

Several studies reported that mtDNA mutations may play important roles in carcinogenesis although the mechanism is not clear yet. Most of the studies compared mtDNA sequences in a tumor with those in normal tissues from different individuals ignoring inter-individual variations. In this study, 271 SNPs, 7 novel SNPs (or SNVs), and 15 somatic mutations were detected in mtDNA of 8 oral cancer tissues with respect to reference (rCRS) and adjacent normal tissues, respectively, using Ion PGM next generation sequencing method. Most of the sequence variations (76 SNPs and 1 somatic) are present in D-loop region followed by CyB (36 SNPs), ATP6 (24 SNPs), ND5 (17 SNPs and 5 somatic), ND4 (18 coding and 2 somatic) and other non-coding and coding DNA sequences. A total of 53 and 8 non-synonymous SNPs and somatic mutations, respectively, were detected in tumor tissues and some of these variations may have deleterious effects on the protein function as predicted by bioinformatic analysis. Moreover, significantly low mtDNA contents and expression of several mitochondrial genes in tumor compared to adjacent normal tissues may have also affected mitochondrial functions. Taken together, this study suggests that mtDNA mutations as well as low expression of mtDNA coded genes may play important roles in tumor growth. Although the sample size is low, an important aspect of the study is the use of adjacent control tissues to find out somatic mutations and a change in the expression of mitochondrial genes, to rule out inter-individual and inter-tissue variations which are important issues in the study of mitochondrial genomics.

Melanin from the nitrogen-fixing bacterium Azotobacter chroococcum: a spectroscopic characterization.

Melanins, the ubiquitous hetero-polymer pigments found widely dispersed among various life forms, are usually dark brown/black in colour. Although melanins have variety of biological functions, including protection against ultraviolet radiation of sunlight and are used in medicine, cosmetics, extraction of melanin from the animal and plant kingdoms is not an easy task. Using complementary physicochemical techniques (i.e. MALDI-TOF, FTIR absorption and cross-polarization magic angle spinning solid-state (13)C NMR), we report here the characterization of melanins extracted from the nitrogen-fixing non-virulent bacterium Azotobacter chroococcum, a safe viable source. Moreover, considering dihydroxyindole moiety as the main constituent, an effort is made to propose the putative molecular structure of the melanin hetero-polymer extracted from the bacterium. Characterization of the melanin obtained from Azotobacter chroococcum would provide an inspiration in extending research activities on these hetero-polymers and their use as protective agent against UV radiation.

The 2.05-helix in hetero-oligopeptides entirely composed of C(α,α)-disubstituted glycines with both side chains longer than methyls.

The existence of the very uncommon, but potentially quite interesting, multiple, consecutive fully-extended conformation (2.05-helix) has been already clearly demonstrated in homo-oligopeptides based on quaternary α-amino acids with both side chains longer than methyls, but not cyclized on the α-carbon atom. To extend the scope of this research, in this work we investigated the occurrence of this flat 3D-structure in hetero-oligopeptides, each composed of two or three different residues of that class. The synthesis of a terminally protected peptide series to the tetrapeptide level was carried out by solution methods. The resulting oligomers were chemically and conformationally characterized. The data obtained point to an overwhelming population of the fully-extended conformation in CDCl3. However, a solvent-driven switch to a predominant 310-helical structure was seen in CD3CN. A delicate, local balance between these two conformations is confirmed to occur in the crystalline state. Molecular dynamics simulations in CHCl3 on a hetero-tetrapeptide converged to the fully-extended conformation even starting from the 310-helical structure.