Protein and Peptide Letters - Volume 14, Issue 6, 2007

Protein and Peptide Informatics offers enormous applications in deciphering of the molecular basis of diseases and in the discovery, design and delivery of biologically active and medicinally effective molecules. The past few decades have witnessed new horizons of research and a paradigm shift in drug designing strategies and technology development with promising achievements to leap ahead in the post-genomic era. This special issue of “Protein Peptide Letters” sketches a panorama of multi-dimensional research at the interface of Protein Peptide Informatics and Drug Designing with special focus on key approaches and innovative contributions emanated from Bioinformatics and Computational Biology. Protein Kinases (PK) are drug-targets for treatment of several dreaded ailments including cancer and are of fundamental importance in modeling optimal drug activities because of their regulatory role in phosphorylation and ATP hydrolysis and hence in many signal transduction pathways in eukaryotes. Experimental studies of their catalytic reactions and inhibition have been integral parts of modern pharmaceutical research and development projects. The review paper by Ms. Anamika and Prof. N. Srinivasan adds a valuable contribution in this area. The bioinformatics and comparative analysis of PK families, their catalytic sequences and structural domains in two strains of Plasmodium presented here elucidate the molecular pathways and growth kinetics of this protozoan parasite which causes Malaria. Statistical datamining of catalytic domain sequences of Protein Kinases carried out in my computational lab (c.f. paper by R. R. Joshi and Chaithanya M.) reveals some interesting facets of ‘selective conservation’ and flexibility of structural genomics of this special family of drug-targets. In the past two decades, trend-setting initiatives have been put forth to understand the biological and pathogenic properties of the human T-cell stimulating viruses and suppressors and T-cell and B-cell epitopes of microorganisms and other antigenic carriers of diseases. Advancement of informatics of Immunogenetics and cellular and molecular Immuno-interactions has boosted emerging applications in anti-viral drug engineering and vaccine discovery. Recent developments focus upon peptide-drug design and computer aided vaccine synthesis. Six papers in this special issue deal with multidimensional research and development applications in this key area. Ms A. D. Ozarkar and Profs. D Prakash, D. N. Deobagkar and D. D. Deobagkar have analyzed the structure of the Duffy binding like (DBLα) domain in Plasmodium falciparum Malaria Vaccine candidate var gene. Their predictions of likely T- and B- cell epitopes in this domain provide interesting implications and applications. A. D. Ghate, B. U. Bhagwat, S. B. Bhosle, S. M. Gadepalli and U. D. Kulkarni-Kale report development of a database and inference tools archiving molecular interactions of protein antigen-antibody. They have also shown good applications of this knowledgebase, named AgAbDb, in modifying the parameters for etimation of B-cell epitopes. Thorough exploration of experiment data and knowledgebase requires mathematically or statistically sound algorithms. My paper on sequential eptiope prediction presents a new, computationally feasible and reliable algorithm of this sort with promising applications in vaccine development by peptide synthesis. The probabilistic model developed for this purpose also computes the binding correspondence between the sequences of predicted epitopes and complementarity determining regions (CDRs) of antibodies and thus provides a possible mode of interpreting the genomics of specificity and affinity of antigen - antibody interactions. Experimental investigations of S.S. Dewasthaly, G.S. Bhonde, V.S. Raman, S. M. Biswas, V.M. Ayachit, and Dr. M.M. Gore identify T-cell stimulating and MAb binding peptide epitopes from envelope glycoprotein (Egp) and NS-1 and Mem proteins of Japanese Encephalitis Virus in mice. Derived from these results the authors report Chimeric T helper- B cell peptides for effective vaccination..........

Phosphorylation by protein kinases is a very common and crucial process in many signal transduction pathways in eukaryotes. This review describes comparative protein kinase analysis of two apicomplexa Plasmodium falciparum (3D7 strain) and Plasmodium yoelii yoelii (17XNL strain) which are causative agents of malaria in human and African rat respectively. Sensitive bioinformatics techniques enable identification of 82 and 60 putative protein kinases in P. falciparum and P. yoelii yoelii respectively and these sequences could be classified into known subfamilies of protein kinases. The most populated kinase subfamilies in both the plasmodium species correspond to CAMK and CMGC groups. Analysis of domain architectures enables detection of uncommon domain organization in kinases of both the organisms such as kinase domain tethered to EF hands as well as PH domain. Components of MAPK signaling pathway is not well conserved in plasmodium organisms. Such observations suggest that plasmodium protein kinases are highly divergent from other eukaryotes. A transmembrane kinase with 6 membrane spanning segments in P. falciparum seems to have no orthologue in P. yoelii yoelii. 19 P. falciparum kinases have been found to cluster separately from P. yoelii yoelii kinases and hence these kinases are unique to P. falciparum genome. Only 28 orthologous pairs of kinases seem to be present between these two plasmodium organisms. Comparative kinome analysis of two plasmodium species has thus provided clues to the function of many protein kinases based upon their classification and domain organization and also implicate marked differences even between two plasmodium organisms.

The catalytic regions of Protein Kinases are known to have similarity in primary chains. However, it is not known whether there is a signature profile specific to a particular catalytic region? Whether the signature profile, if any, is unique to a protein kinases family in a particular species or in a group of species? We have attempted analyzing some of these aspects by statistical data mining using an authentic and exhaustive database of Protein Kinases. The results reveal interesting features and provide some new directions to look at their applications.

P. falciparum encodes PfEMP-1 which is important in pathogenicity and virulence. We have analyzed the structure of the Duffy binding like (DBLα) domain of var genes and predicted the antigenic sites and T and B cell epitopes which present a highly variable picture with major implications in immune interactions and vaccine design.

Immunoinformatics provides tools for reverse vaccinology and encompasses development of knowledge bases and algorithms for prediction of epitopes. AgAbDb, a database archiving molecular interactions of antigen-antibody cocrystal structures, has been developed (http://202.41.70.51:8080/agabdb2/). Analyses of antibody-binding sites on proteins helped to fine-tune the parameters for prediction of sequential and conformational B-cell epitopes.

Design and synthesis of peptide vaccines is of significant pharmaceutical importance. A knowledge based statistical model is fitted here for prediction of binding of an antigenic site of a protein or a B-cell epitope on a CDR (complementarity determining region) of an immunoglobulin. Linear analogues of the 3D structure of the epitopes are computed using this model. Extension for prediction of peptide epitopes from the protein sequence alone is also presented. Validation results show promising potential of this approach in computer-aided peptide vaccine production. The computed probabilities of binding also provide a pioneering approach for ab-initio prediction of ‘potency’ of protein or peptide vaccines modeled by this method.

Virus neutralizing MAb binding and T helper cell stimulating peptide epitopes from structural and nonstructural proteins of Japanese encephalitis virus were delineated. It was observed that priming by T helper peptides potentiated neutralizing antibody response against JE virus. Immunization with chimeric T helper - B cell peptides could thus protect mice from lethal challenge with JE virus.

Major Histocompatibility Complex (MHC) molecules are cell surface glycoproteins that are central to the process of immunity. MHC Class I and II molecules differ in their peptide binding specificity. In this study we have analyzed a non redundant set of MHC binding peptides derived from MHCPEP database, in terms of tripeptides and their positional preference. Results indicate that certain tripeptides have a preference to appear at a particular position for a specific allele. Further, the distribution of rigid tripeptides across all binding sequences was also analyzed and their positions were correlated with anchor residue positions.

A key step in rational vaccine development is to understand how antigens are recognized by their receptors. Several crystal structures of MHC/HLA molecules are now available. We report a structural bioinformatics study of peptide- HLA complexes to derive features that generate recognition specificity, useful for guiding the design process.

We report here the 2.5Å structure of HIV-1 protease tethered-dimer ritonavir complex. The inhibitor bound in the active site has different conformations in the two orientations. There is only one hydrogen bond between the inhibitor and the enzyme. The conserved flap-water is not found in the present complex.

In the search for selectivity, the aspartic proteases are known to be a very difficult case because the enzymes of this family are not only sequentially but structurally also very similar. To gain insight into the selectivity and specificity of the aspartic proteases family we characterized the binding sites of four malarial aspartic protease (plasmepsin I, plasmepsin II, plasmepsin IV, P. vivax plasmepsin) and two human aspartic proteases (cathepsin D and pepsin) with the intention of identifying the regions that could be potential sites for obtaining selectivity using molecular interaction field approach.

Glutathione S-transferase (GST) proteins play vital role in living organism that includes detoxification of exogenous and endogenous chemicals, survivability during stress condition. This paper describes a method developed for predicting GST proteins. We have used a dataset of 107 GST and 107 non-GST proteins for training and the performance of the method was evaluated with five-fold cross-validation technique. First a SVM based method has been developed using amino acid and dipeptide composition and achieved the maximum accuracy of 91.59% and 95.79% respectively. In addition we developed a SVM based method using tripeptide composition and achieved maximum accuracy 97.66% which is better than accuracy achieved by HMM based searching (96.26%). Based on above study a web-server GSTPred has been developed (http://www.imtech.res.in/raghava/gstpred/).

It is important to understand the conformational biases that are present in unfolded states to understand protein folding. In this context, it is surprising that even a short tripeptide like AFA samples folded/ordered conformation as demonstrated recently by NMR experiments of the peptide in aqueous solution at 280 K. In this paper, we present molecular dynamics simulation of the peptide in explicit water using OPLS-AA/L all-atom force field. The results are in overall agreement with NMR results and provide some further insights. The peptide samples turn and extended conformational forms corresponding to minima in free energy landscape. Frequent transitions between the minima are observed due to modest free energy barriers. The turn conformation seems to be stabilized by hydrophobic interactions and possibly by bridging water molecules between backbone donors and acceptors. Thus the peptide does not sample conformations randomly, but samples well defined conformations. The peptide served as a model for folding-unfolding equilibrium in the context of peptide folding. Further, implications for drug design are also discussed.

Gastrin-releasing peptide (GRP) is a member of bombesin-like peptides and bombesin and neuromedin B are other members of this family. They act on receptors that belong to the GPCR superfamily and exert important physiological functions upon binding to their receptors. The biologically active C-terminal decapeptide of GRP (GRP10) was studied in explicit DMPC bilayers using molecular dynamics simulations. In the initial conformation, the peptide was placed perpendicular to the membrane plane and the peptide-membrane complex with ∼20,000 atoms was simulated for a period of 8 ns. After a 5 ns simulation, GRP10 adopted a tilted orientation and the tilt angle with respect to the bilayer normal was ∼60°. Analysis of the interactions of individual residues indicated the role of histidine residues in maintaining a tilted orientation.

The small Tims are chaperones that facilitate insertion of hydrophobic precursors into the inner mitochondrial membrane. We purified Tim12 and found it forms dimers that bind to Tim9. In this interaction, Tim12 undergoes structural changes that may be important for transport of its substrates in the mitochondrial carrier import pathway.

A dataset of experimentally characterized, human Golgi GlyTs with type II membrane topology was created. Based on the experimentally observed acceptor substrate preferences, the GlyTs were classified into five functional categories: biosynthesis of blood group antigens, glycolipids, N-glycans, O-glycans and glycosaminoglycans. The cytoplasmic, transmembrane and stem (CTS) regions were predicted and their length and composition were analyzed. The stem region of GlyTs involved in the biosynthesis of glycolipids and blood group antigens appear to have a shorter stem region compared to those GlyTs which participate in the biosynthesis of N- and O-linked glycans and glycosaminoglycans. The stem regions of all the GlyTs, irrespective of the functional category to which they belong, were found to be rich in disorder- promoting amino acid residues. Thus, the stem region is largely devoid of any regular secondary structure thereby facilitating its tethering role. A higher frequency of occurrence of basic amino acids is observed towards the N-terminus of the transmembrane domain and this is suggested to be important for topogenesis of these enzymes.

CD20, a B-cell-specific protein, is a primary target for immunotherapy of B-cell lymphomas. We used a mimotopes of CD20 to construct vaccines for B-cell-related disorders. The immunogenicity of the vaccines was tested in BALB/c mice. Results of this study suggest that the mimotope may be a useful tool for the construction of a functional vaccine to treat B cell-related disorders.

Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis, suggesting that loss of this factor may be involved in angiogenic eye disease. Here, we show that PEDF inhibits angiotensin II-induced smooth muscle cell proliferation through its anti-oxidative properties. Our present study suggests that PEDF may play a protective role against atherosclerosis.

S14G-humanin (S14G-HN) is one of the latest of a new family of neuropeptides with protective action against Alzheimer's disease insults. The structure of S14G-HN was studied with both spectroscopic techniques and molecular dynamics simulation. Secondary structure predictions and modeling of backbone conformation were carried out. Side chain reconstruction, homology modeling and molecular dynamics (MD) simulations were performed on four different models. A beta strand tendency in residues 5 to 10 and a propensity to adopt turn or irregular conformation in residues 13 to 17 was found. Circular dichroism experimental studies of S14G-HN in aquaeous solution and in different 2,2,2- trifluoroethanol (TFE) concentrations were also performed. In the absence of TFE and at low TFE concentrations, CD spectra are indicative of a small degree of ordering in the peptide. On further increment of TFE concentration, changes occur that indicate the formation of a structured conformation. Both experimental and computational results indicate that S14G-HN has a reduced helical propensity, in contrast with wild type humanin, as well as a higher conformational flexibility.