C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C

Phosphoinositide-specific phospholipase C (PLC) is involved in Ca2+ mediated signalling events that lead to altered cellular status. Using various sequence-analysis methods, we identified two conserved motifs in known PLC sequences. The identified motifs are located in the C2 domain of plant PLCs and are not found in any other protein. These motifs are specifically found in the Ca2+ binding loops and form adjoining beta strands. Further, we identified certain conserved residues that are highly distinct from corresponding residues of animal PLCs. The motifs reported here could be used to annotate plant-specific phospholipase C sequences. Furthermore, we demonstrated that the C2 domain alone is capable of targeting PLC to the membrane in response to a Ca2+ signal. We also showed that the binding event results from a change in the hydrophobicity of the C2 domain upon Ca2+ binding. Bioinformatic analyses revealed that all PLCs from Arabidopsis and rice lack a transmembrane domain, myristoylation and GPI-anchor protein modifications. Our bioinformatic study indicates that plant PLCs are located in the cytoplasm, the nucleus and the mitochondria. Our results suggest that there are no distinct isoforms of plant PLCs, as have been proposed to exist in the soluble and membrane associated fractions. The same isoform could potentially be present in both subcellular fractions, depending on the calcium level of the cytosol. Overall, these data suggest that the C2 domain of PLC plays a vital role in calcium signalling.

Sequence-Structure Similarity: Do Sequentially Identical Peptide Fragments have Similar Three-Dimensional Structures?

The rapidly growing structure databases enhance the probability of finding identical sequences sharing structural similarity. Structure prediction methods are being used extensively to abridge the gap between known protein sequences and the solved structures which is essential to understand its specific biochemical and cellular functions. In this work, we plan to study the ambiguity between sequence-structure relationships and examine if sequentially identical peptide fragments adopt similar three-dimensional structures. Fragments of varying lengths (five to ten residues) were used to observe the behavior of sequence and its three-dimensional structures. The STAMP program was used to superpose the three-dimensional structures and the two parameters (Sequence Structure Similarity Score (Sc) and Root Mean Square Deviation value) were employed to classify them into three categories: similar, intermediate and dissimilar structures. Furthermore, the same approach was carried out on all the three-dimensional protein structures solved in the two organisms, Mycobacterium tuberculosis and Plasmodium falciparum to validate our results.

Repertoire of Protein Kinases Encoded in the Genome of Takifugu rubripes

Takifugu rubripes is teleost fish widely used in comparative genomics to understand the human system better due to its similarities both in number of genes and structure of genes. In this work we survey the fugu genome, and, using sensitive computational approaches, we identify the repertoire of putative protein kinases and classify them into groups and subfamilies. The fugu genome encodes 519 protein kinase-like sequences and this number of putative protein kinases is comparable closely to that of human. However, in spite of its similarities to human kinases at the group level, there are differences at the subfamily level as noted in the case of KIS and DYRK subfamilies which contribute to differences which are specific to the adaptation of the organism. Also, certain unique domain combination of galectin domain and YkA domain suggests alternate mechanisms for immune response and binding to lipoproteins. Lastly, an overall similarity with the MAPK pathway of humans suggests its importance to understand signaling mechanisms in humans. Overall the fugu serves as a good model organism to understand roles of human kinases as far as kinases such as LRRK and IRAK and their associated pathways are concerned.

Cascaded walks in protein sequence space: use of artificial sequences in remote homology detection between natural proteins

Over the past two decades, many ingenious efforts have been made in protein remote homology detection. Because homologous proteins often diversify extensively in sequence, it is challenging to demonstrate such relatedness through entirely sequence-driven searches. Here, we describe a computational method for the generation of `protein-like' sequences that serves to bridge gaps in protein sequence space. Sequence profile information, as embodied in a position-specific scoring matrix of multiply aligned sequences of bona fide family members, serves as the starting point in this algorithm. The observed amino acid propensity and the selection of a random number dictate the selection of a residue for each position in the sequence. In a systematic manner, and by applying a `roulette-wheel' selection approach at each position, we generate parent family-like sequences and thus facilitate an enlargement of sequence space around the family. When generated for a large number of families, we demonstrate that they expand the utility of natural intermediately related sequences in linking distant proteins. In 91% of the assessed examples, inclusion of designed sequences improved fold coverage by 5-10% over searches made in their absence. Furthermore, with several examples from proteins adopting folds such as TIM, globin, lipocalin and others, we demonstrate that the success of including designed sequences in a database positively sensitized methods such as PSI-BLAST and Cascade PSI-BLAST and is a promising opportunity for enormously improved remote homology recognition using sequence information alone.

Sequence and structural basis for chromosomal fragility during translocations in cancer

Chromosomal aberration is considered to be one of the major characteristic features in many cancers. Chromosomal translocation, one type of genomic abnormality, can lead to deregulation of critical genes involved in regulating important physiological functions such as cell proliferation and DNA repair. Although chromosomal translocations were thought to be random events, recent findings suggest that certain regions in the human genome are more susceptible to breakage than others. The possibility of deviation from the usual B-DNA conformation in such fragile regions has been an active area of investigation. This review summarizes the factors that contribute towards the fragility of these regions in the chromosomes, such as DNA sequences and the role of different forms of DNA structures. Proteins responsible for chromosomal fragility, and their mechanism of action are also discussed. The effect of positioning of chromosomes within the nucleus favoring chromosomal translocations and the role of repair mechanisms are also addressed.

Suite of tools for statistical N-gram language modeling for pattern mining in whole genome sequences

Genome sequences contain a number of patterns that have biomedical significance. Repetitive sequences of various kinds are a primary component of most of the genomic sequence patterns. We extended the suffix-array based Biological Language Modeling Toolkit to compute n-gram frequencies as well as n-gram language-model based perplexity in windows over the whole genome sequence to find biologically relevant patterns. We present the suite of tools and their application for analysis on whole human genome sequence.

Genome sequencing unveils a novel sea enterotoxin-carrying PVL phage in staphylococcus aureus ST772 from India

Staphylococcus aureus is a major human pathogen, first recognized as a leading cause of hospital-acquired infections. Community-associated S. aureus (CA-SA) pose a greater threat due to increase in severity of infection and disease among children and healthy adults. CA-SA strains in India are genetically diverse, among which is the sequence type (ST) 772, which has now spread to Australia, Europe and Japan. Towards understanding the genetic characteristics of ST772, we obtained draft genome sequences of five relevant clinical isolates and studied the properties of their PVL-carrying prophages, whose presence is a defining hallmark of CA-SA. We show that this is a novel prophage, which carries the structural genes of the hlb-carrying prophage and includes the sea enterotoxin. This architecture probably emerged early within the ST772 lineage, at least in India. The sea gene, unique to ST772 PVL, despite having promoter sequence characteristics typical of low expression, appears to be highly expressed during early phase of growth in laboratory conditions. We speculate that this might be a consequence of its novel sequence context. The crippled nature of the hlb-converting prophage in ST772. suggests that widespread mobility of the sea enterotoxin might be a selective force behind its `transfer' to the PVL prophage. Wild type ST772 strains induced strong proliferative responses as well as high cytotoxic activity against neutrophils, likely mediated by superantigen SEA and the PVL toxin respectively. Both proliferation and cytotoxicity were markedly reduced in a cured ST772 strain indicating the impact of the phage on virulence. The presence of SEA alongside he genes for the immune system-modulating PVL toxin may contribute to the success and virulence of ST772.

Identification of an exported heat shock protein 70 in Plasmodium falciparum

Host cell remodelling is a hallmark of malaria pathogenesis. It involves protein folding, unfolding and trafficking events and thus participation of chaperones such as Hsp70s and Hsp40s is well speculated. Until recently, only Hsp40s were thought to be the sole representative of the parasite chaperones in the exportome. However, based on the re-annotated Plasmodium falciparum genome sequence, a putative candidate for exported Hsp70 has been reported, which otherwise was known to be a pseudogene. We raised a specific antiserum against a C-terminal peptide uniquely present in PfHsp70-x. Immunoblotting and immunofluorescence-based approaches in combination with sub-cellular fractionation by saponin and streptolysin-O have been taken to determine the expression and localization of PfHsp70-x in infected erythrocyte. The re-annotated sequence of PfHsp70-x reveals it to be a functional protein with an endoplasmic reticulum signal peptide. It gets maximally expressed at the schizont stage of intra-erythrocytic life cycle. Majority of the protein localizes to the parasitophorous vacuole and some of it gets exported to the erythrocyte compartment where it associates with Maurer's clefts. The identification of an exported parasite Hsp70 chaperone presents us with the fact that the parasite has evolved customized chaperones which might be playing crucial roles in aspects of trafficking and host cell remodelling.

Co-evolution of specific amino acid in sigma 1.2 region and nucleotide base in the discriminator to act as sensors of small molecule effectors of transcription initiation in mycobacteria

The transcription from rrn and a number of other promoters is regulated by initiating ribonucleotides (iNTPs) and guanosine tetra/penta phosphate (p)ppGpp], either by strengthening or by weakening of the RNA polymerase (RNAP)-promoter interactions during initiation. Studies in Escherichia coli revealed the importance of a sequence termed discriminator, located between -10 and the transcription start site of the responsive promoters in this mode of regulation. Instability of the open complex at these promoters is attributed to the lack of stabilizing interactions between the suboptimal discriminator and the 1.2 region of sigma 70 (Sig70) in RNAP holoenzyme. We demonstrate a different pattern of interaction between the promoters and sigma A (SigA) of Mycobacterium tuberculosis to execute similar regulation. Instead of cytosine and methionine, thymine at three nucleotides downstream to -10 element and leucine 232 in SigA are found to be essential for iNTPs and pppGpp mediated response at the rrn and gyr promoters of the organism. The specificity of the interaction is substantiated by mutational replacements, either in the discriminator or in SigA, which abolish the nucleotide mediated regulation in vitro or in vivo. Specific yet distinct bases and the amino acids appear to have co-evolved' to retain the discriminator-sigma 1.2 region regulatory switch operated by iNTPs/pppGpp during the transcription initiation in different bacteria.

Type III restriction-modification enzymes: a historical perspective

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction-modification (R-M) systems are classified into four groups. Type III R-M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25-27 bp downstream of one of the recognition sites). Like the Type I R-M enzymes, Type III R-M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R-M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R-M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis.

RNA-dependent RNA polymerase of Japanese encephalitis virus binds the initiator nucleotide GTP to form a mechanistically important pre-initiation state

Flaviviral RNA-dependent RNA polymerases (RdRps) initiate replication of the single-stranded RNA genome in the absence of a primer. The template sequence 5'-CU-3' at the 3'-end of the flaviviral genome is highly conserved. Surprisingly, flaviviral RdRps require high concentrations of the second incoming nucleotide GTP to catalyze de novo template-dependent RNA synthesis. We show that GTP stimulates de novo RNA synthesis by RdRp from Japanese encephalitis virus (jRdRp) also. Crystal structures of jRdRp complexed with GTP and ATP provide a basis for specific recognition of GTP. Comparison of the jRdRp(GTP) structure with other viral RdRp-GTP structures shows that GTP binds jRdRp in a novel conformation. Apo-jRdRp structure suggests that the conserved motif F of jRdRp occupies multiple conformations in absence of GTP. Motif F becomes ordered on GTP binding and occludes the nucleotide triphosphate entry tunnel. Mutational analysis of key residues that interact with GTP evinces that the jRdRp(GTP) structure represents a novel pre-initiation state. Also, binding studies show that GTP binding reduces affinity of RdRp for RNA, but the presence of the catalytic Mn2+ ion abolishes this inhibition. Collectively, these observations suggest that the observed pre-initiation state may serve as a check-point to prevent erroneous template-independent RNA synthesis by jRdRp during initiation.

Analysis of the Genome and Transcriptome of Cryptococcus neoformans var. grubii Reveals Complex RNA Expression and Microevolution Leading to Virulence Attenuation

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

Computational framework to integrate the effect of antigen recognition on disease epidemiology outcome: multi-scale approach

Human Leukocyte Antigen (HLA) plays an important role, in presenting foreign pathogens to our immune system, there by eliciting early immune responses. HLA genes are highly polymorphic, giving rise to diverse antigen presentation capability. An important factor contributing to enormous variations in individual responses to diseases is differences in their HLA profiles. The heterogeneity in allele specific disease responses decides the overall disease epidemiological outcome. Here we propose an agent based computational framework, capable of incorporating allele specific information, to analyze disease epidemiology. This framework assumes a SIR model to estimate average disease transmission and recovery rate. Using epitope prediction tool, it performs sequence based epitope detection for a given the pathogenic genome and derives an allele specific disease susceptibility index depending on the epitope detection efficiency. The allele specific disease transmission rate, that follows, is then fed to the agent based epidemiology model, to analyze the disease outcome. The methodology presented here has a potential use in understanding how a disease spreads and effective measures to control the disease.

Mycobacterium tuberculosis DinG Is a Structure-specific Helicase That Unwinds G4 DNA IMPLICATIONS FOR TARGETING G4 DNA AS A NOVEL THERAPEUTIC APPROACH

The significance of G-quadruplexes and the helicases that resolve G4 structures in prokaryotes is poorly understood. The Mycobacterium tuberculosis genome is GC-rich and contains >10,000 sequences that have the potential to form G4 structures. In Escherichia coli, RecQ helicase unwinds G4 structures. However, RecQ is absent in M. tuberculosis, and the helicase that participates in G4 resolution in M. tuberculosis is obscure. Here, we show that M. tuberculosis DinG (MtDinG) exhibits high affinity for ssDNA and ssDNA translocation with a 5' -> 3' polarity. Interestingly, MtDinG unwinds overhangs, flap structures, and forked duplexes but fails to unwind linear duplex DNA. Our data with DNase I footprinting provide mechanistic insights and suggest that MtDinG is a 5' -> 3' polarity helicase. Notably, in contrast to E. coli DinG, MtDinG catalyzes unwinding of replication fork and Holliday junction structures. Strikingly, we find that MtDinG resolves intermolecular G4 structures. These data suggest that MtDinG is a multifunctional structure-specific helicase that unwinds model structures of DNA replication, repair, and recombination as well as G4 structures. We finally demonstrate that promoter sequences of M. tuberculosis PE_PGRS2, mce1R, and moeB1 genes contain G4 structures, implying that G4 structures may regulate gene expression in M. tuberculosis. We discuss these data and implicate targeting G4 structures and DinG helicase in M. tuberculosis could be a novel therapeutic strategy for culminating the infection with this pathogen.

Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction

Background: mIHF belongs to a subfamily of proteins, distinct from E. coli IHF. Results: Functionally important amino acids of mIHF and the mechanism(s) underlying DNA binding, DNA bending, and site-specific recombination are distinct from that of E. coli IHF. Conclusion: mIHF functions could contribute beyond nucleoid compaction. Significance: Because mIHF is essential for growth, the molecular mechanisms identified here can be exploited in drug screening efforts. The annotated whole-genome sequence of Mycobacterium tuberculosis revealed that Rv1388 (Mtihf) is likely to encode for a putative 20-kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or the organization of the mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg-170, Arg-171, and Arg-173, which might be involved in DNA binding, and a conserved proline (Pro-150) in the tight turn. The phenotypic sensitivity of Escherichia coli ihfA and ihfB strains to UV and methyl methanesulfonate could be complemented with the wild-type Mtihf but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, binds with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHF. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes DNA compaction into nucleoid-like or higher order filamentous structures. We therefore propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins.