Sankaran Krishnaswamy

Functional exploration of the adult ovarian granulosa cell tumor-associated somatic FOXL2 mutation p.Cys134Trp (c.402C>G).

Background The somatic mutation in the FOXL2 gene c.402C>G (p.Cys134Trp) has recently been identified in the vast majority of adult ovarian granulosa cell tumors (OGCTs) studied. In addition, this mutation seems to be specific to adult OGCTs and is likely to be a driver of malignant transformation. However, its pathogenic mechanisms remain elusive. Methodology/Principal Findings We have sequenced the FOXL2 open reading frame in a panel of tumor cell lines (NCI-60, colorectal carcinoma cell lines, JEG-3, and KGN cells). We found the FOXL2 c.402C>G mutation in the adult OGCT-derived KGN cell line. All other cell lines analyzed were negative for the mutation. In order to gain insights into the pathogenic mechanism of the p.Cys134Trp mutation, the subcellular localization and mobility of the mutant protein were studied and found to be no different from those of the wild type (WT). Furthermore, its transactivation ability was in most cases similar to that of the WT protein, including in conditions of oxidative stress. A notable exception was an artificial promoter known to be coregulated by FOXL2 and Smad3, suggesting a potential modification of their interaction. We generated a 3D structural model of the p.Cys134Trp variant and our analysis suggests that homodimer formation might also be disturbed by the mutation. Conclusions/Significance Here, we confirm the specificity of the FOXL2 c.402C>G mutation in adult OGCTs and begin the exploration of its molecular significance. This is the first study demonstrating that the p.Cys134Trp mutant does not have a strong impact on FOXL2 localization, solubility, and transactivation abilities on a panel of proven target promoters, behaving neither as a dominant-negative nor as a loss-of-function mutation. Further studies are required to understand the specific molecular effects of this outstanding FOXL2 mutation.

HNH family subclassification leads to identification of commonality in the His‐Me endonuclease superfamily

The HNHc (SMART ID: SM00507) domain (SCOP nomenclature: HNH family) can be subclassified into at least eight subsets by iterative refinement of HMM profiles. An initial clustering of 323 proteins containing the HNHc domain helped identify the subsets. The subsets could be differentiated on the basis of the pattern of occurrence of seven defining features. Domain association is also different between the subsets. The subsets show organism as well as domain‐based clustering, suggestive of propagation by both duplication and horizontal transfer events. Structure‐based sequence analysis of the subsets led to the identification of common structural and sequence motifs in the HNH family with the other three families under the His‐Me endonuclease superfamily.

Identification of Prophages in Bacterial Genomes by Dinucleotide Relative Abundance Difference

Background Prophages are integrated viral forms in bacterial genomes that have been found to contribute to interstrain genetic variability. Many virulence-associated genes are reported to be prophage encoded. Present computational methods to detect prophages are either by identifying possible essential proteins such as integrases or by an extension of this technique, which involves identifying a region containing proteins similar to those occurring in prophages. These methods suffer due to the problem of low sequence similarity at the protein level, which suggests that a nucleotide based approach could be useful. Methodology Earlier dinucleotide relative abundance (DRA) have been used to identify regions, which deviate from the neighborhood areas, in genomes. We have used the difference in the dinucleotide relative abundance (DRAD) between the bacterial and prophage DNA to aid location of DNA stretches that could be of prophage origin in bacterial genomes. Prophage sequences which deviate from bacterial regions in their dinucleotide frequencies are detected by scanning bacterial genome sequences. The method was validated using a subset of genomes with prophage data from literature reports. A web interface for prophage scan based on this method is available at http://bicmku.in:8082/prophagedb/dra.html. Two hundred bacterial genomes which do not have annotated prophages have been scanned for prophage regions using this method. Conclusions The relative dinucleotide distribution difference helps detect prophage regions in genome sequences. The usefulness of this method is seen in the identification of 461 highly probable loci pertaining to prophages which have not been annotated so earlier. This work emphasizes the need to extend the efforts to detect and annotate prophage elements in genome sequences.

Differential functional effects of novel mutations of the transcription factor FOXL2 in BPES patients.

Mutations of the transcription factor FOXL2, involved in cranio‐facial and ovarian development lead to the Blepharophimosis‐Ptosis‐Epicanthus Inversus Syndrome (BPES) in human. Here, we describe nine mutations in the open reading frame of FOXL2. Six of them are novel: c.292T>A (p.Trp98Arg), c.323T>C (p.Leu108Pro), c.650C>G (p.Ser217Cys) and three frameshifts. We have performed localization and functional studies for three of them. We have observed a strong cytoplasmic mislocalization induced by the missense mutation p.Leu108Pro located in the forkhead (FKH) domain of FOXL2. In line with this, transcriptional activity assays confirmed the loss‐of‐function induced by this variant. Interestingly, the novel mutation p.Ser217Cys, mapping between the FKH and the polyalanine domain of FOXL2 and producing a mild eyelid phenotype, led to normal localization and transactivation. We have also modeled the structure of the FKH domain to explore the potential structural impact of the mutations reported here and other previously reported ones. This analysis shows that mutants can be sorted into two classes: those that potentially alter protein‐protein interactions and those that might disrupt the interactions with DNA. Our findings reveal new insights into the molecular effects of FOXL2 mutations, especially those affecting the FKH binding domain.

Database and Comparative Identification of Prophages

Prophages are integrated viral genomes in bacteria. Prophages are distinct from other genomic segments encoding virulence factors that have been acquired by horizontal gene transfer events. A database for prophages (http://bicmku.in:8082/prophagedb http://ispc.weizmann.ac.il/prophagedb) has been constructed with data available from literature reports. To date other than bacteriophage corner stone genes based iterative searches, no other exhaustive approach unique for identifying prophage elements is available. Here we report detection of prophages based on proteomic signature comparison using a prophage proteome as reference set. This method was tested with using the database and then extended over newly sequenced bacterial genomes with no reported prophages. The approach of using similarity of proteins over a given region helped identify twenty putative prophage regions in nine different bacterial genomes.

Homology Model of Surface Antigen OmpC From Salmonella typhi and its Functional Implications

Abstract Homology based 3D structural model of the immunodominant major surface antigen OmpC from Salmonella typhi, an obligatory human pathogen, was built to understand the possible unique conformational features of its antigenic loops with respect to other immunologically cross reacting porins. The homology model was built based on the known crystal structures of the E. coli porins OmpF and PhoE. Structure based sequence alignment helped to define the structurally conserved regions (SCRs). The SCR regions of OmpC were modelled using the coordinates of corresponding regions from reference proteins. Surface exposed variable regions were modelled based on the sequence similarity and loop search in PDB. Structural refinement based on symmetry restrained energy minimization resulted in an agreeable model for the trimer of OmpC. The resulting model was compared with other porin structures, having b-barrel fold with 16 transmembrane β-strands, and found that the variable regions are unique in terms of sequence and structure. A ranking of the loops taking into account the antigenic index, the sequence variability, the surface accessibility in the context of the trimer, and the structural variability suggests that loop 4 (151–172), loop 5 (194–218) and loop 6 (237–264) are the best ranked B-cell epitopes. The model provides possible explanations for the functional and unique immunological properties associated with the surface exposed regions and outlines the implications for structure based experimental design.

A Protein Similarity Approach For Detecting Prophage Regions In Bacterial Genomes

BackgroundNumerous completely sequenced bacterial genomes harbor prophage elements. These elements have been implicated in increasing the virulence of the host and in phage immunity. The e14 element is a defective lambdoid prophage element present at 25 min in the Escherichia coli K-12 genome. e14 is a well-characterized prophage element and has been subjected to in-depth bioinformatic analysis.ResultsA protein-based comparative approach using BLAST helped identify lambdoid-like prophage elements in a representative set of completely sequenced bacterial genomes. Twelve putative prophage regions were identified in six different bacterial genomes. Examination of the known and newly identified prophage regions suggests that on an average, the prophage elements do not seem to occur either randomly or in a uniform manner along the genome amongst genomes of the selected pathogenic organisms.ConclusionsThe protein based comparative approach can be effectively used to detect lambdoid-like prophage elements in bacterial genomes. It is possible that this method can beextended to all prophage elements and can be made automated.

Analysis of dihedral angle preferences for alanine and glycine residues in alpha and beta transmembrane regions

For the past 50 years, the Ramachandran map has been used effectively to study the protein structure and folding. However, though extensive analysis has been done on dihedral angle preferences of residues in globular proteins, related studies and reports of membrane proteins are limited. It is of interest to explore the conformational preferences of residues in transmembrane regions of membrane proteins which are involved in several important and diverse biological processes. Hence, in the present work, a systematic comparative computational analysis has been made on dihedral angle preferences of alanine and glycine in alpha and beta transmembrane regions (the two major classes of transmembrane proteins) with the aid of the Ramachandran map. Further, the conformational preferences of residues in transmembrane regions were compared with the non-transmembrane regions. We have extracted cation-pi interacting residues present in transmembrane regions and explored the dihedral angle preferences. From our observations, we reveal the higher percentage of occurrences of glycine in alpha and beta transmembrane regions than other hydrophobic residues. Further, we noted a clear shift in ψ-angle preferences of glycine residues from negative bins in alpha transmembrane regions to positive bins in beta transmembrane regions. Also, cation-pi interacting residues in beta transmembrane regions avoid preferring ψ-angles in the range of −59° to −30°. In this article, we insist that the studies on preferences of dihedral angles in transmembrane regions, thorough understanding of structure and folding of transmembrane proteins, can lead to modeling of novel transmembrane regions towards designing membrane proteins.

Expression, purification and molecular modeling of the NIa protease of Cardamom mosaic virus

The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8 M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease.

Functional fragments of disorder in outer membrane β barrel proteins

The traditional view of “sequence–structure–function” has been amended by the discovery of intrinsically disordered proteins. Almost 50% of PDB structures are now known to have one or more regions of disorder, which are involved in diverse functions. These regions typically possess low aromatic content and sequence complexity as well as high net charge and flexibility. In this study, we examined the composition and contribution of intrinsic disorder in outer membrane β barrel protein functions. Our systematic analysis to find the dual personality (DP) fragments, which often function by disorder–order transitions, revealed the presence of 61 DP fragments with 234 residues in β barrel trans membrane protein structures. It was found that though the disorder is more prevalent in the periplasmic regions, most of the residues which undergo disorder–order transitions are found in the extracellular regions. For example, the calcium binding sites in BtuB protein are found to undergo disorder to order transition upon binding calcium. The conformational change in the cell receptor binding site of the OpcA protein, which is important in host cell interactions of N. meningitidis, was also found to be due to the disorder–order transitions occurring in the presence of the ligand. The natively disordered nature of DP fragments makes it more appropriate to call them “functional fragments of disorder.” The present study provides insight into the roles played by intrinsically disordered regions in outer membrane protein functions.