Eswari Pandaranayaka | Madurai Kamaraj University, Madurai, Tamil Nadu, INDIA (original) (raw)

Papers by Eswari Pandaranayaka

Journal of biomolecular structure & dynamics, Jan 18, 2015

Expression of Cardamom mosaic virus (CdMV) coat protein (CP) in E. coli forms virus-like particle... more Expression of Cardamom mosaic virus (CdMV) coat protein (CP) in E. coli forms virus-like particles. In this study, the structure of CdMV CP was predicted and used as a platform to display epitopes of the most abundant surface-associated protein, LipL32 of Leptospira at C, N, and both the termini of CdMV CP. In silico, we have mapped sequential and conformational B-cell epitopes from the crystal structure of LipL32 of Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130 using IEDB Elipro, ABCpred, BCPRED, and VaxiJen servers. Our results show that the epitopes displayed at the N-terminus of CdMV CP are promising vaccine candidates as compared to those displayed at the C-terminus or at both the termini. LipL32 epitopes, EP2, EP3, EP4, and EP6 are found to be promising B-cell epitopes for vaccine development. Based on the type of amino acids, length, surface accessibility, and docking energy with CdMV CP model, the order of antigenicity of the LipL32 epitopes was found to be ...

C3 complement protein of cobra is known to have various essential roles in invitro and in vivo st... more C3 complement protein of cobra is known to have various essential roles in invitro and in vivo studies. The third complement protein plays an important role in the complement system by mediating various biological functions to amplify complement response and stimulate the adaptive immune response. Evolutionary tree for the third complement protein of the complement system infers, C3 an ancient molecule known to evolve with an ordered fashion from lower to higher vertebrates. In the absence of crystal structure for cobra C3 protein, homology modeling was constructed (CoC3). Furthermore a comparative modeling of complement C3 protein of cobra (CoC3), human and bovine deduced, that the cascade of complement activation in cobra occur due to the presence of the eight residues (D726-D727 and D720-D721 in strand, Y698-I699 and D704-E705 present in the helix). Whereas, in human (A734Q-A735Q and A728Y-A729I) and bovine (B704D-B705C and B711D-B710) only four residues are involved in binding of (Factor B) FB, a component of the alternative pathway of the complement system and bringing about complement system activation. Therefore it is hypothesized that bovine and human C3 proteins which are evolutionarily related to CoC3 protein of cobra would have lost the remaining residues involved in complement activation during the due course of evolution. The eight residues involved in the binding of FB in cobra may have some other better role in complement activation and if it is so, this property of CoC3 can be experimentally used for complement activation process.

Human Mutation, 2008

Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development le... more 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.

Purpose: Mutations in the myocilin gene (MYOC) leading to a perturbed outflow of aqueous in the t... more Purpose: Mutations in the myocilin gene (MYOC) leading to a perturbed outflow of aqueous in the trabecular meshwork (TM) has been associated with the pathophysiology of glaucoma. This study examines the expression of normal and mutant myocilin (Gly367Arg) in cultured TM cells. Methods: Normal and mutant MYOC cDNA constructs were used to transfect the TM cells. In order to confirm the method of transfection, reverse trancriptase polymerase chain reaction (RT-PCR) was carried out. Further, confocal microscopic analysis was used to determine the cellular localization of myocilin protein. The extracellular nature of myocilin in the culture supernatant and cell lysates of the transfected cells was analyzed by western blot. Molecular modeling was done earlier using a knowledge based consensus method which involved threading the protein into the identified pentein fold for the COOH-terminal part. Molecular dynamics was carried out using GROMACS for the mutant model which was built using the native myocilin model. Results: The Gly367Arg mutation causes accumulation of myocilin protein within TM cells with extensively reduced secretion contrary to wild type myocilin being characterized by intracellular localization and extracellular secretion. Further, Gly367Arg mutation occurs in a hydrophobic region which leads to burial of a charged residue. The dynamics suggests large conformational change is required to accommodate the mutation favoring aggregation of the protein.

Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development, l... more Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development, lead to the Blepharophimosis Syndrome. Here, we have systematically replaced the amino acids of the helices of the forkhead domain (FHD) of FOXL2 by glycine residues to assess the impact of such substitutions. A number of mutations lead to protein mislocalization, aggregation and to partial or complete loss of trans-activation ability on a series of luciferase reporter systems. To rationalize the results of this glycine mutation scan, we have modeled the structure of the FHD by comparison with crystallographic data available for other FHDs. We failed to detect a clear-cut correlation between protein mislocalization or aggregation and the position of the mutation. However, we found that the localization of the side chain of each amino acid strongly correlated with the impact of its mutation on FOXL2 transactivation capacity. Indeed, when the side chains of the amino acids involved in the helices of the forkhead are supposed to point towards the hydrophobic core formed by the three main helices, a loss of function was observed. On the contrary, if the side chains point outward the hydrophobic core, protein function was preserved. The extension of this analysis to natural mutants shows that a similar correlation can be found for BPES mutations associated or not with ovarian dysfunction. Our findings reveal new insights into the molecular effects of FOXL2 mutations affecting the FHD, which represent two-thirds of intragenic mutations, and provide the first predictive tool of their effects.

Purpose: To examine the possible role of alternate splicing leading to aggregation of myocilin in... more Purpose: To examine the possible role of alternate splicing leading to aggregation of myocilin in primary open-angle glaucoma. Methods: Several single nucleotide variations found in the myocilin (MYOC) genomic region were collected and examined for their possible role in causing splice-site alterations. A model for myocilin built using a knowledge-based consensus method was used to map the altered protein products. A total of 150 open-angle glaucoma patients and 50 normal age-matched control subjects were screened for the predicted polymorphisms, and clustering was performed. Results: A total of 124 genomic variations were screened, and six polymorphisms that lead to altered protein products were detected as possible candidates for the alternative splicing mechanism. Five of these lay in the intronic regions, and the one that lay in the exon region corresponded to the previously identified polymorphism (Tyr347Tyr) implicated in primary open-angle glaucoma. Experimentally screening the intronic region of the MYOC gene showed the presence of the predicted g.14072G>A polymorphism, g.1293C/T heterozygous polymorphism, instead of our predicted g.1293C/-polymorphism. Other than the prediction, two novel SNPs (g.1295G>T and g.1299T>G) and two reported SNPs (g. 1284G>T and g.1286G>T) were also identified. Cluster analysis showed the g.14072G>A homozygous condition was more common in this cohort than the heterozygous condition. Conclusions: We previously proposed that the disruption of dimer or oligomer formation by the C-term region allows greater chances of nucleation for aggregation. Here we suggest that polymorphisms in the myocilin genomic region that cause synonymous codon changes or those that occur in the intron regions can possibly lead to altered myocilin protein products through altered intron–exon splicing.

Journal of Ocular Biology, Diseases, and Informatics, 2011

Molecular vision

To examine the possible role of alternate splicing leading to aggregation of myocilin in primary ... more To examine the possible role of alternate splicing leading to aggregation of myocilin in primary open-angle glaucoma. Several single nucleotide variations found in the myocilin (MYOC) genomic region were collected and examined for their possible role in causing splice-site alterations. A model for myocilin built using a knowledge-based consensus method was used to map the altered protein products. A total of 150 open-angle glaucoma patients and 50 normal age-matched control subjects were screened for the predicted polymorphisms, and clustering was performed. A total of 124 genomic variations were screened, and six polymorphisms that lead to altered protein products were detected as possible candidates for the alternative splicing mechanism. Five of these lay in the intronic regions, and the one that lay in the exon region corresponded to the previously identified polymorphism (Tyr347Tyr) implicated in primary open-angle glaucoma. Experimentally screening the intronic region of the M...

A growing number of diseases seem to be associated with protein aggregation and each disease has ... more A growing number of diseases seem to be associated with protein aggregation and each disease has several proteins involved in it. To obtain a better understanding of the diseases, the proteins involved in it and their primary interaction partners were collected and clustered. A tool is developed to aid in clustering these proteins and all their primary interactors. The tools is used to cluster proteins involved in Primary Open Angle Glaucoma and their primary interactors. A cluster was selected for analysis based on the availability of experimental analysis in literature. The localization of the protein in the chosen cluster was collected. On analyzing four of the proteins in the cluster was found to be heparin binding. Primary open angle glaucoma is known to be associated with loss of retinal vasculature. The tool has helped in finding a cluster of proteins interaction with more experimental data. Also it has helped in finding out the 4 protein associated with the disease that involved in heparin binding among 10500 proteins. This would not have been possible to do manually. Further studying the role of these four proteins based on heparin binding and loss of vasculature in primary open angle glaucoma would give a better understanding of the disease and the molecular mechanism involved in it.

Bioinformation, Jun 30, 2014

ProADD, a database for protein aggregation diseases, is developed to organize the data under a si... more ProADD, a database for protein aggregation diseases, is developed to organize the data under a single platform to facilitate easy access for researchers. Diseases caused due to protein aggregation and the proteins involved in each of these diseases are integrated. The database helps in classification of proteins involved in the protein aggregation diseases based on sequence and structural analysis. Analysis of proteins can be done to mine patterns prevailing among the aggregating proteins.

Journal of Biomolecular Structure & Dynamics, 2012

The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins... more 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.

Purpose: Mutations in the myocilin gene (MYOC) leading to a perturbed outflow of aqueous in the t... more Purpose: Mutations in the myocilin gene (MYOC) leading to a perturbed outflow of aqueous in the trabecular meshwork (TM) has been associated with the pathophysiology of glaucoma. This study examines the expression of normal and mutant myocilin (Gly367Arg) in cultured TM cells. Methods: Normal and mutant MYOC cDNA constructs were used to transfect the TM cells. In order to confirm the method of transfection, reverse trancriptase polymerase chain reaction (RT-PCR) was carried out. Further, confocal microscopic analysis was used to determine the cellular localization of myocilin protein. The extracellular nature of myocilin in the culture supernatant and cell lysates of the transfected cells was analyzed by western blot. Molecular modeling was done earlier using a knowledge based consensus method which involved threading the protein into the identified pentein fold for the COOH-terminal part. Molecular dynamics was carried out using GROMACS for the mutant model which was built using the native myocilin model. Results: The Gly367Arg mutation causes accumulation of myocilin protein within TM cells with extensively reduced secretion contrary to wild type myocilin being characterized by intracellular localization and extracellular secretion. Further, Gly367Arg mutation occurs in a hydrophobic region which leads to burial of a charged residue. The dynamics suggests large conformational change is required to accommodate the mutation favoring aggregation of the protein.

Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development, l... more Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development, lead to the Blepharophimosis Syndrome. Here, we have systematically replaced the amino acids of the helices of the forkhead domain (FHD) of FOXL2 by glycine residues to assess the impact of such substitutions. A number of mutations lead to protein mislocalization, aggregation and to partial or complete loss of transactivation ability on a series of luciferase reporter systems. To rationalize the results of this glycine mutation scan, we have modeled the structure of the FHD by comparison with crystallographic data available for other FHDs. We failed to detect a clear-cut correlation between protein mislocalization or aggregation and the position of the mutation. However, we found that the localization of the side chain of each amino acid strongly correlated with the impact of its mutation on FOXL2 transactivation capacity. Indeed, when the side chains of the amino acids involved in the helices of the forkhead are supposed to point towards the hydrophobic core formed by the three main helices, a loss of function was observed. On the contrary, if the side chains point outward the hydrophobic core, protein function was preserved. The extension of this analysis to natural mutants shows that a similar correlation can be found for BPES mutations associated or not with ovarian dysfunction. Our findings reveal new insights into the molecular effects of FOXL2 mutations affecting the FHD, which represent two-thirds of intragenic mutations, and provide the first predictive tool of their effects.

C3 complement protein of cobra is known to have various essential roles in invitro and in vivo st... more C3 complement protein of cobra is known to have various essential roles in invitro and in vivo studies. The third complement protein plays an important role in the complement system by mediating various biological functions to amplify complement response and stimulate the adaptive immune response. Evolutionary tree for the third complement protein of the complement system infers, C3 an ancient molecule known to evolve with an ordered fashion from lower to higher vertebrates. In the absence of crystal structure for cobra C3 protein, homology modeling was constructed (CoC3). Furthermore a comparative modeling of complement C3 protein of cobra (CoC3), human and bovine deduced, that the cascade of complement activation in cobra occur due to the presence of the eight residues (D726-D727 and D720-D721 in strand, Y698-I699 and D704-E705 present in the helix ). Whereas, in human (A734Q-A735Q and A728Y-A729I) and bovine (B704D-B705C and B711D-B710) only four residues are involved in binding of (Factor B) FB, a component of the alternative pathway of the complement system and bringing about complement system activation. Therefore it is hypothesized that bovine and human C3 proteins which are evolutionarily related to CoC3 protein of cobra would have lost the remaining residues involved in complement activation during the due course of evolution. The eight residues involved in the binding of FB in cobra may have some other better role in complement activation and if it is so, this property of CoC3 can be experimentally used for complement activation process.

Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development le... more 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.

Indian journal of …, Jan 1, 2004

Mol Vis, Jan 1, 2003

To screen for mutations in the MYOC gene of patients with Primary Open Angle Glaucoma (POAG) in I... more To screen for mutations in the MYOC gene of patients with Primary Open Angle Glaucoma (POAG) in India and to better understand the mutations using a possible model of myocilin. Methods: We analyzed DNA for mutations in 107 subjects with POAG and 90 normal control subjects. The exonic sequences of the MYOC gene from all subjects were amplified by Polymerase Chain Reaction (PCR). We carried out Single Strand Conformation Polymorphism (SSCP) for all the PCR products. The DNA samples which showed mobility shift in the banding pattern in SSCP gel were sequenced. We also analyzed the presence of the common mutation Gln368Stop using a specific restriction enzyme Taa 1. The mutations observed here and elsewhere have been mapped onto a possible model built for myocilin using a knowledge-based consensus modeling approach. Results: Two heterozygous mutations Gly367Arg (1099G>A) and Thr377Met (1130C>T) were identified in exon3 of the MYOC gene of probands 40-1 and 51-1 respectively, from material obtained from the 107 unrelated subjects with POAG. These two mutations were not present in the normal controls studied. We identified a Single Nucleotide Polymorphism (SNP) Gly122Gly (366C>T) in exon1 of proband 57-1 as a non-disease causing variation. The common mutation Gln368Stop found in the Western population was not observed in the POAG cases screened in Indian population. The possible structural model for myocilin suggests a predominantly β-strand rich C-terminal region (181-504) which is connected by the α-helical mid-region (111-180) to the N-terminal region (34-110) which has low secondary structure content. Both the mutations, Gly367Arg and Thr377Met identified in our study, map on to the C-terminal region. These mutations disfavor burial of this region during oligomer formation due to the charged or bulky nature of the mutants. Most of the other mutations known for myocilin also are surface exposed on the C-terminal region. Conclusions: Our findings indicate that the mutation frequency of the MYOC gene is 2% in the Indian population affected with POAG, which is not a well-studied ethnic group of the Asian continent. The variations identified in our study have been previously reported in the Western population. The nonsense mutation Gln368Stop was not observed in the present study and thereby suggests that it may not be a common disease-causing mutation in the Indian population. Amongst other Asian populations, studies in Japan also didn't report this nonsense mutation. The location of these mutations suggest that a plausible mode of action could be by disruption of dimer or oligomer formation by the C-terminal region allowing greater chances of nucleation of aggregation by the N-terminal region.

Journal of biomolecular structure & dynamics, Jan 18, 2015

Expression of Cardamom mosaic virus (CdMV) coat protein (CP) in E. coli forms virus-like particle... more Expression of Cardamom mosaic virus (CdMV) coat protein (CP) in E. coli forms virus-like particles. In this study, the structure of CdMV CP was predicted and used as a platform to display epitopes of the most abundant surface-associated protein, LipL32 of Leptospira at C, N, and both the termini of CdMV CP. In silico, we have mapped sequential and conformational B-cell epitopes from the crystal structure of LipL32 of Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130 using IEDB Elipro, ABCpred, BCPRED, and VaxiJen servers. Our results show that the epitopes displayed at the N-terminus of CdMV CP are promising vaccine candidates as compared to those displayed at the C-terminus or at both the termini. LipL32 epitopes, EP2, EP3, EP4, and EP6 are found to be promising B-cell epitopes for vaccine development. Based on the type of amino acids, length, surface accessibility, and docking energy with CdMV CP model, the order of antigenicity of the LipL32 epitopes was found to be ...

C3 complement protein of cobra is known to have various essential roles in invitro and in vivo st... more C3 complement protein of cobra is known to have various essential roles in invitro and in vivo studies. The third complement protein plays an important role in the complement system by mediating various biological functions to amplify complement response and stimulate the adaptive immune response. Evolutionary tree for the third complement protein of the complement system infers, C3 an ancient molecule known to evolve with an ordered fashion from lower to higher vertebrates. In the absence of crystal structure for cobra C3 protein, homology modeling was constructed (CoC3). Furthermore a comparative modeling of complement C3 protein of cobra (CoC3), human and bovine deduced, that the cascade of complement activation in cobra occur due to the presence of the eight residues (D726-D727 and D720-D721 in strand, Y698-I699 and D704-E705 present in the helix). Whereas, in human (A734Q-A735Q and A728Y-A729I) and bovine (B704D-B705C and B711D-B710) only four residues are involved in binding of (Factor B) FB, a component of the alternative pathway of the complement system and bringing about complement system activation. Therefore it is hypothesized that bovine and human C3 proteins which are evolutionarily related to CoC3 protein of cobra would have lost the remaining residues involved in complement activation during the due course of evolution. The eight residues involved in the binding of FB in cobra may have some other better role in complement activation and if it is so, this property of CoC3 can be experimentally used for complement activation process.

Human Mutation, 2008

Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development le... more 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.

Purpose: Mutations in the myocilin gene (MYOC) leading to a perturbed outflow of aqueous in the t... more Purpose: Mutations in the myocilin gene (MYOC) leading to a perturbed outflow of aqueous in the trabecular meshwork (TM) has been associated with the pathophysiology of glaucoma. This study examines the expression of normal and mutant myocilin (Gly367Arg) in cultured TM cells. Methods: Normal and mutant MYOC cDNA constructs were used to transfect the TM cells. In order to confirm the method of transfection, reverse trancriptase polymerase chain reaction (RT-PCR) was carried out. Further, confocal microscopic analysis was used to determine the cellular localization of myocilin protein. The extracellular nature of myocilin in the culture supernatant and cell lysates of the transfected cells was analyzed by western blot. Molecular modeling was done earlier using a knowledge based consensus method which involved threading the protein into the identified pentein fold for the COOH-terminal part. Molecular dynamics was carried out using GROMACS for the mutant model which was built using the native myocilin model. Results: The Gly367Arg mutation causes accumulation of myocilin protein within TM cells with extensively reduced secretion contrary to wild type myocilin being characterized by intracellular localization and extracellular secretion. Further, Gly367Arg mutation occurs in a hydrophobic region which leads to burial of a charged residue. The dynamics suggests large conformational change is required to accommodate the mutation favoring aggregation of the protein.

Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development, l... more Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development, lead to the Blepharophimosis Syndrome. Here, we have systematically replaced the amino acids of the helices of the forkhead domain (FHD) of FOXL2 by glycine residues to assess the impact of such substitutions. A number of mutations lead to protein mislocalization, aggregation and to partial or complete loss of trans-activation ability on a series of luciferase reporter systems. To rationalize the results of this glycine mutation scan, we have modeled the structure of the FHD by comparison with crystallographic data available for other FHDs. We failed to detect a clear-cut correlation between protein mislocalization or aggregation and the position of the mutation. However, we found that the localization of the side chain of each amino acid strongly correlated with the impact of its mutation on FOXL2 transactivation capacity. Indeed, when the side chains of the amino acids involved in the helices of the forkhead are supposed to point towards the hydrophobic core formed by the three main helices, a loss of function was observed. On the contrary, if the side chains point outward the hydrophobic core, protein function was preserved. The extension of this analysis to natural mutants shows that a similar correlation can be found for BPES mutations associated or not with ovarian dysfunction. Our findings reveal new insights into the molecular effects of FOXL2 mutations affecting the FHD, which represent two-thirds of intragenic mutations, and provide the first predictive tool of their effects.

Purpose: To examine the possible role of alternate splicing leading to aggregation of myocilin in... more Purpose: To examine the possible role of alternate splicing leading to aggregation of myocilin in primary open-angle glaucoma. Methods: Several single nucleotide variations found in the myocilin (MYOC) genomic region were collected and examined for their possible role in causing splice-site alterations. A model for myocilin built using a knowledge-based consensus method was used to map the altered protein products. A total of 150 open-angle glaucoma patients and 50 normal age-matched control subjects were screened for the predicted polymorphisms, and clustering was performed. Results: A total of 124 genomic variations were screened, and six polymorphisms that lead to altered protein products were detected as possible candidates for the alternative splicing mechanism. Five of these lay in the intronic regions, and the one that lay in the exon region corresponded to the previously identified polymorphism (Tyr347Tyr) implicated in primary open-angle glaucoma. Experimentally screening the intronic region of the MYOC gene showed the presence of the predicted g.14072G>A polymorphism, g.1293C/T heterozygous polymorphism, instead of our predicted g.1293C/-polymorphism. Other than the prediction, two novel SNPs (g.1295G>T and g.1299T>G) and two reported SNPs (g. 1284G>T and g.1286G>T) were also identified. Cluster analysis showed the g.14072G>A homozygous condition was more common in this cohort than the heterozygous condition. Conclusions: We previously proposed that the disruption of dimer or oligomer formation by the C-term region allows greater chances of nucleation for aggregation. Here we suggest that polymorphisms in the myocilin genomic region that cause synonymous codon changes or those that occur in the intron regions can possibly lead to altered myocilin protein products through altered intron–exon splicing.

Journal of Ocular Biology, Diseases, and Informatics, 2011

Molecular vision

To examine the possible role of alternate splicing leading to aggregation of myocilin in primary ... more To examine the possible role of alternate splicing leading to aggregation of myocilin in primary open-angle glaucoma. Several single nucleotide variations found in the myocilin (MYOC) genomic region were collected and examined for their possible role in causing splice-site alterations. A model for myocilin built using a knowledge-based consensus method was used to map the altered protein products. A total of 150 open-angle glaucoma patients and 50 normal age-matched control subjects were screened for the predicted polymorphisms, and clustering was performed. A total of 124 genomic variations were screened, and six polymorphisms that lead to altered protein products were detected as possible candidates for the alternative splicing mechanism. Five of these lay in the intronic regions, and the one that lay in the exon region corresponded to the previously identified polymorphism (Tyr347Tyr) implicated in primary open-angle glaucoma. Experimentally screening the intronic region of the M...

A growing number of diseases seem to be associated with protein aggregation and each disease has ... more A growing number of diseases seem to be associated with protein aggregation and each disease has several proteins involved in it. To obtain a better understanding of the diseases, the proteins involved in it and their primary interaction partners were collected and clustered. A tool is developed to aid in clustering these proteins and all their primary interactors. The tools is used to cluster proteins involved in Primary Open Angle Glaucoma and their primary interactors. A cluster was selected for analysis based on the availability of experimental analysis in literature. The localization of the protein in the chosen cluster was collected. On analyzing four of the proteins in the cluster was found to be heparin binding. Primary open angle glaucoma is known to be associated with loss of retinal vasculature. The tool has helped in finding a cluster of proteins interaction with more experimental data. Also it has helped in finding out the 4 protein associated with the disease that involved in heparin binding among 10500 proteins. This would not have been possible to do manually. Further studying the role of these four proteins based on heparin binding and loss of vasculature in primary open angle glaucoma would give a better understanding of the disease and the molecular mechanism involved in it.

Bioinformation, Jun 30, 2014

ProADD, a database for protein aggregation diseases, is developed to organize the data under a si... more ProADD, a database for protein aggregation diseases, is developed to organize the data under a single platform to facilitate easy access for researchers. Diseases caused due to protein aggregation and the proteins involved in each of these diseases are integrated. The database helps in classification of proteins involved in the protein aggregation diseases based on sequence and structural analysis. Analysis of proteins can be done to mine patterns prevailing among the aggregating proteins.

Journal of Biomolecular Structure & Dynamics, 2012

The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins... more 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.

Purpose: Mutations in the myocilin gene (MYOC) leading to a perturbed outflow of aqueous in the t... more Purpose: Mutations in the myocilin gene (MYOC) leading to a perturbed outflow of aqueous in the trabecular meshwork (TM) has been associated with the pathophysiology of glaucoma. This study examines the expression of normal and mutant myocilin (Gly367Arg) in cultured TM cells. Methods: Normal and mutant MYOC cDNA constructs were used to transfect the TM cells. In order to confirm the method of transfection, reverse trancriptase polymerase chain reaction (RT-PCR) was carried out. Further, confocal microscopic analysis was used to determine the cellular localization of myocilin protein. The extracellular nature of myocilin in the culture supernatant and cell lysates of the transfected cells was analyzed by western blot. Molecular modeling was done earlier using a knowledge based consensus method which involved threading the protein into the identified pentein fold for the COOH-terminal part. Molecular dynamics was carried out using GROMACS for the mutant model which was built using the native myocilin model. Results: The Gly367Arg mutation causes accumulation of myocilin protein within TM cells with extensively reduced secretion contrary to wild type myocilin being characterized by intracellular localization and extracellular secretion. Further, Gly367Arg mutation occurs in a hydrophobic region which leads to burial of a charged residue. The dynamics suggests large conformational change is required to accommodate the mutation favoring aggregation of the protein.

Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development, l... more Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development, lead to the Blepharophimosis Syndrome. Here, we have systematically replaced the amino acids of the helices of the forkhead domain (FHD) of FOXL2 by glycine residues to assess the impact of such substitutions. A number of mutations lead to protein mislocalization, aggregation and to partial or complete loss of transactivation ability on a series of luciferase reporter systems. To rationalize the results of this glycine mutation scan, we have modeled the structure of the FHD by comparison with crystallographic data available for other FHDs. We failed to detect a clear-cut correlation between protein mislocalization or aggregation and the position of the mutation. However, we found that the localization of the side chain of each amino acid strongly correlated with the impact of its mutation on FOXL2 transactivation capacity. Indeed, when the side chains of the amino acids involved in the helices of the forkhead are supposed to point towards the hydrophobic core formed by the three main helices, a loss of function was observed. On the contrary, if the side chains point outward the hydrophobic core, protein function was preserved. The extension of this analysis to natural mutants shows that a similar correlation can be found for BPES mutations associated or not with ovarian dysfunction. Our findings reveal new insights into the molecular effects of FOXL2 mutations affecting the FHD, which represent two-thirds of intragenic mutations, and provide the first predictive tool of their effects.

C3 complement protein of cobra is known to have various essential roles in invitro and in vivo st... more C3 complement protein of cobra is known to have various essential roles in invitro and in vivo studies. The third complement protein plays an important role in the complement system by mediating various biological functions to amplify complement response and stimulate the adaptive immune response. Evolutionary tree for the third complement protein of the complement system infers, C3 an ancient molecule known to evolve with an ordered fashion from lower to higher vertebrates. In the absence of crystal structure for cobra C3 protein, homology modeling was constructed (CoC3). Furthermore a comparative modeling of complement C3 protein of cobra (CoC3), human and bovine deduced, that the cascade of complement activation in cobra occur due to the presence of the eight residues (D726-D727 and D720-D721 in strand, Y698-I699 and D704-E705 present in the helix ). Whereas, in human (A734Q-A735Q and A728Y-A729I) and bovine (B704D-B705C and B711D-B710) only four residues are involved in binding of (Factor B) FB, a component of the alternative pathway of the complement system and bringing about complement system activation. Therefore it is hypothesized that bovine and human C3 proteins which are evolutionarily related to CoC3 protein of cobra would have lost the remaining residues involved in complement activation during the due course of evolution. The eight residues involved in the binding of FB in cobra may have some other better role in complement activation and if it is so, this property of CoC3 can be experimentally used for complement activation process.

Mutations of the transcription factor FOXL2, involved in cranio-facial and ovarian development le... more 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.

Indian journal of …, Jan 1, 2004

Mol Vis, Jan 1, 2003

To screen for mutations in the MYOC gene of patients with Primary Open Angle Glaucoma (POAG) in I... more To screen for mutations in the MYOC gene of patients with Primary Open Angle Glaucoma (POAG) in India and to better understand the mutations using a possible model of myocilin. Methods: We analyzed DNA for mutations in 107 subjects with POAG and 90 normal control subjects. The exonic sequences of the MYOC gene from all subjects were amplified by Polymerase Chain Reaction (PCR). We carried out Single Strand Conformation Polymorphism (SSCP) for all the PCR products. The DNA samples which showed mobility shift in the banding pattern in SSCP gel were sequenced. We also analyzed the presence of the common mutation Gln368Stop using a specific restriction enzyme Taa 1. The mutations observed here and elsewhere have been mapped onto a possible model built for myocilin using a knowledge-based consensus modeling approach. Results: Two heterozygous mutations Gly367Arg (1099G>A) and Thr377Met (1130C>T) were identified in exon3 of the MYOC gene of probands 40-1 and 51-1 respectively, from material obtained from the 107 unrelated subjects with POAG. These two mutations were not present in the normal controls studied. We identified a Single Nucleotide Polymorphism (SNP) Gly122Gly (366C>T) in exon1 of proband 57-1 as a non-disease causing variation. The common mutation Gln368Stop found in the Western population was not observed in the POAG cases screened in Indian population. The possible structural model for myocilin suggests a predominantly β-strand rich C-terminal region (181-504) which is connected by the α-helical mid-region (111-180) to the N-terminal region (34-110) which has low secondary structure content. Both the mutations, Gly367Arg and Thr377Met identified in our study, map on to the C-terminal region. These mutations disfavor burial of this region during oligomer formation due to the charged or bulky nature of the mutants. Most of the other mutations known for myocilin also are surface exposed on the C-terminal region. Conclusions: Our findings indicate that the mutation frequency of the MYOC gene is 2% in the Indian population affected with POAG, which is not a well-studied ethnic group of the Asian continent. The variations identified in our study have been previously reported in the Western population. The nonsense mutation Gln368Stop was not observed in the present study and thereby suggests that it may not be a common disease-causing mutation in the Indian population. Amongst other Asian populations, studies in Japan also didn't report this nonsense mutation. The location of these mutations suggest that a plausible mode of action could be by disruption of dimer or oligomer formation by the C-terminal region allowing greater chances of nucleation of aggregation by the N-terminal region.