Lipid compositions of cells, tissues, and bio-fluids are complex, with varying concentrations and... more Lipid compositions of cells, tissues, and bio-fluids are complex, with varying concentrations and structural diversity making their identification challenging. Newer methods for comprehensive analysis of lipids are thus necessary. Herein, we propose a targeted-mass spectrometry based lipidomics screening method using a combination of variable retention time window and relative dwell time weightage. Using this method, we identified more than 1000 lipid species within 24-min. The limit of detection varied from the femtomolar to the nanomolar range. About 883 lipid species were detected with a coefficient of variance <30%. We used this method to identify plasma lipids altered due to vitamin B12 deficiency and found a total of 18 lipid species to be altered. Some of the lipid species with ω-6 fatty acid chains were found to be significantly increased while ω-3 decreased in vitamin B12 deficient samples. This method enables rapid screening of a large number of lipid species in a singl...
Long non‐coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here,... more Long non‐coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here, we investigated the role of a novel vascular endothelial‐associated lncRNA (VEAL2) in regulating endothelial permeability. Precise editing of veal2 loci in zebrafish (veal2gib005Δ8/+) induced cranial hemorrhage. In vitro and in vivo studies revealed that veal2 competes with diacylglycerol for interaction with protein kinase C beta‐b (Prkcbb) and regulates its kinase activity. Using PRKCB2 as bait, we identified functional ortholog of veal2 in humans from HUVECs and named it as VEAL2. Overexpression and knockdown of VEAL2 affected tubulogenesis and permeability in HUVECs. VEAL2 was differentially expressed in choroid tissue in eye and blood from patients with diabetic retinopathy, a disease where PRKCB2 is known to be hyperactivated. Further, VEAL2 could rescue the effects of PRKCB2‐mediated turnover of endothelial junctional proteins thus reducing hyperpermeability in hyperglycemic HUVEC model of diabetic retinopathy. Based on evidence from zebrafish and hyperglycemic HUVEC models and diabetic retinopathy patients, we report a hitherto unknown VEAL2 lncRNA‐mediated regulation of PRKCB2, for modulating junctional dynamics and maintenance of endothelial permeability.
Academy of Scientic and Innovative Resea Marg, New Delhi-110001, India CSIR-Institute of Genomic... more Academy of Scientic and Innovative Resea Marg, New Delhi-110001, India CSIR-Institute of Genomics and Integrativ India. E-mail: souvik@igib.res.in; Fax: +91Department of Chemistry, Clemson Univers CSIR-National Chemical Laboratory, Dr. H E-mail: souvik@igib.res.in; dparya@clemso † Electronic supplementary information ( synthesis of compounds. See DOI: 10.103 Cite this: Chem. Sci., 2015, 6, 5837
Activity modulation of proteins is an essential biochemical process in cell. The interplay of the... more Activity modulation of proteins is an essential biochemical process in cell. The interplay of the protein, as receptor, and it’s corresponding ligand dictates the functional effect. An agonist molecule when bound to a receptor produces a response within the cell while an antagonist will block the binding site/produce the opposite effect of that of an agonist. Complexity grows with scenarios where some ligands might act as an agonist in certain conditions while as an antagonist in others [1, 3]. It is imperative to decipher the receptor-ligand functional effect for understanding native biochemical processes as well as for drug discovery. Experimental activity determination is a time extensive process and computational solution towards prediction of activity specific to the receptor-ligand interaction would be of wide interest.
BackgroundViral infections have a history of abrupt and severe eruptions through the years in the... more BackgroundViral infections have a history of abrupt and severe eruptions through the years in the form of pandemics. And yet, definitive therapies or preventive measures are not present.PurposeHerbal medicines have been a source of various antiviral compounds. An accelerated repurposing potential of antiviral herbs can provide usable drugs and identify druggable targets. In this study, we dissect the anti-coronavirus activity of Cissampelos pareira L (Cipa). using an integrative approach.MethodsWe analysed the signature similarities between predicted antiviral agents and Cipa using the connectivity map (https://clue.io/). Next, we tested the anti-SARS-COV-2 activity of Cipa in vitro. A three-way comparative analysis of Cipa transcriptome, COVID-19 BALF transcriptome and CMAP signatures of small compounds was also performed.ResultsSeveral predicted antivirals showed a high positive connectivity score with Cipa such as apcidin, emetine, homoharringtonine etc. We also observed 98% inhi...
ABSTRACT The paper aims to give a present status of knowledge on irradiation-assisted stress corr... more ABSTRACT The paper aims to give a present status of knowledge on irradiation-assisted stress corrosion cracking (IASCC) from the point of view of nuclear power plant life extension. Field experience as well as laboratory test results are considered to illustrate how IASCC already impacts plant operation. IASCC has been differentiated with difficulty from standard integranular stress corrosion cracking (IGSCC), but above a threshold radiation dose, an enhanced portion of intergranular fracture are convincing evidences for IASCC. IASCC cracking in boiling water reactor (BWR) core shrouds started wide studies of materials of irradiated reactor internals. The last cases of IASCC have been found in pressurized water reactor/water-water energy reactor (PWR/WWER) baffle bolts. Laboratory slow strain rate tests as well as constant load and crack growth rate tests in simulated BWR and PWR environments resulted in the discovery of the IASCC threshold dose, the threshold stress, and that the cracking kinetics increase with neutron exposure. The present understanding of the mechanism of IASCC in BWR/PWR systems is given assuming no fundamental differences between the two environments. Localized deformation on grain boundaries affected by segregation and environmental effects is the most likely mechanism. Finally, plant life extensions likely will bring an increase of IASCC risk because of higher accumulated doses. The impact on BWR and PWR internal components is estimated and discussed. It is concluded that further critical experiments and complex data analyses are urgently needed.
Background Employing methods to assess the quality of modeled protein structures is now standard ... more Background Employing methods to assess the quality of modeled protein structures is now standard practice in bioinformatics. In a broad sense, the techniques can be divided into methods relying on consensus prediction on the one hand, and single-model methods on the other. Consensus methods frequently perform very well when there is a clear consensus, but this is not always the case. In particular, they frequently fail in selecting the best possible model in the hard cases (lacking consensus) or in the easy cases where models are very similar. In contrast, single-model methods do not suffer from these drawbacks and could potentially be applied on any protein of interest to assess quality or as a scoring function for sampling-based refinement. Results Here, we present a new single-model method, ProQ2, based on ideas from its predecessor, ProQ. ProQ2 is a model quality assessment algorithm that uses support vector machines to predict local as well as global quality of protein models. ...
India confines more than 17% of the world’s population and has a diverse genetic makeup with seve... more India confines more than 17% of the world’s population and has a diverse genetic makeup with several clinically relevant rare mutations belonging to many sub-group which are undervalued in global sequencing datasets like the 1000 Genome data (1KG) containing limited samples for Indian ethnicity. Such databases are critical for the pharmaceutical and drug development industry where the diversity plays a crucial role in identifying genetic disposition towards adverse drug reactions. A qualitative and comparative sequence and structural study utilizing variant information present in the recently published, largest curated Indian genome database (IndiGen) and the 1000 Genome data was performed for variants belonging to the kinase coding genes,the second most targeted group of drug targets. The sequence level analysis identified similarities and differences among different populations based on the nsSNVs and amino acid exchange frequencies whereas comparative structural analysis of IndiG...
Bioactive fractions obtained from medicinal plants which have been used for the treatment of mult... more Bioactive fractions obtained from medicinal plants which have been used for the treatment of multiple diseases could exert their effects by targeting common pathways. Prior knowledge of their usage could allow us to identify novel molecular links. In this study, we explored the molecular basis of action of one such herbal formulation Cissampelos pareira L. (Cipa), used for the treatment of female hormone disorders and fever. Transcriptomic studies on MCF7 cell lines treated with Cipa extract carried out using Affymetrix arrays revealed a downregulation of signatures of estrogen response potentially modulated through estrogen receptor α (ERα). Molecular docking analysis identified 38 Cipa constituents that potentially bind (ΔG
ATP Binding Cassette Transporter A1 (ABCA1) plays an integral part in Reverse Cholesterol Transpo... more ATP Binding Cassette Transporter A1 (ABCA1) plays an integral part in Reverse Cholesterol Transport (RCT) and is critical for maintaining lipid homeostasis. One theory of lipid efflux by the transporter (alternating access) proposes that ABCA1 harbours two different conformations that provide alternating access for lipid binding and release. This is followed by a sequestration via a direct interaction between ABCA1 and its partner, ApoA1. The alternative theory (lateral access) proposes that ABCA1 obtains lipids laterally from the membrane to form a temporary extracellular “reservoir”. This reservoir contains an isolated lipid monolayer due to the net accumulation of lipids in the exofacial leaflet. Recently, a full-length Cryo-EM structure of this 2,261-residue transmembrane protein showed its discreetly folded domains and have detected the presence of a tunnel enclosed within ECDs but not in theTMDs giving it an outward-facing conformation. This structure was hypothesized to subst...
The complex double-membrane architecture of mitochondria is essential for its ATP synthesis funct... more The complex double-membrane architecture of mitochondria is essential for its ATP synthesis function and divides the organelle into two sub-mitochondrial compartments, inter-membrane space (IMS) and matrix. The folding environments of IMS and matrix are significantly different owing to its dissimilar oxido-reductive environments and distinctly divergent protein quality control (PQC) machineries. Here, by inducing proteotoxic stress restricted to IMS or matrix by targeting three different stressor proteins, we show that the cellular response to IMS or matrix-localized misfolding stress is distinct and unique. IMS and matrix stress response pathways are quite effective in combatting stress despite significant stress-induced alteration in mitochondrial phenotypes. IMS misfolding stress leads to specific upregulation of IMS chaperones and components of TOM complex while matrix chaperones and cytosolic PQC components are upregulated during matrix stress. Notably, the amplitude of upregul...
Internal cavities of proteins are of critical functional importance. Yet, there is a paucity of c... more Internal cavities of proteins are of critical functional importance. Yet, there is a paucity of computational tools that can accurately, and reliably characterize the inner cavities of the proteins, a prerequisite for elucidating their functions. Here we introduce CICLOP a novel computational tool that accurately, reproducibly, and speedily identifies residues lining the inner cavity of a protein, its morphometric dimensions, hydrophobicity as well as evolutionary significance.
ABSTRACTThe folding landscape of proteins can change during evolution with the accumulation of mu... more ABSTRACTThe folding landscape of proteins can change during evolution with the accumulation of mutations that may introduce entropic or enthalpic barriers in the protein folding pathway, making it a possible substrate of molecular chaperones in vivo. Can the nature of such physical barriers of folding dictate the feasibility of chaperone-assistance? To address this, we have simulated the evolutionary step to chaperone-dependence keeping GroEL/ES as the target chaperone and GFP as a model protein in an unbiased screen. We find that the mutation conferring GroEL/ES dependence in vivo and in vitro encode an entropic trap in the folding pathway rescued by the chaperonin. Additionally, GroEL/ES can edit the formation of non-native contacts similar to DnaK/J/E machinery. However, this capability is not utilized by the substrates in vivo. As a consequence, GroEL/ES caters to buffer mutations that predominantly cause entropic traps, despite possessing the capacity to edit both enthalpic and...
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) is mediated via the initial int... more The severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) is mediated via the initial interaction of the virus’s spike (S-) protein with a human receptor protein called angiotensin-converting enzyme 2 (hACE2). Interference in this association can have immense therapeutic importance. We used an in-silico combinatorial approach involving homology-based protein modeling, protein-protein docking, binding energy estimation and virtual screening techniques to probe for genetic variations and drug molecules for disruption of the hACE2-CoV2 interaction. Our results identified Ser19Pro variation on hACE2 showed similar structural stability to the native protein while having a destabilizing effect in the hACE2(Ser19Pro)-S-protein complex. We also found several FDA-approved drug molecules that can potentially induce competition-mediated destabilization of the hACE2-CoV2 complex. In conclusion, these findings provide critical insights for intervention strategies targeting the pathogenic...
Lipid compositions of cells, tissues, and bio-fluids are complex, with varying concentrations and... more Lipid compositions of cells, tissues, and bio-fluids are complex, with varying concentrations and structural diversity making their identification challenging. Newer methods for comprehensive analysis of lipids are thus necessary. Herein, we propose a targeted-mass spectrometry based lipidomics screening method using a combination of variable retention time window and relative dwell time weightage. Using this method, we identified more than 1000 lipid species within 24-min. The limit of detection varied from the femtomolar to the nanomolar range. About 883 lipid species were detected with a coefficient of variance <30%. We used this method to identify plasma lipids altered due to vitamin B12 deficiency and found a total of 18 lipid species to be altered. Some of the lipid species with ω-6 fatty acid chains were found to be significantly increased while ω-3 decreased in vitamin B12 deficient samples. This method enables rapid screening of a large number of lipid species in a singl...
Long non‐coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here,... more Long non‐coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here, we investigated the role of a novel vascular endothelial‐associated lncRNA (VEAL2) in regulating endothelial permeability. Precise editing of veal2 loci in zebrafish (veal2gib005Δ8/+) induced cranial hemorrhage. In vitro and in vivo studies revealed that veal2 competes with diacylglycerol for interaction with protein kinase C beta‐b (Prkcbb) and regulates its kinase activity. Using PRKCB2 as bait, we identified functional ortholog of veal2 in humans from HUVECs and named it as VEAL2. Overexpression and knockdown of VEAL2 affected tubulogenesis and permeability in HUVECs. VEAL2 was differentially expressed in choroid tissue in eye and blood from patients with diabetic retinopathy, a disease where PRKCB2 is known to be hyperactivated. Further, VEAL2 could rescue the effects of PRKCB2‐mediated turnover of endothelial junctional proteins thus reducing hyperpermeability in hyperglycemic HUVEC model of diabetic retinopathy. Based on evidence from zebrafish and hyperglycemic HUVEC models and diabetic retinopathy patients, we report a hitherto unknown VEAL2 lncRNA‐mediated regulation of PRKCB2, for modulating junctional dynamics and maintenance of endothelial permeability.
Academy of Scientic and Innovative Resea Marg, New Delhi-110001, India CSIR-Institute of Genomic... more Academy of Scientic and Innovative Resea Marg, New Delhi-110001, India CSIR-Institute of Genomics and Integrativ India. E-mail: souvik@igib.res.in; Fax: +91Department of Chemistry, Clemson Univers CSIR-National Chemical Laboratory, Dr. H E-mail: souvik@igib.res.in; dparya@clemso † Electronic supplementary information ( synthesis of compounds. See DOI: 10.103 Cite this: Chem. Sci., 2015, 6, 5837
Activity modulation of proteins is an essential biochemical process in cell. The interplay of the... more Activity modulation of proteins is an essential biochemical process in cell. The interplay of the protein, as receptor, and it’s corresponding ligand dictates the functional effect. An agonist molecule when bound to a receptor produces a response within the cell while an antagonist will block the binding site/produce the opposite effect of that of an agonist. Complexity grows with scenarios where some ligands might act as an agonist in certain conditions while as an antagonist in others [1, 3]. It is imperative to decipher the receptor-ligand functional effect for understanding native biochemical processes as well as for drug discovery. Experimental activity determination is a time extensive process and computational solution towards prediction of activity specific to the receptor-ligand interaction would be of wide interest.
BackgroundViral infections have a history of abrupt and severe eruptions through the years in the... more BackgroundViral infections have a history of abrupt and severe eruptions through the years in the form of pandemics. And yet, definitive therapies or preventive measures are not present.PurposeHerbal medicines have been a source of various antiviral compounds. An accelerated repurposing potential of antiviral herbs can provide usable drugs and identify druggable targets. In this study, we dissect the anti-coronavirus activity of Cissampelos pareira L (Cipa). using an integrative approach.MethodsWe analysed the signature similarities between predicted antiviral agents and Cipa using the connectivity map (https://clue.io/). Next, we tested the anti-SARS-COV-2 activity of Cipa in vitro. A three-way comparative analysis of Cipa transcriptome, COVID-19 BALF transcriptome and CMAP signatures of small compounds was also performed.ResultsSeveral predicted antivirals showed a high positive connectivity score with Cipa such as apcidin, emetine, homoharringtonine etc. We also observed 98% inhi...
ABSTRACT The paper aims to give a present status of knowledge on irradiation-assisted stress corr... more ABSTRACT The paper aims to give a present status of knowledge on irradiation-assisted stress corrosion cracking (IASCC) from the point of view of nuclear power plant life extension. Field experience as well as laboratory test results are considered to illustrate how IASCC already impacts plant operation. IASCC has been differentiated with difficulty from standard integranular stress corrosion cracking (IGSCC), but above a threshold radiation dose, an enhanced portion of intergranular fracture are convincing evidences for IASCC. IASCC cracking in boiling water reactor (BWR) core shrouds started wide studies of materials of irradiated reactor internals. The last cases of IASCC have been found in pressurized water reactor/water-water energy reactor (PWR/WWER) baffle bolts. Laboratory slow strain rate tests as well as constant load and crack growth rate tests in simulated BWR and PWR environments resulted in the discovery of the IASCC threshold dose, the threshold stress, and that the cracking kinetics increase with neutron exposure. The present understanding of the mechanism of IASCC in BWR/PWR systems is given assuming no fundamental differences between the two environments. Localized deformation on grain boundaries affected by segregation and environmental effects is the most likely mechanism. Finally, plant life extensions likely will bring an increase of IASCC risk because of higher accumulated doses. The impact on BWR and PWR internal components is estimated and discussed. It is concluded that further critical experiments and complex data analyses are urgently needed.
Background Employing methods to assess the quality of modeled protein structures is now standard ... more Background Employing methods to assess the quality of modeled protein structures is now standard practice in bioinformatics. In a broad sense, the techniques can be divided into methods relying on consensus prediction on the one hand, and single-model methods on the other. Consensus methods frequently perform very well when there is a clear consensus, but this is not always the case. In particular, they frequently fail in selecting the best possible model in the hard cases (lacking consensus) or in the easy cases where models are very similar. In contrast, single-model methods do not suffer from these drawbacks and could potentially be applied on any protein of interest to assess quality or as a scoring function for sampling-based refinement. Results Here, we present a new single-model method, ProQ2, based on ideas from its predecessor, ProQ. ProQ2 is a model quality assessment algorithm that uses support vector machines to predict local as well as global quality of protein models. ...
India confines more than 17% of the world’s population and has a diverse genetic makeup with seve... more India confines more than 17% of the world’s population and has a diverse genetic makeup with several clinically relevant rare mutations belonging to many sub-group which are undervalued in global sequencing datasets like the 1000 Genome data (1KG) containing limited samples for Indian ethnicity. Such databases are critical for the pharmaceutical and drug development industry where the diversity plays a crucial role in identifying genetic disposition towards adverse drug reactions. A qualitative and comparative sequence and structural study utilizing variant information present in the recently published, largest curated Indian genome database (IndiGen) and the 1000 Genome data was performed for variants belonging to the kinase coding genes,the second most targeted group of drug targets. The sequence level analysis identified similarities and differences among different populations based on the nsSNVs and amino acid exchange frequencies whereas comparative structural analysis of IndiG...
Bioactive fractions obtained from medicinal plants which have been used for the treatment of mult... more Bioactive fractions obtained from medicinal plants which have been used for the treatment of multiple diseases could exert their effects by targeting common pathways. Prior knowledge of their usage could allow us to identify novel molecular links. In this study, we explored the molecular basis of action of one such herbal formulation Cissampelos pareira L. (Cipa), used for the treatment of female hormone disorders and fever. Transcriptomic studies on MCF7 cell lines treated with Cipa extract carried out using Affymetrix arrays revealed a downregulation of signatures of estrogen response potentially modulated through estrogen receptor α (ERα). Molecular docking analysis identified 38 Cipa constituents that potentially bind (ΔG
ATP Binding Cassette Transporter A1 (ABCA1) plays an integral part in Reverse Cholesterol Transpo... more ATP Binding Cassette Transporter A1 (ABCA1) plays an integral part in Reverse Cholesterol Transport (RCT) and is critical for maintaining lipid homeostasis. One theory of lipid efflux by the transporter (alternating access) proposes that ABCA1 harbours two different conformations that provide alternating access for lipid binding and release. This is followed by a sequestration via a direct interaction between ABCA1 and its partner, ApoA1. The alternative theory (lateral access) proposes that ABCA1 obtains lipids laterally from the membrane to form a temporary extracellular “reservoir”. This reservoir contains an isolated lipid monolayer due to the net accumulation of lipids in the exofacial leaflet. Recently, a full-length Cryo-EM structure of this 2,261-residue transmembrane protein showed its discreetly folded domains and have detected the presence of a tunnel enclosed within ECDs but not in theTMDs giving it an outward-facing conformation. This structure was hypothesized to subst...
The complex double-membrane architecture of mitochondria is essential for its ATP synthesis funct... more The complex double-membrane architecture of mitochondria is essential for its ATP synthesis function and divides the organelle into two sub-mitochondrial compartments, inter-membrane space (IMS) and matrix. The folding environments of IMS and matrix are significantly different owing to its dissimilar oxido-reductive environments and distinctly divergent protein quality control (PQC) machineries. Here, by inducing proteotoxic stress restricted to IMS or matrix by targeting three different stressor proteins, we show that the cellular response to IMS or matrix-localized misfolding stress is distinct and unique. IMS and matrix stress response pathways are quite effective in combatting stress despite significant stress-induced alteration in mitochondrial phenotypes. IMS misfolding stress leads to specific upregulation of IMS chaperones and components of TOM complex while matrix chaperones and cytosolic PQC components are upregulated during matrix stress. Notably, the amplitude of upregul...
Internal cavities of proteins are of critical functional importance. Yet, there is a paucity of c... more Internal cavities of proteins are of critical functional importance. Yet, there is a paucity of computational tools that can accurately, and reliably characterize the inner cavities of the proteins, a prerequisite for elucidating their functions. Here we introduce CICLOP a novel computational tool that accurately, reproducibly, and speedily identifies residues lining the inner cavity of a protein, its morphometric dimensions, hydrophobicity as well as evolutionary significance.
ABSTRACTThe folding landscape of proteins can change during evolution with the accumulation of mu... more ABSTRACTThe folding landscape of proteins can change during evolution with the accumulation of mutations that may introduce entropic or enthalpic barriers in the protein folding pathway, making it a possible substrate of molecular chaperones in vivo. Can the nature of such physical barriers of folding dictate the feasibility of chaperone-assistance? To address this, we have simulated the evolutionary step to chaperone-dependence keeping GroEL/ES as the target chaperone and GFP as a model protein in an unbiased screen. We find that the mutation conferring GroEL/ES dependence in vivo and in vitro encode an entropic trap in the folding pathway rescued by the chaperonin. Additionally, GroEL/ES can edit the formation of non-native contacts similar to DnaK/J/E machinery. However, this capability is not utilized by the substrates in vivo. As a consequence, GroEL/ES caters to buffer mutations that predominantly cause entropic traps, despite possessing the capacity to edit both enthalpic and...
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) is mediated via the initial int... more The severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) is mediated via the initial interaction of the virus’s spike (S-) protein with a human receptor protein called angiotensin-converting enzyme 2 (hACE2). Interference in this association can have immense therapeutic importance. We used an in-silico combinatorial approach involving homology-based protein modeling, protein-protein docking, binding energy estimation and virtual screening techniques to probe for genetic variations and drug molecules for disruption of the hACE2-CoV2 interaction. Our results identified Ser19Pro variation on hACE2 showed similar structural stability to the native protein while having a destabilizing effect in the hACE2(Ser19Pro)-S-protein complex. We also found several FDA-approved drug molecules that can potentially induce competition-mediated destabilization of the hACE2-CoV2 complex. In conclusion, these findings provide critical insights for intervention strategies targeting the pathogenic...
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