Santosh Kumar Sahu - Academia.edu (original) (raw)

Papers by Santosh Kumar Sahu

Research paper thumbnail of Lipid Flip-Flop-Inducing Antimicrobial Phytochemicals from Gymnema sylvestre are Bacterial Membrane Permeability Enhancers

ACS Omega, 2021

An amphiphilic phytochemical fraction isolated from methanol extract of Gymnema sylvestre leaf po... more An amphiphilic phytochemical fraction isolated from methanol extract of Gymnema sylvestre leaf powder contained six terpenoids, two flavonoids, and one alkaloid that induced rapid flip-flop of fluorescent phospholipid analog in the phosphatidyl choline bilayer. Lipid-flipping activity of the methanol-extracted fraction of G. sylvestre (MEFGS) was dose-dependent and time-dependent with a rate constant k = (12.09 ± 0.94) mg −1 min −1 that was saturable at (40 ± 1) % flipping of the fluorescent lipid analogue. Interactions of MEFGS phytochemicals with large unilamelar vesicles led to time-dependent change in their rounded morphology into irregular shapes, indicating their membranedestabilizing activity. MEFGS exhibited antibacterial activity on Escherichia coli (MTCC-118), Staphylococcus aureus (MTCC-212), and Pseudomonas aeruginosa (MTCC-1035) with IC 50 values 0.5, 0.35, and 0.1 mg/mL, respectively. Phytochemicals in MEFGS increased membrane permeabilization in all three bacteria, as indicated by 23, 17, and 17% increase in the uptake of crystal violet, respectively. MEFGS enhanced membrane damage, resulting in a 3−5 fold increase in leakage of cytosolic ions, 0.5−2 fold increase in leakage of PO 4 − , and 15−20% increase in loss of cellular proteins. MEFGS synergistically increased the efficacy of curcumin, amoxillin, ampicillin, and cefotaxime on S. aureus probably by enhancing their permeability into the bacterium. For the first time, our study reveals that phytochemicals from G. sylvestre enhance the permeability of the bacterial plasma membrane by facilitating flip-flop of membrane lipids. Lipidflipping phytochemicals from G. sylvestre can be used as adjuvant therapeutics to enhance the efficacy of antibacterials by increasing their bioavailability in the target bacteria.

Research paper thumbnail of Evaluation of Biotechnological Potential of Novel Mercury Tolerant Strain of Klebsiella Pneumonia

Journal of Applied Microbiology and Biochemistry, 2018

Research paper thumbnail of Prediction of Sub-cellular Localization of Scramblase Protein Family

International Journal of Computer and Communication Technology, 2010

In the present work, we discuss an anaysis about the localization of different members of scrambl... more In the present work, we discuss an anaysis about the localization of different members of scramblase protein family. Different scramblase sequences were picked up from organisms of all eukaryotic phyla and their localization were predicted using the P-SORT programme. Our analysis showed that the scramblase protein family shows multiple subcellular localization. Most proteins were found to be localized to the cytoplasm, where as others were found to be present in the nucleus or mitochondria. Interestingly, we found that in yeast, all putative scramblases were localized in the nucleus with a reliability of more than 95%. Our analysis shows that scramblases are a family of protein having diversed cellular localization and hence hypothesized to be performing multiple cellular functions in various organisms.

Research paper thumbnail of Electroformation of liposomes and phytosomes using copper electrode

Journal of Liposome Research

Abstract A novel method for electroformation of liposomes and phytosomes using copper electrode i... more Abstract A novel method for electroformation of liposomes and phytosomes using copper electrode is described. Liposomes made at 2 V and 10 Hz AC field from L-α-egg-phosphatidylcholine (egg-PC), K. pneumoniae phosphatidylethanolamine, K. pneumoniae polar lipids and E. coli polar lipids on copper electrode were (777.9 ± 118.4), (370.2 ± 100.5), (825.3 ± 21.54), and (281.3 ± 42.3) nm in diameter, respectively. Giant vesicles were formed at 30 V and 10 Hz AC field from polar lipids of K. pneumoniae and E. coli were (106 ± 29.7) and (86 ± 24.3) µm in diameter, respectively. All liposomes were unilamellar as indicated by their unilamellar indices of 50 ± 2, had surface charge comparable to vesicles made from lipid(s) with similar composition and exhibited only 1–2 mol% of oxidized lipids. Cu concentration in the liposomal samples was <1.5 ppm for large unilamellar vesicles (LUVs) and ˂5 ppm for giant unilamellar vesicles (GUVs). The vesicles were stable for >15 d without loss of their size, charge, or unilamellarity. The method was successfully applied to prepare phytosomes from egg-PC and a phytochemical fraction of Dimorphocalyx glabellus, a medicinal plant with anti-diuretic properties. Phytosomes formed were 1000–1500 nm in diameter and exhibited altered fluorescence and absorbance properties compared to the unencapsulated phytochemical. Phytosomes with phytochemical: egg-PC ratio from 0.15 to 1.5 had encapsulation efficiency ranging 90–30%, respectively, and was stable for 1 month. Our method is easy, inexpensive and convenient that will prove to be useful for preparation of liposomes and phytosomes.

Research paper thumbnail of Copper and iron overload protect Escherichia coli from exogenous H2O2 by modulating membrane phospholipid composition

Environmental Sustainability

Development of green technologies for sustainable development requires growth of bacteria in medi... more Development of green technologies for sustainable development requires growth of bacteria in medium containing H 2 O 2 from 50 to 150 mM that may contain millimolar level of copper (Cu) and iron (Fe). However, the mechanism of oxidative stress involving high concentration of exogenous H 2 O 2 in bacteria and the effect of Cu and Fe overload on the same remain controversial. In the present study, we investigated the effect of millimolar concentration of Cu and Fe on H 2 O 2-induced oxidative stress using the enterotoxigenic Escherichia coli 12566 as a model system. Our results indicate that at millimolar concentration, Fe and Cu protect E. coli from H 2 O 2-induced oxidative stress by (1) suppressing H 2 O 2-induced growth inhibition, (2) reducing H 2 O 2-induced lipid peroxidation, (3) decreasing catalase activity and (4) modulating H 2 O 2-induced release of cytosolic Fe content in a dose-dependent manner. A previous study by our group shows that H 2 O 2 significantly alters the phospholipid (PL) composition in E. coli by a dose-dependent increase in cardiolipin (CL) accompanied by decrease in sum total of phosphatidyl ethanolamine (PE) and phosphatidyl glycerol (PG). In the present study, we show that Cu 2+ and Fe 3+ at 1 mM reduce H 2 O 2-induced change in PL composition of E. coli leading to stabilization of its membrane components. For the first time, our results show that overloading of E. coli with Cu 2+ and Fe 3+ activates a protective mechanism against H 2 O 2-induced oxidative damage by modulating its membrane PL composition. Our study reveals a pivotal regulatory role of these metal ions in the sectors of sustainable technologies such as heavy metal bioremediation, sewage treatment and bioelectrochemical reactors that require microbial growth at high concentration of H 2 O 2 .

Research paper thumbnail of Sequence Analysis of a Subset of Plasma Membrane Raft Proteome Containing CXXC Metal Binding Motifs

International Journal of Knowledge Discovery in Bioinformatics, 2015

In an attempt to identify the metal sensing proteins localized to mammalian plasma membrane, the ... more In an attempt to identify the metal sensing proteins localized to mammalian plasma membrane, the authors screened a list of 300 raft associated proteins that are involved in cellular signaling mechanisms by searching the presence of metal thionin (CXXC) motifs. 50 proteins were found to possess CXXC motifs that could act as potential metal sensing proteins. The authors determined membrane topologies of the above CXXC motif containing proteins using TM-pred and analyzed the positions of their transmembrane (TM) domains using Bio-edit software. Based on the topology of CXXC domains, the authors classified all the raft-associated metal sensing proteins into six categories. They are (i) Exoplasmic tails with CXXC motif, (ii) Exoplasmic loops with CXXC motif, (iii) Cytosolic tails with CXXC motif, (iv) Cytosolic loop with CXXC motif, (v) TM domains with CXXC motifs, (vi) Proteins with multiple topologies of CXXC motif. The authors' study will lead to understanding of the raft-mediate...

Research paper thumbnail of Industrially Important Carbohydrate Degrading Enzymes from Yeasts: Pectinases, Chitinases, and β-1,3-Glucanases

Yeast Biotechnology: Diversity and Applications, 2009

Contents 30.1 Introduction ......................................................................... more Contents 30.1 Introduction .................................................................................................................. 673 30.2 Biological Significance of Carbohydrate Degrading Enzymes ................................... 676 30.2.1 Biological Significance of Pectinases ................................ ... ... T. Satyanarayana, G. ...

Research paper thumbnail of Cholesterol depletion modulates detergent resistant fraction of human serotonin 1A receptors

Molecular Membrane Biology, 2012

Insolubility of membrane components in non-ionic detergents such as Triton X-100 at low temperatu... more Insolubility of membrane components in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion to identify, isolate and characterize membrane domains. In this work, we monitored the detergent insolubility of the serotonin 1A receptor in CHO cell membranes and its modulation by membrane cholesterol. The serotonin 1A receptor is an important member of the G-protein coupled receptor family. It is implicated in the generation and modulation of various cognitive, behavioral and developmental functions and serves as a drug target. Our results show that a significant fraction (~28%) of the serotonin 1A receptor resides in detergent-resistant membranes (DRMs). Interestingly, the fraction of the serotonin 1A receptor in DRMs exhibits a reduction upon membrane cholesterol depletion. In addition, we show that contents of DRM markers such as flotillin-1, caveolin-1 and GM 1 are altered in DRMs upon cholesterol depletion. These results assume significance since the function of the serotonin 1A receptor has previously been shown to be affected by membrane lipids, specifically cholesterol. Our results are relevant in the context of membrane organization of the serotonin 1A receptor in particular, and G-protein coupled receptors in general.

Research paper thumbnail of Flippase Activity in Proteoliposomes Reconstituted with Spinacea oleracea Endoplasmic Reticulum Membrane Proteins: Evidence of Biogenic Membrane Flippase in Plants †

Biochemistry, 2008

Phospholipid translocation (flip-flop) in biogenic (self-synthesizing) membranes such as the endo... more Phospholipid translocation (flip-flop) in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum of eukaryotic cells (rat liver) and bacterial cytoplasmic membranes is a fundamental step in membrane biogenesis. It is known that flip-flop in these membranes occurs without a metabolic energy requirement, bidirectionally with no specificity for phospholipid headgroup. In this study, we demonstrate for the first time ATP-independent flippase activity in endoplasmic reticulum membranes of plants using spinach as a model system. For this, we generated proteoliposomes from a Triton X-100 extract of endoplasmic reticulum membranes of spinach and assayed them for flippase activity using fluorescently labeled phospholipids. The half-time for flipping was found to be 0.7-1.0 min. We also show that (a) proteoliposomes can flip fluorescently labeled analogues of phosphatidylcholine and phosphatidylethanolamine, (b) flipping activity is protein-mediated, (c) more than one class of lipid translocator (flippase) is present in spinach membranes, based on the sensitivity to protease and protein-modifying reagents, and (d) translocation of PC and PE is affected differently upon treatment with protease and protein-modifying reagents. Ca (2+)-dependent scrambling activity was not observed in the vesicles reconstituted from plant ER membranes, ruling out the possibility of the involvement of scramblase in translocation of phospholipids. These results suggest the existence of biogenic membrane flippases in plants and that the mechanism of membrane biogenesis is similar to that found in animals.

Research paper thumbnail of Phospholipid scramblases: An overview

Archives of Biochemistry and Biophysics, 2007

Phospholipid scramblases are a group of homologous proteins that are conserved in all eukaryotic ... more Phospholipid scramblases are a group of homologous proteins that are conserved in all eukaryotic organisms. They are believed to be involved in destroying plasma membrane phospholipid asymmetry at critical cellular events like cell activation, injury and apoptosis. However, a detailed mechanism of phospholipid scrambling still awaits a proper understanding. The most studied member of this family, phospholipid scramblase 1 (PLSCR1) (a 37 kDa protein), is involved in rapid Ca 2+ dependent transbilayer redistribution of plasma membrane phospholipids. Recently the function of PLSCR1 as a phospholipids translocator has been challenged and evidences suggest that PLSCR1 acts as signaling molecule. It has been shown to be involved in protein phosphorylation and as a potential activator of genes in response to interferon and other cytokines. Interferon induced rapid biosynthesis of PLSCR1 targets some of the protein into the nucleus, where it binds to the promoter region of inositol 1,4,5-triphosphate (IP3) receptor type 1 (IP3R1) gene and induces its expression. Palmitoylation of PLSCR1 acts as a switch, controlling its localization either to the PM or inside the nucleus. In the present review, we discuss the current understanding of PLSCR1 in relation to its trafficking, localization and signaling functions.

Research paper thumbnail of Cholesterol depletion modulates detergent resistant fraction of human serotonin1A receptors

Insolubility of membrane components in non-ionic detergents such as Triton X-100 at low temperatu... more Insolubility of membrane components in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion to identify, isolate and characterize membrane domains. In this work, we monitored the detergent insolubility of the serotonin 1A receptor in CHO cell membranes and its modulation by membrane cholesterol. The serotonin 1A receptor is an important member of the G-protein coupled receptor family. It is implicated in the generation and modulation of various cognitive, behavioral and developmental functions and serves as a drug target. Our results show that a significant fraction (~28%) of the serotonin 1A receptor resides in detergent-resistant membranes (DRMs). Interestingly, the fraction of the serotonin 1A receptor in DRMs exhibits a reduction upon membrane cholesterol depletion. In addition, we show that contents of DRM markers such as flotillin-1, caveolin-1 and GM 1 are altered in DRMs upon cholesterol depletion. These results assume significance since the function of the serotonin 1A receptor has previously been shown to be affected by membrane lipids, specifically cholesterol. Our results are relevant in the context of membrane organization of the serotonin 1A receptor in particular, and G-protein coupled receptors in general.

Research paper thumbnail of Industrially Important Carbohydrate Degrading Enzymes from Yeasts: Pectinases, Chitinases, and β-1, 3-Glucanases

… : Diversity and Applications, 2009

Contents 30.1 Introduction ......................................................................... more Contents 30.1 Introduction .................................................................................................................. 673 30.2 Biological Significance of Carbohydrate Degrading Enzymes ................................... 676 30.2.1 Biological Significance of Pectinases ................................ ... ... T. Satyanarayana, G. ...

Research paper thumbnail of GroES and GroEL are essential chaperones for refolding of recombinant human phospholipid scramblase 1 in E. coli

Biotechnology letters, 2009

Human phospholipid scramblase 1(hPL SCR1), when expressed in E. coli (BL-21 DE3), forms inclusion... more Human phospholipid scramblase 1(hPL SCR1), when expressed in E. coli (BL-21 DE3), forms inclusion bodies that are functionally inactive. We studied the effects of various stress inducing agents and chaperones on soluble expression of hPLSCR1 in E. coli (BL-21 ...

Research paper thumbnail of Phospholipid scramblases: an overview

Archives of biochemistry …, 2007

Phospholipid scramblases are a group of homologous proteins that are conserved in all eukaryotic ... more Phospholipid scramblases are a group of homologous proteins that are conserved in all eukaryotic organisms. They are believed to be involved in destroying plasma membrane phospholipid asymmetry at critical cellular events like cell activation, injury and apoptosis. However, a detailed mechanism of phospholipid scrambling still awaits a proper understanding. The most studied member of this family, phospholipid scramblase 1 (PLSCR1) (a 37 kDa protein), is involved in rapid Ca 2+ dependent transbilayer redistribution of plasma membrane phospholipids. Recently the function of PLSCR1 as a phospholipids translocator has been challenged and evidences suggest that PLSCR1 acts as signaling molecule. It has been shown to be involved in protein phosphorylation and as a potential activator of genes in response to interferon and other cytokines. Interferon induced rapid biosynthesis of PLSCR1 targets some of the protein into the nucleus, where it binds to the promoter region of inositol 1,4,5-triphosphate (IP3) receptor type 1 (IP3R1) gene and induces its expression. Palmitoylation of PLSCR1 acts as a switch, controlling its localization either to the PM or inside the nucleus. In the present review, we discuss the current understanding of PLSCR1 in relation to its trafficking, localization and signaling functions.

Research paper thumbnail of Biochemical Characterization of H2O2-Induced Oxidative Stress in E.coli

Journal of Applied Microbiology and Biochemistry, 2018

H 2 O 2-induced toxicity led to remodeling of phospholipid (PL) in E. coli (DH5α) grown in LB med... more H 2 O 2-induced toxicity led to remodeling of phospholipid (PL) in E. coli (DH5α) grown in LB medium at 25°C. A dose-dependent enhancement in conjugated diene content of total cellular lipid extract was observed when E. coli (DH5α) was grown in LB containing 1 to 10 mM H 2 O 2. Elevated conjugated diene content was accompanied by a dose-dependent augmentation of cardiolipin (CL) by two fold with 20% depletion in sum total of phosphatidyl ethanolamine (PE) and phosphatidyl glycerol (PG) [(PE+PG)] content without altering total PL content. A 75% depletion of catalase activity and a dose-dependent variation in cellular free iron content were the oxidative-stress regulatory mechanism in E. coli (DH5α) in response to elevated level of extrinsic H 2 O 2. These findings suggest that E. coli responds to H 2 O 2-induced toxicity by regulating cellular-free iron content and by modulating cellular PL composition. As CL is known to be augmentated in multiple stress conditions, we hypothesize that H 2 O 2-induced augmentation in CL content of E. coli (DH5 α) is a regulatory mechanism to survive oxidative stress. Our findings revel that exogenous H 2 O 2 induces a lipid-mediated oxidative stress regulatory mechanism in E. coli.

Research paper thumbnail of Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell

Biochimica et Biophysica Acta ( …, 2009

Background: Phospholipid scramblases are a group of four homologous proteins conserved from C. el... more Background: Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca 2+ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca 2+ /Mg 2+ binding to human scramblases and conformational changes taking place in them remains unknown. Methods: In the present study, we analyzed the Ca 2+ and Mg 2+ binding to the calcium binding motifs of hPLSCR1-4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry. Results: The results in this study show that (i) affinities of the peptides are in the order hPLSCR1 N hPLSCR3 N hPLSCR2 N hPLSCR4 for Ca 2+ and in the order hPLSCR1 N hPLSCR2 N hPLSCR3 N hPLSCR4 for Mg 2+ , (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca 2+ and Mg 2+ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families. Conclusions: Based on the above results, we hypothesize that the Ca 2+ binding motif of hPLSCR1 is a novel type of Ca 2+ binding motif. General significance: Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.

Research paper thumbnail of Lipid Flip-Flop-Inducing Antimicrobial Phytochemicals from Gymnema sylvestre are Bacterial Membrane Permeability Enhancers

ACS Omega, 2021

An amphiphilic phytochemical fraction isolated from methanol extract of Gymnema sylvestre leaf po... more An amphiphilic phytochemical fraction isolated from methanol extract of Gymnema sylvestre leaf powder contained six terpenoids, two flavonoids, and one alkaloid that induced rapid flip-flop of fluorescent phospholipid analog in the phosphatidyl choline bilayer. Lipid-flipping activity of the methanol-extracted fraction of G. sylvestre (MEFGS) was dose-dependent and time-dependent with a rate constant k = (12.09 ± 0.94) mg −1 min −1 that was saturable at (40 ± 1) % flipping of the fluorescent lipid analogue. Interactions of MEFGS phytochemicals with large unilamelar vesicles led to time-dependent change in their rounded morphology into irregular shapes, indicating their membranedestabilizing activity. MEFGS exhibited antibacterial activity on Escherichia coli (MTCC-118), Staphylococcus aureus (MTCC-212), and Pseudomonas aeruginosa (MTCC-1035) with IC 50 values 0.5, 0.35, and 0.1 mg/mL, respectively. Phytochemicals in MEFGS increased membrane permeabilization in all three bacteria, as indicated by 23, 17, and 17% increase in the uptake of crystal violet, respectively. MEFGS enhanced membrane damage, resulting in a 3−5 fold increase in leakage of cytosolic ions, 0.5−2 fold increase in leakage of PO 4 − , and 15−20% increase in loss of cellular proteins. MEFGS synergistically increased the efficacy of curcumin, amoxillin, ampicillin, and cefotaxime on S. aureus probably by enhancing their permeability into the bacterium. For the first time, our study reveals that phytochemicals from G. sylvestre enhance the permeability of the bacterial plasma membrane by facilitating flip-flop of membrane lipids. Lipidflipping phytochemicals from G. sylvestre can be used as adjuvant therapeutics to enhance the efficacy of antibacterials by increasing their bioavailability in the target bacteria.

Research paper thumbnail of Evaluation of Biotechnological Potential of Novel Mercury Tolerant Strain of Klebsiella Pneumonia

Journal of Applied Microbiology and Biochemistry, 2018

Research paper thumbnail of Prediction of Sub-cellular Localization of Scramblase Protein Family

International Journal of Computer and Communication Technology, 2010

In the present work, we discuss an anaysis about the localization of different members of scrambl... more In the present work, we discuss an anaysis about the localization of different members of scramblase protein family. Different scramblase sequences were picked up from organisms of all eukaryotic phyla and their localization were predicted using the P-SORT programme. Our analysis showed that the scramblase protein family shows multiple subcellular localization. Most proteins were found to be localized to the cytoplasm, where as others were found to be present in the nucleus or mitochondria. Interestingly, we found that in yeast, all putative scramblases were localized in the nucleus with a reliability of more than 95%. Our analysis shows that scramblases are a family of protein having diversed cellular localization and hence hypothesized to be performing multiple cellular functions in various organisms.

Research paper thumbnail of Electroformation of liposomes and phytosomes using copper electrode

Journal of Liposome Research

Abstract A novel method for electroformation of liposomes and phytosomes using copper electrode i... more Abstract A novel method for electroformation of liposomes and phytosomes using copper electrode is described. Liposomes made at 2 V and 10 Hz AC field from L-α-egg-phosphatidylcholine (egg-PC), K. pneumoniae phosphatidylethanolamine, K. pneumoniae polar lipids and E. coli polar lipids on copper electrode were (777.9 ± 118.4), (370.2 ± 100.5), (825.3 ± 21.54), and (281.3 ± 42.3) nm in diameter, respectively. Giant vesicles were formed at 30 V and 10 Hz AC field from polar lipids of K. pneumoniae and E. coli were (106 ± 29.7) and (86 ± 24.3) µm in diameter, respectively. All liposomes were unilamellar as indicated by their unilamellar indices of 50 ± 2, had surface charge comparable to vesicles made from lipid(s) with similar composition and exhibited only 1–2 mol% of oxidized lipids. Cu concentration in the liposomal samples was <1.5 ppm for large unilamellar vesicles (LUVs) and ˂5 ppm for giant unilamellar vesicles (GUVs). The vesicles were stable for >15 d without loss of their size, charge, or unilamellarity. The method was successfully applied to prepare phytosomes from egg-PC and a phytochemical fraction of Dimorphocalyx glabellus, a medicinal plant with anti-diuretic properties. Phytosomes formed were 1000–1500 nm in diameter and exhibited altered fluorescence and absorbance properties compared to the unencapsulated phytochemical. Phytosomes with phytochemical: egg-PC ratio from 0.15 to 1.5 had encapsulation efficiency ranging 90–30%, respectively, and was stable for 1 month. Our method is easy, inexpensive and convenient that will prove to be useful for preparation of liposomes and phytosomes.

Research paper thumbnail of Copper and iron overload protect Escherichia coli from exogenous H2O2 by modulating membrane phospholipid composition

Environmental Sustainability

Development of green technologies for sustainable development requires growth of bacteria in medi... more Development of green technologies for sustainable development requires growth of bacteria in medium containing H 2 O 2 from 50 to 150 mM that may contain millimolar level of copper (Cu) and iron (Fe). However, the mechanism of oxidative stress involving high concentration of exogenous H 2 O 2 in bacteria and the effect of Cu and Fe overload on the same remain controversial. In the present study, we investigated the effect of millimolar concentration of Cu and Fe on H 2 O 2-induced oxidative stress using the enterotoxigenic Escherichia coli 12566 as a model system. Our results indicate that at millimolar concentration, Fe and Cu protect E. coli from H 2 O 2-induced oxidative stress by (1) suppressing H 2 O 2-induced growth inhibition, (2) reducing H 2 O 2-induced lipid peroxidation, (3) decreasing catalase activity and (4) modulating H 2 O 2-induced release of cytosolic Fe content in a dose-dependent manner. A previous study by our group shows that H 2 O 2 significantly alters the phospholipid (PL) composition in E. coli by a dose-dependent increase in cardiolipin (CL) accompanied by decrease in sum total of phosphatidyl ethanolamine (PE) and phosphatidyl glycerol (PG). In the present study, we show that Cu 2+ and Fe 3+ at 1 mM reduce H 2 O 2-induced change in PL composition of E. coli leading to stabilization of its membrane components. For the first time, our results show that overloading of E. coli with Cu 2+ and Fe 3+ activates a protective mechanism against H 2 O 2-induced oxidative damage by modulating its membrane PL composition. Our study reveals a pivotal regulatory role of these metal ions in the sectors of sustainable technologies such as heavy metal bioremediation, sewage treatment and bioelectrochemical reactors that require microbial growth at high concentration of H 2 O 2 .

Research paper thumbnail of Sequence Analysis of a Subset of Plasma Membrane Raft Proteome Containing CXXC Metal Binding Motifs

International Journal of Knowledge Discovery in Bioinformatics, 2015

In an attempt to identify the metal sensing proteins localized to mammalian plasma membrane, the ... more In an attempt to identify the metal sensing proteins localized to mammalian plasma membrane, the authors screened a list of 300 raft associated proteins that are involved in cellular signaling mechanisms by searching the presence of metal thionin (CXXC) motifs. 50 proteins were found to possess CXXC motifs that could act as potential metal sensing proteins. The authors determined membrane topologies of the above CXXC motif containing proteins using TM-pred and analyzed the positions of their transmembrane (TM) domains using Bio-edit software. Based on the topology of CXXC domains, the authors classified all the raft-associated metal sensing proteins into six categories. They are (i) Exoplasmic tails with CXXC motif, (ii) Exoplasmic loops with CXXC motif, (iii) Cytosolic tails with CXXC motif, (iv) Cytosolic loop with CXXC motif, (v) TM domains with CXXC motifs, (vi) Proteins with multiple topologies of CXXC motif. The authors' study will lead to understanding of the raft-mediate...

Research paper thumbnail of Industrially Important Carbohydrate Degrading Enzymes from Yeasts: Pectinases, Chitinases, and β-1,3-Glucanases

Yeast Biotechnology: Diversity and Applications, 2009

Contents 30.1 Introduction ......................................................................... more Contents 30.1 Introduction .................................................................................................................. 673 30.2 Biological Significance of Carbohydrate Degrading Enzymes ................................... 676 30.2.1 Biological Significance of Pectinases ................................ ... ... T. Satyanarayana, G. ...

Research paper thumbnail of Cholesterol depletion modulates detergent resistant fraction of human serotonin 1A receptors

Molecular Membrane Biology, 2012

Insolubility of membrane components in non-ionic detergents such as Triton X-100 at low temperatu... more Insolubility of membrane components in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion to identify, isolate and characterize membrane domains. In this work, we monitored the detergent insolubility of the serotonin 1A receptor in CHO cell membranes and its modulation by membrane cholesterol. The serotonin 1A receptor is an important member of the G-protein coupled receptor family. It is implicated in the generation and modulation of various cognitive, behavioral and developmental functions and serves as a drug target. Our results show that a significant fraction (~28%) of the serotonin 1A receptor resides in detergent-resistant membranes (DRMs). Interestingly, the fraction of the serotonin 1A receptor in DRMs exhibits a reduction upon membrane cholesterol depletion. In addition, we show that contents of DRM markers such as flotillin-1, caveolin-1 and GM 1 are altered in DRMs upon cholesterol depletion. These results assume significance since the function of the serotonin 1A receptor has previously been shown to be affected by membrane lipids, specifically cholesterol. Our results are relevant in the context of membrane organization of the serotonin 1A receptor in particular, and G-protein coupled receptors in general.

Research paper thumbnail of Flippase Activity in Proteoliposomes Reconstituted with Spinacea oleracea Endoplasmic Reticulum Membrane Proteins: Evidence of Biogenic Membrane Flippase in Plants †

Biochemistry, 2008

Phospholipid translocation (flip-flop) in biogenic (self-synthesizing) membranes such as the endo... more Phospholipid translocation (flip-flop) in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum of eukaryotic cells (rat liver) and bacterial cytoplasmic membranes is a fundamental step in membrane biogenesis. It is known that flip-flop in these membranes occurs without a metabolic energy requirement, bidirectionally with no specificity for phospholipid headgroup. In this study, we demonstrate for the first time ATP-independent flippase activity in endoplasmic reticulum membranes of plants using spinach as a model system. For this, we generated proteoliposomes from a Triton X-100 extract of endoplasmic reticulum membranes of spinach and assayed them for flippase activity using fluorescently labeled phospholipids. The half-time for flipping was found to be 0.7-1.0 min. We also show that (a) proteoliposomes can flip fluorescently labeled analogues of phosphatidylcholine and phosphatidylethanolamine, (b) flipping activity is protein-mediated, (c) more than one class of lipid translocator (flippase) is present in spinach membranes, based on the sensitivity to protease and protein-modifying reagents, and (d) translocation of PC and PE is affected differently upon treatment with protease and protein-modifying reagents. Ca (2+)-dependent scrambling activity was not observed in the vesicles reconstituted from plant ER membranes, ruling out the possibility of the involvement of scramblase in translocation of phospholipids. These results suggest the existence of biogenic membrane flippases in plants and that the mechanism of membrane biogenesis is similar to that found in animals.

Research paper thumbnail of Phospholipid scramblases: An overview

Archives of Biochemistry and Biophysics, 2007

Phospholipid scramblases are a group of homologous proteins that are conserved in all eukaryotic ... more Phospholipid scramblases are a group of homologous proteins that are conserved in all eukaryotic organisms. They are believed to be involved in destroying plasma membrane phospholipid asymmetry at critical cellular events like cell activation, injury and apoptosis. However, a detailed mechanism of phospholipid scrambling still awaits a proper understanding. The most studied member of this family, phospholipid scramblase 1 (PLSCR1) (a 37 kDa protein), is involved in rapid Ca 2+ dependent transbilayer redistribution of plasma membrane phospholipids. Recently the function of PLSCR1 as a phospholipids translocator has been challenged and evidences suggest that PLSCR1 acts as signaling molecule. It has been shown to be involved in protein phosphorylation and as a potential activator of genes in response to interferon and other cytokines. Interferon induced rapid biosynthesis of PLSCR1 targets some of the protein into the nucleus, where it binds to the promoter region of inositol 1,4,5-triphosphate (IP3) receptor type 1 (IP3R1) gene and induces its expression. Palmitoylation of PLSCR1 acts as a switch, controlling its localization either to the PM or inside the nucleus. In the present review, we discuss the current understanding of PLSCR1 in relation to its trafficking, localization and signaling functions.

Research paper thumbnail of Cholesterol depletion modulates detergent resistant fraction of human serotonin1A receptors

Insolubility of membrane components in non-ionic detergents such as Triton X-100 at low temperatu... more Insolubility of membrane components in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion to identify, isolate and characterize membrane domains. In this work, we monitored the detergent insolubility of the serotonin 1A receptor in CHO cell membranes and its modulation by membrane cholesterol. The serotonin 1A receptor is an important member of the G-protein coupled receptor family. It is implicated in the generation and modulation of various cognitive, behavioral and developmental functions and serves as a drug target. Our results show that a significant fraction (~28%) of the serotonin 1A receptor resides in detergent-resistant membranes (DRMs). Interestingly, the fraction of the serotonin 1A receptor in DRMs exhibits a reduction upon membrane cholesterol depletion. In addition, we show that contents of DRM markers such as flotillin-1, caveolin-1 and GM 1 are altered in DRMs upon cholesterol depletion. These results assume significance since the function of the serotonin 1A receptor has previously been shown to be affected by membrane lipids, specifically cholesterol. Our results are relevant in the context of membrane organization of the serotonin 1A receptor in particular, and G-protein coupled receptors in general.

Research paper thumbnail of Industrially Important Carbohydrate Degrading Enzymes from Yeasts: Pectinases, Chitinases, and β-1, 3-Glucanases

… : Diversity and Applications, 2009

Contents 30.1 Introduction ......................................................................... more Contents 30.1 Introduction .................................................................................................................. 673 30.2 Biological Significance of Carbohydrate Degrading Enzymes ................................... 676 30.2.1 Biological Significance of Pectinases ................................ ... ... T. Satyanarayana, G. ...

Research paper thumbnail of GroES and GroEL are essential chaperones for refolding of recombinant human phospholipid scramblase 1 in E. coli

Biotechnology letters, 2009

Human phospholipid scramblase 1(hPL SCR1), when expressed in E. coli (BL-21 DE3), forms inclusion... more Human phospholipid scramblase 1(hPL SCR1), when expressed in E. coli (BL-21 DE3), forms inclusion bodies that are functionally inactive. We studied the effects of various stress inducing agents and chaperones on soluble expression of hPLSCR1 in E. coli (BL-21 ...

Research paper thumbnail of Phospholipid scramblases: an overview

Archives of biochemistry …, 2007

Phospholipid scramblases are a group of homologous proteins that are conserved in all eukaryotic ... more Phospholipid scramblases are a group of homologous proteins that are conserved in all eukaryotic organisms. They are believed to be involved in destroying plasma membrane phospholipid asymmetry at critical cellular events like cell activation, injury and apoptosis. However, a detailed mechanism of phospholipid scrambling still awaits a proper understanding. The most studied member of this family, phospholipid scramblase 1 (PLSCR1) (a 37 kDa protein), is involved in rapid Ca 2+ dependent transbilayer redistribution of plasma membrane phospholipids. Recently the function of PLSCR1 as a phospholipids translocator has been challenged and evidences suggest that PLSCR1 acts as signaling molecule. It has been shown to be involved in protein phosphorylation and as a potential activator of genes in response to interferon and other cytokines. Interferon induced rapid biosynthesis of PLSCR1 targets some of the protein into the nucleus, where it binds to the promoter region of inositol 1,4,5-triphosphate (IP3) receptor type 1 (IP3R1) gene and induces its expression. Palmitoylation of PLSCR1 acts as a switch, controlling its localization either to the PM or inside the nucleus. In the present review, we discuss the current understanding of PLSCR1 in relation to its trafficking, localization and signaling functions.

Research paper thumbnail of Biochemical Characterization of H2O2-Induced Oxidative Stress in E.coli

Journal of Applied Microbiology and Biochemistry, 2018

H 2 O 2-induced toxicity led to remodeling of phospholipid (PL) in E. coli (DH5α) grown in LB med... more H 2 O 2-induced toxicity led to remodeling of phospholipid (PL) in E. coli (DH5α) grown in LB medium at 25°C. A dose-dependent enhancement in conjugated diene content of total cellular lipid extract was observed when E. coli (DH5α) was grown in LB containing 1 to 10 mM H 2 O 2. Elevated conjugated diene content was accompanied by a dose-dependent augmentation of cardiolipin (CL) by two fold with 20% depletion in sum total of phosphatidyl ethanolamine (PE) and phosphatidyl glycerol (PG) [(PE+PG)] content without altering total PL content. A 75% depletion of catalase activity and a dose-dependent variation in cellular free iron content were the oxidative-stress regulatory mechanism in E. coli (DH5α) in response to elevated level of extrinsic H 2 O 2. These findings suggest that E. coli responds to H 2 O 2-induced toxicity by regulating cellular-free iron content and by modulating cellular PL composition. As CL is known to be augmentated in multiple stress conditions, we hypothesize that H 2 O 2-induced augmentation in CL content of E. coli (DH5 α) is a regulatory mechanism to survive oxidative stress. Our findings revel that exogenous H 2 O 2 induces a lipid-mediated oxidative stress regulatory mechanism in E. coli.

Research paper thumbnail of Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell

Biochimica et Biophysica Acta ( …, 2009

Background: Phospholipid scramblases are a group of four homologous proteins conserved from C. el... more Background: Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca 2+ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca 2+ /Mg 2+ binding to human scramblases and conformational changes taking place in them remains unknown. Methods: In the present study, we analyzed the Ca 2+ and Mg 2+ binding to the calcium binding motifs of hPLSCR1-4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry. Results: The results in this study show that (i) affinities of the peptides are in the order hPLSCR1 N hPLSCR3 N hPLSCR2 N hPLSCR4 for Ca 2+ and in the order hPLSCR1 N hPLSCR2 N hPLSCR3 N hPLSCR4 for Mg 2+ , (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca 2+ and Mg 2+ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families. Conclusions: Based on the above results, we hypothesize that the Ca 2+ binding motif of hPLSCR1 is a novel type of Ca 2+ binding motif. General significance: Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.