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Kausik Chattopadhyay - One of the best experts on this subject based on the ideXlab platform.
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Physicochemical constraints of elevated pH affect efficient Membrane interaction and arrest an abortive Membrane-bound oligomeric intermediate of the beta-barrel Pore-forming toxin Vibrio cholerae cytolysin.
Archives of biochemistry and biophysics, 2015Co-Authors: Anand Kumar Rai, Nidhi Kundu, Kausik ChattopadhyayAbstract:Vibrio cholerae cytolysin (VCC) is a potent Membrane-damaging cytotoxic protein. VCC causes permeabilization of the target cell Membranes by forming transMembrane oligomeric beta-barrel Pores. Membrane Pore formation by VCC involves following key steps: (i) Membrane binding, (ii) formation of a pre-Pore oligomeric intermediate, (iii) Membrane insertion of the Pore-forming motifs, and (iv) formation of the functional transMembrane Pore. Membrane binding, oligomerization, and subsequent Pore-formation process of VCC appear to be facilitated by multiple regulatory mechanisms that are only partly understood. Here, we have explored the role(s) of the physicochemical constraints, specifically imposed by the elevated pH conditions, on the Membrane Pore-formation mechanism of VCC. Elevated pH abrogates efficient interaction of VCC with the target Membranes, and blocks its Pore-forming activity. Under the elevated pH conditions, Membrane-bound fractions of VCC remain trapped in the form of abortive oligomeric species that fail to generate the functional transMembrane Pores. Such an abortive oligomeric assembly appears to represent a distinct, more advanced intermediate state than the pre-Pore state. The present study offers critical insights regarding the implications of the physicochemical constraints for regulating the efficient Membrane interaction and Pore formation by VCC.
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trapping of vibrio cholerae cytolysin in the Membrane bound monomeric state blocks Membrane insertion and functional Pore formation by the toxin
Journal of Biological Chemistry, 2014Co-Authors: Anand Kumar Rai, Kausik ChattopadhyayAbstract:Vibrio cholerae cytolysin (VCC) is a potent Membrane-damaging cytolytic toxin that belongs to the family of β barrel Pore-forming protein toxins. VCC induces lysis of its target eukaryotic cells by forming transMembrane oligomeric β barrel Pores. The mechanism of Membrane Pore formation by VCC follows the overall scheme of the archetypical β barrel Pore-forming protein toxin mode of action, in which the water-soluble monomeric form of the toxin first binds to the target cell Membrane, then assembles into a prePore oligomeric intermediate, and finally converts into the functional transMembrane oligomeric β barrel Pore. However, there exists a vast knowledge gap in our understanding regarding the intricate details of the Membrane Pore formation process employed by VCC. In particular, the Membrane oligomerization and Membrane insertion steps of the process have only been described to a limited extent. In this study, we determined the key residues in VCC that are critical to trigger Membrane oligomerization of the toxin. Alteration of such key residues traps the toxin in its Membrane-bound monomeric state and abrogates subsequent oligomerization, Membrane insertion, and functional transMembrane Pore-formation events. The results obtained from our study also suggest that the Membrane insertion of VCC depends critically on the oligomerization process and that it cannot be initiated in the Membrane-bound monomeric form of the toxin. In sum, our study, for the first time, dissects Membrane binding from the subsequent oligomerization and Membrane insertion steps and, thus, defines the exact sequence of events in the Membrane Pore formation process by VCC.
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functional mapping of the lectin activity site on the β prism domain of vibrio cholerae cytolysin implications for the Membrane Pore formation mechanism of the toxin
Journal of Biological Chemistry, 2013Co-Authors: Anand Kumar Rai, Karan Paul, Kausik ChattopadhyayAbstract:Vibrio cholerae cytolysin (VCC) is a prominent member in the family of β-barrel Pore-forming toxins. It induces lysis of target eukaryotic cells by forming transMembrane oligomeric β-barrel channels. VCC also exhibits prominent lectin-like activity in interacting with β1-galactosyl-terminated glycoconjugates. Apart from the cytolysin domain, VCC harbors two lectin-like domains: the β-Trefoil and the β-Prism domains; however, precise contribution of these domains in the lectin property of VCC is not known. Also, role(s) of these lectin-like domains in the mode of action of VCC remain obscure. In the present study, we show that the β-Prism domain of VCC acts as the structural scaffold to determine the lectin activity of the protein toward β1-galactosyl-terminated glycoconjugates. Toward exploring the physiological implication of the β-Prism domain, we demonstrate that the presence of the β-Prism domain-mediated lectin activity is crucial for an efficient interaction of the toxin toward the target cells. Our results also suggest that such lectin activity may act to regulate the oligomerization ability of the Membrane-bound VCC toxin. Based on the data presented here, and also consistent with the existing structural information, we propose a novel mechanism of regulation imposed by the β-Prism domain's lectin activity, implicated in the process of Membrane Pore formation by VCC.
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single point mutation in vibrio cholerae cytolysin compromises the Membrane Pore formation mechanism of the toxin
FEBS Journal, 2012Co-Authors: Karan Paul, Kausik ChattopadhyayAbstract:Vibrio cholerae cytolysin (VCC) belongs to the family of β-barrel Pore-forming protein toxins. VCC is secreted by the bacteria as water-soluble monomers, which upon binding to target eukaryotic cells form transMembrane heptameric β-barrel channels. High-resolution 3D structures are described both for the water-soluble monomeric form and the transMembrane oligomeric Pore; albeit that our understanding of the mechanistic details of the Membrane Pore-formation process remains incomplete. Here, we report the characterization of a nonfunctional VCC variant harboring a single point mutation of Ala425Val positioned within a potential Membrane-interacting loop in the VCC structure. The mutation appears to affect interaction of the toxin with erythrocytes as well as cholesterol-containing liposome Membrane, without affecting the oligomerization ability of the Membrane-bound toxin molecules. The Membrane-bound oligomers formed by this VCC mutant do not appear to represent the functional Pore assembly of the toxin; rather, such assembly could be considered as being trapped in an abortive, nonfunctional oligomeric state. Our results suggest that the Ala425Val mutation in VCC critically compromises its cholesterol-dependent Membrane-interaction mechanism and also abrogates the process of functional Membrane Pore formation by the toxin.
Dusan Turk - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the soluble form of equinatoxin ii a Pore forming toxin from the sea anemone actinia equina
Structure, 2001Co-Authors: Alekos Athanasiadis, Peter Macek, Gregor Anderluh, Dusan TurkAbstract:BACKGROUND: Membrane Pore-forming toxins have a remarkable property: they adopt a stable soluble form structure, which, when in contact with a Membrane, undergoes a series of transformations, leading to an active, Membrane-bound form. In contrast to bacterial toxins, no structure of a Pore-forming toxin from an eukaryotic organism has been determined so far, an indication that structural studies of equinatoxin II (EqtII) may unravel a novel mechanism. RESULTS: The crystal structure of the soluble form of EqtII from the sea anemone Actinia equina has been determined at 1.9 A resolution. EqtII is shown to be a single-domain protein based on a 12 strand beta sandwich fold with a hydrophobic core and a pair of alpha helices, each of which is associated with the face of a beta sheet. CONCLUSIONS: The structure of the 30 N-terminal residues is the largest segment that can adopt a different structure without disrupting the fold of the beta sandwich core. This segment includes a three-turn alpha helix that lies on the surface of a beta sheet and ends in a stretch of three positively charged residues, Lys-30, Arg-31, and Lys-32. On the basis of gathered data, it is suggested that this segment forms the Membrane Pore, whereas the beta sandwich structure remains unaltered and attaches to a Membrane as do other structurally related extrinsic Membrane proteins or their domains. The use of a structural data site-directed mutagenesis study should reveal the residues involved in Membrane Pore formation.
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crystal structure of the soluble form of equinatoxin ii a Pore forming toxin from the sea anemone actinia equina
Structure, 2001Co-Authors: Alekos Athanasiadis, Peter Macek, Gregor Anderluh, Dusan TurkAbstract:Abstract Background: Membrane Pore–forming toxins have a remarkable property: they adopt a stable soluble form structure, which, when in contact with a Membrane, undergoes a series of transformations, leading to an active, Membrane-bound form. In contrast to bacterial toxins, no structure of a Pore-forming toxin from an eukaryotic organism has been determined so far, an indication that structural studies of equinatoxin II (EqtII) may unravel a novel mechanism. Results: The crystal structure of the soluble form of EqtII from the sea anemone Actinia equina has been determined at 1.9 A resolution. EqtII is shown to be a single-domain protein based on a 12 strand β sandwich fold with a hydrophobic core and a pair of α helices, each of which is associated with the face of a β sheet. Conclusions: The structure of the 30 N-terminal residues is the largest segment that can adopt a different structure without disrupting the fold of the β sandwich core. This segment includes a three-turn α helix that lies on the surface of a β sheet and ends in a stretch of three positively charged residues, Lys-30, Arg-31, and Lys-32. On the basis of gathered data, it is suggested that this segment forms the Membrane Pore, whereas the β sandwich structure remains unaltered and attaches to a Membrane as do other structurally related extrinsic Membrane proteins or their domains. The use of a structural data site-directed mutagenesis study should reveal the residues involved in Membrane Pore formation.
Long D. Nghiem - One of the best experts on this subject based on the ideXlab platform.
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trace organic contaminant rejection by aquaporin forward osmosis Membrane transport mechanisms and Membrane stability
Water Research, 2018Co-Authors: Long D. Nghiem, Chuyang Y Tang, Stephen GrayAbstract:Abstract We investigated transport mechanisms of trace organic contaminants (TrOCs) through aquaporin thin-film composite forward osmosis (FO) Membrane, and Membrane stability under extreme conditions with respect to TrOC rejections. Morphology and surface chemistry of the aquaporin Membrane were characterised to identify the incorporation of aquaporin vesicles into Membrane active layer. Pore hindrance model was used to estimate aquaporin Membrane Pore size as well as to describe TrOC transport. TrOC transport mechanisms were revealed by varying concentration and type of draw solutions. Experimental results showed that mechanism of TrOC transport through aquaporin-embedded FO Membrane was dominated by solution-diffusion mechanism. Non-ionic TrOC rejections were molecular-weight dependent, suggesting steric hindrance mechanisms. On the other hand, ionic TrOC rejections were less sensitive to molecular size, indicating electrostatic interaction. TrOC transport through aquaporin Membrane was also subjected to retarded forward diffusion where reverse draw solute flux could hinder the forward diffusion of feed TrOC solutes, reducing their permeation through the FO Membrane. Aquaporin Membrane stability was demonstrated by either heat treatment or ethanol solvent challenges. Thermal stability of the aquaporin Membrane was manifested as a relatively unchanged TrOC rejection before and after the heat treatment challenge test. By contrast, ethanol solvent challenge resulted in a decrease in TrOC rejection, which was evident by the disappearance of the lipid tail of the aquaporin vesicles from infrared spectrum and a notable decrease in the Membrane Pore size.
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the effects of feed solution temperature on Pore size and trace organic contaminant rejection by the nanofiltration Membrane nf270
Separation and Purification Technology, 2014Co-Authors: Hai Quang Dang, William E Price, Long D. NghiemAbstract:Abstract This study investigated the effect of feed temperature on Membrane Pore size and the rejection of trace organic contaminants (TrOCs) by the nanofiltration (NF) Membrane NF270. Filtration experiments were conducted using a cross flow Membrane system at 20, 30 and 40 °C. The Membrane Pore radius was estimated using the Pore hindrance transport model at each temperature and the rejection data of three reference organic solutes (i.e. erythritol, xylose and glucose) experimentally obtained in this study. The results suggest that the Pore size of an NF Membrane is dependent on the feed solution temperature. An increase in the feed temperature from 20 to 40 °C led to an increase in the effective Pore radius from 0.39 to 0.44 nm. Consequently, the increase in the feed temperature also caused a considerable drop in the rejection of all TrOCs investigated in this study. The decrease in rejection observed here could be attributed to not only the increase in the solute diffusivity but also the enlargement of the Membrane Pore size. As the feed temperature increased, the decrease in rejection of neutral TrOCs was more severe than that of negatively charged compounds. This is because in addition to size exclusion (or steric hindrance) the rejection of negatively charged TrOCs is also governed by electrostatic interaction given that the Membrane surface is also negatively charged.
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effects of Membrane fouling on the nanofiltration of pharmaceutically active compounds phacs mechanisms and role of Membrane Pore size
Separation and Purification Technology, 2007Co-Authors: Long D. Nghiem, Simon HawkesAbstract:The influence of Membrane fouling on the retention of pharmaceutically active compounds (PhACs) by three nanofiltration Membranes was investigated in this study. Membrane fouling was achieved with a foulant cocktail containing model organic foulant in a background electrolyte solution. The effects of Membrane fouling on the separation process was delineated by comparing the retention values of clean and fouled Membranes and relate them to the Membrane properties as well as physicochemical characteristics of the PhACs. Fouling was more severe for the larger Pore size TFC-SR2 and NF 270 Membranes as compared to the smaller Pore size NF 90 Membrane. More importantly, the influence of Membrane fouling on the retention of PhACs was found largely dependent upon Membrane Pore size. It was hypothesised that such influence was governed by three distinctive mechanisms: modification of the Membrane charge surface, Pore restriction, and cake enhanced concentration polarisation. The presence of the fouling layer could affect the retention behavior of charged solutes by altering the Membrane surface charge density. While the role of this surface charge modification mechanism was clear for inorganic salts, it was less obvious for the negatively charged pharmaceutical species examined in this investigation, possibly due to the interference of the Pore restriction mechanism. Behavior of the very loose TFC-SR2 Membrane was found dominated by Pore restriction and this Membrane consistently showed an increase in retention under fouled conditions. In contrast, evidence of the cake enhanced concentration polarisation effect was observed with the smaller Pore size NF 270 and NF 90 Membranes.
Anand Kumar Rai - One of the best experts on this subject based on the ideXlab platform.
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Physicochemical constraints of elevated pH affect efficient Membrane interaction and arrest an abortive Membrane-bound oligomeric intermediate of the beta-barrel Pore-forming toxin Vibrio cholerae cytolysin.
Archives of biochemistry and biophysics, 2015Co-Authors: Anand Kumar Rai, Nidhi Kundu, Kausik ChattopadhyayAbstract:Vibrio cholerae cytolysin (VCC) is a potent Membrane-damaging cytotoxic protein. VCC causes permeabilization of the target cell Membranes by forming transMembrane oligomeric beta-barrel Pores. Membrane Pore formation by VCC involves following key steps: (i) Membrane binding, (ii) formation of a pre-Pore oligomeric intermediate, (iii) Membrane insertion of the Pore-forming motifs, and (iv) formation of the functional transMembrane Pore. Membrane binding, oligomerization, and subsequent Pore-formation process of VCC appear to be facilitated by multiple regulatory mechanisms that are only partly understood. Here, we have explored the role(s) of the physicochemical constraints, specifically imposed by the elevated pH conditions, on the Membrane Pore-formation mechanism of VCC. Elevated pH abrogates efficient interaction of VCC with the target Membranes, and blocks its Pore-forming activity. Under the elevated pH conditions, Membrane-bound fractions of VCC remain trapped in the form of abortive oligomeric species that fail to generate the functional transMembrane Pores. Such an abortive oligomeric assembly appears to represent a distinct, more advanced intermediate state than the pre-Pore state. The present study offers critical insights regarding the implications of the physicochemical constraints for regulating the efficient Membrane interaction and Pore formation by VCC.
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trapping of vibrio cholerae cytolysin in the Membrane bound monomeric state blocks Membrane insertion and functional Pore formation by the toxin
Journal of Biological Chemistry, 2014Co-Authors: Anand Kumar Rai, Kausik ChattopadhyayAbstract:Vibrio cholerae cytolysin (VCC) is a potent Membrane-damaging cytolytic toxin that belongs to the family of β barrel Pore-forming protein toxins. VCC induces lysis of its target eukaryotic cells by forming transMembrane oligomeric β barrel Pores. The mechanism of Membrane Pore formation by VCC follows the overall scheme of the archetypical β barrel Pore-forming protein toxin mode of action, in which the water-soluble monomeric form of the toxin first binds to the target cell Membrane, then assembles into a prePore oligomeric intermediate, and finally converts into the functional transMembrane oligomeric β barrel Pore. However, there exists a vast knowledge gap in our understanding regarding the intricate details of the Membrane Pore formation process employed by VCC. In particular, the Membrane oligomerization and Membrane insertion steps of the process have only been described to a limited extent. In this study, we determined the key residues in VCC that are critical to trigger Membrane oligomerization of the toxin. Alteration of such key residues traps the toxin in its Membrane-bound monomeric state and abrogates subsequent oligomerization, Membrane insertion, and functional transMembrane Pore-formation events. The results obtained from our study also suggest that the Membrane insertion of VCC depends critically on the oligomerization process and that it cannot be initiated in the Membrane-bound monomeric form of the toxin. In sum, our study, for the first time, dissects Membrane binding from the subsequent oligomerization and Membrane insertion steps and, thus, defines the exact sequence of events in the Membrane Pore formation process by VCC.
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functional mapping of the lectin activity site on the β prism domain of vibrio cholerae cytolysin implications for the Membrane Pore formation mechanism of the toxin
Journal of Biological Chemistry, 2013Co-Authors: Anand Kumar Rai, Karan Paul, Kausik ChattopadhyayAbstract:Vibrio cholerae cytolysin (VCC) is a prominent member in the family of β-barrel Pore-forming toxins. It induces lysis of target eukaryotic cells by forming transMembrane oligomeric β-barrel channels. VCC also exhibits prominent lectin-like activity in interacting with β1-galactosyl-terminated glycoconjugates. Apart from the cytolysin domain, VCC harbors two lectin-like domains: the β-Trefoil and the β-Prism domains; however, precise contribution of these domains in the lectin property of VCC is not known. Also, role(s) of these lectin-like domains in the mode of action of VCC remain obscure. In the present study, we show that the β-Prism domain of VCC acts as the structural scaffold to determine the lectin activity of the protein toward β1-galactosyl-terminated glycoconjugates. Toward exploring the physiological implication of the β-Prism domain, we demonstrate that the presence of the β-Prism domain-mediated lectin activity is crucial for an efficient interaction of the toxin toward the target cells. Our results also suggest that such lectin activity may act to regulate the oligomerization ability of the Membrane-bound VCC toxin. Based on the data presented here, and also consistent with the existing structural information, we propose a novel mechanism of regulation imposed by the β-Prism domain's lectin activity, implicated in the process of Membrane Pore formation by VCC.
Gregor Anderluh - One of the best experts on this subject based on the ideXlab platform.
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Membrane Pore formation at protein lipid interfaces
Trends in Biochemical Sciences, 2014Co-Authors: Robert J C Gilbert, Mauro Dalla Serra, Christopher J Froelich, Mark I Wallace, Gregor AnderluhAbstract:Pore-forming proteins (PFPs) interact with lipid bilayers to compromise Membrane integrity. Many PFPs function by inserting a ring of oligomerized subunits into the bilayer to form a protein-lined hydrophilic channel. However, mounting evidence suggests that PFPs can also generate ‘proteolipidic' Pores by contributing to the fusion of inner and outer bilayer leaflets to form a toroidal structure. We discuss here toroidal Pore formation by peptides including melittin, protegrin, and Alzheimer's Aβ1–41, as well as by PFPs from several evolutionarily unrelated families: the colicin/Bcl-2 grouping including the pro-apoptotic protein Bax, actinoporins derived from sea anemones, and the Membrane attack complex-perforin/cholesterol dependent cytolysin (MACPF/CDC) set of proteins. We also explore how the structure and biological role of toroidal Pores might be investigated further.
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crystal structure of the soluble form of equinatoxin ii a Pore forming toxin from the sea anemone actinia equina
Structure, 2001Co-Authors: Alekos Athanasiadis, Peter Macek, Gregor Anderluh, Dusan TurkAbstract:BACKGROUND: Membrane Pore-forming toxins have a remarkable property: they adopt a stable soluble form structure, which, when in contact with a Membrane, undergoes a series of transformations, leading to an active, Membrane-bound form. In contrast to bacterial toxins, no structure of a Pore-forming toxin from an eukaryotic organism has been determined so far, an indication that structural studies of equinatoxin II (EqtII) may unravel a novel mechanism. RESULTS: The crystal structure of the soluble form of EqtII from the sea anemone Actinia equina has been determined at 1.9 A resolution. EqtII is shown to be a single-domain protein based on a 12 strand beta sandwich fold with a hydrophobic core and a pair of alpha helices, each of which is associated with the face of a beta sheet. CONCLUSIONS: The structure of the 30 N-terminal residues is the largest segment that can adopt a different structure without disrupting the fold of the beta sandwich core. This segment includes a three-turn alpha helix that lies on the surface of a beta sheet and ends in a stretch of three positively charged residues, Lys-30, Arg-31, and Lys-32. On the basis of gathered data, it is suggested that this segment forms the Membrane Pore, whereas the beta sandwich structure remains unaltered and attaches to a Membrane as do other structurally related extrinsic Membrane proteins or their domains. The use of a structural data site-directed mutagenesis study should reveal the residues involved in Membrane Pore formation.
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crystal structure of the soluble form of equinatoxin ii a Pore forming toxin from the sea anemone actinia equina
Structure, 2001Co-Authors: Alekos Athanasiadis, Peter Macek, Gregor Anderluh, Dusan TurkAbstract:Abstract Background: Membrane Pore–forming toxins have a remarkable property: they adopt a stable soluble form structure, which, when in contact with a Membrane, undergoes a series of transformations, leading to an active, Membrane-bound form. In contrast to bacterial toxins, no structure of a Pore-forming toxin from an eukaryotic organism has been determined so far, an indication that structural studies of equinatoxin II (EqtII) may unravel a novel mechanism. Results: The crystal structure of the soluble form of EqtII from the sea anemone Actinia equina has been determined at 1.9 A resolution. EqtII is shown to be a single-domain protein based on a 12 strand β sandwich fold with a hydrophobic core and a pair of α helices, each of which is associated with the face of a β sheet. Conclusions: The structure of the 30 N-terminal residues is the largest segment that can adopt a different structure without disrupting the fold of the β sandwich core. This segment includes a three-turn α helix that lies on the surface of a β sheet and ends in a stretch of three positively charged residues, Lys-30, Arg-31, and Lys-32. On the basis of gathered data, it is suggested that this segment forms the Membrane Pore, whereas the β sandwich structure remains unaltered and attaches to a Membrane as do other structurally related extrinsic Membrane proteins or their domains. The use of a structural data site-directed mutagenesis study should reveal the residues involved in Membrane Pore formation.