Presence Of Cholesterol In Membranes Research Articles

Strategic Design of Tryptophan-Aspartic Acid-Containing Peptide Inhibitors Using Coronin 1 as a Template: Inhibition of Fusion by Enhancing Acyl Chain Order.

Enveloped viruses enter the host cell by fusing at the cell membrane or entering the cell via endocytosis and fusing at the endosome. Conventional inhibitors target the viral fusion protein to inactivate it for inducing fusion. These target-specific vis-à-vis virus-specific inhibitors fail to display their inhibitory efficacy against emerging and remerging viral infections. This necessitates the need to develop broad-spectrum entry inhibitors that are effective irrespective of the virus. Using a broad range of targeting techniques, the fusion inhibitors can modify the physical characteristics of the viral membrane, making it less prone to fusion. We have previously shown that two tryptophan-aspartic acid (WD)-containing hydrophobic peptides, TG-23 and GG-21, from coronin 1, a phagosomal protein, inhibit membrane fusion by modulating membrane organization and dynamics. In the present work, we designed two WD-containing hydrophilic peptides, QG-22 and AG-22, using coronin 1 as a template and evaluated their fusion inhibitory efficacies in the absence and presence of membrane cholesterol. Our results demonstrate that QG-22 and AG-22 inhibit membrane fusion irrespective of the concentration of membrane cholesterol. Our measurements of depth-dependent membrane organization and dynamics reveal that they impede fusion by enhancing the acyl chain order. Overall, our results validate the hypothesis of designing fusion inhibitors by modulating the membrane's physical properties. In addition, it demonstrates that chain hydrophobicity might not be a critical determinant for the development of peptide-based fusion inhibitors.

A Coronin 1-Derived Peptide Inhibits Membrane Fusion by Modulating Membrane Organization and Dynamics.

Membrane fusion is considered the first step in the entry of enveloped viruses into the host cell. Several targeted strategies have been implemented to block viral entry by limiting the fusion protein to form a six-helix bundle, which is a prerequisite for fusion. Nonetheless, the development of broad-spectrum fusion inhibitors is essential to combat emerging and re-emerging viral infections. TG-23, a coronin 1, a tryptophan-aspartate-rich phagosomal protein-derived peptide, demonstrated inhibition of fusion between small unilamellar vesicles (SUVs) by modulating the membrane's physical properties. However, its inhibitory efficacy reduces with an increasing concentration of membrane cholesterol. The present work aims to develop a fusion inhibitor whose efficacy would be unaltered in the presence of membrane cholesterol. A stretch of the tryptophan-aspartic acid-containing peptide with a similar secondary structure and hydrophobicity profile of TG-23 from coronin 1 was synthesized, and its ability to inhibit SUV-SUV fusion with varying concentrations of membrane cholesterol was evaluated. Our results demonstrate that the GG-21 peptide inhibits fusion irrespective of the cholesterol content of the membrane. We have further evaluated the peptide-induced change in the membrane organization and dynamics utilizing arrays of steady-state and time-resolved fluorescence measurements and correlated these results with their effect on fusion. Interestingly, GG-21 displays inhibitory efficacy in a wide variety of lipid compositions despite having a secondary structure and physical properties similar to those of TG-23. Overall, our results advocate that the secondary structure and physical properties of the peptide may not be sufficient to predict its inhibitory efficacy.

Cholesterol Promotes Cytolysin a Activity by Stabilizing the Intermediates during Pore Formation

Pore-forming proteins (PFP) are cell membrane rupturing proteins and form the largest class of proteins that mediate bacterial virulence. PFPs are secreted as water-soluble monomers that bind strongly to the lipid membrane of eukaryotic cells. They do not contain a signature transmembrane motif, but nevertheless adopt structures that traverse the membrane, thereby allowing the passage of molecules from within the cell to the exterior, resulting in host cell lysis. The conformational transitions of a PFP from a water-soluble structure to a distinct membrane-associated form are not understood in mechanistic detail. In this study, we use single molecule tracking and spectroscopy to understand the dynamics of Cytolysin A (ClyA), a prototypical α-PFP from E. coli, on bilayer lipid membranes. Binding of ClyA to PEG-cushioned supported bilayer was rapid and reached saturation within a few seconds. Diffusional analysis of particle trajectories showed existence of mobility states that could be assigned to two distinct structural states that match structures of the protein in the monomer form and the protomer state. In the presence of membrane cholesterol, the population of the slower moving species increased, with the concomitant decrease in the fast mobility population. Analysis of transition probabilities by Hidden Markov Model revealed that the conversion from fast to slower mobility state was due to the conformational transition from a peripherally-associated protein conformation to a membrane-inserted conformation respectively. Furthermore, a hitherto unknown cholesterol interaction domain was discovered in the transmembrane helix of ClyA and validated by mutational studies. Therefore, we propose a molecular mechanism for selective pore formation in eukaryotic membranes which is driven by conformational selectivity in the presence of cholesterol.

Sterol-recognition ability and membrane-disrupting activity of Ornithogalum saponin OSW-1 and usual 3-O-glycosyl saponins

OSW-1 is a structurally unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, and has exhibited highly potent and selective cytotoxicity in tumor cell lines. This study aimed to investigate the molecular mechanism for the membrane-permeabilizing activity of OSW-1 in comparison with those of other saponins by using various spectroscopic approaches. The membrane effects and hemolytic activity of OSW-1 were markedly enhanced in the presence of membrane cholesterol. Binding affinity measurements using fluorescent cholestatrienol and solid-state NMR spectroscopy of a 3-d-cholesterol probe suggested that OSW-1 interacts with membrane cholesterol without forming large aggregates while 3-O-glycosyl saponin, digitonin, forms cholesterol-containing aggregates. The results suggest that OSW-1/cholesterol interaction is likely to cause membrane permeabilization and pore formation without destroying the whole membrane integrity, which could partly be responsible for its highly potent cell toxicity.

Cholesterol Corrects Altered Conformation of MHC-II Protein in Leishmania donovani Infected Macrophages: Implication in Therapy.

BackgroundPreviously we reported that Kala-azar patients show progressive decrease in serum cholesterol as a function of splenic parasite burden. Splenic macrophages (MΦ) of Leishmania donovani (LD) infected mice show decrease in membrane cholesterol, while LD infected macrophages (I-MΦ) show defective T cell stimulating ability that could be corrected by liposomal delivery of cholesterol. T helper cells recognize peptide antigen in the context of class II MHC molecule. It is known that the conformation of a large number of membrane proteins is dependent on membrane cholesterol. In this investigation we tried to understand the influence of decreased membrane cholesterol in I-MΦ on the conformation of MHC-II protein and peptide-MHC-II stability, and its bearing on the antigen specific T-cell activation.Methodology/Principal FindingsMΦ of CBA/j mice were infected with Leishmania donovani (I-MΦ). Two different anti-Aκ mAbs were used to monitor the status of MHC-II protein under parasitized condition. One of them (11.5–2) was conformation specific, whereas the other one (10.2.16) was not. Under parasitized condition, the binding of 11.5–2 decreased significantly with respect to the normal counterpart, whereas that of 10.2.16 remained unaltered. The binding of 11.5–2 was restored to normal upon liposomal delivery of cholesterol in I-MΦ. By molecular dynamics (MD) simulation studies we found that there was considerable conformational fluctuation in the transmembrane domain of the MHC-II protein in the presence of membrane cholesterol than in its absence, which possibly influenced the distal peptide binding groove. This was evident from the faster dissociation of the cognate peptide from peptide-MHC complex under parasitized condition, which could be corrected by liposomal delivery of cholesterol in I-MΦ.ConclusionThe decrease in membrane cholesterol in I-MΦ may lead to altered conformation of MHC II, and this may contribute to a faster dissociation of the peptide. Furthermore, liposomal delivery of cholesterol in I-MΦ restored its normal antigen presenting function. This observation brings strength to our previous observation on host directed therapeutic application of liposomal cholesterol in experimental visceral leishmaniasis.

Elucidating the Mechanism for Sterol Regulation of Chloride Intracellular Ion Channel Protein Interactions with Lipid Membranes

Sterols have been reported to modulate conformation and function of several membrane proteins. One such group is the Chloride Intracellular Ion Channel (CLIC) family. These largely soluble proteins possess the intriguing property of spontaneous insertion into phospholipid bilayers to form integral membrane ion channels. To date, the structure of their membrane-bound form and factors influencing their auto-insertion remains largely unknown. We have performed Langmuir-film, x-ray, and neutron reflectivity experiments to study the interaction of wild-type or mutant versions of the protein CLIC1 with monolayers prepared from 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC) alone, or mixtures of this lipid with either one of the following sterols: cholesterol, β-sitosterol, ergosterol, hydroxyecdysone or cholestane. In lipid monolayers lacking sterols, CLIC1 did not insert. However significant membrane insertion occurred when CLIC1 was added to membranes containing cholesterol. Substitution of membrane cholesterol with different sterols, not only increased CLIC1's membrane interaction but also increased its rate of insertion. However, CLIC1 showed no insertion into monolayers containing cholestane, which lacked the intact sterol 3β-OH group. Unlike CLIC1-wild-type and C24A-CLIC1 mutant protein, G18A and G22A mutants did not insert in monolayers containing cholesterol. These findings for the first time demonstrate that the spontaneous membrane insertion of CLIC1 is dependent on the presence of cholesterol in membranes, along with the presence of an intact 3β-OH group within the sterol structure, which is critical for CLIC1-membrane interactions. Our novel findings also extend to the identification of a cholesterol-binding domain-GXXXG motif within CLIC1 that facilitates the protein's membrane interaction and insertion.

Differential dynamics of the serotonin1A receptor in membrane bilayers of varying cholesterol content revealed by all atom molecular dynamics simulation.

The serotonin1A receptor belongs to the superfamily of G protein-coupled receptors (GPCRs) and is a potential drug target in neuropsychiatric disorders. The receptor has been shown to require membrane cholesterol for its organization, dynamics and function. Although recent work suggests a close interaction of cholesterol with the receptor, the structural integrity of the serotonin1A receptor in the presence of cholesterol has not been explored. In this work, we have carried out all atom molecular dynamics simulations, totaling to 3 μs, to analyze the effect of cholesterol on the structure and dynamics of the serotonin1A receptor. Our results show that the presence of physiologically relevant concentration of membrane cholesterol alters conformational dynamics of the serotonin1A receptor and, on an average lowers conformational fluctuations. Our results show that, in general, transmembrane helix VII is most affected by the absence of membrane cholesterol. These results are in overall agreement with experimental data showing enhancement of GPCR stability in the presence of membrane cholesterol. Our results constitute a molecular level understanding of GPCR-cholesterol interaction, and represent an important step in our overall understanding of GPCR function in health and disease.

Structural Studies of Streptococcus pyogenes Streptolysin O Provide Insights into the Early Steps of Membrane Penetration

Cholesterol-dependent cytolysins (CDCs) are a large family of bacterial toxins that exhibit a dependence on the presence of membrane cholesterol in forming large pores in cell membranes. Significant changes in the three-dimensional structure of these toxins are necessary to convert the soluble monomeric protein into a membrane pore. We have determined the crystal structure of the archetypical member of the CDC family, streptolysin O (SLO), a virulence factor from Streptococcus pyogenes. The overall fold is similar to previously reported CDC structures, although the C-terminal domain is in a different orientation with respect to the rest of the molecule. Surprisingly, a signature stretch of CDC sequence called the undecapeptide motif, a key region involved in membrane recognition, adopts a very different structure in SLO to that of the well-characterized CDC perfringolysin O (PFO), although the sequences in this region are identical. An analysis reveals that, in PFO, there are complementary interactions between the motif and the rest of domain 4 that are lost in SLO. Molecular dynamics simulations suggest that the loss of a salt bridge in SLO and a cation–pi interaction are determining factors in the extended conformation of the motif, which in turn appears to result in a greater flexibility of the neighboring L1 loop that houses a cholesterol-sensing motif. These differences may explain the differing abilities of SLO and PFO to efficiently penetrate target cell membranes in the first step of toxin insertion into the membrane.

Induction of Highly Curved Structures in Relation to Membrane Permeabilization and Budding by the Triterpenoid Saponins, α- and δ-Hederin

The interactions of triterpenoid monodesmosidic saponins, α-hederin and δ-hederin, with lipid membranes are involved in their permeabilizing effect. Unfortunately, the interactions of these saponins with lipid membranes are largely unknown, as are the roles of cholesterol or the branched sugar moieties (two for α-hederin and one for δ-hederin) on the aglycone backbone, hederagenin. The differences in sugar moieties are responsible for differences in the molecular shape of the saponins and the effects on membrane curvature that should be the most positive for α-hederin in a transbilayer direction. In large unilamellar vesicles and monocyte cells, we showed that membrane permeabilization was dependent on the presence of membrane cholesterol and saponin sugar chains, being largest for α-hederin and smallest for hederagenin. In the presence of cholesterol, α-hederin induced the formation of nonbilayer phases with a higher rate of Brownian tumbling or lateral diffusion. A reduction of Laurdan's generalized polarization in relation to change in order of the polar heads of phospholipids was observed. Using giant unilamellar vesicles, we visualized the formation of wrinkled borders, the decrease in liposome size, budding, and the formation of macroscopic pores. All these processes are highly dependent on the sugars linked to the aglycone, with α-hederin showing a greater ability to induce pore formation and δ-hederin being more efficient in inducing budding. Hederagenin induced intravesicular budding but no pore formation. Based on these results, a curvature-driven permeabilization mechanism dependent on the interaction between saponin and sterols and on the molecular shape of the saponin and its ability to induce local spontaneous curvature is proposed.

Influence of cholesterol on the formation of palmitate/Ca2+-activated pores in mitochondria and liposomes

The influenc of cholesterol on the formation of the mitochondrial cyclosporin A (CsA)-insensitive palmitate/Ca(2+)-activated pore has been studied. It has been established that increasing the cholesterol level in mitochondrial membranes results in an increase in the of rate of mitochondrial swelling induced by palmitic acid (> or = 20 microM) and Ca2+ (30 microM). This effect is not related to changes in the functional activity of organelles since cholesterol did not influence the mitochondrial respiration in different metabolic states. At the same time, it was shown that the palmitate/Ca(2+)-induced permeabilization of cholesterol-containing azolectin liposomes was Stronger than that of azolectin liposomes. It was found that, in the liposomal membrane, the Ca(2+)-induced phase separation of palmitic acid into distinct membrane domains takes place. The presence of cholesterol in membranes increases the extent of segregation.

The role of membrane cholesterol in determining bile acid cytotoxicity and cytoprotection of ursodeoxycholic acid

In cholestatic liver diseases, the ability of hydrophobic bile acids to damage membranes of hepatocytes/ductal cells contributes to their cytotoxicity. However, ursodeoxycholic acid (UDC), a hydrophilic bile acid, is used to treat cholestasis because it protects membranes. It has been well established that bile acids associate with and solubilize free cholesterol (CHOL) contained within the lumen of the gallbladder because of their structural similarities. However, there is a lack of understanding of how membrane CHOL, which is a well-established membrane stabilizing agent, is involved in cytotoxicity of hydrophobic bile acids and the cytoprotective effect of UDC. We utilized phospholipid liposomes to examine the ability of membrane CHOL to influence toxicity of individual bile acids, such as UDC and the highly toxic sodium deoxycholate (SDC), as well as the cytoprotective mechanism of UDC against SDC-induced cytotoxicity by measuring membrane permeation and intramembrane dipole potential. The kinetics of bile acid solubilization of phosphatidylcholine liposomes containing various levels of CHOL was also characterized. It was found that the presence of CHOL in membranes significantly reduced the ability of bile acids to damage synthetic membranes. UDC effectively prevented damaging effects of SDC on synthetic membranes only in the presence of membrane CHOL, while UDC enhances the damaging effects of SDC in the absence of CHOL. This further demonstrates that the cytoprotective effects of UDC depend upon the level of CHOL in the lipid membrane. Thus, changes in cell membrane composition, such as CHOL content, potentially influence the efficacy of UDC as the primary drug used to treat cholestasis.

The Endogenous Brain ConstituentN-Arachidonoyl l-Serine Is an Activator of Large Conductance Ca2+-Activated K+Channels

The novel endocannabinoid-like lipid N-arachidonoyl L-serine (ARA-S) causes vasodilation through both endothelium-dependent and -independent mechanisms. We have analyzed the vasorelaxant effect of ARA-S in isolated vascular preparations and its effects on Ca(2+)-activated K(+) currents in human embryonic kidney cells stably transfected with the alpha-subunit of the human, large conductance Ca(+)-activated K(+) (BK(Ca)) channel [human embryonic kidney (HEK) 293hSlo cells]. ARA-S caused relaxation of rat isolated, intact and denuded, small mesenteric arteries preconstricted with (R)-(-)-1-(3-hydroxyphenyl)-2-methylaminoethanol hydrochloride (pEC(50), 5.49 and 5.14, respectively), whereas it caused further contraction of vessels preconstricted with KCl (pEC(50), 5.48 and 4.82, respectively). Vasorelaxation by ARA-S was inhibited by 100 nM iberiotoxin. In human embryonic kidney cells stably transfected with the alpha-subunit of the human BK(Ca) channel cells, ARA-S and its enantiomer, N-arachidonoyl-D-serine, enhanced the whole-cell outward K(+) current with similar potency (pEC(50), 5.63 and 5.32, respectively). The potentiation was not altered by the beta(1) subunit or mediated by ARA-S metabolites, stimulation of known cannabinoid receptors, G proteins, protein kinases, or Ca(2+)-dependent processes; it was lost after patch excision or after membrane cholesterol depletion but was restored after cholesterol reconstitution. BK(Ca) currents were also enhanced by N-arachidonoyl ethanolamide (pEC(50), 5.27) but inhibited by another endocannabinoid, O-arachidonoyl ethanolamine (pIC(50), 6.35), or by the synthetic cannabinoid O-1918 [(-)-1,3-dimethoxy-2-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol] (pIC(50), 6.59), which blocks ARA-S-induced vasodilation. We conclude the following. 1) ARA-S directly activates BK(Ca) channels. 2) This interaction does not involve cannabinoid receptors or cytosolic factors but is dependent on the presence of membrane cholesterol. 3) Direct BK(Ca) channel activation probably contributes to the endothelium-independent component of ARA-S-induced mesenteric vasorelaxation. 4) O-1918 is a BK(Ca) channel inhibitor.

Cholesterol Effects on BAX Pore Activation

The importance of BCL-2 family proteins in the control of cell death has been clearly established. One of the key members of this family, BAX, has soluble, membrane-bound, and membrane-integrated forms that are central to the regulation of apoptosis. Using purified monomeric human BAX, defined liposomes, and isolated human mitochondria, we have characterized the soluble to membrane transition and pore formation by this protein. For the purified protein, activation, but not oligomerization, is required for membrane binding. The transition to the membrane environment includes a binding step that is reversible and distinct from the membrane integration step. Oligomerization and pore activation occur after the membrane integration. In cells, BAX targets several intracellular membranes but notably does not target the plasma membrane while initiating apoptosis. When cholesterol was added to either the liposome bilayer or mitochondrial membranes, we observed increased binding but markedly reduced integration of BAX into both membranes. This cholesterol inhibition of membrane integration accounts for the reduction of BAX pore activation in liposomes and mitochondrial membranes. Our results indicate that the presence of cholesterol in membranes inhibits the pore-forming activity of BAX by reducing the ability of BAX to transition from a membrane-associated protein to a membrane-integral protein.

Redefining cholesterol's role in the mechanism of the cholesterol-dependent cytolysins.

The cholesterol-dependent cytolysins (CDCs) constitute a large family of pore-forming toxins that function exclusively on cholesterol-containing membranes. A detailed analysis of the various stages in the cytolytic mechanism of three members of the CDC family revealed that significant depletion of cholesterol from the erythrocyte membrane stalls these toxins in the prepore complex. Therefore, the depletion of membrane cholesterol prevents the insertion of the transmembrane beta-barrel and pore formation. These unprecedented findings provide a paradigm for the involvement of cholesterol in the CDC cytolytic mechanism and that of other pore-forming toxins whose activity is enhanced by the presence of membrane cholesterol.

Morphological and functional reconsideration of the cytoplasmic bridges which connect male germ cells in snails

Cytoplasmic bridges (CB) between male germ cells of three fresh-water snails have been examined by electron microscopy, using ultrathin sections and freeze-fracture replicas prepared by ordinary methods and after use of filipin for indicating the presence of membrane cholesterol. The bridge plasma membrane, which was formerly considered to be smooth, was found in these snails to be corrugated. The corrugations were periodically parallel and oriented parallel to the axis of the bridge. The mature bridges showed very low densities of intramembranous particles. No filipin-sterol complexes formed on either the P face or the E face of the bridge plasma membrane, in contrast to plasma membranes elsewhere. The numbers of corrugations in each CB varied with the species. The membrane corrugations overlie bundles of electron dense fibers measuring approximately 30 nm in diameter and 60 nm in center-to-center distance, fitting into convexities of the plasma membrane. The present observations lead us to the necessity of reconsidering the morphological and functional aspects of cytoplasmic bridges in vertebrate as well as in invertebrate germ cells.