Anionic Lipids Allosterically Modulate Multiple Nicotinic Acetylcholine Receptor Conformational Equilibria

Anionic lipids influence the ability of the nicotinic acetylcholine receptor to gate open in response to neurotransmitter binding, but the underlying mechanisms are poorly understood. We show here that anionic lipids with relatively small headgroups, and thus the greatest ability to influence lipid packing/bilayer physical properties, are the most effective at stabilizing an agonist-activatable receptor. The differing abilities of anionic lipids to stabilize an activatable receptor stem from differing abilities to preferentially favor resting over both uncoupled and desensitized conformations. Anionic lipids thus modulate multiple acetylcholine receptor conformational equilibria. Our data suggest that both lipids and membrane physical properties act as classic allosteric modulators influencing function by interacting with and thus preferentially stabilizing different native acetylcholine receptor conformational states.     

Myelin-Derived Lipids Modulate Macrophage Activity by Liver X Receptor Activation

Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system in which macrophages and microglia play a central role. Foamy macrophages and microglia, containing degenerated myelin, are abundantly found in active multiple sclerosis lesions. Recent studies have described an altered macrophage phenotype after myelin internalization. However, it is unclear by which mechanisms myelin affects the phenotype of macrophages and how this phenotype can influence lesion progression. Here we demonstrate, by using genome wide gene expression analysis, that myelin-phagocytosing macrophages have an enhanced expression of genes involved in migration, phagocytosis and inflammation. Interestingly, myelin internalization also induced the expression of genes involved in liver-X-receptor signaling and cholesterol efflux. In vitro validation shows that myelin-phagocytosing macrophages indeed have an increased capacity to dispose intracellular cholesterol. In addition, myelin suppresses the secretion of the pro-inflammatory mediator IL-6 by macrophages, which was mediated by activation of liver-X-receptor β. Our data show that myelin modulates the phenotype of macrophages by nuclear receptor activation, which may subsequently affect lesion progression in demyelinating diseases such as multiple sclerosis.     

Cations Mediate Interactions between the Nicotinic Acetylcholine Receptor and Anionic Lipids

Interactions between the nicotinic acetylcholine receptor (nAChR) and phosphatidic acid (PA) are bidirectional in that membranes containing PA are effective at stabilizing an agonist-responsive nAChR, whereas incorporation of the nAChR into the same membranes leads to a substantial increase in lipid lateral packing density. A previous study suggested that the ability of PA to adopt a dianionic ionization state is key. We monitored the ionization state of PA in both reconstituted and protein-free membranes. In model membranes composed of PA and 3:2 (mol/mol) phosphatidylcholine (PC)/PA, the monoanionic-to-dianionic transition of PA was detected with a pKa of 8.7 and 6.5, respectively. In the reconstituted 3:2 PC/PA membranes, however, PA was stabilized in a monoanionic state at pH values up to 10. Although dianionic PA does not play a role in nAChR function, we found that both the stabilization of monoanionic PA and the concentration of other cations at the bilayer surface can account for changes in bilayer physical properties that are observed upon incorporation of the nAChR into 3:2 PC/PA membranes. A nAChR-induced concentration of cations at the bilayer surface likely mediates interactions between the nAChR and the anionic lipids in its membrane environment.     

Charge-Rich Regions Modulate the Anti-Aggregation Activity of Hsp90

Protein aggregation can have dramatic effects on cellular function and plays a causative role in many human diseases. In all cells, molecular chaperones bind to aggregation-prone proteins and hinder aggregation. The ability of a protein to resist aggregation and remain soluble in aqueous solution is linked to the physical properties of the protein. Numerous physical studies demonstrate that charged atoms favor solubility. We note that many molecular chaperones possess a substantial negative charge that may allow them to impart solubility on aggregation-prone proteins. Hsp90 is one such negatively charged molecular chaperone. The charge on Hsp90 is largely concentrated in two highly acidic regions. To investigate the relationship between chaperone charge and protein solubility, we deleted these charge-rich regions and analyzed the resulting Hsp90 constructs for anti-aggregation activity. We found that deletion of both charge-rich regions dramatically impaired Hsp90 anti-aggregation activity. The anti-aggregation role of the deleted charge-rich regions could be due to net charge or sequence-specific features. To distinguish these possibilities, we attached an acid-rich region with a distinct amino acid sequence to our double-deleted Hsp90 construct. This charge rescue construct displayed effective anti-aggregation activity indicating that the net charge of Hsp90 contributes to its anti-aggregation activity.     

Obesity and Alcohol Modulate the Effect of Apolipoprotein E Polymorphism on Lipids and Insulin

Objective: To assess the interaction between apolipoprotein (apo) E polymorphism, alcohol consumption, and BMI on insulin, lipid, and lipoprotein levels in men. Research Methods and Procedures: Cross‐sectional study of 266 healthy men without hypolipidemic or antidiabetic drug treatment. BMI, apo E polymorphisms, insulin, and lipid and lipoprotein levels were assessed. Alcohol consumption was assessed by questionnaire. ?2/?4 carriers were excluded from the analysis. Results: On bivariate analysis, ?2 carriers had lower levels of total and low‐density lipoprotein cholesterol and higher levels of apo E and lipoparticle B:E than ?3 carriers, the opposite being found for ?4 carriers compared with ?3 carriers; ?4 carriers also had significantly higher insulin levels. On multivariate analysis, significant interactions (p < 0.04) between apo E alleles and increased BMI were found for total and low‐density lipoprotein cholesterol and insulin levels, the increase in those parameters with BMI being stronger among ?4 carriers than among ?3 or ?2 carriers. Significant interactions (p < 0.02) between apo E alleles and alcohol consumption were also found for apo B levels, which increased in ?2 carriers but remained relatively stable in ?3 and tended to decrease in ?4 carriers. Discussion: These data suggest that effects of apo E alleles on lipids and insulin levels are partly dependent on environmental variables such as BMI and alcohol intake. These findings highlight the importance of gene × environment interactions on the deleterious effect of obesity on cardiovascular risk factors.     

Biological Activity of Polar Lipids from Bifidobacteria

Fractions of polar lipids have been isolated from bifidobacteria, and the immunoreactivity and serological specificity of glycolipids and phospholipids have been studied. Enzyme immunoassay (dot-EIA) of polar lipids demonstrates that the fractions of glycolipids and phospholipids of bifidobacteria are highly immunoreactive. Pronounced reactions of major glycolipids and phospholipids with a homologous polyvalent antiserum against B. adolescentis 94-BIM have been observed at antigen concentrations of approximately 100 ng. The antiserum contained high titers of specific antibodies against glycolipids and phospholipids of bifidobacteria, as demonstrated by heterogeneous solid-phase enzyme immunoassay (ELISA). Experimental data confirming the presence of subpopulations of specific antibodies (antiglycolipid and antiphospholipid) in the blood sera of immunized animals lead to the conclusion that the major glycolipids and phospholipids of bifidobacteria are specific markers appropriate for serological diagnostics.     

Membrane Lipids Determine the Antibiotic Activity of the Lantibiotic Gallidermin

Lantibiotics, a group of lanthionine-containing peptides, display their antibiotic activity by combining different killing mechanisms within one molecule. The prototype lantibiotic nisin was shown to possess both inhibition of peptidoglycan synthesis and pore formation in bacterial membranes by interacting with lipid II. Gallidermin, which shares the lipid II binding motif with nisin but has a shorter molecular length, differed from nisin in pore formation in several strains of bacteria. To simulate the mode of action, we applied cyclic voltammetry and quartz crystal microbalance to correlate pore formation with lipid II binding kinetics of gallidermin in model membranes. The inability of gallidermin to form pores in DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) (C18/1) and DPoPC (1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine) (C16/1) membranes was related to the membrane thickness. For a better simulation of bacterial membrane characteristics, two different phospholipids with branched fatty acids were incorporated into the DPoPC matrix. Phospholipids with methyl branches in the middle of the fatty acid chains favored a lipid II–independent DPoPC permeabilization by gallidermin, while long-branched phospholipids in which the branch is placed near the hydrophilic region induced an identical lipid II–dependent pore formation of gallidermin and nisin. Obviously, the branched lipids altered lipid packing and reduced the membrane thickness. Therefore, the duality of gallidermin activity (pore formation and inhibition of the cell wall synthesis) seems to be balanced by the bacterial membrane composition.     

Gadolinium Ions Block Mechanosensitive Channels by Altering the Packing and Lateral Pressure of Anionic Lipids

Effects of polyvalent ions on the lateral packing of phospholipids have been known for decades, but the physiological consequences have not been systematically studied. Gd³⁺ is a relatively nonspecific agent that blocks mechano-gated channels with a variable affinity. In this study, we show that the large mechanosensitive channel MscL of Escherichia coli is effectively blocked by Gd³⁺ only when reconstituted with negatively charged phospholipids (e.g., PS). Taking this lead, we studied effects of Gd³⁺ on monolayers and unilamellar vesicles made of natural brain PS, DMPS, and its mixtures with DMPC. In monolayer experiments, we found that μM Gd³⁺ present in the subphase leads to ∼8% lateral compaction of brain PS (at 35 mN/m). Gd³⁺ more strongly shrinks and rigidifies DMPS films causing a spontaneous liquid expanded-to-compact transition to the limiting 40 Ų/mol. Pressure-area isotherms of uncharged DMPC were unaffected by Gd³⁺, and neutralization of DMPS surface by low pH did not produce strong compaction. Upshifts of surface potential isotherms of DMPS monolayers reflected changes in the diffuse double layer due to neutralization of headgroup charges by Gd³⁺, whereas the increased packing density produced up to a 200 mV change in the interfacial dipole potential. The slopes of surface potential versus reciprocal area predicted that Gd³⁺ induced a modest (∼18%) increase in the magnitude of the individual lipid dipoles in DMPS. Isothermal titration calorimetry indicated that binding of Gd³⁺ to DMPS liposomes in the gel state is endothermic, whereas binding to liquid crystalline liposomes produces heat consistent with the isothermal liquid-to-gel phase transition induced by the ion. Both titration curves suggested a K_b of ∼10⁶ M⁻¹. We conclude that anionic phospholipids serve as high-affinity receptors for Gd³⁺ ions, and the ion-induced compaction generates a lateral pressure increase estimated as tens of mN/m. This pressure can “squeeze” the channel and shift the equilibrium toward the closed state.     

N-Methyl-D-Aspartate Recognition Site Ligands Modulate Activity at the Coupled Glycine Recognition Site

In synaptic plasma membranes from rat forebrain, the potencies of glycine recognition site agonists and antagonists for modulating [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding and for displacing strychnine-insensitive [3H]glycine binding are altered in the presence of N-methyl-D-aspartate (NMDA) recognition site ligands. The NMDA competitive antagonist, cis-4-phosphonomethyl-2-piperidine carboxylate (CGS 19755), reduces [3H]glycine binding, and the reduction can be fully reversed by the NMDA recognition site agonist, L-glutamate. Scatchard analysis of [3H]glycine binding shows that in the presence of CGS 19755 there is no change in Bmax (8.81 vs. 8.79 pmol/mg of protein), but rather a decrease in the affinity of glycine (KD of 0.202 microM vs. 0.129 microM). Similar decreases in affinity are observed for the glycine site agonists, D-serine and 1-aminocyclopropane-1-carboxylate, in the presence of CGS 19755. In contrast, the affinity of glycine antagonists, 1-hydroxy-3-amino-2-pyrrolidone and 1-aminocyclobutane-1-carboxylate, at this [3H]glycine recognition site increases in the presence of CGS 19755. The functional consequence of this change in affinity was addressed using the modulation of [3H]TCP binding. In the presence of L-glutamate, the potency of glycine agonists for the stimulation of [3H]TCP binding increases, whereas the potency of glycine antagonists decreases. These data are consistent with NMDA recognition site ligands, through their interactions at the NMDA recognition site, modulating activity at the associated glycine recognition site.     

Sympathetic Neurotransmitters Modulate Osteoclastogenesis and Osteoclast Activity in the Context of Collagen-Induced Arthritis

Excessive synovial osteoclastogenesis is a hallmark of rheumatoid arthritis (RA). Concomitantly, local synovial changes comprise neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze if collagen-induced arthritis (CIA) alters bone marrow-derived macrophage (BMM) osteoclastogenesis and osteoclast activity, and how sympathetic neurotransmitters participate in this process. Therefore, BMMs from Dark Agouti rats at different CIA stages were differentiated into osteoclasts in vitro and osteoclast number, cathepsin K activity, matrix resorption and apoptosis were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA) vasoactive intestinal peptide (VIP) and assay-dependent, adenylyl cyclase activator NKH477. We observed modulation of neurotransmitter receptor mRNA expression in CIA osteoclasts without affecting protein level. CIA stage-dependently altered marker gene expression associated with osteoclast differentiation and activity without affecting osteoclast number or activity. Neurotransmitter stimulation modulated osteoclast differentiation, apoptosis and activity. VIP, NA and adenylyl cyclase activator NKH477 inhibited cathepsin K activity and osteoclastogenesis (NKH477, 10^-6M NA) whereas ACh mostly acted pro-osteoclastogenic. We conclude that CIA alone does not affect metabolism of in vitro generated osteoclasts whereas stimulation with NA, VIP plus specific activation of adenylyl cyclase induced anti-resorptive effects probably mediated via cAMP signaling. Contrary, we suggest pro-osteoclastogenic and pro-resorptive properties of ACh mediated via muscarinic receptors.     

Restricted Feeding Schedules Modulate Free-running Drinking Activity in Malnourished Rats

Low protein malnourished rats held in 12 : 12 light-dark conditions exhibit two bouts of drinking activity, which resemble a “splitting” pattern. These findings have suggested a weak coupling force between the light-(LEO) and the food-entrainable oscillators (FEO). Food restriction to a few hours daily exerts a strong entraining influence on FEO and allows to uncouple both oscillators. To further understand the coupling relation between LEO and FEO, we evaluated the influence of restricted feeding schedules (RFS) on the circadian rhythm of drinking behavior in malnourished rats and their controls. Adult rats were entrained to RFS with a low protein or a regular chow diet in a counterbalanced design. All groups developed drinking anticipatory activity (FAA) to meal time, with similar intensity and onset time. RFS produced lengthening in the period of LEO's free-running rhythm and this effect was significant in MAL rats. Behavioral patterns in control as well as malnourished rats entrained with regular chow indicated independence between LEO and FEO. In contrast, 60% of MAL rats entrained with the low protein diet exhibited phase control by meal time on LEO's free-running. Present data indicate that low protein diets may induce enhanced potency of food as an entraining signal and produce a change in the coupling force between both oscillators, promoting that LEO couples to the FEO. In such conditions FEO seems to override the influence of LEO on the temporal organization of behavior and imposes its phase on the free-running component.     

Restricted Feeding Schedules Modulate Free-running Drinking Activity in Malnourished Rats

Low protein malnourished rats held in 12 : 12 light-dark conditions exhibit two bouts of drinking activity, which resemble a “splitting” pattern. These findings have suggested a weak coupling force between the light-(LEO) and the food-entrainable oscillators (FEO). Food restriction to a few hours daily exerts a strong entraining influence on FEO and allows to uncouple both oscillators. To further understand the coupling relation between LEO and FEO, we evaluated the influence of restricted feeding schedules (RFS) on the circadian rhythm of drinking behavior in malnourished rats and their controls. Adult rats were entrained to RFS with a low protein or a regular chow diet in a counterbalanced design. All groups developed drinking anticipatory activity (FAA) to meal time, with similar intensity and onset time. RFS produced lengthening in the period of LEO’s free-running rhythm and this effect was significant in MAL rats. Behavioral patterns in control as well as malnourished rats entrained with regular chow indicated independence between LEO and FEO. In contrast, 60% of MAL rats entrained with the low protein diet exhibited phase control by meal time on LEO’s free-running. Present data indicate that low protein diets may induce enhanced potency of food as an entraining signal and produce a change in the coupling force between both oscillators, promoting that LEO couples to the FEO. In such conditions FEO seems to override the influence of LEO on the temporal organization of behavior and imposes its phase on the free-running component.     

Thermodynamics of Micelle Formation and Membrane Fusion Modulate Antimicrobial Lipopeptide Activity

Antimicrobial lipopeptides (AMLPs) are antimicrobial drug candidates that preferentially target microbial membranes. One class of AMLPs, composed of cationic tetrapeptides attached to an acyl chain, have minimal inhibitory concentrations in the micromolar range against a range of bacteria and fungi. Previously, we used coarse-grained molecular dynamics simulations and free energy methods to study the thermodynamics of their interaction with membranes in their monomeric state. Here, we extended the study to the biologically relevant micellar state, using, to our knowledge, a novel reaction coordinate based on hydrophobic contacts. Using umbrella sampling along this reaction coordinate, we identified the critical transition states when micelles insert into membranes. The results indicate that the binding of these AMLP micelles to membranes is thermodynamically favorable, but in contrast to the monomeric case, there are significant free energy barriers. The height of these free energy barriers depends on the membrane composition, suggesting that the AMLPs’ ability to selectively target bacterial membranes may be as much kinetic as thermodynamic. This mechanism highlights the importance of considering oligomeric state in solution as criterion when optimizing peptides or lipopeptides as antibiotic leads.     

Diffusion of glycerol through Escherichia coli aquaglyceroporin GlpF

The glycerol uptake facilitator, GlpF, a major intrinsic protein found in Escherichia coli, selectively conducts water and glycerol across the inner membrane. The free energy landscape characterizing the assisted transport of glycerol by this homotetrameric aquaglyceroporin has been explored by means of equilibrium molecular dynamics over a timescale spanning 0.12 μs. To overcome the free energy barriers of the conduction pathway, an adaptive biasing force is applied to the glycerol molecule confined in each of the four channels. The results illuminate the critical role played by intramolecular relaxation on the diffusion properties of the permeant. These free energy calculations reveal that glycerol tumbles and isomerizes on a timescale comparable to that spanned by its adaptive-biasing-force-assisted conduction in GlpF. As a result, reorientation and conformational equilibrium of glycerol in GlpF constitute a bottleneck in the molecular simulations of the permeation event. A profile characterizing the position-dependent diffusion of the permeant has been determined, allowing reaction rate theory to be applied for investigating conduction kinetics based on the measured free energy landscape.     

Penetrating Cations Enhance Uncoupling Activity of Anionic Protonophores in Mitochondria

Protonophorous uncouplers causing a partial decrease in mitochondrial membrane potential are promising candidates for therapeutic applications. Here we showed that hydrophobic penetrating cations specifically targeted to mitochondria in a membrane potential-driven fashion increased proton-translocating activity of the anionic uncouplers 2,4-dinitrophenol (DNP) and carbonylcyanide-p-trifluorophenylhydrazone (FCCP). In planar bilayer lipid membranes (BLM) separating two compartments with different pH values, DNP-mediated diffusion potential of H⁺ ions was enhanced in the presence of dodecyltriphenylphosphonium cation (C₁₂TPP). The mitochondria-targeted penetrating cations strongly increased DNP- and carbonylcyanide m-chlorophenylhydrazone (CCCP)-mediated steady-state current through BLM when a transmembrane electrical potential difference was applied. Carboxyfluorescein efflux from liposomes initiated by the plastoquinone-containing penetrating cation SkQ1 was inhibited by both DNP and FCCP. Formation of complexes between the cation and CCCP was observed spectophotometrically. In contrast to the less hydrophobic tetraphenylphosphonium cation (TPP), SkQ1 and C₁₂TPP promoted the uncoupling action of DNP and FCCP on isolated mitochondria. C₁₂TPP and FCCP exhibited a synergistic effect decreasing the membrane potential of mitochondria in yeast cells. The stimulating action of penetrating cations on the protonophore-mediated uncoupling is assumed to be useful for medical applications of low (non-toxic) concentrations of protonophores.     

Temporal Expectation and Attention Jointly Modulate Auditory Oscillatory Activity in the Beta Band

The neural response to a stimulus is influenced by endogenous factors such as expectation and attention. Current research suggests that expectation and attention exert their effects in opposite directions, where expectation decreases neural activity in sensory areas, while attention increases it. However, expectation and attention are usually studied either in isolation or confounded with each other. A recent study suggests that expectation and attention may act jointly on sensory processing, by increasing the neural response to expected events when they are attended, but decreasing it when they are unattended. Here we test this hypothesis in an auditory temporal cueing paradigm using magnetoencephalography in humans. In our study participants attended to, or away from, tones that could arrive at expected or unexpected moments. We found a decrease in auditory beta band synchrony to expected (versus unexpected) tones if they were unattended, but no difference if they were attended. Modulations in beta power were already evident prior to the expected onset times of the tones. These findings suggest that expectation and attention jointly modulate sensory processing.     

Use of Cationic Lipids to Enhance the Biological Activity of Antisense Oligonucleotides

Abstract

Antisense oligonucleotides bind to specific mRNA or pre-mRNA sequences through Watson-Crick base pairing, resulting in decreased expression of the targeted protein. The use of cationic lipids to enhance cellular uptake of antisense oligonucleotides is reviewed herein. Cationic lipids such as N[1-(2,3-dioleyloxy)propyl]-N, N, N-trimethylammonium chloride (DOTMA) were found to enhance the biological activity of phosphorothioate oligonucleotides by at least 1000-fold in cell culture. Cationic lipid preparations enhanced both the rate and amount of oligonucleotide which associated with cells. In addition, DOTMA markedly changed the subcellular distribution of the oligonucleotide. In the absence of lipid, fluorescein labelled phosphorothioate oligonucleotides accumulated in discrete cytoplasmic structures. In the presence of cationic lipids, the oligonucleotides concentrated within the nucleus, were excluded from nucleoli, and localized in punctate cytoplasmic structures. The accumulation of the oligonucleotide in the nucleus was inhibited by incubation of the cells at 4°C and by monensin, but not by chloroquine, ammonium chloride, or nocodazole. Cell lines, both primary and transformed, differ markedly in their sensitivity to inhibition of gene expression with antisense oligonucleotides in the presence of cationic lipids. The differential sensitivity of the cells correlates with the amount of ³⁵S-labelled oligonucleotide associated with the cells and the number of cells in the population which take up the oligonucleotide. Our studies have demonstrated that several types of cationic lipids markedly enhance the activity of phosphorothioate oligonucleotides in cell culture models. We are currently investigating the ability of cationic lipids to enhance activity of antisense oligonucleotides in more complex systems such as organ cultures and in animals.