Cooperative lipid activation of (Na+ + K+)-ATPase as a consequence of non-cooperative lipid-protein interactions

Lipid activation data for $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ (Ottolenghi, P. (1979) Eur. J. Biochem. 99, 113–131) have been subjected to a regression and fitting analysis based on a recent kinetic model (Sandermann, H. (1982) Eur. J. Biochem, 127, 123–128). The observed kinetic cooperativity could be generated from strictly non-cooperative binding events involving the known number of 30 boundary lipid-binding sites per ATPase monomer. Apparent lipid dissociation equilibrium constants of between 0.3 and 5 μM were obtained, enzyme activity being associated only with the fully lipid-substituted enzyme and enzyme-lipid complexes with less than six unoccupied lipid-binding sites. The enzyme appeared to operate close to a maximum of cooperativity.     

Interactions of the Australian tree frog antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with lipid model membranes: Differential scanning calorimetric and Fourier transform infrared spectroscopic studies

The interactions of the antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylethanolamine (DMPE) were studied by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. The effects of these peptides on the thermotropic phase behavior of DMPC and DMPG are qualitatively similar and manifested by the suppression of the pretransition, and by peptide concentration-dependent decreases in the temperature, cooperativity and enthalpy of the gel/liquid-crystalline phase transition. However, at all peptide concentrations, anionic DMPG bilayers are more strongly perturbed than zwitterionic DMPC bilayers, consistent with membrane surface charge being an important aspect of the interactions of these peptides with phospholipids. However, at all peptide concentrations, the perturbation of the thermotropic phase behavior of zwitterionic DMPE bilayers is weak and discernable only when samples are exposed to high temperatures. FTIR spectroscopy indicates that these peptides are unstructured in aqueous solution and that they fold into α-helices when incorporated into lipid membranes. All three peptides undergo rapid and extensive H-D exchange when incorporated into D₂O-hydrated phospholipid bilayers, suggesting that they are located in solvent-accessible environments, most probably in the polar/apolar interfacial regions of phospholipid bilayers. The perturbation of model lipid membranes by these peptides decreases in magnitude in the order maculatin 1.1 > aurein 1.2 > citropin 1.1, whereas the capacity to inhibit Acholeplasma laidlawii B growth decreases in the order maculatin 1.1 > aurein 1.2 ≅ citropin 1.1. The higher efficacy of maculatin 1.1 in disrupting model and biological membranes can be rationalized by its larger size and higher net charge. However, despite its smaller size and lower net charge, aurein 1.2 is more disruptive of model lipid membranes than citropin 1.1 and exhibits comparable antimicrobial activity, probably because aurein 1.2 has a higher propensity for partitioning into phospholipid membranes.     

FATP1 mediates fatty acid-induced activation of AMPK in 3T3-L1 adipocytes

Fatty acid transport proteins are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. FATP-dependent production of AMP was evaluated using FATP4 proteoliposomes, and fatty acid-dependent activation of AMP-activated protein kinase (AMPK) was assessed in 3T3-L1 adipocytes. Insulin-stimulated fatty acid influx (palmitate or arachidonate) into cultured adipocytes resulted in an increase in the phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase. Consistent with the activation of AMPK, palmitate uptake into 3T3-L1 adipocytes resulted in an increase in intracellular [AMP]/[ATP]. The fatty acid-induced increase in AMPK activation was attenuated in a cell line expressing shRNA targeting FATP1. Taken together, these results demonstrate that, in adipocytes, insulin-stimulated fatty acid influx mediated by FATP1 regulates AMPK and provides a potential regulatory mechanism for balancing de novo production of fatty acids from glucose metabolism with influx of preformed fatty acids via phosphorylation of acetyl-CoA carboxylase.     

Specific lipids supply critical negative spontaneous curvature--an essential component of native Ca2+-triggered membrane fusion

The Ca²⁺-triggered merger of two apposed membranes is the defining step of regulated exocytosis. CHOL is required at critical levels in secretory vesicle membranes to enable efficient, native membrane fusion: CHOL-sphingomyelin enriched microdomains organize the site and regulate fusion efficiency, and CHOL directly supports the capacity for membrane merger by virtue of its negative spontaneous curvature. Specific, structurally dissimilar lipids substitute for CHOL in supporting the ability of vesicles to fuse: diacylglycerol, αT, and phosphatidylethanolamine support triggered fusion in CHOL-depleted vesicles, and this correlates quantitatively with the amount of curvature each imparts to the membrane. Lipids of lesser negative curvature than cholesterol do not support fusion. The fundamental mechanism of regulated bilayer merger requires not only a defined amount of membrane-negative curvature, but this curvature must be provided by molecules having a specific, critical spontaneous curvature. Such a local lipid composition is energetically favorable, ensuring the necessary “spontaneous” lipid rearrangements that must occur during native membrane fusion—Ca²⁺-triggered fusion pore formation and expansion. Thus, different fusion sites or vesicle types can use specific alternate lipidic components, or combinations thereof, to facilitate and modulate the fusion pore.     

The impact of membrane lipid composition on antimicrobial function of an alpha-helical peptide

VP1, a putative alpha-helical antimicrobial peptide (alpha-AMP) inhibited growth of Bacillus subtilis and Escherichia coli at 500microM. The peptide induced stable surface pressure changes in monolayers formed from B. subtilis native lipid extract (circa 4.5mNm(-1)) but transient pressure changes in corresponding E. coli monolayers (circa 1.0mNm(-1)), which led to monolayer disintegration. Synthetic lipid monolayers mimetic of the extracts were used to generate compression isotherms. Thermodynamic analysis of B. subtilis isotherms indicated membrane stabilisation by VP1 (DeltaG(Mix)<0), via a mechanism dependent upon the phosphatidylglycerol to cardiolipin ratio. Corresponding analysis of E. coli isotherms indicated membrane destabilisation by the peptide (DeltaG(Mix)>0). Destabilisation correlated with PE levels present and appeared to involve a mechanism resembling those used by tilted peptides. These data emphasise that structure/function analysis of alpha-AMPs must consider not only their structural characteristics but also the lipid make-up of the target microbial membrane.     

Raft composition at physiological temperature and pH in the absence of detergents

Biological rafts were identified and isolated at 37°C and neutral pH. The strategy for isolating rafts utilized membrane tension to generate large domains. For lipid compositions that led only to microscropically unresolvable rafts in lipid bilayers, membrane tension led to the appearance of large, observable rafts. The large rafts converted back to small ones when tension was relieved. Thus, tension reversibly controls raft enlargement. For cells, application of membrane tension resulted in several types of large domains; one class of the domains was identified as rafts. Tension was generated in several ways, and all yielded raft fractions that had essentially the same composition, validating the principle of tension as a means to merge small rafts into large rafts. It was demonstrated that sphingomyelin-rich vesicles do not rise during centrifugation in sucrose gradients because they resist lysis, necessitating that, contrary to current experimental practice, membrane material be placed toward the top of a gradient for raft fractionation. Isolated raft fractions were enriched in a GPI-linked protein, alkaline phosphatase, and were poor in Na⁺-K⁺ ATPase. Sphingomyelin and gangliosides were concentrated in rafts, the expected lipid raft composition. Cholesterol, however, was distributed equally between raft and nonraft fractions, contrary to the conventional view.     

The influence of phase transitions in phosphatidylethanolamine models on the activity of violaxanthin de-epoxidase

In the present study, the influence of the phospholipid phase state on the activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase (VDE) was analyzed using different phosphatidylethanolamine species as model lipids. By using ³¹P NMR spectroscopy, differential scanning calorimetry and temperature dependent enzyme assays, VDE activity could directly be related to the lipid structures the protein is associated with. Our results show that the gel (L_β) to liquid-crystalline (L_α) phase transition in these single lipid component systems strongly enhances both the solubilization of the xanthophyll cycle pigment violaxanthin in the membrane and the activity of the VDE. This phase transition has a significantly stronger impact on VDE activity than the transition from the L_α to the inverted hexagonal (H_II) phase. Especially at higher temperatures we found increased VDE reaction rates in the presence of the L_α phase compared to those in the presence of H_II phase forming lipids. Our data furthermore imply that the H_II phase is better suited to maintain high VDE activities at lower temperatures.     

The interactions of antimicrobial peptides derived from lysozyme with model membrane systems

Two peptides, RAWVAWR-NH₂ and IVSDGNGMNAWVAWR-NH₂, derived from human and chicken lysozyme, respectively, exhibit antimicrobial activity. A comparison between the L-RAWVAWR, D-RAWVAWR, and the longer peptide has been carried out in membrane mimetic conditions to better understand how their interaction with lipid and detergent systems relates to the reported higher activity for the all L-peptide. Using CD and 2D ¹H NMR spectroscopy, the structures were studied with DPC and SDS micelles. Fluorescence spectroscopy was used to study peptide interactions with POPC and POPG vesicles and DOPC, DOPE, and DOPG mixed vesicle systems. Membrane-peptide interactions were also probed by ITC and DSC. The ability of fluorescein-labeled RAWVAWR to rapidly enter both E. coli and Staphylococcus aureus was visualized using confocal microscopy. Reflecting the bactericidal activity, the long peptide interacted very weakly with the lipids. The RAWVAWR-NH₂ peptides preferred lipids with negatively charged headgroups and interacted predominantly in the solvent-lipid interface, causing significant perturbation of membrane mimetics containing PG headgroups. Peptide structures determined by ¹H NMR indicated a well-ordered coiled structure for the short peptides and the C-terminus of the longer peptide. Using each technique, the two enantiomers of RAWVAWR-NH₂ interacted in an identical fashion with the lipids, indicating that any difference in activity in vivo is limited to interactions not involving the membrane lipids.     

Membrane interactions of hemoglobin variants, HbA, HbE, HbF and globin subunits of HbA: Effects of aminophospholipids and cholesterol

The interaction of hemoglobin with phospholipid bilayer vesicles (liposomes) has been analyzed in several studies to better understand membrane-protein interactions. However, not much is known on hemoglobin interactions with the aminophospholipids, predominantly localized in the inner leaflet of erythrocytes, e.g., phosphatidylserine (PS), phosphatidylethanolamine (PE) in membranes containing phosphatidylcholine (PC). Effects of cholesterol, largely abundant in erythrocytes, have also not been studied in great details in earlier studies. This work therefore describes the study of the interactions of different hemoglobin variants HbA, HbE and HbF and the globin subunits of HbA with the two aminophospholipids in the presence and absence of cholesterol. Absorption measurements indicate preferential oxidative interaction of HbE and alpha-globin subunit with unilamellar vesicles containing PE and PS compared to normal HbA. Cholesterol was found to stabilize such oxidative interactions in membranes containing both the aminophospholipids. HbE and alpha-globin subunits were also found to induce greater leakage of membrane entrapped carboxyfluorescein (CF) using fluorescence measurements. HbE was found to induce fusion of membrane vesicles containing cholesterol and PE when observed under electron microscope. Taken together, these findings might be helpful in understanding the oxidative stress-related mechanism(s) involved in the premature destruction of erythrocytes in peripheral blood, implicated in the hemoglobin disorder, HbE/beta-thalassemia.     

Thermal stability of outer membrane protein porin from Paracoccus denitrificans: FT-IR as a spectroscopic tool to study lipid-protein interaction

Lipid protein interactions play a key role in the stability and function of various membrane proteins. Earlier we have reported the extreme thermal stability of porin from Paracoccus denitrificans reconstituted into liposomes. Here, we used Fourier transform infrared spectroscopy for a label free analysis of the global secondary structural changes and local changes in the tyrosine microenvironment. Our results show that a mixed lipid system (non-uniform bilayer) optimizes the thermal stability of porin as compared to the porin in pure lipids (uniform bilayer) or detergent micelles. This is in line with the fact that the bacterial outer membrane is a dynamic system made up of lipids of varying chain lengths, head groups and the barrel wall height contacting the membrane is uneven.