Huntingtin disrupts lipid bilayers in a polyQ-length dependent manner

Huntington's Disease (HD) is a neurodegenerative disorder that is defined by the accumulation of nanoscale aggregates comprised of the huntingtin (htt) protein. Aggregation is directly caused by an expanded polyglutamine (polyQ) domain in htt, leading to a diverse population of aggregate species, such as oligomers, fibrils, and annular aggregates. Furthermore, the length of this polyQ domain is directly related to onset and severity of disease. The first 17 N-terminal amino acids of htt have been shown to further modulate aggregation. Additionally, these 17 amino acids appear to have lipid binding properties as htt interacts with a variety of membrane-containing structures present in cells, such as organelles, and interactions with these membrane surfaces may further modulate htt aggregation. To investigate the interaction between htt exon1 and lipid bilayers, in situ atomic force microscopy (AFM) was used to directly monitor the aggregation of htt exon1 constructs with varying Q-lengths (35Q, 46Q, 51Q, and myc-53Q) on supported lipid membranes comprised of total brain lipid extract. The exon1 fragments accumulated on the lipid membranes, causing disruption of the membrane, in a polyQ dependent manner. Furthermore, the addition of an N-terminal myc-tag to the htt exon1 fragments impeded the interaction of htt with the bilayer.     

Modulation of Polyglutamine Conformations and Dimer Formation by the N-Terminus of Huntingtin

Polyglutamine expansions within different proteins are associated with nine different neurodegenerative diseases. There is growing interest in understanding the roles of flanking sequences from disease-relevant proteins in the intrinsic conformational and aggregation properties of polyglutamine. We report results from atomistic simulations and circular dichroism experiments that quantify the effect of the N-terminal 17-residue (Nt17) segment of the huntingtin protein on polyglutamine conformations and intermolecular interactions. We show that the Nt17 segment and polyglutamine domains become increasingly disordered as polyglutamine length (N) increases in Nt17-Q_N constructs. Hydrophobic groups within Nt17 become sequestered in intramolecular interdomain interfaces. We also show that the Nt17 segment suppresses the intrinsic propensity of polyglutamine aggregation. This inhibition arises from the incipient micellar structures adopted by monomeric forms of the peptides with Nt17 segments. The degree of intermolecular association increases with increasing polyglutamine length and is governed mainly by associations between polyglutamine domains. Comparative analysis of intermolecular associations for different polyglutamine-containing constructs leads to clearer interpretations of recently published experimental data. Our results suggest a framework for fibril formation and identify roles for flanking sequences in the modulation of polyglutamine aggregation.     

Polyglutamine expansion in huntingtin increases its insertion into lipid bilayers

An expanded polyglutamine (Q) tract (>37Q) in huntingtin (htt) causes Huntington disease. Htt associates with membranes and polyglutamine expansion in htt may alter membrane function in Huntington disease through a mechanism that is not known. Here we used differential scanning calorimetry to examine the effects of polyQ expansion in htt on its insertion into lipid bilayers. We prepared synthetic lipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine and tested interactions of htt amino acids 1-89 with 20Q, 32Q or 53Q with the vesicles. GST-htt1-89 with 53Q inserted into synthetic lipid vesicles significantly more than GST-htt1-89 with 20Q or 32Q. We speculate that by inserting more into cell membranes, mutant huntingtin could increase disorder within the lipid bilayer and thereby disturb cellular membrane function.     

Essential sequence of the N-terminal cytoplasmic localization-related domain of huntingtin and its effect on huntingtin aggregates

Huntington’s disease (HD) is caused by abnormal CAG repeat expansion in the 5′-end of the Huntingtin (HTT) gene. In addition to the canonical C-terminal full-length huntingtin (htt) nuclear export signal, a cytoplasmic localization-related domain (CLRD) in the N-terminus of htt has recently been reported. Here, we analyzed this domain by introducing deletion and substitution mutations in a truncated N-terminal htt protein and subsequently monitored htt expression, aggregation and subcellular localization by immunocytochemistry and Western blot analysis. We demonstrated that Htt_4–17 was the essential sequence for htt cytoplasmic localization. We also found that the subcellular distribution of htt was altered when Htt_1–17 was mutated to contain amino acids of different charges, suggesting a structural requirement of Htt_1–17 for the cytoplasmic localization of htt. Deletion of the first three amino acids did not affect its association with mitochondria. We observed that defective cytoplasmic localization resulted in a reduction of total htt aggregates and increased nuclear aggregates, indicating that the subcellular distribution of the protein might influence the aggregation process. These studies provide new insight into the molecular mechanism of htt aggregation in HD.     

Coenzyme Q distribution in HL-60 human cells depends on the endomembrane system

Coenzyme Q (Q) is an essential factor in the mitochondrial electron chain but also exerts important antioxidant functions in the rest of cell membranes of aerobic organisms. However, the mechanisms of distribution of Q among cell membranes are largely unclear. The aim of the present work is to study the mechanisms of distribution of endogenous Q₁₀ and exogenous Q₉ among cell membranes in human HL-60 cells. Endogenous Q₁₀ synthesized using the radiolabelled precursor [¹⁴C]-pHB was first detected in mitochondria, and it was later incorporated into mitochondria-associated membranes and endoplasmic reticulum (ER). Plasma membrane was the last location to incorporate [¹⁴C]-Q₁₀. Brefeldin A prevented Q₁₀ incorporation in plasma membrane. Exogenous Q₉ was preferably accumulated into the endo-lysosomal fraction but a significant amount was distributed among other cell membranes also depending on the brefeldin-A-sensitive endomembrane system. Our results indicate that mitochondria are the first location for new synthesized Q. Exogenous Q is mainly incorporated into an endo-lysosomal fraction, which is then rapidly incorporated to cell membranes mainly to MAM and mitochondria. We also demonstrate that both endogenous and dietary Q is distributed among endomembranes and plasma membrane by the brefeldin A-sensitive endo-exocytic pathway.     

Obestatin and Nt8U influence glycerolipid metabolism and PPAR gamma signaling in mice

Obestatin, its N-terminal fragment and the N-terminal fragment analog Nt8U were previously shown to reduce food intake, gain in body weight and triglyceride levels in albino mice. To establish their mode of action, mRNA profiling of the epididymal adipose tissue of mice treated with these peptides were performed. The differential expressions were markedly indicative of their involvement in lipid metabolism. Obestatin showed a significant upregulation of the genes patatin-like phospholipase domain containing 3, diacylglycerol O-acyltransferase 2, monoglyceride lipase, aldo–keto reductase family 1, member 7 which are involved in glycerolipid metabolism. It also upregulated peroxisome proliferator-activated receptor gamma, retinoid X receptor gamma, cluster of differentiation 36, adiponectin, C1Q and collagen domain containing, angiopoietin-like 4, lipoprotein lipase, stearoyl-coenzyme A and desaturase 3 involved in the peroxisome proliferator-activated receptor signaling pathway. Nt8U upregulated genes implicated in the same two pathways but with lesser significance and also upregulated APOL2. The N-terminal fragment though differentially regulated a small subset of the genes differentially regulated by obestatin and Nt8U, no conclusive evidence was obtained as to assign a specific pathway for its mode of action. We hypothesize that reduced food intake brought about by obestatin and Nt8U triggers lipid catabolism. The free fatty acids and lysophosphatidic acid thus produced in turn activates peroxisome proliferator-activated receptor gamma and the genes involved in peroxisome proliferator-activated receptor signaling. All of them together lead to reduction in gain in bodyweight, stored fat and circulating lipids. These results also correlate well with the observed efficacy of the peptides.     

The Nt17 Domain and its Helical Conformation Regulate the Aggregation,Cellular Properties and Neurotoxicity of Mutant Huntingtin Exon 1

Converging evidence points to the N-terminal domain comprising the first 17 amino acids of the Huntingtin protein (Nt17) as a key regulator of its aggregation, cellular properties and toxicity. In this study, we further investigated the interplay between Nt17 and the polyQ domain repeat length in regulating the aggregation and inclusion formation of exon 1 of the Huntingtin protein (Httex1). In addition, we investigated the effect of removing Nt17 or modulating its local structure on the membrane interactions, neuronal uptake, and toxicity of monomeric or fibrillar Httex1. Our results show that the polyQ and Nt17 domains synergistically modulate the aggregation propensity of Httex1 and that the Nt17 domain plays important roles in shaping the surface properties of mutant Httex1 fibrils and regulating their poly-Q-dependent growth, lateral association and neuronal uptake. Removal of Nt17 or disruption of its transient helical conformations slowed the aggregation of monomeric Httex1 in vitro, reduced inclusion formation in cells, enhanced the neuronal uptake and nuclear accumulation of monomeric Httex1 proteins, and was sufficient to prevent cell death induced by Httex1 72Q overexpression. Finally, we demonstrate that the uptake of Httex1 fibrils into primary neurons and the resulting toxicity are strongly influenced by mutations and phosphorylation events that influence the local helical propensity of Nt17. Altogether, our results demonstrate that the Nt17 domain serves as one of the key master regulators of Htt aggregation, internalization, and toxicity and represents an attractive target for inhibiting Htt aggregate formation, inclusion formation, and neuronal toxicity.     

Serum withdrawal-induced accumulation of phosphoinositide 3-kinase lipids in differentiating 3T3-L6 myoblasts: distinct roles for Ship2 and PTEN

Phosphoinositide 3-kinase (PI3K) activation and synthesis of phosphatidylinositol-3,4-bisphosphate (PI-3,4-P₂) and phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P₃) lipids mediate growth factor signaling that leads to cell proliferation, migration, and survival. PI3K-dependent activation of Akt is critical for myoblast differentiation induced by serum withdrawal, suggesting that in these cells PI3K signaling is activated in an unconventional manner. Here we investigate the mechanisms by which PI3K signaling and Akt are regulated during myogenesis. We report that PI-3,4-P₂ and PI-3,4,5-P₃ accumulated in the plasma membranes of serum-starved 3T3-L6 myoblasts due to de novo synthesis and increased lipid stability. Surprisingly, only newly synthesized lipids were capable of activating Akt. Knockdown of the lipid phosphatase PTEN moderately increased PI3K lipids but significantly increased Akt phosphorylation and promoted myoblast differentiation. Knockdown of the lipid phosphatase Ship2, on the other hand, dramatically increased the steady-state levels of PI-3,4,5-P₃ but did not affect Akt phosphorylation and increased apoptotic cell death. Together, these results reveal the existence of two distinct pools of PI3K lipids in differentiating 3T3-L6 myoblasts: a pool of nascent lipids that is mainly dephosphorylated by PTEN and is capable of activating Akt and promoting myoblast differentiation and a stable pool that is dephosphorylated by Ship2 and is unable to activate Akt.     

Native mutant huntingtin in human brain: evidence for prevalence of full-length monomer

Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575-850 kDa in control brain and at 650-885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1-17)) and increased when lysates were treated with denaturants (SDS, 8 M urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670-880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43-50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 M urea + DTT. Native full-length mutant htt in embryonic HD(140Q/140Q) mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer.     

Sequestration of glyceraldehyde-3-phosphate dehydrogenase to aggregates formed by mutant huntingtin

Glyceraldehyde-3-phosphate dehydrogenase （GAPDH） has been reported to interact with proteins containing the polyglutamine （polyQ） domain. The present study was undertaken to evaluate the potential contributions of the polyQ and polyproline （polyP） domains to the co-localization of mutant huntingtin （htt） and GAPDH. Overexpression of N-terminal htt （1-969 amino acids） with 100Q and 46Q （httl-969- 100Q and httl-969-46Q, mutant htt） in human mammary gland carcinoma MCF-7 cells formed more htt aggregates than that of httl-969-18Q （wild-type htt）. The co-localization of GAPDH with htt aggregates was found in the cells expressing mutant but not wild-type htt. Deletion of the polyP region in the N-terminal htt had no effect on the co-localization of GAPDH and mutant htt aggregates. These results suggest that the polyQ domain, but not the polyP domain, plays a role in the sequestration of GAPDH to aggregates by mutant htt. This effect might contribute to the dysfunction of neurons caused by mutant htt in Huntington＇s disease.     

Molecular mechanisms of interaction of polycationic peptides with serpentine type receptors and heterotrimeric G-proteins in rat tissues

The key step in the hormonal signal transduction into cell is interaction of receptors with heterotrimeric G-proteins. We and other authors have shown that G-proteins may be activated as a result of their direct interaction with polycationic peptides. The goal of this work was to study molecular mechanisms of effect of hydrophobic peptide I, C-εAhx-WKK(C₁₀)-KKK(C₁₀)-KKKK(C₁₀)-YKK(C₁₀)-KK, and branched peptide II, [(GRGDSGRKKRRQRRRPPQ)₂-K-εAhx-C]₂ including the 48–60 fragment of the HIV-1 TAT-protein, on receptor and G-protein. These two peptides (10^?6?10^?4 M) produced a dose-dependent simulation of the GTP-binding activity of G-proteins in plasma membrane fractions of the brain striatum and cardiac muscle in rats. The effect of peptide I was more pronounced and decreased to a considerable degree in the presence of the C-terminal 385–394 peptide of the G-protein α_s-subunit that selectively disrupts interaction of receptors with G_s-protein. Peptide I reduced markedly affinity of serotonin (agonist) to the serotonin striatum receptors, whereas peptide II inhibited to the significant extent the binding of dihydroalprenolol (antagonist) to β-adrenergic receptors in cardiac muscle. Peptide I, unlike peptide II, decreased essentially the high affinity binding of β-agonist isoproterenol. The obtained data indicate the ability of polycationic peptides to activate G₁-proteins, to disturb their coupling with receptor, and to affect binding properties of the receptor. There are differences in molecular mechanisms of action of peptides with different structures on G-proteins and receptors.     

Twelve pregnane glycosides from Cynanchum atratum

Eleven new 14,15-seco-pregnane-type steroidal glycosides, cynanosides P₁-P₅, Q₁-Q₃, R₁-R₃, and a novel 12,13-seco-14,18-nor-pregnane-type steroidal glycoside, cynanoside S, were isolated from the roots of Cynanchum atratum, together with four known compounds, atratoside C, sublanceoside E₃, chekiangensoside C and cynatroside B. Their structures were determined on the basis of spectroscopic analysis and chemical evidence.     

Effect of the side chain structure of coenzyme Q on the steady state kinetics of bovine heart NADH: coenzyme Q oxidoreductase

Steady state kinetics of bovine heart NADH: coenzyme Q oxidoreductase using coenzyme Q with two isoprenoid unit (Q₂) or with a decyl group (DQ) show an ordered sequential mechanism in which the order of substrate binding and product release is NADH-Q₂ (DQ) -Q₂H₂ (DQH₂)-NAD⁺ in contrast to the order determined using Q₁ (Q₁-NADH-NAD⁺-Q₁H₂) (Nakashima et al., J. Bioenerg. Biomembr. 34, 11–19, 2002). The effect of the side chain structure of coenzyme Q suggests that NADH binding to the enzyme results in a conformational change, in the coenzyme Q binding site, which enables the site to accept coenzyme Q with a side chain significantly larger than one isoprenoid unit. The side chains of Q₂ and DQ bound to the enzyme induce a conformational change in the binding site to stabilize the substrate binding, while the side chain of Q₁ (one isoprenoid unit) is too short to induce the conformational change.     

Interaction of a peptide corresponding to the loop domain of the S2 SARS-CoV virus protein with model membranes

The severe acute respiratory syndrome coronavirus (SARS-CoV) envelope spike (S) glycoprotein is responsible for the fusion between the membranes of the virus and the target cell. In the case of the S2 domain of protein S, it has been found a highly hydrophobic and interfacial domain flanked by the heptad repeat 1 and 2 regions; significantly, different peptides pertaining to this domain have shown a significant leakage effect and an important plaque formation inhibition, which, similarly to HIV-1 gp41, support the role of this region in the fusion process. Therefore, we have carried out a study of the binding and interaction with model membranes of a peptide corresponding to segment 1073–1095 of the SARS-CoV S glycoprotein, peptide SARS_L in the presence of different membrane model systems, as well as the structural changes taking place in both the lipid and the peptide induced by the binding of the peptide to the membrane. Our results show that SARS_L strongly partitions into phospholipid membranes and organizes differently in lipid environments, displaying membrane activity modulated by the lipid composition of the membrane. These data would support its role in SARS-CoV mediated membrane fusion and suggest that the region where this peptide resides could be involved in the merging of the viral and target cell membranes.     

Z-DNA and other non-B-DNA structures are reversed to B-DNA by interaction with netropsin

The interaction between the B-form specific ligands netropsin (Nt) and distamycin-3 (Dst-3) and DNA duplexes has been studied under conditions of salt concentration and low water activity that modify the polymer conformation into a non-B DNA form, putatively a Z-like form. Three polymers with strict alternating purine-pyrimidine sequences and GC content from 100-0% have been tested: poly(dG-dC) . poly(dG-dC), poly(dA-dC) . poly(dG-dT) and poly(dA-dT) . poly(dA-dT). The titrations by Nt and Dst-3 were followed by circular dichroism. Although specific binding of Nt to the Z-form of poly(dG-dC) . poly(dG-dC) does not occur, Nt reverses this Z structure to the B-type conformation; Dst-3 is, however, totally inefficient. The presumed non-B or Z-like structure of poly(dA-dC) . poly(dG-dT) is reversed to the B-form upon interaction with Nt; Dst-3 also induces this reversal but at higher ligand ratios. The modified B-structure of poly(dA-dT) . poly(dA-dT) in low water activity is efficiently reversed to the B-form by interaction with both Nt and Dst-3.     

Flanking polyproline sequences inhibit beta-sheet structure in polyglutamine segments by inducing PPII-like helix structure

Polyglutamine (poly(Q)) expansion is associated with protein aggregation into β-sheet amyloid fibrils and neuronal cytotoxicity. In the mutant poly(Q) protein huntingtin, associated with Huntington's disease, both aggregation and cytotoxicity may be abrogated by a polyproline (poly(P)) domain flanking the C terminus of the poly(Q) region. To understand structural changes that may occur with the addition of the poly(P) sequence, we synthesized poly(Q) peptides with 3-15 glutamine residues and a corresponding set of poly(Q) peptides flanked on the C terminus by 11 proline residues (poly(Q)-poly(P)), as occurs in the huntingtin sequence. The shorter soluble poly(Q) peptides (three or six glutamine residues) showed polyproline type II-like (PPII)-like helix conformation when examined by circular dichroism spectroscopy and were monomers as judged by size-exclusion chromatography (SEC), while the longer poly(Q) peptides (nine or 15 glutamine residues) showed a β-sheet conformation by CD and defined oligomers by SEC. Soluble poly(Q)-poly(P) peptides showed PPII-like content but SEC showed poorly defined, overlapping oligomeric peaks, and as judged by CD these peptides retained significant PPII-like structure with increasing poly(Q) length. More importantly, addition of the poly(P) domain increased the threshold for fibril formation to ≈ 15 glutamine residues. X-ray diffraction, electron microscopy, and film CD showed that, while poly(Q) peptides with ≥ 6 glutamine residues formed β-sheet-rich fibrils, only the longest poly(Q)-poly(P) peptide (15 glutamine residues) did so. From these and other observations, we propose that poly(Q) domains exist in a “tug-of-war” between two conformations, a PPII-like helix and a β-sheet, while the poly(P) domain is conformationally constrained into a proline type II helix (PPII). Addition of poly(P) to the C terminus of a poly(Q) domain induces a PPII-like structure, which opposes the aggregation-prone β-sheet. These structural observations may shed light on the threshold phenomenon of poly(Q) aggregation, and support the hypothesized evolution of “protective” poly(P) tracts adjacent to poly(Q) aggregation domains.     

Interaction of gramicidin S and its aromatic amino-acid analog with phospholipid membranes

To investigate the mechanism of interaction of gramicidin S-like antimicrobial peptides with biological membranes, a series of five decameric cyclic cationic β-sheet-β-turn peptides with all possible combinations of aromatic D-amino acids, Cyclo(Val-Lys-Leu-D-Ar1-Pro-Val-Lys-Leu-D-Ar2-Pro) (Ar ≡ Phe, Tyr, Trp), were synthesized. Conformations of these cyclic peptides were comparable in aqueous solutions and lipid vesicles. Isothermal titration calorimetry measurements revealed entropy-driven binding of cyclic peptides to POPC and POPE/POPG lipid vesicles. Binding of peptides to both vesicle systems was endothermic—exceptions were peptides containing the Trp-Trp and Tyr-Trp pairs with exothermic binding to POPC vesicles. Application of one- and two-site binding (partitioning) models to binding isotherms of exothermic and endothermic binding processes, respectively, resulted in determination of peptide-lipid membrane binding constants (K_b). The K_b1 and K_b2 values for endothermic two-step binding processes corresponded to high and low binding affinities (K_b1 ≥ 100 K_b2). Conformational change of cyclic peptides in transferring from buffer to lipid bilayer surfaces was estimated using fluorescence resonance energy transfer between the Tyr-Trp pair in one of the peptide constructs. The cyclic peptide conformation expands upon adsorption on lipid bilayer surface and interacts more deeply with the outer monolayer causing bilayer deformation, which may lead to formation of nonspecific transient peptide-lipid porelike zones causing membrane lysis.     

Hsp70 and Hsp40 Functionally Interact with Soluble Mutant Huntingtin Oligomers in a Classic ATP-dependent Reaction Cycle

Inclusion bodies of aggregated mutant huntingtin (htt) fragments are a neuropathological hallmark of Huntington disease (HD). The molecular chaperones Hsp70 and Hsp40 colocalize to inclusion bodies and are neuroprotective in HD animal models. How these chaperones suppress mutant htt toxicity is unclear but might involve direct effects on mutant htt misfolding and aggregation. Using size exclusion chromatography and atomic force microscopy, we found that mutant htt fragments assemble into soluble oligomeric species with a broad size distribution, some of which reacted with the conformation-specific antibody A11. Hsp70 associated with A11-reactive oligomers in an Hsp40- and ATP-dependent manner and inhibited their formation coincident with suppression of caspase 3 activity in PC12 cells. Thus, Hsp70 and Hsp40 (DNAJB1) dynamically target specific subsets of soluble oligomers in a classic ATP-dependent reaction cycle, supporting a pathogenic role for these structures in HD.     

Galleria mellonella native and analogue peptides Gm1 and ΔGm1. I) Biophysical characterization of the interaction mechanisms with bacterial model membranes

Natural occurring antimicrobial peptides (AMPs) are important components of the innate immune system of animals and plants. They are considered to be promising alternatives to conventional antibiotics. Here we present a comparative study of two synthetic peptides: Gm1, corresponding to the natural overall uncharged peptide from Galleria mellonella (Gm) and ΔGm1, a modified overall positively charged Gm1 variant. We have studied the interaction of the peptides with lipid membranes composed of different kinds of lipopolysaccharides (LPS) and dimyristoylphosphatidylglycerol (DMPG), in some cases also dimyristoylphosphatidylethanolamine (DMPE) as representative lipid components of Gram-negative bacterial membranes, by applying Fourier-transform infrared spectroscopy (FTIR), Förster resonance energy transfer spectroscopy (FRET), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). Gm1 generates a destabilizing effect on the gel to liquid crystalline phase transition of the acyl chains of the lipids, as deduced from a decrease in the phase transition temperature and enthalpy, suggesting a fluidization, whereas ΔGm1 led to the opposite behavior. Further, FTIR analysis of the functional groups of the lipids participating in the interaction with the peptides indicated a shift in the band position and intensity of the asymmetric PO₂⁻ stretching vibration originating from the lipid phosphate groups, a consequence of the sterical changes in the head group region. Interestingly, FRET spectroscopy showed a similar intercalation of both peptides into the DMPG and LPS, but much less into the DMPE membrane systems. These results are discussed in the light of a possible use of the peptides as antimicrobial and anti-endotoxin drugs.     

Roles of peptide-peptide charge interaction and lipid phase separation in helix-helix association in lipid bilayer

The roles of peptide-peptide charged interaction and lipid phase separation in helix-helix association in lipid bilayers were investigated using a model peptide, P₂₄, as a transmembrane α-helical peptide, and its four analogues. Fluorescence amino acids, tryptophan (P₂₄W) and pyrenylalanine (P₂₄Pya), were introduced into the sequence of P₂₄, respectively. Association of these peptides permits the resonance excitation energy transfer between tryptophan in P₂₄W and pyrenylalanine in P₂₄Pya or excimer formation between P₂₄Pya themselves. To evaluate the effect of charged interaction on the association between α-helical transmembrane segments in membrane proteins, charged amino acids, glutamic acid (P₂₄EW) and lysine (P₂₄KPya), were introduced into P₂₄W and P₂₄Pya, respectively. Energy transfer experiments indicated that the charged interaction between the positive charge of lysine residue in P₂₄KPya and the negative charge of glutamic acid residue in P₂₄EW did not affect the aggregation of transmembrane peptides in lipid membranes. As the content ratio of sphingomyelin (SM) and cholesterol (Ch) was increased in the egg phosphatidylcholine (PC), the stronger excimer fluorescence spectra of P₂₄Pya were observed, indicating that the co-existence of SM and Ch in PC liposomes, that is, the raft of SM and Ch, promotes the aggregation of the α-helical transmembrane peptides in lipid bilayers. Since the increase in the contents of SM and Ch leads to the decrease in the content of liquid crystalline-order phase, the moving area of transmembrane peptides might be limited in the liposomes, resulting in easy formation of the excimer in the presence of the lipid-raft.