The effect of N-terminal acetylation on the structure of an N-terminal tropomyosin peptide and alpha alpha-tropomyosin.

We have used a synthetic peptide consisting of the first 30 residues of striated muscle alpha-tropomyosin, with GlyCys added to the C-terminus, to investigate the effect of N-terminal acetylation on the conformation and stability of the N-terminal domain of the coiled-coil protein. In aqueous buffers at low ionic strength, the reduced, unacetylated 32mer had a very low alpha-helical content (approximately 20%) that was only slightly increased by disulfide crosslinking or N-terminal acetylation. Addition of salt (> 1 M) greatly increased the helical content of the peptide. The CD spectrum, the cooperativity of folding of the peptide, and sedimentation equilibrium ultracentrifugation studies showed that it formed a 2-chained coiled coil at high ionic strength. Disulfide crosslinking and N-terminal acetylation both greatly stabilized the coiled-coil alpha-helical conformation in high salt. Addition of ethanol or trifluoroethanol to solutions of the peptide also increased its alpha-helical content. However, the CD spectra and unfolding behavior of the peptide showed no evidence of coiled-coil formation. In the presence of the organic solvents, N-terminal acetylation had very little effect on the conformation or stability of the peptide. Our results indicate that N-terminal acetylation stabilizes coiled-coil formation in the peptide. The effect cannot be explained by interactions with the "helix-dipole" because the stabilization is observed at very high salt concentrations and is independent of pH. In contrast to the results with the peptide, N-terminal acetylation has only small effects on the overall stability of tropomyosin.     

Lipid-packing perturbation of model membranes by pH-responsive antimicrobial peptides

The indiscriminate use of conventional antibiotics is leading to an increase in the number of resistant bacterial strains, motivating the search for new compounds to overcome this challenging problem. Antimicrobial peptides, acting only in the lipid phase of membranes without requiring specific membrane receptors as do conventional antibiotics, have shown great potential as possible substituents of these drugs. These peptides are in general rich in basic and hydrophobic residues forming an amphipathic structure when in contact with membranes. The outer leaflet of the prokaryotic cell membrane is rich in anionic lipids, while the surface of the eukaryotic cell is zwitterionic. Due to their positive net charge, many of these peptides are selective to the prokaryotic membrane. Notwithstanding this preference for anionic membranes, some of them can also act on neutral ones, hampering their therapeutic use. In addition to the electrostatic interaction driving peptide adsorption by the membrane, the ability of the peptide to perturb lipid packing is of paramount importance in their capacity to induce cell lysis, which is strongly dependent on electrostatic and hydrophobic interactions. In the present research, we revised the adsorption of antimicrobial peptides by model membranes as well as the perturbation that they induce in lipid packing. In particular, we focused on some peptides that have simultaneously acidic and basic residues. The net charges of these peptides are modulated by pH changes and the lipid composition of model membranes. We discuss the experimental approaches used to explore these aspects of lipid membranes using lipid vesicles and lipid monolayer as model membranes.     

A study on the perturbation of model lipid membranes by phenoxazines

The interactions of six newly synthesized phenoxazine derivatives with lipid bilayers were studied by means of calorimetry, fluorescence spectroscopic methods and electron spin resonance. Depending on their structure studied compounds decreased membrane fluidity and increased lipid order in liquid-crystalline bilayers to different degrees. These studies showed also that phenoxazine molecules are located close to the polar/apolar interface of bilayer. The results allow to conclude that phenoxazines rather weakly interact with lipid bilayers.     

Human caspase-7 activity and regulation by its N-terminal peptide

Central to the execution phase of apoptosis are the two closely related caspase-3 and -7. They share common substrate specificity and structure, but differ completely in the sequence of their respective N-terminal regions including their N-peptides, a 23-28 residue segment that are removed during zymogen activation. We show that the N-peptide of caspase-7 plays no role in the fundamental activation or properties of the active protease in vitro. However, the N-peptide modifies the properties of caspase-7 in vivo. In ectopic expression experiments, caspase-7 constructs with no N-peptide are far more lethal than constructs that have an uncleavable peptide. Moreover, the N-peptide of caspase-7 must be removed before efficient activation of the zymogen can occur in vivo. These disparate requirements for the N-peptide argue that it serves to physically sequester the caspase-7 zymogen in a cytosolic location that prevents access by upstream activators (caspase-8, -9, and -10). The N-peptide must first be removed, probably by caspase-3, before efficient conversion and activation of the zymogen can occur in vivo.     

Control of cytomegalovirus lytic gene expression by histone acetylation

Permissiveness for human cytomegalovirus (HCMV) infection is dependent on the state of cellular differentiation and has been linked to repression of the viral major immediate early promoter (MIEP). We have used conditionally permissive cells to analyze differential regulation of the MIEP and possible mechanisms involved in latency. Our data suggest that histone deacetylases (HDACs) are involved in repression of the MIEP in non-permissive cells as inhibition of HDACs induces viral permissiveness and increases MIEP activity. Non-permissive cells contain the class I HDAC, HDAC3; super-expression of HDAC3 in normally permissive cells reduces infection and MIEP activity. We further show that the MIEP associates with acetylated histones in permissive cells, and that in peripheral blood monocytes the MIEP associates with heterochromatin protein 1 (HP1), a chromosomal protein implicated in gene silencing. As monocytes are believed to be a site of viral latency in HCMV carriers and reactivated virus is only observed upon differentiation into macrophages, we propose that chromatin remodeling of the MIEP following cellular differentiation could potentially play a role in reactivation of latent HCMV.     

Increased inhibitory capacity of an anti-C5a complementary peptide following acetylation of N-terminal alanine

Amino acids 37 to 53 (RAARISLGPRCIKAFTE) of C5a anaphylatoxin form an essential region for C5a function. To target this sequence, we generated a complementary peptide (ASGAPAPGPAGPLRPMF) designated PepA which has a potent inhibitory effect on C5a activity. By introducing an acetyl group at the N-terminal alanine of PepA, an acetylated form was generated which was designated AcPepA. The acetylation resulted in increased inhibition of C5a stimulation of neutrophils as determined by Ca influx. Furthermore, AcPepA partially inhibited the lethal shock induced in mice by intravenous administration of Candida albicans water-soluble mannoprotein-beta-glucan complex. In addition, local skin inflammation in rats caused by an anti-Crry monoclonal antibody was suppressed when AcPepA and the antibody were injected together, while PepA had little inhibitory capacity. The potent inhibitory capacity of AcPepA was also confirmed by a skin reaction of guinea pigs inoculated with recombinant human C5a together with AcPepA.     

Imaging the membrane lytic activity of bioactive peptide latarcin 2a

Latarcin 2a (ltc2a, GLFGKLIKKFGRKAISYAVKKARGKH-COOH) is a short linear antimicrobial and cytolytic peptide extracted from the venom of the Central Asian spider, Lachesana tarabaevi, with lytic activity against Gram-positive and Gram-negative bacteria, erythrocytes, and yeast at micromolar concentrations. Ltc2a adopts a helix-hinge-helix structure in membrane mimicking environment, whereas its derivative latarcin 2aG11A (ltc2aG11A, GLFGKLIKKFARKAISYAVKKARGKH-COOH), likely adopts a more rigid structure, demonstrates stronger nonspecific interaction with the zwitterionic membrane, and is potentially more toxic against eukaryotic cells. In this work, interactions of these two ltc2a derivatives with supported "raft" lipid bilayer (1,2-dioleoyl-sn-glycero-3-phosphocholin/egg sphingomyelin/cholesterol 40/40/20mol%) were studied by in situ atomic force microscopy in order to investigate the potential anticancer activity of the peptides since some breast and prostate cancer cell lines contain higher levels of cholesterol-rich lipid rafts than non-cancer cells. Both peptides induced reorganization of the raft model membrane by reducing line tension of the liquid ordered phase. Ltc2aG11A induced membrane thinning likely due to membrane interdigitation. Formation of large pores by the peptides in the bilayer was observed. Cholesterol was found to attenuate membrane disruption by the peptides. Finally, leakage assay showed that both peptides have similar membrane permeability toward various model membrane vesicles.     

Membrane aggregation and perturbation induced by antimicrobial peptide of S-thanatin

Thanatin, a 21-residue peptide, is an inducible insect peptide. In our previous study, we have identified a novel thanatin analog of S-thanatin, which exhibited a broad antimicrobial activity against bacteria and fungi with low hemolytic activity. This study was aimed to delineate the antimicrobial mechanism of S-thanatin and identify its interaction with bacterial membranes. In this study, membrane phospholipid was found to be the target for S-thanatin. In the presence of vesicles, S-thanatin interestingly led to the aggregation of anionic vesicles and sonicated bacteria. Adding S-thanatin to Escherichia coli suspension would result in the collapse of membrane and kill bacteria. The sensitivity assay of protoplast elucidated the importance of outer membrane (OM) for S-thanatin’s antimicrobial activity. Compared with other antimicrobial peptide, S-thanatin produced chaotic membrane morphology and cell debris in electron microscopic appearance. These results supported our hypothesis that S-thanatin bound to negatively charged LPS and anionic lipid, impeded membrane respiration, exhausted the intracellular potential, and released periplasmic material, which led to cell death.     

Viral membrane penetration: lytic activity of a nodaviral fusion peptide

The auto-cleavage product from the C-terminal part of the capsid protein of the flock house virus, namely the ₁ peptide, was used as a model peptide to characterize the initial steps of viral membrane penetration. Monolayers at the air–water interface were used to investigate the phase behaviour of ternary lipid–peptide mixtures, whereas solid-supported membranes were used to visualize the lytic activity of the ₁ peptide. 1,2-Dipalmitoyl-sn-glycero-phospatidylcholine/1,2-dipalmitoyl-sn-glycero-phospatidylserine (4:1) membranes were used as negatively charged model membranes. By means of film balance techniques lipid/peptide discrimination was found resulting in a lipid-rich and a peptide-rich phase. Quartz crystal microbalance and scanning force microscopy experiments led to the conclusion of a detergent-like mechanism of the ₁ peptide resulting in mixed lipid–peptide micelles with a molar ratio of 2.8:1. A monolayer adsorption with an ongoing lysis of membranes was found with ₁ peptide molecules interacting at membrane defects.     

Influence of N-terminal acetylation and C-terminal proteolysis on the analgesic activity of beta-endorphin.

Removal of one, two and four amino-acid residues from the C-terminus of beta-endorphin ('lipotropin C-Fragment', lipotropin residues 61--91) led to the formation of peptides with progressively decreased analgesic potency; there was no change in the persistence of the analgesic effects. The four C-terminal residues are thus important for the activity of beta-endorphin, but not for the duration of action. Removal of eight amino-acid residues from the N-terminus provided a peptide that had no specific affinity for brain opiate receptors in vitro and was devoid of analgesic properties. The N-terminal sequence of beta-endorphin is therefore necessary for the production of analgesia, whereas the C-terminal residues confer potency. The N alpha-acetyl form of beta-endorphin had no specific affinity for brain opiate receptors in vitro and possessed no significant analgesic properties. Since lipotropin C'-Fragment (lipotropin residues 61--87) and the N alpha-acetyl derivative of beta-endorphin occur naturally in brain and pituitary and are only weakly active or inactive as opiates, it is suggested that proteolysis at the C-terminus and acetylation of the N-terminus of beta-endorphin may constitute physiological mechanisms for inactivation of this potent analgesic peptide.     

Fusion-competent state induced by a C-terminal HIV-1 fusion peptide in cholesterol-rich membranes

The replicative cycle of the human immunodeficiency virus type-1 begins after fusion of the viral and target-cell membranes. The envelope glycoprotein gp41 transmembrane subunit contains conserved hydrophobic domains that engage and perturb the merging lipid bilayers. In this work, we have characterized the fusion-committed state generated in vesicles by CpreTM, a synthetic peptide derived from the sequence connecting the membrane-proximal external region (MPER) and the transmembrane domain (TMD) of gp41. Pre-loading cholesterol-rich vesicles with CpreTM rendered them competent for subsequent lipid-mixing with fluorescently-labeled target vesicles. Highlighting the physiological relevance of the lasting fusion-competent state, the broadly neutralizing antibody 4E10 bound to the CpreTM-primed vesicles and inhibited lipid-mixing. Heterotypic fusion assays disclosed dependence on the lipid composition of the vesicles that acted either as virus or cell membrane surrogates. Lipid-mixing exhibited above all a critical dependence on the cholesterol content in those experiments. We infer that the fusion-competent state described herein resembles bona-fide perturbations generated by the pre-hairpin MPER–TMD connection within the viral membrane.     

Towards a functional understanding of protein N-terminal acetylation

Protein N-terminal acetylation is a major modification of eukaryotic proteins. Its functional implications include regulation of protein-protein interactions and targeting to membranes, as demonstrated by studies of a handful of proteins. Fifty years after its discovery, a potential general function of the N-terminal acetyl group carried by thousands of unique proteins remains enigmatic. However, recent functional data suggest roles for N-terminal acetylation as a degradation signal and as a determining factor for preventing protein targeting to the secretory pathway, thus highlighting N-terminal acetylation as a major determinant for the life and death of proteins. These contributions represent new and intriguing hypotheses that will guide the research in the years to come.     

Aggregatation,fusion and aqueous content release from liposomes induced by lysozyme derivatives: Effect on the lytic activity

Chemically modified lysozymes, namely: N-succinyl lysozyme, glycine methyl ester of N-succinyl lysozyme and oxoindole lysozyme have been prepared. Aggregation, fusion and leakage of phospholipid vesicles induced by these derivatives have been studied in comparison with the effect of the unmodified protein. The experiments were carried out with negatively charges 9PC/ PA, 9:1) and uncharged (PC and PC/DOPE/Chol (10:5:5)) lipid vesicles of different packing. Fusion and aggregation of negatively charged phospholipid vesicles is induced by proteins positively charged at pH 7·0 involving electrostatic interactions. a similar pattern on fusion and aggregation of the least stably packed lipid vesicles points also to hydrophobic forces playing a role in the lipid-protein interaction. A conformational change of the protein involved increasing β-turns, loops and unordered structure at the expenses of β-sheet without affecting λhelix content. The conformational effect is necessary to provoke the effects studied, since one of the derivatives (N-succinyl lysozyme) neither changes conformation nor causes aggregation and fusion of vesicles. However, there is no relationship between lysozyme activity and fusion or aggregation of lipid vesicles that catalytic and fusogenci sites of, indicating lysozyme are topographically different     

N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins

N(alpha)-terminal acetylation occurs in the yeast Saccharomyces cerevisiae by any of three N-terminal acetyltransferases (NAT), NatA, NatB, and NatC, which contain Ard1p, Nat3p and Mak3p catalytic subunits, respectively. The N-terminal sequences required for N-terminal acetylation, i.e. the NatA, NatB, and NatC substrates, were evaluated by considering over 450 yeast proteins previously examined in numerous studies, and were compared to the N-terminal sequences of more than 300 acetylated mammalian proteins. In addition, acetylated sequences of eukaryotic proteins were compared to the N termini of 810 eubacterial and 175 archaeal proteins, which are rarely acetylated. Protein orthologs of Ard1p, Nat3p and Mak3p were identified with the eukaryotic genomes of the sequences of model organisms, including Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, Mus musculus and Homo sapiens. Those and other putative acetyltransferases were assigned by phylogenetic analysis to the following six protein families: Ard1p; Nat3p; Mak3p; CAM; BAA; and Nat5p. The first three families correspond to the catalytic subunits of three major yeast NATs; these orthologous proteins were identified in eukaryotes, but not in prokaryotes; the CAM family include mammalian orthologs of the recently described Camello1 and Camello2 proteins whose substrates are unknown; the BAA family comprise bacterial and archaeal putative acetyltransferases whose biochemical activity have not been characterized; and the new Nat5p family assignment was on the basis of putative yeast NAT, Nat5p (YOR253W). Overall patterns of N-terminal acetylated proteins and the orthologous genes possibly encoding NATs suggest that yeast and higher eukaryotes have the same systems for N-terminal acetylation.     

Analysis of the perturbation of phospholipid model membranes by rhodanese and its presequence.

The ability of the cytoplasmically synthesized mitochondrial enzyme rhodanese and its putative import signal sequence to interact with model phospholipid membranes was characterized. Membrane perturbation assays were used to test a current hypothesis that the initial step in protein translocation may involve binding of signal sequences with membrane lipids. Here we show comparative studies on the effect of native and various forms of denatured rhodanese, as well as two peptides, rho(1-23) and rho(11-23), derived from its NH2-terminal sequence, on the perturbation of 6-carboxyfluorescein-containing large unilamellar vesicles composed of either cardiolipin, phosphatidylcholine, or phosphatidylserine. We monitored the degree of perturbation by measuring dye leakage and found differential perturbation by either peptide or protein. Unfolded rhodanese perturbed vesicles in the order phosphatidylserine > cardiolipin > phosphatidylcholine. Denatured rhodanese was approximately 25 times more effective (on a molar basis) than rho(1-23) in the disruption of anionic liposomes. Rho(11-23) was unable to perturb liposomes. We found an inverse correlation between degree of activity of rhodanese folding intermediates and their ability to perturb liposomes. On urea denaturation, enzymatic activity was completely lost before membrane perturbation ability reached significant levels. Analysis of the peptides by circular dichroism showed that anionic liposomes can induce alpha-helical structure only in rho(1-23) and denatured rhodanese. Intrinsic peptide fluorescence studies showed that only rho(1-23) and denatured rhodanese partitioned into these model membranes. Results obtained here imply that peptides from naturally occurring alpha-helical structures may need adjacent motifs for helical structure induction in lipid environments, and the subsequent secondary structure may, in turn, promote partitioning of these segments into the lipid phase and ultimately lead to membrane perturbation.     

Unbinding-binding transition induced by molecular snaps in model membranes

We have used a lamellar phase made of a nonionic surfactant, dodecane and water, as a model membrane to investigate its interactions with macromolecular inclusions bringing together two membranes, i.e., acting as macromolecular snaps. In systems devoid of inclusions, the interlamellar distance depends on the total volume fraction of membranes Phi. We show that, in presence of a transmembrane protein, or of several de novo designed peptides of different length and composition, the lamellar phase undergoes a binding transition. Under such conditions, the interlamellar distance is no longer proportional to Phi(-1), but rather to the surface concentration of snaps within the membrane. It also appears that, in the presence of the hydrophobic segment of peptide snaps, the length of the inclusions must be at least equal to the hydrophobic length of the membrane to be active. Experimental results have been precisely fitted to a model of thermally stabilized membranes, decorated with snaps. However, in the presence of inclusions, the parameter describing the interactions between membranes, has to take into account the length of the inclusion to preserve good predictive capabilities.     

Inactivation of N-TIMP-1 by N-terminal acetylation when expressed in bacteria

The high-affinity binding of tissue inhibitors of metalloproteinases (TIMPs) to matrix metalloproteinases (MMPs) is essential for regulation of the turnover of the extracellular matrix during development, wound healing, and progression of inflammatory diseases, such as cancer, atherosclerosis, and arthritis. Bacterially expressed N-terminal inhibitory domains of TIMPs (N-TIMPs) have been used extensively for biochemical and biophysical study of interactions with MMPs. Titration of N-TIMP-1 expressed in E. coli indicates, however, that only about 42% of the protein is active as an MMP inhibitor. The separation of inactive from fully active N-TIMP-1 has been achieved both by MMP affinity and by high-resolution cation exchange chromatography at an appropriate pH, based on a slight difference of charge. Purification by cation exchange chromatography with a Mono S column enriches the active portion of N-TIMP-1 to >95%, with K(i) of 1.5 nM for MMP-12. Mass spectra reveal that the inactive form differs from active N-TIMP-1 in being N-terminally acetylated, underscoring the importance of the free alpha-NH(2) of Cys1 for MMP inhibition. N(alpha)-acetylation of the CTCVPP sequence broadens the N-terminal sequence motifs reported to be susceptible to alpha-amino acetylation by E. coli N-acetyl transferases. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 960-968, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Effect of channel block on the spiking activity of excitable membranes in a stochastic Hodgkin-Huxley model

The influence of intrinsic channel noise on the spontaneous spiking activity of poisoned excitable membrane patches is studied by use of a stochastic generalization of the Hodgkin-Huxley model. Internal noise stemming from the stochastic dynamics of individual ion channels is known to affect the collective properties of the whole ion channel cluster. For example, there exists an optimal size of the membrane patch for which the internal noise alone causes a regular spontaneous generation of action potentials. In addition to varying the size of ion channel clusters, living organisms may adapt the densities of ion channels in order to optimally regulate the spontaneous spiking activity. The influence of a channel block on the excitability of a membrane patch of a certain size is twofold: first, a variation of ion channel densities primarily yields a change of the conductance level; second, a down-regulation of working ion channels always increases the channel noise. While the former effect dominates in the case of sodium channel block resulting in a reduced spiking activity, the latter enhances the generation of spontaneous action potentials in the case of a tailored potassium channel blocking. Moreover, by blocking some portion of either potassium or sodium ion channels, it is possible to either increase or decrease the regularity of the spike train.     

Conformation and lytic activity of eumenine mastoparan: a new antimicrobial peptide from wasp venom.

Eumenine mastoparan-AF (EMP-AF) is a novel membrane active tetradecapeptide recently isolated from the venom of solitary wasp, Anterhynchium flavomarginatum micado. It was reported previously that EMP-AF peptide presented low cytolytic activities in human erythrocytes and in RBL-2H3 mast cells. In the present work, we observed that this peptide is able to permeate anionic liposomes, and in less extension also the neutral ones. We present evidences showing that the permeation ability is well correlated with the amount of helical conformation assumed by the peptides in these environments. This peptide also showed a broad-spectrum inhibitory activity against Gram-positive and Gram-negative bacteria. The permeability of liposomes and the antibiotic effect showed a significant reduction when C-terminus was deamidated (in acidic form). The removal of the three first amino acid residues from the N-terminus rendered the peptide inactive both in liposomes and in bacteria. The results suggest that the mechanism of action involves a threshold in the accumulation of the peptide at level of cell membrane.