Modulation by nutrients and drugs of liver acyl-CoAs analyzed by mass spectrometry

The profile of liver acyl-CoAs induced by dietary fats of variable compositions or by xenobiotic hypolipidemic amphipathic carboxylates was evaluated in vivo using a novel electrospray ionization tandem mass spectrometry methodology of high resolution, sensitivity, and reliability. The composition of liver fatty acyl-CoAs was found to reflect the composition of dietary fat. Treatment with hypolipidemic carboxylates resulted in liver dominant abundance of their respective acyl-CoAs accompanied by an increase in liver fatty acyl-CoAs. Cellular effects exerted by dietary fatty acids and/or xenobiotic carboxylic drugs may be transduced in vivo by their respective acyl-CoAs.     

Inhibition of rat liver acetyl-CoA carboxylase by beta, beta'-tetramethyl-substituted hexadecanedioic acid (MEDICA 16)

Rat liver acetyl-CoA carboxylase activity was inhibited by the free as well as the CoA monothioester of beta, beta'-methyl-substituted hexadecanedioic acid (MEDICA 16) (Bar-Tana, J., Rose-Kahn, G. and Srebnik, M. (1985) J. Biol. Chem. 260, 8404-8410 (1985). (1) The CoA monothioester of MEDICA 16 served as a dead-end inhibitor with an apparent Ki of 2 microM and 58 microM for the biotin-carboxylated and noncarboxylated enzyme forms, respectively. MEDICA 16-CoA binding was not mutually exclusive with that of citrate and did not affect the avidin-resistance of rat liver acetyl-CoA carboxylase. (2) The free dioic acid of MEDICA 16 was competitive to citrate, having an apparent Ki of about 70 microM, as compared to a Ka of 2-8 mM for the citrate activator. Inhibition of the carboxylase by the free dioic acid of MEDICA 16 was accompanied by an increase in its avidin resistance. The resultant inhibition of acetyl-CoA carboxylase by MEDICA 16 and its CoA thioester, together with the previously reported citrate-competitive inhibition of ATP-citrate lyase by MEDICA 16, may account for the observed hypolipidemic effect of MEDICA 16 under dietary conditions where liver lipogenesis constitutes a major flux of liver lipid synthesis.     

Inhibition of lipid synthesis by beta beta'-tetramethyl-substituted, C14-C22, alpha, omega-dicarboxylic acids in the rat in vivo

beta beta'-Methyl-substituted alpha, omega-dicarboxylic acids (MEDICA) of C14-C18 chain length were found to inhibit liver lipid synthesis in the rat in vivo. Maximum inhibition was observed with MEDICA 16 amounting to a 50% decrease in fatty acid and cholesterol biosynthesis in the presence of 0.07 and 0.015% (w/w) of the drug in the diet, respectively. Inhibition of lipid biosynthesis by MEDICA 16 involved a reduction in cytosolic acetyl-CoA content, while the carbon flux from glucose to glycogen, protein, and carbon dioxide remained unaffected. Inhibition of lipogenesis by MEDICA 16 resulted in a 50% decrease in liver and carcass (but not brain) neutral lipid ester content at 0.25% (w/w) of the drug in the diet, as well as in a dose-dependent hypotriglyceridemic effect, with an up to 3-fold reduction in serum triacylglycerols. Inhibition of cholesterogenesis by MEDICA 16 resulted in a hypocholesterolemic effect, with 60 and 45% reductions in (very low density + low density lipoprotein) cholesterol and high density lipoprotein cholesterol, respectively.     

High-affinity binding of fatty acyl-CoAs and peroxisome proliferator-CoA esters to glutathione S-transferases effect on enzymatic activity.

Acyl-CoAs are present at high concentrations within the cell, yet are strongly buffered by specific binding proteins in order to maintain a low intracellular unbound acyl-CoA concentration, compatible with their metabolic role, their importance in cell signaling, and as protection from their detergent properties. This intracellular regulation may be disrupted by nonmetabolizables acyl-CoA esters of xenobiotics, such as peroxisome proliferators, which are formed at relatively high concentration within the liver cell. The low molecular mass acyl-CoA binding protein (ACBP) and fatty acyl-CoA binding protein (FABP) have been proposed as the buffering system for fatty acyl-CoAs. Whether these proteins also bind xenobiotic-CoA is not known. Here we have identified new liver cytosolic fatty acyl-CoA and xenobiotic-CoA binding sites as glutathione S-transferase (GST), using fluorescent polarization and a acyl-etheno-CoA derivative of the peroxisome proliferator nafenopin as ligand. Rat liver GST and human liver recombinant GSTA1-1, GSTP1-1 and GSTM1-1 were used. Only class alpha rat liver GST and human GSTA1-1 bind xenobiotic-CoAs and fatty acyl-CoAs, with Kd values ranging from 200 nM to 5 microM. One mol of acyl-CoA is bound per mol of dimeric enzyme, and no metabolization or hydrolysis was observed. Binding results in strong inhibition of rat liver GST and human recombinant GSTA1-1 (IC50 at the nanomolar level for palmitoyl-CoA) but not GSTP1-1 and GSTM1-1. Acyl-CoAs do not interact with the GSTA1-1 substrate binding site, but probably with a different domain. Results suggest that under increased acyl-CoA concentration, as occurs after exposure to peroxisome proliferators, acyl-CoA binding to the abundant class alpha GSTs may result in strong inhibition of xenobiotic detoxification. Analysis of the binding properties of GSTs and other acyl-CoA binding proteins suggest that under increased acyl-CoA concentration GSTs would be responsible for xenobiotic-CoA binding whereas ACBP would preferentially bind fatty acyl-CoAs.     

Fatty acylated proteins as components of intracellular signaling pathways

From the studies presented above, it is obvious that fatty acylation is a common modification among proteins involved in cellular regulatory pathways, and in certain cases mutational analyses have demonstrated the importance of covalent fatty acids in the functioning of these proteins. Indeed, certain properties provided by fatty acylation make it an attractive modification for regulatory proteins that might interact with many different substrates, particularly those found at or near the plasma membrane/cytosol interface. In the case of intracellular fatty acylated proteins, the fatty acyl moiety allows tight binding to the plasma membrane without the need for cotranslational insertion through the bilayer. For example, consider the tight, salt-resistant interaction of myristoylated SRC with the membrane, whereas its nonmyristoylated counterpart is completely soluble. Likewise for the RAS proteins, which associate weakly with the membrane in the absence of fatty acylation, while palmitoylation increases their affinity for the plasma membrane and their biological activity. Fatty acylation also permits reversible membrane association in some cases, particularly for several myristoylated proteins, thus conferring plasticity on their interactions with various signaling pathway components. Finally, although this has not been demonstrated, it is conceivable that covalent fatty acid may allow for rapid mobility of proteins within the membrane. Several questions remain to be answered concerning requirements for fatty acylation by regulatory proteins. The identity of the putative SRC "receptor" will provide important clues as to the pathways in which normal SRC functions, as well as into the process of transformation by oncogenic tyrosine kinases. The possibility that other fatty acylated proteins associate with the plasma membrane in an analogous manner also needs to be investigated. An intriguing observation that can be made from the information presented here is that at least three different families of proteins involved in growth factor signaling pathways encode both acylated and nonacylated members, suggesting that selective fatty acylation may provide a means of determining the specificity of their interactions with other regulatory molecules. Further studies of fatty acylated proteins should yield important information concerning the regulation of intracellular signaling pathways utilized during growth and differentiation.     

Exogenous myristic acid acylates proteins in cultured rat hepatocytes

Fatty acid acylation is a functionally important modification of proteins. In the liver, however, acylated proteins remain largely unknown. This work was aimed at investigating fatty acid acylation of proteins in cultured rat hepatocytes. Incubation of these cells with [9,10-3H] myristic acid followed by two-dimensional electrophoresis separation of the delipidated cellular proteins and autoradiography evidenced the reproducible and selective incorporation of radioactivity from the precursor into 18 well-resolved proteins in the 10--120 kDa range and the 4--7 pH range. Radiolabeling of these proteins resulted from covalent linkage to the precursor [9,10-3H] myristic acid or to its elongation product, palmitic acid. The majority of the covalent linkages between the proteins and the fatty acids were broken by base hydrolysis, which indicated that the linkage was of thioester or ester-type. Only one of the studied proteins was attached to myristic acid via an amide linkage which resisted the basic treatment but was broken by acid hydrolysis. After incubation with [9,10-3H] palmitic acid, only two proteins previously detected with myristic acid were radiolabeled. Finally, the identified acylated proteins may be grouped into two classes: proteins involved in signal transduction (the alpha subunit of a heterotrimeric G protein and several small G proteins) and cytoskeletal proteins (cytokeratins, actin).     

Cytosolic epoxide hydrolase

Epoxide hydrolase activity is recovered in the high-speed supernatant fraction from the liver of all mammals so far examined, including man. For some as yet unexplained reason, the rat has a very low level of this activity, so that cytosolic epoxide hydrolase is generally studied in mice. This enzyme selectively hydrolyzes trans epoxides, thereby complementing the activity of microsomal epoxide hydrolase, for which cis epoxides are better substrates. Cytosolic epoxide hydrolase has been purified to homogeneity from the livers of mice, rabbits and humans. Certain of the physicochemical and enzymatic properties of the mouse enzyme have been thoroughly characterized. Neither the primary amino acid, cDNA nor gene sequences for this protein are yet known, but such characterization is presently in progress. Unlike microsomal epoxide hydrolase and most other enzymes involved in xenobiotic metabolism, cytosolic epoxide hydrolase is not induced by treatment of rodents with substances such as phenobarbital, 2-acetylaminofluorene, trans-stilbene oxide, or butylated hydroxyanisole. The only xenobiotics presently known to induce cytosolic epoxide hydrolase are substances which also cause peroxisome proliferation, e.g., clofibrate, nafenopin and phthalate esters. These and other observations indicate that this enzyme may actually be localized in peroxisomes in vivo and is recovered in the high-speed supernatant because of fragmentation of these fragile organelles during homogenization, i.e., recovery of this enzyme in the cytosolic fraction is an artefact. The functional significance of cytosolic epoxide hydrolase is still largely unknown. In addition to deactivating xenobiotic epoxides to which the organism is exposed directly or which are produced during xenobiotic metabolism, primarily by the cytochrome P-450 system, this enzyme may be involved in cellular defenses against oxidative stress.     

Human apolipoprotein A-I. Post-translational modification by fatty acid acylation

The human apolipoproteins are secretory proteins some of which have been shown to undergo proteolytic processing and post-translational addition of carbohydrate. Apolipoprotein A-I (apo-A-I), the predominant protein associated with high density lipoproteins, undergoes co-translational proteolytic processing as well as post-translational conversion of proapo-A-I to mature apo-A-I following cellular secretion. Utilizing the human hepatoma cell line HEP-G2, we have established that, in addition to proteolytic processing, secreted nascent apo-A-I is acylated with palmitate. Uniformly labeled [14C]palmitate and [1-14C]palmitate were each incorporated into apo-A-I when analyzed by sodium dodecyl sulfate gel electrophoresis and autoradiography. The acylation of apo-A-I with palmitate was confirmed by immunoprecipitation and gas chromatography/mass spectrometry. Hydroxylamine treatment resulted in the deacylation of apo-A-I. Although three of the apo-A-I isoforms analyzed by two-dimensional gel electrophoresis were shown to contain radio-labeled palmitate, 80% of acylated apo-A-I was in the proapolipoprotein A-I isoform. [14C]Oleate was not incorporated in secreted apo-A-I, indicating the specificity of the acylation of apo-A-I. Incubation of [14C] palmitate-acylated apo-A-I in serum and plasma under conditions in which proapo-A-I is proteolytically cleaved to mature apo-A-I did not result in deacylation. These data establish that fatty acid acylation occurs in human secretory proteins in addition to the previously reported acylation of cellular membrane proteins. These results suggest that the covalent linkage of lipids to apolipoproteins may play a critical role in apolipoprotein and lipoprotein metabolism.     

Specificity of fatty acid acylation of cellular proteins

Labeling of the BC3H1 muscle cell line with [3H] palmitate and [3H]myristate results in the incorporation of these fatty acids into a broad spectrum of different proteins. The patterns of proteins which are labeled with palmitate and myristate are distinct, indicating a high degree of specificity of fatty acylation with respect to acyl chain length. The protein-linked [3H]palmitate is released by treatment with neutral hydroxylamine or by alkaline methanolysis consistent with a thioester linkage or a very reactive ester linkage. In contrast, only a small fraction of the [3H]myristate which is attached to proteins is released by treatment with hydroxylamine or alkaline methanolysis, suggesting that myristate is linked to proteins primarily through amide bonds. The specificity of fatty acid acylation has also been examined in 3T3 mouse fibroblasts and in PC12 cells, a rat pheochromacytoma cell line. In both cells, palmitate is primarily linked to proteins by a hydroxylamine-labile linkage while the major fraction of the myristic acid (60-70%) is linked to protein via amide linkage and the remainder via an ester linkage. Major differences were noted in the rate of fatty acid metabolism in these cells; in particular in 3T3 cells only 33% of the radioactivity incorporated from myristic acid into proteins is in the form of fatty acids. The remainder is presumably the result of conversion of label to amino acids. In BC3H1 cells, palmitate- and myristate-containing proteins also exhibit differences in subcellular localization. [3H]Palmitate-labeled proteins are found almost exclusively in membranes, whereas [3H]myristate-labeled proteins are distributed in both the soluble and membrane fractions. These results demonstrate that fatty acid acylation is a covalent modification common to a wide range of cellular proteins and is not restricted solely to membrane-associated proteins. The major acylated proteins in the various cell lines examined appear to be different, suggesting that the acylated proteins are concerned with specialized cell functions. The linkages through which fatty acids are attached to proteins also appear to be highly specific with respect to the fatty acid chain length.     

Post-translational modifications of apolipoprotein A-I and Po proteins in the avian peripheral nerve

Apolipoprotein A-I (apo A-I), a soluble lipid transporter, and Po, the major glycoprotein of myelin, are actively synthesized during myelination. To explore the status of post-translational modifications of these proteins in the avian PNS during rapid myelination, endoneurial slices from one day old chick sciatic nerves were incubated with various radioactive precursors that could serve as indicators of such processes. The proteins were isolated from the incubation medium (secreted fraction), the 1% Triton-X-100-soluble intracellular-endoneurial (intracellular) fraction, and myelin-related and purified compact myelin fractions by immunoprecipitation with monospecific anti-apo A-I or anti-Po antisera. Our results demonstrated that secreted apo A-I is fatty acylated, but not phosphorylated or sulfated. Avian Po protein was phosphorylated by a phorbol ester sensitive protein kinase. Sulfation, as well as fatty acylation, of avian Po protein was observed in organ culture using highly sensitive methods of detection. These results indicate that fatty acylation of secreted apo A-I and phosphorylation, sulfation and fatty acylation of Po have been conserved during evolution, and that these post-translational modifications may play a common function in various species.     

Activation of hemolysin toxin: relationship between two internal protein sites of acylation

HlyC, hemolysin-activating lysine acyltransferase, catalyzes the acylation (from acyl-ACP) of Escherichia coli prohemolysin (proHlyA) on the epsilon-amino groups of specific lysine residues, Lys564 and Lys690 of the 1024-amino acid primary structure, to form hemolysin (HlyA). The amino acid sequences flanking the two acylation sites are not homologous except that each has a glycine residue immediately preceding the lysine which is acylated; there are, however, numerous GK sequences throughout proHlyA that are not acylation sites. The substrate specificity of acylation was examined. ProHlyA-derived structures, altered by substantial deletions and separation of the acylation sites into two different peptides and site-directed mutation analyses of acylation sites, often served as internal protein acylation substrates, and the kinetics of the acylations were measured. The two sites of acylation of proHlyA functioned independently of one another with HlyC; there did not appear to be a common HlyC binding site or processivity of the enzyme between the sites. Acyl-HlyC was likely the enzyme form that interacted with the final acylation substrate. In a variety of constructs, the two acylation sites had similar K(m) values, but their V(max) values and catalytic efficiencies as substrates differed. Internal protein acylation was inhibited by specific small peptides mimicking the primary structure of each acylation site except that the crucial lysines were replaced with arginines; similar small peptides containing the crucial lysine, however, were not acylated.     

Regulation of mitochondrial carbamoyl-phosphate synthetase 1 activity by active site fatty acylation

In addition to its role in reversible membrane localization of signal-transducing proteins, protein fatty acylation could play a role in the regulation of mitochondrial metabolism. Previous studies have shown that several acylated proteins exist in mitochondria isolated from COS-7 cells and rat liver. Here, a prominent fatty-acylated 165-kDa protein from rat liver mitochondria was identified as carbamoyl-phosphate synthetase 1 (CPS 1). Covalently attached palmitate was linked to CPS 1 via a thioester bond resulting in an inhibition of CPS 1 activity at physiological concentrations of palmitoyl-CoA. This inhibition corresponds to irreversible inactivation of CPS 1 and occurred in a time- and concentration-dependent manner. Fatty acylation of CPS 1 was prevented by preincubation with N-ethylmaleimide and 5'-p-fluorosulfonylbenzoyladenosine, an ATP analog that reacts with CPS 1 active site cysteine residues. Our results suggest that fatty acylation of CPS 1 is specific for long-chain fatty acyl-CoA and very likely occurs on at least one of the essential cysteine residues inhibiting the catalytic activity of CPS 1. Inhibition of CPS 1 by long-chain fatty acyl-CoAs could reduce amino acid degradation and urea secretion, thereby contributing to nitrogen sparing during starvation.     

Rapid in Vivo Acylation of Acyl Carrier Protein with Exogenous Fatty Acids in Spirodela oligorrhiza

Posttranslational acylation of several chloroplast proteins with palmitic acid was recently demonstrated in Spirodela oligorrhiza (AK Mattoo, M Edelman [1987] Proc Natl Acad Sci USA 84: 1497-1501). We have now identified an in vivo acylated, soluble protein having an apparent M_r of 10 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as an acylated form of acyl carrier protein (ACP). This 10-kilodalton protein is present in low abundance, and its acylation is light-stimulated. Turnover of the acyl moiety but not the apo-protein is rapid in the light. The acylated 10-kilodalton protein coelectrophoreses with in vitro synthesized palmitoyl-acyl carrier protein and is immunoprecipitated from soluble extracts with an antibody raised against spinach ACP. Cerulenin, an inhibitor of β-ketoacyl-ACP synthetase, inhibited in vivo acylation of Spirodela ACP. Cell-free extracts of Spirodela plants were able to catalyze the transfer of palmitate from palmitoyl-CoA to ACP, suggesting the existence in higher plants of a pathway for acylation of ACP that involves transacylation from acyl-CoA.     

Open Biology: overview for special issue on dynamics of protein fatty acylation

Fatty acylation is a widespread form of protein modification that occurs on specific intracellular and secreted proteins. Beyond increasing hydrophobicity and the affinity of the modified protein for lipid bilayers, covalent attachment of a fatty acid exerts effects on protein localization, inter- and intramolecular interactions and signal transduction. As such, research into protein fatty acylation has been embraced by an extensive community of biologists. This special issue highlights advances at the forefront of the field, by focusing on two families of enzymes that catalyse post-translational protein fatty acylation, zDHHC palmitoyl acyltransferases and membrane-bound O-acyl transferases, and signalling pathways regulated by their fatty acylated protein substrates. The collected contributions catalogue the tremendous progress that has been made in enzyme and substrate identification. In addition, articles in this special issue provide insights into the pivotal functions of fatty acylated proteins in immune cell, insulin and EGF receptor-mediated signalling pathways. As selective inhibitors of protein fatty acyltransferases are generated, the future holds great promise for therapeutic targeting of fatty acyltransferases that play key roles in human disease.     

Detection of a nafenopin-binding protein in rat liver cytosol associated with the induction of peroxisome proliferation by hypolipidemic compounds

[3H]nafenopin, a known inducer of liver peroxisomal enzymes, was shown to bind to a specific, saturable pool of binding sites in cytosols from rat liver and kidney cortex. Tissue levels of this binding protein (liver greater than kidney cortex; not detectable in myocardium, skeletal muscle) were seen to correlate with the ability of nafenopin to induce peroxisomal enzymes in these organs. Clofibrate and ciprofibrate, which are structurally similar to nafenopin, competitively blocked the specific binding of [3H]nafenopin. Phenobarbital, a non-inducer of peroxisomes, and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid and 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-beta-hydroxyethyl)acetamide, which are structurally unrelated peroxisome proliferators, did not complete for the specific [3H]nafenopin binding sites. The [3H]nafenopin binding protein is proposed as a mediator of the drug-induced increase in peroxisomes and associated peroxisomal enzymes.     

Proteolipid protein and DM-20 are synthesized by Schwann cells,present in myelin membrane,but they are not fatty acylated

Proteolipid protein (PLP) and DM-20 were intensely labeled after immunoprecipitation of total cellular proteins and myelin proteins labeled with [³⁵S]methionine in nerve slices. These results provided evidence that PLP and DM-20 are incorporated into the myelin membrane following their synthesis in Schwann cells. In contrast, PLP and DM-20 were not fatty acylated after incubation of the nerve slices with [³H]palmitic acid, however, PO glycoprotein and 24kDa protein were heavily fatty acylated. The lack of fatty acylation of PLP and DM-20 in the peripheral nervous system suggests that fatty acyltransferase responsible for their acylation is absent or non-functional in the peripheral nervous system.     

Inhibition of lipid synthesis by beta beta'-tetramethyl-substituted, C14-C22, alpha, omega-dicarboxylic acids in cultured rat hepatocytes

beta beta'-Methyl-substituted, C14-C18, alpha, omega-dicarboxylic acids (MEDICA 14-18) were found to inhibit fatty acids and cholesterol synthesis in cultured rat hepatocytes. Maximum inhibition was observed with MEDICA 16, amounting to a 50% decrease in 3H2O and acetate incorporation into fatty acids and cholesterol in the presence of 0.08 mM of the drug added to the culture medium. Inhibition of lipogenesis was not accompanied by inhibition of palmitate or glycerol esterification into neutral lipids and phospholipids. The respective capacities of MEDICA homologues of varying acyl chain length as inhibitors of fatty acid and cholesterol synthesis in cultured rat hepatocytes and in vivo (Bar-Tana, J., Rose-Kahn, G., and Srebnik, M. (1985) J. Biol. Chem. 260, 8404-8410) correlated well with their respective inhibitory effect on liver ATP-citrate lyase. Thus, MEDICA 16 inhibited liver ATP-citrate lyase competitively to citrate with a Ki of 16 microM as compared to a Km of 0.8 mM for the citrate substrate.     

Novel fatty acid acylation of lens integral membrane protein aquaporin-0

Fatty acid acylation of proteins is a well-studied co- or posttranslational modification typically conferring membrane trafficking signals or membrane anchoring properties to proteins. Commonly observed examples of protein acylation include N-terminal myristoylation and palmitoylation of cysteine residues. In the present study, direct tissue profiling mass spectrometry of bovine and human lens sections revealed an abundant signal tentatively assigned as a lipid-modified form of aquaporin-0. LC/MS/MS proteomic analysis of hydrophobic tryptic peptides from lens membrane proteins revealed both N-terminal and C-terminal peptides modified by 238 and 264 Da which were subsequently assigned by accurate mass measurement as palmitoylation and oleoylation, respectively. Specific sites of modification were the N-terminal methionine residue and lysine 238 revealing, for the first time, an oleic acid modification via an amide linkage to a lysine residue. The specific fatty acids involved reflect their abundance in the lens fiber cell plasma membrane. Imaging mass spectrometry indicated abundant acylated AQP0 in the inner cortical region of both bovine and human lenses and acylated truncation products in the lens nucleus. Additional analyses revealed that the lipid-modified forms partitioned exclusively to a detergent-resistant membrane fraction, suggesting a role in membrane domain targeting.     

Influence of acylation of a Peptide corresponding to the amino-terminal region of endothelial nitric oxide synthase on the interaction with model membranes

Covalent attachment of fatty acids to proteins is a common form of protein modification which has been shown to influence both structure and interaction with membranes. Endothelial nitric oxide synthase (eNOS) is dually acylated by the fatty acids myristate and palmitate. We have synthesized four peptides corresponding to the first 28 amino acids of the N-terminal region of eNOS. Besides the nonacylated eNOS sequence, three additional peptides with different degrees of acylation have been obtained: myristoylated, doubly palmitoylated, and dually myristoylated and doubly palmitoylated. Acylation itself, myristic and/or palmitic, confers the peptide the ability to adopt extended conformations, indicated by the fact that the CD spectrum of all acylated peptides has a minimum at approximately 215 nm characteristic of beta-sheet structure. The nonacylated sequence interacts with model membranes composed of acidic phospholipids probably through ionic interactions with the polar headgroup of the phospholipids. However, the acylated peptides are able to insert deeply into the hydrophobic core of both neutral and acidic phospholipids, maintaining the spectral features of extended conformations. When DMPC vesicles containing cholesterol and sphingomyelin at 10% were used, the insertion of the triacylated peptide almost completely canceled the thermal transition, although the interaction of the other acylated peptides also reduced the transition amplitude but to a much lower extent and affected only the acyl chains in the fluid state.