Fatty Acids Of Different Lengths Research Articles

Plasma Ceramides and Sphingomyelins and Sudden Cardiac Death in the Cardiovascular Health Study

Sphingolipids, including ceramides and sphingomyelins, may influence the pathophysiology and risk of sudden cardiac death (SCD) through multiple biological activities. Whether the length of the fatty acid acylated to plasma sphingolipid species is associated with SCD risk is not known. To determine whether the saturated fatty acid length of plasma ceramides and sphingomyelins influences the association with SCD risk. In this cohort study, multivariable Cox proportional hazards regression models were used to examine the association of sphingolipid species with SCD risk. The study population included 4612 participants in the Cardiovascular Health Study followed up prospectively for a median of 10.2 (IQR, 5.5-11.6) years. Baseline data were collected from January 1992 to December 1995 during annual examinations. Data were analyzed from February 11, 2020, to September 9, 2023. Eight plasma sphingolipid species (4 ceramides and 4 sphingomyelins) with saturated fatty acids of 16, 20, 22, and 24 carbons. Association of plasma ceramides and sphingomyelins with saturated fatty acids of different lengths with SCD risk. Among the 4612 CHS participants included in the analysis (mean [SD] age, 77 [5] years; 2724 [59.1%] women; 6 [0.1%] American Indian; 4 [0.1%] Asian; 718 [15.6%] Black; 3869 [83.9%] White, and 15 [0.3%] Other), 215 SCD cases were identified. In adjusted Cox proportional hazards regression analyses, plasma ceramides and sphingomyelins with palmitic acid (Cer-16 and SM-16) were associated with higher SCD risk per higher SD of log sphingolipid levels (hazard ratio [HR] for Cer-16, 1.34 [95% CI, 1.12-1.59]; HR for SM-16, 1.37 [95% CI, 1.12-1.67]). Associations did not differ by baseline age, sex, race, or body mass index. No significant association of SCD with sphingolipids with very-long-chain saturated fatty acids was observed after correction for multiple testing (HR for ceramide with arachidic acid, 1.06 [95% CI, 0.90-1.24]; HR for ceramide with behenic acid, 0.92 [95% CI, 0.77-1.10]; HR for ceramide with lignoceric acid, 0.92 [95% CI, 0.77-1.09]; HR for sphingomyelin with arachidic acid, 0.83 [95% CI, 0.71-0.98]; HR for sphingomyelin with behenic acid, 0.84 [95% CI, 0.70-1.00]; HR for sphingomyelin with lignoceric acid, 0.86 [95% CI, 0.72-1.03]). The findings of this large, population-based cohort study of SCD identified that higher plasma levels of Cer-16 and SM-16 were associated with higher risk of SCD. Future studies are needed to examine the underlying mechanism of these associations.

Interaction of Lipophilic Cytarabine Derivatives with Biomembrane Model at the Air/Water Interface.

Cell membrane models are useful for obtaining molecular-level information on the interaction of biologically active molecules whose activity is believed to depend also on their effects on the membrane. Cytarabine was conjugated with fatty acids to improve the drug lipophilicity and the interaction with the biomembrane model. Cytarabine was conjugated with fatty acids of different lengths to form the trimyristoyl cytarabine and the tristearoyl cytarabine derivatives. Their interaction with biomembrane models constituted by dimyristoylphosphatidylcholine (DMPC) monolayers was studied by employing the Langmuir–Blodgett technique. DMPC/cytarabine, DMPC/trimyristoyl cytarabine and DMPC/tristearoyl cytarabine mixed monolayers at increasing molar fractions of the compound were prepared and placed on the subphase. The mean molecular area/surface pressure isotherms were recorded at 37 °C. Between the molecules of DMPC and those of cytarabine or prodrugs, repulsive forces act. However, these forces are very weak between DMPC and cytarabine and stronger between DMPC and the cytarabine derivatives, thus avoiding the expulsion of the compounds at higher surface pressure and modifying the stability of the mixed monolayer. The fatty acid moieties could then modulate the affinity of cytarabine for biomembranes.

C-Locked Analogs of the Antimicrobial Peptide BP214.

BP214 is an all-D antimicrobial peptide amide, kklfkkilryl, which shows an excellent activity against colistin-resistant Acinetobacter baumannii and a low hemolytic activity. The aim of the present work was to investigate how C-terminus-to-side chain macrocyclization and fatty acid modification affect the antimicrobial and hemolytic activity of this peptide. In total, 18 analogs of BP214 were synthesized using a combination of Fmoc-based solid-phase peptide synthesis and the submonomer approach. Cyclization was achieved by reacting the ε-amino group of a C-terminal lysine residue with a bromoacetylgroup attached to the Nα amino group of the N-terminal amino acid, generating a secondary amine at which the exocyclic lipopeptide tail was assembled. Three different ring sizes (i.e., 3–5 amino acid residues) of C-locked analogs combined with fatty acids of different lengths (i.e., C10–C14) were investigated. The antimicrobial activity of the analogs was tested against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. The most promising compound was analog 13 (MIC = 4 µg/mL (2.4 µM) against E. coli and 36% hemolysis of red blood cells at 150 µM). In a time-kill assay, this peptide showed a significant, concentration-dependent reduction in viable E. coli cells comparable to that seen for colistin.

Fatty Acids/Tetraphenylethylene Conjugates: Hybrid AIEgens for the Preparation of Peptide-Based Supramolecular Gels.

Aggregation-induced emissive materials are gaining particular attention in the last decades due to their wide application in different fields, from optical devices to biomedicine. In this work, compounds having these kinds of properties, composed of tetraphenylethylene scaffold combined with fatty acids of different lengths, were synthesized and characterized. These molecules were found able to self-assemble into different supramolecular emissive structures depending on the chemical composition and water content. Furthermore, they were used as N-terminus capping agents in the development of peptide-based materials. The functionalization of a 5-mer laminin-derived peptide led to the obtainment of luminescent fibrillary materials that were not cytotoxic and were able to form supramolecular gels in aqueous environment.

Molecular Dynamics of DHHC20 Acyltransferase Suggests Principles of Lipid and Protein Substrate Selectivity.

Lipid modification of viral proteins with fatty acids of different lengths (S-acylation) is crucial for virus pathogenesis. The reaction is catalyzed by members of the DHHC family and proceeds in two steps: the autoacylation is followed by the acyl chain transfer onto protein substrates. The crystal structure of human DHHC20 (hDHHC20), an enzyme involved in the acylation of S-protein of SARS-CoV-2, revealed that the acyl chain may be inserted into a hydrophobic cavity formed by four transmembrane (TM) α-helices. To test this model, we used molecular dynamics of membrane-embedded hDHHC20 and its mutants either in the absence or presence of various acyl-CoAs. We found that among a range of acyl chain lengths probed only C16 adopts a conformation suitable for hDHHC20 autoacylation. This specificity is altered if the small or bulky residues at the cavity’s ceiling are exchanged, e.g., the V185G mutant obtains strong preferences for binding C18. Surprisingly, an unusual hydrophilic ridge was found in TM helix 4 of hDHHC20, and the responsive hydrophilic patch supposedly involved in association was found in the 3D model of the S-protein TM-domain trimer. Finally, the exchange of critical Thr and Ser residues in the spike led to a significant decrease in its S-acylation. Our data allow further development of peptide/lipid-based inhibitors of hDHHC20 that might impede replication of Corona- and other enveloped viruses.

Quaternary ammonium surfactants derived from leucine and methionine: Novel challenging surface active molecules with antimicrobial activity

Surfactants are versatile excipients, which are commonly employed in several pharmaceutical formulations as emulsifiers, stabilizing or solubilizing agents. In this field, current research is aimed at the design or discovery of amphiphilic molecules with improved characteristics in terms of safety (toxicological profile) and biological activities (e.g. antimicrobial activity, interaction with biomembranes). Quaternary ammonium surfactants derived from amino acids are one such class of compounds with significant potential for pharmaceuticals. In this work, quaternary ammonium surfactants derived from the amino acids leucine and methionine and esterified with fatty acids of different lengths (C10, C12 and C14) were synthesized. These amphiphiles were characterized in terms of critical micelle concentration (CMC) by tensiometry and conductivity measurements, cytotoxicity (MTS and LDH assay on Caco-2 and Calu-3 cell lines) and antimicrobial activity on selected Gram-positive (S. aureus and E. faecalis), Gram-negative (E. coli and P. aeruginosa) and the fungus C. albicans. The derivatives bearing a C12 or a C14 chain displayed CMC, EC50 (from cytotoxicity assays) and minimum inhibition concentration (MIC) values that are comparable to those of benzalkonium chloride (used as reference), especially towards Gram-positive and C. albicans. On the contrary, derivatives bearing a C10 chain have higher CMC, EC50 and MIC values, highlighting the crucial role of the hydrophobic tail in determining both physicochemical and biological properties. The C12-C14 synthesized derivatives may represent a promising alternative to benzalkonium chloride as surface active molecules and antimicrobial agents.

Calculation of apparent pKa values of saturated fatty acids with different lengths in DOPC phospholipid bilayers.

We performed all-atom molecular dynamics simulations and calculated free energy profiles and apparent pKa values for neutral and anionic forms of single myristic (C14:0), palmitic (C16:0) and stearic (C18:0) fatty acid embedded in a DOPC bilayer and explicit water solvent. We showed that the neutral forms of the fatty acids are stabilized inside the bilayer by hydrogen bonding of a fatty acid carboxylic group with DOPC phosphate and carbonyl groups. In contrast to the neutral form, the anionic forms of the fatty acids are shifted towards the water-membrane interface and are instead stabilized by hydrogen bonding to interfacial water. By using umbrella sampling simulations, we calculated free energies of stabilization and revealed that the free energy of stabilization inside the bilayer increases with the chain length for both the neutral and deprotonated forms. On the other hand, the free energies of flip-flop of both the neutral and anionic forms are constant upon the prolongation of the fatty acid. Based on the free energy curves, we also calculated apparent fatty acid pKa,app values in the bilayer, which are 7.0, 7.2 and 6.3 for myristic, palmitic and stearic acid and are increased by several pKa units compared to the corresponding pKa values in water. By further analysis of the calculated curves we found that spontaneous protonation of fatty acid anions takes place in the bilayer interior at ca. 1.4 nm from the bilayer center for all studied fatty acids.

Structural evidence for a fatty acid-independent myotoxic mechanism for a phospholipase A2-like toxin

The myotoxic mechanism for PLA2-like toxins has been proposed recently to be initiated by an allosteric change induced by a fatty acid binding to the protein, leading to the alignment of the membrane docking site (MDoS) and membrane disrupting site (MDiS). Previous structural studies performed by us demonstrated that MjTX-II, a PLA2-like toxin isolated from Bothrops moojeni, presents a different mode of ligand-interaction caused by natural amino acid substitutions and an insertion. Herein, we present four crystal structures of MjTX-II, in its apo state and complexed with fatty acids of different lengths. Analyses of these structures revealed slightly different oligomeric conformations but with both MDoSs in an arrangement that resembles an active-state PLA2-like structure. To explore the structural transitions between apo protein and fatty-acid complexes, we performed Normal Mode Molecular Dynamics simulations, revealing that oligomeric conformations of MjTX-II/fatty acid complexes may be reached in solution by the apo structure. Similar simulations with typical PLA2-like structures demonstrated that this transition is not possible without the presence of fatty acids. Thus, we hypothesize that MjTX-II does not require fatty acids to be active, although these ligands may eventually help in its stabilization by the formation of hydrogen bonds. Therefore, these results complement previous findings for MjTX-II and help us understand its particular ligand-binding properties and, more importantly, its particular mechanism of action, with a possible impact on the design of structure-based inhibitors for PLA2-like toxins in general.

Lipid bilayer thickness determines cholesterol's location in model membranes.

Cholesterol is an essential biomolecule of animal cell membranes, and an important precursor for the biosynthesis of certain hormones and vitamins. It is also thought to play a key role in cell signaling processes associated with functional plasma membrane microdomains (domains enriched in cholesterol), commonly referred to as rafts. In all of these diverse biological phenomena, the transverse location of cholesterol in the membrane is almost certainly an important structural feature. Using a combination of neutron scattering and solid-state 2H NMR, we have determined the location and orientation of cholesterol in phosphatidylcholine (PC) model membranes having fatty acids of different lengths and degrees of unsaturation. The data establish that cholesterol reorients rapidly about the bilayer normal in all the membranes studied, but is tilted and forced to span the bilayer midplane in the very thin bilayers. The possibility that cholesterol lies flat in the middle of bilayers, including those made from PC lipids containing polyunsaturated fatty acids (PUFAs), is ruled out. These results support the notion that hydrophobic thickness is the primary determinant of cholesterol's location in membranes.

Molecular simulations of β-lactoglobulin complexed with fatty acids reveal the structural basis of ligand affinity to internal and possible external binding sites.

The interaction of saturated fatty acids of different length (C8:0 to C18:0) with β-lactoglobulin (βLG) was investigated by molecular dynamics simulation and docking approaches. The results show that the presence of such ligands in the hydrophobic central cavity of βLG, known as the protein calyx, determines an enhancement of atomic fluctuations compared with the unliganded form, especially for loops at the entrance of the binding site. Concerted motions are evidenced for protein regions that could favor the binding of ligands. The mechanism of anchoring of fatty acids of different length is similar for the carboxylate head-group, through electrostatic interactions with the side chains of Lys60/Lys69. The key protein residues to secure the hydrocarbon chain are Phe105/Met107, which adapt their conformation upon ligand binding. In particular, Phe105 provides an additional hydrophobic clamp only for the tail of the two fatty acids with the longest chains, palmitic, and stearic acid, which are known to bind βLG with a high affinity. The search of additional external binding sites for fatty acids, distinct from the calyx, was also carried out for palmitic acid. Two external sites with a lower affinity were identified as secondary sites, one consisting in a hydrophobic cavity allowing two distinct binding modes for the fatty acid, and the other corresponding to a surface crevice close to the protein α-helix. The overall results provide a comprehensive picture of the dynamical behavior of βLG in complex with fatty acids, and elucidate the structural basis of the binding of these physiological ligands.

Peroxisomes contribute to biosynthesis of extracellular glycolipids in fungi.

Many microorganisms secrete surface-active glycolipids. The basidiomycetous fungus Ustilago maydis produces two different classes of glycolipids, mannosylerythritol lipids (MEL) and ustilagic acids (UAs). Here we report that biosynthesis of MELs is partially localized in peroxisomes and coupled to peroxisomal fatty acid degradation. The acyltransferases, Mac1 and Mac2, which acylate mannosylerythritol with fatty acids of different length, contain a type 1 peroxisomal targeting signal (PTS1). We demonstrate that Mac1 and Mac2 are targeted to peroxisomes, while other enzymes involved in MEL production reside in different compartments. Mis-targeting of Mac1 and Mac2 to the cytosol did not block MEL synthesis but promoted production of MEL species with altered acylation pattern. This is in contrast to peroxisome deficient mutants that produced MELs similar to the wild type. We could show that cytosolic targeting of Mac1 and Mac2 reduces the amount of UA presumably due to competition for overlapping substrates. Interestingly, hydroxylated fatty acids characteristic for UAs appear in MELs corroborating cross-talk between both biosynthesis pathways. Therefore, peroxisomal localization of MEL biosynthesis is not only prerequisite for generation of the natural spectrum of MELs, but also facilitates simultaneous assembly of different glycolipids in a single cell.

Ca2+-myristoyl switch and membrane binding of chemically acylated neurocalcins.

Neurocalcin is a member of a novel family of neuronal calcium sensors that belongs to the superfamily of EF-hand Ca(2+)-binding proteins. Neurocalcin is myristoylated on its N-terminus in vivo and can associate with biological membranes in a calcium and myristoyl-dependent manner. This process known as "Ca(2+)-myristoyl switch" has been best described for the photoreceptor specific protein, recoverin, as well as for several other neuronal calcium sensors. Here, we used reversed micelles to chemically acylate nonmyristoylated neurocalcin at its N-terminus with fatty acids of different lengths (from C12 to C16). This approach allowed us to prepare neurocalcin derivatives in which a single fatty acid is selectively linked to the N-terminal glycine of the polypeptide chain through an amide bond. The membrane binding properties of the monoacylated neurocalcins were then examined by cosedimentation with phospholipid vesicles and direct binding to lipid monolayers by surface plasmon resonance spectroscopy (Biacore). Our results show that neurocalcins monoacylated with lauric, myristic, or palmitic acid were able to associate with membrane in a calcium-dependent manner. This indicates that the Ca(2+)-myristoyl switch can function with different lipid moieties and is not strictly restricted to myristate. The ability to modify at will the fatty acid linked to the N-terminal glycine should be useful to analyze the contribution of the fatty acid moiety to the biological function of this family of neuronal calcium sensors.

Crosslinked poly(vinyl alcohol) supports for the immobilization of a lipolytic enzyme

Different esters of crosslinked poly(vinyl alcohol) (PVA) were synthetized. They were developed for protein fractionation and immobilization. PVA was crosslinked with epichlorohydrin (CL-PVA) and esterified with linear fatty acids of different length (Cn-CL-PVA). A characterization of the obtained products was made. The swelling behavior, the solubility, and the percentage of esterification were examined. Values of equilibrium water content of about 81% were reached for CL-PVA samples. The polymers' stability and morpholgy were also investigated. Thermal analysis showed an increase in matrices stability, while SEM data showed the superficial development due to crosslinking and esterification reactions. Moreover, evident morphological inhomogeneities, mainly in the commercial and crosslinked products rather than in the final polymer, were present. Finally, immobilization experiments with a commercial crude of Candida rugosa were performed. Experiments showed a greater affinity of the protein for carbon chain length ranging from 8 to 12. Data indicated that compared to Celite 545, C8-CL-PVA was a better support for enzyme immobilization by physical adsorption, confirming the fact that microbial lipases prefer hydrophobic supports. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 74: 1881–1889, 1999

Characterization of lysosomal acid lipase purified from rabbit liver.

Lysosomal acid lipase from rabbit liver was solubilized with digitonin and purified 25,000-fold by Bio-Gel A-1.5 m, DEAE Bio-Gel A and phenyl Sepharose column chromatographies, preparative slab gel electrophoresis and finally Affi-Gel Blue affinity column chromatography. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The molecular weight of the acid lipase was estimated to be 42,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 40,000 by gel filtration on Bio-Gel A-0.5 m. The enzyme was a hydrophobic glycoprotein with an isoelectric point of 5.15-5.90. The purified enzyme hydrolyzed tri-, di-, and monoolein and cholesterol oleate, with apparent Vmax values of 5.41, 56.1, 21.7, and 3.25 mumol/min/mg protein, and Km values of 50, 70, 200, and 40 microM, respectively. It hydrolyzed 4-methylumbelliferyl esters with fatty acids of different lengths in the order, medium length chains greater than long chains much greater than short chains. It did not hydrolyze dipalmitoylphosphatidylcholine. Its activity was inhibited by micromolar concentrations of p-chloromercuriphenyl sulfonic acid and p-bromophenacyl bromide and millimolar concentrations of Cu2+ and diethylpyrocarbonate. The activities of the enzyme towards the five substrates listed above showed almost identical thermal stabilities, mobilities on polyacrylamide gel electrophoresis and inhibition by several inhibitors. These findings support the idea that one enzyme is involved in the hydrolysis of both acylglycerols and cholesterol esters in lysosomes.