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The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) has been extensively investigated as a cancer therapy mainly based on its regulation of membrane lipid composition and structure, activating various cell fate pathways. We discovered, additionally, that 2OHOA can uncouple oxidative phosphorylation, but this has never been demonstrated mechanistically. Here, we explored the effect of 2OHOA on mitochondria isolated by ultracentrifugation from U118MG glioblastoma cells. Mitochondria were analyzed by shotgun lipidomics, molecular dynamic simulations, spectrophotometric assays for determining respiratory complex activity, mass spectrometry for assessing beta oxidation and Seahorse technology for bioenergetic profiling. We showed that the main impact of 2OHOA on mitochondrial lipids is their hydroxylation, demonstrated by simulations to decrease co-enzyme Q diffusion in the liquid disordered membranes embedding respiratory complexes. This decreased co-enzyme Q diffusion can explain the inhibition of disjointly measured complexes I–III activity. However, it doesn’t explain how 2OHOA increases complex IV and state 3 respiration in intact mitochondria. This increased respiration probably allows mitochondrial oxidative phosphorylation to maintain ATP production against the 2OHOA-mediated inhibition of glycolytic ATP production. This work correlates 2OHOA function with its modulation of mitochondrial lipid composition, reflecting both 2OHOA anticancer activity and adaptation to it by enhancement of state 3 respiration.

Antimicrobial activity of many AMPs can be improved by lysine-to-arginine substitution due to a more favourable interaction of arginine guanidinium moiety with bacterial membranes. In a previous work, the structural and functional characterization of an amphipathic antimicrobial peptide named RiLK1, including lysine and arginine as the positively charged amino acids in its sequence, was reported. Specifically, RiLK1 retained its β-sheet structure under a wide range of environmental conditions (temperature, pH, and ionic strength), and exhibited bactericidal activity against Gram-positive and Gram-negative bacteria and fungal pathogens with no evidence of toxicity on mammalian cells. To further elucidate the influence of a lysine-to-arginine replacement on RiLK1 conformational properties, antimicrobial activity and peptide-liposome interaction, a new RiLK1-derivative, named RiLK3, in which the lysine is replaced with an arginine residue, was projected and characterised in comparison with its parental compound. The results evidenced that lysine-to-arginine mutation not only did not assure an improvement in the antimicrobial potency of RiLK1 in terms of bactericidal, virucidal and fungicidal activities, but rather it was completely abolished against the hepatitis A virus. Therefore, RiLK1 exhibited a wide range of antimicrobial activity like other cationic peptides, although the exact mechanisms of action are not completely understood. Moreover, tryptophan fluorescence measurements confirmed that RiLK3 bound to negatively charged lipid vesicles with an affinity lower than that of RiLK1, although no substantial differences from the structural and self-assembled point of view were evidenced. Therefore, our findings imply that antimicrobial efficacy and selectivity are affected by several complex and interrelated factors related to substitution of lysine with arginine, such as their relative proportion and position. In this context, this study could provide a better rationalisation for the optimization of antimicrobial peptide sequences, paving the way for the development of novel AMPs with broad applications.

Pediatric neurological tumors are a heterogeneous group of cancers, many of which carry a poor prognosis and lack a “standard of care” therapy. While they have similar anatomic locations, pediatric neurological tumors harbor specific molecular signatures that distinguish them from adult brain and other neurological cancers. Recent advances through the application of genetics and imaging tools have reshaped the molecular classification and treatment of pediatric neurological tumors, specifically considering the molecular alterations involved. A multidisciplinary effort is ongoing to develop new therapeutic strategies for these tumors, employing innovative and established approaches. Strikingly, there is increasing evidence that lipid metabolism is altered during the development of these types of tumors. Thus, in addition to targeted therapies focusing on classical oncogenes, new treatments are being developed based on a broad spectrum of strategies, ranging from vaccines to viral vectors, and melitherapy. This work reviews the current therapeutic landscape for pediatric brain tumors, considering new emerging treatments and ongoing clinical trials. In addition, the role of lipid metabolism in these neoplasms and its relevance for the development of novel therapies are discussed.

Cell proliferation in pancreatic cancer is determined by a complex network of signaling pathways. Despite the extensive understanding of these protein-mediated signaling processes, there are no significant drug discoveries that could considerably improve a patient’s survival. However, the recent understanding of lipid-mediated signaling gives a new perspective on the control of the physiological state of pancreatic cells. Lipid signaling plays a major role in the induction of cytocidal autophagy and can be exploited using synthetic lipids to induce cell death in pancreatic cancer cells. In this work, we studied the activity of a synthetic lipid, tri-2-hydroxyarachidonein (TGM4), which is a triacylglycerol mimetic that contains three acyl moieties with four double bonds each, on cellular and in vivo models of pancreatic cancer. We demonstrated that TGM4 inhibited proliferation of Mia-PaCa-2 (human pancreatic carcinoma) and PANC-1 (human pancreatic carcinoma of ductal cells) in in vitro models and in an in vivo xenograft model of Mia-PaCa-2 cells. In vitro studies demonstrated that TGM4 induced cell growth inhibition paralleled with an increased expression of PARP and CHOP proteins together with the presence of sub-G0 cell cycle events, indicating cell death. This cytocidal effect was associated with elevated ER stress or autophagy markers such as BIP, LC3B, and DHFR. In addition, TGM4 activated peroxisome proliferator-activated receptor gamma (PPAR-γ), which induced elevated levels of p-AKT and downregulation of p-c-Jun. We conclude that TGM4 induced pancreatic cell death by activation of cytocidal autophagy. This work highlights the importance of lipid signaling in cancer and the use of synthetic lipid structures as novel and potential approaches to treat pancreatic cancer and other neoplasias.

GPCRs receive signals from diverse messengers and activate G proteins that regulate downstream signaling effectors. Efficient signaling is achieved through the organization of these proteins in membranes. Thus, protein–lipid interactions play a critical role in bringing G proteins together in specific membrane microdomains with signaling partners. Significantly, the molecular basis underlying the membrane distribution of each G protein isoform, fundamental to fully understanding subsequent cell signaling, remains largely unclear. We used model membranes with lipid composition resembling different membrane microdomains, and monomeric, dimeric and trimeric Gi proteins with or without single and multiple mutations to investigate the structural bases of G protein–membrane interactions. We demonstrated that cationic amino acids in the N-terminal region of the Gαi1 and C-terminal region of the Gγ2 subunit, as well as their myristoyl, palmitoyl and geranylgeranyl moieties, define the differential G protein form interactions with membranes containing different lipid classes (PC, PS, PE, SM, Cho) and the various microdomains they may form (Lo, Ld, PC bilayer, charged, etc.). These new findings in part explain the molecular basis underlying amphitropic protein translocation to membranes and localization to different membrane microdomains and the role of these interactions in cell signal propagation, pathophysiology and therapies targeted to lipid membranes.

Alzheimer’s disease (AD) is a neurodegenerative disease with as yet no efficient therapies, the pathophysiology of which is still largely unclear. Many drugs and therapies have been designed and developed in the past decade to stop or slow down this neurodegenerative process, although none has successfully terminated a phase-III clinical trial in humans. Most therapies have been inspired by the amyloid cascade hypothesis, which has more recently come under question due to the almost complete failure of clinical trials of anti-amyloid/tau therapies to date. To shift the perspective for the design of new AD therapies, membrane lipid therapy has been tested, which assumes that brain lipid alterations lie upstream in the pathophysiology of AD. A hydroxylated derivative of docosahexaenoic acid was used, 2-hydroxy-docosahexaenoic acid (DHA-H), which has been tested in a number of animal models and has shown efficacy against hallmarks of AD pathology. Here, for the first time, DHA-H is shown to undergo α-oxidation to generate the heneicosapentaenoic acid (HPA, C21:5, n-3) metabolite, an odd-chain omega-3 polyunsaturated fatty acid that accumulates in cell cultures, mouse blood plasma and brain tissue upon DHA-H treatment, reaching higher concentrations than those of DHA-H itself. Interestingly, DHA-H does not share metabolic routes with its natural analog DHA (C22:6, n-3) but rather, DHA-H and DHA accumulate distinctly, both having different effects on cell fatty acid composition. This is partly explained because DHA-H α-hydroxyl group provokes steric hindrance on fatty acid carbon 1, which in turn leads to diminished incorporation into cell lipids and accumulation as free fatty acid in cell membranes. Finally, DHA-H administration to mice elevated the brain HPA levels, which was directly and positively correlated with cognitive spatial scores in AD mice, apparently in the absence of DHA-H and without any significant change in brain DHA levels. Thus, the evidence presented in this work suggest that the metabolic conversion of DHA-H into HPA could represent a key event in the therapeutic effects of DHA-H against AD.

Membranes are mainly composed of a lipid bilayer and proteins, constituting a checkpoint for the entry and passage of signals and other molecules. Their composition can be modulated by diet, pathophysiological processes, and nutritional/pharmaceutical interventions. In addition to their use as an energy source, lipids have important structural and functional roles, e.g., fatty acyl moieties in phospholipids have distinct impacts on human health depending on their saturation, carbon length, and isometry. These and other membrane lipids have quite specific effects on the lipid bilayer structure, which regulates the interaction with signaling proteins. Alterations to lipids have been associated with important diseases, and, consequently, normalization of these alterations or regulatory interventions that control membrane lipid composition have therapeutic potential. This approach, termed membrane lipid therapy or membrane lipid replacement, has emerged as a novel technology platform for nutraceutical interventions and drug discovery. Several clinical trials and therapeutic products have validated this technology based on the understanding of membrane structure and function. The present review analyzes the molecular basis of this innovative approach, describing how membrane lipid composition and structure affects protein-lipid interactions, cell signaling, disease, and therapy (e.g., fatigue and cardiovascular, neurodegenerative, tumor, infectious diseases).

Membrane Lipid Replacement (MLR) uses natural membrane lipid supplements to safely replace damaged, oxidized lipids in membranes in order to restore membrane function, decrease symptoms and improve health. Oral MLR supplements contain mixtures of cell membrane glycerolphospholipids, fatty acids, and other lipids, and can be used to replace and remove damaged cellular and intracellular membrane lipids. Membrane injury, caused mainly by oxidative damage, occurs in essentially all chronic and acute medical conditions, including cancer and degenerative diseases, and in normal processes, such as aging and development. After ingestion, the protected MLR glycerolphospholipids and other lipids are dispersed, absorbed, and internalized in the small intestines, where they can be partitioned into circulating lipoproteins, globules, liposomes, micelles, membranes, and other carriers and transported in the lymphatics and blood circulation to tissues and cellular sites where they are taken in by cells and partitioned into various cellular membranes. Once inside cells, the glycerolphospholipids and other lipids are transferred to various intracellular membranes by lipid carriers, globules, liposomes, chylomicrons, or by direct membrane–membrane interactions. The entire process appears to be driven by ‘bulk flow’ or mass action principles, where surplus concentrations of replacement lipids can stimulate the natural exchange and removal of damaged membrane lipids while the replacement lipids undergo further enzymatic alterations. Clinical studies have demonstrated the advantages of MLR in restoring membrane and organelle function and reducing fatigue, pain, and other symptoms in chronic illness and aging patients.

Adult polyglucosan body disease (APBD) is a neurological disorder characterized by adult-onset neurogenic bladder, spasticity, weakness, and sensory loss. The disease is caused by aberrant glycogen branching enzyme (GBE) (GBE1Y329S) yielding less branched, globular, and soluble glycogen, which tends to aggregate. We explore here whether, despite being a soluble enzyme, GBE1 activity is regulated by protein-membrane interactions. Because soluble proteins can contact a wide variety of cell membranes, we investigated the interactions of purified WT and GBE1Y329S proteins with different types of model membranes (liposomes). Interestingly, both triheptanoin and some triacylglycerol mimetics (TGMs) we have designed (TGM0 and TGM5) markedly enhance GBE1Y329S activity, possibly enough for reversing APBD symptoms. We show that the GBE1Y329S mutation exposes a hydrophobic amino acid stretch, which can either stabilize and enhance or alternatively, reduce the enzyme activity via alteration of protein-membrane interactions. Additionally, we found that WT, but not Y329S, GBE1 activity is modulated by Ca2+ and phosphatidylserine, probably associated with GBE1-mediated regulation of energy consumption and storage. The thermal stabilization and increase in GBE1Y329S activity induced by TGM5 and its omega-3 oil structure suggest that this molecule has a considerable therapeutic potential for treating APBD.

The mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor drug, involves the rapid and specific activation of sphingomyelin synthase (SMS), leading to a 4-fold increase in SM mass in tumor cells. In the present study, we investigated the source of the ceramides required to sustain this dramatic increase in SM. Through radioactive and fluorescent labeling, we demonstrated that sphingolipid metabolism was altered by a 24 h exposure to 2OHOA, and we observed a consistent increase in the number of lysosomes and the presence of unidentified storage materials in treated cells. Mass spectroscopy revealed that different sphingolipid classes accumulated in human glioma U118 cells after exposure to 2OHOA, demonstrating a specific effect on C16-, C20-, and C22-containing sphingolipids. Based on these findings, we propose that the demand for ceramides required to sustain the SMS activation (ca. 200-fold higher than the basal level) profoundly modifies both sphingolipid and phospholipid metabolism. As the treatment is prolonged, tumor cells fail to adequately metabolize sphingolipids, leading to a situation resembling sphingolipidosis, whereby cell viability is compromised.

An experimental and theoretical study on 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes containing fatty acids (FAs) was performed by means of X-ray diffraction analysis and molecular dynamics (MD) simulations. The study was aimed at understanding the interactions of several structurally related FAs with biomembranes, which is necessary for further rational lipid drug design in membrane-lipid therapy. The main effect of FAs was to promote the formation of a HII phase, despite a stabilization of the coexisting Lα + HII phases. Derivatives of OA exhibited a specific density profile in the direction perpendicular to the bilayer that reflects differences in the relative localization of the carboxylate group within the polar region of the membrane as well as in the degree of membrane penetration of the FA acyl chain. Hydroxyl and methyl substituents at carbon-2 in the FA acyl chain were identified as effective modulators of the position of carboxylate group in the lipid bilayer. Our data highlight the specific potential of each FA in modulating the membrane structure properties.

Olive oil consumption leads to high monounsaturated fatty acid intake, especially oleic acid, and has been associated with a reduced risk of hypertension. However, the molecular mechanisms and contribution of its different components to lower blood pressure (BP) require further evaluation. Here, we examined whether a synthetic, non-β-oxidation-metabolizable derivative of oleic acid, 2-hydroxyoleic acid (2-OHOA), can normalize BP in adult spontaneously hypertensive rats (SHRs) and whether its antihypertensive action involves cAMP-dependent protein kinase A (PKA) and Rho kinase, two major regulators of vascular smooth muscle contraction. Oral administration of 2-OHOA to SHRs induced sustained systolic BP decreases in a time-dependent (1–7 days) and dose-dependent (100–900 mg/kg every 12 h) manner. After 7 days of treatment with 2-OHOA (600 mg/kg), the systolic BP of SHRs was similar to that of normotensive Wistar Kyoto rats, returning to its initial hypertensive level after withdrawal of 2-OHOA. This treatment strongly increased the protein expression of the catalytic and regulatory RIα and RIIα PKA subunits as well as PKA activity in aortas from SHRs. Consistently, administration of the PKA inhibitor 8-bromo adenosine-3′,5′-cyclic monophosphorothioate, Rp isomer, to 2-OHOA-treated SHRs induced a pronounced reversal (up to 59%) of the antihypertensive effect of 2-OHOA. Additionally, 2-OHOA completely reversed the pathological overexpression of aortic Rho kinase found in SHRs, suppressing the vasoconstrictory Rho kinase pathway.

Lipid intake in diet regulates the membrane lipid composition, which in turn controls activities of membrane proteins. There is evidence that fatty acids (FAs) and triacylglycerols (TGs) can alter the phospholipid (PL) mesomorphism. However, the molecular mechanisms involved are not fully understood. This study focuses on the effect of the unsaturation degree of the C-18 FAs, oleic acid (OA), linoleic acid and linolenic acid, and their TGs, triolein (TO), trilinolein, and trilinolenin, on the structural properties of phosphoethanolamine PLs. By means of X-ray diffraction and 31P-NMR spectroscopy, it is shown that both types of molecules stabilize the HII phase in 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) liposomes. Several structural factors are considered to explain the correlation between the FA unsaturation degree and the onset temperature of the HII phase.It is proposed that TGs could act as lateral spacers between polar DEPE groups, providing an increase in the effective surface area per lipid molecule that would account for the structural parameters of the HII phase. Fluorescence polarization data indicated a fluidification effect of OA on the lamellar phase. TO increased the viscosity of the hydrophobic core with a high effect on the HII phase.

Fatty acid derivatives are abundant in biological membranes, mainly as components of phospholipids and cholesterol esters. Their presence, free or bound to phospholipids, modulates the lipid membrane behavior. The present study shows the differential influence of the C-18 fatty acids (FAs), oleic, elaidic, and stearic acids on the structural properties of phosphatidylethanolamine (PE). X-ray diffraction of PE-FA systems demonstrated that oleic acid (OA) produced important concentration-dependent alterations of the lipid membrane structure: it induced reductions of up to 20–23°C in the lamellar-to-hexagonal transition temperature of 1-palmitoyl-2-oleoyl PE and dielaidoyl PE and regulated the dimensions of the hexagonal lattice. In contrast, elaidic and stearic acids did not markedly alter the phospholipid mesomorphism. The above effects were attributed to the different “molecular shape” of OA (with a kink at the middle of the molecule) with respect to their congeners, elaidic and stearic acids.The effects of free fatty acids (FFAs) on membrane structure are relevant for several reasons: i) some biological membranes contain very high levels of FFAs. ii) Mediterranean diets with high OA intake have been shown to exert protective effects against tumoral and hypertensive pathologies. iii) FFA derivatives have been developed as antitumoral and antihypertensive drugs.