Effect of the aggregated protein dye YAT2150 on Leishmania parasite viability.

The problems associated with the drugs currently used to treat leishmaniasis, including resistance, toxicity, and the high cost of some formulations, call for the urgent identification of new therapeutic agents with novel modes of action. The aggregated protein dye YAT2150 has been found to be a potent antileishmanial compound, with a half-maximal inhibitory concentration (IC50) of approximately 0.5 µM against promastigote and amastigote stages of Leishmania infantum. The encapsulation in liposomes of YAT2150 significantly improved its in vitro IC50 to 0.37 and 0.19 µM in promastigotes and amastigotes, respectively, and increased the half-maximal cytotoxic concentration in human umbilical vein endothelial cells to >50 µM. YAT2150 became strongly fluorescent when binding intracellular protein deposits in Leishmania cells. This fluorescence pattern aligns with the proposed mode of action of this drug in the malaria parasite Plasmodium falciparum, the inhibition of protein aggregation. In Leishmania major, YAT2150 rapidly reduced ATP levels, suggesting an alternative antileishmanial mechanism. To the best of our knowledge, this first-in-class compound is the only one described so far having significant activity against both Plasmodium and Leishmania, thus being a potential drug for the treatment of co-infections of both parasites.

Disclosure of cinnamic acid/4,9-diaminoacridine conjugates as multi-stage antiplasmodial hits.

4,9-diaminoacridines with reported antiplasmodial activity were coupled to different trans-cinnamic acids, delivering a new series of conjugates inspired by the covalent bitherapy concept. The new compounds were more potent than primaquine against hepatic stages of Plasmodium berghei, although this was accompanied by cytotoxic effects on Huh-7 hepatocytes. Relevantly, the conjugates displayed nanomolar activities against blood stage P. falciparum parasites, with no evidence of hemolytic effects below 100 µM. Moreover, the new compounds were at least 25-fold more potent than primaquine against P. falciparum gametocytes. Thus, the new antiplasmodial hits disclosed herein emerge as valuable templates for the development of multi-stage antiplasmodial drug candidates.

The ESCRT-III machinery participates in the production of extracellular vesicles and protein export during Plasmodium falciparum infection.

Infection with Plasmodium falciparum enhances extracellular vesicle (EV) production in parasitized red blood cells (pRBCs), an important mechanism for parasite-to-parasite communication during the asexual intraerythrocytic life cycle. The endosomal sorting complex required for transport (ESCRT), and in particular the ESCRT-III sub-complex, participates in the formation of EVs in higher eukaryotes. However, RBCs have lost the majority of their organelles through the maturation process, including an important reduction in their vesicular network. Therefore, the mechanism of EV production in P. falciparum-infected RBCs remains to be elucidated. Here we demonstrate that P. falciparum possesses a functional ESCRT-III machinery activated by an alternative recruitment pathway involving the action of PfBro1 and PfVps32/PfVps60 proteins. Additionally, multivesicular body formation and membrane shedding, both reported mechanisms of EV production, were reconstituted in the membrane model of giant unilamellar vesicles using the purified recombinant proteins. Moreover, the presence of PfVps32, PfVps60 and PfBro1 in EVs purified from a pRBC culture was confirmed by super-resolution microscopy and dot blot assays. Finally, disruption of the PfVps60 gene led to a reduction in the number of the produced EVs in the KO strain and affected the distribution of other ESCRT-III components. Overall, our results increase the knowledge on the underlying molecular mechanisms during malaria pathogenesis and demonstrate that ESCRT-III P. falciparum proteins participate in EV production.

The protein aggregation inhibitor YAT2150 has potent antimalarial activity in Plasmodium falciparum in vitro cultures.

BACKGROUND: By 2016, signs of emergence of Plasmodium falciparum resistance to artemisinin and partner drugs were detected in the Greater Mekong Subregion. Recently, the independent evolution of artemisinin resistance has also been reported in Africa and South America. This alarming scenario calls for the urgent development of new antimalarials with novel modes of action. We investigated the interference with protein aggregation, which is potentially toxic for the cell and occurs abundantly in all Plasmodium stages, as a hitherto unexplored drug target in the pathogen. RESULTS: Attempts to exacerbate the P. falciparum proteome's propensity to aggregation by delivering endogenous aggregative peptides to in vitro cultures of this parasite did not significantly affect their growth. In contrast, protein aggregation inhibitors clearly reduced the pathogen's viability. One such compound, the bis(styrylpyridinium) salt YAT2150, exhibited potent antiplasmodial activity with an in vitro IC50 of 90 nM for chloroquine- and artemisinin-resistant lines, arresting asexual blood parasites at the trophozoite stage, as well as interfering with the development of both sexual and hepatic forms of Plasmodium. At its IC50, this compound is a powerful inhibitor of the aggregation of the model amyloid ß peptide fragment 1-40, and it reduces the amount of aggregated proteins in P. falciparum cultures, suggesting that the underlying antimalarial mechanism consists in a generalized impairment of proteostasis in the pathogen. YAT2150 has an easy, rapid, and inexpensive synthesis, and because it fluoresces when it accumulates in its main localization in the Plasmodium cytosol, it is a theranostic agent. CONCLUSIONS: Inhibiting protein aggregation in Plasmodium significantly reduces the parasite's viability in vitro. Since YAT2150 belongs to a novel structural class of antiplasmodials with a mode of action that potentially targets multiple gene products, rapid evolution of resistance to this drug is unlikely to occur, making it a promising compound for the post-artemisinin era.

Adhesion of freshwater sponge cells mediated by carbohydrate-carbohydrate interactions requires low environmental calcium.

Marine ancestors of freshwater sponges had to undergo a series of physiological adaptations to colonize harsh and heterogeneous limnic environments. Besides reduced salinity, river-lake systems also have calcium concentrations far lower than seawater. Cell adhesion in sponges is mediated by calcium-dependent multivalent self-interactions of sulfated polysaccharide components of membrane-bound proteoglycans named aggregation factors. Cells of marine sponges require seawater average calcium concentration (10 mM) to sustain adhesion promoted by aggregation factors. We demonstrate here that the freshwater sponge Spongilla alba can thrive in a calcium-poor aquatic environment and that their cells are able to aggregate and form primmorphs with calcium concentrations 40-fold lower than that required by marine sponges cells. We also find that their gemmules need calcium and other micronutrients to hatch and generate new sponges. The sulfated polysaccharide purified from S. alba has sulfate content and molecular size notably lower than those from marine sponges. Nuclear magnetic resonance analyses indicated that it is composed of a central backbone of non- and 2-sulfated α- and ß-glucose units decorated with branches of α-glucose. Assessments with atomic force microscopy/single-molecule force spectroscopy show that S. alba glucan requires 10-fold less calcium than sulfated polysaccharides from marine sponges to self-interact efficiently. Such an ability to retain multicellular morphology with low environmental calcium must have been a crucial evolutionary step for freshwater sponges to successfully colonize inland waters.

Detection of Protein Aggregation in Live Plasmodium Parasites.

The rapid evolution of resistance in the malaria parasite to every single drug developed against it calls for the urgent identification of new molecular targets. Using a stain specific for the detection of intracellular amyloid deposits in live cells, we have detected the presence of abundant protein aggregates in Plasmodium falciparum blood stages and female gametes cultured in vitro, in the blood stages of mice infected by Plasmodium yoelii, and in the mosquito stages of the murine malaria species Plasmodium berghei Aggregated proteins could not be detected in early rings, the parasite form that starts the intraerythrocytic cycle. A proteomics approach was used to pinpoint actual aggregating polypeptides in functional P. falciparum blood stages, which resulted in the identification of 369 proteins, with roles particularly enriched in nuclear import-related processes. Five aggregation-prone short peptides selected from this protein pool exhibited different aggregation propensity according to Thioflavin-T fluorescence measurements, and were observed to form amorphous aggregates and amyloid fibrils in transmission electron microscope images. The results presented suggest that generalized protein aggregation might have a functional role in malaria parasites. Future antimalarial strategies based on the upsetting of the pathogen's proteostasis and therefore affecting multiple gene products could represent the entry to new therapeutic approaches.

Coupling the Antimalarial Cell Penetrating Peptide TP10 to Classical Antimalarial Drugs Primaquine and Chloroquine Produces Strongly Hemolytic Conjugates.

Recently, we disclosed primaquine cell penetrating peptide conjugates that were more potent than parent primaquine against liver stage Plasmodium parasites and non-toxic to hepatocytes. The same strategy was now applied to the blood-stage antimalarial chloroquine, using a wide set of peptides, including TP10, a cell penetrating peptide with intrinsic antiplasmodial activity. Chloroquine-TP10 conjugates displaying higher antiplasmodial activity than the parent TP10 peptide were identified, at the cost of an increased hemolytic activity, which was further confirmed for their primaquine analogues. Fluorescence microscopy and flow cytometry suggest that these drug-peptide conjugates strongly bind, and likely destroy, erythrocyte membranes. Taken together, the results herein reported put forward that coupling antimalarial aminoquinolines to cell penetrating peptides delivers hemolytic conjugates. Hence, despite their widely reported advantages as carriers for many different types of cargo, from small drugs to biomacromolecules, cell penetrating peptides seem unsuitable for safe intracellular delivery of antimalarial aminoquinolines due to hemolysis issues. This highlights the relevance of paying attention to hemolytic effects of cell penetrating peptide-drug conjugates.

Origin and Evolution of the Sponge Aggregation Factor Gene Family.

Although discriminating self from nonself is a cardinal animal trait, metazoan allorecognition genes do not appear to be homologous. Here, we characterize the Aggregation Factor (AF) gene family, which encodes putative allorecognition factors in the demosponge Amphimedon queenslandica, and trace its evolution across 24 sponge (Porifera) species. The AF locus in Amphimedon is comprised of a cluster of five similar genes that encode Calx-beta and Von Willebrand domains and a newly defined Wreath domain, and are highly polymorphic. Further AF variance appears to be generated through individualistic patterns of RNA editing. The AF gene family varies between poriferans, with protein sequences and domains diagnostic of the AF family being present in Amphimedon and other demosponges, but absent from other sponge classes. Within the demosponges, AFs vary widely with no two species having the same AF repertoire or domain organization. The evolution of AFs suggests that their diversification occurs via high allelism, and the continual and rapid gain, loss and shuffling of domains over evolutionary time. Given the marked differences in metazoan allorecognition genes, we propose the rapid evolution of AFs in sponges provides a model for understanding the extensive diversification of self-nonself recognition systems in the animal kingdom.

Antimalarial Activity of Orally Administered Curcumin Incorporated in Eudragit®-Containing Liposomes.

Curcumin is an antimalarial compound easy to obtain and inexpensive, having shown little toxicity across a diverse population. However, the clinical use of this interesting polyphenol has been hampered by its poor oral absorption, extremely low aqueous solubility and rapid metabolism. In this study, we have used the anionic copolymer Eudragit® S100 to assemble liposomes incorporating curcumin and containing either hyaluronan (Eudragit-hyaluronan liposomes) or the water-soluble dextrin Nutriose® FM06 (Eudragit-nutriosomes). Upon oral administration of the rehydrated freeze-dried nanosystems administered at 25/75 mg curcumin·kg−1·day−1, only Eudragit-nutriosomes improved the in vivo antimalarial activity of curcumin in a dose-dependent manner, by enhancing the survival of all Plasmodium yoelii-infected mice up to 11/11 days, as compared to 6/7 days upon administration of an equal dose of the free compound. On the other hand, animals treated with curcumin incorporated in Eudragit-hyaluronan liposomes did not live longer than the controls, a result consistent with the lower stability of this formulation after reconstitution. Polymer-lipid nanovesicles hold promise for their development into systems for the oral delivery of curcumin-based antimalarial therapies.

Amyloid-ß Peptide Nitrotyrosination Stabilizes Oligomers and Enhances NMDAR-Mediated Toxicity.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the pathological aggregation of the amyloid-ß peptide (Aß). Monomeric soluble Aß can switch from helicoidal to ß-sheet conformation, promoting its assembly into oligomers and subsequently to amyloid fibrils. Oligomers are highly toxic to neurons and have been reported to induce synaptic transmission impairments. The progression from oligomers to fibrils forming senile plaques is currently considered a protective mechanism to avoid the presence of the highly toxic oligomers. Protein nitration is a frequent post-translational modification under AD nitrative stress conditions. Aß can be nitrated at tyrosine 10 (Y10) by peroxynitrite. Based on our analysis of ThT binding, Western blot and electron and atomic force microscopy, we report that Aß nitration stabilizes soluble, highly toxic oligomers and impairs the formation of fibrils. We propose a mechanism by which fibril elongation is interrupted upon Y10 nitration: Nitration disrupts fibril-forming folds by preventing H14-mediated bridging, as shown with an Aß analog containing a single residue (H to E) replacement that mimics the behavior of nitrated Aß related to fibril formation and neuronal toxicity. The pathophysiological role of our findings in AD was highlighted by the study of these nitrated oligomers on mouse hippocampal neurons, where an increased NMDAR-dependent toxicity of nitrated Aß oligomers was observed. Our results show that Aß nitrotyrosination is a post-translational modification that increases Aß synaptotoxicity. SIGNIFICANCE STATEMENT: We report that nitration (i.e., the irreversible addition of a nitro group) of the Alzheimer-related peptide amyloid-ß (Aß) favors the stabilization of highly toxic oligomers and inhibits the formation of Aß fibrils. The nitrated Aß oligomers are more toxic to neurons due to increased cytosolic calcium levels throughout their action on NMDA receptors. Sustained elevated calcium levels trigger excitotoxicity, a characteristic event in Alzheimer's disease.

Triacylglycerol mimetics regulate membrane interactions of glycogen branching enzyme: implications for therapy.

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.

Carbohydrate-Carbohydrate Interactions Mediated by Sulfate Esters and Calcium Provide the Cell Adhesion Required for the Emergence of Early Metazoans.

Early metazoans had to evolve the first cell adhesion mechanism addressed to maintain a distinctive multicellular morphology. As the oldest extant animals, sponges are good candidates for possessing remnants of the molecules responsible for this crucial evolutionary innovation. Cell adhesion in sponges is mediated by the calcium-dependent multivalent self-interactions of sulfated polysaccharides components of extracellular membrane-bound proteoglycans, namely aggregation factors. Here, we used atomic force microscopy to demonstrate that the aggregation factor of the sponge Desmapsamma anchorata has a circular supramolecular structure and that it thus belongs to the spongican family. Its sulfated polysaccharide units, which were characterized via nuclear magnetic resonance analysis, consist preponderantly of a central backbone composed of 3-α-Glc1 units partially sulfated at 2- and 4-positions and branches of Pyr(4,6)α-Gal1→3-α-Fuc2(SO3)1→3-α-Glc4(SO3)1→3-α-Glc→4-linked to the central α-Glc units. Single-molecule force measurements of self-binding forces of this sulfated polysaccharide and their chemically desulfated and carboxyl-reduced derivatives revealed that the sulfate epitopes and extracellular calcium are essential for providing the strength and stability necessary to sustain cell adhesion in sponges. We further discuss these findings within the framework of the role of molecular structures in the early evolution of metazoans.

G protein-membrane interactions II: Effect of G protein-linked lipids on membrane structure and G protein-membrane interactions.

G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (HII) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi1 monomers had a higher affinity for lamellar phases, while Gßγ and Gαßγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

The hydroxylated form of docosahexaenoic acid (DHA-H) modifies the brain lipid composition in a model of Alzheimer's disease, improving behavioral motor function and survival.

We have compared the effect of the commonly used ω-3 fatty acid, docosahexaenoic acid ethyl ester (DHA-EE), and of its 2-hydroxylated DHA form (DHA-H), on brain lipid composition, behavior and lifespan in a new human transgenic Drosophila melanogaster model of Alzheimer's disease (AD). The transgenic flies expressed human Aß42 and tau, and the overexpression of these human transgenes in the CNS of these flies produced progressive defects in motor function (antigeotaxic behavior) while reducing the animal's lifespan. Here, we demonstrate that both DHA-EE and DHA-H increase the longer chain fatty acids (≥18C) species in the heads of the flies, although only DHA-H produced an unknown chromatographic peak that corresponded to a non-hydroxylated lipid. In addition, only treatment with DHA-H prevented the abnormal climbing behavior and enhanced the lifespan of these transgenic flies. These benefits of DHA-H were confirmed in the well characterized transgenic PS1/APP mouse model of familial AD (5xFAD mice), mice that develop defects in spatial learning and in memory, as well as behavioral deficits. Hence, it appears that the modulation of brain lipid composition by DHA-H could have remedial effects on AD associated neurodegeneration. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Role of the C-terminal basic amino acids and the lipid anchor of the Gγ2 protein in membrane interactions and cell localization.

Heterotrimeric G proteins are peripheral membrane proteins that frequently localize to the plasma membrane where their presence in molar excess over G protein coupled receptors permits signal amplification. Their distribution is regulated by protein-lipid interactions, which has a clear influence on their activity. Gßγ dimer drives the interaction between G protein heterotrimers with cell membranes. We focused our study on the role of the C-terminal region of the Gγ2 protein in G protein interactions with cell membranes. The Gγ2 subunit is modified at cysteine (Cys) 68 by the addition of an isoprenyl lipid, which is followed by the proteolytic removal of the last three residues that leaves an isoprenylated and carboxyl methylated Cys-68 as the terminal amino acid. The role of Cys isoprenylation of the CAAX box has been defined for other proteins, yet the importance of proteolysis and carboxyl methylation of isoprenylated proteins is less clear. Here, we showed that not only geranylgeranylation but also proteolysis and carboxyl methylation are essential for the correct localization of Gγ2 in the plasma membrane. Moreover, we showed the importance of electrostatic interactions between the inner leaflet of the plasma membrane and the positively charged C-terminal domain of the Gγ2 subunit (amino acids Arg-62, Lys-64 and Lys-65) as a second signal to reach the plasma membrane. Indeed, single or multiple point mutations at Gγ2 C-terminal amino acids have a significant effect on Gγ2 protein-plasma membrane interactions and its localization to charged Ld (liquid disordered) membrane microdomains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Biophysical characterization of the association of histones with single-stranded DNA.

BACKGROUND: Despite the profound current knowledge of the architecture and dynamics of nucleosomes, little is known about the structures generated by the interaction of histones with single-stranded DNA (ssDNA), which is widely present during replication and transcription. METHODS: Non-denaturing gel electrophoresis, transmission electron microscopy, atomic force microscopy, magnetic tweezers. RESULTS: Histones have a high affinity for ssDNA in 0.15M NaCl ionic strength, with an apparent binding constant similar to that calculated for their association with double-stranded DNA (dsDNA). The length of DNA (number of nucleotides in ssDNA or base pairs in dsDNA) associated with a fixed core histone mass is the same for both ssDNA and dsDNA. Although histone-ssDNA complexes show a high tendency to aggregate, nucleosome-like structures are formed at physiological salt concentrations. Core histones are able to protect ssDNA from digestion by micrococcal nuclease, and a shortening of ssDNA occurs upon its interaction with histones. The purified (+) strand of a cloned DNA fragment of nucleosomal origin has a higher affinity for histones than the purified complementary (-) strand. CONCLUSIONS: At physiological ionic strength histones have high affinity for ssDNA, possibly associating with it into nucleosome-like structures. GENERAL SIGNIFICANCE: In the cell nucleus histones may spontaneously interact with ssDNA to facilitate their participation in the replication and transcription of chromatin.

Functional response of novel bioprotective poloxamer-structured vesicles on inflamed skin.

Resveratrol and gallic acid, a lipophilic and a hydrophilic phenol, were co-loaded in innovative, biocompatible nanovesicles conceived for ensuring the protection of the skin from oxidative- and inflammatory-related affections. The basic vesicles, liposomes and glycerosomes, were produced by a simple, one-step method involving the dispersion of phospholipid and phenols in water or water/glycerol blend, respectively. Liposomes and glycerosomes were modified by the addition of poloxamer, a stabilizer and viscosity enhancer, thus obtaining viscous or semisolid dispersions of structured vesicles. The vesicles were spherical, unilamellar and small in size (~70 nm in diameter). The superior ability of the poloxamer-structured vesicles to promote the accumulation of both phenols in the skin was demonstrated, as well as their low toxicity and great ability to protect fibroblasts from chemically-induced oxidative damage. The in vivo administration of the vesicular phenols on TPA (phorbol ester)-exposed skin led to a significant reduction of oedema and leukocyte infiltration.

Adaptation of targeted nanocarriers to changing requirements in antimalarial drug delivery.

The adaptation of existing antimalarial nanocarriers to new Plasmodium stages, drugs, targeting molecules, or encapsulating structures is a strategy that can provide new nanotechnology-based, cost-efficient therapies against malaria. We have explored the modification of different liposome prototypes that had been developed in our group for the targeted delivery of antimalarial drugs to Plasmodium-infected red blood cells (pRBCs). These new models include: (i) immunoliposome-mediated release of new lipid-based antimalarials; (ii) liposomes targeted to pRBCs with covalently linked heparin to reduce anticoagulation risks; (iii) adaptation of heparin to pRBC targeting of chitosan nanoparticles; (iv) use of heparin for the targeting of Plasmodium stages in the mosquito vector; and (v) use of the non-anticoagulant glycosaminoglycan chondroitin 4-sulfate as a heparin surrogate for pRBC targeting. The results presented indicate that the tuning of existing nanovessels to new malaria-related targets is a valid low-cost alternative to the de novo development of targeted nanosystems.

G protein-membrane interactions I: Gαi1 myristoyl and palmitoyl modifications in protein-lipid interactions and its implications in membrane microdomain localization.

G proteins are fundamental elements in signal transduction involved in key cell responses, and their interactions with cell membrane lipids are critical events whose nature is not fully understood. Here, we have studied how the presence of myristic and palmitic acid moieties affects the interaction of the Gαi1 protein with model and biological membranes. For this purpose, we quantified the binding of purified Gαi1 protein and Gαi1 protein acylation mutants to model membranes, with lipid compositions that resemble different membrane microdomains. We observed that myristic and palmitic acids not only act as membrane anchors but also regulate Gαi1 subunit interaction with lipids characteristics of certain membrane microdomains. Thus, when the Gαi1 subunit contains both fatty acids it prefers raft-like lamellar membranes, with a high sphingomyelin and cholesterol content and little phosphatidylserine and phosphatidylethanolamine. By contrast, the myristoylated and non-palmitoylated Gαi1 subunit prefers other types of ordered lipid microdomains with higher phosphatidylserine content. These results in part explain the mobility of Gαi1 protein upon reversible palmitoylation to meet one or another type of signaling protein partner. These results also serve as an example of how membrane lipid alterations can change membrane signaling or how membrane lipid therapy can regulate the cell's physiology.

The effect of hydroxylated fatty acid-containing phospholipids in the remodeling of lipid membranes.

The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) is an antitumor drug that regulates membrane lipid composition and structure. An important effect of this drug is the restoration of sphingomyelin (SM) levels in cancer cell membranes, where the SM concentration is lower than in non-tumor cells. It is well known that free fatty acid concentration in cell membranes is lower than 5%, and that fatty acid excess is rapidly incorporated into phospholipids. In a recent work, we have considered the effect of free 2OHOA in model membranes in liquid ordered (Lo) and liquid disordered (Ld) phases, by using all-atom molecular dynamics. This study concerns membranes that are modified upon incorporation of 2OHOA into different phospholipids. 2OHOA-containing phospholipids have a permanent effect on lipid membranes, making a Ld membrane surface more compact and less hydrated, whereas the opposite effect is observed in Lo domains. Moreover, the hydroxyl group of fatty acid chains increases the propensity of Ld model membranes to form hexagonal or other non-lamellar structures. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.