Unsaturated Acyl Chains Research Articles

Miscibility of Phosphatidylcholines in Bilayers: Effect of Acyl Chain Unsaturation

The miscibility of phospholipids in a hydrated bilayer is an issue of fundamental importance for understanding the organization of biological membranes. Despite research on lipid miscibility, its molecular basis remains poorly understood. In this study, all-atom MD simulations complemented by Langmuir monolayer and DSC experiments have been performed to investigate the molecular organization and properties of lipid bilayers composed of phosphatidylcholines with saturated (palmitoyl, DPPC) and unsaturated (oleoyl, DOPC) acyl chains. The experimental results showed that the DOPC/DPPC bilayers are systems exhibiting a very limited miscibility (strongly positive values of excess free energy of mixing) at temperatures below the DPPC phase transition. The excess free energy of mixing is divided into an entropic component, related to the ordering of the acyl chains, and an enthalpic component, resulting from the mainly electrostatic interactions between the headgroups of lipids. MD simulations showed that the electrostatic interactions for lipid like-pairs are much stronger than that for mixed pairs and temperature has only a slight influence on these interactions. On the contrary, the entropic component increases strongly with increasing temperature, due to the freeing of rotation of acyl chains. Therefore, the miscibility of phospholipids with different saturations of acyl chains is an entropy-driven process.

Investigation of the Impact of Lipid Acyl Chain Saturation on Fusion Peptide Interactions with Lipid Bilayers

The interaction of many peptides with lipid bilayer membranes strongly depends on the lipid composition. Here, a study of the impact of unsaturated lipid acyl chains on the interaction of a derivative of the HIV-1 fusion peptide with lipid bilayer vesicles is presented. Lipid bilayer vesicles composed of mixtures of lipids with two saturated acyl chains and lipids and one saturated and one unsaturated acyl chain, but identical head groups, were studied. The dependence of the peptide conformation on the unsaturated lipid content was probed by circular dichroism spectroscopy, while the impact of the peptide on the bilayer structure was determined by small-angle neutron scattering. The impact of the peptide on the lipid bilayer vesicle dynamics was investigated using neutron spin echo spectroscopy. Molecular dynamics simulations were used to characterize the behavior of the systems studied to determine if there were clear differences in their physical properties. The results reveal that the peptide–bilayer interaction is not a simple function of the unsaturated lipid acyl chain content of the bilayer. Instead, the peptide behavior is more consistent with that seen for the bilayer containing only unsaturated lipids, which is supported by lipid-specific interactions revealed by the simulations.

How Do Cyclopropane Fatty Acids Protect the Cell Membrane of Escherichia coli in Cold Shock?

The cyclopropanation of unsaturated lipid acyl chains of some bacterial cell membranes is an important survival strategy to protect the same against drastic cooling. To elucidate the role of cyclopropane ring-containing lipids, we have simulated the lipid membrane of Escherichia coli (E. coli) and two modified membranes by replacing the cyclopropane rings with either single or double bonds at widely different temperatures. It has been observed that the cyclopropane rings provide more rigid kinks in the lipid acyl chain compared to the double bonds and therefore further reduce the packing density of the membrane and subsequently enhance the membrane fluidity at low temperatures. They also inhibit the close packing of other lipids and deleterious phase separation by strongly interacting with them. Therefore, this study has explained why E. coli bacterial strain, susceptible to freezing environments, relies on the cyclopropanation of an unsaturated chain.

Packing and elasticity in membrane mechanical properties.

Here we address how membrane properties involving head group size, acyl chain unsaturation, and lipid packing affect their structure and dynamics. Strong out-of-plane proteolipid couplings considered by the flexible surface model (FSM) affect protein function through elastic forces described by the spontaneous curvature and the bending rigidity. We hypothesized that the mechanical properties and the molecular packing resulting from different lipid types are captured by introducing a perdeuterated POPC-d31 probe lipid. Di-monounsaturated phosphatidylcholine and phosphatidylethanolamine (DOPC, DOPE), and different ratios of DOPC/DOPE mixtures were studied using MD simulations combined with experimental solid-state 2H NMR spectroscopy. Residual quadrupolar couplings obtained from de-Paked powder-type spectra yielded order parameter (SCD) profiles for the individual acyl segments giving insights into average membrane structural properties such as area per lipid and bilayer thickness [1]. The corresponding spin-lattice relaxation rates (R1Z) for the resolved peaks followed a theoretical square-law dependence on SCD providing the bending rigidity [2]. Furthermore, MD simulations for the lipid systems were carried out and order parameters were calculated and compared against the NMR-observed SCD values. Utilization of the probe lipid POPC-d31 (1−10 mol%)affected the lamellar to hexagonal phase transition temperature of DOPE lipid systems but did not affect the quadrupolar splittings of DOPE and DOPC systems. Higher bending rigidity was observed for DOPE membranes versus DOPC supporting the idea that the membrane bending rigidity is primarily governed by increased lipid packing and is related to the area-compressibility modulus. Use of a probe lipid to capture membrane mechanical properties of complex lipid systems allow investigation of the relationship of membrane mechanics in vital biological functions.

Investigating the organisation of the asymmetric plasma membrane using a novel solvatochromic reporter.

The plasma membrane (PM) of an animal cell is a heterogenous mixture of lipids and proteins that exhibits both lateral heterogeneity and transverse asymmetry in composition. The complex mixture of lipid species with a range of headgroups and degrees of acyl-chain unsaturation influences the properties of the bilayer such as ordering and consequently the function of embedded membrane proteins. In this study, we have probed the presence of liquid-ordered (lo) domains in each leaflet of the PM and their transbilayer registry in live cells by designing a membrane localised probe that is sensitive to lipid order. This solvatochromic probe, C3L, uses Laurdan as a template and reports on changes in lipid order of complex lipid compositions such as Giant Plasma Membrane Vesicles (GPMVs), while exhibiting a preference for lo-domains. C3L distributes across both leaflets in the live cell membrane, regulated by flippases and scramblases and may be selectively and robustly confined to the inner leaflet after back extraction. C3L indicates the inner leaflet to be significantly disordered compared to the outer leaflet wherein the depletion of sphingomyelin (SM) leads to a more outer-leaflet restricted effect but phosphatidylserine (PS) depletion leads to effects on both leaflets, especially the inner leaflet. This suggests that PS and SM play different roles in building and maintaining domains and coupling them across the bilayer. Further, C3L reported lipid organisation indicates domains of smaller scale and broader order distribution in the PM of live cells at the same temperature compared to homogenous and disordered domains in GPMVs derived from them providing support for active de-mixing in membranes of living cells. Using this tool, we intend to explore the impact of membrane composition and cytoskeletal activity in tuning cellular signalling by regulating transbilayer properties of the PM.

Divergent unsaturated fatty acid synthesis in two highly related model pseudomonads.

The genomes of the best-studied pseudomonads, Pseudomonas aeruginosa and Pseudomonas putida, which share 85% of the predicted coding regions, contain a fabA fabB operon (demonstrated in P. aeruginosa, putative in P. putida). The enzymes encoded by the fabA and fabB genes catalyze the introduction of a double bond into a 10-carbon precursor which is elongated to the 16:1Δ9 and 18:1Δ11 unsaturated fatty acyl chains required for functional membrane phospholipids. A detailed analysis of transcription of the P. putida fabA fabB gene cluster showed that fabA and fabB constitute an operon and disclosed an unexpected and essential fabB promoter located within the fabA coding sequence. Inactivation of the fabA fabB operon fails to halt the growth of P. aeruginosa PAO1 but blocks growth of P. putida F1 unless an exogenous unsaturated fatty acid is provided. We report that the asymmetry between these two species is due to the P. aeruginosa PAO1 desA gene which encodes a fatty acid desaturase that introduces double bonds into the 16-carbon acyl chains of membrane phospholipids. Although P. putida F1 encodes a putative DesA homolog that is 84% identical to the P. aeruginosa PAO1, the protein fails to provide sufficient unsaturated fatty acid synthesis for growth when the FabA FabB pathway is inactivated. We report that the P. putida F1 DesA homolog can functionally replace the P. aeruginosa DesA. Hence, the defect in P. putida F1 desaturation is not due to a defective P. putida F1 DesA protein but probably to a weakly active component of the electron transfer process.

Oxidative lipidomics to elucidate the non-volatile derivatives of four typical triglycerides in vegetable oils under simulated frying conditions

Vegetable oils with different saturations have varied composition of triglycerides (TGs) and produce different non-volatile derivatives during oxidation. Precise characterization of the non-volatile derivatives of TGs is essential for understanding the degradation of TGs and the production pattern of non-volatile derivatives. Oxidative lipidomics was combined with collision-induced dissociation and electron-activated dissociation to elucidate the precise structures of non-volatile derivatives produced under simulated frying conditions by 1,3-dipalmitoyl-2-oleoylglycerol (POP), triolein (OOO), trilinolein (LLL), and trilinolenin (LnLnLn). The results indicate that the unsaturated fatty acyl chains at the sn-2 position were more susceptible to oxidation compared with those at the sn-1/3 position. Species of non-volatile derivatives included epoxy-, hydroperoxy-, hydroxy-, and oxo-TGs, as well as degradation products. The potential reaction pathways of TGs and their non-volatile derivatives were also proposed. This study elucidated oxidative degradation mechanisms of the four typical TGs and provided a theoretical basis for changes of vegetable oils during frying.

Effect of oxidation on POPC lipid bilayers: anionic carboxyl group plays a major role.

Phospholipids with unsaturated acyl chains are major targets of reactive oxygen species leading to formation of oxidized lipids. Oxidized phospholipids have a pronounced role in cell membrane damage. We investigated the effect of oxidation on physiological properties of phospholipid bilayers using atomistic molecular dynamics simulations. We studied phospholipid bilayer systems of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and its two stable oxidized products, 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) and 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC). Structural properties of the POPC lipid bilayer upon the addition of PoxnoPC or PazePC with concentration ranging from 10% to 30% were described. The key finding is that PazePC lipids bend their polar tails toward the bilayer-water interface whereas PoxnoPC lipids orient their tail toward the bilayer interior. The bilayer thickness decreases such that the thickness reduction in bilayers containing PazePC is stronger than in bilayers containing PoxnoPC. The average area per lipid decreases with a stronger effect in bilayers containing PoxnoPC. The addition of PoxnoPC makes both POPC acyl chains slightly more ordered whereas the addition of PazePC reduces the order in the two POPC acyl chains. These structural changes lead to an enhancement in the permeabilities of the bilayers containing these two oxidized products depending on the type, and the amount of oxidation. This enhancement can be achieved with a lower concentration of PazePC (10% or 15%), whereas a higher concentration of PoxnoPC (20%) is required to achieve an apparent enhancement in permeability. While the permeability of bilayers containing PazePC is higher than bilayers containing PoxnoPC in the 10-20% concentration range, by increasing the concentration of the oxidized products to higher than 20%, permeability of the bilayers containing PazePC is reduced such that it is slightly smaller than those containing PoxnoPC.

Short-clustered maltodextrin provides cryoprotection by maintaining cell membrane homeostasis of yeast during frozen storage

Cryoprotective polysaccharides have been extensively explored. We found that short-clustered maltodextrin (SCMD) effectively increased yeast viability. But the specific mechanism of SCMD affecting yeast is still not clear. Here, we detailedly analyze the effects of SCMD addition on cell membrane homeostasis. Firstly, the high-branched structure of SCMD was observed, and the microstructure of SCMD-protecting yeast was more integral. Analysis of membrane physicochemical property and glycerophospholipid metabolism demonstrated the fluidity of cell membrane of SCMD-protecting yeast was enhanced, as the relative abundances of glycerophospholipids with unsaturated acyl chains increased in comparison to Control yeast. Further, molecular dynamics simulations found that simplified SCMD could form more H-bonds with water molecules around lipid bilayers at 250 K to promote the mobility of lipid bilayers. Therefore, the hypothesis of “SCMD existence promotes the membrane fluidity under ice-crystal stress during frozen storage and helps maintain cell membrane homeostasis after thawing, increasing the thawed yeast viability” is proposed.

Dynamic modulations of sphingolipids and glycerophospholipids in COVID-19.

BackgroundA heterogeneous clinical phenotype is a characteristic of coronavirus disease 2019 (COVID‐19). Therefore, investigating biomarkers associated with disease severity is important for understanding the mechanisms responsible for this heterogeneity and for developing novel agents to prevent critical conditions. This study aimed to elucidate the modulations of sphingolipids and glycerophospholipids, which have been shown to possess potent biological properties.MethodsWe measured the serum sphingolipid and glycerophospholipid levels in a total of 887 samples from 215 COVID‐19 subjects, plus 115 control subjects without infectious diseases and 109 subjects with infectious diseases other than COVID‐19.ResultsWe observed the dynamic modulations of sphingolipids and glycerophospholipids in the serum of COVID‐19 subjects, depending on the time course and severity. The elevation of C16:0 ceramide and lysophosphatidylinositol and decreases in C18:1 ceramide, dihydrosphingosine, lysophosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol were specific to COVID‐19. Regarding the association with maximum severity, phosphatidylinositol and phosphatidylcholine species with long unsaturated acyl chains were negatively associated, while lysophosphatidylethanolamine and phosphatidylethanolamine were positively associated with maximum severity during the early phase. Lysophosphatidylcholine and phosphatidylcholine had strong negative correlations with CRP, while phosphatidylethanolamine had strong positive ones. C16:0 ceramide, lysophosphatidylcholine, phosphatidylcholine and phosphatidylethanolamine species with long unsaturated acyl chains had negative correlations with D‐dimer, while phosphatidylethanolamine species with short acyl chains and phosphatidylinositol had positive ones. Several species of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin might serve as better biomarkers for predicting severe COVID‐19 during the early phase than CRP and D‐dimer. Compared with the lipid modulations seen in mice treated with lipopolysaccharide, tissue factor, or histone, the lipid modulations observed in severe COVID‐19 were most akin to those in mice administered lipopolysaccharide.ConclusionA better understanding of the disturbances in sphingolipids and glycerophospholipids observed in this study will prompt further investigation to develop laboratory testing for predicting maximum severity and/or novel agents to suppress the aggravation of COVID‐19.

Cholesterol Stiffening of Lipid Membranes.

Biomembrane order, dynamics, and other essential physicochemical parameters are controlled by cholesterol, a major component of mammalian cell membranes. Although cholesterol is well known to exhibit a condensing effect on fluid lipid membranes, the extent of stiffening that occurs with different degrees of lipid acyl chain unsaturation remains an enigma. In this review, we show that cholesterol locally increases the bending rigidity of both unsaturated and saturated lipid membranes, suggesting there may be a length-scale dependence of the bending modulus. We review our published data that address the origin of the mechanical effects of cholesterol on unsaturated and polyunsaturated lipid membranes and their role in biomembrane functions. Through a combination of solid-state deuterium NMR spectroscopy and neutron spin-echo spectroscopy, we show that changes in molecular packing cause the universal effects of cholesterol on the membrane bending rigidity. Our findings have broad implications for the role of cholesterol in lipid–protein interactions as well as raft-like mixtures, drug delivery applications, and the effects of antimicrobial peptides on lipid membranes.Graphical

Lipid–Inorganic Hybrid Particles with Non-Lamellar Structures

Nanostructured non-lamellar lipid particles are widely studied in various fields of application, although their self-assembled structure is sensitive to internal and external conditions, which may limit their applicability. The aim of this study was to overcome these limitations and create particles with non-lamellar nanostructures which are stable over time, upon drying and heating. This was achieved by the combination of two approaches: self-assembly of lipids and polymerization of alkoxysilanes. Precursors containing one or two unsaturated acyl chains were functionalized with trialkoxysilane headgroups. Contrarily to previous studies, the use of unsaturated acyl chains led to the formation of hybrid particles with non-lamellar internal nanostructures. These particles showed a sponge or a hexagonal arrangement and were named spongosomes and hexosomes. Due to the covalent linking of the precursors, durable structures were obtained. The particles were stable for at least several months and maintained their nanostructures even when they were dried or exposed to high temperatures. The inorganic functionalization of lipids enabled the fixation of the self-assembled nanostructures.

Toy model that explains the regulation of cholesterol on lipid rafts

Cholesterol, as a common lipid on mammalian cell membranes, plays an important role in the formation of lipid rafts. Recent experiments suggest that the strength of cholesterol’s regulation on lipid rafts can be affected by the length of the unsaturated phospholipid acyl chain on the membrane. In order to understand this observation, a simplified toy model containing three different molecules is proposed in this paper, where the tail length of phospholipids is considered. This model shows the regulation of membrane cholesterol on the phase separation of the lipid mixture and the formation of nano-domains, and also suggests that the configuration entropy of phospholipid tails is an essential factor.

Natural crystallisation of tucuma (Astrocaryum vulgare Mart.) pulp olein

Tucuma (Astrocaryum vulgare Mart.) is a native Amazonian fruit rich in lipids, the exploitation of which is performed sustainably by small local producers. This paper investigates the potential of natural crystallisation of tucuma olein to produce fractions that could potentially replace palm oil in food products. The olein crystallisation was performed over 2 and 7 days. The physical, chemical, and rheological characteristics of the tucuma olein and its fractions were evaluated. There was no significant difference in the mass percentage of phase formation between the 2nd and 7th day of cooling. The super olein (upper phase) fraction had a higher total carotenoid content per g of oil than both the olein and the mid-fraction (heavier phase). All fractions presented oleic acid as the major component and triacylglycerols with 52 and 54 carbon atoms, with a predominance of two unsaturated acyl chains and fully unsaturated triacylglycerols. Tucuma mid-fraction showed characteristics of a pseudoplastic fluid behaviour and pseudo-β' crystals as palm stearin and palm mid-fraction. Therefore, tucuma mid-fraction has potential application in the food industry to replace palm and trans-fat oils.

Liquid-crystalline, liquid-ordered, rippled and gel lipid bilayer phases as observed with nile red fluorescence

The basic matrix of cellular membranes consists of a double layer (bilayer) of phospholipids. Semisynthetic lipid bilayers are commonly used in biophysical studies of membranes. According to temperature and composition, lipid bilayers can exist in liquid-crystalline (or liquid-disordered), liquid-ordered, rippled, and gel phases. In the present study, the hydrophobic, solvatochromic molecule Nile red has been used as a fluorescent probe to examine the physical state of bilayers of different compositions in the 15–60 °C range. Phospholipids with saturated or unsaturated acyl chains, in the presence or absence of cholesterol have been studied. Nile red shows absorption maxima at 520–550 nm and emission maxima at 580–640 nm, single photon excitation not being damaging to the system. A red/orange intensity ratio (ROIR) index has been used to normalize the results. ROIR varies clearly and reproducibly with the lipid phase, increasing in the order: liquid-ordered < gel < rippled < liquid-crystalline. It increases with temperature and decreases with cholesterol contents in the bilayers. Nile red allows an unusually clear observation of the rippled-to-liquid crystalline phase transition in saturated phospholipids. FLIM studies with Nile red also show differences between lamellar phases. Rotational relaxation times have been determined for Nile red in liquid-disordered (0.72 ± 0.010 ns), gel (1.16 ± 0.070 ns), and liquid-ordered (1.79 ± 0.14 ns) phases, the large value of the liquid-ordered phase being an indication of the sterol hindering probe tumbling in the hydrophobic matrix.

Effect of lipid saturation on the topology and oligomeric state of helical membrane polypeptides

Natural liquid crystalline membranes are made up of many different lipids carrying a mixture of saturated and unsaturated fatty acyl chains. Whereas in the past considerable attention has been paid to cholesterol content, the phospholipid head groups and the membrane surface charge the detailed fatty acyl composition was often considered less important. However, recent investigations indicate that the detailed fatty acyl chain composition has pronounced effects on the oligomerization of the transmembrane helical anchoring domains of the MHC II receptor or the membrane alignment of the cationic antimicrobial peptide PGLa. In contrast the antimicrobial peptides magainin 2 and alamethicin are less susceptible to lipid saturation. Using histidine-rich LAH4 designer peptides the high energetic contributions of lipid saturation in stabilizing transmembrane helical alignments are quantitatively evaluated. These observations can have important implications for the biological regulation of membrane proteins and should be taken into considerations during biophysical or structural experiments.

Complete Genome Sequence of Paraclostridium bifermentans DSM 14991

ABSTRACTThe complete genome sequence of Paraclostridium bifermentans was obtained by assembly of Illumina and Oxford Nanopore (ONT) reads. The sequence will enable study into the organism’s ability to biohydrogenate unsaturated acyl chains in the transformation of C20 polyunsaturated fatty acids (PUFAs) into the corresponding bioactive non-methylene-interrupted fatty acids (NMIFAs).

Abstract 3018: Targeting lipid metabolism and diet in advanced prostate cancer

Abstract Lipid metabolism reprogramming provides the energetic needs of prostate cancer cells (PCa) to support growth and cell progress through the cell cycle, sustaining a highly proliferative phenotype by increasing the expression levels of several enzymes involved in fatty acid (FAs) synthesis. Fatty acid synthase (FASN), a key enzyme in de novo fatty acid lipogenesis (DNL), is upregulated in metastatic, castration-resistant prostate cancer (CRPC), and its overexpression is related to carcinogenesis, growth, and metastasis. We previously demonstrated that FASN inhibitors show antitumor activity suppressing PCa cell proliferation and slowing tumor growth, both in vitro and in vivo, via rewiring of lipid cancer metabolism and inducing endoplasmic reticulum stress.We observed that impairment of DNL increases polyunsaturated fatty acids (PUFA) uptake as a compensatory mechanism of survival. Exogenously acquired PUFAs cause the remodeling of the prostate cancer cell lipidome, manifesting an enhanced acyl chain unsaturation across several lipid species. Lipid peroxidation and ROS accumulation are also observed, suggesting cell susceptible to oxidative damage following FASN blockade. Alterations in metabolic pathways cause a higher uptake of acetate and glutamine in PCa cells, as assessed by 14C-nutrient incorporation following FASN inhibition. Mitochondrial dysfunction is induced by reduction in several respiratory parameters, including ATP production and basal respiration. This may be, at least in part, the result of a reduction in phosphatidylglycerol, a cardiolipin precursor, altering mitochondrial membrane composition. Supplementation with the PUFA docosahexaenoic acid (DHA) increases phosphatidylcholine levels and total phospholipid acyl chain unsaturation, which can lead to membrane disorganization in prostate cancer cells. Superoxide anion production is enhanced by DHA treatment, as expected, and increased levels of glucose and palmitate oxidation by mitochondria are observed. Interestingly, AR and AR-V7 protein expression, as well as c-MYC and PSA, is reduced with DHA treatment. We then evaluated how de novo lipid synthesis inhibition combined with manipulation of the exogenous supply of fatty acids could impair tumor cell progression. Due to the obligate cell dependence on PUFA following DNL suppression, we combined FASN and Acetyl-CoA carboxylase (ACC) inhibitors with DHA supplementation and observed a significant cell growth inhibition in CRPC cell lines and prostate cancer organoids.Altogether, our results suggest a novel mechanism to inhibit the growth of castration-resistant prostate cancer, combining lipogenesis inhibition with PUFA-enriched diet to overcome advanced disease. Citation Format: Silvia D. Rodrigues, Caroline Fidalgo Ribeiro, Guilherme H. Tamarindo, Hubert Pakula, Massimo Loda. Targeting lipid metabolism and diet in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3018.

Abstract 1513: FASN inhibition-induced BRCAness as a therapeutic option for castration-resistant prostate cancer (CRPC)

Abstract Androgen deprivation therapy (ADT) is the primary treatment for prostate cancer. However, resistance to ADT invariably develops, leading to castration-resistant prostate cancer (CRPC). Prostate cancer progression is marked by overexpression of fatty acid synthase (FASN) and increased de novo synthesis of fatty acids. Importantly, the majority of normal tissues have low expression of FASN and rely mostly on exogenous lipids, making FASN an interesting therapeutic target for prostate cancer. Previously our group evaluated the effects of inhibiting FASN in prostate cancer, with especially promising results in CRPC models. We observed that FASN inhibition decreases DNA damage repair capability by transcriptionally modulating homologous recombination (BRCAness) and non-homologous end joining repair pathways, which leads to increased DNA damage and apoptosis. Key enzymes involved in DNA repair pathways are reduced at the protein level, and the addition of exogenous palmitate, the product of FASN, is able to rescue their expression to normal levels. Interestingly, genes involved in the synthesis of ceramide are upregulated after FASN blockade, with consequent increased cellular levels of ceramides, dihydroceramides and sphingomyelin. Additionally, we observed increased acyl chain unsaturation levels in these lipid species, in line with the uptake of polyunsaturated fatty acids (PUFA) observed when de novo lipogenesis is blocked. The inhibition of ceramide synthesis, both through genetic knock-down with siRNA or pharmacologically with an inhibitor of serine palmitoyltransferase, is capable of partially rescuing the DNA damage induced by FASN inhibition, suggesting a role of ceramides in DNA damage response modulation. Next, we investigated the synergistic effect of combining FASN inhibitor with PARP inhibitor and observed a higher inhibition of cell growth in prostate cancer cell lines in comparison to either drug alone. Human CRPC organoids when treated with PARP and FASN inhibitor in combination showed reduced diameter, as well as reduced cell proliferation, when compared with each agent alone. Overall, our data demonstrate that targeting de novo lipogenesis can increase the therapeutic efficacy of PARP inhibitors and benefit prostate cancer patients that do not harbor BRCA mutations, by pharmacologically downregulating DNA damage repair pathways, particularly HR. Citation Format: Caroline Fidalgo Ribeiro, Silvia D. Rodrigues, Debora C. Bastos, Hubert Pakula, Giorgia Zadra, Marco Foiani, Massimo Loda. FASN inhibition-induced BRCAness as a therapeutic option for castration-resistant prostate cancer (CRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1513.

Natural variation meets synthetic biology: Promiscuous trichome-expressed acyltransferases from Nicotiana.

Acylsugars are defensive, trichome-synthesized sugar esters produced in plants across the Solanaceae (nightshade) family. Although assembled from simple metabolites and synthesized by a relatively short core biosynthetic pathway, tremendous within- and across-species acylsugar structural variation is documented across the family. To advance our understanding of the diversity and the synthesis of acylsugars within the Nicotiana genus, trichome extracts were profiled across the genus coupled with transcriptomics-guided enzyme discovery and in vivo and in vitro analysis. Differences in the types of sugar cores, numbers of acylations, and acyl chain structures contributed to over 300 unique annotated acylsugars throughout Nicotiana. Placement of acyl chain length into a phylogenetic context revealed that an unsaturated acyl chain type was detected in a few closely related species. A comparative transcriptomics approach identified trichome-enriched Nicotiana acuminata acylsugar biosynthetic candidate enzymes. More than 25 acylsugar variants could be produced in a single enzyme assay with four N. acuminata acylsugar acyltransferases (NacASAT1-4) together with structurally diverse acyl-CoAs and sucrose. Liquid chromatography coupled with mass spectrometry screening of in vitro products revealed the ability of these enzymes to make acylsugars not present in Nicotiana plant extracts. In vitro acylsugar production also provided insights into acyltransferase acyl donor promiscuity and acyl acceptor specificity as well as regiospecificity of some ASATs. This study suggests that promiscuous Nicotiana acyltransferases can be used as synthetic biology tools to produce novel and potentially useful metabolites.