Inhibition Of Fatty Acid Biosynthesis Research Articles

Optimizing Archaeal Lipid Biosynthesis in Escherichia coli.

Membrane lipid chemistry is remarkably different in archaea compared with bacteria and eukaryotes. In the evolutionary context, this is also termed the lipid divide and is reflected by distinct biosynthetic pathways. Contemporary organisms have almost without exception only one type of membrane lipid. During early membrane evolution, mixed membrane stages likely occurred, and it was hypothesized that the instability of such mixtures was the driving force for the lipid divide. To examine the compatibility between archaeal and bacterial lipids, the bacterium Escherichia coli has been engineered to contain both types of lipids with varying success. Only limited production of archaeal lipid archaetidylethanolamine was achieved. Here, we substantially increased its production in E. coli by overexpression of an archaeal phosphatidylserine synthase needed for ethanolamine headgroup attachment. Furthermore, we introduced a synthetic isoprenoid utilization pathway to increase the supply of isopentenyl-diphosphate and dimethylallyl diphosphate. This improved archaeal lipid production substantially. The archaeal phospholipids also served as a substrate for the E. coli cardiolipin synthase, resulting in archaeal and novel hybrid archaeal/bacterial cardiolipin species not seen in living organisms before. Growth of the E. coli strain with the mixed membrane shows an enhanced sensitivity to the inhibitor of fatty acid biosynthesis, cerulenin, indicating a critical dependence of the engineered E. coli strain on its native phospholipids.

Development and Antibacterial Properties of 4-[4-(Anilinomethyl)-3-phenylpyrazol-1-yl]benzoic Acid Derivatives as Fatty Acid Biosynthesis Inhibitors.

A number of novel pyrazole derivatives have been synthesized, and several of these compounds are potent antibacterial agents with minimum inhibitory concentrations as low as 0.5 μg/mL. Human cell lines were tolerant to these lead compounds, and they showed negligible hemolytic effects at high concentrations. These bactericidal compounds are very effective against bacterial growth in both planktonic and biofilm contexts. Various techniques were applied to show the inhibition of biofilm growth and eradication of preformed biofilms by lead compounds. Potent compounds are more effective against persisters than positive controls. In vivo studies revealed that lead compounds are effective in rescuing C. elegans from bacterial infections. Several methods were applied to determine the mode of action including membrane permeability assay and SEM micrograph studies. Furthermore, CRISPRi studies led to the determination of these compounds as fatty acid biosynthesis (FAB) inhibitors.

Role of Glycolysis and Fatty Acid Synthesis in the Activation and T Cell-Modulating Potential of Dendritic Cells Stimulated with a TLR5-Ligand Allergen Fusion Protein.

Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune–metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune–metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1β was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1β, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1β). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1β (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.

Expression of AtWRI1 and AtDGAT1 during soybean embryo development influences oil and carbohydrate metabolism.

SummarySoybean oil is one of the most consumed vegetable oils worldwide. Genetic improvement of its concentration in seeds has been historically pursued due to its direct association with its market value. Engineering attempts aiming to increase soybean seed oil presented different degrees of success that varied with the genetic design and the specific variety considered. Understanding the embryo’s responses to the genetic modifications introduced, is a critical step to successful approaches. In this work, the metabolic and transcriptional responses to AtWRI1 and AtDGAT1 expression in soybean seeds were evaluated. AtWRI1 is a master regulator of fatty acid (FA) biosynthesis, and AtDGAT1 encodes an enzyme catalysing the final and rate‐limiting step of triacylglycerides biosynthesis. The events expressing these genes in the embryo did not show an increase in total FA content, but they responded with changes in the oil and carbohydrate composition. Transcriptomic studies revealed a down‐regulation of genes putatively encoding for oil body packaging proteins, and a strong induction of genes annotated as lipases and FA biosynthesis inhibitors. Novel putative AtWRI1 targets, presenting an AW‐box in the upstream region of the genes, were identified by comparison with an event that harbours only AtWRI1. Lastly, targeted metabolomics analysis showed that carbon from sugar phosphates could be used for FA competing pathways, such as starch and cell wall polysaccharides, contributing to the restriction in oil accumulation. These results allowed the identification of key cellular processes that need to be considered to break the embryo’s natural restriction to uncontrolled seed lipid increase.

Removal of peptidoglycan and inhibition of active cellular processes leads to daptomycin tolerance in Enterococcus faecalis.

Daptomycin is a cyclic lipopeptide antibiotic used in the clinic for treatment of severe enterococcal infections. Recent reports indicate that daptomycin targets active cellular processes, specifically, peptidoglycan biosynthesis. Within, we examined the efficacy of daptomycin against Enterococcus faecalis under a range of environmental growth conditions including inhibitors that target active cellular processes. Daptomycin was far less effective against cells in late stationary phase compared to cells in exponential phase, and this was independent of cellular ATP levels. Further, the addition of either the de novo protein synthesis inhibitor chloramphenicol or the fatty acid biosynthesis inhibitor cerulenin induced survival against daptomycin far better than controls. Alterations in metabolites associated with peptidoglycan synthesis correlated with protection against daptomycin. This was further supported as removal of peptidoglycan induced physiological daptomycin tolerance, a synergistic relation between daptomycin and fosfomycin, an inhibitor of the fist committed step peptidoglycan synthesis, was observed, as well as an additive effect when daptomycin was combined with ampicillin, which targets crosslinking of peptidoglycan strands. Removal of the peptidoglycan of Enterococcus faecium, Staphylococcus aureus, and Bacillus subtilis also resulted in significant protection against daptomycin in comparison to whole cells with intact cell walls. Based on these observations, we conclude that bacterial growth phase and metabolic activity, as well as the presence/absence of peptidoglycan are major contributors to the efficacy of daptomycin.

An anticonvulsive drug, valproic acid (valproate), has effects on the biosynthesis of fatty acids and polyketides in microorganisms

Valproic acid or valproate (VPA) is an anticonvulsive drug used for treatments of epilepsy, bipolar disorder, and migraine headaches. VPA is also an epigenetic modulator, inhibiting histone deacetylase, and it has been subjected to clinical study for cancer treatment. During the investigation of VPA on a metabolite profile in a fungus, we found that VPA has significant effects on the production of some fatty acids. Further exploration of VPA on fatty acid profiles of microorganisms, fungi, yeast, and bacteria, as well as representative gut microbiome, revealed that VPA could enhance or reduce the production of some fatty acids. VPA was found to induce the production of trans-9-elaidic acid, a fatty acid that was previously reported to have cellular effects in human macrophages. VPA could also inhibit the production of some polyketides produced by a model fungus. The present work suggests that the induction or inhibition of fatty acid biosynthesis by VPA (100 µM) in gut microbiome could give effects to patients treated with VPA because high doses of VPA oral administration (up to 600 mg to 900 mg) are used by patients; the concentration of VPA in the human gut may reach a concentration of 100 µM, which may give effects to gut microorganisms.

Pyrrolocin C and equisetin inhibit bacterial acetyl-CoA carboxylase.

Antimicrobial resistance is a growing global health and economic concern. Current antimicrobial agents are becoming less effective against common bacterial infections. We previously identified pyrrolocins A and C, which showed activity against a variety of Gram-positive bacteria. Structurally similar compounds, known as pyrrolidinediones (e.g., TA-289, equisetin), also display antibacterial activity. However, the mechanism of action of these compounds against bacteria was undetermined. Here, we show that pyrrolocin C and equisetin inhibit bacterial acetyl-CoA carboxylase (ACC), the first step in fatty acid synthesis. We used transcriptomic data, metabolomic analysis, fatty acid rescue and acetate incorporation experiments to show that a major mechanism of action of the pyrrolidinediones is inhibition of fatty acid biosynthesis, identifying ACC as the probable molecular target. This hypothesis was further supported using purified proteins, demonstrating that biotin carboxylase is the inhibited component of ACC. There are few known antibiotics that target this pathway and, therefore, we believe that these compounds may provide the basis for alternatives to current antimicrobial therapy.

Exogenous Fatty Acids Remodel Staphylococcus aureus Lipid Composition through Fatty Acid Kinase.

Staphylococcus aureus can utilize exogenous fatty acids for phospholipid synthesis. The fatty acid kinase FakA is essential for this utilization by phosphorylating exogenous fatty acids for incorporation into lipids. How FakA impacts the lipid membrane composition is unknown. In this study, we used mass spectrometry to determine the membrane lipid composition and properties of S. aureus in the absence of fakA We found the fakA mutant to have increased abundance of lipids containing longer acyl chains. Since S. aureus does not synthesize unsaturated fatty acids, we utilized oleic acid (18:1) to track exogenous fatty acid incorporation into lipids. We observed a concentration-dependent incorporation of exogenous fatty acids into the membrane that required FakA. We also tested how FakA and exogenous fatty acids impact membrane-related physiology and identified changes in membrane potential, cellular respiration, and membrane fluidity. To mimic the host environment, we characterized the lipid composition of wild-type and fakA mutant bacteria grown in mouse skin homogenate. We show that wild-type S. aureus can incorporate exogenous unsaturated fatty acids from host tissue, highlighting the importance of FakA in the presence of host skin tissue. In conclusion, FakA is important for maintaining the composition and properties of the phospholipid membrane in the presence of exogenous fatty acids, impacting overall cell physiology.IMPORTANCE Environmental fatty acids can be harvested to supplement endogenous fatty acid synthesis to produce membranes and circumvent fatty acid biosynthesis inhibitors. However, how the inability to use these fatty acids impacts lipids is unclear. Our results reveal lipid composition changes in response to fatty acid addition and when S. aureus is unable to activate fatty acids through FakA. We identify concentration-dependent utilization of oleic acid that, when combined with previous work, provides evidence that fatty acids can serve as a signal to S. aureus Furthermore, using mouse skin homogenates as a surrogate for in vivo conditions, we showed that S. aureus can incorporate host fatty acids. This study highlights how exogenous fatty acids impact bacterial membrane composition and function.

Eleutheroside E decreases oxidative stress and NF-κB activation and reprograms the metabolic response against hypoxia-reoxygenation injury in H9c2 cells

Ischemia-reperfusion (I/R) injury causes cardiac dysfunction through several mechanisms including oxidative stress and pro-inflammation. Eleutheroside E (EE) has protective effects in ischemia tissue and anti-inflammatory action. However, the effect of EE on I/R-injured cardiomyocytes is unknown. In this study, we used in vitro H9c2 cell model to investigate the favorable role of EE on myocardial I/R injury. We found that EE administration attenuated the cardiomyocyte apoptosis induced by hypoxia-reoxygenation (H/R) injury. Further, pre-treatment with EE dramatically inhibited mitochondrial oxidative stress, IκBα phosphorylation and nuclear factor kappa B (NF-κB) subunit p65 translocation into nuclei. EE might suppress the MAPK signaling pathway to inhibit the H/R-induced NF-κB activation. Moreover, we had analyzed the metabolomic profile of H/R-injured and H/R + 100 EE-treated H9c2 cells and found that the abundance of most metabolites changed by H/R could be re-modulated by EE treatment. Pathway analysis highlighted the inhibition of fatty acid biosynthesis and alternation of arginine and proline metabolism as two potential links to the favorable effect of EE on H/R-injured cardiomyocytes. The further demonstration showed that nitric oxide (NO), a product that is solely catabolized by l-arginine and has profound anti-oxidative stress activity during H/R in cardiomyocytes, was augmented by EE. Altogether, our results provide evidence that EE may be a potential drug for myocardial I/R injury by reducing oxidative stress, NF-κB activation, and metabolic reprogramming.

Spiro N-methoxy piperidine ring containing aryldiones for the control of sucking insects and mites: discovery of spiropidion.

Crop protection solutions for the control of key economic sucking pests derive essentially from neuronal and muscular acting chemistries, wherein neonicotinoid uses largely dominated for the last two decades. Anticipating likely resistance development of some of those arthropod species to this particular class, we intensified research activities on a non-neuronal site of action targeting insect growth and development some 10 years ago. Our innovation path featured reactivation of a scarcely used and simple building block from the 1960s, namely N-methoxy-4-piperidone 3. Its judicious incorporation into the 2-aryl-1,3-dione scaffold of IRAC group 23 inhibitors of fatty acid biosynthesis resulted in novel tetramic acid derivatives acting on acetyl-coenzyme A carboxylase (ACCase). The optimization campaign focused on modulation of the aryl substitution pattern and understanding substituent options at the lactam nitrogen position of those spiroheterocyclic pyrrolidine-dione derivatives towards an effective control of sucking insects and mites. This work gratifyingly culminated in the discovery of spiro N-methoxy piperidine containing proinsecticide spiropidion 1. Following in planta release, its insecticidally active dione metabolite 2 is translaminar and two-way systemic (both xylem and phloem mobile) for a full plant protection against arthropod pests. Owing to such unique plant systemic properties, growing shoots and roots actually not directly exposed to spiropidion-based chemistry after foliar application nevertheless benefit from its long-lasting efficacy. Spiropidion is for use in field crops, speciality crops and vegetables controlling a broad range of sucking pests. In light of other performance and safety profiles of spiropidion, an IPM fit may be expected. © 2020 Society of Chemical Industry.

Serum Lipidomics Analysis of Classical Swine Fever Virus Infection in Piglets and Emerging Role of Free Fatty Acids in Virus Replication in vitro.

Lipids metabolism plays a significant role in cellular responses to virus pathogens. However, the impact of lipids metabolism in CSFV infection is not yet confirmed. In the present study, for the fist time, we performed serum lipidomics analysis of piglets infected with CSFV based on ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS), and identified 167 differentially expressed lipid metabolites. Interestingly, free fatty acids (FFAs) accumulated significantly in these metabolites, accompanied by an increase in sphingolipids and a decrease in glycerolipids and glycerophospholipids, suggesting that CSFV infection markedly changed the serum lipid metabolism of piglets. FFAs are the principal constituents of many complex lipids and are essential substrates for energy metabolism. Based on this, we focused on whether FFAs play a prominent role in CSFV infection. We found that CSFV infection induced FFAs accumulation in vivo and in vitro, which is due to increased fatty acid biosynthesis. Meanwhile, we discovered that alteration of cellular FFAs accumulation by a mixture of FFAs or inhibitors of fatty acid biosynthesis affects progeny virus production in vitro. Furthermore, in the absence of glucose or glutamine, CSFV still has replication capacity, which is significantly reduced with the addition of fatty acid beta oxidation inhibitors, suggesting that the process of FFAs enter the mitochondria for beta oxidation to produce ATP is necessary for virus replication. Finally, we demonstrated CSFV induced FFAs accumulation results in impaired type I IFN signaling-mediated antiviral responses by down-regulating RIG-I-like receptors (RLRs) signaling molecules, which may represent a mechanism of CSFV replication. Taken together, these findings provide the first data on lipid metabolites during CSFV infection and reveal a new view that CSFV infection requires FFAs to enhance viral replication.

A cell-based high-throughput screen identifies tyrphostin AG 879 as an inhibitor of animal cell phospholipid and fatty acid biosynthesis

Inhibition of animal cell phospholipid biosynthesis has been proposed for anticancer and antiviral therapies. Using CHOK1 derived cell lines, we have developed and used a cell-based high-throughput procedure to screen a 1280 compound, small molecule library for inhibitors of phospholipid biosynthesis. We identified tyrphostin AG 879 (AG879), which inhibited phospholipid biosynthesis by 85–90% at a concentration of 10 μM, displaying an IC50 of 1–3 μM. The synthesis of all phospholipid head group classes was heavily affected. Fatty acid biosynthesis was also dramatically inhibited (90%). AG879 inhibited phospholipid biosynthesis in all additional cell lines tested, including MDCK, HUH7, Vero, and HeLa cell lines. In CHO cells, AG879 was cytostatic; cells survived for at least four days during exposure and were able to divide following its removal. AG879 is an inhibitor of receptor tyrosine kinases (RTK) and inhibitors of signaling pathways known to be activated by RTK's also inhibited phospholipid biosynthesis. We speculate that inhibition of RTK by AG879 results in an inhibition of fatty acid biosynthesis with a resulting decrease in phospholipid biosynthesis and that AG879's effect on fatty acid synthesis and/or phospholipid biosynthesis may contribute to its known capacity as an effective antiviral/anticancer agent.

A pathway-directed positive growth restoration assay to facilitate the discovery of lipid A and fatty acid biosynthesis inhibitors in Acinetobacter baumannii

Acinetobacter baumannii ATCC 19606 can grow without lipooligosaccharide (LOS). Lack of LOS can result from disruption of the early lipid A biosynthetic pathway genes lpxA, lpxC or lpxD. Although LOS itself is not essential for growth of A. baumannii ATCC 19606, it was previously shown that depletion of the lipid A biosynthetic enzyme LpxK in cells inhibited growth due to the toxic accumulation of lipid A pathway intermediates. Growth of LpxK-depleted cells was restored by chemical inhibition of LOS biosynthesis using CHIR-090 (LpxC) and fatty acid biosynthesis using cerulenin (FabB/F) and pyridopyrimidine (acetyl-CoA-carboxylase). Here, we expand on this by showing that inhibition of enoyl-acyl carrier protein reductase (FabI), responsible for converting trans-2-enoyl-ACP into acyl-ACP during the fatty acid elongation cycle also restored growth during LpxK depletion. Inhibition of fatty acid biosynthesis during LpxK depletion rescued growth at 37°C, but not at 30°C, whereas rescue by LpxC inhibition was temperature independent. We exploited these observations to demonstrate proof of concept for a targeted medium-throughput growth restoration screening assay to identify small molecule inhibitors of LOS and fatty acid biosynthesis. The differential temperature dependence of fatty acid and LpxC inhibition provides a simple means by which to separate growth stimulating compounds by pathway. Targeted cell-based screening platforms such as this are important for faster identification of compounds inhibiting pathways of interest in antibacterial discovery for clinically relevant Gram-negative pathogens.

De Novo Lipid Synthesis Facilitates Gemcitabine Resistance through Endoplasmic Reticulum Stress in Pancreatic Cancer.

Pancreatic adenocarcinoma is moderately responsive to gemcitabine-based chemotherapy, the most widely used single-agent therapy for pancreatic cancer. Although the prognosis in pancreatic cancer remains grim in part due to poor response to therapy, previous attempts at identifying and targeting the resistance mechanisms have not been very successful. By leveraging The Cancer Genome Atlas dataset, we identified lipid metabolism as the metabolic pathway that most significantly correlated with poor gemcitabine response in pancreatic cancer patients. Furthermore, we investigated the relationship between alterations in lipogenesis pathway and gemcitabine resistance by utilizing tissues from the genetically engineered mouse model and human pancreatic cancer patients. We observed a significant increase in fatty acid synthase (FASN) expression with increasing disease progression in spontaneous pancreatic cancer mouse model, and a correlation of high FASN expression with poor survival in patients and poor gemcitabine responsiveness in cell lines. We observed a synergistic effect of FASN inhibitors with gemcitabine in pancreatic cancer cells in culture and orthotopic implantation models. Combination of gemcitabine and the FASN inhibitor orlistat significantly diminished stemness, in part due to induction of endoplasmic reticulum (ER) stress that resulted in apoptosis. Moreover, direct induction of ER stress with thapsigargin caused a similar decrease in stemness and showed synergistic activity with gemcitabine. Our in vivo studies with orthotopic implantation models demonstrated a robust increase in gemcitabine responsiveness upon inhibition of fatty acid biosynthesis with orlistat. Altogether, we demonstrate that fatty acid biosynthesis pathway manipulation can help overcome the gemcitabine resistance in pancreatic cancer by regulating ER stress and stemness. Cancer Res; 77(20); 5503-17. ©2017 AACR.

Very Long Chain Fatty Acids Are Functionally Involved in Necroptosis

Necroptosis is a form of regulated cell death that is linked to various human diseases. Distinct membrane-related, thus lipid-dependent, alterations take place during necroptosis. However, little is known about the roles of specific lipids in this process. We used an untargeted LC-MS-based approach to reveal that distinct lipid species are regulated at the molecular level during necroptosis. We found that ceramides and very long chain fatty acids accumulate during this process. Intrigued by the specificity of very long chain fatty acid accumulation, we focused on characterizing their involvement during necroptosis. Biochemical characterizations suggested that activated fatty acid biosynthesis and elongation could be responsible for these accumulations. We further showed that inhibition of fatty acid biosynthesis and depletion of very long chain fatty acids prevented loss of plasma membrane integrity and cell death, strongly suggesting that very long chain fatty acids are functionally involved in necroptosis.

Identification of KasA as the cellular target of an anti-tubercular scaffold.

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis β-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related β-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis.

Prospects of a new antistaphylococcal drug batumin revealed by molecular docking and analysis of the complete genome sequence of the batumin-producer Pseudomonas batumici UCM B-321

Meticillin-resistant Staphylococcus aureus (MRSA) is a serious public health threat causing outbreaks of clinical infection around the world. Mupirocin is a promising anti-MRSA drug, however mupirocin-resistant strains of S. aureus are emerging at an increasing rate. The newly discovered antibiotic batumin may contribute to anti-MRSA therapy. The objective of this work was to identify possible molecular targets for batumin as well as mechanisms of its antistaphylococcal activity using computational molecular docking and by analysing the complete genome sequence of the batumin-producer Pseudomonas batumici UCM B-321. It was found that batumin acted very similarly to mupirocin by inhibiting aminoacyl tRNA synthetases. A previous hypothesis considering the trans-enoyl-CoA reductase FabI as a prime molecular target of batumin was rejected. However, indirect inhibition of fatty acid biosynthesis in sensitive bacteria does take place as a part of stringent response repression triggered by accumulation of uncharged tRNA molecules. Paralogues of diverse leucine-tRNA synthetases in the genome of P. batumici indicated that this protein might be the prime target of batumin. A batumin biosynthesis operon comprising 28 genes was found to be acquired through horizontal gene transfer. It was hypothesised that, in contrast to mupirocin, batumin could inhibit a broader range of aminoacyl tRNA synthetases and that acquired resistance to mupirocin might not endow S. aureus strains with resistance against batumin.

Transformation with oncogenic Ras and the simian virus 40 T antigens induces caspase-dependent sensitivity to fatty acid biosynthetic inhibition.

Oncogenesis is frequently accompanied by the activation of specific metabolic pathways. One such pathway is fatty acid biosynthesis, whose induction is observed upon transformation of a wide variety of cell types. Here, we explored how defined oncogenic alleles, specifically the simian virus 40 (SV40) T antigens and oncogenic Ras(12V), affect fatty acid metabolism. Our results indicate that SV40/Ras(12V)-mediated transformation of fibroblasts induces fatty acid biosynthesis in the absence of significant changes in the concentration of fatty acid biosynthetic enzymes. This oncogene-induced activation of fatty acid biosynthesis was found to be mammalian target of rapamycin (mTOR) dependent, as it was attenuated by rapamycin treatment. Furthermore, SV40/Ras(12V)-mediated transformation induced sensitivity to treatment with fatty acid biosynthetic inhibitors. Pharmaceutical inhibition of acetyl-coenzyme A (CoA) carboxylase (ACC), a key fatty acid biosynthetic enzyme, induced caspase-dependent cell death in oncogene-transduced cells. In contrast, isogenic nontransformed cells were resistant to fatty acid biosynthetic inhibition. This oncogene-induced sensitivity to fatty acid biosynthetic inhibition was independent of the cells' growth rates and could be attenuated by supplementing the medium with unsaturated fatty acids. Both the activation of fatty acid biosynthesis and the sensitivity to fatty acid biosynthetic inhibition could be conveyed to nontransformed breast epithelial cells through transduction with oncogenic Ras(12V). Similar to what was observed in the transformed fibroblasts, the Ras(12V)-induced sensitivity to fatty acid biosynthetic inhibition was independent of the proliferative status and could be attenuated by supplementing the medium with unsaturated fatty acids. Combined, our results indicate that specific oncogenic alleles can directly confer sensitivity to inhibitors of fatty acid biosynthesis. Viral oncoproteins and cellular mutations drive the transformation of normal cells to the cancerous state. These oncogenic alterations induce metabolic changes and dependencies that can be targeted to kill cancerous cells. Here, we find that the cellular transformation resulting from combined expression of the SV40 early region with an oncogenic Ras allele is sufficient to induce cellular susceptibility to fatty acid biosynthetic inhibition. Inhibition of fatty acid biosynthesis in these cells resulted in programmed cell death, which could be rescued by supplementing the medium with nonsaturated fatty acids. Similar results were observed with the expression of oncogenic Ras in nontransformed breast epithelial cells. Combined, our results suggest that specific oncogenic alleles induce metabolic dependencies that can be exploited to selectively kill cancerous cells.

Metabolic switching: synergistic induction of carotenogenesis in the aerial microalga, Vischeria helvetica, under environmental stress conditions by inhibitors of fatty acid biosynthesis.

To investigate the roles of fatty acid biosynthesis in carotenogenesis in the high-lipid accumulating aerial microalga Vischeria helvetica KGU-Y001, we cultured algal cells with fatty acid biosynthesis inhibitors. Under nitrogen-deficient, high-light (200 µmol photons m(-2) s(-1)) conditions, the alga accumulated 6.2 mg carotenoids g(-1) dry weight cells (DWC) after 1 week of culture. The total fatty acid content increased gradually, and reached 290 mg g(-1) DWC after 9 weeks. When algal cells were cultured with a fatty acid biosynthesis inhibitor (molinate) under nitrogen-deficient, high-light conditions for 1 week, carotenoid accumulation was synergistically increased to 2.4 times that in algal cells cultured without the inhibitor in nitrogen-deficient, low-light conditions (40 µmol photons m(-2) s(-1)). The synergistic induction of carotenogenesis was suppressed by an inhibitor of c-jun N-terminal kinase, a mitogen-activated protein kinase-like protein. In a commercial context, carotenoid production could be increased by using fatty acid biosynthesis inhibitors to redirect metabolic flux to carotenoid biosynthesis instead of fatty acid synthesis.

Clickable Analogue of Cerulenin as Chemical Probe to Explore Protein Palmitoylation

Dynamic palmitoylation is an important post-translational modification regulating protein localization, trafficking, and signaling activities. The Asp-His-His-Cys (DHHC) domain containing enzymes are evolutionarily conserved palmitoyl acyltransferases (PATs) mediating diverse protein S-palmitoylation. Cerulenin is a natural product inhibitor of fatty acid biosynthesis and protein palmitoylation, through irreversible alkylation of the cysteine residues in the enzymes. Here, we report the synthesis and characterization of a "clickable" and long alkyl chain analogue of cerulenin as a chemical probe to investigate its cellular targets and to label and profile PATs in vitro and in live cells. Our results showed that the probe could stably label the DHHC-family PATs and enable mass spectrometry studies of PATs and other target proteins in the cellular proteome. Such probe provides a new chemical tool to dissect the functions of palmitoylating enzymes in cell signaling and diseases and reveals new cellular targets of the natural product cerulenin.