RESUMEN
The antibiotic cerulenin is a fungal natural product identified as a covalent inhibitor of ketosynthases within fatty acid and polyketide biosynthesis. Due to its selective and potent inhibitory activity, cerulenin has found significant utility in multidisciplinary biochemical, biomedical, and clinical studies. Although its covalent inhibition profile has been confirmed, cerulenin's mechanism has not been fully determined at a molecular level, frustrating the drug development of related analogues. Herein, we describe the use of stable isotopic tracking with NMR and MS methods to unravel the covalent mechanism of cerulenin against type II fatty acid ketosynthases. We detail the discovery of a unique C2-C3 retro-aldol bond cleavage and a structural rearrangement upon covalent inhibition of cerulenin at the active cysteine residue in E. coli type II fatty acid ketosynthases FabB and FabF.
Asunto(s)
Cerulenina , Cerulenina/farmacología , Cerulenina/química , Escherichia coli/enzimología , Escherichia coli/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Antibacterianos/farmacología , Antibacterianos/química , Acido Graso Sintasa Tipo II/antagonistas & inhibidores , Acido Graso Sintasa Tipo II/metabolismo , Modelos Moleculares , Estructura MolecularRESUMEN
The global impact of emerging viral infections emphasizes the urgent need for effective broad-spectrum antivirals. The cellular organelle, lipid droplet (LD), is utilized by many types of viruses for replication, but its reduction does not affect cell survival. Therefore, LD is a potential target for developing broad-spectrum antivirals. In this study, we found that 2-bromopalmitate (2 BP), a previously defined palmitoylation inhibitor, depletes LD across all studied cell lines and exerts remarkable antiviral effects on different coronaviruses. We comprehensively utilized 2 BP, alongside other palmitoylation inhibitors such as cerulenin and 2-fluoro palmitic acid (2-FPA), as well as the enhancer palmostatin B and evaluated their impact on LD and the replication of human coronaviruses (hCoV-229E, hCoV-Oc43) and murine hepatitis virus (MHV-A59) at non-cytotoxic concentrations. While cerulenin and 2-FPA exhibited moderate inhibition of viral replication, 2 BP exhibited a much stronger suppressive effect on MHV-A59 replication, although they share similar inhibitory effects on palmitoylation. As expected, palmostatin B significantly enhanced viral replication, it failed to rescue the inhibitory effects of 2 BP, whereas it effectively counteracted the effects of cerulenin and 2-FPA. This suggests that the mechanism that 2 BP used to inhibit viral replication is beyond palmitoylation inhibition. Further investigations unveil that 2 BP uniquely depletes LDs, a phenomenon not exhibited by 2-FPA and cerulenin. Importantly, the depletion of LDs was closely associated with the inhibition of viral replication because the addition of oleic acid to 2 BP significantly rescued LD depletion and its inhibitory effects on MHV-A59. Our findings indicate that the inhibitory effects of 2 BP on viral replication primarily stem from LD disruption rather than palmitoylation inhibition. Intriguingly, fatty acid (FA) assays demonstrated that 2 BP reduces the FA level in mitochondria while concurrently increasing FA levels in the cytoplasm. These results highlight the crucial role of LDs in viral replication and uncover a novel biological activity of 2 BP. These insights contribute to the development of broad-spectrum antiviral strategies. IMPORTANCE: In our study, we conducted a comparative investigation into the antiviral effects of palmitoylation inhibitors including 2-bromopalmitate (2-BP), 2-fluoro palmitic acid (2-FPA), and cerulenin. Surprisingly, we discovered that 2-BP has superior inhibitory effects on viral replication compared to 2-FPA and cerulenin. However, their inhibitory effects on palmitoylation were the same. Intrigued by this finding, we delved deeper into the underlying mechanism of 2-BP's potent antiviral activity, and we unveiled a novel biological activity of 2-BP: depletion of lipid droplets (LDs). Importantly, we also highlighted the crucial role of LDs in viral replication. Our insights shed new light on the antiviral mechanism of LD depletion paving the way for the development of broad-spectrum antiviral strategies by targeting LDs.
Asunto(s)
Antivirales , Coronavirus , Virus de la Hepatitis Murina , Palmitatos , Animales , Humanos , Ratones , Antivirales/farmacología , Antivirales/metabolismo , Cerulenina/metabolismo , Cerulenina/farmacología , Coronavirus/efectos de los fármacos , Coronavirus/fisiología , Gotas Lipídicas/efectos de los fármacos , Palmitatos/farmacología , Ácido Palmítico/farmacología , Ácido Palmítico/metabolismo , Propiolactona/análogos & derivados , Replicación Viral/efectos de los fármacos , Virus de la Hepatitis Murina/efectos de los fármacos , Virus de la Hepatitis Murina/fisiologíaRESUMEN
BACKGROUND: Naringenin is an industrially relevant compound due to its multiple pharmaceutical properties as well as its central role in flavonoid biosynthesis. RESULTS: On our way to develop Streptomyces albidoflavus J1074 as a microbial cell factory for naringenin production, we have significantly increased the yields of this flavanone by combining various metabolic engineering strategies, fermentation strategies and genome editing approaches in a stepwise manner. Specifically, we have screened different cultivation media to identify the optimal production conditions and have investigated how the additive feeding of naringenin precursors influences the production. Furthermore, we have employed genome editing strategies to remove biosynthetic gene clusters (BGCs) associated with pathways that might compete with naringenin biosynthesis for malonyl-CoA precursors. Moreover, we have expressed MatBC, coding for a malonate transporter and an enzyme responsible for the conversion of malonate into malonyl-CoA, respectively, and have duplicated the naringenin BGC, further contributing to the production improvement. By combining all of these strategies, we were able to achieve a remarkable 375-fold increase (from 0.06 mg/L to 22.47 mg/L) in naringenin titers. CONCLUSION: This work demonstrates the influence that fermentation conditions have over the final yield of a bioactive compound of interest and highlights various bottlenecks that affect production. Once such bottlenecks are identified, different strategies can be applied to overcome them, although the efficiencies of such strategies may vary and are difficult to predict.
Asunto(s)
Flavanonas , Microbiología Industrial , Streptomyces , Ingeniería Metabólica , Streptomyces/crecimiento & desarrollo , Streptomyces/metabolismo , Flavanonas/biosíntesis , Cerulenina/farmacología , Fenilalanina/farmacología , Tirosina/farmacologíaRESUMEN
BACKGROUND: Fatty acid synthase (FASN) is generally over-expressed in human tumor tissues and catalyzes de novo synthesis of fatty acids on which tumor cells depend. Bestatin, an inhibitor of aminopeptidase/CD13, is one of the dipeptide substrates for the human oligopeptide transporter 1 (PEPT1). OBJECTIVES: In the current study, we aimed to uncover the role of FASN inhibitors in bestatininduced tumor cell apoptosis and the underlying mechanism, extending our understanding of the correlations between FASN and PEPT1 in cancer and providing a new strategy for tumor targeted treatment. METHODS: Cerulenin, orlistat and siRNAs were applied to inhibit FASN. The cell viability and apoptosis were assessed with MTT (thiazolyl blue tetrazolium bromide) assays and annexin VFITC/ PI staining with flow cytometry analysis. Western blot and qRT-PCR analysis were used to detect the protein levels and mRNA levels of the indicated genes in tumor cells, respectively. Protein degradation or stability was examined with cycloheximide chase assays. CD13 activity was detected by gelatin zymography. The HT1080 and C26 xenografts models were conducted to assess the efficacy in vivo. RESULTS: In the current study, we found that inhibiting FASN by cerulenin and orlistat both augmented the effects of bestatin in decreasing tumor cell viability. Cerulenin increased the apoptosis rates and enhanced the cleavage of PARP caused by bestatin. Furthermore, cerulenin, orlistat and siFASNs markedly elevated PEPT1 protein levels. Indeed, cerulenin induced the upregulation of PEPT1 mRNA expression rather than affecting the protein level after the cells were treated with CHX. And Gly-Sar, a typical competitive substrate of PEPT1, could attenuate the augment of bestatin-induced cell killing by cerulenin. Moreover, synergistic restrain of tumor growth accompanied by a reduction of Ki-67 and increment of TUNEL was significantly achieved in the xenograft models. Interestingly, no clear correlation was observed between the CD13 with FASN and/or PEPT1 in tumor cells. CONCLUSION: FASN inhibitors facilitate tumor cells susceptible to bestatin-induced apoptosis involving the up-regulation of PEPT1 at the mRNA translation level and the transport of bestatin by PEPT1, emerging as a promising strategy for tumor targeted therapy.
Asunto(s)
Cerulenina , Neoplasias , Humanos , Cerulenina/farmacología , Orlistat/farmacología , Ácido Graso Sintasas , Neoplasias/tratamiento farmacológico , Apoptosis , ARN Mensajero/genética , Línea Celular Tumoral , Acido Graso Sintasa Tipo IRESUMEN
Cerulenin is a fungal metabolite and a specific inhibitor of fatty acid synthase (FASN), which has shown a potential anticancer activity. 20-25% of breast cancer patients with ErbB2-overexpressing develop resistance to treatment. Therefore, it is urgent to find an effective new target for the treatment of ErbB2-overexpressing breast cancer. Our previous study found that cerulenin inhibits the glycolysis and migration of SK-BR-3 cells, but the effect of cerulenin on other malignant phenotypes of breast cancer is still unknown. Furthermore, the mechanism by which cerulenin displays its inhibitory effects is not fully understood. In this study, we systematically investigate the inhibitory effects of cerulenin on proliferation, migration, invasion and glycolysis of ErbB2-overexpressing breast cancer cells and its molecular mechanism. We found that cerulenin obviously suppresses the proliferation, migration, invasion as well as glycolysis. Through bioinformatic analyses, we found that PKM2 might be a target of cerulenin. In addition, ErbB2 and its signaling pathway upregulated PKM2 protein levels. Furthermore, we demonstrated that cerulenin downregulated the protein levels of ErbB2, PKM2 and EMT markers (MMP9, MMP2 and Snail2) in a dose- and time-dependent manner. Finally, the inhibitory of cerulenin on colony formation, migration, invasion and glycolysis, as well as protein levels of EMT markers were rescued by replenishing with PKM2. These findings illustrated that cerulenin inhibits proliferation, migration, invasion and glycolysis by targeting ErbB2/PKM2 pathway in ErbB2-overexpressing breast cancer cells.
Asunto(s)
Neoplasias de la Mama , Cerulenina , Humanos , Línea Celular Tumoral , Proliferación Celular , Cerulenina/farmacología , Cerulenina/metabolismo , Ácido Graso Sintasas/metabolismo , Glucólisis , Receptor ErbB-2 , Transducción de Señal , Neoplasias de la Mama/metabolismo , Proteínas de Unión a Hormona TiroideRESUMEN
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 1ß was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1ß, 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-1ß). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1ß (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.
Asunto(s)
Receptor Toll-Like 5 , Vacunas , Receptor Toll-Like 5/metabolismo , Alérgenos , Interleucina-10/metabolismo , Flagelina/metabolismo , Hexoquinasa/metabolismo , Glutaminasa/metabolismo , Ligandos , Antimicina A/metabolismo , Antimicina A/farmacología , Cerulenina/metabolismo , Cerulenina/farmacología , Células Dendríticas , Proteínas Recombinantes/metabolismo , Citocinas/metabolismo , Adyuvantes Inmunológicos/farmacología , Vacunas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Glucólisis , Serina-Treonina Quinasas TOR/metabolismo , Desoxiglucosa/farmacología , Oligomicinas/farmacología , Ácidos Grasos/metabolismoRESUMEN
BACKGROUND: Microglia are brain-resident myeloid cells involved in the innate immune response and a variety of neurodegenerative diseases. In macrophages, TonEBP is a transcriptional cofactor of NF-κB which stimulates the transcription of pro-inflammatory genes in response to LPS. Here, we examined the role of microglial TonEBP. METHODS: We used microglial cell line, BV2 cells. TonEBP was knocked down using lentiviral transduction of shRNA. In animals, TonEBP was deleted from myeloid cells using a line of mouse with floxed TonEBP. Cerulenin was used to block the NF-κB cofactor function of TonEBP. RESULTS: TonEBP deficiency blocked the LPS-induced expression of pro-inflammatory cytokines and enzymes in association with decreased activity of NF-κB in BV2 cells. We found that there was also a decreased activity of AP-1 and that TonEBP was a transcriptional cofactor of AP-1 as well as NF-κB. Interestingly, we found that myeloid-specific TonEBP deletion blocked the LPS-induced microglia activation and subsequent neuronal cell death and memory loss. Cerulenin disrupted the assembly of the TonEBP/NF-κB/AP-1/p300 complex and suppressed the LPS-induced microglial activation and the neuronal damages in animals. CONCLUSIONS: TonEBP is a key mediator of microglial activation and neuroinflammation relevant to neuronal damage. Cerulenin is an effective blocker of the TonEBP actions.
Asunto(s)
Mediadores de Inflamación/metabolismo , Lipopolisacáridos/toxicidad , Trastornos de la Memoria/metabolismo , Microglía/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción/metabolismo , Animales , Reacción de Prevención/efectos de los fármacos , Reacción de Prevención/fisiología , Línea Celular , Cerulenina/farmacología , Redes Reguladoras de Genes/efectos de los fármacos , Redes Reguladoras de Genes/fisiología , Masculino , Trastornos de la Memoria/inducido químicamente , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne RNA virus that causes epidemics of debilitating disease in tropical and sub-tropical regions with autochtonous transmission in regions with temperate climate. Currently, there is no licensed vaccine or specific antiviral drug available against CHIKV infection. In this study, we examine the role, in the CHIKV viral cycle, of fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD1), two key lipogenic enzymes required for fatty acid production and early desaturation. We show that both enzymes and their upstream regulator PI3K are required for optimal CHIKV infection. We demonstrate that pharmacologic manipulation of FASN or SCD1 enzymatic activity by non-toxic concentrations of cerulenin or CAY10566 decreases CHIKV genome replication. Interestingly, a similar inhibitory effect was also obtained with Orlistat, an FDA-approved anti-obesity drug that targets FASN activity. These drugs were also effective against Mayaro virus (MAYV), an under-studied arthritogenic Old world Alphavirus endemic in South American countries with potential risk of emergence, urbanization and dispersion to other regions. Altogether, our results identify FASN and SCD1 as conserved druggable cofactors of Alphavirus genome replication and support the broad-spectrum activity of drugs targeting the host fatty acids metabolism.
Asunto(s)
Alphavirus/efectos de los fármacos , Ácido Graso Sintasas/metabolismo , Estearoil-CoA Desaturasa/metabolismo , Replicación Viral/efectos de los fármacos , Alphavirus/genética , Infecciones por Alphavirus/tratamiento farmacológico , Animales , Antivirales/farmacología , Línea Celular , Cerulenina/farmacología , Fiebre Chikungunya/tratamiento farmacológico , Virus Chikungunya/efectos de los fármacos , Virus Chikungunya/genética , Chlorocebus aethiops , Ácido Graso Sintasas/efectos de los fármacos , Genoma Viral , Células HEK293 , Humanos , Orlistat/farmacología , Estearoil-CoA Desaturasa/efectos de los fármacos , Células VeroRESUMEN
Bone and fat cells have an antagonistic relationship. Adipocytes exert a toxic effect on bone cells in vitro through the secretion of fatty acids, which are synthesized by fatty acid synthase (FAS). Inhibition of FAS in vitro rescues osteoblasts from fat-induced toxicity and cell death. In this study, we hypothesized that FAS inhibition would mitigate the loss of bone mass in ovariectomized (OVX) mice. We treated OVX C57BL/6 mice with cerulenin (a known inhibitor of FAS) for 6â¯weeks and compared their bone phenotype with vehicle-treated controls. Cerulenin-treated mice exhibited a significant decrease in body weight, triglycerides, leptin, and marrow and subcutaneous fat without changes in serum glucose or calciotropic hormones. These effects were associated with attenuation of bone loss and normalization of the bone phenotype in the cerulenin-treated OVX group compared to the vehicle-treated OVX group. Our results demonstrate that inhibition of FAS enhances bone formation, induces uncoupling between osteoblasts and osteoclasts, and favors mineralization, thus providing evidence that inhibition of FAS could constitute a new anabolic therapy for osteoporosis.
Asunto(s)
Resorción Ósea/enzimología , Resorción Ósea/patología , Inhibidores Enzimáticos/farmacología , Ácido Graso Sintasas/antagonistas & inhibidores , Ovariectomía , Adiposidad/efectos de los fármacos , Animales , Biomarcadores/sangre , Peso Corporal/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Remodelación Ósea/efectos de los fármacos , Resorción Ósea/sangre , Resorción Ósea/complicaciones , Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Cerulenina/farmacología , Ácido Graso Sintasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoclastos/patología , Osteogénesis/efectos de los fármacos , Osteoporosis/complicaciones , Fenotipo , Células RAW 264.7 , Factores de Transcripción/metabolismoRESUMEN
We have recently developed Corynebacterium glutamicum strains that produce free fatty acids in culture supernatant due to enhanced fatty acid biosynthesis. Of these producing strains, the basic producer PAS-15 has a defect in the gene for a fatty acid biosynthesis repressor protein, and the advanced producer PCC-6 has two additional mutations to augment the production by strain PAS-15. The aim of the present study was to obtain novel genetic traits for improving fatty acid production by these producers. A new mutant with increased production derived from strain PAS-15 had a missense mutation in the accD3 gene (mutation accD3A433T), which is involved in the biosynthesis of mycolic acids that are cell envelope lipids of C. glutamicum, as the causal mutation. Mutation accD3A433T was verified to reduce the AccD3 enzymatic activity and increase fatty acid production in strain PAS-15 by 1.8-fold. Deletion of the accD3 gene in strain PAS-15, which was motivated by the characteristic of mutation accD3A433T, increased fatty acid production by 3.2-fold. Susceptibility of strain PAS-15 to vancomycin was significantly increased by accD3 gene deletion and by mutation accD3A433T to the intermediate level, suggesting that the cell envelope permeability barrier by mycolic acids is weakened by this engineering. Furthermore, mutation accD3A433T also increased fatty acid production in strain PCC-6 by 1.3-fold. These increased production levels were suggested to be involved not only in the redirection of carbon flux from mycolic acid biosynthesis to fatty acid production but also in the permeability of the cell envelope.
Asunto(s)
Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Ácidos Micólicos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cerulenina/farmacología , Corynebacterium glutamicum/efectos de los fármacos , Inhibidores de la Síntesis de Ácidos Grasos/farmacología , Ácidos Grasos/metabolismo , Microorganismos Modificados Genéticamente , Mutación , Vancomicina/farmacologíaRESUMEN
Fatty acids are synthesized in the stroma of plant and algal chloroplasts by the fatty acid synthase complex. Newly synthesized fatty acids are then used to generate plastidial lipids that are essential for chloroplast structure and function. Here, we show that inhibition of fatty acid synthesis in the model alga Chlamydomonas reinhardtii activates autophagy, a highly conserved catabolic process by which cells degrade intracellular material under adverse conditions to maintain cell homeostasis. Treatment of Chlamydomonas cells with cerulenin, a specific fatty acid synthase inhibitor, stimulated lipidation of the autophagosome protein ATG8 and enhanced autophagic flux. We found that inhibition of fatty acid synthesis decreased monogalactosyldiacylglycerol abundance, increased lutein content, down-regulated photosynthesis, and increased the production of reactive oxygen species. Electron microscopy revealed a high degree of thylakoid membrane stacking in cerulenin-treated cells. Moreover, global transcriptomic analysis of these cells showed an up-regulation of genes encoding chloroplast proteins involved in protein folding and oxidative stress and the induction of major catabolic processes, including autophagy and proteasome pathways. Thus, our results uncovered a link between lipid metabolism, chloroplast integrity, and autophagy through a mechanism that involves the activation of a chloroplast quality control system.
Asunto(s)
Autofagia/efectos de los fármacos , Chlamydomonas reinhardtii/fisiología , Ácido Graso Sintasas/antagonistas & inhibidores , Ácidos Grasos/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Cerulenina/farmacología , Chlamydomonas reinhardtii/efectos de los fármacos , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/ultraestructura , Cloroplastos/efectos de los fármacos , Cloroplastos/fisiología , Cloroplastos/ultraestructura , Retículo Endoplásmico/metabolismo , Inhibidores de la Síntesis de Ácidos Grasos/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Estrés Oxidativo , Fotosíntesis , Proteínas de Plantas/antagonistas & inhibidores , Pliegue de Proteína , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia ArribaRESUMEN
It is fully established that the condensing reaction for the initiation of fatty acid synthesis is essential for viability of many bacteria. In model bacteria such as Escherichia coli, this reaction is exclusively catalyzed by ß-ketoacyl-ACP synthase (KAS) III (encoded by fabH) and the FabH loss results in a fatty acid auxotroph. However, such a notion has been under the challenge of recent findings. In an attempt to resolve the conflicting results, in this study, we examined the physiological role of multiple KASIII enzyme homologues in Shewanella oneidensis, an excellent model for researching type II fatty acid synthesis (FASII) and its regulation. We demonstrated that FabH1 and temperature-responsive FabH2 are primarily responsible for initiating synthesis of straight- and branched-chain fatty acids respectively, whereas FabH3 and OleA are dispensable. Cells lacking all these enzymes as a set are viable but carry severe defects in growth. Further analyses revealed that in the absence of KASIII either of FabB (KASI) and FabF2 (KASII) is able to support growth, suggesting that they could initiate FASII. Strikingly, KASIII enzymes and OleA together confer S. oneidensis cells resistance to cerulenin, a selective inhibitor of FabF and FabB. Along with our previous finding that S. oneidensis FabF1 and FabB are fully equivalent with respect to their physiological impacts, these results imply that physiological function promiscuity of bacterial KAS enzymes could be more extensive than previously expected.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cerulenina/farmacología , Ácidos Grasos/biosíntesis , Homología de Secuencia de Aminoácido , Shewanella/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Simulación por Computador , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Bacterianos , Shewanella/efectos de los fármacos , Shewanella/genética , Shewanella/crecimiento & desarrollo , Temperatura , Transcripción Genética/efectos de los fármacosRESUMEN
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.
Asunto(s)
Acinetobacter baumannii/metabolismo , Inhibidores de la Síntesis de Ácidos Grasos/metabolismo , Ácidos Grasos/biosíntesis , Lípido A/metabolismo , Bioensayo/métodos , Cerulenina/farmacología , Enoil-ACP Reductasa (NADH)/metabolismo , Ácido Graso Sintasas/metabolismo , Ácidos Hidroxámicos/farmacología , Treonina/análogos & derivados , Treonina/farmacologíaRESUMEN
Eukaryotic cells store lipids in cytosolic organelles known as lipid droplets (LDs). Lipid droplet bud from the endoplasmic reticulum (ER), and may be harvested by the vacuole for energy during prolonged periods of starvation. How cells spatially coordinate LD production is poorly understood. Here, we demonstrate that yeast ER-vacuole contact sites (NVJs) physically expand in response to metabolic stress, and serve as sites for LD production. NVJ tether Mdm1 demarcates sites of LD budding, and interacts with fatty acyl-CoA synthases at the NVJ periphery. Artificially expanding the NVJ through over-expressing Mdm1 is sufficient to drive NVJ-associated LD production, whereas ablating the NVJ induces defects in fatty acid-to-triglyceride production. Collectively, our data suggest a tight metabolic link between nutritional stress and LD biogenesis that is spatially coordinated at ER-vacuole contact sites.
Asunto(s)
Retículo Endoplásmico/metabolismo , Regulación Fúngica de la Expresión Génica , Gotas Lipídicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico , Vacuolas/metabolismo , Ácido Acético/metabolismo , Ácido Acético/farmacología , Cerulenina/farmacología , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , Medios de Cultivo/química , Medios de Cultivo/farmacología , Citosol/efectos de los fármacos , Citosol/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/ultraestructura , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Ácidos Grasos/biosíntesis , Glucosa/deficiencia , Glucosa/farmacología , Glicerol/metabolismo , Glicerol/farmacología , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Gotas Lipídicas/efectos de los fármacos , Gotas Lipídicas/ultraestructura , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Plásmidos/química , Plásmidos/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagen de Lapso de Tiempo , Transformación Genética , Triglicéridos/biosíntesis , Vacuolas/efectos de los fármacos , Vacuolas/ultraestructuraRESUMEN
BACKGROUND: Although altered membrane physiology has been discussed within the context of cancer, targeting membrane characteristics by drugs being an attractive therapeutic strategy has received little attention so far. METHODS: Various acetyl-CoA carboxylase 1 (ACC1), and fatty acid synthase (FASN) inhibitors (like Soraphen A and Cerulenin) as well as genetic knockdown approaches were employed to study the effects of disturbed phospholipid composition on membrane properties and its functional impact on cancer progression. By using state-of-the-art methodologies such as LC-MS/MS, optical tweezers measurements of giant plasma membrane vesicles and fluorescence recovery after photobleaching analysis, membrane characteristics were examined. Confocal laser scanning microscopy, proximity ligation assays, immunoblotting as well as migration, invasion and proliferation experiments unravelled the functional relevance of membrane properties in vitro and in vivo. RESULTS: By disturbing the deformability and lateral fluidity of cellular membranes, the dimerisation, localisation and recycling of cancer-relevant transmembrane receptors is compromised. Consequently, impaired activation of growth factor receptor signalling cascades results in abrogated tumour growth and metastasis in different in vitro and in vivo models. CONCLUSIONS: This study highlights the field of membrane properties as a promising druggable cellular target representing an innovative strategy for development of anti-cancer agents.
Asunto(s)
Acetil-CoA Carboxilasa/genética , Inhibidores Enzimáticos/administración & dosificación , Acido Graso Sintasa Tipo I/genética , Lipogénesis/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Acetil-CoA Carboxilasa/antagonistas & inhibidores , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular , Cerulenina/administración & dosificación , Cerulenina/farmacología , Inhibidores Enzimáticos/farmacología , Acido Graso Sintasa Tipo I/antagonistas & inhibidores , Técnicas de Silenciamiento del Gen , Humanos , Macrólidos/administración & dosificación , Macrólidos/farmacología , Fluidez de la Membrana/efectos de los fármacos , Terapia Molecular Dirigida , Invasividad Neoplásica , Neoplasias/metabolismo , Fosfolípidos/análisis , Fotoblanqueo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Fatty acyl-CoA synthetase (Faa) activates fatty acid (FA) by converting the FA into the CoA ester in the cell. In the present study, we characterized a FAA homologue (CaFAA4) from the opportunistic pathogen Candida albicans. Most organisms can not only synthesize long-chain fatty acyl-CoAs (LCFA-CoAs) endogenously using a fatty acid synthase (Fas) activity but also can uptake long-chain fatty acids (LCFAs) from the extracellular environment and convert them into LCFA-CoAs via a vectorial acylation system. The budding yeast Saccharomyces cerevisiae possesses two LCFA-CoA synthetases, ScFaa1p and ScFaa4p. The disruption of ScFAA1 and ScFAA4 leads to synthetic lethality in the presence of a fatty acid synthesis inhibitor-cerulenin. The homologue-CaFAA4-rescued the lethality of an S. cerevisiae Scfaa1-Scfaa4 double mutant in the presence of cerulenin. On the other hand, a C. albicans faa4 mutant was unable to grow in the presence of cerulenin even if LCFAs were provided exogenously. Moreover, a biofilm analysis showed that the metabolic activity of the Cafaa4 mutant was approximately 40% lower than that of the wild-type parent, even though there was no significant difference in cell number or cell morphology between these strains. Notably, the Cafaa4 mutant showed increased susceptibility to micafungin during biofilm formation, a phenotype that presumably can be attributed to the impaired metabolism of the mutant strain. These results indicated that CaFaa4p is the unique C. albicans Faa protein responsible for activating LCFAs and is involved in the metabolism of biofilms.
Asunto(s)
Acilcoenzima A/genética , Candida albicans/genética , Coenzima A Ligasas/genética , Ácidos Grasos/genética , Proteínas de Saccharomyces cerevisiae/genética , Biopelículas/crecimiento & desarrollo , Transporte Biológico/genética , Candida albicans/crecimiento & desarrollo , Cerulenina/farmacología , Ácidos Grasos/metabolismo , Mutación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de AminoácidoRESUMEN
The recent outbreak of infection with Zika virus (ZIKV; Flaviviridae) has attracted attention to this previously neglected mosquito-borne pathogen and the need for efficient therapies. Since flavivirus replication is generally known to be dependent on fatty acid biosynthesis, two inhibitors of this pathway, 5-(tetradecyloxyl)-2-furoic acid (TOFA) and cerulenin, were tested for their potentiality to inhibit virus replication. At concentrations previously shown to inhibit the replication of other flaviviruses, neither drug had a significant antiviral affect against ZIKV, but reduced the replication of the non-related mosquito-borne Semliki Forest virus (Togaviridae).
Asunto(s)
Antivirales/farmacología , Cerulenina/farmacología , Inhibidores de la Síntesis de Ácidos Grasos/farmacología , Furanos/farmacología , Hipolipemiantes/farmacología , Virus de los Bosques Semliki/efectos de los fármacos , Virus Zika/efectos de los fármacos , Células A549/virología , Relación Dosis-Respuesta a Droga , Humanos , Replicación Viral/efectos de los fármacosRESUMEN
Omega-3 fatty acids are products of secondary metabolism, essential for growth and important for human health. Although there are numerous reports of bacterial production of omega-3 fatty acids, less information is available on the biotechnological production of these compounds from bacteria. The production of eicosapentaenoic acid (EPA, 20:5ω3) by a new species of marine bacteria Shewanella electrodiphila MAR441T was investigated under different fermentation conditions. This strain produced a high percentage (up to 26%) of total fatty acids and high yields (mg / g of biomass) of EPA at or below the optimal growth temperature. At higher growth temperatures these values decreased greatly. The amount of EPA produced was affected by the carbon source, which also influenced fatty acid composition. This strain required Na+ for growth and EPA synthesis and cells harvested at late exponential or early stationary phase had a higher EPA content. Both the highest amounts (20 mg g-1) and highest percent EPA content (18%) occurred with growth on L-proline and (NH4)2SO4. The addition of cerulenin further enhanced EPA production to 30 mg g-1. Chemical mutagenesis using NTG allowed the isolation of mutants with improved levels of EPA content (from 9.7 to 15.8 mg g-1) when grown at 15°C. Thus, the yields of EPA could be substantially enhanced without the need for recombinant DNA technology, often a commercial requirement for food supplement manufacture.
Asunto(s)
Organismos Acuáticos/metabolismo , Ácido Eicosapentaenoico/biosíntesis , Océanos y Mares , Shewanella/metabolismo , Organismos Acuáticos/efectos de los fármacos , Organismos Acuáticos/crecimiento & desarrollo , Biomasa , Carbono/farmacología , Cerulenina/farmacología , Mutación/genética , Nitrógeno/farmacología , Fosfolípidos/metabolismo , Shewanella/efectos de los fármacos , Shewanella/crecimiento & desarrollo , Factores de TiempoRESUMEN
BACKGROUND: Oleaginous yeasts are fast emerging as a possible feedstock for biodiesel production. Yarrowia lipolytica, a model oleaginous yeast is known to utilize a variety of hydrophobic substrates for lipid accumulation including waste cooking oil (WCO). Approaches to increase lipid content in this yeast include metabolic engineering which requires manipulation of multiple genes in the lipid biosynthesis pathway. A classical and cost-effective approach, namely, random chemical mutagenesis on the yeast can lead to increased production of biodiesel as is explored here. RESULTS: In this study, chemical mutagenesis using the alkylating agent, N- methyl-N'-nitro-N-nitrosoguanidine (MNNG) as well as an additional treatment with cerulenin, a fatty acid synthase inhibitor generated 800 mutants of Y. lipolytica NCIM 3589 (761 MNNG treated and 39 MNNG + cerulenin treated). A three-stage screening using Sudan Black B plate technique, Nile red fluorimetry and total lipid extraction using solvent was performed, which enabled selection of ten high lipid yielding mutants. Time course studies of all the ten mutants were further undertaken in terms of biomass, lipid yield and lipid content to select three stable mutants (YlB6, YlC7 and YlE1) capable of growing and accumulating lipid on WCO, with lipid contents of 55, 60 and 67% as compared to 45% for the wild type. The mutants demonstrated increased volumetric lipid productivities (0.062, 0.044 and 0.041 g L-1 h-1) as compared to the wild type (0.033 g L-1 h-1). The fatty acid profile of the three mutants consisted of a high content of C16 and C18 saturated and monounsaturated fatty acids and was found to be suitable for biodiesel production. The fuel properties, namely, density, kinematic viscosity, total acid number, iodine value of the three mutants were evaluated and found to lie within the limits specified by internationally accepted standards. Additionally, it was noted that the mutants demonstrated better cetane numbers and higher heating values than the wild type strain. CONCLUSION: The chemical mutagenesis strategy adopted in this study resulted in the successful isolation of three stable high SCO yielding mutants. The mutants, namely, YlB6, YlC7 and YlE1 exhibited a 1.22, 1.33 and 1.49-fold increase in lipid contents when grown on 100 g L-1 waste cooking oil than the parental yeast strain. The fatty acid methyl ester (FAME) profiles of all the three mutants was determined to be suitable for biodiesel suggesting their potential applicability while simultaneously addressing the management of waste cooking oil.
Asunto(s)
Biocombustibles/análisis , Grasas Insaturadas en la Dieta/metabolismo , Mutación , Yarrowia/genética , Yarrowia/metabolismo , Biomasa , Cerulenina/farmacología , Culinaria , Ácidos Grasos/metabolismo , Lípidos/análisis , Lípidos/biosíntesis , Metilnitronitrosoguanidina/farmacología , Mutagénesis , Solventes/metabolismo , Yarrowia/efectos de los fármacos , Yarrowia/crecimiento & desarrolloRESUMEN
The type II fatty acid synthesis (FASII) pathway is essential for bacterial lipid biosynthesis and continues to be a promising target for novel antibacterial compounds. Recently, it has been demonstrated that Chlamydia is capable of FASII and this pathway is indispensable for Chlamydia growth. Previously, a high-content screen with Chlamydia trachomatis-infected cells was performed, and acylated sulfonamides were identified to be potent growth inhibitors of the bacteria. C. trachomatis strains resistant to acylated sulfonamides were isolated by serial passage of a wild-type strain in the presence of low compound concentrations. Results from whole-genome sequencing of 10 isolates from two independent drug-resistant populations revealed that mutations that accumulated in fabF were predominant. Studies of the interaction between the FabF protein and small molecules showed that acylated sulfonamides directly bind to recombinant FabF in vitro and treatment of C. trachomatis-infected HeLa cells with the compounds leads to a decrease in the synthesis of Chlamydia fatty acids. This work demonstrates the importance of FASII for Chlamydia development and may lead to the development of new antimicrobials.