ABSTRACT
Background and Objectives: Aberrant upregulation of fatty acid synthase (FASN), catalyzing de novo synthesis of fatty acids, occurs in various tumor types, including human hepatocellular carcinoma (HCC). Although FASN oncogenic activity seems to reside in its pro-lipogenic function, cumulating evidence suggests that FASN's tumor-supporting role might also be metabolic-independent. Materials and Methods: In the present study, we show that FASN inactivation by specific small interfering RNA (siRNA) promoted the downregulation of the S-phase kinase associated-protein kinase 2 (SKP2) and the consequent induction of p27KIP1 in HCC cell lines. Results: Expression levels of FASN and SKP2 directly correlated in human HCC specimens and predicted a dismal outcome. In addition, forced overexpression of SKP2 rendered HCC cells resistant to the treatment with the FASN inhibitor C75. Furthermore, FASN deletion was paralleled by SKP2 downregulation and p27KIP1 induction in the AKT-driven HCC preclinical mouse model. Moreover, forced overexpression of an SKP2 dominant negative form or a p27KIP1 non-phosphorylatable (p27KIP1-T187A) construct completely abolished AKT-dependent hepatocarcinogenesis in vitro and in vivo. Conclusions: In conclusion, the present data indicate that SKP2 is a critical downstream effector of FASN and AKT-dependent hepatocarcinogenesis in liver cancer, envisaging the possibility of effectively targeting FASN-positive liver tumors with SKP2 inhibitors or p27KIP1 activators.
Subject(s)
Carcinoma, Hepatocellular , Cyclin-Dependent Kinase Inhibitor p27 , Liver Neoplasms , S-Phase Kinase-Associated Proteins , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , S-Phase Kinase-Associated Proteins/metabolism , S-Phase Kinase-Associated Proteins/genetics , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Humans , Animals , Mice , Cell Line, Tumor , Fatty Acid Synthases/metabolism , Fatty Acid Synthase, Type I/metabolism , Fatty Acid Synthase, Type I/genetics , Down-Regulation , MaleABSTRACT
Fatty acid synthase (FASN) is a critical enzyme essential for the production of fats in the body. The abnormal expression of FASN is associated with different types of malignancies, including ovarian cancer. FASN plays a crucial role in cell growth and survival as a metabolic oncogene, although the specific processes that cause its dysregulation are still unknown. FASN interacts with signaling pathways linked to the progression of cancer. Pharmacologically inhibiting or inactivating the FASN gene has shown potential in causing the death of cancer cells, offering a possible treatment approach. This review examines the function of FASN in ovarian cancer, namely its level of expression, influence on the advancement of the disease, and its potential as a target for therapeutic interventions.
Subject(s)
Fatty Acid Synthases , Ovarian Neoplasms , Humans , Ovarian Neoplasms/pathology , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Ovarian Neoplasms/enzymology , Female , Fatty Acid Synthases/metabolism , Fatty Acid Synthases/antagonists & inhibitors , Neoplasm Metastasis , Signal Transduction , Animals , Molecular Targeted Therapy , Fatty Acid Synthase, Type IABSTRACT
The small RNA (sRNA) RydC strongly activates cfa, which encodes the cyclopropane fatty acid synthase. Previous work demonstrated that RydC activation of cfa increases the conversion of unsaturated fatty acids to cyclopropanated fatty acids in membrane lipids and changes the biophysical properties of membranes, making cells more resistant to acid stress. The regulators that control RydC synthesis had not previously been identified. In this study, we identify a GntR-family transcription factor, YieP, that represses rydC transcription. YieP positively autoregulates its own transcription and indirectly regulates cfa through RydC. We further identify additional sRNA regulatory inputs that contribute to the control of RydC and cfa. The translation of yieP is repressed by the Fnr-dependent sRNA, FnrS, making FnrS an indirect activator of rydC and cfa. Conversely, RydC activity on cfa is antagonized by the OmpR-dependent sRNA OmrB. Altogether, this work illuminates a complex regulatory network involving transcriptional and post-transcriptional inputs that link the control of membrane biophysical properties to multiple environmental signals. IMPORTANCE: Bacteria experience many environmental stresses that challenge their membrane integrity. To withstand these challenges, bacteria sense what stress is occurring and mount a response that protects membranes. Previous work documented the important roles of small RNA (sRNA) regulators in membrane stress responses. One sRNA, RydC, helps cells cope with membrane-disrupting stresses by promoting changes in the types of lipids incorporated into membranes. In this study, we identified a regulator, YieP, that controls when RydC is produced and additional sRNA regulators that modulate YieP levels and RydC activity. These findings illuminate a complex regulatory network that helps bacteria sense and respond to membrane stress.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Gene Expression Regulation, Bacterial , RNA, Bacterial , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/genetics , RNA, Bacterial/metabolism , RNA, Bacterial/genetics , Transcription, Genetic , RNA, Small Untranslated/genetics , RNA, Small Untranslated/metabolism , Fatty Acid Synthases/metabolism , Fatty Acid Synthases/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Cyclopropanes , Fatty Acids , MethyltransferasesABSTRACT
Under nitrogen deficient conditions, the Aurantiochytrium limacinum strain BL10 greatly increases the production of docosahexaenoic acid (DHA) and n-6 docosapentaenoic acid. Researchers have yet to elucidate the mechanism by which BL10 promotes the activity of polyunsaturated fatty acid synthase (Pfa), which plays a key role in the synthesis of polyunsaturated fatty acid (PUFA). Analysis in the current study revealed that in nitrogen-depleted environments, BL10 boosts the transcription and synthesis of proteins by facilitating the expression of pfa genes via transcriptional regulation. It was also determined that BL10 adjusts the lengths of the 5'- and 3'-untranslated regions (suggesting post-transcriptional regulation) and modifies the ratio of two Pfa1 isoforms to favor PUFA production via post-translational regulation (ubiquitination). These findings clarify the exceptional DHA production of BL10 and provide additional insights into the regulatory mechanisms of PUFA biosynthesis in Aurantiochytrium.
Subject(s)
Fatty Acid Synthases , Fatty Acids, Unsaturated , Nitrogen , Stramenopiles , Nitrogen/metabolism , Fatty Acid Synthases/genetics , Fatty Acid Synthases/metabolism , Fatty Acids, Unsaturated/biosynthesis , Fatty Acids, Unsaturated/metabolism , Stramenopiles/genetics , Stramenopiles/enzymology , Protein Processing, Post-Translational , Transcription, Genetic , Docosahexaenoic Acids/biosynthesis , Docosahexaenoic Acids/metabolismABSTRACT
This research focuses on the target deconvolution of the natural compound myrianthic acid, a triterpenoid characterized by an ursane skeleton isolated from the roots of Myrianthus arboreus and from Oenothera maritima Nutt. (Onagraceae), using MS-based chemical proteomic techniques. Application of drug affinity responsive target stability (DARTS) and targeted-limited proteolysis coupled to mass spectrometry (t-LiP-MS) led to the identification of the enzyme fatty acid synthase (FAS) as an interesting macromolecular counterpart of myrianthic acid. This result, confirmed by comparison with the natural ursolic acid, was thoroughly investigated and validated in silico by molecular docking, which gave a precise picture of the interactions in the MA/FAS complex. Moreover, biological assays showcased the inhibitory activity of myrianthic acid against the FAS enzyme, most likely related to its antiproliferative activity towards tumor cells. Given the significance of FAS in specific pathologies, especially cancer, the myrianthic acid structural moieties could serve as a promising reference point to start the potential development of innovative approaches in therapy.
Subject(s)
Molecular Docking Simulation , Proteomics , Humans , Proteomics/methods , Fatty Acid Synthases/metabolism , Fatty Acid Synthases/chemistry , Fatty Acid Synthases/antagonists & inhibitors , Triterpenes/pharmacology , Triterpenes/chemistry , Triterpenes/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Mass Spectrometry , Cell Line, Tumor , Cell Proliferation/drug effects , Terpenes/chemistry , Terpenes/pharmacology , Terpenes/metabolismABSTRACT
Visual proteomics attempts to build atlases of the molecular content of cells but the automated annotation of cryo electron tomograms remains challenging. Template matching (TM) and methods based on machine learning detect structural signatures of macromolecules. However, their applicability remains limited in terms of both the abundance and size of the molecular targets. Here we show that the performance of TM is greatly improved by using template-specific search parameter optimization and by including higher-resolution information. We establish a TM pipeline with systematically tuned parameters for the automated, objective and comprehensive identification of structures with confidence 10 to 100-fold above the noise level. We demonstrate high-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, fatty acid synthases, lipid membranes and microtubules, and individual subunits inside crowded eukaryotic cells. We provide software tools for the generic implementation of our method that is broadly applicable towards realizing visual proteomics.
Subject(s)
Cryoelectron Microscopy , Electron Microscope Tomography , Proteasome Endopeptidase Complex , Proteomics , Ribosomes , Software , Electron Microscope Tomography/methods , Cryoelectron Microscopy/methods , Ribosomes/ultrastructure , Ribosomes/metabolism , Proteasome Endopeptidase Complex/ultrastructure , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/chemistry , Humans , Proteomics/methods , Nuclear Pore/ultrastructure , Nuclear Pore/metabolism , Microtubules/ultrastructure , Microtubules/metabolism , Fatty Acid Synthases/metabolism , Machine Learning , Imaging, Three-Dimensional/methods , Algorithms , Image Processing, Computer-Assisted/methodsABSTRACT
Desmodium caudatum extracts (DCE) were investigated for their potential therapeutic effects on diabetic nephropathy (DN). In our study, the high-fat diet (HFD) / streptozotocin (STZ)-induced DN model in C57BL/6 mice was treated with 100 mg/kg, 200 mg/kg DCE. The results showed that DCE decreased biochemical parameters and proteinuria levels. The kidney sections staining indicated that DCE treatment recovered glomerular atrophy and alleviated lipid droplets in the glomerular. Additionally, DCE inhibited lipid and glycogen accumulation down-regulated the expression of sterol regulatory element-binding protein 1 (SREBP1) and fatty acid synthase (FAS) proteins. DCE also reduced collagenous fibrous tissue and the expression of transforming growth factor-ß1 (TGF-ß1) and alpha-smooth muscle actin (α-SMA) through Masson's trichrome staining and immunohistochemical analysis. We found that DCE alleviated hydroxyproline content, and epithelial-mesenchymal transition (EMT). Besides, the results shown that DCE enhanced the antioxidant enzymes to mitigate fibrosis by reducing oxidative stress. In conclusion, our study provided evidence of the protective effect of DCE which down-regulated hyperglycemia, hyperlipidemia and inhibition of TGF-ß1 and EMT pathway but elevated antioxidant, suggesting its therapeutic implication for DN.
Subject(s)
Diabetic Nephropathies , Diet, High-Fat , Mice, Inbred C57BL , Oxidative Stress , Plant Extracts , Sterol Regulatory Element Binding Protein 1 , Transforming Growth Factor beta1 , Animals , Diabetic Nephropathies/drug therapy , Diabetic Nephropathies/metabolism , Plant Extracts/pharmacology , Transforming Growth Factor beta1/metabolism , Male , Sterol Regulatory Element Binding Protein 1/metabolism , Mice , Oxidative Stress/drug effects , Diet, High-Fat/adverse effects , Diabetes Mellitus, Experimental/drug therapy , Antioxidants/pharmacology , Epithelial-Mesenchymal Transition/drug effects , Fabaceae/chemistry , Kidney/drug effects , Kidney/metabolism , Kidney/pathology , Actins/metabolism , Fatty Acid Synthases/metabolism , FibrosisABSTRACT
Our previous studies found that Sea Buckthorn polyphenols (SBP) extract inhibits fatty acid synthase (FAS) in vitro. Thus, we continued to explore possible effects and underlying mechanisms of SBP on complicated metabolic disorders in long-term high-fat-diet (HFD)-fed mice. To reveal that, an integrated approach was developed in this study. Targeted quantitative lipidomics with a total of 904 unique lipids mapping contributes to profiling the comprehensive features of disarranged hepatic lipid homeostasis and discovering a set of newfound lipid-based biomarkers to predict the occurrence and indicate the progression of metabolic disorders beyond current indicators. On the other hand, technologies of intermolecular interactions characterization, especially surface plasmon resonance (SPR) assay, contribute to recognizing targeted bioactive constituents present in SBP. Our findings highlight hepatic lipid homeostasis maintenance and constituent-FAS enzyme interactions, to provide new insights that SBP as a functional food alleviates HFD-induced metabolic disorders in mice via reprograming hepatic lipid homeostasis caused by targeting FAS, owing to four polyphenols directly interacting with FAS and cinaroside binding to FAS with good affinity.
Subject(s)
Hippophae , Metabolic Diseases , Mice , Animals , Polyphenols/metabolism , Liver/metabolism , Diet, High-Fat/adverse effects , Fatty Acid Synthases/genetics , Fatty Acid Synthases/metabolism , Lipids/pharmacology , Metabolic Diseases/metabolism , Homeostasis , Mice, Inbred C57BL , Lipid MetabolismABSTRACT
Vascular smooth muscle cells (VSMCs), in their contractile and differentiated state, are fundamental for maintaining vascular function. Upon exposure to cholesterol (CHO), VSMCs undergo dedifferentiation, adopting characteristics of foam cells-lipid-laden, macrophage-like cells pivotal in atherosclerotic plaque formation. CHO uptake by VSMCs leads to two primary pathways: ABCA1-mediated efflux or storage in lipid droplets as cholesterol esters (CEs). CE formation, involving the condensation of free CHO and fatty acids, is catalyzed by sterol O-acyltransferase 1 (SOAT1). The necessary fatty acids are synthesized by the lipogenic enzyme fatty acid synthase (FASN), which we found to be upregulated in atherosclerotic human coronary arteries. This observation led us to hypothesize that FASN-mediated fatty acid biosynthesis is crucial in the transformation of VSMCs into foam cells. Our study reveals that CHO treatment upregulates FASN in human aortic SMCs, concurrent with increased expression of CD68 and upregulation of KLF4, markers associated with the foam cell transition. Crucially, downregulation of FASN inhibits the CHO-induced upregulation of CD68 and KLF4 in VSMCs. Additionally, FASN-deficient VSMCs exhibit hindered lipid accumulation and an impaired transition to the foam cell phenotype following CHO exposure, while the addition of the fatty acid palmitate, the main FASN product, exacerbates this transition. FASN-deficient cells also show decreased SOAT1 expression and elevated ABCA1. Notably, similar effects are observed in KLF4-deficient cells. Our findings demonstrate that FASN plays an essential role in the CHO-induced upregulation of KLF4 and the VSMC to foam cell transition and suggest that targeting FASN could be a novel therapeutic strategy to regulate VSMC phenotypic modulation.
Subject(s)
Foam Cells , Kruppel-Like Factor 4 , Muscle, Smooth, Vascular , Animals , Humans , Atherosclerosis/pathology , Atherosclerosis/metabolism , Cholesterol/metabolism , Fatty Acid Synthases/metabolism , Fatty Acid Synthases/genetics , Fatty Acids/metabolism , Foam Cells/metabolism , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/metabolismABSTRACT
Haloacetaldehyde disinfection by-products (HAL-DBPs) are among the top three unregulated DBPs found in drinking water. The cytotoxicity and genotoxicity of HALs are much higher than that of the regulated trihalomethanes and haloacetic acids. Previous studies have mainly focused on the toxic effects of single HAL, with few examining the toxic effects of mixed exposures to HALs. The study aimed to observe the effects of mixed exposures of 1â¼1000X the realistic level of HALs on the hepatotoxicity and lipid metabolism of C57BL/6J mice, based on the component and concentration of HALs detected in the finished water of Shanghai. Exposure to realistic levels of HALs led to a significant increase in phosphorated acetyl CoA carboxylase 1 (p-ACC1) in the hepatic de novo lipogenesis (DNL) pathway. Additionally, exposure to 100X realistic levels of HALs resulted in significant alterations to key enzymes of DNL pathway, including ACC1, fatty acid synthase (FAS), and diacylglycerol acyltransferase 2 (DGAT2), as well as key proteins of lipid disposal such as carnitine palmitoyltransferase 1 (CPT-1) and peroxisome proliferator activated receptor α (PPARα). Exposure to 1000X realistic levels of HALs significantly increased hepatic and serum triglyceride levels, as well as total cholesterol, low-density lipoprotein, alanine aminotransferase, aspartate transaminase, alkaline phosphatase, and lactate dehydrogenase levels, significantly decreased high-density lipoprotein. Meanwhile, histopathological analysis demonstrated that HALs exacerbated tissue vacuolization and inflammatory cell infiltration in mice livers, which showed the typical phenotypes of non-alcoholic fatty liver disease (NAFLD). These results suggested that the HALs mixture is a critical risk factor for NAFLD and is significantly highly toxic to C57BL/6J mice.
Subject(s)
Acetaldehyde , Lipid Metabolism , Liver , Mice, Inbred C57BL , Animals , Mice , Liver/drug effects , Liver/metabolism , Acetaldehyde/toxicity , Acetaldehyde/analogs & derivatives , Lipid Metabolism/drug effects , Male , Disinfection , Water Pollutants, Chemical/toxicity , Acetyl-CoA Carboxylase/metabolism , PPAR alpha/metabolism , Diacylglycerol O-Acyltransferase/metabolism , Diacylglycerol O-Acyltransferase/genetics , Carnitine O-Palmitoyltransferase/metabolism , Carnitine O-Palmitoyltransferase/genetics , Lipogenesis/drug effects , Disinfectants/toxicity , Fatty Acid Synthases/metabolism , China , Drinking Water/chemistryABSTRACT
Fatty acid de novo biosynthesis in plant plastids is initiated from acetyl-CoA and catalyzed by a series of enzymes, which is required for the vegetative growth, reproductive growth, seed development, stress response, chloroplast development and other biological processes. In this review, we systematically summarized the fatty acid de novo biosynthesis-related genes/enzymes and their critical roles in various plant developmental processes. Based on bioinformatic analysis, we identified fatty acid synthase encoding genes and predicted their potential functions in maize growth and development, especially in anther and pollen development. Finally, we highlighted the potential applications of these fatty acid synthases in male-sterility hybrid breeding, seed oil content improvement, herbicide and abiotic stress resistance, which provides new insights into future molecular crop breeding.
Subject(s)
Fatty Acids , Plastids , Fatty Acids/biosynthesis , Fatty Acids/metabolism , Plastids/metabolism , Plastids/enzymology , Plant Proteins/metabolism , Plant Proteins/genetics , Reproduction , Pollen/genetics , Pollen/metabolism , Pollen/growth & development , Pollen/enzymology , Fatty Acid Synthases/metabolism , Fatty Acid Synthases/genetics , Zea mays/genetics , Zea mays/metabolism , Zea mays/enzymology , Plants/metabolism , Plants/genetics , Plants/enzymologyABSTRACT
Cyclopropane fatty acid synthases (CFAS) are a class of S-adenosylmethionine (SAM) dependent methyltransferase enzymes able to catalyse the cyclopropanation of unsaturated phospholipids. Since CFAS enzymes employ SAM as a methylene source to cyclopropanate alkene substrates, they have the potential to be mild and more sustainable biocatalysts for cyclopropanation transformations than current carbene-based approaches. This work describes the characterisation of E.â coli CFAS (ecCFAS) and its exploitation in the stereoselective biocatalytic synthesis of cyclopropyl lipids. ecCFAS was found to convert phosphatidylglycerol (PG) to methyl dihydrosterculate 1 with up to 58 % conversion and 73 % ee and the absolute configuration (9S,10R) was established. Substrate tolerance of ecCFAS was found to be correlated with the electronic properties of phospholipid headgroups and for the first time ecCFAS was found to catalyse cyclopropanation of both phospholipid chains to form dicyclopropanated products. In addition, mutagenesis and in silico experiments were carried out to identify the enzyme residues with key roles in catalysis and to provide structural insights into the lipid substrate preference of ecCFAS. Finally, the biocatalytic synthesis of methyl dihydrosterculate 1 and its deuterated analogue was also accomplished combining recombinant ecCFAS with the SAM regenerating AtHMT enzyme in the presence of CH3I and CD3I respectively.
Subject(s)
Biocatalysis , Cyclopropanes , Escherichia coli , Cyclopropanes/chemistry , Cyclopropanes/metabolism , Escherichia coli/enzymology , Escherichia coli/metabolism , Stereoisomerism , Methyltransferases/metabolism , Methyltransferases/chemistry , Fatty Acid Synthases/metabolism , Fatty Acid Synthases/chemistry , Methane/analogs & derivatives , Methane/chemistry , Methane/metabolism , Fatty AcidsABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Prinsepia utilis Royle, native to the Himalayan region, has a long history of use in traditional medicine for its heat-clearing, detoxification, anti-inflammatory, and analgesic properties. Oils extracted from P. utilis seeds are also used in cooking and cosmetics. With the increasing market demand, this extraction process generates substantial industrial biowastes. Recent studies have found many health benefits with using aqueous extracts of these biowastes, which are also rich in polysaccharides. However, there is limited research related to the reparative effects of the water extracts of P. utilis oil cakes (WEPUOC) on disruptions of the skin barrier function. AIM OF THE STUDY: This study aimed to evaluate the reparative efficacy of WEPUOC in both acute and chronic epidermal permeability barrier disruptions. Furthermore, the study sought to explore the underlying mechanisms involved in repairing the epidermal permeability barrier. MATERIALS AND METHODS: Mouse models with induced epidermal disruptions, employing tape-stripping (TS) and acetone wiping (AC) methods, were used. The subsequent application of WEPUOC (100 mg/mL) was evaluated through various assessments, with a focus on the upregulation of mRNA and protein expression of Corneocyte Envelope (CE) related proteins, lipid synthase-associated proteins, and tight junction proteins. RESULTS: The polysaccharide was the major phytochemicals of WEPUOC and its content was determined as 32.2% by the anthranone-sulfuric acid colorimetric method. WEPUOC significantly reduced transepidermal water loss (TEWL) and improved the damaged epidermal barrier in the model group. Mechanistically, these effects were associated with heightened expression levels of key proteins such as FLG (filaggrin), INV (involucrin), LOR (loricrin), SPT, FASN, HMGCR, Claudins-1, Claudins-5, and ZO-1. CONCLUSIONS: WEPUOC, obtained from the oil cakes of P. utilis, is rich in polysaccharides and exhibits pronounced efficacy in repairing disrupted epidermal barriers through increased expression of critical proteins involved in barrier integrity. Our findings underscore the potential of P. utilis wastes in developing natural cosmetic prototypes for the treatment of diseases characterized by damaged skin barriers, including atopic dermatitis and psoriasis.
Subject(s)
Epidermis , Fatty Acid Synthases , Plant Extracts , Tight Junction Proteins , Up-Regulation , Animals , Male , Mice , Epidermis/drug effects , Epidermis/metabolism , Fatty Acid Synthases/metabolism , Fatty Acid Synthases/genetics , Permeability/drug effects , Plant Extracts/pharmacology , Plant Extracts/chemistry , Plant Oils/pharmacology , Plant Oils/chemistry , Tight Junction Proteins/metabolism , Up-Regulation/drug effects , Water/chemistryABSTRACT
Marine mangrove vegetation has been traditionally employed in folk medicine to address various ailments. Notably, Rhizophora apiculata Blume has exhibited noteworthy properties, demonstrating efficacy against cancer, viruses, and bacteria. The enzyme fatty acid synthase (FAS) plays a pivotal role in de novo fatty acid synthesis, making it a promising target for combating colon cancer. Our study focused on evaluating the FAS inhibitory effects of both the crude extract and three isolated compounds from R. apiculata. The n-butanol fraction of R. apiculata extract (BFR) demonstrated a significant inhibition of FAS, with an IC50 value of 93.0 µg/mL. For inhibition via lyoniresinol-3α-O-ß-rhamnopyranoside (LR), the corresponding IC50 value was 20.1 µg/mL (35.5 µM). LR competitively inhibited the FAS reaction with acetyl-CoA, noncompetitively with malonyl-CoA, and in a mixed manner with NADPH. Our results also suggest that both BFR and LR reversibly bind to the KR domain of FAS, hindering the reduction of saturated acyl groups in fatty acid synthesis. Furthermore, BFR and LR displayed time-dependent inhibition for FAS, with kobs values of 0.0045 min-1 and 0.026 min-1, respectively. LR also exhibited time-dependent inhibition on the KR domain, with a kobs value of 0.019 min-1. In human colon cancer cells, LR demonstrated the ability to reduce viability and inhibit intracellular FAS activity. Notably, the effects of LR on human colon cancer cells could be reversed with the end product of FAS-catalyzed chemical reactions, affirming the specificity of LR on FAS. These findings underscore the potential of BFR and LR as potent FAS inhibitors, presenting novel avenues for the treatment of human colon cancer.
Subject(s)
Colonic Neoplasms , Rhizophoraceae , Humans , Polyphenols , Fatty Acid Synthases/metabolism , Fatty AcidsABSTRACT
BACKGROUND: ß-Propeller protein-associated neurodegeneration (BPAN) is a genetic neurodegenerative disease caused by mutations in WDR45. The impairment of autophagy caused by WDR45 deficiency contributes to the pathogenesis of BPAN; however, the pathomechanism of this disease is largely unknown. Lipid dyshomeostasis is involved in neurogenerative diseases, but whether lipid metabolism is affected by Wdr45 deficiency and whether lipid dyshomeostasis contributes to the progression of BPAN are unclear. METHODS: We generated Wdr45 knockout SN4741 cell lines using CRISPRâCas9-mediated genome editing, then lipid droplets (LDs) were stained using BODIPY 493/503. Chaperone-mediated autophagy was determined by RT-qPCR and western blotting. The expression of fatty acid synthase (Fasn) was detected by western blot in the presence or absence of the lysosomal inhibitor NH4Cl and the CMA activator AR7. The interaction between Fasn and HSC70 was analyzed using coimmunoprecipitation (Co-IP) assay. Cell viability was measured by a CCK-8 kit after treatment with the Fasn inhibitor C75 or the CMA activator AR7. RESULTS: Deletion of Wdr45 impaired chaperone-mediated autophagy (CMA), thus leading to lipid droplet (LD) accumulation. Moreover, Fasn can be degraded via CMA, and that defective CMA leads to elevated Fasn, which promotes LD formation. LD accumulation is toxic to cells; however, cell viability was not rescued by Fasn inhibition or CMA activation. Inhibition of Fasn with a low concentration of C75 did not affect cell viability but decreases LD density. CONCLUSIONS: These results suggested that Fasn is essential for cell survival but that excessive Fasn leads to LD accumulation in Wdr45 knockout cells.
Subject(s)
Chaperone-Mediated Autophagy , Neurodegenerative Diseases , Humans , Carrier Proteins/genetics , Carrier Proteins/metabolism , Lipid Droplets/metabolism , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/metabolism , Autophagy/genetics , Fatty Acid Synthases/metabolism , LipidsABSTRACT
Recent efforts in genome mining of ribosomally synthesized and post-translationally modified peptides (RiPPs) have expanded the diversity of post-translational modification chemistries. However, RiPPs are rarely reported as hybrid molecules incorporating biosynthetic machinery from other natural product families. Here we report lipoavitides, a class of RiPP/fatty-acid hybrid lipopeptides that display a unique, putatively membrane-targeting 4-hydroxy-2,4-dimethylpentanoyl (HMP)-modified N terminus. The HMP is formed via condensation of isobutyryl-coenzyme A (isobutyryl-CoA) and methylmalonyl-CoA catalysed by a 3-ketoacyl-(acyl carrier protein) synthase III enzyme, followed by successive tailoring reactions in the fatty acid biosynthetic pathway. The HMP and RiPP substructures are then connected by an acyltransferase exhibiting promiscuous activity towards the fatty acyl and RiPP substrates. Overall, the discovery of lipoavitides contributes a prototype of RiPP/fatty-acid hybrids and provides possible enzymatic tools for lipopeptide bioengineering.
Subject(s)
Ribosomes , Acylation , Ribosomes/metabolism , Fatty Acid Synthases/metabolism , Fatty Acid Synthases/chemistry , Fatty Acid Synthases/genetics , Protein Processing, Post-Translational , Peptides/chemistry , Peptides/metabolism , Lipopeptides/chemistry , Lipopeptides/metabolism , Lipopeptides/biosynthesis , Fatty Acids/chemistry , Fatty Acids/metabolismABSTRACT
Incomplete structural details of Mycobacterium tuberculosis (Mtb) fatty acid synthase-I (FAS-I) at near-atomic resolution have limited our understanding of the shuttling mechanism of its mobile acyl carrier protein (ACP). Here, we have performed atomistic molecular dynamics simulation of Mtb FAS-I with a homology-modeled structure of ACP stalled at dehydratase (DH) and identified key residues that mediate anchoring of the recognition helix of ACP near DH. The observed distance between catalytic residues of ACP and DH agrees with that reported for fungal FAS-I. Further, the conformation of the peripheral linker is found to be crucial in stabilizing ACP near DH. Correlated interdomain motion is observed between DH, enoyl reductase, and malonyl/palmitoyl transferase, consistent with prior experimental reports of fungal and Mtb FAS-I.
Subject(s)
Acyl Carrier Protein , Mycobacterium tuberculosis , Acyl Carrier Protein/chemistry , Acyl Carrier Protein/metabolism , Fatty Acid Synthases/chemistry , Fatty Acid Synthases/metabolism , Molecular Dynamics Simulation , CatalysisABSTRACT
Background and objective: Epithelial ovarian cancer (EOC) is associated with latent onset and poor prognosis, with drug resistance being a main concern in improving the prognosis of these patients. The resistance of cancer cells to most chemotherapeutic agents can be related to autophagy mechanisms. This study aimed to assess the therapeutic effect of MK8722, a small-molecule compound that activates AMP-activated protein kinase (AMPK), on EOC cells and to propose a novel strategy for the treatment of EOC. Purpose: To explore the therapeutic effects of MK8722 on EOC cells, and to elucidate the underlying mechanism. Methods and results: It was found that MK8722 effectively inhibited the malignant biological behaviors of EOC cells. In vitro experiments showed that MK8722 targeted and decreased the lipid metabolic pathway-related fatty acid synthase (FASN) expression levels, causing the accumulation of lipid droplets. In addition, transmission electron microscopy revealed the presence of autophagosome-affected mitochondria. Western blotting confirmed that MK8722 plays a role in activating autophagy upstream (PI3K/AKT/mTOR) and inhibiting autophagy downstream via FASN-dependent reprogramming of lipid metabolism. Plasmid transient transfection demonstrated that MK8722 suppressed late-stage autophagy by blocking autophagosome-lysosome fusion. Immunofluorescence and gene silencing revealed that this effect was achieved by inhibiting the interaction of FASN with the SNARE complexes STX17-SNP29-VAMP8. Furthermore, the antitumor effect of MK8722 was verified using a subcutaneous xenograft mouse model. Conclusion: The findings suggest that using MK8722 may be a new strategy for treating EOC, as it has the potential to be a new autophagy/mitophagy inhibitor. Its target of action, FASN, is a molecular crosstalk between lipid metabolism and autophagy, and exploration of the underlying mechanism of FASN may provide a new research direction.
Subject(s)
Lipid Metabolism , Ovarian Neoplasms , Humans , Female , Mice , Animals , Phosphatidylinositol 3-Kinases/metabolism , Autophagy , Fatty Acid Synthases/metabolism , Fatty Acid Synthases/pharmacology , Carcinoma, Ovarian Epithelial , Fatty Acid Synthase, Type I/metabolismABSTRACT
Animals synthesize simple lipids using a distinct fatty acid synthase (FAS) related to the type I polyketide synthase (PKS) enzymes that produce complex specialized metabolites. The evolutionary origin of the animal FAS and its relationship to the diversity of PKSs remain unclear despite the critical role of lipid synthesis in cellular metabolism. Recently, an animal FAS-like PKS (AFPK) was identified in sacoglossan molluscs. Here, we explore the phylogenetic distribution of AFPKs and other PKS and FAS enzymes across the tree of life. We found AFPKs widely distributed in arthropods and molluscs (>6300 newly described AFPK sequences). The AFPKs form a clade with the animal FAS, providing an evolutionary link bridging the type I PKSs and the animal FAS. We found molluscan AFPK diversification correlated with shell loss, suggesting AFPKs provide a chemical defense. Arthropods have few or no PKSs, but our results indicate AFPKs contributed to their ecological and evolutionary success by facilitating branched hydrocarbon and pheromone biosynthesis. Although animal metabolism is well studied, surprising new metabolic enzyme classes such as AFPKs await discovery.
Subject(s)
Polyketides , Animals , Polyketides/metabolism , Fatty Acids , Lipid Metabolism/genetics , Phylogeny , Polyketide Synthases/genetics , Polyketide Synthases/metabolism , Fatty Acid Synthases/genetics , Fatty Acid Synthases/metabolismABSTRACT
The recent scarcity of fishmeal and other resources means that studies on the intrinsic mechanisms of nutrients in the growth and development of aquatic animals at the molecular level have received widespread attention. The target of rapamycin (TOR) pathway has been reported to receive signals from nutrients and environmental stresses, and regulates cellular anabolism and catabolism to achieve precise regulation of cell growth and physiological activities. In this study, we cloned and characterized the full-length cDNA sequence of the TOR gene of Macrobrachium rosenbergii (MrTOR). MrTOR was expressed in all tissues, with higher expression in heart and muscle tissues. In situ hybridization also indicated that MrTOR was expressed in muscle, mainly around the nucleus. RNA interference decreased the expression levels of MrTOR and downstream protein synthesis-related genes (S6K, eIF4E, and eIF4B) (P < 0.05) and the expression and enzyme activity of the lipid synthesis-related enzyme, fatty acid synthase (FAS), and increased enzyme activity of the lipolysis-related enzyme, lipase (LPS). In addition, amino acid injection significantly increased the transcript levels of MrTOR and downstream related genes (S6K, eIF4E, eIF4B, and FAS), as well as triglyceride and total cholesterol tissue levels and FAS activity. Starvation significantly increased transcript levels and enzyme activities of adenylate-activated protein kinase and LPS and decreased transcript levels and enzyme activities of FAS, as well as transcript levels of MrTOR and its downstream genes (P < 0.05), whereas amino acid injection alleviated the starvation-induced decreases in transcript levels of these genes. These results suggested that arginine and leucine activated the TOR signaling pathway, promoted protein and lipid syntheses, and alleviated the pathway changes induced by starvation.