Synthesis Of Long-chain acyl-CoA Research Articles

Regulatory mechanism of plnC in Lactiplantibacillus plantarum B1 on Plantaricin biosynthesis

The PlnC encoded by plnC is an essential response regulator in Plantaricin biosynthesis well-studied in Lactiplantibacillus plantarum. Since the known effects of plnC are not exhaustive, it is crucial to perfect a precise regulatory mechanism for plnC. Overexpression of plnC was found to transiently reduce bacterial activity during the logarithmic growth phase, however, it significantly increased the expression levels of bacteriocin genes within the pln locus by 1.45 to 2.35 times. After 16 hours of incubation, the overexpression of plnC significantly increased the diameter of the inhibition zone from 10.89 ± 0.36 mm to 12.29 ± 0.33 mm, indicating a notable enhancement in bacteriocin activity (P< 0.05). The proteomic analysis further revealed that plnC affected multiple carbohydrate metabolic pathways, QS and HIF-1 signaling pathways, IMP/UMP biosynthesis, and ribosomal proteins, which resulted in promoting the synthesis of nucleotide precursors and exopolysaccharides, and accelerating ribosome translation rate. And the up-regulated synthesis of long-chain acyl-CoA is an important component that may inhibit PlnD (an inhibitory regulator opposite PlnC) during Plantaricin biosynthesis. These offer a critical basis for forward Plantaricin research and application.

Polycyclic aromatic hydrocarbons stress affects fatty acids profile and LacsA (long-chain acyl-CoA synthetize) gene expression in Dunaliella

ABSTRACT This study was conducted to evaluate the effects of different concentrations of two Polycyclic Aromatic Hydrocarbons (PAHs) on the profiles of soluble proteins and fatty acids and on LacsA (long-chain acyl-CoA synthesis) gene expression in Dunaliella tertiolecta and Dunaliella salina. The algae were treated with a control and three experimental concentrations (3000, 6000 and 9000 μg l‒1) of phenanthrene and anthracene. In D. salina treated with 6000 and 9000 μg l‒1 of anthracene, expression of LacsA and α-linolenic acid was higher than in the control treatment. Also, at 3000 and 6000 μg l‒1 of anthracene, there was a sharp increase in hexadecenoic acid and docosahexaenoic acid (omega-3). Dunaliella tertiolecta treated with 9000 μg l‒1of anthracene showed a stronger protein expression profile than other samples which was consistent with LacsA gene expression at the same dose of anthracene. In D. tertiolecta, α-linolenic acid and 11-octadecenoic acid increased significantly at 9000 µg l‒1 of anthracene compared to the control, and there were similar trends in proteins, LacsA gene expression and α-linolenic acid and 11-octadecenoic fatty acids content. Phenanthrene and anthracene have different molecular structures, and it seems that they also have different dose-dependent effects on synthesis/accumulation of fatty acids, SDS-PAGE profiles of proteins and LacsA gene expression in D. salina and D. tertiolecta. According to previous reports, the LacsA gene is mediated by α-linolenic acid, hexadecenoic acid and docosahexaenoic acid (omega-3) synthesis. Since PAHs tend to be accumulated in the cell membrane of the microalgae, microalgae produce various proteins and fatty acids to bioremediate these harmful substances. Our results indicated that Dunaliella spp. may be good candidates for biodegradation purposes as well as an adequate model as a biotechnological accumulator of fatty acids under exposure to PAHs.

Analysis of Wheat Wax Regulation Mechanism by Liposome and Transcriptome.

As a barrier for plants to contact with the outside world, epidermal wax plays an important role in resisting biotic and abiotic stresses. In this study, we analyzed the effect of wax content on leaf permeability by measuring the wax loss rate in the leaf. To further clarify the wax composition of the wheat epidermis and its molecular regulation mechanism, we applied untargeted lipidomic and transcriptome analysis on the leaf epidermis wax of Jimai 22 low-wax mutant (waxless) and multi-wax mutant (waxy). Our research showed that the mutant waxy has a slow loss rate, which can maintain higher leaf water content. 31 lipid subclasses and 1,367 lipid molecules were identified. By analyzing the wax differences of the two mutants, we found that the main lipid components of leaf epidermis wax in Jimai 22 were WE (C19-C50), DG (C27-C53), MG (C31-C35), and OAHFA (C31-C52). Carbon chain length analysis showed that, in wheat epidermis wax, WE was dominated by C44 molecules, DG was mainly concentrated in C47, C45, C37, and C31 molecules, C48 played a leading role in OAHFA, and C35 and C31 played a major role in MG. Among them, DG, MG, and OAHFA were detected in wheat leaf wax for the first time, and they were closely related to stress resistance. Compared with the waxy, 6,840 DEGs were detected in the mutant waxless, 3,181 DEGs were upregulated, and 3,659 DEGs were downregulated. The metabolic pattern of main waxy components in the wheat epidermis was constructed according to KEGG metabolic pathway and 46 related genes were screened, including KSC, TER, FAR, WSD1, CER1, MAH1, ALDH7A1, CYP704B1, ACOT1_2_4, CYP86, MGLL, GPAT, ALDH, DPP1, dgkA, plsC, and E2.3.1.158 related genes. The screened wax-related genes were confirmed to be highly reliable by qRT-PCR. In addition, we found TER gene TraesCS6B03G1132900LC in wheat mutant waxless leaves for the first time, which inhibited the synthesis of long-chain acyl-CoA (n+2) by downregulating its expression. These results provide valuable reference information for further study of wheat epidermis wax heredity and molecular regulation.

Discovery of a benzimidazole series as the first highly potent and selective ACSL1 inhibitors

Long-chain acyl-CoA synthetase-1 (ACSL1), an enzyme that catalyzes the synthesis of long-chain acyl-CoA from the corresponding fatty acids, is believed to play essential roles in lipid metabolism. Structure activity relationship studies based on HTS hit compound 1 delivered the benzimidazole series as the first selective and highly potent ACSL1 inhibitors. Representative compound 13 exhibited not only remarkable inhibitory activity against ACSL1 (IC50 = 0.042 μM) but also excellent selectivity for the other ACSL isoforms. In addition, compound 13 demonstrated an in vivo suppression effect against the production of long-chain acyl-CoAs in mouse.

Normal Flux through ATP-Citrate Lyase or Fatty Acid Synthase Is Not Required for Glucose-stimulated Insulin Secretion

It has been proposed that de novo synthesis of long-chain acyl-CoA (LC-CoA) is a signal for glucose-stimulated insulin secretion (GSIS). Key enzymes involved in synthesis of fatty acids from glucose include ATP-citrate lyase (CL) and fatty acid synthase (FAS). An inhibitor of CL, hydroxycitrate (HC), has been reported to inhibit insulin secretion in some laboratories but not in others. Here we show that high concentrations of NaCl created during preparation of HC by standard methods explain the inhibition of GSIS, and that removal of the excess NaCl prevents the effect. To further investigate the role of CL, two small interfering RNA adenoviruses (Ad-siCL2 and Ad-siCL3) were generated. Ad-siCL3 reduced CL mRNA levels by 92 +/- 6% and CL protein levels by 75 +/- 4% but did not affect GSIS in 832/13 cells compared with cells treated with a control adenovirus (Ad-siControl). Similar results were obtained with Ad-siCL2. Ad-siCL3-treated cells also exhibited a 52 +/- 7% reduction in cytosolic oxaloacetate, an 83 +/- 4% reduction in malonyl-CoA levels, and inhibition of [U-(14)C]glucose incorporation into lipid by 43 +/- 4%, all expected metabolic out-comes of CL suppression. Similarly, treatment of 832/13 cells with a recombinant adenovirus specific to FAS (Ad-siFAS) reduced FAS mRNA levels by 81 +/- 2% in 832/13 cells, resulting in a 59 +/- 4% decrease in [U-(14)C]glucose incorporation into lipid, without affecting GSIS. Finally, treatment of primary rat islets with Ad-siCL3 or Ad-siFAS reduced CL and FAS mRNA levels by 65 +/- 4% and 52 +/- 3%, respectively, but had no effect on GSIS relative to Ad-siControl-treated islets. These findings demonstrate that a normal rate of flux of glucose carbons through CL and FAS is not required for GSIS in insulinoma cell lines or rat islets.

On the Export of Fatty Acids from the Chloroplast

The model for export of fatty acids from plastids proposes that the acyl-ACP (acyl carrier protein) product of de novo fatty acid synthesis is hydrolyzed in the stroma by acyl-ACP thioesterases and the free fatty acid (FFA) released is then transferred to the outer envelope of the plastid where it is reactivated to acyl-CoA for utilization in cytosolic glycerolipid synthesis. Experiments were performed to assess whether the delivery of nascent FFA from the stroma for long chain acyl-CoA synthesis (LACS) occurs via simple diffusion or a more complex mechanism. The flux through the in vivo FFA pool was estimated using kinetic labeling experiments with spinach and pea leaves. The maximum half-life for FFA in the export pool was < or =1 s. Isolated pea chloroplasts incubated in the light with [14C]acetate gave a linear accumulation of FFA. When CoASH and ATP were present there was also a linear accumulation of acyl-CoA thioesters (plus derived polar lipids), with no measurable lag phase (<30 s), indicating that the FFA pool supplying LACS rapidly reached steady state. The LACS reaction was also measured independently in the dark after in situ generated FFA had accumulated yielding estimates of LACS substrate-velocity relationships. Based on these experiments the LACS reaction with in situ generated FFA as substrate is only about 3% of the LACS activity required in vivo at the very low concentrations of the FFA export pool calculated from the in vivo experiment. Furthermore, bovine serum albumin rapidly removed in situ generated FFA from chloroplasts, but could not compete effectively for "nascent" FFA substrates of LACS. Together the data suggest a locally channeled pool of exported FFA that is closely linked to LACS.

Fatty acid synthesis in pea root plastids is inhibited by the action of long-chain acyl- coenzyme as on metabolite transporters.

The uptake in vitro of glucose (Glc)-6-phosphate (Glc-6-P) into plastids from the roots of 10- to 14-d-old pea (Pisum sativum L. cv Puget) plants was inhibited by oleoyl-coenzyme A (CoA) concentrations in the low micromolar range (1--2 microM). The IC(50) (the concentration of inhibitor that reduces enzyme activity by 50%) for the inhibition of Glc-6-P uptake was approximately 750 nM; inhibition was reversed by recombinant rapeseed (Brassica napus) acyl-CoA binding protein. In the presence of ATP (3 mM) and CoASH (coenzyme A; 0.3 mM), Glc-6-P uptake was inhibited by 60%, due to long-chain acyl-CoA synthesis, presumably from endogenous sources of fatty acids present in the preparations. Addition of oleoyl-CoA (1 microM) decreased carbon flux from Glc-6-P into the synthesis of starch and through the oxidative pentose phosphate (OPP) pathway by up to 73% and 40%, respectively. The incorporation of carbon from Glc-6-P into fatty acids was not detected under any conditions. Oleoyl-CoA inhibited the incorporation of acetate into fatty acids by 67%, a decrease similar to that when ATP was excluded from incubations. The oleoyl-CoA-dependent inhibition of fatty acid synthesis was attributable to a direct inhibition of the adenine nucleotide translocator by oleoyl-CoA, which indirectly reduced fatty acid synthesis by ATP deprivation. The Glc-6-P-dependent stimulation of acetate incorporation into fatty acids was reversed by the addition of oleoyl-CoA.

Interaction of acyl-CoA binding protein (ACBP) on processes for which acyl-CoA is a substrate, product or inhibitor.

It is shown that acyl-CoA binding protein (ACBP), in contrast with fatty acid binding protein (FABP), stimulates the synthesis of long-chain acyl-CoA esters by mitochondria. ACBP effectively opposes the product feedback inhibition of the long-chain acyl-CoA synthetase by sequestration of the synthesized acyl-CoA esters. Feedback inhibition of microsomal long-chain acyl-CoA synthesis could not be observed, due to the formation of small acyl-CoA binding vesicles during preparation and/or incubation. Microsomal membrane preparations are therefore unsuitable for studying feedback inhibition of long-chain acyl-CoA synthesis. ACBP was found to have a strong attenuating effect on the long-chain acyl-CoA inhibition of both acetyl-CoA carboxylase and mitochondrial adenine nucleotide translocase. Both processes were unaffected by the presence of long-chain acyl-CoA esters when the ratio of long-chain acyl-CoA to ACBP was below 1, independent of the acyl-CoA concentration used. It is therefore not the acyl-CoA concentration as such which is important from a regulatory point of view, but the ratio of acyl-CoA to ACBP. The cytosolic ratio of long-chain acyl-CoA to ACBP was shown to be well below 1 in the liver of fed rats. ACBP could compete with the triacylglycerol-synthesizing pathway, but not with the phospholipid-synthesizing enzymes, for acyl-CoA esters. Furthermore, in contrast with FABP, ACBP was able to protect long-chain acyl-CoA esters against hydrolysis by microsomal acyl-CoA hydrolases. The results suggest that long-chain acyl-CoA esters synthesized for either triacylglycerol synthesis or beta-oxidation have to pass through the acyl-CoA/ACBP pool before utilization. This means that acyl-CoA synthesized by microsomal or mitochondrial synthetases is uniformly available in the cell. It is suggested that ACBP has a duel function in (1) creating a cytosolic pool of acyl-CoA protected against acyl-CoA hydrolases, and (2) protecting vital cellular processes from being affected by long-chain acyl-CoA esters.

Synthesis of Long-Chain Acyl-CoA in Chloroplast Envelope Membranes

The chloroplast envelope is the site of a very active long-chain acylcoenzyme A (CoA) synthetase. Furthermore, we have recently shown that an acyl CoA thioesterase is also associated with envelope membrane (Joyard J, PK Stumpf 1980 Plant Physiol 65: 1039-1043). To clarify the interacting roles of both the acyl-CoA thioesterase and the acyl-CoA synthetase, the formation of acyl-CoA in envelope membranes was examined with different techniques which permitted the measurement of the actual rates of acyl-CoA formation. Using [(14)C]ATP or [(14)C]oleic acid as labeled substrates, it can be shown that the envelope acyl-CoA synthetase required both Mg(2+) and dithiothreitol. Triton X-100 slightly stimulated the activity. The specificity of the acyl-CoA synthetase was determined either with [(14)C]ATP or with [(3)H]CoA as substrates. The results obtained in both cases were similar, that is, as substrates, the unsaturated fatty acids were more effective than saturated fatty acids, the velocity of the reaction increased from lauric acid to palmitic acid, and the maximum velocity was obtained with unsaturated C(18) fatty acids.The results obtained suggest that the acyl-CoA thioesterase associated with envelope membranes could be an ultimate control to prevent the transport (outside of the chloroplast) or the insertion (into chloroplast lipids) of fatty acids with chains shorter than C(16).

A comparative study of palmitoyl-CoA synthetase activity in rat-liver, heart and gut mitochondrial and microsomal preparations

1. 1. With the aid of marker enzymes, mitochondrial and microsomal fractions of rat-liver, heart and jejunum epithelium were characterized. In heart the palmitoyl-CoA synthetase activity was shown to be localized mainly in the sarcosomes; in intestine the microsomes were the site of long-chain acyl-CoA synthesis, while in liver mitochondria and microsomes were about equally active in catalyzing palmitoyl-CoA synthesis. 2. 2. Nagarse treatment almost completely destroyed the cardiac and hepatic palmitoyl-CoA synthetase activity, in contrast to several other membrane-bound enzymes.