A Peroxisomal Long-Chain Acyl-CoA Synthetase from Glycine max Involved in Lipid Degradation

Seed storage oil, in the form of triacylglycerol (TAG), is degraded to provide carbon and energy during germination and early seedling growth by the fatty acid β-oxidation in the peroxisome. Although the pathways for lipid degradation have been uncovered, understanding of the exact involved enzymes in soybean is still limited. Long-chain acyl-CoA synthetase (ACSL) is a critical enzyme that activates free fatty acid released from TAG to form the fatty acyl-CoA. Recent studies have shown the importance of ACSL in lipid degradation and synthesis, but few studies were focused on soybean. In this work, we cloned a ACSL gene from soybean and designated it as GmACSL2. Sequence analysis revealed that GmACSL2 encodes a protein of 733 amino acid residues, which is highly homologous to the ones in other higher plants. Complementation test showed that GmACSL2 could restore the growth of an ACS-deficient yeast strain (YB525). Co-expression assay in Nicotiana benthamiana indicated that GmACSL2 is located at peroxisome. Expression pattern analysis showed that GmACSL2 is highly expressed in germinating seedling and strongly induced 1 day after imbibition, which indicate that GmACSL2 may take part in the seed germination. GmACSL2 overexpression in yeast and soybean hairy root severely reduces the contents of the lipids and fatty acids, compared with controls in both cells, and enhances the β-oxidation efficiency in yeast. All these results suggest that GmACSL2 may take part in fatty acid and lipid degradation. In conclusion, peroxisomal GmACSL2 from Glycine max probably be involved in the lipid degradation during seed germination.     

Altered Energetics of Exercise Explain Risk of Rhabdomyolysis in Very Long-Chain Acyl-CoA Dehydrogenase Deficiency

Rhabdomyolysis is common in very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and other metabolic myopathies, but its pathogenic basis is poorly understood. Here, we show that prolonged bicycling exercise against a standardized moderate workload in VLCADD patients is associated with threefold bigger changes in phosphocreatine (PCr) and inorganic phosphate (Pi) concentrations in quadriceps muscle and twofold lower changes in plasma acetyl-carnitine levels than in healthy subjects. This result is consistent with the hypothesis that muscle ATP homeostasis during exercise is compromised in VLCADD. However, the measured rates of PCr and Pi recovery post-exercise showed that the mitochondrial capacity for ATP synthesis in VLCADD muscle was normal. Mathematical modeling of oxidative ATP metabolism in muscle composed of three different fiber types indicated that the observed altered energy balance during submaximal exercise in VLCADD patients may be explained by a slow-to-fast shift in quadriceps fiber-type composition corresponding to 30% of the slow-twitch fiber-type pool in healthy quadriceps muscle. This study demonstrates for the first time that quadriceps energy balance during exercise in VLCADD patients is altered but not because of failing mitochondrial function. Our findings provide new clues to understanding the risk of rhabdomyolysis following exercise in human VLCADD.     

Acyl-CoA Synthetase in Maturing Safflower Seeds

Acyl-CoA synthetase in maturing seeds of safflower (Carthamustinctorius) was membranebound, and the highest specific activitywas associated with microsomes. Activity absolutely dependedon the concentrations of fatty acid, CoA, ATP and Mg²⁺. Theapparent K_m values were 4.2 µM for oleate, 24 µMfor CoA, and 250 µM for ATP. The optimum pH of the reactionwas 7.5. Triacsin C, a potent inhibitor of the animal and bacterialacyl-CoA synthetase, was ineffective for the safflower enzyme.The enzyme utilized C₁₆ and C₁₈ long-chain fatty acids preferentially,while medium-chain and very-long-chain fatty acids were poorsubstrates. The order of specificity for native fatty acidswas linoleate > oleate=palmitate > stearate. Althoughactivity per seed varied during seed maturation, it was enoughto account for the rate of triacylglycerol synthesis in vivo. (Received February 2, 1993; Accepted March 3, 1993)     

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 [¹⁴C]ATP or [¹⁴C]oleic acid as labeled substrates, it can be shown that the envelope acyl-CoA synthetase required both Mg²⁺ and dithiothreitol. Triton X-100 slightly stimulated the activity. The specificity of the acyl-CoA synthetase was determined either with [¹⁴C]ATP or with [³H]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 C₁₈ fatty acids.     

Distribution and Formation of Acyl-CoA Synthetase in Microorganisms

The distribution of acyl-CoA synthetase was investigated among microorganisms. High enzyme activity was found in some strains in genera of Pseudomonas, Fusarium, Gibberella and Cylindrocarpon, and in many strains of basidiomycetes. There were two groups in respect to enzyme formation. The enzyme activities of Escherichia, Klebsiella, Enterobacter, Citrobacter and Serratia were detected only when they were grown with fatty acids as the carbon source. On the other hand, the activities of many fungal strains and pseudomonads were easily detected regardless of the carbon source for growth.

Gel filtration on Sephadex G-200 showed that the enzymes of Escherichia coli and Gibberella fujikuroi were mostly present around the void volume of the column and retarded by the gel after treatment with Triton X-100. Pseudomonas aeruginosa produced two kinds of enzymes, one was eluted around the void volume of the column and the other retarded by the gel. This elution pattern did not change upon treatment with Triton X-100. Some catalytic properties of acyl-CoA synthetases from P. aeruginosa and G. fujikuroi were also described.     

Functional Analysis of Long-chain Acyl-CoA Synthetase 1 in 3T3-L1 Adipocytes

ACSL1 (acyl-CoA synthetase 1), the major acyl-CoA synthetase of adipocytes, has been proposed to function in adipocytes as mediating free fatty acid influx, esterification, and storage as triglyceride. To test this hypothesis, ACSL1 was stably silenced (knockdown (kd)) in 3T3-L1 cells, differentiated into adipocytes, and evaluated for changes in lipid metabolism. Surprisingly, ACSL1-silenced adipocytes exhibited no significant changes in basal or insulin-stimulated long-chain fatty acid uptake, lipid droplet size, or tri-, di-, or monoacylglycerol levels when compared with a control adipocyte line. However, ACSL1 kd adipocytes displayed a 7-fold increase in basal and a ∼15% increase in forskolin-stimulated fatty acid efflux without any change in glycerol release, indicating a role for the protein in fatty acid reesterification following lipolysis. Consistent with this proposition, ACSL1 kd cells exhibited a decrease in activation and phosphorylation of AMP-activated protein kinase and its primary substrate acetyl-CoA carboxylase. Moreover, ACSL1 kd adipocytes displayed an increase in phosphorylated protein kinase Cθ and phosphorylated JNK, attenuated insulin signaling, and a decrease in insulin-stimulated glucose uptake. These findings identify a primary role of ACSL1 in adipocytes not in control of lipid influx, as previously considered, but in lipid efflux and fatty acid-induced insulin resistance.Fatty acid influx and efflux mechanisms and their regulation affect lipid storage and metabolism in adipocytes. Imbalances in adipose lipid metabolism have been shown to significantly contribute to the development of obesity and associated metabolic diseases, such as type 2 diabetes, hypertension, and cardiovascular disease (1–3). Although the molecular mechanisms involved in fatty acid efflux are still undefined, several proteins implicated in fatty acid influx have been proposed: CD36 (fatty acid translocase), acyl-CoA synthetases (fatty acid transport protein (FATP)² and acyl-CoA synthetase (ACSL) family members), plasma membrane fatty acid-binding protein, and caveolin-1 (4–9).FATPs and long-chain ACSLs are membrane-bound enzymes that catalyze the ATP-dependent esterification of long chain (ACSL) and very long-chain (FATP) fatty acids to their acyl-CoA derivatives (10, 11). Both types of CoA synthetases have common ATP/AMP binding and fatty acid binding signature motifs. In mammals, six different isoforms of FATP (FATP1–FATP6) and five different isoforms of ACSL (ACSL1, -3, -4, -5, and -6) have been identified with tissue-specific expression patterns (12). White adipose tissue predominantly express FATP1, FATP4, and ACSL1, whereas brown adipose tissue in addition expresses ACSL5. Our recent results have confirmed a major role of FATP1 and CD36, but not FATP4, in insulin-stimulated LCFA uptake in 3T3-L1 adipocytes (6).ACSL1 is a ∼78-kDa intrinsic membrane protein localized to multiple sites in a variety of different cells. In liver, ACSL1 has been shown to be localized to the endoplasmic reticulum and mitochondria-associated membranes, whereas in adipocytes, ACSL1 was also found associated with the plasma membrane, the lipid droplet surface (13), and glucose transporter 4-containing vesicles (14, 15). Recent studies have postulated a cooperative role of FATP1 and ACSL1 in the movement of LCFAs across the plasma membrane via a process termed vectoral acylation (16), in which the CoA- and ATP-dependent esterification of internalized fatty acid provides the thermodynamic force necessary for net lipid influx. Evidence supporting this hypothesis came from a functional cloning strategy that identified mouse ACSL1 along with FATP1 as proteins involved in LCFA transport (17). In contrast to the role of ACSL1 in LCFA uptake and triglyceride synthesis in adipocytes, overexpression of ACSL1 in rat primary hepatocytes channeled fatty acids toward diacylglycerol and phospholipids synthesis and increased reacylation of hydrolyzed fatty acids into triglyceride (18).Since lipid flux is defined by the location and activity of its regulatory enzymes and proteins, overexpression strategies can result in changes in metabolism potentially distinct from the endogenous function. To that end, our laboratory has recently undertaken a gene silencing approach to the evaluation of proteins implicated in adipocyte fatty acid influx and efflux, and prior studies have focused on FATP1, FATP4, and CD36 (6). In this report, we evaluated the adipose-specific role(s) of ACSL1 using stable gene-silencing strategies in 3T3-L1 adipocytes using lentiviral delivery of shRNA. We report herein that, contrary to previous reports, in 3T3-L1 adipocytes, ACSL1 does not facilitate the basal or insulin-stimulated component of LCFA uptake. ACSL1 is, however, involved in the reesterification of hydrolyzed fatty acids released during basal and forskolin-stimulated lipolysis, thereby regulating their availability and efflux from the cell. Additionally, fatty acid reesterification by ACSL1 during lipolysis plays a major role in regulating the AMP-activated protein kinase (AMPK) as well as the PKCθ and JNK pathways leading to insulin resistance. Such findings bring to light a new interpretation of the role of ACSL1 and other acyl-CoA synthetases in the control of intermediary metabolism and lipid-mediated signal transduction.     

Very Long-Chain Fatty Acids in Yeast

Novel fatty acids ranging from 20 to 30 carbons have been found in Saccharomyces cerevisiae. These comprise 1 to 2% of the total fatty acid fraction.     

Acyl-CoA Synthetase in Oilseeds: Fatty Acid Structural Requirements for Activity and Selectivity

The substrate specificities and selectivities of acyl-CoA synthetasesfrom maturing oilseeds were investigated to reveal fatty acidstructures that the enzymes recognize. The synthetases fromrapeseed (Brassica nap us) and castor bean (Ricinus communis)activated palmitic acid 16:0 most rapidly among the saturatedfatty acids tested. Native unsat-urated fatty acids, oleic 18:1cis-9, linoleic 18:2 cis-9,12 and linolenic acid 18:3 m-9,12,15,were all effectively utilized. Palmitoleic acid 16:1 cis-9 wasalso a good substrate, while myristoleic acid 14:1 cis-9 wasa poor substrate. The activation of erucic acid 22:1 cis-13was very slow. Elaidic acid 18:1 trans-9 was utilized at ratessimilar to those of the cis isomer. The efficiencies of petroselinicacid 18:1 cis-6 were half the efficiencies of oleic acid, whilethe rates of activation of m-vaccenic acid 18:1 cw-11 were comparableto those for oleic acid. These findings suggest that acyl-CoAsynthetases of oilseeds producing long-chain fatty acids strictlyrecognize the molecular structures of fatty acids, i.e., thecarbon-chain length between C¹⁶-C¹⁸ and the position of thefirst double bond (     

Overexpression of MTA3 Correlates with Tumor Progression in Non-Small Cell Lung Cancer

The objective of the current study was to investigate the expression pattern and clinicopathological significance of MTA3 in patients with non-small cell lung cancer (NSCLC). The expression profile of MTA3 in NSCLC tissues and adjacent noncancerous lung tissues was detected by immunohistochemistry. MTA3 was overexpressed in 62 of 108 (57.4%) human lung cancer samples and correlated with p-TNM stage (p<0.0001), nodal metastasis (p = 0.0009) and poor prognosis (p<0.05). In addition, the depletion of MTA3 expression with small interfering RNAs inhibited cell growth and colony formation in the A549 and H157 lung cancer cell lines. Moreover, MTA3 depletion induced cell cycle arrest at the G1/S boundary. Western blotting analysis revealed that the knockdown of MTA3 decreased the protein levels of cyclin A, cyclin D1 and p-Rb. These results indicate that MTA3 plays an important role in NSCLC progression.     

Long Chain Fatty Acyl-CoA Synthetase 4 Is a Biomarker for and Mediator of Hormone Resistance in Human Breast Cancer

The purpose of this study was to determine the role of long-chain fatty acyl-CoA synthetase 4 (ACSL4) in breast cancer. Public databases were utilized to analyze the relationship between ACSL4 mRNA expression and the presence of steroid hormone and human epidermal growth factor receptor 2 (HER2) in both breast cancer cell lines and tissue samples. In addition, cell lines were utilized to assess the consequences of either increased or decreased levels of ACSL4 expression. Proliferation, migration, anchorage-independent growth and apoptosis were used as biological end points. Effects on mRNA expression and signal transduction pathways were also monitored. A meta-analysis of public gene expression databases indicated that ACSL4 expression is positively correlated with a unique subtype of triple negative breast cancer (TNBC), characterized by the absence of androgen receptor (AR) and therefore referred to as quadruple negative breast cancer (QNBC). Results of experiments in breast cancer cell lines suggest that simultaneous expression of ACSL4 and a receptor is associated with hormone resistance. Forced expression of ACSL4 in ACSL4-negative, estrogen receptor α (ER)-positive MCF-7 cells resulted in increased growth, invasion and anchorage independent growth, as well as a loss of dependence on estrogen that was accompanied by a reduction in the levels of steroid hormone receptors. Sensitivity to tamoxifen, triacsin C and etoposide was also attenuated. Similarly, when HER2-positive, ACSL4-negative, SKBr3 breast cancer cells were induced to express ACSL4, the proliferation rate increased and the apoptotic effect of lapatinib was reduced. The growth stimulatory effect of ACSL4 expression was also observed in vivo in nude mice when MCF-7 control and ACSL4-expressing cells were utilized to induce tumors. Our data strongly suggest that ACSL4 can serve as both a biomarker for, and mediator of, an aggressive breast cancer phenotype.     

Acyl-CoA Synthetase Is Located in the Outer Membrane and Acyl-CoA Thioesterase in the Inner Membrane of Pea Chloroplast Envelopes

Both acyl-CoA synthetase and acyl-CoA thioesterase activities are present in chloroplast envelope membranes. The functions of these enzymes in lipid metabolism remains unresolved, although the synthetase has been proposed to be involved in either plastid galactolipid synthesis or the export of plastid-synthesized fatty acids to the cytoplasm. We have examined the locations of both enzymes within the two envelope membranes of pea (Pisum sativum var Laxton's Progress No. 9) chloroplasts. Inner and outer envelope membranes were purified from unfractionated envelope preparations by linear density sucrose gradient centrifugation. Acyl-CoA synthetase was located in the outer envelope membrane while acyl-CoA thioesterase was located in the inner envelope membrane. Thus, it seems unlikely that the synthetase is directly involved in galactolipid assembly. Instead, its localization supports the hypothesis that it functions in the transport of plastid-synthesized fatty acids to the endoplasmic reticulum.     

莱茵衣藻酰基辅酶A合成酶cDNA克隆及其酵母表达

旨在预测并克隆莱茵衣藻酰基辅酶A合成酶cDNA(cracs),分析其在酵母中的功能。RT-PCR克隆cracs序列,Clustal W和MEGA6.0软件分别分析其编码蛋白保守序列和进化树,表达并分析其在酵母YB525中的底物偏好性。结果表明,首次在莱茵衣藻中克隆获得一个cracs,测序表明其序列大小为2 004 bp,编码667个氨基酸,编码蛋白crACS的预测分子量为72.3k D,包含酰基辅酶A合成酶的两个保守区:AMP-binding区和FACS区。进化树比对显示,cr ACS与拟南芥的长链酰基辅酶A合成酶LACSs具有较高的同源性。酵母表达显示cracs编码蛋白能互补酵母YB525 LACS的缺陷表型,活化并优先利用C16∶1和C14∶0。莱茵衣藻cracs编码蛋白可活化外源脂肪酸,属于酰基辅酶A合成酶家族。     

Mechanisms Underlying Cancer Growth and Apoptosis by DEK Overexpression in Colorectal Cancer

Our previous study indicated that DEK protein was overexpressed in colorectal carcinoma (CRC) compared with the normal colorectal mucosa. DEK was also significantly correlated with the prognostic characteristics of patients with CRC, demonstrating that DEK played an important role in CRC progression. In this work, we evaluate the effects of DEK on biological behaviors in CRC and explore the related molecular mechanisms. The results showed that DEK was overexpressed in human CRC tissues, and was correlated with the Ki-67 index and the apoptotic index. DEK depletion by RNAi in SW-620 and HCT116 cells significantly decreased cell proliferation, but increased cell apoptosis. Upregulation of DEK was involved in the p53/MDM, Bcl-2 family, and caspase pathways. Our study demonstrates that DEK promotes the growth of CRC, and could be a therapeutic target in CRC.     

Overexpression of Glutathione Synthetase in Escherichia coli

Kohamaic acid A is a potent DNA polymerase inhibitor isolated from the Okinawan marine sponge Ircinia sp. A series of structurally simplified analogs of kohamaic acid A were synthesized with the aim of evaluating structure-activity relationships.     

Overexpression of Arabidopsis Acyl-CoA Binding Protein ACBP3 Promotes Starvation-Induced and Age-Dependent Leaf Senescence

In Arabidopsis thaliana, a family of six genes (ACBP1 to ACBP6) encodes acyl-CoA binding proteins (ACBPs). Investigations on ACBP3 reported here show its upregulation upon dark treatment and in senescing rosettes. Transgenic Arabidopsis overexpressing ACBP3 (ACBP3-OEs) displayed accelerated leaf senescence, whereas an acbp3 T-DNA insertional mutant and ACBP3 RNA interference transgenic Arabidopsis lines were delayed in dark-induced leaf senescence. Acyl-CoA and lipid profiling revealed that the overexpression of ACBP3 led to an increase in acyl-CoA and phosphatidylethanolamine (PE) levels, whereas ACBP3 downregulation reduced PE content. Moreover, significant losses in phosphatidylcholine (PC) and phosphatidylinositol, and gains in phosphatidic acid (PA), lysophospholipids, and oxylipin-containing galactolipids (arabidopsides) were evident in 3-week-old dark-treated and 6-week-old premature senescing ACBP3-OEs. Such accumulation of PA and arabidopsides (A, B, D, E, and G) resulting from lipid peroxidation in ACBP3-OEs likely promoted leaf senescence. The N-terminal signal sequence/transmembrane domain in ACBP3 was shown to be essential in ACBP3-green fluorescent protein targeting and in promoting senescence. Observations that recombinant ACBP3 binds PC, PE, and unsaturated acyl-CoAs in vitro and that ACBP3 overexpression enhances degradation of the autophagy (ATG)-related protein ATG8 and disrupts autophagosome formation suggest a role for ACBP3 as a phospholipid binding protein involved in the regulation of leaf senescence by modulating membrane phospholipid metabolism and ATG8 stability in Arabidopsis. Accelerated senescence in ACBP3-OEs is dependent on salicylic acid but not jasmonic acid signaling.     

An Acyl-CoA Synthetase in Mycobacterium tuberculosis Involved in Triacylglycerol Accumulation during Dormancy

Latent infection with dormant Mycobacterium tuberculosis is one of the major reasons behind the emergence of drug-resistant strains of the pathogen worldwide. In its dormant state, the pathogen accumulates lipid droplets containing triacylglycerol synthesized from fatty acids derived from host lipids. In this study, we show that Rv1206 (FACL6), which is annotated as an acyl-CoA synthetase and resembles eukaryotic fatty acid transport proteins, is able to stimulate fatty acid uptake in E. coli cells. We show that purified FACL6 displays acyl-coenzyme A synthetase activity with a preference towards oleic acid, which is one of the predominant fatty acids in host lipids. Our results indicate that the expression of FACL6 protein in Mycobacterium tuberculosis is significantly increased during in vitro dormancy. The facl6-deficient Mycobacterium tuberculosis mutant displayed a diminished ability to synthesize acyl-coenzyme A in cell-free extracts. Furthermore, during in vitro dormancy, the mutant synthesized lower levels of intracellular triacylglycerol from exogenous fatty acids. Complementation partially restored the lost function. Our results suggest that FACL6 modulates triacylglycerol accumulation as the pathogen enters dormancy by activating fatty acids.     

Ectopic Overexpression of Asparagine Synthetase in Transgenic Tobacco

Here, we monitor the effects of ectopic overexpression of genes for pea asparagine synthetase (AS1) in transgenic tobacco (Nicotiana tabacum). The AS genes of pea and tobacco are normally expressed only during the dark phase of the diurnal growth cycle and specifically in phloem cells. A hybrid gene was constructed in which a pea AS1 cDNA was fused to the cauliflower mosaic virus 35S promoter. The 35S-AS1 gene was therefore ectopically expressed in all cell types in transgenic tobacco and constitutively expressed at high levels in both the light and the dark. Northern analysis demonstrated that the 35S-AS1 transgene was constitutively expressed at high levels in leaves of several independent transformants. Furthermore, amino acid analysis revealed a 10- to 100-fold increase in free asparagine in leaves of transgenic 35S-AS1 plants (construct z127) compared with controls. Plant growth analyses showed increases (although statistically insignificant) in growth phenotype during the vegetative stage of growth in 35S-AS1 transgenic lines. The 35S-AS1 construct was further modified by deletion of the glutamine-binding domain of the enzyme (gln[delta]AS1; construct z167). By analogy to animal AS, we reasoned that inhibition of glutamine-dependent AS activity might enhance the ammonia-dependent AS activity. The 3- to 19-fold increase in asparagine levels in the transgenic plants expressing gln[delta]AS1 compared with wild type suggests that the novel AS holoenzyme present in the transgenic plants (gln[delta]AS1 homodimer) has enhanced ammonia-dependent activity. These data indicate that manipulation of AS expression in transgenic plants causes an increase in nitrogen assimilation into asparagine, which in turn produces effects on plant growth and asparagine biosynthesis.     

Very Long-Chain Fatty Acids in Composition of Plant Membrane Lipids

Literary data on very long-chain fatty acids (VLCFAs) that are present in polar lipids of the plant cell membranes are discussed. Large amounts of VLCFA are found in polar lipids of some cellular organelles as well as in nonextractable lipids from diverse plant objects, where the influence of surface lipids on the relative content of these FAs is excluded. In some plants, the VLCFA fraction in membrane lipids increases under several kinds of stress. Amounts and diversity of VLCFAs are lower in flowering plants as compared with the representatives of more ancient taxons—gymnosperms, ferns, and marine algae. Presence of VLCFAs in the composition of annular lipids of the cell membranes is assumed. Biosynthesis of VLCFAs, enzymes involved in the process, and encoding genes are discussed.