Carboxyl CoA ligases(CCLs) is an important branch of adenylate synthetase gene family, which mainly has two-step catalytic reactions. Firstly, in the presence of adenosine triphosphate, it can catalyze the pyrophosphorylation of carboxylateswith diffe-rent structures to form corresponding acyl adenosine monophosphate intermediates. Secondly, adenosine monophosphate was replaced by free electrons in the mercaptan group of enzyme A or other acyl receptors by nucleophilic attack to form thioesters. In this study, on the basis of the transcriptome database of Arnebia euchroma, two genes were selected, named AeCCL5(XP_019237476.1) and AeCCL7(XP_019237476.1). Bioinformatics analysis showed that their relative molecular weights were 60.569 kDa and 60.928 kDa, theoretical PI were 8.59 and 8.92, respectively. They both have transmembrane domains but without signal peptide. By multiple sequence alignment and phylogenetic tree analysis, we found that the similarity between AeCCLs and other plant homologous proteins was not high, and the substrate binding sites of AeCCLs were not highly conserved. The reasons might be that the sequence and structure need to adapt to the changes of new substrates in the process of evolution. In this study, the full-length of AeCCL5 and AecCCL7 were cloned into the expression vector pCDFDuet-1. The proteins of AeCCL5 and AeCCL7 with His-tag were expressed in Escherichia coli. The proteins of AeCCL5 and AeCCL7 were purified by nickel column. In vitro enzymatic reactions proved that both AeCCL5 and AeCCL7 can participate in the upstream phenylpropane pathway of shikonin biosynthesisby catalyzing 4-coumaric acid to produce 4-coumarin-CoA, and then to synthesis p-hydroxybenzoic acid, which is an important precursor of shikonin biosynthesis in A. euchroma.
Boraginaceae/genetics* ; Cloning, Molecular ; Coenzyme A ; Coenzyme A Ligases/genetics* ; Ligases ; Phylogeny

Raspberry ketones have important therapeutic properties such as anti-influenza and prevention of diabetes. In order to obtain raspberry ketone from Chlamydomonas reinhardtii, two enzymes catalyzing the last two steps of raspberry ketone synthesis, i.e. 4-coumaryl-CoA ligase (4CL) and polyketide synthase (PKS1), were fused using a glycine-serine-glycine (GSG) tripeptide linker to construct an expression vector pChla-4CL-PKS1. The fusion gene 4CL-PKS1 driven by a PSAD promoter was transformed into a wild-type (CC125) and a cell wall-deficient C. reinhardtii (CC425) by electroporation. The results showed the recombinant C. reinhardtii strain CC125 and CC425 with 4CL-PKS1 produced raspberry ketone at a level of 6.7 μg/g (fresh weight) and 5.9 μg/g (fresh weight), respectively, both were higher than that of the native raspberry ketone producing plants (2-4 μg/g).
Acyl Coenzyme A ; Butanones ; Chlamydomonas reinhardtii/genetics* ; Ligases ; Polyketide Synthases

Lipids droplets are organelles that store neutral lipids and are closely related to lipid accumulation. Long chain acyl-coenzyme A synthetase 3 (ACSL3) is a lipid droplet-associated protein mainly distributed in the cell membrane, endoplasmic reticulum, and intracellular lipid droplets, and its distribution depends on cell type and fatty acid supply. ACSL3 is a key regulator of fatty acid metabolism that is closely related to intracellular lipid accumulation, and plays an important role in various pathophysiological processes such as lipid droplet synthesis and lipid metabolism, cellular inflammation, and ferroptosis. This paper mainly reviews the role of ACSL3 in lipid synthesis, ferroptosis, and inflammatory response, with focus on the mechanism of its role in lipid accumulation in atherosclerosis, and provides new ideas for exploring potential therapeutic targets in atherosclerotic diseases.
Humans ; Atherosclerosis ; Coenzyme A Ligases/metabolism* ; Endoplasmic Reticulum/metabolism* ; Fatty Acids/metabolism* ; Lipid Metabolism

4-coumarate coenzyme A ligase is a key enzyme of phenylpropanoid metabolic pathway in higher plant and may regulate the biosynthesis of ferulic acid in Angelica sinensis. In this study, the homology-based cloning and rapid amplification of cDNA ends (RACE) technique were used to clone a full length cDNA encoding 4-coumarate coenzyme A ligase gene (4CL), and then qRT-PCR was taken for analyzing 4CL gene expression levels in the root, stem and root tissue at different growth stages of seedlings of A. sinensis. The results showed that a full-length 4CL cDNA (1,815 bp) was obtained (GenBank accession number: KT880508) which shares an open reading frame (ORF) of 1 632 bp, encodes 544 amino acid polypeptides. We found 4CL gene was expressed in all tissues including leaf, stem and root of seedlings of A. sinensis. The expressions in the leave and stem were increased significantly with the growth of seedlings of A. sinensis (P < 0.05), while it in the root showed little change. It indicates a time-space pattern of 4CL gene expression in seedlings of A. sinensis. These findings will be useful for establishing an experiment basis for studying the structure and function of 4CL gene and elucidating mechanism of ferulic acid biosynthesis and space-time regulation in A. sinensis.
Amino Acid Sequence ; Angelica sinensis ; genetics ; Base Sequence ; Cloning, Molecular ; Coenzyme A Ligases ; genetics ; DNA, Complementary ; chemistry ; Molecular Sequence Data

Stearoyl-CoAdesaturase-1 (SCD-1) is a key regulator of monounsaturated fatty acid synthesis. It plays a vital role in lipid synthesis and metabolism. Ca²⁺ is an important cation in the body and plays an important role in the organism. The aims of this study were to investigate the correlation of SCD-1 gene overexpression with lipid indexes and calcium ion level. The pcDNA3.1 (+) + SCD-1 +Flag eukaryotic expression vector and cultured duck uterine epithelial cells were co-transfected. The overexpression of SCD-1 gene was measured using the Flag Label Detection Kit. Ca ions and lipid contents were detected through Fluo-3/AM Calcium Ion Fluorescence Labeling method and Lipid Measuring Kit, respectively. SCD-1 gene overexpression was negatively correlated with triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C), and positively correlated with Ca ion, total cholesterol (TC), very low-density lipoprotein cholesterol (VLDL-C) and low density lipoprotein cholesterol (LDL-C) levels. Meanwhile, Ca ion was positively correlated with TG, LDL-C and HDL-C contents, and negatively correlated with TC and VLDL-C levels. Overexpression of SCD-1 gene could regulate Ca ion secretion, as well as lipid synthesis and transport in duck uterine epithelial cells.
Animals ; Calcium ; metabolism ; Coenzyme A Ligases ; genetics ; Ducks ; Epithelial Cells ; chemistry ; enzymology ; Gene Expression ; Ions ; Lipids ; genetics ; Triglycerides ; metabolism

Acyl-CoA synthetase long chain family member 5 (ACSL5), is a member of the acyl-CoA synthetases (ACSs) family that activates long chain fatty acids by catalyzing the synthesis of fatty acyl-CoAs. The dysregulation of ACSL5 has been reported in some cancers, such as glioma and colon cancers. However, little is known about the role of ACSL5 in acute myeloid leukemia (AML). We found that the expression of ACSL5 was higher in bone marrow cells from AML patients compared with that from healthy donors. ACSL5 level could serve as an independent prognostic predictor of the overall survival of AML patients. In AML cells, the ACSL5 knockdown inhibited cell growth both in vitro and in vivo. Mechanistically, the knockdown of ACSL5 suppressed the activation of the Wnt/β-catenin pathway by suppressing the palmitoylation modification of Wnt3a. Additionally, triacsin c, a pan-ACS family inhibitor, inhibited cell growth and robustly induced cell apoptosis when combined with ABT-199, the FDA approved BCL-2 inhibitor for AML therapy. Our results indicate that ACSL5 is a potential prognosis marker for AML and a promising pharmacological target for the treatment of molecularly stratified AML.
Humans ; Antineoplastic Agents/therapeutic use* ; Apoptosis ; beta Catenin/metabolism* ; Biomarkers, Tumor/metabolism* ; Cell Line, Tumor ; Coenzyme A Ligases/metabolism* ; Leukemia, Myeloid, Acute/metabolism* ; Lipoylation ; Prognosis ; Wnt Signaling Pathway

OBJECTIVETo establish nonalcoholic fatty liver disease (NAFLD) model induced by free fatty acid (FFA) and iron, and to explore the synergistic effect of FFA and Fe(2+) on the pathogenesis of NAFLD and mechanisms.

METHODSHuman liver carcinoma cell HepG2 was respectively treated with 0.250, 0.500, 1.000 mmol/L oleic acid, 0.500 mmol/L oleic acid+0.125 mmol/L Fe(2+), 0.500 mmol/L oleic acid+0.250 mmol/L Fe(2+), and 0.500 mmol/L oleic acid+0.500 mmol/L Fe(2+). Human liver carcinoma cell HepG2 was normally cultured in the control group. Lipid accumulation of cells were observed by oil red O staining and the determination of the triglyceride (TG) contents by GPO-PAP, then the expression of key genes involved in fatty acid β-oxidation (fatty acyl CoA synthetase-1 (ACSL-1), carnitine acyl transferase 1 (CPT-1a), fatty acid synthetase (FAS)) was determined using RT-PCR. The differences of TG content and ACSL-1, CPT-1a, FAS, mRNA relative value were analyzed among different groups.

RESULTSThe results of oil red O staining indicated that the contents of lipid droplets were obviously elevated with the increase of Fe(2+) concentration in human liver carcinoma cell HepG2 treated with 0.500 mmol/L oleic acid and different concentrations of Fe(2+). The TG contents of HepG2 cell in control group, 0.250, 0.500, 1.000 mmol/L oleic acid groups, 0.500 mmol/L oleic acid+0.125 mmol/L Fe(2+) group, 0.500 mmol/L oleic acid+0.250 mmol/L Fe(2+) group, 0.500 mmol/L oleic acid+0.500 mmol/L Fe(2+) group respectively were (90.0 ± 1.6), (131.7 ± 5.4), (153.7 ± 3.0), (254.1 ± 4.0), (164.5 ± 6.0), (180.1 ± 7.7), (235.6 ± 4.5) nmol/mg (F = 396.00, P < 0.05). The expression levels of ACSL-1 mRNA in 0.500 mmol/L oleic acid group, 0.500 mmol/L oleic acid+0.125 mmol/L Fe(2+) group, 0.500 mmol/L oleic acid +0.250 mmol/L Fe(2+) group, 0.500 mmol/L oleic acid +0.500 mmol/L Fe(2+) group respectively were (0.94 ± 0.02), (0.89 ± 0.04), (0.85 ± 0.02), (0.74 ± 0.04) (F = 50.00, P < 0.05); the mRNA levels of CPT-1a were (0.89 ± 0.03), (0.79 ± 0.05), (0.67 ± 0.04), (0.51 ± 0.05) (F = 79.00, P < 0.05); the mRNA levels of FAS were (1.31 ± 0.05) , (1.44 ± 0.03), (1.51 ± 0.05), (1.56 ± 0.06 ) (F = 79.70, P < 0.05).

CONCLUSIONThe NAFLD liver cell model could be established by oleic acid and Fe(2+) in HepG2 cells. FFA and iron might be involved in the pathogenesis of NAFLD through the intervention of fatty acid β-oxidation.

Carnitine O-Palmitoyltransferase ; Coenzyme A Ligases ; Fatty Acid Synthase, Type I ; Fatty Acids ; Fatty Acids, Nonesterified ; adverse effects ; Hep G2 Cells ; Humans ; Iron ; adverse effects ; Non-alcoholic Fatty Liver Disease ; chemically induced ; Oleic Acid ; RNA, Messenger ; Triglycerides

Fatty acid-CoA ligase 4 (FACL4) is a central enzyme controlling the unesterified free arachidonic acid (AA) level in cells and the free AA is known to induce apoptosis. We have recently reported that expression of FACL4 is upregulated in about 40% of human hepatocellular carcinoma (HCC) and 50% of HCC cell lines, suggesting that FACL4 may be involved in liver carcinogenesis. In this study, we investigated whether HCC cell growth is regulated by FACL4. Immunoblot analysis showed that SNU 398 cells express very low or no detectable level of FACL4. We, therefore, transfected the SNU 398 cells with FACL4 expression vector, and clones expressing FACL4 were pooled and analyzed. We found that forced expression of FACL4 in SNU 398 promotes the growth of cells. In addition, we observed that triacsin C, a FACL4 inhibitor, inhibits the growth of Hep 3B cell line which expresses high level of endogenous FACL4. We also found that the triacsin C-mediated growth inhibition in Hep 3B cells results from the induction of apoptosis with evidence of Bcl-2 reduction. Altogether, our data show that FACL4 affects HCC cell growth and suggest that modulation of FACL4 expression/activity is an approach for treatment of HCC.
Apoptosis ; Carcinoma, Hepatocellular/*enzymology/pathology ; Cell Line, Tumor ; Cell Proliferation ; Coenzyme A Ligases/antagonists & inhibitors/*physiology ; Humans ; Liver Neoplasms/*enzymology/pathology ; Proto-Oncogene Proteins c-bcl-2/metabolism ; Triazenes/pharmacology

AIMTo investigate the roles of the BGR-like gene in testicular development/spermatogenesis.

METHODSA human testis cDNA microarray was hybridized with probes from human adult testes and embryo testes. The differentially expressed clones were sequenced and analyzed. Expression of the BGR-like gene was analyzed by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSA new gene exhibiting 50-fold difference in expression level between adult and fetal human testes was cloned and named the BGR-like gene. The cDNA consisted of 2500 nucleotides and had an open reading frame of 1437 nucleotides encoding a putative protein of 497 amino acid residues. Homologous comparison showed that the BGR-like gene was a new alternative splicing variant of the BGR gene and had sequence homology with the bubblegum gene of human, mouse, rat and Drosophila. Protein motif analysis of the BGR-like gene revealed that it contained a conserved adenosine monophosphate (AMP)-binding domain and a fatty acyl-CoA synthetase signature motif which existed in all acyl-CoA synthetases. The BGR-like gene transcript was imperceptibly expressed in human fetal testes, highly in human adult testes and moderately in elderly testes and human Leydig cells. RT-PCR-based tissue distribution experiments showed that the BGR-like gene was exclusively expressed in testes and was a testes-specific isoform of the BGR gene. A BGR-like gene transcript was not detected in some azoospermic testes.

CONCLUSIONThe BGR-like gene may play an important role in spermatogenesis/testicular development and may be correlated with male infertility.

Adult ; Aged ; Alternative Splicing ; genetics ; Amino Acid Sequence ; Base Sequence ; Coenzyme A Ligases ; genetics ; Drosophila Proteins ; genetics ; Gene Expression Regulation ; Humans ; Infertility, Male ; genetics ; Leydig Cells ; metabolism ; Male ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Spermatogenesis ; genetics ; Testis ; metabolism

OBJECTIVE: To examine the effect of Shenmai Injection (SMJ) on ferroptosis during myocardial ischemia reperfusion (I/R) injury in rats and the underlying mechanism. METHODS: A total of 120 SPF-grade adult male SD rats, weighing 220-250 g were randomly divided into different groups according to a random number table. Myocardial I/R model was established by occluding the left anterior descending artery for 30 min followed by 120 min of reperfusion. SMJ was injected intraperitoneally at the onset of 120 min of reperfusion, and erastin (an agonist of ferroptosis), ferrostatin-1 (Fer-1, an inhibitor of ferroptosis) and ML385 (an inhibitor of nuclear factor erythroid-2 related factor 2 (Nrf2)) were administered intraperitoneally separately 30 min before myocardial ischemia as different pretreatments. Cardiac function before ischemia, after ischemia and after reperfusion was analysed. Pathological changes in the myocardium and the ultrastructure of cardiomyocytes were observed, and the myocardial infarction area was measured. Additionally, the concentration of Fe²⁺ in heart tissues and the levels of creatine kinase-MB (CK-MB), troponin I (cTnl), malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were measured using assay kits, and the expressions of Nrf2, glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase long-chain family member 4 (ACSL4) were examined by Western blot. RESULTS: Compared with the sham group, I/R significantly injured heart tissues, as evidenced by the disordered, ruptured and oedematous myocardial fibres; the increases in infarct size, serum CK-MB, cTnI and MDA levels, and myocardial Fe²⁺ concentrations; and the decreases in SOD activity (P<0.05). These results were accompanied by ultrastructural alterations to the mitochondria, increased expression of ACSL4 and inhibited the activation of Nrf2/GPX4 signalling (P<0.05). Compared with I/R group, pretreatment with 9 mL/kg SMJ and 2 mg/kg Fer-1 significantly reduced myocardial I/R injury, Fe²⁺ concentrations and ACSL4 expression and attenuated mitochondrial impairment, while 14 mg/kg erastin exacerbated myocardial I/R injury (P<0.05). In addition, cardioprotection provided by 9 mL/kg SMJ was completely reversed by ML385, as evidenced by the increased myocardial infarct size, CK-MB, cTnI, MDA and Fe²⁺ concentrations, and the decreased SOD activity (P<0.05). CONCLUSIONS Ferroptosis is involved in myocardial I/R injury. Pretreatment with SMJ alleviated myocardial I/R injury by activating Nrf2/GPX4 signalling-mediated ferroptosis, thereby providing a strategy for the prevention and treatment of ischemic heart diseases.
Animals ; Male ; Rats ; Coenzyme A ; Creatine Kinase ; Ferroptosis ; Ligases ; Malondialdehyde ; Myocardial Infarction/drug therapy* ; Myocardial Ischemia/drug therapy* ; Myocardial Reperfusion Injury/pathology* ; Myocytes, Cardiac/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Rats, Sprague-Dawley ; Superoxide Dismutase/metabolism* ; Troponin I