Terminal differentiation in the avian uropygial gland. Accumulation of fatty acid synthase and malic enzyme in non-dividing cells

Summary The secretory tissue of the uropygial gland is of the holocrine type, containing both dividing progenitor cells and lipid-filled differentiated cells. In this study, we examined the relationship between cell division and differentiation. The location of dividing cells was determined by autoradiography of tissue sections from ducklings injected intra-abdominally with ³H-thymidine. Only cells on the basal lamina of the tubules contained labeled nuclei. Dividing cells were distributed uniformly over the length of the tubules. Over the next five days, most of the labeled cells migrated to the lumen of the tubules and disappeared. Cells containing the lipogenic enzymes, fatty acid synthase and malic enzyme, were localized either immunocytochemically using affinity-purified antibodies or cytochemically using a specific assay for malic enzyme activity. Fatty acid synthase and malic enzyme were undetectable in dividing basal cells but present at high levels in differentiating and differentiated cells. Thus, basal cells lying along the basal lamina of the tubules were replacing lipid-laden cells that were continually sloughed into the lumens of the tubules. The signals for differentiation and enzyme accumulation appear to be linked to one another and to cessation of cell division.     

圆红冬孢酵母新颖脂肪酸合酶的重组表达、纯化与活性检测

脂肪酸合酶(Fatty acid synthase,FAS)催化乙酰辅酶A和丙二酸单酰辅酶A反应生成脂肪酸,是油脂合成代谢途径中最重要的酶之一。在高产油脂的圆红冬孢酵母Rhodosporidium toruloides中发现了一种新颖的FAS,它含两个亚基,与其他物种的FAS相比,具有独特的结构域组成,尤其是含两个酰基载体蛋白(ACP)结构域。由于ACP在脂肪酸合成反应中起辅因子作用,推测多个ACP有利于提高FAS的催化活性,为研究该FAS的生物化学和结构特征,构建了表达FAS两个亚基的载体,并转化大肠杆菌Escherichia coli BL21(DE3),含pET22b-FAS1和pET24-FAS2质粒的重组菌株ZWE06可同时高表达两个亚基,经硫酸铵沉淀、蔗糖密度梯度离心和阴离子交换层析纯化,得到的重组FAS比活力达到548 mU/mg。纯化的FAS复合物可用于后续酶动力学和蛋白结构研究,且表达与纯化方法的建立对研究其他ACP的功能具有参考价值。     

Differential in radiosensitizing potency of enantiomers of the fatty acid synthase inhibitor C75

The elevated activity of fatty acid synthase has been reported in a number of cancer types. Inhibition of this enzyme has been demonstrated to induce cancer cell death and reduce tumor growth. In addition, the fatty acid synthase inhibitor drug C75 has been reported to synergistically enhance the cancer‐killing ability of ionizing radiation. However, clinical use of C75 has been limited due to its producing weight loss, believed to be caused by alterations in the activity of carnitine palmitoyltransferase‐1. C75 is administered in the form of a racemic mixture of (−) and (+) enantiomers that may differ in their regulation of fatty acid synthase and carnitine palmitoyltransferase‐1. Therefore, we assessed the relative cancer‐killing potency of different enantiomeric forms of C75 in prostate cancer cells. These results suggest that (−)‐C75 is the more cytotoxic enantiomer and has greater radiosensitizing capacity than (+)‐C75. These observations will stimulate the development of fatty acid synthase inhibitors that are selective for cancer cells and enhance the tumor‐killing activity of ionizing radiation, while minimizing weight loss in cancer patients.     

Probing the modularity of megasynthases by rational engineering of a fatty acid synthase Type I

Modularity is a fundamental property of megasynthases such as polyketide synthases (PKSs). In this study, we exploit the close resemblance between PKSs and animal fatty acid synthase (FAS) to re‐engineer animal FAS to probe the modularity of the FAS/PKS family. Guided by sequence and structural information, we truncate and dissect animal FAS into its components, and reassemble them to generate new PKS‐like modules as well as bimodular constructs. The novel re‐engineered modules resemble all four common types of PKSs and demonstrate that this approach can be a powerful tool to deliver products with higher catalytic efficiency. Our data exemplify the inherent plasticity and robustness of the overall FAS/PKS fold, and open new avenues to explore FAS‐based biosynthetic pathways for custom compound design.     

Inactivation and conformational changes of fatty acid synthase from chicken liver during unfolding by sodium dodecyl sulfate

Fatty acid synthase is an important enzyme participating in energy metabolism in vivo. The inactivation and conformational changes of the multifunctional fatty acid synthase from chicken liver in SDS solutions have been studied. The results show that the denaturation of this multifunctional enzyme by SDS occurred in three stages. At low concentrations of SDS (less than 0.15 mM) the enzyme was completely inactivated with regard to the overall reaction. For each component of the enzyme, the loss of activity occurred at higher concentrations of SDS. Significant conformational changes (as indicated by the changes of the intrinsic fluorescence emission and the ultraviolet difference spectra) occurred at higher concentrations of SDS. Increasing the SDS concentration caused only slight changes of the CD spectra, indicating that SDS had no significant effect on the secondary structure of the enzyme. The results suggest that the active sites of the multifunctional fatty acid synthase display more conformational flexibility than the enzyme molecule as a whole.     

Polymorphism of the caprine malic enzyme 1 ( ME1) gene and its association with milk quality traits in Murciano-Granadina goats

Malic enzyme 1 plays a fundamental role in lipid metabolism because it yields a significant amount of the NADPH necessary for fatty acid biosynthesis. In ruminants, however, its contribution to this biochemical process seems to be less relevant than in other livestock species. In this study, we have partially sequenced the goat ME1 gene with the aim of identifying polymorphic sites. Structural analysis of the goat ME1 amino acid partial sequence evidenced the existence of two dinucleotide-binding motifs, located at positions 158 to 163 (GLGDLG) and 301 to 306 (GAGEAA), and three amino acid residues (E245, D246 and D269) involved in the binding of Mn2+ and strongly conserved among mammalian species. Moreover, multiple sequence alignment allowed us to identify four single nucleotide polymorphisms at exons 5 (c.483C > T), 6 (c.667G > A), 9 (c.927C > T) and 11 (c.1200G > A). The effects of ME1 genotype on milk production and composition traits were investigated in a Murciano-Granadina goat population. None of the associations found reached statistical significance after applying Benjamini-Hochberg correction. At most, associations with uncorrected P-values below 0.01 were observed for C16:0, C18:1n-9t and total conjugated linoleic acids. These negative results reinforce the notion that ME1 plays an ancillary role in ruminant lipogenesis.     

Association analyses of single nucleotide polymorphisms in bovine stearoyl-CoA desaturase and fatty acid synthase genes with fatty acid composition in commercial cross-bred beef steers

Two previously reported non‐synonymous coding single nucleotide polymorphisms (SNPs) of bovine stearoyl‐CoA desaturase (delta‐9‐desaturase) (SCD) (c.878C>T) and fatty acid synthase (FASN) (g:17924A>G) were assessed for their associations with 72 individual and 12 groups of fatty acids in brisket adipose tissue of 223 Canadian commercial cross‐bred beef steers. It was found that the ‘CC’ genotype of the SCD SNP was significantly associated with lower concentrations of saturated fatty acids (SFA) including 10:0, 14:0 and 20:0, higher concentrations of monounsaturated fatty acids including 9c‐14:1, 12c‐16:1 and 13c‐18:1, higher concentrations of polyunsaturated fatty acids (PUFA) including 9c,15c‐18:2, 10c,12c‐18:2, 11c,13t‐18:2 and 12c,14t‐18:2, but lower concentrations of other PUFA of 9c,13t/8t,12c and 20:2n‐6 (P < 0.05). The ‘AA’ genotype of the FASN SNP was significantly associated with higher concentrations of SFAs of 10:0, 12:0, 13:0, 14:0 and 15:0, lower concentrations of unsaturated fatty acids of 9c‐18:1 and 20:3n‐6, and higher concentrations of unsaturated fatty acids of 9c‐14:1 and 12c‐16:1 (P < 0.05). Significant epistatic effects between the SCD and FASN SNP genotypes were also found for several fatty acids including 10:0, 23:0, 6t/7t/8t‐18:1, 12t‐18:1, 13t/14t‐18:1, 16t‐18:1, total trans18:1 and 9c,13t/8t,12c‐18:2 (P < 0.05). These results further suggest that SCD and FASN are strong candidate genes influencing fatty acid composition in beef cattle.     

人源脂肪酸合酶在酿酒酵母中的表达纯化和结构的初步分析

[背景] 聚酮类化合物在医药领域有重要的应用,相关药物研发依赖聚酮合酶多变的结构认知,人源脂肪酸合酶的组成结构和催化机制与聚酮合酶相近,研究人源脂肪酸合酶结构可为聚酮合酶的研究奠定基础。[目的] 在酿酒酵母中表达纯化人源脂肪酸合酶蛋白,确定合适的体外纯化条件。[方法] 以酿酒酵母BJ5464为表达载体,构建带有His和Strep双亲和层析标签的重组质粒,诱导表达蛋白后用亲和层析方法获取目标蛋白,并结合凝胶电泳和快速蛋白质液相层析技术,确定合适的蛋白纯化条件。[结果] 成功构建重组表达质粒pxw55-hfas-cSHII, 并在体外纯化得到合适浓度和纯度的人源脂肪酸合酶蛋白,筛选不同缓冲液条件并结合电子显微镜观察结果反馈,确定合适的蛋白体外纯化体系。[结论] 蛋白电镜结构分析需要有高纯度、合适浓度并且形成正确构象的蛋白样品,而人源脂肪酸合酶蛋白纯化体系的建立和纯化条件的确定为其电镜结构分析提供了良好的样品,为人源脂肪酸合酶的结构解析及结构相似但更为复杂的聚酮合酶蛋白解析奠定了良好基础。     

Molecular docking study of the interactions between the thioesterase domain of human fatty acid synthase and its ligands

Human fatty acid synthase (hFAS) thioesterase domain (TE) is an attractive drug target to treat obesity and cancer. On the basis of the recently published crystal structure of TE domain of hFAS, we performed molecular surface analysis and docking study to characterize the molecular interactions between the enzyme and its various ligands. Surface analysis identified the ligand-binding pocket of TE domain that encompasses the catalytic triad of Ser2308, His2481, Asp2338. Docking of palmitate, the main biological product of hFAS, into this pocket revealed the ligand-binding mode, in which the hydrophobic interactions are the dominant driving forces. The catalytic mechanism of TE domain can also be well explained based on the generated TE-palmitate complex structure. Moreover, the comparison of the binding modes of five fatty acids with chain lengths ranging from 12 to 20 carbons confirmed that the ligand binding pocket of TE domain is a decisive factor in chain length specificity. In addition, docking of two known TE inhibitors, c75 and orlistat revealed the pharmacophore of these hFAS TE inhibitors, which will prove useful in structure-based drug design against this important target.     

Effect of arachidonic acid,fatty acids,prostaglandins, and leukotrienes on volume regulation in Ehrlich ascites tumor cells

Summary Arachidonic acid inhibits the cell shrinkage observed in Ehrlich ascites tumor cells during regulatory volume decrease (RVD) or after addition of the Ca ionophore A23187 plus Ca. In Na-containing media, arachidonic acid increases cellular Na uptake under isotonic as well as under hypotonic conditions. Arachidonic acid also inhibits KCl and water loss following swelling in Na-free, hypotonic media even when a high K conductance has been ensured by addition of gramicidin. In isotonic, Na-free medium arachidonic acid inhibits A23187 + Ca-induced cell shrinkage in the absence but not in the presence of gramicidin. It is proposed that inhibition of RVD in hypotonic media by arachidonic acid is caused by reduction in the volume-induced Cl and K permeabilities as well as by an increase in Na permeability and that reduction in A23187 + Ca-induced cell shrinkage is due to a reduction in K permeability and an increase in Na permeability. The A23187 + Ca-activated Cl permeability in unaffected by arachidonic acid. PGE₂ inhibits RVD in Na-containing, hypotonic media but not in Na-free, hypotonic media, indicating a PGE₂-induced Na uptake. PGE₂ has no effect on the volume-activated K and Cl permeabilities. LTB₄, LTC₄ and LTE₄ inhibit RVD insignificantly in hypotonically swollen cells. LTD₄, more-over, induces cell shrinkage in steady-state cells and accelerates the RVD following hypotonic exposure. The effect of LTD₄ even reflects a stimulating effect on K and Cl transport pathways. Thus none of the leukotrienes show the inhibitory effect found for arachidonic acid on the K and Cl permeabilities. The RVD response in hypotonic, Na-free media is, on the other hand, also inhibited by addition of the unsaturated oleic, linoleic, linolenic and palmitoleic acid, even in the presence of the cationophor gramicidin. The saturated arachidic and stearic acid had no effect on RVD. It is, therefore, suggested that a minor part of the inhibitory effect of arachidonic acid on RVD in Na-containing media is via an increased synthesis of prostaglandins and that the major part of the arachidonic acid effect on RVD in Na-free media, and most probably also in Na-containing media, is due to the inhibition of the volume-induced K and Cl transport pathways, caused by a nonspecific detergent effect of an unsaturated fatty acid.     

Inhibition of fatty acid synthase by ginkgolic acids from the leaves of Ginkgo biloba and their cytotoxic activity

Fatty acid synthase (FAS) has been proposed to be a new drug target for the development of anticancer agents because of the significant difference in expression of FAS between normal and tumour cells. Since a n-hexane-soluble extract from Ginkgo biloba was demonstrated to inhibit FAS activity in our preliminary test, we isolated active compounds from the n-hexane-soluble extract and evaluated their cytotoxic activity in human cancer cells. Three ginkgolic acids 1–3 isolated from the n-hexane-soluble extract inhibited the enzyme with IC₅₀ values 17.1, 9.2 and 10.5 µM, respectively, and they showed cytotoxic activity against MCF-7 (human breast adenocarcinoma), A549 (human lung adenocarcinoma) and HL-60 (human leukaemia) cells. Our findings suggest that alkylphenol derivatives might be a new type of FAS inhibitor with cytotoxic activity.     

Role of fatty acid synthase in gemcitabine and radiation resistance of pancreatic cancers

Human fatty acid synthase (FASN) is a homo-dimeric protein with multi-enzymatic activity responsible for the synthesis of palmitate. FASN expression has been found to be up-regulated in multiple types of human cancers and its expression correlates with poor prognosis possibly by causing treatment resistance. In this study, we tested if FASN expression is up-regulated in human pancreatic cancers and if its higher expression level in pancreatic cancers causes intrinsic resistance to gemcitabine and radiation. We found that FASN expression is significantly up-regulated in human pancreatic cancer tissues without any correlation to age, sex, race, and tumor stage. Knocking down or over-expressing FASN significantly down- or up-regulate resistance of pancreatic cancer cell lines to both gemcitabine and radiation treatments. These findings imply that the elevated FASN expression in pancreatic cancers may contribute to unsuccessful treatments of pancreatic cancers by causing intrinsic resistance to both chemotherapy and radiation therapy.     

生淀粉糖化酶催化位点氨基酸及酶合成调控的初步研究

通过对Rhizopus OR-1UVN菌种所产生淀粉糖化酶在不同底物不同缓冲溶液条件下酶最适pH的测定,推测出该生淀粉糖化酶活力中心催化位点氨基酸是天冬氨酸(Asp)和谷氨酸(Glu)。实验证明5～50mg/mL浓度葡萄糖对生淀粉糖化酶没有抑制作用。分别以浓度<5mg/mL葡萄糖和淀粉为碳源的培养基进行不同碳源发酵实验,发现以淀粉为碳源的培养基Ⅰ发酵15h开始产生淀粉糖化酶,以葡萄糖为碳源的培养基Ⅱ发酵35h开始产酶(葡萄糖浓度<8mg/mL),而且前者菌体较后者少,由此可知葡萄糖对产酶有阻遏作用。实验还发现解阻遏熟淀粉糖化酶的葡萄糖浓度(15mg/mL)比生淀粉糖化酶的要高。由于葡萄糖的阻遏作用不发生在翻译水平,而发生在转录水平上,而且生淀粉糖化酶(G1)与熟淀粉糖化酶(G2)来自同一条DNA链,可以推测存在mRNA的拼接。通过以生淀粉为碳源的比较实验,发现生淀粉对生淀粉糖化酶形成的诱导作用可能主要是通过mRNA拼接的调节来实现的。     

Insulin-like effects of vanadate and selenate on the expression of glucose-6-phosphate dehydrogenase and fatty acid synthase in diabetic rats

Isulin is capable of regulating cellular and metabolic processes as well as gene expression. In recent years, enthusiasm has surfaced for using insulin mimetics to study the mechanism of action of insulin. Vanadata and selenate are two compounds that have been found to mimic the action of insulin on control to blood glucose levels in vivo. Vanadata has also been shown to regulate the expression of several enzymes both in vivo, however, studies concerning selenate's ability to regulate expression have not been reported. In his study we show that administration of vanadate or selenate to streptozotocin-induced diabetic rats not only normalizes blood glucose levels similarly to insulin but also positively affects the expression of two key metabolic enzymes, glucose-6-phosphate dehydrogenase (G6PDH) and fatty acid synthase (FAS). Both G6PDH and FAS activity are significantly decreased in diabetic animals compared to non-diabetic control. Treatment of the diabetic animals with either insulin, vanadate or selenate restored both activities to about 80–90% of control. All treatment conditions exhibited activities significantly higher than those determined for the diabetic group but did not differ significantly from each other. Increases in GPDH or FAS activity are due to increases in mRNA level. Increase in both G6PDH and FAS mRNA was comparable to the observed increase in activity suggesting that regulation of expression by the mimetics occurs pretranslationally.     

Mitochondrial malic enzyme (ME2) in pancreatic islets of the human, rat and mouse and clonal insulinoma cells: Simple enzyme assay for mitochondrial malic enzyme 2

Despite interest in malic enzyme(ME)s in insulin cells, mitochondrial malic enzyme (ME2) has only been studied with estimates of mRNA or with mRNA knockdown. Because an mRNA’s level does not necessarily reflect the level of its cognate enzyme, we designed a simple spectrophotometric enzyme assay to measure ME2 activity of insulin cells by utilizing the distinct kinetic properties of ME2. Mitochondrial ME2 uses either NAD or NADP as a cofactor, has a high K_m for malate and is allosterically activated by fumarate and inhibited by ATP. Cytosolic ME (ME1) and the other mitochondrial ME (ME3) use only NADP as a cofactor and have lower K_ms for malate. The assay easily showed for the first time that substantial ME2 activity is present in pancreatic islets of humans, rats and mice and INS-1 832/13 cells. ME2’s presence was confirmed with immunoblotting. There was no evidence that ME3 is present in these tissues.     

Mechanisms for elongation in the biosynthesis of fatty acid components of epi-cuticular waxes

It is known that branched-chain amino acids can serve as precursors to iso- and anteiso-branched components of epi-cuticular waxes. Keto acid deamination products of Val, Leu and Ile are thought to serve as primers which are elongated by fatty acid synthase. However, the origin of elongation carbons has not been studied directly. Nor has the mechanism for formation of odd-carbon-length, straight- or branched-chain, cuticular ester fatty acids or free odd-carbon-length, straight fatty acid components of waxes been characterized. It is not known that α-oxidation of even-length precursors or elongation of odd-length primers is involved in these cases. Here, we present evidence which substantiates the expectation that elongation of branched as well as straight-chain precursors to wax ester acids occurs by fatty acid synthase catalyzed by addition of two carbon units via acetate. Also, we present evidence which indicates that odd-carbon-length acids can result from elongation of odd-carbon-length primers (at least branched), rather than even-length acids shortened by α-oxidation.