Chemical Probes to Directly Profile Palmitoleoylation of Proteins

Palmitoleoylation is a unique fatty acylation of proteins in which a monounsaturated fatty acid, palmitoleic acid (C16:1), is covalently attached to a protein. Wnt proteins are known to be palmitoleoylated by cis‐Δ9 palmitoleate at conserved serine residues. O‐palmitoleoylation plays a critical role in regulating Wnt secretion, binding to the receptors, and in the dynamics of Wnt signaling. Therefore, protein palmitoleoylation is important in tissue homeostasis and tumorigenesis. Chemical probes based on saturated fatty acids, such as ω‐alkynyl palmitic acid (Alk‐14 or Alk‐C₁₆), have been used to study Wnt palmitoleoylation. However, such probes require prior conversion to the unsaturated fatty acid by stearoyl‐CoA desaturase (SCD) in cells, significantly decreasing their selectivity and efficiency for studying protein palmitoleoylation. We synthesized and characterized ω‐alkynyl cis‐ and trans‐palmitoleic acids (cis‐ and trans‐Alk‐14:1) as chemical probes to directly study protein palmitoleoylation. We found that cis‐Alk‐14:1 could more efficiently label Wnt proteins in cells. Interestingly, the DHHC family of palmitoyl acyltransferases can charge both saturated and unsaturated fatty acids, potentially using both as acyl donors in protein palmitoylation and palmitoleoylation. Furthermore, proteomic analysis of targets labeled by these probes revealed new cis‐ and trans‐palmitoleoylated proteins. Our studies provided new chemical tools and revealed new insights into palmitoleoylation in cell signaling.     

Characterisation of CYP102A25 from Bacillus marmarensis and CYP102A26 from Pontibacillus halophilus: P450 Homologues of BM3 with Preference towards Hydroxylation of Medium‐Chain Fatty Acids

Cytochrome P450 monooxygenases are highly desired biocatalysts owing to their ability to catalyse a wide variety of chemically challenging C?H activation reactions. The CYP102A subfamily of enzymes are natural catalytically self‐sufficient proteins consisting of a haem and FMN‐FAD reductase domain fused in a single‐component system. They catalyse the oxygenation of saturated and unsaturated fatty acids to produce primarily ω?1, ω?2 and ω?3 hydroxy acids. These monooxygenases have potential applications in biotechnology; however, their substrate range is still limited and there is a continued need to add diversity to this class of biocatalysts. Herein, we present the characterisation of two new members of this class of enzymes, CYP102A25 (BMar) from Bacillus marmarensis and CYP102A26 (PHal) from Pontibacillus halophilus, both of which express readily in a recombinant bacterial host. BMar exhibits the highest activity toward myristic acid and shows moderate activity towards unsaturated fatty acids. PHal exhibits broader activity towards mid‐chain‐saturated (C₁₄–C₁₈) and unsaturated fatty acids. Furthermore, PHal shows good regioselectivity for the hydroxylation of myristic acid, targeting the ω?2 position for C?H activation.     

Fatty Acyl Sulfonyl Fluoride as an Activity-Based Probe for Profiling Fatty Acid-Associated Proteins in Living Cells

Fatty acids play fundamental structural, metabolic, functional, and signaling roles in all biological systems. Altered fatty acid levels and metabolism have been associated with many pathological conditions. Chemical probes have greatly facilitated biological studies on fatty acids. Herein, we report the development and characterization of an alkynyl-functionalized long-chain fatty acid-based sulfonyl fluoride probe for covalent labelling, enrichment, and identification of fatty acid-associated proteins in living cells. Our quantitative chemical proteomics show that this sulfonyl fluoride probe targets diverse classes of fatty acid-associated proteins including many metabolic serine hydrolases that are known to be involved in fatty acid metabolism and modification. We further validate that the probe covalently modifies the catalytically or functionally essential serine or tyrosine residues of its target proteins and enables evaluation of their inhibitors. The sulfonyl fluoride-based chemical probe thus represents a new tool for profiling the expression and activity of fatty acid-associated proteins in living cells.     

Sources and Bioactive Properties of Conjugated Dietary Fatty Acids

The group of conjugated fatty acids known as conjugated linoleic acid (CLA) isomers have been extensively studied with regard to their bioactive potential in treating some of the most prominent human health malignancies. However, CLA isomers are not the only group of potentially bioactive conjugated fatty acids currently undergoing study. In this regard, isomers of conjugated α‐linolenic acid, conjugated nonadecadienoic acid and conjugated eicosapentaenoic acid, to name but a few, have undergone experimental assessment. These studies have indicated many of these conjugated fatty acid isomers commonly possess anti‐carcinogenic, anti‐adipogenic, anti‐inflammatory and immune modulating properties, a number of which will be discussed in this review. The mechanisms through which these bioactivities are mediated have not yet been fully elucidated. However, existing evidence indicates that these fatty acids may play a role in modulating the expression of several oncogenes, cell cycle regulators, and genes associated with energy metabolism. Despite such bioactive potential, interest in these conjugated fatty acids has remained low relative to the CLA isomers. This may be partly attributed to the relatively recent emergence of these fatty acids as bioactives, but also due to a lack of awareness regarding sources from which they can be produced. In this review, we will also highlight the common sources of these conjugated fatty acids, including plants, algae, microbes and chemosynthesis.     

不饱和脂肪酸及其衍生物环氧化研究进展

综述了环氧化不饱和脂肪酸及其衍生物的制备工艺和实验方法,包括过氧酸环氧化法、卤代醇环氧化法、过氧化氢或有机过氧化氢环氧化法、分子氧环氧化法。其中使用过氧酸的方法是工业上使用最为广泛的,可以采用酸或者酶作催化剂;而以过氧化氢为氧化剂的固体催化工艺是目前最有工业化前途的技术。并指出了开发新型、高效、绿色且廉价的催化体系仍是今后的研究方向。     

Volatile fatty acids in flavors of potatoes deep-fried in a beef blend

The volatile free fatty acids were analyzed in commercial french- fried potatoes that had been deep- fried in beef tallow- hydrogenated vegetable oil shortening. The results showed that many of the volatile fatty acids present in beef tallow were transferred to the potatoes. Of the fatty acids derived from beef tallow, butanoic, 2-methylbutanoic, 3- methylbutanoic, heptanoic, 4-methyltanoic, and nonanoic acids were present in concentrations above their respective thresholds, and should contribute to tallow- like flavors. Several unsaturated volatile fatty acids also were identified, and they should contribute to the general deep- fried potato flavor.     

Myeloperoxidase-Derived 2-Chlorohexadecanal Is Generated in Mouse Heart during Endotoxemia and Induces Modification of Distinct Cardiomyocyte Protein Subsets In Vitro

Sepsis is a major cause of mortality in critically ill patients and associated with cardiac dysfunction, a complication linked to immunological and metabolic aberrations. Cardiac neutrophil infiltration and subsequent release of myeloperoxidase (MPO) leads to the formation of the oxidant hypochlorous acid (HOCl) that is able to chemically modify plasmalogens (ether-phospholipids) abundantly present in the heart. This reaction gives rise to the formation of reactive lipid species including aldehydes and chlorinated fatty acids. During the present study, we tested whether endotoxemia increases MPO-dependent lipid oxidation/modification in the mouse heart. In hearts of lipopolysaccharide-injected mice, we observed significantly higher infiltration of MPO-positive cells, increased fatty acid content, and formation of 2-chlorohexadecanal (2-ClHDA), an MPO-derived plasmalogen modification product. Using murine HL-1 cardiomyocytes as in vitro model, we show that exogenously added HOCl attacks the cellular plasmalogen pool and gives rise to the formation of 2-ClHDA. Addition of 2-ClHDA to HL-1 cardiomyocytes resulted in conversion to 2-chlorohexadecanoic acid and 2-chlorohexadecanol, indicating fatty aldehyde dehydrogenase-mediated redox metabolism. However, a recovery of only 40% indicated the formation of non-extractable (protein) adducts. To identify protein targets, we used a clickable alkynyl analog, 2-chlorohexadec-15-yn-1-al (2-ClHDyA). After Huisgen 1,3-dipolar cycloaddition of 5-tetramethylrhodamine azide (N₃-TAMRA) and two dimensional-gel electrophoresis (2D-GE), we were able to identify 51 proteins that form adducts with 2-ClHDyA. Gene ontology enrichment analyses revealed an overrepresentation of heat shock and chaperone, energy metabolism, and cytoskeletal proteins as major targets. Our observations in a murine endotoxemia model demonstrate formation of HOCl-modified lipids in the heart, while pathway analysis in vitro revealed that the chlorinated aldehyde targets specific protein subsets, which are central to cardiac function.     

气相色谱-质谱法分析山楂中的脂肪酸

为了促进山楂及山楂核在药物、精细化工领域的应用,用气相色谱-质谱联用仪,改良脂肪酸甲酯化法检测出山楂果肉中的7种脂肪酸,分别为棕榈酸、亚油酸、油酸、亚麻酸、硬脂酸、花生酸及山嵛酸。用CSASS软件进行峰识别及确定峰面积,峰面积归一化法计算每种脂肪酸的相对质量分数。分析测定了河北、安徽、山东、吉林、辽宁5个产地山楂药材中的脂肪酸相对质量分数,各产地去核山楂药材均以不饱和脂肪酸为主,其相对质量分数分别为59.10%、61.18%、63.08%、59.76%、60.76%。对河北、安徽、辽宁3个产地山楂核中的脂肪酸成分进行了较系统全面的研究,其脂肪酸以不饱和脂肪酸为主,相对质量分数分别为89.80%、61.59%、79.55%,均高于同一产地去核山楂中的不饱和脂肪酸相对质量分数。     

Zeolite‐catalyzed isomerization of oleic acid to branched‐chain isomers

Branched‐chain (bc) saturated fatty acids (SFA) have potential as oleochemical intermediates since they have better oxidative stability than linear unsaturated fatty acids (UFA) and have better low‐temperature properties than linear SFA. Previous studies in converting UFA to bc‐FA using clay catalysts have resulted in only modest yields and conversions. Recent reports, however, have suggested that certain zeolites can be effective catalysts for converting UFA to bc‐FA in higher yields and conversions. In this work, we examined the scope and potential of the zeolite‐catalyzed synthesis of bc‐FA starting from readily available monounsaturated linear FA. Our results show that common UFA such as oleic acid can be converted to bc‐isomers using modified Ferrierite zeolite catalysts with high conversions (98%) and high selectivity (85%) and that the zeolite catalysts are reusable for at least three cycles. The positions of branching (methyl) on the FA chain were determined from the GC‐MS spectra of the picolinyl esters of the bc‐FA.     

Quantification of Fatty Acids and their Regioisomeric Distribution in Triacylglycerols from Porcine and Bovine Sources Using 13C NMR Spectroscopy

The uptake of specific fatty acids in humans is dependent on their position on the glycerol backbone. There is a great interest in methods that can access this information fast and accurately. By way of high-resolution NMR, we have analyzed TAG extracted from pig and beef tissues and obtained quantitative data for the composition and regioisomeric distribution of all major unsaturated fatty acids usually found in these source materials, using a combination of manual integration and deconvolution of ¹³C NMR spectra. In addition, we have developed a method for determining composition and regioisomeric distribution of the two main saturated fatty acids found in pork (16:0, 18:0). The results are discussed in relation to species-specific genetic characteristics of fatty acid and TAG biosynthesis. The developed method could support decisions related to breeding for desired fatty acid profiles, and stimulate further methodology developments using high field NMR.     

Production of hydroxy fatty acids from unsaturated fatty acids byFlavobacterium sp. DS5 hydratase,a C-10 positional- andcis unsaturation-specific enzyme

A new microbial isolate,Flavobacterium sp. DS5, converted oleic and linoleic acids to their corresponding 10-keto-and 10-hydroxy fatty acids. The hydration enzyme seems to be specific to the C-10 position. Conversion products from α- and γ-linolenic acids were identified by gas chromatography/mass spectrometry, Fourier transform infrared, and nuclear magnetic resonance as 10-hydroxy-12(Z),15(Z)-octadecadienoic and 10-hydroxy-6(Z),12(Z)-octadecadienoic acids, respectively. Products from other 9(Z)-unsaturated fatty acids also were identified as their corresponding 10-hydroxy- and 10-keto-fatty acids.Trans unsaturated fatty acid was not converted. From these results, it is concluded that strain DS5 hydratase is indeed a C-10 positional-specific andcis-specific enzyme. DS5 hydratase prefers an 18-carbon monounsaturated fatty acid. Among the C₁₈ unsaturated fatty acids, an additional double bond at either side of the 9,10-position lowers the enzyme hydration activity. Because hydratases from other microbes also convert 9(Z)-unsaturated fatty acids to 10-hydroxy fatty acids, the C-10 positional specificity of microbial hydratases may be universal.     

The cytotoxicity of fatty acid/α‐lactalbumin complexes depends on the amount and type of fatty acid

Complexes of the milk protein, α‐lactalbumin, and the fatty acid, oleic acid, have previously been shown to be cytotoxic. Complexes of α‐lactalbumin and five different fatty acids (vaccenic, linoleic, palmitoleic, stearic, and elaidic acid) were prepared and compared to those formed with oleic acid. All complexes were cytotoxic to human promyelocytic leukemia‐derived (HL‐60) cells but to different degrees depending on the fatty acid. The amount of fatty acid per α‐lactalbumin molecule was found to correlate with the cytotoxicity; the higher the number of fatty acids per protein, the more cytotoxic the complex. Importantly, all the tested fatty acids were also found to be cytotoxic on their own in a concentration dependent manner. The cytotoxic effect of complexes between α‐lactalbumin and linoleic acid, vaccenic acid, or oleic acid was further investigated using flow cytometry and found to induce cell death resembling apoptosis on Jurkat cells. Practical applications: Cytotoxic complexes of α‐lactalbumin and several different fatty acids could be produced. The cytotoxicity of all the variants is similar to that previously determined for α‐lactalbumin/oleic acid complexes.     

Fatty acid profile in baby food products

The fatty acid composition in the lipid phase of 64 commercially available baby food products, of two different batches each, was analyzed. They comprised vegetable products for babies of five, eight, and twelve months and fruit and cereal products of three different brands. The comparison of the composition of the saturated (C18:0, C16:0, C14:0, C12:0, C10:0), the unsaturated monoenoic (C18:1n9 and C16:1n7) and the polyenoic (C18:2n6 and C18:3n3) fatty acids was determined by gas chromatography. All analyzed baby food products provided well‐balanced amounts of saturated fatty acids on the one hand (saturated fatty acids (SFA) 31—37% of total fatty acids) and unsaturated fatty acids on the other hand (monounsaturated fatty acids (MUFA) 23—26% and polyunsaturated fatty acids (PUFA) 38—46% of total fatty acids, respectively). The P/S‐ratio in vegetable products of five months reached a value of 1.5, in all other analyzed products it was around 1. The n‐6:n‐3‐ratio was 10:1 in fruit and cereal products, followed by 11.6:1 in vegetable products of eight and twelve months and 13.5:1 in the group of vegetable products of five months. Since there is a lack of arachidonic acid and docosahexaenoic acid in baby food products, it might be of advantage to consider whether such products should be supplemented by these long‐chain polyunsaturated fatty acids.     

正常小鼠和肿瘤小鼠毛发中长链脂肪酸含量测定及比较

以正十七酸为内标 ,氯化氢 甲醇抽提和酯化 ,对正常、肿瘤、抗肿瘤小鼠毛发中长链脂肪酸——豆蔻酸、棕榈酸、棕榈油酸、油酸、硬脂酸和亚油酸进行毛细管柱气相色谱定量分析和比较。结果显示 :肿瘤小鼠或抗肿瘤小鼠毛发中长链脂肪酸含量明显升高 ,不饱和长链脂肪酸比例升高而饱和长链脂肪酸比例下降 ,提示毛发中不饱和长链脂肪酸尤其亚油酸与生物体内肿瘤的发生和存在有密切关系。本实验方法简单 ,所需样品量少 ,实验重现性好 ,回收率达定量分析要求。     

Gut Microbial Fatty Acid Metabolites Reduce Triacylglycerol Levels in Hepatocytes

Hydroxy and oxo fatty acids were recently found to be produced as intermediates during gut microbial fatty acid metabolism. Lactobacillus plantarum produces these fatty acids from unsaturated fatty acids such as linoleic acid. In this study, we investigated the effects of these gut microbial fatty acid metabolites on the lipogenesis in liver cells. We screened their effect on sterol regulatory element binding protein‐1c (SREBP‐1c) expression in HepG2 cells treated with a synthetic liver X receptor α (LXRα) agonist (T0901317). The results showed that 10‐hydroxy‐12(Z)‐octadecenoic acid (18:1) (HYA), 10‐hydroxy‐6(Z),12(Z)‐octadecadienoic acid (18:2) (γHYA), 10‐oxo‐12(Z)‐18:1 (KetoA), and 10‐oxo‐6(Z),12(Z)‐18:2 (γKetoA) significantly decreased SREBP‐1c mRNA expression induced by T0901317. These fatty acids also downregulated the mRNA expression of lipogenic genes by suppressing LXRα activity and inhibiting SREBP‐1 maturation. Oral administration of KetoA, which effectively reduced triacylglycerol accumulation and acetyl‐CoA carboxylase 2 (ACC2) expression in HepG2 cells, for 2 weeks significantly decreased Srebp‐1c, Scd‐1, and Acc2 expression in the liver of mice fed a high‐sucrose diet. Our findings suggest that the hypolipidemic effect of the fatty acid metabolites produced by L. plantarum can be exploited in the treatment of cardiovascular diseases or dyslipidemia.     

“Dicranin” in the Membrane Phospholipids of a Dicranaceae and Pottiaceae Moss Member of the Eastern Himalayan Biodiversity Hotspot

The phospholipids of two moss samples Oreoweisia laxifolia (Hookf.) Kindb. (family‐Dicranaceae Schimp.) and Leptodontium viticulosoides (P. Beauv.) Wijk & Margad (family‐Pottiaceae Schimp.) of the Eastern Himalayan Biodiversity Hotspot were investigated to find out any peculiarity in their fatty acid profiles. Detailed analysis of phospholipid classes and the respective fatty acids was performed using high‐performance thin‐layer chromatography and gas chromatography‐mass spectrometry. An array of different saturated and unsaturated fatty acids were detected in both the samples. Although it has been proposed previously that acetylenic fatty acids are associated only with triacylglycerol of storage lipids, the most striking observation of the present investigation is the abundance of an acetylenic fatty acid, octadeca‐6‐yn‐9,12,15‐trienoic acid (18:4a), or Dicranin, in the phospholipids of both the mosses. The position of the triple bond in the hydrocarbon chain of the fatty acids was confirmed by dimethyloxazoline derivatization of fatty acids and their characteristic mass fragmentation pattern. The occurrence of Dicranin in phospholipids and in the Pottiaceae family is reported for the first time, with substantial explanations of the observed results. This may raise the issue of rethinking “Dicranin” as a chemotaxonomic marker of Dicranaceae.     

Δ6 Desaturase mRNA Abundance in HepG2 Cells Is Suppressed by Unsaturated Fatty Acids

The effect of unsaturated fatty acids on the abundance of Δ6 desaturase (D6D) mRNA and the fatty acid composition of HepG2 cell membranes was examined. Supplementation of HepG2 cells with oleic acid (18:1n-9, OA), linoleic acid (18:2n-6, LA), α-linolenic acid (18:3n-3, ALA), arachidonic acid (20:4n-6, AA) or eicosapentaenoic acid (20:5n-3, EPA) reduced D6D mRNA abundance by 39 ± 6.6, 40 ± 2.2, 31 ± 5.2, 55 ± 4.8, and 52 ± 5.0%, respectively, compared with control cells. Despite the reduction in D6D mRNA abundance, the level of D6D conversion products (20:3n-9, EPA and AA) in OA, ALA and LA supplemented cells, respectively, was elevated above that in control cells. Our results suggest that although unsaturated fatty acids decrease the abundance of D6D mRNA by as much as 50%, the conversion of polyunsaturated fatty acids and accumulation of long chain polyunsaturated fatty acids (LCPUFA) in HepG2 cell phospholipids continues to occur.     

One-Step Concentration of Highly Unsaturated Fatty Acids from Tuna Oil by Low-Temperature Crystallization

Highly unsaturated fatty acids (HUFA), including eicosapentaenoic acid (EPA, 20:5n‐3), docosapentaenoic acid (DPA, 22:5n‐3 and 22:5n‐6) and docosahexaenoic acid (DHA, 22:6n‐3), play an important role in human health and nutrition. In this study, concentration of HUFA in free fatty acids (FFA) form by low‐temperature crystallization was investigated. For this purpose, tuna oil (7.1% EPA, 26.8% DHA) was first converted into corresponding FFA. Subsequently, crystallization conditions of various solvent types, the ratio of FFA to acetonitrile, operation temperature and crystallization time were optimized at a small scale of 2 g tuna oil fatty acids. Taking purity and yield into account, the optimum conditions were a 1:10 ratio of FFA to acetonitrile (w/v), ?60 °C, and 1 h. The optimal conditions resulted in concentrations of EPA, DHA and HUFA of 15.1, 58.4 and 79.6%, respectively, with corresponding yields of 61.5, 61.8 and 60.7%, respectively. Crystallization was carried out under the optimal conditions at a large scale of 200 g tuna oil FFA, and a similar concentration result was achieved. After evaporating away the solvent, the residual amount of acetonitrile met the US Pharmacopoeia requirement of <410 ppm. The process for enrichment of HUFA is readily scalable, effective and time‐saving.