Global analysis of N-myristoylation and its heterogeneity by combining N-terminomics and nanographite fluoride-based solid-phase extraction.

N-myristoylation is one of the most widespread and important lipidation in eukaryotes and some prokaryotes, which is formed by covalently attaching various fatty acids (predominantly myristic acid C14:0) to the N-terminal glycine of proteins. Disorder of N-myristoylation is critically implicated in numerous physiological and pathological processes. Here, we presented a method for purification and comprehensive characterization of endogenous, intact N-glycine lipid-acylated peptides, which combined the negative selection method for N-terminome and the nanographite fluoride-based solid-phase extraction method (NeS-nGF SPE). After optimizing experimental conditions, we conducted the first global profiling of the endogenous and heterogeneous modification states for N-terminal glycine, pinpointing the precise sites and their associated lipid moieties. Totally, we obtained 76 N-glycine lipid-acylated peptides, including 51 peptides with myristate (C14:0), 10 with myristoleate (C14:1), 6 with tetradecadienoicate (C14:2), 5 with laurate (C12:0) and 4 with lauroleate (C12:1). Therefore, our proteomic methodology could significantly facilitate precise and in-depth analysis of the endogenous N-myristoylome and its heterogeneity.

A metabolic labeling protocol to enrich myristoylated proteins from Caenorhabditis elegans.

Myristoylation is a type of lipidation with important functions. Owing to the lack of high-quality antibodies against myristoylation, developing alternative methods for profiling myristoylated proteins is important. Here, we provide a protocol for metabolic labeling using click chemistry to profile myristoylated proteins in C. elegans. Our approach improves the signal/noise ratio by covalently linking the myristoylated proteins to the beads. This protocol provides a highly specific and reproducible way for enriching myristoylated proteins, which could be modified to analyze other types of lipidations. For complete details on the use and execution of this protocol, please refer to Tang et al. (2021).

Transfer of Lauric and Myristic Acid from Black Soldier Fly Larval Lipids to Egg Yolk Lipids of Hens Is Low.

Implementing insects, such as the black soldier fly larvae (BSFL), as animal feed commonly includes the previous removal of substantial amounts of fat. This fat may represent an as yet underutilized energy source for livestock. However, transfer of lauric and myristic acid, prevalent in BSFL fat and undesired in human nutrition, into animal-source foods like eggs may limit its implementation. To quantify this, a laying hen experiment was performed comprising five different diets (10 hens/diet). These were a control diet with soybean oil and meal and a second diet with soybean oil but with partially defatted BSFL meal as protein source. The other three diets were based on different combinations of partially defatted BSFL meal and fat obtained by two different production methods. Lauric acid made up half of the BSFL fat from both origins. Both BSFL fats also contained substantial amounts of myristic and palmitic acid. However, in the insect-based diets, the net transfer from diet to egg yolk was less than 1% for lauric acid, whereas the net transfer for myristic and palmitic acid was about 30% and 100%, respectively. The net transfer did not vary between BSFL originating from production on different larval feeding substrates. The results illustrate that hens are able to metabolize or elongate very large proportions of ingested lauric acid and myristic acid, which are predominant in the BSFL lipids (together accounting for as much as 37 mol%), such that they collectively account for less than 3.5 mol% of egg yolk fatty acids.

Computational and experimental validation of free radical scavenging properties of high-performance thin-layer chromatography quantified phenyl myristate in Homalium nepalense.

Phenyl myristate was isolated from Homalium nepalense, which is known for its therapeutic virtues in traditional medicine. However, the study of radical scavenging-capacity of phenyl myristate is limited by its relatively low abundance in medicinal plants. We have studied the isolation, structure-elucidation, and bioactivities of high-performance thin-layer chromatography validated phenyl myristate from hydroalcohol-extract of bark of H. nepalense. The chemical structure of phenyl myristate was elucidated by spectroscopic methods. The chromatography was performed on high-performance thin-layer chromatography aluminum plates coated with silica-gel 60 F254 . Determination and quantitation of phenyl myristate were performed by densitometric-scanning at 254 nm (chloroform-methanol, 9:1, v/v; Rf 0.49). The method was validated according to International Council for Harmonisation guidelines in terms of linearity, specificity, sensitivity, accuracy, precision, robustness, and stability. Linearity-range of phenyl myristate was 100-500 ng/5 µL with correlation-coefficient r2  = 0.9997. Limits of detection and quantitation were 3.35 and 10.17 ng, respectively. Phenyl myristate showed significant free-radical-scavenging activities in 2,2-diphenyl-1-picrylhydrazyl, oxygen-radical-absorbance-capacity, and ex vivo cell-based-antioxidant-protection-in-erythrocytes assays. Molecular-docking approach of phenyl myristate showed effective binding at active sites of human serum albumin (HSA) with the lowest binding energy (-8.4 kcal/mol) that was comparable with ascorbic acid (-5.0 kcal/mol). These studies provide mechanistic insight into the potential free radical scavenging activities of phenyl myristate.

Alyssum homolocarpum seed oil (AHSO), containing natural alpha linolenic acid, stearic acid, myristic acid and ß-sitosterol, increases proliferation and differentiation of neural stem cells in vitro.

BACKGROUND: Embryonic neural stem cells (eNSCs) are immature precursors of the central nervous system (CNS), with self-renewal and multipotential differentiation capacities. These are regulated by endogenous and exogenous factors such as alpha-linolenic acid (ALA), a plant-based essential omega-3 polyunsaturated fatty acid. METHODS: In this study, we investigated the effects of various concentrations of Alyssum homolocarpum seed oil (AHSO), containing natural ALA, stearic acid (SA), myristic acid (MA), and ß-sitosterol, on proliferation and differentiation of eNSCs, in comparison to controls and to synthetic pure ALA. RESULTS: Treatment with natural AHSO (25 to 75 µM), similar to synthetic ALA, caused a significant ~ 2-fold increase in eNCSs viability, in comparison to controls. To confirm this proliferative activity, treatment of NSCs with 50 or 75 µM AHSO resulted in a significant increase in mRNA levels of notch1, hes-1 and Ki-67and NICD protein expression, in comparison to controls. Moreover, AHSO administration significantly increased the differentiation of eNSCs toward astrocytes (GFAP+) and oligodendrocytes (MBP+) in a dose dependent manner and was more potent than ALA, at similar concentrations, in comparison to controls. Indeed, only high concentrations of 100 µM AHSO, but not ALA, caused a significant increase in the frequency of neurons (ß-III Tubulin+). CONCLUSION: Our data demonstrated that AHSO, a rich source of ALA containing also other beneficial fatty acids, increased the proliferation and stimulated the differentiation of eNSCs. We suggest that AHSO's effects are caused by ß-sitosterol, SA and MA, present within this oil. AHSO could be used in diet to prevent neurodevelopmental syndromes, cognitive decline during aging, and various psychiatric disorders.

Detection of Heterogeneous Protein S-Acylation in Cells.

The use of synthetically synthesized azide and alkyne fatty acid analogs coupled with bioorthogonal Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction-based detection methods to study protein S-acylation reactions has replaced the traditional method of using in vivo metabolic radiolabeling with tritiated palmitic acid and has greatly facilitated our understanding of this essential cellular process. Here, we describe the chemical synthesis of myristic (C:14), palmitic (C16:0), and stearic (C18:0) acid-azide probes and detail how they may be utilized as chemical reporters for the analysis of S-acylation of exogenously expressed proteins in cells.

Fatty acid profile and oxidation tests of fat extracted from yogurt using rose hip seed oil.

BACKGROUND: Yogurt is a dairy product with a high nutritional value. However, like all milk products, it contains milk fat and is rich in saturated fatty acids. It would be desirable to enrich dairy products in poly- unsaturated fatty acids to increase dietary intake amongst consumers and improve their health. Also, some LAB bacteria are able to produce CLA and CLnA isomers from linoleic and linolenic acids. The aim of this study was to investigate the chemical properties and fatty acid profile of yogurt with the addition of 3.5% of rose hip seed oil. METHODS: Yogurt was made from skimmed milk and yogurt starter culture YC-180 Ch. Hansen (Denmark), with the addition of 3.5% of rose hip seed oil. The peroxide value, acid value, iodine value, TBA rate and fatty acid profile were determined in fat extracted from the yogurt after 1 and 14 days of storage and in fresh rose hip seed oil. The fatty acid profile was determined using gas chromatographic methods with mass spectrometric detectors. RESULTS: Fat extracted from the yogurts had lower levels of peroxides than the fresh oil. It was more acidic and the iodine value was higher than in the fresh oil. Rose hip seed oil enriched the product with polyunsaturated fatty acids. After 14 days of storage, linoleic and linolenic acid levels had increased. Moreover, the content of myristic and palmitic acids had decreased. CONCLUSIONS: The rose hip seed oil added to the yogurt was less susceptible to oxidation. The content of un- saturated fatty acids in the yogurt increased with the addition of the oil, making yogurt with rose hip seed oil an excellent source of Ω-3 and Ω-6 fatty acids. Conjugated linoleic (CLA) and linolenic (CLnA) acids were not detected. However, yogurt manufactured with appropriate adjunct cultures and with the correct oil addition could be a natural source of CLA and CLnA in the human diet.

Evidence for N-Terminal Myristoylation of Tetrahymena Arginine Kinase Using Peptide Mass Fingerprinting Analysis.

In this study, we confirmed N-terminal myristoylation of Tetrahymena pyriformis arginine kinase (AK1) by identifying a myristoylation signal sequence at the N-terminus. A sufficient amount of modified enzyme was synthesized using an insect cell-free protein synthesis system that contains all of the elements necessary for post-transcriptional modification by fatty acids. Subsequent peptide mass fingerprinting (PMF) analyses were performed after digestion with trypsin. The PMF data covered 39 % (143 residues) of internal peptides. The target N-myristoylated peptide had a theoretical mass of 832.4477 and was clearly observed with an experimental mass (m/z-H(+)) of 832.4747. The difference between the two masses was 0.0271, supporting the accuracy of identification and indicating that the synthesized T. pyriformis AK1 is myristoylated. The fixed specimens of T. pyriformis were reacted with an anti-AK1 peptide antibody followed by a secondary antibody with a fluorescent chromophore and were observed using immunofluorescence microscope. In agreement with previous western blotting analyses, microscopic observations suggested that AK1 is localized in the cilia. The present PMF and microscopic analyses indicate that T. pyriformis AK1 may be localized and anchored to ciliary membranes via N-terminal myristoyl groups.

Metabonomic Study of Biochemical Changes in Human Hair of Heroin Abusers by Liquid Chromatography Coupled with Ion Trap-Time of Flight Mass Spectrometry.

Hair analysis is with the advantage of non-invasive collection and long surveillance window. The present study employed a sensitive and reliable liquid chromatography coupled with ion trap-time of flight mass spectrometry method to study the metabonomic characters in the hair of 58 heroin abusers and 72 non-heroin abusers. Results indicated that certain endogenous metabolites, such as sorbitol and cortisol, were accelerated, and the level of arachidonic acid, glutathione, linoleic acid, and myristic acid was decreased in hair of heroin abusers. The metabonomic study is helpful for further understanding of heroin addiction and clinical diagnosis.