Proteomic Analysis of the Function of a Novel Cold-Regulated Multispanning Transmembrane Protein COR413-PM1 in Arabidopsis

The plasma membrane is the first subcellular organ that senses low temperature, and it includes some spanning transmembrane proteins that play important roles in cold regulation. COR413-PM1 is a novel multispanning transmembrane cold-regulated protein; however, the related functions are not clear in Arabidopsis. We found the tolerance to freezing stress of cor413-pm1 was lower than wild-type (WT). A proteomics method was used to analyze the differentially abundant proteins (DAPs) between cor413-pm1 and WT. A total of 4143 protein groups were identified and 3139 were accurately quantitated. The DAPs associated with COR413-PM1 and freezing treatment were mainly involved in the metabolism of fatty acids, sugars, and purine. Quantitative real-time PCR (qRT-PCR) confirmed the proteomic analysis results of four proteins: fatty acid biosynthesis 1 (FAB1) is involved in fatty acid metabolism and might affect the plasma membrane structure; fructokinase 3 (FRK3) and sucrose phosphate synthase A1 (SPSA1) play roles in sugar metabolism and may influence the ability of osmotic adjustment under freezing stress; and GLN phosphoribosyl pyrophosphate amidotransferase 2 (ASE2) affects freezing tolerance through purine metabolism pathways. In short, our results demonstrate that the multispanning transmembrane protein COR413-PM1 regulates plant tolerance to freezing stress by affecting the metabolism of fatty acids, sugars, and purine in Arabidopsis.

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Proteomic analysis of metabolic mechanisms associated with fatty acid biosynthesis during Styrax tonkinensis kernel development

Journal of the Science of Food and Agriculture ◽

10.1002/jsfa.11262 ◽

2021 ◽

Author(s):

Qikui Wu ◽

Chen Chen ◽

Xiaojun Wang ◽

Zihan Zhang ◽

Fangyuan Yu ◽

...

Keyword(s):

Fatty Acid ◽

Proteomic Analysis ◽

Fatty Acid Biosynthesis ◽

Acid Biosynthesis ◽

Kernel Development

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The same rat Δ6-desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis

Biochemical Journal ◽

10.1042/bj3640049 ◽

2002 ◽

Vol 364 (1) ◽

pp. 49-55 ◽

Cited By ~ 83

Author(s):

Sabine D'ANDREA ◽

Hervé GUILLOU ◽

Sophie JAN ◽

Daniel CATHELINE ◽

Jean-Noël THIBAULT ◽

...

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Polyunsaturated Fatty Acid ◽

Unsaturated Fatty Acids ◽

Fatty Acid Biosynthesis ◽

Chain Polyunsaturated Fatty Acid ◽

Acid Biosynthesis ◽

Highly Unsaturated Fatty Acids ◽

Long Chain ◽

Δ6 Desaturase

The recently cloned Δ6-desaturase is known to catalyse the first step in very-long-chain polyunsaturated fatty acid biosynthesis, i.e. the desaturation of linoleic and α-linolenic acids. The hypothesis that this enzyme could also catalyse the terminal desaturation step, i.e. the desaturation of 24-carbon highly unsaturated fatty acids, has never been elucidated. To test this hypothesis, the activity of rat Δ6-desaturase expressed in COS-7 cells was investigated. Recombinant Δ6-desaturase expression was analysed by Western blot, revealing a single band at 45kDa. The putative involvement of this enzyme in the Δ6-desaturation of C24:5n-3 to C24:6n-3 was measured by incubating transfected cells with C22:5n-3. Whereas both transfected and non-transfected COS-7 cells were able to synthesize C24:5n-3 by elongation of C22:5n-3, only cells expressing Δ6-desaturase were also able to produce C24:6n-3. In addition, Δ6-desaturation of [1-14C]C24:5n-3 was assayed invitro in homogenates from COS-7 cells expressing Δ6-desaturase or not, showing that Δ6-desaturase catalyses the conversion of C24:5n-3 to C24:6n-3. Evidence is therefore presented that the same rat Δ6-desaturase catalyses not only the conversion of C18:3n-3 to C18:4n-3, but also the conversion of C24:5n-3 to C24:6n-3. A similar mechanism in the n-6 series is strongly suggested.

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Effect of surface and intracellular pH on hepatocellular fatty acid uptake

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1996.271.6.g1067 ◽

1996 ◽

Vol 271 (6) ◽

pp. G1067-G1073

Author(s):

C. Elsing ◽

A. Kassner ◽

W. Stremmel

Keyword(s):

Fatty Acids ◽

Plasma Membrane ◽

Fatty Acid ◽

Intracellular Ph ◽

Outer Surface ◽

Fatty Acid Uptake ◽

Proton Gradient ◽

Fatty Acid Transport ◽

Acid Uptake ◽

Acid Transport

Fatty acids enter hepatocytes, at least in part, by a carrier-mediated uptake mechanism. The importance of driving forces for fatty acid uptake is still controversial. To evaluate possible driving mechanisms for fatty acid transport across plasma membranes, we examined the role of transmembrane proton gradients on fatty acid influx in primary cultured rat hepatocytes. After hepatocytes were loaded with SNARF-1 acetoxymethyl ester, changes in intracellular pH (pHi) under different experimental conditions were measured and recorded by confocal laser scanning microscopy. Fatty acid transport was increased by 45% during cellular alkalosis, achieved by adding 20 mM NH4Cl to the medium, and a concomitant paracellular acidification was observed. Fatty acid uptake was decreased by 30% during cellular acidosis after withdrawal of NH4Cl from the medium. Cellular acidosis activates the Na+/H+ antiporter to export excessive protons to the outer cell surface. Inhibition of Na+/H+ antiporter activity by amiloride diminishes pHi recovery and thereby accumulation of protons at the outer surface of the plasma membrane. Under these conditions, fatty acid uptake was further inhibited by 57% of control conditions. This suggests stimulation of fatty acid influx by an inwardly directed proton gradient. The accelerating effect of protons at the outer surface of the plasma membrane was confirmed by studies in which pH of the medium was varied at constant pHi. Significantly higher fatty acid influx rates were observed at low buffer pH. Recorded differences in fatty acid uptake appeared to be independent of changes in membrane potential, because BaCl2 did not influence initial uptake velocity during cellular alkalosis and paracellular acidosis. Moreover, addition of oleate-albumin mixtures to the NH4Cl incubation buffer did not change the observed intracellular alkalinization. In contrast, after cells were acid loaded, addition of oleate-albumin solutions to the recovery buffer increased pHi recovery rates from 0.21 +/- 0.02 to 0.36 +/- 0.05 pH units/min (P < 0.05), indicating that fatty acids further stimulate Na+/H+ antiporter activity during pHi recovery from an acid load. It is concluded that carrier-mediated uptake of fatty acids in hepatocytes follows an inwardly directed transmembrane proton gradient and is stimulated by the presence of H+ at the outer surface of the plasma membrane.

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Dietary fat and the diabetic state alter insulin binding and the fatty acyl composition of the adipocyte plasma membrane

Biochemical Journal ◽

10.1042/bj2530417 ◽

1988 ◽

Vol 253 (2) ◽

pp. 417-424 ◽

Cited By ~ 98

Author(s):

C J Field ◽

E A Ryan ◽

A B Thomson ◽

M T Clandinin

Keyword(s):

Fatty Acids ◽

Arachidonic Acid ◽

Plasma Membrane ◽

Fatty Acid ◽

Polyunsaturated Fatty Acids ◽

Insulin Binding ◽

Fatty Acyl ◽

Membrane Composition ◽

Membrane Phospholipids ◽

Diabetic State

Control and diabetic rats were fed on semi-purified high-fat diets providing a polyunsaturated/saturated fatty acid ratio (P/S) of 1.0 or 0.25, to examine the effect of diet on the fatty acid composition of major phospholipids of the adipocyte plasma membrane. Feeding the high-P/S diet (P/S = 1.0) compared with the low-P/S diet (P/S = 0.25) increased the content of polyunsaturated fatty acids in membrane phospholipids in both control and diabetic animals. The diabetic state decreased the content of polyunsaturated fatty acids, particularly arachidonic acid, in adipocyte membrane phospholipids. The decrease in arachidonic acid in membrane phospholipids of diabetic animals tended to be normalized to within the control values when high-P/S diets were given. For control animals, altered plasma-membrane composition was associated with change in insulin binding, suggesting that change in plasma-membrane composition may have physiological consequences for insulin-stimulated functions in the adipocyte.

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EXTH-27. MOLECULAR CHARACTERIZATION AND PRECLINICAL DEVELOPMENT OF NOVEL SMALL MOLECULE INHIBITOR SPECIFIC FOR WILD-TYPE IDH1 FOR FERROPTOSIS INDUCTION IN GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noab196.666 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi169-vi169

Author(s):

Kevin Murnan ◽

Serena Tommasini-Ghelfi ◽

Lisa Hurley ◽

Corey Dussold ◽

Daniel Wahl ◽

...

Keyword(s):

Fatty Acids ◽

Cell Death ◽

Plasma Membrane ◽

Fatty Acid ◽

Unsaturated Fatty Acids ◽

De Novo ◽

De Novo Lipogenesis ◽

Therapeutic Modality ◽

Wild Type ◽

Genetic Ablation

Abstract Increased de novo synthesis, mobilization and uptake of fatty acids are required to provide sufficient lipids for membrane biogenesis in support of rapid tumor cell division and growth. In addition to their structural roles as components of the plasma membrane, fatty acid-derived lipids regulate ferroptotic cell death, a type of programmed cell death, when oxidized by iron-dependent lipoxygenase enzymes. De novo lipogenesis and the defense against oxidative lipid damage require large amounts of cytosolic NADPH. Our group has recently found that HGG up-regulate wild-type Isocitrate dehydrogenase 1 (referred to hereafter as ‘wt-IDH1high HGG’) to generate large quantities of cytosolic NADPH. RNAi-mediated knockdown of wt-IDH1, alone and in combination with radiation therapy (RT), slows the growth of patient-derived HGG xenografts, while overexpression of wt-IDH1 promotes intracranial HGG growth. Isotope tracer and liquid chromatography-based lipidomic studies indicated that wt-IDH1 supports the de novo biosynthesis of mono-unsaturated fatty acids (MUFAs) and promotes the incorporation of monounsaturated phospholipids into the plasma membrane, while displacing polyunsaturated fatty acid (PUFA) phospholipids. In addition, enhanced NADPH production in wt-IDH1high HGG increases glutathione (GSH) level, reduces reactive oxygen species (ROS), activates the phospholipid peroxidase glutathione peroxidase 4 (GPX4)-driven lipid repair pathway, and dampens the accumulation of PUFA-containing lipid peroxides, known executioners of ferroptosis. To pharmacologically target wt-IDH1,we have used and characterized wt-IDH1i-13, a first-in-class competitive α,β-unsaturated enone (AbbVie). wt-IDH1i-13 potently inhibits wt-IDH1 enzymatic activity, by covalently binding to the NADP+ binding pocket. Our data indicate that wt-IDH1i-13 promotes ferroptosis, which can be rescued by pre-treatment of cells with the peroxyl scavenger and ferroptosis inhibitor ferrostatin. wt-IDH1i-13 is brain-penetrant, and similar to genetic ablation, reduces progression and extends the survival of wt-IDH1high HGG bearing mice, alone and in combination with RT. These studies credential to wt-IDH1i-13 as a novel therapeutic modality for the treatment of wt-IDH1 gliomas.

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A kinetic rationale for functional redundancy in fatty acid biosynthesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2013924117 ◽

2020 ◽

Vol 117 (38) ◽

pp. 23557-23564

Author(s):

Alex Ruppe ◽

Kathryn Mains ◽

Jerome M. Fox

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Fatty Acid Synthase ◽

Unsaturated Fatty Acids ◽

Fatty Acid Biosynthesis ◽

Average Length ◽

Functional Redundancy ◽

Experimental Investigations ◽

Total Production

Cells build fatty acids with biocatalytic assembly lines in which a subset of enzymes often exhibit overlapping activities (e.g., two enzymes catalyze one or more identical reactions). Although the discrete enzymes that make up fatty acid pathways are well characterized, the importance of catalytic overlap between them is poorly understood. We developed a detailed kinetic model of the fatty acid synthase (FAS) ofEscherichia coliand paired that model with a fully reconstituted in vitro system to examine the capabilities afforded by functional redundancy in fatty acid synthesis. The model captures—and helps explain—the effects of experimental perturbations to FAS systems and provides a powerful tool for guiding experimental investigations of fatty acid assembly. Compositional analyses carried out in silico and in vitro indicate that FASs with multiple partially redundant enzymes enable tighter (i.e., more independent and/or broader range) control of distinct biochemical objectives—the total production, unsaturated fraction, and average length of fatty acids—than FASs with only a single multifunctional version of each enzyme (i.e., one enzyme with the catalytic capabilities of two partially redundant enzymes). Maximal production of unsaturated fatty acids, for example, requires a second dehydratase that is not essential for their synthesis. This work provides a kinetic, control-theoretic rationale for the inclusion of partially redundant enzymes in fatty acid pathways and supplies a valuable framework for carrying out detailed studies of FAS kinetics.

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SYNTHESIS OF FATTY ACIDS BY TESTICULAR TISSUE IN VITRO

Canadian Journal of Biochemistry and Physiology ◽

10.1139/y63-142 ◽

1963 ◽

Vol 41 (1) ◽

pp. 1267-1274

Author(s):

Peter F. Hall ◽

Edward E. Nishizawa ◽

Kristen B. Eik-Nes

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Fatty Acid Biosynthesis ◽

De Novo ◽

Fatty Acid Fraction ◽

Testicular Tissue ◽

Acid Biosynthesis ◽

Adrenal Tissue ◽

Substrate Fatty Acid

The fatty acids palmitic, palmitoleic, stearic, and oleic have been isolated from rabbit testis and evidence for the synthesis of palmitic and stearic acids de novo from acetate-1-C14is presented. ICSH did not produce demonstrable stimulation of the synthesis of these acids in vitro although the hormone stimulated the production of testosterone-C14by the same tissue. Adrenal tissue was shown to contain palmitic, stearic, and oleic acids, and ACTH did not increase the incorporation of acetate-1-C14into a fatty acid fraction extracted following incubation of adrenal tissue in the presence of this substrate. Fatty acid biosynthesis, therefore, is probably not influenced by the mechanisms by which tropic hormones increase steroid formation.

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Control of Fatty Acid and Lipid Biosynthesis in Euglena gracilis by Ammonia, Light and DCMU

Zeitschrift für Naturforschung B ◽

10.1515/znb-1972-0111 ◽

1972 ◽

Vol 27 (1) ◽

pp. 53-61 ◽

Cited By ~ 37

Author(s):

P. Pohl ◽

H. Wagner

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

White Light ◽

Euglena Gracilis ◽

Fatty Acid Biosynthesis ◽

Neutral Lipids ◽

Fluorescent Light ◽

Low Levels ◽

High Concentrations ◽

Monogalactosyl Diglyceride

When Euglena gracilis was grown under white (fluorescent) light in media containing high concentrations of ammonium chloride (more than 0.005%), the main lipids synthesized were monogalactosyl diglyceride, digalactosyl diglyceride, phosphatidyl glycerol, sulfolipid and the all cis △7,10-16:2, △7,10,13-16:3, △4,7,10,13-16:4, △9,12-18:2 and △9,12,15-18:3 fatty acids. At low levels of ammonium (less than 0.002%) these compounds were produced only in small amounts, while neutral lipids, phosphatidyl choline, phosphatidyl ethanolamine and the 14:0, 16:0 and 16:1 fatty acids predominated.When Euglena gracilis was grown in white light in the presence of dichlorophenyldimethylurea (DCMU) or in the dark, fatty acid and lipid biosyntheses followed the same pattern as in white light at low levels of ammonium. Similar results were obtained when nitrate served as the only nitrogen source in the light and in the dark.The results indicate that in Euglena gracilis there are a light independent and a light and ammonium dependent pathway of fatty acid biosynthesis. Both pathways seem to be in association with specific lipids.

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Ankyrin-bound fatty acid turns over rapidly at the erythrocyte plasma membrane.

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.2981 ◽

1987 ◽

Vol 7 (8) ◽

pp. 2981-2984 ◽

Cited By ~ 48

Author(s):

M Staufenbiel

Keyword(s):

Fatty Acids ◽

Plasma Membrane ◽

Fatty Acid ◽

Long Chain Fatty Acids ◽

Acid Modification ◽

Skeletal Protein ◽

Erythrocyte Plasma Membrane ◽

Polypeptide Backbone ◽

Long Chain ◽

Regulatory Significance

The membrane skeletal protein ankyrin was shown to be continuously acylated and deacylated with long-chain fatty acids in mature erythrocytes. At least a fraction of the lipid bound to ankyrin turned over rapidly (half-life, approximately 50 min) compared with the polypeptide backbone, which was stable throughout the erythrocyte life. This indicates a regulatory significance of the fatty acid modification for the function of ankyrin.

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