Molecular analysis of a bifunctional fatty acid conjugase/desaturase from tung. Implications for the evolution of plant fatty acid diversity.
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The seed oil derived from the tung (Aleurites fordii Hemsl.) tree contains approximately 80% alpha-eleostearic acid (18:3delta(9cis,11trans,13trans)), an unusual conjugated fatty acid that imparts industrially important drying qualities to tung oil. Here, we describe the cloning and functional analysis of two closely related Delta(12) oleate desaturase-like enzymes that constitute consecutive steps in the biosynthetic pathway of eleostearic acid. Polymerase chain reaction screening of a tung seed cDNA library using degenerate oligonucleotide primers resulted in identification of two desaturases, FAD2 and FADX, that shared 73% amino acid identity. Both enzymes were localized to the endoplasmic reticulum of tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells, and reverse transcriptase-polymerase chain reaction revealed that FADX was expressed exclusively within developing tung seeds. Expression of the cDNAs encoding these enzymes in yeast (Saccharomyces cerevisiae) revealed that FAD2 converted oleic acid (18:1delta(9cis)) into linoleic acid (18:2delta(9cis,12cis)) and that FADX converted linoleic acid into alpha-eleostearic acid. Additional characterization revealed that FADX exhibited remarkable enzymatic plasticity, capable of generating a variety of alternative conjugated and delta(12)-desaturated fatty acid products in yeast cells cultured in the presence of exogenously supplied fatty acid substrates. Unlike other desaturases reported to date, the double bond introduced by FADX during fatty acid desaturation was in the trans, rather than cis, configuration. Phylogenetic analysis revealed that tung FADX is grouped with delta(12) fatty acid desaturases and hydroxylases rather than conjugases, which is consistent with its desaturase activity. Comparison of FADX and other lipid-modifying enzymes (desaturase, hydroxylase, epoxygenase, acetylenase, and conjugase) revealed several amino acid positions near the active site that may be important determinants of enzymatic activity. (+info)
Novel targeting signals mediate the sorting of different isoforms of the tail-anchored membrane protein cytochrome b5 to either endoplasmic reticulum or mitochondria.
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Tail-anchored membrane proteins are a class of proteins that are targeted posttranslationally to various organelles and integrated by a single segment of hydrophobic amino acids located near the C terminus. Although the localization of tail-anchored proteins in specific subcellular compartments in plant cells is essential for their biological function, the molecular targeting signals responsible for sorting these proteins are not well defined. Here, we describe the biogenesis of four closely related tung (Aleurites fordii) cytochrome b5 isoforms (Cb5-A, -B, -C, and -D), which are small tail-anchored proteins that play an essential role in many cellular processes, including lipid biosynthesis. Using a combination of in vivo and in vitro assays, we show that Cb5-A, -B, and -C are targeted exclusively to the endoplasmic reticulum (ER), whereas Cb5-D is targeted specifically to mitochondrial outer membranes. Comprehensive mutational analyses of ER and mitochondrial Cb5s revealed that their C termini, including transmembrane domains (TMD) and tail regions, contained several unique physicochemical and sequence-specific characteristics that defined organelle-specific targeting motifs. Mitochondrial targeting of Cb5 was mediated by a combination of hydrophilic amino acids along one face of the TMD, an enrichment of branched beta-carbon-containing residues in the medial portion of the TMD, and a dibasic -R-R/K/H-x motif in the C-terminal tail. By contrast, ER targeting of Cb5 depended primarily upon the overall length and hydrophobicity of the TMD, although an -R/H-x-Y/F- motif in the tail was also a targeting determinant. Collectively, the results presented provide significant insight into the early biogenetic events required for entry of tail-anchored proteins into either the ER or mitochondrial targeting pathways. (+info)
Benefits and side effects of different vegetable oil vectors on apoptosis, oxidative stress, and P2X7 cell death receptor activation.
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PURPOSE: Ocular side effects in patients using eye drops may be due to intolerance to the vector used in eye drops. Castor oil is the commonly used lipophilic vector but has been shown to be cytotoxic. Effects on cells of four oils (olive, camelina, Aleurites moluccana, maize) were compared with those of castor oil in human conjunctival cells. METHODS: Human conjunctival cells were incubated with the oils for 15 minutes. After a 24-hour recovery period, cells were tested for viability, proliferation, apoptosis (P2X7 cell death receptor and caspase 3 activation), intracellular redox potential, and reactive oxygen species production. Fatty acid incorporation in cell membranes was also analyzed. In vivo ocular irritation was assessed using the Draize test. RESULTS: Compared to the four other oils, castor oil was shown to induce significant necrosis and P2X7 cell death receptor and caspase 3 activation and to enhance intracellular reactive oxygen species production. Aleurites moluccana and camelina oils were not cytotoxic and increased cell membrane omega-3 fatty acid content. None of the five tested oils showed any in vivo ocular irritation. CONCLUSIONS: The results demonstrated that castor oil exerts cytotoxic effects on conjunctival cells. This cytotoxicity could explain the side effects observed in some patients using eye drops containing castor oil as a vehicle. The lack of cytotoxic effects observed with the four other oils, Aleurites, camelina, maize, and olive, suggest that they could be chosen to replace castor oil in ophthalmic formulations. (+info)
Tool use by wild New Caledonian crows Corvus moneduloides at natural foraging sites.
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The N termini of Brassica and tung omega-3 fatty acid desaturases mediate proteasome-dependent protein degradation in plant cells.
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The regulation of fatty acid desaturase activity in plants is important for determining the polyunsaturated fatty acid content of cellular membranes, which is often rapidly adjusted in plant cells in response to temperature change. Recent studies have demonstrated that the endoplasmic reticulum (ER)-localized omega-3 desaturases (Fad3s) are regulated extensively at the post-transcriptional level by both temperature-dependent changes in translational efficiency, as well as modulation of protein half-life. While the N-terminal sequences of Fad3 proteins were shown to contain information that mediates their rapid, proteasome-dependent protein turnover in both plant and yeast cells, it is currently unknown whether these sequences alone are sufficient to direct protein degradation. In this report, we fused the N-terminal sequences of two different Fad3 proteins to an ER-localized fluorescent protein reporter, consisting of the green fluorescent protein and the ER integral membrane protein cytochrome b5, and then measured (via microscopy) the degradation of the resulting fusion proteins in plant suspension-cultured cells relative to a second, co-expressed fluorescent reporter protein. Overall, the results demonstrate that the N-termini of both Fad3 proteins are sufficient for conferring rapid, proteasome-dependent degradation to an ER-bound marker protein. (+info)
Expression of tung tree diacylglycerol acyltransferase 1 in E. coli.
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Antinociceptive, anti-inflammatory and wound healing features in animal models treated with a semisolid herbal medicine based on Aleurites moluccana L. Willd. Euforbiaceae standardized leaf extract: semisolid herbal.
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