Unsaturated acyl-CoAs Research Articles

Saccharomyces cerevisiae Δ9-desaturase Ole1 forms a supercomplex with Slc1 and Dga1

Biosynthesis of the various lipid species that compose cellular membranes and lipid droplets depends on the activity of multiple enzymes functioning in coordinated pathways. The flux of intermediates through lipid biosynthetic pathways is regulated to respond to nutritional and environmental demands placed on the cell necessitating that there be extensive flexibility in pathway activity and organization. This flexibility can in part be achieved through the organization of biosynthetic enzymes into metabolon supercomplexes. However, the composition and organization of such supercomplexes remains unclear. Here, we identified protein-protein interactions between acyltransferases Sct1, Gpt2, Slc1, Dga1 and the Δ9 acyl-CoA desaturase Ole1 in Saccharomyces cerevisiae. We further determined that a subset of these acyltransferases interact with each other without Ole1 acting as a scaffold. We show that truncated versions of Dga1 lacking the carboxyl-terminal 20 amino acid residues are non-functional and unable to bind Ole1. Furthermore, charged-to-alanine scanning mutagenesis revealed that a cluster of charged residues near the carboxyl-terminus were required for the interaction with Ole1. Mutation of these charged residues disrupted the interaction between Dga1 and Ole1, but allowed Dga1 to retain catalytic activity and to induce lipid droplet formation. These data support the formation of a complex of acyltransferases involved in lipid biosynthesis that interacts with Ole1, the sole acyl-CoA desaturase in S. cerevisiae, that can channel unsaturated acyl-chains toward phospholipid or triacylglycerol synthesis. This desaturasome complex may provide the architecture that allows for the necessary flux of de novo synthesized unsaturated acyl-CoA to phospholipid or triacylglycerol synthesis as demanded by cellular requirements.

MadR mediates acyl CoA-dependent regulation of mycolic acid desaturation in mycobacteria

Mycobacterium tuberculosis has a lipid-rich cell envelope that is remodeled throughout infection to enable adaptation within the host. Few transcriptional regulators have been characterized that coordinate synthesis of mycolic acids, the major cell wall lipids of mycobacteria. Here, we show that the mycolic acid desaturase regulator (MadR), a transcriptional repressor of the mycolate desaturase genes desA1 and desA2, controls mycolic acid desaturation and biosynthesis in response to cell envelope stress. A madR-null mutant of M. smegmatis exhibited traits of an impaired cell wall with an altered outer mycomembrane, accumulation of a desaturated α-mycolate, susceptibility to antimycobacterials, and cell surface disruption. Transcriptomic profiling showed that enriched lipid metabolism genes that were significantly down-regulated upon madR deletion included acyl-coenzyme A (aceyl-CoA) dehydrogenases, implicating it in the indirect control of β-oxidation pathways. Electromobility shift assays and binding affinities suggest a unique acyl-CoA pool-sensing mechanism, whereby MadR is able to bind a range of acyl-CoAs, including those with unsaturated as well as saturated acyl chains. MadR repression of desA1/desA2 is relieved upon binding of saturated acyl-CoAs of chain length C16 to C24, while no impact is observed upon binding of shorter chain and unsaturated acyl-CoAs. We propose this mechanism of regulation as distinct to other mycolic acid and fatty acid synthesis regulators and place MadR as the key regulatory checkpoint that coordinates mycolic acid remodeling during infection in response to host-derived cell surface perturbation.

Editing of phosphatidic acid and phosphatidylethanolamine by acyl-CoA: lysophospholipid acyltransferases in developing Camelina sativa seeds

Main conclusionsThe main source of polyunsaturated acyl-CoA in cytoplasmic acyl-CoA pool of Camelina sativa seeds are fatty acids derived from phosphatidylcholine followed by phosphatidic acid. Contribution of phosphatidylethanolamine is negligible.While phosphatidylethanolamine (PE) is the second most abundant phospholipid, phosphatidic acid (PA) only constitutes a small fraction of C. sativa seeds’ polar lipids. In spite of this, the relative contribution of PA in providing fatty acids for the synthesis of acyl-CoA, supplying cytosolic acyl-CoA pool seems to be much higher than the contribution of PE. Our data indicate that up to 5% of fatty acids present in mature C. sativa seeds are first esterified with PA, in comparison to 2% first esterified with PE, before being transferred into acyl-CoA pool via backward reactions of either acyl-CoA:lysophosphatidic acid acyltransferases (CsLPAATs) or acyl-CoA:lysophoshatidylethanolamine acyltransferases (CsLPEATs). Those acyl-CoAs are later reused for lipid biosynthesis or remodelling. In the forward reactions both aforementioned acyltransferases display the highest activity at 30 °C. The spectrum of optimal pH differs for both enzymes with CsLPAATs most active between pH 7.5–9.0 and CsLPEATs between pH 9.0 to 10.0. Whereas addition of magnesium ions stimulates CsLPAATs, calcium and potassium ions inhibit them in concentrations of 0.05–2.0 mM. All three types of ions inhibit CsLPEATs activity. Both tested acyltransferases present the highest preferences towards 16:0-CoA and unsaturated 18-carbon acyl-CoAs in forward reactions. However, CsLPAATs preferentially utilise 18:1-CoA and CsLPEATs preferentially utilise 18:2-CoA while catalysing fatty acid remodelling of PA and PE, respectively.

Functional Characterization of Lysophosphatidylcholine: Acyl-CoA Acyltransferase Genes From Sunflower (Helianthus annuus L.).

Lysophosphatidylcholine acyltransferase (LPCAT, EC 2.3.1.23) is an evolutionarily conserved key enzyme in the Lands cycle that catalyzes acylation of lysophosphatidylcholine (LPC) to produce phosphatidylcholine (PC), the main phospholipid in cellular membranes. In this study, three LPCAT genes from sunflower were identified and the corresponding proteins characterized. These HaLPCAT genes encoded functionally active enzymes that were able to complement a deficient yeast mutant. Moreover, enzymatic assays were carried out using microsomal preparations of the yeast cells. When acyl specificities were measured in the forward reaction, these enzymes exhibited a substrate preference for unsaturated acyl-CoAs, especially for linolenoyl-CoA, while in the reverse reaction, linoleoyl or linolenoyl acyl groups were transferred from PC to acyl-CoA to a similar extent. Expression levels of LPCAT genes were studied revealing distinct tissue-specific expression patterns. In summary, this study suggests that the combined forward and reverse reactions catalyzed by sunflower LPCATs facilitate acyl-exchange between the sn-2 position of PC and the acyl-CoA pool. Sunflower LPCATs displayed different characteristics, which could point to different functionalities, favoring the enrichment of seed triacylglycerols (TAGs) with polyunsaturated fatty acid (PUFA).

In vivo synthesis of csypyrone derivatives by exploring the substrate diversity of start units of type Ⅲ polyketide synthase CsyB

As a novel fungal type Ⅲ polyketide synthase, CsyB from Aspergillus oryzae can sequentially accept one molecular short chain fatty acyl CoA as start unit, one molecular malonyl-CoA and one molecular acetoacetyl-CoA as extend unit to produce the short chain csypyrone B1-3. On the basis of crystal structure of CsyB, a fatty acyl CoA binding tunnel of a length of about 16 Å is located in its active center that is proposed to accept diversified start units. In order to examine the substrate diversity of CsyB, CsyB gene was introduced and expressed in Escherichia coli that contained a number of precursors of long chain fatty acyl CoA in vivo. The results of HPLC revealed that a series of long chain csypyrone derivatives were detected in the recombinant strain in comparison with the control strain. These new csypyrone compounds were preliminarily analyzed by UV-visible spectroscopy and LC-HRMS. Three hydroxylated csypyrones were intensively determined by 1D and 2D NMR experiments, especially the position of the hydroxyl group in these compounds. These results demonstrate that CsyB exhibits a broad substrate specificity, which not only can accept the long chain saturated or unsaturated fatty acyl CoA as substrate, but also accept hydroxylated long chain fatty acyl CoA.

The first plant acyl-CoA-binding protein structures: the close homologues OsACBP1 and OsACBP2 from rice.

Acyl-CoA-binding proteins (ACBPs) are a family of proteins that facilitate the binding of long-chain acyl-CoA esters at a conserved acyl-CoA-binding domain. ACBPs act to form intracellular acyl-CoA pools, transport acyl-CoA esters and regulate lipid metabolism. In the model plant Arabidopsis thaliana, a family of six ACBPs has been demonstrated to function in stress and development. Six ACBPs (OsACBPs) have also been identified in Oryza sativa (rice), but they are not as well characterized as those in Arabidopsis thaliana. To understand the need in rice for the two 10 kDa ACBPs, namely OsACBP1 and OsACBP2, which share 79% sequence identity, their crystal structures were elucidated and their affinities toward acyl-CoA esters were compared using isothermal titration calorimetry. OsACBP2 was found to display a higher binding affinity for unsaturated acyl-CoA esters than OsACBP1. A difference between the two proteins is observed at helix 3 and is predicted to lead to different ligand-binding modes in terms of the shape of the binding pocket and the residues that are involved. OsACBP1 thus resembles bovine ACBP, while OsACBP2 is similar to human liver ACBP, in both structure and binding affinity. This is the first time that ACBP structures have been reported from plants, and suggests that OsACBP1 and OsACBP2 are not redundant in function despite their high sequence identity and general structural similarity.

Substrate specificity, kinetic properties and inhibition by fumonisin B1 of ceramide synthase isoforms from Arabidopsis.

Ceramide makes up the acyl-backbone of sphingolipids and plays a central role in determining the function of these essential membrane lipids. In Arabidopsis, the varied chemical composition of ceramide is determined by the specificity of three different isoforms of ceramide synthase, denoted LAG one homologue 1, -2 and -3 (LOH1, LOH2 and LOH3), for a range of long-chain base (LCB) and acyl-CoA substrates. The contribution of each of these isoforms to the synthesis of ceramide was investigated by invitro ceramide synthase assays. The plant LCB phytosphingosine was efficiently used by the LOH1 and LOH3 isoforms, with LOH1 having the lowest Km for the LCB substrate of the three isoforms. In contrast, sphinganine was used efficiently only by the LOH2 isoform. Acyl-CoA specificity was also distinguished between the three isoforms with LOH2 almost completely specific for palmitoyl-CoA whereas the LOH1 isoform showed greatest activity with lignoceroyl- and hexacosanoyl-CoAs. Interestingly, unsaturated acyl-CoAs were not used efficiently by any isoform whereas unsaturated LCB substrates were preferred by LOH2 and 3. Fumonisin B1 (FB1) is a general inhibitor of ceramide synthases but LOH1 was found to have a much lower Ki than the other isoforms pointing towards the origin of FB1 sensitivity in plants. Overall, the data suggest distinct roles and modes of regulation for each of the ceramide synthases in Arabidopsis sphingolipid metabolism.

Possible allostery and oligomerization of recombinant plastidial sn-glycerol-3-phosphate acyltransferase

Plastidial acyl-acyl carrier protein:sn-glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the acyl-acyl carrier protein-dependent sn-1 acylation of sn-glycerol 3-phosphate (G3P) to produce lysophosphatic acid. Functional recombinant Erysimum asperum GPAT (EaGPAT), devoid of transit peptide, was produced in yeast. Analysis of the dependence of EaGPAT activity on increasing G3P concentration resulted in a hyperbolic response. EaGPAT exhibited a preference for 18-carbon unsaturated acyl-CoAs. Assays with concentrations of oleoyl-CoA up to 90μM revealed an exponential response to increasing concentrations of acyl donor, and the introduction of increasing concentrations of unlabeled linoleoyl-CoA into the standard reaction mixture resulted in increased incorporation of radiolabeled oleoyl moieties into lysophosphatidic acid. Collectively, the kinetic results suggest that acyl-CoA may act as both substrate and allosteric effector. EaGPAT was also shown to oligomerize to form higher molecular mass multimers, with the monomer and trimer being the predominant forms of the enzyme. Since most allosteric enzyme exhibit quaternary structure, the self-associating properties of EaGPAT are consistent with those of an allosteric enzyme. These results could have important regulatory implications when plastidial GPAT is introduced into a cytoplasmic environment where acyl-CoA is the acyl donor supporting cytoplasmic glycerolipid assembly.

Mechanical Unloading through Ventricular Assist Devices Increases Myocardial Content of Unsaturated Acyl-CoA Species in Patients with Advanced Heart Failure

Purpose Heart failure (HF) is associated with abnormal metabolism and lipotoxicity. Fatty acids (Fas) and fatty acyl-CoAs may act as important mediators of lipotoxicity but their composition in human HF is unclear. We analyzed acyl-CoA and FA species in patients with HF before and after implantation of a ventricular assist device (LVAD) and controls. Methods and Materials Serum and myocardium were collected from 20 pats before and 15 pats after LVAD placement and 10 controls (con). FA and fatty acyl-CoA content were measured by LC/MS. Results Myocardial total fatty acyl-CoA (2.0±0.4 vs. 4.8±0.4 nmol/g in con, p 0.05) compared to con indicating reduced cardiac lipid uptake. Myocardial fatty acyl-CoA species C14, C16, C18, C16:1, C18:2, C18:1 and C20:4 were lower in HF compared to con (all p Conclusions HF is associated with decreased cardiac Fas and fatty acyl-CoA content. Mechanical unloading improves myocardial and skeletal metabolism with increased fatty acyl-CoAs and increased unsaturated fatty acyl-CoAs, suggesting increased lipid uptake. Thus, impaired lipid metabolism in HF can be corrected by LVAD therapy and potentially with pharmacologic therapies.

Cloning and characterization of a Δ9-desaturase gene of the Antarctic fish Chionodraco hamatus and Trematomus bernacchii

Chionodraco hamatus and Trematomus bernacchii are perciforms, members of the fish suborder Notothenioidei that live in the Antarctic Ocean and experience very cold and persistent environmental temperature. These fish have biochemical and molecular features that allow them to live at these extreme cold temperatures. Fine tuning of the level of unsaturated fatty acids content in membrane is a key mechanism of living organisms to adapt to cold and high temperatures. Desaturases are key enzymes that synthesize unsaturated fatty acyl-CoAs from saturated fatty acids. We cloned and sequenced a Δ(9)-desaturase gene and its cDNA of C. hamatus, and the cDNA of T. bernacchii. The coded proteins are virtually identical and share homology to other Δ(9)-desaturase fish sequences. These proteins contain, in the first trans-membrane domain, two cysteine residues that may form a disulfur bond present in the corresponding membrane region of Δ(9)-desaturase proteins of other Antarctic fish but not in Eleginops maclovinus that experiences higher environmental temperatures and in all other Δ(9)-desaturase genes of mammals present in data bases. C. hamatus Δ(9)-desaturase gene complements a Saccharomyces cerevisiae mutant lacking Δ(9)-desaturase (Ole1) gene. Analysis of sequence homology of the trans-membrane domains of Δ(9)-desaturase and the cytoplasmic region of the same proteins of Antarctic fish, non-Antarctic fish and mammals suggest that the significant differences found in the homologous sequences of the first trans-membrane domain may be due to the specific lipid content of their membrane.

Reversible Lysine Acetylation Regulates Activity of Human Glycine N-Acyltransferase-like 2 (hGLYATL2): IMPLICATIONS FOR PRODUCTION OF GLYCINE-CONJUGATED SIGNALING MOLECULES

Lysine acetylation is a major post-translational modification of proteins and regulates many physiological processes such as metabolism, cell migration, aging, and inflammation. Proteomic studies have identified numerous lysine-acetylated proteins in human and mouse models (Kim, S. C., Sprung, R., Chen, Y., Xu, Y., Ball, H., Pei, J., Cheng, T., Kho, Y., Xiao, H., Xiao, L., Grishin, N. V., White, M., Yang, X. J., and Zhao, Y. (2006) Mol. Cell 23, 607-618). One family of proteins identified in this study was the murine glycine N-acyltransferase (GLYAT) enzymes, which are acetylated on lysine 19. Lysine 19 is a conserved residue in human glycine N-acyltransferase-like 2 (hGLYATL2) and in several other species, showing that this residue may be important for enzyme function. Mutation of lysine 19 in recombinant hGLYATL2 to glutamine (K19Q) and arginine (K19R) resulted in a 50-80% lower production of N-oleoyl glycine and N-arachidonoylglycine, indicating that lysine 19 is important for enzyme function. LC/MS/MS confirmed that Lys-19 is not acetylated in wild-type hGLYATL2, indicating that Lys-19 requires to be deacetylated for full activity. The hGLYATL2 enzyme conjugates medium- and long-chain saturated and unsaturated acyl-CoA esters to glycine, resulting in the production of N-oleoyl glycine and also N-arachidonoyl glycine. N-Oleoyl glycine and N-arachidonoyl glycine are structurally and functionally related to endocannabinoids and have been identified as signaling molecules that regulate functions like the perception of pain and body temperature and also have anti-inflammatory properties. In conclusion, acetylation of lysine(s) in hGLYATL2 regulates the enzyme activity, thus linking post-translational modification of proteins with the production of biological signaling molecules, the N-acyl glycines.

Structural basis for the transcriptional regulation of membrane lipid homeostasis

DesT is a transcriptional repressor that regulates the genes which control the unsaturated:saturated fatty acid ratio available for membrane lipid synthesis. DesT bound to unsaturated acyl-CoA has a high affinity for its cognate palindromic DNA-binding site, whereas DesT bound to saturated acyl-CoA does not bind to this site. Structural analyses of the DesT–oleoyl-CoA–DNA and DesT–palmitoyl-CoA complexes reveal that acyl chain shape directly influences the packing of hydrophobic core residues within the DesT ligand-binding domain. These changes are propagated to the paired DNA-binding domains via conformational changes to modulate DNA binding. These structural interpretations are supported by the in vitro and in vivo characterization of site-directed mutants. The regulation of DesT by the unsaturated:saturated ratio of acyl chains rather than the concentration of a single ligand is a paradigm for understanding transcriptional regulation of membrane lipid homeostasis.

Overexpression of Arabidopsis Acyl-CoA Binding Protein ACBP3 Promotes Starvation-Induced and Age-Dependent Leaf Senescence

In Arabidopsis thaliana, a family of six genes (ACBP1 to ACBP6) encodes acyl-CoA binding proteins (ACBPs). Investigations on ACBP3 reported here show its upregulation upon dark treatment and in senescing rosettes. Transgenic Arabidopsis overexpressing ACBP3 (ACBP3-OEs) displayed accelerated leaf senescence, whereas an acbp3 T-DNA insertional mutant and ACBP3 RNA interference transgenic Arabidopsis lines were delayed in dark-induced leaf senescence. Acyl-CoA and lipid profiling revealed that the overexpression of ACBP3 led to an increase in acyl-CoA and phosphatidylethanolamine (PE) levels, whereas ACBP3 downregulation reduced PE content. Moreover, significant losses in phosphatidylcholine (PC) and phosphatidylinositol, and gains in phosphatidic acid (PA), lysophospholipids, and oxylipin-containing galactolipids (arabidopsides) were evident in 3-week-old dark-treated and 6-week-old premature senescing ACBP3-OEs. Such accumulation of PA and arabidopsides (A, B, D, E, and G) resulting from lipid peroxidation in ACBP3-OEs likely promoted leaf senescence. The N-terminal signal sequence/transmembrane domain in ACBP3 was shown to be essential in ACBP3-green fluorescent protein targeting and in promoting senescence. Observations that recombinant ACBP3 binds PC, PE, and unsaturated acyl-CoAs in vitro and that ACBP3 overexpression enhances degradation of the autophagy (ATG)-related protein ATG8 and disrupts autophagosome formation suggest a role for ACBP3 as a phospholipid binding protein involved in the regulation of leaf senescence by modulating membrane phospholipid metabolism and ATG8 stability in Arabidopsis. Accelerated senescence in ACBP3-OEs is dependent on salicylic acid but not jasmonic acid signaling.

The Cytochrome P450 CYP86A22 Is a Fatty Acyl-CoA ω-Hydroxylase Essential for Estolide Synthesis in the Stigma of Petunia hybrida

The stigmatic estolide is a lipid-based polyester constituting the major component of exudate in solanaceous plants. Although the exudate is believed to play important roles in the pollination process, the biosynthetic pathway of stigmatic estolide, including genes encoding the key enzymes, remains unknown. Here we report the cloning and characterization of the cytochrome P450 gene CYP86A22, which encodes a fatty acyl-CoA omega-hydroxylase involved in estolide biosynthesis in the stigma of Petunia hybrida. A CYP86A22 cDNA was isolated from a developing stigma cDNA library, and the corresponding gene was shown to express predominantly in the developing stigma. Among six P450 genes isolated from this library, only CYP86A22 was implicated in omega-hydroxylation following RNA interference (RNAi)-mediated suppression. Unlike wild-type plants in which omega-hydroxy fatty acids (mainly in the form of 18-hydroxy oleic acid and 18-hydroxy linoleic acid) compose 96% of total stigma fatty acids, the omega-hydroxy fatty acids were essentially absent in the stigmas from 18 of 46 CYP86A22-RNAi transgenic plants and had varying levels of suppression in the remaining 28 plants. Furthermore, lipids in the 18 CYP86A22-RNAi stigmas were predominantly triacylglycerols and diacylglycerols instead of the estolides, which characterize the wild-type stigma. Analyses of recombinant CYP86A22 conclusively demonstrated that this P450 is a omega-hydroxylase with a substrate preference for both saturated and unsaturated acyl-CoAs rather than free fatty acids. We conclude that the cytochrome P450 enzyme CYP86A22 is the key fatty acyl-CoA omega-hydroxylase essential for the production of omega-hydroxy fatty acids and the biosynthesis of triacylglycerol-/diacylglycerol-based estolide polyesters in the petunia stigma.

A transcription factor that senses fatty acid structure

Bacteria regulate their membrane biophysical properties by the coordinated synthesis of saturated and unsaturated fatty acids. In Pseudomonas aeruginosa, unsaturated fatty acids are produced by the de novo fatty acid biosynthetic pathway anaerobically, or by oxidative desaturation of the existing fatty acids catalyzed by Δ9 desaturases DesA and DesB. DesA introduces the double bond into phospholipid acyl chains, whilst DesB desaturates acyl‐CoAs. A transcriptional repressor DesT regulates the expression of desCB operon. The expression level of desCB is strongly and selectively up‐regulated in a ΔdesT deletion strain. Both electrophoresis mobility shift assay and DNase I footprinting analysis demonstrated the existence of two DesT binding sites in the desT‐desCB promoter region, P1 and P2. The binding of purified DesT to P2 was enhanced by unsaturated acyl‐CoAs, whereas saturated acyl‐CoAs prevented DesT interaction with P2. The biological importance of this interaction was verified by the up‐regulation of desCB in cells grown in the presence of stearate and their repression when oleate was present. This unique ligand selectivity allows DesT to sense the physical properties of the cellular acyl‐CoA pool and modulate the expression of the acyl‐CoA Δ9‐desaturase system accordingly. (Supported by GM34496 and ALSAC)

A Pseudomonas aeruginosa transcription factor that senses fatty acid structure

Cells regulate their membrane phospholipid biophysical properties by the co-ordinated synthesis of saturated and unsaturated fatty acids. In bacteria, unsaturated fatty acids are produced by the de novo fatty acid biosynthetic pathway anaerobically, or by oxidative desaturation of the existing fatty acids catalysed by desaturases. A transcriptional repressor in Pseudomonas aeruginosa, DesT (PA4890), regulates the expression of an acyl-CoA desaturase operon (desCB, PA4889 and PA4888). The desCB operon is located adjacent to desT and is transcribed in the opposite direction. The expression level of desCB is strongly and selectively upregulated in a DeltadesT-deletion strain. Both electrophoresis mobility shift assay and DNase I footprinting analysis demonstrated the existence of two DesT binding sites in the desT-desCB promoter region, P1 and P2. The binding of purified DesT to P2 was enhanced by unsaturated acyl-CoAs, whereas saturated acyl-CoAs prevented DesT interaction with P2. The biological importance of this interaction was verified by the upregulation of desCB and desT in cells grown in the presence of stearate and their repression when oleate was present. This unique ligand selectivity allows DesT to sense the physical properties of the cellular acyl-CoA pool and modulate the expression of the acyl-CoA Delta9-desaturase system to adjust fatty acid desaturation activity accordingly.

Identification and Characterization of the Major Lysophosphatidylethanolamine Acyltransferase in Saccharomyces cerevisiae

We recently demonstrated that yeast actively import lysophosphatidylethanolamine (lyso-PtdEtn) through the action of plasma membrane P-type ATPases and rapidly acylate it to form PtdEtn. The predominant lyso-PtdEtn acyltransferase (LPEAT) activity present in cellular extracts is acyl-CoA dependent, but the identity of the gene encoding this activity was unknown. We now demonstrate that a previously uncharacterized open reading frame, YOR175C, encodes the major acyl-CoA-dependent LPEAT activity in yeast and henceforth refer to it as ALE1 (acyltransferase for lyso-PtdEtn). Ale1p is an integral membrane protein and is highly enriched in the mitochondria-associated endoplasmic reticulum membrane. It is a member of the membrane-bound O-acyltransferase family and possesses a dibasic motif at its C terminus that is likely responsible for Golgi retrieval and retention in the endoplasmic reticulum. An ale1Delta strain retains only trace amounts of acyl-CoA-dependent LPEAT activity, and strains lacking the capacity for PtdEtn synthesis via the phosphatidylserine decarboxylase and Kennedy pathways show a stringent requirement for both exogenous lyso-PtdEtn and a functional ALE1 gene for viability. Ale1p catalytic activity has a pH optimum between pH 7 and 7.5 and a strong preference for unsaturated acyl-CoA substrates.

Characterization of Pseudomonas aeruginosa DesT (PA4890), a transcriptional factor sensing long‐chain acyl‐CoAs

Cells regulate their membrane phospholipid biophysical properties by coordinated synthesis of saturated and unsaturated fatty acids. In bacteria, unsaturated fatty acids are produced by the de novo fatty acid biosynthetic pathway anaerobically, or by oxidative desaturation of the existing fatty acids catalyzed by desaturases. In this study, we have identified and characterized a unique transcriptional repressor in Pseudomonas aeruginosa, DesT (PA4890), which regulates the expression of an acyl-CoA desaturase operon (desCB, PA4889 and PA4888). The desCB operon is located immediately upstream of desT and is transcribed to the opposite direction to desT. Both Affymetrix array and quantitative real-time PCR analyses demonstrated that desC and desB expression were strongly and selectively up-regulated in a ΔdesT deletion strain. The purified recombinant DesT bound to the desCB promoter region at two distinct sites with different affinities. The interaction of DesT with the high-affinity binding site (Kd = 0.35 ± 0.03 nM) was enhanced by unsaturated acyl-CoAs such as palmitoleoyl-CoA and oleoyl-CoA (Kd = 0.10 ± 0.01 nM), whereas saturated acyl-CoAs such as myristoyl-CoA and palmitoyl-CoA prevented DesT interaction with the high-affinity site. CoA, short-chain acyl-CoAs, free fatty acids and acyl-ACPs did not influence DesT-DNA interactions in vitro. Binding to the low-affinity site was not regulated. Accordingly, the expression of desB was up-regulated in cells grown in the presence of stearate and repressed when oleate was present. Thus, DesT senses the composition of the cellular acyl-CoA pool to either increase or decrease the expression of the acyl-CoA Δ9 desaturase system to adjust fatty acid metabolism to the composition of the exogenous fatty acid pool.

Unsaturated Fatty Acids Phosphorylate and Destabilize ABCA1 through a Phospholipase D2 Pathway

Abnormal high density lipoprotein (HDL) metabolism among patients with diabetes and insulin resistance may contribute to their increased risk of atherosclerosis. ATP-binding cassette transporter ABCA1 mediates the transport of cholesterol and phospholipids from cells to HDL apolipoproteins and thus modulates HDL levels and atherogenesis. Unsaturated fatty acids, which are elevated in diabetes, impair the ABCA1 pathway in cultured cells by destabilizing ABCA1 protein. Here we examined the cellular pathway that mediates the ABCA1 destabilizing effects of fatty acids. The long-chain acyl-CoA synthetase inhibitor triacsin C completely reversed fatty acid-induced ABCA1 destabilization, indicating that fatty acids need to be activated to their CoA derivatives to enhance ABCA1 degradation. Unsaturated but not saturated fatty acids stimulated phospholipase D (PLD) activity, the PLD inhibitor 1-butanol prevented the unsaturated fatty acid-induced reduction in ABCA1 levels, and the PLD2 activator mastoparan markedly reduced ABCA1 protein levels, implicating a role for PLD2 in the ABCA1 destabilizing effects of fatty acids. Unsaturated fatty acids and mastoparan increased phosphorylation of ABCA1 serines. PLD2 small interfering RNA abolished the ability of unsaturated fatty acids to inhibit lipid transport activity, to reduce protein levels, and to increase serine phosphorylation of ABCA1. The diacylglycerol analog oleoylacetylglycerol also reduced ABCA1 protein levels and increased its serine phosphorylation, suggesting that PLD2-generated diacylglycerols promote the destabilizing phosphorylation of ABCA1. These data provide evidence that intracellular unsaturated acyl-CoA derivatives destabilize ABCA1 by activating a PLD2 signaling pathway.

Human Acyl-CoA Dehydrogenase-9 Plays a Novel Role in the Mitochondrial β-Oxidation of Unsaturated Fatty Acids

Unsaturated fatty acids play an important role in the prevention of human diseases such as diabetes, obesity, cancer, and neurodegeneration. However, their oxidation in vivo by acyl-CoA dehydrogenases (ACADs) that catalyze the first step of each cycle of mitochondrial fatty acid beta-oxidation is not entirely understood. Recently, a novel ACAD (ACAD-9) of unknown function that is highly homologous to human very-long-chain acyl-CoA dehydrogenase was identified by large-scale random sequencing. To characterize its enzymatic role, we have expressed ACAD-9 in Escherichia coli, purified it, and determined its pattern of substrate utilization. The N terminus of the mature form of the enzyme was identified by in vitro mitochondrial import studies of precursor protein. A 37-amino acid leader peptide was cleaved sequentially by two mitochondrial peptidases to yield a predicted molecular mass of 65 kDa for the mature subunit. Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane. Gel filtration analysis indicated that, like very-long-chain acyl-CoA dehydrogenase, ACAD-9 is a dimer, in contrast to the other known ACADs, which are tetramers. Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA). These results suggest a previously unrecognized role for ACAD-9 in the mitochondrial beta-oxidation of long-chain unsaturated fatty acids. Because of the substrate specificity and abundance of ACAD-9 in brain, we speculate that it may play a role in the turnover of lipid membrane unsaturated fatty acids that are essential for membrane integrity and structure.