Abnormal fatty alcohol metabolism in cultured keratinocytes from patients with Sjögren-Larsson syndrome

Sj?gren-Larsson syndrome (SLS) is an inherited neurocutaneous disorder characterized by ichthyosis, mental retardation, spasticity, and deficient activity of fatty aldehyde dehydrogenase (FALDH). FALDH is an enzyme component of fatty alcohol:NAD oxidoreductase (FAO), which is necessary for fatty alcohol metabolism. To better understand the biochemical basis for the cutaneous symptoms in this disease, we investigated lipid metabolism in cultured keratinocytes from SLS patients. Enzyme activities of FALDH and FAO in SLS cells were <10% of normal. SLS keratinocytes accumulated 45-fold more fatty alcohol (hexadecanol, octadecanol, and octadecenol) than normal, whereas wax esters and 1-O-alkyl-2,3-diacylglycerols were increased by 5.6-fold and 7.5-fold, respectively. SLS keratinocytes showed a reduced incorporation of radioactive octadecanol into fatty acid (24% of normal) and triglyceride (13% of normal), but incorporation into wax esters and 1-O-alkyl-2,3-diacylglycerol was increased by 2.5-fold and 2.8-fold, respectively. Our results indicate that FALDH deficiency in SLS keratinocytes causes the accumulation and diversion of fatty alcohol into alternative biosynthetic pathways. The striking lipid abnormalities in cultured SLS keratinocytes are distinct from those seen in fibroblasts and may be related to the stratum corneum dysfunction and ichthyosis in SLS.     

Fatty aldehyde and fatty alcohol metabolism: Review and importance for epidermal structure and function

Normal fatty aldehyde and alcohol metabolism is essential for epidermal differentiation and function. Long-chain aldehydes are produced by catabolism of several lipids including fatty alcohols, sphingolipids, ether glycerolipids, isoprenoid alcohols and certain aliphatic lipids that undergo α- or ω-oxidation. The fatty aldehyde generated by these pathways is chiefly metabolized to fatty acid by fatty aldehyde dehydrogenase (FALDH, alternately known as ALDH3A2), which also functions to oxidize fatty alcohols as a component of the fatty alcohol:NAD oxidoreductase (FAO) enzyme complex. Genetic deficiency of FALDH/FAO in patients with Sjögren–Larsson syndrome (SLS) results in accumulation of fatty aldehydes, fatty alcohols and related lipids (ether glycerolipids, wax esters) in cultured keratinocytes. These biochemical changes are associated with abnormalities in formation of lamellar bodies in the stratum granulosum and impaired delivery of their precursor membranes to the stratum corneum (SC). The defective extracellular SC membranes are responsible for a leaky epidermal water barrier and ichthyosis. Although lamellar bodies appear to be the pathogenic target for abnormal fatty aldehyde/alcohol metabolism in SLS, the precise biochemical mechanisms are yet to be elucidated. Nevertheless, studies in SLS highlight the critical importance of FALDH and normal fatty aldehyde/alcohol metabolism for epidermal function. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Identification of long chain specific aldehyde reductase and its use in enhanced fatty alcohol production in E. coli

Long chain fatty alcohols have wide application in chemical industries and transportation sector. There is no direct natural reservoir for long chain fatty alcohol production, thus many groups explored metabolic engineering approaches for its microbial production. Escherichia coli has been the major microbial platform for this effort, however, terminal endogenous enzyme responsible for converting fatty aldehydes of chain length C14-C18 to corresponding fatty alcohols is still been elusive. Through our in silico analysis we selected 35 endogenous enzymes of E. coli having potential of converting long chain fatty aldehydes to fatty alcohols and studied their role under in vivo condition. We found that deletion of ybbO gene, which encodes NADP⁺ dependent aldehyde reductase, led to >90% reduction in long chain fatty alcohol production. This feature was found to be strain transcending and reinstalling ybbO gene via plasmid retained the ability of mutant to produce long chain fatty alcohols. Enzyme kinetic study revealed that YbbO has wide substrate specificity ranging from C6 to C18 aldehyde, with maximum affinity and efficiency for C18 and C16 chain length aldehyde, respectively. Along with endogenous production of fatty aldehyde via optimized heterologous expression of cyanobaterial acyl-ACP reductase (AAR), YbbO overexpression resulted in 169 mg/L of long chain fatty alcohols. Further engineering involving modulation of fatty acid as well as of phospholipid biosynthesis pathway improved fatty alcohol production by 60%. Finally, the engineered strain produced 1989 mg/L of long chain fatty alcohol in bioreactor under fed-batch cultivation condition. Our study shows for the first time a predominant role of a single enzyme in production of long chain fatty alcohols from fatty aldehydes as well as of modulation of phospholipid pathway in increasing the fatty alcohol production.     

Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) plays a major role in acetaldehyde detoxification. The alcohol sensitivity is associated with a genetic deficiency of ALDH2. We have previously reported that this deficiency influences the risk for late-onset Alzheimer's disease. However, the biological effects of the deficiency on neuronal cells are poorly understood. Thus, we obtained ALDH2-deficient cell lines by introducing mouse mutant Aldh2 cDNA into PC12 cells. The mutant ALDH2 repressed mitochondrial ALDH activity in a dominant negative fashion, but not cytosolic activity. The resultant ALDH2-deficient transfectants were highly vulnerable to exogenous 4-hydroxy-2-nonenal, an aldehyde derivative generated by the reaction of superoxide with unsaturated fatty acid. In addition, the ALDH2-deficient transfectants were sensitive to oxidative insult induced by antimycin A, accompanied by an accumulation of proteins modified with 4-hydroxy-2-nonenal. Thus, these findings suggest that mitochondrial ALDH2 functions as a protector against oxidative stress.     

Sjögren-Larsson syndrome: accumulation of free fatty alcohols in cultured fibroblasts and plasma

Sj?gren-Larsson syndrome (SLS) is an inherited disorder associated with deficient oxidation of long-chain aliphatic alcohols. Previous studies have reported modest elevations in total (free + esterified) fatty alcohols in SLS, but free fatty alcohols have not been selectively measured, in part because of their low concentrations in most tissues and the presence of trace fatty alcohol contaminants in some solvents used for their analysis. We adapted methods to measure free fatty alcohols in cultured cells and plasma that minimize exogenous alcohol contamination. Fatty alcohols were analyzed as acetate derivatives, using capillary column gas chromatography. By this method, cultured skin fibroblasts from SLS patients were found to have 7- and 8-fold elevations in the mean content of hexadecanol (16:0-OH) and octadecanol (18:0-OH), respectively. The mean plasma 16:0-OH and 18:0-OH concentrations in SLS patients (n = 11) were 9- and 22-fold higher than in normal controls, respectively. In SLS fibroblasts, most of the fatty alcohol (59%) that accumulated was free rather than esterified alcohol, whereas free alcohol accounted for 23% of the total alcohol in normal cells. These results indicate that elevations in free fatty alcohols provide a sensitive marker for the enzymatic defect in SLS. The ability to measure free fatty alcohols in cultured cells and plasma should prove useful for investigations of normal fatty alcohol metabolism and the deranged metabolism in SLS.     

Fatty aldehyde dehydrogenase: genomic structure, expression and mutation analysis in Sjögren-Larsson syndrome

Fatty aldehyde dehydrogenase (FALDH) is a microsomal enzyme that catalyzes the oxidation of medium- and long-chain aliphatic aldehydes derived from metabolism of fatty alcohol, phytanic acid, ether glycerolipids and leukotriene B4. The FALDH gene (ALDH3A2) in man and mouse consists of 11 exons and is closely linked to the gene for ALDH3. In both species, alternative splicing results in formation of a second minor protein, FALDHv, that has a unique carboxy-terminal end. The functional significance of this alternate protein is not known. In humans, mutations in the FALDH gene cause Sj?gren-Larsson syndrome (SLS), which is characterized by ichthyosis, mental retardation and spasticity. Missense mutations involving 24 amino acid positions in FALDH have been identified. These amino acids are more highly conserved among related class 3 aldehyde dehydrogenase enzymes than expected, suggesting that they are critically important for protein folding, catalysis or stability. Studies of mutations in SLS should prove useful for understanding structure-function correlations in FALDH and other aldehyde dehydrogenase proteins.     

O2 incorporation into a long-chain fatty acid during bacterial luminescence

The bioluminescence-dependent oxidation of a long-chain fatty aldehyde catalyzed by luciferase from Photobacterium phosphoreum has been studied in ¹⁸O₂ experiments. The results show the incorporation of one atom of molecular oxygen into the product, the corresponding fatty acid. This incorporation is not the result of exchange of ¹⁸O₂ with the aldehyde prior to oxidation to the acid, thereby indicating that the bacterial luciferase catalyzes an aldehyde monooxygenase reaction which is coupled with bioluminescence.     

Fatty aldehyde dehydrogenase: genomic structure,expression and mutation analysis in Sjögren–Larsson syndrome

Fatty aldehyde dehydrogenase (FALDH) is a microsomal enzyme that catalyzes the oxidation of medium- and long-chain aliphatic aldehydes derived from metabolism of fatty alcohol, phytanic acid, ether glycerolipids and leukotriene B4. The FALDH gene (ALDH3A2) in man and mouse consists of 11 exons and is closely linked to the gene for ALDH3. In both species, alternative splicing results in formation of a second minor protein, FALDHv, that has a unique carboxy-terminal end. The functional significance of this alternate protein is not known. In humans, mutations in the FALDH gene cause Sjögren–Larsson syndrome (SLS), which is characterized by ichthyosis, mental retardation and spasticity. Missense mutations involving 24 amino acid positions in FALDH have been identified. These amino acids are more highly conserved among related class 3 aldehyde dehydrogenase enzymes than expected, suggesting that they are critically important for protein folding, catalysis or stability. Studies of mutations in SLS should prove useful for understanding structure–function correlations in FALDH and other aldehyde dehydrogenase proteins.     

Characterisation of recombinant human fatty aldehyde dehydrogenase: Implications for Sjögren-Larsson syndrome

Fatty aldehyde dehydrogenase (FALDH) is an NAD⁺-dependent oxidoreductase involved in the metabolism of fatty alcohols. Enzyme activity has been implicated in the pathology of diabetes and cancer. Mutations in the human gene inactivate the enzyme and cause accumulation of fatty alcohols in Sjögren-Larsson syndrome, a neurological disorder resulting in physical and mental handicaps. Microsomal FALDH was expressed in E. coli and purified. Using an in vitro activity assay an optimum pH of ～9.5 and temperature of ～35°C were determined. Medium- and long-chain fatty aldehydes were converted to the corresponding acids and kinetic parameters determined. The enzyme showed high activity with heptanal, tetradecanal, hexadecanal and octadecanal with lower activities for the other tested substrates. The enzyme was also able to convert some fatty alcohol substrates to their corresponding aldehydes and acids, at 25–30% the rate of aldehyde oxidation. A structural model of FALDH has been constructed, and catalytically important residues have been proposed to be involved in alcohol and aldehyde oxidation: Gln-120, Glu-207, Cys-241, Phe-333, Tyr-410 and His-411. These results place FALDH in a central role in the fatty alcohol/acid interconversion cycle, and provide a direct link between enzyme inactivation and disease pathology caused by accumulation of alcohols.     

A single aldehyde group can serve as a structural element for recognition by transmembrane protein CD36

Transmembrane protein CD36 is considered to bind its distinct ligands such as long-chain fatty acids primarily by recognizing their terminal carboxyl moiety. In this study, we provide evidence that long-chain fatty aldehydes, such as oleic aldehyde, can be recognized by CD36. We suggest that a single aldehyde group may also serve as one of the structural elements recognizable by CD36.     

Transmembrane transport of fatty acids in the heart

Summary Although fatty acid uptake by the myocardium is rapid and efficient, the mechanism of their transmembrane transport has been unclear. Fatty acids are presented to the plasma membrane of cardiomyocytes as albumin complexes within the plasma. Since albumin is not taken up by the cells, it was postulated that specific high affinity binding sites at the sarcolemma may mediate the dissociation of fatty acids from the albumin molecules, before they are transported into the cells. In studies with a representative long-chain fatty acid, oleate, it was in fact shown that fatty acids bind with high affinity to isolated plasma membranes of rat heart myocytes revealing a K_D of 42 nM. Moreover, a specific membrane fatty acid-binding protein (MFABP) was isolated from these membranes. It had a molecular weight of 40 kD, an isoelectric point of 9.0, and lacked carbohydrate or lipid components. Binding to a specific membrane protein might represent the first step of a carrier mediated uptake process. Therefore, the uptake kinetics of oleate by isolated rat heart myocytes was determined under conditions where only cellular influx and not metabolism occurred. Uptake revealed saturation kinetics and was temperature dependent which were considered as specific criteria for a facilitated transport mechanism. For evaluation whether uptake is mediated by MFABP, the effect of a monospecific antibody to this protein on cellular influx of oleate was examined. Inhibition of uptake of fatty acids but not of glucose by the antibody to MFABP indicated the physiologic significance of this protein as transmembrane carrier in the cellular uptake process of fatty acids. Such a transporter might represent an important site for the metabolic regulation of fatty acid influx into the myocardium.     

Gene gun-mediated in vivo analysis of tissue-specific repression of gene transcription driven by the chicken ovalbumin promoter in the liver and oviduct of laying Hens

Carnitine palmitoyltransferase-I (CPT-I) plays a crucial role in regulating cardiac fatty acid oxidation which provides the primary source of energy for cardiac muscle contraction. CPT-I catalyzes the transfer of long chain fatty acids into mitochondria and is recognized as the primary rate controlling step in fatty acid oxidation. Molecular cloning techniques have demonstrated that two CPT-I isoforms exist as genes encoding the 'muscle' and 'liver' enzymes. Regulation of fatty acid oxidation rates depends on both short-term regulation of enzyme activity and long-term regulation of enzyme synthesis. Most early investigations into metabolic control of fatty acid oxidation at the CPT-I step concentrated on the hepatic enzyme which can be inhibited by malonyl-CoA and can undergo dramatic amplification or reduction of its sensitivity to inhibition by malonyl-CoA. The muscle CPT-I is inherently more sensitive to malonyl-CoA inhibition but has not been found to undergo any alteration of its sensitivity. Short-term control of activity of muscle CPT-I is apparently regulated by malonyl-CoA concentration in response to fuel supply (glucose, lactate, pyruvate and ketone bodies). The liver isoform is the only CPT-I enzyme present in the mitochondria of liver, kidney, brain and most other tissues while muscle CPT-I is the sole isoform expressed in skeletal muscle as well as white and brown adipocytes. The heart is unique in that it contains both muscle and liver isoforms. Liver CPT-I is highly expressed in the fetal heart, but at birth its activity begins to decline whereas the muscle isoform, which is very low at birth, becomes the predominant enzyme during postnatal development. In this paper, the differential regulation of the two CPT-I isoforms at the protein and the gene level will be discussed.     

Fatty acid metabolism in human breast cancer cells (MCF7) transfected with heart-type fatty acid binding protein

The human breast cancer cell line MCF7 does not express heart-type fatty acid binding protein (H-FABP), a marker protein for differentiated mammary gland. MCF7 cells transfected with the bovine H-FABP cDNA expressed the corresponding protein and were characterized by growth inhibition and lower tumorgenicity in nude mice [22]. By enzyme linked immunoassay we now determined the amount of bovine H-FABP in these cells as 638 ± 80 ng/mg protein and used the transfected cells to study the role of H-FABP in fatty acid metabolism. Compared to control cells the uptake of radioactively labelled palmitic acid and oleic acid into MCF7 cells after 30 or 60 min was increased by 67% in H-FABP expressing transfectants, demonstrating a stimulatory role for this FABP-type in fatty acid metabolism. However, preferential targeting of [¹⁴C]oleic acid into neutral or phospholipid classes was not observed by the criterion of high performance thin layer chromatography followed by autoradiography. A reason for the modest increase of fatty acid uptake in H-FABP transfected MCF7 cells may be the basal expression of epidermal-type FABP, which was detected for the first time in these cells. It appears that the small amount of E-FABP expressed in MCF7 cells fulfils the need of the cells for a cytosolic fatty acid carrier under culture conditions and that even high concentrations of another FABP do only slightly increase the uptake due to limitations of fatty acid transport through the plasma membrane or of metabolism.     

Substrate preferences of O-methyltransferases in alfalfa suggest new pathways for 3-O-methylation of monolignols

Measurement of relative O-methyltransferase activities against all potential substrates in the monolignol pathway in developing alfalfa stem extracts revealed activities in the order: caffeoyl CoA > caffeoyl alcohol > 5-hydroxyferulic acid > caffeoyl aldehyde > 5-hydroxyconiferyl alcohol > 5-hydroxyferuloyl CoA > 5-hydroxyconiferaldehyde > caffeic acid. Maxima for all activities occurred in the seventh internode. In stem extracts from transgenic alfalfa with antisense downregulated caffeoyl CoA O-methyltransferase (CCoAOMT), activities with all substrates except for the two coenzyme A esters were unaffected. In contrast, downregulation of caffeic acid O-methyltransferase (COMT) reduced activities against the non-esterifed substrates in the order: 5-hydroxyconiferyl alcohol > 5-hydroxyferulic acid and caffeoyl alcohol > caffeoyl aldehyde > caffeic acid > 5-hydroxyconiferaldehyde. Recombinant COMT expressed in Escherichia coli exhibited the highest V(max)/K(m) values with 5-hydroxyconiferaldehyde and caffeoyl aldehyde, and the lowest with caffeic acid. These results indicate that COMT is unlikely to methylate caffeic acid during lignin biosynthesis in vivo, and provide enzymatic evidence for an alternative pathway to monolignols involving methylation of caffeoyl aldehyde and/or caffeoyl alcohol by COMT. The concept of independent pathways to guaiacyl and syringyl monolignols is discussed.     

Whole‐cell biocatalytic and de novo production of alkanes from free fatty acids in Saccharomyces cerevisiae

Rapid global industrialization in the past decades has led to extensive utilization of fossil fuels, which resulted in pressing environmental problems due to excessive carbon emission. This prompted increasing interest in developing advanced biofuels with higher energy density to substitute fossil fuels and bio‐alkane has gained attention as an ideal drop‐in fuel candidate. Production of alkanes in bacteria has been widely studied but studies on the utilization of the robust yeast host, Saccharomyces cerevisiae, for alkane biosynthesis have been lacking. In this proof‐of‐principle study, we present the unprecedented engineering of S. cerevisiae for conversion of free fatty acids to alkanes. A fatty acid α‐dioxygenase from Oryza sativa (rice) was expressed in S. cerevisiae to transform C_12–18 free fatty acids to C_11–17 aldehydes. Co‐expression of a cyanobacterial aldehyde deformylating oxygenase converted the aldehydes to the desired alkanes. We demonstrated the versatility of the pathway by performing whole‐cell biocatalytic conversion of exogenous free fatty acid feedstocks into alkanes as well as introducing the pathway into a free fatty acid overproducer for de novo production of alkanes from simple sugar. The results from this work are anticipated to advance the development of yeast hosts for alkane production. Biotechnol. Bioeng. 2017;114: 232–237. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Characterisation of recombinant human fatty aldehyde dehydrogenase: implications for Sjögren-Larsson syndrome

Fatty aldehyde dehydrogenase (FALDH) is an NAD+-dependent oxidoreductase involved in the metabolism of fatty alcohols. Enzyme activity has been implicated in the pathology of diabetes and cancer. Mutations in the human gene inactivate the enzyme and cause accumulation of fatty alcohols in Sj?gren-Larsson syndrome, a neurological disorder resulting in physical and mental handicaps. Microsomal FALDH was expressed in E. coli and purified. Using an in vitro activity assay an optimum pH of approximately 9.5 and temperature of approximately 35 degrees C were determined. Medium- and long-chain fatty aldehydes were converted to the corresponding acids and kinetic parameters determined. The enzyme showed high activity with heptanal, tetradecanal, hexadecanal and octadecanal with lower activities for the other tested substrates. The enzyme was also able to convert some fatty alcohol substrates to their corresponding aldehydes and acids, at 25-30% the rate of aldehyde oxidation. A structural model of FALDH has been constructed, and catalytically important residues have been proposed to be involved in alcohol and aldehyde oxidation: Gln-120, Glu-207, Cys-241, Phe-333, Tyr-410 and His-411. These results place FALDH in a central role in the fatty alcohol/acid interconversion cycle, and provide a direct link between enzyme inactivation and disease pathology caused by accumulation of alcohols.     

Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase

The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents.     

乙醇胁迫处理对酒酒球菌SD-2a生理特性的影响

为制备高效的葡萄酒乳酸菌发酵剂,以酒酒球菌SD-2a为试验材料,研究了5%、10%不同浓度的乙醇胁迫处理对菌体生长、细胞内MLE活性、冻干存活率及细胞膜脂肪酸组分的影响。试验结果表明乙醇胁迫处理明显降低了菌体的生长速率和生物产量。与对照相比,5%乙醇胁迫处理降低了菌体的冻干存活率,而10%乙醇处理却显著增加了菌体的冻干存活率。细胞膜脂肪酸组分的测定结果表明,前者细胞膜U/S比值为1.12,比对照降低了26.3%,而后者细胞膜U/S比值为2.29,比对照增加了50.6%。故认为酒酒球菌SD-2a为适应不同浓度的乙醇胁迫环境,在细胞膜脂肪酸水平上所采用的机制不同,且菌体的冻干存活率与其细胞膜脂肪酸组分密切相关。     

先进生物燃料导向的脂肪酸途径合成生物学改造

化石能源日益枯竭,迫切需要寻找新型燃料。脂肪族生物燃料由于其热值高、性能好而受到广泛重视。微生物脂肪酸代谢途径是生产先进生物燃料的重要途径。文中综述了近几年基于合成生物学理念改造脂肪酸途径的进展,介绍了合成生物学在微生物柴油、中长链脂肪醇、长链烃类化合物生物合成中的应用,并展望了脂肪族生物燃料的发展方向。