Acyl-coenzyme A synthetases (ACSs) catalyze the fundamental, initial reaction in fatty acid metabolism. "Activation" of fatty acids by thioesterification to CoA allows their participation in both anabolic and catabolic pathways. The availability of the sequenced human genome has facilitated the investigation of the number of ACS genes present. Using two conserved amino acid sequence motifs to probe human DNA databases, 26 ACS family genes/proteins were identified. ACS activity in either humans or rodents was demonstrated previously for 20 proteins, but 6 remain candidate ACSs. For two candidates, cDNA was cloned, protein was expressed in COS-1 cells, and ACS activity was detected. Amino acid sequence similarities were used to assign enzymes into subfamilies, and subfamily assignments were consistent with acyl chain length preference. Four of the 26 proteins did not fit into a subfamily, and bootstrap analysis of phylograms was consistent with evolutionary divergence. Three additional conserved amino acid sequence motifs were identified that likely have functional or structural roles. The existence of many ACSs suggests that each plays a unique role, directing the acyl-CoA product to a specific metabolic fate. Knowing the full complement of ACS genes in the human genome will facilitate future studies to characterize their specific biological functions.-Watkins, P. A., D. Maiguel, Z. Jia, and J. Pevsner. Evidence for 26 distinct acyl-coenzyme A synthetase genes in the human genome. J. Lipid Res.
The family of proteins that includes very long-chain acyl-CoA synthetases (ACSVL) consists of six members. These enzymes have also been designated fatty acid transport proteins. We cloned full-length mouse Acsvl3 cDNA and characterized its protein product ACSVL3/ fatty acid transport protein 3. The predicted amino acid sequence contains two highly conserved motifs characteristic of acyl-CoA synthetases. Northern blot analysis revealed that the mouse Acsvl3 mRNA is highly expressed in adrenal gland, testis, and ovary, with lower expression in the brain of adult mice. A developmental Northern blot revealed that Acsvl3 mRNA levels were significantly higher in embryonic mouse brain (embryonic days 12-14) than in newborn or adult mice, suggesting a possible role in nervous system development. Immunohistochemistry revealed high ACSVL3 expression in adrenal cortical cells, spermatocytes and interstitial cells of the testis, theca cells of the ovary, cerebral cortical neurons, and cerebellar Purkinje cells. Endogenous ACSVL3 was found primarily in mitochondria of MA-10 and Neuro2a cells by both Western blot analysis of subcellular fractions and immunofluorescence analysis. In MA-10 cells, loss-of-function studies using RNA interference confirmed that endogenous ACSVL3 is an acyl-CoA synthetase capable of activating both longchain (C16:0) and very long-chain (C24:0) fatty acids. However, despite decreased acyl-CoA synthetase activity, initial rates of fatty acid uptake were unaffected by knockdown of Acsvl3 expression in MA-10 cells. These studies cast doubt on the designation of ACSVL3 as a fatty acid transport protein.The transport of fatty acids into cells and their subsequent "activation" by thioesterification to CoA are fundamental processes required for entry of fatty acids into the metabolic stream (1). The mechanism of fatty acids entry into cells remains controversial. Some investigators argue that specific proteins are required to transport the fatty acid across the plasma membrane (2-6). Others have provided evidence that proteins are not necessary for translocation of fatty acids through the lipid bilayer (7,8). One group of proteins proposed to mediate fatty acid entry into cells are the fatty acid transport proteins (FATPs) 1 (4). The mammalian FATP family consists of six homologous proteins (FATP1-6) that share 35-58% amino acid identity. 2 Studies with cultured cells overexpressing FATP1-6 have demonstrated increased rates of accretion of fluorescent or radiolabeled fatty acids (3, 9). However, interpretation of fatty acid transport studies is hampered by the fact that, once inside cells, fatty acids are rapidly metabolized. Metabolism will decrease the intracellular concentration of the unesterified fatty acid, shifting the concentration gradient across the plasma membrane to promote entry of additional fatty acids into the cell. The design of most transport studies does not distinguish between transport mechanisms that can occur in a protein-free phospholipid bilayer and transport plus metabolism.Indepe...
Fatty acid transport protein 4 (FATP4) is a fatty acyl-CoA synthetase that preferentially activates very long chain fatty acid substrates, such as C24:0, to their CoA derivatives. To gain better insight into the physiological functions of FATP4, we established dermal fibroblast cell lines from FATP4-deficient wrinkle-free mice and wild type (w.t.) mice. FATP4 ؊/؊ fibroblasts had no detectable FATP4 protein by Western blot. Compared with w.t. fibroblasts, cells lacking FATP4 had an 83% decrease in C24:0 activation. Peroxisomal degradation of C24:0 was reduced by 58%, and rates of C24:0 incorporation into major phospholipid species (54 -64% decrease), triacylglycerol (64% decrease), and cholesterol esters (58% decrease) were significantly diminished. Because these lipid metabolic processes take place in different subcellular organelles, we used immunofluorescence and Western blotting of subcellular fractions to investigate the distribution of FATP4 protein and measured enzyme activity in fractions from w.t. and FATP4 ؊/؊ fibroblasts. FATP4 protein and acyl-CoA synthetase activity localized to multiple organelles, including mitochondria, peroxisomes, endoplasmic reticulum, and the mitochondria-associated membrane fraction. We conclude that in murine skin fibroblasts, FATP4 is the major enzyme producing very long chain fatty acid-CoA for lipid metabolic pathways. Although FATP4 deficiency primarily affected very long chain fatty acid metabolism, mutant fibroblasts also showed reduced uptake of a fluorescent long chain fatty acid and reduced levels of long chain polyunsaturated fatty acids. FATP4-deficient cells also contained abnormal neutral lipid droplets. These additional defects indicate that metabolic abnormalities in these cells are not limited to very long chain fatty acids.Fatty acids containing 22 or more carbon atoms are referred to as very long chain fatty acids (VLCFA).2 VLCFA are normal constituents of membrane lipids but are particularly abundant in brain, testis, and skin (1-3). Fatty acids, including VLCFA, cannot participate in most metabolic processes unless they are first activated to their CoA derivatives (4). Once activated, however, fatty acids can be degraded to release stored energy or incorporated into glycerolipids, phospholipids, sphingolipids, glycolipids, cholesterol esters, and other complex lipids. Certain acyl-CoAs also serve as substrates for protein acylation and/or function as regulatory molecules. Thus, the fatty acid activation reaction is central to normal cellular lipid metabolism.The acyl-CoA synthetase (ACS) enzyme family catalyzes this essential reaction (4). Because fatty acid chain lengths vary from 2 to more than 30 carbon atoms, different ACSs capable of activating short, medium, long, and very long chain fatty acids have evolved. Based on analysis of amino acid sequence homology, we estimate that mammalian genomes encode at least 25 different ACSs.3 Further analysis of highly conserved domains within these sequences allowed us to group the enzymes into families (5). We ...
BackgroundThe urban environment has been criticized for promoting ‘nature-deficit’ and ‘child-nature disconnectedness’. Keeping in mind the importance of nature exposure and its extensive health benefits, many environmental programs around the world hope to (re)connect children with nature. To evaluate the effectiveness of such efforts, valid tools to measure Connectedness to Nature (CN) are needed but do not exist today, especially for use with pre-schoolers.MethodsThe original CN Index was modified and tested among the Parents of Preschool Children (CNI-PPC) in an urban setting (Hong Kong) for its internal consistency (n = 299) and external validity (n = 194). The ‘Strength and Difficulties Questionnaire’ (SDQ) was chosen for divergent and convergent analysis. Conventional recommendations for test adaptation and translation were used.ResultsConfirmatory factor analysis (CFA) revealed that the 16-item scale adequately captured four major dimensions: enjoyment of nature, empathy for nature, responsibility toward nature, and awareness of nature (Cronbach’s α were respectively .86, .87, .75 and .80). When tested against the SDQ, a validated measure for child psychological functioning, and identification of children’s problem behaviours, three CNI-PPC factors influenced the SDQ outcomes: (1) the more enjoyment of nature children displayed the less overall distress and impairment they exhibited (β = -.64); (2) greater responsibility toward nature in children was associated with less hyperactivity (β = -.50), fewer behavioural and peer difficulties (β = -.62 and β = -.65 respectively) and improved prosocial behaviour (β = .77); (3) the more aware children were of nature, the less emotional difficulties they exhibited (β = -.51). The variance explained was large (range R2 = .42 to .80).ConclusionsThus, CNI-PPC factors have meaningful and substantive associations with the strengths and difficulties parents perceive in their children. This indicates that the CNI-PPC is a valid and reliable instrument to measure CN at an age when children cannot respond for themselves. Further, this simple tool can help researchers/practitioners to better understand how connectedness to nature affects child psychological functioning and wellbeing.Trial registrationClinicalTrials.gov Identifier: NCT02715544. Registered 8 March 2016.
The topology of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes, was established by site-directed fluorescence labeling, a simple strategy that generates topological information in the context of the intact protein. Accessibility of cysteine to the fluorescent thiol-directed probe Oregon green maleimide (OGM) was examined for a panel of 34 single-cysteine variants, each generated in a His 9 -tagged cysteine-less host. The reaction with OGM was readily scored by examining the fluorescence profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of material purified by Ni 2؉ -linked affinity chromatography. A position was assigned an external location if its single-cysteine derivative reacted with OGM added to intact cells; a position was designated internal if OGM labeling required cell lysis. We also showed that labeling of external, but not internal, positions was blocked by prior exposure of cells to the impermeable and nonfluorescent thiol-specific agent ethyltrimethylammonium methanethiosulfonate. Of the 34 positions examined in this way, 29 were assigned unambiguously to either an internal or external location; 5 positions could not be assigned, since the target cysteine failed to react with OGM. There was no evidence of false-positive assignment. Our findings document a simple and rapid method for establishing the topology of a membrane protein and show that OxlT has 12 transmembrane segments, confirming inferences from hydropathy analysis.The gram-negative bacterium Oxalobacter formigenes sustains a proton motive force by utilizing a "virtual" proton pump based on the transport and metabolism of oxalate. An electric potential (negative inside) arises from action of the antiporter, OxlT, which links inward transport of divalent oxalate to the outward flow of monovalent formate, the product of oxalate decarboxylation. The net inflow of a single negative charge is then phenomenologically linked to generation of a pH gradient (alkaline inside), because decarboxylation of oxalate consumes a single cytosolic proton. Together, these elements comprise the proton motive force used to drive ATP synthesis in this obligate anaerobe (3,14,20,32). Virtual pumps of equivalent construction have now been observed in a number of microorganisms (14,22,25).It is evident that OxlT occupies a central position in the cell biology of O. formigenes and that study of this transporter is relevant to several aspects of microbial physiology. Added interest in OxlT stems from recent work (8,9,26) suggesting that this protein may also serve as a useful model for biochemical study of other transporters. Accordingly, OxlT may contribute to an understanding of membrane transport at both a functional level and a mechanistic level.Hydropathy analysis of the OxlT amino acid sequence, along with other considerations, suggests the presence of 12 transmembrane segments (TM1 to TM12) (1), consistent with the presumed structure of most other members of the major facilitator superfamily (MFS) (30), the superfami...
Acyl-CoA synthetases play a pivotal role in fatty acid metabolism, providing activated substrates for fatty acid catabolic and anabolic pathways. Acyl-CoA synthetases comprise numerous proteins with diverse substrate specificities, tissue expression patterns, and subcellular localizations, suggesting that each enzyme directs fatty acids toward a specific metabolic fate. We reported that hBG1, the human homolog of the acyl-CoA synthetase mutated in the Drosophila mutant "bubblegum," belongs to a previously unidentified enzyme family and is capable of activating both long-and very longchain fatty acid substrates. We now report that when overexpressed, hBG1 can activate diverse saturated, monosaturated, and polyunsaturated fatty acids. Using in situ hybridization and immunohistochemistry, we detected expression of mBG1, the mouse homolog of hBG1, in cerebral cortical and cerebellar neurons and in steroidogenic cells of the adrenal gland, testis, and ovary. The expression pattern and ability of BG1 to activate very long-chain fatty acids implicates this enzyme in the pathogenesis of X-linked adrenoleukodystrophy. In neuron-derived Neuro2a cells, mBG1 co-sedimented with mitochondria and was found in small vesicular structures located in close proximity to mitochondria. RNA interference was used to decrease mBG1 expression in Neuro2a cells and led to a 30 -35% decrease in activation and -oxidation of the long-chain fatty acid, palmitate. These results suggest that in Neuro2a cells, mBG1-activated long-chain fatty acids are directed toward mitochondrial degradation. mBG1 appears to play a minor role in very long-chain fatty acid activation in these cells, indicating that other acyl-CoA synthetases are necessary for very long-chain fatty acid metabolism in Neuro2a cells.
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