C. Elegans Fatty Acid Two-Hydroxylase Regulates Intestinal Homeostasis by Affecting Heptadecenoic Acid Production

Background/Aims: The hydroxylation of fatty acids at the C-2 position is the first step of fatty acid α-oxidation and generates sphingolipids containing 2-hydroxy fatty acyl moieties. Fatty acid 2-hydroxylation is catalyzed by Fatty acid 2-hydroxylase (FA2H) enzyme. However, the precise roles of FA2H and fatty acid 2-hydroxylation in whole cell homeostasis still remain unclear. Methods: Here we utilize Caenorhabditis elegans as the model and systemically investigate the physiological functions of FATH-1/C25A1.5, the highly conserved worm homolog for mammalian FA2H enzyme. Immunostaining, dye-staining and translational fusion reporters were used to visualize FATH-1 protein and a variety of subcellular structures. The “click chemistry” method was employed to label 2-OH fatty acid in vivo. Global and tissue-specific RNAi knockdown experiments were performed to inactivate FATH-1 function. Lipid analysis of the fath-1 deficient mutants was achieved by mass spectrometry. Results: C. elegans FATH-1 is expressed at most developmental stages and in most tissues. Loss of fath-1 expression results in severe growth retardation and shortened lifespan. FATH-1 function is crucially required in the intestine but not the epidermis with stereospecificity. The “click chemistry” labeling technique showed that the FATH-1 metabolites are mainly enriched in membrane structures preferable to the apical side of the intestinal cells. At the subcellular level, we found that loss of fath-1 expression inhibits lipid droplets formation, as well as selectively disrupts peroxisomes and apical endosomes. Lipid analysis of the fath-1 deficient animals revealed a significant reduction in the content of heptadecenoic acid, while other major FAs remain unaffected. Feeding of exogenous heptadecenoic acid (C17: 1), but not oleic acid (C18: 1), rescues the global and subcellular defects of fath-1 knockdown worms. Conclusion: Our study revealed that FATH-1 and its catalytic products are highly specific in the context of chirality, C-chain length, spatial distribution, as well as the types of cellular organelles they affect. Such an unexpected degree of specificity for the synthesis and functions of hydroxylated FAs helps to regulate protein transport and fat metabolism, therefore maintaining the cellular homeostasis of the intestinal cells. These findings may help our understanding of FA2H functions across species, and offer potential therapeutical targets for treating FA2H-related diseases.

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Malonyl-CoA content and fatty acid oxidation in rat muscle and liver in vivo

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2000.279.2.e259 ◽

2000 ◽

Vol 279 (2) ◽

pp. E259-E265 ◽

Cited By ~ 53

Author(s):

David Chien ◽

David Dean ◽

Asish K. Saha ◽

J. P. Flatt ◽

Neil B. Ruderman

Keyword(s):

Fatty Acid ◽

Fatty Acid Oxidation ◽

Fatty Acyl ◽

Whole Body ◽

Acid Oxidation ◽

Malonyl Coa ◽

Plasma Ffa ◽

Gastrocnemius Muscles ◽

Time Point

Malonyl-CoA acutely regulates fatty acid oxidation in liver in vivo by inhibiting carnitine palmitoyltransferase. Thus rapid increases in the concentration of malonyl-CoA, accompanied by decreases in long-chain fatty acyl carnitine (LCFA-carnitine) and fatty acid oxidation have been observed in liver of fasted-refed rats. It is less clear that it plays a similar role in skeletal muscle. To examine this question, whole body respiratory quotients (RQ) and the concentrations of malonyl-CoA and LCFA-carnitine in muscle were determined in 48-h-starved rats before and at various times after refeeding. RQ values were 0.82 at baseline and increased to 0.93, 1.0, 1.05, and 1.09 after 1, 3, 12, and 18 h of refeeding, respectively, suggesting inhibition of fat oxidation in all tissues. The increases in RQ at each time point correlated closely ( r = 0.98) with increases (50–250%) in the concentration of malonyl-CoA in soleus and gastrocnemius muscles and decreases in plasma FFA and muscle LCFA-carnitine levels. Similar changes in malonyl-CoA and LCFA-carnitine were observed in liver. The increases in malonyl-CoA in muscle during refeeding were not associated with increases in the assayable activity of acetyl-CoA carboxylase (ACC) or decreases in the activity of malonyl-CoA decarboxylase (MCD). The results suggest that, during refeeding after a fast, decreases in fatty acid oxidation occur rapidly in muscle and are attributable both to decreases in plasma FFA and increases in the concentration of malonyl-CoA. They also suggest that the increase in malonyl-CoA in this situation is not due to changes in the assayable activity of either ACC or MCD or an increase in the cytosolic concentration of citrate.

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Elevation of 20-carbon long chain bases due to a mutation in serine palmitoyltransferase small subunit b results in neurodegeneration

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1516733112 ◽

2015 ◽

Vol 112 (42) ◽

pp. 12962-12967 ◽

Cited By ~ 31

Author(s):

Lihong Zhao ◽

Stefka Spassieva ◽

Kenneth Gable ◽

Sita D. Gupta ◽

Lan-Ying Shi ◽

...

Keyword(s):

Chain Length ◽

Small Subunit ◽

Fatty Acyl ◽

Substrate Affinity ◽

Serine Palmitoyltransferase ◽

Membrane Structures ◽

Long Chain Base ◽

Chain Lengths ◽

Long Chain

Sphingolipids typically have an 18-carbon (C18) sphingoid long chain base (LCB) backbone. Although sphingolipids with LCBs of other chain lengths have been identified, the functional significance of these low-abundance sphingolipids is unknown. The LCB chain length is determined by serine palmitoyltransferase (SPT) isoenzymes, which are trimeric proteins composed of two large subunits (SPTLC1 and SPTLC2 or SPTLC3) and a small subunit (SPTssa or SPTssb). Here we report the identification of an Sptssb mutation, Stellar (Stl), which increased the SPT affinity toward the C18 fatty acyl-CoA substrate by twofold and significantly elevated 20-carbon (C20) LCB production in the mutant mouse brain and eye, resulting in surprising neurodegenerative effects including aberrant membrane structures, accumulation of ubiquitinated proteins on membranes, and axon degeneration. Our work demonstrates that SPT small subunits play a major role in controlling SPT activity and substrate affinity, and in specifying sphingolipid LCB chain length in vivo. Moreover, our studies also suggest that excessive C20 LCBs or C20 LCB-containing sphingolipids impair protein homeostasis and neural functions.

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FATTY ACID ETHYL ESTER FORMATION BY PLASMA IN VITRO

Canadian Journal of Biochemistry ◽

10.1139/o66-026 ◽

1966 ◽

Vol 44 (2) ◽

pp. 219-227 ◽

Cited By ~ 10

Author(s):

W. H. Newsome ◽

J. B. M. Rattray

Keyword(s):

Fatty Acid ◽

Acid Ethyl Ester ◽

Fatty Acyl ◽

Ethyl Esters ◽

Acyl Donor ◽

Significant Activity ◽

Fatty Acid Substrate ◽

Ester Formation

The capacity of rat plasma to form ethyl esters when incubated with ethanol and fatty acid was examined. The process was found to be enzymatic and to involve primarily a direct esterification of fatty acid as opposed to a transesterification requiring a fatty acyl donor. Maximal esterification of oleic acid occurred at pH 6.0 but significant activity existed at physiological pH to indicate a capacity of the plasma to utilize ethanol and fatty acid in concentrations that might be expected in vivo. Both normal and post-heparin plasma were found to esterify endogenous free fatty acid. A major factor affecting the esterification process was the availability of fatty acid substrate and the governing role of plasma albumin in this respect is discussed.

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SMb20651 is another acyl carrier protein from Sinorhizobium meliloti

Microbiology ◽

10.1099/mic.0.022079-0 ◽

2009 ◽

Vol 155 (1) ◽

pp. 257-267 ◽

Cited By ~ 9

Author(s):

Ana Laura Ramos-Vega ◽

Yadira Dávila-Martínez ◽

Christian Sohlenkamp ◽

Sandra Contreras-Martínez ◽

Sergio Encarnación ◽

...

Keyword(s):

Fatty Acid ◽

Sinorhizobium Meliloti ◽

Fatty Acid Biosynthesis ◽

Acyl Carrier Protein ◽

Fatty Acyl ◽

Acyl Carrier Proteins ◽

Coa Ligase ◽

Acyl Chains

Acyl carrier proteins (ACPs) are small acidic proteins that carry growing acyl chains during fatty acid or polyketide synthesis. In rhizobia, there are four different and well-characterized ACPs: AcpP, NodF, AcpXL and RkpF. The genome sequence of Sinorhizobium meliloti 1021 reveals two additional ORFs that possibly encode additional ACPs. One of these, smb20651, is located on the plasmid pSymB as part of an operon. The genes of the operon encode a putative asparagine synthetase (AsnB), the predicted ACP (SMb20651), a putative long-chain fatty acyl-CoA ligase (SMb20650) and a putative ammonium-dependent NAD+ synthetase (NadE1). When SMb20651 was overexpressed in Escherichia coli, [3H]β-alanine, a biosynthetic building block of 4′-phosphopantetheine, was incorporated into the protein in vivo. The purified SMb20651 was modified with 4′-phosphopantetheine in the presence of S. meliloti holo-ACP synthase (AcpS). Also, holo-SMb20651 was modified in vitro with a malonyl group by malonyl CoA-ACP transacylase. In E. coli, coexpression of SMb20651 together with other proteins such as AcpS and SMb20650 led to the formation of additional forms of SMb20651. In this bacterium, acylation of SMb20651 with C12 : 0 or C18 : 0 fatty acids was detected, demonstrating that this protein is involved in fatty acid biosynthesis or transfer. Expression of SMb20651 was detected in S. meliloti as holo-SMb20651 and acyl-SMb20651.

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Evolution of moth sex pheromone composition by a single amino acid substitution in a fatty acid desaturase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1514566112 ◽

2015 ◽

Vol 112 (41) ◽

pp. 12586-12591 ◽

Cited By ~ 29

Author(s):

Aleš Buček ◽

Petra Matoušková ◽

Heiko Vogel ◽

Petr Šebesta ◽

Ullrich Jahn ◽

...

Keyword(s):

Fatty Acid ◽

Amino Acid ◽

Amino Acid Substitution ◽

Functional Characterization ◽

Fatty Acid Desaturase ◽

Fatty Acyl ◽

Site Directed Mutagenesis ◽

Single Amino Acid Substitution ◽

Single Amino Acid

For sexual communication, moths primarily use blends of fatty acid derivatives containing one or more double bonds in various positions and configurations, called sex pheromones (SPs). To study the molecular basis of novel SP component (SPC) acquisition, we used the tobacco hornworm (Manduca sexta), which uses a blend of mono-, di-, and uncommon triunsaturated fatty acid (3UFA) derivatives as SP. We identified pheromone-biosynthetic fatty acid desaturases (FADs) MsexD3, MsexD5, and MsexD6 abundantly expressed in the M. sexta female pheromone gland. Their functional characterization and in vivo application of FAD substrates indicated that MsexD3 and MsexD5 biosynthesize 3UFAs via E/Z14 desaturation from diunsaturated fatty acids produced by previously characterized Z11-desaturase/conjugase MsexD2. Site-directed mutagenesis of sequentially highly similar MsexD3 and MsexD2 demonstrated that swapping of a single amino acid in the fatty acyl substrate binding tunnel introduces E/Z14-desaturase specificity to mutated MsexD2. Reconstruction of FAD gene phylogeny indicates that MsexD3 was recruited for biosynthesis of 3UFA SPCs in M. sexta lineage via gene duplication and neofunctionalization, whereas MsexD5 representing an alternative 3UFA-producing FAD has been acquired via activation of a presumably inactive ancestral MsexD5. Our results demonstrate that a change as small as a single amino acid substitution in a FAD enzyme might result in the acquisition of new SP compounds.

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Two functional FAS-I type fatty acid synthases in Corynebacterium glutamicum

Microbiology ◽

10.1099/mic.0.28012-0 ◽

2005 ◽

Vol 151 (7) ◽

pp. 2421-2427 ◽

Cited By ~ 55

Author(s):

Eva Radmacher ◽

Luke J. Alderwick ◽

Gurdyal S. Besra ◽

Alistair K. Brown ◽

Kevin J. C. Gibson ◽

...

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Corynebacterium Glutamicum ◽

Fatty Acid Synthase ◽

Lipid Analysis ◽

Growth Yield ◽

Mycolic Acid ◽

Mycolic Acids ◽

Fatty Acid Synthases

The lipid-rich Corynebacterianeae, to which Corynebacterium glutamicum and Mycobacterium species belong, produce both fatty acids and mycolic acids. Compared with most other bacteria, C. glutamicum possesses two fatty acid synthases, encoded by fasA (8907 kb; FAS-IA) and fasB (8988 kb; FAS-IB). Here, it was shown by mutational analyses that fasA is essential but fasB is not. However, in a fasA background, the fasB mutation results in a slightly reduced growth yield, l-glutamate production is increased, and comparative lipid analysis suggests that in vivo FAS-IB is active primarily to supply palmitate. Transcript quantifications revealed that the fasB transcript contributes 32 % to both fas transcripts during growth on glucose, affirmative for fasB expression, and that fasB is subordinate to fasA. The fasA transcript is downregulated by 8·3-fold during growth on acetate as compared with glucose. The lipid analyses also demonstrate that cells grown on propionate produce a number of uneven fatty acids (e.g. 15 : 0, 17 : 0, 17 : 1), which are not present in cells grown on glucose or acetate, suggesting that fatty acid synthase in vivo may also use propionyl-CoA as the priming unit in fatty acid synthesis. The fatty acid auxotrophic fasAB double mutant was used to determine the suggested incorporation of fatty acids into mycolic acids. Supplementation of this mutant with uniformly labelled [13C]oleate and analysis of isolated mycolic acids confirmed that mature mycolic acids in the mutant consist exclusively of two fused [13C]oleate molecules. In addition to an altered phospholipid profile, the fasB mutant also exhibits differences in its mycolic acid profile. Taken together, the results show that although FAS-IA is the most relevant fatty acid synthase of C. glutamicum and FAS-IB is supplementary, both synthases are necessary to produce the characteristic lipid environment of this organism.

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Host CDK-1 and formin mediate microvillar effacement induced by enterohemorrhagic Escherichia coli

Nature Communications ◽

10.1038/s41467-020-20355-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Cheng-Rung Huang ◽

Cheng-Ju Kuo ◽

Chih-Wen Huang ◽

Yu-Ting Chen ◽

Bang-Yu Liu ◽

...

Keyword(s):

Escherichia Coli ◽

Enterohemorrhagic Escherichia Coli ◽

Intestinal Cell ◽

Intestinal Cells ◽

Cyclin Dependent Kinase ◽

C Elegans ◽

Mitotic Cyclin ◽

Human Intestinal Cells

AbstractEnterohemorrhagic Escherichia coli (EHEC) induces changes to the intestinal cell cytoskeleton and formation of attaching and effacing lesions, characterized by the effacement of microvilli and then formation of actin pedestals to which the bacteria are tightly attached. Here, we use a Caenorhabditis elegans model of EHEC infection to show that microvillar effacement is mediated by a signalling pathway including mitotic cyclin-dependent kinase 1 (CDK1) and diaphanous-related formin 1 (CYK1). Similar observations are also made using EHEC-infected human intestinal cells in vitro. Our results support the use of C. elegans as a host model for studying attaching and effacing lesions in vivo, and reveal that the CDK1-formin signal axis is necessary for EHEC-induced microvillar effacement.

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Orsay δ Protein Is Required for Nonlytic Viral Egress

Journal of Virology ◽

10.1128/jvi.00745-18 ◽

2018 ◽

Vol 92 (14) ◽

Cited By ~ 7

Author(s):

Wang Yuan ◽

Ying Zhou ◽

Yanlin Fan ◽

Yizhi J. Tao ◽

Weiwei Zhong

Keyword(s):

Rna Virus ◽

Nonstructural Protein ◽

Mutant Virus ◽

Host Cells ◽

Apical Surface ◽

Content Type ◽

C Elegans ◽

Apical Side ◽

Orsay Virus

ABSTRACTNonenveloped gastrointestinal viruses, such as human rotavirus, can exit infected cells from the apical surface without cell lysis. The mechanism of such nonlytic exit is poorly understood. The nonenveloped Orsay virus is an RNA virus infecting the intestine cells of the nematodeCaenorhabditis elegans. Dye staining results suggested that Orsay virus exits from the intestine of infected worms in a nonlytic manner. Therefore, the Orsay virus-C. eleganssystem provides an excellentin vivomodel to study viral exit. The Orsay virus genome encodes three proteins: RNA-dependent RNA polymerase, capsid protein (CP), and a nonstructural protein, δ. δ can also be expressed as a structural CP-δ fusion. We generated an ATG-to-CTG mutant virus that had a normal CP-δ fusion but could not produce free δ due to the lack of the start codon. This mutant virus showed a viral exit defect without obvious phenotypes in other steps of viral infection, suggesting that δ is involved in viral exit. Ectopically expressed free δ localized near the apical membrane of intestine cells inC. elegansand colocalized with ACT-5, an intestine-specific actin that is a component of the terminal web. Orsay virus infection rearranged ACT-5 apical localization. Reduction of the ACT-5 level via RNA interference (RNAi) significantly exacerbated the viral exit defect of the δ mutant virus, suggesting that δ and ACT-5 functionally interact to promote Orsay virus exit. Together, these data support a model in which the viral δ protein interacts with the actin network at the apical side of host intestine cells to mediate the polarized, nonlytic egress of Orsay virus.IMPORTANCEAn important step of the viral life cycle is how viruses exit from host cells to spread to other cells. Certain nonenveloped viruses can exit cultured cells in nonlytic ways; however, such nonlytic exit has not been demonstratedin vivo. In addition, it is not clear how such nonlytic exit is achieved mechanisticallyin vivo. Orsay virus is a nonenveloped RNA virus that infects the intestine cells of the nematodeC. elegans. It is currently the only virus known to naturally infectC. elegans. Using thisin vivomodel, we show that the δ protein encoded by Orsay virus facilitates the nonlytic exit of the virus, possibly by interacting with host actin on the apical side of worm intestine cells.

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Bioinformatic Analysis of Leishmania donovani Long-Chain Fatty Acid-CoA Ligase as a Novel Drug Target

Molecular Biology International ◽

10.4061/2011/278051 ◽

2011 ◽

Vol 2011 ◽

pp. 1-14 ◽

Cited By ~ 10

Author(s):

Jaspreet Kaur ◽

Rameshwar Tiwari ◽

Arun Kumar ◽

Neeloo Singh

Keyword(s):

Fatty Acid ◽

Transcriptional Control ◽

Leishmania Donovani ◽

Protein Transport ◽

Molecular Model ◽

Fatty Acyl ◽

Enzyme Activation ◽

Chemical Library ◽

Coa Ligase ◽

Long Chain

Fatty acyl-CoA synthetase (fatty acid: CoA ligase, AMP-forming; (EC 6.2.1.3)) catalyzes the formation of fatty acyl-CoA by a two-step process that proceeds through the hydrolysis of pyrophosphate. Fatty acyl-CoA represents bioactive compounds that are involved in protein transport, enzyme activation, protein acylation, cell signaling, and transcriptional control in addition to serving as substrates for beta oxidation and phospholipid biosynthesis. Fatty acyl-CoA synthetase occupies a pivotal role in cellular homeostasis, particularly in lipid metabolism. Our interest in fatty acyl-CoA synthetase stems from the identification of this enzyme, long-chain fatty acyl-CoA ligase (LCFA) by microarray analysis. We found this enzyme to be differentially expressed by Leishmania donovani amastigotes resistant to antimonial treatment. In the present study, we confirm the presence of long-chain fatty acyl-CoA ligase gene in the genome of clinical isolates of Leishmania donovani collected from the disease endemic area in India. We predict a molecular model for this enzyme for in silico docking studies using chemical library available in our institute. On the basis of the data presented in this work, we propose that long-chain fatty acyl-CoA ligase enzyme serves as an important protein and a potential target candidate for development of selective inhibitors against leishmaniasis.

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Inactivation of Caenorhabditis elegans aminopeptidase DNPP-1 restores endocytic sorting and recycling in tat-1 mutants

Molecular Biology of the Cell ◽

10.1091/mbc.e12-10-0730 ◽

2013 ◽

Vol 24 (8) ◽

pp. 1163-1175 ◽

Cited By ~ 5

Author(s):

Xin Li ◽

Baohui Chen ◽

Sawako Yoshina ◽

Tanxi Cai ◽

Fuquan Yang ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Membrane Surface ◽

Aminopeptidase Activity ◽

Membrane Structures ◽

Recycling Endosomes ◽

C Elegans ◽

Endocytic Sorting ◽

Aspartyl Aminopeptidase

In Caenorhabditis elegans, the P4-ATPase TAT-1 and its chaperone, the Cdc50 family protein CHAT-1, maintain membrane phosphatidylserine (PS) asymmetry, which is required for membrane tubulation during endocytic sorting and recycling. Loss of tat-1 and chat-1 disrupts endocytic sorting, leading to defects in both cargo recycling and degradation. In this study, we identified the C. elegans aspartyl aminopeptidase DNPP-1, loss of which suppresses the sorting and recycling defects in tat-1 mutants without reversing the PS asymmetry defect. We found that tubular membrane structures containing recycling cargoes were restored in dnpp-1 tat-1 double mutants and that these tubules overlap with RME-1–positive recycling endosomes. The restoration of the tubular structures in dnpp-1 tat-1 mutants requires normal functions of RAB-5, RAB-10, and RME-1. In tat-1 mutants, we observed alterations in membrane surface charge and targeting of positively charged proteins that were reversed by loss of dnpp-1. DNPP-1 displays a specific aspartyl aminopeptidase activity in vitro, and its enzymatic activity is required for its function in vivo. Our data reveal the involvement of an aminopeptidase in regulating endocytic sorting and recycling and suggest possible roles of peptide signaling and/or protein metabolism in these processes.

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