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1.
Nucleic Acids Res ; 49(1): 177-189, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33313896

RESUMO

Short-chain acylations of lysine residues in eukaryotic proteins are recognized as essential posttranslational chemical modifications (PTMs) that regulate cellular processes from transcription, cell cycle, metabolism, to signal transduction. Lysine butyrylation was initially discovered as a normal straight chain butyrylation (Knbu). Here we report its structural isomer, branched chain butyrylation, i.e. lysine isobutyrylation (Kibu), existing as a new PTM on nuclear histones. Uniquely, isobutyryl-CoA is derived from valine catabolism and branched chain fatty acid oxidation which is distinct from the metabolism of n-butyryl-CoA. Several histone acetyltransferases were found to possess lysine isobutyryltransferase activity in vitro, especially p300 and HAT1. Transfection and western blot experiments showed that p300 regulated histone isobutyrylation levels in the cell. We resolved the X-ray crystal structures of HAT1 in complex with isobutyryl-CoA that gleaned an atomic level insight into HAT-catalyzed isobutyrylation. RNA-Seq profiling revealed that isobutyrate greatly affected the expression of genes associated with many pivotal biological pathways. Together, our findings identify Kibu as a novel chemical modification mark in histones and suggest its extensive role in regulating epigenetics and cellular physiology.


Assuntos
Código das Histonas , Isobutiratos/metabolismo , Lisina Acetiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Acil Coenzima A/síntese química , Acil Coenzima A/metabolismo , Acilação , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Células HEK293 , Histona Acetiltransferases/química , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Isobutiratos/farmacologia , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem , Valina/metabolismo , Fatores de Transcrição de p300-CBP
2.
Gene ; 764: 145094, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-32860898

RESUMO

Long chain acyl-CoA synthetases (ACSLs), which drive the conversion of long chain fatty acid into acyl-CoA, an ingredient of lipid synthesis, have been well-acknowledged to exert an indispensable role in many metabolic processes in mammals, especially lipid metabolism. However, in chicken, the evolutionary characteristics, expression profiles and regulatory mechanisms of ACSL gene family are rarely understood. Here, we analyzed the genomic synteny, gene structure, evolutionary event and functional domains of the ACSL gene family members using bioinformatics methods. The spatiotemporal expression profiles of ACSL gene family, and their regulatory mechanism were investigated via bioinformatics analysis incorporated with in vivo and in vitro estrogen-treated experiments. Our results indicated that ACSL2 gene was indeed evolutionarily lost in the genome of chicken. Chicken ACSLs shared an AMP-binding functional domain, as well as highly conversed ATP/AMP and FACS signature motifs, and were clustered into two clades, ACSL1/5/6 and ACSL3/4, based on high sequence similarity, similar gene features and conversed motifs. Chicken ACSLs showed differential tissue expression distributions, wherein the significantly decreased expression level of ACSL1 and the significantly increased expression level of ACSL5 were found, respectively, the expression levels of the other ACSL members remained unchanged in the liver of peak-laying hens versus pre-laying hens. Moreover, the transcription activity of ACSL1, ACSL3 and ACSL4 was silenced and ACSL6 was activated by estrogen, but no response to ACSL5. In conclusion, though having highly conversed functional domains, chicken ACSL gene family is organized into two separate groups, ACSL1/5/6 and ACSL3/4, and exhibits varying expression profiles and estrogen effects. These results not only pave the way for better understanding the specific functions of ACSL genes in avian lipid metabolism, but also provide a valuable evidence for gene family characteristics.


Assuntos
Galinhas/genética , Coenzima A Ligases/genética , Evolução Molecular , Metabolismo dos Lipídeos/genética , Família Multigênica/genética , Acil Coenzima A/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Galinhas/crescimento & desenvolvimento , Galinhas/metabolismo , Coenzima A Ligases/metabolismo , Biologia Computacional , Estrogênios/metabolismo , Ácidos Graxos/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hepatócitos , Cultura Primária de Células , Domínios Proteicos/genética , Análise Espaço-Temporal , Sintenia
3.
Nat Commun ; 11(1): 5598, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33154364

RESUMO

Pimelic acid, a seven carbon α,ω-dicarboxylic acid (heptanedioic acid), is known to provide seven of the ten biotin carbon atoms including all those of the valeryl side chain. Distinct pimelate synthesis pathways were recently elucidated in Escherichia coli and Bacillus subtilis where fatty acid synthesis plus dedicated biotin enzymes produce the pimelate moiety. In contrast, the α-proteobacteria which include important plant and mammalian pathogens plus plant symbionts, lack all of the known pimelate synthesis genes and instead encode bioZ genes. Here we report a pathway in which BioZ proteins catalyze a 3-ketoacyl-acyl carrier protein (ACP) synthase III-like reaction to produce pimeloyl-ACP with five of the seven pimelate carbon atoms being derived from glutaryl-CoA, an intermediate in lysine degradation. Agrobacterium tumefaciens strains either deleted for bioZ or which encode a BioZ active site mutant are biotin auxotrophs, as are strains defective in CaiB which catalyzes glutaryl-CoA synthesis from glutarate and succinyl-CoA.


Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Alphaproteobacteria/metabolismo , Biotina/metabolismo , Lisina/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Proteína de Transporte de Acila/metabolismo , Acil Coenzima A/metabolismo , Adipatos/metabolismo , Alphaproteobacteria/enzimologia , Alphaproteobacteria/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Glutaratos/metabolismo , Mutação , Ácidos Pimélicos/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(36): 22080-22089, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32820071

RESUMO

Nonshivering thermogenesis occurs in brown adipose tissue to generate heat in response to cold ambient temperatures. Thioesterase superfamily member 1 (Them1) is transcriptionally up-regulated in brown adipose tissue upon exposure to the cold and suppresses thermogenesis in order to conserve energy reserves. It hydrolyzes long-chain fatty acyl-CoAs that are derived from lipid droplets, preventing their use as fuel for thermogenesis. In addition to its enzymatic domains, Them1 contains a C-terminal StAR-related lipid transfer (START) domain with unknown ligand or function. By complementary biophysical approaches, we show that the START domain binds to long-chain fatty acids, products of Them1's enzymatic reaction, as well as lysophosphatidylcholine (LPC), lipids shown to activate thermogenesis in brown adipocytes. Certain fatty acids stabilize the START domain and allosterically enhance Them1 catalysis of acyl-CoA, whereas 18:1 LPC destabilizes and inhibits activity, which we verify in cell culture. Additionally, we demonstrate that the START domain functions to localize Them1 near lipid droplets. These findings define the role of the START domain as a lipid sensor that allosterically regulates Them1 activity and spatially localizes it in proximity to the lipid droplet.


Assuntos
Ácidos Graxos/metabolismo , Lisofosfatidilcolinas/metabolismo , Palmitoil-CoA Hidrolase/química , Palmitoil-CoA Hidrolase/metabolismo , Acil Coenzima A/metabolismo , Tecido Adiposo Marrom/enzimologia , Tecido Adiposo Marrom/metabolismo , Regulação Alostérica , Ácidos Graxos/química , Humanos , Cinética , Gotículas Lipídicas/enzimologia , Gotículas Lipídicas/metabolismo , Lisofosfatidilcolinas/química , Palmitoil-CoA Hidrolase/genética , Domínios Proteicos
5.
Proc Natl Acad Sci U S A ; 117(28): 16324-16332, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601219

RESUMO

FadE, an acyl-CoA dehydrogenase, introduces unsaturation to carbon chains in lipid metabolism pathways. Here, we report that FadE5 from Mycobacterium tuberculosis (MtbFadE5) and Mycobacterium smegmatis (MsFadE5) play roles in drug resistance and exhibit broad specificity for linear acyl-CoA substrates but have a preference for those with long carbon chains. Here, the structures of MsFadE5 and MtbFadE5, in the presence and absence of substrates, have been determined. These reveal the molecular basis for the broad substrate specificity of these enzymes. FadE5 interacts with the CoA region of the substrate through a large number of hydrogen bonds and an unusual π-π stacking interaction, allowing these enzymes to accept both short- and long-chain substrates. Residues in the substrate binding cavity reorient their side chains to accommodate substrates of various lengths. Longer carbon-chain substrates make more numerous hydrophobic interactions with the enzyme compared with the shorter-chain substrates, resulting in a preference for this type of substrate.


Assuntos
Acil-CoA Desidrogenase/química , Acil-CoA Desidrogenase/metabolismo , Mycobacterium/enzimologia , Acil Coenzima A/metabolismo , Acil-CoA Desidrogenase/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Farmacorresistência Bacteriana/genética , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Modelos Moleculares , Mutação , Mycobacterium/efeitos dos fármacos , Mycobacterium/genética , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
6.
Nat Commun ; 11(1): 3703, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32710080

RESUMO

Mycobacterium tuberculosis is a pathogen with a unique cell envelope including very long fatty acids, implicated in bacterial resistance and host immune modulation. FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis. Here we disclose crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its molecular sensory and switching mechanisms. A long tunnel traverses the entire effector-binding domain, enabling long fatty acyl effectors to bind. Only when the tunnel is entirely occupied, the protein dimer adopts a rigid configuration with its DNA-binding domains in an open state, leading to DNA dissociation. The protein-folding hydrophobic core connects the two domains, and is completed into a continuous spine when the effector binds. Such a transmission spine is conserved in a large number of TetR-like regulators, offering insight into effector-triggered allosteric functional control.


Assuntos
Acil Coenzima A/química , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Mycobacterium tuberculosis/metabolismo , Fatores de Transcrição/química , Acil Coenzima A/metabolismo , Sítio Alostérico , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Cristalografia por Raios X , DNA Bacteriano/química , Proteínas de Ligação a DNA/metabolismo , Ácidos Graxos/metabolismo , Ligantes , Modelos Moleculares , Conformação Proteica , Fatores de Transcrição/metabolismo
7.
Nat Commun ; 11(1): 2882, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32513940

RESUMO

Complex polyketides are typically associated with microbial metabolism. Here, we report that animals also make complex, microbe-like polyketides. We show there is a widespread branch of fatty acid synthase- (FAS)-like polyketide synthase (PKS) proteins, which sacoglossan animals use to synthesize complex products. The purified sacogolassan protein EcPKS1 uses only methylmalonyl-CoA as a substrate, otherwise unknown in animal lipid metabolism. Sacoglossans are sea slugs, some of which eat algae, digesting the cells but maintaining functional chloroplasts. Here, we provide evidence that polyketides support this unusual photosynthetic partnership. The FAS-like PKS family represents an uncharacterized branch of polyketide and fatty acid metabolism, encoding a large diversity of biomedically relevant animal enzymes and chemicals awaiting discovery. The biochemical characterization of an intact animal polyketide biosynthetic enzyme opens the door to understanding the immense untapped metabolic potential of metazoans.


Assuntos
Fotossíntese , Policetídeos/metabolismo , Acil Coenzima A/metabolismo , Animais , Cloroplastos/metabolismo , Escherichia coli/metabolismo , Ácido Graxo Sintases/química , Ácido Graxo Sintases/metabolismo , Gastrópodes/classificação , NADP/metabolismo , Filogenia , Policetídeo Sintases/química , Policetídeo Sintases/metabolismo , Policetídeos/química , Propionatos/química , Propionatos/metabolismo
8.
Enzyme Microb Technol ; 137: 109515, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32423667

RESUMO

Serine palmitoyltransferase (SPTase), the first enzyme of the sphingolipid biosynthesis pathway, produces 3-ketodihydrosphingosine by a Claisen-like condensation/decarboxylation reaction of l-Ser and palmitoyl-CoA (n-C16-CoA). Previous structural analysis of Sphingomonas paucimobilis SPTase (SpSPTase) revealed a dynamic active site loop (RPPATP; amino acids 378-383) in which R378 (underlined) forms a salt bridge with the carboxylic acid group of the PLP : l-Ser external aldimine. We hypothesized that this interaction might play a key role in acyl group substrate selectivity and therefore performed site-saturation mutagenesis at position 378 based on semi-rational design to expand tolerance for shorter acyl-CoA's. The resulting library was initially screened for the reaction between l-Ser and dodecanoyl-CoA (n-C12-CoA). The most interesting mutant (R378 K) was then purified and compared to wild-type SpSPTase against a panel of acyl-CoA's. These data showed that the R378 K substitution shifted the acyl group preference to shorter chain lengths, opening the possibility of using this and other engineered variants for biocatalytic C-C bond-forming reactions.


Assuntos
Acil Coenzima A/metabolismo , Engenharia Metabólica/métodos , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Sphingomonas/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Ensaios de Triagem em Larga Escala , Modelos Moleculares , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Especificidade por Substrato
9.
Nature ; 581(7808): 329-332, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32433610

RESUMO

Diacylglycerol O-acyltransferase 1 (DGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage in humans1. DGAT1 belongs to the membrane-bound O-acyltransferase (MBOAT) superfamily, members of which are found in all kingdoms of life and are involved in the acylation of lipids and proteins2,3. How human DGAT1 and other mammalian members of the MBOAT family recognize their substrates and catalyse their reactions is unknown. The absence of three-dimensional structures also hampers rational targeting of DGAT1 for therapeutic purposes. Here we present the cryo-electron microscopy structure of human DGAT1 in complex with an oleoyl-CoA substrate. Each DGAT1 protomer has nine transmembrane helices, eight of which form a conserved structural fold that we name the MBOAT fold. The MBOAT fold in DGAT1 forms a hollow chamber in the membrane that encloses highly conserved catalytic residues. The chamber has separate entrances for each of the two substrates, fatty acyl-CoA and diacylglycerol. DGAT1 can exist as either a homodimer or a homotetramer and the two forms have similar enzymatic activity. The N terminus of DGAT1 interacts with the neighbouring protomer and these interactions are required for enzymatic activity.


Assuntos
Microscopia Crioeletrônica , Diacilglicerol O-Aciltransferase/química , Diacilglicerol O-Aciltransferase/metabolismo , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Sítios de Ligação , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/ultraestrutura , Diglicerídeos/metabolismo , Humanos , Modelos Moleculares , Multimerização Proteica , Relação Estrutura-Atividade , Triglicerídeos/metabolismo
10.
Nature ; 581(7808): 323-328, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32433611

RESUMO

Triacylglycerols store metabolic energy in organisms and have industrial uses as foods and fuels. Excessive accumulation of triacylglycerols in humans causes obesity and is associated with metabolic diseases1. Triacylglycerol synthesis is catalysed by acyl-CoA diacylglycerol acyltransferase (DGAT) enzymes2-4, the structures and catalytic mechanisms of which remain unknown. Here we determined the structure of dimeric human DGAT1, a member of the membrane-bound O-acyltransferase (MBOAT) family, by cryo-electron microscopy at approximately 3.0 Å resolution. DGAT1 forms a homodimer through N-terminal segments and a hydrophobic interface, with putative active sites within the membrane region. A structure obtained with oleoyl-CoA substrate resolved at approximately 3.2 Å shows that the CoA moiety binds DGAT1 on the cytosolic side and the acyl group lies deep within a hydrophobic channel, positioning the acyl-CoA thioester bond near an invariant catalytic histidine residue. The reaction centre is located inside a large cavity, which opens laterally to the membrane bilayer, providing lipid access to the active site. A lipid-like density-possibly representing an acyl-acceptor molecule-is located within the reaction centre, orthogonal to acyl-CoA. Insights provided by the DGAT1 structures, together with mutagenesis and functional studies, provide the basis for a model of the catalysis of triacylglycerol synthesis by DGAT.


Assuntos
Biocatálise , Microscopia Crioeletrônica , Diacilglicerol O-Aciltransferase/metabolismo , Diacilglicerol O-Aciltransferase/ultraestrutura , Triglicerídeos/biossíntese , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Acil Coenzima A/ultraestrutura , Aciltransferases/química , Aciltransferases/metabolismo , Domínio Catalítico , Membrana Celular/química , Membrana Celular/metabolismo , Diacilglicerol O-Aciltransferase/química , Histidina/química , Histidina/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Multimerização Proteica , Especificidade por Substrato
11.
Proc Natl Acad Sci U S A ; 117(19): 10593-10602, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32332165

RESUMO

A physiological role for long-chain acyl-CoA esters to activate ATP-sensitive K+ (KATP) channels is well established. Circulating palmitate is transported into cells and converted to palmitoyl-CoA, which is a substrate for palmitoylation. We found that palmitoyl-CoA, but not palmitic acid, activated the channel when applied acutely. We have altered the palmitoylation state by preincubating cells with micromolar concentrations of palmitic acid or by inhibiting protein thioesterases. With acyl-biotin exchange assays we found that Kir6.2, but not sulfonylurea receptor (SUR)1 or SUR2, was palmitoylated. These interventions increased the KATP channel mean patch current, increased the open time, and decreased the apparent sensitivity to ATP without affecting surface expression. Similar data were obtained in transfected cells, rat insulin-secreting INS-1 cells, and isolated cardiac myocytes. Kir6.2ΔC36, expressed without SUR, was also positively regulated by palmitoylation. Mutagenesis of Kir6.2 Cys166 prevented these effects. Clinical variants in KCNJ11 that affect Cys166 had a similar gain-of-function phenotype, but was more pronounced. Molecular modeling studies suggested that palmitoyl-C166 and selected large hydrophobic mutations make direct hydrophobic contact with Kir6.2-bound PIP2 Patch-clamp studies confirmed that palmitoylation of Kir6.2 at Cys166 enhanced the PIP2 sensitivity of the channel. Physiological relevance is suggested since palmitoylation blunted the regulation of KATP channels by α1-adrenoreceptor stimulation. The Cys166 residue is conserved in some other Kir family members (Kir6.1 and Kir3, but not Kir2), which are also subject to regulated palmitoylation, suggesting a general mechanism to control the open state of certain Kir channels.


Assuntos
Canais KATP/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Acil Coenzima A/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cisteína/metabolismo , Células HEK293 , Humanos , Canais KATP/genética , Lipoilação/fisiologia , Mutagênese/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp/métodos , Canais de Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Cultura Primária de Células , Ratos , Receptores Sulfonilureia/genética
12.
Gene ; 745: 144647, 2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32247738

RESUMO

AIMS: Post-translational modifications (PTMs) of histones are regulated by the availability of their respective acyl-CoAs. Among these histone PTMs, the metabolic origin of histone butyrylation (Kbu) is still poorly understood. MATERIAL AND METHODS: The impact of starvation on the levels of Kbu was determined by western blotting on histones extracted from the liver of fed and fasted C57BL/6 mice and immunohistochemistry on liver paraffin sections. KEY FINDINGS: Using animal model we provide evidence that the stimulation of ketogenesis following starvation, in addition to histone beta-hydroxybutyrylation (Kbhb), also leads to an increase in histone butyrylation (Kbu). Using an immunohistochemistry (IHC) approach we report first that hepatocytes contained butyrylated histones with important cell-to-cell heterogeneity. More importantly, our investigations based on western blotting and IHC also proposed that the basal levels of Kbu differ between male and female mice, with female mouse hepatocytes containing higher levels of butyrylated histones. Starvation enhanced solely histone Kbu levels in the liver of males but not females. SIGNIFICANCE: This is the first demonstration of a sex-dependent large-scale stimulation of histone acylation. Our data also point to different basal metabolic conditions of the male and female liver cells with a sex-dependent impact on the hepatocytes' epigenome.


Assuntos
Histonas/metabolismo , Fígado/patologia , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Inanição/patologia , Ácido 3-Hidroxibutírico/metabolismo , Acil Coenzima A/metabolismo , Acilação , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Hepatócitos/patologia , Código das Histonas , Humanos , Corpos Cetônicos/metabolismo , Fígado/citologia , Masculino , Camundongos , Fatores Sexuais
13.
Appl Environ Microbiol ; 86(12)2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32303550

RESUMO

Malonyl coenzyme A (malonyl-CoA) and methylmalonyl-CoA are the most common extender units for the biosynthesis of fatty acids and polyketides in Streptomyces, an industrially important producer of polyketides. Carboxylation of acetyl- and propionyl-CoAs is an essential source of malonyl- and methylmalonyl-CoAs; therefore, acyl-CoA carboxylases (ACCases) play key roles in primary and secondary metabolism. The regulation of the expression of ACCases in Streptomyces spp. has not been investigated previously. We characterized a TetR family transcriptional repressor, AccR, that mediates intracellular acetyl-, propionyl-, methylcrotonyl-, malonyl-, and methylmalonyl-CoA levels by controlling the transcription of genes that encode the main ACCase and enzymes associated with branched-chain amino acid metabolism in S. avermitilis AccR bound to a 16-nucleotide palindromic binding motif (GTTAA-N6-TTAAC) in promoter regions and repressed the transcription of the accD1A1-hmgL-fadE4 operon, echA8, echA9, and fadE2, which are involved in the production and assimilation of acetyl- and propionyl-CoAs. Methylcrotonyl-, propionyl-, and acetyl-CoAs acted as effectors to release AccR from its target DNA, resulting in enhanced transcription of target genes by derepression. The affinity of methylcrotonyl- and propionyl-CoAs to AccR was stronger than that of acetyl-CoA. Deletion of accR resulted in increased concentrations of short-chain acyl-CoAs (acetyl-, propionyl-, malonyl-, and methylmalonyl-CoAs), leading to enhanced avermectin production. Avermectin production was increased by 14.5% in an accR deletion mutant of the industrial high-yield strain S. avermitilis A8. Our findings clarify the regulatory mechanisms that maintain the homeostasis of short-chain acyl-CoAs in Streptomyces IMPORTANCE Acyl-CoA carboxylases play key roles in primary and secondary metabolism. However, the regulation of ACCase genes transcription in Streptomyces spp. remains unclear. Here, we demonstrated that AccR responded to intracellular acetyl-, propionyl-, and methylcrotonyl-CoA availability and mediated transcription of the genes related to production and assimilation of these compounds in S. avermitilis When intracellular concentrations of these compounds are low, AccR binds to target genes and represses their transcription, resulting in low production of malonyl- and methylmalonyl-CoAs. When intracellular acetyl-, propionyl-, and methylcrotonyl-CoA concentrations are high, these compounds bind to AccR to dissociate AccR from target DNA, promoting the conversion of these compounds to malonyl- and methylmalonyl-CoAs. This investigation revealed how AccR coordinates short-chain acyl-CoA homeostasis in Streptomyces.


Assuntos
Acil Coenzima A/metabolismo , Proteínas de Bactérias/genética , Carbono-Carbono Ligases/genética , Streptomyces/fisiologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Carbono-Carbono Ligases/metabolismo , Homeostase , Alinhamento de Sequência , Streptomyces/enzimologia , Streptomyces/genética , Transcrição Genética
14.
J Agric Food Chem ; 68(14): 4252-4260, 2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32186186

RESUMO

Ethyl butyrate is one of the most important flavor substances in Chinese Baijiu and is also an ingredient in various daily-use chemical essences and food flavorings. In this study, to produce ethyl butyrate, we first introduced a butyryl-CoA synthesis pathway into Saccharomyces cerevisiae. Subsequently, three different alcohol acyltransferases, SAAT, VAAT, and CmAAT, were separately introduced into S. cerevisiae to catalyze the reaction of butyryl-CoA with ethanol to produce ethyl butyrate, and the results showed that strain EBS with SAAT produced the most ethyl butyrate (20.06 ± 2.23 mg/L). Furthermore, as the reaction catalyzed by Bcd to produce butyryl-CoA from crotonyl-CoA is a rate-limiting step, we replaced Bcd with Ter, and the modified strain EST produced 77.33 ± 4.79 mg/L ethyl butyrate. Finally, the copy numbers of Ter and SAAT were further increased, and the resulting modified strain EST-dST produced 99.65 ± 7.32 mg/L ethyl butyrate.


Assuntos
Butiratos/química , Aromatizantes/química , Saccharomyces cerevisiae/metabolismo , Acil Coenzima A/metabolismo , Bebidas Alcoólicas/microbiologia , Sequência de Bases , Vias Biossintéticas , Escherichia coli/metabolismo , Etanol/metabolismo , Fermentação , Microbiologia Industrial , Cinética , Engenharia Metabólica , Proteínas/metabolismo
15.
Biochem Biophys Res Commun ; 526(1): 246-252, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32204912

RESUMO

Glycerophospholipids, one of the main constituents of biological membranes, are synthesized from glycerol-3-phosphate through the de novo pathway, and are reconstituted through the remodeling pathway. Lysophosphatidylethanolamine acyltransferase 2 (LPEAT2), one of the enzymes that play a role in the remodeling pathway, has been previously reported to have LPEAT, lysophosphatidylcholine acyltransferase (LPCAT) and lysophosphatidylglycerol acyltransferase (LPGAT) activities with 16:0-CoA, 18:0-CoA, and 18:1-CoA as donors. In this study, we found that LPEAT2 is active with 22:6-CoA. Knockdown studies using Neuro 2A cells showed that LPEAT2 has endogenous LPEAT activity with 22:6-CoA, and that LPEAT2 has functions for modulating 22:6/20:4 ratios of phospholipids. In addition, we demonstrated that Neuro 2A cells overexpressing LPEAT2 underwent cell death with necrotic morphology when differentiated into neuron-like cells, with supplementation with 22:6 (DHA). These results suggest that LPEAT2 plays a role in inducing cell death DHA-dependently. This study will lead to better understand how DHA levels are regulated in phospholipids, especially in the brain where LPEAT2 is highly expressed. Our study also provides insight to understand the mechanism of cell death induced by DHA.


Assuntos
Aciltransferases/metabolismo , Ácidos Docosa-Hexaenoicos/metabolismo , Fosfolipídeos/metabolismo , Acil Coenzima A/metabolismo , Animais , Encéfalo/metabolismo , Células CHO , Morte Celular , Cricetinae , Cricetulus , Cinética , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/metabolismo , Distribuição Tecidual
16.
Nucleic Acids Res ; 48(8): 4115-4138, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32182340

RESUMO

Epigenetic regulation of gene expression is tightly controlled by the dynamic modification of histones by chemical groups, the diversity of which has largely expanded over the past decade with the discovery of lysine acylations, catalyzed from acyl-coenzymes A. We investigated the dynamics of lysine acetylation and crotonylation on histones H3 and H4 during mouse spermatogenesis. Lysine crotonylation appeared to be of significant abundance compared to acetylation, particularly on Lys27 of histone H3 (H3K27cr) that accumulates in sperm in a cleaved form of H3. We identified the genomic localization of H3K27cr and studied its effects on transcription compared to the classical active mark H3K27ac at promoters and distal enhancers. The presence of both marks was strongly associated with highest gene expression. Assessment of their co-localization with transcription regulators (SLY, SOX30) and chromatin-binding proteins (BRD4, BRDT, BORIS and CTCF) indicated systematic highest binding when both active marks were present and different selective binding when present alone at chromatin. H3K27cr and H3K27ac finally mark the building of some sperm super-enhancers. This integrated analysis of omics data provides an unprecedented level of understanding of gene expression regulation by H3K27cr in comparison to H3K27ac, and reveals both synergistic and specific actions of each histone modification.


Assuntos
Elementos Facilitadores Genéticos , Epigênese Genética , Código das Histonas , Regiões Promotoras Genéticas , Espermatogênese/genética , Acetilcoenzima A/metabolismo , Acetilação , Acil Coenzima A/metabolismo , Animais , Evolução Biológica , Crotonatos/metabolismo , Genômica , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Masculino , Metabolômica , Camundongos Endogâmicos C57BL , Proteômica , Transcrição Genética , Leveduras/metabolismo , Leveduras/fisiologia
17.
Int J Mol Sci ; 21(5)2020 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-32121266

RESUMO

Lyso-lipid acyltransferases are enzymes involved in various processes such as lipid synthesis and remodelling. Here, we characterized the activity of an acyltransferase from Arabidopsis thaliana (LPIAT). In vitro, this protein, expressed in Escherichia coli membrane, displayed a 2-lyso-phosphatidylinositol acyltransferase activity with a specificity towards saturated long chain acyl CoAs (C16:0- and C18:0-CoAs), allowing the remodelling of phosphatidylinositol. In planta, LPIAT gene was expressed in mature seeds and very transiently during seed imbibition, mostly in aleurone-like layer cells. Whereas the disruption of this gene did not alter the lipid composition of seed, its overexpression in leaves promoted a strong increase in the phosphatidylinositol phosphates (PIP) level without affecting the PIP2 content. The spatial and temporal narrow expression of this gene as well as the modification of PIP metabolism led us to investigate its role in the control of seed germination. Seeds from the lpiat mutant germinated faster and were less sensitive to abscisic acid (ABA) than wild-type or overexpressing lines. We also showed that the protective effect of ABA on young seedlings against dryness was reduced for lpiat line. In addition, germination of lpiat mutant seeds was more sensitive to hyperosmotic stress. All these results suggest a link between phosphoinositides and ABA signalling in the control of seed germination.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Germinação , Osmorregulação , Fosfatos de Fosfatidilinositol/metabolismo , Sementes/crescimento & desenvolvimento , Transdução de Sinais , Ácido Abscísico/farmacologia , Acil Coenzima A/metabolismo , Arabidopsis/efeitos dos fármacos , Germinação/efeitos dos fármacos , Hipocótilo/efeitos dos fármacos , Hipocótilo/crescimento & desenvolvimento , Mutação/genética , Osmorregulação/efeitos dos fármacos , Fenótipo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Salinidade , Sementes/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
18.
Biochemistry ; 59(10): 1113-1123, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32101684

RESUMO

Steroid-degrading bacteria, including Mycobacterium tuberculosis (Mtb), utilize an architecturally distinct subfamily of acyl coenzyme A dehydrogenases (ACADs) for steroid catabolism. These ACADs are α2ß2 heterotetramers that are usually encoded by adjacent fadE-like genes. In mycobacteria, ipdE1 and ipdE2 (formerly fadE30 and fadE33) occur in divergently transcribed operons associated with the catabolism of 3aα-H-4α(3'-propanoate)-7aß-methylhexahydro-1,5-indanedione (HIP), a steroid metabolite. In Mycobacterium smegmatis, ΔipdE1 and ΔipdE2 mutants had similar phenotypes, showing impaired growth on cholesterol and accumulating 5-OH HIP in the culture supernatant. Bioinformatic analyses revealed that IpdE1 and IpdE2 share many of the features of the α- and ß-subunits, respectively, of heterotetrameric ACADs that are encoded by adjacent genes in many steroid-degrading proteobacteria. When coproduced in a rhodococcal strain, IpdE1 and IpdE2 of Mtb formed a complex that catalyzed the dehydrogenation of 5OH-HIP coenzyme A (5OH-HIP-CoA) to 5OH-3aα-H-4α(3'-prop-1-enoate)-7aß-methylhexa-hydro-1,5-indanedione coenzyme A ((E)-5OH-HIPE-CoA). This corresponds to the initial step in the pathway that leads to degradation of steroid C and D rings via ß-oxidation. Small-angle X-ray scattering revealed that the IpdE1-IpdE2 complex was an α2ß2 heterotetramer typical of other ACADs involved in steroid catabolism. These results provide insight into an important class of steroid catabolic enzymes and a potential virulence determinant in Mtb.


Assuntos
Acil-CoA Desidrogenase/metabolismo , Acil-CoA Desidrogenase/fisiologia , Acil Coenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Colesterol/metabolismo , Coenzima A/metabolismo , Coenzima A Ligases/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Esteroides/metabolismo
19.
PLoS One ; 15(2): e0229718, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32108178

RESUMO

Glycine N-myristoylation is an essential acylation modification modulating the functions, stability, and membrane association of diverse cytosolic proteins in human cells. Myristoyl-CoA is the 14-carbon acyl donor of the acyltransferase reaction. Acyl-CoAs of a chain length compatible with the binding site of the N-myristoyltransferase enzymes (NMT) are competitive inhibitors, and the mechanism protecting these enzymes from unwanted acyl-CoA species requires the acyl-CoA binding protein ACBD6. The acyl-CoA binding domain (ACB) and the ankyrin-repeat motifs (ANK) of ACBD6 can perform their functions independently. Interaction of ANK with human NMT2 was necessary and sufficient to provide protection. Fusion of the ANK module to the acyl-CoA binding protein ACBD1 was sufficient to confer the NMT-stimulatory property of ACBD6 to the chimera. The ACB domain is dispensable and sequestration of the competitor was not the basis for NMT2 protection. Acyl-CoAs bound to ACB modulate the function of the ANK module and act as positive effector of the allosteric activation of the enzyme. The functional relevance of homozygous mutations in ACBD6 gene, which have not been associated with a disease so far, is presented. Skin-derived fibroblasts of two unrelated individuals with neurodevelopmental disorder and carrying loss of function mutations in the ACBD6 gene were deficient in protein N-myristoylation. These cells were sensitive to substrate analog competing for myristoyl-CoA binding to NMT. These findings account for the requirement of an ANK-containing acyl-CoA binding protein in the cellular mechanism protecting the NMT enzymes and establish that in human cells, ACBD6 supports the N-myristoylation of proteins.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Acil Coenzima A/metabolismo , Acilação , Aciltransferases/química , Aciltransferases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Fibroblastos/metabolismo , Homozigoto , Humanos , Ligantes , Mutação com Perda de Função , Masculino , Ácidos Mirísticos/química , Ácidos Mirísticos/metabolismo , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Sítios de Splice de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência
20.
Am J Respir Cell Mol Biol ; 62(4): 479-492, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31944822

RESUMO

Idiopathic pulmonary fibrosis is a lung disease with limited therapeutic options that is characterized by pathological fibroblast activation and aberrant lung remodeling with scar formation. YAP (Yes-associated protein) is a transcriptional coactivator that mediates mechanical and biochemical signals controlling fibroblast activation. In this study, we developed a high-throughput small-molecule screen for YAP inhibitors in primary human lung fibroblasts. Multiple HMG-CoA (hydroxymethylglutaryl-coenzyme A) reductase inhibitors (statins) were found to inhibit YAP nuclear localization via induction of YAP phosphorylation, cytoplasmic retention, and degradation. We further show that the mevalonate pathway regulates YAP activation, and that simvastatin treatment reduces fibrosis markers in activated human lung fibroblasts and in the bleomycin mouse model of pulmonary fibrosis. Finally, we show that simvastatin modulates YAP in vivo in mouse lung fibroblasts. Our results highlight the potential of small-molecule screens for YAP inhibitors and provide a mechanism for the antifibrotic activity of statins in idiopathic pulmonary fibrosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fibrose Pulmonar/tratamento farmacológico , Acil Coenzima A/metabolismo , Animais , Biomarcadores/metabolismo , Bleomicina/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Ácido Mevalônico/metabolismo , Camundongos , Fosfoproteínas/metabolismo , Fibrose Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinvastatina/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia
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