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1.
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
2.
Nat Commun ; 11(1): 1132, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111831

RESUMO

The promising drug target N-myristoyltransferase (NMT) catalyses an essential protein modification thought to occur exclusively at N-terminal glycines (Gly). Here, we present high-resolution human NMT1 structures co-crystallised with reactive cognate lipid and peptide substrates, revealing high-resolution snapshots of the entire catalytic mechanism from the initial to final reaction states. Structural comparisons, together with biochemical analysis, provide unforeseen details about how NMT1 reaches a catalytically competent conformation in which the reactive groups are brought into close proximity to enable catalysis. We demonstrate that this mechanism further supports efficient and unprecedented myristoylation of an N-terminal lysine side chain, providing evidence that NMT acts both as N-terminal-lysine and glycine myristoyltransferase.


Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Glicina/metabolismo , Lisina/metabolismo , Aciltransferases/genética , Catálise , Domínio Catalítico , Coenzima A/química , Coenzima A/genética , Coenzima A/metabolismo , Cristalografia por Raios X , Humanos , Cinética , Mutação , Ácido Mirístico/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
3.
Am J Hum Genet ; 106(4): 484-495, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32220290

RESUMO

Glycosylphosphatidylinositol (GPI)-anchored proteins are critical for embryogenesis, neurogenesis, and cell signaling. Variants in several genes participating in GPI biosynthesis and processing lead to decreased cell surface presence of GPI-anchored proteins (GPI-APs) and cause inherited GPI deficiency disorders (IGDs). In this report, we describe 12 individuals from nine unrelated families with 10 different bi-allelic PIGK variants. PIGK encodes a component of the GPI transamidase complex, which attaches the GPI anchor to proteins. Clinical features found in most individuals include global developmental delay and/or intellectual disability, hypotonia, cerebellar ataxia, cerebellar atrophy, and facial dysmorphisms. The majority of the individuals have epilepsy. Two individuals have slightly decreased levels of serum alkaline phosphatase, while eight do not. Flow cytometric analysis of blood and fibroblasts from affected individuals showed decreased cell surface presence of GPI-APs. The overexpression of wild-type (WT) PIGK in fibroblasts rescued the levels of cell surface GPI-APs. In a knockout cell line, transfection with WT PIGK also rescued the GPI-AP levels, but transfection with the two tested mutant variants did not. Our study not only expands the clinical and known genetic spectrum of IGDs, but it also expands the genetic differential diagnosis for cerebellar atrophy. Given the fact that cerebellar atrophy is seen in other IGDs, flow cytometry for GPI-APs should be considered in the work-ups of individuals presenting this feature.


Assuntos
Aciltransferases/genética , Moléculas de Adesão Celular/genética , Doenças Cerebelares/genética , Epilepsia/genética , Variação Genética/genética , Hipotonia Muscular/genética , Transtornos do Neurodesenvolvimento/genética , Anormalidades Múltiplas/genética , Alelos , Feminino , Humanos , Deficiência Intelectual/genética , Masculino , Malformações do Sistema Nervoso/genética , Linhagem , Síndrome
4.
Nat Commun ; 11(1): 1266, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152276

RESUMO

Endophilins-A are conserved endocytic adaptors with membrane curvature-sensing and -inducing properties. We show here that, independently of their role in endocytosis, endophilin-A1 and endophilin-A2 regulate exocytosis of neurosecretory vesicles. The number and distribution of neurosecretory vesicles were not changed in chromaffin cells lacking endophilin-A, yet fast capacitance and amperometry measurements revealed reduced exocytosis, smaller vesicle pools and altered fusion kinetics. The levels and distributions of the main exocytic and endocytic factors were unchanged, and slow compensatory endocytosis was not robustly affected. Endophilin-A's role in exocytosis is mediated through its SH3-domain, specifically via a direct interaction with intersectin-1, a coordinator of exocytic and endocytic traffic. Endophilin-A not able to bind intersectin-1, and intersectin-1 not able to bind endophilin-A, resulted in similar exocytic defects in chromaffin cells. Altogether, we report that two endocytic proteins, endophilin-A and intersectin-1, are enriched on neurosecretory vesicles and regulate exocytosis by coordinating neurosecretory vesicle priming and fusion.


Assuntos
Aciltransferases/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Endocitose/fisiologia , Sistemas Neurossecretores/metabolismo , Aciltransferases/genética , Animais , Células Cromafins/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sistemas Neurossecretores/citologia
5.
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
6.
Proc Natl Acad Sci U S A ; 117(8): 4109-4116, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32041866

RESUMO

The UDP-2,3-diacylglucosamine pyrophosphate hydrolase LpxH is an essential lipid A biosynthetic enzyme that is conserved in the majority of gram-negative bacteria. It has emerged as an attractive novel antibiotic target due to the recent discovery of an LpxH-targeting sulfonyl piperazine compound (referred to as AZ1) by AstraZeneca. However, the molecular details of AZ1 inhibition have remained unresolved, stymieing further development of this class of antibiotics. Here we report the crystal structure of Klebsiella pneumoniae LpxH in complex with AZ1. We show that AZ1 fits snugly into the L-shaped acyl chain-binding chamber of LpxH with its indoline ring situating adjacent to the active site, its sulfonyl group adopting a sharp kink, and its N-CF3-phenyl substituted piperazine group reaching out to the far side of the LpxH acyl chain-binding chamber. Intriguingly, despite the observation of a single AZ1 conformation in the crystal structure, our solution NMR investigation has revealed the presence of a second ligand conformation invisible in the crystalline state. Together, these distinct ligand conformations delineate a cryptic inhibitor envelope that expands the observed footprint of AZ1 in the LpxH-bound crystal structure and enables the design of AZ1 analogs with enhanced potency in enzymatic assays. These designed compounds display striking improvement in antibiotic activity over AZ1 against wild-type K. pneumoniae, and coadministration with outer membrane permeability enhancers profoundly sensitizes Escherichia coli to designed LpxH inhibitors. Remarkably, none of the sulfonyl piperazine compounds occupies the active site of LpxH, foretelling a straightforward path for rapid optimization of this class of antibiotics.


Assuntos
Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Pirofosfatases/antagonistas & inibidores , Pirofosfatases/metabolismo , Aciltransferases/genética , Proteínas de Bactérias/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Metabolismo dos Lipídeos , Testes de Sensibilidade Microbiana , Mutação , Piperazinas/química , Piperazinas/farmacologia , Conformação Proteica , Pirofosfatases/genética
7.
Am J Pathol ; 190(5): 1059-1067, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32084365

RESUMO

Mutations in retinoid isomerase (RPE65) or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal synthesis and cause Leber congenital amaurosis (LCA). Despite the success of recent RPE65 gene therapy, follow-up studies show that patients continue to experience photoreceptor degeneration and lose vision benefit over time. In Lrat-/- mouse model, mislocalized medium (M)-wavelength opsin was degraded, whereas mislocalized short (S)-wavelength opsin accumulated before the onset of cone degeneration. The mechanism for the foveal M/long-wavelength cone photoreceptor degeneration in LCA is unknown. By crossing Lrat-/- mice with a proteasome reporter mouse strain, this study showed that M-opsin-enriched dorsal cones in Lrat-/- mice exhibit proteasome stress because of the degradation of large amounts of M-opsin. Deletion of M-opsin relieves the proteasome stress and completely prevents M cone degeneration in Lrat-/-Opn1sw-/- mice (a pure M cone LCA model, Opn1sw encoding S-opsin) for at least 12 months. These results suggest that M-opsin degradation-associated proteasome stress plays a major role in M cone degeneration in Lrat-/- model. This finding may represent a general mechanism for M cone degeneration in multiple forms of cone degeneration because of M-opsin mislocalization and degradation. These results have important implications for the current gene therapy strategy for LCA that emphasizes the need for combinatorial therapies to both improve vision and slow photoreceptor degeneration.


Assuntos
Opsinas dos Cones/metabolismo , Amaurose Congênita de Leber/metabolismo , Amaurose Congênita de Leber/patologia , Degeneração Neural/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Aciltransferases/deficiência , Aciltransferases/genética , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Células Fotorreceptoras Retinianas Cones/metabolismo
8.
Nat Commun ; 11(1): 870, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054839

RESUMO

Land plants produce diverse flavonoids for growth, survival, and reproduction. Chalcone synthase is the first committed enzyme of the flavonoid biosynthetic pathway and catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC). However, it also produces other polyketides, including p-coumaroyltriacetic acid lactone (CTAL), because of the derailment of the chalcone-producing pathway. This promiscuity of CHS catalysis adversely affects the efficiency of flavonoid biosynthesis, although it is also believed to have led to the evolution of stilbene synthase and p-coumaroyltriacetic acid synthase. In this study, we establish that chalcone isomerase-like proteins (CHILs), which are encoded by genes that are ubiquitous in land plant genomes, bind to CHS to enhance THC production and decrease CTAL formation, thereby rectifying the promiscuous CHS catalysis. This CHIL function has been confirmed in diverse land plant species, and represents a conserved strategy facilitating the efficient influx of substrates from the phenylpropanoid pathway to the flavonoid pathway.


Assuntos
Aciltransferases/metabolismo , Embriófitas/metabolismo , Liases Intramoleculares/metabolismo , Proteínas de Plantas/metabolismo , Aciltransferases/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Biocatálise , Vias Biossintéticas/genética , Chalconas/biossíntese , Embriófitas/genética , Evolução Molecular , Flavonoides/biossíntese , Genes de Plantas , Teste de Complementação Genética , Liases Intramoleculares/genética , Cinética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Policetídeos/metabolismo , Especificidade por Substrato
9.
Nat Commun ; 11(1): 860, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054864

RESUMO

Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids interact with each other in the mammalian plasma membranes, forming dynamic microdomains. How their interaction starts in the cells has been unclear. Here, based on a genome-wide CRISPR-Cas9 genetic screen for genes required for GPI side-chain modification by galactose in the Golgi apparatus, we report that ß1,3-galactosyltransferase 4 (B3GALT4), the previously characterized GM1 ganglioside synthase, additionally functions in transferring galactose to the N-acetylgalactosamine side-chain of GPI. Furthermore, B3GALT4 requires lactosylceramide for the efficient GPI side-chain galactosylation. Thus, our work demonstrates previously unexpected functional relationships between GPI-anchored proteins and glycosphingolipids in the Golgi. Through the same screening, we also show that GPI biosynthesis in the endoplasmic reticulum (ER) is severely suppressed by ER-associated degradation to prevent GPI accumulation when the transfer of synthesized GPI to proteins is defective. Our data demonstrates cross-talks of GPI biosynthesis with glycosphingolipid biosynthesis and the ER quality control system.


Assuntos
Degradação Associada com o Retículo Endoplasmático , Glicoesfingolipídeos/biossíntese , Glicosilfosfatidilinositóis/biossíntese , Aciltransferases/deficiência , Aciltransferases/genética , Aciltransferases/metabolismo , Sistemas CRISPR-Cas , Degradação Associada com o Retículo Endoplasmático/genética , Galactosiltransferases/deficiência , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Técnicas de Inativação de Genes , Glicoesfingolipídeos/genética , Glicosilfosfatidilinositóis/genética , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
10.
Nat Commun ; 11(1): 1067, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103017

RESUMO

Lysine fatty acylation in mammalian cells was discovered nearly three decades ago, yet the enzymes catalyzing it remain unknown. Unexpectedly, we find that human N-terminal glycine myristoyltransferases (NMT) 1 and 2 can efficiently myristoylate specific lysine residues. They modify ADP-ribosylation factor 6 (ARF6) on lysine 3 allowing it to remain on membranes during the GTPase cycle. We demonstrate that the NAD+-dependent deacylase SIRT2 removes the myristoyl group, and our evidence suggests that NMT prefers the GTP-bound while SIRT2 prefers the GDP-bound ARF6. This allows the lysine myrisotylation-demyristoylation cycle to couple to and promote the GTPase cycle of ARF6. Our study provides an explanation for the puzzling dissimilarity of ARF6 to other ARFs and suggests the existence of other substrates regulated by this previously unknown function of NMT. Furthermore, we identified a NMT/SIRT2-ARF6 regulatory axis, which may offer new ways to treat human diseases.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Aciltransferases/metabolismo , Lisina/metabolismo , Sirtuína 2/metabolismo , Acilação/fisiologia , Sequência de Aminoácidos , Linhagem Celular , Cristalografia por Raios X , Células HEK293 , Humanos , Ácido Mirístico/metabolismo
11.
Can J Microbiol ; 66(3): 256-262, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31917612

RESUMO

Optical density (OD) measurement is the standard method used in microbiology for estimating bacterial concentrations in cultures. However, most studies do not compare these measurements with viable cell counts and assume that they reflect the real cell concentration. Burkholderia thailandensis was recently identified as a polyhydroxyalkanoate (PHA) producer. PHA biosynthesis seems to be coded by an orthologue of the Cupriavidus necator phaC gene. When growing cultures of wild-type strain E264 and an isogenic phaC mutant, we noted a difference in their OD600 values, although viable cell counts indicated similar growth. Investigating the cellular morphologies of both strains, we found that under our conditions the wild-type strain was full of PHA granules, deforming the cells, while the mutant contained no granules. These factors apparently affected the light scattering, making the OD600 values no longer representative of cell density. We show a direct correlation between OD600 values and the accumulation of PHA. We conclude that OD measurement is unreliable for growth evaluation of B. thailandensis because of PHA production. This study also suggests that B. thailandensis could represent an excellent candidate for PHA bioproduction. Correlation between OD measurements and viable cell counts should be verified in any study performed with B. thailandensis.


Assuntos
Burkholderia/crescimento & desenvolvimento , Burkholderia/metabolismo , Técnicas Microbiológicas/métodos , Poli-Hidroxialcanoatos/metabolismo , Aciltransferases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Burkholderia/genética
12.
BMC Plant Biol ; 20(1): 14, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31914928

RESUMO

BACKGROUND: The BAHD acyltransferase superfamily exhibits various biological roles in plants, including regulating fruit quality, catalytic synthesizing of terpene, phenolics and esters, and improving stress resistance. However, the copy numbers, expression characteristics and associations with fruit aroma formation of the BAHD genes remain unclear. RESULTS: In total, 717 BAHD genes were obtained from the genomes of seven Rosaceae, (Pyrus bretschneideri, Malus domestica, Prunus avium, Prunus persica, Fragaria vesca, Pyrus communis and Rubus occidentalis). Based on the detailed phylogenetic analysis and classifications in model plants, we divided the BAHD family genes into seven groups, I-a, I-b, II-a, II-b, III-a, IV and V. An inter-species synteny analysis revealed the ancient origin of BAHD superfamily with 78 syntenic gene pairs were detected among the seven Rosaceae species. Different types of gene duplication events jointly drive the expansion of BAHD superfamily, and purifying selection dominates the evolution of BAHD genes supported by the small Ka/Ks ratios. Based on the correlation analysis between the ester content and expression levels of BAHD genes at different developmental stages, four candidate genes were selected for verification as assessed by qRT-PCR. The result implied that Pbr020016.1, Pbr019034.1, Pbr014028.1 and Pbr029551.1 are important candidate genes involved in aroma formation during pear fruit development. CONCLUSION: We have thoroughly identified the BAHD superfamily genes and performed a comprehensive comparative analysis of their phylogenetic relationships, expansion patterns, and expression characteristics in seven Rosaceae species, and we also obtained four candidate genes involved in aroma synthesis in pear fruit. These results provide a theoretical basis for future studies of the specific biological functions of BAHD superfamily members and the improvement of pear fruit quality.


Assuntos
Aciltransferases/genética , Frutas/genética , Pyrus , Rosaceae/genética , Compostos Orgânicos Voláteis/metabolismo , Aciltransferases/metabolismo , Evolução Molecular , Duplicação Gênica , Perfilação da Expressão Gênica , Genoma de Planta , Odorantes , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pyrus/genética , Pyrus/metabolismo , Sintenia/genética
13.
Proc Natl Acad Sci U S A ; 117(3): 1280-1282, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31907319

RESUMO

Nucleic acid vaccines introduce the genetic materials encoding antigenic proteins into host cells. If these proteins are directed into the secretory pathway with a signal/leader sequence, they will be exposed to the host's glycosylation machinery, and, if their amino acid sequences contain consensus sequons for N-linked glycosylation, they may become glycosylated. The presence of host glycans on the proteins of microbial origin may prevent a strong protective immune response either through hindering access to key epitopes by lymphocytes or through altering immune responses by binding to immunoregulatory glycan-binding receptors on immune cells. Ag85A expressed by Mycobacterium tuberculosis (Mtb) is a bacterial surface protein that is commonly used in nucleic acid vaccines in multiple clinical trials. Here we show that, when Ag85A is expressed in mammalian cells, it is glycosylated, does not induce a strong humoral immune response in mice, and does not activate Ag85A-specific lymphocytes as highly as Ag85A natively expressed by the bacterium. Our study indicates that host glycosylation of the vaccine target can impede its antigenicity and immunogenicity. Glycosylation of the antigenic protein targets therefore must be carefully evaluated in designing nucleic acid vaccines.


Assuntos
Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Imunogenicidade da Vacina , Processamento de Proteína Pós-Traducional , Vacinas contra a Tuberculose/imunologia , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Glicosilação , Células HEK293 , Humanos , Linfócitos/imunologia , Camundongos
14.
Gene ; 726: 144148, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31647997

RESUMO

Tafazzin, which is encoded by the TAZ gene, catalyzes transacylation to form mature cardiolipin and shows preference for the transfer of a linoleic acid (LA) group from phosphatidylcholine (PC) to monolysocardiolipin (MLCL) with influence from mitochondrial membrane curvature. The protein contains domains and motifs involved in targeting, anchoring, and an active site for transacylase activity. Tafazzin activity affects many aspects of mitochondrial structure and function, including that of the electron transport chain, fission-fusion, as well as apoptotic signaling. TAZ mutations are implicated in Barth syndrome, an underdiagnosed and devastating disease that primarily affects male pediatric patients with a broad spectrum of disease pathologies that impact the cardiovascular, neuromuscular, metabolic, and hematologic systems.


Assuntos
Aciltransferases/genética , Síndrome de Barth/etiologia , Síndrome de Barth/genética , Síndrome de Barth/metabolismo , Cardiolipinas/genética , Mitocôndrias/genética , Fatores de Transcrição/genética , Animais , Apoptose/genética , Humanos , Transdução de Sinais/genética
15.
Nat Rev Gastroenterol Hepatol ; 17(1): 40-52, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31641249

RESUMO

Nonalcoholic fatty liver disease (NAFLD) affects around a quarter of the global population, paralleling worldwide increases in obesity and metabolic syndrome. NAFLD arises in the context of systemic metabolic dysfunction that concomitantly amplifies the risk of cardiovascular disease and diabetes. These interrelated conditions have long been recognized to have a heritable component, and advances using unbiased association studies followed by functional characterization have created a paradigm for unravelling the genetic architecture of these conditions. A novel perspective is to characterize the shared genetic basis of NAFLD and other related disorders. This information on shared genetic risks and their biological overlap should in future enable the development of precision medicine approaches through better patient stratification, and enable the identification of preventive and therapeutic strategies. In this Review, we discuss current knowledge of the genetic basis of NAFLD and of possible pleiotropy between NAFLD and other liver diseases as well as other related metabolic disorders. We also discuss evidence of causality in NAFLD and other related diseases and the translational significance of such evidence, and future challenges from the study of genetic pleiotropy.


Assuntos
Hepatopatia Gordurosa não Alcoólica/genética , 17-Hidroxiesteroide Desidrogenases/genética , Aciltransferases/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma Hepatocelular/genética , Causalidade , Progressão da Doença , Pleiotropia Genética , Predisposição Genética para Doença , Humanos , Lipase/genética , Cirrose Hepática Alcoólica/genética , Neoplasias Hepáticas/genética , Proteínas de Membrana/genética , Análise da Randomização Mendeliana , Síndrome Metabólica/genética , Biologia de Sistemas
16.
Ann N Y Acad Sci ; 1459(1): 19-37, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31553069

RESUMO

Lipopolysaccharides are complex molecules found in the cell envelop of many Gram-negative bacteria. The toxic activity of these molecules has led to the terminology of endotoxins. They provide bacteria with structural integrity and protection from external environmental conditions, and they interact with host signaling receptors to induce host immune responses. Bacteria have evolved enzymes that act to modify lipopolysaccharides, particularly the lipid A region of the molecule, to enable the circumvention of host immune system responses. These modifications include changes to lipopolysaccharide by the addition of positively charged sugars, such as N-Ara4N, and phosphoethanolamine (pEtN). Other modifications include hydroxylation, acylation, and deacylation of fatty acyl chains. We review the two-component regulatory mechanisms for enzymes that carry out these modifications and provide details of the structures of four enzymes (PagP, PagL, pEtN transferases, and ArnT) that modify the lipid A portion of lipopolysaccharides. We focus largely on the three-dimensional structures of these enzymes, which provide an understanding of how their substrate binding and catalytic activities are mediated. A structure-function-based understanding of these enzymes provides a platform for the development of novel therapeutics to treat antibiotic resistance.


Assuntos
Lipídeo A/química , Lipídeo A/metabolismo , Aciltransferases/química , Aciltransferases/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Humanos , Metiltransferases/química , Metiltransferases/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
17.
J Asian Nat Prod Res ; 22(5): 434-443, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31791147

RESUMO

Various bioactive polyketides have been found in Aloe barbadensis. However, the polyketide synthases (PKSs), which participate in biosynthesis of polyketides in A. barbadensis remain unknown. In this study, two type III PKSs (AbPKS1 and AbPKS2) were identified from A. barbadensis. AbPKS1 and AbPKS2 were able to utilize malonyl-CoA to yield heptaketides (TW93a and aloesone) and octaketides (SEK4 and SEK4b), respectively. AbPKS1 also exhibited catalytic promiscuity in recognizing CoA thioesters of aromatics to produce unusual polyketides. What Is more, a whole cell biocatalysis system with the capability of producing 26.4 mg/L of SEK4/SEK4b and 2.1 mg/L of aloesone was successfully established.


Assuntos
Aloe , Policetídeos , Aciltransferases , Estrutura Molecular , Policetídeo Sintases
18.
Lett Appl Microbiol ; 70(3): 221-229, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31854000

RESUMO

Hard-shelled mussel (Mytilus coruscus) is a popular seafood in Korea. This study aimed to determine the virulence markers and antimicrobial resistance patterns of 33 Aeromonas strains isolated from mussels. The isolates were identified as A. salmonicida (n = 14), A. veronii (n = 9), A. enteropelogenes (n = 4), A. caviae (n = 3), A. allosaccharophila (n = 2) and A. bivalvium (n = 1) by gyrB gene sequencing. The sequence divergence between and within the species ranged from 3·70 to 10·40% and 0-1·50% respectively. Every species formed a distinct group in a neighbour-joining phylogenetic tree. The DNase, gelatinase, caseinase, ß-haemolysis, biofilm and lipase activities were observed in 33 (100·00%), 31 (93·93%), 30 (90·90%), 27 (81·81%), 21 (63·63%) and 17 (51·51%) isolates respectively. The virulence genes were detected by PCR in the following frequencies: fla (90·09%), aer (87·88%), hlyA (87·88%), ahyB (81·19%), gcaT (75·76%), ser (69·70%), lip (66·67%), alt (57·58%), ast (51·51%) and act (21·21%). Every isolate was resistant to at least three of 18 antimicrobials in the disk diffusion test. The multiple antimicrobial resistance index values ranged from 0·11 to 0·44 among the isolates. Our study suggests that mussels can be a potential reservoir of virulent and multidrug-resistant Aeromonas sp. SIGNIFICANCE AND IMPACT OF THE STUDY: Aeromonas sp. are known as common pathogenic bacteria isolated from seafood. The virulence factors and antimicrobial resistance profiles of mussel-borne Aeromonas sp. are poorly understood. This study demonstrated for the first time the existence of virulence markers and antimicrobial resistance of Aeromonas sp. from mussels in Korea. Majority of the isolates were positive for phenotypic virulence characteristics and harboured several virulence genes which reveal the potential virulence of mussel-borne Aeromonas sp. Multiple antimicrobial resistance was also observed among the isolates. Our study highlights the importance of food safety standards in mussel consumption.


Assuntos
Aeromonas/classificação , Aeromonas/isolamento & purificação , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Mytilus/microbiologia , Aciltransferases/genética , Aeromonas/genética , Aeromonas/patogenicidade , Animais , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Infecções por Bactérias Gram-Negativas/microbiologia , Lipase/genética , Filogenia , Reação em Cadeia da Polimerase , República da Coreia , Alimentos Marinhos/microbiologia , Virulência/genética , Fatores de Virulência/genética
19.
Phytochemistry ; 169: 112179, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31669976

RESUMO

Deschampsiaantarctica inhabits the maritime territory of Antarctica and South Patagonia. It grows under very harsh environmental conditions. The survival of this species in low freezing temperatures and under high levels of UV-B radiation may constitute some of the most remarkable adaptive plant responses and suggests that this plant possesses genes associated with cold and UV tolerance. Frequently, increased levels of flavonoids have been linked to highly UV-B irradiated plants. Studies examining the biosynthesis of flavonoids in D. antarctica may provide clues to its success in this extreme environment. In this study, we characterized the family of genes encoding chalcone synthase, a key enzyme of the flavonoid biosynthetic pathway. DaCHS was cloned, sequenced and characterized by using software tools. CHS contains two domains, the N-terminal domain ranges from amino acid 8 to 231 and the C-terminal domain ranges from amino acid 241 to 391. Sequence analysis of the three family members revealed a high degree of identity after comparison with other monocotyledons such as Oryza sativa L., Zea mays L. and Hordeum vulgare L. According to these results, DaCHS can be grouped together with H. vulgare CHS1 in the same branch. The phylogenetic tree was built using MEGA software and the neighbour join method with 1000 bootstrap replicates. A model of DaCHS was constructed by way of structural tools and key amino acid residues were identified at the active motif site.


Assuntos
Aciltransferases/genética , Regulação Enzimológica da Expressão Gênica/genética , Poaceae/enzimologia , Raios Ultravioleta , Aciltransferases/química , Sequência de Aminoácidos , Modelos Moleculares , Filogenia , Alinhamento de Sequência , Software
20.
J Agric Food Chem ; 68(3): 808-817, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31870144

RESUMO

Various Monascus bioactive metabolites used as food or food additives in Asia for centuries are subjected to constant physical and chemical changes and different Monascus genus. With the aim to identify enzymes that participate in or indirectly regulate the pigments and citrinin biosynthesis pathways of Monascus purpureus cultured under high ammonium chloride, the changes of the proteome profile were examined using sequential window acquisition of all theoretical mass spectra-mass spectrometry-based quantitative proteomics approach in combination with bioinformatics analysis. A total of 292 proteins were confidently detected and quantified in each sample, including 163 that increased and 129 that decreased (t-tests, p ≤ 0.05). Pathway analysis indicated that high ammonium chloride in the present study accelerates the carbon substrate utilization and promotes the activity of key enzymes in glycolysis and ß-oxidation of fatty acid catabolism to generate sufficient acetyl-CoA. However, the synthesis of the monascus pigments and citrinin was not enhanced because of inhibition of the polyketide synthase activity. All results demonstrated that the cause of initiation of pigments and citrinin synthesis is mainly due to the apparent inhibition of acyl and acetyl transfer by some acyltransferase and acetyltransferase, likely malony-CoA:ACP transacylase.


Assuntos
Cloreto de Amônio/metabolismo , Citrinina/biossíntese , Monascus/metabolismo , Pigmentos Biológicos/metabolismo , Acetiltransferases/metabolismo , Aciltransferases/metabolismo , Citrinina/química , Proteínas Fúngicas/metabolismo , Espectrometria de Massas , Monascus/química , Pigmentos Biológicos/química , Proteômica
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