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1.
Nature ; 585(7826): 614-619, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879484

RESUMO

Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.


Assuntos
Alcaloides/biossíntese , Alcaloides/provisão & distribução , Hiosciamina/biossíntese , Saccharomyces cerevisiae/metabolismo , Escopolamina/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Atropa belladonna/enzimologia , Derivados da Atropina/metabolismo , Transporte Biológico , Datura/enzimologia , Glucosídeos/biossíntese , Glucosídeos/metabolismo , Hiosciamina/provisão & distribução , Lactatos/metabolismo , Ligases/genética , Ligases/metabolismo , Modelos Moleculares , Doenças do Sistema Nervoso/tratamento farmacológico , Oxirredutases/genética , Oxirredutases/metabolismo , Engenharia de Proteínas , Saccharomyces cerevisiae/genética , Escopolamina/provisão & distribução , Vacúolos/metabolismo
2.
Nat Commun ; 11(1): 4765, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958780

RESUMO

Fatty acids (FAs) are essential nutrients, but how they are transported into cells remains unclear. Here, we show that FAs trigger caveolae-dependent CD36 internalization, which in turn delivers FAs into adipocytes. During the process, binding of FAs to CD36 activates its downstream kinase LYN, which phosphorylates DHHC5, the palmitoyl acyltransferase of CD36, at Tyr91 and inactivates it. CD36 then gets depalmitoylated by APT1 and recruits another tyrosine kinase SYK to phosphorylate JNK and VAVs to initiate endocytic uptake of FAs. Blocking CD36 internalization by inhibiting APT1, LYN or SYK abolishes CD36-dependent FA uptake. Restricting CD36 at either palmitoylated or depalmitoylated state eliminates its FA uptake activity, indicating an essential role of dynamic palmitoylation of CD36. Furthermore, blocking endocytosis by targeting LYN or SYK inhibits CD36-dependent lipid droplet growth in adipocytes and high-fat-diet induced weight gain in mice. Our study has uncovered a dynamic palmitoylation-regulated endocytic pathway to take up FAs.


Assuntos
Antígenos CD36/metabolismo , Endocitose/fisiologia , Ácidos Graxos/metabolismo , Lipoilação , Células 3T3-L1 , Aciltransferases/metabolismo , Adipócitos/metabolismo , Animais , Antígenos CD36/deficiência , Antígenos CD36/genética , Cavéolas/metabolismo , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Humanos , Gotículas Lipídicas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Obesidade/tratamento farmacológico , Fosforilação , Transdução de Sinais , Quinase Syk/antagonistas & inibidores , Quinase Syk/metabolismo , Ganho de Peso/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismo
3.
Adv Exp Med Biol ; 1274: 5-27, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32894505

RESUMO

Biophysical properties of membranes are dependent on their glycerophospholipid compositions. Lysophospholipid acyltransferases (LPLATs) selectively incorporate fatty chains into lysophospholipids to affect the fatty acid composition of membrane glycerophospholipids. Lysophosphatidic acid acyltransferases (LPAATs) of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family incorporate fatty chains into phosphatidic acid during the de novo glycerophospholipid synthesis in the Kennedy pathway. Other LPLATs of both the AGPAT and the membrane bound O-acyltransferase (MBOAT) families further modify the fatty chain compositions of membrane glycerophospholipids in the remodeling pathway known as the Lands' cycle. The LPLATs functioning in these pathways possess unique characteristics in terms of their biochemical activities, regulation of expressions, and functions in various biological contexts. Essential physiological functions for LPLATs have been revealed in studies using gene-deficient mice, and important roles for several enzymes are also indicated in human diseases where their mutation or dysregulation causes or contributes to the pathological condition. Now several LPLATs are emerging as attractive therapeutic targets, and further understanding of the mechanisms underlying their physiological and pathological roles will aid in the development of novel therapies to treat several diseases that involve altered glycerophospholipid metabolism.


Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/antagonistas & inibidores , Aciltransferases/antagonistas & inibidores , Membrana Celular/metabolismo , Desenvolvimento de Medicamentos , Glicerofosfolipídeos/biossíntese , Glicerofosfolipídeos/química , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Aciltransferases/metabolismo , Animais , Membrana Celular/química , Membrana Celular/enzimologia , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Humanos
4.
Gene ; 762: 145104, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32889060

RESUMO

Chalcone synthase (CHS, EC 2.3.1.74) is one of the key and rate-limiting enzymes of phenylpropanoid pathway which plays superior roles in the production of secondary metabolites. In the present study a full-length cDNA of CHS gene was isolated and characterized from Coelogyne ovalis, an orchid of ornamental and medicinal importance. The CHS gene sequence from C. ovalis (CoCHS) was found to be 1445 bp and comprised an open reading frame of 1182 bp, encoding for 394 amino acid residues. Further, the sequence alignment and phylogenetic analysis revealed that CoCHS protein shared high degree of similarity with CHS protein of other orchid species. It also confirmed that it contained all four motifs (I to IV) and signature sequence for the functionality of this gene. Structural modeling of CoCHS based on the crystallographic structure of Freesia hybrida indicated that CoCHS had a similar structure. Quantitative polymerase chain reaction (qPCR) disclosed that CoCHS was expressed in all tissues examined, with the highest transcript being in leaves, followed by pseudobulbs and roots. CoCHS expression was also evaluated in the in vitro-raised plantlets under the abiotic stress (dark, cold, UV-B, wounding, salinity). mRNA transcript expression of CHS gene was found to be positively enhanced and regulated by the different stress types. A correlation between the CoCHS transcript expression with flavonoid and anthocyanin contents revealed that a positive correlation existed between metabolites' content and CoCHS expression within the in vivo as well as in the in vitro-raised plant parts.


Assuntos
Aciltransferases/genética , Regulação da Expressão Gênica de Plantas , Orchidaceae/genética , Proteínas de Plantas/genética , Aciltransferases/química , Aciltransferases/metabolismo , Clonagem Molecular , Orchidaceae/classificação , Orchidaceae/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estresse Fisiológico
5.
Metabolism ; 111: 154341, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32810486

RESUMO

BACKGROUND: Biallelic loss of function variants in AGPAT2, encoding 1-acylglycerol-3-phosphate O-acyltransferase 2, cause congenital generalized lipodystrophy type 1, a disease characterized by near total loss of white adipose tissue and metabolic complications. Agpat2 deficient (Agpat2-/-) mice completely lacks both white and interscapular brown adipose tissue (iBAT). The objective of the present study was to characterize the effects of AGPAT2 deficiency in brown adipocyte differentiation. METHODS: Preadipocytes obtained from newborn (P0.5) Agpat2-/- and wild type mice iBAT were differentiated into brown adipocytes, compared by RNA microarray, RT-qPCR, High-Content Screening (HCS), western blotting and electron microscopy. RESULTS: 1) Differentiated Agpat2-/- brown adipocytes have fewer lipid-laden cells and lower abundance of Pparγ, Pparα, C/ebpα and Pgc1α, both at the mRNA and protein levels, compared those to wild type cells. Prmd16 levels were equivalent in both, Agpat2-/- and wild type, while Ucp1 was only induced in wild type cells, 2) These differences were not due to lower abundance of preadipocytes, 3) Differentiated Agpat2-/- brown adipocytes are enriched in the mRNA abundance of genes participating in interferon (IFN) type I response, whereas genes involved in mitochondrial homeostasis were decreased, 4) Mitochondria in differentiated Agpat2-/- brown adipocytes had altered morphology and lower mass and contacting sites with lipid droplets concomitant with lower levels of Mitofusin 2 and Perlipin 5. CONCLUSION: AGPAT2 is necessary for normal brown adipose differentiation. Its absence results in a lower proportion of lipid-laden cells, increased expression of interferon-stimulated genes (ISGs) and alterations in mitochondrial morphology, mass and fewer mitochondria to lipid droplets contacting sites in differentiated brown adipocytes.


Assuntos
Aciltransferases/metabolismo , Adipócitos Marrons/metabolismo , Adipogenia/fisiologia , Tecido Adiposo Marrom/metabolismo , Expressão Gênica/fisiologia , Interferon Tipo I/metabolismo , Mitocôndrias/metabolismo , Adipócitos Marrons/fisiologia , Tecido Adiposo Marrom/fisiologia , Animais , Diferenciação Celular/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Homeostase/fisiologia , Camundongos , Mitocôndrias/fisiologia , RNA Mensageiro/metabolismo
6.
Plant Mol Biol ; 104(3): 283-296, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32740897

RESUMO

KEY MESSAGE: Differences in FAE1 enzyme affinity for the acyl-CoA substrates, as well as the balance between the different pathways involved in their incorporation to triacylglycerol might be determinant of the different composition of the seed oil in Brassicaceae. Brassicaceae present a great heterogeneity of seed oil and fatty acid composition, accumulating Very Long Chain Fatty Acids with industrial applications. However, the molecular determinants of these differences remain elusive. We have studied the ß-ketoacyl-CoA synthase from the high erucic feedstock Thlaspi arvense (Pennycress). Functional characterization of the Pennycress FAE1 enzyme was performed in two Arabidopsis backgrounds; Col-0, with less than 2.5% of erucic acid in its seed oil and the fae1-1 mutant, deficient in FAE1 activity, that did not accumulate erucic acid. Seed-specific expression of the Pennycress FAE1 gene in Col-0 resulted in a 3 to fourfold increase of erucic acid content in the seed oil. This increase was concomitant with a decrease of eicosenoic acid levels without changes in oleic ones. Interestingly, only small changes in eicosenoic and erucic acid levels occurred when the Pennycress FAE1 gene was expressed in the fae1-1 mutant, with high levels of oleic acid available for elongation, suggesting that the Pennycress FAE1 enzyme showed higher affinity for eicosenoic acid substrates, than for oleic ones in Arabidopsis. Erucic acid was incorporated to triacylglycerol in the transgenic lines without significant changes in their levels in the diacylglycerol fraction, suggesting that erucic acid was preferentially incorporated to triacylglycerol via DGAT1. Expression analysis of FAE1, AtDGAT1, AtLPCAT1 and AtPDAT1 genes in the transgenic lines further supported this conclusion. Differences in FAE1 affinity for the oleic and eicosenoic substrates among Brassicaceae, as well as their incorporation to triacylglycerol might explain the differences in composition of their seed oil.


Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Biocombustíveis , Vias Biossintéticas , Brassicaceae/metabolismo , Thlaspi/enzimologia , Thlaspi/metabolismo , Triglicerídeos/biossíntese , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Aciltransferases/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas/genética , Diacilglicerol O-Aciltransferase/metabolismo , Ácidos Erúcicos/metabolismo , Elongases de Ácidos Graxos/genética , Elongases de Ácidos Graxos/metabolismo , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica de Plantas , Fenótipo , Óleos Vegetais/metabolismo , Plantas Geneticamente Modificadas , Sementes/genética , Análise de Sequência , Thlaspi/genética , Transcriptoma
7.
Food Chem ; 331: 127365, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32619909

RESUMO

Phenolic acids from Salvia miltiorrhiza have been widely used in nutritious, health-promoting products with an increasing demand. In the current study, two biosynthetic genes RAS (rosmarinic acid synthase) and CYP98A14 (a cytochrome P450-dependent monooxygenase) were successfully introduced into S. miltiorrhiza hairy roots. Overexpression of RAS and CYP98A14 resulted in higher content of phenolic acids (up to over 3-fold) in transgenic lines compared to the control. Meanwhile, DPPH results revealed that engineered S. miltiorrhiza hairy roots had stronger antioxidant activities than the control. In addition, phenolic acid crude extracts of the engineered hairy root lines overexpressing RAS or CYP98A14 showed improved antibacterial activities compared to the control lines. Our work exhibits a useful strategy for enhancement of phenolic acid production and bioactivities of S. miltiorrhiza hairy roots by genetic manipulation of RAS and CYP98A14, and also provides a new resource material to obtain active phenolic acids for food and healthy products.


Assuntos
Aciltransferases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Hidroxibenzoatos/metabolismo , Raízes de Plantas/química , Salvia miltiorrhiza/genética , Aciltransferases/genética , Antibacterianos/química , Antibacterianos/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Salmonella/efeitos dos fármacos , Salvia miltiorrhiza/química , Salvia miltiorrhiza/metabolismo
8.
J Vis Exp ; (159)2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32478756

RESUMO

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most γ-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.


Assuntos
Aciltransferases/metabolismo , Química Click/métodos , Ensaios Enzimáticos/métodos , Fluorometria/métodos , Ensaios de Triagem em Larga Escala/métodos , Animais , Ácidos Graxos , Fibroblastos/metabolismo , Fluorescência , Humanos , Lipoproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteobactérias/metabolismo , Especificidade por Substrato
9.
DNA Cell Biol ; 39(9): 1639-1648, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32552000

RESUMO

Colorectal cancer (CRC) patients with KRAS mutation are refractory and usually have poor prognosis. We aimed to identify the hub gene associated with KRAS mutant CRCs. Weighted gene coexpression network analysis (WGCNA) was used to calculate the key module and the hub genes in GSE39582. Combined with the protein-protein interaction (PPI) network and survival analysis, the real hub gene was identified and further validated. With the highest module significance value and correlation coefficient, the blue module was selected as the key module, 19 genes were identified as the hub gene candidates. The above genes were significantly downregulated in KRAS mutant CRCs compared with the wild type. Four genes (AAR2, PSMA7, NELFCD, and PIGU) were further screened as the potential hub genes by the PPI network. Low expression of PIGU for KRAS mutant patients had a poor prognosis. Therefore, PIGU was identified as the hub gene. PIGU expression was also downregulated in other two CRC datasets. "MAPK SIGNALING PATHWAY" was enriched in PIGU lowly expressed samples. PIGU was identified and validated to be closely related to KRAS mutation. It could be a potential prognosis biomarker and a novel treatment target for KRAS mutant CRC patients.


Assuntos
Aciltransferases/genética , Neoplasias Colorretais/genética , Redes Reguladoras de Genes , Proteínas Proto-Oncogênicas p21(ras)/genética , Aciltransferases/metabolismo , Idoso , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação
10.
Protist ; 171(3): 125738, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32544845

RESUMO

This paper represents a comprehensive study of two new thraustochytrids and a marine Rhodotorula red yeast isolated from Australian coastal waters for their abilities to be a potential renewable feedstock for the nutraceutical, food, fishery and bioenergy industries. Mixotrophic growth of these species was assessed in the presence of different carbon sources: glycerol, glucose, fructose, galactose, xylose, and sucrose, starch, cellulose, malt extract, and potato peels. Up to 14g DW/L (4.6gDW/L-day and 2.8gDW/L-day) of biomass were produced by Aurantiochytrium and Thraustochytrium species, respectively. Thraustochytrids biomass contained up to 33% DW of lipids, rich in omega-3 polyunsaturated docosahexaenoic acid (C22:6, 124mg/g DW); up to 10.2mg/gDW of squalene and up to 61µg/gDW of total carotenoids, composed of astaxanthin, canthaxanthin, echinenone, and ß-carotene. Along with the accumulation of these added-value chemicals in biomass, thraustochytrid representatives showed the ability to secrete extracellular polysaccharide matrixes containing lipids and proteins. Rhodotorula sp lipids (26% DW) were enriched in palmitic acid (C16:0, 18mg/gDW) and oleic acid (C18:1, 41mg/gDW). Carotenoids (87µg/gDW) were mainly represented by ß-carotene (up to 54µg/gDW). Efficient growth on organic and inorganic sources of carbon and nitrogen from natural and anthropogenic wastewater pollutants along with intracellular and extracellular production of valuable nutrients makes the production of valuable chemicals from isolated species economical and sustainable.


Assuntos
Biodegradação Ambiental , Quitridiomicetos , Lipídeos/biossíntese , Rhodotorula , Poluentes da Água/metabolismo , Aciltransferases/metabolismo , Biomassa , Carotenoides/metabolismo , Quitridiomicetos/crescimento & desenvolvimento , Quitridiomicetos/isolamento & purificação , Quitridiomicetos/metabolismo , Ácidos Docosa-Hexaenoicos/biossíntese , Ácidos Graxos Insaturados/biossíntese , Nutrientes/metabolismo , Polissacarídeos/biossíntese , Rhodotorula/crescimento & desenvolvimento , Rhodotorula/isolamento & purificação , Rhodotorula/metabolismo , Água do Mar/microbiologia , Águas Residuárias/microbiologia , Áreas Alagadas
11.
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
12.
Nucleic Acids Res ; 48(10): 5294-5305, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32369169

RESUMO

The broad host range bacteriophage Mu employs a novel 'methylcarbamoyl' modification to protect its DNA from diverse restriction systems of its hosts. The DNA modification is catalyzed by a phage-encoded protein Mom, whose mechanism of action is a mystery. Here, we characterized the co-factor and metal-binding properties of Mom and provide a molecular mechanism to explain 'methylcarbamoyl'ation of DNA by Mom. Computational analyses revealed a conserved GNAT (GCN5-related N-acetyltransferase) fold in Mom. We demonstrate that Mom binds to acetyl CoA and identify the active site. We discovered that Mom is an iron-binding protein, with loss of Fe2+/3+-binding associated with loss of DNA modification activity. The importance of Fe2+/3+ is highlighted by the colocalization of Fe2+/3+ with acetyl CoA within the Mom active site. Puzzlingly, acid-base mechanisms employed by >309,000 GNAT members identified so far, fail to support methylcarbamoylation of adenine using acetyl CoA. In contrast, free-radical chemistry catalyzed by transition metals like Fe2+/3+ can explain the seemingly challenging reaction, accomplished by collaboration between acetyl CoA and Fe2+/3+. Thus, binding to Fe2+/3+, a small but unprecedented step in the evolution of Mom, allows a giant chemical leap from ordinary acetylation to a novel methylcarbamoylation function, while conserving the overall protein architecture.


Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Acetilcoenzima A/metabolismo , Bacteriófago mu/fisiologia , Domínio Catalítico , Escherichia coli/genética , Escherichia coli/virologia , Ferro/metabolismo , Conformação Proteica
13.
PLoS One ; 15(4): e0226537, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302307

RESUMO

Enzymes of the chalcone synthase (CHS) family participate in the synthesis of multiple secondary metabolites in plants, fungi and bacteria. CHS showed a significant correlation with the accumulation patterns of anthocyanin. The peel color, which is primarily determined by the content of anthocyanin, is an economically important trait for eggplants that is affected by heat stress. A total of 7 CHS (SmCHS1-7) putative genes were identified in a genome-wide analysis of eggplants (S. melongena L.). The SmCHS genes were distributed on 7 scaffolds and were classified into 3 clusters. Phylogenetic relationship analysis showed that 73 CHS genes from 7 Solanaceae species were classified into 10 groups. SmCHS5, SmCHS6 and SmCHS7 were continuously down-regulated under 38°C and 45°C treatment, while SmCHS4 was up-regulated under 38°C but showed little change at 45°C in peel. Expression profiles of key anthocyanin biosynthesis gene families showed that the PAL, 4CL and AN11 genes were primarily expressed in all five tissues. The CHI, F3H, F3'5'H, DFR, 3GT and bHLH1 genes were expressed in flower and peel. Under heat stress, the expression level of 52 key genes were reduced. In contrast, the expression patterns of eight key genes similar to SmCHS4 were up-regulated at a treatment of 38°C for 3 hour. Comparative analysis of putative CHS protein evolutionary relationships, cis-regulatory elements, and regulatory networks indicated that SmCHS gene family has a conserved gene structure and functional diversification. SmCHS showed two or more expression patterns, these results of this study may facilitate further research to understand the regulatory mechanism governing peel color in eggplants.


Assuntos
Aciltransferases/genética , Flavonoides/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Solanum melongena/genética , Aciltransferases/metabolismo , Antocianinas/genética , Antocianinas/metabolismo , Vias Biossintéticas , Flavonoides/metabolismo , Resposta ao Choque Térmico , Família Multigênica , Filogenia , Proteínas de Plantas/metabolismo , Solanum melongena/fisiologia , Transcriptoma
14.
Artigo em Inglês | MEDLINE | ID: mdl-32244096

RESUMO

Lipoic acid (LA) and its reduced form (dihydrolipoic acid, DHLA) have unique antioxidant properties among such molecules. Moreover, after a process termed lipoylation, LA is an essential prosthetic group covalently-attached to several key multi-subunit enzymatic complexes involved in primary metabolism, including E2 subunits of pyruvate dehydrogenase (PDH). The metabolic pathway of lipoylation has been extensively studied in Escherichia coli and Arabidopsis thaliana in which protein modification occurs via two routes: de novo synthesis and salvage. Common to both pathways, lipoyl synthase (LIP1 in plants, LipA in bacteria, EC 2.8.1.8) inserts sulphur atoms into the molecule in a final, activating step. However, despite the detection of LA and DHLA in other plant species, including tomato (Solanum lycopersicum), no plant LIP1s have been characterised to date from species other than Arabidopsis. In this work, we present the identification and characterisation of two LIPs from tomato, SlLIP1 and SlLIP1p. Consistent with in silico data, both are widely-expressed, particularly in reproductive organs. In line with bioinformatic predictions, we determine that yellow fluorescent protein tagged versions of SlLIP1 and SlLIP1p are mitochondrially- and plastidially-localised, respectively. Both possess the molecular hallmarks and domains of well-characterised bacterial LipAs. When heterologously-expressed in an E. coli lipA mutant, both are capable of complementing specific growth phenotypes and increasing lipoylation levels of E2 subunits of PDH in vivo, demonstrating that they do indeed function as lipoyl synthases.


Assuntos
Aciltransferases , Lipoilação , Lycopersicon esculentum , Mitocôndrias , Plastídeos , Aciltransferases/genética , Aciltransferases/metabolismo , Escherichia coli/genética , Lycopersicon esculentum/enzimologia , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Plastídeos/enzimologia , Ácido Tióctico/metabolismo
15.
Syst Appl Microbiol ; 43(3): 126073, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32139173

RESUMO

Soybean bradyrhizobia (Bradyrhizobium spp.) are bacteria that fix atmospheric nitrogen within the root nodules of soybean, a crop critical for meeting global nutritional protein demand. Members of this group differ in symbiotic effectiveness, and historically both phenotypic and genotypic approaches have been used to assess bradyrhizobial diversity. However, agreement between various approaches of assessment is poorly known. A collection (n=382) of soybean bradyrhizobia (Bradyrhizobium japonicum, B. diazoefficiens, and B. elkanii) were characterized by Internal Transcribed Spacer - Restriction Fragment Length Polymorphism (ITS-RFLP), cellular fatty acid composition (fatty acid methyl esters, FAME), and serological reactions to assess agreement between phenotypic and genotypic methods. Overall, 76% of the accessions demonstrated identical clustering with each of these techniques. FAME was able to identify all 382 accessions, whereas 14% were non-reactive serologically. One ITS-RFLP group, containing 36 Delaware isolates, produced multiple ITS amplicons indicating they possess multiple ribosomal RNA (rrn) operons. Cloning and sequencing revealed that these strains contained as many as three heterogenous rrn operons, a trait previously unknown in bradyrhizobia. A representative subset of 96 isolates was further characterized using 16S rRNA and Internal Transcribed Spacer (ITS) amplicon sequencing. ITS sequences showed better inter- and intra-species discrimination (65-99% identity) than 16S sequences (96-99% identity). This study shows that phenotypic and genotypic approaches are strongly correlated at the species level but should be approached with caution. We also suggest using combined 16S and ITS genotyping data to obtain better inter- and intra-species resolution in bradyrhizobia classification.


Assuntos
Bradyrhizobium/classificação , Bradyrhizobium/fisiologia , Genótipo , Fenótipo , Soja/microbiologia , Aciltransferases/metabolismo , DNA Bacteriano , DNA Espaçador Ribossômico , Filogenia , RNA Bacteriano , RNA Ribossômico 16S , Testes Sorológicos
16.
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
17.
Cell ; 180(6): 1130-1143.e20, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32160528

RESUMO

Fatty acid synthases (FASs) are central to metabolism but are also of biotechnological interest for the production of fine chemicals and biofuels from renewable resources. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the dynamic acyl carrier protein domain to several enzyme active sites. Here, we report the discovery of a γ subunit of the 2.6 megadalton α6-ß6S. cerevisiae FAS, which is shown by high-resolution structures to stabilize a rotated FAS conformation and rearrange ACP domains from equatorial to axial positions. The γ subunit spans the length of the FAS inner cavity, impeding reductase activities of FAS, regulating NADPH turnover by kinetic hysteresis at the ketoreductase, and suppressing off-pathway reactions at the enoylreductase. The γ subunit delineates the functional compartment within FAS. As a scaffold, it may be exploited to incorporate natural and designed enzymatic activities that are not present in natural FAS.


Assuntos
Ácido Graxo Sintases/química , Ácido Graxo Sintases/metabolismo , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/metabolismo , Aciltransferases/metabolismo , Sítios de Ligação , Domínio Catalítico , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X/métodos , Ácidos Graxos/biossíntese , Ácidos Graxos/química , Modelos Moleculares , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade
18.
Biochemistry ; 59(10): 1124-1136, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32125848

RESUMO

ATP:Co(I)rrinoid adenosyltransferases (ACATs) catalyze the transfer of the adenosyl moiety from co-substrate ATP to a corrinoid substrate. ACATs are grouped into three families, namely, CobA, PduO, and EutT. The EutT family of enzymes is further divided into two classes, depending on whether they require a divalent metal ion for activity (class I and class II). To date, a structure has not been elucidated for either class of the EutT family of ACATs. In this work, results of bioinformatics analyses revealed several conserved residues between the C-terminus of EutT homologues and the structurally characterized Lactobacillus reuteri PduO (LrPduO) homologue. In LrPduO, these residues are associated with ATP binding and formation of an intersubunit salt bridge. These residues were substituted, and in vivo and in vitro data support the conclusion that the equivalent residues in the metal-free (i.e., class II) Listeria monocytogenes EutT (LmEutT) enzyme affect ATP binding. Results of in vivo and in vitro analyses of LmEutT variants with substitutions at phenylalanine and tryptophan residues revealed that replacement of the phenylalanine residue at position 72 affected access to the substrate-binding site and replacement of a tryptophan residue at position 238 affected binding of the Cbl substrate to the active site. Unlike the PduO family of ACATs, a single phenylalanine residue is not responsible for displacement of the α-ligand. Together, these data suggest that while EutT enzymes share a conserved ATP-binding motif and an intersubunit salt bridge with PduO family ACATs, class II EutT family ACATs utilize an unidentified mechanism for Cbl lower-ligand displacement and reduction that is different from that of PduO and CobA family ACATs.


Assuntos
Corrinoides/metabolismo , Listeria monocytogenes/enzimologia , Aciltransferases/metabolismo , Trifosfato de Adenosina/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Aldeído Oxirredutases/ultraestrutura , Alquil e Aril Transferases/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Catálise , Domínio Catalítico , Cobalto/química , Cobamidas/metabolismo , Cinética , Lactobacillus reuteri/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Modelos Moleculares , Mutação , Transferases/metabolismo
19.
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
20.
Biomed Res Int ; 2020: 2527643, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32104684

RESUMO

Targeted therapy for kidney cancer has achieved significant clinical results. However, because most patients who use targeted therapy will develop drug resistance, we still need to constantly explore new therapeutic targets. Although porcupine (PORCN) as a palmitoyltransferase plays a crucial role in the activation and secretion of Wnt proteins and affects the activity of the Wnt signaling pathway, little is known about the role of PORCN in clear cell renal cell carcinoma (ccRCC). We found that PORCN is highly expressed in renal cancer cell lines and patients with renal cell carcinoma with high expression of PORCN have a poor prognosis. Pathway analysis of PORCN and its related proteins showed that PORCN played a role through the Wnt signaling pathway, and there was a strong coexpression relationship between PORCN and Wnt proteins. Therefore, PORCN may be a potential and effective target for ccRCC. In the present study, we found that LGK974 could inhibit proliferation and colony formation and induce apoptosis in ccRCC cells. We also found that LGK974 could inhibit the migration and invasion of renal cell carcinoma and reduce the expression of mesenchymal markers. After treatment with LGK974, the expression level of ß-catenin, a key protein in the classical Wnt pathway, was significantly decreased, and the expression levels of the target genes cyclin D1, c-Myc, MMP9, and MMP2 in the Wnt signaling pathway were also significantly decreased, which represented a significant decrease in the activity of the Wnt signaling pathway. At the same time, the cycle of renal cancer cells was significantly blocked. In conclusion, our results indicate that LGK974 could significantly inhibit the progression of renal cancer cells in a safe concentration range, so PORCN may be a safe and effective target for patients with renal cancer.


Assuntos
Aciltransferases/antagonistas & inibidores , Carcinoma de Células Renais/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Renais/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Neoplasias , Pirazinas/farmacologia , Piridinas/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Aciltransferases/metabolismo , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo
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