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1.
Nat Commun ; 12(1): 296, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436600

RESUMO

Nonribosomal peptide synthetases containing starter condensation domains direct the biosynthesis of nonribosomal lipopeptides, which generally exhibit wide bioactivities. The acyl chain has strong impacts on bioactivity and toxicity, but the lack of an in-depth understanding of starter condensation domain-mediated lipoinitiation limits the bioengineering of NRPSs to obtain novel derivatives with desired acyl chains. Here, we show that the acyl chains of the lipopeptides rhizomide, holrhizin, and glidobactin were modified by engineering the starter condensation domain, suggesting a workable approach to change the acyl chain. Based on the structure of the mutated starter condensation domain of rhizomide biosynthetic enzyme RzmA in complex with octanoyl-CoA and related point mutation experiments, we identify a set of residues responsible for the selectivity of substrate acyl chains and extend the acyl chains from acetyl to palmitoyl. Furthermore, we illustrate three possible conformational states of starter condensation domains during the reaction cycle of the lipoinitiation process. Our studies provide further insights into the mechanism of lipoinitiation and the engineering of nonribosomal peptide synthetases.


Assuntos
Lipídeos/química , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Engenharia de Proteínas , Acilação , Sequência de Aminoácidos , Lipopeptídeos/química , Lipopeptídeos/metabolismo , Modelos Moleculares , Mutação Puntual/genética , Domínios Proteicos , Especificidade por Substrato
2.
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
3.
Nucleic Acids Res ; 49(1): 444-457, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33330919

RESUMO

In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA's CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a 'closed' conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an 'open' conformation, which primes its TGS and Helical domains for specific recognition and stabilization of cognate deacylated tRNA on the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Guanosina Pentafosfato/biossíntese , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Acilação , Sítio Alostérico , Bacillus subtilis/genética , Domínio Catalítico , GTP Pirofosfoquinase/metabolismo , Hidrólise , Modelos Genéticos , Modelos Moleculares , Conformação Proteica , Processamento Pós-Transcricional do RNA , Subunidades Ribossômicas Maiores de Bactérias/metabolismo
4.
Food Chem ; 334: 127586, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32707364

RESUMO

It is unknown whether intestinal absorption of acylated anthocyanins occurs in their intact or metabolized form. In this study, with the aid of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging, intestinal absorption of acylated anthocyanins was visually investigated. Anthocyanin extracts from purple carrots were orally administered to Sprague-Dawley rats. Acylated cyanidins were absorbed into portal and circulating blood systems in their intact form, and aglycon; cyanidin 3-O-(6-O-feruloyl-ß-d-glucopyranosyl)-(1 â†’ 6)-[ß-d-xylopyranosyl-(1 â†’ 2)]-ß-d-galactopyranoside (Cy3XFGG), and showed a high absorption of 39.3 ± 0.1 pmol/mL-plasma at 60 min after administration. MALDI-MS imaging analysis of the rat jejunum membranes showed that an organic anion transporting polypeptide (OATP) transporter was involved in Cy3XFGG transport, while deacylated anthocyanins were incorporated through both the glucose transporter 2 and OATP routes. In conclusion, acylated anthocyanin, Cy3XFGG, can be absorbed in its intact form through intestinal OATP.


Assuntos
Antocianinas/análise , Antocianinas/farmacocinética , Imagem Molecular/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Acilação , Administração Oral , Animais , Antocianinas/administração & dosagem , Cor , Daucus carota/química , Absorção Intestinal/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Masculino , Transportadores de Ânions Orgânicos/metabolismo , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacocinética , Ratos Sprague-Dawley
5.
Yakugaku Zasshi ; 140(11): 1313-1322, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-33132266

RESUMO

In total and formal syntheses of dictyodendrins A, B, C, D, E and F, the key step involved the direct construction of the pyrrolo[2,3-c]carbazole core by the gold-catalyzed annulation of a conjugated diyne with a pyrrole to form three bonds and two aromatic rings. The subsequent introduction of substituents at the C1 (Suzuki-Miyaura coupling), C2 (acylation), N3 (alkylation) and C5 positions (Ullmann coupling) provided divergent access to dictyodendrins. Some dictyodendrin analogues exhibited inhibitory activities toward CDK2/CycA2 and GSK3.


Assuntos
Carbazóis/síntese química , Pirróis/síntese química , Acilação , Alquilação , Catálise , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Di-Inos/química , Fenômenos de Química Orgânica , Pirróis/química
6.
Nat Commun ; 11(1): 5003, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024099

RESUMO

Recognition of a start codon by the initiator aminoacyl-tRNA determines the reading frame of messenger RNA (mRNA) translation by the ribosome. In eukaryotes, the GTPase eIF5B collaborates in the correct positioning of the initiator Met-tRNAiMet on the ribosome in the later stages of translation initiation, gating entrance into elongation. Leveraging the long residence time of eIF5B on the ribosome recently identified by single-molecule fluorescence measurements, we determine the cryoEM structure of the naturally long-lived ribosome complex with eIF5B and Met-tRNAiMet immediately before transition into elongation. The structure uncovers an unexpected, eukaryotic specific and dynamic fidelity checkpoint implemented by eIF5B in concert with components of the large ribosomal subunit.


Assuntos
Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/metabolismo , Elongação Traducional da Cadeia Peptídica , Iniciação Traducional da Cadeia Peptídica , Subunidades Ribossômicas Maiores/metabolismo , Acilação , Anticódon , Microscopia Crioeletrônica , Fatores de Iniciação em Eucariotos/genética , Guanosina Difosfato/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/metabolismo , Subunidades Ribossômicas Maiores/química , Subunidades Ribossômicas Maiores/genética , Subunidades Ribossômicas Maiores de Eucariotos , Subunidades Ribossômicas Menores de Eucariotos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo
7.
Yakugaku Zasshi ; 140(10): 1225-1233, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32999201

RESUMO

This article describes our stereoselective and site-selective chemical methods for exploiting cationic heterocycles as electron-withdrawing groups (EWGs). We envisioned that the phosphoramide N-H proton of a pyridyl phosphoramide 3 would be activated by the cationic pyridinium moiety that is formed upon protonation. The resulting imide-like N-H proton and the acidic pyridinium proton of the pyridinium phosphoramide 3⋅HX cooperate together, making 3⋅HX a highly acidic dual Brønsted acid. The catalytic ability of 3⋅HX was demonstrated in the development of the first asymmetric Diels-Alder reaction between 1-amide dienes and maleimides. Focusing on the activation of N-bromosuccinimide (NBS) because of its structural similarity to maleimides, the enantioselective bromolactonization of trisubstituted olefinic acids was accomplished utilizing pyridyl phosphoramide 3f as a Brønsted base catalyst bearing an acidic N-H proton. Lastly, our strategy for the site-selective acylation of polyol compounds is described. In our system, a pyridine aldoxime ester 10, used as a mild acylating reagent, was activated by a catalytic amount of Lewis acid via the inductive effect of the cationic pyridinium moiety. The resulting metal complex preferentially attracted the alcohol with a Lewis basic site, thereby facilitating selective acylation via a template effect. This metal-template-driven strategy allowed for the site-selective acylation of diverse α-hydroxyamides, including unprotected N-glycolyl aminosugars.


Assuntos
Cátions/química , Cátions/síntese química , Química Orgânica/métodos , Desenvolvimento de Medicamentos/métodos , Elétrons , Compostos Heterocíclicos/química , Compostos Heterocíclicos/síntese química , Acilação , Amidas/química , Catálise , Complexos de Coordenação/química , Reação de Cicloadição , Ésteres/química , Compostos de Pralidoxima/química , Estereoisomerismo
8.
Chin J Physiol ; 63(5): 195-203, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33109785

RESUMO

Cancer cachexia is a wasting syndrome resulting from decreased protein synthesis and increased protein degradation. Calpain-dependent cleavage of myofilament is the initial step of myofilament degradation and plays a critical role in muscle atrophy. Ghrelin is a multifunctional hormone known to improve protein synthesis and inhibit protein degradation. However, its mechanism of action is not fully understood. Here we investigated whether acylated ghrelin (AG) and unacylated ghrelin (UnAG) could protect against cancer cachexia in mice bearing CT26 colorectal adenocarcinoma. We found for the first time that both AG and UnAG could inhibit calpain activity in skeletal muscle of cancer cachectic mice. AG and UnAG also improved tumor-free body weight, grip strength, muscle mass, epididymal fat mass, and nutritional state in tumor-bearing (TB) mice. Moreover, AG and UnAG reduced serum tumor necrosis factor-± concentration, increased Akt activity, and downregulated atrogin-1 expression in TB mice. Our results may contribute to the development of an AG/UnAG-based therapy for cancer cachexia.


Assuntos
Adenocarcinoma/patologia , Caquexia , Neoplasias Colorretais/patologia , Grelina/farmacologia , Acilação , Animais , Caquexia/tratamento farmacológico , Caquexia/etiologia , Camundongos , Músculo Esquelético , Transdução de Sinais
9.
PLoS Pathog ; 16(8): e1008776, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32845938

RESUMO

Enteroaggregative Escherichia coli (EAEC) is a diarrheagenic pathotype associated with traveler's diarrhea, foodborne outbreaks and sporadic diarrhea in industrialized and developing countries. Regulation of virulence in EAEC is mediated by AggR and its negative regulator Aar. Together, they control the expression of at least 210 genes. On the other hand, we observed that about one third of Aar-regulated genes are related to metabolism and transport. In this study we show the AggR/Aar duo controls the metabolism of lipids. Accordingly, we show that AatD, encoded in the AggR-regulated aat operon (aatPABCD) is an N-acyltransferase structurally similar to the essential Apolipoprotein N-acyltransferase Lnt and is required for the acylation of Aap (anti-aggregation protein). Deletion of aatD impairs post-translational modification of Aap and causes its accumulation in the bacterial periplasm. trans-complementation of 042aatD mutant with the AatD homolog of ETEC or with the N-acyltransferase Lnt reestablished translocation of Aap. Site-directed mutagenesis of the E207 residue in the putative acyltransferase catalytic triad disrupted the activity of AatD and caused accumulation of Aap in the periplasm due to reduced translocation of Aap at the bacterial surface. Furthermore, Mass spectroscopy revealed that Aap is acylated in a putative lipobox at the N-terminal of the mature protein, implying that Aap is a lipoprotein. Lastly, deletion of aatD impairs bacterial colonization of the streptomycin-treated mouse model. Our findings unveiled a novel N-acyltransferase family associated with bacterial virulence, and that is tightly regulated by AraC/XylS regulators in the order Enterobacterales.


Assuntos
Acetiltransferases/metabolismo , Fator de Transcrição AraC/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/patogenicidade , Regulação Bacteriana da Expressão Gênica , Acetiltransferases/genética , Acilação , Animais , Fator de Transcrição AraC/química , Fator de Transcrição AraC/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óperon , Filogenia , Conformação Proteica , Virulência
10.
J Oleo Sci ; 69(7): 693-701, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32612019

RESUMO

Fatty acid sugar esters are non-ionic surfactant active agents with excellent performance and many uses. This work is devoted to the synthesis of sugar esters by the esterification reaction of sugar with mixed carboxylicpalmitic anhydrides using resin Amberlyst-15 as heterogeneous acid catalyst. These anhydrides should be stable and react as acylating agents. Influence of different reaction parameters, such as the molar ratio (sucrose/anhydride), the type of solvent and the reaction time on the yield of the esterification reaction were studied. The esterification reaction of sucrose with mixed palmitic benzoic anhydride leads to a mixture of sucrose esters of palmitic acid with a good percentage of conversion. The mixed anhydride was both reactive and selective for the preparation of fatty acid ester.


Assuntos
Benzoatos/química , Ácidos Carboxílicos/química , Técnicas de Química Sintética/métodos , Ésteres/síntese química , Ácidos Graxos/síntese química , Ácido Palmítico/química , Sacarose/síntese química , Acilação , Catálise , Esterificação , Solventes , Estirenos , Tensoativos/síntese química , Fatores de Tempo
11.
Food Chem ; 333: 127439, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32653686

RESUMO

Anthocyanin is derived from a flavylium cation structure, and it promotes health in humans and functions in plants as protection against environmental stress. The rapid analysis of anthocyanin structure and content is a critical challenge for improving fruit quality. In this study, the tomato cultivar Indigo Rose, which is a popular purple cultivated tomato used for breeding, was taken as an example for anthocyanin analysis. A rapid analysis method was developed to minimize anthocyanin loss from the fresh fruit. Four new anthocyanins were discovered in the tomato, and the structures of a total of 12 anthocyanins were determined. Among these, petunidin-3-(trans-p-coumaroyl)-rutinoside-5-glucoside and malvidin-3-(trans-p-coumaroyl)-rutinoside-5-glucoside were the main anthocyanins in Indigo Rose. The structural modifications of these anthocyanins were mainly glycosylation and acylation, and there were also hydroxylation and methylation. Our findings provide new insight into the biosynthesis pathway in tomato fruit.


Assuntos
Antocianinas/análise , Antocianinas/química , Lycopersicon esculentum/química , Espectrometria de Massas/métodos , Acilação , Antocianinas/metabolismo , Cromatografia Líquida de Alta Pressão , Análise de Alimentos/métodos , Congelamento , Frutas/química , Glicosilação , Estrutura Molecular
12.
Sci Rep ; 10(1): 10205, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576842

RESUMO

Serine-based ß-lactamases of Class A, C and D all rely on a key water molecule to hydrolyze and inactivate ß-lactam antibiotics. This process involves two conserved catalytic steps. In the first acylation step, the ß-lactam antibiotic forms an acyl-enzyme intermediate (ES*) with the catalytic serine residue. In the second deacylation step, an activated water molecule serves as nucleophile (WAT_Nu) to attack ES* and release the inactivated ß-lactam. The coordination and activation of WAT_Nu is not fully understood. Using time-resolved x-ray crystallography and QM/MM simulations, we analyzed three intermediate structures of Class A ß-lactamase PenP as it slowly hydrolyzed cephaloridine. WAT_Nu is centrally located in the apo structure but becomes slightly displaced away by ES* in the post-acylation structure. In the deacylation structure, WAT_Nu moves back and is positioned along the Bürgi-Dunitz trajectory with favorable energetic profile to attack ES*. Unexpectedly, WAT_Nu is also found to adopt a catalytically incompetent conformation in the deacylation structure forming a hydrogen bond with ES*. Our results reveal that ES* plays a significant role in coordinating and activating WAT_Nu through subtle yet distinct interactions at different stages of the catalytic process. These interactions may serve as potential targets to circumvent ß-lactamase-mediated antibiotic resistance.


Assuntos
Água/química , Água/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismo , Acilação , Antibacterianos/química , Antibacterianos/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X/métodos , Ligação de Hidrogênio , Hidrólise , Cinética , Serina/química , Serina/metabolismo , beta-Lactamas/química , beta-Lactamas/metabolismo
13.
Enzyme Microb Technol ; 137: 109536, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32423673

RESUMO

N-acylated amino acids are widely used as surfactants and/or actives in cosmetics and household formulations. Their industrial production is based on the use of the Schotten-Baumann chemical and unselective reaction. Faced to the growing demand for greener production processes, selective enzymatic synthesis in more environment-friendly conditions starts to be considered as a potential alternative. This study concerns the use of the aminoacylases from Streptomyces ambofaciens to selectively catalyse aminoacid acylation reaction by fatty acids in aqueous medium. The results demonstrated that, when using undecylenoic acid as acyl donor, these aminoacylases properly catalyse the acylation of 14 of the 20 proteogenic l-amino acids tested on their α amino group with a great variability depending on the nature of the amino acid (polar or not, positively/negatively charged, aromatic or not…). More precisely, the following 9 amino acids were shown to be preferentially acylated by S. ambofaciens aminoacylases as follows: lysine > arginine > leucine > methionine > phenylalanine > valine > cysteine > isoleucine > threonine. Different fatty acids were used as acyl donors and, in most cases, the fatty acid length influenced the conversion yield. The kinetic study of α-lauroy-lysine synthesis showed a positive influence of lysine concentration with Vmax and Km of 3.7 mM/h and 76 mM, respectively. Besides, the lauric acid had an inhibitory effect on the reaction with Ki of 70 mM. The addition of cobalt to the reaction medium led to a more than six-fold increase of the reaction rate. These results, achieved with the aminoacylases from S. ambofaciens represent an improved enzyme-based N-acylated amino acids production in order to provide an alternative way to the Schotten-Baumann chemical reaction currently used in the industry.


Assuntos
Amidoidrolases/metabolismo , Aminoácidos/metabolismo , Biocatálise , Streptomyces/enzimologia , Acilação , Cobalto/metabolismo , Cinética
14.
J Biomed Sci ; 27(1): 57, 2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32349769

RESUMO

O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a type of glycosylation that occurs when a monosaccharide, O-GlcNAc, is added onto serine or threonine residues of nuclear or cytoplasmic proteins by O-GlcNAc transferase (OGT) and which can be reversibly removed by O-GlcNAcase (OGA). O-GlcNAcylation couples the processes of nutrient sensing, metabolism, signal transduction and transcription, and plays important roles in development, normal physiology and physiopathology. Cumulative studies have indicated that O-GlcNAcylation affects the functions of protein substrates in a number of ways, including protein cellular localization, protein stability and protein/protein interaction. Particularly, O-GlcNAcylation has been shown to have intricate crosstalk with phosphorylation as they both modify serine or threonine residues. Aberrant O-GlcNAcylation on various protein substrates has been implicated in many diseases, including neurodegenerative diseases, diabetes and cancers. However, the role of protein O-GlcNAcylation in immune cell lineages has been less explored. This review summarizes the current understanding of the fundamental biochemistry of O-GlcNAcylation, and discusses the molecular mechanisms by which O-GlcNAcylation regulates the development, maturation and functions of immune cells. In brief, O-GlcNAcylation promotes the development, proliferation, and activation of T and B cells. O-GlcNAcylation regulates inflammatory and antiviral responses of macrophages. O-GlcNAcylation promotes the function of activated neutrophils, but inhibits the activity of nature killer cells.


Assuntos
Sistema Imunitário/fisiologia , N-Acetilglucosaminiltransferases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Acilação , Animais , Humanos , Sistema Imunitário/crescimento & desenvolvimento
15.
J Vis Exp ; (158)2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32338654

RESUMO

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty acids via a labile thioester bond. S-acylation, which is emerging as a widespread regulatory mechanism, can modulate almost all aspects of the biological activity of proteins, from complex formation to protein trafficking and protein stability. The recent progress in understanding of the biological function of protein S-acylation was achieved largely due to the development of novel biochemical tools allowing robust and sensitive detection of protein S-acylation in a variety of biological samples. Here, we describe acyl resin-assisted capture (Acyl-RAC), a recently developed method based on selective capture of endogenously S-acylated proteins by thiol-reactive Sepharose beads. Compared to existing approaches, Acyl-RAC requires fewer steps and can yield more reliable results when coupled with mass spectrometry for identification of novel S-acylation targets. A major limitation in this technique is the lack of ability to discriminate between fatty acid species attached to cysteines via the same thioester bond.


Assuntos
Acilação/genética , Proteína S/metabolismo
16.
Sci Rep ; 10(1): 6924, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332789

RESUMO

Depression is a devastating mental disorder affected by multiple factors that can have genetic, environmental, or metabolic causes. Although previous studies have reported an association of dysregulated glucose metabolism with depression, its underlying mechanism remains elusive at the molecular level. A small percentage of glucose is converted into uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) via the hexosamine biosynthetic pathway, which serves as an immediate donor for protein O-GlcNAc modification. O-GlcNAcylation is a particularly common post-translational modification (PTM) in the brain, and the functional significance of O-GlcNAcylation in neurodegenerative diseases has been extensively reported. However, whether the degree of O-GlcNAc modification is associated with depressive disorder has not been examined. In this study, we show that increased O-GlcNAcylation levels reduce inhibitory synaptic transmission in the medial prefrontal cortex (mPFC), and that Oga+/- mice with chronically elevated O-GlcNAcylation levels exhibit an antidepressant-like phenotype. Moreover, we found that virus-mediated expression of OGA in the mPFC restored both antidepressant-like behavior and inhibitory synaptic transmission. Therefore, our results suggest that O-GlcNAc modification in the mPFC plays a significant role in regulating antidepressant-like behavior, highlighting that the modulation of O-GlcNAcylation levels in the brain may serve as a novel therapeutic candidate for antidepressants.


Assuntos
Antidepressivos/farmacologia , Inibição Neural , Córtex Pré-Frontal/fisiopatologia , Acilação , Animais , Comportamento Animal , Glicosilação , Heterozigoto , Potenciais Pós-Sinápticos Inibidores , Camundongos Endogâmicos C57BL , Fenótipo , Transmissão Sináptica
17.
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
18.
Chemistry ; 26(43): 9639-9651, 2020 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-32285965

RESUMO

Disseminating antibiotic resistance rendered by bacteria against the widely used ß-lactam antibiotics is a serious concern for public health care. The development of inhibitors for drug-resistant ß-lactamase enzymes is vital to combat this rapidly escalating problem. Recently, the U.S. Food and Drug Administration approved a non-ß-lactam inhibitor called avibactam for the treatment of complicated intra-abdominal and urinary tract infections caused by drug-resistant Gram-negative bacteria. This work sheds light on the molecular origin of the inhibitory effect of avibactam against the drug-resistant CTX-M variant of class A ß-lactamases. In particular, we probed the structural evolution, dynamics features, and energetics along the acylation and deacylation reaction pathways through enhanced sampling molecular dynamics methods and free-energy calculations. We scrutinized the roles of active site residues, the nature of the carbamoyl linkage formed in the inhibitor-enzyme covalent intermediate, and other structural features of the inhibitor molecule. By unraveling the reasons behind the inhibition of all the deacylation routes, we can explain various experimental structural and kinetics data, and propose a way to design new inhibitors based on the ß-lactam framework.


Assuntos
Antibacterianos/química , Compostos Azabicíclicos/química , Bactérias Gram-Negativas/efeitos dos fármacos , Inibidores de beta-Lactamases/química , beta-Lactamases/química , beta-Lactamas/química , Acilação , Antibacterianos/farmacologia , Domínio Catalítico , Bactérias Gram-Negativas/química , Cinética , Simulação de Dinâmica Molecular , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo , beta-Lactamas/farmacologia
19.
Org Biomol Chem ; 18(12): 2219-2222, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32159577

RESUMO

More than 500 siderophores are known to date, but only three were identified to be aryl-containing hydroxamate siderophores, legonoxamines A and B from Streptomyces sp. MA37, and aryl ferrioxamine 2 from Micrococcus luteus KLE1011. Siderophores are produced by microorganisms to scavenge iron from the environment, thereby making this essential metal nutrient available to the microbe. We demonstrate here that LgoC from MA37 is responsible for the key aryl-hydroxamate forming step in legonoxamine biosynthesis. Biochemical characterization established that LgoC displays considerable promiscuity for the acylation between N-hydroxy-cadaverine and SNAC (N-acetylcysteamines) thioester derivatives.


Assuntos
Coenzima A-Transferases/metabolismo , Sideróforos/metabolismo , Acilação , Proteínas de Bactérias/metabolismo , Ácidos Hidroxâmicos/química , Ferro/metabolismo , Micrococcus luteus/química , Sideróforos/biossíntese , Sideróforos/isolamento & purificação , Streptomyces/química , Streptomyces/enzimologia
20.
J Endocrinol ; 245(2): 327-342, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32176867

RESUMO

Circulating growth hormone (GH) concentrations increase during pregnancy in mice and remain pituitary-derived. Whether abundance or activation of the GH secretagogue ghrelin increase during pregnancy, or in response to dietary octanoic acid supplementation, is unclear. We therefore measured circulating GH profiles in late pregnant C57BL/6J mice and in aged-matched non-pregnant females fed with standard laboratory chow supplemented with 5% octanoic or palmitic (control) acid (n = 4-13/group). Serum total and acyl-ghrelin concentrations, stomach and placenta ghrelin mRNA and protein expression, Pcsk1 (encoding prohormone convertase 1/3) and Mboat4 (membrane bound O-acyl transferase 4) mRNA were determined at zeitgeber (ZT) 13 and ZT23. Total and basal GH secretion were higher in late pregnant than non-pregnant mice (P < 0.001), regardless of diet. At ZT13, serum concentrations of total ghrelin (P = 0.004), but not acyl-ghrelin, and the density of ghrelin-positive cells in the gastric antrum (P = 0.019) were higher, and gastric Mboat4 and Pcsk1 mRNA expression were lower in pregnant than non-pregnant mice at ZT23. In the placenta, ghrelin protein was localised mostly to labyrinthine trophoblast cells. Serum acyl-, but not total, ghrelin was lower at mid-pregnancy than in non-pregnant mice, but not different at early or late pregnancy. In conclusion, dietary supplementation with 5% octanoic acid did not increase activation of ghrelin in female mice. Our results further suggest that increases in maternal GH secretion throughout murine pregnancy are not due to circulating acyl-ghrelin acting at the pituitary. Nevertheless, time-dependent increased circulating total ghrelin could potentially increase ghrelin action in tissues that express the acylating enzyme and receptor.


Assuntos
Caprilatos/farmacologia , Suplementos Nutricionais , Grelina/efeitos dos fármacos , Hormônio do Crescimento/efeitos dos fármacos , Acilação , Animais , Feminino , Mucosa Gástrica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Placenta/metabolismo , Gravidez , RNA Mensageiro/metabolismo
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