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1.
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
2.
J Phys Chem A ; 123(43): 9345-9356, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31580071

RESUMO

Pyruvate formate-lyase (PFL) is a glycyl radical enzyme that converts pyruvate and coenzyme A (CoA) into formate and acetyl-CoA in two half-reactions. Recently, we showed that the acetylation of the PFL active site in the first half-reaction induces subtle conformational changes, leading to the opening of a potential channel for CoA entry. Entry of CoA into the active site is crucial for the second half-reaction, involving the acetyl transfer to CoA, and the completion of the catalytic cycle. Using steered molecular dynamics (SMD) simulations, performed on acetylated and nonacetylated monomeric PFL model systems, we first of all investigate the possible entry/exit pathways of CoA with respect to the active site through the previously identified channel. We then perform umbrella sampling simulations on multiple snapshots from SMD trajectories as well as unrestrained molecular dynamics simulations starting from the final structures obtained from entry SMD, with a view to identifying possible bound states of CoA in the near vicinity of the active site. Detailed study of the unrestrained dissociation processes reveals the presence of stable and reactive bound states of CoA close to the active site, one of which is in an ideal position for triggering the second half-reaction. Examination of the spatial distributions associated with the reactive bound states allows us to discuss the free energy barriers. Umbrella sampling, performed on snapshots from unrestrained dynamics confirms the above findings. The significance of the results for the catalysis are discussed for both acetylated and nonacetylated systems.


Assuntos
Acetiltransferases/metabolismo , Coenzima A/metabolismo , Acetiltransferases/química , Sítios de Ligação , Coenzima A/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica
3.
Int J Mol Sci ; 20(19)2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31561575

RESUMO

Acute kidney injury (AKI) refers to an abrupt decrease in kidney function. It affects approximately 7% of all hospitalized patients and almost 35% of intensive care patients. Mortality from acute kidney injury remains high, particularly in critically ill patients, where it can be more than 50%. The primary causes of AKI include ischemia/reperfusion (I/R), sepsis, or nephrotoxicity; however, AKI patients may present with a complicated etiology where many of the aforementioned conditions co-exist. Multiple bio-markers associated with renal damage, as well as metabolic and signal transduction pathways that are involved in the mediation of renal dysfunction have been identified as a result of the examination of models, patient samples, and clinical data of AKI of disparate etiologies. These discoveries have enhanced our ability to diagnose AKIs and to begin to elucidate the mechanisms involved in their pathogenesis. Studies in our laboratory revealed that the expression and activity of spermine/spermidine N1-acetyltransferase (SAT1), the rate-limiting enzyme in polyamine back conversion, were enhanced in kidneys of rats after I/R injury. Additional studies revealed that the expression of spermine oxidase (SMOX), another critical enzyme in polyamine catabolism, is also elevated in the kidney and other organs subjected to I/R, septic, toxic, and traumatic injuries. The maladaptive role of polyamine catabolism in the mediation of AKI and other injuries has been clearly demonstrated. This review will examine the biochemical and mechanistic basis of tissue damage brought about by enhanced polyamine degradation and discuss the potential of therapeutic interventions that target polyamine catabolic enzymes or their byproducts for the treatment of AKI.


Assuntos
Lesão Renal Aguda/etiologia , Lesão Renal Aguda/metabolismo , Poliaminas/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Biomarcadores , Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Redes e Vias Metabólicas , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo
4.
Oncogene ; 38(41): 6794-6800, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31399646

RESUMO

Spermidine/spermine N1-acetyltransferase 1 (SAT1), the rate-limiting enzyme in polyamine catabolism, has broad regulatory roles due to near ubiquitous polyamine binding. We describe a novel function of SAT1 as a gene-specific transcriptional regulator through local polyamine acetylation. SAT1 expression is elevated in aggressive brain tumors and promotes resistance to radiotherapy. Expression profiling in glioma cells identified SAT1 target genes that distinguish high- and low-grade tumors, in support of the prognostic utility of SAT1 expression. We further discovered mechanisms of SAT1-driven tumor aggressiveness through promotion of expression of both DNA damage response pathways as well as cell cycle regulatory genes. Mechanistically, SAT1 associates specifically with the promoter of the MELK gene, which functionally controls other SAT1 targets, and leads biologically to maintenance of neurosphere stemness in conjunction with FOXM1 and EZH2. CRISPR knockin mutants demonstrate the essentiality of the polyamine acetyltransferase activity of SAT1 for its function as a transcriptional regulator. Together, the data demonstrate that gene-specific polyamine removal is a major transcriptional regulatory mechanism active in high-grade gliomas that drives poor outcomes.


Assuntos
Neoplasias Encefálicas/patologia , Glioma/patologia , Transcrição Genética , Acetiltransferases/genética , Acetiltransferases/metabolismo , Neoplasias Encefálicas/enzimologia , Cromatina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Glioma/enzimologia , Humanos , Células-Tronco Neoplásicas/patologia , Proteínas Serina-Treonina Quinases/metabolismo
5.
Environ Mol Mutagen ; 60(9): 792-806, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31374128

RESUMO

Aristolochic acids (AAs) are human nephrotoxins and carcinogens found in concoctions of Aristolochia plants used in traditional medicinal practices worldwide. Genotoxicity of AAs is associated with the formation of active species catalyzed by metabolic enzymes, the full repertoire of which is unknown. Recently, we provided evidence that sulfonation is important for bioactivation of AAs. Here, we employ Salmonella typhimurium umu tester strains expressing human N-acetyltransferases (NATs) and sulfotransferases (SULTs), to study the role of conjugation reactions in the genotoxicities of N-hydroxyaristolactams (AL-I-NOH and AL-II-NOH), metabolites of AA-I and AA-II. Both N-hydroxyaristolactams show stronger genotoxic effects in umu strains expressing human NAT1 and NAT2, than in the parent strain. Additionally, AL-I-NOH displays increased genotoxicity in strains expressing human SULT1A1 and SULT1A2, whereas AL-II-NOH shows enhanced genotoxicity in SULT1A1/2 and SULT1A3 strains. 2,6-Dichloro-4-nitrophenol, SULTs inhibitor, reduced umuC gene expression induced by N-hydroxyaristolactams in SULT1A2 strain. N-hydroxyaristolactams are also mutagenic in parent strains, suggesting that an additional mechanism(s) may contribute to their genotoxicities. Accordingly, using putative SULT substrates and inhibitors, we found that cytosols obtained from human kidney HK-2 cells activate N-hydroxyaristolactams in aristolactam-DNA adducts with the limited involvement of SULTs. Removal of low-molecular-weight reactants in the 3.5-10 kDa range inhibits the formation of aristolactam-DNA by 500-fold, which could not be prevented by the addition of cofactors for SULTs and NATs. In conclusion, our results demonstrate that the genotoxicities of N-hydroxyaristolactams depend on the cell type and involve not only sulfonation but also N,O-acetyltransfer and an additional yet unknown mechanism(s). Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.


Assuntos
Ácidos Aristolóquicos/metabolismo , Ácidos Aristolóquicos/toxicidade , Acetiltransferases/metabolismo , Arilamina N-Acetiltransferase/metabolismo , Arilsulfotransferase/metabolismo , Carcinógenos/toxicidade , Linhagem Celular , DNA/efeitos dos fármacos , Adutos de DNA/genética , Humanos , Mutagênicos/toxicidade , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/metabolismo , Sulfotransferases/metabolismo
6.
J Agric Food Chem ; 67(35): 9851-9857, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31418561

RESUMO

Arachidonic acid (ARA, C20:4) is a typical ω-6 polyunsaturated fatty acid with special functions. Using Yarrowia lipolytica as an unconventional chassis, we previously showed the performance of the Δ-6 pathway in ARA production. However, a significant increase in the Δ-9 pathway has rarely been reported. Herein, the Δ-9 pathway from Isochrysis galbana was constructed via pathway engineering, allowing us to synthesize ARA at 91.5 mg L-1. To further improve the ARA titer, novel enzyme fusions of Δ-9 elongase and Δ-8 desaturase were redesigned in special combinations containing different linkers. Finally, with the integrated pathway engineering and synthetic enzyme fusion, a 29% increase in the ARA titer, up to 118.1 mg/L, was achieved using the reconstructed strain RH-4 that harbors the rigid linker (GGGGS). The results show that the combined pathway and protein engineering can significantly facilitate applications of Y. lipolytica.


Assuntos
Ácido Araquidônico/biossíntese , Engenharia Metabólica , Yarrowia/genética , Yarrowia/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Glucose/metabolismo , Haptófitas/enzimologia
7.
EMBO J ; 38(13): e101414, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31268607

RESUMO

The movement of a molecular motor protein along a cytoskeletal track requires communication between enzymatic, polymer-binding, and mechanical elements. Such communication is particularly complex and not well understood in the dynein motor, an ATPase that is comprised of a ring of six AAA domains, a large mechanical element (linker) spanning over the ring, and a microtubule-binding domain (MTBD) that is separated from the AAA ring by a ~ 135 Å coiled-coil stalk. We identified mutations in the stalk that disrupt directional motion, have microtubule-independent hyperactive ATPase activity, and nucleotide-independent low affinity for microtubules. Cryo-electron microscopy structures of a mutant that uncouples ATPase activity from directional movement reveal that nucleotide-dependent conformational changes occur normally in one-half of the AAA ring, but are disrupted in the other half. The large-scale linker conformational change observed in the wild-type protein is also inhibited, revealing that this conformational change is not required for ATP hydrolysis. These results demonstrate an essential role of the stalk in regulating motor activity and coupling conformational changes across the two halves of the AAA ring.


Assuntos
Acetiltransferases/química , Acetiltransferases/metabolismo , Microtúbulos/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferases/genética , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Nucleic Acids Res ; 47(16): 8439-8451, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31260531

RESUMO

In fission yeast, MBF-dependent transcription is inactivated at the end of S phase through a negative feedback loop that involves the co-repressors, Yox1 and Nrm1. Although this repression system is well known, the molecular mechanisms involved in MBF activation remain largely unknown. Compacted chromatin constitutes a barrier to activators accessing promoters. Here, we show that chromatin regulation plays a key role in activating MBF-dependent transcription. Gcn5, a part of the SAGA complex, binds to MBF-regulated promoters through the MBF co-activator Rep2 in a cell cycle-dependent manner and in a reverse correlation to the binding of the MBF co-repressors, Nrm1 or Yox1. We propose that the co-repressors function as physical barriers to SAGA recruitment onto MBF promoters. We also show that Gcn5 acetylates specific lysine residues on histone H3 in a cell cycle-regulated manner. Furthermore, either in a gcn5 mutant or in a strain in which histone H3 is kept in an unacetylated form, MBF-dependent transcription is downregulated. In summary, Gcn5 is required for the full activation and correct timing of MBF-regulated gene transcription.


Assuntos
Acetiltransferases/genética , Proteínas de Ciclo Celular/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Transativadores/genética , Fatores de Transcrição/genética , Transcrição Genética , Acetilação , Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Retroalimentação Fisiológica , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Tempo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo
9.
Oxid Med Cell Longev ; 2019: 7561879, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354912

RESUMO

Background: N-Acetyltransferase 10 (NAT10) has been reported to be expressed at high levels in hepatocellular carcinoma (HCC); however, its role in chemoresistance is unclear. This study is aimed at investigating whether NAT10 regulates the epithelial-mesenchymal transition (EMT) and chemoresistance in HCC. Methods: HCC cell lines (Huh-7, Bel-7402, SNU387, and SNU449) were treated with remodelin, an inhibitor of NAT10, or transfected with small inhibitory RNAs (siRNAs) targeting NAT10 or Twist. The EMT was induced by hypoxia. The CCK-8 assay was used to quantify cell viability, the EdU incorporation assay to assess cell proliferation. siRNA knockdown efficiency and epithelial/mesenchymal marker expression were assessed by western blotting. Results: Knockdown of NAT10 using siRNA or inhibition of NAT10 using remodelin increased the sensitivity of HCC cell lines to doxorubicin; similar effects were observed in cells transfected with the Twist siRNA. Inhibition of NAT10 using remodelin also reversed the ability of doxorubicin to induce the EMT in HCC cells. Furthermore, inhibiting NAT10 reversed the hypoxia-induced EMT. Finally, we confirmed that combining doxorubicin with remodelin delayed tumor growth and reduced tumor cell proliferation in a mouse xenograft model of HCC. Conclusions: NAT10 may contribute to chemoresistance in HCC by regulating the EMT. The mechanism by which NAT10 regulates the EMT and doxorubicin sensitivity in HCC cells merits further investigation.


Assuntos
Acetiltransferases/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Doxorrubicina/uso terapêutico , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Transição Epitelial-Mesenquimal , Humanos , Transfecção
10.
Int J Mol Sci ; 20(12)2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-31207892

RESUMO

AIM: Primary human hepatocytes (PHHs) undergo dedifferentiation upon the two-dimensional (2D) culture, which particularly hinders their utility in long-term in vitro studies. Lipids, as a major class of biomolecules, play crucial roles in cellular energy storage, structure, and signaling. Here, for the first time, we mapped the alterations in the lipid profile of the dedifferentiating PHHs and studied the possible role of lipids in the loss of the phenotype of PHHs. Simultaneously, differentially expressed miRNAs associated with changes in the lipids and fatty acids (FAs) of the dedifferentiating PHHs were investigated. METHODS: PHHs were cultured in monolayer and their phenotype was monitored morphologically, genetically, and biochemically for five days. The lipid and miRNA profile of the PHHs were analyzed by mass spectrometry and Agilent microarray, respectively. In addition, 24 key genes involved in the metabolism of lipids and FAs were investigated by qPCR. RESULTS: The typical morphology of PHHs was lost from day 3 onward. Additionally, ALB and CYP genes were downregulated in the cultured PHHs. Lipidomics revealed a clear increase in the saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) containing lipids, but a decrease in the polyunsaturated fatty acids (PUFA) containing lipids during the dedifferentiation of PHHs. In line with this, FASN, SCD, ELOVL1, ELOVL3, and ELOVL7 were upregulated but ELOVL2 was downregulated in the dedifferentiated PHHs. Furthermore, differentially expressed miRNAs were identified, and the constantly upregulated miR-27a and miR-21, and downregulated miR-30 may have regulated the synthesis, accumulation and secretion of PHH lipids during the dedifferentiation. CONCLUSION: Our results showed major alterations in the molecular lipid species profiles, lipid-metabolizing enzyme expression as wells as miRNA profiles of the PHHs during their prolonged culture, which in concert could play important roles in the PHHs' loss of phenotype. These findings promote the understanding from the dedifferentiation process and could help in developing optimal culture conditions, which better meet the needs of the PHHs and support their original phenotype.


Assuntos
Desdiferenciação Celular , Hepatócitos/citologia , Metabolismo dos Lipídeos , MicroRNAs/genética , Acetiltransferases/genética , Acetiltransferases/metabolismo , Células Cultivadas , Citocromos/genética , Citocromos/metabolismo , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Hepatócitos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Albumina Sérica Humana/genética , Albumina Sérica Humana/metabolismo , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Regulação para Cima
11.
BMC Med Genet ; 20(1): 101, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31174490

RESUMO

BACKGROUND: N-terminal acetylation is a common protein modification in human cells and is catalysed by N-terminal acetyltransferases (NATs), mostly cotranslationally. The NAA10-NAA15 (NatA) protein complex is the major NAT, responsible for acetylating ~ 40% of human proteins. Recently, NAA10 germline variants were found in patients with the X-linked lethal Ogden syndrome, and in other familial or de novo cases with variable degrees of developmental delay, intellectual disability (ID) and cardiac anomalies. METHODS: Here we report a novel NAA10 (NM_003491.3) c.248G > A, p.(R83H) missense variant in NAA10 which was detected by whole exome sequencing in two unrelated boys with intellectual disability, developmental delay, ADHD like behaviour, very limited speech and cardiac abnormalities. We employ in vitro acetylation assays to functionally test the impact of this variant on NAA10 enzyme activity. RESULTS: Functional characterization of NAA10-R83H by in vitro acetylation assays revealed a reduced enzymatic activity of monomeric NAA10-R83H. This variant is modelled to have an altered charge density in the acetyl-coenzyme A (Ac-CoA) binding region of NAA10. CONCLUSIONS: We show that NAA10-R83H has a reduced monomeric catalytic activity, likely due to impaired enzyme-Ac-CoA binding. Our data support a model where reduced NAA10 and/or NatA activity cause the phenotypes observed in the two patients.


Assuntos
Acetiltransferases/genética , Deficiência Intelectual/genética , Microcefalia/genética , Mutação de Sentido Incorreto , Acetiltransferase N-Terminal A/genética , Acetiltransferase N-Terminal E/genética , Acetilação , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Pré-Escolar , Humanos , Lactente , Masculino , Modelos Moleculares , Acetiltransferase N-Terminal A/química , Acetiltransferase N-Terminal A/metabolismo , Acetiltransferase N-Terminal E/química , Acetiltransferase N-Terminal E/metabolismo , Fenótipo , Domínios Proteicos , Homologia de Sequência de Aminoácidos , Sequenciamento Completo do Exoma
12.
Methods Mol Biol ; 1983: 107-130, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31087295

RESUMO

Posttranslational acetylation modifications of proteins have important consequences for cell biology, including effects on protein trafficking and cellular localization as well as on the interactions of acetylated proteins with other proteins and macromolecules such as DNA. Experiments to uncover and characterize protein acetylation events have historically been more challenging than investigating another common posttranslational modification, protein phosphorylation. More recently, high-quality antibodies that recognize acetylated lysine residues present in acetylated proteins and improved proteomic methodologies have facilitated the discovery that acetylation occurs on numerous cellular proteins and allowed characterization of the dynamics and functional effects of many acetylation events. This article summarizes some established biochemical information about how protein acetylation takes place and is regulated, in order to lay the foundation for subsequent descriptions of strategies used by our lab and others either to directly study acetylation of an individual factor or to identify groups of proteins targeted for acetylation that can then be examined in isolation.


Assuntos
Lisina/metabolismo , Proteínas/metabolismo , Acetilação , Acetiltransferases/metabolismo , Bioensaio/métodos , Histona Desacetilases/metabolismo , Humanos , Lisina/química , Lisina Acetiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteoma , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos
13.
Plant Cell Physiol ; 60(8): 1666-1682, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31058972

RESUMO

We investigated potential biosynthetic pathways of long chain alkenols (LCAs), long chain alkyl diols (LCDs), and long chain hydroxy fatty acids (LCHFAs) in Nannochloropsis oceanica and Nannochloropsis gaditana, by combining culturing experiments with genomic and transcriptomic analyses. Incubation of Nannochloropsis spp. in the dark for 1 week led to significant increases in the cellular concentrations of LCAs and LCDs in both species. Consistently, 13C-labelled substrate experiments confirmed that both LCA and LCD were actively produced in the dark from C14-18 fatty acids by either condensation or elongation/hydroxylation, although no enzymatic evidence was found for the former pathway. Nannochloropsis spp. did, however, contain (i) multiple polyketide synthases (PKSs) including one type (PKS-Clade II) that might catalyze incomplete fatty acid elongations leading to the formation of 3-OH-fatty acids, (ii) 3-hydroxyacyl dehydratases (HADs), which can possibly form Δ2/Δ3 monounsaturated fatty acids, and (iii) fatty acid elongases (FAEs) that could elongate 3-OH-fatty acids and Δ2/Δ3 monounsaturated fatty acids to longer products. The enzymes responsible for reduction of the long chain fatty acids to LCDs and LCAs are, however, unclear. A putative wax ester synthase/acyl coenzyme A (acyl-CoA): diacylglycerol acyltransferase is likely to be involved in the esterification of LCAs and LCDs in the cell wall. Our data thus provide useful insights in predicting the biosynthetic pathways of LCAs and LCDs in phytoplankton suggesting a key role of FAE and PKS enzymes.


Assuntos
Álcoois/metabolismo , Alcenos/metabolismo , Policetídeo Sintases/metabolismo , Acetiltransferases/metabolismo , Álcoois/química , Alcenos/química , Enoil-CoA Hidratase/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Microalgas/enzimologia , Microalgas/metabolismo , Especificidade por Substrato
14.
Nat Struct Mol Biol ; 26(4): 308-314, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30936526

RESUMO

Phages use anti-CRISPR proteins to deactivate the CRISPR-Cas system. The mechanisms for the inhibition of type I and type II systems by anti-CRISPRs have been elucidated. However, it has remained unknown how the type V CRISPR-Cas12a (Cpf1) system is inhibited by anti-CRISPRs. Here we identify the anti-CRISPR protein AcrVA5 and report the mechanisms by which it inhibits CRISPR-Cas12a. Our structural and biochemical data show that AcrVA5 functions as an acetyltransferase to modify Moraxella bovoculi (Mb) Cas12a at Lys635, a residue that is required for recognition of the protospacer-adjacent motif. The AcrVA5-mediated modification of MbCas12a results in complete loss of double-stranded DNA (dsDNA)-cleavage activity. In contrast, the Lys635Arg mutation renders MbCas12a completely insensitive to inhibition by AcrVA5. A cryo-EM structure of the AcrVA5-acetylated MbCas12a reveals that Lys635 acetylation provides sufficient steric hindrance to prevent dsDNA substrates from binding to the Cas protein. Our study reveals an unprecedented mechanism of CRISPR-Cas inhibition and suggests an evolutionary arms race between phages and bacteria.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/fisiologia , Moraxella/metabolismo , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Moraxella/genética , Mutação/genética
15.
Res Microbiol ; 170(4-5): 171-181, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30953691

RESUMO

Living organisms have developed specific defence mechanisms to counteract hostile environmental conditions. Alkylation stress response mechanisms also occur in Mycobacterium tuberculosis (MTB) the pathogen responsible for tuberculosis. The effect of alkylating agents on the cellular growth of MTB was investigated using methyl methanesulfonate (MMS) as methyl donor demonstrating that limited doses of alkylating agents might affect MTB cell viability. A global investigation of Mycobacterium smegmatis response to alkylating stress was then pursued by differential proteomics to identify the most affected cellular pathways. Quantitative analysis of proteomic profiles demonstrated that most of the proteins upregulated in the presence of alkylating agents are involved in biofilm formation and/or cell wall biosynthesis. Tailored experiments confirmed that under stress conditions M. smegmatis elicits physical defence mechanisms by increasing biofilm formation. Among the upregulated proteins, we identified the GlmU bifunctional enzyme as a possible factor involved in biofilm production. Experiments with both conditional deletion and overexpressing glmU mutants demonstrated that down regulation of GlmU decreased M. smegmatis capabilities to produce biofilm whereas overexpression of the enzyme increased biofilm formation. These results were supported by inhibition of GlmU acetyltransferase activity with two different inhibitors, suggesting the involvement of this enzyme in the M. smegmatis defence mechanisms.


Assuntos
Acetiltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Metanossulfonato de Metila/farmacologia , Complexos Multienzimáticos/metabolismo , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium tuberculosis/crescimento & desenvolvimento , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/genética , Alquilação , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Perfilação da Expressão Gênica , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/genética , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Ácido N-Acetilneuramínico/metabolismo , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo
16.
Int J Vitam Nutr Res ; 89(1-2): 62-72, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30957704

RESUMO

The liver is the main site of lipid metabolism and vitamin A storage. Dietary factors are known to affect liver function, thereby leading to metabolic abnormalities. Here, we assessed the impact of long-term feeding of a high-fat diet on hepatic vitamin A status and lipid metabolism. For this purpose, 14 male and 14 female 35-day-old mice (strain C57BL/6J) were each divided into 2 groups of 7 animals and fed either a stock diet or a high-fat (HF) diet for 26 weeks. In addition to increased body weight/weight gain, the HF diet induced hypertriglyceridemia in both (p < 0.01). However, liver triglyceride levels were comparable among groups, which could be partly explained by unaltered expression of various lipogenic pathway proteins such as sterol regulatory element binding protein 1 (SREBP1), fatty acid synthase (FAS), microsomal triglyceride transfer protein (MTTP), and glycerol 3-phosphate acyl transferase (GPAT). On the other hand, hepatic retinol stores increased significantly in both sexes, whereas males displayed elevated circulatory retinol levels. Notably, long-term feeding of a HF diet elevated n-3 polyunsaturated fatty acid (PUFA) and docosahexaenoic acid (DHA, C22:6) levels in the liver (p ≤ 0.001), which is in line with the over-expression of very long-chain fatty acid elongase 2 (ELOVL2) protein in both sexes of mice (p < 0.01). In conclusion, very long-term feeding of a HF diet increased hepatic retinol stores and induced hypertriglyceridemia. However, it had no effect on hepatic triglyceride accumulation, possibly due to increased DHA levels arising from the ELOVL2-mediated elongation pathway.


Assuntos
Acetiltransferases/metabolismo , Dieta Hiperlipídica , Ácidos Docosa-Hexaenoicos , Fígado/fisiologia , Acetiltransferases/química , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL
17.
Nat Commun ; 10(1): 1609, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30962433

RESUMO

Bacterial degradation of organosulfonates plays an important role in sulfur recycling, and has been extensively studied. However, this process in anaerobic bacteria especially gut bacteria is little known despite of its potential significant impact on human health with the production of toxic H2S. Here, we describe the structural and biochemical characterization of an oxygen-sensitive enzyme that catalyzes the radical-mediated C-S bond cleavage of isethionate to form sulfite and acetaldehyde. We demonstrate its involvement in pathways that enables C2 sulfonates to be used as terminal electron acceptors for anaerobic respiration in sulfate- and sulfite-reducing bacteria. Furthermore, it plays a key role in converting bile salt-derived taurine into H2S in the disease-associated gut bacterium Bilophila wadsworthia. The enzymes and transporters in these anaerobic pathways expand our understanding of microbial sulfur metabolism, and help deciphering the complex web of microbial pathways involved in the transformation of sulfur compounds in the gut.


Assuntos
Acetiltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Desulfovibrio/metabolismo , Sulfeto de Hidrogênio/toxicidade , Taurina/metabolismo , Acetiltransferases/genética , Acetiltransferases/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Ácidos e Sais Biliares/metabolismo , Bilophila/metabolismo , Ensaios Enzimáticos , Microbioma Gastrointestinal/fisiologia , Sulfeto de Hidrogênio/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mercaptoetanol/análogos & derivados , Mercaptoetanol/metabolismo , Redes e Vias Metabólicas/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Enxofre/metabolismo
18.
J Ind Microbiol Biotechnol ; 46(7): 1039-1045, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30963326

RESUMO

N-Acetyltransferase Mpr1 was originally discovered as an enzyme that detoxifies L-azetidine-2-carboxylate through its N-acetylation in the yeast Saccharomyces cerevisiae Σ1278b. Mpr1 protects yeast cells from oxidative stresses possibly by activating a novel L-arginine biosynthesis. We recently constructed a stable variant of Mpr1 (N203K) by a rational design based on the structure of the wild-type Mpr1 (WT). Here, we examined the effects of N203K on ethanol fermentation of the sake yeast S. cerevisiae strain lacking the MPR1 gene. When N203K was expressed in the diploid Japanese sake strain, its fermentation performance was improved compared to WT. In a laboratory-scale brewing, a sake strain expressing N203K produced more ethanol than WT. N203K also affected the contents of flavor compounds and organic acids. These results suggest that the stable Mpr1 variant contributes to the construction of new industrial yeast strains with improved fermentation ability and diversity of taste and flavor.


Assuntos
Acetiltransferases/metabolismo , Etanol/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferases/genética , Fermentação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
19.
Nutrients ; 11(4)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991731

RESUMO

The omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) is implicated in theregulation of both lipid and carbohydrate metabolism. Thus, we questioned whether dietary DHAand low or high content of sucrose impact on metabolism in mice deficient for elongation of verylong-chain fatty acids 2 (ELOVL2), an enzyme involved in the endogenous DHA synthesis. Wefound that Elovl2 -/- mice fed a high-sucrose DHA-enriched diet followed by the high sucrose, highfat challenge significantly increased body weight. This diet affected the triglyceride rich lipoproteinfraction of plasma lipoproteins and changed the expression of several genes involved in lipidmetabolism in a white adipose tissue. Our findings suggest that lipogenesis in mammals issynergistically influenced by DHA dietary and sucrose content.


Assuntos
Tecido Adiposo Branco/efeitos dos fármacos , Sacarose na Dieta/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Lipogênese/efeitos dos fármacos , Ganho de Peso/efeitos dos fármacos , Acetiltransferases/genética , Acetiltransferases/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Dieta Hiperlipídica , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/sangue , Ácidos Docosa-Hexaenoicos/deficiência , Lipogênese/genética , Lipoproteínas/sangue , Camundongos Knockout , Triglicerídeos/sangue
20.
Int J Mol Sci ; 20(7)2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30979053

RESUMO

Elongation of very long chain fatty acids protein 6 (Elovl6) is a key enzyme in fatty acid synthesis, which participates in converting palmitate (C16:0) to stearate (C18:0). Although studies of Elovl6 have been carried out in mammals, the nutritional regulation of elovl6 in fish remains poorly understood. In the present study, the cloning and nutritional regulation of elovl6 were determined in large yellow croaker. Sequence and phylogenetic analysis revealed that the full-length cDNA of elovl6 was 1360 bp, including an open reading frame of 810 bp encoding a putative protein of 269 amino acid that possesses the characteristic features of Elovl proteins. The transcript level of elovl6 was significantly increased in the liver of croaker fed the diets with soybean oil (enriched with 18: 2n-6, LA) or linseed oil (enriched with 18: 3n-3, ALA) than that in croaker fed the diet with fish oil (enriched with 20: 5n-3 and 22: 6n-3). Correspondingly, the elovl6 expression in croaker's hepatocytes treated with ALA or LA was remarkably increased compared to the controls. Furthermore, the transcription factors including hepatocyte nuclear factor 1α (HNF1α), CCAAT-enhancer-binding protein ß (CEBPß), retinoid X receptor α (RXRα), and cAMP response element-binding protein 1 (CREB1) greatly enhanced promoter activity of elovl6 in large yellow croaker, and the expression of transcription factors is consistent with the changes of elovl6 expression in response to fatty acids in vivo and in vitro. In conclusion, this study revealed that elovl6 expression in large yellow croaker could be upregulated by dietary ALA or LA via the increased transcriptional expression of transcription factors including hnf1α, cebpß, rxrα, and creb1.


Assuntos
Acetiltransferases/genética , Gorduras na Dieta/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Peixes/genética , Perciformes/genética , Ativação Transcricional , Acetiltransferases/química , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Sequência de Bases , Clonagem Molecular , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Óleo de Semente do Linho/metabolismo , Fígado/fisiologia , Perciformes/fisiologia , Filogenia , Óleo de Soja/metabolismo
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