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1.
Nat Commun ; 12(1): 482, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33473124

RESUMO

DNA ligase 1 (LIG1, Cdc9 in yeast) finalizes eukaryotic nuclear DNA replication by sealing Okazaki fragments using DNA end-joining reactions that strongly discriminate against incorrectly paired DNA substrates. Whether intrinsic ligation fidelity contributes to the accuracy of replication of the nuclear genome is unknown. Here, we show that an engineered low-fidelity LIG1Cdc9 variant confers a novel mutator phenotype in yeast typified by the accumulation of single base insertion mutations in homonucleotide runs. The rate at which these additions are generated increases upon concomitant inactivation of DNA mismatch repair, or by inactivation of the Fen1Rad27 Okazaki fragment maturation (OFM) nuclease. Biochemical and structural data establish that LIG1Cdc9 normally avoids erroneous ligation of DNA polymerase slippage products, and this protection is compromised by mutation of a LIG1Cdc9 high-fidelity metal binding site. Collectively, our data indicate that high-fidelity DNA ligation is required to prevent insertion mutations, and that this may be particularly critical following strand displacement synthesis during the completion of OFM.


Assuntos
Replicação do DNA/fisiologia , DNA Fúngico/metabolismo , DNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferases/metabolismo , DNA Ligase Dependente de ATP/metabolismo , DNA Ligases , Reparo de Erro de Pareamento de DNA/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Endonucleases Flap/metabolismo , Proteínas de Membrana/metabolismo , Mutagênese , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
PLoS Comput Biol ; 16(12): e1007988, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33362253

RESUMO

The enzymes of the GCN5-related N-acetyltransferase (GNAT) superfamily count more than 870 000 members through all kingdoms of life and share the same structural fold. GNAT enzymes transfer an acyl moiety from acyl coenzyme A to a wide range of substrates including aminoglycosides, serotonin, glucosamine-6-phosphate, protein N-termini and lysine residues of histones and other proteins. The GNAT subtype of protein N-terminal acetyltransferases (NATs) alone targets a majority of all eukaryotic proteins stressing the omnipresence of the GNAT enzymes. Despite the highly conserved GNAT fold, sequence similarity is quite low between members of this superfamily even when substrates are similar. Furthermore, this superfamily is phylogenetically not well characterized. Thus functional annotation based on sequence similarity is unreliable and strongly hampered for thousands of GNAT members that remain biochemically uncharacterized. Here we used sequence similarity networks to map the sequence space and propose a new classification for eukaryotic GNAT acetyltransferases. Using the new classification, we built a phylogenetic tree, representing the entire GNAT acetyltransferase superfamily. Our results show that protein NATs have evolved more than once on the GNAT acetylation scaffold. We use our classification to predict the function of uncharacterized sequences and verify by in vitro protein assays that two fungal genes encode NAT enzymes targeting specific protein N-terminal sequences, showing that even slight changes on the GNAT fold can lead to change in substrate specificity. In addition to providing a new map of the relationship between eukaryotic acetyltransferases the classification proposed constitutes a tool to improve functional annotation of GNAT acetyltransferases.


Assuntos
Acetiltransferases/classificação , Anotação de Sequência Molecular , Filogenia , Acetiltransferases/química , Acetiltransferases/genética , Acetiltransferases/metabolismo , Catálise , Cristalografia por Raios X , Conformação Proteica , Especificidade por Substrato
3.
Int J Mol Sci ; 22(1)2020 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-33374407

RESUMO

Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in Escherichia coli at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.


Assuntos
Adjuvantes Imunológicos/farmacocinética , Timalfasina/farmacocinética , Acetilação , Acetiltransferases/metabolismo , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/farmacologia , Animais , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Feminino , Meia-Vida , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Neoplasias/tratamento farmacológico , Peptídeos/química , Proteólise , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/ultraestrutura , Proteínas Ribossômicas/metabolismo , Timalfasina/sangue , Timalfasina/química , Timalfasina/genética , Viroses/tratamento farmacológico
4.
PLoS Genet ; 16(12): e1009219, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33382686

RESUMO

Roberts syndrome (RBS) is a rare developmental disorder that can include craniofacial abnormalities, limb malformations, missing digits, intellectual disabilities, stillbirth, and early mortality. The genetic basis for RBS is linked to autosomal recessive loss-of-function mutation of the establishment of cohesion (ESCO) 2 acetyltransferase. ESCO2 is an essential gene that targets the DNA-binding cohesin complex. ESCO2 acetylates alternate subunits of cohesin to orchestrate vital cellular processes that include sister chromatid cohesion, chromosome condensation, transcription, and DNA repair. Although significant advances were made over the last 20 years in our understanding of ESCO2 and cohesin biology, the molecular etiology of RBS remains ambiguous. In this review, we highlight current models of RBS and reflect on data that suggests a novel role for macromolecular damage in the molecular etiology of RBS.


Assuntos
Acetiltransferases/genética , Proteínas Cromossômicas não Histona/genética , Anormalidades Craniofaciais/genética , Dano ao DNA , Ectromelia/genética , Hipertelorismo/genética , Acetiltransferases/metabolismo , Animais , Proteínas Cromossômicas não Histona/metabolismo , Anormalidades Craniofaciais/metabolismo , Ectromelia/metabolismo , Instabilidade Genômica , Humanos , Hipertelorismo/metabolismo
5.
Nat Commun ; 11(1): 5438, 2020 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-33116145

RESUMO

Toxin-antitoxin systems in bacteria contribute to stress adaptation, dormancy, and persistence. AtaT, a type-II toxin in enterohemorrhagic E. coli, reportedly acetylates the α-amino group of the aminoacyl-moiety of initiator Met-tRNAfMet, thus inhibiting translation initiation. Here, we show that AtaT has a broader specificity for aminoacyl-tRNAs than initially claimed. AtaT efficiently acetylates Gly-tRNAGly, Trp-tRNATrp, Tyr-tRNATyr and Phe-tRNAPhe isoacceptors, in addition to Met-tRNAfMet, and inhibits global translation. AtaT interacts with the acceptor stem of tRNAfMet, and the consecutive G-C pairs in the bottom-half of the acceptor stem are required for acetylation. Consistently, tRNAGly, tRNATrp, tRNATyr and tRNAPhe also possess consecutive G-C base-pairs in the bottom halves of their acceptor stems. Furthermore, misaminoacylated valyl-tRNAfMet and isoleucyl-tRNAfMet are not acetylated by AtaT. Therefore, the substrate selection by AtaT is governed by the specific acceptor stem sequence and the properties of the aminoacyl-moiety of aminoacyl-tRNAs.


Assuntos
Acetiltransferases/metabolismo , Escherichia coli Êntero-Hemorrágica/metabolismo , Proteínas de Escherichia coli/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Aminoacilação de RNA de Transferência , Acetilação , Acetiltransferases/química , Acetiltransferases/genética , Sequência de Bases , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli Êntero-Hemorrágica/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/genética
6.
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
7.
Nucleic Acids Res ; 48(16): 8977-8992, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32710633

RESUMO

The protein kinase Gcn2 is a central transducer of nutritional stress signaling important for stress adaptation by normal cells and the survival of cancer cells. In response to nutrient deprivation, Gcn2 phosphorylates eIF2α, thereby repressing general translation while enhancing translation of specific mRNAs with upstream ORFs (uORFs) situated in their 5'-leader regions. Here we performed genome-wide measurements of mRNA translation during histidine starvation in fission yeast Schizosaccharomyces pombe. Polysome analyses were combined with microarray measurements to identify gene transcripts whose translation was up-regulated in response to the stress in a Gcn2-dependent manner. We determined that translation is reprogrammed to enhance RNA metabolism and chromatin regulation and repress ribosome synthesis. Interestingly, translation of intron-containing mRNAs was up-regulated. The products of the regulated genes include additional eIF2α kinase Hri2 amplifying the stress signaling and Gcn5 histone acetyl transferase and transcription factors, together altering genome-wide transcription. Unique dipeptide-coding uORFs and nucleotide motifs, such as '5'-UGA(C/G)GG-3', are found in 5' leader regions of regulated genes and shown to be responsible for translational control.


Assuntos
Motivos de Nucleotídeos , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Acetiltransferases/metabolismo , Regulação Fúngica da Expressão Gênica , Histidina/metabolismo , Fases de Leitura Aberta , Processamento de Proteína Pós-Traducional , Schizosaccharomyces/genética , eIF-2 Quinase/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(27): 15895-15901, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571932

RESUMO

In eukaryotic cells, the N-terminal amino moiety of many proteins is modified by N-acetyltransferases (NATs). This protein modification can alter the folding of the target protein; can affect binding interactions of the target protein with substrates, allosteric effectors, or other proteins; or can trigger protein degradation. In prokaryotes, only ribosomal proteins are known to be N-terminally acetylated, and the acetyltransferases responsible for this modification belong to the Rim family of proteins. Here, we report that, in Salmonella enterica, the sirtuin deacylase CobB long isoform (CobBL) is N-terminally acetylated by the YiaC protein of this bacterium. Results of in vitro acetylation assays showed that CobBL was acetylated by YiaC; liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to confirm these results. Results of in vitro and in vivo experiments showed that CobBL deacetylase activity was negatively affected when YiaC acetylated its N terminus. We report 1) modulation of a bacterial sirtuin deacylase activity by acetylation, 2) that the Gcn5-related YiaC protein is the acetyltransferase that modifies CobBL, and 3) that YiaC is an NAT. Based on our data, we propose the name of NatA (N-acyltransferase A) in lieu of YiaC to reflect the function of the enzyme.


Assuntos
Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Salmonella enterica/metabolismo , Sirtuínas/metabolismo , Acetilação , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida , Isoformas de Proteínas , Salmonella enterica/enzimologia , Espectrometria de Massas em Tandem
9.
Nat Chem Biol ; 16(9): 964-972, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32514182

RESUMO

Chemical modifications of the nucleosides that comprise transfer RNAs are diverse. However, the structure, location and extent of modifications have been systematically charted in very few organisms. Here, we describe an approach in which rapid prediction of modified sites through reverse transcription-derived signatures in high-throughput transfer RNA-sequencing (tRNA-seq) data is coupled with identification of tRNA modifications through RNA mass spectrometry. Comparative tRNA-seq enabled prediction of several Vibrio cholerae modifications that are absent from Escherichia coli and also revealed the effects of various environmental conditions on V. cholerae tRNA modification. Through RNA mass spectrometric analyses, we showed that two of the V. cholerae-specific reverse transcription signatures reflected the presence of a new modification (acetylated acp3U (acacp3U)), while the other results from C-to-Ψ RNA editing, a process not described before. These findings demonstrate the utility of this approach for rapid surveillance of tRNA modification profiles and environmental control of tRNA modification.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA de Transferência/genética , RNA de Transferência/metabolismo , Vibrio cholerae/genética , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Cólera/microbiologia , Citidina/genética , Escherichia coli/genética , Espectrometria de Massas/métodos , Edição de RNA , RNA de Transferência/química , RNA de Transferência de Tirosina/genética , RNA de Transferência de Tirosina/metabolismo , Coelhos , Vibrio cholerae/patogenicidade
10.
Nucleic Acids Res ; 48(13): 7532-7544, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32501503

RESUMO

Escherichia coli ItaT toxin reportedly acetylates the α-amino group of the aminoacyl-moiety of Ile-tRNAIle specifically, using acetyl-CoA as an acetyl donor, thereby inhibiting protein synthesis. The mechanism of the substrate specificity of ItaT had remained elusive. Here, we present functional and structural analyses of E. coli ItaT, which revealed the mechanism of ItaT recognition of specific aminoacyl-tRNAs for acetylation. In addition to Ile-tRNAIle, aminoacyl-tRNAs charged with hydrophobic residues, such as Val-tRNAVal and Met-tRNAMet, were acetylated by ItaT in vivo. Ile-tRNAIle, Val-tRNAVal and Met-tRNAMet were acetylated by ItaT in vitro, while aminoacyl-tRNAs charged with other hydrophobic residues, such as Ala-tRNAAla, Leu-tRNALeu and Phe-tRNAPhe, were less efficiently acetylated. A comparison of the structures of E. coli ItaT and the protein N-terminal acetyltransferase identified the hydrophobic residues in ItaT that possibly interact with the aminoacyl moiety of aminoacyl-tRNAs. Mutations of the hydrophobic residues of ItaT reduced the acetylation activity of ItaT toward Ile-tRNAIlein vitro, as well as the ItaT toxicity in vivo. Altogether, the size and shape of the hydrophobic pocket of ItaT are suitable for the accommodation of the specific aminoacyl-moieties of aminoacyl-tRNAs, and ItaT has broader specificity toward aminoacyl-tRNAs charged with certain hydrophobic amino acids.


Assuntos
Acetiltransferases/química , Toxinas Bacterianas/química , Proteínas de Escherichia coli/química , Aminoacilação de RNA de Transferência , Acetiltransferases/genética , Acetiltransferases/metabolismo , Motivos de Aminoácidos , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Especificidade por Substrato
11.
Nihon Yakurigaku Zasshi ; 155(3): 140-144, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32378630

RESUMO

Shati/Nat8l and TMEM168 were identified from nucleus accumbens (NAcc), which received continuous methamphetamine treatments. Shati/Nat8l is a synthetic enzyme that produces N-acetylaspartate (NAA) from L-aspartate and acetyl-coenzyme NAA is converted into N-acetylaspartylglutamate (NAAG) by NAAG synthetase (NAAGS). NAAG works as a highly selective endogenous agonist for the metabotropic glutamate type 3 receptor (mGluR3). We attempted to microinjection of adeno associated virus (AAV) including Shati/Nat8l into mice NAcc. These NAcc-Shati/Nat8l mice showed attenuation of the pharmacological effects of methamphetamine. NAcc-Shati or TMEM168 mice were also produced by AAV strategy and these mice also attenuated the methamphetamine-induced hyper locomotion and place preference test. TMEM168 interacts with osteopontin in NAcc of mice and cultured cells. Further, osteopontin it self has suppressive effects of methamphetamine. TMEM168 enhances anxiety in the elevated-plus maze and light-dark box test. The anxiety is recovered by the treatment of antianxiety drug diazepam. There our serial studies demonstrate that investigation of drug dependence-related molecule could lead to new pathway for new target for psychiatric disease.


Assuntos
Acetiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Metanfetamina , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Animais , Camundongos , Núcleo Accumbens , Osteopontina
12.
Mol Cell ; 78(4): 725-738.e4, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32277910

RESUMO

Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Several replication-fork-associated "cohesion establishment factors," including the multifunctional Ctf18-RFC complex, aid this process in as yet unknown ways. Here, we show that Ctf18-RFC's role in sister chromatid cohesion correlates with PCNA loading but is separable from its role in the replication checkpoint. Ctf18-RFC loads PCNA with a slight preference for the leading strand, which is dispensable for DNA replication. Conversely, the canonical Rfc1-RFC complex preferentially loads PCNA onto the lagging strand, which is crucial for DNA replication but dispensable for sister chromatid cohesion. The downstream effector of Ctf18-RFC is cohesin acetylation, which we place toward a late step during replication maturation. Our results suggest that Ctf18-RFC enriches and balances PCNA levels at the replication fork, beyond the needs of DNA replication, to promote establishment of sister chromatid cohesion and possibly other post-replicative processes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/fisiologia , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Acetiltransferases/genética , Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
13.
Nat Struct Mol Biol ; 27(5): 450-460, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32341533

RESUMO

Leading-strand template aberrations cause helicase-polymerase uncoupling and impede replication fork progression, but the details of how uncoupled forks are restarted remain uncertain. Using purified proteins from Saccharomyces cerevisiae, we have reconstituted translesion synthesis (TLS)-mediated restart of a eukaryotic replisome following collision with a cyclobutane pyrimidine dimer. We find that TLS functions 'on the fly' to promote resumption of rapid replication fork rates, despite lesion bypass occurring uncoupled from the Cdc45-MCM-GINS (CMG) helicase. Surprisingly, the main lagging-strand polymerase, Pol δ, binds the leading strand upon uncoupling and inhibits TLS. Pol δ is also crucial for efficient recoupling of leading-strand synthesis to CMG following lesion bypass. Proliferating cell nuclear antigen monoubiquitination positively regulates TLS to overcome Pol δ inhibition. We reveal that these mechanisms of negative and positive regulation also operate on the lagging strand. Our observations have implications for both fork restart and the division of labor during leading-strand synthesis generally.


Assuntos
Replicação do DNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Acetiltransferases/genética , Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinação
14.
Nat Commun ; 11(1): 1727, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32265440

RESUMO

Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesis. During de novo fatty acid and polyketide biosynthesis, ß-ketoacyl-acyl carrier protein (ACP) synthases (KS), catalyze this process via a decarboxylative Claisen-like condensation reaction. KSs must recognize multiple chemically distinct ACPs and choreograph a ping-pong mechanism, often in an iterative fashion. Here, we report crystal structures of substrate mimetic bearing ACPs in complex with the elongating KSs from Escherichia coli, FabF and FabB, in order to better understand the stereochemical features governing substrate discrimination by KSs. Complemented by molecular dynamics (MD) simulations and mutagenesis studies, these structures reveal conformational states accessed during KS catalysis. These data taken together support a gating mechanism that regulates acyl-ACP binding and substrate delivery to the KS active site. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features in elongating KSs.


Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , Acetiltransferases/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Ácido Graxo Sintase Tipo II/química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/isolamento & purificação , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Acetiltransferases/metabolismo , Sequência de Aminoácidos/genética , Sítios de Ligação/genética , Catálise , Domínio Catalítico/genética , Cristalografia por Raios X , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintase Tipo II/metabolismo , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese , Mutação , Conformação Proteica , Proteínas Recombinantes
15.
J Fish Biol ; 97(1): 83-99, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32222967

RESUMO

The biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA), a process to convert C18 polyunsaturated fatty acids into eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or arachidonic acid (ARA), requires the concerted activities of two enzymes, the fatty acyl desaturase (Fads) and elongase (Elovl). This study highlights the cloning, functional characterisation and tissue expression pattern of a Fads and an Elovl from the Boddart's goggle-eyed goby (Boleophthalmus boddarti), a mudskipper species widely distributed in the Indo-Pacific region. Phylogenetic analysis revealed that the cloned fads and elovl are clustered with other teleost orthologs, respectively. The investigation of the genome of several mudskipper species, namely Boleophthalmus pectinirostris, Periophthalmus schlosseri and Periophthalmus magnuspinnatus, revealed a single Fads2 and two elongases, Elovl5 and Elovl4 for each respective species. A heterologous yeast assay indicated that the B. boddarti Fads2 possessed low desaturation activity on C18 PUFA and no desaturation on C20 and C22 PUFA substrates. In comparison, the Elovl5 showed a wide range of substrate specificity, with a capacity to elongate C18, C20 and C22 PUFA substrates. An amino acid residue that affects the capacity to elongate C22:5n-3 was identified in the B. boddarti Elovl5. Both genes are highly expressed in brain tissue. Among all tissues, DHA is highly concentrated in neuron-rich tissues, whereas EPA is highly deposited in gills. Taken together, the results showed that due to the inability to perform desaturation steps, B. boddarti is unable to biosynthesise LC-PUFA, relying on dietary intake to acquire these nutrients.


Assuntos
Ácidos Graxos Dessaturases/metabolismo , Elongases de Ácidos Graxos/metabolismo , Ácidos Graxos Insaturados/biossíntese , Proteínas de Peixes/metabolismo , Perciformes/metabolismo , Acetiltransferases/química , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Ácidos Graxos Dessaturases/química , Ácidos Graxos Dessaturases/genética , Elongases de Ácidos Graxos/química , Elongases de Ácidos Graxos/genética , Proteínas de Peixes/genética , Regulação Enzimológica da Expressão Gênica , Filogenia , Especificidade por Substrato
16.
J Appl Microbiol ; 129(2): 345-355, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32091657

RESUMO

AIMS: Paclitaxel is a type of broad-spectrum anticancer drug in short supply. The price of acetyl-CoA (17 709 677·4 USD mol-1 ), which is the acetyl group donor for the enzymatic synthesis of the intermediate, baccatin Ⅲ, is still the bottleneck of the mass production of paclitaxel. This study reports a novel acetyl group donor, which could substantially reduce the cost of production. METHODS AND RESULTS: In this study, a substrate spectrum with 14 kinds of representative acetyl-donor substitutes predicted by computer-aided methods was tested in a 10-deacetylbaccatin Ⅲ-10-O-acetyltransferase (DBAT) heterogeneous-expressed open-whole-cell catalytic system. The results of computer prediction and experimental analysis revealed the rule of the acetyl-donor compounds based on this substrate spectrum. N-acetyl-d-glucosamine (30·95 USD mol-1 , about 572 202-fold cheaper than acetyl-CoA) is selected as a suitable substitute under the rule. The yield when using N-acetyl-d-glucosamine as acetyl donor in open-whole-cell catalytic system was 2·13-fold of that when using acetyl-CoA. In the in vivo system, the yield increased 24·17%, which may indicate its cooperation with acetyl-CoA. CONCLUSION: The success of open-whole-cell synthesis and in vivo synthesis of baccatin Ⅲ by adding N-acetyl-d-glucosamine as acetyl substrate demonstrates that it is a useful substrate to improve the yield of baccatin Ⅲ. SIGNIFICANCE AND IMPACT OF THE STUDY: All these findings provided a potential acetyl-donor substitute for acetyl-CoA, as well as a low cost and efficient method of preparing paclitaxel through baccatin Ⅲ semi-synthesis.


Assuntos
Acetilglucosamina/metabolismo , Alcaloides/biossíntese , Acetilcoenzima A/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Alcaloides/economia , Antineoplásicos Fitogênicos/biossíntese , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/economia , Biocatálise , Paclitaxel/biossíntese , Paclitaxel/química , Paclitaxel/economia , Especificidade por Substrato , Taxoides/economia
17.
Int J Mol Sci ; 21(3)2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-32050518

RESUMO

High temperature at anthesis is one of the most serious stress factors for rice (Oryza sativa L.) production, causing irreversible yield losses and reduces grain quality. Illustration of thermotolerance mechanism is of great importance to accelerate rice breeding aimed at thermotolerance improvement. Here, we identified a new thermotolerant germplasm, SDWG005. Microscopical analysis found that stable anther structure of SDWG005 under stress may contribute to its thermotolerance. Dynamic transcriptomic analysis totally identified 3559 differentially expressed genes (DEGs) in SDWG005 anthers at anthesis under heat treatments, including 477, 869, 2335, and 2210 for 1, 2, 6, and 12 h, respectively; however, only 131 were regulated across all four-time-points. The DEGs were divided into nine clusters according to their expressions in these heat treatments. Further analysis indicated that some main gene categories involved in heat-response of SDWG005 anthers, such as transcription factors, nucleic acid and protein metabolisms related genes, etc. Comparison with previous studies indicates that a core gene-set may exist for thermotolerance mechanism. Expression and polymorphic analysis of agmatine-coumarin-acyltransferase gene OsACT in different accessions suggested that it may involve in SDWG005 thermotolerance. This study improves our understanding of thermotolerance mechanisms in rice anthers during anthesis, and also lays foundation for breeding thermotolerant varieties via molecular breeding.


Assuntos
Oryza/genética , Termotolerância , Transcriptoma , Acetiltransferases/genética , Acetiltransferases/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
18.
Mol Cell ; 77(6): 1279-1293.e4, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32032532

RESUMO

Cohesin, a member of the SMC complex family, holds sister chromatids together but also shapes chromosomes by promoting the formation of long-range intra-chromatid loops, a process proposed to be mediated by DNA loop extrusion. Here we describe the roles of three cohesin partners, Pds5, Wpl1, and Eco1, in loop formation along either unreplicated or mitotic Saccharomyces cerevisiae chromosomes. Pds5 limits the size of DNA loops via two different pathways: the canonical Wpl1-mediated releasing activity and an Eco1-dependent mechanism. In the absence of Pds5, the main barrier to DNA loop expansion appears to be the centromere. Our data also show that Eco1 acetyl-transferase inhibits the translocase activity that powers loop formation and contributes to the positioning of loops through a mechanism that is distinguishable from its role in cohesion establishment. This study reveals that the mechanisms regulating cohesin-dependent chromatin loops are conserved among eukaryotes while promoting different functions.


Assuntos
Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/química , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferases/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Mitose , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
19.
Nat Commun ; 11(1): 52, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911608

RESUMO

Prostatic luminal epithelial cells secrete high levels of acetylated polyamines into the prostatic lumen, sensitizing them to perturbations of connected metabolic pathways. Enhanced flux is driven by spermidine/spermine N1-acetyltransferase (SSAT) activity, which acetylates polyamines leading to their secretion and drives biosynthetic demand. The methionine salvage pathway recycles one-carbon units lost to polyamine biosynthesis to the methionine cycle to overcome stress. Prostate cancer (CaP) relies on methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme, to relieve strain. Here, we show that inhibition of MTAP alongside SSAT upregulation is synergistic in androgen sensitive and castration recurrent CaP models in vitro and in vivo. The combination treatment increases apoptosis in radical prostatectomy ex vivo explant samples. This unique high metabolic flux through polyamine biosynthesis and connected one carbon metabolism in CaP creates a metabolic dependency. Enhancing this flux while simultaneously targeting this dependency in prostate cancer results in an effective therapeutic approach potentially translatable to the clinic.


Assuntos
Metionina/metabolismo , Poliaminas/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Acetiltransferases/genética , Acetiltransferases/metabolismo , Adenina/administração & dosagem , Adenina/análogos & derivados , Animais , Apoptose , Linhagem Celular Tumoral , Quimioterapia Combinada , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Pirrolidinas/administração & dosagem , Terapia de Salvação , Espermina/administração & dosagem , Espermina/análogos & derivados , Espermina/metabolismo
20.
PLoS One ; 15(1): e0220348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935221

RESUMO

In a process linked to DNA replication, duplicated chromosomes are entrapped in large, circular cohesin complexes and functional sister chromatid cohesion (SCC) is established by acetylation of the SMC3 cohesin subunit. Roberts Syndrome (RBS) and Warsaw Breakage Syndrome (WABS) are rare human developmental syndromes that are characterized by defective SCC. RBS is caused by mutations in the SMC3 acetyltransferase ESCO2, whereas mutations in the DNA helicase DDX11 lead to WABS. We found that WABS-derived cells predominantly rely on ESCO2, not ESCO1, for residual SCC, growth and survival. Reciprocally, RBS-derived cells depend on DDX11 to maintain low levels of SCC. Synthetic lethality between DDX11 and ESCO2 correlated with a prolonged delay in mitosis, and was rescued by knockdown of the cohesin remover WAPL. Rescue experiments using human or mouse cDNAs revealed that DDX11, ESCO1 and ESCO2 act on different but related aspects of SCC establishment. Furthermore, a DNA binding DDX11 mutant failed to correct SCC in WABS cells and DDX11 deficiency reduced replication fork speed. We propose that DDX11, ESCO1 and ESCO2 control different fractions of cohesin that are spatially and mechanistically separated.


Assuntos
Acetiltransferases/genética , Proteínas de Ciclo Celular/genética , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/genética , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Células Epiteliais/enzimologia , Fibroblastos/enzimologia , Acetiltransferases/metabolismo , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Proliferação de Células , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Quebra Cromossômica , Segregação de Cromossomos , Anormalidades Craniofaciais/enzimologia , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/patologia , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Ectromelia/enzimologia , Ectromelia/genética , Ectromelia/patologia , Células Epiteliais/patologia , Fibroblastos/patologia , Expressão Gênica , Humanos , Hipertelorismo/enzimologia , Hipertelorismo/genética , Hipertelorismo/patologia , Camundongos , Mitose , Modelos Biológicos , Mutação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
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