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1.
Sheng Wu Gong Cheng Xue Bao ; 36(10): 2171-2180, 2020 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-33169581

RESUMO

In most insects, polyunsaturated fatty acids (PUFAs) are mainly polyunsaturated fatty acids with a carbon-chain length less than 18 carbon atoms, hardly any long-chain polyunsaturated fatty acids such as C20 and C22 that are more valuable and bioactive. This study, by using Drosophila melanogaster (Fruit fly) as a model organism, optimized the Δ6-fatty acid elongase enzyme Elovl5 gene from mice and transferred it to fruit flies for expression. Vectors containing Elovl5 gene were successfully injected into drosophila embryo through the microscopic injection. There were enhanced green fluorescent proteins expressed in the whole developmental stage of Drosophila be means of fluorescence microscope. At the same time, expression of Elovl5 gene significantly contributed to the transformation of fruit flies C18-polyunsaturated fatty acids in the body towards the biosynthesis of longer-chain polyunsaturated fatty acids. The transgenic fruit fly model rich in long-chain polyunsaturated fatty acids such as C20 and C22 were obtained, providing a basis for further research on biosynthesis of polyunsaturated fatty acids in fruit flies.


Assuntos
Acetiltransferases , Drosophila melanogaster , Elongases de Ácidos Graxos , Ácidos Graxos , Acetiltransferases/genética , Animais , Drosophila melanogaster/genética , Elongases de Ácidos Graxos/genética , Elongases de Ácidos Graxos/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/genética , Técnicas de Transferência de Genes , Camundongos
2.
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
3.
Folia Biol (Praha) ; 66(3): 91-103, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33069188

RESUMO

The most recent genome-editing system called CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat system with associated protein 9-nuclease) was employed to delete four non-essential genes (i.e., Caeco1, Caidh1, Carom2, and Cataf10) individually to establish their gene functionality annotations in pathogen Candida albicans. The biological roles of these genes were investigated with respect to the cell wall integrity and biogenesis, calcium/calcineurin pathways, susceptibility of mutants towards temperature, drugs and salts. All the mutants showed increased vulnerability compared to the wild-type background strain towards the cell wall-perturbing agents, (antifungal) drugs and salts. All the mutants also exhibited repressed and defective hyphal growth and smaller colony size than control CA14. The cell cycle of all the mutants decreased enormously except for those with Carom2 deletion. The budding index and budding size also increased for all mutants with altered bud shape. The disposition of the mutants towards cell wall-perturbing enzymes disclosed lower survival and more rapid cell wall lysis events than in wild types. The pathogenicity and virulence of the mutants was checked by adhesion assay, and strains lacking rom2 and eco1 were found to possess the least adhesion capacity, which is synonymous to their decreased pathogenicity and virulence.


Assuntos
Candida albicans/fisiologia , Proteínas Fúngicas/fisiologia , Genes Fúngicos , Acetiltransferases/deficiência , Acetiltransferases/genética , Acetiltransferases/fisiologia , Antifúngicos/farmacologia , Sistemas CRISPR-Cas , Cálcio/fisiologia , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/patogenicidade , Cátions/farmacologia , Adesão Celular , Ciclo Celular , Parede Celular/efeitos dos fármacos , Quitinases/farmacologia , Dano ao DNA , Proteínas Fúngicas/genética , Deleção de Genes , Glucana Endo-1,3-beta-D-Glucosidase/farmacologia , Hifas/crescimento & desenvolvimento , Isocitrato Desidrogenase/deficiência , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/fisiologia , Fases de Leitura Aberta , Reprodução Assexuada , Fatores Associados à Proteína de Ligação a TATA/deficiência , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/fisiologia , Virulência/genética
4.
Proc Natl Acad Sci U S A ; 117(40): 24947-24956, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32968016

RESUMO

The acquisition of mutations plays critical roles in adaptation, evolution, senescence, and tumorigenesis. Massive genome sequencing has allowed extraction of specific features of many mutational landscapes but it remains difficult to retrospectively determine the mechanistic origin(s), selective forces, and trajectories of transient or persistent mutations and genome rearrangements. Here, we conducted a prospective reciprocal approach to inactivate 13 single or multiple evolutionary conserved genes involved in distinct genome maintenance processes and characterize de novo mutations in 274 diploid Saccharomyces cerevisiae mutation accumulation lines. This approach revealed the diversity, complexity, and ultimate uniqueness of mutational landscapes, differently composed of base substitutions, small insertions/deletions (InDels), structural variants, and/or ploidy variations. Several landscapes parallel the repertoire of mutational signatures in human cancers while others are either novel or composites of subsignatures resulting from distinct DNA damage lesions. Notably, the increase of base substitutions in the homologous recombination-deficient Rad51 mutant, specifically dependent on the Polζ translesion polymerase, yields COSMIC signature 3 observed in BRCA1/BRCA2-mutant breast cancer tumors. Furthermore, "mutome" analyses in highly polymorphic diploids and single-cell bottleneck lineages revealed a diverse spectrum of loss-of-heterozygosity (LOH) signatures characterized by interstitial and terminal chromosomal events resulting from interhomolog mitotic cross-overs. Following the appearance of heterozygous mutations, the strong stimulation of LOHs in the rad27/FEN1 and tsa1/PRDX1 backgrounds leads to fixation of homozygous mutations or their loss along the lineage. Overall, these mutomes and their trajectories provide a mechanistic framework to understand the origin and dynamics of genome variations that accumulate during clonal evolution.


Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Mutação/genética , Saccharomyces cerevisiae/genética , Acetiltransferases/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/patologia , Dano ao DNA/genética , DNA Polimerase Dirigida por DNA , Diploide , Feminino , Endonucleases Flap/genética , Genoma Fúngico/genética , Humanos , Perda de Heterozigosidade/genética , Proteínas de Membrana/genética , Peroxirredoxinas/genética , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Sequenciamento Completo do Genoma
5.
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
6.
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
7.
Nucleic Acids Res ; 48(15): 8617-8625, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32597957

RESUMO

Type II toxin-antitoxins systems are widespread in prokaryotic genomes. Typically, they comprise two proteins, a toxin, and an antitoxin, encoded by adjacent genes and forming a complex in which the enzymatic activity of the toxin is inhibited. Under stress conditions, the antitoxin is degraded liberating the active toxin. Though thousands of various toxin-antitoxins pairs have been predicted bioinformatically, only a handful has been thoroughly characterized. Here, we describe the AtaT2 toxin from a toxin-antitoxin system from Escherichia coli O157:H7. We show that AtaT2 is the first GNAT (Gcn5-related N-acetyltransferase) toxin that specifically targets charged glycyl tRNA. In vivo, the AtaT2 activity induces ribosome stalling at all four glycyl codons but does not evoke a stringent response. In vitro, AtaT2 acetylates the aminoacyl moiety of isoaccepting glycyl tRNAs, thus precluding their participation in translation. Our study broadens the known target specificity of GNAT toxins beyond the earlier described isoleucine and formyl methionine tRNAs, and suggest that various GNAT toxins may have evolved to specificaly target other if not all individual aminoacyl tRNAs.


Assuntos
Acetiltransferases/genética , Escherichia coli O157/genética , Glicina-tRNA Ligase/genética , Biossíntese de Proteínas/genética , Antitoxinas/genética , Toxinas Bacterianas/genética , Escherichia coli O157/patogenicidade , Sistemas Toxina-Antitoxina/genética
8.
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
9.
Nucleic Acids Res ; 48(12): 6824-6838, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32432721

RESUMO

RNA-seq experiments previously performed by our laboratories showed enrichment in intronic sequences and alterations in alternative splicing in dengue-infected human cells. The transcript of the SAT1 gene, of well-known antiviral action, displayed higher inclusion of exon 4 in infected cells, leading to an mRNA isoform that is degraded by non-sense mediated decay. SAT1 is a spermidine/spermine acetyl-transferase enzyme that decreases the reservoir of cellular polyamines, limiting viral replication. Delving into the molecular mechanism underlying SAT1 pre-mRNA splicing changes upon viral infection, we observed lower protein levels of RBM10, a splicing factor responsible for SAT1 exon 4 skipping. We found that the dengue polymerase NS5 interacts with RBM10 and its sole expression triggers RBM10 proteasome-mediated degradation. RBM10 over-expression in infected cells prevents SAT1 splicing changes and limits viral replication, while its knock-down enhances the splicing switch and also benefits viral replication, revealing an anti-viral role for RBM10. Consistently, RBM10 depletion attenuates expression of interferon and pro-inflammatory cytokines. In particular, we found that RBM10 interacts with viral RNA and RIG-I, and even promotes the ubiquitination of the latter, a crucial step for its activation. We propose RBM10 fulfills diverse pro-inflammatory, anti-viral tasks, besides its well-documented role in splicing regulation of apoptotic genes.


Assuntos
Acetiltransferases/genética , Dengue/genética , Imunidade Inata/genética , Proteínas de Ligação a RNA/genética , Processamento Alternativo/genética , Apoptose/genética , Dengue/virologia , Vírus da Dengue/genética , Vírus da Dengue/patogenicidade , Éxons/genética , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Isoformas de Proteínas/genética , Processamento de RNA/genética , RNA-Seq , Replicação Viral/genética
10.
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
11.
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
12.
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
13.
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
14.
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
15.
Int J Mol Sci ; 21(1)2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31935967

RESUMO

The expression and biological functions of oncofetal markers GD2 and GD3 were extensively studied in neuroectoderm-derived cancers in order to characterize their potential as therapeutic targets. Using immunological approaches, we previously identified GD3, GD2, and OAcGD2 expression in breast cancer (BC) cell lines. However, antibodies specific for O-acetylated gangliosides are not exempt of limitations, as they only provide information on the expression of a limited set of O-acetylated ganglioside species. Consequently, the aim of the present study was to use structural approaches in order to apprehend ganglioside diversity in melanoma, neuroblastoma, and breast cancer cells, focusing on O-acetylated species that are usually lost under alkaline conditions and require specific analytical procedures. We used purification and extraction methods that preserve the O-acetyl modification for the analysis of native gangliosides by MALDI-TOF. We identified the expression of GM1, GM2, GM3, GD2, GD3, GT2, and GT3 in SK-Mel28 (melanoma), LAN-1 (neuroblastoma), Hs 578T, SUM 159PT, MDA-MB-231, MCF-7 (BC), and BC cell lines over-expressing GD3 synthase. Among O-acetylated gangliosides, we characterized the expression of OAcGM1, OAcGD3, OAcGD2, OAcGT2, and OAcGT3. Furthermore, the experimental procedure allowed us to clearly identify the position of the sialic acid residue that carries the O-acetyl group on b- and c-series gangliosides by MS/MS fragmentation. These results show that ganglioside O-acetylation occurs on both inner and terminal sialic acid residue in a cell type-dependent manner, suggesting different O-acetylation pathways for gangliosides. They also highlight the limitation of immuno-detection for the complete identification of O-acetylated ganglioside profiles in cancer cells.


Assuntos
Acetiltransferases/metabolismo , Gangliosídeos/metabolismo , Placa Neural/citologia , Acetilação , Acetiltransferases/genética , Neoplasias da Mama/metabolismo , Feminino , Gangliosídeos/química , Humanos , Células MCF-7 , Melanoma/metabolismo , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Placa Neural/metabolismo , Neuroblastoma/metabolismo
16.
J Biol Chem ; 295(10): 3247-3256, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-31996374

RESUMO

Snyder-Robinson syndrome (SRS) is an X-linked intellectual disability syndrome caused by a loss-of-function mutation in the spermine synthase (SMS) gene. Primarily affecting males, the main manifestations of SRS include osteoporosis, hypotonic stature, seizures, cognitive impairment, and developmental delay. Because there is no cure for SRS, treatment plans focus on alleviating symptoms rather than targeting the underlying causes. Biochemically, the cells of individuals with SRS accumulate excess spermidine, whereas spermine levels are reduced. We recently demonstrated that SRS patient-derived lymphoblastoid cells are capable of transporting exogenous spermine and its analogs into the cell and, in response, decreasing excess spermidine pools to normal levels. However, dietary supplementation of spermine does not appear to benefit SRS patients or mouse models. Here, we investigated the potential use of a metabolically stable spermine mimetic, (R,R)-1,12-dimethylspermine (Me2SPM), to reduce the intracellular spermidine pools of SRS patient-derived cells. Me2SPM can functionally substitute for the native polyamines in supporting cell growth while stimulating polyamine homeostatic control mechanisms. We found that both lymphoblasts and fibroblasts from SRS patients can accumulate Me2SPM, resulting in significantly decreased spermidine levels with no adverse effects on growth. Me2SPM administration to mice revealed that Me2SPM significantly decreases spermidine levels in multiple tissues. Importantly, Me2SPM was detectable in brain tissue, the organ most affected in SRS, and was associated with changes in polyamine metabolic enzymes. These findings indicate that the (R,R)-diastereomer of 1,12-Me2SPM represents a promising lead compound in developing a treatment aimed at targeting the molecular mechanisms underlying SRS pathology.


Assuntos
Retardo Mental Ligado ao Cromossomo X/patologia , Espermidina/metabolismo , Espermina/análogos & derivados , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Retardo Mental Ligado ao Cromossomo X/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Poliaminas/análise , Poliaminas/metabolismo , Espermina/administração & dosagem , Espermina/metabolismo , Espermina/farmacologia , Espermina Sintase/genética , Células Tumorais Cultivadas
17.
Genome ; 63(3): 179-187, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31917611

RESUMO

Hair follicle (HF) growth and cycling is a complex biological process that occurs in most mammals. As HF growth and cycling directly impacts rabbit wool yield, it is important to better understand the potential regulation pattern of HF development. Our previous study demonstrated that HTATIP2 may participate in regulating rabbit HF cycles, but the molecular mechanism of HTATIP2 remained unclear. In this study, the coding sequence of the HTATIP2 gene in Angora rabbit was cloned. The length of the coding region sequence was 840 bp, which could code 279 amino acids, and exhibited high homology in different mammals. Bioinformatics analyses indicated that the HTATIP2 protein is stable, hydrophilic, located around the cytoplasm, and has a putative signal peptide. Moreover, we verified that HTATIP2 is highly expressed during catagen and telogen of the HF cycle. The overexpression vector was constructed and siRNAs were designed. Overexpression and knockdown of HTATIP2 appeared to regulate JAK-STAT pathway genes, such as BCL2, CCND1, c-Myc, and STAT2. It is therefore likely that HTATIP2 promotes cell apoptosis and inhibits cell proliferation. Our results indicate that HTATIP2 is highly expressed during catagen and telogen and may play an important role in JAK-STAT signaling. This study provides a theoretical foundation for investigating HTATIP2 in biological processes.


Assuntos
Acetiltransferases/genética , Acetiltransferases/metabolismo , Clonagem Molecular/métodos , Folículo Piloso/citologia , Animais , Apoptose , Linhagem Celular , Proliferação de Células , Folículo Piloso/metabolismo , Coelhos , Análise de Sequência de DNA/veterinária , Transdução de Sinais , Regulação para Cima ,
18.
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
19.
Nat Genet ; 52(2): 167-176, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31959995

RESUMO

The kidneys integrate information from continuous systemic processes related to the absorption, distribution, metabolism and excretion (ADME) of metabolites. To identify underlying molecular mechanisms, we performed genome-wide association studies of the urinary concentrations of 1,172 metabolites among 1,627 patients with reduced kidney function. The 240 unique metabolite-locus associations (metabolite quantitative trait loci, mQTLs) that were identified and replicated highlight novel candidate substrates for transport proteins. The identified genes are enriched in ADME-relevant tissues and cell types, and they reveal novel candidates for biotransformation and detoxification reactions. Fine mapping of mQTLs and integration with single-cell gene expression permitted the prioritization of causal genes, functional variants and target cell types. The combination of mQTLs with genetic and health information from 450,000 UK Biobank participants illuminated metabolic mediators, and hence, novel urinary biomarkers of disease risk. This comprehensive resource of genetic targets and their substrates is informative for ADME processes in humans and is relevant to basic science, clinical medicine and pharmaceutical research.


Assuntos
Biotransformação/genética , Rim/metabolismo , Locos de Características Quantitativas , Insuficiência Renal Crônica/urina , Acetiltransferases/genética , Acetiltransferases/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Biomarcadores/urina , Estudos de Coortes , Citocromo P-450 CYP2D6/genética , Estudo de Associação Genômica Ampla , Humanos , Inativação Metabólica , Rim/citologia , Metoprolol/farmacocinética , Polimorfismo de Nucleotídeo Único , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/metabolismo , Urina/fisiologia , Xenobióticos/farmacocinética , Xenobióticos/urina
20.
Curr Pharm Des ; 26(2): 260-264, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31924153

RESUMO

BACKGROUND: Methamphetamine (METH) is one of the most widely distributed psychostimulants worldwide. Despite active counter measures taken by different countries, neither overall usage of METH nor the frequency of repeat users has reduced over the past decade. METH induces abuse and dependence as it acts on the central nervous system and temporarily stimulates the brain. The recidivism rate for abuse of stimulants in Japan is very high and therefore prevention of repeated usage is paramount. However, we lack information about the relationship between METH users and genomic changes in humans in Japan, which would provide important information to aid such efforts. OBJECTIVE: Shati/Nat8l is a METH-inducible molecule and its overexpression has protective effects on the brain upon METH usage. Here we investigated the effect of METH usage on DNA methylation rates at the promoter site of SHATI/NAT8L. We used DNA samples from human METH users, who are usually difficult to recruit in Japan. METHODS: We measured DNA methylation at SHATI/NAT8L promoter sites by pyrosequencing method using 193 samples of METH users and 60 samples of healthy subjects. In this method, DNA methylation is measured by utilizing the property that only non-methylated cytosine changes to urasil after bisulfite conversion. RESULTS: We found that the rate of DNA methylation at six CpG islands of SHATI/NAT8L promoter sites is significantly higher in METH users when compared to healthy subjects. CONCLUSION: These results suggest that the DNA methylation rate of SHATI/NAT8L promotor regions offers a new diagnostic method for METH usage.


Assuntos
Acetiltransferases/genética , Transtornos Relacionados ao Uso de Anfetaminas/diagnóstico , Metilação de DNA , Regiões Promotoras Genéticas , Transtornos Relacionados ao Uso de Anfetaminas/genética , Estimulantes do Sistema Nervoso Central , Humanos , Japão , Metanfetamina
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