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1.
Cell ; 177(7): 1781-1796.e25, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31104845

RESUMO

DNA N6-adenine methylation (6mA) has recently been described in diverse eukaryotes, spanning unicellular organisms to metazoa. Here, we report a DNA 6mA methyltransferase complex in ciliates, termed MTA1c. It consists of two MT-A70 proteins and two homeobox-like DNA-binding proteins and specifically methylates dsDNA. Disruption of the catalytic subunit, MTA1, in the ciliate Oxytricha leads to genome-wide loss of 6mA and abolishment of the consensus ApT dimethylated motif. Mutants fail to complete the sexual cycle, which normally coincides with peak MTA1 expression. We investigate the impact of 6mA on nucleosome occupancy in vitro by reconstructing complete, full-length Oxytricha chromosomes harboring 6mA in native or ectopic positions. We show that 6mA directly disfavors nucleosomes in vitro in a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a diverged DNA N6-adenine methyltransferase and defines the role of 6mA in chromatin organization.


Assuntos
Complexos Multienzimáticos/metabolismo , Nucleossomos/enzimologia , Oxytricha/enzimologia , Proteínas de Protozoários/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Tetrahymena thermophila/enzimologia , Complexos Multienzimáticos/genética , Nucleossomos/genética , Oxytricha/genética , Proteínas de Protozoários/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Tetrahymena thermophila/genética
2.
Annu Rev Biochem ; 87: 187-216, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29925259

RESUMO

How individual enzymes evolved is relatively well understood. However, individual enzymes rarely confer a physiological advantage on their own. Judging by its current state, the emergence of metabolism seemingly demanded the simultaneous emergence of many enzymes. Indeed, how multicomponent interlocked systems, like metabolic pathways, evolved is largely an open question. This complexity can be unlocked if we assume that survival of the fittest applies not only to genes and enzymes but also to the metabolites they produce. This review develops our current knowledge of enzyme evolution into a wider hypothesis of pathway and network evolution. We describe the current models for pathway evolution and offer an integrative metabolite-enzyme coevolution hypothesis. Our hypothesis addresses the origins of new metabolites and of new enzymes and the order of their recruitment. We aim to not only survey established knowledge but also present open questions and potential ways of addressing them.


Assuntos
Enzimas/genética , Enzimas/metabolismo , Evolução Molecular , Redes e Vias Metabólicas/genética , Enzimas/química , Cinética , Modelos Biológicos , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Filogenia , Especificidade por Substrato/genética
3.
Annu Rev Biochem ; 86: 1-19, 2017 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-28125288

RESUMO

After an undergraduate degree in biology at Harvard, I started graduate school at The Rockefeller Institute for Medical Research in New York City in July 1965. I was attracted to the chemical side of biochemistry and joined Fritz Lipmann's large, hierarchical laboratory to study enzyme mechanisms. That work led to postdoctoral research with Robert Abeles at Brandeis, then a center of what, 30 years later, would be called chemical biology. I spent 15 years on the Massachusetts Institute of Technology faculty, in both the Chemistry and Biology Departments, and then 26 years on the Harvard Medical School Faculty. My research interests have been at the intersection of chemistry, biology, and medicine. One unanticipated major focus has been investigating the chemical logic and enzymatic machinery of natural product biosynthesis, including antibiotics and antitumor agents. In this postgenomic era it is now recognized that there may be from 105 to 106 biosynthetic gene clusters as yet uncharacterized for potential new therapeutic agents.


Assuntos
Antibacterianos/metabolismo , Antineoplásicos/metabolismo , Bioquímica/história , Produtos Biológicos/metabolismo , Pesquisa Biomédica/história , Indústria Farmacêutica/história , Antibacterianos/química , Antineoplásicos/química , Bioquímica/tendências , Produtos Biológicos/química , Pesquisa Biomédica/tendências , Indústria Farmacêutica/tendências , Regulação da Expressão Gênica , História do Século XX , História do Século XXI , Humanos , Ligases/genética , Ligases/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Resistência a Vancomicina/genética , Recursos Humanos
4.
Mol Cell ; 81(11): 2445-2459.e13, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33905682

RESUMO

How are E3 ubiquitin ligases configured to match substrate quaternary structures? Here, by studying the yeast GID complex (mutation of which causes deficiency in glucose-induced degradation of gluconeogenic enzymes), we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryoelectron microscopy (cryo-EM) structures show that, to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two minimally functional GID E3s assemble into the 20-protein Chelator-GIDSR4, which resembles an organometallic supramolecular chelate. The Chelator-GIDSR4 assembly avidly binds multiple Fbp1 degrons so that multiple Fbp1 protomers are simultaneously ubiquitylated at lysines near the allosteric and substrate binding sites. Importantly, key structural and biochemical features, including capacity for supramolecular assembly, are preserved in the human ortholog, the CTLH E3. Based on our integrative structural, biochemical, and cell biological data, we propose that higher-order E3 ligase assembly generally enables multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Moléculas de Adesão Celular/química , Frutose-Bifosfatase/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Complexos Multienzimáticos/química , Proteínas de Saccharomyces cerevisiae/química , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sítios de Ligação , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Microscopia Crioeletrônica , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/metabolismo , Expressão Gênica , Gluconeogênese/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células K562 , Cinética , Modelos Moleculares , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Células Sf9 , Spodoptera , Homologia Estrutural de Proteína , Especificidade por Substrato , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação
5.
Mol Cell ; 78(4): 670-682.e8, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32343944

RESUMO

Biomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here, we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA sequencing (RNA-seq). We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved non-coding RNAs (ncRNAs) are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the buildup of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.


Assuntos
Caulobacter crescentus/metabolismo , Endorribonucleases/metabolismo , Escherichia coli/metabolismo , Complexos Multienzimáticos/metabolismo , Organelas/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Caulobacter crescentus/genética , Caulobacter crescentus/crescimento & desenvolvimento , Endorribonucleases/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Humanos , Complexos Multienzimáticos/genética , Organelas/genética , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Helicases/genética , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Mensageiro/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo
6.
Annu Rev Microbiol ; 76: 533-552, 2022 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-35671533

RESUMO

RNA degradosomes are multienzyme complexes composed of ribonucleases, RNA helicases, and metabolic enzymes. RNase E-based degradosomes are widespread in Proteobacteria. The Escherichia coli RNA degradosome is sequestered from transcription in the nucleoid and translation in the cytoplasm by localization to the inner cytoplasmic membrane, where it forms short-lived clusters that are proposed to be sites of mRNA degradation. In Caulobacter crescentus, RNA degradosomes localize to ribonucleoprotein condensates in the interior of the cell [bacterial ribonucleoprotein-bodies (BR-bodies)], which have been proposed to drive the concerted degradation of mRNA to nucleotides. The turnover of mRNA in growing cells is important for maintaining pools of nucleotides for transcription and DNA replication.Membrane attachment of the E. coli RNA degradosome is necessary to avoid wasteful degradation of intermediates in ribosome assembly. Sequestering RNA degradosomes to C. crescentus BR-bodies, which exclude structured RNA, could have a similar role in protecting intermediates in ribosome assembly from degradation.


Assuntos
Caulobacter crescentus , Endorribonucleases , Escherichia coli , Complexos Multienzimáticos , Nucleotídeos , Polirribonucleotídeo Nucleotidiltransferase , RNA Helicases , Estabilidade de RNA , RNA Mensageiro , Caulobacter crescentus/enzimologia , Caulobacter crescentus/genética , Endorribonucleases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Nucleotídeos/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo
7.
Mol Cell ; 73(4): 749-762.e5, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30661981

RESUMO

The introduction of azole heterocycles into a peptide backbone is the principal step in the biosynthesis of numerous compounds with therapeutic potential. One of them is microcin B17, a bacterial topoisomerase inhibitor whose activity depends on the conversion of selected serine and cysteine residues of the precursor peptide to oxazoles and thiazoles by the McbBCD synthetase complex. Crystal structures of McbBCD reveal an octameric B4C2D2 complex with two bound substrate peptides. Each McbB dimer clamps the N-terminal recognition sequence, while the C-terminal heterocycle of the modified peptide is trapped in the active site of McbC. The McbD and McbC active sites are distant from each other, which necessitates alternate shuttling of the peptide substrate between them, while remaining tethered to the McbB dimer. An atomic-level view of the azole synthetase is a starting point for deeper understanding and control of biosynthesis of a large group of ribosomally synthesized natural products.


Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Bacteriocinas/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Complexos Multienzimáticos/metabolismo , Ribossomos/enzimologia , Inibidores da Topoisomerase II/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bacteriocinas/química , Bacteriocinas/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Difração de Raios X
8.
Nature ; 583(7814): 122-126, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32461692

RESUMO

The cellular NADH/NAD+ ratio is fundamental to biochemistry, but the extent to which it reflects versus drives metabolic physiology in vivo is poorly understood. Here we report the in vivo application of Lactobacillus brevis (Lb)NOX1, a bacterial water-forming NADH oxidase, to assess the metabolic consequences of directly lowering the hepatic cytosolic NADH/NAD+ ratio in mice. By combining this genetic tool with metabolomics, we identify circulating α-hydroxybutyrate levels as a robust marker of an elevated hepatic cytosolic NADH/NAD+ ratio, also known as reductive stress. In humans, elevations in circulating α-hydroxybutyrate levels have previously been associated with impaired glucose tolerance2, insulin resistance3 and mitochondrial disease4, and are associated with a common genetic variant in GCKR5, which has previously been associated with many seemingly disparate metabolic traits. Using LbNOX, we demonstrate that NADH reductive stress mediates the effects of GCKR variation on many metabolic traits, including circulating triglyceride levels, glucose tolerance and FGF21 levels. Our work identifies an elevated hepatic NADH/NAD+ ratio as a latent metabolic parameter that is shaped by human genetic variation and contributes causally to key metabolic traits and diseases. Moreover, it underscores the utility of genetic tools such as LbNOX to empower studies of 'causal metabolism'.


Assuntos
Fígado/metabolismo , NAD/metabolismo , Estresse Fisiológico , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Citosol/metabolismo , Modelos Animais de Doenças , Fatores de Crescimento de Fibroblastos/sangue , Variação Genética , Teste de Tolerância a Glucose , Humanos , Resistência à Insulina , Levilactobacillus brevis/enzimologia , Levilactobacillus brevis/genética , Masculino , Camundongos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Triglicerídeos/sangue
9.
Nature ; 585(7824): 288-292, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32641834

RESUMO

The mitochondrial electron transport chain (ETC) is necessary for tumour growth1-6 and its inhibition has demonstrated anti-tumour efficacy in combination with targeted therapies7-9. Furthermore, human brain and lung tumours display robust glucose oxidation by mitochondria10,11. However, it is unclear why a functional ETC is necessary for tumour growth in vivo. ETC function is coupled to the generation of ATP-that is, oxidative phosphorylation and the production of metabolites by the tricarboxylic acid (TCA) cycle. Mitochondrial complexes I and II donate electrons to ubiquinone, resulting in the generation of ubiquinol and the regeneration of the NAD+ and FAD cofactors, and complex III oxidizes ubiquinol back to ubiquinone, which also serves as an electron acceptor for dihydroorotate dehydrogenase (DHODH)-an enzyme necessary for de novo pyrimidine synthesis. Here we show impaired tumour growth in cancer cells that lack mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX)12, which also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II or DHODH diminished the tumour growth of AOX-expressing cancer cells deficient in mitochondrial complex III, which highlights the necessity of ubiquinone as an electron acceptor for tumour growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX)13 targeted to the mitochondria or cytosol were still unable to grow tumours. This suggests that regeneration of NAD+ is not sufficient to drive tumour growth in vivo. Collectively, our findings indicate that tumour growth requires the ETC to oxidize ubiquinol, which is essential to drive the oxidative TCA cycle and DHODH activity.


Assuntos
Mitocôndrias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Ubiquinona/análogos & derivados , Animais , Linhagem Celular Tumoral , Proliferação de Células , Ciona intestinalis/enzimologia , Ciclo do Ácido Cítrico , Citosol/metabolismo , Di-Hidro-Orotato Desidrogenase , Transporte de Elétrons , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/deficiência , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Levilactobacillus brevis/enzimologia , Masculino , Camundongos , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NAD/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Neoplasias/enzimologia , Fosforilação Oxidativa , Oxirredutases/genética , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ubiquinona/metabolismo
10.
Nucleic Acids Res ; 52(10): 5852-5865, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38742638

RESUMO

Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.


Assuntos
Cobamidas , Proteínas de Escherichia coli , Escherichia coli , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano , RNA Mensageiro , Riboswitch , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Cobamidas/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Iniciação Traducional da Cadeia Peptídica , RNA Helicases/genética , RNA Helicases/metabolismo , Endorribonucleases/metabolismo , Endorribonucleases/genética , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Proteínas da Membrana Bacteriana Externa , Polirribonucleotídeo Nucleotidiltransferase , Proteínas de Membrana Transportadoras
11.
Nucleic Acids Res ; 52(15): 9161-9173, 2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-38842944

RESUMO

The ompD transcript, encoding an outer membrane porin in Salmonella, harbors a controlling element in its coding region that base-pairs imperfectly with a 'seed' region of the small regulatory RNA (sRNA) MicC. When tagged with the sRNA, the ompD mRNA is cleaved downstream of the pairing site by the conserved endoribonuclease RNase E, leading to transcript destruction. We observe that the sRNA-induced cleavage site is accessible to RNase E in vitro upon recruitment of ompD into the 30S translation pre-initiation complex (PIC) in the presence of the degradosome components. Evaluation of substrate accessibility suggests that the paused 30S PIC presents the mRNA for targeted recognition and degradation. Ribonuclease activity on PIC-bound ompD is critically dependent on the recruitment of RNase E into the multi-enzyme RNA degradosome, and our data suggest a process of substrate capture and handover to catalytic sites within the degradosome, in which sequential steps of seed matching and duplex remodelling contribute to cleavage efficiency. Our findings support a putative mechanism of surveillance at translation that potentially terminates gene expression efficiently and rapidly in response to signals provided by regulatory RNA.


Assuntos
Endorribonucleases , Complexos Multienzimáticos , Polirribonucleotídeo Nucleotidiltransferase , Porinas , RNA Helicases , Estabilidade de RNA , RNA Mensageiro , Endorribonucleases/metabolismo , Endorribonucleases/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Helicases/metabolismo , RNA Helicases/genética , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Estabilidade de RNA/genética , Porinas/metabolismo , Porinas/genética , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , Pequeno RNA não Traduzido/metabolismo , Pequeno RNA não Traduzido/genética , Regulação Bacteriana da Expressão Gênica
12.
Exp Cell Res ; 440(1): 114118, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38852763

RESUMO

Autophagy phenomenon in the cell maintains proteostasis balance by eliminating damaged organelles and protein aggregates. Imbalance in autophagic flux may cause accumulation of protein aggregates in various neurodegenerative disorders. Regulation of autophagy by either calcium or chaperone play a key role in the removal of protein aggregates from the cell. The neuromuscular rare genetic disorder, GNE Myopathy, is characterized by accumulation of rimmed vacuoles having protein aggregates of ß-amyloid and tau that may result from altered autophagic flux. In the present study, the autophagic flux was deciphered in HEK cell-based model for GNE Myopathy harbouring GNE mutations of Indian origin. The refolding activity of HSP70 chaperone was found to be reduced in GNE mutant cells compared to wild type controls. The autophagic markers LC3II/I ratio was altered with increased number of autophagosome formation in GNE mutant cells compared to wild type cells. The cytosolic calcium levels were also increased in GNE mutant cells of Indian origin. Interestingly, treatment of GNE mutant cells with HSP70 activator, BGP-15, restored the expression and refolding activity of HSP70 along with autophagosome formation. Treatment with calcium chelator, BAPTA-AM restored the cytoplasmic calcium levels and autophagosome formation but not LC3II/I ratio significantly. Our study provides insights towards GNE mutation specific response for autophagy regulation and opens up a therapeutic advancement area in calcium signalling and HSP70 function for GNE related Myopathy.


Assuntos
Autofagia , Cálcio , Miopatias Distais , Proteínas de Choque Térmico HSP70 , Complexos Multienzimáticos , Mutação , Humanos , Autofagia/genética , Autofagia/efeitos dos fármacos , Mutação/genética , Cálcio/metabolismo , Miopatias Distais/genética , Miopatias Distais/metabolismo , Miopatias Distais/patologia , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Células HEK293 , Autofagossomos/metabolismo , Autofagossomos/efeitos dos fármacos , Índia
13.
Nature ; 570(7760): 194-199, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31142841

RESUMO

Serine hydroxymethyltransferase 2 (SHMT2) regulates one-carbon transfer reactions that are essential for amino acid and nucleotide metabolism, and uses pyridoxal-5'-phosphate (PLP) as a cofactor. Apo SHMT2 exists as a dimer with unknown functions, whereas PLP binding stabilizes the active tetrameric state. SHMT2 also promotes inflammatory cytokine signalling by interacting with the deubiquitylating BRCC36 isopeptidase complex (BRISC), although it is unclear whether this function relates to metabolism. Here we present the cryo-electron microscopy structure of the human BRISC-SHMT2 complex at a resolution of 3.8 Å. BRISC is a U-shaped dimer of four subunits, and SHMT2 sterically blocks the BRCC36 active site and inhibits deubiquitylase activity. Only the inactive SHMT2 dimer-and not the active PLP-bound tetramer-binds and inhibits BRISC. Mutations in BRISC that disrupt SHMT2 binding impair type I interferon signalling in response to inflammatory stimuli. Intracellular levels of PLP regulate the interaction between BRISC and SHMT2, as well as inflammatory cytokine responses. These data reveal a mechanism in which metabolites regulate deubiquitylase activity and inflammatory signalling.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Glicina Hidroximetiltransferase/metabolismo , Interferon Tipo I/imunologia , Complexos Multienzimáticos/imunologia , Complexos Multienzimáticos/metabolismo , Transdução de Sinais/imunologia , Microscopia Crioeletrônica , Enzimas Desubiquitinantes/antagonistas & inibidores , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/ultraestrutura , Glicina Hidroximetiltransferase/ultraestrutura , Células HEK293 , Humanos , Inflamação/imunologia , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mutação , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Fosfato de Piridoxal/metabolismo
14.
Nucleic Acids Res ; 51(4): 1971-1983, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36762482

RESUMO

The uridine at the 34th position of tRNA, which is able to base pair with the 3'-end codon on mRNA, is usually modified to influence many aspects of decoding properties during translation. Derivatives of 5-methyluridine (xm5U), which include methylaminomethyl (mnm-) or carboxymethylaminomethyl (cmnm-) groups at C5 of uracil base, are widely conserved at the 34th position of many prokaryotic tRNAs. In Gram-negative bacteria such as Escherichia coli, a bifunctional MnmC is involved in the last two reactions of the biosynthesis of mnm5(s2)U, in which the enzyme first converts cmnm5(s2)U to 5-aminomethyl-(2-thio)uridine (nm5(s2)U) and subsequently installs the methyl group to complete the formation of mnm5(s2)U. Although mnm5s2U has been identified in tRNAs of Gram-positive bacteria and plants as well, their genomes do not contain an mnmC ortholog and the gene(s) responsible for this modification is unknown. We discovered that MnmM, previously known as YtqB, is the methyltransferase that converts nm5s2U to mnm5s2U in Bacillus subtilis through comparative genomics, gene complementation experiments, and in vitro assays. Furthermore, we determined X-ray crystal structures of MnmM complexed with anticodon stem loop of tRNAGln. The structures provide the molecular basis underlying the importance of U33-nm5s2U34-U35 as the key determinant for the specificity of MnmM.


Assuntos
Proteínas de Escherichia coli , Metiltransferases , Complexos Multienzimáticos , Escherichia coli/genética , Complexos Multienzimáticos/genética , RNA de Transferência/genética , Tiouridina , Uridina/química
15.
Trends Biochem Sci ; 45(1): 42-57, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31679841

RESUMO

Bacterial RNA degradosomes are multienzyme molecular machines that act as hubs for post-transcriptional regulation of gene expression. The ribonuclease activities of these complexes require tight regulation, as they are usually essential for cell survival while potentially destructive. Recent studies have unveiled a wide variety of regulatory mechanisms including autoregulation, post-translational modifications, and protein compartmentalization. Recently, the subcellular organization of bacterial RNA degradosomes was found to present similarities with eukaryotic messenger ribonucleoprotein (mRNP) granules, membraneless compartments that are also involved in mRNA and protein storage and/or mRNA degradation. In this review, we present the current knowledge on the composition and targets of RNA degradosomes, the most recent developments regarding the regulation of these machineries, and their similarities with the eukaryotic mRNP granules.


Assuntos
Endorribonucleases/metabolismo , Complexos Multienzimáticos/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , RNA Bacteriano/metabolismo , Endorribonucleases/genética , Complexos Multienzimáticos/genética , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Helicases/genética
16.
Muscle Nerve ; 69(6): 708-718, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38558464

RESUMO

INTRODUCTION/AIMS: GNE myopathy is a rare autosomal recessive disorder caused by pathogenic variants in the GNE gene, which is essential for the sialic acid biosynthesis pathway. Although over 300 GNE variants have been reported, some patients remain undiagnosed with monoallelic pathogenic variants. This study aims to analyze the entire GNE genomic region to identify novel pathogenic variants. METHODS: Patients with clinically compatible GNE myopathy and monoallelic pathogenic variants in the GNE gene were enrolled. The other GNE pathogenic variant was verified using comprehensive methods including exon 2 quantitative polymerase chain reaction and nanopore long-read single-molecule sequencing (LRS). RESULTS: A deep intronic GNE variant, c.862+870C>T, was identified in nine patients from eight unrelated families. This variant generates a cryptic splice site, resulting in the activation of a novel pseudoexon between exons 5 and 6. It results in the insertion of an extra 146 nucleotides into the messengerRNA (mRNA), which is predicted to result in a truncated humanGNE1(hGNE1) protein. Peanut agglutinin(PNA) lectin staining of muscle tissues showed reduced sialylation of mucin O-glycans on sarcolemmal glycoproteins. Notably, a third of patients with the c.862+870C>T variant exhibited thrombocytopenia. A common core haplotype harboring the deep intronic GNE variant was found in all these patients. DISCUSSION: The transcript with pseudoexon activation potentially affects sialic acid biosynthesis via nonsense-mediated mRNA decay, or resulting in a truncated hGNE1 protein, which interferes with normal enzyme function. LRS is expected to be more frequently incorporated in genetic analysis given its efficacy in detecting hard-to-find pathogenic variants.


Assuntos
Éxons , Íntrons , Complexos Multienzimáticos , Trombocitopenia , Humanos , Masculino , Feminino , Complexos Multienzimáticos/genética , Éxons/genética , Íntrons/genética , Adulto , Trombocitopenia/genética , Miopatias Distais/genética , Adulto Jovem , Adolescente , Criança , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Linhagem , Pessoa de Meia-Idade
17.
Proc Natl Acad Sci U S A ; 118(28)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34260408

RESUMO

How noncoding transcription influences chromatin states is still unclear. The Arabidopsis floral repressor gene FLC is quantitatively regulated through an antisense-mediated chromatin silencing mechanism. The FLC antisense transcripts form a cotranscriptional R-loop that is dynamically resolved by RNA 3' processing factors (FCA and FY), and this is linked to chromatin silencing. Here, we investigate this silencing mechanism and show, using single-molecule DNA fiber analysis, that FCA and FY are required for unimpeded replication fork progression across the Arabidopsis genome. We then employ the chicken DT40 cell line system, developed to investigate sequence-dependent replication and chromatin inheritance, and find that FLC R-loop sequences have an orientation-dependent ability to stall replication forks. These data suggest a coordination between RNA 3' processing of antisense RNA and replication fork progression in the inheritance of chromatin silencing at FLC.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cromatina/genética , Replicação do DNA/genética , Inativação Gênica , Proteínas de Domínio MADS/genética , Processamento Pós-Transcricional do RNA/genética , RNA Antissenso/genética , Animais , Proteínas de Arabidopsis/metabolismo , Linhagem Celular , Galinhas , DNA de Plantas/química , DNA de Plantas/genética , DNA Polimerase Dirigida por DNA/genética , Complexos Multienzimáticos/genética , Conformação de Ácido Nucleico
18.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34544865

RESUMO

Bats are responsible for the zoonotic transmission of several major viral diseases, including those leading to the 2003 SARS outbreak and likely the ongoing COVID-19 pandemic. While comparative genomics studies have revealed characteristic adaptations of the bat innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the viral tolerance in bats. Here we report the establishment of genome-wide RNA interference (RNAi) and CRISPR libraries for the screening of the model megabat, Pteropus alecto. We used the complementary RNAi and CRISPR libraries to interrogate P. alecto cells for infection with two different viruses: mumps virus and influenza A virus, respectively. Independent screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells and human cells. The MTHFD1 inhibitor, carolacton, potently blocked replication of several RNA viruses, including SARS-CoV-2. We also discovered that bats have lower expression levels of MTHFD1 than humans. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad-spectrum antiviral therapy.


Assuntos
Aminoidrolases/genética , COVID-19/genética , Formiato-Tetra-Hidrofolato Ligase/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Complexos Multienzimáticos/genética , Pandemias , Aminoidrolases/antagonistas & inibidores , Animais , Antivirais/uso terapêutico , COVID-19/virologia , Linhagem Celular , Quirópteros/genética , Quirópteros/virologia , Formiato-Tetra-Hidrofolato Ligase/antagonistas & inibidores , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Antígenos de Histocompatibilidade Menor , Complexos Multienzimáticos/antagonistas & inibidores , Vírus de RNA/genética , SARS-CoV-2/patogenicidade , Replicação Viral/genética , Tratamento Farmacológico da COVID-19
19.
Plant J ; 109(4): 844-855, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34807484

RESUMO

l-Tyrosine is an essential amino acid for protein synthesis and is also used in plants to synthesize diverse natural products. Plants primarily synthesize tyrosine via TyrA arogenate dehydrogenase (TyrAa or ADH), which are typically strongly feedback inhibited by tyrosine. However, two plant lineages, Fabaceae (legumes) and Caryophyllales, have TyrA enzymes that exhibit relaxed sensitivity to tyrosine inhibition and are associated with elevated production of tyrosine-derived compounds, such as betalain pigments uniquely produced in core Caryophyllales. Although we previously showed that a single D222N substitution is primarily responsible for the deregulation of legume TyrAs, it is unknown when and how the deregulated Caryophyllales TyrA emerged. Here, through phylogeny-guided TyrA structure-function analysis, we found that functionally deregulated TyrAs evolved early in the core Caryophyllales before the origin of betalains, where the E208D amino acid substitution in the active site, which is at a different and opposite location from D222N found in legume TyrAs, played a key role in the TyrA functionalization. Unlike legumes, however, additional substitutions on non-active site residues further contributed to the deregulation of TyrAs in Caryophyllales. The introduction of a mutation analogous to E208D partially deregulated tyrosine-sensitive TyrAs, such as Arabidopsis TyrA2 (AtTyrA2). Moreover, the combined introduction of D222N and E208D additively deregulated AtTyrA2, for which the expression in Nicotiana benthamiana led to highly elevated accumulation of tyrosine in planta. The present study demonstrates that phylogeny-guided characterization of key residues underlying primary metabolic innovations can provide powerful tools to boost the production of essential plant natural products.


Assuntos
Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mutagênese , Plantas/genética , Plantas/metabolismo , Tirosina/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis , Betalaínas/biossíntese , Caryophyllales/genética , Caryophyllales/metabolismo , Fabaceae , Complexos Multienzimáticos/classificação , Oxirredutases/genética , Oxirredutases/metabolismo , Filogenia , Prefenato Desidrogenase/genética , Prefenato Desidrogenase/metabolismo
20.
RNA ; 27(11): 1339-1352, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34341070

RESUMO

The conserved endoribonuclease RNase E dominates the dynamic landscape of RNA metabolism and underpins control mediated by small regulatory RNAs in diverse bacterial species. We explored the enzyme's hydrolytic mechanism, allosteric activation, and interplay with partner proteins in the multicomponent RNA degradosome assembly of Escherichia coli. RNase E cleaves single-stranded RNA with preference to attack the phosphate located at the 5' nucleotide preceding uracil, and we corroborate key interactions that select that base. Unexpectedly, RNase E activity is impeded strongly when the recognized uracil is isomerized to 5-ribosyluracil (pseudouridine), from which we infer the detailed geometry of the hydrolytic attack process. Kinetics analyses support models for recognition of secondary structure in substrates by RNase E and for allosteric autoregulation. The catalytic power of the enzyme is boosted when it is assembled into the multienzyme RNA degradosome, most likely as a consequence of substrate capture and presentation. Our results rationalize the origins of substrate preferences of RNase E and illuminate its catalytic mechanism, supporting the roles of allosteric domain closure and cooperation with other components of the RNA degradosome complex.


Assuntos
Endorribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Complexos Multienzimáticos/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Pseudouridina/metabolismo , RNA Helicases/metabolismo , RNA Bacteriano/metabolismo , Sítios de Ligação , Endorribonucleases/química , Endorribonucleases/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Complexos Multienzimáticos/genética , Conformação de Ácido Nucleico , Polirribonucleotídeo Nucleotidiltransferase/genética , Conformação Proteica , RNA Helicases/genética , RNA Bacteriano/genética
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