Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Commun ; 12(1): 3440, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103529

RESUMO

The multi-subunit translation initiation factor eIF2B is a control node for protein synthesis. eIF2B activity is canonically modulated through stress-responsive phosphorylation of its substrate eIF2. The eIF2B regulatory subcomplex is evolutionarily related to sugar-metabolizing enzymes, but the biological relevance of this relationship was unknown. To identify natural ligands that might regulate eIF2B, we conduct unbiased binding- and activity-based screens followed by structural studies. We find that sugar phosphates occupy the ancestral catalytic site in the eIF2Bα subunit, promote eIF2B holoenzyme formation and enhance enzymatic activity towards eIF2. A mutant in the eIF2Bα ligand pocket that causes Vanishing White Matter disease fails to engage and is not stimulated by sugar phosphates. These data underscore the importance of allosteric metabolite modulation for proper eIF2B function. We propose that eIF2B evolved to couple nutrient status via sugar phosphate sensing with the rate of protein synthesis, one of the most energetically costly cellular processes.


Assuntos
Fator de Iniciação 2B em Eucariotos/metabolismo , Estresse Fisiológico , Fosfatos Açúcares/metabolismo , Regulação Alostérica , Sítios de Ligação , Sequência Conservada , Microscopia Crioeletrônica , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2B em Eucariotos/ultraestrutura , Evolução Molecular , Guanosina Difosfato/metabolismo , Células HEK293 , Humanos , Leucoencefalopatias/patologia , Ligantes , Metaboloma , Modelos Moleculares , Mutação/genética , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade por Substrato , Fosfatos Açúcares/química
2.
Biochimie ; 183: 100-107, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33476699

RESUMO

The folate and methionine cycles, constituting one-carbon metabolism, are critical pathways for cell survival. Intersecting these two cycles, 5,10-methylenetetrahydrofolate reductase (MTHFR) directs one-carbon units from the folate to methionine cycle, to be exclusively used for methionine and S-adenosylmethionine (AdoMet) synthesis. MTHFR deficiency and upregulation result in diverse disease states, rendering it an attractive drug target. The activity of MTHFR is inhibited by the binding of AdoMet to an allosteric regulatory domain distal to the enzyme's active site, which we have previously identified to constitute a novel fold with a druggable pocket. Here, we screened 162 AdoMet mimetics using differential scanning fluorimetry, and identified 4 compounds that stabilized this regulatory domain. Three compounds were sinefungin analogues, closely related to AdoMet and S-adenosylhomocysteine (AdoHcy). The strongest thermal stabilisation was provided by (S)-SKI-72, a potent inhibitor originally developed for protein arginine methyltransferase 4 (PRMT4). Using surface plasmon resonance, we confirmed that (S)-SKI-72 binds MTHFR via its allosteric domain with nanomolar affinity. Assay of MTHFR activity in the presence of (S)-SKI-72 demonstrates inhibition of purified enzyme with sub-micromolar potency and endogenous MTHFR from HEK293 cell lysate in the low micromolar range, both of which are lower than AdoMet. Nevertheless, unlike AdoMet, (S)-SKI-72 is unable to completely abolish MTHFR activity, even at very high concentrations. Combining binding assays, kinetic characterization and compound docking, this work indicates the regulatory domain of MTHFR can be targeted by small molecules and presents (S)-SKI-72 as an excellent candidate for development of MTHFR inhibitors.


Assuntos
Inibidores Enzimáticos/química , Metilenotetra-Hidrofolato Redutase (NADPH2)/antagonistas & inibidores , Metilenotetra-Hidrofolato Redutase (NADPH2)/química , S-Adenosilmetionina/química , Regulação Alostérica , Humanos , Domínios Proteicos
3.
IUCrJ ; 7(Pt 4): 693-706, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32695416

RESUMO

DHTKD1 is a lesser-studied E1 enzyme among the family of 2-oxoacid de-hydrogenases. In complex with E2 (di-hydro-lipo-amide succinyltransferase, DLST) and E3 (dihydrolipo-amide de-hydrogenase, DLD) components, DHTKD1 is involved in lysine and tryptophan catabolism by catalysing the oxidative de-carboxyl-ation of 2-oxoadipate (2OA) in mitochondria. Here, the 1.9 Šresolution crystal structure of human DHTKD1 is solved in complex with the thi-amine diphosphate co-factor. The structure reveals how the DHTKD1 active site is modelled upon the well characterized homologue 2-oxoglutarate (2OG) de-hydrogenase but engineered specifically to accommodate its preference for the longer substrate of 2OA over 2OG. A 4.7 Šresolution reconstruction of the human DLST catalytic core is also generated by single-particle electron microscopy, revealing a 24-mer cubic scaffold for assembling DHTKD1 and DLD protomers into a megacomplex. It is further demonstrated that missense DHTKD1 variants causing the inborn error of 2-amino-adipic and 2-oxoadipic aciduria impact on the complex formation, either directly by disrupting the interaction with DLST, or indirectly through destabilizing the DHTKD1 protein. This study provides the starting framework for developing DHTKD1 modulators to probe the intricate mitochondrial energy metabolism.

4.
Elife ; 4: e06792, 2015 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-26002083

RESUMO

Salmonella PhoQ is a histidine kinase with a periplasmic sensor domain (PD) that promotes virulence by detecting the macrophage phagosome. PhoQ activity is repressed by divalent cations and induced in environments of acidic pH, limited divalent cations, and cationic antimicrobial peptides (CAMP). Previously, it was unclear which signals are sensed by salmonellae to promote PhoQ-mediated virulence. We defined conformational changes produced in the PhoQ PD on exposure to acidic pH that indicate structural flexibility is induced in α-helices 4 and 5, suggesting this region contributes to pH sensing. Therefore, we engineered a disulfide bond between W104C and A128C in the PhoQ PD that restrains conformational flexibility in α-helices 4 and 5. PhoQ(W104C-A128C) is responsive to CAMP, but is inhibited for activation by acidic pH and divalent cation limitation. phoQ(W104C-A128C) Salmonella enterica Typhimurium is virulent in mice, indicating that acidic pH and divalent cation sensing by PhoQ are dispensable for virulence.


Assuntos
Proteínas de Bactérias/metabolismo , Cátions Bivalentes/metabolismo , Regulação Bacteriana da Expressão Gênica , Concentração de Íons de Hidrogênio , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/patogenicidade , Fatores de Virulência/biossíntese , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica/efeitos dos fármacos , Salmonelose Animal/microbiologia , Salmonelose Animal/patologia , Virulência
5.
Structure ; 20(7): 1177-88, 2012 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-22705207

RESUMO

pH sensing is crucial for survival of most organisms, yet the molecular basis of such sensing is poorly understood. Here, we present an atomic resolution structure of the periplasmic portion of the acid-sensing chemoreceptor, TlpB, from the gastric pathogen Helicobacter pylori. The structure reveals a universal signaling fold, a PAS domain, with a molecule of urea bound with high affinity. Through biophysical, biochemical, and in vivo mutagenesis studies, we show that urea and the urea-binding site residues play critical roles in the ability of H. pylori to sense acid. Our signaling model predicts that protonation events at Asp114, affected by changes in pH, dictate the stability of TlpB through urea binding.


Assuntos
Proteínas de Bactérias/química , Helicobacter pylori/metabolismo , Prótons , Receptores de Superfície Celular/química , Ureia/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Escherichia coli , Helicobacter pylori/genética , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Plasmídeos , Ligação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Ureia/metabolismo
6.
Mol Microbiol ; 82(5): 1277-90, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22017253

RESUMO

Productive intercellular delivery of cargo by secretory systems requires exquisite temporal and spatial choreography. Our laboratory has demonstrated that the haemolysin co-regulated secretion island I (HSI-I)-encoded type VI secretion system (H1-T6SS) of Pseudomonas aeruginosa transfers effector proteins to other bacterial cells. The activity of these effectors requires cell contact-dependent delivery by the secretion apparatus, and thus their export is highly repressed under planktonic growth conditions. Here we define regulatory pathways that orchestrate efficient secretion by this system. We identified a T6S-associated protein, TagF, as a posttranslational repressor of the H1-T6SS. Strains activated by TagF derepression or stimulated through a previously identified threonine phosphorylation pathway (TPP) share the property of secretory ATPase recruitment to the T6S apparatus, yet display different effector output levels and genetic requirements for their export. We also found that these two pathways respond to distinct stimuli; we identified surface growth as a physiological cue that activates the H1-T6SS exclusively through the TPP. Coordination of posttranslational triggering with cell contact-promoting growth conditions provides a mechanism for the T6SS to avoid wasteful release of effectors.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Pseudomonas aeruginosa/fisiologia , Ilhas Genômicas , Fosforilação , Transporte Proteico , Pseudomonas aeruginosa/genética
7.
Microbiology (Reading) ; 157(Pt 9): 2445-2455, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21602215

RESUMO

Helicobacter pylori moves in response to environmental chemical cues using a chemotaxis two-component signal-transduction system. Autoinducer-2 (AI-2) is a quorum-sensing signal produced by the LuxS protein that accumulates in the bacterial environment in a density-dependent manner. We showed previously that a H. pylori luxS mutant was defective in motility on soft agar plates. Here we report that deletion of the luxS gene resulted in swimming behaviour with a reduced frequency of stops as compared to the wild-type strain. Stopping frequency was restored to wild-type levels by genetic complementation of the luxS mutation or by addition of synthetic 4,5-dihydroxy-2,3-pentanedione (DPD), which cyclizes to form AI-2. Synthetic DPD also increased the frequency of stops in wild-type H. pylori, similar to the behaviour induced by the known chemorepellent HCl. We found that whereas mutants lacking the chemoreceptor genes tlpA, tlpC or tlpD responded to an exogenous source of synthetic DPD, the chemoreceptor mutant tlpB was non-responsive to a gradient or uniform distribution of the chemical. Furthermore, a double mutant lacking both tlpB and luxS exhibited chemotactic behaviour similar to the tlpB single mutant, whereas a double mutant lacking both tlpB and the chemotransduction gene cheA behaved like a nonchemotactic cheA single mutant, supporting the model that tlpB functions in a signalling pathway downstream of luxS and upstream of cheA. We conclude that H. pylori perceives LuxS-produced AI-2 as a chemorepellent via the chemoreceptor TlpB.


Assuntos
Proteínas de Bactérias/metabolismo , Helicobacter pylori/metabolismo , Homosserina/análogos & derivados , Lactonas/metabolismo , Percepção de Quorum , Proteínas de Bactérias/genética , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/metabolismo , Quimiotaxia/efeitos dos fármacos , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Homosserina/metabolismo , Pentanos/metabolismo , Pentanos/farmacologia , Transdução de Sinais
8.
Cell Host Microbe ; 7(1): 25-37, 2010 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-20114026

RESUMO

The functional spectrum of a secretion system is defined by its substrates. Here we analyzed the secretomes of Pseudomonas aeruginosa mutants altered in regulation of the Hcp Secretion Island-I-encoded type VI secretion system (H1-T6SS). We identified three substrates of this system, proteins Tse1-3 (type six exported 1-3), which are coregulated with the secretory apparatus and secreted under tight posttranslational control. The Tse2 protein was found to be the toxin component of a toxin-immunity system and to arrest the growth of prokaryotic and eukaryotic cells when expressed intracellularly. In contrast, secreted Tse2 had no effect on eukaryotic cells; however, it provided a major growth advantage for P. aeruginosa strains, relative to those lacking immunity, in a manner dependent on cell contact and the H1-T6SS. This demonstration that the T6SS targets a toxin to bacteria helps reconcile the structural and evolutionary relationship between the T6SS and the bacteriophage tail and spike.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Antibacterianos/metabolismo , Antibacterianos/toxicidade , Antibiose , Proteínas de Bactérias/genética , Toxinas Bacterianas/toxicidade , Bacteriófagos/genética , Contagem de Colônia Microbiana , Evolução Molecular , Ordem dos Genes , Ilhas Genômicas , Proteínas de Membrana Transportadoras/genética , Pseudomonas aeruginosa/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...