Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
Mais filtros










Base de dados
Tipo de estudo
Intervalo de ano de publicação
1.
Nature ; 594(7861): 111-116, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34012115

RESUMO

Ubiquitylation is a widespread post-translational protein modification in eukaryotes and marks bacteria that invade the cytosol as cargo for antibacterial autophagy1-3. The identity of the ubiquitylated substrate on bacteria is unknown. Here we show that the ubiquitin coat on Salmonella that invade the cytosol is formed through the ubiquitylation of a non-proteinaceous substrate, the lipid A moiety of bacterial lipopolysaccharide (LPS), by the E3 ubiquitin ligase ring finger protein 213 (RNF213). RNF213 is a risk factor for moyamoya disease4,5, which is a progressive stenosis of the supraclinoid internal carotid artery that causes stroke (especially in children)6,7. RNF213 restricts the proliferation of cytosolic Salmonella and is essential for the generation of the bacterial ubiquitin coat, both directly (through the ubiquitylation of LPS) and indirectly (through the recruitment of LUBAC, which is a downstream E3 ligase that adds M1-linked ubiquitin chains onto pre-existing ubiquitin coats8). In cells that lack RNF213, bacteria do not attract ubiquitin-dependent autophagy receptors or induce antibacterial autophagy. The ubiquitylation of LPS on Salmonella that invade the cytosol requires the dynein-like core of RNF213, but not its RING domain. Instead, ubiquitylation of LPS relies on an RZ finger in the E3 shell. We conclude that ubiquitylation extends beyond protein substrates and that ubiquitylation of LPS triggers cell-autonomous immunity, and we postulate that non-proteinaceous substances other than LPS may also become ubiquitylated.


Assuntos
Adenosina Trifosfatases/metabolismo , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo , Salmonella typhimurium , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Autofagia , Linhagem Celular , Células HeLa , Humanos , Camundongos , Domínios RING Finger , Infecções por Salmonella/microbiologia , Ubiquitina/metabolismo
2.
Mol Cell ; 81(1): 88-103.e6, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33220178

RESUMO

The small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B's nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB's efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (cryo-EM) showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(αP) and ISRIB reciprocally opposed each other's binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.


Assuntos
Acetamidas/química , Cicloexilaminas/química , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/química , Regulação Alostérica , Animais , Sítios de Ligação , Células CHO , Cricetulus , Microscopia Crioeletrônica , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/genética , Fator de Iniciação 2B em Eucariotos/metabolismo , Células HeLa , Humanos , Fosforilação
3.
J Biol Chem ; 294(36): 13478-13486, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31337709

RESUMO

The aminoguanidine compound robenidine is widely used as an antibiotic for the control of coccidiosis, a protozoal infection in poultry and rabbits. Interestingly, robenidine is structurally similar to guanabenz (analogs), which are currently undergoing clinical trials as cytoprotective agents for the management of neurodegenerative diseases. Here we show that robenidine and guanabenz protect cells from a tunicamycin-induced unfolded protein response to a similar degree. Both compounds also reduced the tumor necrosis factor α-induced activation of NF-κB. The cytoprotective effects of guanabenz (analogs) have been explained previously by their ability to maintain eIF2α phosphorylation by allosterically inhibiting protein phosphatase PP1:PPP1R15A. However, using a novel split-luciferase-based protein-protein interaction assay, we demonstrate here that neither robenidine nor guanabenz disrupt the interaction between PPP1R15A and either PP1 or eIF2α in intact cells. Moreover, both drugs also inhibited the unfolded protein response in cells that expressed a nonphosphorylatable mutant (S51A) of eIF2α. Our results identify robenidine as a PP1:PPP1R15A-independent cytoprotective compound that holds potential for the management of protein misfolding-associated diseases.


Assuntos
Antibacterianos/farmacologia , Substâncias Protetoras/farmacologia , Proteína Fosfatase 1/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Robenidina/farmacologia , Animais , Células CHO , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cricetulus , Relação Dose-Resposta a Droga , Células HEK293 , Células HeLa , Humanos , Relação Estrutura-Atividade
4.
J Biol Chem ; 293(20): 7766-7776, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29618508

RESUMO

The integrated stress response (ISR) is regulated by kinases that phosphorylate the α subunit of translation initiation factor 2 and phosphatases that dephosphorylate it. Genetic and biochemical observations indicate that the eIF2αP-directed holophosphatase, a therapeutic target in diseases of protein misfolding, is comprised of a regulatory subunit, PPP1R15, and a catalytic subunit, protein phosphatase 1 (PP1). In mammals, there are two isoforms of the regulatory subunit, PPP1R15A and PPP1R15B, with overlapping roles in the essential function of eIF2αP dephosphorylation. However, conflicting reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific dephosphorylation by PPP1R15-containing PP1 holoenzymes. An additional concern relates to the sensitivity of the holoenzyme to the [(o-chlorobenzylidene)amino]guanidines Sephin1 or guanabenz, putative small-molecule proteostasis modulators. It has been suggested that the source and method of purification of the PP1 catalytic subunit and the presence or absence of an N-terminal repeat-containing region in the PPP1R15A regulatory subunit might influence the requirement for G-actin and sensitivity of the holoenzyme to inhibitors. We found that eIF2αP dephosphorylation by PP1 was moderately stimulated by repeat-containing PPP1R15A in an unphysiological low ionic strength buffer, whereas stimulation imparted by the co-presence of PPP1R15A and G-actin was observed under a broad range of conditions, low and physiological ionic strength, regardless of whether the PPP1R15A regulatory subunit had or lacked the N-terminal repeat-containing region and whether it was paired with native PP1 purified from rabbit muscle or recombinant PP1 purified from bacteria. Furthermore, none of the PPP1R15A-containing holophosphatases tested were inhibited by Sephin1 or guanabenz.


Assuntos
Actinas/metabolismo , Resistência a Medicamentos , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Guanabenzo/análogos & derivados , Proteína Fosfatase 1/antagonistas & inibidores , Animais , Domínio Catalítico , Guanabenzo/farmacologia , Células HeLa , Humanos , Fosforilação , Isoformas de Proteínas , Proteólise , Coelhos
5.
Science ; 359(6383): 1533-1536, 2018 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-29599245

RESUMO

The integrated stress response (ISR) is a conserved translational and transcriptional program affecting metabolism, memory, and immunity. The ISR is mediated by stress-induced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, reverses the attenuation of eIF2B by phosphorylated eIF2α, protecting mice from neurodegeneration and traumatic brain injury. We describe a 4.1-angstrom-resolution cryo-electron microscopy structure of human eIF2B with an ISRIB molecule bound at the interface between the ß and δ regulatory subunits. Mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB analogs in vivo and ISRIB binding in vitro. Our findings point to a site in eIF2B that can be exploited by ISRIB to regulate translation.


Assuntos
Acetamidas/química , Cicloexilaminas/química , Fator de Iniciação 2B em Eucariotos/química , Acetamidas/farmacologia , Animais , Microscopia Crioeletrônica , Cicloexilaminas/farmacologia , Fator de Iniciação 2B em Eucariotos/genética , Células HeLa , Humanos , Camundongos , Mutagênese , Fosforilação , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Conformação Proteica , Estresse Fisiológico/efeitos dos fármacos
6.
Elife ; 62017 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-28447936

RESUMO

Dephosphorylation of translation initiation factor 2 (eIF2α) terminates signalling in the mammalian integrated stress response (ISR) and has emerged as a promising target for modifying the course of protein misfolding diseases. The [(o-chlorobenzylidene)amino]guanidines (Guanabenz and Sephin1) have been proposed to exert protective effects against misfolding by interfering with eIF2α-P dephosphorylation through selective disruption of a PP1-PPP1R15A holophosphatase complex. Surprisingly, they proved inert in vitro affecting neither stability of the PP1-PPP1R15A complex nor substrate-specific dephosphorylation. Furthermore, eIF2α-P dephosphorylation, assessed by a kinase shut-off experiment, progressed normally in Sephin1-treated cells. Consistent with its role in defending proteostasis, Sephin1 attenuated the IRE1 branch of the endoplasmic reticulum unfolded protein response. However, repression was noted in both wildtype and Ppp1r15a deleted cells and in cells rendered ISR-deficient by CRISPR editing of the Eif2s1 locus to encode a non-phosphorylatable eIF2α (eIF2αS51A). These findings challenge the view that [(o-chlorobenzylidene)amino]guanidines restore proteostasis by interfering with eIF2α-P dephosphorylation.


Assuntos
Inibidores Enzimáticos/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Guanabenzo/análogos & derivados , Guanabenzo/metabolismo , Proteína Fosfatase 1/antagonistas & inibidores , Processamento de Proteína Pós-Traducional
7.
PLoS One ; 11(11): e0166278, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27812215

RESUMO

The eukaryotic translation initiation factor eIF2B promotes mRNA translation as a guanine nucleotide exchange factor (GEF) for translation initiation factor 2 (eIF2). Endoplasmic reticulum (ER) stress-mediated activation of the kinase PERK and the resultant phosphorylation of eIF2's alpha subunit (eIF2α) attenuates eIF2B GEF activity thereby inducing an integrated stress response (ISR) that defends against protein misfolding in the ER. Mutations in all five subunits of human eIF2B cause an inherited leukoencephalopathy with vanishing white matter (VWM), but the role of the ISR in its pathogenesis remains unclear. Using CRISPR-Cas9 genome editing we introduced the most severe known VWM mutation, EIF2B4A391D, into CHO cells. Compared to isogenic wildtype cells, GEF activity of cells with the VWM mutation was impaired and the mutant cells experienced modest enhancement of the ISR. However, despite their enhanced ISR, imposed by the intrinsic defect in eIF2B, disrupting the inhibitory effect of phosphorylated eIF2α on GEF by a contravening EIF2S1/eIF2αS51A mutation that functions upstream of eIF2B, selectively enfeebled both EIF2B4A391D and the related severe VWM EIF2B4R483W cells. The basis for paradoxical dependence of cells with the VWM mutations on an intact eIF2α genotype remains unclear, as both translation rates and survival from stressors that normally activate the ISR were not reproducibly affected by the VWM mutations. Nonetheless, our findings support an additional layer of complexity in the development of VWM, beyond a hyperactive ISR.


Assuntos
Estresse do Retículo Endoplasmático/genética , Fator de Iniciação 2B em Eucariotos/genética , Mutação , Substância Branca/citologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Fator de Iniciação 2B em Eucariotos/química , Humanos , Recombinação Genética , Resposta a Proteínas não Dobradas/genética , Substância Branca/metabolismo
8.
Elife ; 4: e08961, 2015 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-26473973

RESUMO

DnaK/Hsp70 chaperones form oligomers of poorly understood structure and functional significance. Site-specific proteolysis and crosslinking were used to probe the architecture of oligomers formed by the endoplasmic reticulum (ER) Hsp70, BiP. These were found to consist of adjacent protomers engaging the interdomain linker of one molecule in the substrate binding site of another, attenuating the chaperone function of oligomeric BiP. Native gel electrophoresis revealed a rapidly-modulated reciprocal relationship between the burden of unfolded proteins and BiP oligomers and slower equilibration between oligomers and inactive, covalently-modified BiP. Lumenal ER calcium depletion caused rapid oligomerization of mammalian BiP and a coincidental diminution in substrate binding, pointing to the relative inertness of the oligomers. Thus, equilibration between inactive oligomers and active monomeric BiP is poised to buffer fluctuations in ER unfolded protein load on a rapid timescale attainable neither by inter-conversion of active and covalently-modified BiP nor by the conventional unfolded protein response.


Assuntos
Proteínas de Choque Térmico/metabolismo , Multimerização Proteica , Animais , Cricetinae , Eletroforese , Retículo Endoplasmático/enzimologia , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
9.
Science ; 348(6238): 1027-30, 2015 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-25858979

RESUMO

The integrated stress response (ISR) modulates messenger RNA translation to regulate the mammalian unfolded protein response (UPR), immunity, and memory formation. A chemical ISR inhibitor, ISRIB, enhances cognitive function and modulates the UPR in vivo. To explore mechanisms involved in ISRIB action, we screened cultured mammalian cells for somatic mutations that reversed its effect on the ISR. Clustered missense mutations were found at the amino-terminal portion of the delta subunit of guanine nucleotide exchange factor (GEF) eIF2B. When reintroduced by CRISPR-Cas9 gene editing of wild-type cells, these mutations reversed both ISRIB-mediated inhibition of the ISR and its stimulatory effect on eIF2B GEF activity toward its substrate, the translation initiation factor eIF2, in vitro. Thus, ISRIB targets an interaction between eIF2 and eIF2B that lies at the core of the ISR.


Assuntos
Acetamidas/farmacologia , Cicloexilaminas/farmacologia , Resistência a Medicamentos/genética , Fator de Iniciação 2B em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Memória/efeitos dos fármacos , Nootrópicos/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Células CHO , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cricetulus , Fator de Iniciação 2B em Eucariotos/metabolismo , Testes Genéticos , Mutação de Sentido Incorreto , Biossíntese de Proteínas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/imunologia
10.
Elife ; 42015 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-25774600

RESUMO

Dephosphorylation of eukaryotic translation initiation factor 2a (eIF2a) restores protein synthesis at the waning of stress responses and requires a PP1 catalytic subunit and a regulatory subunit, PPP1R15A/GADD34 or PPP1R15B/CReP. Surprisingly, PPP1R15-PP1 binary complexes reconstituted in vitro lacked substrate selectivity. However, selectivity was restored by crude cell lysate or purified G-actin, which joined PPP1R15-PP1 to form a stable ternary complex. In crystal structures of the non-selective PPP1R15B-PP1G complex, the functional core of PPP1R15 made multiple surface contacts with PP1G, but at a distance from the active site, whereas in the substrate-selective ternary complex, actin contributes to one face of a platform encompassing the active site. Computational docking of the N-terminal lobe of eIF2a at this platform placed phosphorylated serine 51 near the active site. Mutagenesis of predicted surface-contacting residues enfeebled dephosphorylation, suggesting that avidity for the substrate plays an important role in imparting specificity on the PPP1R15B-PP1G-actin ternary complex.


Assuntos
Actinas/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteína Fosfatase 1/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Domínio Catalítico , Bovinos , Sequência Conservada , Cricetinae , Cricetulus , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Fosforilação , Coelhos , Especificidade por Substrato
11.
BMC Biol ; 13: 2, 2015 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-25575667

RESUMO

BACKGROUND: Endoplasmic reticulum (ER) lumenal protein thiol redox balance resists dramatic variation in unfolded protein load imposed by diverse physiological challenges including compromise in the key upstream oxidases. Lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting a rapid and reversible shift towards a more reducing poise. Calcium depletion induced ER redox alterations are relevant to physiological conditions associated with calcium signaling, such as the response of pancreatic cells to secretagogues and neuronal activity. The core components of the ER redox machinery are well characterized; however, the molecular basis for the calcium-depletion induced shift in redox balance is presently obscure. RESULTS: In vitro, the core machinery for generating disulfides, consisting of ERO1 and the oxidizing protein disulfide isomerase, PDI1A, was indifferent to variation in calcium concentration within the physiological range. However, ER calcium depletion in vivo led to a selective 2.5-fold decline in PDI1A mobility, whereas the mobility of the reducing PDI family member, ERdj5 was unaffected. In vivo, fluorescence resonance energy transfer measurements revealed that declining PDI1A mobility correlated with formation of a complex with the abundant ER chaperone calreticulin, whose mobility was also inhibited by calcium depletion and the calcium depletion-mediated reductive shift was attenuated in cells lacking calreticulin. Measurements with purified proteins confirmed that the PDI1A-calreticulin complex dissociated as Ca(2+) concentrations approached those normally found in the ER lumen ([Ca(2+)]K(0.5max) = 190 µM). CONCLUSIONS: Our findings suggest that selective sequestration of PDI1A in a calcium depletion-mediated complex with the abundant chaperone calreticulin attenuates the effective concentration of this major lumenal thiol oxidant, providing a plausible and simple mechanism for the observed shift in ER lumenal redox poise upon physiological calcium depletion.


Assuntos
Cálcio/deficiência , Difusão , Retículo Endoplasmático/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Animais , Células COS , Cálcio/metabolismo , Calreticulina/metabolismo , Chlorocebus aethiops , Dissulfetos/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Camundongos , Chaperonas Moleculares/metabolismo , Oxirredução , Ligação Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...