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1.
Nat Struct Mol Biol ; 28(10): 835-846, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34625748

RESUMO

Many regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits engage the surface of PP1c, locally affecting flexible access of the phosphopeptide to the active site. However, catalytic efficiency of holophosphatases towards their phosphoprotein substrates remains unexplained. Here we present a cryo-EM structure of the tripartite PP1c-PPP1R15A-G-actin holophosphatase that terminates signaling in the mammalian integrated stress response (ISR) in the pre-dephosphorylation complex with its substrate, translation initiation factor 2α (eIF2α). G-actin, whose essential role in eIF2α dephosphorylation is supported crystallographically, biochemically and genetically, aligns the catalytic and regulatory subunits, creating a composite surface that engages the N-terminal domain of eIF2α to position the distant phosphoserine-51 at the active site. Substrate residues that mediate affinity for the holophosphatase also make critical contacts with eIF2α kinases. Thus, a convergent process of higher-order substrate recognition specifies functionally antagonistic phosphorylation and dephosphorylation in the ISR.


Assuntos
Proteína Fosfatase 1/química , Proteína Fosfatase 1/metabolismo , Estresse Fisiológico/fisiologia , eIF-2 Quinase/metabolismo , Actinas/química , Actinas/metabolismo , Animais , Células CHO , Domínio Catalítico , Cricetulus , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Modelos Moleculares , Fosforilação , Fosfosserina/metabolismo , Proteína Fosfatase 1/genética , Reprodutibilidade dos Testes , eIF-2 Quinase/genética
2.
Cell Rep ; 35(7): 109144, 2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34010647

RESUMO

Circulating polymers of α1-antitrypsin (α1AT) are neutrophil chemo-attractants and contribute to inflammation, yet cellular factors affecting their secretion remain obscure. We report on a genome-wide CRISPR-Cas9 screen for genes affecting trafficking of polymerogenic α1ATH334D. A CRISPR enrichment approach based on recovery of single guide RNA (sgRNA) sequences from phenotypically selected fixed cells reveals that cells with high-polymer content are enriched in sgRNAs targeting genes involved in "cargo loading into COPII-coated vesicles," where "COPII" is coat protein II, including the cargo receptors lectin mannose binding1 (LMAN1) and surfeit protein locus 4 (SURF4). LMAN1- and SURF4-disrupted cells display a secretion defect extending beyond α1AT monomers to polymers. Polymer secretion is especially dependent on SURF4 and correlates with a SURF4-α1ATH334D physical interaction and with their co-localization at the endoplasmic reticulum (ER). These findings indicate that ER cargo receptors co-ordinate progression of α1AT out of the ER and modulate the accumulation of polymeric α1AT not only by controlling the concentration of precursor monomers but also by promoting secretion of polymers.

3.
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
5.
Nature ; 578(7795): 444-448, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31875646

RESUMO

Metformin, the world's most prescribed anti-diabetic drug, is also effective in preventing type 2 diabetes in people at high risk1,2. More than 60% of this effect is attributable to the ability of metformin to lower body weight in a sustained manner3. The molecular mechanisms by which metformin lowers body weight are unknown. Here we show-in two independent randomized controlled clinical trials-that metformin increases circulating levels of the peptide hormone growth/differentiation factor 15 (GDF15), which has been shown to reduce food intake and lower body weight through a brain-stem-restricted receptor. In wild-type mice, oral metformin increased circulating GDF15, with GDF15 expression increasing predominantly in the distal intestine and the kidney. Metformin prevented weight gain in response to a high-fat diet in wild-type mice but not in mice lacking GDF15 or its receptor GDNF family receptor α-like (GFRAL). In obese mice on a high-fat diet, the effects of metformin to reduce body weight were reversed by a GFRAL-antagonist antibody. Metformin had effects on both energy intake and energy expenditure that were dependent on GDF15, but retained its ability to lower circulating glucose levels in the absence of GDF15 activity. In summary, metformin elevates circulating levels of GDF15, which is necessary to obtain its beneficial effects on energy balance and body weight, major contributors to its action as a chemopreventive agent.


Assuntos
Peso Corporal/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Fator 15 de Diferenciação de Crescimento/metabolismo , Metformina/farmacologia , Administração Oral , Adulto , Idoso , Animais , Glicemia/análise , Glicemia/metabolismo , Dieta Hiperlipídica , Método Duplo-Cego , Ingestão de Energia/efeitos dos fármacos , Enterócitos/citologia , Enterócitos/efeitos dos fármacos , Feminino , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/antagonistas & inibidores , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/deficiência , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator 15 de Diferenciação de Crescimento/sangue , Fator 15 de Diferenciação de Crescimento/deficiência , Fator 15 de Diferenciação de Crescimento/genética , Homeostase/efeitos dos fármacos , Humanos , Intestinos/citologia , Intestinos/efeitos dos fármacos , Masculino , Metformina/administração & dosagem , Camundongos , Camundongos Obesos , Pessoa de Meia-Idade , Perda de Peso/efeitos dos fármacos
6.
Elife ; 82019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31749445

RESUMO

The eukaryotic translation initiation factor 2α (eIF2α) kinase GCN2 is activated by amino acid starvation to elicit a rectifying physiological program known as the Integrated Stress Response (ISR). A role for uncharged tRNAs as activating ligands of yeast GCN2 is supported experimentally. However, mouse GCN2 activation has recently been observed in circumstances associated with ribosome stalling with no global increase in uncharged tRNAs. We report on a mammalian CHO cell-based CRISPR-Cas9 mutagenesis screen for genes that contribute to ISR activation by amino acid starvation. Disruption of genes encoding components of the ribosome P-stalk, uL10 and P1, selectively attenuated GCN2-mediated ISR activation by amino acid starvation or interference with tRNA charging without affecting the endoplasmic reticulum unfolded protein stress-induced ISR, mediated by the related eIF2α kinase PERK. Wildtype ribosomes isolated from CHO cells, but not those with P-stalk lesions, stimulated GCN2-dependent eIF2α phosphorylation in vitro. These observations support a model whereby lack of a cognate charged tRNA exposes a latent capacity of the ribosome P-stalk to activate GCN2 in cells and help explain the emerging link between ribosome stalling and ISR activation.


Assuntos
Aminoácidos/metabolismo , Ribossomos/metabolismo , Inanição/metabolismo , Animais , Células CHO , Sistemas CRISPR-Cas , Cricetulus , Retículo Endoplasmático/metabolismo , Regulação Enzimológica da Expressão Gênica , Células HeLa , Humanos , Cinética , Ligantes , Camundongos , Modelos Moleculares , Mutagênese , Fosforilação , Ligação Proteica , Conformação Proteica , /genética , Desdobramento de Proteína , RNA de Transferência/metabolismo , Ribossomos/química , Transdução de Sinais , Transcriptoma , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
7.
Cell Metab ; 29(3): 707-718.e8, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30639358

RESUMO

GDF15 is an established biomarker of cellular stress. The fact that it signals via a specific hindbrain receptor, GFRAL, and that mice lacking GDF15 manifest diet-induced obesity suggest that GDF15 may play a physiological role in energy balance. We performed experiments in humans, mice, and cells to determine if and how nutritional perturbations modify GDF15 expression. Circulating GDF15 levels manifest very modest changes in response to moderate caloric surpluses or deficits in mice or humans, differentiating it from classical intestinally derived satiety hormones and leptin. However, GDF15 levels do increase following sustained high-fat feeding or dietary amino acid imbalance in mice. We demonstrate that GDF15 expression is regulated by the integrated stress response and is induced in selected tissues in mice in these settings. Finally, we show that pharmacological GDF15 administration to mice can trigger conditioned taste aversion, suggesting that GDF15 may induce an aversive response to nutritional stress.


Assuntos
Ingestão de Energia/fisiologia , Fator 15 de Diferenciação de Crescimento/metabolismo , Adulto , Animais , Linhagem Celular , Dieta Hiperlipídica/métodos , Fator 15 de Diferenciação de Crescimento/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Adulto Jovem
9.
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
10.
Cell ; 171(7): 1625-1637.e13, 2017 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-29198525

RESUMO

When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response (UPR) increases ER-protein-folding capacity to restore protein-folding homeostasis. Unfolded proteins activate UPR signaling across the ER membrane to the nucleus by promoting oligomerization of IRE1, a conserved transmembrane ER stress receptor. However, the coupling of ER stress to IRE1 oligomerization and activation has remained obscure. Here, we report that the ER luminal co-chaperone ERdj4/DNAJB9 is a selective IRE1 repressor that promotes a complex between the luminal Hsp70 BiP and the luminal stress-sensing domain of IRE1α (IRE1LD). In vitro, ERdj4 is required for complex formation between BiP and IRE1LD. ERdj4 associates with IRE1LD and recruits BiP through the stimulation of ATP hydrolysis, forcibly disrupting IRE1 dimers. Unfolded proteins compete for BiP and restore IRE1LD to its default, dimeric, and active state. These observations establish BiP and its J domain co-chaperones as key regulators of the UPR.


Assuntos
Endorribonucleases/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Resposta a Proteínas não Dobradas , Animais , Cricetinae , Retículo Endoplasmático/metabolismo , Humanos , Dobramento de Proteína
11.
J Exp Med ; 214(2): 401-422, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28082357

RESUMO

ATG16L1T300A, a major risk polymorphism in Crohn's disease (CD), causes impaired autophagy, but it has remained unclear how this predisposes to CD. In this study, we report that mice with Atg16l1 deletion in intestinal epithelial cells (IECs) spontaneously develop transmural ileitis phenocopying ileal CD in an age-dependent manner, driven by the endoplasmic reticulum (ER) stress sensor IRE1α. IRE1α accumulates in Paneth cells of Atg16l1ΔIEC mice, and humans homozygous for ATG16L1T300A exhibit a corresponding increase of IRE1α in intestinal epithelial crypts. In contrast to a protective role of the IRE1ß isoform, hyperactivated IRE1α also drives a similar ileitis developing earlier in life in Atg16l1;Xbp1ΔIEC mice, in which ER stress is induced by deletion of the unfolded protein response transcription factor XBP1. The selective autophagy receptor optineurin interacts with IRE1α, and optineurin deficiency amplifies IRE1α levels during ER stress. Furthermore, although dysbiosis of the ileal microbiota is present in Atg16l1;Xbp1ΔIEC mice as predicted from impaired Paneth cell antimicrobial function, such structural alteration of the microbiota does not trigger ileitis but, rather, aggravates dextran sodium sulfate-induced colitis. Hence, we conclude that defective autophagy in IECs may predispose to CD ileitis via impaired clearance of IRE1α aggregates during ER stress at this site.


Assuntos
Proteínas Relacionadas à Autofagia/fisiologia , Doença de Crohn/etiologia , Endorribonucleases/fisiologia , Ileíte/etiologia , /fisiologia , Fatores Etários , Animais , Autofagia , Estresse do Retículo Endoplasmático , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microbiota
12.
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
13.
Oncotarget ; 7(39): 64124-64135, 2016 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-27802424

RESUMO

In response to endoplasmic reticulum (ER) stress, activation of pancreatic ER kinase (PERK) coordinates an adaptive program known as the integrated stress response (ISR) by phosphorylating translation initiation factor 2α (eIF2α). Phosphorylated eIF2α is quickly dephosphorylated by the protein phosphatase 1 and growth arrest and DNA damage 34 (GADD34) complex. Data indicate that the ISR can either promote or suppress tumor development. Our previous studies showed that the ISR is activated in medulloblastoma in both human patients and animal models, and that the decreased ISR via PERK heterozygous deficiency attenuates medulloblastoma formation in Patched1 heterozygous deficient (Ptch1+/-) mice by enhancing apoptosis of pre-malignant granule cell precursors (GCPs) during cell transformation. We showed here that GADD34 heterozygous mutation moderately enhanced the ISR and noticeably increased the incidence of medulloblastoma in adult Ptch1+/- mice. Surprisingly, GADD34 homozygous mutation strongly enhanced the ISR, but significantly decreased the incidence of medulloblastoma in adult Ptch1+/- mice. Intriguingly, GADD34 homozygous mutation significantly enhanced pre-malignant GCP apoptosis in cerebellar hyperplastic lesions and reduced the lesion numbers in young Ptch1+/- mice. Nevertheless, neither GADD34 heterozygous mutation nor GADD34 homozygous mutation had a significant effect on medulloblastoma cells in adult Ptch1+/- mice. Collectively, these data imply the dual role of the ISR, promoting and inhibiting, in medulloblastoma tumorigenesis by regulating apoptosis of pre-malignant GCPs during the course of malignant transformation.


Assuntos
Transformação Celular Neoplásica/metabolismo , Neoplasias Cerebelares/enzimologia , Estresse do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/metabolismo , Meduloblastoma/enzimologia , Proteína Fosfatase 1/metabolismo , eIF-2 Quinase/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Homozigoto , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica , Receptor Patched-1/deficiência , Receptor Patched-1/genética , Fenótipo , Fosforilação , Proteína Fosfatase 1/deficiência , Proteína Fosfatase 1/genética , Transdução de Sinais , Fatores de Tempo
14.
Am J Pathol ; 186(7): 1939-1951, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27181404

RESUMO

Evidence suggests that activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum stress negatively or positively influences cell transformation by regulating apoptosis. Patched1 heterozygous deficient (Ptch1(+/-)) mice reproduce human Gorlin's syndrome and are regarded as the best animal model to study tumorigenesis of the sonic hedgehog subgroup of medulloblastomas. It is believed that medulloblastomas in Ptch1(+/-) mice results from the transformation of granule cell precursors (GCPs) in the developing cerebellum. Here, we determined the role of PERK signaling on medulloblastoma tumorigenesis by assessing its effects on premalignant GCPs and tumor cells. We found that PERK signaling was activated in both premalignant GCPs in young Ptch1(+/-) mice and medulloblastoma cells in adult mice. We demonstrated that PERK haploinsufficiency reduced the incidence of medulloblastomas in Ptch1(+/-) mice. Interestingly, PERK haploinsufficiency enhanced apoptosis of premalignant GCPs in young Ptch1(+/-) mice but had no significant effect on medulloblastoma cells in adult mice. Moreover, we showed that the PERK pathway was activated in medulloblastomas in humans. These results suggest that PERK signaling promotes medulloblastoma tumorigenesis by attenuating apoptosis of premalignant GCPs during the course of malignant transformation.


Assuntos
Transformação Celular Neoplásica/metabolismo , Neoplasias Cerebelares/patologia , Meduloblastoma/patologia , Células-Tronco Neurais/patologia , eIF-2 Quinase/metabolismo , Adulto , Animais , Apoptose , Western Blotting , Carcinogênese/metabolismo , Carcinogênese/patologia , Transformação Celular Neoplásica/patologia , Neoplasias Cerebelares/enzimologia , Criança , Pré-Escolar , Modelos Animais de Doenças , Ativação Enzimática/fisiologia , Feminino , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Lactente , Masculino , Meduloblastoma/enzimologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neurônios/patologia , Reação em Cadeia da Polimerase em Tempo Real
15.
FASEB J ; 30(2): 798-812, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26487695

RESUMO

The eukaryotic translation initiation factor 2α (eIF2α) phosphorylation-dependent integrated stress response (ISR), a component of the unfolded protein response, has long been known to regulate intermediary metabolism, but the details are poorly worked out. We report that profiling of mRNAs of transgenic mice harboring a ligand-activated skeletal muscle-specific derivative of the eIF2α protein kinase R-like ER kinase revealed the expected up-regulation of genes involved in amino acid biosynthesis and transport but also uncovered the induced expression and secretion of a myokine, fibroblast growth factor 21 (FGF21), that stimulates energy consumption and prevents obesity. The link between the ISR and FGF21 expression was further reinforced by the identification of a small-molecule ISR activator that promoted Fgf21 expression in cell-based screens and by implication of the ISR-inducible activating transcription factor 4 in the process. Our findings establish that eIF2α phosphorylation regulates not only cell-autonomous proteostasis and amino acid metabolism, but also affects non-cell-autonomous metabolic regulation by induced expression of a potent myokine.


Assuntos
Aminoácidos/metabolismo , Metabolismo Energético/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Crescimento de Fibroblastos/biossíntese , Regulação da Expressão Gênica/fisiologia , Músculo Esquelético/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Aminoácidos/genética , Animais , Fator de Iniciação 2 em Eucariotos/genética , Fatores de Crescimento de Fibroblastos/genética , Humanos , Camundongos , Camundongos Transgênicos , Músculo Esquelético/citologia , Fosforilação/genética
16.
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
17.
Diabetes ; 64(11): 3951-62, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26159176

RESUMO

Dysregulated endoplasmic reticulum stress and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) are associated with pancreatic ß-cell failure and diabetes. Here, we report the first homozygous mutation in the PPP1R15B gene (also known as constitutive repressor of eIF2α phosphorylation [CReP]) encoding the regulatory subunit of an eIF2α-specific phosphatase in two siblings affected by a novel syndrome of diabetes of youth with short stature, intellectual disability, and microcephaly. The R658C mutation in PPP1R15B affects a conserved amino acid within the domain important for protein phosphatase 1 (PP1) binding. The R658C mutation decreases PP1 binding and eIF2α dephosphorylation and results in ß-cell apoptosis. Our findings support the concept that dysregulated eIF2α phosphorylation, whether decreased by mutation of the kinase (EIF2AK3) in Wolcott-Rallison syndrome or increased by mutation of the phosphatase (PPP1R15B), is deleterious to ß-cells and other secretory tissues, resulting in diabetes associated with multisystem abnormalities.


Assuntos
Diabetes Mellitus/genética , Transtornos do Crescimento/genética , Microcefalia/genética , Mutação de Sentido Incorreto , Proteína Fosfatase 1/genética , Adolescente , Adulto , Feminino , Humanos , Masculino , Síndrome
18.
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
19.
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
20.
Biophys J ; 108(5): 999-1002, 2015 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-25762312

RESUMO

FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (Kd) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the complete set of interaction parameters, including fractions of bound donors and acceptors, local protein concentrations, and dissociation constants, in each image pixel. The method makes use of fluorescence lifetime information from both donor and acceptor molecules and takes advantage of the linear properties of the phasor plot approach. We demonstrate the capability of our method in vitro in a microfluidic device and also in cells, via the determination of the binding affinity between tagged versions of glutathione and glutathione S-transferase, and via the determination of competitor concentration. The potential of the method is explored with simulations.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Microfluídica/métodos , Células HEK293 , Humanos , Proteínas Luminescentes/metabolismo
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