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1.
Sci Adv ; 7(30)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34290093

RESUMO

The dedicator of cytokinesis (DOCK) family of guanine nucleotide exchange factors (GEFs) promotes cell motility, phagocytosis, and cancer metastasis through activation of Rho guanosine triphosphatases. Engulfment and cell motility (ELMO) proteins are binding partners of DOCK and regulate Rac activation. Here, we report the cryo-electron microscopy structure of the active ELMO1-DOCK5 complex bound to Rac1 at 3.8-Å resolution. The C-terminal region of ELMO1, including the pleckstrin homology (PH) domain, aids in the binding of the catalytic DOCK homology region 2 (DHR-2) domain of DOCK5 to Rac1 in its nucleotide-free state. A complex α-helical scaffold between ELMO1 and DOCK5 stabilizes the binding of Rac1. Mutagenesis studies revealed that the PH domain of ELMO1 enhances the GEF activity of DOCK5 through specific interactions with Rac1. The structure provides insights into how ELMO modulates the biochemical activity of DOCK and how Rac selectivity is achieved by ELMO.

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.
FEBS Lett ; 594(10): 1532-1549, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32017069

RESUMO

Activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor, a central player in immune response regulation, is based on phosphorylation of inhibitor of kappaB alpha (IκBα) by the Inhibitor of kappaB kinase (IKK) that triggers IκBα degradation. Although inhibitor of kappaB beta (IκBß) is structurally similar to IκBα, its precise characteristics remain undefined. Herein, we report that the molecular interactivity of IκBß with the kinase-active region of IKK subunit 2 (IKK2), as well as its phosphorylation status, differs markedly from those of IκBα. A mass spectrometry analysis revealed that IκBß phosphorylation sites are distributed in its C-terminal region, whereas IκBα phosphorylation sites are located in the N-terminal region. Furthermore, IKK2 phosphorylation sites in IκBß are found in a region distinct from typical degradation signals, such as phosphodegron and proline/glutamic acid/serine/threonine-rich sequence (PEST) motifs. Mutation of the IκBß phosphorylation sites enhances its resistance to homeostatic proteasomal degradation. These findings contribute a novel concept in NF-κB/IKK signalling research.


Assuntos
Biocatálise , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/química , Proteínas I-kappa B/metabolismo , Inibidor de NF-kappaB alfa/química , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Motivos de Aminoácidos , Homeostase , Humanos , Modelos Moleculares , Mutação , Fosforilação/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Transdução de Sinais
4.
Science ; 364(6439): 495-499, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31048492

RESUMO

A core event in the integrated stress response, an adaptive pathway common to all eukaryotic cells in response to various stress stimuli, is the phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Normally, unphosphorylated eIF2 transfers the methionylated initiator tRNA to the ribosome in a guanosine 5'-triphosphate-dependent manner. By contrast, phosphorylated eIF2 inhibits its specific guanine nucleotide exchange factor, eIF2B. To elucidate how the eIF2 phosphorylation status regulates the eIF2B activity, we determined cryo-electron microscopic and crystallographic structures of eIF2B in complex with unphosphorylated or phosphorylated eIF2. The unphosphorylated and phosphorylated forms of eIF2 bind to eIF2B in completely different manners: the nucleotide exchange-active and -inactive modes, respectively. These structures explain how phosphorylated eIF2 dominantly inhibits the nucleotide exchange activity of eIF2B.


Assuntos
Fator de Iniciação 2B em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/química , Estresse Fisiológico , Motivos de Aminoácidos , Microscopia Crioeletrônica , Fator de Iniciação 2B em Eucariotos/metabolismo , Humanos , Fosforilação
5.
Mol Cell ; 74(6): 1205-1214.e8, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31080011

RESUMO

Translation initiation of hepatitis C virus (HCV) genomic RNA is induced by an internal ribosome entry site (IRES). Our cryoelectron microscopy (cryo-EM) analysis revealed that the HCV IRES binds to the solvent side of the 40S platform of the cap-dependently translating 80S ribosome. Furthermore, we obtained the cryo-EM structures of the HCV IRES capturing the 40S subunit of the IRES-dependently translating 80S ribosome. In the elucidated structures, the HCV IRES "body," consisting of domain III except for subdomain IIIb, binds to the 40S subunit, while the "long arm," consisting of domain II, remains flexible and does not impede the ongoing translation. Biochemical experiments revealed that the cap-dependently translating ribosome becomes a better substrate for the HCV IRES than the free ribosome. Therefore, the HCV IRES is likely to efficiently induce the translation initiation of its downstream mRNA with the captured translating ribosome as soon as the ongoing translation terminates.


Assuntos
Fatores de Iniciação em Eucariotos/química , Hepacivirus/genética , Iniciação Traducional da Cadeia Peptídica , RNA Viral/química , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Células HEK293 , Hepacivirus/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Sítios Internos de Entrada Ribossomal , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Viral/genética , RNA Viral/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo
6.
Protein Expr Purif ; 150: 92-99, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29793032

RESUMO

Since phosphorylation is involved in various physiological events, kinases and interacting factors can be potential targets for drug discovery. For the development and improvement of inhibitors from the point of view of mechanistic enzymology, a cell-free protein synthesis system would be advantageous, since it could prepare mutant proteins easily. However, especially in the case of protein kinase, product solubility remains one of the major challenges. To overcome this problem, we prepared a chaperone-supplemented extract from Escherichia coli BL21 cells harboring a plasmid encoding a set of chaperone genes, dnaK, dnaJ, and grpE. We explored cell-disruption procedures and constructed an efficient protein synthesis system. Employing this system, we produced the kinase domain of human hematopoietic cell kinase (HCK) to obtain further structural information about its molecular interaction with one of its inhibitors, previously developed by our group (RK-20449). Lower reaction temperature improved the solubility, and addition of a protein phosphatase (YpoH) facilitated the homogeneous production of the non-phosphorylated kinase domain. Crystals of the purified product were obtained and the kinase-inhibitor complex structure was solved at 1.7 Šresolution. In addition, results of kinase activity measurement, using a synthetic substrate, showed that the kinase activity was facilitated by autophosphorylation at Tyr416, as confirmed by the peptide mass mapping.


Assuntos
Expressão Gênica , Proteínas Proto-Oncogênicas c-hck , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Humanos , Fosforilação , Domínios Proteicos , Proteínas Proto-Oncogênicas c-hck/biossíntese , Proteínas Proto-Oncogênicas c-hck/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
7.
J Biochem ; 162(5): 357-369, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28992119

RESUMO

Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity. The developed procedure comprises cell cultivation using a fermentor, harvesting and washing of cells by tangential flow filtration, cell disruption with high-pressure homogenizer and continuous diafiltration. By optimizing and combining these methods, ∼100 ml of the cell extract was prepared from 150 g of Escherichia coli cells. The protein synthesis activities, defined as the yield of protein per unit of absorbance at 260 nm of the cell extract, were shown to be reproducible, and the average activity of several batches was twice that obtained using a previously reported method. In addition, combinatorial use of the high-pressure homogenizer and diafiltration increased the scalability, indicating that the cell concentration at disruption varies from 0.04 to 1 g/ml. Furthermore, addition of Gam protein and examinations of the N-terminal sequence rendered the extract prepared here useful for rapid screening with linear DNA templates.


Assuntos
Extratos Celulares/química , Sistema Livre de Células , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/química , Proteínas de Fluorescência Verde/metabolismo , Biossíntese de Proteínas/fisiologia , Proteínas Virais/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Fluorescência Verde/genética , Reprodutibilidade dos Testes , Proteínas Virais/genética
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