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1.
J Biol Chem ; 289(46): 31960-31971, 2014 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-25258322

RESUMO

The genomic RNA of encephalomyocarditis virus (EMCV) encodes a single polyprotein, and the primary scission of the polyprotein occurs between nonstructural proteins 2A and 2B by an unknown mechanism. To gain insight into the mechanism of 2A-2B processing, we first translated the 2A-2B region in vitro with eukaryotic and prokaryotic translation systems. The 2A-2B processing occurred only in the eukaryotic systems, not in the prokaryotic systems, and the unprocessed 2A-2B protein synthesized by a prokaryotic system remained uncleaved when incubated with a eukaryotic cell extract. These results suggest that 2A-2B processing is a eukaryote-specific, co-translational event. To define the translation factors required for 2A-2B processing, we constituted a protein synthesis system with eukaryotic elongation factors 1 and 2, eukaryotic release factors 1 and 3 (eRF1 and eRF3), aminoacyl-tRNA synthetases, tRNAs, ribosome subunits, and a plasmid template that included the hepatitis C virus internal ribosome entry site. We successfully reproduced 2A-2B processing in the reconstituted system even without eRFs. Our results indicate that this unusual event occurs in the elongation phase of translation.


Assuntos
Vírus da Encefalomiocardite/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Alanina/genética , Aminoacil-tRNA Sintetases/metabolismo , Sítios de Ligação , Sistema Livre de Células , DNA Complementar/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Células HeLa , Hepacivirus/metabolismo , Humanos , Mutação , Fases de Leitura Aberta , Plasmídeos/metabolismo , Dobramento de Proteína , Ribossomos/química
2.
Protein Expr Purif ; 87(1): 5-10, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23063735

RESUMO

Many biologically important factors are composed of multiple subunits. To study the structure and function of the protein complexes and the role of each subunit, a rapid and efficient method to prepare recombinant protein complexes is needed. In this work, we established an in vitro reconstitution system of eukaryotic translation initiation factor (eIF) 3, a protein complex consisting of 11 distinct subunits. A HeLa cell-derived in vitro coupled transcription/translation system was programmed with multiple plasmids encoding the 11 eIF3 subunits in total. After incubation for several hours, the eIF3 complex was purified through tag-dependent affinity chromatography. When eIF3l, one of the nonessential subunits of eIF3, was not expressed, the eIF3 complex that was devoid of eIF3l was still obtained. Both the 11 subunits complex and the eIF3l-less complex were as active as native eIF3 as observed by a reconstituted translation initiation assay system. In conclusion, the cell-free co-expression system should be a feasible and rapid system to reconstitute protein complexes.


Assuntos
Fator de Iniciação 3 em Eucariotos/biossíntese , Biossíntese de Proteínas , Subunidades Proteicas/biossíntese , Sistema Livre de Células , Cromatografia de Afinidade , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/isolamento & purificação , Células HeLa , Humanos , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Deleção de Sequência , Transcrição Gênica
3.
Biotechnol Lett ; 35(3): 309-14, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23143176

RESUMO

Virus particles are promising vehicles and templates for vaccination, drug delivery and material sciences. Although infectious picornaviruses can be synthesized from genomic or synthetic RNA by cell-free protein expression systems derived from mammalian cell extract, there has been no direct evidence that authentic viral particles are indeed synthesized in the absence of living cells. We purified encephalomyocarditis virus (EMCV) synthesized by a HeLa cell extract-derived, cell-free protein expression system, and visualized the viral particles by transmission electron-microscopy. The in vitro-synthesized EMCV particles were indistinguishable from the in vivo-synthesized particles. Our results validate the use of the cell-free technique for the synthesis of EMCV particles.


Assuntos
Biotecnologia/métodos , Extratos Celulares/isolamento & purificação , Vírus da Encefalomiocardite/isolamento & purificação , Proteínas Virais/biossíntese , Sistema Livre de Células , Vírus da Encefalomiocardite/ultraestrutura , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Vírion/isolamento & purificação , Vírion/ultraestrutura
4.
Protein Expr Purif ; 82(1): 61-9, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22133715

RESUMO

The eukaryotic cytosolic chaperonin CCT (chaperonin-containing TCP-1) assists folding of newly synthesized polypeptides. The fully functional CCT is built from two identical rings, each composed of single copies of eight distinct subunits. To study the structure and function of the CCT complex and the role of each subunit, a rapid and efficient method for preparing a recombinant CCT complex is needed. In this work, we established an efficient expression and purification method to obtain human recombinant CCT. BHK-21 cells were infected with a vaccinia virus expressing T7 RNA polymerase and transfected with eight plasmids, each encoding any one of the eight CCT subunits in the T7 RNA polymerase promoter/terminator unit. The CCT1 subunit was engineered to carry a hexa-histidine tag or FLAG tag in the internal loop region. Three days later, cells were harvested for purification of the CCT complex through tag-dependent affinity chromatography and gel filtration. The purified recombinant CCT complexes were indistinguishable from the endogenous CCT purified from HeLa cells in terms of morphology and function. In conclusion, the co-expression system established in this study should be a simple and powerful tool for reconstitution of a large multi-subunit complex.


Assuntos
Chaperonina com TCP-1/química , Chaperonina com TCP-1/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Chaperonina com TCP-1/isolamento & purificação , Expressão Gênica , Humanos , Dados de Sequência Molecular , Plasmídeos/genética , Dobramento de Proteína , Subunidades Proteicas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Vaccinia virus/genética
5.
Biotechnol Lett ; 34(1): 67-73, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21952914

RESUMO

Virus particles are used in vaccination, drug delivery, and material sciences. Here we devised a system where the RNA virus encephalomyocarditis virus (EMCV) is synthesized from DNA templates in vitro. When a plasmid or a PCR product harboring the full-length cDNA of EMCV in the T7 promoter/terminator unit was incubated in a HeLa cell extract supplemented with T7 RNA polymerase, EMCV was produced within 4 h at an efficiency of over 10-fold compared with the system programmed with the EMCV RNA. The EMCV RNA transcribed by the virally encoded RNA-dependent RNA polymerase was predominantly incorporated into the EMCV particle even in the presence of a larger amount of the EMCV RNA transcribed by T7 RNA polymerase from the plasmid.


Assuntos
Vírus da Encefalomiocardite/genética , RNA Viral/biossíntese , Proteínas Virais/biossíntese , Montagem de Vírus , Biotecnologia/métodos , Extratos Celulares , Sistema Livre de Células , Células HeLa , Humanos , Plasmídeos , Virologia/métodos
6.
J Clin Invest ; 131(16)2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34185705

RESUMO

The Western pattern diet is rich not only in fat and calories but also in phosphate. The negative effects of excessive fat and calorie intake on health are widely known, but the potential harms of excessive phosphate intake are poorly recognized. Here, we show the mechanism by which dietary phosphate damages the kidney. When phosphate intake was excessive relative to the number of functioning nephrons, circulating levels of FGF23, a hormone that increases the excretion of phosphate per nephron, were increased to maintain phosphate homeostasis. FGF23 suppressed phosphate reabsorption in renal tubules and thus raised the phosphate concentration in the tubule fluid. Once it exceeded a threshold, microscopic particles containing calcium phosphate crystals appeared in the tubule lumen, which damaged tubule cells through binding to the TLR4 expressed on them. Persistent tubule damage induced interstitial fibrosis, reduced the number of nephrons, and further boosted FGF23 to trigger a deterioration spiral leading to progressive nephron loss. In humans, the progression of chronic kidney disease (CKD) ensued when serum FGF23 levels exceeded 53 pg/mL. The present study identified calcium phosphate particles in the renal tubular fluid as an effective therapeutic target to decelerate nephron loss during the course of aging and CKD progression.


Assuntos
Fosfatos de Cálcio/metabolismo , Túbulos Renais/metabolismo , Insuficiência Renal Crônica/metabolismo , Animais , Líquidos Corporais/química , Fosfatos de Cálcio/química , Linhagem Celular , Cristalização , Dieta Ocidental/efeitos adversos , Progressão da Doença , Endocitose , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Homeostase , Humanos , Túbulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatos/administração & dosagem , Fosfatos/efeitos adversos , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/patologia , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
7.
Biotechnol Lett ; 32(7): 897-902, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20349333

RESUMO

Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.


Assuntos
Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Biossíntese de Proteínas , Proteínas/metabolismo , Antígenos de Diferenciação/isolamento & purificação , Proteínas de Ciclo Celular/isolamento & purificação , Extratos Celulares , Escherichia coli/genética , Expressão Gênica , Humanos , Proteína Fosfatase 1 , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Deleção de Sequência
8.
FEBS Lett ; 594(18): 3020-3031, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32608065

RESUMO

Testis-expressed gene 101 (TEX101) is a glycosyl-phosphatidylinositol-anchored glycoprotein essential for sperm fertility and spermatogenesis. TEX101 interacts with lymphocyte antigen 6 complex, locus K (Ly6k) as well as a disintegrin and metallopeptidase domain 3 (ADAM3). Although these proteins are considered essential for fertility, the associated mechanisms remain uncharacterized. Herein, we determined the crystal structure of human and mouse TEX101, revealing that TEX101 contains two tandem Ly6/uPAR (LU) domains. Detailed structural analyses revealed characteristic surfaces of TEX101 that may be involved in the interactions with other proteins or membranes. These results provide the structural basis for the role of TEX101 in fertilization and could contribute to developing diagnostic methods and treatments for infertility or developing male contraceptives.


Assuntos
Fertilidade , Proteínas de Membrana/química , Animais , Linhagem Celular , Cristalografia por Raios X , Drosophila melanogaster , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Domínios Proteicos , Espermatogênese , Espermatozoides/metabolismo
9.
Protein Expr Purif ; 62(2): 190-8, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18814849

RESUMO

The aim of this study was to develop an efficient cell-free protein expression system derived from mammalian cells. We established a HeLa cell-based in vitro coupled transcription/translation system with T7 RNA polymerase and a plasmid that harbored a T7 promoter/terminator unit. To enhance protein synthesis in the coupled system, we placed the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) or the hepatitis C virus (HCV) IRES between the T7 promoter and the coding region of the plasmid. Remarkably, we found that these IRES-dependent systems were able to produce large proteins including GCN2 (160 kD), Dicer (200 kD) and mTOR (260 kD) to levels detectable on SDS-PAGE by Comassie Brilliant Blue-staining. We purified the synthesized proteins to near homogeneity, and validated their functionalities in the appropriate biochemical assays. In conclusion, the HeLa cell-based in vitro coupled transcription/translation system using the EMCV or HCV IRES is a convenient tool, particularly for the production of large recombinant proteins.


Assuntos
Biossíntese de Proteínas , Proteínas Recombinantes/biossíntese , Transcrição Gênica , Regiões 5' não Traduzidas/genética , Vírus da Encefalomiocardite/genética , Células HeLa , Hepacivirus/genética , Humanos , Sequências Reguladoras de Ácido Ribonucleico
10.
J Biochem ; 150(4): 423-30, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21622667

RESUMO

To study the relationship between translation and replication of encephalomyocarditisvirus (EMCV) RNA, we established a cell-free RNA replication system by employing a human cell extracts-based in vitro translation system. In this system, a cis-EMCV RNA replicon encoding the Renilla luciferase (R-luc) or GFP and the viral regulatory proteins efficiently replicated with simultaneous translation of the encoded protein. To examine how translation of the replicon RNA, but not the translated products, affected replication, a trans-EMCV RNA replicon encoding R-luc and the RNA replication elements was next constructed. The trans-replicon RNA replicated only in the presence of the regulatory proteins pre-expressed in trans. Incubation with cycloheximide, puromycin or a dominant-negative eukaryotic translation initiation factor 4A following expression of the regulatory proteins almost completely inhibited not only translation of the trans-replicon RNA but also replication of the RNA, suggesting that EMCV RNA translation promotes replication of the RNA. In conclusion, the cell-free RNA replication systems should become useful tools for the study of the viral RNA replication.


Assuntos
Extratos Celulares/farmacologia , Vírus da Encefalomiocardite/efeitos dos fármacos , Vírus da Encefalomiocardite/genética , RNA Viral/biossíntese , Replicação Viral/efeitos dos fármacos , Animais , Sistema Livre de Células , Células Cultivadas , Cricetinae , Cicloeximida/farmacologia , Vírus da Encefalomiocardite/metabolismo , Células HeLa , Humanos , Puromicina/farmacologia , RNA Viral/genética , Replicação Viral/genética
11.
EMBO J ; 26(14): 3373-83, 2007 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-17581632

RESUMO

Eukaryotic translation initiation factor (eIF)3 is the largest eIF ( approximately 650 kDa), consisting of 10-13 different polypeptide subunits in mammalian cells. To understand the role of each subunit, we successfully reconstituted a human eIF3 complex consisting of 11 subunits that promoted the recruitment of the 40S ribosomal subunit to mRNA. Strikingly, the eIF3g and eIF3i subunits, which are evolutionarily conserved between human and the yeast Saccharomyces cerevisiae are dispensable for active mammalian eIF3 complex formation. Extensive deletion analyses suggest that three evolutionarily conserved subunits (eIF3a, eIF3b, and eIF3c) and three non-conserved subunits (eIF3e, eIF3f, and eIF3h) comprise the functional core of mammalian eIF3.


Assuntos
Fator de Iniciação 3 em Eucariotos/metabolismo , Subunidades Proteicas/metabolismo , Sequência Conservada , Células HeLa , Humanos , Proteínas Recombinantes/metabolismo
12.
Protein Expr Purif ; 46(2): 348-57, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16289705

RESUMO

Development of an efficient cell-free translation system from mammalian cells is an important goal. We examined whether supplementation of HeLa cell extracts with any translation initiation factor or translational regulator could enhance protein synthesis. eIF2 (eukaryotic translation initiation factor 2) and eIF2B augmented translation of capped, uncapped and encephalomyocarditis virus-internal ribosome entry site-promoted mRNAs. eIF4E specifically stimulated capped mRNA translation, while p97, a homologue to the C-terminal two-thirds of eIF4G, increased uncapped mRNA translation. When the HeLa cell extract was supplemented with a combination of eIF2, eIF2B, and p97, the capacity to synthesize a protein from an uncapped mRNA became comparable to that from the capped counterpart stimulated with a combination of eIF2, eIF2B, and eIF4E. A dialysis method rendered the HeLa cell extract capable of synthesizing proteins for 36h, and the yield was augmented when supplemented with initiation factors. In contrast, the productivity of a rabbit reticulocyte lysate was not enhanced by this method. Collectively, the translation factor-supplemented HeLa cell extract should become an important tool for the production of recombinant proteins.


Assuntos
Fatores de Iniciação em Procariotos , Biossíntese de Proteínas , Capuzes de RNA , Proteínas Recombinantes/biossíntese , Sistema Livre de Células , Células HeLa , Humanos
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