RESUMEN
"Undruggable" targets such as KRAS are particularly challenging in the development of drugs. We devised a novel chemical knockdown strategy, CANDDY (Chemical knockdown with Affinity aNd Degradation DYnamics) technology, which promotes protein degradation using small molecules (CANDDY molecules) that are conjugated to a degradation tag (CANDDY tag) modified from proteasome inhibitors. We demonstrated that CANDDY tags allowed for direct proteasomal target degradation independent of ubiquitination. We synthesized a KRAS-degrading CANDDY molecule, TUS-007, which induced degradation in KRAS mutants (G12D and G12V) and wild-type KRAS. We confirmed the tumor suppression effect of TUS-007 in subcutaneous xenograft models of human colon cells (KRAS G12V) with intraperitoneal administrations and in orthotopic xenograft models of human pancreatic cells (KRAS G12D) with oral administrations. Thus, CANDDY technology has the potential to therapeutically target previously undruggable proteins, providing a simpler and more practical drug targeting approach and avoiding the difficulties in matchmaking between the E3 enzyme and the target.
Asunto(s)
Proteínas , Proteínas Proto-Oncogénicas p21(ras) , Animales , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Modelos Animales de Enfermedad , Ubiquitinación , MutaciónRESUMEN
To clarify the pathogenic mechanism of disease and establish effective therapies, animal disease models that can be dynamically analyzed are urgently required. Knockout mouse models and conditional genetically engineered mouse models were developed to analyze genes and proteins involved in disease. However, these methods have drawbacks, including embryonic lethality, side effects and low efficiency. To address this issue, we created a novel transgenic mouse model in which the YB1 gene was fused with a destabilizing domain (DD), named the YB1-DD mouse. YB-1 is widely expressed throughout development and has been implicated as a cell survival factor. Newly synthesized DD proteins are degraded through the proteasome pathway, but their degradation can be blocked with trimethoprim (TMP). In this study, we established a novel conditional knockdown mouse model that enables targeting of protein degradation directly; this model resulted in dose-dependent regulation of the target protein YB-1 by the ligand TMP in YB1 heterozygous mice. Since this conditional knockdown mouse model appears to be functional, it has potential as a useful disease model based on direct protein degradation control.
Asunto(s)
Factores de Transcripción/genética , Factores de Transcripción/fisiología , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados/genética , Ratones Transgénicos/genética , Ingeniería de Proteínas/métodos , Proteolisis , TrimetoprimRESUMEN
We have developed a simple in vitro virus (IVV) selection system based on cell-free co-translation, using a highly stable and efficient mRNA display method. The IVV system is applicable to the high-throughput and comprehensive analysis of proteins and protein-ligand interactions. Huge amounts of genomic sequence data have been generated over the last decade. The accumulated genetic alterations and the interactome networks identified within cells represent a universal feature of a disease, and knowledge of these aspects can help to determine the optimal therapy for the disease. The concept of the "integrome" has been developed as a means of integrating large amounts of data. We have developed an interactome analysis method aimed at providing individually-targeted health care. We also consider future prospects for this system.
Asunto(s)
Virus/metabolismo , Sistema Libre de Células , Genómica , Humanos , Medicina de Precisión , Mapas de Interacción de Proteínas , Proteínas/metabolismo , ARN Mensajero/metabolismoRESUMEN
UNLABELLED: Protein-protein interactions (PPIs) are mediated through specific regions on proteins. Some proteins have two or more protein interacting regions (IRs) and some IRs are competitively used for interactions with different proteins. IRView currently contains data for 3417 IRs in human and mouse proteins. The data were obtained from different sources and combined with annotated region data from InterPro. Information on non-synonymous single nucleotide polymorphism sites and variable regions owing to alternative mRNA splicing is also included. The IRView web interface displays all IR data, including user-uploaded data, on reference sequences so that the positional relationship between IRs can be easily understood. IRView should be useful for analyzing underlying relationships between the proteins behind the PPI networks. AVAILABILITY: IRView is publicly available on the web at http://ir.hgc.jp/
Asunto(s)
Bases de Datos de Proteínas , Mapeo de Interacción de Proteínas , Proteínas/análisis , Programas Informáticos , Empalme Alternativo , Animales , Humanos , Internet , Ratones , Estructura Terciaria de ProteínaRESUMEN
Bcl-X(L), an antiapoptotic member of the Bcl-2 family, is a mitochondrial protein that inhibits activation of Bax and Bak, which commit the cell to apoptosis, and it therefore represents a potential target for drug discovery. Peptides have potential as therapeutic molecules because they can be designed to engage a larger portion of the target protein with higher specificity. In the present study, we selected 16-mer peptides that interact with Bcl-X(L) from random and degenerate peptide libraries using mRNA display. The selected peptides have sequence similarity with the Bcl-2 family BH3 domains, and one of them has higher affinity (IC(50)=0.9 microM) than Bak BH3 (IC(50)=11.8 microM) for Bcl-X(L) in vitro. We also found that GFP fusions of the selected peptides specifically interact with Bcl-X(L), localize in mitochondria, and induce cell death. Further, a chimeric molecule, in which the BH3 domain of Bak protein was replaced with a selected peptide, retained the ability to bind specifically to Bcl-X(L). These results demonstrate that this selected peptide specifically antagonizes the function of Bcl-X(L) and overcomes the effects of Bcl-X(L) in intact cells. We suggest that mRNA display is a powerful technique to identify peptide inhibitors with high affinity and specificity for disease-related proteins.
Asunto(s)
Péptidos/farmacología , Proteína bcl-X/antagonistas & inhibidores , Muerte Celular/efectos de los fármacos , Biblioteca de Genes , Humanos , Mitocondrias , Biblioteca de Péptidos , Péptidos/metabolismo , Unión Proteica , ARN Mensajero , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
In vitro antibody-display technologies are powerful approaches for isolating monoclonal antibodies from recombinant antibody libraries. However, these display techniques require several rounds of affinity selection which is time-consuming. Here, we combined mRNA display with a microfluidic system for in vitro selection and evolution of antibodies and achieved ultrahigh enrichment efficiency of 10(6)- to 10(8)-fold per round. After only one or two rounds of selection, antibodies with high affinity and specificity were obtained from naive and randomized single-chain Fv libraries of approximately 10(12) molecules. Furthermore, we confirmed that not only protein-protein (antigen-antibody) interactions, but also protein-DNA and protein-drug interactions were selected with ultrahigh efficiencies. This method will facilitate high-throughput preparation of antibodies and identification of protein interactions in proteomic and therapeutic fields.
Asunto(s)
Evolución Molecular Dirigida , Región Variable de Inmunoglobulina/genética , Técnicas Analíticas Microfluídicas/métodos , ARN Mensajero/biosíntesis , Animales , Línea Celular , Biblioteca de Genes , Humanos , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/inmunología , Ratones , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/inmunologíaRESUMEN
BACKGROUND: High-throughput methods for detecting protein-protein interactions enable us to obtain large interaction networks, and also allow us to computationally identify the associations of proteins as protein complexes. Although there are methods to extract protein complexes as sets of proteins from interaction networks, the extracted complexes may include false positives because they do not account for the structural limitations of the proteins and thus do not check that the proteins in the extracted complex can simultaneously bind to each other. In addition, there have been few searches for deeper insights into the protein complexes, such as of the topology of the protein-protein interactions or into the domain-domain interactions that mediate the protein interactions. RESULTS: Here, we introduce a combinatorial approach for prediction of protein complexes focusing not only on determining member proteins in complexes but also on the DDI/PPI organization of the complexes. Our method analyzes complex candidates predicted by the existing methods. It searches for optimal combinations of domain-domain interactions in the candidates based on an assumption that the proteins in a candidate can form a true protein complex if each of the domains is used by a single protein interaction. This optimization problem was mathematically formulated and solved using binary integer linear programming. By using publicly available sets of yeast protein-protein interactions and domain-domain interactions, we succeeded in extracting protein complex candidates with an accuracy that is twice the average accuracy of the existing methods, MCL, MCODE, or clustering coefficient. Although the configuring parameters for each algorithm resulted in slightly improved precisions, our method always showed better precision for most values of the parameters. CONCLUSIONS: Our combinatorial approach can provide better accuracy for prediction of protein complexes and also enables to identify both direct PPIs and DDIs that mediate them in complexes.
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Algoritmos , Complejos Multiproteicos/química , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Proteínas/metabolismo , Análisis por Conglomerados , Programación Lineal , Técnicas del Sistema de Dos HíbridosRESUMEN
To what extent has alternative splicing contributed to the evolution of protein-function diversity? We previously constructed a pool of block-deletion mutants of the human estrogen receptor alpha ligand binding domain by random multi-recombinant PCR. Here we performed iterative in vitro selection of GTP-binding proteins by using the library of mRNA-displayed proteins and GTP-affinity chromatography combined with quantitative real-time PCR. We obtained a novel GTP-binding protein with moderate affinity and substrate-specificity. The results of our in vitro simulation imply that alternative splicing may have contributed substantially to the diversification of protein function during evolution.
Asunto(s)
Empalme Alternativo , Evolución Molecular Dirigida/métodos , Receptor alfa de Estrógeno/genética , Evolución Molecular , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/aislamiento & purificación , Secuencia de Aminoácidos , Técnicas Químicas Combinatorias , Receptor alfa de Estrógeno/química , Proteínas de Unión al GTP/genética , Biblioteca de Genes , Humanos , Ligandos , Datos de Secuencia Molecular , Estructura Terciaria de ProteínaRESUMEN
Comprehensive analysis of DNA-protein interactions is important for mapping transcriptional regulatory networks on a genome-wide level. Here we present a new application of mRNA display for in vitro selection of DNA-binding protein heterodimeric complexes. Under improved selection conditions using a TPA-responsive element (TRE) as a bait DNA, known interactors c-fos and c-jun were simultaneously enriched about 100-fold from a model library (a 1:1:20 000 mixture of c-fos, c-jun and gst genes) after one round of selection. Furthermore, almost all kinds of the AP-1 family genes including c-jun, c-fos, junD, junB, atf2 and b-atf were successfully selected from an mRNA display library constructed from a mouse brain poly A(+) RNA after six rounds of selection. These results indicate that the mRNA display selection system can identify a variety of DNA-binding protein complexes in a single experiment. Since almost all transcription factors form heterooligomeric complexes to bind with their target DNA, this method should be most useful to search for DNA-binding transcription factor complexes.
Asunto(s)
Proteínas de Unión al ADN/aislamiento & purificación , Biblioteca de Genes , ARN Mensajero/análisis , Factores de Transcripción/aislamiento & purificación , Animales , Sitios de Unión , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ratones , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/aislamiento & purificación , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/aislamiento & purificación , Proteínas Proto-Oncogénicas c-jun/metabolismo , Elementos de Respuesta , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Here we describe the application of the in vitro virus mRNA display method, which involves covalent linkage of an in vitro-synthesized antibody (phenotype) to its encoding mRNA (genotype) through puromycin, for in vitro evolution of single-chain Fv (scFv) antibody fragments. To establish the validity of this approach to directed antibody evolution, we used random mutagenesis by error-prone DNA shuffling and off-rate selection to improve the affinity of an anti-fluorescein scFv as a model system. After four rounds of selection of the library of mRNA-displayed scFv mutants, we obtained six different sequences encoding affinity-matured mutants with five consensus mutations. Kinetic analysis of the mutant scFvs revealed that the off-rates have been decreased by more than one order of magnitude and the dissociation constants were improved approximately 30-fold. The antigen-specificity was not improved by affinity maturation, but remained similar to that of the wild type. Although the five consensus mutations of the high-affinity mutants were scattered over the scFv sequence, analysis by site-directed mutagenesis demonstrated that the critical mutations for improving affinity were the two that lay within the complementarity determining regions (CDRs). Thus, mRNA display is expected to be useful for rapid artificial evolution of high-affinity diagnostic and therapeutic antibodies by optimizing their CDRs.
Asunto(s)
Evolución Molecular Dirigida/métodos , Región Variable de Inmunoglobulina/genética , ARN Mensajero/química , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Unión Competitiva , Barajamiento de ADN , Ensayo de Inmunoadsorción Enzimática , Fluoresceína/metabolismo , Biblioteca de Genes , Región Variable de Inmunoglobulina/análisis , Región Variable de Inmunoglobulina/biosíntesis , Mutagénesis , Biosíntesis de Proteínas , Resonancia por Plasmón de Superficie , Transcripción GenéticaRESUMEN
Here, we describe novel puromycin derivatives conjugated with iminobiotin and a fluorescent dye that can be linked covalently to the C-terminus of full-length proteins during cell-free translation. The iminobiotin-labeled proteins can be highly purified by affinity purification with streptavidin beads. We confirmed that the purified fluorescence-labeled proteins are useful for quantitative protein-protein interaction analysis based on fluorescence cross-correlation spectroscopy (FCCS). The apparent dissociation constants of model protein pairs such as proto-oncogenes c-Fos/c-Jun and archetypes of the family of Ca2+-modulated calmodulin/related binding proteins were in accordance with the reported values. Further, detailed analysis of the interactions of the components of polycomb group complex, Bmi1, M33, Ring1A and RYBP, was successfully conducted by means of interaction assay for all combinatorial pairs. The results indicate that FCCS analysis with puromycin-based labeling and purification of proteins is effective and convenient for in vitro protein-protein interaction assay, and the method should contribute to a better understanding of protein functions by using the resource of available nucleotide sequences.
Asunto(s)
Colorantes Fluorescentes/química , Mapeo de Interacción de Proteínas/métodos , Espectrometría de Fluorescencia/métodos , Calmodulina/análisis , Calmodulina/química , Calmodulina/aislamiento & purificación , Cromatografía de Afinidad , Proteínas Sensoras del Calcio Intracelular/análisis , Proteínas Sensoras del Calcio Intracelular/química , Proteínas Sensoras del Calcio Intracelular/aislamiento & purificación , Proteínas del Grupo Polycomb , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-fos/análisis , Proteínas Proto-Oncogénicas c-fos/química , Proteínas Proto-Oncogénicas c-fos/aislamiento & purificación , Proteínas Proto-Oncogénicas c-jun/análisis , Proteínas Proto-Oncogénicas c-jun/química , Proteínas Proto-Oncogénicas c-jun/aislamiento & purificación , Puromicina/análogos & derivados , Proteínas Represoras/análisis , Proteínas Represoras/química , Proteínas Represoras/aislamiento & purificaciónRESUMEN
Many genes with important roles in development and disease contain exceptionally long introns, but special mechanisms for their expression have not been investigated. We present bioinformatic, phylogenetic, and experimental evidence in Drosophila for a mechanism that subdivides many large introns by recursive splicing at nonexonic elements and alternative exons. Recursive splice sites predicted with highly stringent criteria are found at much higher frequency than expected in the sense strands of introns >20 kb, but they are found only at the expected frequency on the antisense strands, and they are underrepresented within introns <10 kb. The predicted sites in long introns are highly conserved between Drosophila melanogaster and Drosophila pseudoobscura, despite extensive divergence of other sequences within the same introns. These patterns of enrichment and conservation indicate that recursive splice sites are advantageous in the context of long introns. Experimental analyses of in vivo processing intermediates and lariat products from four large introns in the unrelated genes kuzbanian, outspread, and Ultrabithorax confirmed that these introns are removed by a series of recursive splicing steps using the predicted nonexonic sites. Mutation of nonexonic site RP3 within Ultrabithorax also confirmed that recursive splicing is the predominant processing pathway even with a shortened version of the intron. We discuss currently known and potential roles for recursive splicing.
Asunto(s)
Drosophila melanogaster/genética , Intrones , Empalme Alternativo , Animales , Secuencia de Bases , Línea Celular , Biología Computacional , Análisis Mutacional de ADN , Cartilla de ADN/genética , Drosophila , Proteínas de Drosophila , Exones , Eliminación de Gen , Proteínas de Homeodominio , Insectos , Modelos Genéticos , Datos de Secuencia Molecular , Método de Montecarlo , Mutación , Filogenia , ARN/química , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Factores de TranscripciónRESUMEN
Our high-throughput in vitro virus (IVV) method for selection of protein-protein interactions (PPI) and complexes, based on a simple cell-free co-translation and selection followed by computational sequence data analysis, was previously used to identify 31 Fos and Jun interactors. Here, in silico analyses of biological function, localization and phenotype of these AP-1 (Fos/Jun) interactors were performed. The results suggest that Fos and Jun do not necessarily work together, but also interact separately with novel interactors, including products of disease-related genes. Fos showed transcription-related activities, while Jun interacted with motor-related and structural proteins. The reliability of the IVV selection for the Fos interactors was further confirmed by means of in vitro reciprocal prey and bait protein experiments and co-immunoprecipitation. Further study of these novel interactors may provide clues to new pathways or mechanisms of biological functions and diseases.
Asunto(s)
Biología Computacional/métodos , Ingeniería de Proteínas/métodos , Mapeo de Interacción de Proteínas/métodos , Animales , Células COS , Chlorocebus aethiops , Electroforesis en Gel de Poliacrilamida , Inmunoprecipitación , Ratones , Plásmidos/genética , Unión Proteica , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Reproducibilidad de los Resultados , TransfecciónRESUMEN
Although yeast two-hybrid assay and biochemical methods combined with mass spectrometry have been successfully employed for the analyses of protein-protein interactions in the field of proteomics, these methods encounter various difficulties arising from the usage of living cells, including inability to analyze toxic proteins and restriction of testable interaction conditions. Totally in vitro display technologies such as ribosome display and mRNA display are expected to circumvent these difficulties. In this study, we applied an mRNA display technique to screening for interactions of a basic leucine zipper domain of Jun protein in a mouse brain cDNA library. By performing iterative affinity selection and sequence analyses, we selected 16 novel Jun-associated protein candidates in addition to four known interactors. By means of real-time PCR and pull-down assay, 10 of the 16 newly discovered candidates were confirmed to be direct interactors with Jun in vitro. Furthermore, interaction of 6 of the 10 proteins with Jun was observed in cultured cells by means of co-immunoprecipitation and observation of subcellular localization. These results demonstrate that this in vitro display technology is effective for the discovery of novel protein-protein interactions and can contribute to the comprehensive mapping of protein-protein interactions.
Asunto(s)
Biosíntesis de Proteínas , Mapeo de Interacción de Proteínas/métodos , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Animales , Línea Celular , Biblioteca de Genes , Inmunoprecipitación , Leucina Zippers , Ratones , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-jun/química , ARN Mensajero/metabolismo , Transcripción GenéticaRESUMEN
For high-throughput in vitro protein selection using genotype (mRNA)-phenotype (protein) fusion formation and C-terminal protein labeling as a post-selection analysis, it is important to improve the stability and efficiency of mRNA templates for both technologies. Here we describe an efficient single-strand ligation (90% of the input mRNAs) using a fluorescein-conjugated polyethylene glycol puromycin (Fluor-PEG Puro) spacer. This ligation provides a stable c-jun mRNA with a flexible Fluor-PEG Puro spacer for efficient fusion formation (70% of the input mRNA with the PEG spacer) in a cell-free wheat germ translation system. When using a 5' untranslated region including SP6 promoter and Omega29 enhancer (a part of tobacco mosaic virus Omega), an A(8) sequence (eight consecutive adenylate residues) at the 3' end is suitable for fusion formation, while an XA(8) sequence (XhoI and the A(8) sequence) is suitable for C-terminal protein labeling. Further, we report that Fluor-PEG N-t-butyloxycarbonylpuromycin [Puro(Boc)] spacer enhances the stability and efficiency of c-jun mRNA template for C-terminal protein labeling. These mRNA templates should be useful for puromycin-based technologies (fusion formation and C-terminal protein labeling) to facilitate high-throughput in vitro protein selection for not only evolutionary protein engineering, but also proteome exploration.
Asunto(s)
Ingeniería de Proteínas/métodos , Proteínas/análisis , ARN Mensajero/química , Regiones no Traducidas 3' , Secuencia de Bases , ADN Ligasas/metabolismo , Fluoresceína/química , Colorantes Fluorescentes/química , Polietilenglicoles/química , Biosíntesis de Proteínas , Proteínas/química , Proteínas/genética , Proteínas Proto-Oncogénicas c-jun/genética , Puromicina/química , Estabilidad del ARN , Moldes GenéticosRESUMEN
Cell-free translation systems facilitate rapid production of specific proteins and are particularly suited as high-throughput methods for whole-genome protein synthesis. Moreover, these systems do not rely on living cells, thereby allowing the synthesis of unstable or cytotoxic proteins in vitro. In this review, we describe the principles and potential applications of cell-free protein translation systems and the future prospects of proteomics approaches using next-generation sequencing and cell-free expression technologies.
Asunto(s)
Sistema Libre de Células/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas/metabolismo , Ingeniería de Proteínas/métodos , Proteómica/métodosRESUMEN
The development of high-speed analytical techniques such as next-generation sequencing and microarrays allows high-throughput analysis of biological information at a low cost. These techniques contribute to medical and bioscience advancements and provide new avenues for scientific research. Here, we outline a variety of new innovative techniques and discuss their use in omics research (e.g., genomics, transcriptomics, metabolomics, proteomics, and interactomics). We also discuss the possible applications of these methods, including an interactome sequencing technology that we developed, in future medical and life science research.
Asunto(s)
Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mapeo de Interacción de Proteínas/métodos , Humanos , Metabolómica , Proteómica , TranscriptomaRESUMEN
Nuclear factor (NF)-κB-inducing kinase (NIK) is a serine/threonine kinase that activates NF-κB pathways, thereby regulating a wide variety of immune systems. Aberrant NIK activation causes tumor malignancy, suggesting a requirement for precise regulation of NIK activity. To explore novel interacting proteins of NIK, we performed in vitro virus screening and identified the catalytic subunit Aα isoform of serine/threonine phosphatase calcineurin (CnAα) as a novel NIK-interacting protein. The interaction of NIK with CnAα in living cells was confirmed by co-immunoprecipitation. Calcineurin catalytic subunit Aß isoform (CnAß) also bound to NIK. Experiments using domain deletion mutants suggested that CnAα and CnAß interact with both the kinase domain and C-terminal region of NIK. Moreover, the phosphatase domain of CnAα is responsible for the interaction with NIK. Intriguingly, we found that TRAF3, a critical regulator of NIK activity, also binds to CnAα and CnAß. Depletion of CnAα and CnAß significantly enhanced lymphotoxin-ß receptor (LtßR)-mediated expression of the NIK-dependent gene Spi-B and activation of RelA and RelB, suggesting that CnAα and CnAß attenuate NF-κB activation mediated by LtßR-NIK signaling. Overall, these findings suggest a possible role of CnAα and CnAß in modifying NIK functions.
Asunto(s)
Calcineurina/metabolismo , Regulación de la Expresión Génica , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Dominio Catalítico , Línea Celular/metabolismo , Citocina TWEAK , Humanos , Isoenzimas , Receptor beta de Linfotoxina/metabolismo , Ratones , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-ets/genética , Transducción de Señal , Factor 3 Asociado a Receptor de TNF/metabolismo , Factor de Transcripción ReIA/metabolismo , Factor de Transcripción ReIB/metabolismo , Factores de Transcripción/genética , Factores de Necrosis Tumoral/metabolismo , Quinasa de Factor Nuclear kappa BRESUMEN
Next-generation sequencing (NGS) has been applied to various kinds of omics studies, resulting in many biological and medical discoveries. However, high-throughput protein-protein interactome datasets derived from detection by sequencing are scarce, because protein-protein interaction analysis requires many cell manipulations to examine the interactions. The low reliability of the high-throughput data is also a problem. Here, we describe a cell-free display technology combined with NGS that can improve both the coverage and reliability of interactome datasets. This in vitro method is suitable for exploring the interactome networks of transcription factors.