IC-Tagging and Protein Relocation to ARV muNS Inclusions: A Method to Study Protein-Protein Interactions in the Cytoplasm or Nucleus of Living Cells
| Autor: | Javier Benavente, Alberto Brandariz-Nuñez, Rebeca Menaya-Vargas, José Martínez-Costas |
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| Přispěvatelé: | Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular |
| Rok vydání: | 2010 |
| Předmět: |
Cytoplasm
Cytoplasmic inclusion lcsh:Medicine Chick Embryo Plasma protein binding Immunostaining Viral Nonstructural Proteins Biochemistry/Protein Folding Inclusion Bodies Viral Cytoplasmic inclusions SV40 Chlorocebus aethiops Protein Interaction Mapping lcsh:Science Biochemistry/Biomacromolecule-Ligand Interactions Cells Cultured Multidisciplinary Transfection Transport protein Cell biology Protein Transport medicine.anatomical_structure COS Cells Biotechnology/Protein Chemistry and Proteomics Biotechnology/Bioengineering Research Article Protein Binding Protein-protein interactions Orthoreovirus Avian Recombinant Fusion Proteins Green Fluorescent Proteins Biology Plasmid construction Protein–protein interaction Birds Virology medicine Animals Cell Nucleus lcsh:R Protein interactions Fibroblasts Molecular biology Peptide Fragments Cell nucleus Microscopy Fluorescence Protein expression lcsh:Q Tumor Suppressor Protein p53 Protein ligand |
| Zdroj: | Minerva. Repositorio Institucional de la Universidad de Santiago de Compostela instname PLoS ONE, Vol 5, Iss 11, p e13785 (2010) PLoS ONE |
| Popis: | Background: Characterization of protein-protein interactions is essential for understanding cellular functions. Although there are many published methods to analyze protein-protein interactions, most of them present serious limitations. In a different study we have characterized a novel avian reovirus muNS-based protein tagging and inclusion targeting method, and demonstrated its validity to purify free an immobilized protein. Methodology/Principal Findings: Here we present a method to identify protein-protein interactions inside living eukaryotic cells (tested in primate and avian cells). When p53 was tagged with Intercoil (IC; muNS residues 477–542), it not only got integrated into muNS cytoplasmic inclusions, but also attracted its known ligand SV40 large T antigen (TAg) to these structures. We have also adapted this system to work within the cell nucleus, by creating muNS-related protein chimeras that form nuclear inclusions. We show that nuclear muNS-derived inclusions are as efficient as cytoplasmic ones in capturing IC-tagged proteins, and that the proteins targeted to nuclear inclusions are able to interact with their known ligands. Conclusions/Significance: Our protein redistribution method does not present the architectural requirement of reconstructing a transcription factor as any of the two-hybrid systems do. The method is simple and requires only cell transfection and a fluorescence microscope. Our tagging method can be used either in the cytoplasm or the nucleus of living cells to test protein-protein interactions or to perform functional studies by protein ligand sequestration. This work was supported by grants from the European Commission under contracts ERAS-CT-2003-980409 (as part of the European Science Foundation EUROCORES Programme EuroSCOPE, web: http://www.esf.org/euroscope); the Spanish Ministerio de Ciencia y Tecnología (BFU2007-61330, BFU 205- 24982-E, web: http://www.mityc.es/) and Xunta de Galicia (08CSA009203PR, web: http://www.conselleriaiei.org/ga/dxidi/index.php). ABN was the recipient of a predoctoral FPI fellowship from the Spanish Ministerio de Ciencia y Tecnología SI |
| Databáze: | OpenAIRE |
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