IC-Tagging and Protein Relocation to ARV muNS Inclusions: A Method to Study Protein-Protein Interactions in the Cytoplasm or Nucleus of Living Cells
Por favor, use este identificador para citas ou ligazóns a este ítem:
http://hdl.handle.net/10347/22843
Ficheiros no ítem
Metadatos do ítem
Título: | 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/a: | Brandariz Núñez, Alberto Menaya Vargas, Rebeca Benavente Martínez, Francisco Javier Martínez Costas, José Manuel |
Centro/Departamento: | Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular |
Palabras chave: | Cytoplasmic inclusions | Immunostaining | Protein expression | Protein interactions | Birds | Plasmid construction | SV40 | Protein-protein interactions | |
Data: | 2010 |
Editor: | PLOS |
Cita bibliográfica: | Brandariz-Nuñez A, Menaya-Vargas R, Benavente J, Martinez-Costas J (2010) IC-Tagging and Protein Relocation to ARV muNS Inclusions: A Method to Study Protein-Protein Interactions in the Cytoplasm or Nucleus of Living Cells. PLoS ONE 5(11): e13785. https://doi.org/10.1371/journal.pone.0013785 |
Resumo: | 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. |
Versión do editor: | https://doi.org/10.1371/journal.pone.0013785 |
URI: | http://hdl.handle.net/10347/22843 |
DOI: | 10.1371/journal.pone.0013785 |
E-ISSN: | 1932-6203 |
Dereitos: | © 2010 Brandariz-Nuñez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited |
Coleccións
-
- BBM-Artigos [91]
A licenza do ítem descríbese como
© 2010 Brandariz-Nuñez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
© 2010 Brandariz-Nuñez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Items relacionados
Mostrando items relacionados por título, autor e materia.
-
The Major Storage Protein in Potato Tuber Is Mobilized by a Mechanism Dependent on Its Phosphorylation Status
Bernal Pampín, Javier; Mouzo Calzadilla, Daniel; López Pedrouso, María Dolores; Franco Ruiz, Daniel; García Calvo, Lucio; Zapata Babío, José Carlos (MDPI, 2019)The role of the protein phosphorylation mechanism in the mobilization of vegetative storage proteins (VSPs) is totally unknown. Patatin is the major VSP of the potato (Solanum tuberosum L.) tuber that encompasses multiple ... -
Pore size is a critical parameter for obtaining sustained protein release from electrochemically synthesized mesoporous silicon microparticles
Pastor, Ester L.; Reguera-Nuñez, Elaine; Matveeva, Eugenia; García-Fuentes, Marcos (PeerJ, 2015)Mesoporous silicon has become a material of high interest for drug delivery due to its outstanding internal surface area and inherent biodegradability. We have previously reported the preparation of mesoporous silicon ... -
Hydration in Deep Eutectic Solvents Induces Non-monotonic Changes in the Conformation and Stability of Proteins
Sánchez Fernández, Adrián; Basic, Medina; Xiang, Jenny; Prevost, Sylvain; Jackson, Andrew J.; Dicko, Cedric (ACS Publications, 2022)The preservation of labile biomolecules presents a major challenge in chemistry, and deep eutectic solvents (DESs) have emerged as suitable environments for this purpose. However, how the hydration of DESs impacts the ...