In vitro selection of Jun-associated proteins using mRNA display

Abstract

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.

Modulation of Glucose Metabolism by CD44 Contributes to Antioxidant Status and Drug Resistance in Cancer Cells

“…IVV screening is a powerful tool for identifying biologic macromolecules that participate in protein-protein interactions (15)(16)(17). By using this approach, we identified PKM2 as a mediator of the promotion of the glycolytic phenotype of cancer cells by CD44.…”

“…IVV screening and GST pull-down assay IVV selection and glutathione S-transferase (GST) pulldown assay were conducted as described previously (15)(16)(17). The cDNA library for screening was obtained from U251MG cells.…”

Musashi Proteins in Neural Stem/Progenitor Cells

“…This result indicates that currently unknown molecular machinery may exist that differs from the translational suppression machinery (Kawahara et al, 2008). Components of this machinery, e.g., binding proteins of Msi1, need to be comprehensively clarified using high-throughput techniques (Rigaut et al, 1999;Horisawa et al, 2004Horisawa et al, , 2008.…”

Notch Signaling in Hair Follicle Development

“…These results appear to be in opposition to previous findings that Msi1 represses the translation of its targets (32) and imply the existence of another molecular machinery that activates translation of targets. Components of this machinery, e.g., binding proteins of Msi1, need to be clarified comprehensively by means of high-throughput techniques (77)(78)(79).…”