Yeast two-hybrid screening for proteins interacting with the anti-apoptotic protein A20.
The yeast two-hybrid system is a powerful technique for identifying proteins that interact with a specific protein of interest. The rationale of the yeast two-hybrid system relies on the physical separation of the DNA-binding domain from the transcriptional activation domain of several transcription factors. Therefore, the protein of interest (bait) is fused to a DNA-binding domain, and complimentary DNA (cDNA) library-encoded proteins are fused to a transcriptional activation domain. When a protein encoded by the cDNA library binds to the bait, both activities of the transcription factor are rejoined and transcription from a reporter gene is started. Here, we will give a comprehensive guide for the GAL4-based two-hybrid system, exemplified by the detection of binding partners for the zinc finger protein A20. The latter is an inducible cellular inhibitor of tumor necrosis factor (TNF)-induced apoptosis and nuclear factor (NF)-kappaB-dependent gene expression. Yeast two-hybrid screening with A20 as bait revealed several A20-binding proteins, including A20 itself, members of the 14-3-3 family, as well as three novel proteins ABIN-1, ABIN-2, and TXBP151. The latter protein was subsequently shown to mediate at least part of the anti-apoptotic activities of A20, whereas ABIN-1 and -2 are more likely to be involved in the NF-kappaB inhibitory effects of A20.
Heyninck
Karen
K
Department of Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, Belgium.
Van Huffel
Sofie
S
Kreike
Marja
M
Beyaert
Rudi
R
D000818:Animals; D003546:Cysteine Endopeptidases; D015723:Gene Library; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D009687:Nuclear Proteins; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics / Q000502:physiology*
D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D003546:Cysteine Endopeptidases; C513261:Tnfaip3 protein, mouse