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A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions
Published on: July 18, 2013
A new method for the selection of protein interactions in mammalian cells
E Rojo-Niersbach1, D Morley, S Heck
1Max-Delbrück-Laboratorium in der Max-Planck-Gesellschaft, Carl-von-Linné-Weg 10, 50829 Köln, Germany.
Insights
This study introduces a novel method for detecting protein interactions in mammalian cells. The system successfully confirmed interactions in vivo, offering an alternative to the yeast two-hybrid system.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Understanding protein-protein interactions is crucial for deciphering cellular processes.
- Existing methods like the yeast two-hybrid system have limitations in mammalian cell contexts.
- Verifying interactions in vivo is essential for biological relevance.
Purpose of the Study:
- To develop and validate a new method for selecting protein interactions within mammalian cells.
- To confirm previously characterized in vitro protein interactions in a live-cell setting.
- To provide a broadly applicable alternative to existing interaction screening techniques.
Main Methods:
- A novel system utilizing reconstituted ubiquitin and a reporter gene (gpt2) modified with an arginine residue.
- Proteins of interest were fused to complementary ubiquitin halves.
- Selection and counterselection in a human fibroblast cell line (HT1080HPRT(-)) using specific culture media.
Main Results:
- Demonstrated the in vivo occurrence of interactions between human TATA-binding protein 1 and nuclear factor kappaB.
- Confirmed the in vivo association of Homo sapiens nuclear autoantigen SP100B with human heterochromatin protein 1alpha.
- Successfully selected for cells exhibiting these protein interactions using the developed system.
Conclusions:
- The novel method enables the selection of protein interactions directly within mammalian cells.
- This system validates in vitro findings by demonstrating in vivo interactions.
- The approach offers a viable and generalizable alternative to the yeast two-hybrid system for studying protein interactions.
Abstract:
In the present study we present a new method that allows for the selection of protein interactions in mammalian cells. We have used this system to verify two interactions previously characterized in vitro. (1) The interaction between human TATA-binding protein 1 and nuclear factor kappaB and (2) the association of Homo sapiens nuclear autoantigen SP100B with human heterochromatin protein 1alpha, a protein implicated in chromatin remodelling. We observe for the first time that these interactions also occur in vivo. One protein was fused to the N-terminal half of ubiquitin, while the interacting partner was fused to the C-terminal half of ubiquitin, that was itself linked to guanine phosphoryltransferase 2 (gpt2) modified to begin with an arginine residue. Upon interaction of both proteins, ubiquitin is reconstituted, and its association with the Rgpt2 reporter is subsequently cleaved off by ubiquitin-processing enzymes. The presence of arginine in the Rgpt2 gene product leads to the degradation of the product by the N-end rule pathway. In the human fibroblast cell line HT1080HPRT(-) (that is deficient in the enzyme for hypoxanthine-guanine phosphoribosyltransferase) cells in which interaction between both proteins of interest occurs can then be selected for by hypoxanthine/aminopterin/thymine medium and counterselected against by 6-thioguanine medium. This method provides a suitable alternative to the yeast two-hybrid system and is generally applicable.
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