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Designed Ankyrin Repeat Proteins as a tool box for analyzing p63
Alexander Strubel1, Philipp Münick1, Apirat Chaikuad2,3
1Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, 60438, Frankfurt, Germany.
Abstract:
The function of the p53 transcription factor family is dependent on several folded domains. In addition to a DNA-binding domain, members of this family contain an oligomerization domain. p63 and p73 also contain a C-terminal Sterile α-motif domain. Inhibition of most transcription factors is difficult as most of them lack deep pockets that can be targeted by small organic molecules. Genetic knock-out procedures are powerful in identifying the overall function of a protein, but they do not easily allow one to investigate roles of individual domains. Here we describe the characterization of Designed Ankyrin Repeat Proteins (DARPins) that were selected as tight binders against all folded domains of p63. We determine binding affinities as well as specificities within the p53 protein family and show that DARPins can be used as intracellular inhibitors for the modulation of transcriptional activity. By selectively inhibiting DNA binding of the ΔNp63α isoform that competes with p53 for the same promoter sites, we show that p53 can be reactivated. We further show that inhibiting the DNA binding activity stabilizes p63, thus providing evidence for a transcriptionally regulated negative feedback loop. Furthermore, the ability of DARPins to bind to the DNA-binding domain and the Sterile α-motif domain within the dimeric-only and DNA-binding incompetent conformation of TAp63α suggests a high structural plasticity within this special conformation. In addition, the developed DARPins can also be used to specifically detect p63 in cell culture and in primary tissue and thus constitute a very versatile research tool for studying the function of p63.
Insights
Designed Ankyrin Repeat Proteins (DARPins) target p63 protein domains, enabling selective inhibition of transcription factors. This research reactivates p53 and reveals a negative feedback loop, offering a versatile tool for p63 research.
Area of Science:
- Molecular Biology
- Protein Engineering
- Transcription Factor Regulation
Background:
- The p53 transcription factor family, including p63 and p73, relies on specific folded domains for function.
- Targeting transcription factors for inhibition is challenging due to the lack of suitable binding pockets for small molecules.
- Investigating the roles of individual protein domains is difficult using traditional genetic methods.
Purpose of the Study:
- To characterize Designed Ankyrin Repeat Proteins (DARPins) selected for binding to p63 folded domains.
- To assess the potential of DARPins as intracellular inhibitors for modulating transcriptional activity.
- To explore the utility of DARPins in studying p63 function and regulation.
Main Methods:
- Selection and characterization of DARPins binding to p63 folded domains.
- Determination of DARPin binding affinities and specificities within the p53 family.
- In vitro and cellular assays to evaluate DARPin-mediated inhibition of transcriptional activity.
Main Results:
- DARPins were identified as tight binders for all folded domains of p63.
- DARPins effectively inhibited the DNA-binding activity of the ΔNp63α isoform, leading to p53 reactivation.
- Inhibition of p63 DNA binding stabilized p63, indicating a negative feedback mechanism.
- DARPins demonstrated binding to various p63 conformations and could detect p63 in biological samples.
Conclusions:
- DARPins serve as potent intracellular inhibitors for p63, modulating transcriptional activity.
- Selective inhibition of ΔNp63α by DARPins reactivates p53 and provides insights into regulatory feedback loops.
- The developed DARPins are versatile tools for p63 research, including detection and functional studies.
