Investigation of TAp63a with conformation-selective DARPins

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Designed Ankyrin Repeat Proteins (DARPins) are modular proteins consisting of 3-5 modules that can be used as specific binders similar to antibodies. Through ribosome display DARPins recognizing specific proteins or specific conformations can be created. The protein p63 is a member of the family of the p53 tumor suppressor, however, is not a classical tumor suppressor itself. It serves important developmental functions as a factor controlling the proliferative potential in the basal layer of epithelial tissue and as a quality control factor in oocytes. In oocytes a specific isoform of p63 is expressed that can adopt an only dimeric and closed conformation that is activated into the open, active and tetrameric state through phosphorylation. We have created DARPins that selectively can identify either the closed dimeric or the open and tetrameric state. In addition, we have created DARPins against all folded domains of all p53 family members. We now want to validate the selectivity of these DARPins in a cellular context and then use them to search mouse tissues for the different conformational states. In particular, it has been proposed that the isoform found in oocytes also exists in certain dermal precursor cells and plays a role during embryonic development of the skin. We will investigate which of the different conformations of this protein is adopted in these tissues and developmental stages. In addition, we could show in vitro that DARPins can stabilize the mutated SAM domain of p63. Mutations in this domain result in humans in the Hay-Wells Syndrome, due to aggregation of the destabilized SAM domain. We want to test in cell culture if the DARPins can stabilize the SAM domain and thus prevent aggregation.