Investigation of TAp63a with conformation-selective DARPins

设计的锚蛋白重复蛋白（DARPins）是由3-5个模块组成的模块蛋白，可用作类似于抗体的特异性结合剂。通过核糖体展示，可以产生识别特定蛋白质或特定构象的DARPin。p63蛋白是p53肿瘤抑制因子家族的一员，然而，它本身并不是典型的肿瘤抑制因子。作为控制上皮组织基底层增殖潜力的因子和作为卵母细胞质量控制因子，它具有重要的发育功能。在卵母细胞中，表达p63的特定同种型，其可以采用仅二聚体和闭合构象，其通过磷酸化活化成开放、活性和四聚体状态。我们已经创造了DARPin，它可以选择性地识别封闭的二聚体或开放的四聚体状态。此外，我们已经创建了针对所有p53家族成员的所有折叠结构域的DARPin。我们现在想验证这些DARPin在细胞环境中的选择性，然后用它们来搜索小鼠组织的不同构象状态。特别地，已经提出在卵母细胞中发现的同种型也存在于某些真皮前体细胞中，并且在皮肤的胚胎发育期间起作用。我们将研究这种蛋白质的不同构象中的哪一种在这些组织和发育阶段被采用。此外，我们可以在体外证明DARPin可以稳定p63的突变SAM结构域。由于不稳定的SAM结构域的聚集，该结构域中的突变导致人类发生Hay-Wells综合征。我们想在细胞培养中测试DARPin是否可以稳定SAM结构域，从而防止聚集。

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.