Investigation of TAp63a with conformation-selective DARPins

用构象选择性 DARPins 研究 TAp63a

• 批准号: 367436894
• 负责人: Professor Dr. Volker Dötsch
• 金额: --
• 依托单位: Institut für Biophysikalische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/367436894?language=en
• 关键词: Investigation; TAp63a; conformation; selective; DARPins

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.

设计锚蛋白重复蛋白 (DARPins) 是由 3-5 个模块组成的模块化蛋白，可用作类似于抗体的特异性结合剂。通过核糖体展示，可以创建识别特定蛋白质或特定构象的 DARPins。 p63 蛋白是 p53 肿瘤抑制蛋白家族的成员，但其本身并不是经典的肿瘤抑制蛋白。它作为控制上皮组织基底层增殖潜力的因素和卵母细胞的质量控制因素发挥着重要的发育功能。在卵母细胞中，表达 p63 的特定亚型，它只能采用二聚体和闭合构象，通过磷酸化被激活为开放、活性和四聚体状态。我们创建了 DARPins，它可以选择性地识别封闭二聚体或开放四聚体状态。此外，我们还针对所有 p53 家族成员的所有折叠域创建了 DARPins。我们现在想要验证这些 DARPins 在细胞环境中的选择性，然后使用它们来搜索小鼠组织的不同构象状态。特别是，有人提出，在卵母细胞中发现的同工型也存在于某些真皮前体细胞中，并在皮肤的胚胎发育过程中发挥作用。我们将研究该蛋白质的哪些不同构象在这些组织和发育阶段被采用。此外，我们可以在体外证明 DARPins 可以稳定 p63 的突变 SAM 结构域。由于不稳定的 SAM 结构域聚集，该结构域的突变会导致人类患海威尔斯综合症。我们想要在细胞培养中测试 DARPins 是否可以稳定 SAM 结构域，从而防止聚集。