An inducible protein knockout strategy based on an orthogonal, ligand-activated E3 ubiquitin ligase

 描述（由申请人提供）：DNA水平的遗传操作和RNA干扰（RNAi）已成为蛋白质功能研究的主流工具。然而，诱导型蛋白质敲除与这些传统方法相比具有许多优点，包括通过配体诱导快速和可逆反应的潜力、可调谐性（剂量依赖性定量耗尽）、仅从特定细胞库耗尽靶蛋白以及耗尽靶蛋白的特异性修饰形式。然而，一个通用的可诱导的蛋白质敲除系统，可以耗尽特定的内源性蛋白质（S）仍然有待开发。最近，我们已经确定了E3泛素连接酶变构激活的第一个分子机制。RNF 146在不存在配体的情况下是本质上无活性的E3，并且在其配体（iso-ADPr，聚（ADP-核糖）的结构单元）的存在下变得稳健地活化。我们的目标是利用RNF 146/iso-ADPr作为模板，开发一种可诱导的、可调节的蛋白质敲除系统。该开发的瓶颈是设计细胞可渗透的iso-ADPr直向同源物并相应地工程化RNF 146蛋白，使得工程化蛋白响应于PAR聚合物中的直向同源物化合物而不响应于内源性iso-ADPr部分。在这个项目中，我们将克服这个瓶颈，并验证我们的共同工程化的正交蛋白/配体对所需的结合和激活特异性，使用内源性β-连环蛋白，Wnt信号传导的中央效应子的耗竭作为模型系统。开发和优化通用的诱导型蛋白质敲除系统不仅为生物学研究提供了强有力的工具，而且可能在医学上具有深远的意义。

 DESCRIPTION (provided by applicant): Genetic manipulations at the DNA level and RNA interference (RNAi) have been the mainstream tools for functional studies of proteins. However, inducible protein knockout has a number of advantages over these traditional approaches, including the potential to induce fast and reversible response by the ligand, tunability (dosage-dependent quantitative depletion), depletion of the target protein from a specific cellular pool only, and depletion of a specifically-modified form of the target protein. However, a general inducible protein knockout system that can deplete specific endogenous protein(s) remains to be developed. Most recently, we have defined the first molecular mechanism for allosteric activation of E3 ubiquitin ligases. RNF146 is an intrinsically inactive E3 in the absent of a ligan, and becomes robustly activated in the presence of its ligand (iso-ADPr, a structural unit of poly (ADP-ribose)). With well-defined molecular mechanism and structural basis for this allosteric switch, we aim to use RNF146/iso-ADPr as the template to develop an inducible, tunable protein knockout system. The bottle neck for this development is to design cell-permeable iso-ADPr orthologue(s) and engineer the RNF146 protein accordingly, so that the engineered protein responds to the orthologue compound but not to the endogenous iso-ADPr moiety in PAR polymers. In this project, we will overcome this bottleneck and validate the desired binding and activation specificities for our co-engineered orthogonal protein/ligand pairs, using the depletion of endogenous -catenin, a central effector of Wnt signaling, as a model system. Development and optimization of a general inducible protein knockout system will not only provide a powerful tool for biological research, but may also have profound implications in medicine.