Development of proteasome adaptors to catalytically deplete specific proteins from cells

 描述(申请人提供)：这个项目的长期目标是开发一种通用的工具，快速有效地从真核细胞的胞浆和细胞核中去除特定的蛋白质。该项目将测试一种基于蛋白酶体适配器的策略的可行性，我们称之为降解子，建立在重组抗体平台上。蛋白酶体适配器或降解物将针对蛋白质，以被细胞泛素-蛋白酶体系统(UPS)破坏。降解物应该是一种有用的研究工具，它允许研究人员从细胞中移除特定的蛋白质来确定它们的功能。它们还可以作为一种治疗方法来消耗有毒蛋白质，如细胞中的癌基因产物。Degradons代表了一种控制细胞蛋白质浓度的新策略，它是对RNAi技术的补充，可以单独使用，也可以与RNAi方法结合使用。在RNAi干扰蛋白质合成的地方，降解物会导致蛋白质的破坏。因此，降解物不受自然蛋白质周转率的限制，可以专用于翻译后修饰形式的蛋白质。Degradon设计建立在对蛋白酶体生物化学的详细机制理解的基础上，这是过去十年来发展起来的，以及开发高亲和力和特异性相互作用剂的实验策略的成熟，最重要的是抗体技术。降解策略的灵感来自UPS的天然成分，充当底物适配器的UBL-UBA蛋白质，以及通过颠覆UPS从细胞中清除肿瘤抑制蛋白的病毒蛋白和癌基因。Degradons将由两个相互作用区域组成，一个是来自天然底物受体、病毒蛋白或癌基因的蛋白酶体结合域，另一个是主要来自重组抗体试剂的靶标识别域。这两个领域将由一个灵活的、抗降解的手臂连接起来。降解子会将结合的目标定位在最适合破坏的位置，但他们自己会逃脱蛋白分解而起催化作用。降解物将使用为基于抗体的药物开发的方法被输送到细胞。我们将在代表主要调控蛋白和重要癌基因的三个目标蛋白上测试降解设计。它们是激酶bcr-Abl、转录因子Myc和控制P53浓度的泛素连接酶MDM2。一整套系统工程的适配器将首先在体外降解系统中进行生化表征，然后在哺乳动物培养细胞和癌细胞系中对最佳设计进行优化。与RNAi相比，降解物将自行进行测试，RNAi和降解物的组合将探索两种蛋白质耗尽方法之间的协同效应。最后，将测试通过受体介导的摄取作为蛋白质药物的降解物的输送。如果降解物显示出作为蛋白质耗尽剂的前景，未来的实验将在原代细胞培养和动物模型中测试它们的有效性。

 DESCRIPTION (provided by applicant): The long-term goal of this project is to develop a versatile tool to deplete specific proteins from the cytosol and nucleus of eukaryotic cells rapidl and effectively. The project will test the feasibility of a strategy based on proteasome adaptors, which we call degradons, built on a recombinant antibody platform. The proteasome adaptors or degradons will target proteins for destruction by the cellular Ubiquitin-Proteasome-System (UPS). Degradons should be useful as a research tool by allowing the researcher to remove specific proteins from cells to determine their function. They could also serve as a therapeutic approach to deplete toxic proteins such as oncogene products from cells. Degradons represents a new strategy to control cellular protein concentrations that is complementary to RNAi technology and could be used independently or in combination with RNAi approaches. Where RNAi interferes with protein synthesis, degradons induce protein destruction. Thus, degradons are not limited by natural protein turnover rates and can be specific for post-translationally modified forms of proteins. Degradon design builds on the detailed mechanistic understanding of proteasome biochemistry that has been developed over the past ten years and the maturation of experimental strategies to develop high affinity and specificity interaction agents, most importantly antibody technology. The degradon strategy is inspired by natural components of the UPS, the UbL-UBA proteins that serve as substrate adaptors, as well as viral proteins and oncogenes that act by subverting the UPS to purge tumor suppressor proteins from cells. Degradons will consist of two interaction domains, a proteasome binding domain derived from natural substrate receptors, viral proteins or oncogenes, and a target recognition domain, mostly derived from a recombinant antibody reagent. The two domains will be linked by a flexible, degradation-resistant arm. Degradons will position the bound target optimally for destruction but themselves escapes proteolysis to act catalytically. Degradons will be delivered to cells using methods developed for antibody-based drugs. We will test degradon design on three target proteins representing major classes of regulatory proteins and important oncogens. They are the kinase Bcr-Abl, the transcription factor Myc, and the ubiquitin ligase Mdm2, which controls p53 concentrations. Sets of systematically engineered adaptors will first be characterized biochemically in an in vitro degradation system and the best designs will then be optimized in mammalian culture cells and cancer cell lines. Degradons will be tested by themselves in comparison to RNAi and combination of RNAi and degradons will explore synergistic effects between both methods of protein depletion. Finally, delivery of degradons as a protein drug by receptor-mediated uptake will be tested. If degradons show promise as a protein depletion agent, future experiments will test their effectiveness in primary cell cultures and animal models.