Conditional Protein Targeting

项目描述：本研究项目的广泛、长期目标是开发在蛋白质分子水平上有条件地调节蛋白质功能的一般方法，而不是通过靶向编码特定蛋白质的前体DNA或mRNA序列。这项新技术应该完全针对目标蛋白，并且应该使用细胞渗透性小分子提供快速和剂量依赖性的蛋白质功能控制。细胞通常通过平衡蛋白质分子的产生和降解来调节蛋白质的表达水平。现有的大多数扰动方法，如转录开关和RNAi，都集中在蛋白质合成上，但是当合成被关闭时，不可避免地会有实验延迟，因为现有的蛋白质必须被降解。这项研究计划采取了一种完全不同的方法，专注于有条件地针对特定蛋白质进行降解的方法。目标是设计小的蛋白质结构域，当在哺乳动物细胞中表达时，这些结构域可以快速和组成性地降解。重要的是，不稳定结构域的不稳定性忠实地传递给融合到这些小结构域的其他蛋白质。添加一个细胞渗透性配体，与不稳定结构域紧密结合，保护它们免受降解，使融合蛋白能够发挥其细胞功能。不稳定域与感兴趣基因的遗传融合确保了特异性，随之而来的小分子控制为该方法提供了速度、可逆性和剂量依赖性。本提案的具体目标集中在开发新的不稳定结构域，为哺乳动物细胞中表达的膜结合蛋白以及酵母中表达的必需蛋白的蛋白表达提供条件控制。这项技术将用于有条件地调节参与分化细胞表观遗传重塑的酶。相关性：本研究的目的是开发通用的新方法来快速和可逆地调节真核细胞中特定蛋白质的表达。这种方法将有助于开发许多新的人类疾病模型（包括培养细胞或敲除小鼠），这些模型系统对正在进行的寻找新的和改进的药物来治疗人类疾病有很大的帮助。

DESCRIPTION (provided by applicant): Project Description: The broad, long-term objective of this research program is to develop general methods to conditionally regulate protein function at the level of the protein molecules rather than by targeting the precursor DNA or mRNA sequences that encode a particular protein of interest. This new technology should be completely specific for the targeted protein and should provide rapid and dose-dependent control of protein function using cell-permeable small molecules. Cells typically regulate protein expression levels by balancing the creation and degradation of protein molecules. The majority of existing perturbation methods such as transcriptional switches and RNAi focus on protein synthesis, however when synthesis is switched off there is inevitably an experimental delay as the existing protein must be degraded. This research proposal takes a fundamentally different approach by focusing on methods to conditionally target specific proteins for degradation. The goal is to engineer small protein domains that are rapidly and constitutively degraded when expressed in mammalian cells. Importantly, the instability of a destabilizing domain is faithfully conferred to other proteins fused to these small domains. Addition of a cell-permeable ligand that binds tightly to the destabilizing domains shields them from degradation, allowing the fused proteins to perform their cellular functions. The genetic fusion of the destabilizing domain to the gene-of-interest ensures specificity, and the attendant small-molecule control confers speed, reversibility and dose-dependence to this method. The specific aims of this proposal focus on developing new destabilizing domains that provide conditional control of protein expression to membrane-bound proteins expressed in mammalian cells as well as essential proteins expressed in yeast. This technology will be used to conditionally regulate enzymes involved in epigenetic remodeling of differentiated cells. Relevance: The goal of this research is to develop general new methods to rapidly and reversibly regulate the expression of specific proteins in eukaryotic cells. This methodology would enable the development of many new models for human diseases (including cultured cells or knock-out mice), and these model systems are enormously helpful in the ongoing search for new and improved drugs to treat human diseases.