Novel Molecules as In Vivo Biological Probes

描述(由申请人提供)：这项研究计划的广泛、长期目标是开发通用技术，在蛋白质分子水平上有条件地调节蛋白质功能，而不是通过瞄准编码目标蛋白质的DNA或mRNA前体。这项技术对目标蛋白质具有高度特异性，并使用细胞通透性小分子提供对蛋白质功能的快速和可调控制。其目标是设计被称为不稳定结构域的小蛋白结构域，当在哺乳动物细胞中表达时，这些结构域会迅速和有条件地降解。不稳定结构域的不稳定性被忠实地赋予与这些小结构域融合的其他蛋白质。与不稳定区域紧密结合的细胞通透性配体调节它们的稳定性。不稳定结构域与目标基因的基因融合确保了特异性，随之而来的小分子控制赋予了这种方法速度、可逆性和剂量依赖性。到目前为止，开发的试剂涉及在没有配体时不稳定的结构域，而在配体结合时稳定的结构域。这一提议的具体目的是为了提供补充的新结构域，这些结构域由于添加了细胞可渗透配体而不是稳定的，而是不稳定的。从机制上理解这些结构域在哺乳动物细胞中是如何识别和降解的，将使这项技术对用户更有用。这些研究还可能揭示细胞用来识别和降解未折叠或错误折叠的蛋白质的一般机制，这些机制可能与重要的人类疾病有关。 公共卫生相关性：该研究计划的目标是开发新的方法来快速和可逆地调节真核细胞和活的哺乳动物中特定蛋白质的稳定性。这种方法将使许多新的人类疾病模型得以开发，这些模型系统对正在进行的治疗人类疾病的新的和改进的药物的寻找有极大的帮助。

DESCRIPTION (provided by applicant): The broad, long-term objective of this research program is to develop general technology to conditionally regulate protein function at the level of the protein molecules rather than by targeting the DNA or mRNA precursors that encode a protein-of-interest. This technology is highly specific for the targeted protein and provides rapid and tunable control of protein function using cell-permeable small molecules. The goal is to engineer small protein domains called destabilizing domains that are rapidly and conditionally degraded when expressed in mammalian cells. The instability of a destabilizing domain is faithfully conferred to other proteins fused to these small domains. A cell-permeable ligand that binds tightly to the destabilizing domains regulates their stability. 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 reagents developed to date involve domains that are unstable in the absence of ligand and stabilized when ligand binds. The specific aims of this proposal are designed to deliver complementary new domains that are destabilized rather than stabilized by the addition of the cell-permeable ligand. A mechanistic understanding of how these domains are recognized and degraded in mammalian cells will make this technology more useful to users. These studies may also reveal general mechanisms that cells use to recognize and degrade unfolded or misfolded proteins, and these mechanisms are likely relevant to important human diseases. PUBLIC HEALTH RELEVANCE: The goal of this research program is to develop novel methods to rapidly and reversibly regulate the stability of specific proteins in eukaryotic cells and living mammals. This methodology would enable the development of many new models for human diseases, and these model systems are enormously helpful in the ongoing search for new and improved drugs to treat human diseases.