Novel Molecules as In Vivo Biological Probes

描述（由申请人提供）：本研究计划的广泛、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，而不是通过靶向编码目标蛋白质的DNA或mRNA前体。该技术对靶蛋白具有高度特异性，并使用细胞可渗透的小分子提供对蛋白质功能的快速和可调控制。其目标是设计称为去稳定结构域的小蛋白质结构域，当在哺乳动物细胞中表达时，这些结构域会快速且有条件地降解。去稳定化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。一个细胞可渗透的配体，紧密结合到不稳定的结构域调节其稳定性。去稳定结构域与感兴趣的基因的遗传融合确保了特异性，并且伴随的小分子控制赋予该方法速度、可逆性和剂量依赖性。迄今为止开发的试剂涉及在配体不存在下不稳定并且在配体结合时稳定的结构域。该提议的具体目的是提供互补的新结构域，其通过加入细胞可渗透配体而不稳定。对这些结构域如何在哺乳动物细胞中被识别和降解的机械理解将使这项技术对用户更有用。这些研究还可能揭示细胞用于识别和降解未折叠或错误折叠蛋白质的一般机制，这些机制可能与重要的人类疾病有关。

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.