Novel Molecules as In Vivo Biological Probes

作为体内生物探针的新型分子

• 批准号: 8470176
• 负责人: THOMAS James WANDLESS
• 金额: $ 30.45万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2014-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8470176
• 关键词: Affinity; Behavior; Biological; Biological Models; Cell physiology; Cells; Complement; Cultured Cells; DNA; Dependence; Development; Disease model; Dose; Engineering; Ensure; Equilibrium; Eukaryotic Cell; Funding; Genes; Genetic; Goals; Hour; Human; Life; Ligand Binding; Ligands; Mammalian Cell; Mammals; Mass Spectrum Analysis; Methodology; Methods; Modeling; Mus; Pharmaceutical Preparations; Proteins; RNA Interference; Reagent; Research; Research Personnel; Small Interfering RNA; Specificity; Speed; System; Tacrolimus Binding Proteins; Techniques; Technology; Tertiary Protein Structure; Testing; Tetracyclines; Withdrawal; base; design; human disease; improved; in vivo; interest; knock-down; mRNA Precursor; mutant; novel; programs; prospective; protein degradation; protein function; protein misfolding; public health relevance; screening; small molecule; transgene expression

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.

描述（由申请人提供）：该研究计划的广泛、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感兴趣蛋白质的 DNA 或 mRNA 前体。该技术对目标蛋白质具有高度特异性，并使用细胞渗透性小分子对蛋白质功能进行快速且可调节的控制。目标是设计称为不稳定结构域的小蛋白质结构域，这些结构域在哺乳动物细胞中表达时会快速、有条件地降解。不稳定结构域的不稳定性忠实地赋予了与这些小结构域融合的其他蛋白质。与不稳定结构域紧密结合的细胞渗透性配体可调节其稳定性。不稳定结构域与感兴趣基因的基因融合确保了特异性，并且伴随的小分子控制赋予了该方法速度、可逆性和剂量依赖性。迄今为止开发的试剂涉及在没有配体的情况下不稳定并且在配体结合时稳定的结构域。该提案的具体目标是提供互补的新结构域，这些结构域通过添加细胞渗透性配体而变得不稳定而不是稳定。从机制上理解这些结构域如何在哺乳动物细胞中被识别和降解将使该技术对用户更有用。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蛋白质的一般机制，并且这些机制可能与重要的人类疾病相关。