Development of a Photo-cleavable Agent for Reversible Protein Dimerization

用于可逆蛋白质二聚化的光裂解剂的开发

• 批准号: 8684027
• 负责人: Thomas L. Schwarz
• 金额: $ 26.3万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8684027
• 关键词: Acute; Adverse effects; Affinity; Agonist; Axonal Transport; Binding; Biological Assay; Cell Surface Receptors; Cell membrane; Cells; Chemicals; Cleaved cell; Couples; Cytolysis; Defect; Dependence; Development; Dihydrofolate Reductase; Dimerization; Dynein ATPase; Escherichia coli; Excision; Exposure to; Financial compensation; Future; Genes; Kinesin; Kinetics; Lasers; Life; Light; Lighting; Measures; Membrane; Methods; Mitochondria; Motor; Movement; Mutation; Names; Nervous system structure; Neurons; Pharmaceutical Preparations; Photochemistry; Property; Protein Engineering; Protein Isoforms; Proteins; RNA Interference; Reporter; Specificity; Structure; Synapses; Tacrolimus Binding Proteins; Techniques; Testing; Time; Variant; Work; base; crosslink; design; imaging modality; mutant; novel; novel strategies; protein activation; protein function; public health relevance; reagent testing; receptor; transcription factor

DESCRIPTION (provided by applicant): This proposal seeks to develop a novel method for controlling the activity of engineered proteins within cells. The use of chemical dimerizing agents such as rapalog have been very valuable as a means of activating cell surface receptors or disabling proteins in a cell by crosslinking them. We seek to create a Chemically-Induced Dimerization agent (CID) that is cleaved by light, thereby making the dimerization rapidly reversible. Such a molecule needs to lack endogenous binding partners in cells, be readily membrane permeant, have no toxic side effects, and be efficiently cleaved by light of a wavelength that will not cause cellular damage or interfere with standard imaging methods. We propose to characterize a candidate photocleavable CID to determine its kinetics and concentration dependence and thereby validate its suitability for work in cells in culture. We propose methods to optimize its affinity and efficacy, and we propose to test the reagent in a "split kinesin" assay of axonal transport to determine if it can be used to rapidly uncouple a cargo from its anterograde motor in a live neuron.

描述（由申请人提供）：该提案寻求开发一种用于控制细胞内工程化蛋白质活性的新方法。化学二聚剂的使用 例如雷帕霉素作为活化细胞表面受体或通过交联使细胞中的蛋白质失活的手段是非常有价值的。我们试图创造一种化学诱导的二聚化剂（CID），它被光切割，从而使二聚化迅速可逆。这样的分子需要在细胞中缺乏内源性结合配偶体，容易透过膜，没有毒副作用，并且被不会引起细胞损伤或干扰标准成像方法的波长的光有效地切割。我们建议表征候选光可裂解CID以确定其动力学和浓度依赖性，从而验证其在培养细胞中工作的适用性。我们提出了优化其亲和力和功效的方法，并且我们建议在轴突运输的“分裂驱动蛋白”测定中测试该试剂，以确定它是否可用于快速地将货物从其在活神经元中的顺行马达中解偶联。