Enzymatic Protein Labeling

描述(由申请人提供)：蛋白质在生物系统中执行一系列特别不同的任务。为了研究这些分子在体外和体内的功能，并创造具有治疗和诊断能力的新分子实体，研究人员开发了一系列广泛的方法来对蛋白质进行化学修饰。虽然所有这些方法都很有用，但它们也都有限制其实用性的缺点。最近，我们开发了一种利用蛋白质法尼基转移酶(PFTase)对蛋白质进行酶修饰的方法。这种方法允许叠氮化物和含炔的底物转移到含有四个小残基的蛋白质上，并在其C-末端测序。叠氮化或炔功能化蛋白的后续生物正交反应可用于制备各种蛋白质偶联物。这种方法的独特之处在于，它允许用最低限度的标签实现选择性的共价蛋白质修饰。在这项应用中，我们建议利用这一蛋白质修饰策略的效用，将其应用于治疗学中的几个重要问题，同时我们继续改进和完善它。本项目的具体目标是：(1)设计和合成简化的叠氮基、炔基和其他含功能基团的PFTase底物(2)开发一个通用的定向进化系统，以产生有用的PFTase新突变体，包括具有松弛和/或替代异戊二烯和肽/蛋白质底物专一性的酶。(3)采用PFTase催化的酶促蛋白质修饰方法，制备稳定的聚乙二醇化形式的促红细胞生成素(EPO)和脑源性神经营养因子(BDNF)，并评价其对大鼠鼻内缺氧模型的神经保护作用。(4)用类似的方法制备抗体-RNA结合物，可用于靶向T细胞的siRNAs。如果成功，本申请中描述的工作可能导致改进的基于蛋白质的药物，可用于治疗中风、阿尔茨海默氏症和其他神经退行性疾病，以及用于治疗自身免疫性疾病和T细胞白血病的新的、更具选择性的药物。此外，这里开发的蛋白质修饰方法学应该有助于制备各种各样的其他蛋白质结合物，这些结合物可以用于大量的治疗和诊断应用。 公共卫生相关性：如果成功，本申请中描述的工作可能导致改进的基于蛋白质的药物，可用于治疗中风、阿尔茨海默氏症和其他神经退行性疾病以及自身免疫性疾病和癌症。还将开发新的方法，这些方法也可以转化为其他疾病的新治疗剂的开发。

DESCRIPTION (provided by applicant): Proteins perform a particularly diverse range of tasks in biological systems. To study how these molecules function in vitro and in vivo and to create new molecular entities with therapeutic and diagnostic capabilities, researchers have developed a broad range of methods to chemically modify proteins. While all of these methods are useful, they also all have disadvantages that limit their utility. Recently, we developed a method for enzymatically modifying proteins using the enzyme protein farnesyltransferase (PFTase). This method allows azide and alkyne-containing substrates to be transferred to proteins containing a small, four residue, and sequence at their C-terminus. Subsequent bio- orthogonal reaction of the azide or alkyne-functionalized protein can be used to prepare a wide variety of protein conjugates. What makes this approach unique is the fact that it allows selective covalent protein modification to be achieved with a minimalist tag. In this application we propose to capitalize on the utility of this protein modification strategy by applying it to several important problems in therapeutics while we continue to improve and refine it. The specific aims of this project are: (1) Design and synthesize simplified azide-, alkyne-, and other functional group-containing substrates for PFTase (2) Develop a general directed evolution system to produce useful new mutants of PFTase including enzymes with relaxed and/or alternative isoprenoid and peptide/protein substrate specificity. (3) Use the PFTase catalyzed enzymatic protein modification method to prepare PEGylated forms of erythropoietin (EPO) and brain derived neurotrophic factor (BDNF) with increased stability and evaluate their neuroprotective efficacy following intranasal application in a rat hypoxia model. (4) Use a similar approach to prepare antibody-RNA conjugates that can be used to target siRNAs to T cells. If successful, the work described in this application could lead to improved protein- based drugs that could be used for the treatment of stroke, Alzheimer's and other neurodegenerative conditions as well as new, more selective agents for the treatment of autoimmune diseases and T cell leukemias. Moreover, the protein modification methodology developed here should be useful for the preparation of a wide variety of other protein conjugates that could be employed for a plethora of therapeutic and diagnostic applications. PUBLIC HEALTH RELEVANCE: If successful, the work described in this application could lead to improved protein-based drugs that could be used for the treatment of stroke, Alzheimer's and other neurodegenerative conditions as well as autoimmune diseases and cancer. New methods will also be developed that could be translated into the development of novel therapeutic agents for other diseases as well.