Van Fossen Elise M, Grutzius Sonia, Ruby Carl E, Mourich Dan V, Cebra Chris, Bracha Shay, Karplus P Andrew, Cooley Richard B, Mehl Ryan A
Oregon State University, Department of Biochemistry and Biophysics, Agricultural and Life Sciences, Corvallis, OR, United States.
Oregon State University, Department of Clinical Sciences, College of Veterinary Medicine, Corvallis, OR, United States.
Front Chem. 2022 Feb 21;10:835229. doi: 10.3389/fchem.2022.835229. eCollection 2022.
A critical step in developing therapeutics for oxidative stress-related pathologies is the ability to determine which specific modified protein species are innocuous by-products of pathology and which are causative agents. To achieve this goal, technologies are needed that can identify, characterize and quantify oxidative post translational modifications (oxPTMs). Nanobodies (Nbs) represent exquisite tools for intracellular tracking of molecules due to their small size, stability and engineerability. Here, we demonstrate that it is possible to develop a selective Nb against an oxPTM protein, with the key advance being the use of genetic code expansion (GCE) to provide an efficient source of the large quantities of high-quality, homogenous and site-specific oxPTM-containing protein needed for the Nb selection process. In this proof-of-concept study, we produce a Nb selective for a 3-nitrotyrosine (nitroTyr) modified form of the 14-3-3 signaling protein with a lesser recognition of nitroTyr in other protein contexts. This advance opens the door to the GCE-facilitated development of other anti-PTM Nbs.
开发针对氧化应激相关病症的治疗方法的关键一步是能够确定哪些特定的蛋白质修饰产物是无害的病理副产物，哪些是致病因子。为了实现这一目标，需要能够识别、表征和量化氧化后翻译修饰（oxPTM）的技术。纳米抗体（Nb）由于其体积小、稳定性高和可工程化，是用于细胞内分子追踪的绝佳工具。在这里，我们证明了开发一种针对oxPTM蛋白的选择性纳米抗体是可行的，关键进展在于利用遗传密码扩展（GCE）来提供大量高质量、同质且位点特异性含oxPTM蛋白的有效来源，这是纳米抗体筛选过程所必需的。在这项概念验证研究中，我们制备了一种对14-3-3信号蛋白的3-硝基酪氨酸（硝基酪氨酸）修饰形式具有选择性的纳米抗体，而在其他蛋白质环境中对硝基酪氨酸的识别较少。这一进展为GCE促进的其他抗PTM纳米抗体的开发打开了大门。
