Cellular Degradation Biology Center and Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, South Korea.
Cellular Degradation Biology Center and Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, South Korea; AUTOTAC Bio Inc., Seoul, South Korea.
Methods Enzymol. 2023;686:165-203. doi: 10.1016/bs.mie.2023.05.009. Epub 2023 Jun 7.
As defined by the N-degron pathway, single N-terminal (Nt) amino acids can function as N-degrons that induce the degradation of proteins and other biological materials. Central to this pathway is the selective recognition of N-degrons by cognate N-recognins that direct the substrates to either the ubiquitin (Ub)-proteasome system (UPS) or autophagy-lysosome pathway (ALP). Eukaryotic cells have developed diverse pathways to utilize all 20 amino acids in the genetic code as pro-N-degrons or N-degrons which can be generated through endoproteolytic cleavage or post-translational modifications. Amongst these, the arginine (Arg) N-degron plays a key role in both cis- and trans-degradation of a large spectrum of cellular materials by the proteasome or lysosome. In mammals, Arg/N-degrons can be generated through endoproteolytic cleavage or post-translational conjugation of the amino acid L-Arg by ATE1-encoded R-transferases (EC 2.3.2.8), which requires Arg-tRNA as a cofactor. Arg/N-degrons of short-lived substrates are recognized by a family of N-recognins characterized by the UBR box for polyubiquitination and proteasomal degradation. Under stresses, however, the same degrons can be recognized for autophagic degradation by the ZZ domain of the N-recognin p62/SQSTSM-1/Sequestosome-1 or KCMF1. Biochemical tools were developed to monitor the interaction of Arg/N-degrons with its cognate N-recognins. These assays were employed to identify new N-recognins and to characterize their biochemical properties and physiological functions. The principles of these assays may be applied for other types of N-degron pathways. Below, we describe the methods that analyze the interaction of Arg/N-degrons and their chemical mimics to N-recognins.
根据 N 去泛素化途径的定义，单个 N 端（Nt）氨基酸可以作为 N 去泛素化基序，诱导蛋白质和其他生物材料的降解。该途径的核心是通过识别 N 去泛素化基序的同源 N 识别因子，将底物定向到泛素（Ub）-蛋白酶体系统（UPS）或自噬-溶酶体途径（ALP）。真核细胞已经开发了多种途径，以利用遗传密码子中的所有 20 种氨基酸作为原 N 去泛素化基序或 N 去泛素化基序，这些基序可以通过内切蛋白酶切割或翻译后修饰产生。其中，精氨酸（Arg）N 去泛素化基序在蛋白酶体或溶酶体对大量细胞物质的顺式和反式降解中发挥关键作用。在哺乳动物中，Arg/N 去泛素化基序可以通过内切蛋白酶切割或通过 ATE1 编码的 R 转移酶（EC 2.3.2.8）对氨基酸 L-Arg 的翻译后缀合产生，这需要 Arg-tRNA 作为辅助因子。短寿命底物的 Arg/N 去泛素化基序被一系列 N 识别因子识别，这些 N 识别因子的特征是多泛素化和蛋白酶体降解的 UBR 盒。然而，在应激下，相同的去泛素化基序也可以被 N 识别因子 p62/SQSTSM-1/Sequestosome-1 或 KCMF1 的 ZZ 结构域识别，用于自噬降解。已经开发了生化工具来监测 Arg/N 去泛素化基序与其同源 N 识别因子的相互作用。这些测定法用于鉴定新的 N 识别因子，并表征其生化特性和生理功能。这些测定法的原理可应用于其他类型的 N 去泛素化途径。下文描述了分析 Arg/N 去泛素化基序及其化学模拟物与 N 识别因子相互作用的方法。
