Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom; Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
Methods Enzymol. 2023;686:267-295. doi: 10.1016/bs.mie.2023.02.004. Epub 2023 Mar 10.
2-Aminoethanethiol dioxygenase (ADO) is the mammalian orthologue of the plant cysteine oxidases and together these enzymes are responsible for catalysing dioxygenation of N-terminal cysteine residues of certain proteins. This modification creates an N-degron motif that permits arginylation and subsequent proteasomal degradation of such proteins via the Arg-branch of the N-degron pathway. In humans 4 proteins have been identified as substrates of ADO; regulators of G-protein signalling (RGS) 4, 5 and 16, and interleukin-32 (IL-32). Nt-cysteine dioxygenation of these proteins occurs rapidly under normoxic conditions, but ADO activity is very sensitive to O availability and as such the stability of substrate proteins is inversely proportional to cellular O levels. Much is still to understand about the biochemistry and physiology of this pathway in vitro and in vivo, and Cys N-degron targeted fluorescent proteins can provide a simple and effective tool to study this at both subcellular and high-throughput scales. This chapter describes the design, production and implementation of a fluorescent fusion protein proteolytically regulated by ADO and the N-degron pathway.
2-氨基乙硫醇双加氧酶（ADO）是植物半胱氨酸氧化酶的哺乳动物同源物，这些酶共同负责催化某些蛋白质 N 端半胱氨酸残基的双加氧作用。这种修饰产生了一个 N 降解基序，允许通过 N 降解基序的精氨酸分支对这些蛋白质进行精氨酸化和随后的蛋白酶体降解。在人类中，已经鉴定出 4 种蛋白质为 ADO 的底物；G 蛋白信号调节剂（RGS）4、5 和 16，以及白细胞介素-32（IL-32）。在常氧条件下，这些蛋白质的 Nt-半胱氨酸双加氧作用迅速发生，但 ADO 活性对 O 可用性非常敏感，因此底物蛋白的稳定性与细胞内 O 水平成反比。关于该途径在体外和体内的生物化学和生理学仍有许多需要了解，而 Cys N 降解基序靶向荧光蛋白可以提供一种简单有效的工具，用于在亚细胞和高通量尺度上研究该途径。本章描述了一种受 ADO 和 N 降解基序调控的荧光融合蛋白的设计、生产和实施。
