SUMOylation and Ubiquitination

Ubiquitination, a post-translational modification, regulates a vast array of fundamental biological processes with dysregulation of the dedicated enzymes giving rise to pathologies such as cancer and neurodegenerative diseases. Assembly and its ensuing removal of this post-translational modification, determining a large variety of biological functions, is executed by a number of enzymes sequentially activating, conjugating, ligating, as well as deubiquitinating. Considering the vast impact of ubiquitination on regulating cellular homeostasis, understanding the function of these vast enzyme networks merits the development and innovation of tools. Thus, advances in synthetic strategies for generating ubiquitin, permitted the development of a plethora of ubiquitin assay reagents and numerous activity-based probes (ABPs) enable the study of enzymes involved in the complex system of ubiquitination. With ubiquitination playing such a pivotal role in the pathogenesis of a multitude of diseases, the identification of inhibitors for ubiquitin enzymes as well as the development of ABPs and high-throughput assay reagents is of utmost importance. Accordingly, this chapter will review the current state-of-the-art activity-based probes, reporter substrates, and other relevant tools based on Ub as a recognition element while highlighting the need of innovative technologies and unique concepts to study emerging facets of ubiquitin biology. Introduction One of the most versatile post-translational modifications is the attachment of the small protein ubiquitin (Ub) or its polymeric chains to target substrates. The attachment of the 76 amino acid long protein Ub to a nucleophilic functionality in the amino acid side chain of substrate proteins alters the fate of the modified protein, thereby regulating the vast majority of fundamental cellular processes such as DNA damage response (Muratani and Tansey, 2003), cell cycle progression (Kernan et al., 2018), transcription (Hicke, 2001), endocytosis (McCann et al., 2016), as well as apoptosis ( Jackson and Durocher, 2013) and autophagy (Kwon and Ciechanover, 2017). Covalent attachment of Ub to its substrate proteins is orchestrated by the sequential action of three specialized enzyme classes – E1, E2, and E3 enzymes (Fig. 2.1A). However, the combination of E2 and E3 enzymes dictates what type of ubiquitin chain is formed and which substrate protein becomes ubiquitinated. To date, 2 human E1’s, about 40 E2’s and over 600 E3 enzymes are known. Adenylation of the C-terminus of Ub at the expense of ATP yields a high-energy E1-Ub-thioester. Upon activation, Ub is transferred unto the active-site cysteine residue of the E2-enzyme, poising it for transfer unto the lysine residue of its substrates by the cooperation of an E3 enzyme. This final step in Ub-transfer through the E3 enzyme can occur via three main classes of E3 ligases: the homologous to the E6-APC terminus (HECT), the really interesting new gene (RING),