Post-translational modification of proteins with ubiquitin (Ub) and Ub chains controls protein breakdown by the proteasome, cellular localization of proteins, transcriptional activity, and DNA repair.[1] Ubiquitin is a highly conserved 76 amino acid protein that can be linked to target proteins through an isopeptide bond between the C-terminal carboxylate of Ub and the ɛ-amine of a lysine residue or N terminus of the target protein. Ubiquitin is able to form chains by self-conjugation onto any of its seven lysine residues (namely, K6, K11, K33, K27, K29, K48, and K63). Although all the linkages have been identified in cells,[2] only K48 and K63 linkages have been thoroughly studied so far. The conjugation of ubiquitin requires the concerted action of E1, E2, and E3 enzymes, defined combinations of which provide specificity for the protein target and the nature of the Ub chain topoisomers. The E1 enzyme initiates the cascade by activating Ub at the expense of ATP to form an E1-Ub thioester between the cysteine residue of the E1 active site and the C-terminal carboxylate of Ub. This E1-Ub thioester serves as a donor of activated Ub that then enters the complex enzymatic conjugation cascade.