Edited by Michael IbbaUbiquitination is a post-translational modification (PTM) capable of being regulated by other PTMs, including acetylation. However, the biological consequences of acetylated ubiquitin (acUb) variants are poorly understood, due to their transient nature in vivo and poor characterization in vitro. Since Ub is known to be acetylated in human cells, we produced all possible acUb variants using genetic code expansion. We also developed a protocol that optimizes acetyl-lysine addition to minimize mistranslated proteins and maximize site-specific acUb protein production. Purified acUb proteins were used in pilot ubiquitination assays and found to be competent with IpaH3CT and RNF8 E3 ligases. Overall, this work provides an optimized method to express and purify all acetyl-lysine variants for ubiquitin and shows these proteins can be used to identify potential unique ubiquitination patterns.Covalent attachment of ubiquitin (Ub) to a substrate serves as a signaling event that determines the fate of the target protein. A series of E1, E2, and E3 enzymes specify the position of ubiquitination and the types of linkage between ubiquitin molecules in the case of polyubiquitinated substrates. In this latter case, K48-linked polyubiquitin chains target the substrate for proteasomal degradation [1], while other linkages are signals for DNA damage repair [2], cell cycle progression [3], or downstream cascades such as the MEK-ERK pathway [4]. In addition to ubiquitination, protein function can also be attenuated by a variety of other post-translational modifications (PTMs) such as phosphorylation and acetylation, and there is often cooperativity between these PTMs. For example, current knowledge holds that phosphorylation of Ub at Ser65 by PINK1 is required for the activation of the E3 ligase parkin [5]. Ser65 is just one of the eleven residues in Ub that can be phosphorylated and at least one other phosphorylation site has been identified in vivo [6] and can exhibit similar activation levels for parkin [7]. Additionally, acetylation could occur at any of the seven Lys residues with unknown downstream effects.With the recent advances in enrichment methods and improvements to mass spectrometry, there has been a significant shift in modified proteome analysis. As a result, the acetylome has been mapped in great detail with various acetyl-proteome datasets demonstrating ubiquitin acetylation. Many of these datasets demonstrate that fluctuations in acetylated peptides can be dependent on different cell stimuli such as deacetylase (HDAC, SIRT) inhibition [8-10] and/or knockout [11], or cell stress. For example, ionizing radiation [12] or induction of autophagy [13] results in

Ubiquitination is controlled by a series of E1, E2, and E3 enzymes that can ligate ubiquitin to cellular proteins and dictate the turnover of a substrate and the outcome of signalling events such as DNA damage repair and cell cycle. This process is complex due to the combinatorial power of ~35 E2 and ~1000 E3 enzymes involved and the multiple lysine residues on ubiquitin that can be used to assemble polyubiquitin chains. Recently, mass spectrometric methods have identified that most enzymes in the ubiquitination cascade can be further modified through acetylation or phosphorylation under particular cellular conditions and altered modifications have been noted in different cancers and neurodegenerative diseases. This review provides a cohesive summary of ubiquitination, acetylation, and phosphorylation sites in ubiquitin, the human E1 enzyme UBA1, all E2 enzymes, and some representative E3 enzymes. The potential impacts these post-translational modifications might have on each protein function are highlighted, as well as the observations from human disease.

The ubiquitin (Ub) proteolysis pathway uses an E1, E2, and E3 enzyme cascade to label substrate proteins with ubiquitin and target them for degradation. The mechanisms of ubiquitin chain formation remain unclear and include a sequential addition model, in which polyubiquitin chains are built unit by unit on the substrate, or a preassembly model, in which polyubiquitin chains are preformed on the E2 or E3 enzyme and then transferred in one step to the substrate. The E2 conjugating enzyme UBE2K has a 150-residue catalytic core domain and a C-terminal ubiquitin-associated (UBA) domain. Polyubiquitin chains anchored to the catalytic cysteine and free in solution are formed by UBE2K supporting a preassembly model. To study how UBE2K might assemble polyubiquitin chains, we synthesized UBE2K-Ub and UBE2K-Ub 2 covalent complexes and analyzed E2 interactions with the covalently attached Ub and Ub 2 moieties using NMR spectroscopy. The UBE2K-Ub complex exists in multiple conformations, including the catalytically competent closed state independent of the UBA domain. In contrast, the UBE2K-Ub 2 complex takes on a more extended conformation directed by interactions between the classic I44 hydrophobic face of the distal Ub and the conserved MGF hydrophobic patch of the UBA domain. Our results indicate there are distinct differences between the UBE2K-Ub and UBE2K-Ub 2 complexes and show how the UBA domain can alter the position of a polyubiquitin chain attached to the UBE2K active site. These observations provide structural insights into the unique Ub chain-building capacity for UBE2K.

Ubiquitin (Ub) signaling requires the covalent passage of Ub among E1, E2, and E3 enzymes. The choice of E2 and E3 enzymes combined with multiple rounds of the cascade leads to the formation of polyubiquitin chains linked through any one of the seven lysines on Ub. The linkage type and length act as a signal to trigger important cellular processes such as protein degradation or the DNA damage response. Recently, proteomics studies have identified that Ub can be acetylated at six of its seven lysine residues under various cell stress conditions. To understand the potential differences in Ub signaling caused by acetylation, we synthesized all possible acetylated ubiquitin (acUb) variants and examined the E1-mediated formation of the corresponding E2∼acUb conjugates in vitro using kinetic methods. A Forster resonance energy transfer assay was optimized in which the Ub constructs were labeled with a CyPet fluorophore and the E2 UBE2D1 was labeled with a YPet fluorophore to monitor the formation of E2∼Ub conjugates. Our methods enable the detection of small differences that may otherwise be concealed in steady-state ubiquitination experiments. We determined that Ub, acetylated at K11, K27, K33, K48, or K63, has altered turnover numbers for E2∼Ub conjugate formation by the E1 enzyme Uba1. This work provides evidence that acetylation of Ub can alter the catalysis of ubiquitination early on in the pathway.