Programmed ubiquitin acetylation using genetic code expansion reveals altered ubiquitination patterns

Abstract

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

SCF FBXW17 E3 ubiquitin ligase regulates FBXL19 stability and cell migration

“…The lysine residue is an important amino acid in posttranslational protein modifications 26‐28 . Acetylation and ubiquitination of lysine residues determine enzyme activity, protein–protein interaction, and stability 29‐31 . We have shown that K141 in FBXL19 can be acetylated by CBP 20 .…”

Acetylated Ubiquitin Modulates the Catalytic Activity of the E1 Enzyme Uba1

“…Acetylated ubiquitin acUbK63 CyPet was expressed in E. coli BW25141­(λDE3) as previously established . All acetylated ubiquitin proteins were expressed like His-TEV constructs expressed previously . Briefly, after a fresh co-transformation of the orthogonal translation system and the acUbKx CyPet construct of interest, a colony was transferred into selective Luria broth (LB) (30 μg/mL kanamycin, 34 μg/mL chloramphenicol, and 0.2% glucose) and grown at 37 °C overnight.…”

“…However, the acetyl-lysine side chain is longer and less polar and has the potential to form more extensive hydrophobic contacts than glutamine, suggesting glutamine substitution may not be indicative of the full effects of acetylation. Recently, many acetylated proteins, , including Ub, , have been produced in Escherichia coli using genetic code expansion. Reassignment of the amber stop codon (UAG) and the use of tRNA pyl‑opt enable a fast and accurate way to produce homogeneous acetylated proteins in vitro and have been applied in vivo .…”

Carbon Dioxide and the Carbamate Post-Translational Modification

“…Ub activity can be further regulated by additional PTMs, e.g. acetylation, phosphorylation and SUMOylation ( Koyano et al, 2014 ; Morimoto and Shirakawa, 2016 ; Lamoliatte et al, 2017 ; Lacoursiere et al, 2020 ). For example, all Ub lysine residues (except K29) are acetylated under differing cellular conditions ( Choudhary et al, 2009 ; Elia et al, 2015 ; Lacoursiere et al, 2020 ).…”

“…acetylation, phosphorylation and SUMOylation ( Koyano et al, 2014 ; Morimoto and Shirakawa, 2016 ; Lamoliatte et al, 2017 ; Lacoursiere et al, 2020 ). For example, all Ub lysine residues (except K29) are acetylated under differing cellular conditions ( Choudhary et al, 2009 ; Elia et al, 2015 ; Lacoursiere et al, 2020 ). Ohtake et al, 2015 demonstrated that when endogenous Ub is acetylated at K6 and K48, this does not affect the ability of Ub to conjugate with substrate protein but inhibits the elongation of K11, K48 and K63-linked Ub-chains by several E2 enzymes (by neutralising the lysine residue positive charge involved in non-covalent interaction of Ub with specific E2s).…”