Using Ubiquitin Binders to Decipher the Ubiquitin Code

Abstract

Post-translational modifications (PTMs) by ubiquitin (Ub) are versatile, highly dynamic, and involved in nearly all aspects of eukaryote biological function. The reversibility and heterogeneity of Ub chains attached to protein substrates have complicated their isolation, quantification, and characterization. Strategies have emerged to isolate endogenous ubiquitylated targets, including technologies based on the use of Ub-binding peptides, such as TUBEs (Tandem-repeated Ubiquitin-Binding Entities). TUBEs allow the identification and characterization of ubiquitin chains, novel substrates for deubiquitylases (DUBs) and Ub ligases (E3s). Here we review their impact on purification, analysis of pan or chain-selective polyubiquitylated proteins and underline the biological relevance of this information. Together with peptide aptamers and other Ub affinity-based approaches, TUBEs will contribute to unravelling the secrets of the Ub-code. The Complexity of the Ubiquitin CodeUbiquitination of proteins is a significant regulatory process that affects almost all cellular functions. Ubiquitin (Ub) is a small, compact, and highly conserved 76 amino acid protein. The C-terminus of Ub is attached by an isopeptide bond to the Ɛ-amino group of lysine residues on target proteins. After Ub is attached to a protein, this proximal Ub can act as a substrate for additional Ubs, which are conjugated to any of its seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) or to its N-terminal methionine (M1) [1]. Protein ubiquitylation was first described as a signal for proteasomal degradation. The so called Ub-proteasome system (UPS) (see Glossary) uses mainly K48 and K11 polyubiquitylation as signals for substrate recognition [2,3]. The 26S proteasome include subunits that contain Ub-binding domains (UBDs) (Box 1) that participate in the binding of ubiquitin chains [4,5]. The binding and disassembly of many different types of Ub chains vary in both length and linkage specificity [6,7]. Although the presence of one Ub chain type is not by itself indicative of one function, accumulated evidence supports the existence of certain generic roles. For instance, proteasome inhibition leads to accumulation of ubiquitylated proteins containing all seven different types of Ub linkages except for K63 [8], strongly suggesting that K63 chains do not primarily target proteins to proteasomes. K63 Ub chains drive proteolysis primarily by the autophagylysosome system (ALS) [2] and might mediate DNA repair and other signaling pathways. Not much is known about the functions and mechanisms of more atypical linkages such Lys6, Lys27, Lys29, Lys33, and Met1 [9]. This Ub chain complexity known as the "Ub-code" includes mono-and multi-mono-Ub modifications, chains mixing multiple Ub linkages and other Ub-Like molecules (UbL). The biological relevance of most of these complex chains remains to be elucidated [10,11].

The ubiquitin ligase Hecw controls oogenesis and neuronal homeostasis by promoting the liquid state of ribonucleoprotein particles

“…6d). We confirmed this result in vivo by isolating endogenous ubiquitinated targets using tandem-repeated Ub-binding entities (TUBEs), a sensitive method that demonstrates the covalent attachment of polyubiquitin chains to proteins modified at low stoichiometry 36 . While Fmrp is clearly present in the pull-down from wild-type ovaries, the lane corresponding to the Hecw KO ovaries displays a remarkably reduced amount of protein ( Fig.…”

Resolving the Complexity of Ubiquitin Networks

“…Due to typically low binding affinity of UBDs toward Ub, synthetic multiple repeats of UBDs (tandem Ub-binding entities, TUBEs) were engineered (Figure 7A). Expressed as recombinant epitope-tagged fusions, those tools are characterized by high overall Ub-binding avidity and enable efficient capturing of both high and low abundant Ub targets from cellular lysates (Hjerpe et al, 2009;Mattern et al, 2019). A large number of available Ub traps differs in number and types of UBDs, length of linkers and type of epitope tags.…”

Emerging Roles and Research Tools of Atypical Ubiquitination

“…The expected background, however, comes from proteins that contain natural HIS or are biotinylated in the cell. Other epitope tags, such as FLAG, MYC, HA, GST, and GFP, require immunoprecipitation under mild conditions to isolate ubiquitinated substrates and will copurify nonubiquitinated interactors . GFP and its derivatives, which can be observed directly by fluorescence microscopy, are used to visualize the process of ubiquitination and deubiquitination in living cells or intact animal models .…”

“…More importantly, TUBEs originated from UBDs with binding specificity can be used to enrich linkage specific ubiquitinated proteins. Today, TUBEs that have been developed and commercialized include: non‐selective‐specific TUBE1, TUBE2, and TUBE3, and selective‐specific: Anti‐K63 TUBEs, Anti‐K48 TUBEs, and Anti‐M1 (Linear) TUBEs ( Table ). In addition, peptide aptamers such as affimers and adnectins are other recently established affinity‐based technologies.…”