The Length of a Ubiquitin Chain: A General Factor for Selective Recognition by Ubiquitin‐Binding Proteins

Abstract

The attachment of ubiquitin (Ub) chains of various length to proteins is a prevalent posttranslational modification in eukaryotes. The fate of a modified protein is determined by Ub‐binding proteins (UBPs), which interact with Ub chains in a linkage‐selective manner. However, the impact and functional consequences of chain length on the binding selectivity of UBPs remain mostly elusive. We have generated Ub chains of defined length and linkage by using click chemistry and GELFrEE fractionation. These defined polymers were used in affinity‐based enrichment assays to identify length‐ and linkage‐selective interaction partners on a proteome‐wide scale. For the first time, it is revealed that the length of a Ub chain generally has a major impact on its ability to be selectively recognized by UBPs.

Site-specific ubiquitylation acts as a regulator of linker histone H1

“…With sufficient amounts of non-hydrolyzable site-specifically ubiquitylated H1 in hand, we set out to dissect the ubiquitylation-dependent modular interactome. Building on previous efforts 53 , 54 , we adapted an affinity purification-mass spectrometry (AP-MS) based approach to identify Ub-dependent interaction partners of H1 using its N-terminal Strep-tag II for enrichment (Fig. 2a , Supplementary Fig.…”

Why structure and chain length matter: on the biological significance underlying the structural heterogeneity of poly(ADP-ribose)

“…dimers/tetramers) rather interacted with enzymes involved in protein modification (e.g. kinases, proteases, and also E3 ligases) ( 170 ). In line with this, previous studies already demonstrated that different deubiquitinating enzymes are influenced by different chain lengths.…”

Development and application of ubiquitin-based chemical probes

“…The authors also used gel eluted liquid fraction entrapment electrophoresis to separate the Ubs into Ub 2, Ub 4 , Ub 6+ to examine their interactors separately. 39 Signicant differences were observed in the interactors of Ubs with different lengths. For example, Ub-associated domaincontaining protein 1 (Ubac1), RING nger 123 (RNF123), and USP15 only interact with long Ub chains (Ub 4 , Ub 6+ ).…”

“…Such probes usually contain one or more Ub units and an enrichment/purication tag. [33][34][35][36][37][38][39][40] Ub units can be either natural or mimetics that do not impact the interaction with linkage-specic interactors ( Fig. 4a).…”

“…Cu-catalysed azidealkyne cycloaddition (CuAAC) reactions can be used to construct isopeptide bond mimics. 38,39,91 Introduction of the azide into the Ub C-terminal was accomplished through unnatural amino acid insertion, and then the alkyne was introduced into the substrate Lys through bio-orthogonal reactions. The Ub and substrate were ligated in the form of triazole via CuAAC reaction.…”