Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked Ubiquitin Signaling

Abstract

SummarySeveral ubiquitin chain types have remained unstudied, mainly because tools and techniques to detect these posttranslational modifications are scarce. Linkage-specific antibodies have shaped our understanding of the roles and dynamics of polyubiquitin signals but are available for only five out of eight linkage types. We here characterize K6- and K33-linkage-specific “affimer” reagents as high-affinity ubiquitin interactors. Crystal structures of affimers bound to their cognate chain types reveal mechanisms of specificity and a K11 cross-reactivity in the K33 affimer. Structure-guided improvements yield superior affinity reagents suitable for western blotting, confocal fluorescence microscopy and pull-down applications. This allowed us to identify RNF144A and RNF144B as E3 ligases that assemble K6-, K11-, and K48-linked polyubiquitin in vitro. A protocol to enrich K6-ubiquitinated proteins from cells identifies HUWE1 as a main E3 ligase for this chain type, and we show that mitofusin-2 is modified with K6-linked polyubiquitin in a HUWE1-dependent manner.

Hepatocyte TNF Receptor–Associated Factor 6 Aggravates Hepatic Inflammation and Fibrosis by Promoting Lysine 6–Linked Polyubiquitination of Apoptosis Signal‐Regulating Kinase 1

“…C) ubiquitination assays. In addition, by using a K6‐specific affimer, a linkage‐specific tool for the study of atypical K6 ubiquitin linkages, we found that the level of endogenous K6‐linked ASK1 polyubiquitination was almost undetectable under resting conditions in TRAF6‐deficient L02 hepatocytes, but was enhanced by TRAF6 reconstitution and increased much higher upon PA stimulation (Fig. D).…”

Association between blooming time and climatic adaptation in Prunus mume

“…Abscisic acid‐insensitive 5‐like protein 1 (Wang, Li, Mao, Li, & Jing, ), ethylene‐responsive transcription factor RAP2‐12 (Kosmacz et al, ), auxin‐induced protein AUX28‐like (Xie et al, ), and auxin‐induced protein 22D‐like (Han et al, ) take part in plant growth and development regulated by plant hormones. E3 ubiquitin‐protein ligase CIP8‐like (Wang et al, ), E3 ubiquitin‐protein ligase RNF144B (Michel, Swatek, Hospenthal, & Komander, ), and ERAD‐associated E3 ubiquitin‐protein ligase HRD1B‐like are associated with ubiquitination (Wang, Ye, Lencer, & Hansen, ). Serine/threonine‐protein kinase (Dudek et al, ), 1‐phosphatidylinositol‐3‐phosphate 5‐kinase FAB1B (Hirano, Sato, & behavior, ), G‐type lectin S‐receptor‐like serine/threonine‐protein kinase (Sun et al, ), phosphatidylinositol 4‐phosphate 5‐kinase α (Honda et al, ), and so on are all genes in the selective sweep region that might allow plants to better adapt to the environment and have potential value for future studies.…”

Mechanisms of up-regulation of Ubiquitin-Proteasome activity in the absence of NatA dependent N-terminal acetylation

“…This indicates that Tom1 recognizes oligoubiquitinated UFD substrates and either extends pre-formed chains or synthesizes new chains conjugated directly to the substrate, but using a residue distinct from K48 of the N-terminal ubiquitin moiety for chain attachment. Since HUWE1, the mammalian homologue, was shown to synthesize K6-and K11-linked chains (Michel et al, 2017;Yau et al, 2017), it is possible that Tom1 can use those lysine residues of the N-terminal ubiquitin moiety to initiate new chains. Moreover, detailed analysis of the banding pattern revealed that in the presence of Tom1 tri-ubiquitinated species of different apparent molecular weight were generated ( Fig 4E), indicating that ubiquitin conjugates synthesized by Tom1 and Ufd4 are clearly distinct.…”