Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked Ubiquitin Signaling.
Mol Cell
; 68(1): 233-246.e5, 2017 Oct 05.
Article
in En
| MEDLINE
| ID: mdl-28943312
ABSTRACT
Several 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.
Key words
Full text:
1
Database:
MEDLINE
Main subject:
Molecular Probes
/
Signal Transduction
/
Protein Processing, Post-Translational
/
Mitochondrial Proteins
/
Ubiquitin
/
Ubiquitin-Protein Ligases
/
GTP Phosphohydrolases
Type of study:
Prognostic_studies
Language:
En
Journal:
Mol Cell
Journal subject:
BIOLOGIA MOLECULAR
Year:
2017
Type:
Article
Affiliation country:
United kingdom