Ubiquitin linkage-specific affimers reveal Insights into K6-linked ubiquitin signaling

 

Martin A. Michel, Kirby N. Swatek, Manuela K. Hospenthal, and David Komander

Molecular Cell
2017 vol: 68 issue: 1 pp: 233-246.E5 doi: 10.1016/j.molcel.2017.08.020

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.

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Topics: Lys6-linked ubiquitin chains, Affimer, Mitophagy, Parkin, HUWE1, Mfn2, X-ray crystallography, Monolith – MicroScale Thermophoresis, MST, Proteins, Publications

 

 

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