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

Mol Cell. 2017 Oct 5;68(1):233-246.e5. doi: 10.1016/j.molcel.2017.08.020. Epub 2017 Sep 21.

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.

Keywords: HUWE1; Lys6-linked ubiquitin chains; Mfn2; Parkin; X-ray crystallography; affimer; microscale thermophoresis; mitophagy.

MeSH terms

  • Amino Acid Motifs
  • Binding Sites
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Cell Line, Tumor
  • Cloning, Molecular
  • Crystallography, X-Ray
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • GTP Phosphohydrolases / chemistry*
  • GTP Phosphohydrolases / genetics
  • GTP Phosphohydrolases / metabolism
  • Gene Expression
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Kinetics
  • Lysine / chemistry
  • Lysine / metabolism
  • Mitochondrial Proteins / chemistry*
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism
  • Models, Molecular
  • Molecular Probes / chemistry*
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Processing, Post-Translational*
  • Protein Structure, Secondary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Signal Transduction*
  • Substrate Specificity
  • Tumor Suppressor Proteins
  • Ubiquitin / chemistry*
  • Ubiquitin / genetics
  • Ubiquitin / metabolism
  • Ubiquitin-Protein Ligases / chemistry*
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitination

Substances

  • Carrier Proteins
  • Mitochondrial Proteins
  • Molecular Probes
  • Recombinant Proteins
  • Tumor Suppressor Proteins
  • Ubiquitin
  • HUWE1 protein, human
  • RNF144A protein, human
  • RNF144B protein, human
  • Ubiquitin-Protein Ligases
  • GTP Phosphohydrolases
  • MFN2 protein, human
  • Lysine