Insights into ubiquitin chain architecture using Ub-clipping

Nature. 2019 Aug;572(7770):533-537. doi: 10.1038/s41586-019-1482-y. Epub 2019 Aug 15.

Abstract

Protein ubiquitination is a multi-functional post-translational modification that affects all cellular processes. Its versatility arises from architecturally complex polyubiquitin chains, in which individual ubiquitin moieties may be ubiquitinated on one or multiple residues, and/or modified by phosphorylation and acetylation1-3. Advances in mass spectrometry have enabled the mapping of individual ubiquitin modifications that generate the ubiquitin code; however, the architecture of polyubiquitin signals has remained largely inaccessible. Here we introduce Ub-clipping as a methodology by which to understand polyubiquitin signals and architectures. Ub-clipping uses an engineered viral protease, Lbpro∗, to incompletely remove ubiquitin from substrates and leave the signature C-terminal GlyGly dipeptide attached to the modified residue; this simplifies the direct assessment of protein ubiquitination on substrates and within polyubiquitin. Monoubiquitin generated by Lbpro∗ retains GlyGly-modified residues, enabling the quantification of multiply GlyGly-modified branch-point ubiquitin. Notably, we find that a large amount (10-20%) of ubiquitin in polymers seems to exist as branched chains. Moreover, Ub-clipping enables the assessment of co-existing ubiquitin modifications. The analysis of depolarized mitochondria reveals that PINK1/parkin-mediated mitophagy predominantly exploits mono- and short-chain polyubiquitin, in which phosphorylated ubiquitin moieties are not further modified. Ub-clipping can therefore provide insight into the combinatorial complexity and architecture of the ubiquitin code.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Glycine / chemistry
  • Glycine / metabolism
  • HCT116 Cells
  • HeLa Cells
  • Humans
  • Mitophagy
  • Peptide Hydrolases / metabolism*
  • Polyubiquitin / chemistry
  • Polyubiquitin / metabolism
  • Protein Kinases / metabolism
  • Ubiquitin / chemistry*
  • Ubiquitin / metabolism*
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitination

Substances

  • Ubiquitin
  • Polyubiquitin
  • Ubiquitin-Protein Ligases
  • parkin protein
  • Protein Kinases
  • PTEN-induced putative kinase
  • Peptide Hydrolases
  • Glycine