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Journal Article

Insights into ubiquitin chain architecture using Ub-clipping


Swatek,  Kirby N.
Schulman, Brenda / Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Max Planck Society;

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Swatek, K. N., Usher, J. L., Kueck, A. F., Gladkova, C., Mevissen, T. E. T., Pruneda, J. N., et al. (2019). Insights into ubiquitin chain architecture using Ub-clipping. NATURE, 572(7770), 533-537. doi:10.1038/s41586-019-1482-y.

Cite as: https://hdl.handle.net/21.11116/0000-0005-7A41-9
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 acetylation(1-3). 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, Lb(pro)*, 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 Lb(pro)* 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.