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

Insights into ubiquitin chain architecture using Ub-clipping.

Author information

Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany.
Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, USA.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR, USA.
Department of Medical Biochemistry, Max Perutz Labs, Vienna Biocenter, Medical University of Vienna, Vienna, Austria.
Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
Ubiquitin Signalling Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
Department of Medical Biology, The University of Melbourne, Melbourne, Victoria, Australia.


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.


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