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Updated: May 17, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
Cyclisation of Lys48-linked diubiquitin in vitro and in vivo
Kleitos Sokratous1, Joanna Strachan, Lucy V Roach
1School of Chemistry, University of Nottingham, Nottingham, UK.
Abstract:
Ubiquitin (Ub) is able to form polymeric isopeptide-linked chains through condensation of any of its seven lysine (Lys) residues with the C-terminus of an adjacent Ub monomer. Electrospray ionisation mass spectrometry (ESI-MS) of commercial in vitro-generated Lys48-linked di-Ub (Lys48-Ub(2)) revealed a major population of cyclised dimer. The absence of a free C-terminus in this population was confirmed by an inability to bind the zinc finger ubiquitin-binding domain (ZnF-UBP) of USP5/isopeptidase-T. Endogenous Ub(2) purified from skeletal muscle and cultured mammalian cells was found to contain cyclic Lys48-Ub(2), demonstrating that cyclisation of poly-Ub can also occur in vivo.
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