Cyclisation of Lys48-linked diubiquitin in vitro and in vivo.

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.

Probing affinity and ubiquitin linkage selectivity of ubiquitin-binding domains using mass spectrometry.

Non-covalent interactions between ubiquitin (Ub)-modified substrates and Ub-binding domains (UBDs) are fundamental to signal transduction by Ub receptor proteins. Poly-Ub chains, linked through isopeptide bonds between internal Lys residues and the C-terminus of Ub, can be assembled with varied topologies to mediate different cellular processes. We have developed and applied a rapid and sensitive electrospray ionization-mass spectrometry (ESI-MS) method to determine isopeptide linkage-selectivity and affinity of poly-Ub·UBD interactions. We demonstrate the technique using mono-Ub and poly-Ub complexes with a number of α-helical and zinc-finger (ZnF) UBDs from proteins with roles in neurodegenerative diseases and cancer. Affinities in the 2-200 µM range were determined to be in excellent agreement with data derived from other biophysical techniques, where available. Application of the methodology provided further insights into the poly-Ub linkage specificity of the hHR23A-UBA2 domain, confirming its role in Lys48-linked poly-Ub signaling. The ZnF UBP domain of isopeptidase-T showed no linkage specificity for poly-Ub chains, and the Rabex-5 MIU also exhibited little or no specificity. The discovery that a number of domains are able to bind cyclic Lys48 di-Ub with affinities similar to those for the acyclic form indicates that cyclic poly-Ub may be capable of playing a role in Ub-signaling. Detection of a ternary complex involving Ub interacting simultaneously with two different UBDs demonstrated the co-existence of multi-site interactions, opening the way for the study of crosstalk between individual Ub-signaling pathways.

Insights into the molecular composition of endogenous unanchored polyubiquitin chains.

The diverse influences of ubiquitin, mediated by its post-translational covalent modification of other proteins, have been extensively investigated. However, more recently roles for unanchored (nonsubstrate linked) polyubiquitin chains have also been proposed. Here we describe the use of ubiquitin-binding domains to affinity purify endogenous unanchored polyubiquitin chains and their subsequent characterization by mass spectrometry (MS). Using the A20 Znf domain of the ubiquitin receptor ZNF216 we isolated a protein from skeletal muscle shown by a combination of nanoLC-MS and LC-MS/MS to represent an unmodified and unanchored K48-linked ubiquitin dimer. Selective purification of unanchored polyubiquitin chains using the Znf UBP (BUZ) domain of USP5/isopeptidase-T allowed the isolation of K48 and K11-linked ubiquitin dimers, as well as revealing longer chains containing as many as 15 ubiquitin moieties, which include the K48 linkage. Top-down nanoLC-MS/MS of the A20 Znf-purified ubiquitin dimer generated diagnostic ions consistent with the presence of the K48 linkage, illustrating for the first time the potential of this approach to probe connectivity within endogenous polyubiquitin modifications. As well as providing initial proteomic insights into the molecular composition of endogenous unanchored polyubiquitin chains, this work also represents the first definition of polyubiquitin chain length in vivo.