Purification and characterisation of endogenous unanchored polyubiquitin

Abstract

The 76 amino-acid protein ubiquitin is peculiar in its ability to covalently modify substrate proteins. The complexity and diversity in structure and function, of this posttranslational modification is increasingly evident and has been extensively scrutinized. However, the existing notion for ubiquitin to be functionally relevant when covalently linked to substrates was recently found to be dispensable. Pioneering work by Chen’s group has shown that unanchored (non-substrate bound) polyubiquitin chains are just as effective in regulating various cellular processes and presented compelling evidence about the influence of unanchored polyubiquitin chains in regulating cell signalling. That said, not enough emphasis has been placed in developing tools for the direct isolation and characterisation of endogenous unanchored polyubiquitin chains. Our group had previously developed a platform, which employs the Znf-UBP (BUZ) ubiquitin-binding domain of human Isopeptidase T enzyme, to (for the first time) directly purify cellular unanchored polyubiquitin chains of up to 15 ubiquitin moieties long from rat skeletal muscle tissue, also confirming the presence of K48 isopeptide linkages in such chains. Following on, the work described here details further refinement and optimisation of the purification protocol specifically from eukaryotic cell line (HEK293T). We could confirm by western blotting, the presence of K48 and K63 linked endogenous unanchored polyubiquitin chains, some 10 or more ubiquitin moieties long. Our next step was to optimise existing standard protein processing protocols specifically for the study of cellular unanchored ubiquitome. Considering ubiquitin being resistant to trypsin digestion, we found a general lack of consensus in protocols best suited for processing and analysis of the cellular ubiquitome. Extensive optimisations of various protein digestion conditions were performed to arrive on the protocol best suited for unanchored polyubiquitin. The protocol was designed to address the specific problem of maximising (efficient) digestion of ubiquitin, minimising losses in the process to produce samples for quantitative analysis. The purchase of a QqQ mass spectrometer by the group enabled quantitative estimation of polyubiquitin linkages by using AQUA standards although this required significant optimisations as well. Subsequently, we could successfully detect and estimate the abundance of isopeptide linkages purified from HEK293T cell line to be K63 (83%)>>K11 (9%)>K48, K27 (3.7-4.0%) at basal conditions. Moreover, during the course of trying to attain self-sufficiency in this area of work, our knowledge about the pitfalls and caveats in the area also improved. Following the development of a robust in-house platform for the isolation and characterisation of unanchored polyubiquitin chains, we were eager to apply the protocol to study the endogenous unanchored polyubiquitin makeup of cells at basal conditions, and take a step further to determine the change in their levels upon activation of signalling, manipulation of possible regulators and in response to stress. While our attempt at absolute quantification of linkage abundance was not entirely successful, we present data which confirmed increase in K48 and K11 (but not K63) linked unanchored polyubiquitin chains upon proteasomal inhibition and also a strong indication of accumulation of all three linkage types upon suppression of IsoT. Finally, the study has improved our understanding of the regulation of the cellular unanchored ubiquitome