Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry (UbiChEM-MS) Reveals Cell-Cycle Dependent Formation of Lys11/Lys48 Branched Ubiquitin Chains

Abstract

The dynamics of cellular signaling events are tightly regulated by a diverse set of ubiquitin chains. Recent work has suggested that branched ubiquitin chains composed of Lys11 and Lys48 isopeptide linkages play a critical role in regulating cell cycle progression. Yet, endogenous Lys11/Lys48 branched chains could not be detected. By combining a Lys11 linkage specific antibody with high-resolution middle-down mass spectrometry (an approach termed UbiChEM-MS) we sought to identify endogenous Lys11/Lys48 branched ubiquitin chains in cells. Using asynchronous cells, we find that Lys11-linked branched chains can only be detected upon cotreatment with a proteasome and nonselective deubiquitinase inhibitor. Releasing cells from mitotic arrest results in a marked accumulation of Lys11/Lys48 branched chains in which branch points represent ~3–4% of the total ubiquitin population. This report highlights the utility of UbiChEM-MS in characterizing the architecture of Lys11 Ub chains under various cellular conditions and corroborates the formation of Lys11/Lys48 branched chains during mitosis.

The Role of Electron Transfer Dissociation in Modern Proteomics

“…We direct readers to recent reviews that cover broader technologies for analysis of phosphorylation, 102 − 104 glycosylation, 105 − 107 and PTMs in general 100 , 101 for further insights. We also note that ETD has been valuable for PTM analysis beyond the scope discussed here, including ubiquitylation, 108 − 110 ADP-ribosylation, 111 − 113 and arginine methylation. 114 − 118 …”

Who guards the guardian? Mechanisms that restrain APC/C during the cell cycle

“…Notably, UBE2S assembles non-canonical ubiquitin chains, linked through K11 in ubiquitin [38–41]. In addition, more recent evidence points to an ability of APC/C to form branched or heterotypic chains, and these unique chain topologies could provide strong signals for organizing the temporal ordering of substrate degradation at mitotic exit [42–44].…”

The Deubiquitinating Enzyme Inhibitor PR-619 is a Potent DNA Topoisomerase II Poison

“…PR-619 has become a useful tool in order to probe the role of ubiquitination in various cell biologic systems (Balut et al, 2011; Seiberlich et al, 2012; Pandey and Kumar, 2015; Crowder et al, 2016; Rana et al, 2017; Wang et al, 2017); however, the work here suggests that some caution should be applied when using this inhibitor in cell line–based studies. We have observed that at concentrations 20 µ M and above, PR-619 induces TOP2-covalent DNA complexes in K562 cells that are converted to DNA double-strand breaks.…”

“…In cell-based assays PR-619 was shown to substantially inhibit the activity of ubiquitin-specific protease, ubiquitin carboxy-terminal hydrolase, OTU, and MJD class DUBs when applied at 20–50 µ M, and exhibited a clear effect at a concentration as low as 5 µ M (Altun et al, 2011). PR-619 has been employed as a tool to investigate the role of ubiquitination in cellular processes including lysosomal degradation (Balut et al, 2011), caspase activation (Crowder et al, 2016), the stability of sirtuin-7 (Pandey and Kumar, 2015), protein aggregate formation (Seiberlich et al, 2012), human immunodeficiency virus replication (Setz et al, 2017), and oocyte maturation (Wang et al, 2017), as well as in the analysis of ubiquitin chain structure (Rana et al, 2017). In addition, PR-619 also inhibited the de-SUMOylating enzyme sentrin-specific protease (SENP) 6 in vitro and leads to accumulation of SUMOylated proteins in cells (Altun et al, 2011; Barry et al, 2018).…”