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Covalently attaching ubiquitin (Ub) to cellular proteins as a post-translational modification can result in altered function of modified proteins. Enzymes regulating Ub as a post-translational modification, such as ligases and deubiquitinases, are challenging to characterize in part due to the low throughput of in-vitro assays. Single-molecule nanopore based assays have the advantage of detecting proteins with high specificity and resolution, and in a label-free, real-time fashion. Here we demonstrate the use of a MspA nanopore for discriminating and quantifying Ub proteins. We further applied the MspA pore to measure the Ub-chain disassembly activity of UCH37, a proteasome associated deubiquitinase. The implementation of this MspA system into nanopore arrays could enable high throughput characterizations of unknown deubiquitinases as well as drug screening against disease related enzymes., (© 2021 Wiley-VCH GmbH.)

Access to well-defined ubiquitin conjugates has been key to elucidating the biochemical functions of proteins in the ubiquitin signaling network. Yet, we have a poor understanding of how deubiquitinases and ubiquitin-binding proteins respond to ubiquitin modifications when anchored to a protein other than ubiquitin or a ubiquitin-like protein. This is due to the difficulty of synthesizing ubiquitinated proteins comprised of native isopeptide bonds. Here we report on the evolution of a deubiquitinase capable of site-specifically modifying itself with defined ubiquitin chains. Following mutagenesis and yeast display screening, we identify a variant of the yeast ubiquitin C-terminal hydrolase Yuh1 that has a 28-fold improvement in the transamidation to hydrolysis ratio relative to the wild type enzyme. The switch in activity enables robust autoubiquitination of a lysine in the crossover loop to form an isopeptide bond. We demonstrate the utility of autoubiquitinating the evolved Yuh1 variant by investigating the consequences of ubiquitin chain anchoring on the activities of other deubiquitinases. Much to our surprise, we find that certain deubiquitinases are exquisitely sensitive to chain anchoring. These results highlight the importance of investigating the biochemical activities of deubiquitinases with both substrate-anchored and unanchored ubiquitin chains.

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.

Information embedded in different ubiquitin chains is transduced by proteins with ubiquitin-binding domains (UBDs) and erased by a set of hydrolytic enzymes referred to as deubiquitinases (DUBs). Understanding the selectivity of UBDs and DUBs is necessary for decoding the functions of different ubiquitin chains. Critical to these efforts is the access to chemically defined ubiquitin chains bearing site-specific fluorescent labels. One approach toward constructing such molecules involves peptide ligation by sortase (SrtA), a bacterial transpeptidase responsible for covalently attaching cell surface proteins to the cell wall. Here, we demonstrate the utility of SrtA in modifying individual subunits of ubiquitin chains. Using ubiquitin derivatives in which an N-terminal glycine is unveiled after protease-mediated digestion, we synthesized ubiquitin dimers, trimers, and tetramers with different isopeptide linkages. SrtA was then used in combination with fluorescent depsipeptide substrates to effect the modification of each subunit in a chain. By constructing branched ubiquitin chains with individual subunits tagged with a fluorophore, we provide evidence that the ubiquitin-specific protease USP15 prefers ubiquitin trimers but has little preference for a particular isopeptide linkage. Our results emphasize the importance of subunit-specific labeling of ubiquitin chains when studying how DUBs process these chains., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)