Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analyzing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAMM DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs

Molecular basis of Lys11-polyubiquitin specificity in the deubiquitinase Cezanne

The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types in polyubiquitin co-exist and are independently regulated in cells. This ‘ubiquitin code’ determines the fate of the modified protein1. Deubiquitinating enzymes of the Ovarian Tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin2, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system. We here reveal how the deubiquitinase Cezanne/OTUD7B specifically targets Lys11-linked polyubiquitin. Crystal structures of Cezanne alone and in complex with mono- and Lys11-linked diubiquitin, in combination with hydrogen-deuterium exchange mass spectrometry, enable reconstruction of the enzymatic cycle in exquisite detail. An intricate mechanism of ubiquitin-assisted conformational changes activate the enzyme, and while all chain types interact with the enzymatic S1 site, only Lys11-linked chains can bind productively across the active site and stimulate catalytic turnover. Our work highlights the fascinating plasticity of deubiquitinases, and indicates that new conformational states can occur when a true substrate, such as diubiquitin, is bound at the active site

The Spallation Neutron Source in Oak Ridge: A powerful tool for materials research

When completed in 2006, the Spallation Neutron Source (SNS) will use an accelerator to produce the most intense beams of pulsed neutrons in the world. This unique facility is being built by a collaboration of six US Department of Energy laboratories and will serve a diverse community of users drawn from academia, industry, and government labs. The project continues on schedule and within budget, with commissioning and installation of all systems going well. Installation of 14 state-of-the-art instruments is under way, and design work is being completed for several others. These new instruments will enable inelastic and elastic-scattering measurements across a broad range of science such as condensed-matter physics, chemistry, engineering materials, biology, and beyond. Neutron Science at SNS will be complemented by research opportunities at several other facilities tinder way at Oak Ridge National Laboratory. (c) 2006 Elsevier B.V. All rights reserved

Dot1 promotes H2B ubiquitination by a methyltransferase-independent mechanism

The histone methyltransferase Dot1 is conserved from yeast to human and methylates lysine 79 of histone H3 (H3K79) on the core of the nucleosome. H3K79 methylation by Dot1 affects gene expression and the response to DNA damage, and is enhanced by monoubiquitination of the C-terminus of histone H2B (H2Bub1). To gain more insight into the functions of Dot1, we generated genetic interaction maps of increased-dosage alleles of DOT1. We identified a functional relationship between increased Dot1 dosage and loss of the DUB module of the SAGA co-activator complex, which deubiquitinates H2Bub1 and thereby negatively regulates H3K79 methylation. Increased Dot1 dosage was found to promote H2Bub1 in a dose-dependent manner and this was exacerbated by the loss of SAGA-DUB activity, which also caused a negative genetic interaction. The stimulatory effect on H2B ubiquitination was mediated by the N-terminus of Dot1, independent of methyltransferase activity. Our findings show that Dot1 and H2Bub1 are subject to bi-directional crosstalk and that Dot1 possesses chromatin regulatory functions that are independent of its methyltransferase activity

On terminal alkynes that can react with active-site cysteine nucleophiles in proteases

Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins bound to an immobilized Ub alkyne probe confirmed the selectivity toward de-ubiquitinating enzymes. The observed reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate

Orthogonal thiol functionalization at a single atomic center for profiling transthiolation activity of E1 activating enzymes

Transthiolation is a fundamental biological reaction and is utilized by many enzymes involved in the conjugation of ubiquitin and ubiquitin-like proteins. However, tools that enable selective profiling of this activity are lacking. Transthiolation requires cysteine–cysteine juxtaposition; therefore a method that enables irreversible “stapling” of proximal thiols would facilitate the development of novel probes that could be used to profile this activity. Herein, we characterize biocompatible chemistry that enables sequential functionalization of cysteines within proteins at a single atomic center. We use our method to develop a new class of activity-based probe that profiles transthiolation activity of human E1 activating enzymes. We demonstrate use <i>in vitro</i> and <i>in situ</i> and compatibility with competitive activity-based protein profiling. We also use the probe to gain insight into the determinants of transthiolation between E2 and a RING-in-between-RING (RBR) E3 ligase. Furthermore, we anticipate that this method of thiol functionalization will have broad utility by enabling simple redox-stable cross-linking of proximal cysteines in general