Dataset Information

A linear diubiquitin-based probe for efficient and selective detection of the deubiquitinating enzyme OTULIN

ABSTRACT: The methionine1 (M1)-specific deubiquitinase OTULIN acts as a negative regulator of NF-ΚB signaling and immune homeostasis. By replacing G76 in distal ubiquitin by dehydroalanine we designed the diubiquitin (diUb) probe UbG76Dha-Ub (OTULIN ABP) that couples to the catalytic site of OTULIN and thereby captures OTULIN in its active conformation. OTULIN ABP displays high selectivity for OTULIN and is not labeling to other M1-specific DUB including CYLD. In fact, labeling of USP5/IsopeptidaseT was the only detectable cross-reactivity. Further, ATP-dependent association of UbG76Dha-Ub probe with E1 activating enzymes induces assembly of polyOTULIN ABP chains, but USP5 labelling and E1 binding was abolished by deleting the C-terminal glycine in proximal ubiquitin (UbG76Dha-UbΔG76: OTULIN ABPΔG76). Pull-downs demonstrate that substrate-bound OTULIN associates with the linear ubiquitin chain assembly complex (LUBAC). Thus, we present a highly selective DUB for OTULIN that will facilitate studying the cellular function of this critical deubiquitinase.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell

SUBMITTER: Philip Charles

LAB HEAD: Benedikt M Kessler

PROVIDER: PXD006868 | Pride | 2017-09-19

REPOSITORIES: Pride

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Publications

A Linear Diubiquitin-Based Probe for Efficient and Selective Detection of the Deubiquitinating Enzyme OTULIN.

Weber Aurelia A Elliott Paul R PR Pinto-Fernandez Adan A Bonham Sarah S Kessler Benedikt M BM Komander David D El Oualid Farid F Krappmann Daniel D

Cell chemical biology 20170914 10

The methionine 1 (M1)-specific deubiquitinase (DUB) OTULIN acts as a negative regulator of nuclear factor κB signaling and immune homeostasis. By replacing Gly76 in distal ubiquitin (Ub) by dehydroalanine we designed the diubiquitin (diUb) activity-based probe Ub<sub>G76Dha</sub>-Ub (OTULIN activity-based probe [ABP]) that couples to the catalytic site of OTULIN and thereby captures OTULIN in its active conformation. The OTULIN ABP displays high selectivity for OTULIN and does not label other M1 ...[more]

PMID: 28919039

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Project description:Proctor2007 - Age related decline of proteolysis, ubiquitin-proteome system This is a stochastic model of the ubiquitin-proteasome system for a generic pool of native proteins (NatP), which have a half-life of about 10 hours under normal conditions. It is assumed that these proteins are only degraded after they have lost their native structure due to a damage event. This is represented in the model by the misfolding reaction which depends on the level of reactive oxygen species (ROS) in the cell. Misfolded proteins (MisP) are first bound by an E3 ubiquitin ligase. Ubiquitin (Ub) is activated by E1 (ubiquitin-activating enzyme) and then passed to E2 (ubiquitin-conjugating enzyme). The E2 enzyme then passes the ubiquitin molecule to the E3/MisP complex with the net effect that the misfolded protein is monoubiquitinated and both E2 and E3 are released. Further ubiquitin molecules are added in a step-wise manner. When the chain of ubiquitin molecules is of length 4 or more, the polyubiquitinated misfolded protein may bind to the proteasome. The model also includes de-ubiquitinating enzymes (DUB) which cleave ubiquitin molecules from the chain in a step-wise manner. They work on chains attached to misfolded proteins both unbound and bound to the proteasomes. Misfolded proteins bound to the proteasome may be degraded releasing ubiquitin. Misfolded proteins including ubiquitinated proteins may also aggregate. Aggregates (AggP) may be sequestered (Seq_AggP) which takes them out of harm's way or they may bind to the proteasome (AggP_Proteasome). Proteasomes bound by aggregates are no longer available for protein degradation. Figure 2 and Figure 3 has been simulated using Gillespie2. This model is described in the article: An in silico model of the ubiquitin-proteasome system that incorporates normal homeostasis and age-related decline. Proctor CJ, Tsirigotis M, Gray DA. BMC Syst Biol 2007; 1: 17 Abstract: BACKGROUND: The ubiquitin-proteasome system is responsible for homeostatic degradation of intact protein substrates as well as the elimination of damaged or misfolded proteins that might otherwise aggregate. During ageing there is a decline in proteasome activity and an increase in aggregated proteins. Many neurodegenerative diseases are characterised by the presence of distinctive ubiquitin-positive inclusion bodies in affected regions of the brain. These inclusions consist of insoluble, unfolded, ubiquitinated polypeptides that fail to be targeted and degraded by the proteasome. We are using a systems biology approach to try and determine the primary event in the decline in proteolytic capacity with age and whether there is in fact a vicious cycle of inhibition, with accumulating aggregates further inhibiting proteolysis, prompting accumulation of aggregates and so on. A stochastic model of the ubiquitin-proteasome system has been developed using the Systems Biology Mark-up Language (SBML). Simulations are carried out on the BASIS (Biology of Ageing e-Science Integration and Simulation) system and the model output is compared to experimental data wherein levels of ubiquitin and ubiquitinated substrates are monitored in cultured cells under various conditions. The model can be used to predict the effects of different experimental procedures such as inhibition of the proteasome or shutting down the enzyme cascade responsible for ubiquitin conjugation. RESULTS: The model output shows good agreement with experimental data under a number of different conditions. However, our model predicts that monomeric ubiquitin pools are always depleted under conditions of proteasome inhibition, whereas experimental data show that monomeric pools were depleted in IMR-90 cells but not in ts20 cells, suggesting that cell lines vary in their ability to replenish ubiquitin pools and there is the need to incorporate ubiquitin turnover into the model. Sensitivity analysis of the model revealed which parameters have an important effect on protein turnover and aggregation kinetics. CONCLUSION: We have developed a model of the ubiquitin-proteasome system using an iterative approach of model building and validation against experimental data. Using SBML to encode the model ensures that it can be easily modified and extended as more data become available. Important aspects to be included in subsequent models are details of ubiquitin turnover, models of autophagy, the inclusion of a pool of short-lived proteins and further details of the aggregation process. This model is hosted on BioModels Database and identified by: BIOMD0000000105. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:Post-translational control by ubiquitin regulates various aspects of cellular biology. This chemical modification on proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Among the various types of poly-ubiquitin constructed are untethered species that comprise a series of ubiquitin moieties linked to one another, but free from another protein. The current notion is that these unanchored poly-ubiquitin species are deleterious to the cell and are rapidly deconstructed. We recently examined the toxicity and utilization of untethered poly-ubiquitin in an intact organism by using the fruit fly, Drosophila melanogaster. We found that these ubiquitin species are largely innocuous to flies and that free poly-Ub can be controlled by being degraded by the proteasome or being conjugated onto another protein as a single unit. However, whether the fly is mounting an organismal defense against untethered chains was not explored in detail. Here, we conducted RNA-seq analyses to examine at the transcriptome level the impact of unanchored poly-Ub in the fly. We found ~90 transcripts whose expression is altered in the presence of different types of unanchored poly-ubiquitin. The set of genes identified was mostly devoid of ubiquitin-, proteasome- or autophagy-related components. The largest gene ontology category identified, housing ~15 of the altered genes, was proteolysis. The seeming absence of a large and multipronged response to unanchored ubiquitin chains in the fly supports the conclusion that these species need not be toxic in vivo and underscores the need to reexamine the role of free ubiquitin chains in the cell. We designed two types of poly-Ub chains with six Ub molecules in tandem. They cannot be cleaved by DUBs (there are no "GG" motifs linking the Ub molecules). Ub6-GG can be conjugated in toto onto other proteins; Ub6-Stop cannot. We drove ubiquitous expression using sqh-Gal4, and then performed RNA-Seq on adult flies to determine any cellular response to the presence of our unanchored Ub chains.

Dataset Information

A linear diubiquitin-based probe for efficient and selective detection of the deubiquitinating enzyme OTULIN

Publications

A Linear Diubiquitin-Based Probe for Efficient and Selective Detection of the Deubiquitinating Enzyme OTULIN.

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