Characterization of ubiquitin and ubiquitin‐like‐protein isopeptidase activities

Nicholson, Benjamin; Leach, Craig A.; Goldenberg, Seth J.; Francis, Dana M.; Kodrasov, Matthew P.; Tian, Xufan; Shanks, John; Sterner, David E.; Bernal, Alejandro Tejerina; Mattern, Michael R.; Wilkinson, Keith D.; Butt, Tauseef R.

doi:10.1110/ps.083450408

Cited by 125 publications

(132 citation statements)

References 43 publications

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“…CHOP Reporter Assay-DUB isopeptidase activity was measured by CHOP assay as previously described (31). Used in this study were LifeSensors' UB-CHOP (PR1001), ISG15-CHOP (PR1005), and ISG15-CHOP2 (PR1105).…”

Section: Methodsmentioning

confidence: 99%

“…We chose as our substrate the CHOP reporter assay (31), which consists of a linear fusion of UB (or other UBLs) and phospholipase-A2 (PLA 2 ). Because PLA 2 is catalytically inactive and absent of a free, native N terminus, incubation of this substrate with DUB in the presence of 1-acyl-2-[6-(7-nitro-1,3-benzoxadiazol-4-yl)amino]caproyl-NBD, a quenched fluorescent substrate for PLA 2 -provides a sensitive and quantifiable readout of PLA 2 cleavage from Ubiquitin, i.e.…”

Section: Methodsmentioning

confidence: 99%

“…Because PLA 2 is catalytically inactive and absent of a free, native N terminus, incubation of this substrate with DUB in the presence of 1-acyl-2-[6-(7-nitro-1,3-benzoxadiazol-4-yl)amino]caproyl-NBD, a quenched fluorescent substrate for PLA 2 -provides a sensitive and quantifiable readout of PLA 2 cleavage from Ubiquitin, i.e. DUB activity (31). Raw assay data are shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

“…5). Of these USP8, USP2, PLPro, USP14, and USP18 (5/13) have been previously shown to have de-ISGylase activity (31,38). Also shown to have de-ISGylase activity and present on the array was PLP2 (31), though it did not display array activity.…”

Section: Table I Protein Contents Of the Arrays Used In This Studymentioning

confidence: 99%

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Deubiquitylase, DeSUMOylase, and DeISGylase Activity Microarrays for Assay of Substrate Preference and Functional Modifiers

Loc’h

Cuccherini

Leach

et al. 2011

Molecular & Cellular Proteomics

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Microarray-based proteomics expanded the information potential of DNA arrays to the level of protein translation and interaction, but so far, not much beyond. Although enzymatic activity from immobilized proteins has been reliably studied using surface plasmon resonance, a microarray of catalytically competent enzymes would facilitate high throughput, parallel study of their function. The ability to localize activity from soluble substrates has frustrated development of such an array. Here, we report the novel use of previously developed, highly specific suicide substrates for three families of enzymes: deubiquitylases, deSUMOylases, and deISGylases. We show specificity of each family to its cognate substrate, and demonstrate utility of the array in a secondary screen of small molecule inhibitors.Molecular & Cellular Proteomics 10: 10.1074/ mcp.M110.002402, 1-11, 2011.Microarray-based proteomics has typically sought to accomplish the same primary objectives as its accomplice, mass spectrometry (MS): cataloging protein expression, defining protein-protein interactions, and defining types and sites of post-translational modification (1). Although the presence of specific enzymatic reaction products can be monitored by MS, a more typical proteomic approach to the study of enzymatic function has been transfer of a detectable moiety to array-immobilized proteins, as in identification of kinase substrates, for example (2-4). Although this approach to functional proteomics excels in multiplexed identification of substrate targets, it does not permit multiplexed study of the enzymes themselves. Enzymatic activity following immobilization has been demonstrated for individual proteins on resin or gold surfaces (5, 6); using multiple spotting technology in which substrate was immobilized and reactions were initiated upon spotting a glycerol/enzyme solution (7); and in microarray format using small molecule fluorescent activity labels to profile enzyme activity from immobilized cysteine proteases, phosphatases, and serine hydrolases (8, 9). Such an array of immobilized, enzymatically active proteins can facilitate multiplexed study of modulators of that activity, be they chemical, polypeptide, or other (reaction conditions, for example). Here, we utilize a similar strategy to extend this seminal work to the ubiquitin pathway.Ubiquitin is a small protein (8.5 kDa) conjugated to a wide range of protein substrates in all eukaryotic cells. The ubiquitin pathway consists of ligases that conjugate, and proteases that remove ubiquitin from target proteins. Over 90% of the proteins in a cell will be ubiquitylated at some point during their life cycle (10), the consequences of which include modulating enzymatic activity (11), cell signaling (12, 13), affecting protein-protein interaction (14), controlling histone-DNA interaction (15) transcription (16), DNA repair (17), and target protein degradation (18). Once dismissed as the "garbage can" of the cell, this pathway has established itself to be as multifunctional as it is essential...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Table I Protein Contents Of the Arrays Used In This Studymentioning

confidence: 99%

See 2 more Smart Citations

Deubiquitylase, DeSUMOylase, and DeISGylase Activity Microarrays for Assay of Substrate Preference and Functional Modifiers

Loc’h

Cuccherini

Leach

et al. 2011

Molecular & Cellular Proteomics

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show abstract

“…These compounds are nonselective isopeptidase inhibitors; indeed, in vitro, they are also able to block the deconjugation of Ubl proteins such as SUMO. 2,3 …”

Section: Ups Inhibitorsmentioning

confidence: 99%

Inhibitors of the ubiquitin-proteasome system are not all alike: Identification of a new necrotic pathway

Foti

Brancolini²

2009

Autophagy

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Medicinal Chemistry in the Context of the Human Genome

Brunschweiger

Hall

2013

Methods and Principles in Medicinal Chemistry

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Characterization of ubiquitin and ubiquitin‐like‐protein isopeptidase activities

Cited by 125 publications

References 43 publications

Deubiquitylase, DeSUMOylase, and DeISGylase Activity Microarrays for Assay of Substrate Preference and Functional Modifiers

Deubiquitylase, DeSUMOylase, and DeISGylase Activity Microarrays for Assay of Substrate Preference and Functional Modifiers

Inhibitors of the ubiquitin-proteasome system are not all alike: Identification of a new necrotic pathway

Medicinal Chemistry in the Context of the Human Genome

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