Characterising plant deubiquitinases with in vitro activity-based labelling and ubiquitin chain disassembly assays

Michael Skelly; Steven H Spoel

doi:10.21769/BioProtoc.4015

Characterising plant deubiquitinases with in vitro activity-based labelling and ubiquitin chain disassembly assays

Michael Skelly, Steven H Spoel

School of Biological Sciences

Research output: Contribution to journal › Article › peer-review

Abstract

Post-translational modification of proteins by ubiquitin is an essential cellular signaling mechanism in all eukaryotes. Ubiquitin is removed from target proteins by a wide range of deubiquitinase (DUB) enzymes with different activities and substrate specificities. Understanding how DUBs function in vitro is a vital first step to uncovering their cellular roles. Here we provide protocols for rapidly analyzing DUB activity in vitro by activity-based labelling with the suicide probe HA-ubiquitin vinyl sulfone (HA-UbVS) and by ubiquitin chain disassembly assays. We have previously used these methods to analyses the activity of the Arabidopsis thaliana DUB, UBP6, but these protocols are in principle applicable to any DUB of interest.

Original language	English
Article number	e4015
Number of pages	5
Journal	Bio-protocol
Volume	11
Issue number	9
DOIs	https://doi.org/10.21769/BioProtoc.4015
Publication status	Published - 5 May 2021

Keywords

ubiquitin
deubiquitinase
ubiquitin vinyl sulfone
proteasome
cell signaling
proteostasis

Access to Document

10.21769/BioProtoc.4015Licence: Creative Commons: Attribution (CC-BY)

SpoelEtalBP2021CharacterisingPlantDeubiquitinasesWithInVitroActivitYBasedLabellingFinal published version, 240 KBLicence: Creative Commons: Attribution (CC-BY)

IMMUNE-EXPRESS: Proteasome-Mediated Gene Expression in Plant Immunity
Spoel, S.
EU government bodies
1/03/16 → 31/08/21
Project: Research
Licensing Transcription Activator Activity with Ubiquitin Time Clocks
Spoel, S.
BBSRC
1/05/14 → 30/04/17
Project: Research
Protein modification as a tool for reprogramming gene activity
Spoel, S.
Other (Learned Society)
1/10/10 → 30/09/15
Project: Research

Cite this

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title = "Characterising plant deubiquitinases with in vitro activity-based labelling and ubiquitin chain disassembly assays",

abstract = "Post-translational modification of proteins by ubiquitin is an essential cellular signaling mechanism in all eukaryotes. Ubiquitin is removed from target proteins by a wide range of deubiquitinase (DUB) enzymes with different activities and substrate specificities. Understanding how DUBs function in vitro is a vital first step to uncovering their cellular roles. Here we provide protocols for rapidly analyzing DUB activity in vitro by activity-based labelling with the suicide probe HA-ubiquitin vinyl sulfone (HA-UbVS) and by ubiquitin chain disassembly assays. We have previously used these methods to analyses the activity of the Arabidopsis thaliana DUB, UBP6, but these protocols are in principle applicable to any DUB of interest. ",

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language = "English",

volume = "11",

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AU - Skelly, Michael

AU - Spoel, Steven H

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N2 - Post-translational modification of proteins by ubiquitin is an essential cellular signaling mechanism in all eukaryotes. Ubiquitin is removed from target proteins by a wide range of deubiquitinase (DUB) enzymes with different activities and substrate specificities. Understanding how DUBs function in vitro is a vital first step to uncovering their cellular roles. Here we provide protocols for rapidly analyzing DUB activity in vitro by activity-based labelling with the suicide probe HA-ubiquitin vinyl sulfone (HA-UbVS) and by ubiquitin chain disassembly assays. We have previously used these methods to analyses the activity of the Arabidopsis thaliana DUB, UBP6, but these protocols are in principle applicable to any DUB of interest.

AB - Post-translational modification of proteins by ubiquitin is an essential cellular signaling mechanism in all eukaryotes. Ubiquitin is removed from target proteins by a wide range of deubiquitinase (DUB) enzymes with different activities and substrate specificities. Understanding how DUBs function in vitro is a vital first step to uncovering their cellular roles. Here we provide protocols for rapidly analyzing DUB activity in vitro by activity-based labelling with the suicide probe HA-ubiquitin vinyl sulfone (HA-UbVS) and by ubiquitin chain disassembly assays. We have previously used these methods to analyses the activity of the Arabidopsis thaliana DUB, UBP6, but these protocols are in principle applicable to any DUB of interest.

KW - ubiquitin

KW - deubiquitinase

KW - ubiquitin vinyl sulfone

KW - proteasome

KW - cell signaling

KW - proteostasis

U2 - 10.21769/BioProtoc.4015

DO - 10.21769/BioProtoc.4015

M3 - Article

VL - 11

JO - Bio-protocol

JF - Bio-protocol

SN - 2331-8325

IS - 9

M1 - e4015

ER -

Characterising plant deubiquitinases with in vitro activity-based labelling and ubiquitin chain disassembly assays

Abstract

Keywords

Access to Document

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Projects

IMMUNE-EXPRESS: Proteasome-Mediated Gene Expression in Plant Immunity

Licensing Transcription Activator Activity with Ubiquitin Time Clocks

Protein modification as a tool for reprogramming gene activity

Cite this