Activity Based Profiling of Deubiquitylating Enzymes and Inhibitors in Animal Tissues.
The attachment of ubiquitin or ubiquitin-like modifiers to proteins is an important signal for the regulation of a variety of biological processes including the targeting of substrates for degradation, receptor internalization, regulation of gene expression, and DNA repair. Posttranslational modification of proteins by ubiquitin controls many cellular processes, and aberrant ubiquitylation can contribute to cancer, immunopathologies, and neurodegeneration. Thus, deubiquitylating enzymes (DUBs) that remove ubiquitin from proteins have become attractive therapeutic targets. Monitoring the activity of DUBs in cells or in tissues is critical for understanding the biological function of DUBs in particular pathways and is essential for determining the physiological specificity and potency of small-molecule DUB inhibitors. Here, we describe a method for the homogenization of animal tissues and incubation of tissue lysates with ubiquitin-based activity probes to monitor DUB activity in mouse tissues and target engagement following treatment of animals with small-molecule DUB inhibitors.
MISSION Therapeutics Limited, Moneta, Babraham Research Campus, Cambridge, CB22 3AT, UK.
Lauren
L
McLellan
MISSION Therapeutics Limited, Moneta, Babraham Research Campus, Cambridge, CB22 3AT, UK.
Cassie
C
Forder
MISSION Therapeutics Limited, Moneta, Babraham Research Campus, Cambridge, CB22 3AT, UK.
Aaron
A
Cranston
MISSION Therapeutics Limited, Moneta, Babraham Research Campus, Cambridge, CB22 3AT, UK.
Jeanine
J
Harrigan
MISSION Therapeutics Limited, Moneta, Babraham Research Campus, Cambridge, CB22 3AT, UK. xjacq@missiontherapeutics.com.
Xavier
X
Jacq
D000818:Animals; D000072017:Deubiquitinating Enzymes / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination / Q000235:genetics / Q000502:physiology
D025801:Ubiquitin; D000072017:Deubiquitinating Enzymes
Activity-based probe; Deubiquitylating enzyme; Target engagement; Tissue; Ubiquitin