RNAi-Based Screening for the Identification of Specific Substrate-Deubiquitinase Pairs

Nagore Elu; Natalia Presa; Ugo Mayor

doi:10.1007/978-1-0716-2859-1_7

RNAi-Based Screening for the Identification of Specific Substrate-Deubiquitinase Pairs

Methods Mol Biol. 2023:2602:95-105. doi: 10.1007/978-1-0716-2859-1_7.

Authors

Nagore Elu¹, Natalia Presa¹, Ugo Mayor^{2

3}

Affiliations

¹ Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Leioa, Spain.
² Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Leioa, Spain. ugo.mayor@ehu.eus.
³ Ikerbasque, Basque Foundation for Science, Bilbao, Spain. ugo.mayor@ehu.eus.

PMID: 36446969
DOI: 10.1007/978-1-0716-2859-1_7

Abstract

Ubiquitination signals are regulated in time and space due to the coordinated action of E3s and DUBs, which enables the precise control of cellular function and homeostasis. Mutations in all types of ubiquitin-proteasome system (UPS) components are related to pathological conditions. The identification of E3/DUBs' ubiquitinated substrates can provide a clearer view of the molecular mechanisms underlying those diseases. However, the analysis of ubiquitinated proteins is not trivial. Here, we propose a protocol to identify DUB/substrate pairs, by combining DUB silencing, specific pull-down of the substrate, and image analysis of its ubiquitinated fraction.

Keywords: DUBs; Deubiquitinating enzymes; Deubiquitination; GFP pull-down; RNAi silencing; Substrate-DUB pairs.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Deubiquitinating Enzymes / genetics
RNA Interference
Research*
Ubiquitin* / genetics
Ubiquitinated Proteins

Substances

Ubiquitin
Ubiquitinated Proteins
Deubiquitinating Enzymes