A Proteomic Approach for Systematic Mapping of Substrates of Human Deubiquitinating Enzymes.

Juanma Ramirez,Anne Olazabal-Herrero,Javier Beaskoetxea,Gorka Prieto,Benoit Lectez,Alberto Paradela,Fernando Corrales,Elvira Fernandez-Vigo,Kerman Aloria,Jesus M Arizmendi,Eva Borràs,Ugo Mayor,Nerea Osinalde,Jose Antonio Rodriguez,Eduard Sabidó,Unai Alduntzin,Javier Muñoz

doi:10.3390/ijms22094851

Abstract

The human genome contains nearly 100 deubiquitinating enzymes (DUBs) responsible for removing ubiquitin moieties from a large variety of substrates. Which DUBs are responsible for targeting which substrates remain mostly unknown. Here we implement the bioUb approach to identify DUB substrates in a systematic manner, combining gene silencing and proteomics analyses. Silencing of individual DUB enzymes is used to reduce their ubiquitin deconjugating activity, leading to an increase of the ubiquitination of their substrates, which can then be isolated and identified. We report here quantitative proteomic data of the putative substrates of 5 human DUBs. Furthermore, we have built a novel interactive database of DUB substrates to provide easy access to our data and collect DUB proteome data from other groups as a reference resource in the DUB substrates research field.

Highlights

Cells are very dynamic entities that are constantly synthesizing and degrading proteins in order to ensure their proper functioning
deubiquitinating enzymes (DUBs) are encoded by ~100 genes in humans that, based on sequence and structural similarities of their catalytic domain, are classified into seven families: Ubiquitin-specific proteases (USPs), Ubiquitin C-terminal Hydrolases (UCHs), Ovarian Tumour proteases (OTUs), MachadoJoseph disease proteases or Josephins (MJDs), MIU-containing novel DUBs (MINDYs), Zinc finger containing Ubiquitin Peptidase 1 (ZUP1) and JAB1/MPN/MOV34 (JAMMs) proteases [6]
Cells are first transfected with a siRNA to silence a certain DUB, and supplied with a biotinylated ubiquitin by transfection of the bioUb construct [22], a precursor polypeptide composed of six biotinylatable versions of ubiquitin and the biotin ligase enzyme of E. coli, BirA (Figure S1A)

Summary

Introduction

Cells are very dynamic entities that are constantly synthesizing and degrading proteins in order to ensure their proper functioning. Ubiquitinated proteins are directed to the proteasome, a multi-catalytic enzyme complex, where they are subjected to degradation [1]. This is not the only fate of ubiquitinated proteins [2]. To other post-translational modifications, the UPS pathway has a mechanism to reverse the effect of E1, E2 and E3 enzymes through the activity of a fourth family of enzymes, the deubiquitinating (DUB) enzymes [6]. To E3s [12,13], DUB enzymes show substrate specificity, and particular DUBs may exhibit a preference for certain ubiquitin chain linkages [6]. Our hypothesis is that each DUB will have its own repertoire of target substrates on specific cell types

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Conclusion