Target Identification

BACKGROUND

Ubiquitin Proteasome System, UPS

The Ubiquitin Proteasome System (UPS) drives the degradation of ubiquitinated target proteins. Modification of proteins by members of the ubiquitin family is regulated by E3 ligases and deubiquitinases (DUBs). Dysregulation of this balance leads to pathologies, including neurodegeneration, cancer, and rare diseases.

Targeted Protein Degradation, TPD

The research on UPS, E3 ligases and DUBs is key for the emerging strategies of targeted protein degradation or TPD. TPD uses molecular degraders (i.e. molecular glues and proteolysis targeting chimeras or PROTACs) to re-route the cellular protein degradation machinery towards disease-relevant proteins of interest.

PORTFOLIO

PROCEDURE

ZbioX will 1) assess needs and propose best approach among the offered technologies, 2) conduct the necessary experiments in transfected cultured cells, 3) follow the required treatments for each strategy and collect cell extracts, 4) isolate modified proteins by affinity chromatography, 5) identify isolated proteins using liquid chromatography – mass spectrometry, 6) provide bioinformatic analysis, and 7) if appropriate, archive data into dedicated databases.

The E3 ligase of interest is fused to the E. coli biotinylating enzyme BirA. BirA-E3 fusion is transfected in cells expressing a ubiquitin-like protein (UbL) fused to AviTagGEF (a specific proximity-dependent biotinylation target of BirA).

TO KNOW MORE

• Barroso-Gomila et al 2023 Nat Commun. doi: 10.1038/s41467-023-43326-8
• Merino Cacho et al 2024 ChemBioChem. doi: 10.1002/cbic.202300746

TurboID is an optimized version of promiscuous BirA that allows the identification of interactomes by proximity-dependent biotinylating of any lysine in proximity. By fusing TurboID to a protein of interest and transfecting into cells, its surrounding interactors will be biotinylated.

TO KNOW MORE

• Branon et al 2018 Nat Biotechnol. doi: 10.1038/nbt.4201
• Roux te al 2012 J Cell Biol. doi: 10.1083/jcb.201112098.

Molecular traps, like TUBEs (tandem ubiquitin binding entities) and SUBEs (SUMO binding entities) are able to capture endogenous proteins modified by ubiquitin or by the Small ubiquitin-modifier, SUMO, respectively, from animal tissues and organs. Capture can be followed by LC/MS analysis to identify modified targets.

TO KNOW MORE

• Mattern, Sutherland at al 2019 TIBs. doi: 10.1016/j.tibs.2019.01.011.
• Hjerpe et al 2009 EMBO Rep. doi: 10.1038/embor.2009.192
• Aillet et al 2012 Methods Mol Biol. doi: 10.1007/978-1-61779-474-2_12.
• Lang et al 2016 Methods Mol Biol. doi: 10.1007/978-1-4939-6358-4_8.

Based on protein fragment complementation of TurboID, UbL-ID is designed to identify interactors of a protein of interest which are dependent on modification by (or interaction with) Ubiquitin-like family members (UbL). The protein of interest is fused to one part of TurboID, while the desired UbL is fused to the other part of TurboID.

TO KNOW MORE

• Barroso-Gomila et al 2023 Methods Mol Biol. doi: 10.1007/978-1-0716-2859-1_13.
• Barroso-Gomila et al 2021 Nat Commun. doi: 10.1038/s41467-021-26807-6.

By fusing Ubiquitin-like family members (UbLs) to the AviTag sequence, which is biotinylated by the E. coli biotin ligase BirA, we can identify UbL-modified proteins from cells or in vivo.

TO KNOW MORE

• Pirone et al 2017 Sci Rep. doi: 10.1038/srep40756.
• Pirone et al 2016 Methods Mol Biol. doi: 10.1007/978-1-4939-6358-4_12.
• Lectez et al 2014 J Proteome Res. doi: 10.1021/pr5001913.