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Ubiquitin-specific proximity labeling for the identification of E3 ubiquitin ligase substrates

ABSTRACT: Protein ubiquitination controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in diseases1. Moreover, protein degraders that redirect ubiquitination activities toward disease targets are an emerging and promising therapeutic class2. Over 600 E3 ubiquitin ligases are expressed in humans3,4, but their substrates remain largely elusive due to a lack of robust methods to identify E3 ligase substrates. Here we report the development of E-STUB (E3 substrate tagging by ubiquitin biotinylation), a ubiquitin-specific proximity labeling method that biotinylates ubiquitinated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitinated targets of protein degraders, including collateral targets and ubiquitylation events that do not exhibit a degradative outcome. It also detects known substrates of E3 ligase cereblon (CRBN) and von Hippel-Lindau (VHL) with high precision. With the ability to elucidate proximal ubiquitination events, E-STUB may facilitate the development of proximity-inducing drugs and act as a generalizable method for E3 substrate mapping.

INSTRUMENT(S): timsTOF Pro 2

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Eric Fischer

LAB HEAD: Eric Fischer

PROVIDER: PXD042700 | Pride | 2024-03-20

REPOSITORIES: Pride

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			Action	DRS
	esf4_4670_Slot1-7_1_8598.d.zip	Other
	esf4_4671_Slot1-8_1_8599.d.zip	Other
	esf4_4672_Slot1-9_1_8600.d.zip	Other
	esf4_4676_Slot1-13_1_8606.d.zip	Other
	esf4_4677_Slot1-14_1_8607.d.zip	Other

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Ubiquitin-specific proximity labeling for the identification of E3 ubiquitin ligase substrates

Project description:Protein ubiquitination controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in diseases1. Moreover, protein degraders that redirect ubiquitination activities toward disease targets are an emerging and promising therapeutic class2. Over 600 E3 ubiquitin ligases are expressed in humans3,4, but their substrates remain largely elusive due to a lack of robust methods to identify E3 ligase substrates. Here we report the development of E-STUB (E3 substrate tagging by ubiquitin biotinylation), a ubiquitin-specific proximity labeling method that biotinylates ubiquitinated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitinated targets of protein degraders, including collateral targets and ubiquitylation events that do not exhibit a degradative outcome. It also detects known substrates of E3 ligase cereblon (CRBN) and von Hippel-Lindau (VHL) with high precision. With the ability to elucidate proximal ubiquitination events, E-STUB may facilitate the development of proximity-inducing drugs and act as a generalizable method for E3 substrate mapping.

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