PXD045521

PXD045521 is an original dataset announced via ProteomeXchange.

Title	A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates
Description	Ubiquitination of proteins is central to protein homeostasis and other cellular processes including DNA repair, vesicular transport, cell-division etc. The process of ubiquitination is conserved from yeast to humans and is carried out by the sequential action of three enzymes: E1, E2 and E3. There are an estimated >600 E3 ligases in humans that execute ubiquitination of specific target proteins in a spatio-temporal manner to elicit desired signaling effects. Here, we developed a simple, proximity-based labeling method to selectively biotinylate substrates of a given ubiquitin ligase. Our method exploits the proximity and the relative orientation of the E3-ligase catalytic domain with respect to ubiquitin observed in the enzymatic intermediate-state structures of E3-E2~Ub. By fusing the biotin ligase BirA and an Avi-tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich the bona fide substrates and potential new substrates of the ligase using a one-step Streptavidin pulldown under denaturing conditions. As proof-of-principle, we applied our method, which we named Ub-POD, to the RING E3 ligase Rad18. Rad18 ubiquitinates DNA-sliding clamp PCNA upon UV-induced DNA damage. We identified PCNA and several other critical players in the DNA damage repair pathway in a single Ub-POD experiment. We further discovered an unintended but important application of Ub-POD, where we were able to pin down the cellular localization of Rad18- mediated ubiquitination to the damaged DNA nuclear puncta through Streptavidin immunofluorescence. We also applied our method to TRAF6, another RING ubiquitin ligase involved in TNF signaling, successfully identifying known and potentially new substrates. Finally, we adapted our method to the U-box type E3 ubiquitin ligase CHIP to demonstrate that we can identify substrates of two major classes of mammalian ubiquitin ligases. We anticipate that our method and principle could be widely adapted to all classes of ubiquitin ligases to identify substrates and localize the cellular site(s) of ubiquitination.
HostingRepository	PRIDE
AnnounceDate	2024-11-26
AnnouncementXML	Submission_2024-11-26_06:18:59.729.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Sagar Bhogaraju
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2023-09-20 06:20:38	ID requested
⏵ 1	2024-11-26 06:19:00	announced

10.1126/sciadv.adp3000;

Mukhopadhyay U, Levantovsky S, Carusone TM, Gharbi S, Stein F, Behrends C, Bhogaraju S, A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates. Sci Adv, 10(32):eadp3000(2024) [pubmed]

submitter keyword: BirA, substrate identification, U box, RING, E3 ligase,ubiquitin

Sagar Bhogaraju
contact affiliation	EMBL Grenoble, France
contact email	bhogaraju@embl.fr
lab head
Sagar Bhogaraju
contact affiliation	EMBL Grenoble
contact email	bhogaraju@embl.fr
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2024/11/PXD045521

PRIDE project URI

• PRIDE
  1. PXD045521
     1. Label: PRIDE project
     2. Name: A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates