PXD049050-1

PXD049050 is an original dataset announced via ProteomeXchange.

Title	Structural basis of PROTAC-induced target ubiquitination mechanism - verification of ubiquitin UBE2R1 (C93K) loading assay
Description	Proteolysis Targeting Chimeras (PROTACs) hijack the ubiquitin proteasome system to ubiquitinate target proteins, targeting them for degradation by the proteasome. An understudied process that is essential to the PROTAC mechanism of action is ubiquitination of the target protein and ubiquitin (Ub) chain elongation. These phenomena are difficult to study structurally due the instability of the thioester-linked ubiquitin (Ub)-E2 conjugates that rapidly discharges ubiquitin onto the target protein, catalysed by a Cullin-RING E3 Ubiquitin Ligase (CRL). To overcome this, stable isopeptide-linked Ub-E2 conjugates were generated using an active site mutant of the chain elongation E2 enzyme UBE2R1 and used in structural studies of a model PROTAC system consisting of neddylated CRL2VHL, MZ1 and BRD4(BD2)-Ub. To generate the stable isopeptide-linked Ub-E2 conjugates, the active site cysteine (C93) of UBE2R1 was mutated to a lysine (C93K). Then in a reaction using recombinant human E1 at high pH, ubiquitin was conjugated to UBE2R1. As the ubiquitin E1 transfers ubiquitin to the active site cysteine of UBE2R1 (in unmodified systems), the preferred site of ubiquitin modification should theoretically be on K93. However, other lysine residues can also be modified so to ensure that the primary site of ubiquitin modification occurred at the active site (residue K93), ubiquitin-modified UBE2R1 was analysed by mass spectrometry and a search for GG-K modified peptides was performed. This search provided good evidence that the main site of modification occurred on residue K93 of the UBE2R1 C93K mutant. Verifying this as a suitable structural mimetic of the thioester-linker ubiquitin loaded UBE2R1.
HostingRepository	PRIDE
AnnounceDate	2025-05-06
AnnouncementXML	Submission_2025-05-06_12:53:33.496.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Sarah Chandler
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	ubiquitinylated lysine
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2024-01-31 08:17:37	ID requested
⏵ 1	2025-05-06 12:53:34	announced

Crowe C, Nakasone MA, Chandler S, Craigon C, Sathe G, Tatham MH, Makukhin N, Hay RT, Ciulli A, Mechanism of degrader-targeted protein ubiquitinability. Sci Adv, 10(41):eado6492(2024) [pubmed]

10.1126/sciadv.ado6492;

submitter keyword: structural, mimetic,UBE2R1, ubiquitin, E2

Ronald T Hay
contact affiliation	University of Dundee, Scool of Life Sciences, Department of Molecular, Cellular andDevelopmental biology
contact email	r.t.hay@dundee.ac.uk
lab head
Sarah Chandler
contact affiliation	University of Dundee
contact email	2431160@dundee.ac.uk
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD049050
     1. Label: PRIDE project
     2. Name: Structural basis of PROTAC-induced target ubiquitination mechanism - verification of ubiquitin UBE2R1 (C93K) loading assay