Dataset Information

Structural basis of PROTAC-induced target ubiquitination mechanism - identification of BRD4 ubiquitination sites

ABSTRACT: 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. To elucidate the mechanism of PROTAC-induced ubiquitination of BRD4(BD2) ubiquitination sites on Brd4BD2 were mapped by mass spectrometry following an in vitro ubiquitination assay containing neddylated-CRL2VHL, Brd4BD2, MZ1, Uba1 and either UBE2R1 or UBE2D2. Time points were taken at 0 minutes, 10 minutes, 0.5 hours, 1 hour and 3 hours. Proteins were separated by SDS-page analysis. To visualize proteins the gel was stained with Coomassie blue and gel slices containing ubiquitin modified Brd4BD2 across timepoints and increasing molecular weight were excised from the gel and were analyzed by mass spectrometry. To identify sites of ubiquitination a search for GG-K peptides was performed in MaxQuant. Based on existing structures the modified lysine residues were all situated on the same face of BD2.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Sarah Chandler

LAB HEAD: Ronald T Hay

PROVIDER: PXD049043 | Pride | 2025-05-06

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	BRD4_UBIQUITINATION_SITES_ALL_PROTEINS.fasta	Fasta
	SChandler_20230802_D2_01.raw	Raw
	SChandler_20230802_D2_02.raw	Raw
	SChandler_20230802_D2_03.raw	Raw
	SChandler_20230802_D2_04.raw	Raw

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Publications

Mechanism of degrader-targeted protein ubiquitinability.

Crowe Charlotte C Nakasone Mark A MA Chandler Sarah S Craigon Conner C Sathe Gajanan G Tatham Michael H MH Makukhin Nikolai N Hay Ronald T RT Ciulli Alessio A

Science advances 20241011 41

Small-molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and potential to tackle previously undrugged targets. Stable and long-lived degrader-mediated ternary complexes drive fast and profound target degradation; however, the mechanisms by which they affect target ubiquitination remain elusive. Here, we show cryo-EM structures of the VHL Cullin 2 RING E3 ligase with the degrader MZ1 directing target protein Brd4<sup>BD2</sup> towa ...[more]

PMID: 39392888

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Project 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. This approach resulted in multiple novel cryo-EM structures of the active CRL in complex with the model substrate and MZ1. To validate the conformation of the Ub-Ube2R1-Rbx1 within these structures, a ubiquitin-directed photoreactive probe (UDPRP) was designed. The UDPRP consists of a stable-isopeptide linked Ub-Ube2R1 conjugate as used in the except the ubiquitin is site-specifically labelled with the photocrosslinker, N-maleimido-diazirine. The diazirine based photocrosslinker is site-specifically labelled to ubiquitin through cysteine-maleimide chemistry. Upon exposure to UV light (365 nM) forms a reactive carbene intermediate which either rapidly inserts into X-H bonds (including O-H, N-H, S-H and C-H bonds) and can therefore covalently trap proteins in close-proximity. In a photocrosslinking assay containing neddylated-CRL2VHL and the UDPRP. The UDPRP crosslinked to Rbx1, the RING portion of the multi-subunit CRL E3. To map the site of crosslinking the photo-crosslinked product was excised from the gel and analysed by mass-spectrometry. Four inter-protein crosslinked peptides from ubiquitin-rbx1 were detected, with an andromeda score >90, linking ubiquitin E34C to the last three C-terminal residues of Rbx1.

Project description:Translation elongation rates are regulated to ensure proper conformation and biological function of proteins. Translation of either non-stop mRNA or transcripts coding for poly-basic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control system (RQC). During this process, the stalled ribosome is dissociated into subunits, and the polypeptide is ubiquitinated by the E3 ubiquitin ligase Listerin on the 60S large ribosomal subunit (LSU) leading to subsequent proteasomal degradation. However, it is largely unknown how stalled ribosomes are recognized and dissociated into subunits. Here we report that ubiquitination of the ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 is required for the production of the RQC substrate. RQC-trigger (RQT) factors, a RNA helicase-family protein Slh1/Rqt2, ubiquitin binding protein Cue3/Rqt3 and yKR023W/Rqt4, were also required for the primary steps of RQC, and associated with Hel2-ribosome complexes. Rqt2-4 factors were dispensable for the ubiquitination of uS10 by Hel2/Rqt1 and associated with ribosomes independent of the ubiquitination of uS10. However, the ubiquitin-binding activity of Rqt3 were crucial to trigger RQC. Cryo-electron microscopy (cryo-EM) analysis revealed that Hel2 bound ribosomes are in an rotated state containing hybrid state AP- and PE-tRNAs. Furthermore, ribosome profiling revealed that short footprints, hallmarks of hybrid state ribosomes18, were accumulated at tandem CGA rare codons at the beginning of the poly arginine stalling sequence and long footprints at subsequent codons, respectively. Short footprints at CGA codons were decreased in rqt1 mutant but drastically increased in uS10 mutants defective in the ubiquitination or rqt2 mutant. Collectively, our results demonstrate that Hel2 stabilizes ratcheted ribosomes leading to ubiquitination of uS10. Subsequently, Rqt2-4 factors target these hybrid state ribosomes specifically, allowing subsequent RQC reactions.