Dataset Information

Ubiquitination of a small molecule

ABSTRACT: Ubiquitin is a small, highly conserved protein that acts as a post-translational modification in eukaryotes. Ubiquitination of proteins frequently serves as a degradation signal, marking them for disposal by the proteasomal. Here, we report a novel small molecule from a diversity- oriented synthesis library, BRD1732, that is directly ubiquitinated in cells, resulting in dramatic accumulation of unproductive ubiquitin monomer and polyubiquitin chains and broad inhibition of the ubiquitin-proteasome system. Ubiquitination of BRD1732 and its associated cytotoxicity are stereospecific and dependent upon two homologous E3 ubiquitin ligases, RNF19A and RNF19B. Our finding opens a possibility for indirect ubiquitination of a target through a ubiquitinated bifunctional small molecule, and more broadly raises the potential for post- translational modification in trans.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Dylan Mitchell

LAB HEAD: Dylan Mitchell

PROVIDER: PXD065168 | Pride | 2025-09-11

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	485236_ee00041_DCM2077.mzid	Mzid
	485282_ee00044_DCM2077.mzid	Mzid
	485283_ee00043_DCM2077.mzid	Mzid
	485284_ee00042_DCM2077.mzid	Mzid
	485946_ee00060_DCM2077.mzid	Mzid

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Publications

Highly specific intracellular ubiquitination of a small molecule.

Li Weicheng W Garcia-Rivera Enrique M EM Mitchell Dylan C DC Chick Joel M JM Maetani Micah M Bhadoria Rohit R Knapp John M JM Misu Ryosuke R Matthews Geoffrey M GM Shirasaki Ryosuke R de Matos Simoes Ricardo R Viswanathan Vasanthi V Pulice John L JL Rees Matthew G MG Roth Jennifer A JA Gygi Steven P SP Mitsiades Constantine S CS Kadoch Cigall C Schreiber Stuart L SL Ostrem Jonathan M L JML

Nature chemical biology 20250821

Ubiquitin is a small, highly conserved protein that acts as a posttranslational modification in eukaryotes. Ubiquitination of proteins frequently serves as a degradation signal, marking them for disposal by the proteasome. Here we report a novel small molecule from a diversity-oriented synthesis library, BRD1732, that is directly ubiquitinated in cells, resulting in dramatic accumulation of inactive ubiquitin monomers and polyubiquitin chains, which causes broad inhibition of the ubiquitin-prote ...[more]

PMID: 40841494

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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.