A modular turn-on strategy to profile E2-specific ubiquitination events in living cells
Ontology highlight
ABSTRACT: This dataset describes label-free quantification of E2-specific ubiquitinated species identified by a chemically-induced proximity approach, tCUbE.
Project description:The E2 ubiquitin (Ub)-conjugating enzyme primarily determines Ub conjugation as Ub-isopeptide (lysine), Ub-oxyester (serine/threonine) or Ub-thioester (cysteine). However, E2-specific Ub conjugation profiles within cells remain elusive. Here, we developed the fusion E2–Ub-R74G profiling (FUSEP) strategy to access E2-specific Ub conjugation profiles in cells with amino acid resolution. The probe-specific leucine-glycine-glycine-glycine-modified Ub remnant enables systematic studies of nonlysine Ub conjugation and provides site-specific information. Multiple E2 enzymes were found to be involved in nonlysine ubiquitination. Profiling with UBE2D3–Ub-R74G probes identified the existence of tyrosine ubiquitination in human Cullin-1, a scaffold protein for Cullin-RING E3 Ub ligases. This modification is distinct from lysine ubiquitination. A single-pass membrane-bound E3 ligase, RNF149, was identified to pair with UBE2D3 to regulate pyroptosis by ubiquitinating apoptosis-associated speck-like protein ASC. The availability of this toolbox paves the way for uncovering E2-specific Ub conjugation profiles and identifying previously unknown E3 Ub ligases for potential therapeutic applications.
Project description:Transcriptional responses of the CD-1 mouse strain following treatments with E2 or the short acting estrogen estriol (E3) were evaluated by microarray and compared to our previous profiles done in C57Bl6 mice. The pattern of response was similar in both strains, with early (2h) responses to E2 or E3 appearing very similar. The later (24h) response revealed that E2 exhibits a more robust response than E3, illustrating the short-acting nature of E3.
Project description:HeLa/16E6-16E7 cells after expression of wild-type E2 compared to an E2 DNA-binding mutant Keywords: Wild-type E2 Infection vs. Mutant E2 Infection
Project description:We here apply the COmbined FRActional DIagonal Chromatography (COFRADIC) technology to enrich for ubiquitinated peptides and identify sites of ubiquitination by mass spectrometry. Our technology bypasses the need to ectopically overexpress tagged variants of ubiquitin and the use of sequence-biased antibodies recognizing ubiquitin remnants. In brief, all protein primary amino groups are blocked by chemical acetylation after which ubiquitin chains are proteolytically and specifically removed by the catalytic core domain of the USP2 deubiquitinase (USP2cc). As USP2cc cleaves the isopeptidyl bond between the ubiquitin C-terminus and the ?-amino group of the ubiquitinated lysine, this enzyme re-introduces primary ?-amino groups in proteins. These amino groups are then chemically modified with a handle that allows specific isolation of ubiquitinated peptides during subsequent COFRADIC chromatographic runs. Our method allowed us to identify over 8,200 endogenous ubiquitination sites in more than 3,600 different proteins in a native human Jurkat cell lysate.
Project description:Protein ubiquitination orchestrates nearly all eukaryotic cellular events.1 It starts by attaching ubiquitin through isopeptide bonds to a single or multiple lysine residues of a target protein via a coordinated enzymatic reaction involving activating (E1), conjugating (E2), and ligating (E3) enzymes to form mono- or multi-mono-ubiquitinated products. There is a need for a technique that can facilitate the high-yield production of monoubiquitinated proteins. Here we present an efficient approach that fills this gap.