The ubiquitin-conjugating enzyme (E2) Ube2w ubiquitinates the N terminus of substrates

Abstract

Attachment of ubiquitin to substrate is typically thought to occur via formation of an isopeptide bond between the C-terminal glycine residue of ubiquitin and a lysine residue in the substrate. In vitro, Ube2w is nonreactive with free lysine yet readily ubiquitinates substrate. Ube2w also contains novel residues within its active site that are important for its ability to ubiquitinate substrate. To identify the site of modification, we analyzed ubiquitinated substrates by mass spectrometry and found the N-terminal -NH2 group as the site of conjugation. To confirm N-terminal ubiquitination, we generated lysine-less and N-terminally blocked versions of one substrate, the polyglutamine disease protein ataxin-3, and showed that Ube2w can ubiquitinate a lysine-less, but not N-terminally blocked, ataxin-3. This was confirmed with a second substrate, the neurodegenerative disease protein Tau. Finally, we directly sequenced the N terminus of unmodified and ubiquitinated ataxin-3, demonstrating that Ube2w attaches ubiquitin to the N terminus of its substrates. Together these data demonstrate that Ube2w has novel enzymatic properties that direct ubiquitination of the N terminus of substrates.

Keywords: N-terminal Ubiquitination; Neurodegeneration; Post-translational Modification; Ubiquitin; Ubiquitin-conjugating Enzyme (Ubc); Ubiquitination.

FIGURE 1.

Ube2w has novel enzymatic properties. A, amino acid reactivity of Ube2w∼Ub and UbcH5c∼Ub. Ube2w or UbcH5c was charged with ubiquitin for 5 min prior to the addition of the indicated free amino acids for 25 min. Samples were analyzed by Coomassie Blue stain. B, sequence alignment of key active site residues for select E2s. Green indicates the active cysteine residue, blue indicates conserved key active site residues, and red indicates differences in Ube2w. H. Sapiens, Homo sapiens; M. Mulatta, Macaca mulatta; B. Taurus, Bos taurus; G. Gallus, Gallus gallus; D. Rerio, Danio rerio; C. Elegans, Caenorhabditis elegans. C, alignment of Ube2w from different species. Red indicates residues that are nonidentical residues across species. D, thioester formation by Ube2w and Ube2w^H94N. Ube2w or Ube2w^H94N was incubated with E1, ubiquitin, and ATP/MgCl₂ for either 0 or 5 min prior to the addition of SDS sample buffer with or without βME (as indicated). Samples were analyzed by Coomassie Blue stain. E, Ube2w^H94N has reduced capacity to ubiquitinate substrate. Tau ubiquitination reactions were performed with Ube2w or Ube2w^H94N for the indicated lengths of time. Samples were analyzed by Western blot for either Tau or CHIP, as indicated.

FIGURE 2.

Ube2w modifies the N terminus of substrates. A, Ube2w ubiquitinates the N terminus of ataxin-3. Ataxin-3 was ubiquitinated in vitro by Ube2w, run on SDS-PAGE, and stained with Coomassie Blue. After in-gel digestion with trypsin, peptides were subjected to LC-MS/MS analysis using an orbitrap mass spectrometer. The resulting MS/MS spectra were searched against the Swiss-Prot human protein database appended with synthetic ataxin-3 using the x!Tandem/TPP software suite, considering N-terminal ubiquitination as both a fixed and a variable modification. All peptide-to-spectral matches (PSMs) with a PeptideProphet probability of >0.9 were considered correct assignments. PSMs of modified peptides were manually verified, and a representative spectrum is shown. Observed b- and y-ion series are indicated. The majority (46/50) of PSMs assigned to N-terminal peptide were ubiquitinated, indicating modification of the ataxin-3 N terminus. B, Ube2w modifies lysine-less ataxin-3 but not N-terminal GST-tagged ataxin-3. In vitro ubiquitination reactions were performed for the indicated lengths of time with ataxin-3, ^K0ataxin-3, or ^GSTataxin-3. Samples were analyzed by Western blot with anti-ataxin-3 antibody. C, as in B except employing UbcH5c as the E2 instead of Ube2w. D, chemical modification of the N terminus of ataxin-3 prevents Ube2w-mediated ubiquitination. Ubiquitination reactions were performed for the indicated lengths of time with ataxin-3 or carbamylated ataxin-3 as substrate. E, as in D but employing Tau or carbamylated Tau as substrate. F, as in E except employing UbcH5c as the E2 instead of Ube2w. G, results of Edman sequencing are consistent with N-terminal modification of substrates by Ube2w. Unmodified ataxin-3 or ataxin-3 ubiquitinated by Ube2w were transferred to PVDF and Ponceau-stained, and bands corresponding to unmodified or ubiquitinated ataxin-3 were subjected to Edman sequencing. The predicted sequences for unmodified or ubiquitinated ataixn-3 are shown. Blank and standard cycles were run prior to each run, and pmols of amino acid observed from each run were quantified.