A Novel Strategy to Isolate Ubiquitin Conjugates Reveals Wide Role for Ubiquitination during Neural Development

Abstract

Ubiquitination has essential roles in neuronal development and function. Ubiquitin proteomics studies on yeast and HeLa cells have proven very informative, but there still is a gap regarding neuronal tissue-specific ubiquitination. In an organism context, direct evidence for the ubiquitination of neuronal proteins is even scarcer. Here, we report a novel proteomics strategy based on the in vivo biotinylation of ubiquitin to isolate ubiquitin conjugates from the neurons of Drosophila melanogaster embryos. We confidently identified 48 neuronal ubiquitin substrates, none of which was yet known to be ubiquitinated. Earlier proteomics and biochemical studies in non-neuronal cell types had identified orthologs to some of those but not to others. The identification here of novel ubiquitin substrates, those with no known ubiquitinated ortholog, suggests that proteomics studies must be performed on neuronal cells to identify ubiquitination pathways not shared by other cell types. Importantly, several of those newly found neuronal ubiquitin substrates are key players in synaptogenesis. Mass spectrometry results were validated by Western blotting to confirm that those proteins are indeed ubiquitinated in the Drosophila embryonic nervous system and to elucidate whether they are mono- or polyubiquitinated. In addition to the ubiquitin substrates, we also identified the ubiquitin carriers that are active during synaptogenesis. Identifying endogenously ubiquitinated proteins in specific cell types, at specific developmental stages, and within the context of a living organism will allow understanding how the tissue-specific function of those proteins is regulated by the ubiquitin system.

Using in Vivo Biotinylated Ubiquitin to Describe a Mitotic Exit Ubiquitome from Human Cells

“…With this goal in mind we adopted a system for in vivo tagging of ubiquitin chains with biotin, previously used to identify ubiquitin-conjugated proteins from the Drosophila neural system (30), and applied it to a human cell line (U2OS) that can be tightly synchronized at mitosis. In contrast to several recent studies that employed antibodies specific to the diGly-Lys remnant that marks ubiquitination sites following trypsin digestion (19,25), an in vivo ubiquitin tagging strategy allows direct validation of candidate ubiquitinated proteins (whether mono-or polyubiquitinated) through immunoblotting of samples.…”

Quantitative Proteomic Analysis of Cellular Protein Modulation upon Inhibition of the NEDD8-Activating Enzyme by MLN4924

“…Proteomic experiments designed to identify ubiquitinated proteins have primarily used epitope-tagged ubiquitin (15)(16)(17)(18)(19)(20)(21)(22) or ubiquitin affinity methods (23)(24)(25)(26)(27). However, because NAE inhibition blocks the ubiquitination of a minor subset of pro-teasome substrates, approaches relying on changes in global ubiquitination are unlikely to sufficiently enrich NAE-dependent changes.…”

Proteomic techniques to probe the ubiquitin landscape

“…The birAubiquitin is then incorporated into the ubiquitin-chains allowing the pull-down of biotinubiqutinated substrates using streptavidin or avidin-beads. Coupled to mass-spectrometric analysis, this study identified several hundred ubiquitinated substrates [85][86][87].…”