PhD Candidate Western University London, Ontario, Canada
Gabriel Onea (Western University)| Matthew Maitland (Western University)| Gilles Lajoie (Western University)| Caroline Schild-Poulter (Western University)
The C-terminal to LisH (CTLH) complex is a tumour-suppressive E3 Really Interesting New Gene (RING) ubiquitin ligase that regulates intracellular signaling pathways and maintains cellular homeostasis. It is comprised of 8 individual proteins that have been shown to mediate a variety of intracellular protein-protein interactions; of these proteins, Required for Meiotic Division 5 Homolog A (RMND5A) and Macrophage Erythroblast Attacher (MAEA) contain the RING domains required for the CTLH complex to mediate ubiquitin transfer to specific protein substrates. Although its E3 ligase activity has been recently characterized, the endogenous subcellular localization of the complex and compartment-specific protein interaction network – which encompasses many unidentified E3 ligase substrates – has not been identified. In this study, we hypothesized that the CTLH complex is present in the cytoplasm and nucleus, and that the CTLH complex mediates compartment-specific protein-protein interactions with cytoplasmic and nuclear proteins. To test this hypothesis, we used a chemical-based subcellular fractionation method combined with low- and high-throughput proteomics. We first optimized the chemical fractionation method and used Western blot to detect each endogenous CTLH complex member in HeLa cell cytoplasmic and nuclear protein extracts. We then identified compartment-specific CTLH complex interactors using Affinity Purification Coupled to Mass Spectrometry (AP-MS) using the main CTLH complex scaffold protein – Ran Binding Protein M (RanBPM) – as bait in cytoplasmic and nuclear HeLa protein extracts. Following Liquid Chromatography Electrospray Ionizing Tandem Mass Spectrometry (LC-ESI-MS/MS) and subsequent in silico filtering using the MiST algorithm, we identified 27 cytoplasmic, 155 nuclear, and 31 nucleocytoplasmic high-confidence (MiST Score ≥ 0.75) RanBPM interactors. STRING enrichment analysis of the high-confidence interactors revealed that RanBPM and its associated CTLH complex may be involved in a variety of conserved Gene Ontology (GO) processes including Epigenetic Regulation of Gene Expression and Chromatin Assembly. As such, we validated a number of newly-identified compartment-specific RanBPM interactors associated with the enriched GO terms by co-immunoprecipitation – using RanBPM as bait – and resulting Western blot. Furthermore, we focussed on an interesting novel putative interactor that is present in the two aforementioned GO processes – histone variant MacroH2A1 – and further validated its interaction with the CTLH complex in vivo using Proximity Ligation Assay (PLA), suggesting that this interaction may be physiologically relevant. Overall, by using this workflow, we identified and validated a number of novel compartment-specific potential CTLH complex interactors in vitro, further validated a promising novel interaction between the CTLH complex and MacroH2A1 in vivo, and successfully implicated the CTLH complex in a number of previously unexplored cellular mechanisms.