Substrate profiling of non-canonical ubiquitinase and structural and functional characterisation of nucleosome-binding proteins

Abstract

Proteins are a class of biomolecules essential to living organisms involved in virtually all biological processes. Protein-protein interactions cause the formation of multi-protein complexes through the formation of covalent linkages or non-covalent interactions and facilitate their functions. Characterisation of protein-protein interactions is therefore crucial to understanding protein function and their biological roles, especially for proteins involved in vast interaction networks. Ubiquitin is a protein with broad tissue and cellular distribution, characterised by its covalent conjugation to other proteins, ubiquitination. Recently, a novel form of ubiquitination was identified, catalysed by Legionella pneumophila effector protein family SidE, independent of host ubiquitination machinery, but the identities of its substrates remain largely unknown. In Chapter 2, I sought to identify substrates of the SidE protein SdeA in the human proteome. I designed and synthesised a biotinylated ubiquitin probe and demonstrated its exclusive utilisation by SdeA for protein ubiquitination. An affinity purification-mass spectrometry-based approach identified over 1000 putative SdeA substrates in the human proteome and selected putative substrates were validated, revealing biological processes potentially influenced by SdeA. Genomic DNA of eukaryotic cells are compacted into the nucleus as chromatin through the formation of nucleosomes. Interactions between nucleosomes and proteins mediate transcription regulation and maintenance of genome integrity by modulating activity of nucleosome-binding proteins or by regulating nucleosome dynamics. Activity of histone deacetylase SIRT6 is dependent on interaction with the nucleosome, but the mechanism of activation is unknown due to a lack of structural data. In Chapter 3, I sought to elucidate the mechanism of nucleosome-dependent SIRT6 activation. I presented the cryo-EM structure of the SIRT6-nucleosome complex at 3.07 Å resolution and identified putative interactions between the SIRT6 ZBD and the nucleosome acidic patch, which were validated by electrophoretic mobility shift assays using mutant SIRT6 and nucleosomes. I performed in vitro SIRT6 deacetylation assays to demonstrate that nucleosome-dependent SIRT6 activation was not a result of SIRT6-substrate proximity, to show a reduction in SIRT6 activation by perturbing SIRT6-nucleosome interaction, and to perform kinetic analysis of nucleosome-dependent SIRT6 deacetylation, providing insights on its mechanisms. HMGN5 is a nucleosome-binding protein that promotes chromatin decompaction. It contains a distinct long acidic C-terminal region associated with counteracting H1-mediated chromatin compaction, but its molecular functions are unclear. In Chapter 4, I sought to characterise the role of the C-terminal region of human HMGN5 on nucleosome dynamics. Cross-linking mass spectrometry revealed interactions of HMGN5 C-terminal region with HMGN5 nucleosome-binding and H1.4 C-terminal domain. Electrophoretic mobility shift assays and mass spectrometry showed the formation of a HMGN5-chromatosome complex. Förster resonance energy transfer and protein-induced fluorescence enhancement assays showed disruption of H1-DNA interaction and increased nucleosome linker DNA flexibility by HMGN5 mediated by the C-terminal region.