COI Project 4: Exploring non-canonical ubiquitylation in innate immune pathways

About the Project

Introduction: Covalent modification by ubiquitin via Lysine isopeptide bonds is fundamental for regulating protein turnover and function. Additionally, ubiquitin esterification occurs on Serine/Threonine/Tyrosine residues in proteins and on non-proteinaceous substrates including ribose, saccharides, lipids, and small molecule drugs [1,2]. Ubiquitin posttranslational modifications may therefore be much more widespread across cell biological pathways. Recent literature reflects the increased interest in analytical methods for mapping of non-canonical substrates modified by ubiquitin and ubiquitin-like (UBL) proteins [2,3]. We intend to explore non-canonical ubiquitin modifications on proteins and non-protein substrates in the context of innate immune pathways such as interleukin-1beta and type-I interferon signalling.

Experimental approach: Mass spectrometry (MS)-based methodologies involve advanced proteomic techniques to identify ubiquitin modifications on amino acids other than Lysine, such as Serine, Threonine, Tyrosine and Cysteine as well as protein N-termini. After digestion, standard MS workflows identify canonical ubiquitination by detecting a ubiquitin C-terminal tag attached to the amine side chains of Lys residues of substrate-derived peptides suitable for MS/MS sequencing. For non-canonical modifications on proteins and substrates other than proteins, specialized strategies are required, such as using antibodies to enrich N-terminally modified peptides in combination with using high-resolution MS/MS based on softer fragmentation technologies to detect esterification and possibly other types of substrate modifications. We shall explore the application and subsequent optimisation of the UbSite approach [3,4] and also extend this beyond Ubiquitin to ISG15, critical for interferon signalling pathway regulation.

Expected outcomes: Enabling such technologies will reveal a previously unrecognized angle of the ubiquitin code's complexity in cells. In particular, these studies will uncover novel molecular connections between metabolic and innate immune signalling pathways that modulate inflammation linked to type-I interferonopathies and to interleukin-1beta [5].

Supervisors

1. Prof. Benedikt Kessler
2. Dr Iolanda Vendrell
3. Dr Andreas Damianou

Funding

The Nuffield Department of Medicine (NDM) is one of the largest and most highly regarded departments of medicine in Europe and noted for the wide range and excellence of its basic and clinical research. The CAMS Oxford Institute is embedded within NDM, and has two fully-funded DPhil Studentships on offer for the 2026 student intake, supported by funding from COI and AbbVie