Comprehensive approach to study branched ubiquitin chains reveals roles for K48-K63 branches in VCP/p97-related processes

Branched ubiquitin (Ub) chains make up a significant proportion of Ub polymers in human cells and are formed when two or more sites on a single Ub molecule are modified with Ub creating bifurcated architectures. Despite their abundance, we have a poor understanding of the cellular functions of branched Ub signals that stems from a lack of facile tools and methods to study them. Here we develop a comprehensive pipeline to define branched Ub function, using K48-K63-branched chains as a case study. We discover branch-specific binders and, by developing a method that monitors cleavage of linkages within complex polyUb, we discover the VCP/p97-associated ATXN3, and MINDY family deubiquitinases to act as debranching enzymes. By engineering and utilizing a branched K48-K63-Ub chain-specific nanobody, we reveal roles for these chains in VCP/p97-related processes. In summary, we provide a blueprint to investigate branched Ub function that can be readily applied to study other branched chain types.<br/

Discovery and characterization of non-canonical E2 conjugating enzymes

E2 conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation is fundamental for maintaining cellular homeostasis and impacts almost all cellular process. By interacting with multiple E3 ligases, E2s direct the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a post-translational modification that specifically targets lysine side chains (canonical ubiquitylation). We used MALDI-TOF Mass Spectrometry to discover and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We employed protein modelling and prediction tools to identify the catalytic determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results join a stream of recent literature that challenges the definition of ubiquitylation as an exquisitely lysine-specific modification and provide crucial insights into the missing E2 element responsible for non-canonical ubiquitylation.<br/

Sweeping away barriers to interdisciplinary research:recommendations based on X-Net project outcomes - March 2024

X-Net is an interdisciplinary research network whose main aim is to understand barriers to interdisciplinary research, before offering solutions to overcome them. X-Net recommends a 13-step programme of targeted multi-level interventions drawn from evidence gathered by the network in 2022- 2023. The 13 interventions would deeply weave interdisciplinarity into UK scientific research culture and free the flow of ideas and expertise across traditional disciplinary boundaries and sectors

Chemo-phosphoproteomic profiling with ATR inhibitors berzosertib and gartisertib uncovers new biomarkers and DNA damage response regulators

The ATR kinase protects cells against DNA damage and replication stress and represents a promising anti-cancer drug target. The ATR inhibitors (ATRi) berzosertib and gartisertib are both in clinical trials for the treatment of advanced solid tumours as monotherapy or in combination with genotoxic agents. We carried out quantitative phospho-proteomic screening for ATR biomarkers that are highly sensitive to berzosertib and gartisertib, using an optimized mass spectrometry pipeline. Screening identified a range of novel ATR-dependent phosphorylation events, which were grouped into three broad classes: i) targets whose phosphorylation is highly sensitive to ATRi and which could be the next generation of ATR biomarkers; ii) proteins with known genome maintenance roles not previously known to be regulated by ATR; iii) novel targets whose cellular roles are unclear. Class iii targets represent candidate DNA damage response proteins and, with this in mind, proteins in this class were subjected to secondary screening for recruitment to DNA damage sites. We show that one of the proteins recruited, SCAF1, interacts with RNAPII in a phospho-dependent manner and recruitment requires PARP activity and interaction with RNAPII. We also show that SCAF1 deficiency partly rescues RAD51 loading in cells lacking the BRCA1 tumour suppressor. Taken together these data reveal potential new ATR biomarkers and new genome maintenance factors