Defining mechanisms of cellular stress responses driven by heterotypic ubiquitin chains

Project Information

StressHUb

Grant agreement ID: 101002428

DOI

10.3030/101002428

EC signature date 21 April 2021

Start date 1 January 2022

End date 31 December 2026

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 185 859,00

EU contribution

€ 2 185 859,00

Coordinated by

UNIVERSITY OF DUNDEE
United Kingdom

Periodic Reporting for period 3 - StressHUb (Defining mechanisms of cellular stress responses driven by heterotypic ubiquitin chains)

Reporting period: 2025-01-01 to 2026-06-30

Summary of the context and overall objectives of the project

This ERC Consolidator Grant, StressHUb, aims to explore the role of branched ubiquitin (HUb) chains, complex ubiquitin modifications, in cellular stress responses. Ubiquitin, a small regulatory protein, is known for its ability to modify other proteins post-translationally, forming either monoubiquitin or polyubiquitin chains that regulate diverse cellular processes. Recent discoveries suggest that ubiquitin can also form branched heterotypic chains, which may adopt unique conformations and convey specific intracellular signals vital for maintaining cellular homeostasis, particularly during stress.

The central hypothesis of this research is that branched ubiquitin chains are formed in response to specific cellular stresses and act as specialized signals that trigger cellular stress responses. We hypothesise that branched chains function as priority signals that ensure prompt and appropriate cellular reactions to stress, thereby preserving cellular homeostasis. Despite their abundance, studying these branched ubiquitin chains is challenging. This is due to their complex nature, the scarcity of tools to analyze them effectively and easily, and their relatively low abundance within cells. This project seeks to overcome these challenges by developing novel tools, methods, and approaches that will enable detailed investigation of the role of branched chains in stress signalling.

Broadly, the main objectives of the project include:
• Establishing methods to generate branched Ub chains of defined architectures
• Determining the crystal structures of various branched ubiquitin chains to understand their unique interaction interfaces and conformations.
• Define how branched ubiquitin is decoded in cells
• Tool & Method Development: Creating innovative tools to study branched ubiquitin chains in cells
• Identifying cellular machinery responsible for making and disassembling branched chains
• Defining the conditions and stress stimuli that lead to the formation of specific branched ubiquitin chains and uncovering their roles in cellular stress response pathways

By focusing on selected branched ubiquitin chains, this project aims to establish a foundational understanding of how branched ubiquitin chains function as unique stress signals. The outcomes are expected to provide novel insights into intracellular signalling mechanisms and offer new strategies for therapeutic intervention in diseases linked to protein misfolding and degradation.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

This project focuses on understanding the role of branched ubiquitin (Ub) chains in cellular stress responses. The complexity and low abundance of these branched chains in unperturbed cells, combined with the lack of specific analytical tools, have hindered their study.
The project's primary goals are to develop innovative tools and methods to study branched ubiquitin chains, elucidate the molecular mechanisms of their formation, and characterize their functional roles in stress response pathways. Our initial efforts focussed on K48-K63 linked branched ubiquitin, with the goal of establishing a toolkit and blueprint to study other branched chain types.

First, we established methods to produce large amounts of K48-K63 branched chains. We show that this method can be used to assemble other branched chain types. The branched chains of defined architectures that were assembled, were used to identify cellular proteins that bind to them. This revealed for the first time that specific binders or readers to branched ubiquitin exist in cells. To understand how branched chains are processed or cleaved by deubiquitinating enzymes (DUBs), we pioneered the development of a novel DUB assay that is quantitative and can monitor the linkage cleaved within polyUb. Using this approach, we discover ATXN3 and MINDY DUBs to be debranching enzymes. Importantly, the identification of branched ubiquitin as the substrate of ATXN3 sets the stage for understanding the cellular substrates and function of ATXN3.

Another major achievement of the project to date is the engineering of a K48–K63 branch-specific nanobody. We revealed the molecular basis of this nanobody's specificity for branched Ub through crystal structures of nanobody-branched ubiquitin chain complexes. Utilizing this nanobody, we detected increased K48–K63 branched ubiquitin accumulation following inhibition of the valosin-containing protein (VCP)/p97 and after DNA damage. Our results suggest that K48–K63-branched chains play a role in DNA damage responses and VCP/p97-related processes, and we are working to uncover the molecular players and underlying mechanisms.
In summary, our findings represent a significant advance in understanding the role of branched ubiquitin chains in cellular signalling. By revealing the enzymes involved in branched chain formation and debranching, as well as identifying them to be involved in DNA damage responses and p97-mediated processes, the project has laid the groundwork for further exploration of how branched ubiquitin chains regulate cellular stress responses.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The project has significantly advanced the understanding of branched ubiquitin (Ub) chains, particularly the K48–K63-branched Ub chains, which were previously underexplored due to a lack of effective tools and methods. Prior to this work, the complexity and low abundance of branched Ub chains in cells made them difficult to study, leaving their roles in cellular signalling largely speculative. The project has made several key innovations that push the boundaries of current knowledge. These include the development of novel tools and techniques that will be used to set up cellular assays and screens to monitor formation of branched chains in living cells and identify the cellular stresses that trigger them. We are now using the blueprint we have established to chart the molecular players involved in other branched ubiquitin chains. We are using the nanobodies to get in depth insights into branched chain function in cellular stress responses. Furthermore, we are establishing methodologies to identify E3 ligases capable of making branched ubiquitin chains. The identification of molecular players regulating K48-K63 branched chain disassembly and decoding now provides us a handle to study the biology of this chain type. We anticipate that our multipronged strategy will reveal insights into how branched ubiquitin chains are formed, decoded and utilized as priority signals in cellular stress responses.