Cells rely on a molecular tagging system called ubiquitylation to mark proteins for destruction or to change their behaviour, but one particular type of tag—K11-linked chains—remains poorly understood because researchers lack the tools to detect it reliably. This project aims to build a set of nanobodies—tiny antibody-like proteins derived from camels—that can recognise each of the eight distinct forms of ubiquitin chains with high precision. Current detection tools, such as conventional antibodies, are often unreliable, expensive, or unavailable for certain chain types, which has stalled progress in understanding how these signals control processes like cell division and protein quality control. If successful, the nanobody toolkit will give cell biologists a cheap, scalable, and highly specific way to track ubiquitin signals in living cells, using techniques from microscopy to mass spectrometry. The researchers will then apply these tools to map the cellular pathways that depend on K11-linked chains, identifying the proteins that build, read, and erase these signals. This is fundamental science—it will not produce a drug or device directly—but ubiquitylation touches nearly every aspect of cell biology, so a clearer picture of how these signals work could eventually inform treatments for diseases where protein regulation goes awry, such as cancer or neurodegeneration.
View original technical description
Ubiquitylation is a key regulator of eukaryotic cell signalling. At its core, ubiquitylation typically involves attaching a ubiquitin molecule onto a lysine residue of a target protein, altering the fate of the modified protein. Ubiquitin moieties can also be linked together through one of their seven lysine residues (K6, K11, K27, K29, K33, K48 and K63) or the N-terminal methionine to form polyubiquitin chains. Importantly, each polyubiquitin linkage type encodes a distinct cellular signal, triggering specific cellular outcomes. Specialised enzymes - E3 ligases, ubiquitin binding proteins (UBDs) and deubiquitinases (DUBs) – are known to write, read, and erase these signals, demonstrating remarkable specificity in the ubiquitin system. Among the eight types of polyubiquitin, K48-linked chains are well known for targeting modified proteins for degradation. In contrast, K11-linked chains, which play important roles in cell cycle regulation and protein quality control, remain less understood. Specifically, the molecular players underlying the assembly and decoding of K11 chains are not well elucidated. A key goal of our research is to uncover the mechanisms by which K11-linked polyubiquitin control various cellular processes. A major challenge in ubiquitylation research is the lack of high-quality, linkage-specific tools. Current detection methods, such as linkage-specific antibodies, are limited by incomplete coverage, variable specificity, and restricted availability, severely hindering research. To address this gap, we aim to develop a robust toolkit of nanobody reagents targeting seven homotypic chain types. Importantly, nanobodies are smaller and more versatile than conventional antibodies, are easier to produce at scale and offer high specificity. We will employ a yeast surface display library that contains over a billion different synthetic and natural camelid nanobodies to select for nanobodies that bind specifically to each chain type. Importantly, we will use a rigorous negative selection process to eliminate non-specific binders that bind to any of the other linkage types, ensuring the development of highly selective linkage-specific reagents. We will employ protein engineering approaches to enhance the binding affinity of the top candidates. To understand the molecular basis of linkage-specific recognition of the nanobodies, we will determine crystal structures of nanobodies bound to their target polyubiquitin chains. We will rigorously establish the linkage specificity of the nanobodies using biophysical, biochemical and mass spectrometry-based approaches. This nanobody toolkit will enable the detection and visualisation of polyubiquitin of different linkage types in various cellular contexts and through different approaches, including biochemistry, live cell imaging, flow cytometry, and mass-spectrometry based techniques. We will apply this nanobody-based toolkit for a detailed investigation of the function of K11-linked ubiquitin chains in cell signalling. By pioneering a high-throughput screening method, we aim to identify cellular pathways that rely on K11 linkages for information transfer. Subsequently, we will identify and characterise the substrates and interacting proteins that mediate cell signalling through K11-linkages. Since ubiquitylation impinges on all aspects of cell biology, we anticipate that this nanobody-based toolkit will become an invaluable resource for the research community, advancing our understanding of ubiquitylation and its role in cellular function.
Plain English summaries and category classifications on this site are generated by AI and may not perfectly reflect the original research.
Is something wrong? Let us know