Cellular Functions of Proteasome-Associated Ubiquitin Ligase Activity

The ubiquitin-26S proteasome has vital roles in cellular signalling by selectively degrading proteins that are short-lived or damaged. In eukaryotes attachment of the conserved small protein ubiquitin marks substrates for recruitment to the proteasome where they are proteolysed into small peptides. Failure to degrade ubiquitin-marked substrates causes severe cellular stress and is a leading cause of developmental defects across different eukaryotes, including human pathologies such as neurodegenerative diseases, autoimmunity, cardiomyopathy, and genetic disorders like cystic fibrosis. Understanding the conserved regulatory mechanisms that govern healthy proteasome functioning therefore has the potential to impact diverse fields in biomedicine and biotechnology. Recent findings indicate that upon arrival at the proteasome, substrates may undergo further modification by ubiquitin. But why do substrates require further polyubiquitination if they have already been recruited to the proteasome? Although it has been suggested that such 'last-minute' polyubiquitination facilitates proteasome processivity, the mechanisms underpinning this activity remain elusive. We recently demonstrated that ubiquitin ligases from the HECT-type family physically associate with the proteasome, thereby governing universal substrate polyubiquitination and associated phenotypical traits. Intriguingly, HECT-type ligases interact with multiple E3 ubiquitin ligases from distinct signalling pathways. Based on these findings we propose to elucidate the substrate repertoires of HECT-type ligases and explore new hypotheses for their role in signalling: (1) does healthy proteasome function require substrate relay from multiple E3 ligases to proteasome-associated HECT-type ligases?; (2) do proteasome-associated HECT-type ligases modify the activity of interacting E3 ligases, thereby enabling proteasomes to feedback regulate ubiquitin-mediated signalling pathways? Understanding how HECT-type ligases promote proteasome function will aid in targeted design of new strategies to improve proteasome function during disease.

The ubiquitin-26S proteasome has vital roles in cellular signalling by degrading proteins that are short-lived or damaged. In eukaryotes attachment of the conserved protein ubiquitin marks substrates for recruitment to the proteasome where they are proteolysed. Failure to degrade ubiquitin-marked substrates causes proteotoxic stress, which is associated with developmental defects across eukaryotes, including human pathologies such as neurodegenerative diseases, autoimmunity, cardiomyopathy, and genetic disorders like cystic fibrosis. Understanding the conserved regulatory mechanisms that govern healthy proteasome functioning impact both biomedicine and biotechnology. Recent findings indicate that upon arrival at the proteasome, substrates may undergo further modification by ubiquitin. But why do substrates require further polyubiquitination if they have already been recruited to proteasomes? Although such 'last-minute' polyubiquitination may facilitate proteasome processivity, the mechanisms underpinning this activity remain elusive. We recently demonstrated that ubiquitin ligases from the HECT-type family physically associate with the proteasome, thereby governing universal substrate polyubiquitination and associated phenotypical traits. Intriguingly, HECT-type ligases interact with multiple E3 ubiquitin ligases from distinct signalling pathways. Based on these findings we propose to elucidate the substrate repertoires of HECT-type ligases and explore new hypotheses for their role in signalling: (1) does healthy proteasome function require substrate relay from multiple E3 ligases to proteasome-associated HECT-type ligases?; (2) do proteasome-associated HECT-type ligases modify the activity of interacting E3 ligases, thereby enabling proteasomes to feedback regulate ubiquitin-mediated signalling pathways? Understanding how HECT-type ligases promote proteasome function will aid in targeted design of new strategies to improve proteasome function during disease.

The proposed research will have far reaching impacts for a variety of sectors and the wider public. Human activities increasingly impose constraints on agriculture and threaten future food security. These constraints result in crops having to face a multitude of abiotic and biotic stresses. Consequently, crops either engage in a lengthy and costly battle against disease or succumb under disease pressure, both of which result in severely reduced crop yield. Indeed, a reduction in marketable crop yields cost the UK and global economies billions every year and still renders much of agriculture subsidized. Resistance or tolerance against stressors is provided by sophisticated proteasome-mediated signalling pathways that prioritize stress defences over normal cellular functions. A greater understanding of the mechanisms controlling effective proteasome signalling that allows plants to cope with stresses will therefore offer academic scientists and the commercial private sectors novel avenues for exploration of methods to significantly increase crop yields under human-imposed agricultural constraints. Increasing yields is not just desirable but a necessity for a rapidly growing population that demands food security, while avoiding increases in food prices as experienced in recent times. Moreover, our proposed research will offer indications how to directly engineer and improve the plant's existing defence mechanisms. This is a particularly attractive approach, because it meets the sustainable standards that are now appropriately demanded for agriculture by both policy makers and the public. Indeed, timely enhancement of the plant's own defences to optimize crop yields avoids the use of harmful chemicals and pesticides that may pose significant hazards to local biodiversity and even public health.

Our work and that of others show that the fundamental principles that underpin proteasome signalling are strongly conserved within the eukaryotic domain. Indeed, dysregulation of proteasome function in humans is associated with a variety of pathologies, including neurodegenerative diseases, autoimmunity, cardiomyopathy, and genetic disorders such as cystic fibrosis. This indicates the potential for impacts from our findings to cross over into biomedical research advances, the direct beneficiaries of which will be located in both the biomedical academic community and the private or commercial pharmaceutical sector, while ultimately the design of new medicines and therapies will improve quality of life and promote healthy aging of the wider public.

A variety of pathways will be used to ensure the engagement of all beneficiaries. During the project existing and new contacts with agricultural, agrichemical and biomedical industries will be exploited to promote the translation of fundamental findings into applicable products. Besides continued web and media coverage of our research through established channels, knowledge transfer and public engagement events will engage the wider public.