26S-NanoLuc: a transformational assay of proteasome assembly

Almost all fundamental cellular processes depend on the activity of the ubiquitin-proteasome (UPS) degradation system to control the abundance of regulatory proteins or destroy aberrant proteins. The UPS is critical to life and the ability to adapt to stress conditions. Unsurprisingly, mutations in components of the UPS underly many age-related diseases (cancers and neurodegenerative disorders). Better understanding of proteasome regulation will provide novel insights into the fundamental principles of how living organisms work.
A core element of the UPS is the 26S proteasome, a proteolytic complex in eukaryotes that consists of two different subcomplexes: the 20S core particle (CP) and the 19S regulatory particle (RP). The CP is flanked at one or both ends by the RP to form the functional forms of the 26S proteasome. Protein degradation occurs inside the narrow proteolytic chamber of the CP while the RP drives substrate recognition, unfolding and translocation into the CP. One key aspect of proteasome regulation is the assembly of its 33 distinct subunits into a functional complex. We recently showed that proteasome assembly is a stress-regulated process important for stress survival. However, how dynamic proteasome assembly is in responding to signalling pathway activation and stresses, and what the underlying mechanisms are, remain key questions to be addressed in proteasome biology. One important limitation to solving these questions is the lack of technology to measure proteasome assembly efficiently and rapidly. We propose to develop a novel cell-based assay to quantify assembly of proteasomes that can be used in high-throughput screening. The assay will be transformative for answering the above questions and for understanding the regulation of proteasome assembly.
To achieve this, we will adapt the highly quantitative and extremely sensitive split NanoLuc technology to monitor the association between the RP and the CP (here called "26S-NanoLuc assay"), which correspond to one of the latest steps in 26S proteasome assembly. Different pairs of proteasome subunit termini that are in close proximity, one from the CP and one from the RP, will be tagged with HiBiT and LgBiT, respectively. When the CP associates with the RP to form a functional 26S proteasome, HiBiT will bind to LgBiT and generate a bright luminescence signal that is directly proportional to the amount of assembled 26S proteasome. Using available proteasome structures, we will first select three pairs of proteasome subunit termini in close proximity to be tagged in human Rpe1 cells (genetically stable near-diploid cells widely used as a cellular model) using CRISPR-Cas9 genome editing: Rpn5/Alpha1, Rpn6/Alpha2 and Rpn1/Alpha4. After the tagging of proteasome subunits, we will verify that a luminescence signal is generated without impacting proteasome integrity. The integrity of the proteasome in engineered cells will be carefully verified by in vitro and in-gel peptidase assays, and by using proteasome reporter substrates. This will ensure that tagging is not altering proteasome assembly and activity, and that the 26S-NanoLuc assay is physiologically relevant.
Success will put us in a position to ask for further funding to use our 26S-NanoLuc assay to better study the role and kinetics of stress responses and signalling pathways in regulating proteasome assembly in cells. This new technology will be made available to the research community. In addition, cell-based small-molecule high-throughput screening could be performed to identify molecules modulating 26S proteasome assembly as research tools or potential drugs. This original and pioneering work will help push the boundaries of our understanding of how proteasome homeostasis is regulated in cells, so that damaged and misfolded proteins are cleared before they become deleterious, a key aspect of understanding the rules of life.