| Zusammenfassung |
Protein-protein interactions (PPIs) commonly occur in biology with ~650,000 contributing to processes critical to human life. From cancer to dementia, their dysregulation contributes to many disease states. PPIs occur when one protein interacts with another resulting in functional changes that have repercussions on the cell. Their importance means PPIs are studied across the different bioscience fields and many tools developed to investigate them. Fluorescence-based methods allow researchers to study these innately dynamic processes in a proteins' natural environment, the cell, in real time leading to many new insights. Genetically-encoded probes called fluorescent proteins have proved pivotal to investigating PPIs in the cell but the underlying fluorescent methods are currently limited to monitoring interactions between two different proteins; self-association of the same proteins cannot be easily measured. This is major issue as many PPIs, arguably the majority, involve the self-association of identical proteins. Thus, researchers cannot investigate this vital piece of the PPI story within a protein's natural cellular context. The aim of this project is to develop new genetically encoded tools to allow researchers to monitor protein self-association (homo-oligomerisation) for use in the cell. Building on recent work in the Jones lab, we plan to generate new fluorescent protein variants optimised for use in the cell to functionally respond when fused to a self-associating protein target. We will demonstrate their usefulness with the gene regulation complex NF-kappaB, which is responsible for controlling many important cellular processes including immune response, replication and cell death; its dysregulation is linked to many diseases including autoimmune disease and cancer. NF-kappaB is also an archetypical protein complex undergoing dynamic exchange between identical and different protein components thus making it an excellent model implementation system and providing new insights into this important biological complex. The potential applications of our genetically encoded homo-oligomerisation monitoring tools are vast, allowing researchers across the breadth of biology to access new knowledge on how PPIs dictate biological processes. Importantly, it will be compatible with existing fluorescence microscopy approaches so will not need associated equipment development and can be quickly implemented by the research community. Our probes can also feed into existing approaches for monitoring PPIs in cells allowing dynamic events such as exchange between protein components (e.g. self-associating versus mixed PPIs) and more complex systems (e.g. combination of self and mixed PPIs) that routinely form protein complexes. |
