Amyotrophic Lateral Sclerosis (ALS) is a progressive and devastating neurodegenerative disease with poor prognosis. Our team has identified novel missense mutations in an FBX gene (manuscript submitted), some of which lead to aggressive ALS pathology. At the protein level, FBX is a component of an E3-ubiquitin ligase and is responsible for recognition of specific substrates for ubiquitination [1]. Our hypothesis is that disease-variants of FBX may have altered substrate recognition properties. To date, there are only three known FBX substrates. One of the main challenges of identifying substrates of E3 ligases is the transient and low binding affinity between the ligase and its substrates. Accordingly, one aim of this project is to identify novel substrates of FBX using proximity-dependant biotin identification (BioID) and LC-MS/MS. BioID is a new methodology that involves fusing mutant biotin ligase in frame with the enzyme of interest in order to biotinylate proteins that come into close proximity [2]. This approach has advantages in that it enables the selective isolation and identification of weak and transient protein-protein interactions using standard biotin-affinity capture. Furthermore, it provides a list of candidates for further interaction studies. In addition to identifying novel substrates of FBX, this project will also work toward producing recombinant FBX for structural studies. The structure will help us rationalise the changes in E3-ligase activity caused by mutations, and will provide a basis for structure-based drug design. Overall, this project will investigate the link between, and the degree to which, FBX affects ALS disease progression. It will also work toward identifying specific structural features of FBX that might be therapeutically targetable.