Cyclin F - a contributing factor to protein aggregation in Motor Neuron Disease
Stephanie L. Rayner1, Albert Lee1, Kelly L. Williams1, Ian P. Blair1, Mark P. Molloy2 and Roger S. Chung1 1Department of Biomedical Sciences, Macquarie University, NSW 2109, Australia; 2Department of Chemistry and Biomolecular Sciences, Macquarie University, NSW, 2109, Australia
Background: Recently, our team identified novel missense mutations in CCNF in ALS/FTD patients . At the protein level, cyclin F is a component of an E3 ubiquitin ligase and recruits substrates for ubiquitylation. In over 95% of ALS cases, ubiquitylated TDP-43 inclusions form in affected neurons however, the enzymes responsible for this ubiquitylation are largely unknown. In this study, our overall aim was to determine the link between cyclin F and TDP-43 hyper-ubiquitylation.
Objectives: The specific aims of this study were to: i. Identify potential ubiquitylation substrates of cyclin F using proximity-based biotinylation (BioID), mass spectrometry (MS) and immunoprecipitations (IPs). ii. Validate substrates that can be ubiquitylated by cyclin F, using an in vitro ubiquitylation assay. iii. Determine the biological consequence of cyclin F ubiquitylation of novel substrates.
Method: BioID methodology involves fusing mutant biotin ligase (BirA*) in frame with cyclin F such that cyclin F-BirA* biotinylates proteins that come into close proximity to cyclin F in live cells. Gene expression in HEK293 Flp-In T-Rex cells was induced using tetracycline and cells were treated with 50 μM biotin for 24 hours to enable biotinylation. After cell lysis, biotinylated proteins were enriched by streptavidin-bead pull downs. Biotinylated proteins were digested in-solution with trypsin and analysed by LC-MS/MS and bioinfomatic methods. In addition to this, standard IP of cyclin F from Neuro-2a/SHSY5Y cells, followed by immunoblotting was used to validate interaction partners. To validate ubiquitylated substrates of cyclin F, an in vitro ubiquitylation assay was developed. Here, E1, E2, ubiquitin and ATP was added to the Cyclin F-substrate complex to enable cyclin F mediated ubiquitylation. For protein aggregation assays, mCherry-cyclin F and GFP-TDP-43 were transfected into HEK293 or NSC34 cells. TDP-43 aggregates were counted using florescence.
Results/Discussion: Our results indicate that TDP-43 is a binding partner and substrate of cyclin F as shown by BioID, IP and an in vitro ubiquitylation assay. Notably, our in vitro ubiquitylation assay confirms that cyclin F ubiquitylates TDP-43, with disease-variant cyclin F preferentially generating K48-linked ubiquitin chains to TDP-43. Interestingly, the overexpression of cyclin F led to the aggregation of TDP-43, with mutant cyclin F leading to a significant increase in TDP-43 aggregation. This is the first ALS-linked E3 ligase that has been shown to ubiquitylate TDP-43, generating a mechanism by which TDP-43 may be ubiquitylated during the disease course of ALS/FTD.
Acknowledgements:This research was supported by grants from MNDRIA (GIA1728), and NHMRC (APP1107644 and APP1095215)
References: Williams, K.L. et al., Nat Commun. 2016 Apr 15;7:11253