Fbxo7 is a clinically relevant F-box protein, associated with both cancer and Parkinson's disease (PD). Additionally, SNPs within FBXO7 are correlated with alterations in red blood cell parameters. Point mutations within FBXO7 map within specific functional domains, including near its F-box domain and its substrate recruiting domains, suggesting that deficiencies in SCFFbxo7/PARK15 ubiquitin ligase activity are mechanistically linked to early-onset PD. To date, relatively few substrates of the ligase have been identified. These include HURP, whose ubiquitination results in proteasome-mediated degradation, and c-IAP1, TRAF2 and NRAGE which are not destabilized as a result of ubiquitination. None of these substrates has been linked directly to PD, nor has it not been determined whether they would directly engage neuronal cell death pathways. To discover ubiquitinated substrates of SCFFbxo7 implicated more directly in PD aetiology, we conducted a high-throughput screen using protein arrays to identify new candidates. A total of 338 new targets were identified and from these we validated Gsk3β, which can phosphorylate α-synuclein, and Tomm20, a mitochondrial translocase that, when ubiquitinated, promotes mitophagy, as SCFFbxo7substrates both in vitro and in vivo . Ubiquitin chain restriction (UbiCRest) analyses revealed Fbxo7 modified Gsk3β using K63 linkages. Our results indicate that Fbxo7 negatively regulates Gsk3β activity, rather than its levels or localisation. In addition, Fbxo7 ubiquitinated Tomm20, and its levels correlated with Fbxo7 expression, indicating a stabilizing effect. None of the PD-associated mutations in Fbxo7 impaired Tomm20 ubiquitination. Our findings demonstrate that SCFFbxo7 impacts directly on two proteins implicated in pathological processes leading to PD.
- ubiquitin ligases
- glycogen synthase kinase
- protein array
- ©2016 The Author(s)
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