Gsk3β and Tomm20 are substrates of the SCFFbxo7/PARK15 ubiquitin ligase associated with Parkinson's disease

Abstract

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 (hepatoma up-regulated protein), whose ubiquitination results in proteasome-mediated degradation, and c-IAP1 (inhibitor of apoptosis protein 1), TNF receptor-associated factor 2 (TRAF2), and NRAGE, which are not destabilized as a result of ubiquitination. None of these substrates have been linked directly to PD, nor has it 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 glycogen synthase kinase 3β (Gsk3β), which can phosphorylate α-synuclein, and translocase of outer mitochondrial membrane 20 (Tomm20), a mitochondrial translocase that, when ubiquitinated, promotes mitophagy, as SCFFbxo7 substrates both in vitro and in vivo. Ubiquitin chain restriction analyses revealed that Fbxo7 modified Gsk3β using K63 linkages. Our results indicate that Fbxo7 negatively regulates Gsk3β activity, rather than its levels or localization. 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 has an impact directly on two proteins implicated in pathological processes leading to PD.

Parkinson’s Disease: From Pathogenesis to Pharmacogenomics

“…FBXO7 negatively regulates glycogen synthase kinase 3 β (GSK3β) activity. FBXO7 ubiquitinates translocase of outer mitochondrial membrane 20 (Tomm20), and its levels correlate with Fbxo7 expression, indicating a stabilizing effect [69]. DJ-1, the product of the causative gene of a familial form of PD, plays a significant role in anti-oxidative defense to protect cells from oxidative stress.…”

The Key Roles of GSK-3β in Regulating Mitochondrial Activity

“…SCF Fbxo7 ubiquitinates GSK-3β in a K63-dependent manner and Fbxo7 represses its activity. In contrast, Fbxo7 stabilizes TOM20 and correlates with its levels [20]. These indicate a connective network that is a novel field for further investigation.…”

Mechanistic contributions of FBXO7 to Parkinson disease

“…Teixeira and colleagues identified the mitochondrial protein translocase of outer mitochondrial membrane 20 (TOM20) in a ubiquitination target screen and validated TOM20 as a bona fide substrate of FBXO7 (Teixeira et al . ). The authors reported ubiquitin modification of TOM20 by FBXO7 (Teixeira et al .…”

“…The authors reported ubiquitin modification of TOM20 by FBXO7 (Teixeira et al . ), but TOM20 also responds to the presence of the E3 ligase parkin upon CCCP treatment (Chan et al . ).…”

“…While FBXO7‐mediated ubiquitination appears to have a stabilizing, nonproteolytic effects on TOM20 (Teixeira et al . ), parkin promotes the proteasome‐facilitated turnover of TOM20 prior to mitophagy (Chan et al . ).…”