Novel insights into FAT10 biology : Parkin as the first FAT10-E3 ligase and the regulation of FAT10 activity by phosphorylation

Abstract

Summary FAT10 (HLA-F adjacent transcript 10) is an 18 kDa protein which consists of two ubiquitin-like domains and bears a free diglycine-motif at its C-terminus through which it covalently conjugates to target proteins. FAT10 is activated by the E1 enzyme UBA6 and conjugated by the E2 enzyme USE1. Among all the ubiquitin-like modifiers, FAT10 is the only one that targets proteins for degradation via the 26S proteasome. The expression of FAT10 is synergistically inducible by the pro-inflammatory cytokines IFNγ and TNFα. Overall, it has been shown that this ubiquitin-like modifier has 569 interacting partners involved in different functional pathways such as autophagy, cell cycle regulation, apoptosis and cancer progression. Nevertheless, even though a number of studies have shed light on the diverse biological functions of FAT10, the biochemical regulation of this ubiquitin-like modifier has not been fully elucidated. For instance, until today no FAT10 E3 ligases have been identified, and the post-translational regulation of FAT10 function is unknown. In this study, we described the interplay between FAT10 and the E3 ligase Parkin. Parkin is an E3 ubiquitin ligase belonging to the RING-between-RING family, and mutations in the Parkin-encoding gene Park2 are associated with the familial Parkinson’s disease. Here, we showed that FAT10 gets conjugated to Parkin and mediates its degradation in a proteasome-dependent manner. Parkin binds to the E2 enzyme of FAT10 (USE1), auto-FAT10ylates itself, and facilitates the FAT10ylation of the Parkin substrate Mitofusin2 in vitro and in cellulo, thus unravelling Parkin as the first ever described FAT10 E3 ligase. Upon mitochondrial depolarization, FAT10ylation of Parkin inhibits its activation and ubiquitin-ligase activity causing the impairment of mitophagy and the subsequent aggravation of the rotenone-induced death of dopaminergic neuronal cells. Overall, FAT10ylation inhibits Parkin and mitophagy rendering FAT10 a likely inflammation-induced exacerbating factor and a potential drug target for Parkinson’s disease. Furthermore, this study also showed that a fraction of FAT10 is phosphorylated at multiple sites in presence of the inflammatory cytokine TNFα and during Influenza virus infection. In addition, our in vitro and in cellulo studies indicated that specific kinases of the IKK family are able to phosphorylate FAT10. We also demonstrated that the phosphorylation of FAT10 can selectively regulate its substrate selection and its biological function as a regulator of antiviral immunity, providing insights on a fine biochemical regulation of FAT10 activity.