Biotechnology Institute Thurgau at the University of Konstanz, Kreuzlingen, Switzerland.
Division of Immunology, Department of Biology, University of Konstanz, Kontstanz, Germany.
PLoS One. 2023 Aug 14;18(8):e0290002. doi: 10.1371/journal.pone.0290002. eCollection 2023.
The ubiquitin-like modifier FAT10 is highly upregulated under inflammatory conditions and targets its conjugation substrates to the degradation by the 26S proteasome. This process termed FAT10ylation is mediated by an enzymatic cascade and includes the E1 activating enzyme ubiquitin-like modifier activating enzyme 6 (UBA6), the E2 conjugating enzyme UBA6-specific E2 enzyme 1 (USE1) and E3 ligases, such as Parkin. In this study, the function of the HECT-type ubiquitin E3 ligase HUWE1 was investigated as a putative E3 ligase and/or conjugation substrate of FAT10. Our data provide strong evidence that HUWE1 is FAT10ylated in a UBA6 and FAT10 diglycine-dependent manner in vitro and in cellulo and that the HUWE1-FAT10 conjugate is targeted to proteasomal degradation. Since the mutation of all relevant cysteine residues within the HUWE1 HECT domain did not abolish FAT10 conjugation, a role of HUWE1 as E3 ligase for FAT10ylation is rather unlikely. Moreover, we have identified the autophagy-related protein AMBRA1 as a new FAT10 interaction partner. We show that the HUWE1-FAT10 conjugate formation is diminished in presence of AMBRA1, while the interaction between AMBRA1 and HUWE1 is strengthened in presence of FAT10. This implies a putative interplay of all three proteins in cellular processes such as mitophagy.
泛素样修饰物 FAT10 在炎症条件下高度上调，并将其缀合底物靶向 26S 蛋白酶体降解。这个过程称为 FAT10ylation，由酶级联介导，包括 E1 激活酶泛素样修饰物激活酶 6（UBA6）、E2 缀合酶 UBA6 特异性 E2 酶 1（USE1）和 E3 连接酶，如 Parkin。在这项研究中，研究了 HECT 型泛素 E3 连接酶 HUWE1 作为潜在的 E3 连接酶和/或 FAT10 的缀合底物的功能。我们的数据提供了强有力的证据，表明 HUWE1 在体外和细胞内以 UBA6 和 FAT10 二甘氨酸依赖性方式被 FAT10 化，并且 HUWE1-FAT10 缀合物被靶向到蛋白酶体降解。由于 HUWE1 HECT 结构域内所有相关半胱氨酸残基的突变并没有消除 FAT10 缀合，因此 HUWE1 作为 FAT10 化的 E3 连接酶的作用不太可能。此外，我们已经鉴定出自噬相关蛋白 AMBRA1 是 FAT10 的一个新的相互作用伙伴。我们表明，在 AMBRA1 存在的情况下，HUWE1-FAT10 缀合物的形成减少，而在 FAT10 存在的情况下，AMBRA1 和 HUWE1 之间的相互作用增强。这意味着这三种蛋白质在细胞过程（如线粒体自噬）中可能存在相互作用。
