Characterisation of human RING finger protein TRIM69, a novel testis E3 ubiquitin ligase and its subcellular localisation.

The E3 ubiquitin ligase activity and subcellular localisation of human TRIM69 (hTRIM69) gene were studied. It was found that hTRIM69 mediated ubiquitination in an E2 conjugating enzyme selective fashion in vitro and an intact RING finger domain was indispensible for the process. Further evidences showed that hTRIM69 could mediate ubiquitination in vivo, which could be enhanced by a proteasome inhibitor. hTRIM69 was found to localise in both the cytoplasm and the nucleus in a speckled aggregating pattern, which also required an intact RING finger domain. Collectively, hTRIM69 is a novel E3 ubiquitin ligase identified from human testis and may function to ubiquitinate its particular substrates during spermatogenesis.

Han Y ，Li R ，Gao J ，Miao S ，Wang L ... -  《-》

Characterization of TRIM62 as a RING finger E3 ubiquitin ligase and its subcellular localization.

TRIM62, also named DEAR1, is a member of the TRIM/RBCC family, which includes proteins with conserved RING finger, B-box and coiled-coil domains. Several reports have identified a role for this family in cancer, retroviral infection and innate immunity. In this study, the E3 ubiquitin ligase activity and subcellular localization of TRIM62 were characterized. TRIM62, in association with the E2 enzyme UbcH5b, was found to catalyze self-ubiquitination in vitro, a process that required an intact RING finger domain. A ubiquitination assay performed in HEK293T cells further confirmed the E3 ubiquitin ligase activity and self-ubiquitination activity of TRIM62 and the requirement of the RING finger domain. Importantly, the treatment of HEK293T cells with a proteasome inhibitor stabilized poly-ubiquitinated TRIM62, indicating that self-ubiquitination promoted the proteasomal degradation of TRIM62. Additionally, TRIM62 and its two mutants were distinctly localized in the cytoplasm in both HEK293T and HeLa cells. Collectively, our data indicate that TRIM62, a cytoplasmic protein, is a RING finger domain-dependent E3 ubiquitin ligase that catalyzes self-ubiquitination both in vitro and in vivo.

Huang F ，Xiao H ，Sun BL ，Yang RG ... -  《-》

Identification of TRIM22 as a RING finger E3 ubiquitin ligase.

Duan Z ，Gao B ，Xu W ，Xiong S ... -  《-》

Mulan E3 ubiquitin ligase interacts with multiple E2 conjugating enzymes and participates in mitophagy by recruiting GABARAP.

Mulan is an E3 ubiquitin ligase embedded in the outer mitochondrial membrane (OMM) with its RING finger facing the cytoplasm and a large domain located in the intermembrane space (IMS). Mulan is known to have an important role in cell growth, cell death, and more recently in mitophagy. The mechanism of its function is poorly understood; but as an E3 ligase it is expected to interact with specific E2 ubiquitin conjugating enzymes and these complexes will bind and ubiquitinate specific substrates. The unique topology of Mulan can provide a direct link of communicating mitochondrial signals to the cytoplasm. Our studies identified four different E2 conjugating enzymes (Ube2E2, Ube2E3, Ube2G2 and Ube2L3) as specific interactors of Mulan. Each of these E2 conjugating enzymes was fused to the RING finger domain of Mulan and used in a modified yeast two-hybrid screen. Several unique interactors for each Mulan-E2 complex were isolated. One such specific interactor of Mulan-Ube2E3 was the GABARAP (GABAA receptor-associated protein). GABARAP is a member of the Atg8 family of proteins that plays a major role in autophagy/mitophagy. The interaction of GABARAP with Mulan-Ube2E3 required an LC3-interacting region (LIR) located in the RING finger domain of Mulan as well as the presence of Ube2E3. The isolation of four different E2 conjugating enzymes, as specific partners of Mulan E3 ligase, suggests that Mulan is involved in multiple biological pathways. In addition, the interaction of GABARAP with Mulan-Ube2E3 supports the role of Mulan as an important regulator of mitophagy and provides a plausible mechanism for its function in this process.

Ambivero CT ，Cilenti L ，Main S ，Zervos AS ... -  《-》

Secretory protein with RING finger domain (SPRING) specific to Trypanosoma cruzi is directed, as a ubiquitin ligase related protein, to the nucleus of host cells.

Hashimoto M ，Murata E ，Aoki T 《-》