Identification And Functional Study Of A Novel Ubiquitin Ligase TRIM69/HSD34 In Human Testis

A large number of cell-specific and developmental stage-specific proteins are accurately degraded through the ubiquitin-dependent proteasome pathway, which plays a key role in spermatogenesis. In this paper, we characterized a protein from human testis as an E3 ubiquitin ligase, tried to find several substrates of its, and studied the possible cell events of these substrates involved, especially when they were ubiquitinated by TRIM69. Indirectly, we learned about the potential roles of TRIM69, which would be a great help to understand the functions of ubiquitin-dependent proteasome system in spermatogenesis.We identified a novel gene of Trim69 from human testis. TRIM69 coded by this gene, a member of TRIM (tripartite motif) family, consists of an N-terminal RING-finger domain, followed by one B-box motif, a coiled-coil region, and a c-terminal B30.2 domain. It was reported that many members of TRIM family had E3 ubiquitin ligase property.On the basis of work done by our lab, TRIM69 demonstrated a part of characteristics of E3 ubiquitin ligase. To further confirm TRIM69 as an E3 ubiquitin ligase, we carried out an in-vitro ubiquitination experiment. TRIM69 functioned as E3 ubiquitin ligase in the presence of five ubiquitin conjugating enzymes including UbcH2, UbcH6, UbcH5a, UbcH5c and UbcH13/Uevla.C61A/C64A mutant of TRIM69 in RING-finger domain or loss of RING-finger domain would obviously decrease its E3 ubiquitin ligase. RING finger domain was the active domain of TRIM69 and C61 and C64 sites were the key sites of this domain.In vivo ubiquitination experiment showed that the ubiquitylation of TRIM69 was increased by the proteasome inhibitor, which suggested that TRIM69 could target itself for proteasomal degradation through the poly-ubquitylation,.Immunostaining experiment showed the localization and distribution of both GFP-tagged-Trim69 and Flag-tagged-Trim69 were in common with aggregation and diffusion forms in cell cytoplasm and in cell nucleus.To further elucidate the function of TRIM69, we need to find proteins association with it. So a yeast two-hybrid assay was performed with a bait of the truncate of TRIM69 wihtout the N-terminal RING finger domain (246bp). Also Co-IP and pull down experiments were furehr performed and confirmed the protein HUS1 association with TRIM69. In vitro and in vivo ubiquitination experiments showed that TRIM69 could promote the ubiquitylation of HUS1. Also HUS1 protein stability experiment demonstrated that TRIM69 could accelerate the degradation of HUS1 and the process could be inhibited partly by MG132, an inhibitor of proteasome.It was reported that JAB1(c-jun-activation-domain binding protein) could mediated 9-1-1 complex degradation. But it was still not clear which E3 ubiquitin ligase involved in this process.Immunoprecipitation experiment by antibody against RAD1 demonstrated that TRIM69, JAB1, HUS1, and RAD1 protein could form a complex in cells.The interaction between TRIM69 and JAB 1 was confirmed by Co-IP and pull down experiments. Also their interaction was dependent on the C-terminal of TRIM69. We also verified overexpression of JAB1 decreased endogenous HUS1 protein stability in a dose-dependent tendency.Then we transfected HA-Jab1 and Flag-Trim69 plasmids into HeLa cells respectively or togehter and observed the endogenousHUS1 protein level change. Endogenous HUS1 level decreased obviously by overepression of JAB 1 respectively but resumed in part after cooverexpression of TRIM69. Correspondingly, co-overexpression of TRIM69 also made JAB1 protein level decrease, which might due to the decrease of endogenous HUS1 protein level. So JAB1 stability experiment was carried out and the results showed that TRIM69 did promote the degradation of JAB1 and the degradation could be inhibited by MG132. Also in vivo ubiquitination experiment showed TRIM69 could promote the ubiquitination of JAB 1, which was also dependent on its RING-finger domain.It was known that JAB1 could activate AP-1 pathway by interacting with c-Jun. We used dual luciferase reporter gene assay to verify the effect of TRIM69 on AP-1 transcriptional activity. The results indicated that overexpression of TRIM69 could inhibit the c-Jun and JAB1 mediated activation of AP-1 reporter activity in a dose-dependent manner. Western blot assay also showed the expression levels of JAB1, c-Jun, and c-Jun phosphorylation were decreased by TRIM69 in a dose-dependent manner. The stable cell lines with overexpression of TRIM69 are still in the process of screening and will be used in study the effect of TRIM69 on cell proliferation.