The melanoma-associated antigen 1 (MAGEA1) protein stimulates the E3 ubiquitin-ligase activity of TRIM31 within a TRIM31-MAGEA1-NSE4 complex

The MAGE (Melanoma-associated antigen) protein family members are structurally related to each other by a MAGE-homology domain comprised of 2 winged helix motifs WH/A and WH/B. This family specifically evolved in placental mammals although single homologs designated NSE3 (non-SMC element) exist in most eukaryotes. NSE3, together with its partner proteins NSE1 and NSE4 form a tight subcomplex of the structural maintenance of chromosomes SMC5–6 complex. Previously, we showed that interactions of the WH/B motif of the MAGE proteins with their NSE4/EID partners are evolutionarily conserved (including the MAGEA1-NSE4 interaction). In contrast, the interaction of the WH/A motif of NSE3 with NSE1 diverged in the MAGE paralogs. We hypothesized that the MAGE paralogs acquired new RING-finger-containing partners through their evolution and form MAGE complexes reminiscent of NSE1-NSE3-NSE4 trimers. In this work, we employed the yeast 2-hybrid system to screen a human RING-finger protein library against several MAGE baits. We identified a number of potential MAGE-RING interactions and confirmed several of them (MDM4, PCGF6, RNF166, TRAF6, TRIM8, TRIM31, TRIM41) in co-immunoprecipitation experiments. Among these MAGE-RING pairs, we chose to examine MAGEA1-TRIM31 in detail and showed that both WH/A and WH/B motifs of MAGEA1 bind to the coiled-coil domain of TRIM31 and that MAGEA1 interaction stimulates TRIM31 ubiquitin-ligase activity. In addition, TRIM31 directly binds to NSE4, suggesting the existence of a TRIM31-MAGEA1-NSE4 complex reminiscent of the NSE1-NSE3-NSE4 trimer. These results suggest that MAGEA1 functions as a co-factor of TRIM31 ubiquitin-ligase and that the TRIM31-MAGEA1-NSE4 complex may have evolved from an ancestral NSE1-NSE3-NSE4 complex.

黑色素瘤相关抗原（Melanoma-associated antigen, MAGE）蛋白家族的各成员结构同源，均拥有由两个翼螺旋基序（winged helix motif）WH/A和WH/B构成的MAGE同源结构域。该家族仅在胎盘哺乳动物中特异性演化，但在绝大多数真核生物（eukaryotes）中均存在被命名为NSE3（non-SMC element，非SMC元件）的单拷贝同源物。NSE3与其搭档蛋白NSE1、NSE4共同构成染色体结构维持SMC5–6复合物（structural maintenance of chromosomes SMC5–6 complex）的紧密亚复合物。此前本研究团队表明，MAGE蛋白的WH/B基序与其NSE4/EID搭档之间的相互作用在演化上具有保守性，其中包括MAGEA1-NSE4的相互作用。与之相反，NSE3的WH/A基序与NSE1的相互作用在MAGE旁系同源物（paralogs）中发生了分化。我们提出假说：MAGE旁系同源物在演化过程中获得了新的含环指结构域（RING-finger）的搭档蛋白，并形成了类似NSE1-NSE3-NSE4三聚体的MAGE复合物。本研究中，我们采用酵母双杂交系统（yeast 2-hybrid system），以多种MAGE蛋白为诱饵，筛选人类环指蛋白（RING-finger protein）文库。我们鉴定出多组潜在的MAGE-环指蛋白相互作用对，并通过免疫共沉淀（co-immunoprecipitation）实验验证了其中数对，包括MDM4、PCGF6、RNF166、TRAF6、TRIM8、TRIM31、TRIM41。在上述MAGE-环指蛋白配对中，我们选择深入研究MAGEA1-TRIM31配对，并证实MAGEA1的WH/A和WH/B两个基序均结合TRIM31的卷曲螺旋结构域（coiled-coil domain），且MAGEA1的相互作用可增强TRIM31的泛素连接酶活性（ubiquitin-ligase activity）。此外，TRIM31可直接结合NSE4，这提示存在类似NSE1-NSE3-NSE4三聚体的TRIM31-MAGEA1-NSE4复合物。上述结果表明，MAGEA1可作为TRIM31泛素连接酶的辅因子发挥功能，且TRIM31-MAGEA1-NSE4复合物可能由祖先的NSE1-NSE3-NSE4复合物演化而来。