Ufmylation是翻译后修饰,其中修饰剂UFM1与靶蛋白连接。这种缀合需要三种酶的协同工作,分别称为UBA5,UFC1和UFL1。最初,UBA5在一个过程中激活UFM1,该过程以UFM1连接到UBA5的活动站点Cys结束。然后,在反式巯基化反应中,UFM1从UBA5转移到UFC1,与后者形成硫酯键。最后,在UFL1的帮助下,UFM1被转移到最终目的地-目标蛋白上的赖氨酸残基。因此,毫不奇怪,这些酶之一的缺失废除了缀合过程。然而,这些酶的过表达如何影响这一过程尚不清楚。在这里我们发现,出乎意料的是,UBA5的过表达,而不是UFC1的过表达,会损害细胞的迁移能力,以类似于缺乏UBA5或UFC1的细胞的方式。在机械层面,我们发现UBA5的过表达逆转了反式巯基化反应,从而导致UFM1从UFC1向UBA5的反向转移。这个,如在缺乏UBA5的细胞中所见,降低带电UFC1的水平并因此损害缀合过程。相比之下,UBA5与UFM1的共表达消除了这种作用,表明UFM1从UFC1到UBA5的反向转移取决于游离UFM1的水平。总的来说,我们的结果表明,必须严格调节UFM1接合酶的细胞表达水平,以确保UFM1转移的正确方向性。
Ufmylation is a posttranslational modification in which the modifier UFM1 is attached to target proteins. This conjugation requires the concerted work of three enzymes named UBA5, UFC1, and UFL1. Initially, UBA5 activates UFM1 in a process that ends with UFM1 attached to UBA5\'s active site Cys. Then, in a trans-thiolation reaction, UFM1 is transferred from UBA5 to UFC1, forming a thioester bond with the latter. Finally, with the help of UFL1, UFM1 is transferred to the final destination-a lysine residue on a target protein. Therefore, not surprisingly, deletion of one of these enzymes abrogates the conjugation process. However, how overexpression of these enzymes affects this process is not yet clear. Here we found, unexpectedly, that overexpression of UBA5, but not UFC1, damages the ability of cells to migrate, in a similar way to cells lacking UBA5 or UFC1. At the mechanistic level, we found that overexpression of UBA5 reverses the trans-thiolation reaction, thereby leading to a back transfer of UFM1 from UFC1 to UBA5. This, as seen in cells lacking UBA5, reduces the level of charged UFC1 and therefore harms the conjugation process. In contrast, co-expression of UBA5 with UFM1 abolishes this effect, suggesting that the reverse transfer of UFM1 from UFC1 to UBA5 depends on the level of free UFM1. Overall, our results propose that the cellular expression level of the UFM1 conjugation enzymes has to be tightly regulated to ensure the proper directionality of UFM1 transfer.