STK40 inhibits trophoblast fusion by mediating COP1 ubiquitination to degrade P57Kip2

Background:<bold> </bold>The syncytiotrophoblast (SCT) layer in the placenta serves as a crucial physical barrier separating maternal-fetal circulation, facilitating essential signal and substance exchange between the mother and fetus. Any abnormalities in its formation or function can result in various maternal syndromes, such as preeclampsia. The transition of proliferative villous cytotrophoblasts (VCT) from the mitotic cell cycle to the G0 phase is a prerequisite for VCT differentiation and their fusion into SCT. The imprinting gene P57(Kip2), specifically expressed in intermediate VCT capable of fusion, plays a pivotal role in driving this key event. Moreover, aberrant expression of P57(Kip2) has been linked to pathological placental conditions and adverse fetal outcomes. Methods:<bold> </bold>Validation of STK40 interaction with P57(Kip2) using rigid molecular simulation docking and co-immunoprecipitation. STK40 expression was modulated by lentivirus in BeWo cells, and the effect of STK40 on trophoblast fusion was assessed by real-time quantitative PCR, western blot, immunofluorescence, and cell viability and proliferation assays. Co-immunoprecipitation, transcriptome sequencing, and western blot were used to determine the potential mechanisms by which STK40 regulates P57(Kip2). Results:<bold> </bold>In this study, STK40 has been identified as a novel interacting protein with P57(Kip2), and its expression is down-regulated during the fusion process of trophoblast cells. Overexpressing STK40 inhibited cell fusion in BeWo cells while stimulating mitotic cell cycle activity. Further experiments indicated that this effect is attributed to its specific binding to the CDK-binding and the Cyclin-binding domains of P57(Kip2), mediating the E3 ubiquitin ligase COP1-mediated ubiquitination and degradation of P57(Kip2). Moreover, abnormally high expression of STK40 might significantly contribute to the occurrence of preeclampsia. Conclusions: This study offers new insights into the role of STK40 in regulating the protein-level homeostasis of P57(Kip2) during placental development.