Ankyrin-repeat and SOCS box-containing proteins (ASB) interact with the elongin B-C adapter via their SOCS box domain and with the cullin and ring box proteins to form E3 ubiquitin ligase complexes within the protein ubiquitination pathway. ASB9 in particular is a differentially expressed gene in ovulatory follicles (OFs) induced by the luteinizing hormone (LH) surge or hCG injection in ovarian granulosa cells (GC) while downregulated in growing dominant follicles. Although ASB9 has been involved in biological processes such as protein modification, the signaling network associated with ASB9 in GC is yet to be fully defined. We previously identified and reported ASB9 interactions and binding partners in GC including PAR1, TAOK1, and TNFAIP6/TSG6. Here, we further investigate ASB9 effects on target binding partners regulation and signaling in GC. CRISPR/Cas9-induced inhibition of ASB9 revealed that ASB9 regulates PAR1, TAOK1, TNFAIP6 as well as genes associated with proliferation and cell cycle progression such as PCNA, CCND2, and CCNE2 while CCNA2 was not affected. Inhibition of ASB9 was also associated with increased GC number and decreased caspase3/7 activity, CASP3 expression, and BAX/BCL2 ratio. Furthermore, ASB9 induction in OF in vivo 24 h post-hCG is concomitant with a significant decrease in phosphorylation levels of MAPK3/1 while pMAPK3/1 levels increased following ASB9 inhibition in GC in vitro. Together, these results provide strong evidence for ASB9 as a regulator of GC activity and function by modulating MAPK signaling likely through specific binding partners such as PAR1, therefore controlling GC proliferation and contributing to GC differentiation into luteal cells.

中文翻译：

---

锚蛋白重复和含 SOCS 盒的蛋白 9 (ASB9) 调节卵巢颗粒细胞功能和 MAPK 信号

锚蛋白重复和含 SOCS 盒的蛋白 (ASB) 通过其 SOCS 盒结构域与细长蛋白 BC 接头相互作用，并与 cullin 和环盒蛋白相互作用，在蛋白质泛素化途径中形成 E3 泛素连接酶复合物。特别是 ASB9 是排卵卵泡 (OFs) 中的差异表达基因，由卵巢颗粒细胞 (GC) 中的促黄体生成素 (LH) 激增或 hCG 注射诱导，同时在生长的优势卵泡中下调。尽管 ASB9 已参与蛋白质修饰等生物学过程，但与 ASB9 在 GC 中相关的信号网络尚未完全确定。我们之前在 GC 中发现并报告了 ASB9 相互作用和结合伙伴，包括 PAR1、TAOK1 和 TNFAIP6/TSG6。在这里，我们进一步研究了 ASB9 对 GC 中目标结合伙伴调控和信号传导的影响。PAR1、TAOK1、TNFAIP6以及与增殖和细胞周期进程相关的基因，如PCNA、CCND2和CCNE2，而CCNA2不受影响。ASB9 的抑制也与增加的 GC 数量和降低的 caspase3/7 活性、CASP3表达和BAX / BCL2比率有关。此外，hCG 后 24 小时体内OF 中的 ASB9 诱导伴随着 MAPK3/1 磷酸化水平的显着降低，而体外GC 中 ASB9 抑制后 pMAPK3/1 水平增加. 总之，这些结果为 ASB9 作为 GC 活性和功能的调节剂提供了强有力的证据，通过调节 MAPK 信号传导可能通过 PAR1 等特异性结合伙伴，因此控制 GC 增殖并有助于 GC 分化为黄体细胞。