MEIOB and SPATA22 are meiosis-specific proteins, interact with each other, and are essential for meiotic recombination and fertility. Aspartic acid 383 (D383) in MEIOB is critical for its interaction with SPATA22 in biochemical studies. Here we report that genetic studies validate the requirement of D383 for the function of MEIOB in mice. The Meiob^D383A/D383A mice display meiotic arrest due to depletion of both MEIOB and SPATA22 proteins in the testes. We developed a cell-based bimolecular fluorescence complementation (BiFC) assay, in which MEIOB and SPATA22 are fused to split YFP moieties and their co-expression in cultured cells leads to the MEIOB–SPATA22 dimerization and reconstitution of the fluorophore. As expected, the interaction-disrupting D383A substitution results in the absence of YFP fluorescence in the BiFC assay. A high-throughput screen of small molecule libraries identified candidate hit compounds at a rate of 0.7%. Isocotoin, a hit compound from the natural product library, inhibits the MEIOB–SPATA22 interaction and promotes their degradation in HEK293 cells in a dose-dependent manner. Therefore, the BiFC assay can be employed to screen for small molecule inhibitors that disrupt protein–protein interactions or promote degradation of meiosis-specific proteins.

中文翻译：

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基于细胞的高内涵筛选将isocotoin 鉴定为减数分裂特异性MEIOB-SPATA22 复合物的小分子抑制剂†。

MEIOB 和 SPATA22 是减数分裂特异性蛋白质，彼此相互作用，对减数分裂重组和生育至关重要。MEIOB 中的天冬氨酸 383 (D383) 对其在生化研究中与 SPATA22 的相互作用至关重要。在这里，我们报告遗传研究验证了 D383 对小鼠 MEIOB 功能的要求。该Meiob ^{D383A / D383A}由于睾丸中 MEIOB 和 SPATA22 蛋白的消耗，小鼠表现出减数分裂停滞。我们开发了一种基于细胞的双分子荧光互补 (BiFC) 测定，其中 MEIOB 和 SPATA22 融合到分裂的 YFP 部分，它们在培养细胞中的共表达导致 MEIOB-SPATA22 二聚化和荧光团的重建。正如预期的那样，破坏相互作用的 D383A 替换导致 BiFC 检测中没有 YFP 荧光。小分子文库的高通量筛选以 0.7% 的比率识别出候选命中化合物。Isocotoin 是天然产物库中的一种命中化合物，可抑制 MEIOB-SPATA22 相互作用并以剂量依赖性方式促进它们在 HEK293 细胞中的降解。所以，