Kawaguchi Shinichi, Xu Xin, Soga Takashi, Yamaguchi Kenta, Kawasaki Ryuuya, Shimouchi Ryota, Date Susumu, Kai Toshie
Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
D3 Center, Osaka University, Osaka, Japan.
Elife. 2025 Apr 22;13:RP101967. doi: 10.7554/eLife.101967.
Protein-protein interactions are fundamental to understanding the molecular functions and regulation of proteins. Despite the availability of extensive databases, many interactions remain uncharacterized due to the labor-intensive nature of experimental validation. In this study, we utilized the AlphaFold2 program to predict interactions among proteins localized in the nuage, a germline-specific non-membrane organelle essential for piRNA biogenesis in . We screened 20 nuage proteins for 1:1 interactions and predicted dimer structures. Among these, five represented novel interaction candidates. Three pairs, including Spn-E_Squ, were verified by co-immunoprecipitation. Disruption of the salt bridges at the Spn-E_Squ interface confirmed their functional importance, underscoring the predictive model's accuracy. We extended our analysis to include interactions between three representative nuage components-Vas, Squ, and Tej-and approximately 430 oogenesis-related proteins. Co-immunoprecipitation verified interactions for three pairs: Mei-W68_Squ, CSN3_Squ, and Pka-C1_Tej. Furthermore, we screened the majority of proteins (~12,000) for potential interaction with the Piwi protein, a central player in the piRNA pathway, identifying 164 pairs as potential binding partners. This in silico approach not only efficiently identifies potential interaction partners but also significantly bridges the gap by facilitating the integration of bioinformatics and experimental biology.
蛋白质-蛋白质相互作用是理解蛋白质分子功能和调控的基础。尽管有大量数据库可用，但由于实验验证的劳动密集型性质，许多相互作用仍未得到表征。在本研究中，我们利用AlphaFold2程序预测定位于生殖质（nuage）中的蛋白质之间的相互作用，生殖质是一种种系特异性非膜细胞器，对果蝇中的piRNA生物发生至关重要。我们筛选了20种生殖质蛋白进行1:1相互作用并预测二聚体结构。其中，有五种代表了新的相互作用候选物。包括Spn-E_Squ在内的三对通过免疫共沉淀得到验证。Spn-E_Squ界面处盐桥的破坏证实了它们的功能重要性，强调了预测模型的准确性。我们将分析扩展到包括三种代表性生殖质成分——Vas、Squ和Tej——与大约430种卵子发生相关蛋白之间的相互作用。免疫共沉淀验证了三对相互作用：Mei-W68_Squ、CSN3_Squ和Pka-C1_Tej。此外，我们筛选了大多数蛋白质（约12000种）与Piwi蛋白（piRNA途径中的核心参与者）的潜在相互作用，确定了164对作为潜在结合伙伴。这种计算机模拟方法不仅有效地识别了潜在的相互作用伙伴，还通过促进生物信息学和实验生物学的整合显著缩小了差距。
