Coiled-coil domain-containing 124 (CCDC124) is a recently discovered ribosome-binding protein conserved in eukaryotes. CCDC124 has regulatory functions on the mediation of reversible ribosomal hibernation and translational recovery by direct attachment to large subunit ribosomal protein uL5, 25S rRNA backbone, and tRNA-binding P/A-site major groove. Moreover, it independently mediates cell division and cellular stress response by facilitating cytokinetic abscission and disulfide stress-dependent transcriptional regulation, respectively. However, the structural characterization and intracellular physiological status of CCDC124 remain unknown. In this study, we employed advanced in silico protein modeling and characterization tools to generate a native-like tertiary structure of CCDC124 and examine the disorder, low sequence complexity, and aggregation propensities, as well as high-order dimeric/oligomeric states. Subsequently, dimerization of CCDC124 was investigated with co-immunoprecipitation (CO-IP) analysis, immunostaining, and a recent live-cell protein–protein interaction method, bimolecular fluorescence complementation (BiFC). Results revealed CCDC124 as a highly disordered protein consisting of low complexity regions at the N-terminus and an aggregation sequence (151-IAVLSV-156) located in the middle region. Molecular docking and post-docking binding free energy analyses highlighted a potential involvement of V153 residue on the generation of high-order dimeric/oligomeric structures. Co-IP, immunostaining, and BiFC analyses were used to further confirm the dimeric state of CCDC124 predominantly localized at the cytoplasm. In conclusion, our findings revealed in silico structural characterization and in vivo subcellular physiological state of CCDC124, suggesting low-complexity regions located at the N-terminus of disordered CCDC124 may regulate the formation of aggregates or high-order dimeric/oligomeric states.

中文翻译：

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二聚化是本征无序卷曲螺旋结构域 124 聚集倾向的基础

包含卷曲螺旋结构域的 124 (CCDC124) 是最近发现的一种在真核生物中保守的核糖体结合蛋白。CCDC124 通过直接附着于大亚基核糖体蛋白 uL5、25S rRNA 骨架和 tRNA 结合 P/A 位点大沟，对可逆核糖体冬眠和翻译恢复具有调节作用。此外，它分别通过促进细胞因子脱落和二硫键依赖性转录调节来独立介导细胞分裂和细胞应激反应。然而，CCDC124 的结构特征和细胞内生理状态仍然未知。在这项研究中，我们采用先进的计算机蛋白质建模和表征工具来生成 CCDC124 的类似天然的三级结构并检查无序、低序列复杂性、和聚集倾向，以及高阶二聚体/寡聚体状态。随后，通过免疫共沉淀 (CO-IP) 分析、免疫染色和最近的活细胞蛋白质-蛋白质相互作用方法双分子荧光互补 (BiFC) 研究了 CCDC124 的二聚化。结果显示 CCDC124 是一种高度无序的蛋白质，由 N 端的低复杂性区域和位于中间区域的聚集序列 (151-IAVLSV-156) 组成。分子对接和对接后结合自由能分析强调了 V153 残基可能参与高阶二聚体/寡聚体结构的产生。Co-IP、免疫染色和附设分析用于进一步确认 CCDC124 主要位于细胞质的二聚体状态。综上所述，