Background Members of the family of NEK protein kinases (NIMA-related kinases) were described to have crucial roles in regulating different aspects of the cell cycle. NEK10 was reported to take part in the maintenance of the G2/M checkpoint after exposure to ultraviolet light. NEK1, NEK5, NEK2 and NEK4 proteins on the other hand have been linked to mitochondrial functions. Methods HEK293T cells were transfected with FLAG empty vector or FLAG-NEK10 and treated or not with Zeocin. For proteomic analysis, proteins co-precipitated with the FLAG constructs were digested by trypsin, and then analyzed via LC-MS/MS. Proteomic data retrieved were next submitted to Integrated Interactome System analysis and differentially expressed proteins were attributed to Gene Ontology biological processes and assembled in protein networks by Cytoscape. For functional, cellular and molecular analyses two stable Nek10 silenced HeLa cell clones were established. Results Here, we discovered the following possible new NEK10 protein interactors, related to mitochondrial functions: SIRT3, ATAD3A, ATAD3B, and OAT. After zeocin treatment, the spectrum of mitochondrial interactors increased by the proteins: FKBP4, TXN, PFDN2, ATAD3B, MRPL12, ATP5J, DUT, YWHAE, CS, SIRT3, HSPA9, PDHB, GLUD1, DDX3X, and APEX1. We confirmed the interaction of NEK10 and GLUD1 by proximity ligation assay and confocal microscopy. Furthermore, we demonstrated that NEK10-depleted cells showed more fragmented mitochondria compared to the control cells. The knock down of NEK10 resulted further in changes in mitochondrial reactive oxygen species (ROS) levels, decreased citrate synthase activity, and culminated in inhibition of mitochondrial respiration, affecting particularly ATP-linked oxygen consumption rate and spare capacity. NEK10 depletion also decreased the ratio of mtDNA amplification, possibly due to DNA damage. However, the total mtDNA content increased, suggesting that NEK10 may be involved in the control of mtDNA content. Conclusions Taken together these data place NEK10 as a novel regulatory player in mitochondrial homeostasis and energy metabolism.

中文翻译：

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NEK10 相互作用组和耗竭揭示了线粒体中的新作用。

背景 NEK 蛋白激酶（NIMA 相关激酶）家族的成员被描述在调节细胞周期的不同方面具有关键作用。据报道NEK10在暴露于紫外线后参与了G2/M检查站的维护。另一方面，NEK1、NEK5、NEK2 和 NEK4 蛋白与线粒体功能有关。方法用FLAG空载体或FLAG-NEK10转染HEK293T细胞，用Zeocin处理或不处理。对于蛋白质组学分析，与 FLAG 构建体共沉淀的蛋白质被胰蛋白酶消化，然后通过 LC-MS/MS 进行分析。检索到的蛋白质组学数据接下来提交给 Integrated Interactome System 分析，差异表达的蛋白质归因于 Gene Ontology 生物过程，并由 Cytoscape 组装在蛋白质网络中。对于功能性，细胞和分子分析 建立了两个稳定的 Nek10 沉默 HeLa 细胞克隆。结果 在这里，我们发现了以下可能与线粒体功能相关的新 NEK10 蛋白相互作用物：SIRT3、ATAD3A、ATAD3B 和 OAT。zeocin 处理后，线粒体相互作用的谱因以下蛋白质而增加：FKBP4、TXN、PFDN2、ATAD3B、MRPL12、ATP5J、DUT、YWHAE、CS、SIRT3、HSPA9、PDHB、GLUD1、DDX3X 和 APEX1。我们通过邻近连接测定和共聚焦显微镜证实了 NEK10 和 GLUD1 的相互作用。此外，我们证明了与对照细胞相比，NEK10 耗尽的细胞显示出更多碎片化的线粒体。NEK10 的敲除导致线粒体活性氧 (ROS) 水平的进一步变化，柠檬酸合酶活性降低，并最终抑制线粒体呼吸，尤其影响与 ATP 相关的耗氧率和备用能力。NEK10 消耗也降低了 mtDNA 扩增的比率，可能是由于 DNA 损伤。然而，总 mtDNA 含量增加，表明 NEK10 可能参与了 mtDNA 含量的控制。结论 综合这些数据，NEK10 成为线粒体稳态和能量代谢的新型调节参与者。