NEK10 interactome and depletion reveal new roles in mitochondria.

IF 2.1 3区生物学 Q3 BIOCHEMICAL RESEARCH METHODS Proteome Science Pub Date : 2020-04-28 eCollection Date: 2020-01-01 DOI:10.1186/s12953-020-00160-w

Andressa Peres de Oliveira, Fernanda Luisa Basei, Priscila Ferreira Slepicka, Camila de Castro Ferezin, Talita D Melo-Hanchuk, Edmarcia Elisa de Souza, Tanes I Lima, Valquiria Tiago Dos Santos, Davi Mendes, Leonardo Reis Silveira, Carlos Frederico Martins Menck, Jörg Kobarg

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引用次数: 15

Abstract

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.

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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蛋白激酶家族成员(nima相关激酶)在调节细胞周期的不同方面具有重要作用。据报道，NEK10在紫外线照射后参与G2/M检查点的维护。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，并用Zeocin处理或不处理HEK293T细胞。对于蛋白质组学分析，与FLAG构建体共沉淀的蛋白质用胰蛋白酶消化，然后用LC-MS/MS分析。随后，将获得的蛋白质组学数据提交到Integrated Interactome System进行分析，并将差异表达的蛋白质归因于Gene Ontology生物学过程，并由Cytoscape将其组装到蛋白质网络中。为了进行功能、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的NEK10蛋白相互作用因子:SIRT3, ATAD3A, ATAD3B和OAT。zeocin处理后，线粒体相互作用蛋白谱增加，包括FKBP4、TXN、PFDN2、ATAD3B、MRPL12、ATP5J、DUT、YWHAE、CS、SIRT3、HSPA9、PDHB、GLUD1、DDX3X和APEX1。我们通过近距离结扎实验和共聚焦显微镜证实了NEK10和GLUD1的相互作用。此外，我们证明，与对照细胞相比，nek10缺失的细胞显示出更多的线粒体碎片化。NEK10基因的敲低进一步导致线粒体活性氧(ROS)水平的变化，柠檬酸合酶活性的降低，并最终抑制线粒体呼吸，特别是影响atp相关的氧气消耗率和备用容量。NEK10缺失也降低了mtDNA扩增率，可能是由于DNA损伤。但总mtDNA含量增加，提示NEK10可能参与mtDNA含量的控制。综上所述，这些数据表明NEK10在线粒体稳态和能量代谢中起着新的调节作用。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Proteome Science 生物-生化研究方法

CiteScore

2.90

自引率

0.00%

发文量

审稿时长

4.5 months

期刊介绍： Proteome Science is an open access journal publishing research in the area of systems studies. Proteome Science considers manuscripts based on all aspects of functional and structural proteomics, genomics, metabolomics, systems analysis and metabiome analysis. It encourages the submissions of studies that use large-scale or systems analysis of biomolecules in a cellular, organismal and/or environmental context. Studies that describe novel biological or clinical insights as well as methods-focused studies that describe novel methods for the large-scale study of any and all biomolecules in cells and tissues, such as mass spectrometry, protein and nucleic acid microarrays, genomics, next-generation sequencing and computational algorithms and methods are all within the scope of Proteome Science, as are electron topography, structural methods, proteogenomics, chemical proteomics, stem cell proteomics, organelle proteomics, plant and microbial proteomics. In spite of its name, Proteome Science considers all aspects of large-scale and systems studies because ultimately any mechanism that results in genomic and metabolomic changes will affect or be affected by the proteome. To reflect this intrinsic relationship of biological systems, Proteome Science will consider all such articles.