NEK10 interactome and depletion reveal new roles in mitochondria

Proteome Science

Tanes I. Lima¹  Leonardo Reis Silveira¹  Davi Mendes²  Carlos Frederico Martins Menck²  Valquiria Tiago dos Santos²  Talita D. Melo-Hanchuk³  Fernanda Luisa Basei³  Camila de Castro Ferezin³  Andressa Peres de Oliveira³  Jörg Kobarg³  Priscila Ferreira Slepicka⁴  Edmarcia Elisa de Souza⁴ 

[1] Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas;Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo;Instituto de Biologia, Departamento de Bioquímica e Biologia Tecidual, Universidade Estadual de Campinas;Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas;

关键词: NEK10;    Interactomics;    Mitochondrial dynamics and metabolism;    mtDNA;   

DOI  :  10.1186/s12953-020-00160-w

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown