NEK10 interactome and depletion reveal new roles in mitochondria

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Peres de Oliveira, Andressa ^{[1, 2]} ; Basei, Fernanda Luisa ^{[2, 3]} ; Slepicka, Priscila Ferreira ^[4] ; de Castro Ferezin, Camila ^{[2, 3]} ; Melo-Hanchuk, Talita D. ^[2] ; de Souza, Edmarcia Elisa ^[4] ; Lima, Tanes I. ^{[5, 6]} ; dos Santos, Valquiria Tiago ^[1] ; Mendes, Davi ^[1] ; Silveira, Leonardo Reis ^[5] ; Menck, Carlos Frederico Martins ^[1] ; Kobarg, Jorg ^{[2, 3]}

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Inst Ciencias Biomed, Dept Microbiol, Sao Paulo - Brazil

^[2] Univ Estadual Campinas, Inst Biol, Dept Bioquim & Biol Tecidual, Campinas, SP - Brazil

^[3] Univ Estadual Campinas, Fac Ciencias Farmaceut, Rua Candido Portinari 200, BR-13083871 Campinas, SP - Brazil

^[4] Ctr Nacl Pesquisa Energia & Mat, Lab Nacl Biociencias, Campinas, SP - Brazil

^[5] Univ Estadual Campinas, Inst Biol, Dept Biol Estrutural & Func, Campinas, SP - Brazil

^[6] Univ Sao Paulo, Dept Bioquim & Imunol, Ribeirao Preto - Brazil

• Tipo de documento: Artigo Científico
• Fonte: PROTEOME SCIENCE; v. 18, n. 1 APR 28 2020.
• Citações Web of Science: 0

Resumo

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. (AU)

• Processo FAPESP: 14/15982-6 - Consequências de deficiências de reparo de lesões no genoma
• Beneficiário: Carlos Frederico Martins Menck
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 08/57906-3 - Instituto Nacional de Fotônica Aplicada à Biologia Celular - INFABIC
• Beneficiário: Hernandes Faustino de Carvalho
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 18/05350-3 - Investigação do papel da interação entre Mitofusinas e Nek4 na sinalização mitocôndria- núcleo após estresse celular
• Beneficiário: Fernanda Luisa Basei
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 17/03489-1 - De estudos funcionais à busca de novos inibidores anticâncer: explorando cinases reguladores do ciclo celular da família de NEK humana
• Beneficiário: Jörg Kobarg
• Modalidade de apoio: Auxílio à Pesquisa - Temático