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Analysis of the role of NEK1 in the regulation of the response to nuclear and mitochondrial DNA damage

Grant number: 17/21067-7
Support type:Scholarships abroad - Research Internship - Post-doctor
Effective date (Start): February 05, 2018
Effective date (End): February 04, 2019
Field of knowledge:Biological Sciences - Biochemistry - Molecular Biology
Principal researcher:Jörg Kobarg
Grantee:Mariana Bonjiorno Martins
Supervisor abroad: David M. Wilson III
Home Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
Research place: National Institutes of Health, Bethesda (NIH), United States  
Associated to the scholarship:16/02040-8 - FUNCTIONAL ANALYSIS OF MUTATIONS IN NEK1 AND NEK7 IN CANCER AND IDENTIFICATION OF THEIR PHYSIOLOGICAL SUBSTRATES, BP.PD

Abstract

Nek1 protein belongs to the NIMA-related kinases or "Neks" family, which consists of eleven proteins named Nek1 to Nek 11. These paralogs have been functionally associated with mitosis, cilium regulation and the DNA damage response (DDR). Defects in DNA repair is suggested in Nek1-deficient cells by the persistence of DNA double-strand breaks (DSBs) following exposure to ionizing radiation (IR) or ultraviolet (UV) light. Using the regulatory and catalytic domain of Nek1 as bait, we identified interacting protein partners that are involved in cell cycle regulation and repair of DSBs. Among those identified were the DNA exonuclease Mre11, the chromatin remodeler ATRX and a binding partner of the tumor suppressor P53, 53BP1. Involvement of Nek1 in DSB repair was further demonstrated by artificially increasing kinase activity, an event that resulted in the redistribution and phosphorylation of Nek1 from the cytoplasm to the nucleus, where it colocalized with the DDR factors³-H2AX and Nfbd1/Mdc1in nuclear foci .This finding implies thatNek1 kinase activity is rapidly activated after genotoxic injury. Our interaction studies also revealed that partners of Nek1 are involved in homology-directed DNA repair, nucleotide excision repair (NER) and mismatch repair (MMR). Furthermore, our recent work indicates involvement of Nek1 in the DDR to the DNA crosslinking agent cisplatin. In addition, it has been demonstrated that Nek1 participates in cell death responses by phosphorylating and altering the conformation of a voltage-dependent anion channel (VDAC1) that is responsible for regulating the mitochondrial membrane potential (MMP). Cells deficient in or silenced for endogenous Nek1 fail to phosphorylate VDAC1 and consequently die early after exposure to UV. In addition, such mutant cells have an increase in mitochondrial membrane permeability, and ectopic expression of a mutant Nek1 similarly results in the loss of VDAC1 phosphorylation and consequent cell death. Thus, in the basal state and in response to injury, which includes DNA damage, Nek1 phosphorylates VDAC1 to limit mitochondrial cell death. The employment of functional studies is necessary to clarify the role of Nek1 in response to nuclear and mitochondrial DNA (mtDNA) damage, as well as its participation in DNA repair pathways. To achieve this goal, we will use a set of available cell lines in our group: wildtype HEK293T cells; HEK293T cells that overexpress wildtype Nek1 flag-tagged and the vector control, and HEK293T cells deficient for Nek1 via shRNA silencing and the scramble control. These cells will be treated with a range of genotoxic agents in an attempt to evaluate the global contribution of Nek1 in the DDR. We will use techniques, such as measurement of DNA damage and mtDNA copy number, to assess the levels of DNA damage following exposure of Nek1 mutant cells to different genotoxic agents. Laser microirradiation and confocal microscopy will be used to introduce localized DNA damage of different types to visualize recruitment/retention ofNek1 and other proteins related to DDR. We will also evaluate the effect of silencing Nek1 on the progression of the cell cycle after exposure to genotoxic agents using flow cytometry methods. Immunodetection assays will be performed to evaluate the interaction of Nek1 kinase with proteins related to DNA repair and the DDR. Mitochondrial functionality will be measured in cells deficient in Nek1 via a collection of assays, such as mtDNA damage, ROS levels, mitophagy and oxidative phosphorylation. These experiments together will elucidate the molecular mechanisms involving Nek1 that regulate cell cycle, the DDR and mitochondrial activity, potentially revealing a crosstalk between the nuclear and mitochondrial compartments. Given that Nek1 is up-regulated in many human cancers, our studies will uncover novel molecular strategies that can be pursued in the future as part of a new therapeutic paradigm. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
MARTINS, MARIANA BONJIORNO; PEREZ, ARINA MARINA; BOHR, VILHELM A.; WILSON, III, DAVID M.; KOBARG, JORG. NEK1 deficiency affects mitochondrial functions and the transcriptome of key DNA repair pathways. MUTAGENESIS, v. 36, n. 3, p. 223-236, . (16/02040-8, 14/15982-6, 17/21067-7, 17/03489-1)
BETIM PAVAN, ISADORA CAROLINA; DE OLIVEIRA, ANDRESSA PERES; FIRMINO DIAS, PEDRO RAFAEL; BASEI, FERNANDA LUISA; ISSAYAMA, LUIDY KAZUO; FEREZIN, CAMILA DE CASTRO; SILVA, FERNANDO RIBACK; RODRIGUES DE OLIVEIRA, ANA LUISA; ALVES DOS REIS MOURA, LIVIA; MARTINS, MARIANA BONJIORNO; et al. On Broken Ne(c)ks and Broken DNA: The Role of Human NEKs in the DNA Damage Response. CELLS, v. 10, n. 3, . (16/02040-8, 17/21067-7, 17/03489-1)

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