Study of the role of the interaction between mitofusin and Nek4 in the signaling between mitochondria and nucleus after cellular stress

• Grant number: 18/05350-3
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: June 01, 2018
• End date: April 01, 2023
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Jörg Kobarg
• Grantee: Fernanda Luisa Basei
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 17/03489-1 - From functional studies to searching for new inhibitors for cancer: exploring kinases that regulate the cell cycle of the human NEK family, AP.TEM
• Associated scholarship(s): 21/07025-5 - Nek4 and mitofusins interaction and its impact in mitochondria/ER communication and cancer cell fate, BE.EP.PD

Abstract

Neks are serine-threonine kinases that are similar to NIMA, a protein found in Aspergillus nidulans which is essential for cell division. Eleven human Neks have been identified (1-11) and there is growing evidence pointing Neks role in diverse cellular process, such as cell cycle control, including cell division, mitotic spindle assembly, furrow ingression, primary cilium formation and maintenance, besides DNA damage response (DDR) and apoptosis control. Nek4 have already been associated with these process, although its role in these processes remains to be elucidated. In DNA damage response, its known that Nek4 interacts with proteins from Non-Homologous End Join (NHEJ) pathway and cells knockdown to Nek4 are etoposide-resistant. In an IP/MS assay performed by our group, several proteins related to DDR, mitochondrial metabolism and apoptosis were identified. The mitochondrial related function has already been described to Nek1 and Nek5, such as apoptosis and reactive oxygen species (ROS) levels regulation. Among the mitochondrial related proteins found in the Nek4 IP/MS assay, Mitofusin was identified. The GTPases Mitofusin 1 (Mfn1) and Mitofusin 2 (Mfn2) play a crucial role on the fusion process and the maintenance of the contacts between the mitochondria and endoplasmic reticulum. In this sense, Mfn2 plays a key role in the unfolded protein response (UPR) after endoplasmic reticulum stress. In the absence of Mfn2, the UPR is increased along with ROS and apoptosis activation deficiency. Our previous results have shown that both Nek4 isoforms interact with Mfn1 and Mfn2 as well as with the Mfn2 truncated version without the transmembrane domain, indicating that the interaction occurs through the N-terminus portion of Mfn2, the cytoplasmic region. We also verified that this interaction does not depend on the Nek4 kinase activity. Furthermore, surprisingly, we observe the presence of Nek4 in the mitochondria and endoplasmic reticulum contacting region, denominated MAMs (mitochondria-associated membranes), suggesting that Nek4 could be involved in UPR too. Corroborating with these results, we observed that Nek4 knockdown leads to alterations in the mitochondrial dynamics, indicated by the reduction in the ATP levels, increase in the ROS levels and alterations in the mitochondrial morphology. The involvement of Nek4 in DNA damage response has already been described, as well as the resistance to anticancer drugs in Nek4 depleted cells. Recently has been shown that Nek4 shows an anti-apoptotic role in TRAIL induced apoptosis in cancer cells. Considering this, the aim of this project is understanding the role of Nek4 in the mitochondria and verify if the Nek4/ Mfns interaction is component of nuclear mitochondria crosstalk after cellular stress. In this context we intend to investigate if Nek4, as a kinase, could regulate the Mfns activity. The cell cycle control and DDR, ROS levels regulation are process closely connected, and in this way, a better understanding of the crosstalk between nucleus-mitochondria signalling is essential to understanding cancer development.

Scientific publications

(5)

(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)

BASEI, FERNANDA L.; MOURA, LIVIA A. R.; KOBARG, JORG. Using the E1A Minigene Tool to Study mRNA Splicing Changes. JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, n. 170, APR 2021. (17/03489-1, 18/05350-3)

PERES DE OLIVEIRA, ANDRESSA; BASEI, FERNANDA LUISA; SLEPICKA, PRISCILA FERREIRA; DE CASTRO FEREZIN, CAMILA; MELO-HANCHUK, TALITA D.; DE SOUZA, EDMARCIA ELISA; LIMA, TANES I.; DOS SANTOS, VALQUIRIA TIAGO; MENDES, DAVI; SILVEIRA, LEONARDO REIS; et al. NEK10 interactome and depletion reveal new roles in mitochondria. PROTEOME SCIENCE, v. 18, n. 1, APR 28 2020. (14/15982-6, 18/05350-3, 17/03489-1)

BASEI, FERNANDA LUISA; FEREZIN, CAMILA DE CASTRO; RODRIGUES DE OLIVEIRA, ANA LUISA; MUNOZ, JUAN PABLO; ZORZANO, ANTONIO; KOBARG, JORG. Nek4 regulates mitochondrial respiration and morphology. FEBS Journal, v. 289, n. 11, p. 18-pg., 2022-01-24. (18/05350-3, 17/03489-1)

FEREZIN, CAMILA DE CASTRO; BASEI, FERNANDA LUISA; MELO-HANCHUK, TALITA D.; DE OLIVEIRA, ANA LUISA; PERES DE OLIVEIRA, ANDRESSA; MORI, MATEUS P.; DE SOUZA-PINTO, NADJA C.; KOBARG, JORG. NEK5 interacts with LonP1 and its kinase activity is essential for the regulation of mitochondrial functions and mtDNA maintenance. FEBS OPEN BIO, v. 11, n. 3, FEB 2021. (16/10530-5, 17/03489-1, 18/05350-3, 14/50938-8)

BASEI, FERNANDA L.; MOURA, LIVIA A. R.; KOBARG, JORG. Using the E1A Minigene Tool to Study mRNA Splicing Changes. JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, v. N/A, n. 170, p. 13-pg., 2021-04-01. (17/03489-1, 18/05350-3)