Characterization of functional effects of point mutations in NEK1 detected in amyotrophic lateral sclerosis on mitochondria and DNA damage response

Author(s)

Luidy Kasuo Issayama

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Farmacêuticas
• Defense date: 2023-08-17

Examining board members

Jörg Kobarg; Nadja Cristhina de Souza Pinto; Guido Lenz; Katlin Brauer Massirer; Daniel Fábio Kawano

• Advisor: Jörg Kobarg

Abstract

NEKs are proteins that play various biological roles in cells. Like their orthologs, the NIMA protein, they are also responsible for the entry of the cell into cell division, but they have other specific biological roles for each. In humans, there are 11 described members that differ predominantly through their regulatory domain and share a high homology in their catalytic domain, which is likely related to the different functions performed by members of this family. NEK1, the first ortholog of the NIMA protein described, is a serine/threonine and tyrosine kinase, whose function is mainly related to DNA damage repair, primary cilium regulation, and cell cycle control. DNA damage repair signaling is through phosphorylation by TLK1 (Tousled-Like Kinase), at Threonine 141, signaling to homology repair pathways, stabilizing the ATR-ATRIP complex (TLK1>NEK1>ATR). In addition, it has been shown that NEK1 interacts with and phosphorylates the voltage-dependent anion channel 1 (VDAC1) present in the mitochondria. This interaction has been shown to be important for the prevention of apoptosis by closing this channel. Interestingly, this regulation may be important for the regulation of mitochondrial activity, and therefore deleterious defects in NEK1 activity may have consequences for cell metabolism. In fact, recent studies have shown that loss of NEK1 function has consequences on mitochondrial activity in cells, leading to an increase in reactive oxygen species production and cell death. Taking this into consideration, NEK1 mutants found in patients with familial amyotrophic lateral sclerosis (ALS) may have defects in mitochondrial activity, causing an increase in reactive oxygen species, accumulation of DNA damage, and susceptibility to cell death, known hallmarks of neurodegenerative diseases such as ALS. Therefore, the work aimed to explore the molecular mechanisms affected by NEK1 deletion and study the effect of loss-of-function point mutations of NEK1 found in ALS patients, comparing with NEK1-silenced cells. For this, two mutations were selected through bioinformatics that showed the greatest deleterious effects on kinase activity, one in the catalytic domain (Q132R) and another near the hypothetical dimerization site of NEK1 present in one of the coiled-coils (R615G). NEK1 deletion results showed an increase in nuclear and cytoplasmic reactive oxygen species production and double-strand break (DSB) signaling, which were reduced when treated with N-acetyl-cysteine, a known scavenger of reactive oxygen species. When transfected with wild-type NEK1, DSB signaling also reduced, which did not occur when transfected with ALS mutants, revealing that both are related to this mechanism. The main peculiarity of these mutants is in the formation of a possible NEK1 dimer for its autophosphorylation, in which only the R615G mutant showed a reduced interaction with NEK1 WT in the co-immunoprecipitation assay. Therefore, studying point mutation variants is essential for understanding the molecular mechanisms involved in the progression of diseases such as ALS, and NEK1 has gained significant prominence in the search for more effective treatments (AU)

• FAPESP's process: 18/13775-4 - The human protein kinase NEK1: the characterization of its physiological activation by DNA damage and its use as a biological marker and potential therapeutic target in Thyroid Cancer
• Grantee: Luidy Kazuo Issayama
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)