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Oxidative stress-mediated post-translational regulation of Dicer: structural and functional characterization of 4-HNE-DICER interaction

Grant number: 17/14426-0
Support type:Scholarships abroad - Research Internship - Master's degree
Effective date (Start): October 15, 2017
Effective date (End): April 14, 2018
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Julio Cesar Batista Ferreira
Grantee:Ligia Akemi Kiyuna
Supervisor abroad: Ian Macrae
Home Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo, SP, Brazil
Local de pesquisa : Scripps Research Institute, San Diego, United States  
Associated to the scholarship:16/00900-0 - Impact of aldehydes on Dicer activity and expression profile: benefits of ALDH2 activation, BP.MS

Abstract

4-hydroxy-2-nonenal (4-HNE) is a major by-product of lipid peroxidation, a process that is exacerbated under oxidative stress conditions. This aldehyde is highly reactive with proteins, lipids and DNA, causing target loss of function and degradation in most cases. For this reason, accumulation of 4-HNE has been correlated with the establishment and progression of many diseases, including cardiovascular diseases. We recently demonstrated using proteomics that 4-HNE directly targets DICER during heart failure in rats, a critical enzyme for miRNA biology (unpublished data). Using mass spectrometry analysis we identified three residues from DICER as 4-HNE targets (K1324, H1325, K1339) in heart failure. Of interest, these residues are located into the catalytic domain RNAse IIIA and close to the active site motif EXXXD1320. Considering that targeted deletion of Dicer in the heart leads to dilated cardiomyopathy and heart failure, we hypothesized that 4-HNE-Dicer interaction could compromise its function and drive cardiac dysfunction. Here we plan to study the impact of each individual 4-HNE-Dicer interaction on Dicer function and cell response to oxidative stress. Our preliminary results in animal and cell models demonstrate that Dicer activity is reduced under both chronic and acute aldehyde stress conditions. However, the molecular mechanisms involved in this response are still unclear. Neither the aldehyde-Dicer adduction, nor its effect on protein stability and activity has been previously reported. Of interest, Dicer activity dropped approximately 47% in failing hearts from rats and humans (cardiac specimens). A substantial reduction of mature microRNA expression was also observed in failing hearts. Notably, increasing cardiac 4-HNE clearance by activating aldehyde dehydrogenase 2 (ALDH2) prevented Dicer loss of function (~27%) in heart failure. Consistent with our in vivo data, H9C2 cells treated with 4-HNE (50µM) showed a 40% increase in 4-HNE-protein adducts followed by a 32% reduction in Dicer activity. These preliminary findings suggest that 4-HNE has a negative impact on DICER function. However, to test its direct impact and establish causality, we need to express recombinant DICER (WT and mutants) and perform some in vitro assays to measure DICER activity and stability in the presence of 4-HNE. It is also critical to express WT and mutant DICER in DICER KO MEF cells to validate the biological impact of these individual interactions. For that, we will collaborate with Dr. Ian J. MacRae from the Scripps Research Institute, CA-USA, who has wide experience with the aforementioned strategies. (AU)