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Impact of aldehydes on Dicer activity and expression profile: benefits of ALDH2 activation

Grant number: 16/00900-0
Support type:Scholarships in Brazil - Master
Effective date (Start): July 01, 2016
Effective date (End): September 01, 2018
Field of knowledge:Biological Sciences - Physiology
Principal Investigator:Julio Cesar Batista Ferreira
Grantee:Ligia Akemi Kiyuna
Home Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo, SP, Brazil
Associated scholarship(s):17/14426-0 - Oxidative stress-mediated post-translational regulation of Dicer: structural and functional characterization of 4-HNE-DICER interaction, BE.EP.MS

Abstract

Oxidative stress contributes to the establishment and progression of a wide variety of pathological conditions thorough accumulation of reactive oxygen species (ROS), which can act directly on the cellular components. Additionally, they are responsible for the generation of different pro-oxidant molecules, such as the aldehyde 4-hydroxi- 2- nonenal (4-HNE), which is the main product of lipid peroxidation. This aldehyde is highly reactive and forms Michaelis adducts with proteins, lipids and DNA, causing target loss of function and degradation. For this reason, accumulation of 4-HNE has been correlated to the development of many diseases, such as cancer, neurodegenerative and cardiovascular diseases. Previous studies from our group found a significant increase in cardiac 4-HNE adducts following myocardium infarction in rats, which was associated to the development of heart failure. As a result of a proteomic study, the endonuclease DICER was identified as one of the main targets of 4-HNE in this pathological condition. DICER has an important role in microRNAs biogenesis and post-transcriptional gene regulation. More recently, DICER levels were shown to be affected by oxidative stress. However, neither the mechanisms involved in oxidative stress-mediated DICER post-translational regulation nor its effects on the miRNA biogenesis have been elucidated. This study aims to characterize 4-HNE as a regulator of DICER in oxidative stress. The first step of this project consists on validating the initial proteomic findings, which first described 4-HNE interaction with DICER in heart failure. In addition, we intend to evaluate its effects on DICER activity and expression profile in different experimental models including rats, cell culture and the nematode Caenorhabditis elegans. The C. elegans model will allow in vivo analysis of DICER expression in response to different experimental conditions. Finally, we will validate the potential role of selective activation of the aldehyde dehydrogenase 2 (ALDH2), the main enzyme that remove 4-HNE, in preventing and/or reducing 4-HNE-DICER interaction. Oxidative stress will be induced using two chemical stressors independently: Paraquat (superoxide anion generator) and 4-HNE. DICER activity will be assessed by two fluorescence based assays: for tests with modified lineage of HEK 293 (RNAi-293-EGFP / RFP) we will employ a real time imaging method; in addition, a DICER specific molecular beacon will be used for cell and tissue lysate analysis. DICER expression levels will be assessed by qPCR and immunoblotting in cell and tissue lysates; real time imaging will be also used for a C. elegans fluorescence based in vivo assay. Our hypothesis is that 4-HNE accumulation in oxidative stress conditions alters DICER activity and/or expression profile through the Michaelis adducts formation. Our preliminary results seem promising once it demonstrated that 4-HNE impairs siRNA biogenesis in HEK cells.