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Impact of 4-hydroxinonenal on Dicer regulation: a translational approach

Grant number: 17/16694-2
Support type:Regular Research Grants
Duration: April 01, 2018 - March 31, 2020
Field of knowledge:Biological Sciences - Physiology
Principal Investigator:Julio Cesar Batista Ferreira
Grantee:Julio Cesar Batista Ferreira
Home Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo, SP, Brazil

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using proteomics that 4-HNE directly targets DICER during heart failure in rats, a critical enzyme for miRNA biology (unpublished data).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. Dicer is an essential endonuclease in the biogenesis of miRNAs, and consequently, in post-transcriptional gene regulation. Of interest, oxidative stress reduces the expression of DICER. However, the mechanisms involved in this process, as well as its contribution to miRNA biogenesis, post-transcriptional gene regulation and cellular functioning have not yet been elucidated. In this context, the objective of the present research proposal is to characterize 4-HNE as the regulating agent of DICER under oxidative stress. For this, we will validate our initial findings of interaction between 4-HNE and DICER and their impact on the miRNA profile in heart failure. In addition, we will characterize the impact of this interaction on the activity and expression of DICER in cell culture (HEK 293 and H9C2 cells), in tissue lysate and in vivo using the nematode Caenorhabditis elegans that constitutively expresses the DICER-EGFP construct. The direct effect of this interaction will be studied in vitro using wild-type recombinant DICER and with point mutations that neutralize the action of 4-HNE on the respective amino acids. Furthermore, we will test the pharmacological potential of the selective activation of the enzyme aldehyde dehydrogenase 2 (ALDH2) in the prevention or reduction of the 4-HNE-DICER interaction in vivo and in the cell culture. ALDH2 is the major enzyme involved in the degradation of 4-HNE. Finally, these findings will be validated in cardiac biopsies from donors and patients with ischemic heart failure, strengthening the translational character of the present research proposal. Our hypothesis is that the accumulation of 4-HNE in oxidative stress directly impairs the activity and/or expression of DICER through the 4-HNE-DICER adduct, affecting the biogenesis of miRNAs. Our preliminary results are promising, demonstrating that 4-HNE treatment compromises the biogenesis of siRNA in HEK cells over time. Considering the current relevance of the miRNA profile in cell biology and physiopathology, a more detailed understanding of possible intermolecular interactions affecting the activity of limiting enzymes in the biogenesis process of miRNAs, as well as their regulation by oxidative stress, will be of great value for the future use of pharmacological and non-pharmacological therapies that act on key mechanisms involved in the pathophysiology of degenerative diseases, such as heart failure. (AU)