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Genetic and metabolic interactions among lactate dehydrogenase, glycerol phosphate dehydrogenase and alternative oxidase

Grant number: 22/05632-4
Support type:Scholarships in Brazil - Post-Doctorate
Effective date (Start): June 01, 2022
Effective date (End): May 31, 2024
Field of knowledge:Biological Sciences - Biochemistry - Metabolism and Bioenergetics
Principal researcher:Marcos Túlio de Oliveira
Grantee:Carlos Antonio Couto Lima
Home Institution: Faculdade de Ciências Agrárias e Veterinárias (FCAV). Universidade Estadual Paulista (UNESP). Campus de Jaboticabal. Jaboticabal , SP, Brazil
Associated research grant:21/06711-2 - Modulation of tissue growth and biomass accumulation by the mitochondrial alternative oxidase, AP.JP2

Abstract

The xenotopic expression of mitochondrial alternative oxidase (AOX) has been related to a partial or total reversal of mitochondrial dysfunction in several animal models, whether exclusive mitochondrial dysfunction or diseases where ROS production may be a supporting factor. In this way, AOX is increasingly described as a candidate for human therapies. AOX is a mitochondrial inner membrane enzyme that can bypass complexes III and IV of the respiratory chain, providing an additional pathway for oxygen reduction and coenzyme Q reoxidation, and allowing continued metabolic flux in situations where the electron transfer is compromised. Therefore, the analysis of the alterations caused by the insertion of AOX in the human respiratory chain is important for the understanding of metabolic events. Under conditions of nutritional stress, we will perform high-resolution respirometry assays using various larval tissues to demonstrate how mitochondria function in the presence of varying levels of AOX. We will combine mitochondrial oxygen consumption in different states (OXPHOS, leak, and uncoupled) with measurements of membrane potential, ATP production, and ROS. Data from our laboratory suggest that the presence of AOX can create parallel electron transfer pathways in larval mitochondria. Identifying the nutritional preferences of different tissues will certainly help us to interpret and infer the tissue-specific metabolic rearrangements caused by AOX. We will also associate AOX expression with the overexpression, depletion, or abolition of one of the paralogs encoding mGPDH in Drosophila, GPO1. We postulate that mGPDH overexpression will increase mGPDH-AOX uncoupling and decrease AOX-induced larval biomass accumulation and pupal viability. Increased metabolic flux through mGPDH-AOX under conditions of high membrane potential, as suggested, perhaps forces the larval production of glycerol-3-phosphate (G3P), the substrate for para mGPDH. Previous work has shown that cytosolic glycerol-3-phosphate dehydrogenase (cGPDH) works in concert with lactate dehydrogenase (LDH), coordinating the cellular redox environment and the growth of Drosophila larvae. The production of G3P from dihydroxyacetone phosphate (DHAP) is catalyzed by cGPDH in an NADH-dependent manner. The fact that we observed altered metabolic flux through mGPDH-AOX and elevated levels of LDH, lactate, and 2-hydroxyglutarate raises the question of whether and how LDH is contributing directly to the metabolic rearrangements necessary for the development of AOX-expressing larvae. We intend to overexpress, reduce or abrogate LDH expression in the presence of varying levels of AOX and allow the larvae to develop under nutritional stress. Furthermore, the combination of mGPDH overexpression, knockdown, and knockout with the LDH and AOX genotypes will give us a broad view of the interactions between these candidates, in addition to allowing the analysis at the metabolic level. Studying this specific balance between lactate and G3P metabolism is important for understanding how growing tissues can reprogram their metabolism by inhibiting a specific pathway, such as the switch to G3P oxidation and cell survival that occurs when cancer cells are treated with LDH-inhibiting drugs. (AU)

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