Determining the nutrient oxidation preferences by mitochondria with the alternative oxidase

• Grant number: 24/13345-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: August 01, 2024
• End date: July 31, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal Investigator: Marcos Túlio de Oliveira
• Grantee: Juliana Chibana Alves
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 21/06711-2 - Modulation of tissue growth and biomass accumulation by the mitochondrial alternative oxidase, AP.JP2

Abstract

Alternative oxidase (AOX) is a mitochondrial enzyme that catalyzes reactions analogous to those of complexes III and IV, oxidizing coenzyme Q and reducing oxygen. Although it does not contribute to the proton-motive force utilized in ATP synthesis, AOX is capable of maintaining the flow of electrons when complexes III and IV are compromised. Despite its potential therapeutic applications, the expression of AOX in Drosophila melanogaster grown on a low-nutrient medium results in lethality during the pupal stage. This phenomenon does not occur in the standard medium or in control individuals (devoid of AOX), which develop normally. The standard medium appears to contain essential nutrients for the development of AOX flies, as the accumulation of mass during the larval stage is necessary for the completion of metamorphosis. The objective of our laboratory is to ascertain which nutritional components of the standard medium permit AOX flies to undergo complete development. To this end, our objective is to gain insight into the nutritional preferences of D. melanogaster larval mitochondria with and without AOX. To achieve this, we will analyze the mitochondrial physiology of growing larvae using the O2k high-resolution respirometer. The experiment will utilize L2 larvae from the crossing of virgin females of the UAS-AOXF6 strain with a daGAL4 male and UAS-AOXF6 females with w1118 males (control animals). In order to test the malate-citrate and malate-aspartate shuttles, as well as the direct contribution of mitochondrial lactate dehydrogenase, a series of inhibitors will be employed, including those targeting lactate dehydrogenase, glutamate-oxaloacetate transaminase, and the malate/±-ketoglutarate translocator. Furthermore, oligomycin will be employed with the same substrates and inhibitors to assess leak respiration.