Analysis of the formation of aggregates of the metabolic enzymes GS and ALDH2 and their impact on mitochondrial function

• Grant number: 23/17617-2
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: March 01, 2024
• End date: December 31, 2024
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal Investigator: Raquel Arminda Martinez Machado
• Grantee: Thaís Fontes Milz Casal
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
• Associated research grant: 21/05726-6 - Metabolism in the microenvironment and the role of metabolic exchanges in tumor progression, AP.TEM

Abstract

Unlike traditional studies focused on reactions delimited by membrane and organelle compartments, new work has been evaluating the dynamics of proteins in non-membranous compartments, which can play critical roles in the regulation of metabolic networks. In this context, several proteins with the ability to form biomolecular and/or filamentous condensates have been described, but the connection between these structures and the regulation of enzymatic activity is still poorly understood. Recent work has identified 60 yeast metabolic enzymes with the ability to form aggregates under nutritional stress. Based on this study, and on preliminary results obtained in our laboratory, we selected two mammalian metabolic enzymes, glutamine synthetase (GS) and aldehyde dehydrogenase 2 (ALDH2), to be studied for their ability to form aggregates in cells in response to changes in the cultivation conditions, and its consequent impact on mitochondrial function. To do this, we will use the human prostate carcinoma cell line, PC3, and perform the ectopic expression of these enzymes in fusion with the mKO2 fluorescent protein. After transfection, cells will be subjected to different nutritional stress conditions and will proceed to screening, where we will be able to observe changes in the fluorescence pattern using the Operetta CLS" automatic imaging system. Possible changes in mitochondrial respiration will be measured by the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XF24. We believe this work may contribute to a better understanding of the molecular mechanisms involved in the condensation of metabolic enzymes and their implications for the regulation of cellular metabolism.