Identification of new enzymes capable of aggregating under conditions of nutrient deprivation and their role in tumor progression.

• Grant number: 23/01388-4
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: May 01, 2023
• End date: September 30, 2024
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Sandra Martha Gomes Dias
• Grantee: Raquel Arminda Martinez Machado
• 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

One of the central problems of cell biology is how cells organize biochemical reactions in space and time. Traditionally, studies of this problem have focused on reactions concentrated within membrane compartments and organelles. Recently, however, there has been a growing appreciation that the dynamic partitioning of proteins into novel non-membranous compartments can be used to regulate cytoplasmic processes such as signal transduction and RNA metabolism. It is currently believed that the process of formation of non-membranous compartments may also play a critical role in the regulation of metabolic networks. Several studies have identified metabolic enzymes that are capable of forming biomolecular and/or filamentous condensates; however, the connections between these structures and the regulation of enzymatic activity are only known for a limited number of cases. Recent work has identified 60 yeast metabolic enzymes capable of forming structures, focus or filament (detected by fluorescence microscopy), under certain growth conditions and metabolic states. The study showed that the association often happened at metabolic enzymes at connection points of a given major pathway with its metabolic arms. In this way, it is speculated that the aggregates may be important for, in times of growth or stress, promoting or inactivating the activity of a given enzyme and, in this way, guaranteeing the metabolic flow to different destinations. Several of the enzymes detected in yeast as being capable of changing the fluorescence profile from diffuse to aggregate (focus or filament), due to changes in the metabolic state, are conserved in mammals. For few of them the same phenomenon has already been shown in mammalian cells, and even smaller is the number of enzymes for which the structure in these aggregated states has been defined by electron microscopy. In this sense, in this project, we will evaluate 14 mammalian metabolic enzymes (glycolytic pathway, purine biosynthesis pathway, glutamine metabolism pathway and methionine/homocysteine metabolization pathway) homologous to those of yeast, regarding their ability to aggregate, in situ , in response to changes in growing conditions. The enzymes will be ectopically expressed in fusion with the fluorescent protein mKO2 and we will manipulate the culture medium in order to remove glutamine keeping the other amino acids (+aa -Gln), glutamine and all others (-aa -Gln), all amino acids keeping glutamine (-aa + Gln) and glucose, the main nutrients involved in the described pathways. Enzymes that change the aggregation pattern in the cell as a result of nutrient manipulation will be cloned into expression vectors in E. coli, expressed and purified in vitro. Its oligomerization state will be evaluated in function of the addition of molecules that can potentially change its state as substrates, products and cofactors. At least 1 of the enzymes that show oligomerization capacity in vitro and for which we found the conditions for this will be evaluated by electron microscopy with negative satinning and cryo-microscopy. Studies with selected enzymes will be performed intwocollaboration with Prof. Andre Ambrósio (Physics Institute, USP - São Carlos). For this enzyme, if we have the necessary information to understand the molecular mechanisms of polymerization, we will use syngeneic implantation techniques in immunocompetent mice, followed by analysis of the profile of immune infiltrates and metabolites. Tumor cells will be modified in order to modify polymerization (constitutive formation or breakage) by introducing mutations in the enzyme gene. These cells will also be studied in vitro to verify the impact of enzyme polymerization on oxygen consumption and lactate secretion (cellular acidification).