Molecular and Structural Determinants of Glutaminase C Filament Formation: Implications for Mitochondrial Biology in Tumor Cells

• Grant number: 24/22806-1
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: June 01, 2025
• End date: May 31, 2029
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Andre Luis Berteli Ambrosio
• Grantee: Lorgio Victor Bautista Samaniego
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Associated research grant: 21/05726-6 - Metabolism in the microenvironment and the role of metabolic exchanges in tumor progression, AP.TEM

Abstract

Cancer cells exhibit increased glycolytic and glutaminolytic pathways, consuming glucose and glutamine at high rates. The intensification of glycolysis (Warburg effect) and glutaminolysis provides essential intermediates for the synthesis of amino acids, nucleic acids, and lipids, as well as energy, glutathione, and NADPH for redox homeostasis. Glutamine also contributes to maintaining the undifferentiated and metastatic phenotype, with its metabolism being associated with tumor aggressiveness. Glutaminase (EC 3.5.1.2) is the key enzyme in the degradation pathway of this amino acid in cancer.Our group determined the structure of Glutaminase C (GAC) using single-particle Cryo-EM in the presence of the activator inorganic phosphate (Pi), revealing that it forms elongated filaments composed of tetrameric protomers. Using confocal fluorescence microscopy, transmission electron microscopy, and cryogenic tomography, we demonstrated that these filaments form inside mitochondria when cells exhibit low glutamine levels. The formation of these mitochondrial filaments renders the organelles more elongated (by resisting fission) and resistant to mitophagy.The main objective of this project is to advance the understanding of the molecular determinants of filament formation by resolving the structure of GAC mutants that stabilize these filaments using Cryo-EM and Cryo-ET. Specifically, the human variants K320hA (associated with the activation loop) and S482hC (related to the catalytic triad), which exhibit Pi-independent filamentation and enzymatic hyperactivation, will be analyzed. Additionally, the near-inactive variant R382hE, associated with the filamentation interface (unable to form filaments), will also be studied. Another complementary objective is to compare the filamentation capacity of these variants in a mitochondrial context using advanced fluorescence microscopy techniques.