**Determination of the In Situ Structure of Filamentous Glutaminase by Cryo-Electron Tomography of Cellular Lamellae**

• Grant number: 24/13840-1
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: September 01, 2024
• End date: June 30, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Glaucius Oliva
• Grantee: Carlos Fabiano Capato
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Associated research grant: 13/07600-3 - CIBFar - Center for Innovation in Biodiversity and Drug Discovery, AP.CEPID

Abstract

Tumor cells exhibit increased glycolytic and glutaminolytic pathways, consuming glucose and glutamine at accelerated rates. The intensification of the glycolytic pathway, known as the Warburg effect, and the glutaminolytic pathway provide various intermediates for different synthetic routes and are hallmarks of malignant transformation. Glutamine, besides providing biosynthetic intermediates for the three main cellular blocks (amino acids, nucleic acids, and lipids), also supplies energy, glutathione, and NADPH for redox balance. Glutamine is crucial for maintaining an undifferentiated environment and metastatic phenotype, with its metabolism directly linked to tumor aggressiveness. Glutaminase is the main enzyme in the degradation pathway of this amino acid in cancer. Several levels of regulation of this enzyme are known to date, including transcriptional, post-translational (through covalent modifications), and allosteric regulation. Our group determined the structure of glutaminase (specifically the glutaminase C isoform) by single-particle cryo-electron microscopy (Cryo-EM) in the presence of the activator inorganic phosphate (Pi) and showed that it forms elongated filaments composed of tetrameric protomers. We demonstrated by confocal fluorescence microscopy, transmission electron microscopy, and tomography under cryogenic conditions of cellular lamellae that these filaments are formed inside mitochondria when cells reach low levels of glutamine. The in situ structure, through subtomogram averaging, achieved a maximum global resolution of 28 Å. The formation of these filaments within mitochondria makes them more elongated (by resistance to fission) and resistant to mitophagy. This postdoctoral project aims to advance the understanding of the molecular determinants of these filament formations by resolving the in situ structure of glutaminase using Cryo-FIB ET at higher resolutions (> 5 Å) while gaining technical and experimental knowledge in cutting-edge structural biology methods to be made available and conducted in Brazil. For this, we will count on the continued collaboration with Dr. Simone Mattei's group from EMBL Heidelberg, Germany. The chances of success of this proposal are enhanced by the recent grant from FAPESP for two electron microscopes to our institute (IFSC/USP), one of which is the Aquilos 2 (ThermoFisher Scientific), essential for the preparation of cryo-lamellae by correlative light-electron microscopy.