Produção heteróloga, purificação, caracterização funcional e predição estrutural do carregador mitochondrial de piruvato humano

Author(s)

José Edwin Neciosup Quesñay

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2020-11-18

Examining board members

Andre Luís Berteli Ambrosio; Richard Charles Garratt; Maria Cristina Nonato; Shaker Chuck Farah; Germán Gustavo Sgro

• Advisor: Andre Luís Berteli Ambrosio

Abstract

Pyruvate is considered the central hub in the cellular metabolism, key for the generation of both energy and biosynthetic blocks. When the oxidation of pyruvate by the citric acid cycle is required, cytosolic pyruvate must gain access to the mitochondrial matrix in a process thought to actively involve two mitochondrial pyruvate carrier (MPC) subunits assembled into heterotypic oligomers. Since 2012, when molecular identities of mammalian MPC1 and MPC2 (and MPC3 in yeast) were presented, range of in vitro and in vivo studies has since revealed an unexpected interplay between two or even three protein subunits that define different functional assemblies on a metabolic context-specific basis; these have clear implications on the physiology of homeostasis and diseases. However, the structure-based functional mechanism of MPC remains elusive, despite intensive efforts by different research groups that employ state-of-the-art computational tools and experimental techniques. In this thesis, the recombinant production of human MPC through a co-expression strategy is first described; nevertheless, substantial complex formation was not observed, and predominantly individual subunits were purified. In contrast to MPC1, which co-purifies with a host chaperone, we demonstrated that MPC2 homo-oligomers promote efficient pyruvate transport into proteoliposomes. The derived functional requirements and kinetic features of MPC2 resemble those previously demonstrated for MPC in the literature. Distinctly, chemical inhibition of transport is observed only for a thiazolidinedione derivative. The autonomous transport role for MPC2 is validated in cells when the ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth and increased oxygen consumption. Multiple oligomeric species of MPC2 across mitochondrial isolates, purified protein and artificial lipid bilayers suggest functional high-order complexes. Significant changes in the secondary structure content of MPC2, as probed by synchrotron radiation circular dichroism, further supports the interaction between the protein and ligands. X-ray crystallography was hampered by the inability to grow suitable crystals by vapor diffusion and in lipidic cubic phase, despite the successful obtention of promising hit conditions. Lastly, cryo-electron microscopy analysis of MPC2 reconstituted into synthetic copolymer nanodiscs allowed for the proposition of a stoichiometric assembly. Collectively, our results provide the initial framework for the independent role of MPC2 in homeostasis and diseases related to dysregulated pyruvate metabolism and may represent a promising route for the determination of a high-resolution atomic model for human MPC2. (AU)

• FAPESP's process: 15/02734-7 - Functional and structural studies of four homologous recombinants of the mitochondrial pyruvate carrier complex
• Grantee: José Edwin Neciosup Quesñay
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)