Heterologous production, purification, functional characterization, and structural prediction of the human mitochondrial pyruvate carrier

Abstract

El piruvato es un metabolito clave para la generación de energía y procesos biosintéticos en las células. Se oxida en el ciclo del ácido cítrico que tiene lugar en la matriz mitocondrial. Para que el piruvato citosólico sea transportado a la matriz mitocondrial, se requiere un proceso que involucra subunidades del transportador mitocondrial de piruvato (MPC) ensambladas en oligómeros heterotípicos. Desde 2012, se conocen las identidades moleculares de MPC1 y MPC2 en mamíferos, y MPC3 en levaduras. Sin embargo, el mecanismo funcional basado en la estructura de MPC sigue siendo desconocido, a pesar de los esfuerzos realizados por diferentes grupos de investigación. Esta tesis describe la producción recombinante de MPC humano utilizando una estrategia de coexpresión, que resultó en la purificación de subunidades individuales predominantemente. Se encontró que los homo-oligómeros de MPC2 promueven un transporte eficiente de piruvato a proteoliposomas, mientras que la inhibición química del transporte solo se observó para un derivado de tiazolidinediona. El papel autónomo de transporte para MPC2 se validó en células cuando la expresión ectópica de MPC2 humano en levaduras sin MPC endógeno estimuló el crecimiento y aumentó el consumo de oxígeno. El análisis de criomicroscopía electrónica de MPC2 reconstituido en nanodiscos de copolímero sintético permitió la propuesta de un ensamblaje estequiométrico. Estos resultados proporcionan el marco inicial para el papel independiente de MPC2 en la homeostasis y las enfermedades relacionadas con el metabolismo del piruvato desregulado, y pueden representar una ruta prometedora para la determinación de un modelo atómico de alta resolución para MPC2 humano.

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.