Functional characterization of CAD, an antitumoral target controlling the de novo pyrimidine biosynthesis

• Autores: Francisco del Caño Ochoa
• Directores de la Tesis: Santiago Ramon Maiques (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2019
• Idioma: inglés
• Número de páginas: 156
• Programa de doctorado: Programa de Doctorado en Biociencias Moleculares por la Universidad Autónoma de Madrid
• Materias:
  • Física
    • Física molecular
      • Estructura molecular
    • Física del estado sólido
      • Cristalografía
  • Química
    • Bioquímica
      • Enzimología
      • Proteínas
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Pyrimidine nucleotides are essential compounds for the synthesis of nucleic acids and other key cellular processes. The cells obtain the pyrimidines through two different metabolic pathways depending on their developmental stage. In differentiated cells, pyrimidines are obtained mainly by recycling through salvage pathways, and the de novo synthesis of pyrimidines is low. In contrast, when cells grow and proliferate, the activation of de novo synthesis is necessary to fuel replication and to manufacture of other essential macromolecules. In animals, three of the six enzymatic activities that constitute the de novo synthesis pathway, carbamoyl phosphate synthetase (CPS), aspartate transcarbamoylase (ATC) and dihydroorotase (DHO) are fused into a single multifunctional protein called CAD.

    This multienzyme protein initiates and controls the de novo synthesis of pyrimidines and is overexpressed in different types of cancer, which makes it a potential target for the development of antitumoral compounds. In recent years, our group has characterized the DHO and ATC enzymatic domains of human CAD, but beyond knowing the atomic structure and kinetic properties, it is necessary to study CAD in a cellular context to better understand its functioning and move towards designing compounds that regulate their activity and may have a therapeutic value. In the course of this thesis, we have addressed the study of the subcellular localization of CAD using fluorescent chimeras and generating, through CRISPR/Cas9 technology, the first human CAD knockout and GFP-CAD-knockin cell lines.

    Our results show that CAD is a protein present exclusively in the cytosol that, contradicting results published by other groups, is not transported to the nucleus during the cell cycle.

    Until recently, it was thought that due to the central role of CAD in the synthesis of pyrimidines, mutations that compromised its activity would have a lethal effect, explaining that no diseases were associated with this gene. However, since 2015, it is known that CAD-deficit is a serious metabolic disease in children at an early age who die if they are not diagnosed in time. Until now, patients are diagnosed by exome sequencing, with the associated difficulty of distinguishing between possible pathogenic mutations and undescribed variants of the protein. Thanks to the molecular tools developed in this thesis, we set up a simple cell assay that allows the identification of pathogenic mutations, helping in the correct diagnosis and treatment of patients. In addition, we have studied the effect of the pathogenic mutations on the structure and activity of the isolated CAD domains. These clinical mutations have helped us to discover key elements for the functioning of the protein.

    This detailed study of the mechanisms of CAD has led us to characterize in detail a flexible loop in the DHO domain of human CAD, and to describe its participation in the catalytic mechanism of the enzyme.