Estudio de los efectos de mutaciones naturales y modificaciones postraduccionales sobre la estabilidad y funcionalidad de la enzima asociada a cáncer NQO1

Abstract

The stability and function of proteins can be affected by mutations that result in the substitution of one amino acid for another (missense mutation) as well as by posttranslational modifications. When this effect is hereditary and leads to the loss of protein stability and function, it often results in diseases known as loss-of-function diseases. For example, flavoproteins, which have multiple functions, are associated with various hereditary diseases due to mutations that lead to the loss of their function. Specifically, in this thesis, we are going to study the effect of natural mutations and post-translational modifications in the NAD(P)H:quinone oxidoreductase 1 (NQO1) protein, a multifunctional flavoprotein, whose loss of function due to the P187S polymorphism has been linked to a potential increase in the risk of cancer. First, we have studied various natural variants that have been detected in population and cancer sample massive sequencing studies, but whose effect on the stability and function of NQO1 was unknown. These variants had missense mutations at the Nterminal end and in the active site of the protein, and the effect of these mutations propagated to distant regions from the mutated site, causing, in some cases, decreased thermal stability, reduced affinity for FAD, and reduced catalytic activity. Secondly, we compared how the destabilization caused by phosphorylation resembled that caused by the disease-associated P187S polymorphism, using the phosphomimetic mutation S82D. To do this, we studied how both mutations affected the structural stability of the protein in different regions using hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), combined with limited proteolysis studies, enzymatic assays, and cell culture experiments. The results showed that both S82D and P187S had various effects on NQO1, such as reduced catalytic activity or intracellular stability, which occurred due to the structural propagation of their destabilizing effect to various functional sites. Lastly, we compared the consequences of site-specific phosphorylation at three residues (S40, S82, and T128) with varying solvent exposure following the methodology used with S82. In this case, we observed that each phosphomimetic mutation (S40D, S82D, and T128D) had different effects. S82D caused the most destabilization, significantly affecting FAD affinity, enzymatic activity, and conformational and intracellular 8 stability. While S40D mainly affected conformational and intracellular stability, and T128D had no appreciable effect on the stability of NQO1 but did impact enzymatic activity.