The role of protein dynamics in loss-of-function disease mechanisms: A NQO1 cancer-associated polymorphism as model
- Autores: Encarnación Medina Carmona
- Directores de la Tesis: Ángel Luis Pey Rodríguez (dir. tes.)
- Lectura: En la Universidad de Granada ( España ) en 2018
- Idioma: inglés
- ISBN: 9788491639176
- Número de páginas: 161
- Tribunal Calificador de la Tesis: Francisco Conejero Lara (presid.), Ana Isabel Azuaga Fortes (secret.), Salvador Ventura Zamora (voc.), José Luis Ortega Roldán (voc.), Milagros Medina Trullenque (voc.)
- Programa de doctorado: Programa de Doctorado en Química por la Universidad de Granada
- Materias:
- Enlaces
- Tesis en acceso abierto en: DIGIBUG
- Resumen
Mutations causing single amino acids replacements, also known as missense mutations, can noticeably affect diverse protein properties such as the ability to fold or the stability inside cells which often lead to disease. Specifically, disease-causing missense mutations that bring about a decreased functionality and/or stability produce the so-called loss-of-function diseases. There are numerous diseases associated with loss-of-function phenotype, involving from a large number of inherited monogenic diseases to more complex multifactorial diseases like cancer [1,2]. In particular, this doctoral thesis focuses on the study of a NAD(P)H: quinone-oxidoreductase 1 (NQO1) polymorphism, P187S, which has been associated with increased cancer risk [3]. P187S is a paradigm of loss-of-function mutation that causes significant defects on stability and function of NQO1. On the one hand, this polymorphism causes a severe reduction of enzymatic activity, possibly as a consequence of its low affinity for FAD, while its intracellular instability is linked to an enhanced proteasomal degradation [4–7]. Several researches have focused on P187S showing a X-ray structure cristallographic virtually identical compared to the structure of wild-type NQO1. In addition, Nuclear Magnetic Resonance (NMR) spectroscopy and proteolysis experiments confirmed the presence of partially unfolded states in solution although these results cannot totally explain the destructive effects of P187S in vitro and in vivo [5]. In this context, we suggest that the pathogenic mechanisms of this polymorphism could be deciphered with an in-depth analysis of the protein dynamics.
We used a combination of experimental and computational techniques to investigate the P187S effects on protein dynamics. Interestingly, the first section of our results showed that P187S seems to affect the local dynamics of two functionally and structurally distant sites of NQO1: the N-terminal domain (NTD), related to enzyme inactivation; and the C-terminal domain (CTD) which appears to play a key role in the low intracellular stability of P187S. In addition, the treatment of P187S with its natural ligand FAD may rescue in vitro activity of P187S but the combination with a second ligand (i.e. dicoumarol) that acts as stabilizer of the still dynamic CTD, is required to correct the degradation through this domain. With the goal of evaluating the structural and functional role of CTD, this domain was exhaustively evaluated. Our results provided evidence for the existence of a multifunctional allosteric network between the CTD, the FAD binding site and the P187S site. For wild-type NQO1, we suggest that this network contributes to a very high affinity for FAD and dicoumarol, whereas P187S seems to disturb the network by dynamic and structural changes in the FAD binding site and the CTD affecting the binding affinity of P187S for both ligands.
In the third section, we tried to identify hot spots that would correct the NQO1 alterations caused by P187S by consensus approach [8,9]. Specifically, we found a suppressor mutation H80R which reactivates P187S by local and dynamic stabilization of FAD binding site. The deep characterization of the changes exerted by H80R suggested that this suppressor mutation mainly acts by shifting the conformational equilibrium of the apo-state towards more competent states for the binding of FAD. A second consensus mutation E247Q showed a stabilizing effect on the CTD and this effect is propagated to the distal NTD supporting the existence of the previously proposed network. In addition, the combination of these two suppressor mutations strongly protected NQO1 from the loss-of-function caused by P187S.
In summary, the results of this doctoral thesis show that protein dynamics plays a fundamental role in the loss-of-function mechanisms associated with P187S, and its understanding is crucial to find alternatives that correct the deleterious effects of this polymorphism. We propose that our multi-disciplinary strategy could help to decipher complex mutational effects related to disease in many other loss-of-function genetic diseases, and to identify structural hot spot as targets for pharmacological correction.
