Phosphorylation compromises FAD binding and intracellular stability of wild-type and cancer-associated NQO1: Insights into flavo-proteome stability Medina Carmona, Encarnación Rizzuti, Bruno Martín Escolano, Rubén Pacheco García, Juan Luis Mesa Torres, Noel Neira, José L. Guzzi, Rita Pey Rodríguez, Ángel Luis Over a quarter million of protein phosphorylation sites have been identified so far, although the effects of site-specific phosphorylation on protein function and stability, as well as their possible impact in the phenotypic manifestation in genetic diseases are vastly unknown. We investigated here the effects of phosphorylating S82 in human NADP(H):quinone oxidoreductase 1, a representative example of disease-associated flavoprotein in which protein stability is coupled to the intracellular flavin levels. Additionally, the cancer-associated P187S polymorphism causes inactivation and destabilization of the enzyme. By using extensive in vitro and in silico characterization of phosphomimetic S82D mutations, we showed that S82D locally affected the flavin binding site of the wild-type (WT) and P187S proteins thus altering flavin binding affinity, conformational stability and aggregation propensity. Consequently, the phosphomimetic S82D may destabilize the WT protein intracellularly by promoting the formation of the degradation-prone apo-protein. Noteworthy, WT and P187S proteins respond differently to the phosphomimetic mutation in terms of intracellular stability, further supporting differences in molecular recognition of these two variants by the proteasomal degradation pathway. We propose that phosphorylation could have critical consequences on stability and function of human flavoproteins, important for our understanding of genotype-phenotype relationships in their related genetic diseases. 2025-05-12T06:29:18Z 2025-05-12T06:29:18Z 2019-03-15 journal article Medina-Carmona E, Rizzuti B, Martín-Escolano R, Pacheco-García JL, Mesa-Torres N, Neira JL, Guzzi R, Pey AL. Phosphorylation compromises FAD binding and intracellular stability of wild-type and cancer-associated NQO1: Insights into flavo-proteome stability. Int J Biol Macromol. 2019 Mar 15;125:1275-1288. doi: 10.1016/j.ijbiomac.2018.09.108. Epub 2018 Sep 19. PMID: 30243998. https://hdl.handle.net/10481/104053 10.1016/j.ijbiomac.2018.09.108 eng http://creativecommons.org/licenses/by-nc-nd/4.0/ open access Attribution-NonCommercial-NoDerivatives 4.0 Internacional Elsevier