Phosphorylation compromises FAD binding and intracellular stability of wild-type and cancer-associated NQO1: Insights into flavo-proteome stability

• Published in: International journal of biological macromolecules Vol. 125; pp. 1275 - 1288
• Main Authors: Medina-Carmona, Encarnación, Rizzuti, Bruno, Martín-Escolano, Rubén, Pacheco-García, Juan Luis, Mesa-Torres, Noel, Neira, José L., Guzzi, Rita, Pey, Angel L.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.03.2019
• Subjects: Amino Acid Sequence; Binding Sites; Cell Line; Computational Biology - methods; Conformational disease; Flavin-Adenine Dinucleotide - chemistry; Flavin-Adenine Dinucleotide - metabolism; Flavoprotein; Humans; Kinetics; Molecular Dynamics Simulation; Mutation; NAD(P)H Dehydrogenase (Quinone) - chemistry; NAD(P)H Dehydrogenase (Quinone) - genetics; NAD(P)H Dehydrogenase (Quinone) - metabolism; Neoplasms - metabolism; Phosphorylation; Protein Binding; Protein Denaturation; Protein phosphorylation; Protein Stability; Proteome; Proteomics - methods; Protein phosphorylation; Conformational disease; Flavoprotein
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2018.09.108

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. •Flavoproteins are often phosphorylated but their consequences are not known well.•We investigated phosphorylation in NQO1 using a phosphomimetic S82D mutant.•S82D affects protein structure and dynamics of the FAD binding site.•S82D increases the population of the apo-state and may destabilize NQO1 in cells.•Phosphorylation can be critical for the stability/function of the flavoproteome.