Departamento de Química Física, Universidad de Granada, Av. Fuentenueva s/n, 18071, Granada, Spain.
Departamento de Bioquímica y Biología Molecular y Cellular, Facultad de Ciencias, Instituto de Biocomputación y Física de Sistemas Complejos (BIFI) (GBsC-CSIC Joint Unit), Universidad de Zaragoza, 50009, Zaragoza, Spain.
Arch Biochem Biophys. 2022 Oct 30;729:109392. doi: 10.1016/j.abb.2022.109392. Epub 2022 Sep 9.
Protein phosphorylation is a common phenomenon in human flavoproteins although the functional consequences of this site-specific modification are largely unknown. Here, we evaluated the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects as well as in the structural stability of the antioxidant and disease-associated human flavoprotein NQO1 using biophysical and biochemical methods. In vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by H/D exchange in different ligation states provided structural insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. We show that site-specific phosphorylation of human NQO1 may cause pleiotropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease. Our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scale.
蛋白质磷酸化是人类黄素蛋白中的一种常见现象，尽管这种特定部位修饰的功能后果在很大程度上是未知的。在这里，我们使用模拟磷酸化的突变（S40、S82 和 T128 位点）评估了其对多种功能方面的影响，以及对抗氧化和与疾病相关的人类黄素蛋白 NQO1 的结构稳定性的影响，使用了生物物理和生化方法。体外生物物理研究揭示了磷酸化在不同部位的影响，例如降低 FAD 的结合亲和力和其结合位点（S82）的结构稳定性、构象稳定性（S40 和 S82）以及降低催化效率和功能协同性（T128）。不同连接状态下的 H/D 交换局部稳定性测量提供了这些影响的结构见解。真核细胞的转染表明，S40 和 S82 位点的磷酸化可能通过增强蛋白酶体诱导的降解来降低 NQO1 蛋白的稳定水平。我们表明，人类 NQO1 的位点特异性磷酸化可能会对这种多功能蛋白产生多效和违反直觉的影响，这可能对其与人类疾病的关系产生影响。我们的方法允许在体外和细胞内建立特定部位磷酸化、功能和结构稳定性之间的关系，为在黄素蛋白组范围内进行更详细的磷酸化分析铺平了道路。
