Different phenotypic outcome due to site-specific phosphorylation in the cancer-associated NQO1 enzyme studied by phosphomimetic mutations

作     者：Pacheco-Garcia, Juan Luis Anoz-Carbonell, Ernesto Loginov, Dmitry S. Vankova, Pavla Salido, Eduardo Man, Petr Medina, Milagros Palomino-Morales, Rogelio Pey, Angel L. 

作者机构：Univ Granada Dept Quim Fis Ave Fuentenueva S-N Granada 18071 Spain Univ Zaragoza Fac Ciencias Dept Bioquim & Biol Mol & Cellular Inst Biocomp & Fis Sistemas Complejos BIFIGBsC CS Zaragoza 50009 Spain Acad Sci Czech Republ Inst Microbiol BioCeV Prumyslova 595 Vestec 25250 Czech Republic Acad Sci Czech Republ Inst Biotechnol BioCeV Prumyslova 595 Vestec 25250 Czech Republic Univ La Laguna Hosp Univ Canarias Ctr Rare Dis CIBERER Tenerife 38320 Spain Univ Granada Fac Sci Dept Biochem & Mol Biol 1 Granada Spain Univ Granada Biomed Res Ctr CIBM Granada Spain Univ Granada Dept Quim Fis Un Excelencia Quim Aplicada Biomed & Medioambiente Ave Fuentenueva S-N Granada 18071 Spain Univ Granada Inst Biotecnol Ave Fuentenueva S-N Granada 18071 Spain 

出 版 物：《ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS》 (生物化学与生物物理学集刊)

年 卷 期：2022年第729卷第0期

页      面：000-000页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 07[理学] 

基　　金：ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency [RTI2018-096246-B-I00] Consejeria de Economia, Conocimiento, Empresas y Universidad, Junta de Andalucia [P18-RT-2413] ERDF/Counseling of Economic transformation, Industry, Knowledge and Universities, Junta de Andalucia [B-BIO-84-UGR20] MCIN/AEI [PID2019-103901 GB-I00] Government of Arag on-FEDER [E35_20R] Horizon 2020 EU_FT-ICR_MS project EU/MEYS projects BioCeV [CZ.1.05/1.1.00/02.0109] CIISB [LM2018127] Universidad de Granada/CBUA 

主　　题：Flavoprotein Phosphorylation Structure-function relationships 

摘      要：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.