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Different phenotypic outcome due to site-specific phosphorylation in the cancer-associated NQO1 enzyme studied by phosphomimetic mutations
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SYSNO ASEP 0564242 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Different phenotypic outcome due to site-specific phosphorylation in the cancer-associated NQO1 enzyme studied by phosphomimetic mutations Author(s) Pacheco-Garcia, J. L. (ES)
Anoz-Carbonell, E. (ES)
Loginov, Dmitry Sergej (MBU-M) RID
Vaňková, Pavla (BTO-N)
Salido, E. (ES)
Man, Petr (MBU-M) RID, ORCID
Medina, M. (ES)
Palomino-Morales, R. (ES)
Pey, A. L. (ES)Article number 109392 Source Title Archives of Biochemistry and Biophysics. - : Elsevier - ISSN 0003-9861
Roč. 729, OCT 30 2022 (2022)Number of pages 12 s. Language eng - English Country US - United States Keywords Flavoprotein ; Phosphorylation ; Structure-function relationships Subject RIV CE - Biochemistry OECD category Biochemistry and molecular biology Subject RIV - cooperation Institute of Biotechnology R&D Projects ED1.1.00/02.0109 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) Research Infrastructure CIISB II - 90127 - Masarykova univerzita Method of publishing Open access Institutional support MBU-M - RVO:61388971 ; BTO-N - RVO:86652036 UT WOS 000867178000002 EID SCOPUS 85137903560 DOI 10.1016/j.abb.2022.109392 Annotation 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. Workplace Institute of Microbiology Contact Eliška Spurná, eliska.spurna@biomed.cas.cz, Tel.: 241 062 231 Year of Publishing 2023 Electronic address https://www.sciencedirect.com/science/article/pii/S0003986122002764?via%3Dihub
Number of the records: 1