title: FAD binding overcomes defects in activity and stability displayed by cancer-associated variants of human NQO1.
abstract: NAD(P)H quinone oxidoreductase 1 is involved in antioxidant defence and protection from cancer, stabilizing the apoptosis regulator p53 towards degradation. Here, we studied the enzymological, biochemical and biophysical properties of two cancer-associated variants (p.R139W and p.P187S). Both variants (especially p.187S) have lower thermal stability and greater susceptibility to proteolysis compared to the wild-type. p.P187S also has reduced activity due to a lower binding affinity for the FAD cofactor as assessed by activity measurements and direct titrations. Native gel electrophoresis and dynamic light scattering also suggest that p.P187S has a higher tendency to populate unfolded states under native conditions. Detailed thermal stability studies showed that all variants irreversibly denature causing dimer dissociation, while addition of FAD restores the stability of the polymorphic forms to wild-type levels. The kinetic destabilization induced by polymorphisms as well as the kinetic protection exerted by FAD was confirmed by measuring denaturation kinetics at temperatures close to physiological. Our data suggest that the main molecular mechanisms associated with these cancer-related variants are their low binding affinity for FAD and/or kinetic instability. Thus, pharmacological chaperones may be useful in the treatment of patients bearing these polymorphisms.
authors.affiliation: Department of Physical Chemistry, Faculty of Sciences, University of Granada, Av. Fuentenueva s/n, 18071, Spain. Electronic address: angelpey@ugr.es.
authors.forename: Angel L
authors.initials: AL
authors.lastname: Pey
authors.affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK.
authors.forename: Clare F
authors.initials: CF
authors.lastname: Megarity
authors.affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK; Institute for Global Food Security, Queen's University Belfast, 18-30 Malone Road, Belfast BT9 5BN, UK. Electronic address: d.timson@qub.ac.uk.
authors.forename: David J
authors.initials: DJ
authors.lastname: Timson
keywords: Cancer-associated polymorphism; Kinetic instability; NAD(P)H quinone oxidoreductase 1; NQO1*2; NQO1*3