A small molecule chaperone rescues the stability and activity of a cancer-associated variant of NAD(P)H:quinone oxidoreductase 1 invitro.

Emilia Strandback,Tobias Madl,Wolf‐Dieter Lienhart,Altijana Hromic‐Jahjefendic,Klaus Zangger,Andreas Winkler,Benjamin Bourgeois,Peter Macheroux,Anja Högler,Karl Gruber,Daniel Waltenstorfer

doi:10.1002/1873-3468.13636

Abstract

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a human FAD‐dependent enzyme that plays a crucial role in the antioxidant defense system. A naturally occurring single‐nucleotide polymorphism (NQO1*2) in the NQO1 gene leads to an amino acid substitution (P187S), which severely compromises the activity and stability of the enzyme. The NQO1*2 genotype has been linked to a higher risk for several types of cancer and poor survival rate after anthracycline‐based chemotherapy. In this study, we show that a small molecular chaperone (N‐(2‐bromophenyl)pyrrolidine‐1‐sulfonamide) repopulates the native wild‐type conformation. As a consequence of the stabilizing effect, the enzymatic activity of the P187S variant protein is strongly improved in the presence of the molecular chaperone in vitro.

Highlights

Emilia Strandback1, Wolf-Dieter Lienhart1, Altijana Hromic-Jahjefendic2,3, Benjamin Bourgeois4, Anja Ho€gler1, Daniel Waltenstorfer1, Andreas Winkler1, Klaus Zangger5, Tobias Madl4,6, Karl Gruber2 and Peter Macheroux1
Abbreviations BPPSA, N-(2-bromophenyl)pyrrolidine-1-sulfonamide; Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), hydrogen-deuterium exchange coupled to mass spectrometry; HSQC, heteronuclear single quantum coherence; Isothermal titration calorimetry (ITC), isothermal titration calorimetry; NQO1, NAD(P)H:quinone oxidoreductase 1; PDB, protein data bank; Small-angle X-ray scattering (SAXS), small-angle X-ray scattering; SNP, single-nucleotide polymorphism; WT, wild-type
To further investigate how the ligand BPPSA influences the stability of NQO1, limited proteolysis experiments were performed in the presence and absence of BPPSA

Summary

Experimental procedures

All chemicals and reagents were of highest quality available either from Sigma-Aldrich 800 lL of NQO1 WT or NQO1 P187S variant in 50 mM HEPES, pH 7.0, at a concentration of 40 lM in the measurement cell, was titrated with BPPSA (in 2-lL steps from a 2 mM stock, dissolved in 50% v/v EtOH). The reaction mixture for the assay performed with variation of the concentration of NADH contained 2.5 nM NQO1 WT or 20 nM NQO1 P187S variant, 200 lM menadione (e333 nm = 2450 MÀ1ÁcmÀ1, prepared in EtOH with a final concentration of 1% v/v), 1–10 mM NADH (e340 nm = 6220 MÀ1ÁcmÀ1), and 10 times the KD of FAD equal to 0.6 lM for NQO1 WT and 4.2 lM for the NQO1 P187S variant, respectively. Analogous dilutions were prepared with wild-type and variant samples preincubated with 750 lM BPPSA and 4.9 lM FAD to achieve the same final protein concentrations. The receptor was kept rigid, while the ligand BPPSA was flexible

Results

Discussion

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