A redox-mediated conformational change in NQO1 controls binding to microtubules and α-tubulin acetylation

Abstract

The localization of NQO1 near acetylated microtubules has led to the hypothesis that NQO1 may work in concert with the NAD + -dependent deacetylase SIRT2 to regulate acetyl α-tubulin (K 40 ) levels on microtubules. NQO1 catalyzes the oxidation of NADH to NAD + and may supplement levels of NAD + near microtubules to aid SIRT2 deacetylase activity. While HDAC6 has been shown to regulate the majority of microtubule acetylation at K 40 , SIRT2 is also known to modulate microtubule acetylation (K 40 ) in the perinuclear region. In this study we examined the potential roles NQO1 may play in modulating acetyl α-tubulin levels. Knock-out or knock-down of NQO1 or SIRT2 did not change the levels of acetyl α-tubulin in 16HBE human bronchial epithelial cells and 3T3-L1 fibroblasts; however, treatment with a mechanism-based inhibitor of NQO1 (MI2321) led to a short-lived temporal increase in acetyl α-tubulin levels in both cell lines without impacting the intracellular pools of NADH or NAD + . Inactivation of NQO1 by MI2321 resulted in lower levels of NQO1 immunostaining on microtubules, consistent with redox-dependent changes in NQO1 conformation as evidenced by the use of redox-specific, anti-NQO1 antibodies in immunoprecipitation studies. Given the highly dynamic nature of acetylation-deacetylation reactions at α-tubulin K 40 and the crowded protein environment surrounding this site, disruption in the binding of NQO1 to microtubules may temporally disturb the physical interactions of enzymes responsible for maintaining the microtubule acetylome.

The diverse functionality of NQO1 and its roles in redox control

“…These microtubule structures contain high levels of acetylated α-tubulin (K40) and NQO1 was shown to co-localize with these acetylated structures in double-immunostaining studies [ 177 ]. Binding of NQO1 dimer to microtubules likely occurs via its positively charged C-terminal tails which are exposed when the enzyme is in the oxidized state [ 177 , 186 ]. A number of NAD + -dependent enzymes including SIRT2 and PARP have also been observed to co-localize with these acetylated microtubule structures suggesting that NQO1 may be providing NAD + for these enzymes [ [187] , [188] , [189] ].…”

“…The distinct reduced and oxidized conformations of NQO1 have not been structurally characterized but non-denaturing gels clearly show different migratory properties for the reduced and oxidized conformations of the enzyme [ 177 ]. In subsequent studies [ 186 ], it was demonstrated that NQO1 can exist in at least three different conformational forms, a reduced conformation, an oxidized conformation and an inactivated conformation which have different reactivities with antibodies and therefore different implications for reacting with downstream proteins.

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“…Oxidized and reduced NQO1 have different conformations but the addition of NQO1 inhibitors (dicoumarol or indolequinone mechanism based inhibitors) also induced a change in the conformation of NQO1 [ 177 , 186 ]. siRNA-mediated knockdown or CRISPR/Cas9 knockout of NQO1 in 16-HBE human lung epithelial cells or 3T3L1 mouse fibroblasts had little effect on acetyl α-tubulin K40 levels suggesting NQO1 was not providing NAD + for SIRT2 mediated deacetylation in this system.…”

“…siRNA-mediated knockdown or CRISPR/Cas9 knockout of NQO1 in 16-HBE human lung epithelial cells or 3T3L1 mouse fibroblasts had little effect on acetyl α-tubulin K40 levels suggesting NQO1 was not providing NAD + for SIRT2 mediated deacetylation in this system. Altering the conformation of NQO1 using an indolequinone mechanism-based inhibitor, however, resulted in decreased binding of NQO1 to microtubules and, as observed when the oxidized conformation of NQO1 was generated intracellularly, an increase in the levels of acetyl α-tubulin K40 [ 186 ]. No changes in the cellular levels of reduced and oxidized pyridine nucleotides were detected as a result of treatment of cells with the indolequinone inhibitor of NQO1 suggesting the change in acetyl α-tubulin K40 levels was dependent on the conformational change in NQO1 and decreased microtubule binding [ 186 ].…”

“…Altering the conformation of NQO1 using an indolequinone mechanism-based inhibitor, however, resulted in decreased binding of NQO1 to microtubules and, as observed when the oxidized conformation of NQO1 was generated intracellularly, an increase in the levels of acetyl α-tubulin K40 [ 186 ]. No changes in the cellular levels of reduced and oxidized pyridine nucleotides were detected as a result of treatment of cells with the indolequinone inhibitor of NQO1 suggesting the change in acetyl α-tubulin K40 levels was dependent on the conformational change in NQO1 and decreased microtubule binding [ 186 ]. Our interpretation of these results was that altered conformations of NQO1 regulate its binding to microtubules and disrupt the crowded protein environment in the lumen and the microtubule acetylome resulting in either increased acetylation or decreased deacetylation of α-tubulin K40 ( Fig.…”