Co-localization of NQO1, Sirt2 and acetyl tubulin in 16HBE cells.

<p>(A) Co-immunostaining for NQO1 (green) and acetyl α-tubulin (red) showing co-localization on mitotic structures. (B) Co-immunostaining for NQO1 (green) and Sirt2 (red) showing co-localization on centrosome(s). Arrows indicate co-localization between high intensity immunostaining for NQO1, acetyl α-tubulin and Sirt2 in different stages of the centriole cycle. (C, centrosome(s); MS, mitotic spindles; MB, midbody).</p

Redox modulation of NQO1

<div><p>NQO1 is a FAD containing NAD(P)H-dependent oxidoreductase that catalyzes the reduction of quinones and related substrates. In cells, NQO1 participates in a number of binding interactions with other proteins and mRNA and these interactions may be influenced by the concentrations of reduced pyridine nucleotides. NAD(P)H can protect NQO1 from proteolytic digestion suggesting that binding of reduced pyridine nucleotides results in a change in NQO1 structure. We have used purified NQO1 to demonstrate the addition of NAD(P)H induces a change in the structure of NQO1; this results in the loss of immunoreactivity to antibodies that bind to the C-terminal domain and to helix 7 of the catalytic core domain. Under normal cellular conditions NQO1 is not immunoprecipitated by these antibodies, however, following treatment with β-lapachone which caused rapid oxidation of NAD(P)H NQO1 could be readily pulled-down. Similarly, immunostaining for NQO1 was significantly increased in cells following treatment with β-lapachone demonstrating that under non-denaturing conditions the immunoreactivity of NQO1 is reflective of the NAD(P)⁺/NAD(P)H ratio. In untreated human cells, regions with high intensity immunostaining for NQO1 co-localize with acetyl α-tubulin and the NAD⁺-dependent deacetylase Sirt2 on the centrosome(s), the mitotic spindle and midbody during cell division. These data provide evidence that during the centriole duplication cycle NQO1 may provide NAD⁺ for Sirt2-mediated deacetylation of microtubules. Overall, NQO1 may act as a redox-dependent switch where the protein responds to the NAD(P)⁺/NAD(P)H redox environment by altering its structure promoting the binding or dissociation of NQO1 with target macromolecules.</p></div

Description of study sample.

<p>Data are represented as means ± SEM. Mini-Mental-State Examination (MMSE) normal range: 0–30. Activities of Daily Living (ADL) normal range: 0–6.</p

Co-localization of NQO1 with microtubules/acetylated microtubules in TrHBMEC.

<p>Co-immunostaining for NQO1 (A180) and α-tubulin showing co-localization on microtubules.(A) Co-localization of NQO1 with α-tubulin using fluorescently-labeled secondary antibodies. (B) Co-localization of NQO1 with α-tubulin/acetyl α-tubulin using PLA-based detection. Immunostaining was performed as described in <i>Materials and methods</i>.</p

Increased immunostaining for NQO1 in 16HBE cells following treatment with β-lapachone.

<p>16HBE cells were treated with DMSO or β-lapachone (10μM) for the indicated times then processed for immunocytochemistry and confocal analysis as described in <i>Materials and Methods</i>. Immunostaining for NQO1 was performed under non-denaturing conditions using the A180 antibody combined with DAPI nuclear staining.</p

Reduced pyridine nucleotides and dicumarol induce a conformational change in NQO1.

<p>(A) Comparison of the ability of antibodies which bind to helix 7 (A180) and antibodies which bind to the C-terminal domain (C-Term) to immunoprecipitate rhNQO1 in the absence and presence of NADH. (B, C) Immunoprecipitation of rhNQO1 with the A180 antibody in the absence and presence of NADH or NADPH. (D) Immunoprecipitation of rhNQO1 by C-Term and A180 antibodies in the absence and presence of dicumarol. (E) The effect of NADH and dicumarol on the migration of rhNQO1 in non-denaturing PAGE. Reaction conditions for immunoprecipitation studies and non-denaturing PAGE are described in <i>Materials and methods</i>.</p

Co-localization of NQO1, acetyl α-tubulin and Sirt2 in TrHBMEC.

<p>(A) Co-immunostaining for NQO1 (A180, green) and acetyl α-tubulin (red) in TrHBMEC cells showing co-localization on centrosomes (arrows). (B) Co-immunostaining for NQO1 (A180, green) and acetyl α-tubulin (red) in TrHBMEC cells showing co-localization on mitotic spindles. Co-immunostaining for NQO1 (A180, green) and Sirt2 (red) in 16HBE cells. Arrows indicate co-localization between high intensity immunostaining for NQO1, acetyl α-tubulin and Sirt2 on the centrosomes.</p

Relative positioning of the C-term and A180 antibody epitopes on human NQO1.

<p>Alternating viewpoints of the human NQO1 homodimer (PDB ID: 1D4A) with each monomer colored separately (blue and green) and the locations of the C-terminal epitopes (CT, yellow) and A180 epitopes (magenta) highlighted.</p

<i>faah</i> DNA methylation levels <i>vs</i> MMSE score.

<p>a: Amount of methylated DNA at <i>faah</i> gene in LOAD subjects subgrouped on the basis of MMSE score; b: Correlation between changes in DNA methylation at <i>faah</i> gene and LOAD subjects with severe AD, based on MMSE score. Data were compared by Pearson's rank correlation coefficient (p<0.05, r = −0.6240).</p

Genetic and epigenetic regulation of <i>faah</i> gene.

<p>a: Levels of FAAH mRNA in PBMCs from LOAD patients (n = 13) and controls (n = 12); b: Amount of methylated DNA at <i>faah</i> gene in controls (n = 33 ) and LOAD subjects (n = 33); c: Correlation between <i>faah</i> gene expression and % change of DNA methylation in LOAD subjects. Data were compared by Spearman's rank correlation coefficient (p<0.05, r = −0.5326). Scatter dots represent 2^−DDCt values calculated by Delta-Delta Ct (DDCt) method, as described in the Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039186#s2" target="_blank">Methods</a> section.</p