Quinone-induced protein handling changes: implications for major protein handling systems in quinone-mediated toxicity

Abstract

Para-quinones such as 1,4-Benzoquinone (BQ) and menadione (MD) and ortho-quinones including the oxidation products of catecholamines, are derived from xenobiotics as well as endogenous molecules. The effects of quinones on major protein handling systems in cells; the 20/26S proteasome, the ER stress response, autophagy, chaperone proteins and aggresome formation, have not been investigated in a systematic manner. Both BQ and aminochrome (AC) inhibited proteasomal activity and activated the ER stress response and autophagy in rat dopaminergic N27 cells. AC also induced aggresome formation while MD had little effect on any protein handling systems in N27 cells. The effect of NQO1 on quinone induced protein handling changes and toxicity was examined using N27 cells stably transfected with NQO1 to generate an isogenic NQO1-overexpressing line. NQO1 protected against BQ-induced apoptosis but led to a potentiation of AC- and MD-induced apoptosis. Modulation of quinone-induced apoptosis in N27 and NQO1-overexpressing cells correlated only with changes in the ER stress response and not with changes in other protein handling systems. These data suggested that NQO1 modulated the ER stress response to potentiate toxicity of AC and MD, but protected against BQ toxicity. We further demonstrated that NQO1 mediated reduction to unstable hydroquinones and subsequent redox cycling was important for the activation of the ER stress response and toxicity for both AC and MD. In summary, our data demonstrate that quinone-specific changes in protein handling are evident in N27 cells and the induction of the ER stress response is associated with quinone-mediated toxicity.

Keywords: Aminochrome; Autophagy; ER stress responses; Oxidative stress; Proteasomal inhibition; Quinones.

Figure 1. Chemical structures of quinones utilized in this study

Figure 2. Inhibition of proteasomal activity by quinones

(A–C) The effect of quinones on purified 20S proteasomal activity. BQ and AC inhibited the chymotrypsin-like active site of purified human 20S proteasome in a dose-dependent manner after incubation for 30 min. MD had no effect on 20S proteasomal activity. DQ and DMNQ, the fully substituted analogs of BQ and MD respectively, did not inhibit 20S proteasomal activity. (D–F) The effect of quinones on 20/26S proteasomal activity in N27 cells. BQ and AC, but not MD, significantly inhibited the chymotrypsin-like active site of intracellular 20/26S proteasomal activity in N27 cells at 24h. These data are presented as mean ± SD, (n=3); *p<0.05, **p<0.01 and ***p<0.001 are considered significant by one-way ANOVA using Dunnet’s multiple comparison test.

Figure 3. BQ inhibited proteasomal activity and further activated ER stress responses and autophagy

(A) Growth inhibition in N27 cells upon treatment with BQ. Cells were incubated with increasing concentration of BQ (0–80µM) for 24h and cell viability was assessed by the MTT assay. (B) The inhibition of proteasomal activity by BQ (20µM) occurred at early time points in N27 cells. (C) The representative immunoblot blot shows that as early as 10min BQ induced accumulation of higher molecular weight polyubiquitinated proteins. (D) Time course of the ER stress response and autophagy following treatment with BQ. Treatment (24h) with tunicamycin (Tn, 3µM) and MG132 (1µM) were included as positive controls. β-actin was included as a loading control. Values in (A) and (B) are presented as mean ± SD, (n=3); **p<0.01 is considered significant by one-way ANOVA using Dunnet’s post test.

Figure 4. Dopamine derived AC triggered ER stress responses, turnover of autophagic flux and formation of aggresome-like inclusion bodies in N27 cells

(A) Growth inhibition in N27 cells upon treatment with AC. Cells were incubated with increasing concentration of AC (0–80µM) for 24h and cell viability was assessed using the MTT assay. (B) AC (80µM) inhibited proteasomal activity in a time-dependent manner in N27 cells. Values in (A) and (B) are expressed as mean ± SD of 3 determinations. *p<0.05, **p<0.01 and ***p<0.001 are considered significant by one-way ANOVA using Dunnet’s post test. (C) The representative immunoblot blot shows AC induced the time-dependent accumulation of polyubiquitinated proteins. (D) Time course of the ER stress response and autophagy following treatment with AC (80µM). Treatment (24h) with tunicamycin (Tn, 3µM) and MG132 (1µM) were included as positive controls. β-actin was included as a loading control. Untreated cells in complete medium is labeled as C(S+), or in serum free medium as C(S−). (E) AC induced formation of ubiquitin positive aggresome-like inclusion bodies in N27 cells. After exposure to AC (80µM) for 24h, cells were fixed and immunostained for ubiquitin (red) and nuclei (DAPI, blue). Note that aggregates of ubiquitinated proteins are observed near the perinuclear region (white arrows, bottom left and right).

Figure 5. Overexpression of NQO1 in N27 cells (clone 4 cells) after stable transfection with wild type human NQO1

(A) NQO1 overexpression in clone 4 cells was confirmed by immunoblot analysis (rhNQO1, 35ng purified recombinant human NQO1 standard). (B) Cytosolic NQO1 enzymatic activity was measured in parental N27 and clone 4 cells as described in Materials and Methods. (C) Confocal analysis using immunostaining for NQO1 (red) in N27 cells (left) and clone 4 cells (right) confirmed cytosolic overexpression of NQO1 in clone 4 cells. Values in (B) are presented as mean ± SD, (n=3); ***p<0.001 are considered significant by student t-test.

Figure 6. Overexpression of NQO1 in N27 cells modulated the ER stress response to protect against BQ induced toxicity while potentiated toxicity induced by AC and MD

(A) Overexpression of NQO1 protected N27 cells against BQ induced apoptosis. (B, C) Clone 4 cells were more susceptible to AC (80µM) and MD (16µM) induced apoptosis. Apoptosis was measured in N27 and clone 4 cells after treatment with the indicated quinones for 24h using annexin V/PI cell staining in combination with flow cytometry. These data are presented as mean ± SD, (n=3); ***p< 0.001 are considered significant by one-way ANOVA using Tukey’s multiple comparison test. (D–F) Time course of the ER stress response and the ER stress mediated apoptosis pathway following treatment with BQ (20µM), AC (80µM) and MD (16µM). Note that cleaved caspase-12 (C-Caps 12) was activated indicating that quinones can induce ER stress mediated apoptosis.

Figure 7. Overexpression of NQO1 increased AC and MD but not BQ, induced oxidative stress in clone 4 cells

(A) NQO1 overexpression increased the rate of oxygen consumption following treatments with AC or MD in N27 cells. Oxygen consumption was measured in N27 cells and clone 4 cells in the absence (control) or presence of the indicated quinones. Measurements were made over 10min using a Clark electrode at 37°C. Overexpression of NQO1 in clone 4 cells resulted in significantly increased oxygen consumption after treatment with either AC or MD suggesting unstable hydroquinones of AC or MD were generated and undergo autoxidation. These data are presented as mean ± SD, (n=3); *p<0.05 and ***p<0.001 are considered significant by one-way ANOVA using Tukey’s post test, # p<0.05 is significant comparing to untreated control. (B) Quinone-induced oxidative stress was measured using CellROX Green^® reagent in combination with confocal microscopy. Note the intense green staining in the nucleus of clone 4 cells compared to N27 cells after treatment with AC and MD respectively, suggesting NQO1 increased the level of oxidative stress induced by AC and MD.

Figure 8. Schematic representation of quinone-mediated protein handling changes and toxicity