Changes in endoplasmic reticulum stress proteins and aldolase A in cells exposed to dopamine

Abstract

In Parkinson's disease, oxidative stress is implicated in protein misfolding and aggregation, which may activate the unfolded protein response by the endoplasmic reticulum (ER). Dopamine (DA) can initiate oxidative stress via H(2)O(2) formation by DA metabolism and by oxidation into DA quinone. We have previously shown that DA quinone induces oxidative protein modification, mitochondrial dysfunction in vitro, and dopaminergic cell toxicity in vivo and in vitro. In this study, we used cysteine- and lysine-reactive fluorescent dyes with 2D difference in-gel electrophoresis, mass spectrometry, and peptide mass fingerprint analysis to identify proteins in PC12 cell mitochondrial-enriched fractions that were altered in abundance following DA exposure (150 muM, 16 h). Quantitative changes in proteins labeled with fluorescent dyes indicated increases in a subset of proteins after DA exposure: calreticulin, ERp29, ERp99, Grp58, Grp78, Grp94 and Orp150 (149-260%), and decreased levels of aldolase A (39-42%). Changes in levels of several proteins detected by 2D difference in-gel electrophoresis were confirmed by western blot. Using this unbiased proteomics approach, our findings demonstrated that in PC12 cells, DA exposure leads to a cellular response indicative of ER stress prior to the onset of cell death, providing a potential link between DA and the unfolded protein response in the pathogenesis of Parkinson's disease.

Figure 1. 2D-DIGE of PC12 cell mitochondrial-enriched fractions using cysteine-reactive dyes with insets of sample proteins

(A.) Mitochondrial-enriched fractions from control and 16h, 150 μM DA-exposed PC12 cells were isolated by differential centrifugation, and equal protein amounts were reacted with either Cy3 (cyan scan; control) and Cy5 (pink scan; DA-exposed) maleimide dyes. Pink spots designate proteins in which more cysteine labeling occurred in the DA-exposed sample, indicating increased protein levels following DA exposure. Cyan spots designate proteins in which more cysteine labeling occurred in control, indicating cysteine modification or decreased protein levels following DA exposure. Dark blue spots indicate proteins in which equal cysteine labeling occurred in the control and DA-exposed mitochondrial-enriched fraction. MS-identified proteins are indicated on the gel and listed in Figure 3B. In cases where more than one spot with the same protein identification was determined by MS, letter (−A, −B, −C, etc) or numerical (−1, −2, and −3) notations were used. All spots identified corresponded to only one protein and related isoforms. Identification of each spot was replicated in 2 or more experiments. The gel is representative of n=8 cysteine 2D-DIGE gels. Boxes outline inset pictures in B-D. (B.) Black and white representation of the control protein, Cy3 fluorescent scan and the DA-treated protein, Cy5 fluorescent scan with Orp150 and Grp78 spots indicated. (C.) Black and white representation of the control protein, Cy3 fluorescent scan and the DA-treated protein, Cy5 fluorescent scan with the Grp58 spot indicated. (D.) Black and white representation of the control protein, Cy3 fluorescent scan and the DA-treated protein, Cy5 fluorescent scan with the aldolase A-3 spot indicated.

Figure 2. 2D-DIGE of PC12 cell mitochondrial-enriched fraction using lysine-reactive dyes with insets of sample proteins

(A.) Mitochondrial-enriched fraction from control and 16h, 150 μM DA-exposed differentiated PC12 cells were isolated by differential centrifugation. Mitochondrial protein was reacted with either Cy5 (pink scan; control) and Cy3 (cyan scan; DA-exposed) NHS-ester dyes to label lysine residues. Cyan (blue) spots designate proteins in which more lysine labeling occurred in the DA-exposed sample, indicating increased protein levels. Pink spots designate proteins in which more lysine labeling occurred in the control sample, indicating decreased protein levels induced by DA exposure. Dark blue spots indicate proteins in which equal lysine labeling occurred in the control and DA-exposed samples. MS-identified proteins are indicated on the gel and listed in Figure 3B. When multiple spots were determined by MS with the same protein identification, notations with letters (−A, −B, −C, etc) or numbers (−1, −2, and −3) were used. All listed identified protein spots corresponded to only one protein and related isoforms, and was replicated in at least 2 experiments. The gel is representative of n=5 lysine 2D-DIGE gels. Boxes outline inset pictures in figures B-D. (B.) Black and white representation of the control protein (Cy5 fluorescent scan) and the DA-treated protein (Cy3 fluorescent scan) with Orp150 and Grp78 spots indicated. (C.) Black and white representation of the control protein (Cy5 fluorescent scan) and the DA-treated protein (Cy3 fluorescent scan) with the Grp58 spot indicated. (D.) Black and white representation of the control protein (Cy5 fluorescent scan) and the DA-treated protein (Cy3 fluorescent scan) with the aldolase A-3 spot indicated.

Figure 3. Changes in protein spot intensity of PC12 cell in the mitochondrial fraction following DA-exposure

Mitochondrial-enriched fractions from control and DA-exposed (150 μM, 16 h) differentiated PC12 cells were reacted with Cy3 or Cy5 maleimide or NHS-ester dyes. Proteins listed above were identified by MALDI peptide mass fingerprint. Multiple spots with the same protein identification are denoted by letters (−A, −B, −C, etc) or numbers (−1, −2, and −3). (A.) The density of protein spots was analyzed using Decyder DIA software, and changes in DA exposed compared to control protein were determined in both maleimide (cysteine) and NHS-ester (lysine) reacted protein for most spots shown. Proteins spots that appear to correspond between cysteine- and lysine-labeling experiments, but were not identified according to our MS criteria, are labeled as NI (no identification). Proteins that do not have corresponding spots in both labeling experiments, are labeled with ND (no spot detected). DA treated PC12 cell protein intensity is measured as average % control ± SEM, n=5-8. *, significance p<0.05. (B.) Proteins identified that were significantly changed from control as quantified by DeCyder analysis and that were changed at least ± 1.2-fold from control in cysteine (maleimide) and/or lysine (NHS-ester) DIGE experiments are grouped by function. Actual molecular weight (MW in kDa) and isoelectric point (pI), best protein score and % confidence for each identified protein are also listed. † MASCOT probability-based protein MOWSE scores provided represent the highest protein score obtained across all experiments.

Figure 4. Western blot analysis of Grp78, Grp58, ERp29, and Grp94 in PC12 mitochondrial-enriched fraction and Grp78 and Grp58 in whole cell lysate following DA-exposure

Mitochondria-enriched protein and PC12 whole-cell lysate from control and 150 μM DA-exposed PC12 cells were collected. (A.) Representative blots for proteins Grp78, Grp58, ERp29, and Grp94 are shown for PC12 mitochondrial fractions isolated from 16h control and 150 μM DA-treated cells. (B.) Grp78/actin, Grp58/actin, ERp29/actin, and Grp94/actin ratios for PC12 mitochondrial fraction DA treated cells were quantified and compared to control. Values for (C.) Grp78/actin and (D.) Grp58/actin ratios were measured in whole cell lysates at various time-points and expressed as % Control ± SEM, n=3 separate experiments, each measured in duplicate. *, significance p<0.05.

Figure 5. Western blot analysis of aldolase A in PC12 mitochondrial-enriched fraction and whole cell lysate following DA-exposure

Mitochondria-enriched protein and PC12 whole-cell lysate from PC12 cells were collected after exposure to control media or media containing 150 μM DA. (A.) Representative blots for aldolase A are shown for PC12 mitochondrial-enriched fractions isolated from 16h control and 150 μM DA-treated cells. (B.) The quantification of control and DA exposed PC12 cell mitochondrial-enriched protein are reported as % control ± SEM of the average immunoblot intensity of aldolase A/actin. (C.) Aldolase A levels in PC12 whole cell lysate following DA exposure were examined at various time-points compared to control cells. Aldolase A/tubulin ratios were reported as % time-matched control ± SEM. n=3 separate experiments, each measured in duplicate. *, significance p<0.05.