OP-A induced PINK1 accumulation, Bax and Bak translocation and cytochrome <i>c</i> release.

<p>HaCaT, CHL-1 and A375 cell lines were incubated with 0.3, 0.6 and 1.2 μM OP-A for 24 h and whole cell extracts (WCE), mitochondrial-enriched and cytosolic fractions were analysed by western blot (A) with antibodies directed against PINK1, Bak, Bax, and cytochrome <i>c</i>. Panels B, C and D, densitometric analysis of PINK1, Bak and Bax levels present in the mitochondrial fraction. β-tubulin was used as loading control and standard for densitometry. Data, expressed as %, or fold variation with respect to β-tubulin, were analysed with FlowJo software. *, p<0.05, **, p<0.01, ***, p<0.001.</p

OP-A induced cell cycle alteration.

<p>HaCaT, CHL-1 and A375 cell lines, incubated with 0.3, 0.6 and 1.2 μM OP-A for 24 h, were stained with propidium iodide and, after over-night incubation at 4°C, the percentage of cells in each phase of the cycle measured by flow cytometry. *, p<0.05, **, p<0.01, ***, p<0.001.</p

OP-A effect on mitochondrial membrane potential, mitochondrial mass, lysosome content and mitochondrial ROS production.

<p>HaCaT, CHL-1 and A375 cell lines incubated with 0.3, 0.6 and 1.2 μM OP-A for 24 h, were analysed to evaluate: A, mitochondrial membrane potential (MMP), (2 μM JC-1 staining); B, mitochondrial mass (100 nM MitoTracker Green staining); C, lysosome content (100 nM LysoTracker Red staining); D, mitochondrial ROS (5 μM MitoSox Red staining). Cells were incubated for 30 min at 37°C and then acquired by means of a FACSCalibur. Data, expressed as %, or fold increase with respect to controls, were analysed with FlowJo software. *, p<0.05, **, p<0.01, ***, p<0.001.</p

OP-A induced autophagy and cell death by apoptosis.

<p>A, flow cytometry analysis of cell death. HaCaT, CHL-1 and A375 cell lines were incubated with 0.3, 0.6 and 1.2 μM OP-A for 24 h and cell death analysis performed by propidium iodide staining and flow cytometry evaluation of the sub-G1 population. Where indicated, 20 μM necrostatin-1 was added to evaluate the occurrence of necrotic cell death. B, western blot of PARP, caspase-9 and -3, and LC3II proteins. C, D and E. Densitometric analysis of immunorecognized protein bands of caspase-3, caspase-9 and LC3II, respectively. Data were analysed with FlowJo software. *, p<0.05, **, p<0.01, ***, p<0.001.</p

OP-A reduced A375 melanoma cells viability.

<p>Cells were incubated with 0.3, 0.6 and 1.2 μM OP-A for 24 and 48 h. Panel A: metabolic activity assayed by MTS test. Data are expressed as % of cell survival with respect to control. ***, p<0.0001. Panel B: mitochondrial and nuclear morphology. Mitochondrial network imaging was performed by incubating untreated and treated cells for 20 min at 37°C, with 1 μM MitoTracker Red CMXRos reagent in RPMI medium and nuclei counterstained with 1 μM HOECHST 33342. Images were captured by means of a Floid instrument. Arrowheads indicated picnotic and fragmented nuclei.</p

Identification of proteins differentially represented in OP-A-treated and control A375 melanoma cells as revealed by 2-DE and nanoLC-ESI-LIT-MS/MS analysis.

<p>Spot numbering corresponds to that reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167672#pone.0167672.g006" target="_blank">Fig 6</a>. Spot number, NCBI accession, protein name, MASCOT score, theoretical mass and pI values, peptides and unique peptides identified during analysis, sequence coverage, constitutive amino acids, emPAI score and fold change in treated cells with respect to control are shown.</p

Image_1.TIF

<p>The deglycase and chaperone protein DJ-1 is pivotal for cellular oxidative stress responses and mitochondrial quality control. Mutations in PARK7, encoding DJ-1, are associated with early-onset familial Parkinson’s disease and lead to pathological oxidative stress and/or disrupted protein degradation by the proteasome. The aim of this study was to gain insights into the pathogenic mechanisms of selected DJ-1 missense mutations, by characterizing protein–protein interactions, core parameters of mitochondrial function, quality control regulation via autophagy, and cellular death following dopamine accumulation. We report that the DJ-1^M26I mutant influences DJ-1 interactions with SUMO-1, in turn enhancing removal of mitochondria and conferring increased cellular susceptibility to dopamine toxicity. By contrast, the DJ-1^D149A mutant does not influence mitophagy, but instead impairs Ca²⁺ dynamics and free radical homeostasis by disrupting DJ-1 interactions with a mitochondrial accessory protein known as DJ-1-binding protein (DJBP/EFCAB6). Thus, individual DJ-1 mutations have different effects on mitochondrial function and quality control, implying mutation-specific pathomechanisms converging on impaired mitochondrial homeostasis.</p

Image_2.TIF

<p>The deglycase and chaperone protein DJ-1 is pivotal for cellular oxidative stress responses and mitochondrial quality control. Mutations in PARK7, encoding DJ-1, are associated with early-onset familial Parkinson’s disease and lead to pathological oxidative stress and/or disrupted protein degradation by the proteasome. The aim of this study was to gain insights into the pathogenic mechanisms of selected DJ-1 missense mutations, by characterizing protein–protein interactions, core parameters of mitochondrial function, quality control regulation via autophagy, and cellular death following dopamine accumulation. We report that the DJ-1^M26I mutant influences DJ-1 interactions with SUMO-1, in turn enhancing removal of mitochondria and conferring increased cellular susceptibility to dopamine toxicity. By contrast, the DJ-1^D149A mutant does not influence mitophagy, but instead impairs Ca²⁺ dynamics and free radical homeostasis by disrupting DJ-1 interactions with a mitochondrial accessory protein known as DJ-1-binding protein (DJBP/EFCAB6). Thus, individual DJ-1 mutations have different effects on mitochondrial function and quality control, implying mutation-specific pathomechanisms converging on impaired mitochondrial homeostasis.</p