NO Stress Regulates The Apoptosis In The Cells Of Parkinson’s Disease Paradigm

Nitric oxide (NO) is a highly reactive free-radical in organisms. The NO in vivo can modify the protein cysteine residues, called S-nitrosylation. S-nitrosylation of a protein may cause the changes of the protein spatial structure and functions, subsequently leading to impacts on many biological processes, such as vasodilatation, neurotransmission and immunoregulation. Parkinson’s disease (PD) is a typical neurodegenerative disorder and a public health problem in the aged population. An increased of NO stress and protein oxidation is commonly observed in the brain tissues of PD patients. It is generally accepted NO stress is tightly correlated with the apoptosis of dopaminergic neuron in PD; nevertheless the molecular mechanism has yet to be elucidated. We herein have employed a common cell model of dopaminergic neurodegeneration in PD study, SH-SY5Y cells with apoptosis rotenone-induced, monitored NO stress and the status of the S-nitrosylated proteins within the cells with/without rotenone treatment, and explored the mechanism of how the protein S-nitrosylation brought the changes of apoptotic pathway in such cell model.With multiple approaches to examine cellular behavior and biological indicators, we found the NO level in SH-SY5Y cells was positively correlated with the rotenone doses and the duration of drug treatment. Moreover, the SH-SY5Y cells exhibited a typically apoptotic characteristics after the treatment of rotenone. We developed a method of S-nitrosylated proteomics, CyDye switch/2D-DIGE, which integrated biotin switch assay with differential in gel electrophoresis (DIGE). With this method, the drawbacks in traditional S-nitrosylated proteomics such the instability of labeling S-nitrosylated cysteine by biotinylation and low throughput in detection of the S-nitrosylated proteins could be avoided, whereas the intact proteins with the S-nitrosylated signs could be globally identified. Upon the fluorescent gel images of CyDye switch/2D-DIGE, a total of 213 spots were detected through fluorescence scanning, including 7 spots with significantly increased spot volumes (>1.5 folds) responding rotenone treatment. After tryptic in-gel digestion and peptide detection with mass spectrometry, all the 7 spots were identified as the proteins that were not shared with similar functions, but most of them were reported as potentially S-nitrosylated.Of the S-nitrosylated proteins with up-regulation responsive to rotenone, proliferating cell nuclear antigen (PCNA) is a well-known protein of which is closely associated with cell proliferation and apoptosis. PCNA is considered as a nuclear protein that regulates apoptosis through controlling DNA replication and repairing. We observed, however, PCNA was mainly localized in cytoplasm but not in nuclei in SH-SY5Y cells. This prompted us to inquire to how the cytoplasm and the S-nitrosylated PCNA could impact the apoptotic pathway in such cell line. By measurement of the interactome with cytoplasmic PCNA, we found that PCNA was able to interact with caspase9, a key component at upstream of apoptotic pathway in cytoplasm.Analysis to the recombinant PCNA treated with NO-donor using high resolution mass spectrometry demonstrated that PCNA had two cysteine residues, Cys81 and Cys162, sensitive to S-nitrosylation induced by NO. Furthermore, through site-directed mutagenesis and pull-down assay, the S-nitrosylation at PCNA Cys81 was found to weaken the interaction between PCNA and caspase9. Generally the caspase9 cleavage prompts activation of apoptotic pathway. Our data indicated that the interaction of PCNA and caspase9 inhibited the caspase9 cleavage, whereas the S-nitrosylated PCNA lost the inhibitive effect. The bioinformatics analysis to the PCNA protein structure predicted that the S-nitrosylation at PCNA Cys81 could result in the conformation changes of PCNA, whereas S-nitrosylation at PCNA Cys162 would not exert any spatial effect. The interaction of PCNA and caspase9 is deduced on the motif nearby Cys81, thus S-nitrosylation at this site could distort the interactive surface between the two proteins.We propose a novel mechanism of which NO stress regulates dopaminergic neurodegeneration in PD. In the rotenone-induced cell model, the SH-SY5Y proteins are extensively S-nitrosylated due to an increase of endogenous NO stress. PCNA located at cytoplasm is a key protein attacked by NO. The S-nitrosylation at PCNA Cys81 brings about the protein configuration changes leading to weakening the interaction of PCNA and caspase9 and activation of apoptotic pathways.