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Proteomic Analyses for the Global S-Nitrosylated Proteins in the Brain Tissues of Different Human Prion Diseases

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Abstract

Human prion diseases are fatal neurodegenerative disorders characterized by neuronal damage in brain. Protein S-nitrosylation, the covalent adduction of a NO to cysteine, plays a role in human brain biology, and brain dysfunction is a prominent feature of prion disease, yet the direct brain targets of S-nitrosylation are largely unknown. We described the first proteomic analysis of global S-nitrosylation in brain tissues of sporadic Creutzfeldt–Jakob disease (sCJD), fatal familial insomnia (FFI), and genetic CJD with a substitution of valine for glycine at codon 114 of the prion protein gene (G114V gCJD) accompanying with normal control with isobaric tags for relative and absolute quantitation (iTRAQ) combined with a nano-HPLC/Q-Exactive mass spectrometry platform. In parallel, we used several approaches to provide quality control for the experimentally defined S-nitrosylated proteins. A total of 1509 S-nitrosylated proteins (SNO-proteins) were identified, and data are available via ProteomeXchange with identifier PXD002813. The cerebellum tissues appeared to contain more commonly differentially expressed SNO-proteins (DESPs) than cortex of sCJD, FFI, and gCJD. Three selected SNO-proteins were verified by Western blots, consistent with proteomics assays. Gene ontology analysis showed that more up-regulated DESPs were involved in metabolism, cell cytoskeleton/structure, and immune system both in the cortex and cerebellum, while more down-regulated ones in both regions were involved in cell cytoskeleton/structure, cell-cell communication, and miscellaneous function protein. Pathway analysis suggested that systemic lupus erythematosus, pathogenic Escherichia coli infection, and extracellular matrix-receptor interaction were the most commonly affected pathways, which were identified from at least two different diseases. Using STRING database, the network of immune system and cell cytoskeleton and structure were commonly identified in the context of the up-regulated and down-regulated DESPs, respectively, both in the cortex and cerebellum. Our study thus have implications for understanding the molecular mechanisms of human prion diseases related to abnormal protein S-nitrosylation and pave the way for future studies focused on potential biomarkers for the diagnosis and therapy of human prion diseases.

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Acknowledgments

This work was supported by Chinese National Natural Science Foundation Grants (81301429), China Mega-Project for Infectious Disease (2011ZX10004-101, 2012ZX10004215), and SKLID Development Grant (2012SKLID102).

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The authors declare that they have no competing interests.

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    Authors and Affiliations

    1. State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, 310003, China

      Li-Na Chen, Qi Shi, Bao-Yun Zhang, Xiao-Mei Zhang, Jing Wang, Kang Xiao, Yan Lv, Jing Sun, Xiao-Dong Yang, Cao Chen, Wei Zhou, Jun Han & Xiao-Ping Dong

    2. National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Chang-Bai Rd 155, Beijing, 102206, People’s Republic of China

      Li-Na Chen, Qi Shi, Bao-Yun Zhang, Xiao-Mei Zhang, Jing Wang, Kang Xiao, Yan Lv, Jing Sun, Xiao-Dong Yang, Cao Chen, Wei Zhou, Jun Han & Xiao-Ping Dong

    3. Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China

      Xiao-Ping Dong

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    Correspondence to Xiao-Ping Dong.

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    Li-Na Chen and Qi Shi contributed equally to this work.

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    Supplementary Figure 1

    a PrP-specific Western blots of the cortical and cerebella tissues of sCJD, FFI, G114V gCJD, and normal control. The individual β-actin are shown below. PK+ with treatment of PK; PK− without treatment of PK. b Biotin-specific dot blots. In total, 2 μl of the final elution products was separately spotted on the membrane and immunoblotted with anti-biotin antibody. c Biotin-specific Western blots. In total, 2 μl of the final elution products and streptavidin-coated magnetic beads after elution was separated in 12 % SDS-PAGE. The immunoblots were incubated with HRP-labeled streptavidin and visualized using an ECL kit. (PPTX 523 kb)

    Supplementary Figure 2

    a Determination of the elution products from the brain extracts of human prion diseases and normal control with 12 % SDS-PAGE with Coomassie blue staining. M represents protein markers. b The distribution of the digested peptides in the length of amino acid after searching in the Swiss-Prot database. The peptide’s length in amino acid is shown in x-axis and the frequency of the peptides is shown in y-axis. (PPTX 1601 kb)

    Supplementary Figure 3

    The complete blots from which the graphs in Fig. 3a were derived. a Total-AQP1 and SNO-AQP1. b Total-Neurogranin and SNO-Neurogranin. c Total-Opalin and SNO-Opalin. Arrows indicate the individual blots. (PPTX 498 kb)

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    Chen, LN., Shi, Q., Zhang, BY. et al. Proteomic Analyses for the Global S-Nitrosylated Proteins in the Brain Tissues of Different Human Prion Diseases. Mol Neurobiol 53, 5079–5096 (2016). https://doi.org/10.1007/s12035-015-9440-7

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