Proteomic Analyses For The Global S-nitrosylated Proteins In The Brain Tissues Of Different Human Prion Diseases

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders characterized by the neuronal damage that may be caused by the aggregation of toxic proteins and encompass Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI), Gerstmann-Straussler-Scheinker syndrome and Kuru in human, scrapie in sheep, chronic wasting disease in deer and elk, bovine spongiform encephalopathy (BSE) in cattle and so on. Etiologically, CJD can also be classified into four subtypes, sporadic CJD (sCJD), iatrogenic CJD (iCJD), genetic (gCJD) and variant CJD (vCJD). It is characterized by the accumulation of a pathological form of a host coded glycoprotein, designated PrPSc (scrapie isoform of prion protein), from its normal isoform PrPc (cellular isoform of prion protein). Protein S-nitrosylation, the covalent adduction of a nitric oxide (NO) to cysteine, like O-Phosphorylation, plays a key role in human brain of many neurodegenerative diseases. NO is a redox signal molecule, which regulates the activity and function of proteins by protein S’-nitrosylation. Currently, the pathogenesis of many neurodegenerative diseases is believed to relate with the redox status in the brains. The brain dysfunction is a prominent feature of prion diseases, but the direct brain targets of S-nitrosylation are largely unknown yet.In this study, firstly, using brain samples from 263K-infected hamsters and normal hamsters, we described a modified method for the isolation and enrichment of S’-nitrosylated (SNO) proteins from brain tissues based on the biotin switch technique (BST). Various working conditions were comparatively evaluated and optimized, such as the parameters for the incubation of biotin-labeled samples with streptavidin beads and for the elution of SNO proteins from streptavidin beads. The main results included as follows:(1). The incubation parameters were set as at a ratio of 1:3 (streptavidin beads/brain homogenates) at 25℃ for 120 min. (2). The optimized elution buffer was the buffer containing 0.5% SDS. (3). Under these conditions,12 rounds of successive incubation were required in order to recover all the SNO proteins in human and rodent brain homogenates. Subsequent Western blots and iTRAQ-based Mass spectrometry (MS) analyses confirmed that the eluted SNO products possessed the reliable immunoreactivity and can be directly used in proteomic assays.Based on the newly established method for the isolation of S’-nitrosylated proteins described above, we proposed, for the first time, the proteomic analysis of global S-nitrosylation in cortex and cerebellum of brain tissues of sCJD (one case, male, 80-year-old), FFI (pool of three cases, a 48-year-old male; a 26-year-old female and a 55-year-old man) and gCJD with a substitution of valine for glycine at codon 114 of the prion protein gene (one case, female,47-year-old) accompanying with normal control (pool of three controls, a 50-year-old male, a 63-year-old male and a 42-year-old female) with isobaric tags for relative and absolute quantitation (iTRAQ) technique combined with a nano-high-performance liquid chromatography/Q Exactive mass spectrometry platform. In parallel, we used several approaches to provide quality control for the experimentally defined S-nitrosylated proteins. The results of this study are as follows: (1). Total 1509 S-nitrosylated proteins were identified, and cerebellum tissues appeared to contain more commonly differentially expressed SNO-proteins (n= 220) than cortex regions (n= 159) of sCJD, FFI and G114V gCJD. (2). Three select commonly differentially expressed SNO-proteins were verified by the Western blots, showing the same changing patterns as in iTRAQ assays. (3). Gene ontology functional category analysis showed that more up-regulated S-nitrosylated proteins were involved in metabolism, cell cytoskeleton/structure and immune system both in cortex and cerebellum, while more down-regulated ones in both regions were involved in cell cytoskeleton/structure, cell-cell communication and miscelaneous function protein. (4). KEGG Pathway analysis suggested that systemic lupus erythematosus, pathogenic Escherichia coli infection, extracellular matrix-receptor interaction were the most commonly affected pathways, which were identified from at least two different diseases. (5). Using search tool for the retrieval of interacting genes database, the network of immune system and cell cytoskeleton and structure were commonly identified in the context of the up-regulated and down-regulated S-nitrosylated proteins, respectively, both in cortex and cerebellum.In summary, we presented the first comprehensive proteomic analysis of global S-nitrosylation data set of several subtypes of human prion diseases that will have implications for understanding the molecular mechanisms of human prion diseases related to abnormal protein S-nitrosylation and pave the way for future studies on potential biomarkers for the diagnosis and therapy of human prion diseases.