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Proteomic study on neurite responses to oxidative stress: search for differentially expressed proteins in isolated neurites of N1E-115 cells.

ABSTRACT: Reactive oxygen species attack several living organs and induce cell death. Previously, we found axonal/dendrite degeneration before the induction of cell death in hydrogen peroxide-treated neuroblastoma: N1E-115 cells and primary neurons. This phenomenon may be connected with membrane oxidation, microtubule destabilization and disruption of intracellular calcium homeostasis. However, its detailed mechanisms are not fully understood. Here, we identified proteins after treatment with hydrogen peroxide using isolated neurites by liquid chromatography-matrix-assisted laser desorption/ionization-time of flight/time of flight analysis. Twenty-one proteins were increased after treatment with hydrogen peroxide. Specifically, 5 proteins which were secretogranin-1, heat shock protein family D member 1, Brain acid soluble protein 1, heat shock 70-kDa protein 5 and superoxide dismutase 1, were identified of all experiments and increased in isolated neurites of hydrogen peroxide-treated cells compared to the controls. Furthermore, secretogranin-1 and heat shock protein family D member 1 protein expressions were significantly increased in normal aged and Alzheimer's transgenic mice brains. These results indicate that secretogranin-1 and heat shock protein family D member 1 might contribute to reactive oxygen species-induced neurite degeneration. Both proteins have been related to neurodegenerative disorders, so their study may shed light on neurite dysfunction.

SUBMITTER: Fukui K

PROVIDER: S-EPMC6348415 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Researches have been conducted for the identification of differentially expressed genes (DEGs) by generating and mining of cDNA expressed sequence tags (ESTs) for more than a decade. Although the availability of public databases make possible the comprehensive mining of DEGs among the ESTs from multiple tissue types, existing studies usually employed statistics suitable only for two categories. Multi-class test has been developed to enable the finding of tissue specific genes, but subsequent search for cancer genes involves separate two-category test only on the ESTs of the tissue of interest. This constricts the amount of data used. On the other hand, simple pooling of cancer and normal genes from multiple tissue types runs the risk of Simpson's paradox. Here we presented a different approach which searched for multi-cancer DEG candidates by analyzing all pertinent ESTs in all categories and narrowing down the cancer biomarker candidates via integrative analysis with microarray data and selection of secretory and membrane protein genes as well as incorporation of network analysis. Finally, the differential expression patterns of three selected cancer biomarker candidates were confirmed by real-time qPCR analysis. RESULTS:Seven hundred and twenty three primary DEG candidates (p-value < 0.05 and lower bound of confidence interval of odds ratio ? 1.65) were selected from a curated EST database with the application of Cochran-Mantel-Haenszel statistic (CMH). GeneGO analysis results indicated this set as neoplasm enriched. Cross-examination with microarray data further narrowed the list down to 235 genes, among which 96 had membrane or secretory annotations. After examined the candidates in protein interaction network, public tissue expression databases, and literatures, we selected three genes for further evaluation by real-time qPCR with eight major normal and cancer tissues. The higher-than-normal tissue expression of COL3A1, DLG3, and RNF43 in some of the cancer tissues is in agreement with our in silico predictions. CONCLUSIONS:Searching digitized transcriptome using CMH enabled us to identify multi-cancer differentially expressed gene candidates. Our methodology demonstrated simultaneously analysis for cancer biomarkers of multiple tissue types with the EST data. With the revived interest in digitizing the transcriptomes by NGS, cancer biomarkers could be more precisely detected from the ESTs. The three candidates identified in this study, COL3A1, DLG3, and RNF43, are valuable targets for further evaluation with a larger sample size of normal and cancer tissue or serum samples.

Project description:A large number of previous clinical studies have reported a delayed graft function for brain dead donors, when compared with living relatives or cadaveric organ transplantations. However, there is no accurate method for the quality evaluation of kidneys from brain‑dead donors. In the present study, two‑dimensional gel electrophoresis and MALDI‑TOF MS‑based comparative proteomic analysis were conducted to profile the differentially‑expressed proteins between brain death and the control group renal tissues. A total of 40 age‑ and sex‑matched rabbits were randomly divided into donation following brain death (DBD) and control groups. Following the induction of brain death via intracranial progressive pressure, the renal function and the morphological alterations were measured 2, 6 and 8 h afterwards. The differentially expressed proteins were detected from renal histological evidence at 6 h following brain death. Although 904±19 protein spots in control groups and 916±25 in DBD groups were identified in the two‑dimensional gel electrophoresis, >2‑fold alterations were identified by MALDI‑TOF MS and searched by NCBI database. The authors successfully acquired five downregulated proteins, these were: Prohibitin (isoform CRA_b), beta-1,3‑N-acetylgalactosaminyltransferase 1, Annexin A5, superoxide dismutase (mitochondrial) and cytochrome b‑c1 complex subunit 1 (mitochondrial precursor). Conversely, the other five upregulated proteins were: PRP38 pre‑mRNA processing factor 38 (yeast) domain containing A, calcineurin subunit B type 1, V‑type proton ATPase subunit G 1, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 and peroxiredoxin‑3 (mitochondrial). Immunohistochemical results revealed that the expressions of prohibitin (PHB) were gradually increased in a time‑dependent manner. The results indicated that there were alterations in levels of several proteins in the kidneys of those with brain death, even if the primary function and the morphological changes were not obvious. PHB may therefore be a novel biomarker for primary quality evaluation of kidneys from brain‑dead donors.

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Proteomic study on neurite responses to oxidative stress: search for differentially expressed proteins in isolated neurites of N1E-115 cells.

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