Abstract
The reversible phosphorylation of proteins regulates most biological processes, while abnormal phosphorylation is a cause or consequence of many diseases including Alzheimer's disease (AD). One of the hallmarks of AD is the formation of neurofibrillary tangles (NFTs), which is composed of hyperphosphorylated tau proteins. Sodium selenate has been recently found to reduce tau hyperphosphorylation and NFTs formation, and to improve spatial learning and motor performance in AD mice. In the current study, the phosphoproteomics of N2aSW cells treated with selenate were investigated. To avoid missing low-abundance phosphoproteins, both the total proteins of cells and the phosphor-enriched proteins were extracted and subjected to the two-dimensional gel electrophoresis with Pro-Q diamond staining and then LC-MS/MS analysis. A total of 65 proteins were altered in phosphorylation level, of which 39 were up-regulated and 26 were down-regulated. All identified phosphoproteins were bioinformatically annotated according to their physiochemical features, subcellular location, and biological function. Most of these significantly changed phosphoproteins are involved in crucial neural processes such as protesome activity, oxidative stress, cysteine and methionine metabolism, and energy metabolism. Furthermore, decreases were found in homocysteine, phosphor-tau and amyloid β upon selenate treatment. Our results suggest that selenate may intervene in the pathological process of AD by altering the phosphorylation of some key proteins involved in oxidative stress, energy metabolism and protein degradation, thus play important roles in maintaining redox homeostasis, generating ATP, and clearing misfolded proteins and aggregates. The present paper provides some new clues to the mechanism of selenate in AD prevention.
Highlights
Protein phosphorylation is one of the most ubiquitous post-translational modifications involved in regulating a majority of biological processes
Primary antibodies against b-actin, Tau, Tau, Tau, Tau, Tau, BACE1, amyloid precursor protein (APP) and horseradish peroxidase (HRP)-conjugated mouse or rabbit IgG were purchased from Abcam Biochemicals (Cambridge, UK)
Phosphoproteins altered in the total proteins of cells treated with selenate To study the effect of sodium selenate on protein phosphorylation in N2aSW cells, comparative phosphoproteomic analysis was implemented through a series of steps, including 2DE separation, image analysis, in-gel digestion, MS identification and database searching
Summary
Protein phosphorylation is one of the most ubiquitous post-translational modifications involved in regulating a majority of biological processes. It is required for proper protein folding, and functions as a signal for further protein modifications such as ubiquitination. Phosphorylation may alter protein subcellular localization, induce conformational changes, alter catalytic activity, and modify protein-protein interactions. Protein phosphorylation is regulated by a highly dynamic network of kinases and phosphatases. At least one-third of eukaryotic proteins are phosphorylated [1], among them only a subset are modified by any given stimulus. Abnormal phosphorylation is a cause or consequence of many human diseases including Alzheimer’s disease (AD) [2, 3]
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