Alzheimer’s disease (AD) is a neurodegenerative disorder that is characterized by peptide and protein misfolding and aggregation, in part due to the presence of excess metal ions such as copper. Aggregation and cytotoxicity of amyloid-β (Aβ) peptide with copper ion have been investigated extensively; however, the effects of metalation on tau are less known. Here, we presented the effects of Cu+ and Cu2+ on aggregation and neurotoxicity of the second repeat unit of the microtubule-binding domain of tau (tau-R2). Tau-R2 was demonstrated to bind 0.44 Cu2+ and 0.34 Cu+ per monomer with dissociation constants of 1.1 nM and 0.2 pM, respectively. Copper in both oxidation states stimulated the aggregation, ROS production, and neuronal cytotoxicity of tau-R2. We showed that copper-associated tau-R2 aggregates, decreased protein levels of microtubule-associated protein 2 (MAP-2), and synaptophysin in the primarily cultured cortical neurons, reduced mitochondrial density and mobility in the axon and, as a consequence, impaired the growth and probably also the function of neurons. Previously, we reported that the His-rich domain of selenoprotein P (SelP-H) inhibited metal-induced aggregation and toxicity of Aβ, due to its metal chelation ability. Here we demonstrated that SelP-H not only inhibited copper-mediated tau aggregation but also interfered with the ongoing aggregation and reversed the already formed aggregates. More intriguing, SelP-H significantly attenuated Cu2+/Cu+-tau-R2-induced intracellular ROS production and the impairments of synapse and mitochondrial movement in neurons. This work implies that the surface-exposed His-rich domain of SelP makes it capable of modulating Cu+/Cu2+-mediated aggregation and neurotoxicity of both Aß and tau and may play important roles in the prevention of AD progression.

Aggregation and cytotoxicity of the amyloid-β (Aβ) peptide with transition metal ions in neuronal cells have been suggested to be involved in the progression of Alzheimer's disease (AD). A therapeutic strategy to combat this incurable disease is to design chemical agents to target metal–Aβ species. Selenoproteins are a group of special proteins that contain the 21st amino acid Sec in their sequence. Due to the presence of Sec, studies of this group of proteins are basically focused on their roles in regulating redox potential and scavenging reactive oxygen species. Here, we reported that the His-rich domain of selenoprotein P (SelP-H) and the Sec-to-Cys mutant selenoprotein M (SelM′) are capable of binding transition metal ions and modulating the Zn2+-mediated Aβ aggregation, ROS production and neurotoxicity. SelM′ (U48C) and SelP-H were found to coordinate 0.5 and 2 molar equivalents of Zn2+/Cd2+ with micromolar and submicromolar affinities, respectively. Metal binding induced the structural changes in SelP-H and SelM′ according to the circular dichorism spectra. Zn2+ binding to Aβ42 almost completely suppressed Aβ42 fibrillization, which could be significantly restored by SelP-H and SelM′, as observed by thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM). Interestingly, both SelP-H and SelM′ inhibited Zn2+–Aβ42-induced neurotoxicity and the intracellular ROS production in living cells. These studies suggest that SelP and SelM may play certain roles in regulating redox balance as well as metal homeostasis.

Selenium is an important trace mineral necessary for human health. Clinical trials have shown potential inhibitory effects of selenium in advanced or aggressive prostate cancer. However, its mechanism of action remains unclear. This study investigated the mechanism of action of sodium selenite in human prostate cancer PC-3 cells using proteomics. CCK-8 assays were used to detect cell viability and the inhibitory rate. Cell apoptosis was detected by annexin V-FITC and propidium iodide double staining using flow cytometry. Selenite inhibited the growth of PC-3 cells causing them to display morphological changes typical of apoptosis. The rate of cell apoptosis also increased. Proteomics identified a variety of differentially expressed proteins in PC-3 cells exposed to selenite. Eighteen protein spots were identified by MALDI-TOF mass spectrometry. These proteins were separated into those involved in redox balance, protein degradation and cellular energy metabolism. Three differently expressed proteins (SOD1, Stathmin and Erp29) were chosen for Western blot verification, together with several apoptosis-related proteins. Western blot analyses showed that selenite-induced apoptosis was accompanied by activation of caspase-8 and specific proteolytic cleavage of PARP. This led to an increase in the pro-apoptotic protein Bax, and to a decrease in the anti-apoptotic protein Bcl-2 and in hypoxia inducible factor-1α. Increased ROS generation and decreased mitochondrial membrane potential were consistent with reduced expression of antioxidative proteins identified by comparative proteomics. We therefore propose that sodium selenite induces the apoptosis of PC-3 cells mainly through the mitochondrial pathway, but also via ER stress and HIF-1α mediated pathways.

Selenium (Se), an essential trace element in vivo, is present mainly as selenocystein (Sec) in various selenoproteins. The Sec residue is translated from an in-frame TGA codon, which traditionally functions as a stop codon. Prediction of selenoprotein genes is difficult due to the lack of an effective method for distinguishing the dual function of the TGA codon in the open reading frame of a selenoprotein gene. In this article a eukaryotic bioinformatic prediction system that we have developed was used to predict selenoprotein genes from the genome of the common bottlenose dolphin, Tursiops truncatus. Sixteen selenoprotein genes were predicted, including selenoprotein P and glutathione peroxidase. In particular, a type II iodothyronine deiodinase was found to have two Sec residues, while the type I iodothyronine deiodinase gene has two alternative splice forms. These results provide important information for the investigation of the relationship between a variety of selenoproteins and the evolution of the marine-living dolphin.

Lanthanides (Lns) compounds have been reported to possess contrary effects on cell activity, i.e., promoting cell cycle progression and cell growth by lower concentration treatment, but suppressing cell proliferation and inducing cell apoptosis at higher dosing. However, the cellular processes during the intervention and the possible underlying mechanisms are still not well clarified. Using a combination of high-throughput liquid chromatography (LC) with mass spectrometry (MS), we have investigated the metabolomic profiles of Hela cells following gadolinium chloride (GdCl3) treatment in time- and concentration- dependent manners. A total of 48 metabolites released by Hela cells are identified to be differentially expressed (P < 0.05) in different states. Metabolic pathways analyses reveal that the differential metabolites are mainly characterized by increased lipid and amino acid metabolisms and by decreased lipid, amino acid, and carbohydrate metabolisms for cells treated with GdCl3 at lower and higher concentrations, respectively. Notably, in the higher level GdCl3 case, the down-expressions of metabolites are predominantly in the glycolytic and the redox pathways. The above results, obtained by using a metabolomic strategy for the first time, disclose that different cell signaling pathways are activated by GdCl3 treatment with different concentrations, leading to inhibitory or promotional effect on Hela cells.

A modified method for multi-site-directed mutagenesis was developed here based on polymerase chain reaction (PCR), DpnI digestion, and overlap extension. It needs only methylated plasmids obtained by Dam methyltransferase or plasmids from dam+Escherichia coli containing target gene. The procedure consists of PCR, DpnI digestion, overlap extension PCR, and plasmid transformation. The method was developed for multi-site-directed mutagenesis, including close proximity of mutation sites. It does not require 5′-phosphorylated primers and ligation and, thus, significantly simplifies the routine work and reduces the experimental cost for multi-site-directed mutagenesis.