Selenium (Se), as a nutritionally essential trace element, has been shown to decrease with age and is closely related to Alzheimer's disease (AD). To probe the effects of Se on AD pathology, two-dimensional fluorescence difference gel electrophoresis was applied to the serum samples collected from the wild-type (WT) mice and the triple transgenic (PS1M146V/AβPPSwe/TauP301L) AD mice (3xTg-AD), treated with or without sodium selenate in drinking water for 4 months beginning at 2 months of age. Proteomics results revealed 17 differentially expressed proteins between WT and 3xTg-AD mice. It was found that the administration of selenate reversed the alterations of the differentially expressed serum proteins by up-regulating 13 proteins and down-regulating 2 proteins which were reported to be involved in the key pathogenesis of AD, including regulation of Aβ production, lipid metabolism regulation, and anti-inflammation. These results suggested that a dietary supplement with selenate is effective for prevention and treatment of AD, and the mechanism was maybe related to its role in Aβ regulation, lipid metabolism, and anti-inflammation. Moreover, we also presented that α-2 macroglobulin, transthyretin, haptoglobin, alpha-2-HS-glycoprotein, and alpha-1-antitrypsin in the serum can be used to evaluate the effect of selenate on AD pathology.

Selenoprotien W (SelW) plays a key role in brain development, although the exact biological function and mechanisms remain unclear. We performed a yeast two-hybrid screen on a human fetal brain cDNA library and identified FAM96B as a novel binding partner of SelW. FRET analyses confirmed the interaction between SelW' and FAM96B. The mutated SelW' construct was cloned and overexpressed in E. coli, and a pull-down assay verified a direct interaction between SelW' and FAM96B. Finally, Co-Immunoprecipitation on murine brain tissue proteins demonstrated an endogenous interaction between the two proteins in the brain. Taken together, our findings prove a direct interaction between SelW and FAM96B, which may provide new insights into the role of SelW in brain development and neurodegenerative diseases.

Amyloid-β (Aβ) peptides have taken a central role in AD research, the aggregation of Aβ peptide is involved in the progression of Alzheimer's disease (AD). The 35th amino acid was methionine (Met) in Aβ peptides and it's redox state is critical in determining the biological activity of Aβ. It has been suggested that oxidation of Met35 (Met35O) plays a key role in the formation of paranuclei and in the control of oligomerization pathway choice. As an antioxidative selenoenzyme, Selenoprotein R (SelR) plays important roles in reducing the R-form of MetO to Met to maintain intracellular redox balance. However, the relationship between SelR and Aβ was little investigated. Here, we found that SelR can directly interact with Aβ42, and the interaction between SelR and Aβ42 was verified by fluorescence resonance energy transfer (FRET), co-immunoprecipitation (co-IP), and pull-down assays. SelR is closely related to AD, its biological functions in human brain become a research focus. This work implies that SelR makes it capable of modulating Aβ42 aggregation and provides a novel avenue for further study on the mechanism of SelR in AD prevention.

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.

Selenoprotien W (SelW) is an important selenoprotein that has the priority to be stored in brain when selenium is deficiency. However, the biological function and mechanisms of SelW in brain remain unclear. In this study, human SelW gene was cloned, site-directedly mutated and inserted into the "bait" plasmid. An interactive protein of SelW, prosaposin (P SAP), was discovered by screening the human fetal brain cDNA library using the yeast two-hybrid system. To verify the protein-protein interaction, two FRET methods, sensitized emission and acceptor bleaching, were performed respectively in HEK293T cells. Both assays confirmed the interaction between SelW' and PSAP. Then the expression vector of SelW' was constructed and SelW was overexpressed in E. coli. Pull-down assay was carried out by using the purified SelW', and exogenously direct interaction between SelW' and PSAP was also verified. Finally, co-IP method was applied to successfully verify the endogenous interaction between the two proteins in the brain tissues of Kunming mice. All these results show that SelW interacts with PSAP directly both in vivo and in vitro. SelW may play a key role in the brain development and neurodegenerative disease formation by interacting with PASP.

Selenoprotein R (SelR) plays an important role in maintaining intracellular redox balance by reducing the R-form of methionine sulfoxide to methionine. As SelR is highly expressed in brain and closely related to Alzheimer′s disease (AD), its biological functions in human brain become a research focus. In this paper, the selenocysteine-coding TGA of SelR gene was mutated to cysteine-coding TGC and used to screen the human fetal brain cDNA library with a yeast two-hybrid system. Our results demonstrated that SelR interacts with clusterin (Clu), a chaperone protein. This protein interaction was further verified by fluorescence resonance energy transfer (FRET), coimmunoprecipitation (co-IP), and pull-down assays. The interacting domain of Clu was determined by co-IP to be a dynamic, molten globule structure spanning amino acids 315 to 381 with an amphipathic-helix. The interacting domain of SelR was investigated by gene manipulation, ligand replacement, protein over-expression, and enzyme activity measurement to be a tetrahedral complex consisting of a zinc ion binding with four Cys residues. Study on the mutual effect of SelR and Clu showed synergic property between the two proteins. Cell transfection with SelR gene increased the expression of Clu, while cell transfection with Clu promoted the enzyme activity of SelR. Co-overexpression of SelR and Clu in N2aSW cells, an AD model cell line, significantly decreased the level of intracellular reactive oxygen species. Furthermore, FRET and co-IP assays demonstrated that Clu interacted with β-amyloid peptide, a pathological protein of AD, which suggested a potential effect of SelR and Aβ with the aid of Clu. The interaction between SelR and Clu provides a novel avenue for further study on the mechanism of SelR in AD prevention.

Selenoprotein M (SelM), one of the executants of selenium in vivo, is highly expressed in human brain and most probably involved in antioxidation, neuroprotection, and intracellular calcium regulation, which are the key factors for preventing the onset and progression of Alzheimer’s disease (AD). In this paper, human SelM was successfully overexpressed in human embryonic kidney cells HEK293T. Sodium selenite (Na2SeO3 0.5 μmol/L) increased the expression of full-length SelM and inhibited the expression of truncated SelM. The full-length SelM exhibited higher antioxidant activity than its selenocysteine-to-cysteine mutation form SelM', whereas the truncated SelM had an adverse effect that increased the oxidative stress level of cells. When β-amyloid (Aβ42, an AD relevant peptide) was cotransfected with the empty expression vector, SelM, or SelM' under the induction of 0.5 μmol/L Na2SeO3, the intracellular Aβ42 aggregation rates were detected to be 57.9% ± 5.5%, or 22.3% ± 2.6%, or 26.3% ± 2.1%, respectively, showing the inhibitory effects on Aβ aggregation by the full-length SelM and SelM'. Meanwhile, the intumescentia of mitochondria caused by Aβ42 transfection was significantly mitigated by the cotransfection of SelM or SelM′ with Aβ42 under the induction of 0.5 μmol/L Na2SeO3. On the contrary, cotransfection of SelM and Aβ42 without the induction of Na2SeO3 increased Aβ42 aggregation rate to 65.1% ± 3.2%, and it could not inhibit the Aβ-induced intumescent mitochondria. In conclusion, full-length SelM and SelM¢ might prevent Aβ aggregation by resisting oxidative stress generated during the formation of Aβ oligomers in cells.

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.

评述了阿尔茨海默病(Alzheimer's disease,AD)的多种病因学说,包括胆碱能学说、β-淀粉样级联学说、ABC学说(aging,beta-amyloid,channel)、tau蛋白过度磷酸化学说、氧化应激学说、神经细胞凋亡学说和基因突变学说等.指出AD是多因素引起的病变,其发病机制也必然有多种.由于该病与人体衰老过程密切相关,发病机理复杂,且神经细胞丢失后不具再生能力,这就决定了研究治疗AD有效药物的艰巨性.还指出AD防治工作应重视早期预报和早期干预,发展早期诊断方法包括正电子发射计算机断层扫描、磁共振成像,及血液和尿液中寻找灵敏的生物标志物等技术方法.目前有关AD防治药物包括一线治疗药物、疫苗、尚在研究阶段的药物、辅助性治疗药物、中药以及医疗保健品.前3类药物因疗效或研究结果长期未取得突破性进展,人们开始关注后3类药物对延缓AD的作用及机理.课题组重点开展后3种药物防治AD的作用和机理研究,发现硒代蛋氨酸、辅酶Q-10和中药组分淫羊藿甙对防治AD有好的疗效.综述了目前抗AD药物的研究现状和面临的挑战,提出AD的防治策略是:摒弃针对单一致病因素的治疗思路,从多因素及内在相互联系的系统生物学角度,开展AD防治机制及新药研发.

Selenium, an essential trace element for human health, mainly exerts its biological function through selenoproteins. Selenoprotein M (SelM) is one of the highly expressed selenoproteins in the brain, but its biological effect and molecular mechanism remain unclear. Thus, the interactive protein of SelM was investigated in this paper to guide further study. In order to avoid protein translational stop, the selenocysteine-encoding UGA inside the open reading frame of SelM was site-directly changed to the cysteine-encoding UGC to generate the SelM' mutant. Meanwhile, its N terminal transmembrane signal peptide was also cut off. This truncated SelM' was used to screen a human fetal brain cDNA library by the yeast two-hybrid system. A new interactive protein of SelM' was found to be galectin-1 (Gal-1). This protein-protein interaction was further verified by the results of fluorescence resonance energy transfer techniques, glutathione S-transferase pull-down and co-immunoprecipitation assays. As Gal-1 plays important roles in preventing neurodegeneration and promoting neuroprotection in the brain, the interaction between SelM' and Gal-1 displays a new direction for studying the biological function of SelM in the human brain.

Selenoprotein M (SelM) was discovered in 2002 by bioinformatics analysis. SelM is located in the endoplasmic reticulum, containing a common redox motif cysteine-X-X-selenocysteine. SelM attracts great attention due to its high expression in brain and its potential roles in antioxidative defense, neuroprotective function and cytosolic calcium regulation. This paper reviews recent research progress in SelM, especially its biological function and its relation with diseases, together with the author's work on SelM. The prospect of SelM research is also discussed in this paper.

Valosin‐containing protein (VCP) is a type‐II adenosine triphosphatase (ATPase) wih extensive biological function in organisms. Silkworm is the second insect model for genetic studies and a bioreactor for proteinaceous drugs and biomaterials. In this paper, a new VCP‐like gene was amplified from the fat body of silkworm following genome prediction and spliced expressed sequence tag sequences, using both reverse transcription polymerase chain reaction (RT‐PCR) and 3′‐RACE (rapid amplification of complementary DNA ends) methods. Bioinformatical analysis showed that the translated amino acid sequence contained a highly conserved domain of VCPs similar to that of many insects. This domain consists of the conserved structure motifs of the ATP binding site and the catalytical center, which is closely related to the insect VCPs in a phylogenetic tree. The silkworm VCP‐like gene was successfully inserted into the plasmid and transformed into Escherichia coli cells to express VCP‐like protein with ATPase activity. The expression of silkworm VCP‐like protein was also confirmed by Western blotting and mass spectrometric analyses. Distribution of the VCP‐like gene in various tissues of the silkworm was also studied by real‐time PCR. Results showed that the messenger RNA (mRNA) of VCP‐like protein is widely expressed in fat body, reproductive organs (testis or ovary), silk gland, head, Malpighian tubule, epidermis and midgut. Among them, fat body has the highest mRNA expression level of the VCP‐like gene, while the midgut has the lowest expression level. This study provides groundwork for further study on the structure and function of the new VCP‐like protein.

Selenium (Se) is an essential trace element in vivo involved in the defense against oxidative stress. Se deficiency is associated with many human diseases. The bioactivity of Se is dose- and species-dependent. Silkworm pupa has been reported to accumulate Se mainly in proteins. Thus the characterization of major Se-containing proteins is very important in the application of Se-rich silkworm pupas in food and drugs. In this study, crude proteins were extracted from Se-rich silkworm pupas, followed by DEAE-Sepharose and Sephedex G-75 chromatography. Se content was measured after each step to determine the highest Se-containing fraction for the next step of separation. The proteins obtained were analyzed using SDS-PAGE, followed by in-gel digestion with trypsin, and were characterized by MALDI-TOF MS and ESI-MS/MS. These data showed two proteins mainly accumulated Se in the silkworm pupas. Those two proteins were proven by mass spectrometry to be arylphorin and sex-specific storage-protein 2 precursor (SP-2), respectively. Both of them belong to the storage proteins of amino acids during metamorphosis and the non-feeding pupal stage. The results suggest that Se could be enriched by storage proteins and be supplied to silkworm pupas in accompany with amino acids for the synthesis of new Se-containing proteins and peptides.

Selenoprotein is biosynthesized by the incorporation of selenocysteine into proteins, where the TGA codon in the open reading frame does not act as a stop signal but is translated into selenocysteine. The dual functions of TGA result in mis-annotation or lack of selenoproteins in the sequenced genomes of many species. Available computational tools fail to correctly predict selenoproteins. Thus, we developed a new method to identify selenoproteins from the genome of Anopheles gambiae computationally. Based on released genomic information, several programs were edited with PERL language to identify selenocysteine insertion sequence (SECIS) element, the coding potential of TGA codons, and cysteine-containing homologs of selenoprotein genes. Our results showed that 11365 genes were terminated with TGA codons, 918 of which contained SECIS elements. Similarity search revealed that 58 genes contained Sec/Cys pairs and similar flanking regions around in-frame TGA codons. Finally, 7 genes were found to fully meet requirements for selenoproteins, although they have not been annotated as selenoproteins in NCBI databases. Deduced from their basic properties, the newly found selenoproteins in the genome of Anopheles gambiae are possibly related to in vivo oxidation tolerance and protein regulation in order to interfere with anopheles’ vectorial capacity of Plasmodium . This study may also provide theoretical bases for the prevention of malaria from anopheles transmission.

硒是一种生物必需微量元素,主要以硒蛋白形式发挥生物学功能.硒蛋白的生物合成取决于硒代半胱氨酸插入蛋白质的合成过程.TGA码既是终止码,又可翻译成硒代半胱氨酸,这使普通基因注释软件无法正确预测硒蛋白,导致现有数据库中许多物种的硒蛋白被错误注释或丢失.本研究基于已公布的家蚕基因组预测信息、采用PERL语言编程,对家蚕基因组中硒蛋白进行了计算机检索与分析.结果表明,在家蚕数据库18510个已注释基因中,以TGA码终止的基因有6348条,其中含有SECIS结构的基因249条,兼含半胱氨酸同源类似物的基因52条.再经硒代半胱氨酸(Sec)侧翼序列比对,最终检索到完全具备硒蛋白特点的基因5条,其中谷胱甘肽硫转移酶(GST)是一种已知微生物硒蛋白,而其他4种则是新硒蛋白,分别在已有基因表中被注释为CG6024蛋白,CG5195蛋白,ATP-结合盒转运蛋白A型(ABCA)和核VCP相似蛋白.通过对GST,ABCA和VCP主要性质的分析,推测家蚕硒蛋白在氧化调节、硒储存运输和细胞凋亡等过程中起重要作用.