Liming Shen

Redox proteomics identification of specifically carbonylated proteins in the hippocampi of triple transgenic Alzheimer's disease mice at its earliest pathological stage

UNLABELLED Alzheimers disease (AD) is the most common cause of dementia in the elderly population. Attempts to develop therapies for the treatment of the late stage AD have been unsuccessful. Increasing evidences indicate that oxidative stress is an early event of neurodegeneration, however the pathogenic mechanism of AD remains unclarified. In the present study, slot-blot analysis was used to determine the levels of protein carbonyls in the hippocampi of 3-month-old triple transgenic AD mice (3 × Tg-AD). The increased levels of protein carbonyls were observed in the hippocampi of 3 × Tg-AD mice as compared to the non-transgenic controls (non-Tg). Using a redox-proteomic approach, twelve proteins were found to be significantly altered in the levels of protein carbonyls in the hippocampus. These proteins are crucial in energy metabolism, protein folding, cell structure, signal transduction and excitotoxicity. Immunoprecipitation and Western blot were used to validate two proteins identified by the redox proteomics. In addition, increased expression level of carbonyl reductase 1 (CBR1) was observed in the hippocampi of 3 × Tg-AD mice. These results demonstrate that significant protein carbonylation occurs early in the 3-month-old 3 × Tg-AD mice, which support the viewpoint that oxidative stress is an early event in AD progression. BIOLOGICAL SIGNIFICANCE In this study, we have observed increased levels of protein carbonyls in the hippocampi of 3 × Tg-AD mice before the appearance of Aβ plaques and neurofibrillary tangles (NFTs). By redox proteomics, twelve specifically carbonylated proteins were identified. Among them, alpha-enolase (ENO1) and glutamine synthetase (GS) were identified as the common targets of oxidation in the brains of 3 × Tg-AD mice, mild cognitive impairment (MCI) sufferers and AD patients. For the first time, the oxidation of t-complex protein 1 subunit epsilon (CCT5) and protein disulfide-isomerase A3 (PDIA3) were reported to be associated with AD. These results indicated that the combination of monoclonal anti-DNP antibody with digital imaging system could enhance the specificity and accuracy of redox proteomics analysis. Those data support the viewpoint that oxidative stress occurs at the early pathological stage of AD. In addition, this paper provides new information for understanding the pathological process of AD and for developing more appropriate therapies to intervene AD progression.

A proteomic investigation into the human cervical cancer cell line HeLa treated with dicitratoytterbium (III) complex.

Lanthanides have been reported to induce apoptosis in cancer cell lines. Human cervical cancer cell line HeLa was found to be more sensitive to dicitratolanthanum (III) complex ([LaCit2](3-)) than other cancer cell lines. However, the effect and mechanism of dicitratoytterbium (III) complex ([YbCit2](3-)) on HeLa cells is unknown. Using biochemical and comparative proteomic analyses, [YbCit2](3-) was found to inhibit HeLa cell growth and induce apoptosis. Similar to the effects of [LaCit2](3-), proteomics results from [YbCit2](3-)-treated cells revealed profound changes in proteins relating to mitochondria and oxidative stress, suggesting that mitochondrial dysfunction plays a key role in [YbCit2](3-)-induced apoptosis. This was confirmed by the decreased mitochondrial transmembrane potential and the increased generation of reactive oxygen species in [YbCit2](3-)-treated cells. Western blot analysis showed that [YbCit2](3-)-induced apoptosis was accompanied by the activation of caspase-9 and specific proteolytic cleavage of PARP, leading to an increase in the pro-apoptotic protein Bax and a decrease in the anti-apoptotic protein Bcl-2. These results suggest a mitochondrial pathway of cell apoptosis in [YbCit2](3-)-treated cells, which will help us understand the molecular mechanisms of lanthanide-induced apoptosis in tumor cells.

Proteomic study on sodium selenite-induced apoptosis of human cervical cancer HeLa cells.

Sodium selenite can induce the apoptosis of cancer cells, however its mechanism has seldom been studied via proteomics. In this paper, human cervical cancer HeLa cells were investigated by MTT assay and morphological observation to get appropriate selenite concentrations for proteomic study. Results showed that selenite at concentrations larger than 10 μmol/L significantly inhibited the viability of HeLa cells. 40 μmol/L selenite was in the appropriate range for proteomic study. After 24 h treatment with 40 μmol/L selenite, total proteins were extracted from the cells and applied to two-dimensional gel electrophoresis (2DE). Those proteins with their expression levels altered at least 2-fold comparing to the control were picked up for protein identification via MALDI-TOF mass spectrometry and further confirmed by Western blot analysis. About 1000 spots were detected by the software in each 2DE gel, among which 13 differentially expressed proteins were identified by mass spectrometry and most of them are relevant to oxidative stress, such as peroxiredoxins, superoxide dismutase, quinolinate phosphoribosyl transferase, and D-dopachrome tautomerase. Meanwhile, reactive oxygen species (ROS) and mitochondrial membrane potential were also detected by flow cytometry and laser confocal scanning microscope. An increase in ROS generation and a decrease in mitochondrial membrane potential were detected in the selenite-treated cells compared with the control, which are consistent with the down-expression of antioxidative proteins in proteomics. Those results indicate that selenite induces the apoptosis of HeLa cells via ROS-mediated mitochondrial pathway. The present study also implies the potentiality of selenium in cervical cancer treatment.

Proteomic analysis of lanthanum citrate-induced apoptosis in human cervical carcinoma SiHa cells

Lanthanides possess diverse biological effect and have been shown to promote cell proliferation and induce apoptosis. Our previous studies showing that lanthanide citrate complex has significant antitumor activity in human cervical cancer HeLa cells. This study aims at determining if [LaCit2]3− have the activity against another type of human cervical cancer cell line SiHa and the changes in protein expression that contribute to the mechanism(s) of [LaCit2]3−-mediated apoptosis in SiHa cells. Cell growth inhibition was measured by MTT method, and apoptosis was detected by means of Hoechst 33258 staining and flow cytometry analysis. After [LaCit2]3−-treatment the results show that the growth of SiHa cells was inhibited, the cells displayed typical apoptosis morphological changes, and increase in the rates of apoptosis. Using proteomics approaches, a variety of differentially expressed proteins were identified in SiHa cells before and after treatment with [LaCit2]3−. There were profound changes in 10 proteins relating to mitochondrial function and oxidative stress, suggesting that mitochondrial dysfunction plays a key role in [LaCit2]3−-induced apoptosis. This was confirmed by a decrease in the mitochondrial transmembrane potential (Δψm), and increases in H2O2 generation in [LaCit2]3−-treated cells. Among them the alerted proteins, Prx I, ANXA1 and TRAF5 were validated by western blotting analyses. These results suggest that there is an intrinsic molecular pathway of cell apoptosis in [LaCit2]3−-treated SiHa cells. This observation is in accordance with our previous reports about the effects of [LaCit2]3− and [YbCit2]3− on HeLa cells and it provide a molecular mechanism underlying lanthanide citrate complex-mediated cell apoptosis.

Redox Proteomic Profiling of Specifically Carbonylated Proteins in the Serum of Triple Transgenic Alzheimer’s Disease Mice

Oxidative stress is a key event in the onset and progression of neurodegenerative diseases, including Alzheimer’s disease (AD). To investigate the role of oxidative stress in AD and to search for potential biomarkers in peripheral blood, serums were collected in this study from the 3-, 6-, and 12-month-old triple transgenic AD mice (3×Tg-AD mice) and the age- and sex-matched non-transgenic (non-Tg) littermates. The serum oxidized proteins were quantified by slot-blot analysis and enzyme-linked immunosorbent assay (ELISA) to investigate the total levels of serum protein carbonyl groups. Western blotting, in conjunction with two-dimensional gel electrophoresis (2D-Oxyblot), was employed to identify and quantify the specifically-carbonylated proteins in the serum of 3×Tg-AD mice. The results showed that the levels of serum protein carbonyls were increased in the three month old 3×Tg-AD mice compared with the non-Tg control mice, whereas no significant differences were observed in the six and 12 months old AD mice, suggesting that oxidative stress is an early event in AD progression. With the application of 2D-Oxyblot analysis, (immunoglobin) Ig gamma-2B chain C region (IGH-3), Ig lambda-2 chain C region (IGLC2), Ig kappa chain C region (IGKC), and Ig kappa chain V-V region HP R16.7 were identified as significantly oxidized proteins compared with the control. Among them IGH-3 and IGKC were validated via immunoprecipitation and Western blot analysis. Identification of oxidized proteins in the serums of 3×Tg-AD mice can not only reveal potential roles of those proteins in the pathogenesis of AD but also provide potential biomarkers of AD at the early stage.

Phosphoproteomic Profiling of Selenate-Treated Alzheimer's Disease Model Cells

The reversible phosphorylation of proteins regulates most biological processes, while abnormal phosphorylation is a cause or consequence of many diseases including Alzheimers 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.

Gadolinium promoted proliferation in mouse embryo fibroblast NIH3T3 cells through Rac and PI3K/Akt signaling pathways

Nephrogenic systemic fibrosis (NSF) is a fibrosing disorder disease developed in patients with underlying renal insufficiency following exposure to gadolinium-based contrast agents (GBCAs). Previous studies have demonstrated that GdCl3 can promote NIH3T3 fibroblast cell proliferation, which provide a new clue to the role of GBCAs in the development of NSF. In the present study, we further clarify the molecular mechanism of Gd-promoted proliferation. The results showed that intervention with the Rac inhibitor NSC23766 abrogated Gd-promoted proliferation. The levels of active Rac1 significantly increased in Gd-treated cells detected by pull-down assays. In addition, the phosphorylation of Akt was significantly elevated in the treatment group, which was blocked by NSC23766. NSC23766 also reduced the migration of NIH3T3 cells enhanced by Gd. Moreover, the F-actin cytoskeleton was strengthened and the mitotic cell numbers was significantly increased after exposure to Gd. These results suggest that Rac and PI3K/Akt signaling pathways, as well as integrin-mediated signal pathway may play important roles in Gd-induced cell proliferation. In addition, under serum-free condition, Gd could decrease ROS accumulation and increase NIH3T3 cell survival.

Redox proteomic identification of carbonylated proteins in autism plasma: insight into oxidative stress and its related biomarkers in autism

BackgroundAutism is a severe childhood neurological disorder with poorly understood etiology and pathology. Currently, there is no authentic laboratory test to confirm the diagnosis of autism. Oxidative damage may play a central role in the pathogenesis of autism. Present study is an effort to search for possible biomarkers of autism and further clarify the molecular changes associated with oxidative stress that occurs in the plasma of autistic children.MethodsWe performed redox proteomics analysis to compare carbonylated proteins in the plasma of autistic subjects and healthy controls. Immunoprecipitation and Western blot analysis were used to validate carbonylated proteins identified by the redox proteomics.ResultsProtein carbonylation levels in two proteins, complement component C8 alpha chain and Ig kappa chain C were found to be significantly increased in autistic patients compared with controls. These two proteins were successfully validated via immunoprecipitation and Western blot analysis.ConclusionsThe results further highlight the role of oxidative stress in the pathogenesis of autism and provide some information for the diagnosis and/or monitoring of autism.

iTRAQ‐Based Proteomic Analysis Reveals Protein Profile in Plasma from Children with Autism

Autism is a childhood neurological disorder with poorly understood etiology and pathology. This study is designed to identify differentially expressed proteins that might serve as potential biomarkers for autism.

Effect of Sodium Selenate on Hippocampal Proteome of 3×Tg-AD Mice—Exploring the Antioxidant Dogma of Selenium against Alzheimer’s Disease

Selenium (Se), an antioxidant trace element, is an important nutrient for maintaining brain functions and is reported to be involved in Alzheimers disease (AD) pathologies. The present study has been designed to elucidate the protein changes in hippocampus of 3×Tg-AD mice after supplementing sodium selenate as an inorganic source of selenium. By using iTRAQ proteomics technology, 113 differentially expressed proteins (DEPs) are found in AD/WT mice with 37 upregulated and 76 downregulated proteins. Similarly, in selenate-treated 3×Tg-AD (ADSe/AD) mice, 115 DEPs are found with 98 upregulated and 17 downregulated proteins. The third group of mice (ADSe/WT) showed 75 DEPs with 46 upregulated and 29 downregulated proteins. Among these results, 42 proteins (40 downregulated and 2 upregulated) in the diseased group showed reverse expression when treated with selenate. These DEPs are analyzed with different bioinformatics tools and are found associated with various AD pathologies and pathways. Based on their functions, selenate-reversed proteins are classified as structural proteins, metabolic proteins, calcium regulating proteins, synaptic proteins, signaling proteins, stress related proteins, and transport proteins. Six altered AD associated proteins are successfully validated by Western blot analysis. This study shows that sodium selenate has a profound effect on the hippocampus of the triple transgenic AD mice. This might be established as an effective therapeutic agent after further investigation.