Selenium-binding Protein Research Articles

Identification of an argpyrimidine-modified protein in human red blood cells from schizophrenic patients: A possible biomarker for diseases involving carbonyl stress

Argpyrimidine (ARP) is an advanced glycation end product thought to be generated from a reaction between methylglyoxal and arginine residues in proteins. In this study, we observed marked accumulation of an approximately 56 kD protein, reactive to anti-ARP antibodies, in the red blood cells (RBCs) of some patients with refractory schizophrenia. This ARP-modified protein was purified from the blood of schizophrenic patients and identified as selenium binding protein 1 (SBP1) by LC-MS/MS. This is the first report of ARP-modified proteins accumulating in RBCs of patients with diseases involving carbonyl stress. We also observed high accumulation of ARP-modified SBP1 in the RBCs of patients with chronic kidney disease. Therefore, this modified protein may be a novel marker of carbonyl stress.

Acute toxicity of functionalized single wall carbon nanotubes: A biochemical, histopathologic and proteomics approach

Recently carbon nanotubes (CNTs) showed promising potentials in different biomedical applications but their safe use in humans and probable toxicities are still challenging. The aim of this study was to determine the acute toxicity of functionalized single walled carbon nanotubes (SWCNTs). In this project, PEGylated and Tween functionalized SWCNTs were prepared. BALB/c mice were randomly divided into nine groups, including PEGylated SWCNTs (75,150μg/mouse) and PEG, Tween80 suspended SWCNTs, Tween 80 and a control group (intact mice). One or 7 days after intravenous injection, the mice were killed and serum and livers were collected. The oxidative stress markers, biochemical and histopathological changes were studied. Subsequently, proteomics approach was used to investigate the alterations of protein expression profiles in the liver.Results showed that there were not any significant differences in malondealdehyde (MDA), glutathione (GSH) levels and biochemical enzymes (ALT and AST) between groups, while the histopathological observations of livers showed some injuries. The results of proteomics analysis revealed indolethylamine N-Methyltransferase (INMT), glycine N-Methyltransferase (GNMT), selenium binding protein (Selenbp), thioredoxin peroxidase (TPx), TNF receptor associated protein 1(Trap1), peroxiredoxin-6 (Prdx6), electron transport flavoprotein (Etf-α), regucalcin (Rgn) and ATP5b proteins were differentially expressed in functionalized SWCNTs groups. Western blot analyses confirmed that the changes in Prdx6 were consistent with 2-DE gel analysis. In summary, acute toxicological study on two functionalized SWCNTs did not show any significant toxicity at selected doses. Proteomics analysis also showed that following exposure to functionalized SWCNTs, the expression of some proteins with antioxidant activity and detoxifying properties were increased in liver tissue.

Assessment of Plasma Selenium-Binding Protein-1 Level in Geriatric Population

Geriatric physiologically undergoes aging process that can cause decreasing cell function and increasing risk of degenerative diseases caused by the accumulation of reactive oxygen species (ROS) in the body. Selenium (Se) is an antioxidant, which is needed for maintaining the balance of ROS. The aim of this study was to observe the selenium-binding protein (SELENBP1) level in relation with the geriatric patients charcateristics including sex, age, body mass index (BMI), activity of daily living (ADL), instrumental activity of daily living (IADL), cognitive function, nutrition, depression, and insomnia status. The study used cross-sectional quantitative descriptive study design on 14 geriatric patients in Geriatric Outpatients Clinics, at a hospital in Bandung. The data was obtained by interviewing the patients and then blood samples were taken. The analysis of SELENBP1 was done using ELISA kit. The average level (SD) of SELENBP1 from all of the characteristics group was 2.68 (0.69) ng/ml. The highest SELENBP1 level was identified in female geriatric patients and followed by male and pre-obese groups. The lowest SELENBP1 level was identified in geriatric patients aged 70-79 years. Keywords: elderly, SELENBP1, selenium.

Abstract 387: Weighted Gene Co-Expression Network Analysis Identification and Experimental Validation of Genetic Determinants of Right Ventricular Function

Right ventricular dysfunction (RVD) portends worse outcomes in chronic heart failure (HF), irrespective of left ventricular function. Despite the significance and prevalence of RVD, its molecular pathobiology is poorly understood. Gene networks can identify and explore complex relationships between genes, gene pathways, and phenotypes. We used weighted gene co-expression network analysis (WGNA) of human heart samples to identify genetic determinants of RV function and validated candidate genes in animal models of RV versus LV dysfunction. We isolated and sequenced total RNA from explanted left and right ventricles of ischemic cardiomyopathy (ICM) patients (n=10) who underwent cardiac transplantation. Non-failing donor hearts that were declined for transplant were used as controls (n=5). Half the ICM patients had severe RV failure (RVF) based upon hemodynamic indices (mean±SEM) with mean RA 25.8±1.7 mmHg, RA:PCWP ratio 1.05±0.11, and PA pulsatility index 1.6±0.2. Using direct gene membership comparison and z-score analysis, we found that more than half the modules in RV-specific networks are shared by combined RV-LV gene networks. We used principle component analysis to examine modules that correlated with RVF-related phenotypes. Two modules unique to the RV network identified selenium-binding protein gene Selenbp1 as a potential driver of RVF. We also identified a strong association between anti-apoptotic gene Mif and vesicle fusion gene Snap47 with RA:PCWP and PA pulsatility indices, suggesting potential important roles of these genes in RVF. Finally, peroxiredoxin 5 PRDX5 and E3 ubiquitin-protein ligase SHPRH were associated with protection against RVF. We sought to validate these candidate gene drivers of RV function in mouse models of RV versus LV-specific dysfunction induced by pulmonary artery banding (PAB) and transverse aortic constriction (TAC), respectively. PRDX5 expression was markedly reduced in RV of PAB versus Sham but unchanged in LV of TAC versus Sham. Selenbp1 was diminished in both RV of PAB and LV of TAC mice, relative to Sham. Expression of other candidate genes did not vary significantly with PAB or TAC. Our preliminary findings suggest that cardiac PRDX5 may play an important role in regulating RV function.

P 172 - Two putative selenium binding proteins as modulators of C. elegans stress response and life span

Selenium-binding proteins do not contain selenium in the form of selenocysteine or selenomethionine but as inorganic selenium bound to the protein. The C. elegans genome encodes only one selenocysteine-containing selenoprotein, TrxR-1. However, at least two ORFs encode putative selenium-binding proteins, CeSELENBP1 and CeSELENBP2. These are 54% and 36% homologous to human selenium-binding protein-1 (SELENBP1), respectively, and both contain a cysteine residue hypothesized to bind selenite as in SELENBP1. Considering the role of selenium in antioxidant defence, we hypothesized that CeSELENBP1 and CeSELENBP2 may modulate the response of C. elegans to oxidative stress. Unexpectedly, life-long knock-down of either of the genes significantly increased life span and stress resistance to the redox cycler paraquat. DAF-16 and SKN-1 are key players in the C. elegans stress response that are known to mediate many of the published life-extending effects of chemical compounds, phytochemicals and nutrition regimens. However, RNAi against CeSELENBP1 and CeSELENBP2 significantly increased life span of the mutants to an extent similar to that found in wild type worms exposed to RNAi targeting either of the putative selenium binding proteins. This suggests that neither DAF-16 nor SKN-1 are involved in life span extension elicited by knock-down of CeSELENBP1 and CeSELENBP2.

Identification of a sensitive urinary biomarker, selenium-binding protein 1, for early detection of acute kidney injury

ABSTRACTAcute kidney injury (AKI) is associated with increased mortality rate in patients but clinically available biomarkers for disease detection are currently not available. Recently, a new biomarker, selenium-binding protein 1 (SBP1), was identified for detection of nephrotoxicity using proteomic analysis. The aim of this study was to assess the sensitivity of urinary SBP1 levels as an early detection of AKI using animal models such as cisplatin or ischemia/reperfusion (I/R). Sprague–Dawley rats were injected with cisplatin (6 mg/kg, once i.p.) and sacrificed at 1, 3, or 5 days after treatment. Ischemia was achieved by bilaterally occluding both kidneys with a microvascular clamp for 45 min and verified visually by a change in tissue color. After post-reperfusion, urine samples were collected at 9, 24, and 48 hr intervals. Urinary excretion of protein-based biomarkers was measured by Western blot analysis. In cisplatin-treated rats, mild histopathologic alterations were noted at day 1 which became severe at day 3. Blood urea nitrogen (BUN) and serum creatinine (SCr) levels were significantly increased at day 3. Levels of urinary excretion of SBP1, neutrophil gelatinase-associated lipocalin (NGAL), and a tissue inhibitor of metalloproteinase-1 (TIMP-1) were markedly elevated at day 3 and 5 following drug treatment. In the vehicle-treated I/R group, serum levels of BUN and SCr and AST activity were significantly increased compared to sham. Urinary excretion of SBP1 and NGAL rose markedly following I/R. The urinary levels of SBP1, NGAL, TIMP-1, and KIM-1 proteins excreted by AKI patients and normal subjects were compared. Among these proteins, a marked rise in SBP1 was observed in urine of patients with AKI compared to normal subjects. Based upon receiver-operator curves (ROC), SBP1 displayed a higher area under the curve (AUC) scores than levels of SCr, BUN, total protein, and glucose. In particular, SBP1 protein was readily detected in small amounts of urine without purification. Data thus indicate that urinary excretion of SBP1 may be useful as a reliable biomarker for early diagnosis of AKI in patients.

Selenium-Binding Protein 1 (SBP1) autoantibodies in ovarian disorders and ovarian cancer.

Infertility is a risk factor for ovarian cancer (OvCa). The goal was to determine if antibodies to selenium-binding protein 1 (SBP1), an autoantibody we identified in patients with premature ovarian failure (POF), occurs in both infertility and OvCa patients, and thus could be associated with preneoplasia. Anti-SBP1 was measured by immunoassay against recombinant SBP1, in sera from OvCa (n = 41), infertility (n = 92) and control (n = 87) patients. Infertility causes were POF, unexplained, irregular ovulation or endometriosis. The percent of anti-SBP1-positive sera was higher in POF (P = 0.02), irregular ovulation (P = 0.001), unexplained causes (P = 0.02), late (III-IV)-stage OvCa (P = 0.02) but was not significant in endometriosis, benign ovarian tumors/cysts, early stage (I-II) OvCa or uterine cancer compared to healthy controls. Anti-SBP1 was significantly higher in women with serous (P = 0.04) but not non-serous (P = 0.33) OvCa compared to controls. Also, we determined if anti-SBP1 was associated with CA125 or anti-TP53, markers often studied in OvCa. Anti-TP53 and CA125 were measured by established immunoassays. The ability of anti-SBP1 alone to discriminate infertility or OvCa from controls or when combined with anti-TP53 and CA125, to identify OvCa was evaluated by comparing the area under the curve (AUC) in ROC analysis. Anti-SBP1 alone discriminated infertility (AUC = 0.7; P = 0.001) or OvCa (AUC = 0.67; P = 0.03) from controls. The sensitivity and specificity of OvCa identification was increased by combining CA125, anti-TP53 and anti-SBP1 (AUC = 0.96). Therefore, anti-SBP1 occurs in infertile women with POF, ovulatory disturbances or unexplained infertility and in serous OvCa. This suggests an autoimmune process is associated with the development of serous OvCa.

Analysis of protein profiling studies of β-thalassemia/Hb E disease.

A number of studies have used global protein profiling technologies on a range of patient samples to detect proteins that are differentially expressed in β-thalassemia/Hb E as an aid for understanding the physiopathology of this disease. Seven studies have identified a total of 111 unique, differentially expressed proteins. Seven proteins (prothrombin, alpha-1-antichymotrypsin, fibrinogen beta chain, hemoglobin beta, selenium-binding protein, microtubule-actin cross-linking factor and adenomatous polyposis coli protein 2) have been identified in two independent studies, whereas two proteins (carbonic anhydrase 1 and peroxiredoxin-2) have been identified in three independent studies. Both of these latter two proteins were consistently upregulated in the studies that identified them. Ontological analysis of all differentially regulated proteins identified "response to inorganic substances" as the most significant functional annotation cluster, which is consistent with iron overload being a major pathological consequence of this disease. Despite the range of samples investigated and the relatively small number of studies undertaken, a coherent picture of the mediators of the pathological consequences of β-thalassemia/Hb E disease is starting to emerge.

Selenium-binding protein 1: a sensitive urinary biomarker to detect heavy metal-induced nephrotoxicity.

Identifying novel biomarkers to detect nephrotoxicity is clinically important. Here, we attempted to identify new biomarkers for mercury-induced nephrotoxicity and compared their sensitivity to that of traditional biomarkers in animal models. Comparative proteomics analysis was performed in kidney tissues of Sprague-Dawley rats after oral treatment with HgCl2 (0.1, 1, or 5mg/kg/day) for 21days. Kidney cortex tissues were analyzed by two-dimensional gel electrophoresis/matrix-assisted laser desorption/ionization, and differentially expressed proteins were identified. The corresponding spots were quantitated by RT-PCR. Selenium-binding protein 1 (SBP1) was found to be the most markedly upregulated protein in the kidney cortex of rats after HgCl2 administration. However, blood urea nitrogen, serum creatinine, and glucose levels increased significantly only in the 1 or 5mg/kg HgCl2-treated groups. A number of urinary excretion proteins, including kidney injury molecule-1, clusterin, monocyte chemoattractant protein-1, and β-microglobulin, increased dose-dependently. Histopathological examination revealed severe proximal tubular damage in high-dose (5mg/kg) HgCl2-exposed groups. In addition, urinary excretion of SBP1 significantly increased in a dose-dependent manner. To confirm the critical role of SBP1 as a biomarker for nephrotoxicity, normal kidney proximal tubular cells were treated with HgCl2, CdCl2, or cisplatin for 24h. SBP1 levels significantly increased in conditioned media exposed to nephrotoxicants, but decreased in cell lysates. Our investigations suggest that SBP1 may play a critical role in the pathological processes underlying chemical-induced nephrotoxicity. Thus, urinary excretion of SBP1 might be a sensitive and specific biomarker to detect early stages of kidney injury.

Selenium-binding protein 1 in head and neck cancer is low-expression and associates with the prognosis of nasopharyngeal carcinoma.

Background:Selenium-binding protein 1 (SELENBP1) expression is reduced markedly in many types of cancers and low SELENBP1 expression levels are associated with poor patient prognosis.Methods:SELENBP1 gene expression in head and neck squamous cell carcinoma (HNSCC) was analyzed with GEO dataset and characteristics of SELENBP1 expression in paraffin embedded tissue were summarized. Expression of SELENBP1 in nasopharyngeal carcinoma (NPC), laryngeal cancer, oral cancer, tonsil cancer, hypopharyngeal cancer and normal tissues were detected using immunohistochemistry, at last, 99 NPC patients were followed up more than 5 years and were analyzed the prognostic significance of SELENBP1.Results:Analysis of GEO dataset concluded that SELENBP1 gene expression in HNSCC was lower than that in normal tissue (P < 0.01), but there was no significant difference of SELENBP1 gene expression in different T-stage and N-stage (P > 0.05). Analysis of pathological section concluded that SELENBP1 in the majority of HNSCC is low expression and in cancer nests is lower expression than surrounding normal tissue, even associated with the malignant degree of tumor. Further study indicated the low SELENBP1 expression group of patients with NPC accompanied by poor overall survival and has significantly different comparing with the high expression group.Conclusion:SELENBP1 expression was down-regulated in HNSCC, but has no associated with T-stage and N-stage of tumor. Low expression of SELENBP1 in patients with NPC has poor over survival, so SELENBP1 could be a novel biomarker for predicting prognosis.

Effect of Aerobic Exercise on Selenium Level in Plasma

Moderate and high aerobic exercises are the activities that can induce high oxidant. Demand of endogen and exogen antioxidant for neutralizing oxidant induced by exercise will play important role in maintaining body performance. Microtrace elements, such as selenium, may able to balance high oxidant formation. However, limited research and data have been collected with regards to the exercise and selenium. Therefore, this research was conducted to investigate the effect of aerobic exercise on selenium level in plasma. An analytic study with experimental method of data collection was performed from October -November 2016. 17 male students with the age between 19-25 years old and without medically compromised disease from Universitas Padjadjaran, Jatinangor, were selected as subjects. Blood samples were taken pre- and post-aerobic exercise, i.e., brisk walking with the intensity of 4.5 mi/h. Selenium level in plasma was then examined by enzyme linked immunosorbent assay (ELISA) kit as selenium binding protein (SELENBP1). This study found that 11 (65%) subjects had a decrease in their selenium plasma after exercise (3.1 ± 2.58 ng/ml), 5 (29%) subjects had an increase in their selenium plasma after exercise (2.08 ± 2.8 ng/ml) and 1 (6%) subject had unchanged selenium (SELENBP1) plasma (1.4 ± 1.4 ng/ml) level. Effect of aerobic exercise on selenium level in plasma varied among individuals. Keywords: aerobic exercise, selenium, young adult

Loss of Selenium-Binding Protein 1 Decreases Sensitivity to Clastogens and Intracellular Selenium Content in HeLa Cells.

Selenium-binding protein 1 (SBP1) is not a selenoprotein but structurally binds selenium. Loss of SBP1 during carcinogenesis usually predicts poor prognosis. Because genome instability is a hallmark of cancer, we hypothesize that SBP1 sequesters cellular selenium and sensitizes cancer cells to DNA-damaging agents. To test this hypothesis, we knocked down SBP1 expression in HeLa cervical cancer cells by employing a short hairpin RNA (shRNA) approach. Reduced sensitivity to hydrogen peroxide, paraquat and camptothecin, reactive oxygen species content, and intracellular retention of selenium after selenomethionine treatment were observed in SBP1 shRNA HeLa cells. Results from Western analyses showed that treatment of HeLa cells with selenomethionine resulted in increased SBP1 protein expression in a dose-dependent manner. Knockdown of SBP1 rendered HeLa cells increased expression of glutathione peroxidase-1 but not glutathione peroxidase-4 protein levels and accelerated migration from a wound. Altogether, SBP1 retains supplemental selenium and sensitizes HeLa cancer cells to clastogens, suggesting a new cancer treatment strategy by sequestering selenium through SBP1.

Plasma Selenium Level based on Dietary Intake in Geriatric Clinic Population at Dr. Hasan Sadikin General Hospital Bandung

Low plasma selenium level was associated with the increasing risk of death in geriatric patients, particularly those with multiple comorbidities. The sufficient level of selenium intake as an antioxidant is necessary for this population. This study aimed to investigate the plasma selenium level based on dietary intake in geriatric clinic population at Dr. Hasan Sadikin General Hospital Bandung.This study was a descriptive study using cross-sectional method. Fourteen geriatric patients were selected by consecutive sampling technique. Semiquantitative food frequency questionnaire (SQ-FFQ) was used as a tool to assess dietary intake. Plasma selenium level was measured as selenium binding protein 1 (SELENBP1) using enzyme-linked immunosorbent assay kit. Overall, mean of plasma selenium level of the subjects was 2,68 µg/L and mean of selenium intake was 62,85 µg/day. Selenium level of the subjects with sufficient selenium intake (85,7%) was 2,62 µg/L and selenium level of the subjects with deficient selenium intake (14,3%) was 3,05 µg/L. In conclusion, plasma selenium level among geriatric patients was varied and not dependent on dietary intake. Keywords: antioxidant, dietary intake, geriatric, plasma selenium level

Downregulation of selenium-binding protein 1 is associated with poor prognosis in lung squamous cell carcinoma.

BackgroundWe found that selenium-binding protein 1 (SBP1) was progressively decreased in the human bronchial epithelial carcinogenic processes. Knockdown of SBP1 in immortalized human bronchial epithelial cell line 16HBE cells significantly increased the efficiency of B[a]P-induced cell transformation. However, the relationship between SBP1 expression and clinicopathological factors of patients has not been defined completely. The specific role of SBP1 in prognosis of lung squamous cell carcinoma (LSCC) is still unknown.MethodsTissue samples from 82 patients treated by pulmonary lobectomy for LSCC were used. Immunohistochemistry and western blotting were used to detect the expressions of SBP1 protein. The relationships between the expression level of SBP1 and the clinicopathological features of patients were analyzed. Cox proportional hazard regression analysis and Kaplan–Meier method were used to perform survival analysis.ResultsExpressions of SBP1 proteins were significantly lower in LSCC tissues than that in the corresponding normal bronchial epithelium (NBE) tissues (P = 0.000). In LSCC, The expression levels of SBP1 had not correlated with patients’ age, gender, smoking state, primary tumor stages (T), TNM clinical stages, and distant metastasis (M) (P > 0.05). However, downregulation of SBP1 was significantly associated with higher lymph node metastasis and lower overall survival rate (P < 0.05). Cox regression analysis indicated low expressions of SBP1 can be an independent prognostic factor for poor overall survival in LSCC patients (P = 0.002).ConclusionsDownregulation of SBP1 may play a key role in the tumorigenic process of LSCC. SBP1 may be a novel potential prognostic factor of LSCC.

Supernatant protein biomarkers of red blood cell storage hemolysis as determined through an absolute quantification proteomics technology.

Laboratory technologies have highlighted the progressive accumulation of the so-called "storage lesion," a wide series of alterations to stored red blood cells (RBCs) that may affect the safety and effectiveness of the transfusion therapy. New improvements in the field are awaited to ameliorate this lesion, such as the introduction of washing technologies in the cell processing pipeline. Laboratory studies that have tested such technologies so far rely on observational qualitative or semiquantitative techniques. A state-of-the-art quantitative proteomics approach utilizing quantitative concatamers (QconCAT) was used to simultaneously monitor fluctuations in the abundance of 114 proteins in AS-3 RBC supernatants (n = 5; 11 time points, including before and after leukoreduction, at 3 hours, on Days 1 and 2, and weekly sampling from Day 7 through Day 42). Leukoreduction-dependent depletion of plasma proteins was observed at the earliest time points. A subset of proteins showed very high linear correlation (r(2) > 0.9) not only with storage time, but also with absolute levels of hemoglobin α1 and β, a proxy for RBC hemolysis and vesiculation. Linear regression was performed to describe the temporal relationship between these proteins. Our findings suggest a role for supernatant glyceraldehyde-3-phosphate dehydrogenase; peroxiredoxin-1, -2, and -6; carbonic anhydrase-1 and -2; selenium binding protein-1; biliverdin reductase; aminolevulinate dehydratase; and catalase as potential biomarkers of RBC quality during storage. A targeted proteomics technology revealed novel biomarkers of the RBC storage lesion and promises to become a key analytical readout for the development and testing of alternative cell processing strategies.

Sodium Selenite Inhibits Proliferation of Gastric Cancer Cells by Inducing SBP1 Expression.

Selenium is an essential trace element with an inhibitory effect on many types of human cancers, including gastric cancer. Selenium-binding protein 1 (SBP1) has been shown as a possible mediator of selenium's anti-cancer functions. Indeed, SBP1 was downregulated in gastric cancer, which is related with poor prognosis. However, the molecular mechanisms underlying the anti-tumor effects of SBP1 remain poorly understood. In this study, we aimed to assess the effects of selenium and/or SBP1 on the proliferation of gastric cancer cells. We used SGC7901 and N87 human gastric cancer cell lines and nude mice carrying subcutaneously implanted SGC7901 cells. Treatment with sodium selenite for 48 h caused the inhibition of cell proliferation and the increase in apoptosis of SGC7901 and N87 cells. Furthermore, sodium selenite increased the expression level of SBP1 and decreased the levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and the Wnt pathway components and its downstream targets, including β-catenin, GSK-3β, c-myc and cyclinD1 in these cell lines. However, these effects of sodium selenite were attenuated in SGC7901 and N87 cells by knockdown of SBP1 expression. Thus, the sodium selenite-induced SBP1 expression is associated with the inhibition of cell proliferation and with the induced apoptosis. Importantly, sodium selenite treatment retarded the growth of the transplanted SGC7901 cells in nude mice, with the induction of SBP1 expression, which was associated with the decrease of Nrf2 expression and the inactivation of the Wnt/β-catenin signaling pathway. We suggest that sodium selenite may have a potential application in gastric cancer treatment.

A Comprehensive Analysis of Selenium-Binding Proteins in the Brain Using Its Reactive Metabolite.

The intracellular metabolism of selenium in the brain currently remains unknown, although the antioxidant activity of this element is widely acknowledged to be important in maintaining brain functions. In this study, a comprehensive method for identifying the selenium-binding proteins using PenSSeSPen as a model of the selenium metabolite, selenotrisulfide (RSSeSR, STS), was applied to a complex cell lysate generated from the rat brain. Most of the selenium from L-penicillamine selenotrisulfide (PenSSeSPen) was captured by the cytosolic protein thiols in the form of STS through the thiol-exchange reaction (R-SH+PenSSeSPen→R-SSeSPen+PenSH). The cytosolic protein species, which reacted with the PenSSeSPen mainly had a molecular mass of less than 20 kDa. A thiol-containing protein at m/z 15155 in the brain cell lysate was identified as the cystatin-12 precursor (CST12) from a rat protein database search and a tryptic fragmentation experiment. CST12 belongs to the cysteine proteinase inhibitors of the cystatin superfamily that are of interest in mechanisms regulating the protein turnover and polypeptide production in the central nervous system and other tissues. Consequently, CST12 is suggested to be one of the cytosolic proteins responsible for the selenium metabolism in the brain.

A Critical Role for Cysteine 57 in the Biological Functions of Selenium Binding Protein-1.

The concentration of selenium-binding protein1 (SBP1) is often lower in tumors than in the corresponding tissue and lower levels have been associated with poor clinical outcomes. SBP1 binds tightly selenium although what role selenium plays in its biological functions remains unknown. Previous studies indicated that cysteine 57 is the most likely candidate amino acid for selenium binding. In order to investigate the role of cysteine 57 in SBP1, this amino acid was altered to a glycine and the mutated protein was expressed in human cancer cells. The SBP1 half-life, as well as the cellular response to selenite cytotoxicity, was altered by this change. The ectopic expression of SBP1GLY also caused mitochondrial damage in HCT116 cells. Taken together, these results indicated that cysteine 57 is a critical determinant of SBP1 function and may play a significant role in mitochondrial function.

Protein Oxidation in the Lungs of C57BL/6J Mice Following X-Irradiation.

Damage to normal lung tissue is a limiting factor when ionizing radiation is used in clinical applications. In addition, radiation pneumonitis and fibrosis are a major cause of mortality following accidental radiation exposure in humans. Although clinical symptoms may not develop for months after radiation exposure, immediate events induced by radiation are believed to generate molecular and cellular cascades that proceed during a clinical latent period. Oxidative damage to DNA is considered a primary cause of radiation injury to cells. DNA can be repaired by highly efficient mechanisms while repair of oxidized proteins is limited. Oxidized proteins are often destined for degradation. We examined protein oxidation following 17 Gy (0.6 Gy/min) thoracic X-irradiation in C57BL/6J mice. Seventeen Gy thoracic irradiation resulted in 100% mortality of mice within 127–189 days postirradiation. Necropsy findings indicated that pneumonitis and pulmonary fibrosis were the leading cause of mortality. We investigated the oxidation of lung proteins at 24 h postirradiation following 17 Gy thoracic irradiation using 2-D gel electrophoresis and OxyBlot for the detection of protein carbonylation. Seven carbonylated proteins were identified using mass spectrometry: serum albumin, selenium binding protein-1, alpha antitrypsin, cytoplasmic actin-1, carbonic anhydrase-2, peroxiredoxin-6, and apolipoprotein A1. The carbonylation status of carbonic anhydrase-2, selenium binding protein, and peroxiredoxin-6 was higher in control lung tissue. Apolipoprotein A1 and serum albumin carbonylation were increased following X-irradiation, as confirmed by OxyBlot immunoprecipitation and Western blotting. Our findings indicate that the profile of specific protein oxidation in the lung is altered following radiation exposure.

SELENBP1 expression in the prefrontal cortex of subjects with schizophrenia.

Selenium binding protein 1 (SELENBP1) messenger RNA (mRNA) has previously been shown to be upregulated in the brain and blood from subjects with schizophrenia. We aimed to validate these findings in a new cohort using real-time PCR in Brodmann's Area (BA) 9, and to determine the disease specificity of increased SELENBP1 expression by measuring SELENBP1 mRNA in subjects with major depressive disorder and bipolar disorder. We then extended the study to include other cortical regions such as BA8 and BA44. SELENBP1 mRNA was higher in BA9 (P=0.001), BA8 (P=0.003) and BA44 (P=0.0007) from subjects with schizophrenia. Conversely, in affective disorders, there was no significant difference in SELENBP1 mRNA in BA9 (P=0.67), suggesting that the upregulation may be diagnosis specific. Measurement of SELENBP1 protein levels showed that changes in mRNA did not translate to changes in protein. In addition, chronic treatment of rats with antipsychotics did not significantly affect the expression of Selenbp1 in the cortex (P=0.24). Our data show that elevated SELENBP1 transcript expression is widespread throughout the prefrontal cortex in schizophrenia, and confirm that this change is a consistent feature of schizophrenia and not a simple drug effect.