RSSFibulin-3 peptides (Fib3-1 and Fib3-2) are potential biomarkers of osteoarthritis.
Source
Bone and Cartilage Research Unit, University of Liege, avenue de l'Hôpital 3 (B23), 4000 Liège, Belgium. yhenrotin@ulg.ac.be.
Abstract
OBJECTIVES:
The aim of this study was to identify new biomarkers of osteoarthritis by proteomic analysis and to develop specific immunoassays to detect and quantify them.
METHODS:
Proteomic analysis was performed in urine from ten women (76.0 ± 5.0 years) undergoing knee replacement surgery due to severe osteoarthritis and five healthy women (25.6 ± 2.6 years). Protein content from each sample group was analyzed by differential in gel electrophoresis (DIGE). Protein spots that exhibit an abundance ratio greater than 1.5 between groups were identified by mass spectrometry. Specific ELISAs were developed and validated in serum population collected from 236 healthy subjects aged from 20 to 64 years and from 76 patients with severe radiological knee OA (68.8 ± 11.9 years). Immunohistochemistry analysis was performed on articular cartilage from tibial plateaus.
RESULTS:
Thirteen proteins within spots significantly modified between groups were identified. Particular interest was focused on peptides of fibulin-3 named Fib3-1 and Fib3-2. Two antisera directed against these peptides were used to develop immunoassays. By comparison with age-matched healthy subjects, Fib3-1 (median, 54.6 pM vs 85.1 pM; p < 0.0001) and Fib3-2 (median, 144.4 pM vs 191.4 pM; p < 0.0001) serum levels were elevated in OA patients. Using ROC-AUC analysis, we have demonstrated that Fib3-1 and Fib3-2 discriminate OA and normal population. Immunostaining revealed the presence of Fib3-1 and Fib3-2 into chondrocytes and in the extracellular matrix of the superficial layer of the fibrillated cartilage.
CONCLUSIONS:
Fib3-1 and Fib3-2 are potential biochemical markers for the diagnosis of OA.
Copyright © 2012 by the American College of Rheumatology.
- PMID:
- 22275171
- [PubMed - as supplied by publisher]
Rapid characterization of N-linked glycosylation site using non-specific protease digestion of gel-separated glycoproteins and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry.
Source
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Abstract
Rapid identification of glycosylation sites of glycoproteins is urgently needed in glycoproteomics study. In the present work, a rapid and simple method based on non-specific digestion of gel-separated glycoproteins and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry was described, which can efficiently identify the N-linked glycosylation sites. One-step in-gel digestion of Ribonuclease B (RNase B) by proteinase K was employed to generate glycopeptides with short and discrepant peptide composition. When compared with glycopeptides prepared by two-step in gel-digestion using trypsin-proteinase K or trypsin-pronase, the direct proteinase K treatment showed obvious superiority in both glycopeptide recovery and preparation simplicity. Most importantly, it helps to generate greater variety of glycopeptide series with rich information for glycosylation site identification. In addition, binary matrices 5-chloro-2-mercaptobenzothiazole (CMBT) /2,5-dihydroxybenzoic acid (DHB) were found to form homogeneous microcrystal on the target with the purified glycopeptides, leading to improved detection sensitivity. Thus, the present work provides an optimized solution to speed up the characterization of N-linked glycosylation sites in glycoproteins.
- PMID:
- 22274947
- [PubMed - in process]
Review: Formation of peptide radical ions through dissociative electron transfer in ternary metal-ligand-peptide complexes.
Source
Department of Chemistry, The University of Hong Kong, Hong Kong, China. Ivankchu@hku.hk.
Abstract
The formation and fragmentation of odd-electron ions of peptides and proteins is of interest to applications in biologicalmass spectrometry. Gas-phase redox chemistry occurring during collision-induced dissociation of ternary metal-ligand-peptide complexes enables the formation of a variety of peptide radicals, including the canonical radical cations, M(+•), radical dications, [M+H](2+•), radical anions, [M-2H](-•) and phosphorylated radical cations. In addition, odd-electron peptide ions with well-defined initial location of the radical site are produced through side-chain losses from the radical ions. Subsequent fragmentation of these species provides information regarding the role of charge and location of the radical site on the competition between radical-induced and proton-driven fragmentation of odd-electron peptide ions. This account summarizes current understanding of the factors that control the efficiency of the intramolecular electron transfer (ET) in ternary metal-ligand-peptide complexes resulting in formation of odd-electron peptide ions. Specifically, we discuss the effect of the metal center, the ligand and the peptide structure on the competition between the ET, proton transfer (PT) and loss of neutral peptide and neutral peptide fragments from the complex. Fundamental studies of the structures, stabilities and the energetics and dynamics of fragmentation of these complexes are also important for detailed molecular-level understanding of photosynthesis and respiration in biological systems.
- PMID:
- 22274945
- [PubMed - in process]
Organic anion transporters involved in the excretion of bestatin in the kidney.
Source
Department of Clinical Pharmacology, College of Pharmacy, Dalian Medical University, China.
Abstract
Bestatin, a dipeptide, a low molecular weight aminopeptidase inhibitor, has been demonstrated to be an immunomodulator with an antitumor activity. However, the transporter-mediated renal excretion of bestatin is not fully understood. The purpose of this study was to elucidate the transporter-mediated renal excretion mechanism for bestatin. The plasma concentration of bestatin was increased markedly and both the accumulative renal excretion and renal clearance of bestatin were decreased significantly after intravenous administration of bestatin in combination with probenecid. p-Aminohippuric acid (PAH), a substrate of organic anion transporter (OAT) 1, benzylpenicillin (PCG), a substrate of OAT3 and JBP485, a substrate of OAT1 and OAT3, reduced the uptake of bestatin in rat kidney slices and in hOAT1- or hOAT3-HEK 293 cells. The accumulation of bestatin in hOAT1-HEK and hOAT3-HEK 293 cells was significantly greater than that in vector-HEK, and the K(m) and V(max) were 0.679±0.007mM and 0.807±0.006nmol/mgprotein/30s for OAT1, 0.632±0.014mM and 1.303±0.015nmol/mgprotein/30s for OAT3 respectively. PAH and JBP485 inhibited significantly the uptake of bestatin in hOAT1-HEK with the K(i) values of 92±9μM and 197±21μM; and PCG, JBP485 inhibited significantly the uptake of bestatin in hOAT3-HEK 293 cells with the K(i) values of 88±12μM and 160±16μM. Our results are novel in demonstrating for the first time that OAT1 and OAT3 are involved in the renal excretion of bestatin.
Copyright © 2012. Published by Elsevier Inc.
- PMID:
- 22273603
- [PubMed - as supplied by publisher]
Trehalose protects from aggravation of amyloid pathology induced by isoflurane anesthesia in APPswe mutant mice.
Source
Department of Neurobiology, Hospital Ramón y Cajal , Ctra. de Colmenar Km. 9 , Madrid 28034. Spain. maria.a.mena@hrc.es.
Abstract
There is an open controversy about the role of surgery and anesthesia in the pathogenesis of Alzheimer's disease (AD). Clinical studies have shown a high prevalence of these procedures in subjects with AD but the interpretation of these studies is difficult because of the co-existence of multiple variables. Experimental studies in vitro and in vivo have shown that small molecular weight volatile anesthetics enhance amyloidogenesis in vitro and produce behavioral deficits and brain lesions similar to those found in patients with AD. We examined the effect of co-treatment with trehalose on isoflurane-induced amyloidogenesis in mice. WT and APPswe mice, of 11 months of age, were exposed to 1% isoflurane, 3 times, for 1.5 hours each time and sacrificed 24 hours after their last exposure to isoflurane. The right hemi-brain was used for histological analysis and the contra-lateral hemi-brain used for biochemical studies. In this study, we have shown that repetitive exposure to isoflurane in pre-symptomatic mature APPswe mice increases apoptosis in hippocampus and cerebral cortex, enhances astrogliosis and the expression of GFAP and that these effects are prevented by co-treatment with trehalose, a disaccharide with known effects as enhancer of autophagy. We have also confirmed that in our model the co-treatment with trehalose increases the expression of autophagic markers as well as the expression of chaperones. Co-treatment with trehalose reduces the levels of β amyloid peptide aggregates, tau plaques and levels of phospho-tau. Our study, therefore, provides new therapeutic avenues that could help to prevent the putative pro-amyloidogenic properties of small volatile anesthetics.
- PMID:
- 22272607
- [PubMed - as supplied by publisher]
Penetration of endothelial cell coated multicellular tumor spheroids by iron oxide nanoparticles.
Source
1. Department of Chemistry, Brown University, Providence, RI 02912.
Abstract
Iron oxide nanoparticles are a useful diagnostic contrast agent and have great potential for therapeutic applications. Multiple emerging diagnostic and therapeutic applications and the numerous versatile parameters of the nanoparticle platform require a robust biological model for characterization and assessment. Here we investigate the use of iron oxide nanoparticles that target tumor vasculature, via the tumstatin peptide, in a novel three-dimensional tissue culture model. The developed tissue culture model more closely mimics the in vivo environment with a leaky endothelium coating around a glioma tumor mass. Tumstatin-iron oxide nanoparticles showed penetration and selective targeting to endothelial cell coating on the tumor in the three-dimensional model, and had approximately 2 times greater uptake in vitro and 2.7 times tumor neo-vascularization inhibition. Tumstatin provides targeting and therapeutic capabilities to the iron oxide nanoparticle diagnostic contrast agent platform. And the novel endothelial cell-coated tumor model provides an in vitro microtissue environment to evaluate nanoparticles without moving into costly and time-consuming animal models.
Seasonal Variation in the Hepatoproteome of the Dehydrationand Freeze-Tolerant Wood Frog, Rana sylvatica.
Source
Laboratory for Ecophysiological Cryobiology, Department of Zoology, Miami University, Oxford, OH 45056, USA; E-Mails: timmuir@augustana.edu (T.J.M.); leere@muohio.edu (R.E.L.); costanjp@muohio.edu (J.P.C.).
Abstract
Winter's advent invokes physiological adjustments that permit temperate ectotherms to cope with stresses such as food shortage, water deprivation, hypoxia, and hypothermia. We used liquid chromatography (LC) in combination with tandemmass spectrometry (MS/MS) quantitative isobaric (iTRAQ™) peptide mapping to assess variation in the abundance of hepatic proteins in summer- and winter-acclimatized wood frogs (Rana sylvatica), a northerly-distributed species that tolerates extreme dehydration and tissue freezing during hibernation. Thirty-three unique proteins exhibited strong seasonal lability. Livers of winter frogs had relatively high levels of proteins involved in cytoprotection, including heat-shock proteins and an antioxidant, and a reduced abundance of proteins involved in cell proliferation, protein synthesis, and mitochondrial function. They also exhibited altered levels of certain metabolic enzymes that participate in the biochemical reorganization associated with aphagia and reliance on energy reserves, as well as the freezing mobilization and post-thaw recovery of glucose, an important cryoprotective solute in freezing adaptation.
- PMID:
- 22272080
- [PubMed - in process]
Mass spectrometric detection of CYP450 adducts following oxidative desulfuration of methyl parathion.
Source
Department of Pathology, University of Mississippi Medical Center, Jackson, Mississippi, USA. pkyle@umc.edu.
Abstract
Cytochrome P450 (CYP)-mediated desulfuration of methyl parathion results in mechanism-based inhibition of the enzyme. Although previous data suggest that reactive sulfur is released and binds to the apoprotein, the identities of neither the adduct(s) nor the affected amino acid(s) have been clearly determined. In this study, nanospray tandemmass spectroscopy was used to analyze peptide digests of CYP resolved by SDS-PAGE from liver microsomes of male rats following incubation in the absence or presence of methyl parathion. Oxidative desulfuration was confirmed by measurement of methyl paraoxon, and inhibition of specific CYP isozymes was determined by measurement of testosterone hydroxylation. Total CYP content was quantified spectrophotometrically. Incubation of microsomes with methyl parathion decreased CYP content by 58%. This effect was not associated with a comparable increase in absorbance at 420 nm, suggesting the displacement of heme from the apoprotein. Rates of testosterone 2β- and 6β-hydroxylation, respectively, were reduced to 8 and 2%, implicating CYP3A and CYP2C11 in the oxidative desulfuration of methyl parathion. Mass spectrometric analysis identified 96 amu adducts to cysteines 64 and 378 of CYP3A1. In addition, a peptide containing cysteine 433 that coordinates with heme was possibly modified as it was detected in control, but not methyl parathion samples. A comparison of rat CYP3A1 with human CYP3A4 suggests that cysteines 64 and 378 reside along the substrate channel, remote from the active site. Alteration of these residues might modulate substrate entry to the binding pocket of the enzyme. Copyright © 2012 John Wiley & Sons, Ltd.
Copyright © 2012 John Wiley & Sons, Ltd.
Appetite hormones and the transition to hyperphagia in children with Prader-Willi syndrome.
Source
Metabolic and Molecular Imaging Group, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital, London, UK.
Abstract
Objective:Prader-Willi syndrome (PWS) is a genetic neurodevelopmental disorder with several nutritional phases during childhood proceeding from poor feeding, through normal eating without and with obesity, to hyperphagia and life-threatening obesity, with variable ages of onset. We investigated whether differences in appetite hormones may explain the development of abnormal eating behaviour in young children with PWS.Subjects:In this cross-sectional study, children with PWS (n=42) and controls (n=9) aged 7 months-5 years were recruited. Mothers were interviewed regarding eating behaviour, and body mass index (BMI) was calculated. Fasting plasma samples were assayed for insulin, leptin, glucose, peptide YY (PYY), ghrelin and pancreatic polypeptide (PP).Results:There was no significant relationship between eating behaviour in PWS subjects and the levels of any hormones or insulin resistance, independent of age. Fasting plasma leptin levels were significantly higher (mean±s.d.: 22.6±12.5 vs 1.97±0.79 ng ml(-1), P=0.005), and PP levels were significantly lower (22.6±12.5 vs 69.8±43.8 pmol l(-1), P<0.001) in the PWS group compared with the controls, and this was independent of age, BMI, insulin resistance or IGF-1 levels. However, there was no significant difference in plasma insulin, insulin resistance or ghrelin levels between groups, though PYY declined more rapidly with age but not BMI in PWS subjects.Conclusion:Even under the age of 5 years, PWS is associated with low levels of anorexigenic PP, as in older children and adults. Hyperghrelinaemia or hypoinsulinaemia was not seen in these young children with PWS. Change in these appetite hormones was not associated with the timing of the transition to the characteristic hyperphagic phase. However, abnormal and/or delayed development or sensitivity of the effector pathways of these appetitive hormones (for example, parasympathetic and central nervous system) may interact with low PP levels, and later hyperghrelinaemia or hypoinsulinaemia, to contribute to hyperphagia in PWS.International Journal of Obesity advance online publication, 24 January 2012; doi:10.1038/ijo.2011.274.
Proteomics Analysis of Plasma for Early Diagnosis of Endometriosis.
Source
From the Department of Obstetrics and Gynaecology, Leuven University Fertility Centre, University Hospital Gasthuisberg, the Department ofMolecular Cell Biology, Campus Gasthuisberg, ProMeta, Interfaculty Centre for Proteomics and Metabolomics, O&N2, and the IBBT-KU Leuven Future Health Department, Leuven, Belgium; the Division of Reproductive Biology, Institute of Primate Research, Karen, Nairobi, Kenya; the Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary; and the Department of Electrical Engineering, ESAT-SCD, Katholieke Universiteit Leuven, Leuven, Belgium.
Abstract
OBJECTIVE:
To test the hypothesis that differential surface-enhanced laser desorption/ionization time-of-flight massspectrometry protein or peptide expression in plasma can be used in infertile women with or without pelvic pain to predict the presence of laparoscopically and histologically confirmed endometriosis, especially in the subpopulation with a normal preoperative gynecologic ultrasound examination.
METHODS:
Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry analysis was performed on 254 plasma samples obtained from 89 women without endometriosis and 165 women with endometriosis (histologically confirmed) undergoing laparoscopies for infertility with or without pelvic pain. Data were analyzed using least squares support vector machines and were divided randomly (100 times) into a training data set (70%) and a test data set (30%).
RESULTS:
Minimal-to-mild endometriosis was best predicted (sensitivity 75%, 95% confidence interval [CI] 63-89; specificity 86%, 95% CI 71-94; positive predictive value 83.6%, negative predictive value 78.3%) using a model based on five peptide and protein peaks (range 4.898-14.698 m/z) in menstrual phase samples. Moderate-to-severe endometriosis was best predicted (sensitivity 98%, 95% CI 84-100; specificity 81%, 95% CI 67-92; positive predictive value 74.4%, negative predictive value 98.6%) using a model based on five other peptide and protein peaks (range 2.189-7.457 m/z) in luteal phase samples. The peak with the highest intensity (2.189 m/z) was identified as a fibrinogen β-chain peptide. Ultrasonography-negative endometriosis was best predicted (sensitivity 88%, 95% CI 73-100; specificity 84%, 95% CI 71-96) using a model based on five peptide peaks (range 2.058-42.065 m/z) in menstrual phase samples.
CONCLUSION:
A noninvasive test using proteomic analysis of plasma samples obtained during the menstrual phase enabled the diagnosis of endometriosis undetectable by ultrasonography with high sensitivity and specificity.
LEVEL OF EVIDENCE:
II.
- PMID:
- 22270279
- [PubMed - as supplied by publisher]
A NOVEL LECTIN FROM AGROCYBE AEGERITA SHOWS HIGH BINDING SELECTIVITY FOR TERMINAL N-ACETYLGLUCOSAMINE.
Abstract
A novel lectin was isolated from the mushroom Agrocybe aegerita (designated AAL-2) by affinity chromatography with N-acetylglucosamine (GlcNAc) coupled Sepharose 6B after (NH4)2SO4 precipitation. The AAL-2 coding sequence (1224 bp) was identified by performing a homologous search of the five tryptic peptides identified by mass spectrometry against the translated transcriptome of A. aegerita. The molecular weight of AAL-2 was calculated to be 43.175 kDa from mass spectrometry (MS), which was consistent with the data calculated from the amino acid sequence. To analyze the sugar binding properties of AAL-2, a glycan array composed of 465 glycan candidates was employed and the result showed that AAL-2 bound with high selectivity to terminal, nonreducing GlcNAc residues, and further analysis revealed that AAL-2 bound to terminal, nonreducing GlcNAc residues with higher affinity than previously well-known GlcNAc-binding lectins such as wheat germ agglutinin (WGA) and Griffonia simplicifolia lectin-II (GSL-II). Isothermal titration calorimetry (ITC) further showed that GlcNAc bound to AAL-2 in a sequential manner with moderate affinity. In the current study, we also evaluated the antitumor activity of AAL-2. The results showed that AAL-2 could bind to the surface of hepatoma cells, leading to induced cell apoptosis in vitro. Furthermore, AAL-2 exerted an anti-hepatoma effect via inhibition of tumor growth and prolongation of survival time of tumor bearing mice in vivo.
Comparative analysis of virulence factors secreted by Bacillus anthracis Sterne at host body temperature.
Source
Division of Molecular and Life Science, Hanyang University, Ansan, Republic of Korea Institute of Natural Science and Technology, Hanyang University, Ansan, Republic of Korea.
Abstract
Aims: For the analysis of virulence factors produced and secreted by Bacillus anthracis vegetative cells during mammalian host infection, we evaluated the secretome of B. anthracis Sterne exposed to host-specific factors specifically to host body temperature. Methods and Results: We employed a comparative proteomics-based approach to analyze the proteins secreted by B. anthracis Sterne under host-specific body temperature conditions. A total of 17 proteins encoded on a single chromosome and the pXO1 plasmid were identified by peptide mass fingerprinting. Multiple algorithms were used to predict the secretion mechanisms of the detected proteins in B. anthracis. Conclusions: Several putative virulence factors and known factors responsible for sporulation were differentially regulated, including CodY, pXO1-130 and BA1952, revealing insights into temperature cues in the B. anthracis secretome. Significance and Impact of the Study: This study identified temperature-regulated proteins. Further studies aimed at understanding the physical and functional roles of these proteins in infection and control by elevated temperatures will contribute to detection, diagnostics and prophylaxis. © 2012 The Authors Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.
© 2012 The Authors Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.
Human osteoblastic cells discriminate between 20-kDa amelogenin isoforms.
Source
Department of Biomaterials, Faculty of Dentistry, University of Oslo, Oslo, Norway Department of Oral Biology, Leeds Dental Institute, University of Leeds, Leeds, UK.
Abstract
Riksen EA, Petzold C, Brookes S, Lyngstadaas SP, Reseland JE. Human osteoblastic cells discriminate between 20-kDa amelogenin isoforms. Eur J Oral Sci 2011; 119 (Suppl. 1): 357-365. © 2011 Eur J Oral Sci Enamel matrix derivative (EMD) is used to stimulate healing of alveolar bone after destructive marginal periodontitis; however, the roles of the different EMD constituents are unclear. The aim here was to compare the effect of two EMD fractions (A1 and A2) on primary human osteoblasts cultured in the presence of 50 μg ml(-1) of A1, A2, or EMD. SDS-PAGE showed that A1 and A2 were comprised of amelogenins migrating at around 20 kDa. Fourier transform infrared (FTIR) analysis revealed that A1 and A2 had different secondary structures, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) identified different peptide mass values. Osteoblasts responded differently to A1 and A2. Whereas A1 enhanced the proliferation [measured by the incorporation of 5-bromo-2'-deoxyuridine (BrdU)] of osteoblasts, the expression of runt-related transcription factor-2 (RUNX2) mRNA, and the secretion of interleukin 6 (IL-6) into the cell culture medium, exposure to A2 resulted in increased alkaline phosphatase (ALP) activity, increased expression of CD44 mRNA, and increased secretion of osteoprotegrin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL). The level of osteocalcin in the cell culture medium was increased after all treatments, while A2 stimulated the expression of dentin matrix protein 1 (DMP1) mRNA. The results suggest that both A1 and A2 participate in the observed effect of EMD, but have different effects on the expression of osteoblast mRNA and the secretion of osteoblast protein, and thus might facilitate the differentiation of a different phenotype.
© 2011 Eur J Oral Sci.
Glucagon-like peptide-1 (GLP-1) receptor agonism improves metabolic, biochemical and histopathological indices of nonalcoholic steatohepatitis (NASH) in mice.
Source
1Novartis Institutes for BioMedical Research.
Abstract
These preclinical studies aimed to (1) increase our understanding the dietary induction of NASH, and, (2) further explore the utility and mechanisms of GLP-1 receptor (GLP-1R) agonism in treating NASH. We compared the effects of a high trans-fat (HTF) or high lard fat (HLF) diet on key facets of NAFLD/NASH in Lep(ob)/Lep(ob) and C57BL6J (B6) mice. Although HLF-fed mice experienced overall greater gains in weight and adiposity, the addition of trans-fat better mirrored pathophysiologic features of NASH (e.g., hepatic steatosis and fibrosis). Administration of AC3174, an exenatide analog and GLP-1R agonist, to Lep(ob)/Lep(ob) and B6 mice attenuated hepatic endpointsin both dietary models. Next, we assessed whether AC3174-mediated improvements in HTF diet-induced NASH were solely due to weight loss. AC3174-treatment significantly reduced body weight (8.3%), liver mass (14.2%), liver lipid (12.9%), plasma ALT and triglycerides, whereas a calorie-restricted weight-matched group demonstrated only modest non-significant reductions in liver mass(9%) and liver lipid (5.1%) relative to controls. Treatment of GLP-1R-deficient (GLP-1RKO) mice with AC3174 had no effect on body weight, adiposity, liver or plasma indices pointing to the GLP-1R-dependence of AC3174's effects. Interestingly, the role of endogenous GLP-1R's in NASH merits further exploration as the GLP-1RKO model was protected from the deleterious hepatic effects of HTF. Our pharmacological data further support clinical evaluation of the utility of GLP-1R agonists for treatment of NASH.
Hydrogen exchange mass spectrometry as an analytical tool for the analysis of amyloid fibrillogenesis.
Source
Center for Insoluble Protein Structure and Interdisciplinary Nanoscience Center (iNANO) at the Department of Molecular Biology, Science Park, University of Aarhus, Gustav Wieds Vej 10c, 8000 Aarhus C, Denmark.
Abstract
In this study, we have used glucagon as a model system for analyzing amyloid fibrillogenesis by hydrogen exchange MALDI mass spectrometry (HXMS). The hydrogen exchange mass spectrometry data correlated well with the traditional method based on Thioflavin T fluorescence and provided quantitative information by measuring the fibrillating molecules directly. The hydrogen exchange mass spectrometry data collected during fibrillogenesis revealed that glucagon fibrillation was a two component system showing an on/off type of interaction where only monomeric and fibrils were present without any substantial amount of intermediate species. This was evident by the extensive deuteration of the monomer and protection of the entire 29 residue glucagon peptide upon fibrillation.. The method complements the traditional procedures and has the potential to provide new information with respect to the nature of transient species, the structure of the growing fibrils and the mechanism of formation.
- PMID:
- 22267952
- [PubMed]
- PMCID: PMC3261750
- [Available on 2012/4/30]
Tyrosines in the Carboxy-terminus of Syk once Phosphorylated Interact with Signaling Proteins including TULA-2, a Negative Regulator of Mast Cell Degranulation.
Source
NIDCR, United States.
Abstract
Activation of the high affinity IgE receptor (FcεRI) results in the tyrosine phosphorylation of two conserved tyrosines located close to the carboxy terminus of the protein tyrosine kinase Syk. Synthetic peptides representing the last ten amino acids of the tail of Syk with these two tyrosines either nonphosphorylated or phosphorylated were used to precipitate proteins from mast cell lysates. Proteins specifically precipitated by the phosphorylated peptide were identified by mass spectrometry. These included the adaptor proteins SLP-76, Nck-1, Grb2 and GADS and the proteins phosphatases SHIP-1 and UBASH3B (also known as TULA-2). The presence of these in the precipitates was further confirmed by immunoblotting. Using the peptides as probes in far westerns showed direct binding of the phosphorylatedpeptide to Nck-1 and SHIP-1. Immunoprecipitations suggested that there were complexes of these proteins associated with Syk especially after receptor activation; in these complexes are Nck, SHIP-1, SLP-76, Grb2 and TULA-2 (UBASH3B or STS-1). The decreased expression of TULA-2 by treatment of mast cells with siRNA increased the FcεRI-induced tyrosine phosphorylation of the activation loop tyrosines of Syk and the phosphorylation of phospholipase C-2γ. There was parallel enhancement of the receptor-induced degranulation and NFAT or NFκB activation indicating that TULA-2 like SHIP-1 functions as a negative regulator of FcεRI signaling in mast cells. Therefore the terminal tyrosines of Syk once phosphorylated bind complexes of proteins that are positive and negative regulators of signaling in mast cells.
Phosphorylation of the amyloid-β peptide at serine 8 attenuates its clearance via insulin degrading and angiotensin converting enzymes.
Source
University of Bonn, Germany;
Abstract
Accumulation of amyloid β-peptides (Aβ) in the brain is a common pathological feature of Alzheimer's disease (AD). Aggregates of Aβ are neurotoxic and appear to be critically involved in the neurodegeneration during AD pathogenesis. Accumulation of Aβ could be caused by increased production as indicated by several mutations in the amyloid precursor protein or the γ-secretase components presenilin-1 or -2 that cause familial early onset AD. However, recent data also indicate a decreased clearance rate of Aβ in AD brains. We recently demonstrated that Aβ undergoes phosphorylation by extracellular or cell surface localized protein kinase A, leading to increased aggregation. Here we provide evidence that phosphorylation of monomeric Aβ at serine residue 8 also decreases its clearance by microglial cells. By using mass spectrometry, we demonstrate that phosphorylation at serine 8 inhibits the proteolytic degradation of monomeric Aβ by the insulin degrading enzyme (IDE), a major Aβ degrading enzyme released from microglial cells. Phosphorylation also decreased the degradation of Aβ by the angiotensin converting enzyme (ACE). In contrast, Aβ degradation by plasmin was largely unaffected by phosphorylation. Thus, phosphorylation of Aβ could play a dual role in Aβ metabolism. It decreases its proteolytic clearance and also promotes its aggregation. The inhibition of extracellular Aβ phosphorylation, stimulation of cell-surface protease expression and/or increasing their activity could be explored to promote Aβ degradation in AD therapy or prevention.
Pyroglutamate A(beta) aggravates behavioral deficits in 5XFAD mice.
Source
University of Goettingen, Germany;
Abstract
Pyroglutamate-modified Abeta peptides at amino acid position three (AβpE3-42) are gaining considerable attention as potential key players in the pathogenesis of Alzheimer disease (AD). AβpE3-42 is abundant in AD brain and has a high aggregation propensity, stability and cellular toxicity. The aim of the present work was to study the direct effect of elevated AβpE3-42 levels on ongoing AD pathology using transgenic mouse models. To this end, we generated a novel mouse model (TBA42) that produces AβpE3-42. TBA42 mice showed age-dependent behavioral deficits and AβpE3-42- accumulation. The Aβ profile of an established AD mouse model, 5XFAD, was characterized using immunoprecipitation followed by mass spectrometry. Brains from 5XFAD mice demonstrated a heterogeneous mixture of full-length, N-truncated and modified Aβ peptides: Aβ1-42, Aβ1-40, AβpE3-40, AβpE3-42, Aβ3-42, Aβ4-42 and Aβ5-42. 5XFAD and TBA42 mice were then crossed to generate bigenic FAD42 mice. At six months of age, FAD42 mice showed an aggravated behavioral phenotype compared to single transgenic 5XFAD or TBA42 mice. ELISA and plaque load measurements revealed that AβpE3 levels were elevated in FAD42 mice. No change in Aβx-42 or other Aβ isoforms was discovered by ELISA and mass spectrometry. These observations argue for a seeding effect of AβpE-42 in FAD42 mice.
Cu(II) Coordination Chemistry of Patellamide Derivatives: Possible Biological Functions of Cyclic Pseudopeptides.
Source
Universität Heidelberg, Anorganisch-Chemisches Institut, Im Neuenheimer Feld 270, 69120 Heidelberg (Germany). peter.comba@aci.uni-heidelberg.de.
Abstract
Two synthetic derivatives of the naturally occurring cyclic pseudooctapeptides patellamide A-F and ascidiacyclamide, that is, H(4) pat(2) , H(4) pat(3) , as well as their Cu(II) complexes are described. These cyclic peptide derivatives differ from the naturally occurring macrocycles by the variation of the incorporated heterocyclic donor groups and the configuration of the amino acids connecting the heterocycles. The exchange of the oxazoline and thiazole groups by dimethylimidazoles or methyloxazoles leads to more rigid macrocycles, and the changes in the configuration of the side chains leads to significant differences in the folding of the cyclic peptides. These variations allow a detailed study of the various possible structural changes on the chemistry of the Cu(II) complexes formed. The coordination of Cu(II) with these macrocyclic species was monitored by high-resolution electrospray mass spectrometry (ESI-MS), spectrophotometric (UV/Vis) and circular dichroic (CD) titrations, and electron paramagnetic resonance (EPR) spectroscopy. Density functional theory (DFT) calculations and molecular mechanics (MM) simulations have been used to model the structures of the Cu(II) complexes and provide a detailed understanding of their geometric preferences and conformational flexibility. This is related to the Cu(II) coordination chemistry and the reactivity of the dinuclear Cu(II) complexes towards CO(2) fixation. The variation observed between the natural and various synthetic peptide systems enables conclusions about structure-reactivity correlations, and our results also provide information on why nature might have chosen oxazolines and thiazoles as incorporated heterocycles.
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Cloning of the surface layer gene sllB from Bacillus sphaericus ATCC 14577 and its heterologous expression and purification.
Source
Department of Laboratory Medicine, Yancheng Health Vocational and Technical College, Yancheng 224006, Jiangsu Province, P.R. China.
Abstract
A cDNA fragment encoding the S-layer protein SllB cloned from Bacillus sphaericus ATCC 14577 was expressed on the surface of E. coli BL21 (DE3) cells and confirmed by the square lattice structure at the nanoscale level. The amplified gene fragment designed with PCR primers from a specified reference sequence (GenBank accession no. AJ849550) showed a high degree of sequence identity with the known sequences for S-layer protein. The best alignment scores were seen in B. sphaericus strains JG-A12 and NCTC9602, which code for a pre-form protein with a predicted cleavage site located between the two alanine residues 31 and 32. After this signal peptide sequence was removed, the mature protein had a molecular mass of 116.2613 kDa and a theoretical pI of 5.40. Further bioinformatic analysis revealed three S-layer homology (SLH) domains in the N-terminus of the mature protein, positioned at the 1-61, 63-128 and 137-197 residues. The mature S-layer protein was composed of alpha helices (24.86%), extended strands (27.01%), and rich random coils (48.13%). Bioinformatics-driven characterization of SllB may provide scientific evidence for further application of this gene in the fields of nanobiotechnology and biomimetics in the future.
No comments:
Post a Comment