Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
BBA - Proteins and Proteomics (v.1784, #6)
Globular and pre-fibrillar prion aggregates are toxic to neuronal cells and perturb their electrophysiology by Narinder Sanghera; Mark Wall; Venien-Bryan Catherine Vénien-Bryan; Teresa J.T. Pinheiro (pp. 873-881).
Prion diseases are characterised at autopsy by neuronal loss and accumulation of amorphous protein aggregates and/or amyloid fibrils in the brains of humans and animals. These protein deposits result from the conversion of the cellular, mainly α-helical prion protein (PrPC) to the β-sheet-rich isoform (PrPSc). Although the pathogenic mechanism of prion diseases is not fully understood, it appears that protein aggregation is itself neurotoxic and not the product of cell death. The precise nature of the neurotoxic species and mechanism of cell death are yet to be determined, although recent studies with other amyloidogenic proteins suggest that ordered pre-fibrillar or oligomeric forms may be responsible for cellular dysfunction. In this study we have refolded recombinant prion protein (rPrP) to two distinct forms rich in β-sheet structure with an intact disulphide bond. Here we report on the structural properties of globular aggregates and pre-fibrils of rPrP and show that both states are toxic to neuronal cells in culture. We show that exogenous rPrP aggregates are internalised by neuronal cells and found in the cytoplasm. We also measured the changes in electrophysiological properties of cultured neuronal cells on exposure to exogenous prion aggregates and discuss the implications of these findings.
Keywords: Abbreviations; ATR FTIR; attenuated total reflection Fourier transform infrared; CD; circular dichroism; CNS; central nervous system; EM; electron microscopy; PrP; prion protein; MTT; 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; SHaPrP; Syrian hamster prion protein; ThT; thioflavin-T; TSE; transmissible spongiform encephalopathyPrion; Fibrillisation; Toxic molecule; Amyloid globular aggregate; Membrane potential
Large-scale identification of novel mitosis-specific phosphoproteins by Ma Xiang; Cao Xue; Li Huicai; Liu Jin; Li Hong; He Dacheng (pp. 882-890).
Systematic identification of phosphoproteins is essential for understanding cellular signalling pathways since phosphorylation plays important roles in cellular regulation. Monoclonal antibody MPM-2 recognizes a discrete set of mitosis-specific phosphoproteins and constitutes a specific tool to investigate the significance of phosphorylation in cell cycle. However, due to the difficulties in identifying antigens revealed on immunoblot membrane, only minority of MPM-2 antigens have been identified. Here we originated proteomics approaches for large-scale identification of MPM-2 phosphoproteins. Mitotic extracts were run on several two-dimensional gel electrophoresis (2D) in parallel, and stained by Coomassie Blue. Each individual spot on one of the gels was excised, and proteins in it were further resolved by regular SDS-electrophoresis and blotted on membrane for MPM-2 stain. Counterparts of the positive proteins were selected on another parallel 2D gel and identified by mass-spectrometry. Using this strategy, 100 spots were excised from Coomassie-stained 2D gel and screened by 1D immunoblots for MPM-2 reactivity, and 22 proteins containing potential MPM-2 epitope were identified in addition to a known MPM-2 antigen, laminin-binding protein. These results were further validated by immunofluorescence, co-immunoprecipitation and in vitro phosphorylation assay. The identification of an unprecedented number of potential MPM-2 phosphoprotein antigens gives new insight into the range of proteins involved in the regulation of the early stages of cell division. Meanwhile, this strategy could be used wherever unknown antigens are explored, especially for antibodies that can recognize more than one antigen.
Keywords: Proteomics; Phosphoproteins; MPM-2; Cell cycle; Mitosis
Interaction of the major protein from bovine seminal plasma, PDC-109 with phospholipid membranes and soluble ligands investigated by fluorescence approaches by V. Anbazhagan; Rajani S. Damai; Aniruddha Paul; Musti J. Swamy / (pp. 891-899).
The major protein from bovine seminal plasma, PDC-109 binds selectively to choline phospholipids on the sperm plasma membrane and plays a crucial role in priming spermatozoa for fertilization. The microenvironment and accessibility of tryptophans of PDC-109 in the native state, in the presence of phosphorylcholine (PrC) and phospholipid membranes as well as upon denaturation have been investigated by fluorescence approaches. Quenching of the protein intrinsic fluorescence by different quenchers decreased in the order: acrylamide>succinimide≫Cs+>I−. Ligand binding afforded considerable protection from quenching, with shielding efficiencies following the order: dimyristoylphosphatidylcholine (DMPC)>lysophosphatidylcholine (Lyso-PC)>PrC. This has been attributed to a partial penetration of the protein into the DMPC membranes and Lyso-PC micelles, as well as a further stabilization of the binding due to the interaction of PDC-109 with lipid acyl chains and the resulting tightening of the protein structure, leading to a decreased accessibility of the tryptophan residues. Red-edge excitation shift (REES) studies yielded REES values of 4 nm for both native and denatured PDC-109, whereas reduced and denatured protein gave a REES of only 0.5 nm, clearly indicating that the structural and dynamic features of the microenvironment around the tryptophan residues are retained even after denaturation, presumably due to the constraints imposed on the protein structure by disulfide bonds. Upon binding of PDC-109 to DMPC membranes and Lyso-PC micelles the REES values were reduced to 2.5 and 1.0 nm, respectively, which could be due to the penetration of some parts of the protein, especially the segment containing Trp-90 into the membrane interior, where the red-edge effects are considerably reduced.
Keywords: Abbreviations; PrC; phosphorylcholine; DMPC; dimyristoylphosphatidylcholine; Lyso-PC; lysophosphatidylcholine; REES; Red-edge excitation shift; FnII; fibronectin type II; ESR; electron spin resonance; FTIR; Fourier transform infrared; PC; phosphatidylcholine; Trp; tryptophan; DMPE; dimyristoylphosphatidylethanolamine; DMPG; dimyristoylphosphatidylglycerol; DMPA; dimyristoylphosphatidic acid; EDTA; ethylenediamine tetraacetic acid; SUV; small unilamellar vesicles; TBS-I; 50 mM Tris–HCl buffer, pH 7.4, containing 0.15 M NaCl, 5 mM EDTA and 0.025% sodium azide; Gdn.HCl; guanidinium hydrochloride; β-ME; β-mercaptoethanolBSP-A1/A2; Capacitation; Cholesterol efflux; Major protein; Fluorescence quenching; Red-edge excitation shift
The kinetic properties producing the perfunctory pH profiles of catalase-peroxidases by Robert L. Moore; Luke J. Powell; Douglas C. Goodwin (pp. 900-907).
Many structure–function relationship studies performed on the catalase-peroxidase enzymes are based on limited kinetic data. To provide a more substantive understanding of catalase-peroxidase function, we undertook a more exhaustive evaluation of catalase-peroxidase catalysis as a function of pH. Kinetic parameters across a broad pH range for the catalase and peroxidase activities of E. coli catalase peroxidase (KatG) were obtained, including the separate analysis of the oxidizing and reducing substrates of the peroxidase catalytic cycle. This investigation identified ABTS-dependent inhibition of peroxidase activity, particularly at low pH, unveiling that previously reported pH optima are clearly skewed. We show that turnover and efficiency of peroxidase activity increases with decreasing pH until the protein unfolds. The data also suggest that the catalase pH optimum is more complex than it is often assumed to be. The apparent optimum is in fact the intersection of the optimum for binding (7.00) and the optimum for activity (5.75). We also report the apparent p Kas for binding and catalysis of catalase activity as well as approximate values for certain peroxidatic and catalatic steps.
Keywords: Abbreviations; Kat; catalase-peroxidases; KatG; Escherichia coli; catalase peroxidase; ABTS; 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid); Por; porphyrin; K; M; Michaelis constant; k; cat; turnover number; CD; Circular Dichroism; BpKatG; Burkholderia pseudomallei; catalase-peroxidase; SynKatG; Synechocystis; PCC 6803 catalase-peroxidase; MtbKatG; Mycobacterium tuberculosis KatGCatalase-peroxidase; KatG; Kinetics; pH-dependence; Substrate-dependent inhibition; Heme
Determinants for substrate phosphorylation by Dictyostelium myosin II heavy chain kinases A and B and eukaryotic elongation factor-2 kinase by Scott W. Crawley; Cote Graham P. Côté (pp. 908-915).
The α kinases are a widespread family of atypical protein kinases characterized by a novel type of catalytic domain. In this paper the peptide substrate recognition motifs for three α kinases, Dictyostelium discoideum myosin heavy chain kinase (MHCK) A and MHCK B and mammalian eukaryotic elongation factor-2 kinase (eEF-2K), were characterized by incorporating amino acid substitutions into a previously identified MHCK A peptide substrate (YAYDTRYRR) (Luo X. et al. (2001) J. Biol. Chem. 276, 17836–43). A lysine or arginine in the P+1 position on the C-terminal side of the phosphoacceptor threonine (P site) was found to be critical for peptide substrate recognition by MHCK A, MHCK B and eEF-2K. Phosphorylation by MHCK B was further enhanced 8-fold by a basic residue in the P+2 position whereas phosphorylation by MHCK A was enhanced 2- to 4-fold by basic residues in the P+2, P+3 and P+4 positions. eEF-2K required basic residues in both the P+1 and P+3 positions to recognize peptide substrates. eEF-2K, like MHCK A and MHCK B, exhibited a strong preference for threonine as the phosphoacceptor amino acid. In contrast, the Dictyostelium VwkA and mammalian TRPM7 α kinases phosphorylated both threonine and serine residues. The results, together with a phylogenetic analysis of the α kinase catalytic domain, support the view that the metazoan eEF-2Ks and the Dictyostelium MHCKs form a distinct subgroup of α kinases with conserved properties.
Keywords: Abbreviations; ePK; eukaryotic protein kinase; MHCK; myosin II heavy chain kinase; A-CAT; catalytic domain of MHCK A; B-CAT; catalytic domain of MHCK B; eEF-2K; eukaryotic elongation factor-2 kinase; vWFA; von Willebrand factor A-like motif kinase; TRPM; melastatin-related transient receptor potential; GST; glutathione; S; -transferase; TES; 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acidMyosin; Protein kinase; α kinase; Peptide substrate specificity; Dictyostelium
Folding behavior of a backbone-reversed protein: Reversible polyproline type II to β-sheet thermal transitions in retro-GroES multimers with GroES-like features by Shubbir Ahmed; Anshuman Shukla; Purnananda Guptasarma (pp. 916-923).
The structural consequences of the reversal of polypeptide backbone direction (retro modification) remain insufficiently explored. Here, we describe the behavior of an engineered, backbone-reversed form of the 97 residues-long GroES co-chaperonin of Escherichia coli. FTIR and far-UV CD spectroscopy suggest that retro-GroES adopts a mixed polyproline type II (PPII)-beta-strand structure with a βII type CD spectrum similar to that of GroES. Gel-filtration chromatography reveals that the protein adopts trimeric and/or pentameric quaternary structures, with solubility retained up to concentrations of 5.0–5.5 mg/ml in aqueous solutions. Mutations inserting a single tryptophan residue as a spectroscopic probe at three different sites cause no perturbation in the protein's CD spectral characteristics, or in its quaternary structural status. The protein is cooperatively dissociated, and non-cooperatively unfolded, by both guanidine hydrochloride and urea. Intriguingly, unlike with GroES, retro-GroES is not unfolded by heat. Instead, there is a reversible structural transition involving conversion of PPII structure to β sheet structure, upon heating, with no attendant aggregation even at 90 °C. Retro-GroES does not bind GroEL. In summary, some structure-forming characteristics of GroES appear to be conserved through the backbone reversal process, although the differential conformational behavior upon heating also indicates differences.
Keywords: Retro-protein; Protein folding; Protein engineering; Beta strand; Polyproline type II structure
Conformational analysis of the broad-spectrum antibacterial peptide, ranatuerin-2CSa: Identification of a full length helix–turn–helix motif by Anusha P. Subasinghage; J. Michael Conlon; Chandralal M. Hewage (pp. 924-929).
Design of clinically valuable antibacterial agents based upon naturally occurring peptides requires the use of spectroscopic methods, particularly NMR, to determine the three-dimensional structure of the native peptide so that analogues with improved therapeutic properties can be made. Ranatuerin-2CSa (GILSSFKGVAKGVAKDLAG KLLETLKCKITGC), first isolated from skin secretions of the Cascades frog, Rana cascadae, represents a promising candidate for drug development. The peptide shows potent growth inhibitory activity against Escherichia coli (MIC=5 μM) and Staphylococcus aureus (MIC=10 μM) but displays haemolytic activity against human erythrocytes (LC50=160 μM). The solution structure of ranatuerin-2CSa was investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, the peptide lacks secondary structure but, in a 2,2,2-trifluoroethanol (TFE- d 3)-H2O solvent mixture, the structure is characterised by a full length helix–turn–helix conformation between residues I2–L21, L22–L25 and K26–T30 respectively. This structural information will facilitate the design of novel therapeutic agents based upon the ranatuerin-2CSa structure with improved antimicrobial potencies but decreased cytolytic activities against mammalian cells.
Keywords: Abbreviations; CD; circular dichroism; DQF-COSY; double-quantum filtered correlation spectroscopy; NMR; nuclear magnetic resonance; NOE; nuclear Overhauser effect; NOESY; nuclear Overhauser effect spectroscopy; TFE; trifluoroethanol; TOCSY; total correlation spectroscopy; RMSD; root mean square deviationRanatuerin-2; Antimicrobial; NMR; Molecular modelling
Formation of amyloid fibrils by bovine carbonic anhydrase by Anshul Rana; Teemish Praveen Gupta; Saurabh Bansal; Bishwajit Kundu (pp. 930-935).
Amyloids are typically characterized by extensive aggregation of proteins where the participating polypeptides are involved in formation of intermolecular cross β-sheet structures. Alternate structure attainment and amyloid formation has been hypothesized to be a generic property of a polypeptide, the propensities of which vary widely depending on the polypeptide involved and the physicochemical conditions it encounters. Many proteins that exist in the normal form in-vivo have been shown to form amyloid when incubated in partially denaturing conditions. The protein bovine carbonic anhydrase II (BCA II) when incubated in mildly denaturing conditions showed that the partially unfolded conformers assemble together and form ordered amyloid aggregates. The properties of these aggregates were tested using the traditional Congo-Red (CR) and Thioflavin-T (ThT) assays along with fluorescence microscopy, transmission electron microscopy (TEM), and circular dichroism (CD) spectroscopy. The aggregates were found to possess most of the characteristics ascribed to amyloid fibers. Thus, we report here that the single-domain globular protein, BCA II, is capable of forming amyloid fibrils. The primary sequence of BCA II was also analyzed using recurrence quantification analysis in order to suggest the probable residues responsible for amyloid formation.
Keywords: Amyloidogenesis; Recurrence quantification analysis; Aggregation; Bovine carbonic anhydrase
High glucose downregulates endothelial progenitor cell number via SIRT1 by Maria Luisa Balestrieri; Monica Rienzo; Francesca Felice; Raffaele Rossiello; Vincenzo Grimaldi; Lara Milone; Amelia Casamassimi; Luigi Servillo; Bartolomeo Farzati; Alfonso Giovane; Claudio Napoli (pp. 936-945).
Increasing evidence indicates that mammalian SIRT1 mediates calorie restriction and influences lifespan regulating a number of biological molecules such as FoxO1. SIRT1 controls the angiogenic activity of endothelial cells via deacetylation of FoxO1. Endothelial dysfunction and reduced new blood vessel growth in diabetes involve a decreased bioactivity of endothelial progenitor cells (EPCs) via repression of FoxO1 transcriptional activity. The relative contribution of SIRT1 with respect to the direct effects of high glucose on EPC number is poorly understood. We report that treatment of EPCs with high glucose for 3 days determined a consistent downregulation of EPC positive to DiLDL/lectin staining and, interestingly, this was associated with reduced SIRT1 expression levels and enzyme activity, and increased acetyl-FoxO1 expression levels. Moreover, EPCs responded to high glucose with major changes in the expression levels of cell metabolism-, cell cycle-, and oxidative stress-related genes or proteins. Proteomic analysis shows increased expression of nicotinamide phosphorybosyl transferase and mitochondrial superoxide dismutase whereas a glucose-related heat shock protein is reduced. These findings show that SIRT1 is a critical modulator of EPCs dysfunction during alteration of glucose metabolism.
Keywords: Endothelial progenitor cells; SIRT1; Glucose
Structural and stability characteristics of a monothiol glutaredoxin: Glutaredoxin-like protein 1 from Plasmodium falciparum by Timir Tripathi; Stefan Rahlfs; Katja Becker; Vinod Bhakuni (pp. 946-952).
Recently discovered monothiol glutaredoxins with CXXS-active site sequence share a common structural motif and biochemical mechanism of action and are involved in multiple cellular functions. Here we report first studies on the structural and stability characterization of a monothiol glutaredoxin, in particular — PfGLP1. Our results demonstrate that in the native conformation, the enzyme has a compact core structure with a relatively flexible N-terminal portion having an open configuration. Comparative functional studies with the full-length and N-terminal truncated protein demonstrate that the flexible N-terminal portion does not play any significant role in functional activity of the protein. In contrast to other Grxs, PfGLP1 does not contain a Fe–S cluster. The pH dependent studies demonstrate that the protein is resistant to alkaline pH but highly sensitive to acidic pH and undergoes significant unfolding between pH 4 and 5. However, acidic conditions also do not induce complete unfolding of the enzyme. The protein is stabilized with a conformational free energy of about 3.2±0.1 kcal mol−1. The protein is a highly cooperative molecule as during denaturant-induced equilibrium unfolding a simultaneous unfolding of the protein without stabilization of any partially folded intermediate is observed.
Keywords: Abbreviations; Grx(s); Glutaredoxin(s); GLP(s); Glutaredoxin-like protein(s); PfGLP1; Plasmodium falciparum; glutaredoxin-like protein 1; ESI-MS; electrospray ionization mass spectrometry; SEC; size exclusion chromatography; GdnHCl; guanidine hydrochloride; GSH; reduced glutathioneCooperative; Denaturation; Equilibrium; Proteolysis; Stability
Biochemical and structural characterization of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from Pyrococcus horikoshii by Deyse de Souza Dantas; Camila Ramos dos Santos; Guimaraes Pereira Gonçalo Amarante Guimarães Pereira; Francisco Javier Medrano (pp. 953-960).
The 6-oxopurine phosphoribosyltransferase (HPRT, EC 2.4.2.8) from the hyperthermophile Pyrococcus horikoshii was expressed in Escherichia coli and purified. Steady-state kinetic studies indicated that the enzyme is able to use hypoxanthine, guanine and xanthine. The first two substrates showed similar catalytic efficiencies, and xanthine presented a much lower value (around 20 times lower), but the catalytic constant was comparable to that of hypoxanthine. The enzyme was not able to bind to GMP-agarose, but was able to bind the other reverse reaction substrate, inorganic pyrophosphate, with low affinity ( Kd of 4.7±0.1 mM). Dynamic light scattering and analytical gel filtration suggested that the enzyme exists as a homohexamer in solution.
Keywords: Hypoxanthine-guanine-xanthine phosphoribosyltransferase; HGXPRT; Pyrococcus horikoshii
Kinetic study of the thermal inactivation of cholinesterase enzymes immobilized in solid matrices by Avraham Bromberg; Sharon Marx; Gad Frishman (pp. 961-966).
The thermal inactivation of immobilized cholinesterase enzymes (ChE) in solid matrices where the protein unfolding is blocked was studied, thus enabling investigation of the kinetics of the inactivation process directly from the native structure to the inactivated state. The thermal inactivation of butyrylcholinesterase (BChE), recombinant human acetylcholinesterase (rHuAChE), and eel acetylcholinesterase (AChE) enzymes was studied in dry films composed of poly(vinyl pyrollidone) (PVP), bovine serum albumin (BSA) and trehalose at 60°–120 °C. The kinetics follows a bi-exponential decay equation representing a combination of fast and slow processes. The activation enthalpy Δ H# and the activation entropy Δ S# for each of the three enzymes have been evaluated. The values of Δ H# for the fast process and for the slow process of BChE are 33±3, and 28±2 kcal/mol, respectively, and the values of Δ S# are 0.84±0.04, and −18.2±0.5 cal/deg, respectively. The appropriate value of Δ H# for rHuAChE is 26±2 Kcal/mol, for both processes and the values of Δ S# are −17.6±0.9, and −23.0±0.9 cal/deg, respectively. Similarly, the values of Δ H# for eelAChE are 30±3, 31±1 kcal/mol, and the values of Δ S# are −6.7±0.5, −9.1±0.2 cal/deg respectively.
Keywords: Enzyme; Kinetics; Thermal stability; Acetylcholinesterase; Butyrylcholinesterase; Poly(vinyl pyrrolidone)
Molecular and kinetic characterization of two UDP-glucose pyrophosphorylases, products of distinct genes, from Arabidopsis by Meng Meng; Malgorzata Wilczynska; Leszek A. Kleczkowski (pp. 967-972).
UDP-glucose pyrophosphorylase (UGPase) is an important enzyme in the production (and conversions) of UDP-glucose, a key precursor for carbohydrate biosynthesis. cDNAs corresponding to two UGPase isozymes in Arabidopsis were overexpressed in Escherichia coli and, subsequently, the recombinant proteins were purified and characterized. Both proteins were highly conserved, sharing 93% identity. Based on crystal structure-derived images, the main amino acid differences mapped to N- and C-termini domains, but not to central active site region. The two proteins existed mainly as monomers, and they had similar molecular masses of ca. 53 kDa. However, comparison of molecular masses of UGPases from Arabidopsis root and leaf extracts revealed that the root protein was slightly larger, suggesting a post-translational modification. Specific activity of the purified UGPase-1 was ca. 10–30% lower than that of UGPase-2, depending on direction of the reaction, whereas its Km values with all substrates in both directions of the reaction were consistently ca. twice lower than those of UGPase-2 (0.03–0.14 mM vs. 0.07–0.36 mM, respectively). Both proteins were “true” UGPases, and had no activity with ADP-glucose/ATP or galactose-1-P. Equilibrium constant for both proteins was ca. 0.3, suggesting preference for the pyrophosphorolysis direction of the reaction. The data are discussed with respect to potential roles of UGPase in carbohydrate synthesis/metabolism in Arabidopsis.
Keywords: Abbreviations; aa; amino acid(s); AGPase; ADP-glucose pyrophosphorylase; GDH; glucose-6-P dehydrogenase; PGM; phosphoglucomutase; SuSy; sucrose synthase; Ugp; cDNA for UGPase; UGP; gene for UGPase; UGPase; UDP-glucose pyrophosphorylaseADP-glucose pyrophosphorylase; Heterologous expression; Homologous genes; Isozyme evolution; Sucrose synthase