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Sample records for phosphorylated peptide cations

  1. Cell Penetrating Peptides and Cationic Antibacterial Peptides

    PubMed Central

    Rodriguez Plaza, Jonathan G.; Morales-Nava, Rosmarbel; Diener, Christian; Schreiber, Gabriele; Gonzalez, Zyanya D.; Lara Ortiz, Maria Teresa; Ortega Blake, Ivan; Pantoja, Omar; Volkmer, Rudolf; Klipp, Edda; Herrmann, Andreas; Del Rio, Gabriel

    2014-01-01

    Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs. PMID:24706763

  2. Enrichment of phosphorylated peptides and proteins by selective precipitation methods.

    PubMed

    Rainer, Matthias; Bonn, Günther K

    2015-01-01

    Protein phosphorylation is one of the most prominent post-translational modifications involved in the regulation of cellular processes. Fundamental understanding of biological processes requires appropriate bioanalytical methods for selectively enriching phosphorylated peptides and proteins. Most of the commonly applied enrichment approaches include chromatographic materials including Fe(3+)-immobilized metal-ion affinity chromatography or metal oxides. In the last years, the introduction of several non-chromatographic isolation technologies has increasingly attracted the interest of many scientists. Such approaches are based on the selective precipitation of phosphorylated peptides and proteins by applying various metal cations. The excellent performance of precipitation-based enrichment methods can be explained by the absence of any stationary phase, resin or sorbent, which usually leads to unspecific binding. This review provides an overview of recently published methods for the selective precipitation of phosphorylated peptides and proteins. PMID:25587840

  3. Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles

    NASA Astrophysics Data System (ADS)

    Aumiller, William M.; Keating, Christine D.

    2016-02-01

    Biological cells are highly organized, with numerous subcellular compartments. Phosphorylation has been hypothesized as a means to control the assembly/disassembly of liquid-like RNA- and protein-rich intracellular bodies, or liquid organelles, that lack delimiting membranes. Here, we demonstrate that charge-mediated phase separation, or complex coacervation, of RNAs with cationic peptides can generate simple model liquid organelles capable of reversibly compartmentalizing biomolecules. Formation and dissolution of these liquid bodies was controlled by changes in peptide phosphorylation state using a kinase/phosphatase enzyme pair. The droplet-generating phase transition responded to modification of even a single serine residue. Electrostatic interactions between the short cationic peptides and the much longer polyanionic RNAs drove phase separation. Coacervates were also formed on silica beads, a primitive model for localization at specific intracellular sites. This work supports phosphoregulation of complex coacervation as a viable mechanism for dynamic intracellular compartmentalization in membraneless organelles.

  4. Antiendotoxin activity of cationic peptide antimicrobial agents.

    PubMed Central

    Gough, M; Hancock, R E; Kelly, N M

    1996-01-01

    The endotoxin from gram-negative bacteria consists of a molecule lipopolysaccharide (LPS) which can be shed by bacteria during antimicrobial therapy. A resulting syndrome, endotoxic shock, is a leading cause of death in the developed world. Thus, there is great interest in the development of antimicrobial agents which can reverse rather than promote sepsis, especially given the recent disappointing clinical performance of antiendotoxin therapies. We describe here two small cationic peptides, MBI-27 and MBI-28, which have both antiendotoxic and antibacterial activities in vitro and in vivo in animal models. We had previously demonstrated that these peptides bind to LPS with an affinity equivalent to that of polymyxin B. Consistent with this, the peptides blocked the ability of LPS and intact cells to induce the endotoxic shock mediator, tumor necrosis factor (TNF), upon incubation with the RAW 264.7 murine macrophage cell line. MBI-28 was equivalent to polymyxin B in its ability to block LPS induction of TNF by this cell line, even when added 60 min after the TNF stimulus. Furthermore, MBI-28 offered significant protection in a galactosamine-sensitized mouse model of lethal endotoxic shock. This protection correlated with the ability of MBI-28 to reduce LPS-induced circulating TNF by nearly 90% in this mouse model. Both MBI-27 and MBI-28 demonstrated antibacterial activity against gram-negative bacteria in vitro and in vivo against Pseudomonas aeruginosa infections in neutropenic mice. PMID:8945527

  5. Data on the peptide mapping and MS identification for phosphorylated peptide.

    PubMed

    Wang, Hui; Tu, Zong-Cai; Liu, Guang-Xian; Zhang, Lu; Chen, Yuan

    2016-09-01

    This article contains peptides mapping, mass spectrometry and processed data related to the research "Identification and quantification of the phosphorylated ovalbumin by high resolution mass spectrometry under dry-heating treatment" [1]. Fourier transform ion cyclotron mass spectrometry (FTICR MS) was used to investigate the specific phosphorylation sites and the degree of phosphorylation (DSP) at each site. Specifically, phosphorylated peptides were monitored through mass shift on the FTICR MS spectrum. DSP was evaluated through the relative abundance levels of the FTICR MS spectrometry. From these data, the calculation method of DSP was exemplified. PMID:27274527

  6. Current scenario of peptide-based drugs: the key roles of cationic antitumor and antiviral peptides

    PubMed Central

    Mulder, Kelly C. L.; Lima, Loiane A.; Miranda, Vivian J.; Dias, Simoni C.; Franco, Octávio L.

    2013-01-01

    Cationic antimicrobial peptides (AMPs) and host defense peptides (HDPs) show vast potential as peptide-based drugs. Great effort has been made in order to exploit their mechanisms of action, aiming to identify their targets as well as to enhance their activity and bioavailability. In this review, we will focus on both naturally occurring and designed antiviral and antitumor cationic peptides, including those here called promiscuous, in which multiple targets are associated with a single peptide structure. Emphasis will be given to their biochemical features, selectivity against extra targets, and molecular mechanisms. Peptides which possess antitumor activity against different cancer cell lines will be discussed, as well as peptides which inhibit virus replication, focusing on their applications for human health, animal health and agriculture, and their potential as new therapeutic drugs. Moreover, the current scenario for production and the use of nanotechnology as delivery tool for both classes of cationic peptides, as well as the perspectives on improving them is considered. PMID:24198814

  7. Quality control of cationic cell-penetrating peptides.

    PubMed

    Stalmans, Sofie; Gevaert, Bert; Verbeke, Frederick; D'Hondt, Matthias; Bracke, Nathalie; Wynendaele, Evelien; De Spiegeleer, Bart

    2016-01-01

    During fundamental research, it is recommended to evaluate the test compound identity and purity in order to obtain reliable study outcomes. For peptides, quality control (QC) analyses are routinely performed using reversed-phase liquid chromatography coupled to an ultraviolet (UV) detector system. These traditional QC methods, using a C18 column and a linear gradient with formic acid (FA) as acidic modifier in the mobile phase, might not result in optimal chromatographic performance for basic peptides due to their cationic nature and hence may lead to erroneous results. Therefore, the influence of the used chromatographic system on the final QC results of basic peptides was evaluated using five cationic cell-penetrating peptides and five C18-chromatographic systems, differing in the column particle size (high performance liquid chromatography (HPLC) versus ultra-high performance liquid chromatography (UHPLC)), the acidic modifier (FA versus trifluoroacetic acid (TFA)), and the column temperature (30 °C versus 60 °C). Our results indicate that a UHPLC system with the C18 column thermostated at 30 °C and a mobile phase containing TFA, provides the most suitable routine QC analysis method for cationic peptides, outperforming in sensitivity and resolution compared to the other systems. We also demonstrate the use of a single quad mass spectrometry (MS) detector system during QC analysis of (cationic) peptides, allowing identification of the peptide and its impurities, as well as the evaluation of the peak purity. PMID:26397208

  8. Doubling down on peptide phosphorylation as a variable mass modification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Some mass spectrometrists believe that searching for variable post-translational modifications like phosphorylation of serine or threonine when using database-search algorithms to interpret peptide tandem mass spectra will increase false positive rates. The basis for this is the premise that the al...

  9. Insights into the Unique Phosphorylation of the Lasso Peptide Paeninodin.

    PubMed

    Zhu, Shaozhou; Hegemann, Julian D; Fage, Christopher D; Zimmermann, Marcel; Xie, Xiulan; Linne, Uwe; Marahiel, Mohamed A

    2016-06-24

    Lasso peptides are a new class of ribosomally synthesized and post-translationally modified peptides and thus far are only isolated from proteo- and actinobacterial sources. Typically, lasso peptide biosynthetic gene clusters encode enzymes for biosynthesis and export but not for tailoring. Here, we describe the isolation of the novel lasso peptide paeninodin from the firmicute Paenibacillus dendritiformis C454 and reveal within its biosynthetic cluster a gene encoding a kinase, which we have characterized as a member of a new class of lasso peptide-tailoring kinases. By employing a wide variety of peptide substrates, it was shown that this novel type of kinase specifically phosphorylates the C-terminal serine residue while ignoring those located elsewhere. These experiments also reveal that no other recognition motif is needed for efficient enzymatic phosphorylation of the C-terminal serine. Furthermore, through comparison with homologous HPr kinases and subsequent mutational analysis, we confirmed the essential catalytic residues. Our study reveals how lasso peptides are chemically diversified and sets the foundation for rational engineering of these intriguing natural products. PMID:27151214

  10. Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides.

    PubMed

    Bertran-Vicente, Jordi; Penkert, Martin; Nieto-Garcia, Olaia; Jeckelmann, Jean-Marc; Schmieder, Peter; Krause, Eberhard; Hackenberger, Christian P R

    2016-01-01

    In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods. Here we report on the development of a chemoselective synthetic method to phosphorylate Cys side-chains in unprotected peptides. This approach makes use of a reaction between nucleophilic phosphites and electrophilic disulfides accessible by standard methods. We achieve the stereochemically defined phosphorylation of a Cys residue and verify the modification using electron-transfer higher-energy dissociation (EThcD) mass spectrometry. To demonstrate the use of the approach in resolving biological questions, we identify an endogenous Cys phosphorylation site in IICB(Glc), which is known to be involved in the carbohydrate uptake from the bacterial phosphotransferase system (PTS). This new chemical and analytical approach finally allows further investigating the functions and significance of Cys phosphorylation in a wide range of crucial cellular processes. PMID:27586301

  11. Immune modulation by multifaceted cationic host defense (antimicrobial) peptides.

    PubMed

    Hilchie, Ashley L; Wuerth, Kelli; Hancock, Robert E W

    2013-12-01

    Cationic host defense (antimicrobial) peptides were originally studied for their direct antimicrobial activities. They have since been found to exhibit multifaceted immunomodulatory activities, including profound anti-infective and selective anti-inflammatory properties, as well as adjuvant and wound-healing activities in animal models. These biological properties suggest that host defense peptides, and synthetic derivatives thereof, possess clinical potential beyond the treatment of antibiotic-resistant infections. In this Review, we provide an overview of the biological activities of host defense and synthetic peptides, their mechanism(s) of action and new therapeutic applications and challenges that are associated with their clinical use. PMID:24231617

  12. Enzymatic phosphorylation of hair keratin enhances fast adsorption of cationic moieties.

    PubMed

    Volkov, Vadim; Cavaco-Paulo, Artur

    2016-04-01

    The current study describes the in vitro phosphorylation of a human hair keratin, using protein kinase for the first time. Phosphorylation of keratin was demonstrated by (31)P NMR (Nuclear Magnetic Resonance) and Diffuse Reflectance Infrared Fourier Transform (DRIFT) techniques. Phosphorylation induced a 2.5 fold increase of adsorption capacity in the first 10 min for cationic moiety like methylene blue (MB). Thorough description of MB adsorption process was performed by several isothermal models. Reconstructed fluorescent microscopy images depict distinct amounts of dye bound to the differently treated hair. The results of this work suggest that the enzymatic phosphorylation of keratins might have significant implications in hair shampooing and conditioning, where short application times of cationic components are of prime importance. PMID:26756110

  13. Development of the affinity materials for phosphorylated proteins/peptides enrichment in phosphoproteomics analysis.

    PubMed

    Wang, Zhi-Gang; Lv, Nan; Bi, Wen-Zhi; Zhang, Ji-Lin; Ni, Jia-Zuan

    2015-04-29

    Reversible protein phosphorylation is a key event in numerous biological processes. Mass spectrometry (MS) is the most powerful analysis tool in modern phosphoproteomics. However, the direct MS analysis of phosphorylated proteins/peptides is still a big challenge because of the low abundance and insufficient ionization of phosphorylated proteins/peptides as well as the suppression effects of nontargets. Enrichment of phosphorylated proteins/peptides by affinity materials from complex biosamples is the most widely used strategy to enhance the MS detection. The demand of efficiently enriching phosphorylated proteins/peptides has spawned diverse affinity materials based on different enrichment principles (e.g., electronic attraction, chelating). In this review, we summarize the recent development of various affinity materials for phosphorylated proteins/peptides enrichment. We will highlight the design and fabrication of these affinity materials, discuss the enrichment mechanisms involved in different affinity materials, and suggest the future challenges and research directions in this field. PMID:25845677

  14. Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies.

    PubMed Central

    Safran, A; Neumann, D; Fuchs, S

    1986-01-01

    Three peptides corresponding to residues 354-367, 364-374, 373-387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP-dependent protein kinase, the tyrosine-specific protein kinase and the calcium/phospholipid-dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase it was shown that only peptides 354-367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP-dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non-phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Antibodies to peptide 354-367 were found to react preferentially with non-phosphorylated receptor whereas the two other anti-peptide antibodies bound equally to phosphorylated and non-phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms. Images Fig. 2. Fig. 4. Fig. 5. PMID:3816758

  15. N-Acetylglycine Cation Tautomerization Enabled by the Peptide Bond.

    PubMed

    Kocisek, Jaroslav; Piekarski, Dariusz Grzegorz; Delaunay, Rudy; Huber, Bernd A; Adoui, Lamri; Martín, Fernando; Alcamí, Manuel; Rousseau, Patrick; Domaracka, Alicja; Kopyra, Janina; Díaz-Tendero, Sergio

    2015-09-17

    We present a combined experimental and theoretical study of the ionization of N-acetylglycine molecules by 48 keV O(6+) ions. We focus on the single ionization channel of this interaction. In addition to the prompt fragmentation of the N-acetylglycine cation, we also observe the formation of metastable parent ions with lifetimes in the microsecond range. On the basis of density functional theory calculations, we assign these metastable ions to the diol tautomer of N-acetylglycine. In comparison with the simple amino acids, the tautomerization rate is higher because of the presence of the peptide bond. The study of a simple biologically relevant molecule containing a peptide bond allows us to demonstrate how increasing the complexity of the structure influences the behavior of the ionized molecule. PMID:26243533

  16. The role of phosphorylation in dentin phosphoprotein peptide absorption to hydroxyapatite surfaces: a molecular dynamics study

    PubMed Central

    Villarreal-Ramirez, Eduardo; Garduño-Juarez, Ramon; Gericke, Arne; Boskey, Adele

    2015-01-01

    Dentin phosphoprotein (DPP) is a protein expressed mainly in dentin and to a lesser extent in bone. DPP has a disordered structure, rich in glutamic acid, aspartic acid and phosphorylated serine/threonine residues. It has a high capacity for binding to calcium ions and to hydroxyapatite (HA) crystal surfaces. We used molecular dynamics (MD) simulations as a method for virtually screening interactions between DPP motifs and HA. The goal was to determine which motifs are absorbed to HA surfaces. For these simulations, we considered five peptides from the human DPP sequence. All-atom MD simulations were performed using GROMACS, the peptides were oriented parallel to the {100} HA crystal surface, the distance between the HA and the peptide was 3 nm. The system was simulated for 20 ns. Preliminary results show that for the unphosphorylated peptides, the acidic amino acids present an electrostatic attraction where their side chains are oriented towards HA. This attraction, however, is slow to facilitate bulk transport to the crystal surface. On the other hand, the phosphorylated (PP) peptides are rapidly absorbed on the surface of the HA with their centers of mass closer to the HA surface. More importantly, the root mean square fluctuation (RMSF) indicates that the average structures of the phosphorylated peptides are very inflexible and elongate, while that of the unphosphorylated peptides are flexible. Radius of gyration (Rg) analysis showed the compactness of un-phosphorylated peptides is lower than phosphorylated peptides. Phosphorylation of the DPP peptides is necessary for binding to HA surfaces. PMID:25158198

  17. Photosensitizing effect of cations on amino acids and peptides.

    PubMed

    Bogdanova, N P; Khenokh, M A

    1969-01-01

    In connection with a study of the chemical evolution of abiogenically synthesized organic compounds on primitive Earth and the physical conditions of other planets, this paper reports the experimental results obtained by the photolysis of solutions of aliphatic amino acids (glycine, alanine, valine, leucine, n. leucine) and peptides in the atmosphere of the air, N2, Ar and CO2 in the presence of the most simple photocatalyzers-cations of sulphates. The evidence shows that the photochemical conversion of NH2 acids depends on the content of the atmosphere. The decay of NH2-group is most active in air. N2 and Ar exert no significant influence on deamination, whereas in the atmosphere of CO2 the formation of ammonia in valine, for example, was only 29 per cent of its total amount during photolysis in the air. Cu2+ and Fe2+ catalyzed while Al3+ inhibited the ammonia excretion. The formation of acetaldehyde during alanine photolysis was actually independent from the atmosphere of N2 and was inhibited in Ar and CO2. Oxydative processes inducing the formation of glyoxalic acid and formaldehyde were sharply inhibited in Ar, N2 and CO2. Under the influence of ultraviolet light of the decay of NH2-acids is also accompanied by the formation of new NH2-acids. The photosensitizing effect of cations induces a rupture of -CO-NH-bonds in peptides and, provided heavy radiation doses, prevents the formation of new NH2-acids. The longer the dipeptide chain, the more significant the quantum yield of its decomposition. The photolysis of dipeptides, leading to their decay, does not necessarily induce a hydrolytic rupture of -CO-NH-bonds resulting in the formation of three amino acids. The results obtained permit approaching problems concerning the effect of the gas content of the atmosphere and various cations (photocatalyzers) on photolytic conversion of abiogenically synthesized and biogenically significant substances, amino acids for example, at the action of ultraviolet light. PMID

  18. Collectins and Cationic Antimicrobial Peptides of the Respiratory Epithelia

    PubMed Central

    Grubor, B.; Meyerholz, D. K.; Ackermann, M. R.

    2009-01-01

    The respiratory epithelium is a primary site for the deposition of microorganisms that are acquired during inspiration. The innate immune system of the respiratory tract eliminates many of these potentially harmful agents preventing their colonization. Collectins and cationic antimicrobial peptides are antimicrobial components of the pulmonary innate immune system produced by respiratory epithelia, which have integral roles in host defense and inflammation in the lung. Synthesis and secretion of these molecules are regulated by the developmental stage, hormones, as well as many growth and immunoregulatory factors. The purpose of this review is to discuss antimicrobial innate immune elements within the respiratory tract of healthy and pneumonic lung with emphasis on hydrophilic surfactant proteins and β-defensins. PMID:16966437

  19. Cationic Antimicrobial Peptides Disrupt the Streptococcus pyogenes ExPortal

    PubMed Central

    Vega, Luis Alberto; Caparon, Michael G.

    2012-01-01

    Summary Although they possess a well-characterized ability to porate the bacterial membrane, emerging research suggests that cationic antimicrobial peptides (CAPs) can influence pathogen behavior at levels that are sub-lethal. In this study, we investigated the interaction of polymyxin B and human neutrophil peptide (HNP-1) with the human pathogen Streptococcus pyogenes. At sub-lethal concentrations, these CAPs preferentially targeted the ExPortal, a unique microdomain of the S. pyogenes membrane, specialized for protein secretion and processing. A consequence of this interaction was the disruption of ExPortal organization and a redistribution of ExPortal components into the peripheral membrane. Redistribution was associated with inhibition of secretion of certain toxins, including the SpeB cysteine protease and the Streptolysin O (SLO) cytolysin, but not SIC, a protein that protects S. pyogenes from CAPs. These data suggest a novel function for CAPs in targeting the ExPortal and interfering with secretion of factors required for infection and survival. This mechanism may prove valuable for the design of new types of antimicrobial agents to combat the emergence of antibiotic-resistant pathogens. PMID:22780862

  20. Phosphorylation-dependent mineral-type specificity for apatite-binding peptide sequences.

    PubMed

    Addison, William N; Miller, Sharon J; Ramaswamy, Janani; Mansouri, Ahmad; Kohn, David H; McKee, Marc D

    2010-12-01

    Apatite-binding peptides discovered by phage display provide an alternative design method for creating functional biomaterials for bone and tooth tissue repair. A limitation of this approach is the absence of display peptide phosphorylation--a post-translational modification important to mineral-binding proteins. To refine the material specificity of a recently identified apatite-binding peptide, and to determine critical design parameters (net charge, charge distribution, amino acid sequence and composition) controlling peptide affinity for mineral, we investigated the effects of phosphorylation and sequence scrambling on peptide adsorption to four different apatites (bone-like mineral, and three types of apatite containing initially 0, 5.6 and 10.5% carbonate). Phosphorylation of the VTKHLNQISQSY peptide (VTK peptide) led to a 10-fold increase in peptide adsorption (compared to nonphosphorylated peptide) to bone-like mineral, and a 2-fold increase in adsorption to the carbonated apatite, but there was no effect of phosphorylation on peptide affinity to pure hydroxyapatite (without carbonate). Sequence scrambling of the nonphosphorylated VTK peptide enhanced its specificity for the bone-like mineral, but scrambled phosphorylated VTK peptide (pVTK) did not significantly alter mineral-binding suggesting that despite the importance of sequence order and/or charge distribution to mineral-binding, the enhanced binding after phosphorylation exceeds any further enhancement by altered sequence order. Osteoblast culture mineralization was dose-dependently inhibited by pVTK and to a significantly lesser extent by scrambled pVTK, while the nonphosphorylated and scrambled forms had no effect, indicating that inhibition of osteoblast mineralization is dependent on both peptide sequence and charge. Computational modeling of peptide-mineral interactions indicated a favorable change in binding energy upon phosphorylation that was unaffected by scrambling. In conclusion

  1. Electron capture dissociation mass spectrometric analysis of lysine-phosphorylated peptides

    PubMed Central

    Kowalewska, Karolina; Stefanowicz, Piotr; Ruman, Tomasz; Frączyk, Tomasz; Rode, Wojciech; Szewczuk, Zbigniew

    2010-01-01

    Phosphorylation of proteins is an essential signalling mechanism in eukaryotic and prokaryotic cells. Although N-phosphorylation of basic amino acid is known for its importance in biological systems, it is still poorly explored in terms of products and mechanisms. In the present study, two MS fragmentation methods, ECD (electron-capture dissociation) and CID (collision-induced dissociation), were tested as tools for analysis of N-phosphorylation of three model peptides, RKRSRAE, RKRARKE and PLSRTLSVAAKK. The peptides were phosphorylated by reaction with monopotassium phosphoramidate. The results were confirmed by 1H NMR and 31P NMR studies. The ECD method was found useful for the localization of phosphorylation sites in unstable lysine-phosphorylated peptides. Its main advantage is a significant reduction of the neutral losses related to the phosphoramidate moiety. Moreover, the results indicate that the ECD–MS may be useful for analysis of regioselectivity of the N-phosphorylation reaction. Stabilities of the obtained lysine-phosphorylated peptides under various conditions were also tested. PMID:20144148

  2. Phosphorylation-dependent mineral type specificity for apatite-binding peptide sequences

    PubMed Central

    Addison, William N.; Miller, Sharon J.; Ramaswamy, Janani; Mansouri, Ahmad; Kohn, David H.; McKee, Marc D.

    2010-01-01

    Apatite-binding peptides discovered by phage display provide an alternative design method for creating functional biomaterials for bone and tooth tissue repair. A limitation of this approach is the absence of display peptide phosphorylation – a post-translational modification important to mineral-binding proteins. To refine the material specificity of a recently identified apatite-binding peptide, and to determine critical design parameters (net charge, charge distribution, amino acid sequence and composition) controlling peptide affinity for mineral, we investigated the effects of phosphorylation and sequence scrambling on peptide adsorption to four different apatites (bone-like mineral, and three types of apatite containing initially 0, 5.6 and 10.5% carbonate). Phosphorylation of peptide VTKHLNQISQSY (pVTK) led to a 10-fold increase in peptide adsorption (compared to nonphosphorylated peptide) to bone-like mineral, and a 2-fold increase in adsorption to the carbonated apatite, but there was no effect of phosphorylation on peptide affinity to pure hydroxyapatite (without carbonate). Sequence scrambling of the nonphosphorylated VTK peptide enhanced its specificity for the bone-like mineral, but scrambled pVTK peptide did not significantly alter mineral-binding suggesting that despite the importance of sequence order and/or charge distribution to mineral binding, the enhanced binding after phosphorylation exceeds any further enhancement by altered sequence order. Osteoblast culture mineralization was dose-dependently inhibited by pVTK and to a significantly lesser extent by scrambled pVTK, while the nonphosphorylated and scrambled forms had no effect, indicating that inhibition of osteoblast mineralization is dependent on both peptide sequence and charge. Computational modeling of peptide-mineral interactions indicated a favorable change in binding energy upon phosphorylation that was unaffected by scrambling. In conclusion, phosphorylation of serine residues

  3. Coordination of trivalent metal cations to peptides: results from IRMPD spectroscopy and theory.

    PubMed

    Prell, James S; Flick, Tawnya G; Oomens, Jos; Berden, Giel; Williams, Evan R

    2010-01-21

    Structures of trivalent lanthanide metal cations La(3+), Ho(3+), and Eu(3+) with deprotonated Ala(n) (n = 2-5) or Leu-enk (Tyr-Gly-Gly-Phe-Leu) are investigated with infrared multiple photon dissociation (IRMPD) spectroscopy between 900 and 1850 cm(-1) and theory. In all of these complexes, a salt bridge is formed in which the metal cation coordinates to the carboxylate group of the peptide, resulting in a limited conformational space and many sharp IRMPD spectral bands. The IRMPD spectra clearly indicate that all carbonyl groups solvate the metal cation in each of the Ala(n) complexes. Due to strong vibrational coupling between the carbonyl groups, a sharp, high-energy amide I band due to in-phase stretching of all of the amide carbonyl groups bound to the metal cation is observed that is separated by approximately 50 cm(-1) from a strong, lower-energy amide I band. This extent of carbonyl coupling, which is sometimes observed in condensed-phase peptide and protein IR spectroscopy, has not been reported in IRMPD spectroscopy studies of other cationized peptide complexes. Intense bands due to carbonyl groups not associated with the metal cation are observed for Leu-enk complexes, indicating that a side chain group, such as the Tyr or Phe aromatic ring, prevents complete carbonyl coordination of the metal cation. Substitution of smaller lanthanide cations for La(3+) in these peptide complexes results only in minor structural changes consistent with the change in metal cation size. These are the first IRMPD spectra reported for lanthanide metal cationized peptides, and comparison to previously reported protonated and alkali metal or alkaline earth metal cationized peptide complexes reveals many trends consistent with the higher charge state of the lanthanide cations. PMID:19950916

  4. Cationic uncouplers of oxidative phosphorylation are inducers of mitochondrial permeability transition.

    PubMed

    Shinohara, Y; Bandou, S; Kora, S; Kitamura, S; Inazumi, S; Terada, H

    1998-05-22

    To determine whether cationic uncouplers of oxidative phosphorylation induce permeability transition in mitochondria, the effects of the divalent cationic sulfhydryl cross-linker copper-o-phenanthroline (Cu(OP)2) and the cyanine dye tri-S-C4(5) on rat liver mitochondria were examined. Like Ca2+, they accelerated mitochondrial respiration with succinate and induced mitochondrial swelling when inorganic phosphate (Pi) was present in the incubation medium. The acceleration of respiration and swelling were inhibited by the SH-reagent N-ethylmaleimide, and by the specific permeability transition inhibitor cyclosporin A (CsA). In addition, these cations, like Ca2+, induced release of ADP entrapped in the mitochondrial matrix space, and the morphological change of mitochondria induced by these cations was essentially the same as that induced by Ca2+. It is concluded that the uncoupling actions of Cu(OP)2 and tri-S-C4(5) are due to induction of permeability transition in the inner mitochondrial membrane. PMID:9645482

  5. Examining the Influence of Phosphorylation on Peptide Ion Structure by Ion Mobility Spectrometry-Mass Spectrometry.

    PubMed

    Glover, Matthew S; Dilger, Jonathan M; Acton, Matthew D; Arnold, Randy J; Radivojac, Predrag; Clemmer, David E

    2016-05-01

    Ion mobility spectrometry-mass spectrometry (IMS-MS) techniques are used to study the general effects of phosphorylation on peptide structure. Cross sections for a library of 66 singly phosphorylated peptide ions from 33 pairs of positional isomers, and unmodified analogues were measured. Intrinsic size parameters (ISPs) derived from these measurements yield calculated collision cross sections for 85% of these phosphopeptide sequences that are within ±2.5% of experimental values. The average ISP for the phosphoryl group (0.64 ± 0.05) suggests that in general this moiety forms intramolecular interactions with the neighboring residues and peptide backbone, resulting in relatively compact structures. We assess the capability of ion mobility to separate positional isomers (i.e., peptide sequences that differ only in the location of the modification) and find that more than half of the isomeric pairs have >1% difference in collision cross section. Phosphorylation is also found to influence populations of structures that differ in the cis/trans orientation of Xaa-Pro peptide bonds. Several sequences with phosphorylated Ser or Thr residues located N-terminally adjacent to Pro residues show fewer conformations compared to the unmodified sequences. Graphical Abstract ᅟ. PMID:26860087

  6. Examining the Influence of Phosphorylation on Peptide Ion Structure by Ion Mobility Spectrometry-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Glover, Matthew S.; Dilger, Jonathan M.; Acton, Matthew D.; Arnold, Randy J.; Radivojac, Predrag; Clemmer, David E.

    2016-05-01

    Ion mobility spectrometry-mass spectrometry (IMS-MS) techniques are used to study the general effects of phosphorylation on peptide structure. Cross sections for a library of 66 singly phosphorylated peptide ions from 33 pairs of positional isomers, and unmodified analogues were measured. Intrinsic size parameters (ISPs) derived from these measurements yield calculated collision cross sections for 85% of these phosphopeptide sequences that are within ±2.5% of experimental values. The average ISP for the phosphoryl group (0.64 ± 0.05) suggests that in general this moiety forms intramolecular interactions with the neighboring residues and peptide backbone, resulting in relatively compact structures. We assess the capability of ion mobility to separate positional isomers (i.e., peptide sequences that differ only in the location of the modification) and find that more than half of the isomeric pairs have >1% difference in collision cross section. Phosphorylation is also found to influence populations of structures that differ in the cis/ trans orientation of Xaa-Pro peptide bonds. Several sequences with phosphorylated Ser or Thr residues located N-terminally adjacent to Pro residues show fewer conformations compared to the unmodified sequences.

  7. Examining the Influence of Phosphorylation on Peptide Ion Structure by Ion Mobility Spectrometry-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Glover, Matthew S.; Dilger, Jonathan M.; Acton, Matthew D.; Arnold, Randy J.; Radivojac, Predrag; Clemmer, David E.

    2016-02-01

    Ion mobility spectrometry-mass spectrometry (IMS-MS) techniques are used to study the general effects of phosphorylation on peptide structure. Cross sections for a library of 66 singly phosphorylated peptide ions from 33 pairs of positional isomers, and unmodified analogues were measured. Intrinsic size parameters (ISPs) derived from these measurements yield calculated collision cross sections for 85% of these phosphopeptide sequences that are within ±2.5% of experimental values. The average ISP for the phosphoryl group (0.64 ± 0.05) suggests that in general this moiety forms intramolecular interactions with the neighboring residues and peptide backbone, resulting in relatively compact structures. We assess the capability of ion mobility to separate positional isomers (i.e., peptide sequences that differ only in the location of the modification) and find that more than half of the isomeric pairs have >1% difference in collision cross section. Phosphorylation is also found to influence populations of structures that differ in the cis/trans orientation of Xaa-Pro peptide bonds. Several sequences with phosphorylated Ser or Thr residues located N-terminally adjacent to Pro residues show fewer conformations compared to the unmodified sequences.

  8. Dynamic Light Scattering Analysis of the Effect of Phosphorylated Osteopontin Peptides on Mineral Formation

    NASA Astrophysics Data System (ADS)

    Mozaffari, Maryam; Goiko, Maria; de Bruyn, John; Goldberg, Harvey

    2015-03-01

    Biomineralization is the process by which living organisms synthesize minerals. Osteopontin (OPN), a mineral-associated protein, has been shown to be a potent inhibitor of mineral formation, a process that is dependent on phosphorylation. To gain a better understanding of the mechanism of inhibition, dynamic light scattering (DLS) was used to monitor the initial stages of nucleation, providing information about the size and relative concentration of the growing crystals as a function of time. DLS was used to investigate the effect of phosphorylated (P3, pOPAR) and non-phosphorylated (P0, OPAR) OPN peptides on the formation and growth of hydroxyapatite (HA) crystals from supersaturated solutions of calcium and phosphate ions. The non-phosphorylated P0 had a limited effect on HA nucleation and growth, while its thrice-phosphorylated isoform, P3, was a potent inhibitor of HA nucleation. The aspartic acid-rich OPAR was found to moderately inhibit nucleation but not growth, while its singly-phosphorylated isoform, pOPAR, inhibited HA nucleation more effectively, with some effect on HA crystal growth. The order of the inhibitory potential of these peptides was pOPAR>OPAR>P3>P0. This work confirms that highly acidic and phosphorylated peptides can inhibit the nucleation of HA more effectively.

  9. Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria

    PubMed Central

    Rodríguez-Rojas, Alexandro; Makarova, Olga; Müller, Uta; Rolff, Jens

    2015-01-01

    Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection. PMID:26430769

  10. Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria.

    PubMed

    Rodríguez-Rojas, Alexandro; Makarova, Olga; Müller, Uta; Rolff, Jens

    2015-10-01

    Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection. PMID:26430769

  11. Extended Coverage of Singly and Multiply Phosphorylated Peptides from a Single Titanium Dioxide Microcolumn.

    PubMed

    Wakabayashi, Masaki; Kyono, Yutaka; Sugiyama, Naoyuki; Ishihama, Yasushi

    2015-10-20

    We developed a novel approach to enlarge phosphoproteome coverage by selective elution depending on the number of phosphoryl group of peptides from a single titanium dioxide (TiO2) microcolumn using hydrophilic interaction chromatography (HILIC). In this approach, acidic methylphosphonate buffer including organic solvent is used for selective elution of singly phosphorylated peptides from an aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) microcolumn and basic elution conditions with phosphate, ammonium hydroxide, and pyrrolidine are then employed for eluting multiply phosphorylated peptides retained by the HAMMOC microcolumn. Finally, we successfully identified 11 300 nonredundant phosphopeptides from triplicate analyses of 100 μg of HeLa cell lysates using this approach. This simple strategy made it possible to accomplish comprehensive and efficient phosphoproteome analysis from limited sample amounts loaded onto a single HAMMOC microcolumn without additional fractionation or enrichment approaches. PMID:26402220

  12. Vasoactive intestinal peptide stimulates protein phosphorylation in a colonic epithelial cell line

    SciTech Connect

    Cohn, J.A.

    1987-09-01

    The T/sub 84/ colonic epithelial cell line was used to examine protein phosphorylation during neurohumoral stimulation of ion transport. T/sub 84/ cell monolayers grown on collagen-coated filters were mounted in Ussing chambers to measure ion transport stimulated by vasoactive intestinal peptide. Maximal stimulation of active secretion occurred after 8-10 min of stimulation. Protein phosphorylation events accompanying stimulated secretion were detected using two-dimensional gel electrophoresis to resolve phosphoproteins from monolayers previously labeled using /sup 32/P/sub i/. Within 8 min of exposure to vasoactive intestinal peptide, several phosphorylation events were detected, including a two- to fivefold increase in /sup 32/P incorporation into four soluble proteins with apparent molecular weights of 17,000, 18,000, 23,000, and 37,000. The same phosphorylation response occurs in monolayers stimulated by dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), suggesting that cAMP mediates these intracellular events. This study indicates that changes in protein phosphorylation accompany the secretory action of vasocactive intestinal peptide and suggests that T/sub 84/ cells offer a useful model for studying the possibility that such phosphorylation events regulate enterocyte ion transport.

  13. Phosphorylated Peptide Functionalization of Lanthanide Upconversion Nanoparticles for Tuning Nanomaterial-Cell Interactions.

    PubMed

    Yao, Chi; Wei, Caiyi; Huang, Zhi; Lu, Yiqing; El-Toni, Ahmed Mohamed; Ju, Dianwen; Zhang, Xiangmin; Wang, Wenning; Zhang, Fan

    2016-03-23

    Peptide modification of nanoparticles with high efficiency is critical in determining the properties and bioapplications of nanoparticles, but the methodology remains a challenging task. Here, by using the phosphorylated linear and cyclic peptide with the arginine-glycine-aspartic acid (RGD) targeting motifs as typical examples, the peptides binding efficiency for the inorganic metal compound nanoparticles was increased significantly after the phosphorylation treatment, and the modification allowed for improving the selectivity and signal-to-noise ratio for cancer targeting and reduced the toxicity derived from nonspecific interactions of nanoparticles with cells owing to the higher amount of phosphopeptide binding. In addition, molecular dynamics (MD) simulations of various peptides on inorganic metal compound surfaces revealed that the peptide adsorption on the surface is mainly driven by electrostatic interactions between phosphate oxygen and the polarized interfacial water layer, consistent with the experimental observation of the strong binding propensity of phosphorylated peptides. Significantly, with the RGD phosphopeptide surface modification, these nanoparticles provide a versatile tool for tuning material-cell interactions to achieve the desired level of autophagy and may prove useful for various diagnostic and therapeutic applications. PMID:26927957

  14. Small cationic antimicrobial peptides delocalize peripheral membrane proteins

    PubMed Central

    Wenzel, Michaela; Chiriac, Alina Iulia; Otto, Andreas; Zweytick, Dagmar; May, Caroline; Schumacher, Catherine; Gust, Ronald; Albada, H. Bauke; Penkova, Maya; Krämer, Ute; Erdmann, Ralf; Metzler-Nolte, Nils; Straus, Suzana K.; Bremer, Erhard; Becher, Dörte; Brötz-Oesterhelt, Heike; Sahl, Hans-Georg; Bandow, Julia Elisabeth

    2014-01-01

    Short antimicrobial peptides rich in arginine (R) and tryptophan (W) interact with membranes. To learn how this interaction leads to bacterial death, we characterized the effects of the minimal pharmacophore RWRWRW-NH2. A ruthenium-substituted derivative of this peptide localized to the membrane in vivo, and the peptide also integrated readily into mixed phospholipid bilayers that resemble Gram-positive membranes. Proteome and Western blot analyses showed that integration of the peptide caused delocalization of peripheral membrane proteins essential for respiration and cell-wall biosynthesis, limiting cellular energy and undermining cell-wall integrity. This delocalization phenomenon also was observed with the cyclic peptide gramicidin S, indicating the generality of the mechanism. Exogenous glutamate increases tolerance to the peptide, indicating that osmotic destabilization also contributes to antibacterial efficacy. Bacillus subtilis responds to peptide stress by releasing osmoprotective amino acids, in part via mechanosensitive channels. This response is triggered by membrane-targeting bacteriolytic peptides of different structural classes as well as by hypoosmotic conditions. PMID:24706874

  15. Peptide phosphorylation by calcium-dependent protein kinase from maize seedlings.

    PubMed

    Loog, M; Toomik, R; Sak, K; Muszynska, G; Järv, J; Ek, P

    2000-01-01

    Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings, and its substrate specificity studied using a set of synthetic peptides derived from the phosphorylatable sequence RVLSRLHS15VRER of maize sucrose synthase 2. The decapeptide LARLHSVRER was found to be efficiently phosphorylated as a minimal substrate. The same set of peptides were found to be phosphorylated by mammalian protein kinase Cbeta (PKC), but showed low reactivity with protein kinase A (PKA). Proceeding from the sequence LARLHSVRER, a series of cellulose-membrane-attached peptides of systematically modified structure was synthesised. These peptides had hydrophobic (Ala, Leu) and ionic (Arg, Glu) amino acids substituted in each position. The phosphorylation of these substrates by CDPK-1 was measured and the substrate specificity of the maize protein kinase characterised by the consensus sequence motif A/L-5X-4R-3X-2X-1SX+1R+2Z+3R+4, where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine compared with the other three varied amino acids. This motif had a characteristic sequence element RZR at positions +2 to +4 and closely resembled the primary structure of the sucrose synthase phosphorylation site. The sequence surrounding the phosphorylatable serine in this consensus motif was similar to the analogous sequence K/RXXS/TXK/R proposed for mammalian PKC, but different from the consensus motif RRXS/TX for PKA. PMID:10632703

  16. Tuning the conformation properties of a peptide by glycosylation and phosphorylation

    SciTech Connect

    Liang, F.-C.; Chen, Rita P.-Y.; Lin, C.-C.; Huang, K.-T.; Chan, S.I. . E-mail: SunneyChan@yahoo.com

    2006-04-07

    We have deployed the {alpha}-helical hairpin peptide ({alpha}-helix/turn/{alpha}-helix) and used it as a model system to explore how glycosylation and phosphorylation might affect the conformational properties of the peptide. The native conformations of the modified peptides in buffer solution have been compared with that of the wild-type peptide by nuclear magnetic resonance spectroscopy. Circular dichroism spectroscopy was used to probe the effects of an O-linked {beta}-GlcNAc and a phosphate group on the overall folding stability of the peptide. Finally, the rate of fibrillogenesis was used to infer the effects of these chemical modifications on the {alpha}-to-{beta} transition as well as the rate of nucleation of amyloidogenesis.

  17. Nanomechanical Response of Bacterial Cells to Cationic Antimicrobial Peptides

    NASA Astrophysics Data System (ADS)

    Lu, Shun; Walters, Grant; Parg, Richard; Dutcher, John

    2014-03-01

    The effectiveness of antimicrobial compounds can be easily screened, however their mechanism of action is much more difficult to determine. Many compounds act by compromising the mechanical integrity of the bacterial cell envelope, and our study introduces an atomic force microscopy (AFM)-based creep deformation technique to evaluate changes in the time-dependent mechanical properties of Pseudomonas aeruginosa PAO1 bacterial cells upon exposure to two different but structurally related antimicrobial peptides: polymyxin B and polymyxin B nonapeptide. We observed a distinctive signature for the loss of integrity of the bacterial cell envelope following exposure to the peptides. Measurements performed before and after exposure, as well as time-resolved measurements and those performed at different concentrations, revealed large changes to the viscoelastic parameters that are consistent with differences in the membrane permeabilizing effects of the peptides. The AFM creep deformation measurement provides new, unique insight into the kinetics and mechanism of action of antimicrobial peptides on bacteria.

  18. Solution Versus Gas-Phase Modification of Peptide Cations with NHS-Ester Reagents

    NASA Astrophysics Data System (ADS)

    Mentinova, Marija; Barefoot, Nathan Z.; McLuckey, Scott A.

    2012-02-01

    A comparison between solution and gas phase modification of primary amine sites in model peptide cations with N-hydroxysuccinimide (NHS) ester reagents is presented. In all peptides, the site of modification in solution was directed to the N-terminus by conducting reactions at pH = 5, whereas for the same peptides, a lysine residue was preferentially modified in the gas phase. The difference in pKa values of the N-terminus and ɛ-amino group of the lysine allows for a degree of control over sites of protonation of the peptides in aqueous solution. With removal of the dielectric and multiple charging of the peptide ions in the gas phase, the accommodation of excess charge can affect the preferred sites of reaction. Interaction of the lone pair of the primary nitrogen with a proton reduces its nucleophilicity and, as a result, its reactivity towards NHS-esters. While no evidence for reaction of the N-terminus with sulfo-NHS-acetate was noted in the model peptide cations, a charge inversion experiment using bis[sulfosuccinimidyl] suberate, a cross-linking reagent with two sulfo-NHS-ester functionalities, showed modification of the N-terminus. Hence, an unprotonated N-terminus can serve as a nucleophile to displace NHS, which suggests that its lack of reactivity with the peptide cations is likely due to the participation of the N-terminus in solvating excess charge.

  19. Cation-halide transport through peptide pores containing aminopicolinic acid.

    PubMed

    Basak, Debajyoti; Sridhar, Sucheta; Bera, Amal K; Madhavan, Nandita

    2016-05-18

    Synthetic pores that selectively transport ions of biological significance through membranes could be potentially used in medical diagnostics or therapeutics. Herein, we report cation-selective octapeptide pores derived from alanine and aminopicolinic acid. The ion transport mechanism through the pores has been established to be a cation-chloride symport. The cation-chloride co-transport is biologically essential for the efficient functioning of the central nervous system and has been implicated in diseases such as epilepsy. The pores formed in synthetic lipid bilayers do not exhibit any closing events. The ease of synthesis as well as infinite lifetimes of these pores provides scope for modifying their transport behaviour to develop sensors. PMID:27137995

  20. Cationic Peptide Exposure Enhances Pulsed-Electric-Field-Mediated Membrane Disruption

    PubMed Central

    Kennedy, Stephen M.; Aiken, Erik J.; Beres, Kaytlyn A.; Hahn, Adam R.; Kamin, Samantha J.; Hagness, Susan C.; Booske, John H.; Murphy, William L.

    2014-01-01

    Background The use of pulsed electric fields (PEFs) to irreversibly electroporate cells is a promising approach for destroying undesirable cells. This approach may gain enhanced applicability if the intensity of the PEF required to electrically disrupt cell membranes can be reduced via exposure to a molecular deliverable. This will be particularly impactful if that reduced PEF minimally influences cells that are not exposed to the deliverable. We hypothesized that the introduction of charged molecules to the cell surfaces would create regions of enhanced transmembrane electric potential in the vicinity of each charged molecule, thereby lowering the PEF intensity required to disrupt the plasma membranes. This study will therefore examine if exposure to cationic peptides can enhance a PEF’s ability to disrupt plasma membranes. Methodology/Principal Findings We exposed leukemia cells to 40 μs PEFs in media containing varying concentrations of a cationic peptide, polyarginine. We observed the internalization of a membrane integrity indicator, propidium iodide (PI), in real time. Based on an individual cell’s PI fluorescence versus time signature, we were able to determine the relative degree of membrane disruption. When using 1–2 kV/cm, exposure to >50 μg/ml of polyarginine resulted in immediate and high levels of PI uptake, indicating severe membrane disruption, whereas in the absence of peptide, cells predominantly exhibited signatures indicative of no membrane disruption. Additionally, PI entered cells through the anode-facing membrane when exposed to cationic peptide, which was theoretically expected. Conclusions/Significance Exposure to cationic peptides reduced the PEF intensity required to induce rapid and irreversible membrane disruption. Critically, peptide exposure reduced the PEF intensities required to elicit irreversible membrane disruption at normally sub-electroporation intensities. We believe that these cationic peptides, when coupled with

  1. UV/Vis Action Spectroscopy and Structures of Tyrosine Peptide Cation Radicals in the Gas Phase.

    PubMed

    Viglino, Emilie; Shaffer, Christopher J; Tureček, František

    2016-06-20

    We report the first application of UV/Vis photodissociation action spectroscopy for the structure elucidation of tyrosine peptide cation radicals produced by oxidative intramolecular electron transfer in gas-phase metal complexes. Oxidation of Tyr-Ala-Ala-Ala-Arg (YAAAR) produces Tyr-O radicals by combined electron and proton transfer involving the phenol and carboxyl groups. Oxidation of Ala-Ala-Ala-Tyr-Arg (AAAYR) produces a mixture of cation radicals involving electron abstraction from the Tyr phenol ring and N-terminal amino group in combination with hydrogen-atom transfer from the Cα positions of the peptide backbone. PMID:27159034

  2. Nanomechanical Response of Pseudomonas aeruginosa PAO1 Bacterial Cells to Cationic Antimicrobial Peptides

    NASA Astrophysics Data System (ADS)

    Lu, Shun; Walters, Grant; Dutcher, John

    2013-03-01

    We have used an atomic force microscopy (AFM)-based creep deformation technique to study changes to the viscoelastic properties of individual Gram-negative Pseudomonas aeruginosa PAO1 cells as a function of time of exposure to two cationic peptides: polymyxin B (PMB), a cyclic antimicrobial peptide, and the structurally-related compound, polymyxin B nonapeptide (PMBN). The measurements provide a direct measure of the mechanical integrity of the bacterial cell envelope, and the results can be understood in terms of simple viscoelastic models of arrangements of springs and dashpots, which can be ascribed to different components within the bacterial cell. Time-resolved creep deformation experiments reveal abrupt changes to the viscoelastic properties of P. aeruginosa bacterial cells after exposure to both PMB and PMBN, with quantitatively different changes for the two cationic peptides. These measurements provide new insights into the kinetics and mechanism of action of antimicrobial peptides on bacterial cells.

  3. Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides.

    PubMed

    Kaconis, Yani; Kowalski, Ina; Howe, Jörg; Brauser, Annemarie; Richter, Walter; Razquin-Olazarán, Iosu; Iñigo-Pestaña, Melania; Garidel, Patrick; Rössle, Manfred; Martinez de Tejada, Guillermo; Gutsmann, Thomas; Brandenburg, Klaus

    2011-06-01

    Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS. PMID:21641310

  4. Sequential enrichment of singly- and multiply-phosphorylated peptides with zwitterionic hydrophilic interaction chromatography material.

    PubMed

    Sheng, Qianying; Yang, Kaiya; Xue, Xingya; Li, Xiuling; Guo, Zhimou; Shen, Aijin; Ke, Yanxiong; Lan, Minbo; Liang, Xinmiao

    2015-09-25

    An interesting and novel method for the selective and sequential enrichment of singly- and multiply-phosphorylated peptides with a zwitterionic material "Click TE-Cys" is presented. Retention mechanisms between phosphopeptides and Click TE-Cys are systematically investigated by checking the influence of acetonitrile content, pH value, and buffer concentration on the retention of phosphopeptides. Both hydrophilic interaction and electrostatic interaction are involved in retention between phosphopeptides and Click TE-Cys. Based on these results, an optimized method is established for selective enrichment of phosphopeptides using Click TE-Cys. This method not only exhibits high selectivity for phosphopeptides, but also fractionates singly- and multiply-phosphorylated peptides into two fractions. This method was evaluated using relatively complex samples, including peptide mixtures of α-casein and bovine serum albumin (BSA) at a molar ratio of 1:10 and skim milk. This efficient and optimized protocol has great potential for enriching multiply-phosphorylated peptides and could be a valuable tool for specific enrichment of phosphopeptides in phosphoproteome analysis. PMID:26298604

  5. Structure and dynamics of cationic membrane peptides and proteins: Insights from solid-state NMR

    PubMed Central

    Hong, Mei; Su, Yongchao

    2011-01-01

    Many membrane peptides and protein domains contain functionally important cationic Arg and Lys residues, whose insertion into the hydrophobic interior of the lipid bilayer encounters significant energy barriers. To understand how these cationic molecules overcome the free energy barrier to insert into the lipid membrane, we have used solid-state NMR spectroscopy to determine the membrane-bound topology of these peptides. A versatile array of solid-state NMR experiments now readily yields the conformation, dynamics, orientation, depth of insertion, and site-specific protein–lipid interactions of these molecules. We summarize key findings of several Arg-rich membrane peptides, including β-sheet antimicrobial peptides, unstructured cell-penetrating peptides, and the voltage-sensing helix of voltage-gated potassium channels. Our results indicate the central role of guanidinium-phosphate and guanidinium-water interactions in dictating the structural topology of these cationic molecules in the lipid membrane, which in turn account for the mechanisms of this functionally diverse class of membrane peptides. PMID:21344534

  6. Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation.

    PubMed

    Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Delangle, Pascale; Guilloreau, Luc; Adriano, Jean-Marc; Berthomieu, Catherine

    2012-01-01

    To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T(9)TKE(12) sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K(d) = 25±6 nM to K(d) = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (K(d) = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν(as)(P-O) and ν(s)(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν(as)(UO(2))(2+) vibration (from 923 cm(-1) to 908 cm(-1)) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. PMID:22870263

  7. Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

    PubMed Central

    Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Delangle, Pascale; Guilloreau, Luc; Adriano, Jean-Marc; Berthomieu, Catherine

    2012-01-01

    To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T9TKE12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from Kd = 25±6 nM to Kd = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the νas(P-O) and νs(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in νas(UO2)2+ vibration (from 923 cm−1 to 908 cm−1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. PMID:22870263

  8. Cationic Bioactive Peptide from the Seeds of Benincasa hispida

    PubMed Central

    Sharma, Sunayana; Verma, Hirday Narain

    2014-01-01

    A designated bioactive peptide “Hispidalin” purified from the seeds of Benincasa hispida, which is a medicinal plant, belongs to Cucurbitaceae family. Purification was achieved by using a procedure consisting of extraction from potassium phosphate buffer followed by FPLC and HPLC steps. Based on amino acid residue, this peptide is amphipathic and basic with one net positive charge having isoelectric pH 8.1. This peptide is without sulphur containing amino acid suggesting its extended conformation lacking double bond secondary structure. The results obtained from MALDI-TOF suggested that Hispidalin is of molecular mass 5.7 KDa with 49 amino acid residues and confirmed SDS-PAGE resolved ∼6.0 KDa protein band. This novel and unknown peptide “Hispidalin” showed broad and potent inhibitory effects against various human bacterial and fungal pathogens; its growth inhibition was significantly comparable with commercial antibacterial and antifungal drugs. The Hispidalin at 40 μg/mL concentration exhibited 70.8% DPPH free radical-scavenging activity and 69.5% lipid peroxide inhibition. Thus, in the present study, Hispidalin demonstrated remarkable antimicrobial and antioxidant potentials from the seeds of B. hispida. PMID:24834076

  9. Biophysical Mechanisms of Endotoxin Neutralization by Cationic Amphiphilic Peptides

    PubMed Central

    Kaconis, Yani; Kowalski, Ina; Howe, Jörg; Brauser, Annemarie; Richter, Walter; Razquin-Olazarán, Iosu; Iñigo-Pestaña, Melania; Garidel, Patrick; Rössle, Manfred; Martinez de Tejada, Guillermo; Gutsmann, Thomas; Brandenburg, Klaus

    2011-01-01

    Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS. PMID:21641310

  10. Electrogenerated Chemiluminescence Bioassay of Two Protein Kinases Incorporating Peptide Phosphorylation and Versatile Probe.

    PubMed

    Liu, Xia; Dong, Manman; Qi, Honglan; Gao, Qiang; Zhang, Chengxiao

    2016-09-01

    A sensitive electrogenerated chemiluminescence (ECL) bioassay was developed for the detection of two protein kinases incorporating the peptide phosphorylation and a versatile ECL probe. Cyclic adenosine monophosphate-dependent protein kinase (PKA) and casein kinase II (CK2) were used as proof-of-concept targets while a PKA-specific peptide (CLRRASLG) and a CK2-specific peptide (CRRRADDSDDDDD) were used as the recognition substrates. Taking advantage of the ability of protein A binding with the Fc region of a variety of antibodies with high affinity, a ruthenium derivative-labeled protein A was utilized as a versatile ECL probe for bioassay of multiple protein kinases. A specific peptide substrate toward target protein kinase was first self-assembled on the surface of gold electrode and then serine in the specific peptide on the electrode was phosphorylated by target protein kinase in the presence of adenosine-5'-triphosphate. After recognition of the phosphorylated peptide by monoclonal antiphosphoserine antibody, the versatile ECL probe was specifically bound to the antiphosphoserine antibody on the electrode surface. The ECL bioassay was developed successfully in the individual detection of PKA and CK2 with detection limit of 0.005 U/mL and 0.004 U/mL, respectively. In addition, the ECL bioassay was applied to quantitative analysis of the kinase inhibitors and monitoring drug-triggered kinase activation in cell lysates. Moreover, an ECL imaging bioassay using electron-multiplying charged coupled device as detector on the gold electrode array was developed for the simultaneous detection of PKA and CK2 activity from 0.01 U/mL to 0.4 U/mL, respectively, at one time. This work demonstrates that the ingenious design and use of a versatile ECL probe are promising to simultaneous detection of multiple protein kinases and screening of kinase inhibitor. PMID:27518533

  11. Cationic polymethacrylates with covalently linked membrane destabilizing peptides as gene delivery vectors.

    PubMed

    Funhoff, Arjen M; van Nostrum, Cornelus F; Lok, Martin C; Kruijtzer, John A W; Crommelin, Daan J A; Hennink, Wim E

    2005-01-01

    A membrane-disrupting peptide derived from the influenza virus was covalently linked to different polymethacrylates (pDMAEMA, pDAMA and the degradable pHPMA-DMAE, monomers depicted in Fig. 1) using N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) as coupling agent to increase the transfection efficiency of polyplexes based on these polymers. It was shown by circular dichroism (CD) measurements that the polymer-conjugated peptide was, as the free peptide, able to undergo a conformational change of a random coil to an alpha helix upon lowering the pH to 5.0. This indicates that the property of the peptide to destabilize the endosomal membrane was preserved after its conjugation to the cationic polymers. In line herewith, a liposome leakage assay revealed that the polymer-bound peptide has comparable activity as the free peptide. The DNA condensing properties of the synthesized polymer-peptide conjugates were studied with dynamic light scattering and zeta-potential measurements, and it was shown that small (100 to 250 nm), positively charged (+15 to +20 mV) particles were formed. In vitro transfection and toxicity was tested in COS-7 cells, and these experiments showed that the polyplexes with grafted peptide had a substantially higher transfection activity than the control polyplexes, while the toxicity remained unchanged. Cellular uptake of the polyplexes was visualized with confocal laser scanning microscopy, and no differences in cellular uptake could be determined between the peptide containing systems and the control formulation. This shows that the increased transfection activity is indeed due to a better endosomal escape of the peptide grafted polyplexes. This study demonstrates that it is possible to covalently conjugate an endosome disruptive peptide to cationic gene delivery polymers with preservation of its membrane destabilization activity, making these conjugates suitable for in vivo DNA delivery. PMID:15588908

  12. Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III) Complexes.

    PubMed

    Sumaoka, Jun; Akiba, Hiroki; Komiyama, Makoto

    2016-01-01

    Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclear Tb(III) complexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr), have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the Tb(III) ion as the emission center. Even in the coexistence of phosphoserine (pSer) and phosphothreonine (pThr), pTyr can be efficintly detected with high selectivity. Simply by adding these Tb(III) complexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates. PMID:27375742

  13. Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III) Complexes

    PubMed Central

    Sumaoka, Jun; Akiba, Hiroki; Komiyama, Makoto

    2016-01-01

    Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclear TbIII complexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr), have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the TbIII ion as the emission center. Even in the coexistence of phosphoserine (pSer) and phosphothreonine (pThr), pTyr can be efficintly detected with high selectivity. Simply by adding these TbIII complexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates. PMID:27375742

  14. Photo-induced reversible structural transition of cationic diphenylalanine peptide self-assembly.

    PubMed

    Ma, Hongchao; Fei, Jinbo; Li, Qi; Li, Junbai

    2015-04-17

    The photo-induced self-assembly of a cationic diphenylalanine peptide (CDP) is investigated using a photoswitchable sulfonic azobenzene as the manipulating unit. A reversible structural transition between a branched structure and a vesicle-like structure is observed by alternating between UV and visible light irradiation. PMID:25405602

  15. Cation-exchange chromatography of peptides on poly(2-sulfoethyl aspartamide)-silica.

    PubMed

    Alpert, A J; Andrews, P C

    1988-06-29

    A strong cation-exchange material, poly(2-sulfoethyl aspartamide)-silica (PolySULFOETHYL Aspartamide) was developed for purification and analysis of peptides by high-performance liquid chromatography. All peptides examined were retained at pH 3, even when the amino terminus was the only basic group. Peptides were eluted in order of increasing number of basic residues with a salt gradient. Capacity was high, as was selectivity and column efficiency. This new column material displays modest mixed-mode effects, allowing the resolution of peptides having identical charges at a given pH. The selectivity can be manipulated by the addition of organic solvent to the mobile phases; this increases the retention of some peptides and decreases the retention of others. The retention in any given case may reflect a combination of steric factors and non-electrostatic interactions. Selectivity was complementary to that of reversed-phase chromatography (RPC) materials. Excellent purifications were obtained by sequential use of PolySULFOETHYL Aspartamide and RPC columns for purification of peptides from crude tissue extracts. The new cation exchanger is quite promising as a supplement to RPC for general peptide chromatography. PMID:2844843

  16. Synthetic cationic peptide IDR-1018 modulates human macrophage differentiation.

    PubMed

    Pena, Olga M; Afacan, Nicole; Pistolic, Jelena; Chen, Carol; Madera, Laurence; Falsafi, Reza; Fjell, Christopher D; Hancock, Robert E W

    2013-01-01

    Macrophages play a critical role in the innate immune response. To respond in a rapid and efficient manner to challenges in the micro-environment, macrophages are able to differentiate towards classically (M1) or alternatively (M2) activated phenotypes. Synthetic, innate defense regulators (IDR) peptides, designed based on natural host defence peptides, have enhanced immunomodulatory activities and reduced toxicity leading to protection in infection and inflammation models that is dependent on innate immune cells like monocytes/macrophages. Here we tested the effect of IDR-1018 on macrophage differentiation, a process essential to macrophage function and the immune response. Using transcriptional, protein and systems biology analysis, we observed that differentiation in the presence of IDR-1018 induced a unique signature of immune responses including the production of specific pro and anti-inflammatory mediators, expression of wound healing associated genes, and increased phagocytosis of apoptotic cells. Transcription factor IRF4 appeared to play an important role in promoting this IDR-1018-induced phenotype. The data suggests that IDR-1018 drives macrophage differentiation towards an intermediate M1-M2 state, enhancing anti-inflammatory functions while maintaining certain pro-inflammatory activities important to the resolution of infection. Synthetic peptides like IDR-1018, which act by modulating the immune system, could represent a powerful new class of therapeutics capable of treating the rising number of multidrug resistant infections as well as disorders associated with dysregulated immune responses. PMID:23308112

  17. Effectiveness, against tuberculosis, of pseudo-ternary complexes: peptide-DNA-cationic liposome.

    PubMed

    Rosada, Rogério Silva; Silva, Célio Lopes; Santana, Maria Helena Andrade; Nakaie, Clóvis Ryuichi; de la Torre, Lucimara Gaziola

    2012-05-01

    We report the effects of a synthetic peptide designed to act as a nuclear localization signal on the treatment of tuberculosis. The peptide contains 21 amino acid residues with the following specific domains: nuclear localization signal from SV 40T, cationic shuttle sequence, and cysteamide group at the C-terminus. The peptide was complexed with the plasmid DNAhsp65 and incorporated into cationic liposomes, forming a pseudo-ternary complex. The same cationic liposomes, composed of egg chicken L-α-phosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane, and 1,2-dioleoyl-3-trimethylammonium-propane (2:1:1M), were previously evaluated as a gene carrier for tuberculosis immunization protocols with DNAhsp65. The pseudo-ternary complex presented a controlled size (250 nm), spherical-like shape, and various lamellae in liposomes as evaluated by transmission electron microscopy. An assay of fluorescence probe accessibility confirmed insertion of the peptide/DNA into the liposome structure. Peptide addition conferred no cytotoxicity in vitro, and similar therapeutic effects against tuberculosis were seen with four times less DNA compared with naked DNA treatment. Taken together, the results indicate that the pseudo-ternary complex is a promising gene vaccine for tuberculosis treatment. This work contributes to the development of multifunctional nanostructures in the search for strategies for in vivo DNA delivery. PMID:21999959

  18. Ground and Excited-Electronic-State Dissociations of Hydrogen-Rich and Hydrogen-Deficient Tyrosine Peptide Cation Radicals

    NASA Astrophysics Data System (ADS)

    Viglino, Emilie; Lai, Cheuk Kuen; Mu, Xiaoyan; Chu, Ivan K.; Tureček, František

    2016-09-01

    We report a comprehensive study of collision-induced dissociation (CID) and near-UV photodissociation (UVPD) of a series of tyrosine-containing peptide cation radicals of the hydrogen-rich and hydrogen-deficient types. Stable, long-lived, hydrogen-rich peptide cation radicals, such as [AAAYR + 2H]+● and several of its sequence and homology variants, were generated by electron transfer dissociation (ETD) of peptide-crown-ether complexes, and their CID-MS3 dissociations were found to be dramatically different from those upon ETD of the respective peptide dications. All of the hydrogen-rich peptide cation radicals contained major (77%-94%) fractions of species having radical chromophores created by ETD that underwent photodissociation at 355 nm. Analysis of the CID and UVPD spectra pointed to arginine guanidinium radicals as the major components of the hydrogen-rich peptide cation radical population. Hydrogen-deficient peptide cation radicals were generated by intramolecular electron transfer in CuII(2,2 ':6 ',2 ″-terpyridine) complexes and shown to contain chromophores absorbing at 355 nm and undergoing photodissociation. The CID and UVPD spectra showed major differences in fragmentation for [AAAYR]+● that diminished as the Tyr residue was moved along the peptide chain. UVPD was found to be superior to CID in localizing Cα-radical positions in peptide cation radical intermediates.

  19. Ground and Excited-Electronic-State Dissociations of Hydrogen-Rich and Hydrogen-Deficient Tyrosine Peptide Cation Radicals.

    PubMed

    Viglino, Emilie; Lai, Cheuk Kuen; Mu, Xiaoyan; Chu, Ivan K; Tureček, František

    2016-09-01

    We report a comprehensive study of collision-induced dissociation (CID) and near-UV photodissociation (UVPD) of a series of tyrosine-containing peptide cation radicals of the hydrogen-rich and hydrogen-deficient types. Stable, long-lived, hydrogen-rich peptide cation radicals, such as [AAAYR + 2H](+●) and several of its sequence and homology variants, were generated by electron transfer dissociation (ETD) of peptide-crown-ether complexes, and their CID-MS(3) dissociations were found to be dramatically different from those upon ETD of the respective peptide dications. All of the hydrogen-rich peptide cation radicals contained major (77%-94%) fractions of species having radical chromophores created by ETD that underwent photodissociation at 355 nm. Analysis of the CID and UVPD spectra pointed to arginine guanidinium radicals as the major components of the hydrogen-rich peptide cation radical population. Hydrogen-deficient peptide cation radicals were generated by intramolecular electron transfer in Cu(II)(2,2':6',2″-terpyridine) complexes and shown to contain chromophores absorbing at 355 nm and undergoing photodissociation. The CID and UVPD spectra showed major differences in fragmentation for [AAAYR](+●) that diminished as the Tyr residue was moved along the peptide chain. UVPD was found to be superior to CID in localizing Cα-radical positions in peptide cation radical intermediates. Graphical Abstract ᅟ. PMID:27278824

  20. Ground and Excited-Electronic-State Dissociations of Hydrogen-Rich and Hydrogen-Deficient Tyrosine Peptide Cation Radicals

    NASA Astrophysics Data System (ADS)

    Viglino, Emilie; Lai, Cheuk Kuen; Mu, Xiaoyan; Chu, Ivan K.; Tureček, František

    2016-06-01

    We report a comprehensive study of collision-induced dissociation (CID) and near-UV photodissociation (UVPD) of a series of tyrosine-containing peptide cation radicals of the hydrogen-rich and hydrogen-deficient types. Stable, long-lived, hydrogen-rich peptide cation radicals, such as [AAAYR + 2H]+● and several of its sequence and homology variants, were generated by electron transfer dissociation (ETD) of peptide-crown-ether complexes, and their CID-MS3 dissociations were found to be dramatically different from those upon ETD of the respective peptide dications. All of the hydrogen-rich peptide cation radicals contained major (77%-94%) fractions of species having radical chromophores created by ETD that underwent photodissociation at 355 nm. Analysis of the CID and UVPD spectra pointed to arginine guanidinium radicals as the major components of the hydrogen-rich peptide cation radical population. Hydrogen-deficient peptide cation radicals were generated by intramolecular electron transfer in CuII(2,2':6',2″-terpyridine) complexes and shown to contain chromophores absorbing at 355 nm and undergoing photodissociation. The CID and UVPD spectra showed major differences in fragmentation for [AAAYR]+● that diminished as the Tyr residue was moved along the peptide chain. UVPD was found to be superior to CID in localizing Cα-radical positions in peptide cation radical intermediates.

  1. Phosphorylation of the synthetic octapeptide pyroGlu-ASP-ASP-SER-ASP-GLU-GLU-ASN and binding to DNA in presence of divalent cations.

    PubMed

    Cardellini, E; Ponti, D; Gianfranceschi, G L

    1999-12-01

    Small acidic peptides involved in gene expression have been isolated from prokaryotic and eukaryotic cells. Synthetic peptides, designed on the basis of native peptides characteristics, show a biological activity similar to that of native peptides in in vitro reconstituted systems. These synthetic peptides are able to bind to DNA in presence of divalent cations (Cu2+, Fe2+, Mg2+) and salt/ethanol. PMID:10634508

  2. Complexes of DNA with cationic peptides: conditions of formation and factors effecting internalization by mammalian cells.

    PubMed

    Dizhe, E B; Ignatovich, I A; Burov, S V; Pohvoscheva, A V; Akifiev, B N; Efremov, A M; Perevozchikov, A P; Orlov, S V

    2006-12-01

    This work was devoted to the study of conditions of the formation of DNA/K8 complex and analysis of factors effecting the entry of DNA/K8 complex into mammalian cells in comparison with DNA complexes with arginine-rich fragment (47-57) of human immunodeficiency virus (type 1) transcription factor Tat (Tat peptide). The stoichiometry of positively charged DNA/K8 complexes has been studied for the first time. Non-cooperative character of DNA-K8 interaction was revealed. It has been shown that along with the positive charge of such complexes, the presence of an excess of free K8 peptide in the culture medium is a necessary condition for maximal efficiency of cell transfection with DNA/K8 complexes. A stimulatory effect of free K8 peptide on the efficiency of mammalian cell transfection by DNA/K8 complexes is likely to be mediated by the interactions of cationic peptide K8 with negatively charged proteoglycans on the cell surface, which leads to protection of DNA/K8 complexes from disruption by cellular heparan sulfates. However, the protective role of free cationic peptides depends not only on their positive charge, but also on the primary structure of the peptide. In contrast with the results obtained for DNA complexes with molecular conjugates based on poly-L-lysine, the aggregation of DNA/K8 complexes leads to a significant increase in the expression of transferred gene. PMID:17223788

  3. Infrared Multiphoton Dissociation of Peptide Cations in a Dual Pressure Linear Ion Trap Mass Spectrometer

    PubMed Central

    Gardner, Myles W.; Smith, Suncerae I.; Ledvina, Aaron R.; Madsen, James A.; Coon, Joshua J.; Schwartz, Jae C.; Stafford, George C.; Brodbelt, Jennifer S.

    2009-01-01

    A dual pressure linear ion trap mass spectrometer was modified to permit infrared multiphoton dissociation (IRMPD) in each of the two cells - the first a high pressure cell operated at nominally 5 × 10-3 Torr and the second a low pressure cell operated at nominally 3 × 10-4 Torr. When IRMPD was performed in the high pressure cell, most peptide ions did not undergo significant photodissociation; however, in the low pressure cell peptide cations were efficiently dissociated with less than 25 ms of IR irradiation regardless of charge state. IRMPD of peptide cations allowed the detection of low m/z product ions including the y1 fragments and immonium ions which are not typically observed by ion trap collision induced dissociation (CID). Photodissociation efficiencies of ~100% and MS/MS (tandem mass spectrometry) efficiencies of greater than 60% were observed for both multiply and singly protonated peptides. In general, higher sequence coverage of peptides was obtained using IRMPD over CID. Further, greater than 90% of the product ion current in the IRMPD mass spectra of doubly charged peptide ions was composed of singly charged product ions compared to the CID mass spectra in which the abundances of the multiply and singly charged product ions were equally divided. Highly charged primary product ions also underwent efficient photodissociation to yield singly charged secondary product ions, thus simplifying the IRMPD product ion mass spectra. PMID:19739654

  4. Natriuretic peptides induce weak VASP phosphorylation at Serine 239 in platelets

    PubMed Central

    Borgognone, Alessandra; Lowe, Kate L; Watson, Stephen P; Madhani, Melanie

    2013-01-01

    Cyclic guanosine-3′,5′-monophoshate (cGMP) is the common second messenger for the cardiovascular effects of nitric oxide (NO) and natriuretic peptides (NP; for example, atrial natriuretic peptide [ANP]), which activate soluble and particulate guanylyl cyclases (sGC and pGC), respectively. The role of NO in regulating cGMP and platelet function is well documented, whereas there is little evidence supporting a role for NPs in regulating platelet reactivity. By studying platelet aggregation and secretion in response to a PAR-1 peptide, collagen and ADP, and phosphorylation of the cGMP-dependent protein kinase (PKG) substrate VASP at serine 239, we evaluated the effects of NPs in the absence or presence of the non-selective cGMP and cAMP phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylanxthine (IBMX). Our results show that NPs, possibly through the clearance receptor (natriuretic peptide receptor-C, NPR-C) expressed on platelet membranes, increase VASP phosphorylation but only following PDE inhibition, indicating a small, localised cGMP synthesis. As platelet aggregation and secretion measured under the same conditions were not affected, we conclude that the magnitude of PKG activation achieved by NPs in platelets per se is not sufficient to exert functional inhibition of platelet involvement in haemostasis. PMID:23469931

  5. The effects of BT/TAMUS 2032 cationic peptide on innate immunity and susceptibility of young chickens to extraintestinal Salmonella enterica serovar Enteritidis infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The BT/TAMUS 2032 cationic peptides are a group of related cationic peptides produced by a Gram-positive bacterium. Cationic amphiphilic peptides have been found to stimulate or prime the innate immune responses in mammals. The innate immune system of poultry is functionally inefficient during the ...

  6. Phosphorylation of synthetic peptides by a tyrosine protein kinase from the particulate fraction of a lymphoma cell line.

    PubMed Central

    Casnellie, J E; Harrison, M L; Pike, L J; Hellström, K E; Krebs, E G

    1982-01-01

    The particulate fraction from a lymphoma cell line, LSTRA, was found to contain an apparent high level of tyrosine protein kinase activity. When this fraction was incubated with [gamma-32P]ATP in the presence of 10 mM MnCl2, hydrolyzed, and assayed, 70--80% of the radioactivity recovered in phosphoamino acids was in phosphotyrosine. Gel electrophoresis of the proteins showed that a large portion of the 32P was in a single protein with a molecular weight of approximately 58,000. The phosphorylated residue in this protein was identified as phosphotyrosine. Detergent extracts of the particulate fraction from LSTRA cells contained both the Mr 58,000 protein and the enzyme responsible for its phosphorylation. These extracts were found to catalyze the phosphorylation of the tyrosine residue in the synthetic peptide, Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly, corresponding to the sequence around the tyrosine that is phosphorylated in pp60src; the Km for the peptide in this reaction was 5 mM. High-performance liquid chromatography was used to assay for this phosphorylation. A second peptide was synthesized that contained two additional arginine residues whose presence permitted the phosphorylation of the peptide to be measured by a simple assay using phosphocellulose paper. The Km for this peptide was 3--4 mM, indicating that the presence of the additional arginine residues did not alter the apparent affinity of the kinase for the peptide. Images PMID:6804939

  7. Reversible and irreversible effects of basic peptides on the mitochondrial cationic channel.

    PubMed Central

    Fèvre, F; Henry, J P; Thieffry, M

    1994-01-01

    We have previously shown that a 13-residue basic peptide, derived from the presequence of a mitochondrial precursor, blocked the cationic channel of the outer mitochondrial membrane. The properties of the blockade suggested that the peptide could go through the pore in the presence of a sufficient driving force. In an attempt to evaluate more precisely the relevance of such an interpretation, we have examined the effect on the same channel of basic peptides from 16 to 34 residues, most of which are parts of or derive from mitochondrial presequences. Two peptides were found to induce a reversible voltage-dependent blockade, the properties of which were the same as those of the blockade induced by the 13-residue peptide. The others had a similar effect, but triggered in addition a modification of the voltage gating that persisted after washing the peptide out. The modification was in turn abolished by trypsin added to the side of the channel previously exposed to the peptide. The protease acted on the bound peptide and not on the channel itself. The irreversible modification of the voltage gating, the mechanism of which remains obscure, was not specific for mitochondrial-addressing sequences. PMID:7521225

  8. Self-assembled cationic peptide nanoparticles as an efficient antimicrobial agent

    NASA Astrophysics Data System (ADS)

    Liu, Lihong; Xu, Kaijin; Wang, Huaying; Jeremy Tan, P. K.; Fan, Weimin; Venkatraman, Subbu S.; Li, Lanjuan; Yang, Yi-Yan

    2009-07-01

    Antimicrobial cationic peptides are of interest because they can combat multi-drug-resistant microbes. Most peptides form α-helices or β-sheet-like structures that can insert into and subsequently disintegrate negatively charged bacterial cell surfaces. Here, we show that a novel class of core-shell nanoparticles formed by self-assembly of an amphiphilic peptide have strong antimicrobial properties against a range of bacteria, yeasts and fungi. The nanoparticles show a high therapeutic index against Staphylococcus aureus infection in mice and are more potent than their unassembled peptide counterparts. Using Staphylococcus aureus-infected meningitis rabbits, we show that the nanoparticles can cross the blood-brain barrier and suppress bacterial growth in infected brains. Taken together, these nanoparticles are promising antimicrobial agents that can be used to treat brain infections and other infectious diseases.

  9. Interaction of linear cationic peptides with phospholipid membranes and polymers of sialic acid.

    PubMed

    Kuznetsov, A S; Dubovskii, P V; Vorontsova, O V; Feofanov, A V; Efremov, R G

    2014-05-01

    Polysialic acid (PSA) is a natural anionic polymer typically occurring on the outer surface of cell membranes. PSA is involved in cell signaling and intermolecular interactions with proteins and peptides. The antimicrobial potential of peptides is usually evaluated in model membranes consisting of lipid bilayers but devoid of either PSA or its analogs. The goal of this work was to investigate the possible effect of PSA on the structure of melittin (Mlt) and latarcins Ltc1K, Ltc2a, and the activity of these peptides with respect to model membranes. These peptides are linear cationic ones derived from the venom of bee (Mlt) and spider (both latarcins). The length of each of the peptides is 26 amino acid residues, and they all have antimicrobial activity. However, they differ with respect to conformational mobility, hydrophobic characteristics, and overall charge. In this work, using circular dichroism spectroscopy, we show that the peptides adopt an α-helical conformation upon interaction with either PSA or phospholipid liposomes formed of either zwitterionic or anionic phospholipids or their mixtures. The extent of helicity depends on the amino acid sequence and properties of the medium. Based on small angle X-ray scattering data and the analysis of the fluorescence spectrum of the Trp residue in Mlt, we conclude that the peptide forms an oligomeric complex consisting of α-helical Mlt and several PSA molecules. Both latarcins, unlike Mlt, the most hydrophobic of the peptides, interact weakly with zwitterionic liposomes. However, they bind anionic liposomes or those composed of anionic/zwitterionic lipid mixtures. Latarcin Ltc1K forms associates on liposomes composed of zwitterionic/anionic lipid mixture. The structure of the peptide associates is either disordered or of β-sheet conformation. In all other cases the studied peptides adopt predominately α-helical conformation. In addition, we demonstrate that PSA inhibits membranolytic activity of Mlt and latarcin

  10. Production of a cytotoxic cationic antibacterial peptide in Escherichia coli using SUMO fusion partner.

    PubMed

    Li, Jian Feng; Zhang, Jie; Song, Ren; Zhang, Jia Xin; Shen, Yang; Zhang, Shuang Quan

    2009-08-01

    Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and tumor cells, which may possibly be used as an antimicrobial agent. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of cationic antibacterial peptide ABP-CM4. The fusion protein expressed in a soluble form was purified to a purity of 90% by Ni-IDA chromatography and 112 mg protein of interest was obtained per liter of fermentation culture. After the SUMO-CM4 fusion protein was cleaved by the SUMO protease at 30 degrees C for 1 h, the cleaved sample was re-applied to a Ni-IDA. Finally, about 24 mg recombinant CM4 was obtained from 1 l fermentation culture with no less than 96% purity and the recombinant CM4 had similar antimicrobial properties to the synthetic CM4. Thus, the SUMO-mediated peptide expression and purification system potentially could be employed for the production of recombinant cytotoxic peptides. PMID:19582446

  11. Nanoparticles of cationic chimeric peptide and sodium polyacrylate exhibit striking antinociception activity at lower dose.

    PubMed

    Gupta, Kshitij; Singh, Vijay P; Kurupati, Raj K; Mann, Anita; Ganguli, Munia; Gupta, Yogendra K; Singh, Yogendra; Saleem, Kishwar; Pasha, Santosh; Maiti, Souvik

    2009-02-20

    The current study investigates the performance of polyelectrolyte complexes based nanoparticles in improving the antinociceptive activity of cationic chimeric peptide-YFa at lower dose. Size, Zeta potential and morphology of the nanoparticles were determined. Size of the nanoparticles decreases and zeta potential increases with concomitant increase in charge ratio (Z(+/-)). The nanoparticles at Z(+/-)12 are spherical with 70+/-7 nm diameter in AFM and displayed positive surface charge and similar sizes (83+/-8 nm) by Zetasizer. The nanoparticles of Z(+/-) 12 are used in this study. Cytotoxicity by MTT assay on three different mammalian cell lines (liver, neuronal and kidney) revealed lower toxicity of nanoparticles. Hematological parameters were also not affected by nanoparticles compared to normal counts of water treated control group. Nanoparticles containing 10 mg/kg YFa produced increased antinociception, approximately 36%, in tail-flick latency test in mice, whereas the neat peptide at the same concentration did not show any antinociception activity. This enhancement in activity is attributed to the nanoparticle associated protection of peptide from proteolytic degradation. In vitro peptide release study in plasma also supported the antinociception profile of nanoparticles. Thus, our results suggest of a potential nanoparticle delivery system for cationic peptide drug candidates for improving their stability and bioavailability. PMID:19014986

  12. Phosphorylated peptides occur in a non-helical portion of the tail of a catch muscle myosin

    SciTech Connect

    Castellani, L.; Elliott, B.W. Jr.; Cohen, C.

    1987-05-01

    Myosin from a molluscan catch muscle (the Anterior Byssus Retractor (ABRM) of Mytilus edulis) is unusual in being phosphorylated in the rod by an endogenous heavy-chain kinase. This phosphorylation enhances myosin solubility at low ionic strength and induces molecular folding of the myosin tail. Papain and chymotryptic cleavage of this myosin, phosphorylated with (..gamma..-/sup 32/P)ATP, indicates that the phosphorylated residues are associated with the carboxy-terminal end of the light meromyosin. Ion-exchange and reverse-phase HPLC of radiolabeled chymotryptic peptides allow the isolation of two different peptides with high specific activity. One of these peptides is rich in lysine and arginine residues, a finding consistent with the observation that basic residues often determine the substrate specificity of protein kinases. The second peptide contains proline residues. Taken together, these results suggest that, as in the case of Acanthamoeba myosin, phosphorylation occurs in a nonhelical portion of the rod that may also control solubility. Identification of the residues that are phosphorylated and their location in the rod may reveal how the phosphorylation-dependent changes observed in the myosin in vitro are related to changes in intermolecular interactions in the thick filaments in vivo.

  13. Tandem Mass Spectrometric Characterization of Thiol Peptides Modified by the Chemoselective Cationic Sulfhydryl Reagent (4-Iodobutyl)Triphenylphosphonium—. Effects of a Cationic Thiol Derivatization on Peptide Fragmentation

    NASA Astrophysics Data System (ADS)

    Wang, Jing; Zhang, Jie; Arbogast, Brian; Maier, Claudia S.

    2011-10-01

    Fixed charge chemical modifications on peptides and proteins can impact fragmentation behaviors in tandem mass spectrometry (MS/MS). In this study, we employed a thiol-specific cationic alkylation reagent, (4-iodobutyl)triphenylphosphonium (IBTP), to selectively modify cysteine thiol groups in mitochondrial proteome samples. Tandem mass spectrometric characteristics of butyltriphenylphosphonium (BTP)-modified peptides were evaluated by comparison to their carbamidomethylated (CAM) analogues using a quadrupole time-of-flight (Q-TOF) instrument under low energy collision-induced dissociation (CID) conditions. Introduction of the fixed charge modification resulted in the observation of peptide and fragment (bn and yn) ions with higher charge states than those observed for CAM-modified analogues. The charged BTP moiety had a significant effect on the neighboring amide bond fragmentation products. A decrease in relative abundances of the product ions at the corresponding cleavage sites was observed compared with those from the CAM-modified derivatives. This effect was particularly noticeable when an Xxx-Pro bond was in the vicinity of a BTP group. We hypothesized that the presence of a phosphonium moiety will reduce the tendency for protonation of the proximal amide bonds in the peptide backbone. Indeed, calculations indicated that proton affinities of backbone amide bonds close to the modified cysteine residues were generally 20-50 kcal/mol lower for BTP-modified peptides than for the unmodified or CAM-modified analogues with the sequence motif -Ala-Cys-Alan-Ala2-, -Ala-Cys-Alan-Pro-Ala-, and -Ala-Pro-Alan-Cys-Ala-, n = 0-3.

  14. Amphipathicity Determines Different Cytotoxic Mechanisms of Lysine- or Arginine-Rich Cationic Hydrophobic Peptides in Cancer Cells.

    PubMed

    Liu, Xiaoli; Cao, Rui; Wang, Sha; Jia, Junli; Fei, Hao

    2016-06-01

    Cationic amphipathic peptides (CAPs) are known to be able to cause membrane destabilization and induce cell death, yet how the hydrophobicity, amphipathicity, and lysine (K)/arginine (R) composition synergistically affect the peptide activity remains incompletely understood. Here, we designed a panel of peptides based on the well-known anticancer peptide KLA. Increasing hydrophobicity enhanced the cytotoxicities of both the K- and R-rich peptides. Peptides with an intact amphipathic helical interface can cause instant cell death through a membrane lysis mechanism. Interestingly, rearranging the residue positions to minimize amphipathicity caused a great decrease of cytotoxicity to the K-rich peptides but not to the R-rich peptides. The amphipathicity-minimized R-rich peptide 6 (RL2) (RLLRLLRLRRLLRL-NH2) penetrated the cell membrane and induced caspase-3-dependent apoptotic cell death. We found that the modulation of hydrophobicity, amphipathicity, and K/R residues leads to distinct mechanisms of action of cationic hydrophobic peptides. Amphipathicity-reduced, arginine-rich cationic hydrophobic peptides (CHPs) may represent a new class of peptide therapeutics. PMID:27195657

  15. Cationic amphipathic peptides accumulate sialylated proteins and lipids in the plasma membrane of eukaryotic host cells

    PubMed Central

    Weghuber, Julian; Aichinger, Michael C.; Brameshuber, Mario; Wieser, Stefan; Ruprecht, Verena; Plochberger, Birgit; Madl, Josef; Horner, Andreas; Reipert, Siegfried; Lohner, Karl; Henics, Tamás; Schütz, Gerhard J.

    2011-01-01

    Cationic antimicrobial peptides (CAMPs) selectively target bacterial membranes by electrostatic interactions with negatively charged lipids. It turned out that for inhibition of microbial growth a high CAMP membrane concentration is required, which can be realized by the incorporation of hydrophobic groups within the peptide. Increasing hydrophobicity, however, reduces the CAMP selectivity for bacterial over eukaryotic host membranes, thereby causing the risk of detrimental side-effects. In this study we addressed how cationic amphipathic peptides—in particular a CAMP with Lysine–Leucine–Lysine repeats (termed KLK)—affect the localization and dynamics of molecules in eukaryotic membranes. We found KLK to selectively inhibit the endocytosis of a subgroup of membrane proteins and lipids by electrostatically interacting with negatively charged sialic acid moieties. Ultrastructural characterization revealed the formation of membrane invaginations representing fission or fusion intermediates, in which the sialylated proteins and lipids were immobilized. Experiments on structurally different cationic amphipathic peptides (KLK, 6-MO-LF11-322 and NK14-2) indicated a cooperation of electrostatic and hydrophobic forces that selectively arrest sialylated membrane constituents. PMID:21718688

  16. Ammonium Ion Exchanged Zeolite for Laser Desorption/Ionization Mass Spectrometry of Phosphorylated Peptides

    PubMed Central

    Yang, Mengrui; Fujino, Tatsuya

    2015-01-01

    α-Cyano-4-hydroxycinnamic acid (CHCA), an organic matrix molecule for matrix-assisted laser desorption/ionization mass spectrometry, was adsorbed to NH4+-type zeolite surface, and this new matrix was used for the detection of low-molecular-weight compounds. It was found that this matrix could simplify the mass spectrum in the low-molecular-weight region and prevent interference from fragments and alkali metal ion adducted species. CHCA adsorbed to NH4+-type ZSM5 zeolite (CHCA/NH4ZSM5) was used to measure atropine and aconitine, two toxic alkaloids in plants. In addition, CHCA/NH4ZSM5 enabled us to detect phosphorylated peptides; peaks of the protonated peptides had higher intensities than the peaks observed using CHCA only. PMID:26448749

  17. Preconcentration and detection of the phosphorylated forms of cardiac troponin I in a cascade microchip by cationic isotachophoresis.

    PubMed

    Bottenus, Danny; Hossan, Mohammad Robiul; Ouyang, Yexin; Dong, Wen-Ji; Dutta, Prashanta; Ivory, Cornelius F

    2011-11-21

    This paper describes the detection of a cardiac biomarker, cardiac troponin I (cTnI), spiked into depleted human serum using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic channel. The microfluidic chip incorporates a 100× cross-sectional area reduction, including a 10× depth reduction and a 10× width reduction, to increase sensitivity during ITP. The cross-sectional area reductions in combination with ITP allowed visualization of lower concentrations of fluorescently labeled cTnI. ITP was performed in both "peak mode" and "plateau mode" and the final concentrations obtained were linear with initial cTnI concentration. We were able to detect and quantify cTnI at initial concentrations as low as 46 ng mL(-1) in the presence of human serum proteins and obtain cTnI concentrations factors as high as ~ 9000. In addition, preliminary ITP experiments including both labeled cTnI and labeled protein kinase A (PKA) phosphorylated cTnI were performed to visualize ITP migration of different phosphorylated forms of cTnI. The different phosphorylated states of cTnI formed distinct ITP zones between the leading and terminating electrolytes. To our knowledge, this is the first attempt at using ITP in a cascade microchip to quantify cTnI in human serum and detect different phosphorylated forms. PMID:21935555

  18. Preconcentration and detection of the phosphorylated forms of cardiac troponin I in a cascade microchip by cationic isotachophoresis†

    PubMed Central

    Bottenus, Danny; Hossan, Mohammad Robiul; Ouyang, Yexin; Dong, Wen-Ji; Dutta, Prashanta; Ivory, Cornelius F.

    2011-01-01

    This paper describes the detection of a cardiac biomarker, cardiac troponin I (cTnI), spiked into depleted human serum using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic channel. The microfluidic chip incorporates a 100x cross-sectional area reduction, including a 10x depth reduction and a 10x width reduction, to increase sensitivity during ITP. The cross-sectional area reductions in combination with ITP allowedvisualization of lower concentrations of fluorescently labeled cTnI. ITP was performed in both “peak mode” and “plateau mode” and the final concentrations obtained were linear with initial cTnI concentration. We were able to detect and quantify cTnI at initial concentrations as low as 46 ng mL−1 in the presence of human serum proteins and obtain cTnI concentrations factors as high as ~ 9000. In addition, preliminary ITP experiments including both labeled cTnI and labeled protein kinase A (PKA) phosphorylated cTnI were performed to visualize ITP migration of different phosphorylated forms ofcTnI. The different phosphorylated states of cTnI formed distinct ITP zones between the leading and terminating electrolytes. To our knowledge, this is the first attempt at using ITP in a cascade microchip to quantify cTnI in human serum and detect different phosphorylated forms. PMID:21935555

  19. Cationic Peptide Conjugation Enhances the Activity of Peroxidase-Mimicking DNAzymes.

    PubMed

    Xiao, Lu; Zhou, Zhaojuan; Feng, Mengli; Tong, Aijun; Xiang, Yu

    2016-03-16

    Peroxidase-mimicking DNAzymes containing G-quadruplex structures are widely applied in chemistry as catalysts and signal amplification for biosensing. Enhancing the catalytic activity of these DNAzymes can therefore improve the performance of many catalysts and biosensors using them. In this work, we synthesized cationic peptide conjugates of peroxidase-mimicking DNAzymes, which were found to exhibit both enhanced peroxidase and oxidase activities up to 4-fold and 3-fold compared with the original DNAzymes, respectively. Further investigation suggested that the enhanced activity was ascribed to the stabilization of parallel DNA G-quadruplex structures and hemin binding by the cationic peptide covalently attached to the DNAzyme. Such a mechanism of activity enhancement was successfully utilized for biosensing applications with improved sensitivity and broadened target range. Hydrogen peroxide (H2O2) detection in K(+)-free solutions by the DNAzyme-peptide conjugate showed 2-fold sensitivity enhancement over the unmodified DNAzyme under the same condition, and the activity switch by target-induced cleavage of the DNAzyme-peptide conjugate was also used for the detection of caspase 3 protease with enzymatic amplification in homogeneous solutions. PMID:26751843

  20. Heteropoly acids triggered self-assembly of cationic peptides into photo- and electro-chromic gels.

    PubMed

    Li, Jingfang; Xu, Jing; Li, Xiaodong; Gao, Wenmei; Wang, Liyan; Wu, Lixin; Lee, Myongsoo; Li, Wen

    2016-07-01

    A series of cationic peptides with alternating lysines and hydrophobic residues were designed and synthesized. These kinds of short peptides with protonated lysines can complex with anionic heteropoly acids (HPAs) to form a stable gel in water/ethanol mixed solution. Circular dichroism spectroscopy showed that the short peptides adopted a mixed conformation (β-sheet and random-coil) within the gel matrix. Scanning and transmission electron microscopy revealed that the heteropoly acids, acting as nanosized cross-linkers, first initiated the self-assembly of the cationic peptides into spherical nanostructures. Then these nanospheres accumulated with each other through hydrogen bonds and hydrophobic interactions to form large sheet-like assemblies, which further interconnected with each other forming continuous 3D network structures. Fourier-transform infrared spectroscopy showed that the structural integrity of the HPAs was maintained during the gelation process. The resultant hybrid gels showed reversible photo- and elecrtro-chromic properties. X-ray photoelectron spectroscopy revealed that the hybrid gels, capable of persistent and reversible changes of their colour, are attributed to the intervalence charge-transfer transition of the HPAs. Reversible information writing and erasing were demonstrated through a repeated photo-lithograph or electric stimuli without significant loss of the gel performance. PMID:27240759

  1. Self-assembly of cationic multidomain peptide hydrogels: supramolecular nanostructure and rheological properties dictate antimicrobial activity†

    PubMed Central

    Jiang, Linhai; Xu, Dawei; Sellati, Timothy J.

    2016-01-01

    Hydrogels are an important class of biomaterials that have been widely utilized for a variety of biomedical/medical applications. The biological performance of hydrogels, particularly those used as wound dressing could be greatly advanced if imbued with inherent antimicrobial activity capable of staving off colonization of the wound site by opportunistic bacterial pathogens. Possessing such antimicrobial properties would also protect the hydrogel itself from being adversely affected by microbial attachment to its surface. We have previously demonstrated the broad-spectrum antimicrobial activity of supramolecular assemblies of cationic multi-domain peptides (MDPs) in solution. Here, we extend the 1-D soluble supramolecular assembly to 3-D hydrogels to investigate the effect of the supramolecular nanostructure and its rheological properties on the antimicrobial activity of self-assembled hydrogels. Among designed MDPs, the bactericidal activity of peptide hydrogels was found to follow an opposite trend to that in solution. Improved antimicrobial activity of self-assembled peptide hydrogels is dictated by the combined effect of supramolecular surface chemistry and storage modulus of the bulk materials, rather than the ability of individual peptides/peptide assemblies to penetrate bacterial cell membrane as observed in solution. The structure–property–activity relationship developed through this study will provide important guidelines for designing biocompatible peptide hydrogels with built-in antimicrobial activity for various biomedical applications. PMID:26524425

  2. Scolopendin 2, a cationic antimicrobial peptide from centipede, and its membrane-active mechanism.

    PubMed

    Lee, Heejeong; Hwang, Jae-Sam; Lee, Jaeho; Kim, Jae Il; Lee, Dong Gun

    2015-02-01

    Scolopendin 2 is a 16-mer peptide (AGLQFPVGRIGRLLRK) derived from the centipede Scolopendra subspinipes mutilans. We observed that this peptide exhibited antimicrobial activity in a salt-dependent manner against various fungal and bacterial pathogens and showed no hemolytic effect in the range of 1.6 μM to 100 μM. Circular dichroism analysis showed that the peptide has an α-helical properties. Furthermore, we determined the mechanism(s) of action using flow cytometry and by investigating the release of intracellular potassium. The results showed that the peptide permeabilized the membranes of Escherichia coli O157 and Candida albicans, resulting in loss of intracellular potassium ions. Additionally, bis-(1,3-dibutylbarbituric acid) trimethine oxonol and 3,3'-dipropylthiacarbocyanine iodide assays showed that the peptide caused membrane depolarization. Using giant unilamellar vesicles encapsulating calcein and large unilamellar vesicles containing fluorescein isothiocyanate-dextran, which were similar in composition to typical E. coli O157 and C. albicans membranes, we demonstrated that scolopendin 2 disrupts membranes, resulting in a pore size between 4.8 nm and 5.0 nm. Thus, we have demonstrated that a cationic antimicrobial peptide, scolopendin 2, exerts its broad-spectrum antimicrobial effects by forming pores in the cell membrane. PMID:25462167

  3. Electrostatic Localization of RNA to Protocell Membranes by Cationic Hydrophobic Peptides

    PubMed Central

    Kamat, Neha P; Tobé, Sylvia; Hill, Ian T; Szostak, Jack W

    2015-01-01

    Cooperative interactions between RNA and vesicle membranes on the prebiotic earth may have led to the emergence of primitive cells. The membrane surface offers a potential platform for the catalysis of reactions involving RNA, but this scenario relies upon the existence of a simple mechanism by which RNA could become associated with protocell membranes. Here, we show that electrostatic interactions provided by short, basic, amphipathic peptides can be harnessed to drive RNA binding to both zwitterionic phospholipid and anionic fatty acid membranes. We show that the association of cationic molecules with phospholipid vesicles can enhance the local positive charge on a membrane and attract RNA polynucleotides. This phenomenon can be reproduced with amphipathic peptides as short as three amino acids. Finally, we show that peptides can cross bilayer membranes to localize encapsulated RNA. This mechanism of polynucleotide confinement could have been important for primitive cellular evolution. PMID:26223820

  4. Electrostatic Localization of RNA to Protocell Membranes by Cationic Hydrophobic Peptides.

    PubMed

    Kamat, Neha P; Tobé, Sylvia; Hill, Ian T; Szostak, Jack W

    2015-09-28

    Cooperative interactions between RNA and vesicle membranes on the prebiotic earth may have led to the emergence of primitive cells. The membrane surface offers a potential platform for the catalysis of reactions involving RNA, but this scenario relies upon the existence of a simple mechanism by which RNA could become associated with protocell membranes. Here, we show that electrostatic interactions provided by short, basic, amphipathic peptides can be harnessed to drive RNA binding to both zwitterionic phospholipid and anionic fatty acid membranes. We show that the association of cationic molecules with phospholipid vesicles can enhance the local positive charge on a membrane and attract RNA polynucleotides. This phenomenon can be reproduced with amphipathic peptides as short as three amino acids. Finally, we show that peptides can cross bilayer membranes to localize encapsulated RNA. This mechanism of polynucleotide confinement could have been important for primitive cellular evolution. PMID:26223820

  5. Absorptive-mediated endocytosis of cationized albumin and a beta-endorphin-cationized albumin chimeric peptide by isolated brain capillaries. Model system of blood-brain barrier transport

    SciTech Connect

    Kumagai, A.K.; Eisenberg, J.B.; Pardridge, W.M.

    1987-11-05

    Cationized albumin (pI greater than 8), unlike native albumin (pI approximately 4), enters cerebrospinal fluid (CSF) rapidly from blood. This suggests that a specific uptake mechanism for cationized albumin may exist at the brain capillary wall, i.e. the blood-brain barrier. Isolated bovine brain capillaries rapidly bound cationized (/sup 3/H)albumin and approximately 70% of the bound radioactivity was resistant to mild acid wash, which is assumed to represent internalized peptide. Binding was saturable and a Scatchard plot gave a maximal binding capacity (Ro) = 5.5 +/- 0.7 micrograms/mgp (79 +/- 10 pmol/mgp), and a half-saturation constant (KD) = 55 +/- 8 micrograms/ml (0.8 +/- 0.1 microM). The binding of cationized (/sup 3/H)albumin (pI = 8.5-9) was inhibited by protamine, protamine sulfate, and polylysine (molecular weight = 70,000) with a Ki of approximately 3 micrograms/ml for all three proteins. The use of cationized albumin in directed delivery of peptides through the blood-brain barrier was examined by coupling (/sup 3/H)beta-endorphin to unlabeled cationized albumin (pI = 8.5-9) using the bifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)proprionate. The (/sup 3/H)beta-endorphin-cationized albumin chimeric peptide was rapidly bound and endocytosed by isolated bovine brain capillaries, and this was inhibited by unlabeled cationized albumin but not by unconjugated beta-endorphin or native bovine albumin. Cationized albumin provides a new tool for studying absorptive-mediated endocytosis at the brain capillary and may also provide a vehicle for directed drug delivery through the blood-brain barrier.

  6. Reaction of phosphorylated and O-glycosylated peptides by chemically targeted identification at ambient temperature.

    PubMed

    Rusnak, Felicia; Zhou, Jie; Hathaway, Gary M

    2004-12-01

    Conditions for carrying out chemically targeted identification of peptides containing phosphorylated or glycosylated serine residues have been investigated. Ba(OH)2 was used at ambient temperature to catalyze the beta-elimination reaction at 25 degrees C. Nucleophilic addition of 2-aminoethanethiol was performed in both parallel and tandem experiments. The method was demonstrated by the reaction of beta-casein tryptic digest phosphopeptides and an O-glycosylated peptide. Contrary to an earlier report by others, the glycopeptide was found to react with essentially the same kinetics as phosphopeptides. Conversion of four phosphoserines in residues 15, 17, 18, and 19 from bovine beta-casein N-terminal tryptic phosphopeptides were followed by monitoring the time course of the addition reaction. The chemistry proceeded rapidly at room temperature with a half-reaction time of 15 min. No side-reaction products were observed; however, care was taken to minimize all counter ions that either precipitate barium or neutralize the base. Digestion of the converted peptides with lysine endopeptidase identified all five phosphoserines in the beta-casein tryptic digest. Alternatively, preincubation with base followed by nucleophilic addition of the thiol was found to work satisfactorily. The use of the water-soluble hydrochloride of 2-aminoethanethiol allowed beta-elimination, nucleophilic addition, and desalting to be carried out on a micro C18 reverse phase pipette tip. PMID:15585826

  7. Facile Peptides Functionalization of Lanthanide-Based Nanocrystals through Phosphorylation Tethering for Efficient in Vivo NIR-to-NIR Bioimaging.

    PubMed

    Yao, Chi; Wang, Peiyuan; Wang, Rui; Zhou, Lei; El-Toni, Ahmed Mohamed; Lu, Yiqing; Li, Xiaomin; Zhang, Fan

    2016-02-01

    Peptide modification of nanoparticles is a challenging task for bioapplications. Here, we show that noncovalent surface engineering based on ligand exchange of peptides for lanthanide based upconversion and downconversion near-infrared (NIR) luminescent nanoparticles can be efficiently realized by modifying the hydroxyl functional group of a side grafted serine of peptides into a phosphate group (phosphorylation). By using the phosphorylated peptide with the arginine-glycine-aspartic acid (RGD) targeting motifs as typical examples, the modification allows improving the selectivity, sensitivity, and signal-to-noise ratio for the cancer targeting and bioimaging and reducing the toxicity derived from nonspecific interactions of nanoparticles with cells. The in vivo NIR bioimaging signal could even be detected at low injection amounts down to 20 μg per animal. PMID:26750555

  8. Asp-Gly based peptides confined at the surface of cationic gemini surfactant aggregates.

    PubMed

    Brizard, Aurélie; Dolain, Christel; Huc, Ivan; Oda, Reiko

    2006-04-11

    Cationic gemini surfactants complexed with anionic oligoglycine-aspartate (called gemini peptides hereafter) were synthesized, and their aggregation behaviors were studied. The effects of the hydrophobic chain length (C10-C22) and the length of the oligoglycine (0-4) were investigated, and it was clearly shown by critical micellar concentration, Krafft temperature, and isothermal surface pressure measurements that the hydrophobic effect and interpeptidic interaction influence the aggregation behavior in a cooperative manner. Below their Krafft temperatures, some of them formed both hydro- and organogels with three-dimensional networks and the Fourier transform infrared measurements show the presence of interpeptidic hydrogen bonds. PMID:16584231

  9. Self-assembly of cationic multidomain peptide hydrogels: supramolecular nanostructure and rheological properties dictate antimicrobial activity

    NASA Astrophysics Data System (ADS)

    Jiang, Linhai; Xu, Dawei; Sellati, Timothy J.; Dong, He

    2015-11-01

    Hydrogels are an important class of biomaterials that have been widely utilized for a variety of biomedical/medical applications. The biological performance of hydrogels, particularly those used as wound dressing could be greatly advanced if imbued with inherent antimicrobial activity capable of staving off colonization of the wound site by opportunistic bacterial pathogens. Possessing such antimicrobial properties would also protect the hydrogel itself from being adversely affected by microbial attachment to its surface. We have previously demonstrated the broad-spectrum antimicrobial activity of supramolecular assemblies of cationic multi-domain peptides (MDPs) in solution. Here, we extend the 1-D soluble supramolecular assembly to 3-D hydrogels to investigate the effect of the supramolecular nanostructure and its rheological properties on the antimicrobial activity of self-assembled hydrogels. Among designed MDPs, the bactericidal activity of peptide hydrogels was found to follow an opposite trend to that in solution. Improved antimicrobial activity of self-assembled peptide hydrogels is dictated by the combined effect of supramolecular surface chemistry and storage modulus of the bulk materials, rather than the ability of individual peptides/peptide assemblies to penetrate bacterial cell membrane as observed in solution. The structure-property-activity relationship developed through this study will provide important guidelines for designing biocompatible peptide hydrogels with built-in antimicrobial activity for various biomedical applications.Hydrogels are an important class of biomaterials that have been widely utilized for a variety of biomedical/medical applications. The biological performance of hydrogels, particularly those used as wound dressing could be greatly advanced if imbued with inherent antimicrobial activity capable of staving off colonization of the wound site by opportunistic bacterial pathogens. Possessing such antimicrobial properties would

  10. Increased Diversity of the HLA-B40 Ligandome by the Presentation of Peptides Phosphorylated at Their Main Anchor Residue*

    PubMed Central

    Marcilla, Miguel; Alpízar, Adán; Lombardía, Manuel; Ramos-Fernandez, Antonio; Ramos, Manuel; Albar, Juan Pablo

    2014-01-01

    Human leukocyte antigen (HLA) class I molecules bind peptides derived from the intracellular degradation of endogenous proteins and present them to cytotoxic T lymphocytes, allowing the immune system to detect transformed or virally infected cells. It is known that HLA class I–associated peptides may harbor posttranslational modifications. In particular, phosphorylated ligands have raised much interest as potential targets for cancer immunotherapy. By combining affinity purification with high-resolution mass spectrometry, we identified more than 2000 unique ligands bound to HLA-B40. Sequence analysis revealed two major anchor motifs: aspartic or glutamic acid at peptide position 2 (P2) and methionine, phenylalanine, or aliphatic residues at the C terminus. The use of immobilized metal ion and TiO2 affinity chromatography allowed the characterization of 85 phosphorylated ligands. We further confirmed every sequence belonging to this subset by comparing its experimental MS2 spectrum with that obtained upon fragmentation of the corresponding synthetic peptide. Remarkably, three phospholigands lacked a canonical anchor residue at P2, containing phosphoserine instead. Binding assays showed that these peptides bound to HLA-B40 with high affinity. Together, our data demonstrate that the peptidome of a given HLA allotype can be broadened by the presentation of peptides with posttranslational modifications at major anchor positions. We suggest that ligands with phosphorylated residues at P2 might be optimal targets for T-cell-based cancer immunotherapy. PMID:24366607

  11. The outer membranes of Brucella spp. are resistant to bactericidal cationic peptides.

    PubMed Central

    Martínez de Tejada, G; Pizarro-Cerdá, J; Moreno, E; Moriyón, I

    1995-01-01

    The actions of polymyxin B, rabbit polymorphonuclear lysosome extracts, 14 polycationic peptides (including defensin NP-2, cecropin P1, lactoferricin B, and active peptides from cationic protein 18 and bactenecin), EDTA, and Tris on Brucella spp. were studied, with other gram-negative bacteria as controls. Brucella spp. were comparatively resistant to all of the agents listed above and bound less polymyxin B, and their outer membranes (OMs) were neither morphologically altered nor permeabilized to lysozyme by polymyxin B concentrations, although both effects were observed for controls. EDTA and peptides increased or accelerated the partition of the hydrophobic probe N-phenyl-naphthylamine into Escherichia coli and Haemophilus influenzae OMs but had no effect on Brucella OMs. Since Brucella and H. influenzae OMs are permeable to hydrophobic compounds (G. Martínez de Tejada and I. Moriyón, J. Bacteriol. 175:5273-5275, 1993), the results show that such unusual permeability is not necessarily related to resistance to polycations. Although rough (R) B. abortus and B. ovis were more resistant than the controls were, there were qualitative and quantitative differences with smooth (S) brucellae; this may explain known host range and virulence differences. Brucella S-lipopolysaccharides (LPSs) had reduced affinities for polycations, and insertion of Brucella and Salmonella montevideo S-LPSs into the OM of a Brucella R-LPS mutant increased and decreased, respectively, its resistance to cationic peptides. The results show that the core lipid A of Brucella LPS plays a major role in polycation resistance and that O-chain density also contributes significantly. It is proposed that the features described above contribute to Brucella resistance to the oxygen-independent systems of phagocytes. PMID:7622230

  12. Cationic antimicrobial peptides in psoriatic skin cooperate to break innate tolerance to self-DNA.

    PubMed

    Lande, Roberto; Chamilos, Georgios; Ganguly, Dipyaman; Demaria, Olivier; Frasca, Loredana; Durr, Sophie; Conrad, Curdin; Schröder, Jens; Gilliet, Michel

    2015-01-01

    Psoriasis is a T-cell-mediated skin autoimmune disease characterized by the aberrant activation of dermal dendritic cells (DCs) and the sustained epidermal expression of antimicrobial peptides. We have previously identified a link between these two events by showing that the cathelicidin antimicrobial peptide LL37 has the ability to trigger self-nucleic acid mediated activation of plasmacytoid DCs (pDCs) in psoriatic skin. Whether other cationic antimicrobial peptides exert similar activities is unknown. By analyzing heparin-binding HPLC fractions of psoriatic scales, we found that human beta-defensin (hBD)2, hBD3, and lysozyme are additional triggers of pDC activation in psoriatic skin lesions. Like LL37, hBD2, hBD3, and lysozyme are able to condense self-DNA into particles that are endocytosed by pDCs, leading to activation of TLR9. In contrast, other antimicrobial peptides expressed in psoriatic skin including elafin, hBD1, and psoriasin (S100A7) did not show similar activities. hBD2, hBD3, and lysozyme were detected in psoriatic skin lesions in the vicinity of pDCs and found to cooperate with LL37 to induce high levels of IFN production by pDCs, suggesting their concerted role in the pathogenesis of psoriasis. PMID:25332209

  13. The specificity of protection against cationic antimicrobial peptides by lactoferrin binding protein B.

    PubMed

    Morgenthau, Ari; Partha, Sarathy K; Adamiak, Paul; Schryvers, Anthony B

    2014-10-01

    A variety of Gram-negative pathogens possess host-specific lactoferrin (Lf) receptors that mediate the acquisition of iron from host Lf. The integral membrane protein component of the receptor, lactoferrin binding protein A specifically binds host Lf and is required for acquisition of iron from Lf. In contrast, the role of the bi-lobed surface lipoprotein, lactoferrin binding protein B (LbpB), in Lf binding and iron acquisition is uncertain. A common feature of LbpBs from most species is the presence of clusters of negatively charged amino acids in the protein's C-terminal lobe. Recently it has been shown that the negatively charged regions from the Neisseria meningitidis LbpB are responsible for protecting against an 11 amino acid cationic antimicrobial peptide (CAP), lactoferricin (Lfcin), derived from human Lf. In this study we investigated whether the LbpB confers resistance to other CAPs since N. meningitidis is likely to encounter other CAPs from the host. LbpB provided protection against the cathelicidin derived peptide, cathelicidin related antimicrobial peptide (mCRAMP), but did not confer protection against Tritrp 1 or LL37 under our experimental conditions. When tested against a range of rationally designed synthetic peptides, LbpB was shown to protect against IDR-1002 and IDR-0018 but not against HH-2 or HHC10. PMID:25038734

  14. Interaction between a Cationic Surfactant-like Peptide and Lipid Vesicles and Its Relationship to Antimicrobial Activity

    PubMed Central

    2013-01-01

    We investigate the properties of an antimicrobial surfactant-like peptide (Ala)6(Arg), A6R, containing a cationic headgroup. The interaction of this peptide with zwitterionic (DPPC) lipid vesicles is investigated using a range of microscopic, X-ray scattering, spectroscopic, and calorimetric methods. The β-sheet structure adopted by A6R is disrupted in the presence of DPPC. A strong effect on the small-angle X-ray scattering profile is observed: the Bragg peaks from the DPPC bilayers in the vesicle walls are eliminated in the presence of A6R and only bilayer form factor peaks are observed. All of these observations point to the interaction of A6R with DPPC bilayers. These studies provide insight into interactions between a model cationic peptide and vesicles, relevant to understanding the action of antimicrobial peptides on lipid membranes. Notably, peptide A6R exhibits antimicrobial activity without membrane lysis. PMID:24156610

  15. Unique translational modification of an invertebrate neuropeptide: a phosphorylated member of the adipokinetic hormone peptide family

    PubMed Central

    2005-01-01

    Separation of an extract of corpora cardiaca from the protea beetle, Trichostetha fascicularis, by single-step RP (reverse-phase)-HPLC and monitoring of tryptophan fluorescence resulted in two distinctive peaks, the material of which mobilized proline and carbohydrates in a bioassay performed using the beetle. Material from one of these peaks was; however, inactive in the classical bioassays of locusts and cockroaches that are used for detecting peptides belonging to the AKH (adipokinetic hormone) family. After enzymatically deblocking the N-terminal pyroglutamic acid (pGlu) residue in the peptide material and sequencing by Edman degradation, a partial sequence was obtained: (pGlu)-Ile-Asn-Met-Thr-Xaa-Gly-Trp. The complete sequence was deduced from ESI-MSn (electrospray ionization multi-stage-MS); position six was identified as a phosphothreonine residue and the C-terminus is amidated. The peptide, code-named Trifa-CC, was chemically synthesized and used in confirmatory experiments to show that the primary structure had been correctly assigned. To our knowledge, this is the first report of a phosphorylated invertebrate neuropeptide. Synthetic Trifa-CC co-elutes with the natural peptide, found in the gland of the protea beetle, after RP-HPLC. Moreover, the natural peptide can be dephosphorylated by alkaline phosphatase and the product of that reaction has the same retention time as a synthetic nonphosphorylated octapeptide which has the same sequence as Trifa-CC. Finally, synthetic Trifa-CC has hypertrehalosaemic and hyperprolinaemic biological activity in the protea beetle, but even high concentrations of synthetic Trifa-CC are inactive in locusts and cockroaches. Hence, the correct peptide structure has been assigned. Trifa-CC of the protea beetle is an unusual member of the AKH family that is unique in its post-translational modification. Since it increases the concentration of carbohydrates and proline in the haemolymph when injected into the protea beetle, and

  16. Synthetic cationic antimicrobial peptides bind with their hydrophobic parts to drug site II of human serum albumin

    PubMed Central

    2014-01-01

    Background Many biologically active compounds bind to plasma transport proteins, and this binding can be either advantageous or disadvantageous from a drug design perspective. Human serum albumin (HSA) is one of the most important transport proteins in the cardiovascular system due to its great binding capacity and high physiological concentration. HSA has a preference for accommodating neutral lipophilic and acidic drug-like ligands, but is also surprisingly able to bind positively charged peptides. Understanding of how short cationic antimicrobial peptides interact with human serum albumin is of importance for developing such compounds into the clinics. Results The binding of a selection of short synthetic cationic antimicrobial peptides (CAPs) to human albumin with binding affinities in the μM range is described. Competitive isothermal titration calorimetry (ITC) and NMR WaterLOGSY experiments mapped the binding site of the CAPs to the well-known drug site II within subdomain IIIA of HSA. Thermodynamic and structural analysis revealed that the binding is exclusively driven by interactions with the hydrophobic moieties of the peptides, and is independent of the cationic residues that are vital for antimicrobial activity. Both of the hydrophobic moieties comprising the peptides were detected to interact with drug site II by NMR saturation transfer difference (STD) group epitope mapping (GEM) and INPHARMA experiments. Molecular models of the complexes between the peptides and albumin were constructed using docking experiments, and support the binding hypothesis and confirm the overall binding affinities of the CAPs. Conclusions The biophysical and structural characterizations of albumin-peptide complexes reported here provide detailed insight into how albumin can bind short cationic peptides. The hydrophobic elements of the peptides studied here are responsible for the main interaction with HSA. We suggest that albumin binding should be taken into careful

  17. Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B

    PubMed Central

    Ndeboko, Bénédicte; Lemamy, Guy Joseph; Nielsen, Peter. E; Cova, Lucyna

    2015-01-01

    Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Because current anti-HBV treatments are only virostatic, there is an urgent need for development of alternative antiviral approaches. In this context, cell-penetrating peptides (CPPs) and cationic polymers, such as chitosan (CS), appear of particular interest as nonviral vectors due to their capacity to facilitate cellular delivery of bioactive cargoes including peptide nucleic acids (PNAs) or DNA vaccines. We have investigated the ability of a PNA conjugated to different CPPs to inhibit the replication of duck hepatitis B virus (DHBV), a reference model for human HBV infection. The in vivo administration of PNA-CPP conjugates to neonatal ducklings showed that they reached the liver and inhibited DHBV replication. Interestingly, our results indicated also that a modified CPP (CatLip) alone, in the absence of its PNA cargo, was able to drastically inhibit late stages of DHBV replication. In the mouse model, conjugation of HBV DNA vaccine to modified CS (Man-CS-Phe) improved cellular and humoral responses to plasmid-encoded antigen. Moreover, other systems for gene delivery were investigated including CPP-modified CS and cationic nanoparticles. The results showed that these nonviral vectors considerably increased plasmid DNA uptake and expression. Collectively promising results obtained in preclinical studies suggest the usefulness of these safe delivery systems for the development of novel therapeutics against chronic hepatitis B. PMID:26633356

  18. Mechanism of Cationic Nanoparticles and Cell-Penetrating Peptides Direct Translocate Across Cell Membranes

    NASA Astrophysics Data System (ADS)

    Lin, Jiaqi; Alexander-Katz, Alfredo

    2014-03-01

    Cationic Nanoparticles (NPs) and cell-penetrating peptides (CPPs) are known effective intracellular delivery agents. These positively charged particles can bypass traditional endocytosis route to enter the cytosol, which is known as direct translocation. However, mechanism of direct translocation of both NPs and CPPs is not well understood. Using Coarse-grained (CG) molecular dynamics simulation, we found that gold nanoparticles (AuNPs) as well as HIV-1 Tat peptides can translocate across model biological membranes through nanoscale holes under a transmembrane (TM) potential. After the translocation, the TM is strongly weakened and the holes gradually reseal themselves, while the NPs/CPPs roam freely in the ``intracellular region.'' Both size and shape of the NPs/ CPPs are found to be a determine factor of their translocation behaviour, and the relationship between direct translocation and endocytosis is also discussed. The results provided here establish fundamental rules of direct translocation entry of NPs/CPPs, which may guide the rational design of cationic intracellular nanocarriers.

  19. Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B.

    PubMed

    Ndeboko, Bénédicte; Lemamy, Guy Joseph; Nielsen, Peter E; Cova, Lucyna

    2015-01-01

    Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Because current anti-HBV treatments are only virostatic, there is an urgent need for development of alternative antiviral approaches. In this context, cell-penetrating peptides (CPPs) and cationic polymers, such as chitosan (CS), appear of particular interest as nonviral vectors due to their capacity to facilitate cellular delivery of bioactive cargoes including peptide nucleic acids (PNAs) or DNA vaccines. We have investigated the ability of a PNA conjugated to different CPPs to inhibit the replication of duck hepatitis B virus (DHBV), a reference model for human HBV infection. The in vivo administration of PNA-CPP conjugates to neonatal ducklings showed that they reached the liver and inhibited DHBV replication. Interestingly, our results indicated also that a modified CPP (CatLip) alone, in the absence of its PNA cargo, was able to drastically inhibit late stages of DHBV replication. In the mouse model, conjugation of HBV DNA vaccine to modified CS (Man-CS-Phe) improved cellular and humoral responses to plasmid-encoded antigen. Moreover, other systems for gene delivery were investigated including CPP-modified CS and cationic nanoparticles. The results showed that these nonviral vectors considerably increased plasmid DNA uptake and expression. Collectively promising results obtained in preclinical studies suggest the usefulness of these safe delivery systems for the development of novel therapeutics against chronic hepatitis B. PMID:26633356

  20. The Use of Titanium Dioxide for Selective Enrichment of Phosphorylated Peptides.

    PubMed

    Thingholm, Tine E; Larsen, Martin R

    2016-01-01

    Titanium dioxide (TiO2) has very high affinity for phosphopeptides and in recent years it has become one of the most popular methods for phosphopeptide enrichment from complex biological samples. Peptide loading onto TiO2 resin in a highly acidic environment in the presence of 2,5-dihydroxybenzoic acid (DHB), phthalic acid, lactic acid, or glycolic acid has been shown to improve selectivity significantly by reducing unspecific binding of non-phosphorylated peptides. The phosphopeptides bound to the TiO2 are subsequently eluted from the chromatographic material using an alkaline buffer. TiO2 chromatography is extremely tolerant towards most buffers used in biological experiments, highly robust and as such it has become the method of choice in large-scale phosphoproteomics. Here we describe a batch mode protocol for phosphopeptide enrichment using TiO2 chromatographic material followed by desalting and concentration of the sample by reversed phase micro-columns prior to downstream MS and LC-MS/MS analysis. PMID:26584923

  1. Production of phytotoxic cationic α-helical antimicrobial peptides in plant cells using inducible promoters.

    PubMed

    Company, Nuri; Nadal, Anna; Ruiz, Cristina; Pla, Maria

    2014-01-01

    Synthetic linear antimicrobial peptides with cationic α-helical structures, such as BP100, have potent and specific activities against economically important plant pathogenic bacteria. They are also recognized as valuable therapeutics and preservatives. However, highly active BP100 derivatives are often phytotoxic when expressed at high levels as recombinant peptides in plants. Here we demonstrate that production of recombinant phytotoxic peptides in transgenic plants is possible by strictly limiting transgene expression to certain tissues and conditions, and specifically that minimization of this expression during transformation and regeneration of transgenic plants is essential to obtain viable plant biofactories. On the basis of whole-genome transcriptomic data available online, we identified the Os.hsp82 promoter that fulfilled this requirement and was highly induced in response to heat shock. Using this strategy, we generated transgenic rice lines producing moderate yields of severely phytotoxic BP100 derivatives on exposure to high temperature. In addition, a threshold for gene expression in selected tissues and stages was experimentally established, below which the corresponding promoters should be suitable for driving the expression of recombinant phytotoxic proteins in genetically modified plants. In view of the growing transcriptomics data available, this approach is of interest to assist promoter selection for specific purposes. PMID:25387106

  2. Production of Phytotoxic Cationic α-Helical Antimicrobial Peptides in Plant Cells Using Inducible Promoters

    PubMed Central

    Company, Nuri; Nadal, Anna; Ruiz, Cristina; Pla, Maria

    2014-01-01

    Synthetic linear antimicrobial peptides with cationic α-helical structures, such as BP100, have potent and specific activities against economically important plant pathogenic bacteria. They are also recognized as valuable therapeutics and preservatives. However, highly active BP100 derivatives are often phytotoxic when expressed at high levels as recombinant peptides in plants. Here we demonstrate that production of recombinant phytotoxic peptides in transgenic plants is possible by strictly limiting transgene expression to certain tissues and conditions, and specifically that minimization of this expression during transformation and regeneration of transgenic plants is essential to obtain viable plant biofactories. On the basis of whole-genome transcriptomic data available online, we identified the Os.hsp82 promoter that fulfilled this requirement and was highly induced in response to heat shock. Using this strategy, we generated transgenic rice lines producing moderate yields of severely phytotoxic BP100 derivatives on exposure to high temperature. In addition, a threshold for gene expression in selected tissues and stages was experimentally established, below which the corresponding promoters should be suitable for driving the expression of recombinant phytotoxic proteins in genetically modified plants. In view of the growing transcriptomics data available, this approach is of interest to assist promoter selection for specific purposes. PMID:25387106

  3. Cationic Membrane Peptides: Atomic-Level Insight of Structure-Activity Relationships from Solid-State NMR

    PubMed Central

    Su, Yongchao; Li, Shenhui; Hong, Mei

    2012-01-01

    Many membrane-active peptides, such as cationic cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs), conduct their biological functions by interacting with the cell membrane. The interactions of charged residues with lipids and water facilitate membrane insertion, translocation or disruption of these highly hydrophobic species. In this mini-review we will summarize high-resolution structural and dynamic findings towards the understanding of the structure-activity relationship of lipid membrane-bound CPPs and AMPs, as examples of the current development of solid-state NMR (SSNMR) techniques for studying membrane peptides. We will present the most recent atomic-resolution structure of the guanidinium-phosphate complex, as constrained from experimentally measured site-specific distances. These SSNMR results will be valuable specifically for understanding the intracellular translocation pathway of CPPs and antimicrobial mechanism of AMPs, and more generally broaden our insight into how cationic macromolecules interact with and cross the lipid membrane. PMID:23108593

  4. Effects of Cationic Antimicrobial Peptides on Liquid-Preserved Boar Spermatozoa

    PubMed Central

    Schulze, Martin; Junkes, Christof; Mueller, Peter; Speck, Stephanie; Ruediger, Karin; Dathe, Margitta; Mueller, Karin

    2014-01-01

    Antibiotics are mandatory additives in semen extenders to control bacterial contamination. The worldwide increase in resistance to conventional antibiotics requires the search for alternatives not only for animal artificial insemination industries, but also for veterinary and human medicine. Cationic antimicrobial peptides are of interest as a novel class of antimicrobial additives for boar semen preservation. The present study investigated effects of two synthetic cyclic hexapeptides (c-WFW, c-WWW) and a synthetic helical magainin II amide derivative (MK5E) on boar sperm during semen storage at 16°C for 4 days. The standard extender, Beltsville Thawing Solution (BTS) containing 250 µg/mL gentamicin (standard), was compared to combinations of BTS with each of the peptides in a split-sample procedure. Examination revealed peptide- and concentration-dependent effects on sperm integrity and motility. Negative effects were more pronounced for MK5E than in hexapeptide-supplemented samples. The cyclic hexapeptides were partly able to stimulate a linear progressive sperm movement. When using low concentrations of cyclic hexapeptides (4 µM c-WFW, 2 µM c-WWW) sperm quality was comparable to the standard extender over the course of preservation. C-WFW-supplemented boar semen resulted in normal fertility rates after AI. In order to investigate the interaction of peptides with the membrane, electron spin resonance spectroscopic measurements were performed using spin-labeled lipids. C-WWW and c-WFW reversibly immobilized an analog of phosphatidylcholine (PC), whereas MK5E caused an irreversible increase of PC mobility. These results suggest testing the antimicrobial efficiency of non-toxic concentrations of selected cyclic hexapeptides as potential candidates to supplement/replace common antibiotics in semen preservation. PMID:24940997

  5. NOVEL CONTINUOUS PH/SALT GRADIENT AND PEPTIDE SCORE FOR STRONG CATION EXCHANGE CHROMATOGRAPHY IN 2D-NANO-LC/MSMS PEPTIDE IDENTIFICATION FOR PROTEOMICS

    EPA Science Inventory

    Tryptic digests of human serum albumin (HSA) and human lung epithelial cell lysates were used as test samples in a novel proteomics study. Peptides were separated and analyzed using 2D-nano-LC/MSMS with strong cation exchange (SCX) and reverse phase (RP) chromatography and contin...

  6. Structures of the DfsB Protein Family Suggest a Cationic, Helical Sibling Lethal Factor Peptide.

    PubMed

    Taylor, Jonathan D; Taylor, Gabrielle; Hare, Stephen A; Matthews, Steve J

    2016-02-13

    Bacteria have developed a variety of mechanisms for surviving harsh environmental conditions, nutrient stress and overpopulation. Paenibacillus dendritiformis produces a lethal protein (Slf) that is able to induce cell death in neighbouring colonies and a phenotypic switch in more distant ones. Slf is derived from the secreted precursor protein, DfsB, after proteolytic processing. Here, we present new crystal structures of DfsB homologues from a variety of bacterial species and a surprising version present in the yeast Saccharomyces cerevisiae. Adopting a four-helix bundle decorated with a further three short helices within intervening loops, DfsB belongs to a non-enzymatic class of the DinB fold. The structure suggests that the biologically active Slf fragment may possess a C-terminal helix rich in basic and aromatic residues that suggest a functional mechanism akin to that for cationic antimicrobial peptides. PMID:26804569

  7. Stimuli Response of Cationic Polymer Brush Prepared by ATRP: Application in Peptide Fractionation.

    PubMed

    Scott, Colleen; Mitrovic, Bojan; Eastwood, Stephanie; Kinsel, Gary

    2014-08-01

    Random cationic copolymer brushes composed of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and N-isopropylacrylamide (NIPAAm) were synthesized using the atom transfer radical polymerization (ATRP) method. The effects of varying the monomer feed ratios (30:70 and 70:30 DMAEMA:NIPAAm) and polymerization times on the film height, morphology and stimuli response to pH of the brush were evaluated. While the polymerization time was found to have little influence on the properties of the brushes, the monomer feed ratios had a great impact. The 70 % DMAEMA polymer brush had similar height as the 30 % DMAEMA brush after 45 min; however, it had a greater response to pH and morphological change compared to the 30 % DMAEMA. The 70 % DMAEMA brush was used to demonstrate an efficient approach to alleviate the ion suppression effect in MALDI analysis of complex mixtures by effectively fractionating a binary mixture of peptides prior to MALDI-MS analysis. PMID:25253913

  8. Stimuli Response of Cationic Polymer Brush Prepared by ATRP: Application in Peptide Fractionation

    PubMed Central

    Scott, Colleen; Mitrovic, Bojan; Eastwood, Stephanie; Kinsel, Gary

    2014-01-01

    Random cationic copolymer brushes composed of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and N-isopropylacrylamide (NIPAAm) were synthesized using the atom transfer radical polymerization (ATRP) method. The effects of varying the monomer feed ratios (30:70 and 70:30 DMAEMA:NIPAAm) and polymerization times on the film height, morphology and stimuli response to pH of the brush were evaluated. While the polymerization time was found to have little influence on the properties of the brushes, the monomer feed ratios had a great impact. The 70 % DMAEMA polymer brush had similar height as the 30 % DMAEMA brush after 45 min; however, it had a greater response to pH and morphological change compared to the 30 % DMAEMA. The 70 % DMAEMA brush was used to demonstrate an efficient approach to alleviate the ion suppression effect in MALDI analysis of complex mixtures by effectively fractionating a binary mixture of peptides prior to MALDI-MS analysis. PMID:25253913

  9. Intracellular delivery of fluorescent protein into viable wheat microspores using cationic peptides

    PubMed Central

    Bilichak, Andriy; Luu, Justin; Eudes, François

    2015-01-01

    Microspores are specialized generative cells with haploid genome that demonstrate the amenability toward embryogenesis under certain conditions. The induced microspore culture technique is largely exploited by the breeding programs of wheat and other crops due to its high efficiency for generation of the large number of haploid plants in the relatively short period of time. The ability to produce mature double haploid plant from a single cell has also attracted attention of the plant biotechnologists in the past few years. More importantly, the possibility to deliver proteins for improvement of embryogenesis and the genome modification purposes holds great potential for transgene-free wheat biotechnology. In the present study, we examined the ability of cationic and amphipathic cell penetrating peptides (CPPs) to convey a covalently-linked mCherry protein inside the viable microspores. We demonstrate that the affinity of CPPs to the microspore cells dependents on their charge with the highest efficiency of CPP-mCherry binding to the cells achieved by cationic CPPs (penetratin and R9). Additionally, due to overall negative charge of the microspore cell wall, the successful uptake of the protein cargo by live microspore cells is attained by utilization of a reversible disulfide bond between the R9 CPP and mCherry protein. Overall, the approach proposed herein can be applied by the other biotechnology groups for the fast and efficient screening of the different CPP candidates for their ability to deliver proteins inside the viable plant cells. PMID:26379691

  10. Antifungal activity of a synthetic cationic peptide against the plant pathogens Colletotrichum graminicola and three Fusarium species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A small cationic peptide (JH8944) was tested for activity against a number of pathogens of agricultural crops. JH8944 inhibited conidium growth in most of the tested plant pathogens with a dose of 50 µg ml 1, although one isolate of Fusarium oxysporum was inhibited at 5 µg ml 1. Most conidia of Fusa...

  11. Modulation of chicken intestinal immune gene expression by small cationic peptides as feed additives during the first week posthatch

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have been investigating modulation strategies tailored around the selective stimulation of the host’s immune system as an alternative to direct targeting of microbial pathogens by antibiotics. One such approach is the use of a group of small cationic peptides (BT) produced by a Gram-positive soi...

  12. Autolytic system of Staphylococcus simulans 22: influence of cationic peptides on activity of N-acetylmuramoyl-L-alanine amidase.

    PubMed Central

    Bierbaum, G; Sahl, H G

    1987-01-01

    Pep 5 and nisin are cationic peptide antibiotics which in addition to their membrane-disruptive action induce autolysis in staphylococci. To investigate the mechanism of lysis induction, the influence of the peptides on the activity of the N-acetylmuramoyl-L-alanine amidase of Staphylococcus simulans 22 was studied. In experiments with isolated cell walls at low ionic strength, the amidase activity was stimulated by the addition of Pep 5 and nisin, as well as by polylysine, streptomycin, and mono- and divalent cations. The concentrations necessary for activation depended on the nature of the cation and ranged from 5 microM for poly-L-lysine (n = 17) to 150 mM for Na+ at a cell wall concentration of 100 micrograms of cell walls per ml. No effect was observed if the cell walls were devoid of polyanionic constituents. Kinetic data suggested that the amidase bound to the teichoic and teichuronic acids of the cell wall and was thereby inhibited. Cationic molecules reversed this inhibition, most likely by displacing the enzyme from the polyanions. If the concentrations of the larger peptides were high in relation to cell wall concentration, the activation turned into inhibition, presumably by interfering with the access of the enzyme to its substrate. These experiments demonstrate that the activity of the amidase is modulated by basic peptides in vitro and help to explain how Pep 5 and nisin may cause lysis of treated cells. Images PMID:2890620

  13. Petasis-Ugi ligands: New affinity tools for the enrichment of phosphorylated peptides.

    PubMed

    Batalha, Íris L; Roque, Ana C A

    2016-09-15

    Affinity chromatography is a widespread technique for the enrichment and isolation of biologics, which relies on the selective and reversible interaction between affinity ligands and target molecules. Small synthetic affinity ligands are valuable alternatives due to their robustness, low cost and fast ligand development. This work reports, for the first time, the use of a sequential Petasis-Ugi multicomponent reaction to generate rationally designed solid-phase combinatorial libraries of small synthetic ligands, which can be screened for the selection of new affinity adsorbents towards biological targets. As a proof of concept, the Petasis-Ugi reaction was here employed in the discovery of affinity ligands suitable for phosphopeptide enrichment. A combinatorial library of 84 ligands was designed, synthesized on a chromatographic solid support and screened in situ for the specific binding of phosphopeptides binding human BRCA1C-terminal domains. The success of the reaction on the chromatographic matrix was confirmed by both inductively coupled plasma atomic emission spectroscopy and fluorescence microscopy. Three lead ligands were identified due to their superior performance in terms of binding capacity and selectivity towards the phosphorylated moiety on peptides, which showed the feasibility of the Petasis-Ugi reaction for affinity ligand development. PMID:27469904

  14. Covalent modification of a ten-residue cationic antimicrobial peptide with levofloxacin

    NASA Astrophysics Data System (ADS)

    Rodriguez, Carlos; Papanastasiou, Emilios; Juba, Melanie; Bishop, Barney

    2014-09-01

    The rampant spread of antibiotic resistant bacteria has spurred interest in alternative strategies for developing next-generation antibacterial therapies. As such, there has been growing interest in cationic antimicrobial peptides (CAMPs) and their therapeutic applications. Modification of CAMPs via conjugation to auxiliary compounds, including small molecule drugs, is a new approach to developing effective, broad-spectrum antibacterial agents with novel physicochemical properties and versatile antibacterial mechanisms. Here, we’ve explored design parameters for engineering CAMPs conjugated to small molecules with favorable physicochemical and antibacterial properties by covalently affixing a fluoroquinolone antibiotic, levofloxacin, to the ten-residue CAMP Pep-4. Relative to the unmodified Pep-4, the conjugate was found to demonstrate substantially increased antibacterial potency under high salt concentrations. Historically, it has been observed that most CAMPs lose antibacterial effectiveness in such high ionic strength environments, a fact that has presented a challenge to their development as therapeutics. Physicochemical studies revealed that P4LC was more hydrophobic than Pep-4, while mechanistic findings indicated that the conjugate was more effective at disrupting bacterial membrane integrity. Although the inherent antibacterial effect of the incorporated levofloxacin molecules did not appear to be substantially realized in this conjugate, these findings nevertheless suggest that covalent attachment of small molecule antibiotics with favorable physicochemical properties to CAMPs could be a promising strategy for enhancing peptide performance and overall therapeutic potential. These results have broader applicability to the development of future CAMP-antibiotic conjugates for potential therapeutic applications.

  15. Development of polymeric–cationic peptide composite nanoparticles, a nanoparticle-in-nanoparticle system for controlled gene delivery

    PubMed Central

    Jain, Arvind K; Massey, Ashley; Yusuf, Helmy; McDonald, Denise M; McCarthy, Helen O; Kett, Vicky L

    2015-01-01

    We report the formulation of novel composite nanoparticles that combine the high transfection efficiency of cationic peptide-DNA nanoparticles with the biocompatibility and prolonged delivery of polylactic acid–polyethylene glycol (PLA-PEG). The cationic cell-penetrating peptide RALA was used to condense DNA into nanoparticles that were encapsulated within a range of PLA-PEG copolymers. The composite nanoparticles produced exhibited excellent physicochemical properties including size <200 nm and encapsulation efficiency >80%. Images of the composite nanoparticles obtained with a new transmission electron microscopy staining method revealed the peptide-DNA nanoparticles within the PLA-PEG matrix. Varying the copolymers modulated the DNA release rate >6 weeks in vitro. The best formulation was selected and was able to transfect cells while maintaining viability. The effect of transferrin-appended composite nanoparticles was also studied. Thus, we have demonstrated the manufacture of composite nanoparticles for the controlled delivery of DNA. PMID:26648722

  16. Inhibition of protein phosphorylation by synthetic peptides from the Fc region of human IgG during the mixed lymphocyte response

    SciTech Connect

    McClurg, M.R.; Hahn, G.S.; Plummer, J.M.

    1986-03-01

    Certain synthetic peptides derived from the Fc region of human IgG suppressed protein, RNA, and DNA synthesis during mixed lymphocyte reactions. Responder mononuclear cells were incubated with medium or agents that alter phosphorylation of cellular proteins before immunomodulatory Fc peptides and stimulator cells were added. Incubating cells with trifluoperazine which inhibits calcium binding to calmodulin and inhibits protein kinase C (PKC) increased inhibition of the MLR induced by Fc peptides. Conversely, incubating cells with dubutyryl cyclic AMP (DBcAMP), calmodulin, 1,2-diolein, or phorbol myristate acetate (PMA) abolished inhibition of the MLR induced by Fc peptides. Inhibition of the MLR by Fc ..gamma.. peptides was not affected when DBcAMP or PMA was added after peptide addition. The PKC activity of cell homogenates was decreased by 69% when Fc..gamma.. peptides were present during the MLR. The in vitro phosphorylation of histone Hl by partially purified PKC from lymphocytes was inhibited 74% in the presence of Fc..gamma.. peptides. These results indicate that suppression of the MLR induced by Fc..gamma.. peptides is dependent on inhibition of protein phosphorylation by kinases including protein kinase C. The inhibition of phosphorylation may be related to the ability of Fc..gamma.. peptides to reverse animal models of autoimmune disease.

  17. Synthesis of linear and cyclic peptide-PEG-lipids for stabilization and targeting of cationic liposome-DNA complexes.

    PubMed

    Ewert, Kai K; Kotamraju, Venkata Ramana; Majzoub, Ramsey N; Steffes, Victoria M; Wonder, Emily A; Teesalu, Tambet; Ruoslahti, Erkki; Safinya, Cyrus R

    2016-03-15

    Because nucleic acids (NAs) have immense potential value as therapeutics, the development of safe and effective synthetic NA vectors continues to attract much attention. In vivo applications of NA vectors require stabilized, nanometer-scale particles, but the commonly used approaches of steric stabilization with a polymer coat (e.g., PEGylation; PEG=poly(ethylene glycol)) interfere with attachment to cells, uptake, and endosomal escape. Conjugation of peptides to PEG-lipids can improve cell attachment and uptake for cationic liposome-DNA (CL-DNA) complexes. We present several synthetic approaches to peptide-PEG-lipids and discuss their merits and drawbacks. A lipid-PEG-amine building block served as the common key intermediate in all synthetic routes. Assembling the entire peptide-PEG-lipid by manual solid phase peptide synthesis (employing a lipid-PEG-carboxylic acid) allowed gram-scale synthesis but is mostly applicable to linear peptides connected via their N-terminus. Conjugation via thiol-maleimide or strain-promoted (copper-free) azide-alkyne cycloaddition chemistry is highly amenable to on-demand preparation of peptide-PEG-lipids, and the appropriate PEG-lipid precursors are available in a single chemical step from the lipid-PEG-amine building block. Azide-alkyne cycloaddition is especially suitable for disulfide-bridged peptides such as iRGD (cyclic CRGDKGPDC). Added at 10 mol% of a cationic/neutral lipid mixture, the peptide-PEG-lipids stabilize the size of CL-DNA complexes. They also affect cell attachment and uptake of nanoparticles in a peptide-dependent manner, thereby providing a platform for preparing stabilized, affinity-targeted CL-DNA nanoparticles. PMID:26874401

  18. Minimum requirements of hydrophobic and hydrophilic features in cationic peptide antibiotics (CPAs): pharmacophore generation and validation with cationic steroid antibiotics (CSAs).

    PubMed

    Sundriyal, Sandeep; Sharma, Rohit K; Jain, Rahul; Bharatam, Prasad V

    2008-04-01

    Cationic peptide antibiotics (CPAs) are known to possess amphiphilic structure, by virtue of which they display lytic activity against bacterial cell membranes. Naturally occurring antimicrobial peptides contain a large number of amino acid residues, which limits their clinical applicability. Recent studies indicate that it is possible to decrease the chain-length of these peptides without loss of activity, and suggest that a minimum of two positive ionizable (hydrophilic) and two bulky groups (hydrophobic) are required for antimicrobial activity. By employing the HipHop module of the software package CATALYST, we have translated these experimental findings into 3-D pharmacophore models by finding common features among active peptides. Positively ionizable (PI) and hydrophobic (HYD) features are the important characteristics of compounds used for pharmacophore model development. Based on the highest score and the presence of amphiphilic structure, two separate hypothesis, Ec-2 and Sa-6 for Escherichia coli and Staphylococcus aureus, respectively, were selected for mapping analysis of active and inactive peptides against these organisms. The resulting models not only provided information on the minimum requirement of PI and HYD features but also indicated the importance of their relative arrangement in space. The minimum requirement for PI features was two in both cases but the number of HYD features required in the case of E. coli was four while for S. aureus it was found to be three. These hypotheses were able to differentiate between active and inactive CPAs against both organisms and were able to explain the experimental results. The hypotheses were further validated using cationic steroid antibiotics (CSAs), a different class of facial amphiphiles with same mechanism of antimicrobial action as that of CPAs. The results showed that CSAs also require similar minimum features to be active against both E. coli and S. aureus. These studies also indicate that the

  19. Design and synthesis of cationic antibacterial peptide based on Leucrocin I sequence, antibacterial peptide from crocodile (Crocodylus siamensis) white blood cell extracts.

    PubMed

    Yaraksa, Nualyai; Anunthawan, Thitiporn; Theansungnoen, Tinnakorn; Daduang, Sakda; Araki, Tomohiro; Dhiravisit, Apisak; Thammasirirak, Sompong

    2014-03-01

    Leucrocin I is an antibacterial peptide isolated from crocodile (Crocodylus siamensis) white blood cell extracts. Based on Leucrocin I sequence, cationic peptide, NY15, was designed, synthesized and evaluated for antibacterial activity against Bacillus sphaericus TISTR 678, Bacillus megaterium (clinical isolate), Vibrio cholerae (clinical isolate), Salmonella typhi (clinical isolate), Salmonella typhi ATCC 5784 and Escherichia coli 0157:H7. The efficacy of the peptide made from all L-amino acids was also compared with all D-amino acids. The peptide made from all D-amino acids was more active than the corresponding L-enantiomer. In our detailed study, the interaction between peptides and the cell membrane of Vibrio cholerae as part of their killing mechanism was studied by fluorescence and electron microscopy. The results show that the membrane was the target of action of the peptides. Finally, the cytotoxicity assays revealed that both L-NY15 and D-NY15 peptides are non-toxic to mammalian cells at bacteriolytic concentrations. PMID:24192554

  20. Short, Synthetic Cationic Peptides Have Antibacterial Activity against Mycobacterium smegmatis by Forming Pores in Membrane and Synergizing with Antibiotics

    PubMed Central

    Gupta, Kajal; Singh, Sameer; van Hoek, Monique L.

    2015-01-01

    Multicellular organisms are constantly exposed to a multitude of pathogenic microbes. Infection is inhibited in vivo by the innate and adaptive immune system. Mycobacterium species have emerged that are resistant to most antibiotics. We identified several naturally occurring cationic antimicrobial peptides that were active at low micromolar concentrations against Mycobacterium smegmatis. Human-derived cathelicidin LL-37 is well characterized and studied against M. smegmatis; we compared LL-37 with Chinese cobra-derived cathelicidin NA-CATH and mouse cathelicidin (mCRAMP). Two synthetic 11-residue peptides (ATRA-1A and ATRA-2) containing variations of a repeated motif within NA-CATH were tested for their activity against M. smegmatis along with a short synthetic peptide derivative from the human beta-defensin hBD3 (hBD3-Pep4). We hypothesized that these smaller synthetic peptides may demonstrate antimicrobial effectiveness with shorter length (and at less cost), making them strong potential candidates for development into broad-spectrum antimicrobial compounds or use in combination with antibiotics. These peptides have antimicrobial activity with EC50 ranging from 0.05 to 1.88 μg/mL against Mycobacterium smegmatis. The ATRA-1A short peptide was found to be the most effective antimicrobial peptide (AMP) (EC50 = 0.05 μg/mL). High bactericidal activity correlated with bacterial membrane depolarization and permeabilization activities. The efficacy of the peptides was further analyzed through Minimal Inhibitory Concentration (MIC) assays. The MICs were determined by the microdilution method. The peptide mCRAMP showed the best MIC activity at 15.6 μg/mL. Neither of the effective short synthetic peptides demonstrated synergy with the antibiotic rifampicin, although both demonstrated synergy with the cyclic peptide antibiotic polymyxin B. The peptides LL-37 and mCRAMP displayed synergism with rifampicin in MIC assays, whereas antibiotic polymyxin B displayed synergism

  1. Molecular Basis of Resistance to Muramidase and Cationic Antimicrobial Peptide Activity of Lysozyme in Staphylococci

    PubMed Central

    Herbert, Silvia; Bera, Agnieszka; Nerz, Christiane; Kraus, Dirk; Peschel, Andreas; Goerke, Christiane; Meehl, Michael; Cheung, Ambrose; Götz, Friedrich

    2007-01-01

    It has been shown recently that modification of peptidoglycan by O-acetylation renders pathogenic staphylococci resistant to the muramidase activity of lysozyme. Here, we show that a Staphylococcus aureus double mutant defective in O-acetyltransferase A (OatA), and the glycopeptide resistance-associated two-component system, GraRS, is much more sensitive to lysozyme than S. aureus with the oatA mutation alone. The graRS single mutant was resistant to the muramidase activity of lysozyme, but was sensitive to cationic antimicrobial peptides (CAMPs) such as the human lysozyme-derived peptide 107R-A-W-V-A-W-R-N-R115 (LP9), polymyxin B, or gallidermin. A comparative transcriptome analysis of wild type and the graRS mutant revealed that GraRS controls 248 genes. It up-regulates global regulators (rot, sarS, or mgrA), various colonization factors, and exotoxin-encoding genes, as well as the ica and dlt operons. A pronounced decrease in the expression of the latter two operons explains why the graRS mutant is also biofilm-negative. The decrease of the dlt transcript in the graRS mutant correlates with a 46.7% decrease in the content of esterified d-alanyl groups in teichoic acids. The oatA/dltA double mutant showed the highest sensitivity to lysozyme; this mutant completely lacks teichoic acid–bound d-alanine esters, which are responsible for the increased susceptibility to CAMPs and peptidoglycan O-acetylation. Our results demonstrate that resistance to lysozyme can be dissected into genes mediating resistance to its muramidase activity (oatA) and genes mediating resistance to CAMPs (graRS and dlt). The two lysozyme activities act synergistically, as the oatA/dltA or oatA/graRS double mutants are much more susceptible to lysozyme than each of the single mutants. PMID:17676995

  2. A Novel Cell-Penetrating Peptide Derived from Human Eosinophil Cationic Protein

    PubMed Central

    Fang, Shun-lung; Fan, Tan-chi; Fu, Hua-Wen; Chen, Chien-Jung; Hwang, Chi-Shin; Hung, Ta-Jen; Lin, Lih-Yuan; Chang, Margaret Dah-Tsyr

    2013-01-01

    Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP32–41, derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP32–41 entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP32–41 to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP32–41 internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP32–41 attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP32–41 internalization routes. ECP32–41 was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases. PMID:23469189

  3. Identification of phosphorylated peptides from complex mixtures using negative-ion orifice-potential stepping and capillary liquid chromatography/electrospray ionization mass spectrometry.

    PubMed

    Ding, J; Burkhart, W; Kassel, D B

    1994-01-01

    A rapid method for identifying and characterizing sites of phosphorylation of peptides and proteins is described. High-performance capillary liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) is used to distinguish non-phosphorylated and phosphorylated peptides originating from mixtures as complex as enzyme digests. The method relies on the ability to produce a fragment ion characteristic and unique to phosphopeptides (m/z 79, PO3) by stepping the orifice potential of the mass spectrometer as a function of mass. At low m/z values, a high orifice potential is applied to induce extensive fragmentation of the peptide, leading to the formation of the m/z 79 phosphate-derived ion. This method is analogous to that described by Carr et al. for the identification of glycopeptides from enzymatic digestion of glycoproteins (S.A. Carr, M.J. Huddleston, M.F. Bean, Protein Science 2, 183 (1993)). The method was first evaluated and validated for a mixture of non-, mono- and di-phosphorylated synthetic peptides. Both mono- and di-phosphorylated peptides were found to generate fragment ions characteristic of PO3 whereas the non-phosphorylated peptide did not. Application of the method was extended to identifying phosphopeptides generated from an endoprotease Lys-C digestion of beta-casein. Both the expected mono- and tetra-phosphorylated Lys-C peptides were observed and identified rapidly in the LC/SEI-MS analysis. The procedure was used additionally to identify the site(s) of phosphorylation of the cytosolic non-receptor tyrosine kinase, pp60(c-src). PMID:8118063

  4. Antimicrobial potential of lycosin-I, a cationic and amphiphilic peptide from the venom of the spider Lycosa singorensis.

    PubMed

    Tan, H; Ding, X; Meng, S; Liu, C; Wang, H; Xia, L; Liu, Z; Liang, S

    2013-07-01

    Antimicrobial peptides (AMPs) are significant components of the innate immune system and play indispensable roles in the resistance to bacterial infection. Here, we investigated the antimicrobial activity of lycosin-I, a 24-residue cationic anticancer peptide derived from Lycosa singorensis with high structural similarity to several cationic and amphiphilic antimicrobial peptides. The antimicrobial activity of lycosin-I against 27 strains of microbes including bacteria and fungi was examined and compared with that of the Xenopus-derived AMP magainin 2 using a microdilution assay. Lycosin-I inhibited the growth of most microorganisms at low micromolar concentrations, and was a more potent inhibitor than magainin 2. Lycosin-I showed rapid, selective and broad-spectrum bactericidal activity and a synergistic effect with traditional antibiotics. In vivo, it showed potent bactericidal activity in a mouse thigh infection model. High Mg2+ concentrations reduced the antibacterial effect of lycosin-I, implying that the peptide might directly interact with the bacterial cell membrane. Uptake of the fluorogenic dye SYTOX and changes in the surface of lycosin-Itreated bacterial cells observed by scanning electron microscopy confirmed that lycosin-I permeabilized the cell membrane, resulting in the rapid bactericidal effect. Taken together, our findings indicate that lycosin-I is a promising peptide with the potential for the development of novel antibacterial agents. PMID:23638903

  5. In vitro susceptibility tests for cationic peptides: comparison of broth microdilution methods for bacteria that grow aerobically.

    PubMed

    Giacometti, A; Cirioni, O; Barchiesi, F; Del Prete, M S; Fortuna, M; Caselli, F; Scalise, G

    2000-06-01

    The in vitro susceptibilities of 90 clinical isolates of gram-positive and gram-negative aerobic bacteria to six cationic peptides, buforin II, cecropin P1, indolicidin, magainin II, nisin, and ranalexin, were evaluated by two broth microdilution methods. The first method was performed according to the procedures outlined by the National Committee for Clinical Laboratory Standards for bacteria that grow aerobically, while the second was performed according to the procedures recently proposed by the R. E. W. Hancock laboratory for testing antimicrobial peptides. Overall, the first method produced MICs two- and fourfold higher than the second method. PMID:10817731

  6. Novel Cβ-Cγ Bond Cleavages of Tryptophan-Containing Peptide Radical Cations

    NASA Astrophysics Data System (ADS)

    Song, Tao; Hao, Qiang; Law, Chun-Hin; Siu, Chi-Kit; Chu, Ivan K.

    2012-02-01

    In this study, we observed unprecedented cleavages of the Cβ-Cγ bonds of tryptophan residue side chains in a series of hydrogen-deficient tryptophan-containing peptide radical cations (M•+) during low-energy collision-induced dissociation (CID). We used CID experiments and theoretical density functional theory (DFT) calculations to study the mechanism of this bond cleavage, which forms [M - 116]+ ions. The formation of an α-carbon radical intermediate at the tryptophan residue for the subsequent Cβ-Cγ bond cleavage is analogous to that occurring at leucine residues, producing the same product ions; this hypothesis was supported by the identical product ion spectra of [LGGGH - 43]+ and [WGGGH - 116]+, obtained from the CID of [LGGGH]•+ and [WGGGH]•+, respectively. Elimination of the neutral 116-Da radical requires inevitable dehydrogenation of the indole nitrogen atom, leaving the radical centered formally on the indole nitrogen atom ([Ind]•-2), in agreement with the CID data for [WGGGH]•+ and [W1-CH3GGGH]•+; replacing the tryptophan residue with a 1-methyltryptophan residue results in a change of the base peak from that arising from a neutral radical loss (116 Da) to that arising from a molecule loss (131 Da), both originating from Cβ-Cγ bond cleavage. Hydrogen atom transfer or proton transfer to the γ-carbon atom of the tryptophan residue weakens the Cβ-Cγ bond and, therefore, decreases the dissociation energy barrier dramatically.

  7. Cytotoxicity and the effect of cationic peptide fragments against cariogenic bacteria under planktonic and biofilm conditions.

    PubMed

    Kreling, Paula Fernanda; Aida, Kelly Limi; Massunari, Loiane; Caiaffa, Karina Sampaio; Percinoto, Célio; Bedran, Telma Blanca Lombardo; Spolidorio, Denise Madalena Palomari; Abuna, Gabriel Flores; Cilli, Eduardo Maffud; Duque, Cristiane

    2016-10-01

    This study evaluated the cytotoxicity and effect of fragments derived from three oral cationic peptides (CP): LL-37, D6-17 and D1-23 against cariogenic bacteria under planktonic and biofilm conditions. For cytotoxicity analysis, two epithelial cell lines were used. The minimum inhibitory concentration and the minimal bactericidal concentration were determined for the CP fragments and the control (chlorhexidine-CHX) against cariogenic bacteria. The fractional inhibitory concentration was obtained for the combinations of CP fragments on Streptococcus mutans. Biofilm assays were conducted with the best antimicrobial CP fragment against S. mutans. The results indicated that D6-17 was not cytotoxic. D1-23, LL-37 and CHX were not cytotoxic in low concentrations. D1-23 presented the best bactericidal activity against S. mutans, S. mitis and S. salivarius. Combinations of CP fragments did not show a synergic effect. D1-23 presented a higher activity against S. mutans biofilm than CHX. It was concluded that D1-23 showed a substantial effect against cariogenic bacteria and low cytotoxicity. PMID:27538256

  8. Homologous desensitization of guanylyl cyclase A, the receptor for atrial natriuretic peptide, is associated with a complex phosphorylation pattern

    PubMed Central

    Schröter, Juliane; Zahedi, René P; Hartmann, Michael; Gaßner, Birgit; Gazinski, Alexandra; Waschke, Jens; Sickmann, Albert; Kuhn, Michaela

    2010-01-01

    Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and intracellular guanosine 3′,5′-cyclic monophosphate production, is critically involved in the regulation of blood pressure. In patients with chronic heart failure, the plasma levels of ANP are increased, but the cardiovascular actions are severely blunted, indicating a receptor or postreceptor defect. Studies on metabolically labelled GC-A-overexpressing cells have indicated that GC-A is extensively phosphorylated, and that ANP-induced homologous desensitization of GC-A correlates with receptor dephosphorylation, a mechanism which might contribute to a loss of function in vivo. In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK293, revealed unambiguously that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513. MS quantification based on multiple reaction monitoring demonstrated that ANP-provoked desensitization was accompanied by a complex pattern of receptor phosphorylation and dephosphorylation. The population of completely phosphorylated GC-A was diminished. However, intriguingly, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP. The functional relevance of this observation was analysed by site-directed mutagenesis. The substitution of Ser487 by glutamate (which mimics phosphorylation) blunted the activation of the GC-A receptor by ANP, but prevented further desensitization. Our data corroborate previous studies suggesting that the responsiveness of GC-A to ANP is regulated by phosphorylation. However, in addition to the dephosphorylation of the previously postulated sites (Ser497, Thr500, Ser502, Ser506, Ser510), homologous desensitization seems to involve the phosphorylation of GC-A at Ser487, a newly identified site of phosphorylation. The identification and further characterization of the

  9. Dissection of Binding between a Phosphorylated Tyrosine Hydroxylase Peptide and 14-3-3ζ: A Complex Story Elucidated by NMR

    PubMed Central

    Hritz, Jozef; Byeon, In-Ja L.; Krzysiak, Troy; Martinez, Aurora; Sklenar, Vladimir; Gronenborn, Angela M.

    2014-01-01

    Human tyrosine hydroxylase activity is regulated by phosphorylation of its N-terminus and by an interaction with the modulator 14-3-3 proteins. We investigated the binding of singly or doubly phosphorylated and thiophosphorylated peptides, comprising the first 50 amino acids of human tyrosine hydroxylase, isoform 1 (hTH1), that contain the critical interaction domain, to 14-3-3ζ, by 31P NMR. Single phosphorylation at S19 generates a high affinity 14-3-3ζ binding epitope, whereas singly S40-phosphorylated peptide interacts with 14-3-3ζ one order-of-magnitude weaker than the S19-phosphorylated peptide. Analysis of the binding data revealed that the 14-3-3ζ dimer and the S19- and S40-doubly phosphorylated peptide interact in multiple ways, with three major complexes formed: 1), a single peptide bound to a 14-3-3ζ dimer via the S19 phosphate with the S40 phosphate occupying the other binding site; 2), a single peptide bound to a 14-3-3ζ dimer via the S19 phosphorous with the S40 free in solution; or 3), a 14-3-3ζ dimer with two peptides bound via the S19 phosphorous to each binding site. Our system and data provide information as to the possible mechanisms by which 14-3-3 can engage binding partners that possess two phosphorylation sites on flexible tails. Whether these will be realized in any particular interacting pair will naturally depend on the details of each system. PMID:25418103

  10. Modeling the Interaction between Integrin-Binding Peptide (RGD) and Rutile Surface: The Effect of Cation Mediation on Asp Adsorption

    SciTech Connect

    Wu, Chunya; Skelton, Adam; Chen, Mingjun; Vlcek, Lukas; Cummings, Peter T

    2012-01-01

    The binding of a negatively charged residue, aspartic acid (Asp) in tripeptide arginine-glycine-aspartic acid, onto a negatively charged hydroxylated rutile (110) surface in aqueous solution, containing divalent (Mg{sup 2+}, Ca{sup 2+}, or Sr{sup 2+}) or monovalent (Na{sup +}, K{sup +}, or Rb{sup +}) cations, was studied by molecular dynamics (MD) simulations. The results indicate that ionic radii and charges will significantly affect the hydration, adsorption geometry, and distance of cations from the rutile surface, thereby regulating the Asp/rutile binding mode. The adsorption strength of monovalent cations on the rutile surface in the order Na{sup +} > K{sup +} > Rb{sup +} shows a 'reverse' lyotropic trend, while the divalent cations on the same surface exhibit a 'regular' lyotropic behavior with decreasing crystallographic radii (the adsorption strength of divalent cations: Sr{sup 2+} > Ca{sup 2+} > Mg{sup 2+}). The Asp side chain in NaCl, KCl, and RbCl solutions remains stably H-bonded to the surface hydroxyls and the inner-sphere adsorbed compensating monovalent cations act as a bridge between the COO{sup -} group and the rutile, helping to 'trap' the negatively charged Asp side chain on the negatively charged surface. In contrast, the mediating divalent cations actively participate in linking the COO{sup -} group to the rutile surface; thus the Asp side chain can remain stably on the rutile (110) surface, even if it is not involved in any hydrogen bonds with the surface hydroxyls. Inner- and outer-sphere geometries are all possible mediation modes for divalent cations in bridging the peptide to the rutile surface.

  11. CASEIN KINASE-MEDIATED PHOSPHORYLATION OF SERINE 839 IS NECESSARY FOR BASOLATERAL LOCALIZATION OF THE Ca2+-ACTIVATED NON-SELECTIVE CATION CHANNEL TRPM4

    PubMed Central

    Cerda, Oscar; Cáceres, Mónica; Park, Kang-Sik; Leiva-Salcedo, Elías; Romero, Aníbal; Varela, Diego

    2014-01-01

    TRPM4 is a Ca2+-activated non-selective cation channel expressed in a wide range of human tissues. TRPM4 participates in a variety of physiological processes such as T cell activation, myogenic vasoconstriction and allergic reactions. TRPM4 Ca2+ sensitivity is enhanced by calmodulin (CaM) and phosphathydilinositol 4, 5-biphosphate (PI(4,5)P2) binding, as well as, under certain conditions, PKC activation. However, information as to the mechanisms of modulation of this channel remain unknown, including direct identification of phosphorylation sites on TRPM4 and their role in channel features. Here, we use mass-spectrometric-based proteomic approaches (immunoprecipitation and tandem mass spectrometry), to unambiguously identify S839 as a phosphorylation site present on human TRPM4 expressed in a human cell line. Site-directed mutagenesis employing a serine to alanine mutation to eliminate phosphorylation, and a phospho-mimetic aspartate mutation, as well as biochemical and immunocytochemical experiments, revealed a role for S839 phosphorylation in the basolateral expression of TRPM4 channels in epithelial cells. Moreover, we demonstrated that casein kinase 1 (CK1) phosphorylates S839 and is responsible for the basolateral localization of TRPM4. PMID:25231975

  12. Bioinspired superoxide-dismutase mimics: The effects of functionalization with cationic polyarginine peptides.

    PubMed

    Ching, H Y Vincent; Kenkel, Isabell; Delsuc, Nicolas; Mathieu, Emilie; Ivanović-Burmazović, Ivana; Policar, Clotilde

    2016-07-01

    Continuing a bio-mimetic approach, we have prepared peptide conjugates of a superoxide dismutase (SOD) mimic [MnL](+) (where HL=N-(2-hydroxybenzyl)-N,N'-bis[2-(N-methylimidazolyl)methyl]ethane-1,2-diamine), namely [MnL'-Arg(n-1)](n+) (where n=2, 4, 7 and 10) and [MnL'-Gly1](+). [MnL'-Arg(n-1)](n+) contained cationic residue(s) that emulate the electrostatic channel of the enzyme. Physicochemical methods showed that functionalization at the secondary amine of HL did not impair coordination to Mn(II) with association constants (Kassoc) between 1.6 and 3.3×10(6)M(-1). The Mn(III)/Mn(II) redox potential of the conjugates was between 0.27 and 0.30V vs SCE, slightly higher than [MnL](+) under the same conditions, but remain at a value that facilitates O2(-) dismutation. The catalytic rate constant (kcat) of the dismutation for the series was studied using a direct stopped-flow method, which showed that for compounds with the same overall charge, the alkylation of the secondary amine of [MnL](+) (kcat=5.0±0.1×10(6)M(-1)s(-1)) led to a lower value (i.e. for [MnL'Gly](+), kcat=4.2±0.1×10(6)M(-1)s(-1)). However, under the same conditions, kcat values between 5.0±0.4×10(6)M(-1)s(-1) and 6.6±0.1×10(6)M(-1)s(-1) were determined for [MnL'-Arg(n-1)](n+) conjugates, indicating that the cationic residue(s) compensated for the loss in activity. Analysis of the effect of ionic strength on the kcat strongly suggested that not all the charges were involved, but only the closest ones electrostatically influenced the SOD active metal centre. PMID:26916739

  13. Neuropilin-1 and heparan sulfate proteoglycans cooperate in cellular uptake of nanoparticles functionalized by cationic cell-penetrating peptides

    PubMed Central

    Pang, Hong-Bo; Braun, Gary B.; Ruoslahti, Erkki

    2015-01-01

    Cell-penetrating peptides (CPPs) have been widely used to deliver nanomaterials and other types of macromolecules into mammalian cells for therapeutic and diagnostic use. Cationic CPPs that bind to heparan sulfate (HS) proteoglycans on the cell surface induce potent endocytosis; however, the role of other surface receptors in this process is unclear. We describe the convergence of an HS-dependent pathway with the C-end rule (CendR) mechanism that enables peptide ligation with neuropilin-1 (NRP1), a cell surface receptor known to be involved in angiogenesis and vascular permeability. NRP1 binds peptides carrying a positive residue at the carboxyl terminus, a feature that is compatible with cationic CPPs, either intact or after proteolytic processing. We used CPP and CendR peptides, as well as HS- and NRP1-binding motifs from semaphorins, to explore the commonalities and differences of the HS and NRP1 pathways. We show that the CendR-NRP1 interaction determines the ability of CPPs to induce vascular permeability. We also show at the ultrastructural level, using a novel cell entry synchronization method, that both the HS and NRP1 pathways can initiate a macropinocytosis-like process and visualize these CPP-cargo complexes going through various endosomal compartments. Our results provide new insights into how CPPs exploit multiple surface receptor pathways for intracellular delivery. PMID:26601141

  14. In-vitro activity of cationic peptides alone and in combination with clinically used antimicrobial agents against Pseudomonas aeruginosa.

    PubMed

    Giacometti, A; Cirioni, O; Barchiesi, F; Fortuna, M; Scalise, G

    1999-11-01

    The in-vitro activity of cecropin P1, indolicidin, magainin II, nisin and ranalexin alone and in combination with nine clinically used antimicrobial agents was investigated against a control strain, Pseudomonas aeruginosa ATCC 27853 and 40 clinical isolates of P. aeruginosa. Antimicrobial activities were measured by MIC, MBC and viable count. In the combination study, the clinically used antibiotics were used at concentrations close to their mean serum level in humans in order to establish the clinical relevance of the results. To select peptide-resistant mutants, P. aeruginosa ATCC 27853 was treated with consecutive cycles of exposure to each peptide at 1 x MIC. The peptides had a varied range of inhibitory values: all isolates were more susceptible to cecropin P1, while ranalexin showed the lowest activity. Nevertheless, synergy was observed when the peptides were combined with polymyxin E and clarithromycin. Consecutive exposures to each peptide at 1 x MIC resulted in the selection of stable resistant mutants. Cationic peptides might be valuable as new antimicrobial agents. Our findings show that they are effective against P. aeruginosa, and that their activity is enhanced when they are combined with clinically used antimicrobial agents, particularly with polymyxin E and clarithromycin. PMID:10552980

  15. Cationic Antimicrobial Peptides Promote Microbial Mutagenesis and Pathoadaptation in Chronic Infections

    PubMed Central

    Limoli, Dominique H.; Rockel, Andrea B.; Host, Kurtis M.; Jha, Anuvrat; Kopp, Benjamin T.; Hollis, Thomas; Wozniak, Daniel J.

    2014-01-01

    Acquisition of adaptive mutations is essential for microbial persistence during chronic infections. This is particularly evident during chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) patients. Thus far, mutagenesis has been attributed to the generation of reactive species by polymorphonucleocytes (PMN) and antibiotic treatment. However, our current studies of mutagenesis leading to P. aeruginosa mucoid conversion have revealed a potential new mutagen. Our findings confirmed the current view that reactive oxygen species can promote mucoidy in vitro, but revealed PMNs are proficient at inducing mucoid conversion in the absence of an oxidative burst. This led to the discovery that cationic antimicrobial peptides can be mutagenic and promote mucoidy. Of specific interest was the human cathelicidin LL-37, canonically known to disrupt bacterial membranes leading to cell death. An alternative role was revealed at sub-inhibitory concentrations, where LL-37 was found to induce mutations within the mucA gene encoding a negative regulator of mucoidy and to promote rifampin resistance in both P. aeruginosa and Escherichia coli. The mechanism of mutagenesis was found to be dependent upon sub-inhibitory concentrations of LL-37 entering the bacterial cytosol and binding to DNA. LL-37/DNA interactions then promote translesion DNA synthesis by the polymerase DinB, whose error-prone replication potentiates the mutations. A model of LL-37 bound to DNA was generated, which reveals amino termini α-helices of dimerized LL-37 bind the major groove of DNA, with numerous DNA contacts made by LL-37 basic residues. This demonstrates a mutagenic role for antimicrobials previously thought to be insusceptible to resistance by mutation, highlighting a need to further investigate their role in evolution and pathoadaptation in chronic infections. PMID:24763694

  16. Novel Cβ-Cγ bond cleavages of tryptophan-containing peptide radical cations.

    PubMed

    Song, Tao; Hao, Qiang; Law, Chun-Hin; Siu, Chi-Kit; Chu, Ivan K

    2012-02-01

    In this study, we observed unprecedented cleavages of the C(β)-C(γ) bonds of tryptophan residue side chains in a series of hydrogen-deficient tryptophan-containing peptide radical cations (M(•+)) during low-energy collision-induced dissociation (CID). We used CID experiments and theoretical density functional theory (DFT) calculations to study the mechanism of this bond cleavage, which forms [M - 116](+) ions. The formation of an α-carbon radical intermediate at the tryptophan residue for the subsequent C(β)-C(γ) bond cleavage is analogous to that occurring at leucine residues, producing the same product ions; this hypothesis was supported by the identical product ion spectra of [LGGGH - 43](+) and [WGGGH - 116](+), obtained from the CID of [LGGGH](•+) and [WGGGH](•+), respectively. Elimination of the neutral 116-Da radical requires inevitable dehydrogenation of the indole nitrogen atom, leaving the radical centered formally on the indole nitrogen atom ([Ind](•)-2), in agreement with the CID data for [WGGGH](•+) and [W(1-CH3)GGGH](•+); replacing the tryptophan residue with a 1-methyltryptophan residue results in a change of the base peak from that arising from a neutral radical loss (116 Da) to that arising from a molecule loss (131 Da), both originating from C(β)-C(γ) bond cleavage. Hydrogen atom transfer or proton transfer to the γ-carbon atom of the tryptophan residue weakens the C(β)-C(γ) bond and, therefore, decreases the dissociation energy barrier dramatically. PMID:22135037

  17. Effects of a cationic and hydrophobic peptide, KL4, on model lung surfactant lipid monolayers.

    PubMed

    Ma, J; Koppenol, S; Yu, H; Zografi, G

    1998-04-01

    We report on the surface behavior of a hydrophobic, cationic peptide, [lysine-(leucine)4]4-lysine (KL4), spread at the air/water interface at 25 degrees C and pH 7.2, and its effect at very low molar ratios on the surface properties of the zwitterionic phospholipid 1,2-dipalmitoylphosphatidylcholine (DPPC), and the anionic forms of 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) and palmitic acid (PA), in various combinations. Surface properties were evaluated by measuring equilibrium spreading pressures (pi(e)) and surface pressure-area isotherms (pi-A) with the Wilhelmy plate technique. Surface phase separation was observed with fluorescence microscopy. KL4 itself forms a single-phase monolayer, stable up to a surface pressure pi of 30 mN/m, and forms an immiscible monolayer mixture with DPPC. No strong interaction was detected between POPG and KL4 in the low pi region, whereas a stable monolayer of the PA/KL4 binary mixture forms, which is attributed to ionic interactions between oppositely charged PA and KL4. KL4 has significant effects on the DPPC/POPG mixture, in that it promotes surface phase separation while also increasing pi(e) and pi(max), and these effects are greatly enhanced in the presence of PA. In the model we have proposed, KL4 facilitates the separation of DPPC-rich and POPG/PA-rich phases to achieve surface refinement. It is these two phases that can fulfill the important lung surfactant functions of high surface pressure stability and efficient spreading. PMID:9545051

  18. Mild uncoupling of respiration and phosphorylation as a mechanism providing nephro- and neuroprotective effects of penetrating cations of the SkQ family.

    PubMed

    Plotnikov, E Y; Silachev, D N; Jankauskas, S S; Rokitskaya, T I; Chupyrkina, A A; Pevzner, I B; Zorova, L D; Isaev, N K; Antonenko, Y N; Skulachev, V P; Zorov, D B

    2012-09-01

    It is generally accepted that mitochondrial production of reactive oxygen species is nonlinearly related to the value of the mitochondrial membrane potential with significant increment at values exceeding 150 mV. Due to this, high values of the membrane potential are highly dangerous, specifically under pathological conditions associated with oxidative stress. Mild uncoupling of oxidative phosphorylation is an approach to preventing hyperpolarization of the mitochondrial membrane. We confirmed data obtained earlier in our group that dodecylrhodamine 19 (C(12)R1) (a penetrating cation from SkQ family not possessing a plastoquinone group) has uncoupling properties, this fact making it highly potent for use in prevention of pathologies associated with oxidative stress induced by mitochondrial hyperpolarization. Further experiments showed that C(12)R1 provided nephroprotection under ischemia/reperfusion of the kidney as well as under rhabdomyolysis through diminishing of renal dysfunction manifested by elevated level of blood creatinine and urea. Similar nephroprotective properties were observed for low doses (275 nmol/kg) of the conventional uncoupler 2,4-dinitrophenol. Another penetrating cation that did not demonstrate protonophorous activity (SkQR4) had no effect on renal dysfunction. In experiments with induced ischemic stroke, C(12)R1 did not have any effect on the area of ischemic damage, but it significantly lowered neurological deficit. We conclude that beneficial effects of penetrating cation derivatives of rhodamine 19 in renal pathologies and brain ischemia may be at least partially explained by uncoupling of oxidation and phosphorylation. PMID:23157263

  19. Phosphorylation by protein kinase C and cyclic AMP-dependent protein kinase of synthetic peptides derived from the linker region of human P-glycoprotein.

    PubMed Central

    Chambers, T C; Pohl, J; Glass, D B; Kuo, J F

    1994-01-01

    Specific sites in the linker region of human P-glycoprotein phosphorylated by protein kinase C (PKC) were identified by means of a synthetic peptide substrate, PG-2, corresponding to residues 656-689 from this region of the molecule. As PG-2 has several sequences of the type recognized by the cyclic AMP-dependent protein kinase (PKA), PG-2 was also tested as a substrate for PKA. PG-2 was phosphorylated by purified PKC in a Ca2+/phospholipid-dependent manner, with a Km of 1.3 microM, and to a maximum stoichiometry of 2.9 +/- 0.1 mol of phosphate/mol of peptide. Sequence analysis of tryptic fragments of PG-2 phosphorylated by PKC identified Ser-661, Ser-667 and Ser-671 as the three sites of phosphorylation. PG-2 was also found to be phosphorylated by purified PKA in a cyclic AMP-dependent manner, with a Km of 21 microM, and to a maximum stoichiometry of 2.6 +/- 0.2 mol of phosphate/mol of peptide. Ser-667, Ser-671 and Ser-683 were phosphorylated by PKA. Truncated peptides of PG-2 were utilized to confirm that Ser-661 was PKC-specific and Ser-683 was PKA-specific. Further studies showed that PG-2 acted as a competitive substrate for the P-glycoprotein kinase present in membranes from multidrug-resistant human KB cells. The membrane kinase phosphorylated PG-2 mainly on Ser-661, Ser-667 and Ser-671. These results show that human P-glycoprotein can be phosphorylated by at least two protein kinases, stimulated by different second-messenger systems, which exhibit both overlapping and unique specificities for phosphorylation of multiple sites in the linker region of the molecule. Images Figure 3 Figure 5 PMID:7909431

  20. Studies of Peptide:N-glycnase-p97 Interaction Suggest that p97 Phosphorylation Modulates Endoplasmic Reticulum-Associated Degradation

    SciTech Connect

    Zhao,G.; Zhou, X.; Wang, L.; Li, G.; Schindelin, H.; Lennarz, W.

    2007-01-01

    During endoplasmic reticulum-associated degradation, the multifunctional AAA ATPase p97 is part of a protein degradation complex. p97 associates via its N-terminal domain with various cofactors to recruit ubiquitinated substrates. It also interacts with alternative substrate-processing cofactors, such as Ufd2, Ufd3, and peptide:N-glycanase (PNGase) in higher eukaryotes. These cofactors determine different fates of the substrates and they all bind outside of the N-terminal domain of p97. Here, we describe a cofactor-binding motif of p97 contained within the last 10 amino acid residues of the C terminus, which is both necessary and sufficient to mediate interactions of p97 with PNGase and Ufd3. The crystal structure of the N-terminal domain of PNGase in complex with this motif provides detailed insight into the interaction between p97 and its substrate-processing cofactors. Phosphorylation of p97's highly conserved penultimate tyrosine residue, which is the main phosphorylation site during T cell receptor stimulation, completely blocks binding of either PNGase or Ufd3 to p97. This observation suggests that phosphorylation of this residue modulates endoplasmic reticulum-associated protein degradation activity by discharging substrate-processing cofactors.

  1. Cationic Cell-Penetrating Peptide Binds to Planar Lipid Bilayers Containing Negatively Charged Lipids but does not Induce Conductive Pores

    PubMed Central

    Gurnev, Philip A.; Yang, Sung-Tae; Melikov, Kamran C.; Chernomordik, Leonid V.; Bezrukov, Sergey M.

    2013-01-01

    Using a cation-selective gramicidin A channel as a sensor of the membrane surface charge, we studied interactions of oligoarginine peptide R9C, a prototype cationic cell-penetrating peptide (CPP), with planar lipid membranes. We have found that R9C sorption to the membrane depends strongly on its lipid composition from virtually nonexistent for membranes made of uncharged lipids to very pronounced for membranes containing negatively charged lipids, with charge overcompensation at R9C concentrations exceeding 1 μM. The sorption was reversible as it was removed by addition of polyanionic dextran sulfate to the membrane bathing solution. No membrane poration activity of R9C (as would be manifested by increased bilayer conductance) was detected in the charged or neutral membranes, including those with asymmetric negative/neutral and negative/positive lipid leaflets. We conclude that interaction of R9C with planar lipid bilayers does not involve pore formation in all studied lipid combinations up to 20 μM peptide concentration. However, R9C induces leakage of negatively charged but not neutral liposomes in a process that involves lipid mixing between liposomes. Our findings suggest that direct traversing of CPPs through the uncharged outer leaflet of the plasma membrane bilayer is unlikely and that permeabilization necessarily involves both anionic lipids and CPP-dependent fusion between opposing membranes. PMID:23663836

  2. [Expression, purification of recombinant cationic peptide AIK in Escherichia coli and its antitumor activity].

    PubMed

    Fan, Fangfang; Sun, Huiying; Xu, Hui; Liu, Jiawei; Zhang, Haiyuan; Li, Yilan; Ning, Xuelian; Sun, Yue; Bai, Jing; Fu, Songbin; Zhou, Chunshui

    2015-12-01

    AIK is a novel cationic peptide with potential antitumor activity. In order to construct the AIK expression vector by Gateway technology, and establish an optimal expression and purification method for recombinant AIK, a set of primers containing AttB sites were designed and used to create the AttB-TEV-FLAG-AIR fusion gene by overlapping PCR. The resulting fusion gene was cloned into the donor vector pDONR223 by attB and attP mediated recombination (BP reaction), then, transferred into the destination vector pDESTl 5 by attL and attR mediated recombination (LR reaction). All the cloning was verified by both colony PCR and DNA sequencing. The BL21 F. coli transformed by the GST-AIR expression plasmid was used to express the GST-AIK fusion protein with IPTG induction and the induction conditions were optimized. GST-AIR fusion protein was purified by glutathione magnetic beads, followed by rTEV cleavage to remove GST tag and MTS assay to test the growth inhibition activity of the recombinant AIR on human leukemia HL-60 cells. We found that a high level of soluble expression of GST-AIK protein (more than 30% out of the total bacterial proteins) was achieved upon 0.1 mmol/L ITPG induction for 4 h at 37 °C in the transformed BL21 F. coli with starting OD₆₀₀ at 1.0. Through GST affinity purification and rTEV cleavage, the purity of the resulting recombinant AIK was greater than 95%. And the MTS assays on HL-60 cells confirmed that the recombinant AIK retains an antitumor activity at a level similar to the chemically synthesized AIK. Taken together, we have established a method for expression and purification of recombinant AIK with a potent activity against tumor cells, which will be beneficial for the large-scale production and application of recombinant AIK in the future. PMID:27093838

  3. Phosphorylation of Alzheimer disease amyloid precursor peptide by protein kinase C and Ca sup 2+ /calmodulin-dependent protein kinase II

    SciTech Connect

    Gandy, S.; Czernik, A.J.; Greengard, P. )

    1988-08-01

    The amino acid sequence of the Alzheimer disease amyloid precursor (ADAP) has been deduced from the corresponding cDNA, and hydropathy analysis of the sequence suggest a receptor-like structure with a single transmembrane domain. The putative cytoplasmic domain of ADAP contains potential sites for serine and threonine phosphorylation. In the present study, synthetic peptides derived from this domain were used as model substrates for various purified protein kinases. Protein kinase C rapidly catalyzed the phosphorylation of a peptide corresponding to amino acid residues 645-661 of ADAP. Ca{sup 2+}/calmodulin-dependent protein kinase II phosphorylated ADAP peptide (645-661) on Thr-654 and Ser-655. Using rat cerebral cortex synaptosomes prelabeled with {sup 32}P{sub i}, a {sup 32}P-labeled phosphoprotein of {approx}135 kDa was immunoprecipitated by using antisera prepared against ADAP peptide(597-624), consistent with the possibility that the holoform of ADAP in rat brain is a phosphoprotein. Based on analogy with the effect of phosphorylation by protein kinase C of juxtamembrane residues in the cytoplasmic domain of the epidermal growth factor receptor and the interleukin 2 receptor, phosphorylation of ADAP may target it for internalization.

  4. Influence of Amino Acid Composition and Phosphorylation on the Ion Yields of Peptides in MALDI-MS

    NASA Astrophysics Data System (ADS)

    Asakawa, Daiki; Moriguchi, Shohey; Takayama, Mitsuo

    2012-01-01

    The influence of arginine (Arg), lysine (Lys), and phenylalanine (Phe) residues and phosphorylation on the molecular ion yields of model peptides have been quantitatively studied using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in both positive- and negative-ion mode. The results obtained from these experiments have been interpreted from the standpoint of two different components, namely, desorption and ionization, on the basis of the physicochemical properties of constituent amino acids of the model peptides. The presence of basic residues such as Arg and Lys enhanced the ion yields of protonated molecules [M + H]+. An N-terminal rather than a C-terminal Arg residue was advantageous for the formation of both [M + H]+ and [M - H]-. The presence of the Phe residue resulted in the increase of the ion yields of both [M + H]+ and [M - H]-. In contrast, the presence of phosphate group(s) contributed to the suppression of the yields of both [M + H]+ and [M - H]- due to the loss of phosphate group. The detection limits for both [M + H]+ and [M - H]- of model peptides have been evaluated.

  5. Identification of a Peptide Toxin from Grammostola spatulata Spider Venom That Blocks Cation-Selective Stretch-Activated Channels

    PubMed Central

    Suchyna, Thomas M.; Johnson, Janice H.; Hamer, Katherine; Leykam, Joseph F.; Gage, Douglas A.; Clemo, Henry F.; Baumgarten, Clive M.; Sachs, Frederick

    2000-01-01

    We have identified a 35 amino acid peptide toxin of the inhibitor cysteine knot family that blocks cationic stretch-activated ion channels. The toxin, denoted GsMTx-4, was isolated from the venom of the spider Grammostola spatulata and has <50% homology to other neuroactive peptides. It was isolated by fractionating whole venom using reverse phase HPLC, and then assaying fractions on stretch-activated channels (SACs) in outside-out patches from adult rat astrocytes. Although the channel gating kinetics were different between cell-attached and outside-out patches, the properties associated with the channel pore, such as selectivity for alkali cations, conductance (∼45 pS at −100 mV) and a mild rectification were unaffected by outside-out formation. GsMTx-4 produced a complete block of SACs in outside-out patches and appeared specific since it had no effect on whole-cell voltage-sensitive currents. The equilibrium dissociation constant of ∼630 nM was calculated from the ratio of association and dissociation rate constants. In hypotonically swollen astrocytes, GsMTx-4 produces ∼40% reduction in swelling-activated whole-cell current. Similarly, in isolated ventricular cells from a rabbit dilated cardiomyopathy model, GsMTx-4 produced a near complete block of the volume-sensitive cation-selective current, but did not affect the anion current. In the myopathic heart cells, where the swell-induced current is tonically active, GsMTx-4 also reduced the cell size. This is the first report of a peptide toxin that specifically blocks stretch-activated currents. The toxin affect on swelling-activated whole-cell currents implicates SACs in volume regulation. PMID:10779316

  6. Translocation of cationic amphipathic peptides across the membranes of pure phospholipid giant vesicles.

    PubMed

    Wheaten, Sterling A; Ablan, Francis D O; Spaller, B Logan; Trieu, Julie M; Almeida, Paulo F

    2013-11-01

    The ability of amphipathic polypeptides with substantial net positive charges to translocate across lipid membranes is a fundamental problem in physical biochemistry. These peptides should not passively cross the bilayer nonpolar region, but they do. Here we present a method to measure peptide translocation and test it on three representative membrane-active peptides. In samples of giant unilamellar vesicles (GUVs) prepared by electroformation, some GUVs enclose inner vesicles. When these GUVs are added to a peptide solution containing a membrane-impermeant fluorescent dye (carboxyfluorescein), the peptide permeabilizes the outer membrane, and dye enters the outer GUV, which then exhibits green fluorescence. The inner vesicles remain dark if the peptide does not cross the outer membrane. However, if the peptide translocates, it permeabilizes the inner vesicles as well, which then show fluorescence. We also measure translocation, simultaneously on the same GUV, by the appearance of fluorescently labeled peptides on the inner vesicle membranes. All three peptides examined are able to translocate, but to different extents. Peptides with smaller Gibbs energies of insertion into the membrane translocate more easily. Further, translocation and influx occur broadly over the same period, but with very different kinetics. Translocation across the outer membrane follows approximately an exponential rise, with a characteristic time of 10 min. Influx occurs more abruptly. In the outer vesicle, influx happens before most of the translocation. However, some peptides cross the membrane before any influx is observed. In the inner vesicles, influx occurs abruptly sometime during peptide translocation across the membrane of the outer vesicle. PMID:24152283

  7. Peptide-lanthanide cation equilibria in aqueous phase. I. Bound shifts for L-carnosine-praseodymium complexes

    NASA Astrophysics Data System (ADS)

    Mossoyan, J.; Asso, M.; Benlian, D.

    L-Carnosine complexes of Pr 3+ were characterized in aqueous solution by 1H NMR and potentiometric titration. A rigorous treatment of chemical shifts and pH variation data with lanthanide concentration is presented. Two different forms of the peptide ligand, forming simultaneously two complexes, were taken into account. At low pH values the cation is only coordinated at the carboxylate site of the ligand in a weak complex ( β2 = 6) whereas in neutral solution a stronger complex ( β1 = 37) is present as a consequence of the deprotonation of the imidazole ring. The computation of induced bound shifts † 2 and Δ1 for resonating nuclei of the peptide in both forms yields consistent figures. These provide the experimental basis for a conformational model which is usually not obtainable for labile complexes with low stability constants.

  8. Stability and efficacy of synthetic cationic antimicrobial peptides nebulized using high frequency acoustic waves.

    PubMed

    Wang, Ying; Rezk, Amgad R; Khara, Jasmeet Singh; Yeo, Leslie Y; Ee, Pui Lai Rachel

    2016-05-01

    Surface acoustic wave (SAW), a nanometer amplitude electroelastic wave generated and propagated on low-loss piezoelectric substrates (such as LiNbO3), is an extremely efficient solid-fluid energy transfer mechanism. The present study explores the use of SAW nebulization as a solution for effective pulmonary peptide delivery. In vitro deposition characteristics of the nebulized peptides were determined using a Next Generation Cascade Impactor. 70% of the peptide-laden aerosols generated were within a size distribution favorable for deep lung distribution. The integrity of the nebulized peptides was found to be retained, as shown via mass spectrometry. The anti-mycobacterial activity of the nebulized peptides was found to be uncompromised compared with their non-nebulized counterparts, as demonstrated by the minimum inhibition concentration and the colony forming inhibition activity. The peptide concentration and volume recoveries for the SAW nebulizer were significantly higher than 90% and found to be insensitive to variation in the peptide sequences. These results demonstrate the potential of the SAW nebulization platform as an effective delivery system of therapeutic peptides through the respiratory tract to the deep lung. PMID:27375820

  9. Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

    SciTech Connect

    Berbís, M. Álvaro; André, Sabine; Cañada, F. Javier; Pipkorn, Rüdiger; Ippel, Hans; Mayo, Kevin H.; Kübler, Dieter; Gabius, Hans-Joachim; Jiménez-Barbero, Jesús

    2014-01-03

    Highlights: •Galectin-3 is composed of a carbohydrate recognition domain and an N-terminal tail. •Synthetic peptides derived from the tail are shown to interact with the CRD. •This interaction is modulated by Ser- and Tyr-phosphorylation of the peptides. -- Abstract: Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with {sup 15}N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.

  10. Melanoma cell surface-expressed phosphatidylserine as a therapeutic target for cationic anticancer peptide, temporin-1CEa.

    PubMed

    Wang, Che; Chen, Yin-Wang; Zhang, Liang; Gong, Xian-Ge; Zhou, Yang; Shang, De-Jing

    2016-07-01

    We have previously reported that temporin-1CEa, a cationic antimicrobial peptide, exerts preferential cytotoxicity toward cancer cells. However, the exact molecular mechanism for this cancer-selectivity is still largely unknown. Here, we found that the negatively charged phosphatidylserine (PS) expressed on cancer cell surface serves as a target for temporin-1CEa. Our results indicate that human A375 melanoma cells express 50-fold more PS than non-cancerous HaCaT cells. The expression of cell surface PS in various cancer cell lines closely correlated with their ability to be recognized, bound and killed by temporin-1CEa. Additionally, the cytotoxicity of temporin-1CEa against A375 cells can be ameliorated by annexin V, which binds to cell surface PS with high affinity. Moreover, the data of isothermal titration calorimetry assay further confirmed a direct binding of temporin-1CEa to PS, at a ratio of 1:5 (temporin-1CEa:PS). Interestingly, the circular dichroism spectra analysis using artificial biomembrane revealed that PS not only provides electrostatic attractive sites for temporin-1CEa but also confers the membrane-bound temporin-1CEa to form α-helical structure, therefore, enhances the affinity and membrane disrupting ability of temporin-1CEa. In summary, these findings suggested that the melanoma cells expressed PS may serve as a promising target for temporin-1CEa or other cationic anticancer peptides. PMID:26596643

  11. Improved detection of multi-phosphorylated peptides in the presence of phosphoric acid in liquid chromatography/mass spectrometry

    SciTech Connect

    Kim, Jeongkwon; Camp, David G.; Smith, Richard D.

    2004-02-18

    In contrast to lower phosphorylation states (e.g., the tryptic monophosphopeptide FQpSEEQQQTEDELQDK from bovine -casein), the specific detection of multi-phosphorylated peptides (e.g. the tetraphosphopeptide RELEELNVPGEIVEpSLpSpSpSEESITR from tryptic digestion of bovine -casein) has often been problematic for liquid chromatography-mass spectrometry analysis due to their high affinity for adsorption to exposed surfaces. We observed an enhancement in the overall detection of phosphopeptides upon addition of phosphoric acid (0.1% to 1.0%) to the sample solution; a 10-fold increase in sensitivity was measured for the detection of two tryptic phosphopeptides as well as a significant improvement in the detection of the tetraphosphopeptide. Using capillary LC with an ion trap tandem mass spectrometer for detection and identification, the achievable detection limits were 50 fmol and 50 pmol for the monophosphopeptide and the tetraphosphopeptide, respectively. Phosphoric acid is believed to act as a blocking agent to available silanol groups on both the silica capillary surface and the C-18-bonded silica surface.

  12. Chicken NK-lysin is an alpha-helical cationic peptide that exerts its antibacterial activity through damage of bacterial cell membranes.

    PubMed

    Lee, Mi Ok; Jang, Hyun-Jun; Han, Jae Yong; Womack, James E

    2014-04-01

    The antimicrobial peptides (AMP) are important elements of the first line of defense against pathogens in animals, and an important constituent of innate immunity. Antimicrobial peptides act on a broad spectrum of microbial organisms. NK-Lysin is a cationic antibacterial peptide that was originally isolated from porcine intestinal tissue based on its antibacterial activity. We synthesized peptides corresponding to each helical region of chicken NK-lysin and analyzed their secondary structures in addition to their antimicrobial activity. Circular dichroism spectroscopy of the synthetic chicken NK-lysin (cNK-78) and 4 small peptides in negatively charged liposomes demonstrated transition in the conformation of α-helical peptides relative to the charged environment. Chicken NK-lysin inhibits the growth of a representative gram-negative bacterium, Escherichia coli. The antimicrobial activity of 2 peptides designated H23 and H34 was similar to that of mature NK-lysin, cNK-78. Microscopic analyses revealed the death of bacterium with disrupted membranes after peptide treatment, suggesting that chicken NK-lysin, an alpha-helical cationic peptide, exerts its antimicrobial activity by damaging the bacterial cell membrane. PMID:24706963

  13. Conformational Flexibility Determines Selectivity and Antibacterial, Antiplasmodial, and Anticancer Potency of Cationic α-Helical Peptides*

    PubMed Central

    Vermeer, Louic S.; Lan, Yun; Abbate, Vincenzo; Ruh, Emrah; Bui, Tam T.; Wilkinson, Louise J.; Kanno, Tokuwa; Jumagulova, Elmira; Kozlowska, Justyna; Patel, Jayneil; McIntyre, Caitlin A.; Yam, W. C.; Siu, Gilman; Atkinson, R. Andrew; Lam, Jenny K. W.; Bansal, Sukhvinder S.; Drake, Alex F.; Mitchell, Graham H.; Mason, A. James

    2012-01-01

    We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens. PMID:22869378

  14. C-type natriuretic peptide activates a non-selective cation current in acutely isolated rat cardiac fibroblasts via natriuretic peptide C receptor-mediated signalling.

    PubMed

    Rose, R A; Hatano, N; Ohya, S; Imaizumi, Y; Giles, W R

    2007-04-01

    In the heart, fibroblasts play an essential role in the deposition of the extracellular matrix and they also secrete a number of hormonal factors. Although natriuretic peptides, including C-type natriuretic peptide (CNP) and brain natriuretic peptide, have antifibrotic effects on cardiac fibroblasts, the effects of CNP on fibroblast electrophysiology have not been examined. In this study, acutely isolated ventricular fibroblasts from the adult rat were used to measure the effects of CNP (2 x 10(-8) M) under whole-cell voltage-clamp conditions. CNP, as well as the natriuretic peptide C receptor (NPR-C) agonist cANF (2 x 10(-8) M), significantly increased an outwardly rectifying non-selective cation current (NSCC). This current has a reversal potential near 0 mV. Activation of this NSCC by cANF was abolished by pre-treating fibroblasts with pertussis toxin, indicating the involvement of G(i) proteins. The cANF-activated NSCC was inhibited by the compounds Gd(3+), SKF 96365 and 2-aminoethoxydiphenyl borate. Quantitative RT-PCR analysis of mRNA from rat ventricular fibroblasts revealed the expression of several transient receptor potential (TRP) channel transcripts. Additional electrophysiological analysis showed that U73122, a phospholipase C antagonist, inhibited the cANF-activated NSCC. Furthermore, the effects of CNP and cANF were mimicked by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG), independently of protein kinase C activity. These are defining characteristics of specific TRPC channels. More detailed molecular analysis confirmed the expression of full-length TRPC2, TRPC3 and TRPC5 transcripts. These data indicate that CNP, acting via the NPR-C receptor, activates a NSCC that is at least partially carried by TRPC channels in cardiac fibroblasts. PMID:17204501

  15. A new cryptic cationic antimicrobial peptide from human apolipoprotein E with antibacterial activity and immunomodulatory effects on human cells.

    PubMed

    Pane, Katia; Sgambati, Valeria; Zanfardino, Anna; Smaldone, Giovanni; Cafaro, Valeria; Angrisano, Tiziana; Pedone, Emilia; Di Gaetano, Sonia; Capasso, Domenica; Haney, Evan F; Izzo, Viviana; Varcamonti, Mario; Notomista, Eugenio; Hancock, Robert E W; Di Donato, Alberto; Pizzo, Elio

    2016-06-01

    Cationic antimicrobial peptides (AMPs) possess fast and broad-spectrum activity against both Gram-negative and Gram-positive bacteria, as well as fungi. It has become increasingly evident that many AMPs, including those that derive from fragments of host proteins, are multifunctional and able to mediate various immunomodulatory functions and angiogenesis. Among these, synthetic apolipoprotein-derived peptides are safe and well tolerated in humans and have emerged as promising candidates in the treatment of various inflammatory conditions. Here, we report the characterization of a new AMP corresponding to residues 133-150 of human apolipoprotein E. Our results show that this peptide, produced either by chemical synthesis or by recombinant techniques in Escherichia coli, possesses a broad-spectrum antibacterial activity. As shown for several other AMPs, ApoE (133-150) is structured in the presence of TFE and of membrane-mimicking agents, like SDS, or bacterial surface lipopolysaccharide (LPS), and an anionic polysaccharide, alginate, which mimics anionic capsular exo-polysaccharides of several pathogenic microorganisms. Noteworthy, ApoE (133-150) is not toxic toward several human cell lines and triggers a significant innate immune response, assessed either as decreased expression levels of proinflammatory cytokines in differentiated THP-1 monocytic cells or by the induction of chemokines released from PBMCs. This novel bioactive AMP also showed a significant anti-inflammatory effect on human keratinocytes, suggesting its potential use as a model for designing new immunomodulatory therapeutics. PMID:27028511

  16. Antifungal Activity of a Synthetic Cationic Peptide against the Plant Pathogens Colletotrichum graminicola and Three Fusarium Species.

    PubMed

    Johnson, Eric T; Evans, Kervin O; Dowd, Patrick F

    2015-09-01

    A small cationic peptide (JH8944) was tested for activity against a number of pathogens of agricultural crops. JH8944 inhibited conidium growth in most of the tested plant pathogens with a dose of 50 μg/ml, although one isolate of Fusarium oxysporum was inhibited at 5 μg/ml of JH8944. Most conidia of Fusarium graminearum were killed within 6 hours of treatment with 50 μg/ml of JH8944. Germinating F. graminearum conidia required 238 μg/ml of JH8944 for 90% growth inhibition. The peptide did not cause any damage to tissues surrounding maize leaf punctures when tested at a higher concentration of 250 μg/ml even after 3 days. Liposomes consisting of phosphatidylglycerol were susceptible to leakage after treatment with 25 and 50 μg/ml of JH8944. These experiments suggest this peptide destroys fungal membrane integrity and could be utilized for control of crop fungal pathogens. PMID:26361481

  17. Antifungal Activity of a Synthetic Cationic Peptide against the Plant Pathogens Colletotrichum graminicola and Three Fusarium Species

    PubMed Central

    Johnson, Eric T.; Evans, Kervin O.; Dowd, Patrick F.

    2015-01-01

    A small cationic peptide (JH8944) was tested for activity against a number of pathogens of agricultural crops. JH8944 inhibited conidium growth in most of the tested plant pathogens with a dose of 50 μg/ml, although one isolate of Fusarium oxysporum was inhibited at 5 μg/ml of JH8944. Most conidia of Fusarium graminearum were killed within 6 hours of treatment with 50 μg/ml of JH8944. Germinating F. graminearum conidia required 238 μg/ml of JH8944 for 90% growth inhibition. The peptide did not cause any damage to tissues surrounding maize leaf punctures when tested at a higher concentration of 250 μg/ml even after 3 days. Liposomes consisting of phosphatidylglycerol were susceptible to leakage after treatment with 25 and 50 μg/ml of JH8944. These experiments suggest this peptide destroys fungal membrane integrity and could be utilized for control of crop fungal pathogens. PMID:26361481

  18. The effect of thiol functional group incorporation into cationic helical peptides on antimicrobial activities and spectra.

    PubMed

    Wiradharma, Nikken; Khan, Majad; Yong, Lin-Kin; Hauser, Charlotte A E; Seow, See Voon; Zhang, Shuguang; Yang, Yi-Yan

    2011-12-01

    Antimicrobial peptides (AMP) have been proposed as blueprints for the development of new antimicrobial agents for the treatment of drug resistant infections. A series of synthetic AMPs capable of forming α-helical structures and containing free-sulfhydryl groups are designed in this study ((LLKK)(2)C, C(LLKK)(2)C, (LLKK)(3)C, C(LLKK)(3)C). In particular, the AMP with 2 cysteine residues at the terminal ends of the peptide and 2 repeat units of LLKK, i.e., C(LLKK)(2)C, has been demonstrated to have high selectivity towards a wide range of microbes from Gram-positive Bacillus subtilis, Gram-negative Escherichia coli, Pseudomonas aerogenosa, and yeast Candida albicans over red blood cells. At the MIC levels, this peptide does not induce significant hemolysis, and its MIC values occur at the concentration of more than 10 times of their corresponding 50% hemolysis concentrations (HC(50)). Microscopy studies suggest that this peptide kills microbial cells by inducing pores of ∼20-30 nm in size in microbial membrane on a short time scale, which further develops to grossly damaged membrane envelope on a longer time scale. Multiple treatments of microbes with this peptide at sub MIC concentration do not induce resistance, even up to passage 10. However, the same treatment with conventional antibiotics penicillin G or ciprofloxacin easily develop resistance in the treated microbes. In addition, the peptides are shown not to induce secretion of IFN-γ and TNF-α in human monocytes as compared to lipopolysaccharide, which implies additional safety aspects of the peptides to be used as both systemic and topical antimicrobial agents. Therefore, this study provides an excellent basis to develop promising antimicrobial agents that possess a broad range of antimicrobial activities with less susceptibility for development of drug resistance. PMID:21906803

  19. Human neutrophil formyl peptide receptor phosphorylation and the mucosal inflammatory response

    PubMed Central

    Leoni, Giovanna; Gripentrog, Jeannie; Lord, Connie; Riesselman, Marcia; Sumagin, Ronen; Parkos, Charles A.; Nusrat, Asma; Jesaitis, Algirdas J.

    2015-01-01

    Bacterial/mitochondrial fMLF analogs bind FPR1, driving accumulation/activation of PMN at sites of infection/injury, while promoting wound healing in epithelia. We quantified levels of UFPR1 and TFPR1 in isolated PMN by use of phosphosensitive NFPRb and phosphorylation-independent NFPRa antibodies. UFPR1 and total TFPR were assessed inflamed mucosa, observed in human IBD. In isolated PMN after fMLF stimulation, UFPR1 declined 70% (fMLFEC50 = 11 ± 1 nM; t1/2 = 15 s) and was stable for up to 4 h, whereas TFPR1 changed only slightly. Antagonists (tBoc-FLFLF, CsH) and metabolic inhibitor NaF prevented the fMLF-dependent UFPR1 decrease. Annexin A1 fragment Ac2-26 also induced decreases in UFPR1 (Ac2-26EC50 ∼ 3 µM). Proinflammatory agents (TNF-α, LPS), phosphatase inhibitor (okadaic acid), and G-protein activator (MST) modestly increased fMLFEC50, 2- to 4-fold, whereas PTX, Ca2+ chelators (EGTA/BAPTA), H2O2, GM-CSF, ENA-78, IL-1RA, and LXA4 had no effect. Aggregation-inducing PAF, however, strongly inhibited fMLF-stimulated UFPR1 decreases. fMLF-driven PMN also demonstrated decreased UFPR1 after traversing monolayers of cultured intestinal epithelial cells, as did PMN in intestinal mucosal samples, demonstrating active inflammation from UC patients. Total TFPR remained high in PMN within inflamed crypts, migrating through crypt epithelium, and in the lamina propria-adjoining crypts, but UFPR1 was only observed at some peripheral sites on crypt aggregates. Loss of UFPR1 in PMN results from C-terminal S/T phosphorylation. Our results suggest G protein–insensitive, fMLF-dependent FPR1 phosphorylation in isolated suspension PMN, which may manifest in fMLF-driven transmigration and potentially, in actively inflamed tissues, except at minor discrete surface locations of PMN-containing crypt aggregates. PMID:25395303

  20. Improved intra-array and interarray normalization of peptide microarray phosphorylation for phosphorylome and kinome profiling by rational selection of relevant spots.

    PubMed

    Scholma, Jetse; Fuhler, Gwenny M; Joore, Jos; Hulsman, Marc; Schivo, Stefano; List, Alan F; Reinders, Marcel J T; Peppelenbosch, Maikel P; Post, Janine N

    2016-01-01

    Massive parallel analysis using array technology has become the mainstay for analysis of genomes and transcriptomes. Analogously, the predominance of phosphorylation as a regulator of cellular metabolism has fostered the development of peptide arrays of kinase consensus substrates that allow the charting of cellular phosphorylation events (often called kinome profiling). However, whereas the bioinformatical framework for expression array analysis is well-developed, no advanced analysis tools are yet available for kinome profiling. Especially intra-array and interarray normalization of peptide array phosphorylation remain problematic, due to the absence of "housekeeping" kinases and the obvious fallacy of the assumption that different experimental conditions should exhibit equal amounts of kinase activity. Here we describe the development of analysis tools that reliably quantify phosphorylation of peptide arrays and that allow normalization of the signals obtained. We provide a method for intraslide gradient correction and spot quality control. We describe a novel interarray normalization procedure, named repetitive signal enhancement, RSE, which provides a mathematical approach to limit the false negative results occuring with the use of other normalization procedures. Using in silico and biological experiments we show that employing such protocols yields superior insight into cellular physiology as compared to classical analysis tools for kinome profiling. PMID:27225531

  1. Improved intra-array and interarray normalization of peptide microarray phosphorylation for phosphorylome and kinome profiling by rational selection of relevant spots

    PubMed Central

    Scholma, Jetse; Fuhler, Gwenny M.; Joore, Jos; Hulsman, Marc; Schivo, Stefano; List, Alan F.; Reinders, Marcel J. T.; Peppelenbosch, Maikel P.; Post, Janine N.

    2016-01-01

    Massive parallel analysis using array technology has become the mainstay for analysis of genomes and transcriptomes. Analogously, the predominance of phosphorylation as a regulator of cellular metabolism has fostered the development of peptide arrays of kinase consensus substrates that allow the charting of cellular phosphorylation events (often called kinome profiling). However, whereas the bioinformatical framework for expression array analysis is well-developed, no advanced analysis tools are yet available for kinome profiling. Especially intra-array and interarray normalization of peptide array phosphorylation remain problematic, due to the absence of “housekeeping” kinases and the obvious fallacy of the assumption that different experimental conditions should exhibit equal amounts of kinase activity. Here we describe the development of analysis tools that reliably quantify phosphorylation of peptide arrays and that allow normalization of the signals obtained. We provide a method for intraslide gradient correction and spot quality control. We describe a novel interarray normalization procedure, named repetitive signal enhancement, RSE, which provides a mathematical approach to limit the false negative results occuring with the use of other normalization procedures. Using in silico and biological experiments we show that employing such protocols yields superior insight into cellular physiology as compared to classical analysis tools for kinome profiling. PMID:27225531

  2. Unblocking the Sink: Improved CID-Based Analysis of Phosphorylated Peptides by Enzymatic Removal of the Basic C-Terminal Residue

    NASA Astrophysics Data System (ADS)

    Lanucara, Francesco; Chi Hoo Lee, Dave; Eyers, Claire E.

    2013-12-01

    A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H3PO4), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H3PO4 elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H3PO4 upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available.

  3. Mechanistic Examination of Cβ–Cγ Bond Cleavages of Tryptophan Residues during Dissociations of Molecular Peptide Radical Cations

    SciTech Connect

    Song, Tao; Ma, Ching-Yung; Chu, Ivan K.; Siu, Chi-Kit; Laskin, Julia

    2013-02-14

    In this study, we used collision-induced dissociation (CID) to examine the gas-phase fragmentations of [GnW]•+ (n = 2-4) and [GXW]•+ (X = C, S, L, F, Y, Q) species. The Cβ–Cγ bond cleavage of a C-terminal decarboxylated tryptophan residue ([M - CO2]•+) can generate [M - CO2 - 116]+, [M - CO2 - 117]•+, and [1H-indole]•+ (m/z 117) species as possible product ions. Competition between the formation of [M - CO2 - 116]+ and [1H-indole]•+ systems implies the existence of a proton-bound dimer formed between the indole ring and peptide backbone. Formation of such a proton-bound dimer is facile via a protonation of the tryptophan γ-carbon atom as suggested by density functional theory (DFT) calculations. DFT calculations also suggested the initially formed ion 2--the decarboxylated species that is active against Cβ–Cγ bond cleavage -can efficiently isomerize to form a more-stable -radical isomer (ion 9) as supported by Rice-Ramsperger-Kassel-Marcus (RRKM) modeling. The Cβ–Cγ bond cleavage of a tryptophan residue also can occur directly from peptide radical cations containing a basic residue. CID of [WGnR]•+ (n = 1-3) radical cations consistently resulted in predominant formation of [M-116]+ product ions. It appears that the basic arginine residue tightly sequesters the proton and allows the charge-remote Cβ–Cγ bond cleavage to prevail over the charge-directed one. DFT calculations predicted the barrier for the former is 6.2 kcal mol -1 lower than that of the latter. Furthermore, the pathway involving a salt-bridge intermediate also was accessible during such a bond cleavage event.

  4. Atrial natriuretic peptide degradation by CPA47 cells - Evidence for a divalent cation-independent cell-surface proteolytic activity

    NASA Technical Reports Server (NTRS)

    Frost, S. J.; Chen, Y. M.; Whitson, P. A.

    1992-01-01

    Atrial natriuretic peptide (ANP) is rapidly cleared and degraded in vivo. Nonguanylate-cyclase receptors (C-ANPR) and a metalloproteinase, neutral endopeptidase (EC 3.4.24.11) (NEP 24.11), are thought to be responsible for its metabolism. We investigated the mechanisms of ANP degradation by an endothelial-derived cell line, CPA47. CPA47 cells degraded 88 percent of 125I-ANP after 1 h at 37 degrees C as determined by HPLC. Medium preconditioned by these cells degraded 41 percent of the 125I-ANP, and this activity was inhibited by a divalent cation chelator, EDTA. Furthermore, a cell-surface proteolytic activity degraded 125I-ANP in the presence of EDTA when receptor-mediated endocytosis was inhibited either by low temperature (4 degrees C) or by hyperosmolarity at 37 degrees C. The metalloproteinase, NEP 24.11, is unlikely to be the cell-surface peptidase because 125I-ANP is degraded by CPA47 cells at 4 degrees C in the presence of 5 mM EDTA. These data indicate that CPA47 cells can degrade ANP by a novel divalent cation-independent cell-surface proteolytic activity.

  5. Studies on the autophosphorylation of the insulin receptor from human placenta. Analysis of the sites phosphorylated by two-dimensional peptide mapping.

    PubMed Central

    Tavaré, J M; Denton, R M

    1988-01-01

    1. A partially purified preparation of human placental insulin receptors was incubated with [gamma-32P]ATP in the presence or absence of insulin. The 32P-labelled insulin-receptor beta-subunits were then isolated, cleaved with trypsin followed by protease V8 and the [32P]phosphopeptides generated were analysed by thin layer electrophoresis and chromatography. This approach revealed that insulin stimulates autophosphorylation of the insulin-receptor beta-subunit in vitro on at least seven tyrosine residues distributed among three distinct domains. 2. One domain (domain 2), containing tyrosine residues 1146, 1150 and 1151 was the most rapidly phosphorylated and could be recovered as mono-, di- and triphosphorylated peptides cleaved by trypsin at Arg-1143 and either Lys-1153 or Lys-1156. Multiple phosphorylation of this domain appears to partially inhibit the cleavage at Lys-1153 by trypsin. 3. In a second domain (domain 3) containing two phosphorylated tyrosine residues at positions 1316 and 1322 the tyrosines were phosphorylated more slowly than those in domain 2. This domain is close to the C-terminus of the beta-subunit polypeptide chain. 4. At least two further tyrosine residues appeared to be phosphorylated after those in domains 2 and 3. These residues probably residue within a domain lying in close proximity to the inner face of the plasma membrane containing tyrosines 953, 960 and 972, but conclusive evidence is still required. 5. The two-dimensional thin-layer analysis employed in this study to investigate insulin-receptor phosphorylation has several advantages over previous methods based on reverse-phase chromatography. It allows greater resolution of 32P-labelled tryptic peptides and, when coupled to radioautography, is considerably more sensitive. The approach can be readily adapted to study phosphorylation of the insulin receptor within intact cells. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:3166375

  6. LL37 and Cationic Peptides Enhance TLR3 Signaling by Viral Double-stranded RNAs

    PubMed Central

    Lai, Yvonne; Adhikarakunnathu, Sreedevi; Bhardwaj, Kanchan; Ranjith-Kumar, C. T.; Wen, Yahong; Jordan, Jarrat L.; Wu, Linda H.; Dragnea, Bogdan; Mateo, Lani San; Kao, C. Cheng

    2011-01-01

    Background Toll-like Receptor 3 (TLR3) detects viral dsRNA during viral infection. However, most natural viral dsRNAs are poor activators of TLR3 in cell-based systems, leading us to hypothesize that TLR3 needs additional factors to be activated by viral dsRNAs. The anti-microbial peptide LL37 is the only known human member of the cathelicidin family of anti-microbial peptides. LL37 complexes with bacterial lipopolysaccharide (LPS) to prevent activation of TLR4, binds to ssDNA to modulate TLR9 and ssRNA to modulate TLR7 and 8. It synergizes with TLR2/1, TLR3 and TLR5 agonists to increase IL8 and IL6 production. This work seeks to determine whether LL37 enhances viral dsRNA recognition by TLR3. Methodology/Principal Findings Using a human bronchial epithelial cell line (BEAS2B) and human embryonic kidney cells (HEK 293T) transiently transfected with TLR3, we found that LL37 enhanced poly(I:C)-induced TLR3 signaling and enabled the recognition of viral dsRNAs by TLR3. The presence of LL37 also increased the cytokine response to rhinovirus infection in BEAS2B cells and in activated human peripheral blood mononuclear cells. Confocal microscopy determined that LL37 could co-localize with TLR3. Electron microscopy showed that LL37 and poly(I:C) individually formed globular structures, but a complex of the two formed filamentous structures. To separate the effects of LL37 on TLR3 and TLR4, other peptides that bind RNA and transport the complex into cells were tested and found to activate TLR3 signaling in response to dsRNAs, but had no effect on TLR4 signaling. This is the first demonstration that LL37 and other RNA-binding peptides with cell penetrating motifs can activate TLR3 signaling and facilitate the recognition of viral ligands. Conclusions/Significance LL37 and several cell-penetrating peptides can enhance signaling by TLR3 and enable TLR3 to respond to viral dsRNA. PMID:22039520

  7. Concatemerization increases the inhibitory activity of short, cell-penetrating, cationic and tryptophan-rich antifungal peptides.

    PubMed

    López-García, Belén; Harries, Eleonora; Carmona, Lourdes; Campos-Soriano, Lidia; López, José Javier; Manzanares, Paloma; Gandía, Mónica; Coca, María; Marcos, Jose F

    2015-10-01

    There are short cationic and tryptophan-rich antifungal peptides such as the hexapeptide PAF26 (RKKWFW) that have selective toxicity and cell penetration properties against fungal cells. This study demonstrates that concatemeric peptides with tandem repeats of the heptapeptide PAF54 (which is an elongated PAF26 sequence) show increased fungistatic and bacteriostatic activities while maintaining the absence of hemolytic activity of the monomer. The increase in antimicrobial activity of the double-repeated PAF sequences (diPAFs), compared to the nonrepeated PAF, was higher (4-8-fold) than that seen for the triple-repeated sequences (triPAFs) versus the diPAFs (2-fold). However, concatemerization diminished the fungicidal activity against quiescent spores of the filamentous fungus Penicillium digitatum. Peptide solubility and sensitivity to proteolytic degradation were affected by the design of the concatemers: incorporation of the AGPA sequence hinge to separate PAF54 repeats increased solubility while the C-terminal addition of the KDEL sequence decreased in vitro stability. These results led to the design of the triPAF sequence PAF102 of 30 amino acid residues, with increased antimicrobial activity and minimal inhibitory concentration (MIC) value of 1-5 μM depending on the fungus. Further characterization of the mode-of-action of PAF102 demonstrated that it colocalizes first with the fungal cell wall, it is thereafter internalized in an energy dependent manner into hyphal cells of the filamentous fungus Fusarium proliferatum, and finally kills hyphal cells intracellularly. Therefore, PAF102 showed mechanistic properties against fungi similar to the parental PAF26. These observations are of high interest in the future development of PAF-based antimicrobial molecules optimized for their production in biofactories. PMID:25846331

  8. Conjugation of fatty acids with different lengths modulates the antibacterial and antifungal activity of a cationic biologically inactive peptide.

    PubMed

    Malina, Amir; Shai, Yechiel

    2005-09-15

    Many studies have shown that an amphipathic structure and a threshold of hydrophobicity of the peptidic chain are crucial for the biological function of AMPs (antimicrobial peptides). However, the factors that dictate their cell selectivity are not yet clear. In the present study, we show that the attachment of aliphatic acids with different lengths (10, 12, 14 or 16 carbon atoms) to the N-terminus of a biologically inactive cationic peptide is sufficient to endow the resulting lipopeptides with lytic activity against different cells. Mode-of-action studies were performed with model phospholipid membranes mimicking those of bacterial, mammalian and fungal cells. These include determination of the structure in solution and membranes by using CD and ATR-FTIR (attenuated total reflectance Fourier-transform infrared) spectroscopy, membrane leakage experiments and by visualizing bacterial and fungal damage via transmission electron microscopy. The results obtained reveal that: (i) the short lipopeptides (10 and 12 carbons atoms) are non-haemolytic, active towards both bacteria and fungi and monomeric in solution. (ii) The long lipopeptides (14 and 16 carbons atoms) are highly antifungal, haemolytic only at concentrations above their MIC (minimal inhibitory concentration) values and aggregate in solution. (iii) All the lipopeptides adopt a partial alpha-helical structure in 1% lysophosphatidylcholine and bacterial and mammalian model membranes. However, the two short lipopeptides contain a significant fraction of random coil in fungal membranes, in agreement with their reduced antifungal activity. (iv) All the lipopeptides have a membranolytic effect on all types of cells assayed. Overall, the results reveal that the length of the aliphatic chain is sufficient to control the pathogen specificity of the lipopeptides, most probably by controlling both the overall hydrophobicity and the oligomeric state of the lipopeptides in solution. Besides providing us with basic

  9. Reduced cytotoxicity and enhanced bioactivity of cationic antimicrobial peptides liposomes in cell cultures and 3D epidermis model against HSV.

    PubMed

    Ron-Doitch, Sapir; Sawodny, Beate; Kühbacher, Andreas; David, Mirjam M Nordling; Samanta, Ayan; Phopase, Jaywant; Burger-Kentischer, Anke; Griffith, May; Golomb, Gershon; Rupp, Steffen

    2016-05-10

    Cationic antimicrobial peptides (AMPs) are part of the innate immunity, and act against a wide variety of pathogenic microorganisms by perturbation of the microorganism's plasma membrane. Although attractive for clinical applications, these agents suffer from limited stability and activity in vivo, as well as non-specific interaction with host biological membranes, leading to cytotoxic adverse effects. We hypothesized that encapsulation of AMPs within liposomes could result in reduced cytotoxicity, and with enhanced stability as well as bioactivity against herpes simplex virus 1 (HSV-1). We formulated nano-sized liposomal formulations of LL-37 and indolicidin, and their physicochemical properties, cellular uptake, in vitro cytotoxicity and antiviral efficacy have been determined. Lower cytotoxicity of LL-37 liposomes was found in comparison to indolicidin liposomes attributed to the superior physicochemical properties, and to the different degree of interaction with the liposomal membrane. The disc-like shaped LL-37 liposomes (106.8±10.1nm, shelf-life stability of >1year) were taken up more rapidly and to a significantly higher extent than the free peptide by human keratinocyte cell line (HaCaT), remained intact within the cells, followed by release of the active peptide within the cytoplasm and migration of the vesicles' lipids to the plasma membrane. LL-37 liposomes were found significantly less toxic than both the free agent and liposomal indolicidin. In the new 3D epidermis model (immortalized primary keratinocytes) liposomal LL-37 treatment (>20μM), but not free LL-37, efficiently protected the epidermis, inhibiting HSV-1 infection. This positive antiviral effect was obtained with no cytotoxicity even at very high concentrations (400μM). Thus, the antiviral activity of encapsulated LL-37 was significantly improved, expanding its therapeutic window. Liposomal LL-37 appears to be a promising delivery system for HSV therapy. PMID:27012977

  10. Doubly Phosphorylated Peptide Vaccines to Protect Transgenic P301S Mice against Alzheimer’s Disease Like Tau Aggregation

    PubMed Central

    Richter, Monique; Mewes, Agneta; Fritsch, Manuela; Krügel, Ute; Hoffmann, Ralf; Singer, David

    2014-01-01

    Intracellular neurofibrillary tangles and extracellular senile plaques are potential targets for active and passive immunotherapies. In this study we used the transgenic mouse model P301S for active immunizations with peptide vaccines composed of a double phosphorylated tau neoepitope (pSer202/pThr205, pThr212/pSer214, pThr231/pSer235) and an immunomodulatory T cell epitope from the tetanus toxin or tuberculosis antigen Ag85B. Importantly, the designed vaccine combining Alzheimer’s disease (AD) specific B cell epitopes with foreign (bacterial) T cell epitopes induced fast immune responses with high IgG1 titers after prophylactic immunization that subsequently decreased over the observation period. The effectiveness of the immunization was surveyed by evaluating the animal behavior, as well as the pathology in the brain by biochemical and histochemical techniques. Immunized mice clearly lived longer with reduced paralysis than placebo-treated mice. Additionally, they performed significantly better in rotarod and beam walk tests at the age of 20 weeks, indicating that the disease development was slowed down. Forty-eight weeks old vaccinated mice passed the beam walk test significantly better than control animals, which together with the increased survival rates undoubtedly prove the treatment effect. In conclusion, the data provide strong evidence that active immune therapies can reduce toxic effects of deposits formed in AD. PMID:26344748

  11. Nitrilotriacetic acid-coated magnetic nanoparticles as affinity probes for enrichment of histidine-tagged proteins and phosphorylated peptides.

    PubMed

    Li, Yi-Cheng; Lin, Ya-Shiuan; Tsai, Pei-Jane; Chen, Cheng-Tai; Chen, Wei-Yu; Chen, Yu-Chie

    2007-10-01

    We herein demonstrate superparamagnetic Fe3O4 nanoparticles coated with nitrilotriacetic acid derivative (NTA) that can bind with different immobilized metal ions are capable of probing diverse target species. Immobilized Ni(II) on the surfaces of the NTA-magnetic nanoparticles have the capability of selectively trapping histidine (His)-tagged proteins such as a mutated streptopain tagged with 6x His, i.e., C192S (MW approximately 42 kDa), from cell lysates. Enrichment was achieved by vigorously mixing the sample solution and the nanoparticles by pipetting in and out of a sample vial for only 30 s. After enrichment, the probe-target species could be readily isolated by magnetic separation. We also characterized the proteins enriched on the affinity probes using on-probe tryptic digestion under microwave irradiation for only 2 min, followed by matrix-assisted laser desorption/ionization mass spectrometry analysis. Using this enrichment and tryptic digestion, the target species can be rapidly enriched and characterized, reducing the time required for carrying out the complete analysis to less than 10 min. Furthermore, when either Zr(IV) or Ga (III) ions are immobilized on the surfaces of the NTA-magnetic nanoparticles, the nanoparticles have the capability of selectively enriching phosphorylated peptides from tryptic digests of alpha-, beta-caseins, and diluted milk. The detection limit for the tryptic digests of alpha- and beta-caseins is approximately 50 fmol. PMID:17784733

  12. A Lack of Synergy Between Membrane-permeabilizing Cationic Antimicrobial Peptides and Conventional Antibiotics

    PubMed Central

    He, Jing; Starr, Charles G.; Wimley, William C.

    2014-01-01

    The rapid rise in morbidity and mortality from drug-resistant pathogenic bacteria has generated elevated interest in combination therapy using antimicrobial agents. Antimicrobial peptides (AMPs) are a candidate drug class to advance the development of combination therapies. Although the literature is ambiguous, the generic membrane disrupting activity of AMPs could enable them to synergize with conventional small molecule antibiotics by increasing access to the cell and by triggering membrane damage mediators. We used a novel assay to measure interactions, expressed as fractional inhibitory concentration (FIC), between four conventional antibiotics in combination with four well-characterized, membrane permeabilizing AMPs, against three species of Gram negative and Gram positive bacteria, giving 40 total pair-wise measurements of FIC with statistical uncertainties. We chose a set of AMPs that are known to dramatically disrupt the membranes of both Gram negative and Gram positive bacteria. Yet none of the membrane permeabilizing antimicrobial peptides interacted synergistically with any of the conventional antibiotic drugs in any organism. Large-scale membrane disruption and permeabilization by AMPs is not sufficient to drive them to act synergistically with chemical antibiotics in either Gram negative or Gram positive microbes. PMID:25268681

  13. Microinjection of CART peptide 55-102 into the nucleus accumbens blocks both the expression of behavioral sensitization and ERK phosphorylation by cocaine.

    PubMed

    Yoon, Hyung Shin; Kim, Seungwoo; Park, Hye Kyung; Kim, Jeong-Hoon

    2007-08-01

    The role of the biologically active CART 55-102 peptide in the nucleus accumbens (NAcc) in the expression of cocaine-induced behavioral sensitization was investigated. Rats were divided into four groups: one for saline and the other three for cocaine pre-exposures (15 mg/kg, i.p., once daily for 7 days). After 3 weeks of withdrawal, rats were microinjected into the NAcc either saline or CART 55-102 (1.0, or 2.5 microg/0.5 microl/side) followed by cocaine challenge (10 mg/kg, i.p.). Microinjection into the NAcc of CART 55-102 peptide dose-dependently blocked the expression of locomotor sensitization produced by repeated cocaine pre-exposures. Next, we further examined the effect of CART 55-102 microinjection on extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation levels in the NAcc. Additional four groups of rats were all cocaine pre-exposed and, after 3 weeks of withdrawal, they were either saline or cocaine challenged systemically following microinjection into the NAcc of either saline, CART 55-102 (2.5 microg/0.5 microl/side), or the equivalent mole amount of inactive CART 1-27 peptide. The increase of ERK1/2 phosphorylation levels in the NAcc by cocaine was completely blocked by CART 55-102 microinjection in this site, while it remains unaffected by inactive CART 1-27 peptide. These results suggest that CART 55-102 peptide in the NAcc may play a compensatory inhibitory role in the expression of behavioral sensitization by cocaine and these effects may be mediated by its inhibition of ERK1/2 phosphorylation in this site. PMID:17610912

  14. Large Scale Discovery and De Novo-Assisted Sequencing of Cationic Antimicrobial Peptides (CAMPs) by Microparticle Capture and Electron-Transfer Dissociation (ETD) Mass Spectrometry.

    PubMed

    Juba, Melanie L; Russo, Paul S; Devine, Megan; Barksdale, Stephanie; Rodriguez, Carlos; Vliet, Kent A; Schnur, Joel M; van Hoek, Monique L; Bishop, Barney M

    2015-10-01

    The identification and sequencing of novel cationic antimicrobial peptides (CAMPs) have proven challenging due to the limitations associated with traditional proteomics methods and difficulties sequencing peptides present in complex biomolecular mixtures. We present here a process for large-scale identification and de novo-assisted sequencing of newly discovered CAMPs using microparticle capture followed by tandem mass spectrometry equipped with electron-transfer dissociation (ETD). This process was initially evaluated and verified using known CAMPs with varying physicochemical properties. The effective parameters were then applied in the analysis of a complex mixture of peptides harvested from American alligator plasma using custom-made (Bioprospector) functionalized hydrogel particles. Here, we report the successful sequencing process for CAMPs that has led to the identification of 340 unique peptides and the discovery of five novel CAMPs from American alligator plasma. PMID:26327436

  15. Optical tweezers reveal a dynamic mechanical response of cationic peptide-DNA complexes

    NASA Astrophysics Data System (ADS)

    Lee, Amy; Zheng, Tai; Sucayan, Sarah; Chou, Szu-Ting; Tricoli, Lucas; Hustedt, Jason; Kahn, Jason; Mixson, A. James; Seog, Joonil

    2013-03-01

    Nonviral carriers have been developed to deliver nucleic acids by forming nanoscale complexes; however, there has been limited success in achieving high transfection efficiency. Our hypothesis is that a factor affecting gene delivery efficiency is the mechanical response of the condensed complex. To begin to test this hypothesis, we directly measured the mechanical properties of DNA-carrier complexes using optical tweezers. Histidine-lysine (HK) polymer, Asparagine-lysine (NK) polymer and poly-L-lysine were used to form complexes with a single DNA molecule. As carriers were introduced, a sudden decrease in DNA extension occurrs at a force level which is defined as critical force (Fc). Fc is carrier and concentration dependent. Pulling revealed reduction in DNA extension length for HK-DNA complexes. The characteristics of force profiles vary by agent and can be dynamically manipulated by changes in environmental conditions such as ionic strength of the buffer as well as pH. Heparin can remove cationic reagents which are otherwise irreversibly bound to DNA. The implications for optimizing molecular interactions to enhance transfection efficiency will be discussed.

  16. Key Residues of Outer Membrane Protein OprI Involved in Hexamer Formation and Bacterial Susceptibility to Cationic Antimicrobial Peptides

    PubMed Central

    Chang, Ting-Wei; Wang, Chiu-Feng; Huang, Hsin-Jye; Wang, Iren; Hsu, Shang-Te Danny

    2015-01-01

    Antimicrobial peptides (AMPs) are important components of the host innate defense mechanism against invading pathogens. Our previous studies have shown that the outer membrane protein, OprI from Pseudomonas aeruginosa or its homologue, plays a vital role in the susceptibility of Gram-negative bacteria to cationic α-helical AMPs (Y. M. Lin, S. J. Wu, T. W. Chang, C. F. Wang, C. S. Suen, M. J. Hwang, M. D. Chang, Y. T. Chen, Y. D. Liao, J Biol Chem 285:8985–8994, 2010, http://dx.doi.org/10.1074/jbc.M109.078725; T. W. Chang, Y. M. Lin, C. F. Wang, Y. D. Liao, J Biol Chem 287:418–428, 2012, http://dx.doi.org/10.1074/jbc.M111.290361). Here, we obtained two forms of recombinant OprI: rOprI-F, a hexamer composed of three disulfide-bridged dimers, was active in AMP binding, while rOprI-R, a trimer, was not. All the subunits predominantly consisted of α-helices and exhibited rigid structures with a melting point centered around 76°C. Interestingly, OprI tagged with Escherichia coli signal peptide was expressed in a hexamer, which was anchored on the surface of E. coli, possibly through lipid acids added at the N terminus of OprI and involved in the binding and susceptibility to AMP as native P. aeruginosa OprI. Deletion and mutation studies showed that Cys1 and Asp27 played a key role in hexamer formation and AMP binding, respectively. The increase of OprI hydrophobicity upon AMP binding revealed that it undergoes conformational changes for membrane fusion. Our results showed that OprI on bacterial surfaces is responsible for the recruitment and susceptibility to amphipathic α-helical AMPs and may be used to screen antimicrobials. PMID:26248382

  17. Identification of EnvC and Its Cognate Amidases as Novel Determinants of Intrinsic Resistance to Cationic Antimicrobial Peptides

    PubMed Central

    Oguri, Tamiko; Yeo, Won-Sik; Bae, Taeok

    2016-01-01

    Cationic antimicrobial peptides (CAMPs) are an essential part of the innate immune system. Some Gram-negative enteric pathogens, such as Salmonella enterica, show intrinsic resistance to CAMPs. However, the molecular basis of intrinsic resistance is poorly understood, largely due to a lack of information about the genes involved. In this study, using a microarray-based genomic technique, we screened the Keio collection of 3,985 Escherichia coli mutants for altered susceptibility to human neutrophil peptide 1 (HNP-1) and identified envC and zapB as novel genetic determinants of intrinsic CAMP resistance. In CAMP killing assays, an E. coli ΔenvCEc or ΔzapBEc mutant displayed a distinct profile of increased susceptibility to both LL-37 and HNP-1. Both mutants, however, displayed wild-type resistance to polymyxin B and human β-defensin 3 (HBD3), suggesting that the intrinsic resistance mediated by EnvC or ZapB is specific to certain CAMPs. A corresponding Salmonella ΔenvCSe mutant showed similarly increased CAMP susceptibility. The envC mutants of both E. coli and S. enterica displayed increased surface negativity and hydrophobicity, which partly explained the increased CAMP susceptibility. However, the ΔenvCEc mutant, but not the ΔenvCSe mutant, was defective in outer membrane permeability, excluding this defect as a common factor contributing to the increased CAMP susceptibility. Animal experiments showed that the Salmonella ΔenvCSe mutant had attenuated virulence. Taken together, our results indicate that the role of envC in intrinsic CAMP resistance is likely conserved among Gram-negative enteric bacteria, demonstrate the importance of intrinsic CAMP resistance for full virulence of S. enterica, and provide insight into distinct mechanisms of action of CAMPs. PMID:26810659

  18. Antimicrobial activity of four cationic peptides immobilised to poly-hydroxyethylmethacrylate.

    PubMed

    Dutta, Debarun; Kumar, Naresh; D P Willcox, Mark

    2016-04-01

    The objective of this study was to immobilise and characterise a variety of antimicrobial peptides (AMPs) onto poly-hydroxyethylmethacrylate (pHEMA) surfaces to achieve an antibacterial effect. Four AMPs, viz. LL-37, melimine, lactoferricin and Mel-4 were immobilised on pHEMA by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) which assisted covalent attachment. Increasing concentrations of AMPs were immobilised to determine the effect on the adhesion of Pseudomonas aeruginosa and Staphylococcus aureus. The AMP immobilised pHEMAs were characterised by X-ray photoelectron spectroscopy (XPS) to determine the surface elemental composition and by amino acid analysis to determine the total amount of AMP attached. In vitro cytotoxicity of the immobilised pHEMA samples to mouse L929 cells was investigated. Melimine and Mel-4 when immobilised at the highest concentrations showed 3.1 ± 0.6 log and 1.3 ± 0.2 log inhibition against P. aeruginosa, and 3.9 ± 0.6 log and 2.4 ± 0.5 log inhibition against S. aureus, respectively. Immobilisation of LL-37 resulted in up to 2.6 ± 1.0 log inhibition against only P. aeruginosa, but no activity against S. aureus. LFc attachment showed no antibacterial activity. Upon XPS analysis, immobilised melimine, LL-37, LFc and Mel-4 had 1.57 ± 0.38%, 1.13 ± 1.36%, 0.66 ± 0.47% and 0.73 ± 0.32% amide nitrogen attached to pHEMA compared to 0.12 ± 0.14% in the untreated controls. Amino acid analysis determined that the total amount of AMP attachment to pHEMA was 44.3 ± 7.4 nmol, 3.8 ± 0.2 nmol, 6.5 ± 0.6 nmol and 48.9 ± 2.3 nmol for the same peptides respectively. None of the AMP immobilised pHEMA surfaces showed any toxicity towards mouse L929 cells. The immobilisation of certain AMPs at nanomolar concentration to pHEMA is an effective option to develop a stable antimicrobial surface. PMID:26934297

  19. The two-component system CprRS senses cationic peptides and triggers adaptive resistance in Pseudomonas aeruginosa independently of ParRS.

    PubMed

    Fernández, Lucía; Jenssen, Håvard; Bains, Manjeet; Wiegand, Irith; Gooderham, W James; Hancock, Robert E W

    2012-12-01

    Cationic antimicrobial peptides pass across the outer membrane by interacting with negatively charged lipopolysaccharide (LPS), leading to outer membrane permeabilization in a process termed self-promoted uptake. Resistance can be mediated by the addition of positively charged arabinosamine through the action of the arnBCADTEF operon. We recently described a series of two-component regulators that lead to the activation of the arn operon after recognizing environmental signals, including low-Mg(2+) (PhoPQ, PmrAB) or cationic (ParRS) peptides. However, some peptides did not activate the arn operon through ParRS. Here, we report the identification of a new two-component system, CprRS, which, upon exposure to a wide range of antimicrobial peptides, triggered the expression of the LPS modification operon. Thus, mutations in the cprRS operon blocked the induction of the arn operon in response to several antimicrobial peptides independently of ParRS but did not affect the response to low Mg(2+). Distinct patterns of arn induction were identified. Thus, the responses to polymyxins were abrogated by either parR or cprR mutations, while responses to other peptides, including indolicidin, showed differential dependency on the CprRS and ParRS systems in a concentration-dependent manner. It was further demonstrated that, following exposure to inducing antimicrobial peptides, cprRS mutants did not become adaptively resistant to polymyxins as was observed for wild-type cells. Our microarray studies demonstrated that the CprRS system controlled a quite modest regulon, indicating that it was quite specific to adaptive peptide resistance. These findings provide greater insight into the complex regulation of LPS modification in Pseudomonas aeruginosa, which involves the participation of at least 4 two-component systems. PMID:23006746

  20. Transformation of [M + 2H](2+) Peptide Cations to [M - H](+), [M + H + O](+), and M(+•) Cations via Ion/Ion Reactions: Reagent Anions Derived from Persulfate.

    PubMed

    Pilo, Alice L; Bu, Jiexun; McLuckey, Scott A

    2015-07-01

    The gas-phase oxidation of doubly protonated peptides is demonstrated here using ion/ion reactions with a suite of reagents derived from persulfate. Intact persulfate anion (HS2O8(-)), peroxymonosulfate anion (HSO5(-)), and sulfate radical anion (SO4(-•)) are all either observed directly upon negative nanoelectrospray ionization (nESI) or easily obtained via beam-type collisional activation of persulfate into the mass spectrometer. Ion/ion reactions between each of these reagents and doubly protonated peptides result in the formation of a long-lived complex. Collisional activation of the complex containing a peroxymonosulfate anion results in oxygen transfer from the reagent to the peptide to generate the [M + H + O](+) species. Activation of the complex containing intact persulfate anion either results in oxygen transfer to generate the [M + H + O](+) species or abstraction of two hydrogen atoms and a proton to generate the [M - H](+) species. Activation of the complex containing sulfate radical anion results in abstraction of one hydrogen atom and a proton to form the peptide radical cation, [M](+•). This suite of reagents allows for the facile transformation of the multiply protonated peptides obtained via nESI into a variety of oxidized species capable of providing complementary information about the sequence and structure of the peptide. PMID:25944366

  1. Transformation of [M+2H]2+ Peptide Cations to [M - H]+, [M+H+O]+, and M+• Cations via Ion/Ion Reactions: Reagent Anions Derived from Persulfate

    NASA Astrophysics Data System (ADS)

    Pilo, Alice L.; Bu, Jiexun; McLuckey, Scott A.

    2015-07-01

    The gas-phase oxidation of doubly protonated peptides is demonstrated here using ion/ion reactions with a suite of reagents derived from persulfate. Intact persulfate anion (HS2O8 -), peroxymonosulfate anion (HSO5 -), and sulfate radical anion (SO4 -•) are all either observed directly upon negative nanoelectrospray ionization (nESI) or easily obtained via beam-type collisional activation of persulfate into the mass spectrometer. Ion/ion reactions between each of these reagents and doubly protonated peptides result in the formation of a long-lived complex. Collisional activation of the complex containing a peroxymonosulfate anion results in oxygen transfer from the reagent to the peptide to generate the [M+H+O]+ species. Activation of the complex containing intact persulfate anion either results in oxygen transfer to generate the [M+H+O]+ species or abstraction of two hydrogen atoms and a proton to generate the [M - H]+ species. Activation of the complex containing sulfate radical anion results in abstraction of one hydrogen atom and a proton to form the peptide radical cation, [M]+•. This suite of reagents allows for the facile transformation of the multiply protonated peptides obtained via nESI into a variety of oxidized species capable of providing complementary information about the sequence and structure of the peptide.

  2. Novel engineered cationic antimicrobial peptides display broad-spectrum activity against Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei.

    PubMed

    Abdelbaqi, Suha; Deslouches, Berthony; Steckbeck, Jonathan; Montelaro, Ronald; Reed, Douglas S

    2016-02-01

    Broad-spectrum antimicrobials are needed to effectively treat patients infected in the event of a pandemic or intentional release of a pathogen prior to confirmation of the pathogen's identity. Engineered cationic antimicrobial peptides (eCAPs) display activity against a number of bacterial pathogens including multi-drug-resistant strains. Two lead eCAPs, WLBU2 and WR12, were compared with human cathelicidin (LL-37) against three highly pathogenic bacteria: Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. Both WLBU2 and WR12 demonstrated bactericidal activity greater than that of LL-37, particularly against F. tularensis and Y. pestis. Only WLBU2 had bactericidal activity against B. pseudomallei. WLBU2, WR12 and LL-37 were all able to inhibit the growth of the three bacteria in vitro. Because these bacteria can be facultative intracellular pathogens, preferentially infecting macrophages and dendritic cells, we evaluated the activity of WLBU2 against F. tularensis in an ex vivo infection model with J774 cells, a mouse macrophage cell line. In that model WLBU2 was able to achieve greater than 50% killing of F. tularensis at a concentration of 12.5 μM. These data show the therapeutic potential of eCAPs, particularly WLBU2, as a broad-spectrum antimicrobial for treating highly pathogenic bacterial infections. PMID:26673248

  3. In vitro pharmacokinetics of antimicrobial cationic peptides alone and in combination with antibiotics against methicillin resistant Staphylococcus aureus biofilms.

    PubMed

    Dosler, Sibel; Mataraci, Emel

    2013-11-01

    Antibiotic therapy for methicillin-resistant Staphylococcus aureus (MRSA) infections is becoming more difficult in hospitals and communities because of strong biofilm-forming properties and multidrug resistance. Biofilm-associated MRSA is not affected by therapeutically achievable concentrations of antibiotics. Therefore, we investigated the in vitro pharmacokinetic activities of antimicrobial cationic peptides (AMPs; indolicidin, cecropin [1-7]-melittin A [2-9] amide [CAMA], and nisin), either alone or in combination with antibiotics (daptomycin, linezolid, teicoplanin, ciprofloxacin, and azithromycin), against standard and 2 clinically obtained MRSA biofilms. The minimum inhibitory concentrations (MIC) and minimum biofilm-eradication concentrations (MBEC) were determined by microbroth dilution technique. The time-kill curve (TKC) method was used to determine the bactericidal activities of the AMPs alone and in combination with the antibiotics against standard and clinically obtained MRSA biofilms. The MIC values of the AMPs and antibiotics ranged between 2 to 16 and 0.25 to 512 mg/L, and their MBEC values were 640 and 512 to 5120 mg/L, respectively. The TKC studies demonstrated that synergistic interactions occurred most frequently when using nisin+daptomycin/ciprofloxacin, indolicidin+teicoplanin, and CAMA+ciprofloxacin combinations. No antagonism was observed with any combination. AMPs appear to be good candidates for the treatment of MRSA biofilms, as they act as both enhancers of anti-biofilm activities and help to prevent or delay the emergence of resistance when used either alone or in combination with antibiotics. PMID:23988790

  4. The human Vps29 retromer component is a metallo-phosphoesterase for a cation-independent mannose 6-phosphate receptor substrate peptide

    PubMed Central

    Damen, Ester; Krieger, Elmar; Nielsen, Jens E.; Eygensteyn, Jelle; Van Leeuwen, Jeroen E. M.

    2006-01-01

    The retromer complex is involved in the retrograde transport of the CI-M6PR (cation-independent mannose 6-phosphate receptor) from endosomes to the Golgi. It is a hetero-trimeric complex composed of Vps26 (vacuolar sorting protein 26), Vps29 and Vps35 proteins, which are conserved in eukaryote evolution. Recently, elucidation of the crystal structure of Vps29 revealed that Vps29 contains a metallo-phosphoesterase fold [Wang, Guo, Liang, Fan, Zhu, Zang, Zhu, Li, Teng, Niu et al. (2005) J. Biol. Chem. 280, 22962–22967; Collins, Skinner, Watson, Seaman and Owen (2005) Nat. Struct. Mol. Biol. 12, 594–602]. We demonstrate that recombinant hVps29 (human Vps29) displays in vitro phosphatase activity towards a serine-phosphorylated peptide, containing the acidic-cluster dileucine motif of the cytoplasmatic tail of the CI-M6PR. Efficient dephosphorylation required the additional presence of recombinant hVps26 and hVps35 proteins, which interact with hVps29. Phosphatase activity of hVps29 was greatly decreased by alanine substitutions of active-site residues that are predicted to co-ordinate metal ions. Using inductively coupled plasma MS, we demonstrate that recombinant hVps29 binds zinc. Moreover, hVps29-dependent phosphatase activity is greatly reduced by non-specific and zinc-specific metal ion chelators, which can be completely restored by addition of excess ZnCl2. The binuclear Zn2+ centre and phosphate group were modelled into the hVps29 catalytic site and pKa calculations provided further insight into the molecular mechanisms of Vps29 phosphatase activity. We conclude that the retromer complex displays Vps29-dependent in vitro phosphatase activity towards a serinephosphorylated acidic-cluster dileucine motif that is involved in endosomal trafficking of the CI-M6PR. The potential significance of these findings with respect to regulation of transport of cycling trans-Golgi network proteins is discussed. PMID:16737443

  5. Graphene oxide-peptide nanocomplex as a versatile fluorescence probe of protein kinase activity based on phosphorylation protection against carboxypeptidase digestion.

    PubMed

    Zhou, Jiang; Xu, Xiahong; Liu, Wei; Liu, Xin; Nie, Zhou; Qing, Meng; Nie, Lihua; Yao, Shouzhuo

    2013-06-18

    The research on complicated kinomics and kinase-target drug discovery requires the development of simple, cost-effective, and multiplex kinase assays. Herein, we propose a novel and versatile biosensing platform for the detection of protein kinase activity based on graphene oxide (GO)-peptide nanocomplex and phosphorylation-induced suppression of carboxypeptidase Y (CPY) cleavage. Kinase-catalyzed phosphorylation protects the fluorophore-labeled peptide probe against CPY digestion and induces the formation of a GO/peptide nanocomplex resulting in fluorescence quenching, while the nonphosphopeptide is degraded by CPY to release free fluorophore as well as restore fluorescence. This GO-based nanosensor has been successfully applied to sensitively detect two model kinases, casein kinase (CKII) and cAMP-dependent protein kinase (PKA) with low detection limits of 0.0833 mU/μL and 0.134 mU/μL, respectively. The feasibility of this GO-based sensor was further demonstrated by the assessment of kinase inhibition by staurosporine and H-89, in vitro kinase assay in cell lysates, and simultaneous detection of CKII and PKA activity. Moreover, the GO-based fluorescence anisotropy (FA) kinase assay has been also developed using GO as a FA signal amplifier. The proposed sensor is homogeneous, facile, universal, label-free, and applicable for multiplexed kinase assay, presenting a promising method for kinase-related biochemical fundamental research and inhibitor screening. PMID:23734972

  6. Chemoattraction of macrophages by secretory molecules derived from cells expressing the signal peptide of eosinophil cationic protein

    PubMed Central

    2012-01-01

    Background Eosinophil cationic protein is a clinical asthma biomarker that would be released into blood, especially gathered in bronchia. The signal peptide of eosinophil cationic protein (ECPsp) plays an important role in translocating ECP to the extracellular space. We previously reported that ECPsp inhibits microbial growth and regulates the expression of mammalian genes encoding tumor growth factor-α (TGF-α) and epidermal growth factor receptor (EGFR). Results In the present study, we first generated a DNA microarray dataset, which showed that ECPsp upregulated proinflammatory molecules, including chemokines, interferon-induced molecules, and Toll-like receptors. The levels of mRNAs encoding CCL5, CXCL10, CXCL11, CXCL16, STAT1, and STAT2 were increased in the presence of ECPsp by 2.07-, 4.21-, 7.52-, 2.6-, 3.58-, and 1.67-fold, respectively. We then constructed a functional linkage network by integrating the microarray dataset with the pathway database of Kyoto Encyclopedia of Genes and Genomes (KEGG). Follow-up analysis revealed that STAT1 and STAT2, important transcriptional factors that regulate cytokine expression and release, served as hubs to connect the pathways of cytokine stimulation (TGF-α and EGFR pathways) and inflammatory responses. Furthermore, integrating TGF-α and EGFR with the functional linkage network indicated that STAT1 and STAT2 served as hubs that connect two functional clusters, including (1) cell proliferation and survival, and (2) inflammation. Finally, we found that conditioned medium in which cells that express ECPsp had been cultured could chemoattract macrophages. Experimentally, we also demonstrated that the migration of macrophage could be inhibited by the individual treatment of siRNAs of STAT1 or STAT2. Therefore, we hypothesize that ECPsp may function as a regulator for enhancing the migration of macrophages through the upregualtion of the transcriptional factors STAT1 and STAT2. Conclusion The increased expression and

  7. The production of recombinant cationic α-helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum.

    PubMed

    Company, Nuri; Nadal, Anna; La Paz, José-Luis; Martínez, Sílvia; Rasche, Stefan; Schillberg, Stefan; Montesinos, Emilio; Pla, Maria

    2014-01-01

    Synthetic linear antimicrobial peptides with cationic α-helical structures, such as BP100, are valuable as novel therapeutics and preservatives. However, they tend to be toxic when expressed at high levels as recombinant peptides in plants, and they can be difficult to detect and isolate from complex plant tissues because they are strongly cationic and display low extinction coefficient and extremely limited immunogenicity. We therefore expressed BP100 with a C-terminal tag which preserved its antimicrobial activity and demonstrated significant accumulation in plant cells. We used a fluorescent tag to trace BP100 following transiently expression in Nicotiana benthamiana leaves and showed that it accumulated in large vesicles derived from the endoplasmic reticulum (ER) along with typical ER luminal proteins. Interestingly, the formation of these vesicles was induced by BP100. Similar vesicles formed in stably transformed Arabidopsis thaliana seedlings, but the recombinant peptide was toxic to the host during latter developmental stages. This was avoided by selecting active BP100 derivatives based on their low haemolytic activity even though the selected peptides remained toxic to plant cells when applied exogenously at high doses. Using this strategy, we generated transgenic rice lines producing active BP100 derivatives with a yield of up to 0.5% total soluble protein. PMID:24102775

  8. Development of Online pH Gradient-Eluted Strong Cation Exchange Nanoelectrospray-Tandem Mass Spectrometry for Proteomic Analysis Facilitating Basic and Histidine-Containing Peptides Identification.

    PubMed

    Xu, Jingjing; Gao, Jing; Yu, Chengli; He, Han; Yang, Yiming; Figeys, Daniel; Zhou, Hu

    2016-01-01

    A novel one-dimensional online pH gradient-eluted strong cation exchange-nanoelectrospray ionization-tandem mass spectrometry (SCX-nano-ESI-MS/MS) method was developed for protein identification and tested with a mixture of six standard proteins, total lysate of HuH7 and N2a cells, as well as membrane fraction of N2a cells. This method utilized an online nanoflow SCX column in a nano-LC system coupled with a nanoelectrospray high-resolution mass spectrometer. Protein digests were separated on a nanoflow SCX column with a pH gradient and directly introduced into a mass spectrometer through nanoelectrospray ionization. More than five thousand unique peptides were identified in each 90 min LC-MS/MS run using 500 nanogram of protein digest either from total cell lysate or from membrane fraction. The unique peptide overlap between online strong cation exchange nano-ESI-MS/MS (SCXLC-MS/MS) and reverse phase nano-ESI-MS/MS (RPLC-MS/MS) is only ≤30%, which indicated these two methods were complementary to each other. The correlation coefficient of retention time and theoretical isoelectric point (pI) of identified peptides in SCXLC-MS/MS was higher than 0.4, which showed that peptides elution in SCXLC-MS/MS was dependent on their charge states. Furthermore, SCXLC-MS/MS showed identification capability for a higher proportion of basic peptides compared to the RPLC-MS/MS method, especially for histidine-containing peptides. Our SCXLC-MS/MS method is an excellent alternative method to the RPLC-MS/MS method for analysis of standard proteins, total cell and membrane proteomes. PMID:26646553

  9. Atrial Natriuretic Peptide Stimulates Dopamine Tubular Transport by Organic Cation Transporters: A Novel Mechanism to Enhance Renal Sodium Excretion

    PubMed Central

    Kouyoumdzian, Nicolás M.; Rukavina Mikusic, Natalia L.; Kravetz, María C.; Lee, Brenda M.; Carranza, Andrea; Del Mauro, Julieta S.; Pandolfo, Marcela; Gironacci, Mariela M.; Gorzalczany, Susana; Toblli, Jorge E.; Fernández, Belisario E.

    2016-01-01

    The aim of this study was to demonstrate the effects of atrial natriuretic peptide (ANP) on organic cation transporters (OCTs) expression and activity, and its consequences on dopamine urinary levels, Na+, K+-ATPase activity and renal function. Male Sprague Dawley rats were infused with isotonic saline solution during 120 minutes and randomized in nine different groups: control, pargyline plus tolcapone (P+T), ANP, dopamine (DA), D-22, DA+D-22, ANP+D-22, ANP+DA and ANP+DA+D-22. Renal functional parameters were determined and urinary dopamine concentration was quantified by HPLC. Expression of OCTs and D1-receptor in membrane preparations from renal cortex tissues were determined by western blot and Na+, K+-ATPase activity was determined using in vitro enzyme assay. 3H-DA renal uptake was determined in vitro. Compared to P+T group, ANP and dopamine infusion increased diuresis, urinary sodium and dopamine excretion significantly. These effects were more pronounced in ANP+DA group and reversed by OCTs blockade by D-22, demonstrating that OCTs are implied in ANP stimulated-DA uptake and transport in renal tissues. The activity of Na+, K+-ATPase exhibited a similar fashion when it was measured in the same experimental groups. Although OCTs and D1-receptor protein expression were not modified by ANP, OCTs-dependent-dopamine tubular uptake was increased by ANP through activation of NPR-A receptor and protein kinase G as signaling pathway. This effect was reflected by an increase in urinary dopamine excretion, natriuresis, diuresis and decreased Na+, K+-ATPase activity. OCTs represent a novel target that links the activity of ANP and dopamine together in a common mechanism to enhance their natriuretic and diuretic effects. PMID:27392042

  10. Overcoming barriers in Pseudomonas aeruginosa lung infections: Engineered nanoparticles for local delivery of a cationic antimicrobial peptide.

    PubMed

    d'Angelo, Ivana; Casciaro, Bruno; Miro, Agnese; Quaglia, Fabiana; Mangoni, Maria Luisa; Ungaro, Francesca

    2015-11-01

    Cationic antimicrobial peptides (CAMPs) are very promising in the treatment of multi-drug resistant Pseudomonas aeruginosa lung infections experienced by cystic fibrosis (CF) patients. Nevertheless, there is an urgent need of inhalable formulations able to deliver the intact CAMP in conductive airways and to shield its interactions with airway mucus/bacterial biofilm, thus enhancing CAMP/bacteria interactions. Along these lines, the aim of this work was the design and development of nano-embedded microparticles (NEM) for sustained delivery of CAMPs in the lung. To this purpose, nanoparticles (NPs) made of poly(lactide-co-glycolide) (PLGA) containing a model CAMP, colistin (Col), were produced by emulsion/solvent diffusion technique. Engineering NPs with chitosan (CS) and poly(vinyl alcohol) (PVA) allowed to modulate surface properties and, in so doing, to improve NP transport through artificial CF mucus. In order to achieve a long-term stable dosage form useful for NP inhalation, NPs were spray-dried in different carriers (lactose or mannitol), thus producing NEM. The most promising NEM formulations were selected on the basis of bulk and flow properties, distribution of NPs in the carrier and aerosolization performance upon delivery through a breath-actuated dry powder inhaler. Of note, selected Col-loaded NEM were found to kill P. aeruginosa biofilm and to display a prolonged efficacy in biofilm eradication compared to the free Col. This effect was likely ascribable to the ability of NPs to penetrate into bacterial biofilm, as demonstrated by confocal analysis, and to sustain Col release inside it. Taken all together, our results indicate that adequate engineering of PLGA NPs represents an enticing technological approach to harness novel antimicrobials for P. aeruginosa lung infection, such as CAMPs, especially in CF. PMID:26340361

  11. The MisR Response Regulator Is Necessary for Intrinsic Cationic Antimicrobial Peptide and Aminoglycoside Resistance in Neisseria gonorrhoeae.

    PubMed

    Kandler, Justin L; Holley, Concerta L; Reimche, Jennifer L; Dhulipala, Vijaya; Balthazar, Jacqueline T; Muszyński, Artur; Carlson, Russell W; Shafer, William M

    2016-08-01

    During infection, the sexually transmitted pathogen Neisseria gonorrhoeae (the gonococcus) encounters numerous host-derived antimicrobials, including cationic antimicrobial peptides (CAMPs) produced by epithelial and phagocytic cells. CAMPs have both direct and indirect killing mechanisms and help link the innate and adaptive immune responses during infection. Gonococcal CAMP resistance is likely important for avoidance of host nonoxidative killing systems expressed by polymorphonuclear granulocytes (e.g., neutrophils) and intracellular survival. Previously studied gonococcal CAMP resistance mechanisms include modification of lipid A with phosphoethanolamine by LptA and export of CAMPs by the MtrCDE efflux pump. In the related pathogen Neisseria meningitidis, a two-component regulatory system (2CRS) termed MisR-MisS has been shown to contribute to the capacity of the meningococcus to resist CAMP killing. We report that the gonococcal MisR response regulator but not the MisS sensor kinase is involved in constitutive and inducible CAMP resistance and is also required for intrinsic low-level resistance to aminoglycosides. The 4- to 8-fold increased susceptibility of misR-deficient gonococci to CAMPs and aminoglycosides was independent of phosphoethanolamine decoration of lipid A and the levels of the MtrCDE efflux pump and seemed to correlate with a general increase in membrane permeability. Transcriptional profiling and biochemical studies confirmed that expression of lptA and mtrCDE was not impacted by the loss of MisR. However, several genes encoding proteins involved in membrane integrity and redox control gave evidence of being MisR regulated. We propose that MisR modulates the levels of gonococcal susceptibility to antimicrobials by influencing the expression of genes involved in determining membrane integrity. PMID:27216061

  12. Reinforcing Lipid A Acylation on the Cell Surface of Acinetobacter baumannii Promotes Cationic Antimicrobial Peptide Resistance and Desiccation Survival

    PubMed Central

    Boll, Joseph M.; Tucker, Ashley T.; Klein, Dustin R.; Beltran, Alexander M.; Brodbelt, Jennifer S.; Davies, Bryan W.

    2015-01-01

    ABSTRACT Acinetobacter baumannii is an emerging Gram-negative pathogen found in hospitals and intensive care units. In order to persist in hospital environments, A. baumannii withstands desiccative conditions and can rapidly develop multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the conserved lipid A component of the Gram-negative outer membrane to lyse the bacterial cell. However, many Gram-negative pathogenic bacteria, including A. baumannii, fortify their outer membrane with hepta-acylated lipid A to protect the cell from CAMP-dependent cell lysis. Whereas in Escherichia coli and Salmonella, increased production of the outer membrane acyltransferase PagP results in formation of protective hepta-acylated lipid A, which reinforces the lipopolysaccharide portion of the outer membrane barrier, A. baumannii does not carry a gene that encodes a PagP homolog. Instead, A. baumannii has evolved a PagP-independent mechanism to synthesize protective hepta-acylated lipid A. Taking advantage of a recently adapted A. baumannii genetic recombineering system, we characterized two putative acyltransferases in A. baumannii designated LpxLAb (A. baumannii LpxL) and LpxMAb (A. baumannii LpxM), which transfer one and two lauroyl (C12:0) acyl chains, respectively, during lipid A biosynthesis. Hepta-acylation of A. baumannii lipid A promoted resistance to vertebrate and polymyxin CAMPs, which are prescribed as last-resort treatment options. Intriguingly, our analysis also showed that LpxMAb-dependent acylation of lipid A is essential for A. baumannii desiccation survival, a key resistance mechanism for survival in hospital environments. Compounds that inhibit LpxMAb-dependent hepta-acylation of lipid A could act synergistically with CAMPs to provide innovative transmission prevention strategies and treat multidrug-resistant infections. PMID:25991684

  13. In vitro activities of antibiotics and antimicrobial cationic peptides alone and in combination against methicillin-resistant Staphylococcus aureus biofilms.

    PubMed

    Mataraci, Emel; Dosler, Sibel

    2012-12-01

    Methicillin-resistant Staphylococcus aureus (MRSA) strains are most often found as hospital- and community-acquired infections. The danger of MRSA infections results from not only the emergence of multidrug resistance but also the occurrence of bacteria that form strong biofilms. We investigated the in vitro activities of antibiotics (daptomycin, linezolid, teichoplanine, azithromycin, and ciprofloxacin) and antimicrobial cationic peptides {AMPs; indolicidin, CAMA [cecropin (1-7)-melittin A (2-9) amide], and nisin} alone or in combination against MRSA ATCC 43300 biofilms. The MICs and minimum biofilm eradication concentrations (MBECs) were determined by the broth microdilution technique. Antibiotic and AMP combinations were assessed using the checkerboard technique. For MRSA planktonic cells, MICs of antibiotics and AMPs ranged between 0.125 and 512 and 8 and 16 mg/liter, respectively, and the MBEC values were between 512 and 5,120 and 640 mg/liter, respectively. With a fractional inhibitory concentration of ≤0.5 as the borderline, synergistic interactions against MRSA biofilms were frequent with almost all antibiotic-antibiotic and antibiotic-AMP combinations. Against planktonic cells, they generally had an additive effect. No antagonism was observed. All of the antibiotics, AMPs, and their combinations were able to inhibit the attachment of bacteria at 1/10 MIC and biofilm formation at 1× MIC. Biofilm-associated MRSA was not affected by therapeutically achievable concentrations of antimicrobial agents. Use of a combination of antimicrobial agents can provide a synergistic effect, which rapidly enhances antibiofilm activity and may help prevent or delay the emergence of resistance. AMPs seem to be good candidates for further investigations in the treatment of MRSA biofilms, alone or in combination with antibiotics. PMID:23070152

  14. Oral delivery of oil-based formulation for a novel synthetic cationic peptide of GnRH (gonadotropin-releasing hormone) antagonist for prostate cancer treatment.

    PubMed

    Zhang, Guiying; Wang, Tao; Gao, Lijun; Quan, Dongqin

    2013-06-25

    LXT-101, a cationic peptide is a novel antagonist of gonadotropin-releasing hormone (GnRH) for prostate cancer treatment. However, effective delivery of peptide drugs into the body by the oral route remains a major challenge due to their origin properties with high molecular weights, strong polarity and low stability in the gastrointestinal (GI) tract. In this study, we have developed a novel oral delivery of oil-based formulation in which therapeutic peptide LXT-101 are solubilized in oils and with this solution as oil phase, an optimum formulation of self-microemulsifying drug delivery system (SMEDDS) was developed. The peptide stability with the SMEDDS formulation in artificial gastric and intestinal fluid was tested in vitro. On the other hand, the testosterone level and plasma concentration of LXT-101 in rats after oral administration of the SMEDDS formulation were investigated in vivo. The data in vitro indicated that LXT-101 in the SMEDDS formulation was stable over 8 h in artificial gastric and intestinal fluid. LXT-101 can be absorbed in vivo and suppression of testosterone maintained in castration level within 12 h can be achieved effectively after SMEDDS formulation administered orally at a dose of 3.5 mg/kg. The approach can provide a potential way for delivery peptides by oral. PMID:23623791

  15. The anti-cancer activity of a cationic anti-microbial peptide derived from monomers of polyhydroxyalkanoate.

    PubMed

    O'Connor, Stephen; Szwej, Emilia; Nikodinovic-Runic, Jasmina; O'Connor, Aisling; Byrne, Annette T; Devocelle, Marc; O'Donovan, Norma; Gallagher, William M; Babu, Ramesh; Kenny, Shane T; Zinn, Manfred; Zulian, Qun Ren; O'Connor, Kevin E

    2013-04-01

    The biodegradable polymer medium chain length polyhydroxyalkanoate (mclPHA), produced by Pseudomonas putida CA-3, was depolymerised and the predominant monomer (R)-3-hydroxydecanoic acid (R10) purified. R10 was conjugated to a d-peptide DP18 and its derivatives. All peptides conjugated with R10 exhibited greater anti-cancer activity compared to the unconjugated peptides. Unconjugated and conjugated peptides were cytocidal for cancer cells. Conjugation of R10 to peptides was essential for enhanced anti-proliferation activity, as unconjugated mixes did not result in enhancement of anti-cancer activity. The conjugation of R10 resulted in more rapid uptake of peptides into HeLa and MiaPaCa cells compared to unconjugated peptide. Both unconjugated and R10 conjugated peptides localized to the mitochondria of HeLa and MiaPaCa cells and induced apoptosis. Peptide conjugated with a terminally hydroxylated decanoic acid (ω-hydroxydecanoic acid) exhibited 3.3 and 6.3 fold higher IC(50) values compared to R10 conjugated peptide indicating a role for the position of the hydroxyl moiety in enhancement of anti-cancer activity. Conjugation of decanoic acid (C10) to peptides resulted in similar or higher IC(50) values compared to R10 conjugates but C10 conjugates did not exhibit any cancer selectivity. Combination studies showed that R10DP18L exhibited synergy with cisplatin, gemcitabine, and taxotere with IC(50) values in the nanomolar range. PMID:23343631

  16. The soybean-derived peptide lunasin inhibits non-small cell lung cancer cell proliferation by suppressing phosphorylation of the retinoblastoma protein

    PubMed Central

    McConnell, Elizabeth J.; Devapatla, Bharat; Yaddanapudi, Kavitha; Davis, Keith R.

    2015-01-01

    Lunasin, a soybean bioactive peptide, has both chemopreventive and chemotherapeutic activities. The aim of this study was to determine the chemotherapeutic potential of lunasin against human lung cancer. Treatment of non-small cell lung cancer (NSCLC) cells with highly purified soybean-derived lunasin caused limited, cell-line specific anti-proliferative effects on anchorage-dependent growth whereas two normal bronchial epithelial cell lines were unaffected. Lunasin's antiproliferative effects were potentiated upon utilization of anchorage-independent conditions. Furthermore, NSCLC cell lines that were unaffected by lunasin in anchorage-dependent assays exhibited a dose-dependent inhibition in colony formation or colony size. Mouse xenograft studies revealed that 30 mg lunasin/kg body weight per day decreased NSCLC H1299 tumor volume by 63.0% at day 32. Mechanistic studies using cultured NSCLC H661 cells showed that lunasin inhibited cell cycle progression at the G1/S phase interface without inducing apoptosis. Immunoblot analyses of key cell-cycle proteins demonstrated that lunasin altered the expression of the G1 specific cyclin-dependent kinase complex components, increased levels of p27Kip1, reduced levels of phosphorylated Akt, and ultimately inhibited the sequential phosphorylation of the retinoblastoma protein (RB). These results establish for the first time that lunasin can inhibit NSCLC proliferation by suppressing cell-cycle dependent phosphorylation of RB. PMID:25609198

  17. Spinal neurons that contain gastrin-releasing peptide seldom express Fos or phosphorylate extracellular signal-regulated kinases in response to intradermal chloroquine

    PubMed Central

    Gutierrez-Mecinas, Maria; Polgár, Erika; Todd, Andrew J

    2016-01-01

    Background Gastrin-releasing peptide (GRP) is thought to play a role in the itch evoked by intradermal injection of chloroquine. Although some early studies suggested that GRP was expressed in pruriceptive primary afferents, it is now thought that GRP in the spinal cord is derived mainly from a population of excitatory interneurons in lamina II, and it has been suggested that these are involved in the itch pathway. To test this hypothesis, we used the transcription factor Fos and phosphorylation of extracellular signal-regulated kinases (ERK) to look for evidence that interneurons expressing GRP were activated following intradermal injection of chloroquine into the calf, in mice that express enhanced green fluorescent protein (EGFP) in these cells. Results Injection of chloroquine resulted in numerous Fos- or phospho-ERK (pERK) positive cells in the somatotopically appropriate part of the superficial dorsal horn. The proportion of all neurons in this region that showed Fos or pERK was 18% and 21%, respectively. However, among the GRP–EGFP, only 7% were Fos-positive and 3% were pERK-positive. As such, GRP–EGFP cells were significantly less likely than other neurons to express Fos or to phosphorylate ERK. Conclusions Both expression of Fos and phosphorylation of ERK can be used to identify dorsal horn neurons activated by chloroquine injection. However, these results do not support the hypothesis that interneurons expressing GRP are critical components in the itch pathway. PMID:27270268

  18. Antigenic epitopes fused to cationic peptide bound to oligonucleotides facilitate Toll-like receptor 9-dependent, but CD4+ T cell help-independent, priming of CD8+ T cells.

    PubMed

    Schirmbeck, Reinhold; Riedl, Petra; Zurbriggen, Rinaldo; Akira, Shizuo; Reimann, Jörg

    2003-11-15

    A priority in current vaccine research is the development of adjuvants that support the efficient priming of long-lasting, CD4(+) T cell help-independent CD8(+) T cell immunity. Oligodeoxynucleotides (ODN) with immune-stimulating sequences (ISS) containing CpG motifs facilitate the priming of MHC class I-restricted CD8(+) T cell responses to proteins or peptides. We show that the adjuvant effect of ISS(+) ODN on CD8(+) T cell priming to large, recombinant Ag is enhanced by binding them to short, cationic (arginine-rich) peptides that themselves have no adjuvant activity in CD8(+) T cell priming. Fusing antigenic epitopes to cationic (8- to 10-mer) peptides bound to immune-stimulating ISS(+) ODN or nonstimulating NSS(+) ODN (without CpG-containing sequences) generated immunogens that efficiently primed long-lasting, specific CD8(+) T cell immunity of high magnitude. Different MHC class I-binding epitopes fused to short cationic peptides of different origins showed this adjuvant activity. Quantitative ODN binding to cationic peptides strikingly reduced the toxicity of the latter, suggesting that it improves the safety profile of the adjuvant. CD8(+) T cell priming supported by this adjuvant was Toll-like receptor 9 dependent, but required no CD4(+) T cell help. ODN (with or without CpG-containing sequences) are thus potent Th1-promoting adjuvants when bound to cationic peptides covalently linked to antigenic epitopes, a mode of Ag delivery prevailing in many viral nucleocapsids. PMID:14607920

  19. Discovery and Mechanistic Studies of Facile N-Terminal Cα–C Bond Cleavages in the Dissociation of Tyrosine-Containing Peptide Radical Cations

    SciTech Connect

    Mu, Xiaoyan; Song, Tao; Xu, Minjie; Lai, Cheuk-Kuen; Siu, Chi-Kit; Laskin, Julia; Chu, Ivan K.

    2014-03-28

    Gas phase fragmentations of protein and peptide (M) ions in a mass spectrometer—induced by, for example, electron-capture dissociation1-2 and electron-transfer dissociation3-422 —form the foundation for top-down amino acid sequencing approaches for the rapid identification of protein components in complex biological samples. During these processes, protonated protein and peptide radicals ([M + nH]•(n – 1)+)5–8 are generated; their fragmentations are governed largely by the properties of the unpaired electron. Because of their importance in modern bioanalytical chemistry, considerable attention has been drawn recently toward understanding the radical cation chemistry behind the fragmentations of these odd-electron biomolecular ions in the gas phase.

  20. How Cation-Pi Interactions Enhance and Structure the Binding of Metal Ions to Amino Acids and Peptides. Dialanine Probed by Irmpd Spectroscopy as a Prime Example

    NASA Astrophysics Data System (ADS)

    Dunbar, Robert C.; Steill, Jeffrey; Oomens, Jos

    2010-06-01

    Spectroscopic examination of metalated amino acids and model peptides in the infrared region gives incisive conformational information. The role of cation-pi interactions of the metal ions with aromatic amino acids in structuring the complexes and enforcing particular architectures is being clarified by such experiments using IRMPD action spectroscopy as the experimental probe. The presence of multiple aromatic groups as in dialanine gives particularly stringent conformational stabilization. Comparing spectroscopic peak shifts across a range of alkali and alkaline earth metal ions, ranging from lithium to cesium, and from calcium to barium, allows us to view the systematic relations between normal mode frequencies and ion/peptide interactions. The spectra of the ions were acquired by irradiating the cell of the Fourier-transform ion cyclotron resonance mass spectrometer with infrared light from the FELIX free electron laser at wavelengths in the approximate range 500 to 1900 cm-1.

  1. Determining in vivo Phosphorylation Sites using Mass Spectrometry

    PubMed Central

    Breitkopf, Susanne B.; Asara, John M.

    2012-01-01

    Phosphorylation is the most studied protein post-translational modification (PTM) in biological systems since it controls cell growth, proliferation, survival, etc. High resolution/high mass accuracy mass spectrometers are used to identify protein phosphorylation sites due to their speed, sensitivity, selectivity and throughput. The protocol described here focuses on two common strategies: 1) Identifying phosphorylation sites from individual proteins and small protein complexes, and 2) Identifying global phosphorylation sites from whole cell and tissue extracts. For the first, endogenous or epitope tagged proteins are typically immunopurified (IP) from cell lysates, purified via gel electrophoresis or precipitation and enzymatically digested into peptides. Samples can be optionally enriched for phosphopeptides using immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2) and then analyzed by microcapillary liquid chromatography/tandem mass spectrometry (LC-MS/MS). Global phosphorylation site analyses that capture pSer/pThr/pTyr sites from biological sources sites are more resource and time-consuming and involve digesting the whole cell lysate, followed by peptide fractionation by strong cation exchange chromatography (SCX), phosphopeptide enrichment by IMAC or TiO2 and LC-MS/MS. Alternatively, one can fractionate the protein lysate by SDS-PAGE, followed by digestion, phosphopeptide enrichment and LC-MS/MS. One can also IP only phospho-tyrosine peptides using a pTyr antibody followed by LC-MS/MS. PMID:22470061

  2. Effects of linear cationic x-helical antimicrobial peptides on immune-relevant genes in trout macrophages.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is increasing evidence of the potential role of antimicrobial peptides in the regulation of immune responses in mammalian species. However, the effects of these peptides in fish have yet to be investigated. In this study, we examined the transcriptional expression profile of representative i...

  3. Antibacterial activity of novel cationic peptides against clinical isolates of multi-drug resistant Staphylococcus pseudintermedius from infected dogs.

    PubMed

    Mohamed, Mohamed F; Hammac, G Kenitra; Guptill, Lynn; Seleem, Mohamed N

    2014-01-01

    Staphylococcus pseudintermedius is a major cause of skin and soft tissue infections in companion animals and has zoonotic potential. Additionally, methicillin-resistant S. pseudintermedius (MRSP) has emerged with resistance to virtually all classes of antimicrobials. Thus, novel treatment options with new modes of action are required. Here, we investigated the antimicrobial activity of six synthetic short peptides against clinical isolates of methicillin-susceptible and MRSP isolated from infected dogs. All six peptides demonstrated potent anti-staphylococcal activity regardless of existing resistance phenotype. The most effective peptides were RRIKA (with modified C terminus to increase amphipathicity and hydrophobicity) and WR-12 (α-helical peptide consisting exclusively of arginine and tryptophan) with minimum inhibitory concentration50 (MIC50) of 1 µM and MIC90 of 2 µM. RR (short anti-inflammatory peptide) and IK8 "D isoform" demonstrated good antimicrobial activity with MIC50 of 4 µM and MIC90 of 8 µM. Penetratin and (KFF)3K (two cell penetrating peptides) were the least effective with MIC50 of 8 µM and MIC90 of 16 µM. Killing kinetics revealed a major advantage of peptides over conventional antibiotics, demonstrating potent bactericidal activity within minutes. Studies with propidium iodide and transmission electron microscopy revealed that peptides damaged the bacterial membrane leading to leakage of cytoplasmic contents and consequently, cell death. A potent synergistic increase in the antibacterial effect of the cell penetrating peptide (KFF)3K was noticed when combined with other peptides and with antibiotics. In addition, all peptides displayed synergistic interactions when combined together. Furthermore, peptides demonstrated good therapeutic indices with minimal toxicity toward mammalian cells. Resistance to peptides did not evolve after 10 passages of S. pseudintermedius at sub-inhibitory concentration. However, the MICs of amikacin and

  4. Antibacterial Activity of Novel Cationic Peptides against Clinical Isolates of Multi-Drug Resistant Staphylococcus pseudintermedius from Infected Dogs

    PubMed Central

    Mohamed, Mohamed F.; Hammac, G. Kenitra; Guptill, Lynn; Seleem, Mohamed N.

    2014-01-01

    Staphylococcus pseudintermedius is a major cause of skin and soft tissue infections in companion animals and has zoonotic potential. Additionally, methicillin-resistant S. pseudintermedius (MRSP) has emerged with resistance to virtually all classes of antimicrobials. Thus, novel treatment options with new modes of action are required. Here, we investigated the antimicrobial activity of six synthetic short peptides against clinical isolates of methicillin-susceptible and MRSP isolated from infected dogs. All six peptides demonstrated potent anti-staphylococcal activity regardless of existing resistance phenotype. The most effective peptides were RRIKA (with modified C terminus to increase amphipathicity and hydrophobicity) and WR-12 (α-helical peptide consisting exclusively of arginine and tryptophan) with minimum inhibitory concentration50 (MIC50) of 1 µM and MIC90 of 2 µM. RR (short anti-inflammatory peptide) and IK8 “D isoform” demonstrated good antimicrobial activity with MIC50 of 4 µM and MIC90 of 8 µM. Penetratin and (KFF)3K (two cell penetrating peptides) were the least effective with MIC50 of 8 µM and MIC90 of 16 µM. Killing kinetics revealed a major advantage of peptides over conventional antibiotics, demonstrating potent bactericidal activity within minutes. Studies with propidium iodide and transmission electron microscopy revealed that peptides damaged the bacterial membrane leading to leakage of cytoplasmic contents and consequently, cell death. A potent synergistic increase in the antibacterial effect of the cell penetrating peptide (KFF)3K was noticed when combined with other peptides and with antibiotics. In addition, all peptides displayed synergistic interactions when combined together. Furthermore, peptides demonstrated good therapeutic indices with minimal toxicity toward mammalian cells. Resistance to peptides did not evolve after 10 passages of S. pseudintermedius at sub-inhibitory concentration. However, the MICs of amikacin and

  5. Modulation of Calcium Oxalate Dihydrate Growth by Selective Crystal-face Binding of Phosphorylated Osteopontin and Polyaspartate Peptide Showing Occlusion by Sectoral (Compositional) Zoning*

    PubMed Central

    Chien, Yung-Ching; Masica, David L.; Gray, Jeffrey J.; Nguyen, Sarah; Vali, Hojatollah; McKee, Marc D.

    2009-01-01

    Calcium oxalate dihydrate (COD) mineral and the urinary protein osteopontin/uropontin (OPN) are commonly found in kidney stones. To investigate the effects of OPN on COD growth, COD crystals were grown with phosphorylated OPN or a polyaspartic acid-rich peptide of OPN (DDLDDDDD, poly-Asp86–93). Crystals grown with OPN showed increased dimensions of the {110} prismatic faces attributable to selective inhibition at this crystallographic face. At high concentrations of OPN, elongated crystals with dominant {110} faces were produced, often with intergrown, interpenetrating twin crystals. Poly-Asp86–93 dose-dependently elongated crystal morphology along the {110} faces in a manner similar to OPN. In crystal growth studies using fluorescently tagged poly-Asp86–93 followed by imaging of crystal interiors using confocal microscopy, sectoral (compositional) zoning in COD was observed resulting from selective binding and incorporation (occlusion) of peptide exclusively into {110} crystal sectors. Computational modeling of poly-Asp86–93 adsorption to COD {110} and {101} surfaces also suggests increased stabilization of the COD {110} surface and negligible change to the natively stable {101} surface. Ultrastructural, colloidal-gold immunolocalization of OPN by transmission electron microscopy in human stones confirmed an intracrystalline distribution of OPN. In summary, OPN and its poly-Asp86–93 sequence similarly affect COD mineral growth; the {110} crystallographic faces become enhanced and dominant attributable to {110} face inhibition by the protein/peptide, and peptides can incorporate into the mineral phase. We, thus, conclude that the poly-Asp86–93 domain is central to the OPN ability to interact with the {110} faces of COD, where it binds to inhibit crystal growth with subsequent intracrystalline incorporation (occlusion). PMID:19581305

  6. A secreted peptide acts on BIN2-mediated phosphorylation of ARFs to potentiate auxin response during lateral root development.

    PubMed

    Cho, Hyunwoo; Ryu, Hojin; Rho, Sangchul; Hill, Kristine; Smith, Stephanie; Audenaert, Dominique; Park, Joonghyuk; Han, Soeun; Beeckman, Tom; Bennett, Malcolm J; Hwang, Daehee; De Smet, Ive; Hwang, Ildoo

    2014-01-01

    The phytohormone auxin is a key developmental signal in plants. So far, only auxin perception has been described to trigger the release of transcription factors termed Auxin Response Factors (ARFs) from their auxin/indole-3-acetic acid (AUX/IAA) repressor proteins. Here, we show that phosphorylation of ARF7 and ARF19 by BRASSINOSTEROID-insensitive2 (BIN2) can also potentiate auxin signalling output during lateral root organogenesis. BIN2-mediated phosphorylation of ARF7 and ARF19 suppresses their interaction with AUX/IAAs, and subsequently enhances the transcriptional activity to their target genes lateral organ boundaries-domain16 (LBD16) and LBD29. In this context, BIN2 is under the control of the Tracheary element differentiation inhibitory factor (TDIF)-TDIF receptor (TDR) module. TDIF-initiated TDR signalling directly acts on BIN2-mediated ARF phosphorylation, leading to the regulation of auxin signalling during lateral root development. In summary, this study delineates a TDIF-TDR-BIN2 signalling cascade that controls regulation of ARF and AUX/IAA interaction independent of auxin perception during lateral root development. PMID:24362628

  7. Disruption of parathyroid hormone and parathyroid hormone-related peptide receptor phosphorylation prolongs ERK1/2 MAPK activation and enhances c-fos expression

    PubMed Central

    Abou-Samra, Abdul B.

    2012-01-01

    Previous studies have demonstrated that parathyroid hormone (PTH) binding to the PTH/PTH-related peptide receptor (PPR) stimulates G protein coupling, receptor phosphorylation, β-arrestin translocation, and internalization of the ligand/receptor complex. The extracellular signal-regulated mitogen-activated protein kinases 1/2 (ERK1/2 MAPK) are downstream effectors of PPR. In the current study, we investigated the role of PPR phosphorylation in the PTH regulation of the ERK1/2 MAPK pathway. Short treatment with PTH (0–40 min) of LLCP-K1 cells stably expressing a wild-type (WT) or a phosphorylation-deficient (PD) PPR (WT-PPR or PD-PPR cells, respectively) results in similar activation of ERK1/2. Interestingly, PTH stimulation of ERK1/2 in the WT-PPR cells then decreases as a result of longer PTH (60 min) treatment, and inhibition of ERK1/2 by PTH is observed at 90 min. Strikingly, the PD-PPR cells exhibit prolonged ERK1/2 activation up to 90 min of PTH treatment. An ERK1/2-dependent increase in c-fos expression is observed in the PD-PPR cells. Subsequently, c-fos expression in the WT-PPR and PD-PPR cells was markedly attenuated by a specific ERK1/2 pathway inhibitor. Further investigations revealed that PTH treatment causes a robust recruitment of a green fluorescent protein-tagged β-arrestin2 (β-arrestin2-GFP) in the WT-PPR cells. In contrast, β-arrestin2 recruitment was reduced in the PD-PPR cells. Importantly, expression of a receptor phosphorylation-independent β-arrestin2 (R169E) in the PD-PPR cells restored the biphasic effect of PTH on ERK1/2 as in the WT-PPR cells. The study reports a novel role for receptor phosphorylation and β-arrestin2 in the subsequent inhibition of the ERK1/2 pathway and in control of gene expression. PMID:22414806

  8. Neuroprotective effect of synthetic chalcone derivatives as competitive dual inhibitors against μ-calpain and cathepsin B through the downregulation of tau phosphorylation and insoluble Aβ peptide formation.

    PubMed

    Jeon, Kyung-Hwa; Lee, Eunyoung; Jun, Kyu-Yeon; Eom, Ji-Eun; Kwak, Soo Yeon; Na, Younghwa; Kwon, Youngjoo

    2016-10-01

    A series of chalcone derivatives were synthesized and evaluated for their μ-calpain and cathepsin B inhibitory activities. Among the tested chalcone derivatives, two compounds, 7 and 11, showed potent inhibitory activities against μ-calpain and cathepsin B and were selected for further evaluation. Compounds 7 and 11 showed enzyme inhibitory activities at the cellular level and displayed neuroprotective effects against oxidative stress-induced apoptosis in SH-SY5Y cells, a human neuroblastoma cell line. Moreover, compounds 7 and 11 reduced p25 formation, tau phosphorylation and insoluble Aβ peptide formation. Enzyme kinetic experiments and docking studies revealed that compounds 7 and 11 competitively inhibited both μ-calpain and cathepsin B enzymes. PMID:27318120

  9. A neuroligin-1-derived peptide stimulates phosphorylation of the NMDA receptor NR1 subunit and rescues MK-801-induced decrease in long-term potentiation and memory impairment.

    PubMed

    Korshunova, Irina; Gjørlund, Michelle D; Owczarek, Sylwia; Petersen, Anders V; Perrier, Jean-François; Gøtzsche, Casper René; Berezin, Vladimir

    2015-03-01

    Neuroligins (NLs) are postsynaptic adhesion molecules, interacting with presynaptic neurexins (NXs), which determine the differential formation of excitatory (glutamatergic, NL1) and inhibitory (GABAergic, NL2) synapses. We have previously demonstrated that treatment with a NL2-derived peptide, neurolide-2, reduces sociability and increase animal aggression. We hypothesized that interfering with NL1 function at the excitatory synapses might regulate synaptic plasticity and learning, and counteract memory deficits induced by N-methyl-d-aspartate (NMDA) receptor inhibition. First, neuronal NMDA receptor phosphorylation after treatment with NL1 or a mimetic peptide, neurolide-1, was quantified by immunoblotting. Subsequently, we investigated effects of neurolide-1 on long-term potentiation (LTP) induction in hippocampal slices compromised by NMDA receptor inhibitor MK-801. Finally, we investigated neurolide-1 effects on short- and long-term social and spatial memory in social recognition, Morris water-maze, and Y-maze tests. We found that subcutaneous neurolide-1 administration, restored hippocampal LTP compromised by NMDA receptor inhibitor MK-801. It counteracted MK-801-induced memory deficit in the water-maze and Y-maze tests after long-term treatment (24 h and 1-2 h before the test), but not after short-term exposure (1-2 h). Long-term exposure to neurolide-1 also facilitated social recognition memory. In addition, neurolide-1-induced phosphorylation of the NMDA receptor NR1 subunit on a site important for synaptic trafficking, potentially favoring synaptic receptor retention. Our findings emphasize the role of NL1-NMDA receptor interaction in cognition, and identify neurolide-1, as a valuable pharmacological tool to examine the in vivo role of postsynaptic NL1 in cognitive behavior in physiological and pathological conditions. PMID:26038702

  10. Analysis and optimization of the cationic lipid component of a lipid/peptide vector formulation for enhanced transfection in vitro and in vivo.

    PubMed

    Writer, Michele; Hurley, Christopher A; Sarkar, Supti; Copeman, Danielle M; Wong, John B; Odlyha, Marianne; Jayne Lawrence, M; Tabor, Alethea B; McAnulty, Robin J; Ayazi Shamlou, Parviz; Hailes, Helen C; Hart, Stephen L

    2006-01-01

    We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K16GACRRETAWACG) and Lipofectin, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization. PMID:17162579

  11. Cationic amphipathic D-enantiomeric antimicrobial peptides with in vitro and ex vivo activity against drug-resistant Mycobacterium tuberculosis.

    PubMed

    Lan, Yun; Lam, Jason T; Siu, Gilman K H; Yam, Wing Cheong; Mason, A James; Lam, Jenny K W

    2014-12-01

    Tuberculosis (TB) is the leading cause of bacterial death worldwide. Due to the emergence of multi-drug resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB), and the persistence of latent infections, a safe and effective TB therapy is highly sought after. Antimicrobial peptides (AMPs) have therapeutic potential against infectious diseases and have the ability to target microbial pathogens within eukaryotic cells. In the present study, we investigated the activity of a family of six AMPs containing all-D amino acids (D-LAK peptides) against MDR and XDR clinical strains of Mycobacterium tuberculosis (Mtb) both in vitro and, using THP-1 cells as a macrophage model, cultured ex vivo. All the D-LAK peptides successfully inhibited the growth of Mtb in vitro and were similarly effective against MDR and XDR strains. D-LAK peptides effectively broke down the heavy clumping of mycobacteria in broth culture, consistent with a 'detergent-like effect' that could reduce the hydrophobic interactions between the highly lipidic cell walls of the mycobacteria, preventing bacteria cell aggregation. Furthermore, though not able to eradicate the intracellular mycobacteria, D-LAK peptides substantially inhibited the intracellular growth of drug-resistant Mtb clinical isolates at concentrations that were well tolerated by THP-1 cells. Finally, combining D-LAK peptide with isoniazid could enhance the anti-TB efficacy. D-LAK peptide, particularly D-LAK120-A, was effective as an adjunct agent at non-toxic concentration to potentiate the efficacy of isoniazid against drug-resistant Mtb in vitro, possibly by facilitating the access of isoniazid into the mycobacteria by increasing the surface permeability of the pathogen. PMID:25154927

  12. Cationic Antimicrobial Peptides Derived from Crocodylus siamensis Leukocyte Extract, Revealing Anticancer Activity and Apoptotic Induction on Human Cervical Cancer Cells.

    PubMed

    Theansungnoen, Tinnakorn; Maijaroen, Surachai; Jangpromma, Nisachon; Yaraksa, Nualyai; Daduang, Sakda; Temsiripong, Theeranan; Daduang, Jureerut; Klaynongsruang, Sompong

    2016-06-01

    Known antimicrobial peptides KT2 and RT2 as well as the novel RP9 derived from the leukocyte extract of the freshwater crocodile (Crocodylus siamensis) were used to evaluate the ability in killing human cervical cancer cells. RP9 in the extract was purified by a combination of anion exchange column and reversed-phase HPLC, and its sequence was analyzed by mass spectrometry. The novel peptide could inhibit Gram-negative Vibrio cholerae (clinical isolation) and Gram-positive Bacillus pumilus TISTR 905, and its MIC values were 61.2 µM. From scanning electron microscopy, the peptide was seen to affect bacterial surfaces directly. KT2 and RT2, which are designed antimicrobial peptides using the C. siamensis Leucrocin I template, as well as RP9 were chemically synthesized for investigation of anticancer activity. By Sulforhodamine B colorimetric assay, these antimicrobial peptides could inhibit both HeLa and CaSki cancer cell lines. The IC50 values of KT2 and RT2 for HeLa and CaSki cells showed 28.7-53.4 and 17.3-30.8 µM, while those of RP9 were 126.2 and 168.3 µM, respectively. Additionally, the best candidate peptides KT2 and RT2 were used to determine the apoptotic induction on cancer cells by human apoptosis array assay. As a result, KT2 and RT2 were observed to induce apoptotic cell death in HeLa cells. Therefore, these results indicate that KT2 and RT2 with antimicrobial activity have a highly potent ability to kill human cervical cancer cells. PMID:27129462

  13. Cancer-preventive peptide lunasin from Solanum nigrum L. inhibits acetylation of core histones H3 and H4 and phosphorylation of retinoblastoma protein (Rb).

    PubMed

    Jeong, Jin Boo; Jeong, Hyung Jin; Park, Jae Ho; Lee, Sun Hee; Lee, Jeong Rak; Lee, Hee Kyeong; Chung, Gyu Young; Choi, Jeong Doo; de Lumen, Ben O

    2007-12-26

    Lunasin, a unique 43 amino acid, 4.8 kDa cancer-chemopreventive peptide initially reported in soybean and now found in barley and wheat, has been shown to be cancer-chemopreventive in mammalian cells and in a skin cancer mouse model against oncogenes and chemical carcinogens. To identify bioactive components in traditional herbal medicines and in search for new sources of lunasin, we report here the properties of lunasin from Solanum nigrum L. (SNL), a plant indigenous to northeast Asia. Lunasin was screened in the crude extracts of five varieties of the medicinal plants of Solanaceae origin and seven other major herbal plants. An in vitro digestion stability assay for measuring bioavailability was carried out on SNL crude protein and autoclaved SNL using pepsin and pancreatin. A nonradioactive histone acetyltransferase (HAT) assay and HAT activity colorimetric assay were used to measure the inhibition of core histone acetylation. The inhibitory effect of lunasin on the phosphorylation of retinoblastoma protein (Rb) was determined by immunoblotting against phospho-Rb. Lunasin isolated from autoclaved SNL inhibited core histone H3 and H4 acetylation, the activities of the HATs, and the phosphorylation of the Rb protein. Lunasin in the crude protein and in the autoclaved crude protein was very stable to pepsin and pancreatin in vitro digestion, while the synthetic pure lunasin was digested at 2 min after the reaction. We conclude that lunasin is a bioactive and bioavailable component in SNL and that consumption of SNL may play an important role in cancer prevention. PMID:18038993

  14. A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1.

    PubMed Central

    Mohammadi, M; Honegger, A M; Rotin, D; Fischer, R; Bellot, F; Li, W; Dionne, C A; Jaye, M; Rubinstein, M; Schlessinger, J

    1991-01-01

    Phospholipase C-gamma (PLC-gamma) is a substrate of the fibroblast growth factor receptor (FGFR; encoded by the flg gene) and other receptors with tyrosine kinase activity. It has been demonstrated that the src homology region 2 (SH2 domain) of PLC-gamma and of other signalling molecules such as GTPase-activating protein and phosphatidylinositol 3-kinase-associated p85 direct their binding toward tyrosine-autophosphorylated regions of the epidermal growth factor or platelet-derived growth factor receptor. In this report, we describe the identification of Tyr-766 as an autophosphorylation site of flg-encoded FGFR by direct sequencing of a tyrosine-phosphorylated tryptic peptide isolated from the cytoplasmic domain of FGFR expressed in Escherichia coli. The same phosphopeptide was found in wild-type FGFR phosphorylated either in vitro or in living cells. Like other growth factor receptors, tyrosine-phosphorylated wild-type FGFR or its cytoplasmic domain becomes associated with intact PLC-gamma or with a fusion protein containing the SH2 domain of PLC-gamma. To delineate the site of association, we have examined the capacity of a 28-amino-acid tryptic peptide containing phosphorylated Tyr-766 to bind to various constructs containing SH2 and other domains of PLC-gamma. It is demonstrated that the tyrosine-phosphorylated peptide binds specifically to the SH2 domain but not to the SH3 domain or other regions of PLC-gamma. Hence, Tyr-766 and its flanking sequences represent a major binding site in FGFR for PLC-gamma. Alignment of the amino acid sequences surrounding Tyr-766 with corresponding regions of other FGFRs revealed conserved tyrosine residues in all known members of the FGFR family. We propose that homologous tyrosine-phosphorylated regions in other FGFRs also function as binding sites for PLC-gamma and therefore are involved in coupling to phosphatidylinositol breakdown. Images PMID:1656221

  15. Quantum chemical studies of a model for peptide bond formation. 3. Role of magnesium cation in formation of amide and water from ammonia and glycine

    NASA Technical Reports Server (NTRS)

    Oie, T.; Loew, G. H.; Burt, S. K.; MacElroy, R. D.

    1984-01-01

    The SN2 reaction between glycine and ammonia molecules with magnesium cation Mg2+ as a catalyst has been studied as a model reaction for Mg(2+)-catalyzed peptide bond formation using the ab initio Hartree-Fock molecular orbital method. As in previous studies of the uncatalyzed and amine-catalyzed reactions between glycine and ammonia, two reaction mechanisms have been examined, i.e., a two-step and a concerted reaction. The stationary points of each reaction including intermediate and transition states have been identified and free energies calculated for all geometry-optimized reaction species to determine the thermodynamics and kinetics of each reaction. Substantial decreases in free energies of activation were found for both reaction mechanisms in the Mg(2+)-catalyzed amide bond formation compared with those in the uncatalyzed and amine-catalyzed amide bond formation. The catalytic effect of the Mg2+ cation is to stabilize both the transition states and intermediate, and it is attributed to the neutralization of the developing negative charge on the electrophile and formation of a conformationally flexible nonplanar five-membered chelate ring structure.

  16. Diagnostic model of saliva peptide finger print analysis of oral squamous cell carcinoma patients using weak cation exchange magnetic beads

    PubMed Central

    Jiang, Wei-Peng; Wang, Zhen; Xu, Li-Xin; Peng, Xin; Chen, Feng

    2015-01-01

    Saliva diagnostics utilizing nanotechnology and molecular technologies to detect oral squamous cell carcinoma (OSCC) has become an attractive field of study. However, no specific methods have been established. To refine the diagnostic power of saliva peptide fingerprints for the early detection of OSCC, we screened the expression spectrum of salivary peptides in 40 T1 stage OSCC patients (and healthy controls) using MALDI-TOF-MS combined with magnetic beads. Fifty proteins showed significantly different expression levels in the OSCC samples (P<0.05). Potential biomarkers were also predicted. The novel diagnostic proteomic model with m/z peaks of 1285.6 Da and 1432.2 Da are of certain value for early diagnosis of OSCC. PMID:26182373

  17. A Cationic Peptide, TAT-Cd0, Inhibits Herpes Simplex Virus Type 1 Ocular Infection In Vivo

    PubMed Central

    Jose, Gilbert G.; Larsen, Inna V.; Gauger, Joshua; Carballo, Erica; Stern, Rebecca; Brummel, Rachel; Brandt, Curtis R.

    2013-01-01

    Purpose. To test the in vivo activity of a peptide derived from the protein transducing domain of the human immunodeficiency virus (HIV) Tat protein, TAT-Cd0, in a murine herpes simplex type 1 (HSV-1) keratitis model. Methods. The efficacy of TAT-Cd0 was assessed in a postinfection treatment model with different concentrations (1 mg/mL, 0.1 mg/mL, 0.01 mg/mL) of the peptide in one of four delivery vehicles: artificial tears, PBS, methylcellulose, and aquaphor cream. Treatment began within 4 or 24 hours postinfection. Viral titers in the tear film were determined by plaque assay. Results. TAT-Cd0 reduced the severity of keratitis in all of the delivery vehicles tested when treatment started, 4 hours postinfection. Peptide in the tears or PBS delivery vehicle had the most significant reduction in disease severity and delayed the onset of vascularization and stromal keratitis. The percentage of mice presenting with disease was also significantly reduced and viral titers were reduced by 1 log at 24 hours postinfection in mice treated with 1 mg/mL TAT-Cd0, suggesting that inhibiting replication early is sufficient to achieve clinical effects. Lower concentrations were not effective and delaying treatment by 24 hours was also not effective. Conclusions. This study shows that TAT-Cd0 is an effective antiviral against HSV-1 strain KOS when applied shortly postinfection and that aqueous-based formulations are more suitable. PMID:23341013

  18. Immobilization of cationic antimicrobial peptides and natural cashew gum in nanosheet systems for the investigation of anti-leishmanial activity.

    PubMed

    Bittencourt, Clicia Ramos; de Oliveira Farias, Emanuel Airton; Bezerra, Karla Costa; Véras, Leiz Maria Costa; Silva, Vladimir Costa; Costa, Carlos Henrique Nery; Bemquerer, Marcelo P; Silva, Luciano Paulino; de Souza de Almeida Leite, José Roberto; Eiras, Carla

    2016-02-01

    This report details the development of thin films containing an antimicrobial peptide, specifically, dermaseptin 01 (GLWSTIKQKGKEAAIAAA-KAAGQAALGAL-NH2, [DRS 01]), and a natural polysaccharide, for a novel application in detecting the presence of Leishmania cells and maintaining anti-leishmanial activity. The peptide DRS 01 was immobilized in conjunction with natural cashew gum (CG) onto an indium tin oxide (ITO) substrate using the Layer-by-Layer (LbL) deposition technique. The LbL film ITO/CG/DRS 01, containing DRS 01 as the outer layer, was capable of detecting the presence of Leishmania cells and acting as an anti-leishmanial system. Detection was performed using cyclic voltammetry (CV) in phosphate buffer (pH7.2) in the presence of promastigote cells (0-10(7)cells/mL). The results showed a linear and inversely proportional relation between the concentration of Leishmania infantum protozoan cells and the measured current values obtained for the films, which was attributed to the effect of peptide-induced lysis of the cell membrane, and resulted in freed residues that were adsorbed on the electrode surface. With this, the paper shows a method using thin films with this new material to demonstrate the anti-leishmanial activity in vitro models of carpet-like mechanisms. PMID:26652407

  19. Radiolabeling of DOTA-like conjugated peptides with generator-produced (68)Ga and using NaCl-based cationic elution method.

    PubMed

    Mueller, Dirk; Breeman, Wouter A P; Klette, Ingo; Gottschaldt, Michael; Odparlik, Andreas; Baehre, Manfred; Tworowska, Izabela; Schultz, Michael K

    2016-06-01

    Gallium-68 ((68)Ga) is a generator-produced radionuclide with a short half-life (t½ = 68 min) that is particularly well suited for molecular imaging by positron emission tomography (PET). Methods have been developed to synthesize (68)Ga-labeled imaging agents possessing certain drawbacks, such as longer synthesis time because of a required final purification step, the use of organic solvents or concentrated hydrochloric acid (HCl). In our manuscript, we provide a detailed protocol for the use of an advantageous sodium chloride (NaCl)-based method for radiolabeling of chelator-modified peptides for molecular imaging. By working in a lead-shielded hot-cell system,(68)Ga(3+) of the generator eluate is trapped on a cation exchanger cartridge (100 mg, ∼8 mm long and 5 mm diameter) and then eluted with acidified 5 M NaCl solution directly into a sodium acetate-buffered solution containing a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or DOTA-like chelator-modified peptide. The main advantages of this procedure are the high efficiency and the absence of organic solvents. It can be applied to a variety of peptides, which are stable in 1 M NaCl solution at a pH value of 3-4 during reaction. After labeling, neutralization, sterile filtration and quality control (instant thin-layer chromatography (iTLC), HPLC and pH), the radiopharmaceutical can be directly administered to patients, without determination of organic solvents, which reduces the overall synthesis-to-release time. This procedure has been adapted easily to automated synthesis modules, which leads to a rapid preparation of (68)Ga radiopharmaceuticals (12-16 min). PMID:27172166

  20. Poly (I:C)-DOTAP cationic nanoliposome containing multi-epitope HER2-derived peptide promotes vaccine-elicited anti-tumor immunity in a murine model.

    PubMed

    Alipour Talesh, Ghazal; Ebrahimi, Zahra; Badiee, Ali; Mansourian, Mercedeh; Attar, Hossein; Arabi, Leila; Jalali, Seyed Amir; Jaafari, Mahmoud Reza

    2016-08-01

    In the current study we aimed at developing a vaccine delivery/adjuvant system to enhance anti-tumor immunity against the natural multi-epitope HER2/Neu-derived P5 peptide. Polyriboinosinic: polyribocytidylic acid [Poly (I:C)] is a strong immunoadjuvant able to enhance specific antitumor immunity induced by peptide-based vaccines. Nevertheless, delivering the peptide and adjuvant intracellularly into their target site remains a challenging issue. We hypothesized this barrier could be overcome through the use of a cationic nanoliposome carrier system which can carry and protect the antigen and adjuvant in the extracellular environment and augment the induction of antitumor immunity. P5 was encapsulated in cationic nanoliposomes composed of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)-Cholesterol either alone or complexed with Poly (I:C). Immunocompetent BALB/c mice were immunized with the formulations 3 times in two-week intervals and the efficiency and type of immune response were then evaluated both in vitro and in vivo. The groups immunized with Lip-P5+PIC (DOTAP-Cholestrol-P5+Poly (I:C)) and Lip+PIC (DOTAP-Cholestrol+Poly (I:C)) enhanced the release of Interferon (IFN)-γ in comparison with other groups. Flow cytometry analysis revealed that Lip-P5+PIC formulation induced the highest level of IFN-γ in CD8(+) lymphocytes. Lip-P5+PIC, Lip+PIC and Lip-P5 (DOTAP-Cholestrol-P5) provided some extent of protection in terms of tumor regression in TUBO tumor mice model during the first 65days post tumor challenge but at the end only the tumors of mice immunized with Lip-P5+PIC were significantly smaller than all other groups. Furthermore, tumors of mice receiving Lip-P5+PIC grew at a significantly slower rate throughout the observation period. Our results showed that the combination of Poly (I:C) and DOTAP with the tumor antigen and without applying additional T-helper epitope induced strong antitumor responses. The observations presented here are of great interest

  1. Evaluation of column carryover of phosphorylated peptides and fumonisins by duplicated solvent gradient method in liquid chromatography/tandem mass spectrometry.

    PubMed

    Sakamaki, Hiroshi; Uchida, Takeharu; Lim, Lee Wah; Takeuchi, Toyohide

    2015-01-01

    Columns made of three different materials were evaluated with regard to the carryover of phosphorylated peptides and fumonisins in liquid chromatography/tandem mass spectrometry (LC/MS/MS). In order to eliminate carryover caused by the injection operation in the autosampler, the column carryover was calculated using the duplicated solvent gradient method. A column made of a glass-lined stainless-steel tube and polyethylene frits (GL-PE column) yielded the most significant improvements in the peak shape and the carryover as compared to the other columns. The carryover of fumonisin B1 (FB1) and HLADLSpK (T19p) in the GL-PE column could be reduced; the lower limit of quantitation of T19p, and the range of the calibration curve were also improved. Since carryover peaks with the GL-PE column were symmetrical peaks of the samples, carryover in the column did not occur. The carryover calculated by the duplicated solvent gradient method corresponded to those in the flow path from the injection port to the inlet frit of the column. The carryover value of FB1 in the column with a stainless-steel tube and stainless-steel frits (S-S column) was 1.70%, and that of the flow path was 0.23%. We found that the majority of the carryover in our system occurred in the S-S column. PMID:25746806

  2. Inhibition of RelA-Ser536 Phosphorylation by a Competing Peptide Reduces Mouse Liver Fibrosis Without Blocking the Innate Immune Response

    PubMed Central

    Moles, Anna; Sanchez, Ana M; Banks, Paul S; Murphy, Lindsay B; Luli, Saimir; Borthwick, Lee; Fisher, Andrew; O’Reilly, Steven; van Laar, Jacob M; White, Steven A; Perkins, Neil D; Burt, Alastair D; Mann, Derek A; Oakley, Fiona

    2013-01-01

    Phosphorylation of the RelA subunit at serine 536 (RelA-P-Ser536) is important for hepatic myofibroblast survival and is mechanistically implicated in liver fibrosis. Here, we show that a cell-permeable competing peptide (P6) functions as a specific targeted inhibitor of RelA-P-Ser536 in vivo and exerts an antifibrogenic effect in two progressive liver disease models, but does not impair hepatic inflammation or innate immune responses after lipopolysaccharide challenge. Using kinase assays and western blotting, we confirm that P6 is a substrate for the inhibitory kappa B kinases (IKKs), IKKα and IKKβ, and, in human hepatic myofibroblasts, P6 prevents RelA-P-Ser536, but does not affect IKK activation of IκBα. We demonstrate that RelA-P-Ser536 is a feature of human lung and skin fibroblasts, but not lung epithelial cells, in vitro and is present in sclerotic skin and diseased lungs of patients suffering from idiopathic pulmonary fibrosis. Conclusion: RelA-P-Ser536 may be a core fibrogenic regulator of fibroblast phenotype. (Hepatology 2013) PMID:22996371

  3. Differential Effects of Penicillin Binding Protein Deletion on the Susceptibility of Enterococcus faecium to Cationic Peptide Antibiotics

    PubMed Central

    Kumaraswamy, Monika; Nonejuie, Poochit; Werth, Brian J.; Rybak, Micahel J.; Pogliano, Joseph; Rice, Louis B.; Nizet, Victor

    2015-01-01

    Beta-lactam antibiotics sensitize Enterococcus faecium to killing by endogenous antimicrobial peptides (AMPs) of the innate immune system and daptomycin through mechanisms yet to be elucidated. It has been speculated that beta-lactam inactivation of select E. faecium penicillin binding proteins (PBPs) may play a pivotal role in this sensitization process. To characterize the specific PBP inactivation that may be responsible for these phenotypes, we utilized a previously characterized set of E. faecium PBP knockout mutants to determine the effects of such mutations on the activity of daptomycin and the AMP human cathelicidin (LL-37). Enhanced susceptibility to daptomycin was dependent more on a cumulative effect of multiple PBP deletions than on inactivation of any single specific PBP. Selective knockout of PBPZ rendered E. faecium more vulnerable to killing by both recombinant LL-37 and human neutrophils, which produce the antimicrobial peptide in high quantities. Pharmacotherapy targeting multiple PBPs may be used as adjunctive therapy with daptomycin to treat difficult E. faecium infections. PMID:26195528

  4. Computational Study of the Cation-Modified GSH Peptide Interactions With Perovskite-Type BFO-(111) Membranes Under Aqueous Conditions.

    PubMed

    Bian, Liang; Dong, Fa-Qin; Song, Mian-Xin; Xu, Jin-Bao; Zhang, Xiao-Yan

    2015-12-01

    We elucidated a number of facets regarding glutathione (GSH)-bismuth ferrite (BiFeO3, BFO) interactions and reactivity that have previously remained unexplored on a molecular level. In this approach, the cation-modified reduced GSH (or oxidised glutathione (GS·)) formed on the (111)-oriented BiFeO3 membrane (namely BFO-(111)) can serve as an efficient quencher, and the luminescence mechanism is explained in aqueous conditions. Notably, we suggest the use of Fe(2+)↓ ion as an electron donor and K(+) ion as an electron acceptor to exert a "gluing" effect on the glutamic acid (Glu) and glycine (Gly) side chains, producing an exposed sulfhydryl (-SH) configuration. This method may enable the rational design of a convenient platform for biosensors. PMID:26061445

  5. Computational Study of the Cation-Modified GSH Peptide Interactions With Perovskite-Type BFO-(111) Membranes Under Aqueous Conditions

    NASA Astrophysics Data System (ADS)

    Bian, Liang; Dong, Fa-qin; Song, Mian-xin; Xu, Jin-bao; Zhang, Xiao-yan

    2015-06-01

    We elucidated a number of facets regarding glutathione (GSH)-bismuth ferrite (BiFeO3, BFO) interactions and reactivity that have previously remained unexplored on a molecular level. In this approach, the cation-modified reduced GSH (or oxidised glutathione (GS·)) formed on the (111)-oriented BiFeO3 membrane (namely BFO-(111)) can serve as an efficient quencher, and the luminescence mechanism is explained in aqueous conditions. Notably, we suggest the use of Fe2+↓ ion as an electron donor and K+ ion as an electron acceptor to exert a "gluing" effect on the glutamic acid (Glu) and glycine (Gly) side chains, producing an exposed sulfhydryl (-SH) configuration. This method may enable the rational design of a convenient platform for biosensors.

  6. Evaluation of separation properties of a modified strong cation exchange material named MEX and its application in 2D-MEX × C18 system to separate peptides from scorpion venom.

    PubMed

    Chen, Bo; Xu, Junyan; Fu, Qing; Dong, Xuefang; Guo, Zhimou; Jin, Yu; Liang, Xinmiao

    2015-07-01

    Peptides from scorpion venom represent one of the most promising drug sources for drug discovery for some specific diseases. Current challenges in their separation include high complexity, high homologies and the huge range of peptides. In this paper, a modified strong cation exchange material, named MEX, was utilised for the two-dimensional separation of peptides from complex scorpion venom. The silica-based MEX column was bonded with two functional groups; benzenesulfonic acid and cyanopropyl. To better understand its separation mechanisms, seven standard peptides with different properties were employed in an evaluation study, the results of which showed that two interactions were involved in the MEX column: electrostatic interactions based on benzenesulfonic acid groups dominated the separation of peptides; weak hydrophobic interactions introduced by cyanopropyl groups increased the column's selectivity for peptides with the same charge. This characteristic allowed the MEX column to overcome some of the drawbacks of traditional strong cation exchange (SCX) columns. Furthermore, the study showed the great effects of the acetonitrile (ACN) content, the sodium perchlorate (NaClO4) concentration and the buffer pH in the mobile phase on the peptides' retention and separation selectivity on the MEX column. Subsequently, the MEX column was combined with a C18 column to establish an off-line 2D-MEX × C18 system to separate peptides from scorpion Buthus martensi Karsch (BmK) venom. Due to complementary separation mechanisms in each dimension, a high orthogonality of 47.62% was achieved. Moreover, a good loading capacity, excellent stability and repeatability were exhibited by the MEX column, which are beneficial for its use in future preparation experiments. Therefore, the MEX column could be an alternative to the traditional SCX columns for the separation of peptides from scorpion venom. PMID:25996445

  7. Design and Synthesis of Amphiphilic and Luminescent Tris-Cyclometalated Iridium(III) Complexes Containing Cationic Peptides as Inducers and Detectors of Cell Death via a Calcium-Dependent Pathway.

    PubMed

    Hisamatsu, Yosuke; Shibuya, Ai; Suzuki, Nozomi; Suzuki, Toshihiro; Abe, Ryo; Aoki, Shin

    2015-05-20

    Cationic amphiphilic peptides have the potential to function as agents for the treatment of microbial infections and cancer therapy. The cationic and hydrophobic parts of these molecules allow them to associate strongly with negatively charged bacterial or cancer cell membranes, thus exerting antimicrobial and anticancer activities through membrane disruption. Meanwhile, cyclometalated iridium(III) complexes such as fac-Ir(ppy)3 (ppy = 2-phenylpyridine) and fac-Ir(tpy)3 (tpy = 2-(4'-tolyl)pyridine) possess C3-symmetric structures and excellent photophysical properties as phosphorescence materials, which make them important candidates for use in biological applications such as chemosensors, biolabeling, living cell staining, in vivo tumor imaging, and anticancer agents. We recently reported on some regioselective substitution reactions of Ir(tpy)3 and Ir(ppy)3 at the 5'-position (p-position with respect to the C-Ir bond) on the 2-phenylpyridine ligands and their subsequent conversions to a variety of functional groups. We report here on the design and synthesis of amphiphilic and luminescent tris-cyclometalated Ir complexes in which cationic peptides are attached through alkyl chain linkers that work as inducers and detectors of cell death. Ir complexes containing cationic peptides such as a KKGG sequence and alkyl chain linkers of adequate length (C6 and C8) exhibit considerable cytotoxicity against cancer cells such as Jurkat, Molt-4, HeLa-S3, and A549 cells, and that dead cells are well stained with these Ir complexes. Furthermore, an Ir complex in which the KKGG peptide is attached through a C6 linker displayed lower cytotoxicity against normal mouse lymphocytes. Mechanistic studies suggest that Ir complexes containing the KKGG peptide interact with anionic molecules on the cell surface and/or membrane receptors to trigger the Ca(2+) dependent pathway and intracellular Ca(2+) response, resulting in necrosis accompanied by membrane disruption. PMID:25875312

  8. Latent Ice Recrystallization Inhibition Activity in Nonantifreeze Proteins: Ca2+-Activated Plant Lectins and Cation-Activated Antimicrobial Peptides

    PubMed Central

    2015-01-01

    Organisms living in polar regions have evolved a series of antifreeze (glyco) proteins (AFGPs) to enable them to survive by modulating the structure of ice. These proteins have huge potential for use in cellular cryopreservation, ice-resistant surfaces, frozen food, and cryosurgery, but they are limited by their relatively low availability and questions regarding their mode of action. This has triggered the search for biomimetic materials capable of reproducing this function. The identification of new structures and sequences capable of inhibiting ice growth is crucial to aid our understanding of these proteins. Here, we show that plant c-type lectins, which have similar biological function to human c-type lectins (glycan recognition) but no sequence homology to AFPs, display calcium-dependent ice recrystallization inhibition (IRI) activity. This IRI activity can be switched on/off by changing the Ca2+ concentration. To show that more (nonantifreeze) proteins may exist with the potential to display IRI, a second motif was considered, amphipathicity. All known AFPs have defined hydrophobic/hydrophilic domains, rationalizing this choice. The cheap, and widely used, antimicrobial Nisin was found to have cation-dependent IRI activity, controlled by either acid or addition of histidine-binding ions such as zinc or nickel, which promote its amphipathic structure. These results demonstrate a new approach in the identification of antifreeze protein mimetic macromolecules and may help in the development of synthetic mimics of AFPs. PMID:26407233

  9. Latent Ice Recrystallization Inhibition Activity in Nonantifreeze Proteins: Ca2+-Activated Plant Lectins and Cation-Activated Antimicrobial Peptides.

    PubMed

    Mitchell, Daniel E; Gibson, Matthew I

    2015-10-12

    Organisms living in polar regions have evolved a series of antifreeze (glyco) proteins (AFGPs) to enable them to survive by modulating the structure of ice. These proteins have huge potential for use in cellular cryopreservation, ice-resistant surfaces, frozen food, and cryosurgery, but they are limited by their relatively low availability and questions regarding their mode of action. This has triggered the search for biomimetic materials capable of reproducing this function. The identification of new structures and sequences capable of inhibiting ice growth is crucial to aid our understanding of these proteins. Here, we show that plant c-type lectins, which have similar biological function to human c-type lectins (glycan recognition) but no sequence homology to AFPs, display calcium-dependent ice recrystallization inhibition (IRI) activity. This IRI activity can be switched on/off by changing the Ca2+ concentration. To show that more (nonantifreeze) proteins may exist with the potential to display IRI, a second motif was considered, amphipathicity. All known AFPs have defined hydrophobic/hydrophilic domains, rationalizing this choice. The cheap, and widely used, antimicrobial Nisin was found to have cation-dependent IRI activity, controlled by either acid or addition of histidine-binding ions such as zinc or nickel, which promote its amphipathic structure. These results demonstrate a new approach in the identification of antifreeze protein mimetic macromolecules and may help in the development of synthetic mimics of AFPs. PMID:26407233

  10. C-terminal tail phosphorylation of N-formyl peptide receptor: differential recognition of two neutrophil chemoattractant receptors by monoclonal antibodies NFPR1 and NFPR2.

    PubMed

    Riesselman, Marcia; Miettinen, Heini M; Gripentrog, Jeannie M; Lord, Connie I; Mumey, Brendan; Dratz, Edward A; Stie, Jamal; Taylor, Ross M; Jesaitis, Algirdas J

    2007-08-15

    The N-formyl peptide receptor (FPR), a G protein-coupled receptor that binds proinflammatory chemoattractant peptides, serves as a model receptor for leukocyte chemotaxis. Recombinant histidine-tagged FPR (rHis-FPR) was purified in lysophosphatidyl glycerol (LPG) by Ni(2+)-NTA agarose chromatography to >95% purity with high yield. MALDI-TOF mass analysis (>36% sequence coverage) and immunoblotting confirmed the identity as FPR. The rHis-FPR served as an immunogen for the production of 2 mAbs, NFPR1 and NFPR2, that epitope map to the FPR C-terminal tail sequences, 305-GQDFRERLI-313 and 337-NSTLPSAEVE-346, respectively. Both mAbs specifically immunoblotted rHis-FPR and recombinant FPR (rFPR) expressed in Chinese hamster ovary cells. NFPR1 also recognized recombinant FPRL1, specifically expressed in mouse L fibroblasts. In human neutrophil membranes, both Abs labeled a 45-75 kDa species (peak M(r) approximately 60 kDa) localized primarily in the plasma membrane with a minor component in the lactoferrin-enriched intracellular fractions, consistent with FPR size and localization. NFPR1 also recognized a band of M(r) approximately 40 kDa localized, in equal proportions to the plasma membrane and lactoferrin-enriched fractions, consistent with FPRL1 size and localization. Only NFPR2 was capable of immunoprecipitation of rFPR in detergent extracts. The recognition of rFPR by NFPR2 is lost after exposure of cellular rFPR to f-Met-Leu-Phe (fMLF) and regained after alkaline phosphatase treatment of rFPR-bearing membranes. In neutrophils, NFPR2 immunofluorescence was lost upon fMLF stimulation. Immunoblotting approximately 60 kDa species, after phosphatase treatment of fMLF-stimulated neutrophil membranes, was also enhanced. We conclude that the region 337-346 of FPR becomes phosphorylated after fMLF activation of rFPR-expressing Chinese hamster ovary cells and neutrophils. PMID:17675514

  11. Cationic peptide mR18L with lipid lowering properties inhibits LPS-induced systemic and liver inflammation in rats.

    PubMed

    Sharifov, Oleg F; Nayyar, Gaurav; Ternovoy, Vladimir V; Mishra, Vinod K; Litovsky, Silvio H; Palgunachari, Mayakonda N; Garber, David W; Anantharamaiah, G M; Gupta, Himanshu

    2013-07-12

    The cationic single domain peptide mR18L has demonstrated lipid-lowering and anti-atherogenic properties in different dyslipidemic mouse models. Lipopolysaccharide (LPS)-mediated inflammation is considered as one of the potential triggers for atherosclerosis. Here, we evaluated anti-inflammatory effects of mR18L peptide against LPS-mediated inflammation. First, we tested the efficacy and tolerance of 1, 2.5 and 5mg/kg mR18L in normolipidemic rats stimulated with 5mg/kg LPS. LPS and then mR18L were injected in different intraperitoneal regions. By 2h post LPS, mR18L inhibited LPS-mediated plasma TNF-α elevation at all doses, with the effect being stronger for 2.5mg/kg (P<0.05 vs. 1mg/kg, non-significant vs. 5mg/kg). In a similar model, 2.5mg/kg mR18L reduced LPS-mediated inflammation in the liver, as assessed by microscopic examination of liver sections and measurements of iNOS expression in the liver tissue. In plasma, 2.5mg/kg mR18L decreased levels of TNF-α and IL-6, decreased endotoxin activity and enhanced HDL binding to LPS. In another similar experiment, mR18L administered 1h post LPS, prevented elevation of plasma triglycerides by 6h post LPS and increased plasma activity of anti-oxidant enzyme paraoxonase 1, along with noted trends in reducing plasma levels of endotoxin and IL-6. Surface plasmon resonance study revealed that mR18L readily binds LPS. We conclude that mR18L exerts anti-endotoxin activity at least in part due to direct LPS-binding and LPS-neutralizing effects. We suggest that anti-endotoxin activity of mR18L is an important anti-inflammatory property, which may increase anti-atherogenic potential of this promising orally active lipid-lowering peptide. PMID:23791744

  12. Interaction of the cationic peptide bactenecin with phospholipid monolayers at the air-water interface: i interaction with 1,2-dipalmitoyl-sn-glycero-3-phosphatidilcholine.

    PubMed

    López-Oyama, A B; Flores-Vázquez, A L; Burboa, M G; Gutiérrez-Millán, L E; Ruiz-García, J; Valdez, M A

    2009-07-23

    In this work we have investigated the influence of NaCl on the adsorption of the antimicrobial cationic peptide bactenecin in the monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at the air-water interface, as a function of NaCl concentrations in the subphase. We show that the effect of the salt concentration on DPPC monolayers is a monotonic decrease of the liquid-condensed-liquid-expanded (LC-LE) coexistence region. By contrast, the effect of the bactenecin adsorption at the DPPC monolayer not only removed the LC-LE coexistence region plateau, but also shifted the DPPC isotherms to higher pressures and increased the compressibility of the DPPC/bactenecin monolayers with respect to the pure DPPC monolayer around the LC phase. Analysis of the domain structure, obtained by Brewster angle and atomic force microscopes, indicates that the salt concentration in the subphase builds an electrostatic barrier, increasing the rigidity of DPPC monolayers and limiting the bactenecin adsorption at the LC-LE phase coexistence. PMID:19569630

  13. Cationicity-enhanced analogues of the antimicrobial peptides, AcrAP1 and AcrAP2, from the venom of the scorpion, Androctonus crassicauda, display potent growth modulation effects on human cancer cell lines.

    PubMed

    Du, Qiang; Hou, Xiaojuan; Ge, Lilin; Li, Renjie; Zhou, Mei; Wang, Hui; Wang, Lei; Wei, Minjie; Chen, Tianbao; Shaw, Chris

    2014-01-01

    The non disulphide-bridged peptides (NDBPs) of scorpion venoms are attracting increased interest due to their structural heterogeneity and broad spectrum of biological activities. Here, two novel peptides, named AcrAP1 and AcrAP2, have been identified in the lyophilised venom of the Arabian scorpion, Androctonus crassicauda, through "shotgun" molecular cloning of their biosynthetic precursor-encoding cDNAs. The respective mature peptides, predicted from these cloned cDNAs, were subsequently isolated from the same venom sample using reverse phase HPLC and their identities were confirmed by use of mass spectrometric techniques. Both were found to belong to a family of highly-conserved scorpion venom antimicrobial peptides - a finding confirmed through the biological investigation of synthetic replicates. Analogues of both peptides designed for enhanced cationicity, displayed enhanced potency and spectra of antimicrobial activity but, unlike the native peptides, these also displayed potent growth modulation effects on a range of human cancer cell lines. Thus natural peptide templates from venom peptidomes can provide the basis for rational analogue design to improve both biological potency and spectrum of action. The diversity of such templates from such natural sources undoubtedly provides the pharmaceutical industry with unique lead compounds for drug discovery. PMID:25332684

  14. Crystallization and preliminary X-ray diffraction studies on the human Plk1 Polo-box domain in complex with an unphosphorylated and a phosphorylated target peptide from Cdc25C

    SciTech Connect

    García-Álvarez, Begoña; Ibañez, Sonia; Montoya, Guillermo

    2006-04-01

    Crystals of the human Plk1 Polo-box domain in complex with a Cdc25C target peptide in an unphosphorylated and a phosphorylated state have been obtained in orthorhombic and monoclinic forms that diffract to 2.1 and 2.85 Å, respectively, using synchrotron radiation. Polo-like kinase (Plk1) is crucial for cell-cycle progression via mitosis. Members of the Polo-like kinase family are characterized by the presence of a C-terminal domain termed the Polo-box domain (PBD) in addition to the N-terminal kinase domain. The PBD of Plk1 was cloned and overexpressed in Escherichia coli. Crystallization experiments of the protein in complex with an unphosphorylated and a phosphorylated target peptide from Cdc25C yield crystals suitable for X-ray diffraction analysis. Crystals of the PBD in complex with the phosphorylated peptide belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.23, b = 67.35, c = 88.25 Å, α = γ = β = 90°, and contain one molecule per asymmetric unit. Crystals of the PBD in complex with the unphosphorylated peptide belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 40.18, b = 49.17, c = 56.23 Å, α = γ = 90, β = 109.48°, and contain one molecule per asymmetric unit. The crystals diffracted to resolution limits of 2.1 and 2.85 Å using synchrotron radiation at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS), respectively.

  15. Insights into the Phosphoryl Transfer Catalyzed by cAMP-Dependent Protein Kinase: An X-ray Crystallographic Study of Complexes with Various Metals and Peptide Substrate SP20

    PubMed Central

    2013-01-01

    X-ray structures of several ternary substrate and product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with different bound metal ions. In the PKAc complexes, Mg2+, Ca2+, Sr2+, and Ba2+ metal ions could bind to the active site and facilitate the phosphoryl transfer reaction. ATP and a substrate peptide (SP20) were modified, and the reaction products ADP and the phosphorylated peptide were found trapped in the enzyme active site. Finally, we determined the structure of a pseudo-Michaelis complex containing Mg2+, nonhydrolyzable AMP-PCP (β,γ-methyleneadenosine 5′-triphosphate) and SP20. The product structures together with the pseudo-Michaelis complex provide snapshots of different stages of the phosphorylation reaction. Comparison of these structures reveals conformational, coordination, and hydrogen bonding changes that might occur during the reaction and shed new light on its mechanism, roles of metals, and active site residues. PMID:23672593

  16. Detachable strong cation exchange monolith, integrated with capillary zone electrophoresis and coupled with pH gradient elution, produces improved sensitivity and numbers of peptide identifications during bottom-up analysis of complex proteomes.

    PubMed

    Zhang, Zhenbin; Yan, Xiaojing; Sun, Liangliang; Zhu, Guijie; Dovichi, Norman J

    2015-04-21

    A detachable sulfonate-silica hybrid strong cation-exchange monolith was synthesized in a fused silica capillary, and used for solid phase extraction with online pH gradient elution during capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) proteomic analysis. Tryptic digests were prepared in 50 mM formic acid and loaded onto the strong cation-exchange monolith. Fractions were eluted using a series of buffers with lower concentration but higher pH values than the 50 mM formic acid background electrolyte. This combination of elution and background electrolytes results in both sample stacking and formation of a dynamic pH junction and allows use of relatively large elution buffer volumes while maintaining reasonable peak efficiency and resolution. A series of five pH bumps were applied to elute E. coli tryptic peptides from the monolith, followed by analysis using CZE coupled to an LTQ-Orbitrap Velos mass spectrometer; 799 protein groups and 3381 peptides were identified from 50 ng of the digest in a 2.5 h analysis, which approaches the identification rate for this organism that was obtained with an Orbitrap Fusion. We attribute the improved numbers of peptide and protein identifications to the efficient fractionation by the online pH gradient elution, which decreased the complexity of the sample in each elution step and improved the signal intensity of low abundance peptides. We also performed a comparative analysis using a nanoACQUITY UltraPerformance LCH system. Similar numbers of protein and peptide identifications were produced by the two methods. Protein identifications showed significant overlap between the two methods, whereas peptide identifications were complementary. PMID:25822566

  17. Cell-penetrating peptides do not cross mitochondrial membranes even when conjugated to a lipophilic cation: evidence against direct passage through phospholipid bilayers

    PubMed Central

    2004-01-01

    CPPs (cell-penetrating peptides) facilitate the cellular uptake of covalently attached oligonucleotides, proteins and other macromolecules, but the mechanism of their uptake is disputed. Two models are proposed: direct movement through the phospholipid bilayer and endocytic uptake. Mitochondria are a good model system to distinguish between these possibilities, since they have no vesicular transport systems. Furthermore, CPP-mediated delivery of macromolecules to the mitochondrial matrix would be a significant breakthrough in the study of mitochondrial function and dysfunction, and could also lead to new therapies for diseases caused by mitochondrial damage. Therefore we investigated whether two CPPs, penetratin and Tat, could act as mitochondrial delivery vectors. We also determined whether conjugation of the lipophilic cation TPP (triphenylphosphonium) to penetratin or Tat facilitated their uptake into mitochondria, since TPP leads to uptake of attached molecules into mitochondria driven by the membrane potential. Neither penetratin nor Tat, nor their TPP conjugates, are internalized by isolated mitochondria, indicating that these CPPs cannot cross mitochondrial phospholipid bilayers. Tat and TPP–Tat are taken up by cells, but they accumulate in endosomes and do not reach mitochondria. We conclude that CPPs cannot cross mitochondrial phospholipid bilayers, and therefore cannot deliver macromolecules directly to mitochondria. Our findings shed light on the mechanism of uptake of CPPs by cells. The lack of direct movement of CPPs through mitochondrial phospholipid bilayers, along with the observed endosomal accumulation of Tat and TPP–Tat in cells, makes it unlikely that CPPs enter cells by direct membrane passage, and instead favours cellular uptake via an endocytic pathway. PMID:15270716

  18. Cationic antimicrobial peptides serve as activation signals for the Salmonella Typhimurium PhoPQ and PmrAB regulons in vitro and in vivo

    PubMed Central

    Richards, Susan M.; Strandberg, Kristi L.; Conroy, Megan; Gunn, John S.

    2012-01-01

    Salmonella enterica serovar Typhimurium uses two-component regulatory systems (TCRSs) to respond to environmental stimuli. Upon infection, the TCRSs PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB) are activated by environmental signals detected in the lumen of the intestine and within host cells. TCRS-mediated gene expression leads to upregulation of genes involved in lipopolysaccharide (LPS) modification and cationic antimicrobial peptide (CAMP) resistance. This research expands on previous studies which have shown that CAMPs can activate Salmonella TCRSs in vitro. The focus of this work was to determine if CAMPs can act as environmental signals for PhoPQ- and PmrAB-mediated gene expression in vitro, during infection of macrophages and in a mouse model of infection. Monitoring of PhoPQ and PmrAB activation using recombinase-based in vivo expression technology (RIVET), alkaline phosphtase and β-galactosidase reporter fusion constructs demonstrated that S. Typhimurium PhoQ can sense CAMPs in vitro. In mouse macrophages, the cathelecidin CRAMP does not activate the PhoPQ regulon. Acidification of the Salmonella-containing vacuole activates PhoP- and PmrA-regulated loci but blocking acidification still does not reveal a role for CRAMP in TCRS activation in mouse macrophages. However, assays performed in susceptible wild type (WT), CRAMP knockout (KO), and matrilysin (a metalloproteinase necessary for activating murine α-defensins) KO mice suggest CRAMP, but not α-defensins, serve as a putative direct TCRS activation signal in the mouse intestine. These studies provide a better understanding of the in vivo environments that result in activation of these virulence-associated TCRSs. PMID:22919691

  19. Phosphoryl transfer reaction snapshots in crystals: Insights into the mechanism of protein kinase a catalytic subunit

    DOE PAGESBeta

    Das, Amit; Gerlits, Oksana O.; Heller, William T.; Kovalevskyi, Andrii Y.; Langan, Paul; Tian, Jianhui

    2015-06-19

    To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca2+ ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex, themore » thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca2+ cations with Mg2+ ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. As a result, the present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date.« less

  20. Phosphoryl transfer reaction snapshots in crystals: Insights into the mechanism of protein kinase a catalytic subunit

    SciTech Connect

    Das, Amit; Gerlits, Oksana O.; Heller, William T.; Kovalevskyi, Andrii Y.; Langan, Paul; Tian, Jianhui

    2015-06-19

    To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca2+ ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex, the thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca2+ cations with Mg2+ ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. As a result, the present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date.

  1. Predikin and PredikinDB: a computational framework for the prediction of protein kinase peptide specificity and an associated database of phosphorylation sites

    PubMed Central

    Saunders, Neil FW; Brinkworth, Ross I; Huber, Thomas; Kemp, Bruce E; Kobe, Bostjan

    2008-01-01

    Background We have previously described an approach to predicting the substrate specificity of serine-threonine protein kinases. The method, named Predikin, identifies key conserved substrate-determining residues in the kinase catalytic domain that contact the substrate in the region of the phosphorylation site and so determine the sequence surrounding the phosphorylation site. Predikin was implemented originally as a web application written in Javascript. Results Here, we describe a new version of Predikin, completely revised and rewritten as a modular framework that provides multiple enhancements compared with the original. Predikin now consists of two components: (i) PredikinDB, a database of phosphorylation sites that links substrates to kinase sequences and (ii) a Perl module, which provides methods to classify protein kinases, reliably identify substrate-determining residues, generate scoring matrices and score putative phosphorylation sites in query sequences. The performance of Predikin as measured using receiver operator characteristic (ROC) graph analysis equals or surpasses that of existing comparable methods. The Predikin website has been redesigned to incorporate the new features. Conclusion New features in Predikin include the use of SQL queries to PredikinDB to generate predictions, scoring of predictions, more reliable identification of substrate-determining residues and putative phosphorylation sites, extended options to handle protein kinase and substrate data and an improved web interface. The new features significantly enhance the ability of Predikin to analyse protein kinases and their substrates. Predikin is available at . PMID:18501020

  2. Actinide cation-cation complexes

    SciTech Connect

    Stoyer, N.J.; Seaborg, G.T.

    1994-12-01

    The +5 oxidation state of U, Np, Pu, and Am is a linear dioxo cation (AnO{sub 2}{sup +}) with a formal charge of +1. These cations form complexes with a variety of other cations, including actinide cations. Other oxidation states of actinides do not form these cation-cation complexes with any cation other than AnO{sub 2}{sup +}; therefore, cation-cation complexes indicate something unique about AnO{sub 2}{sup +} cations compared to actinide cations in general. The first cation-cation complex, NpO{sub 2}{sup +}{center_dot}UO{sub 2}{sup 2+}, was reported by Sullivan, Hindman, and Zielen in 1961. Of the four actinides that form AnO{sub 2}{sup +} species, the cation-cation complexes of NpO{sub 2}{sup +} have been studied most extensively while the other actinides have not. The only PuO{sub 2}{sup +} cation-cation complexes that have been studied are with Fe{sup 3+} and Cr{sup 3+} and neither one has had its equilibrium constant measured. Actinides have small molar absorptivities and cation-cation complexes have small equilibrium constants; therefore, to overcome these obstacles a sensitive technique is required. Spectroscopic techniques are used most often to study cation-cation complexes. Laser-Induced Photacoustic Spectroscopy equilibrium constants for the complexes NpO{sub 2}{sup +}{center_dot}UO{sub 2}{sup 2+}, NpO{sub 2}{sup +}{center_dot}Th{sup 4+}, PuO{sub 2}{sup +}{center_dot}UO{sub 2}{sup 2+}, and PuO{sub 2}{sup +}{center_dot}Th{sup 4+} at an ionic strength of 6 M using LIPAS are 2.4 {plus_minus} 0.2, 1.8 {plus_minus} 0.9, 2.2 {plus_minus} 1.5, and {approx}0.8 M{sup {minus}1}.

  3. Phosphorylation of RS1 (RSC1A1) Steers Inhibition of Different Exocytotic Pathways for Glucose Transporter SGLT1 and Nucleoside Transporter CNT1, and an RS1-Derived Peptide Inhibits Glucose Absorption.

    PubMed

    Veyhl-Wichmann, Maike; Friedrich, Alexandra; Vernaleken, Alexandra; Singh, Smriti; Kipp, Helmut; Gorboulev, Valentin; Keller, Thorsten; Chintalapati, Chakravarthi; Pipkorn, Rüdiger; Pastor-Anglada, Marçal; Groll, Jürgen; Koepsell, Hermann

    2016-01-01

    Cellular uptake adapts rapidly to physiologic demands by changing transporter abundance in the plasma membrane. The human gene RSC1A1 codes for a 67-kDa protein named RS1 that has been shown to induce downregulation of the sodium-D-glucose cotransporter 1 (SGLT1) and of the concentrative nucleoside transporter 1 (CNT1) in the plasma membrane by blocking exocytosis at the Golgi. Injecting RS1 fragments into Xenopus laevis oocytes expressing SGLT1 or CNT1 and measuring the expressed uptake of α-methylglucoside or uridine 1 hour later, we identified a RS1 domain (RS1-Reg) containing multiple predicted phosphorylation sites that is responsible for this post-translational downregulation of SGLT1 and CNT1. Dependent on phosphorylation, RS1-Reg blocks the release of SGLT1-containing vesicles from the Golgi in a glucose-dependent manner or glucose-independent release of CNT1-containing vesicles. We showed that upregulation of SGLT1 in the small intestine after glucose ingestion is promoted by glucose-dependent disinhibition of the RS1-Reg-blocked exocytotic pathway of SGLT1 between meals. Mimicking phosphorylation of RS1-Reg, we obtained a RS1-Reg variant that downregulates SGLT1 in the brush-border membrane at high luminal glucose concentration. Because RS1 mediates short-term regulation of various transporters, we propose that the RS1-Reg-navigated transporter release from Golgi represents a basic regulatory mechanism of general importance, which implies the existence of receptor proteins that recognize different phosphorylated forms of RS1-Reg and of complex transporter-specific sorting in the trans-Golgi. RS1-Reg-derived peptides that downregulate SGLT1 at high intracellular glucose concentrations may be used for downregulation of glucose absorption in small intestine, which has been proposed as strategy for treatment of type 2 diabetes. PMID:26464324

  4. Histone phosphorylation

    PubMed Central

    Rossetto, Dorine; Avvakumov, Nikita; Côté, Jacques

    2012-01-01

    Histone posttranslational modifications are key components of diverse processes that modulate chromatin structure. These marks function as signals during various chromatin-based events, and act as platforms for recruitment, assembly or retention of chromatin-associated factors. The best-known function of histone phosphorylation takes place during cellular response to DNA damage, when phosphorylated histone H2A(X) demarcates large chromatin domains around the site of DNA breakage. However, multiple studies have also shown that histone phosphorylation plays crucial roles in chromatin remodeling linked to other nuclear processes. In this review, we summarize the current knowledge of histone phosphorylation and describe the many kinases and phosphatases that regulate it. We discuss the key roles played by this histone mark in DNA repair, transcription and chromatin compaction during cell division and apoptosis. Additionally, we describe the intricate crosstalk that occurs between phosphorylation and other histone modifications and allows for sophisticated control over the chromatin remodeling processes. PMID:22948226

  5. Binding of cationic peptides (KX)4K to DPPG bilayers. Increasing the hydrophobicity of the uncharged amino acid X drives formation of membrane bound β-sheets: A DSC and FT-IR study.

    PubMed

    Hädicke, André; Blume, Alfred

    2016-06-01

    The binding of cationic peptides of the sequence (KX)4K to lipid vesicles of negatively charged dipalmitoyl-phosphatidylglycerol (DPPG) was investigated by differential scanning calorimetry (DSC) and temperature dependent Fourier-transformed infrared (FT-IR) spectroscopy. The hydrophobicity of the uncharged amino acid X was changed from G (glycine) over A (alanine), Abu (α-aminobutyric acid), V (valine) to L (leucine). The binding of the peptides caused an increase of the phase transition temperature (Tm) of DPPG by up to 20°C. The shift depended on the charge ratio and on the hydrophobicity of the amino acid X. Unexpectedly, the upward shift of Tm increased with increasing hydrophobicity of X. FT-IR spectroscopy showed a shift of the CH2 stretching vibrations of DPPG to lower frequency, particularly for bilayers in the liquid-crystalline phase, indicating an ordering of the hydrocarbon chains when the peptides were bound. Changes in the lipid C=O vibrational band indicated a dehydration of the lipid headgroup region after peptide binding. (KG)4K was bound in an unordered structure at all temperatures. All other peptides formed intermolecular antiparallel β-sheets, when bound to gel phase DPPG. However, for (KA)4K and (KAbu)4K, the β-sheets converted into an unordered structure above Tm. In contrast, the β-sheet structures of (KV)4K and (KL)4K remained stable even at 80°C when bound to the liquid-crystalline phase of DPPG. Strong aggregation of DPPG vesicles occurred after peptide binding. For the aggregates, we suggest a structure, where aggregated single β-sheets are sandwiched between opposing DPPG bilayers with a dehydrated interfacial region. PMID:26903220

  6. Binding of the Cationic Peptide (KL)4K to Lipid Monolayers at the Air-Water Interface: Effect of Lipid Headgroup Charge, Acyl Chain Length, and Acyl Chain Saturation.

    PubMed

    Hädicke, André; Blume, Alfred

    2016-04-28

    The binding of the cationic peptide (KL)4K to monolayers of different anionic lipids was determined by adsorption experiments. The chemical structure of the anionic phospholipids was changed in different ways. First, the hydrophobic region of phosphatidylglycerols was altered by elongation of the acyl chain length. Second, an unsaturated chain was introduced. Third, lipids with negatively charged headgroups of different chemical structure were compared. (KL)4K itself shows no surface activity and does not bind to monolayers of zwitterionic lipids. Analysis of (KL)4K binding to anionic lipid monolayers reveals a competition between two binding processes: (i) incorporation of the peptide into the acyl chain region (surface pressure increase) and (ii) electrostatic interaction screening the negative charges with reduction of charge repulsion (surface pressure decrease due to monolayer condensation). The lipid acyl chain length and the chemical structure of the headgroup have minor effects on the binding properties. However, a strong dependence on the phase state of the monolayer was observed. In the liquid-expanded (LE) phase, the fluid monolayer provides enough space, so that peptide insertion due to hydrophobic interactions dominates. For monolayers in the liquid-condensed (LC) phase, peptide binding followed by monolayer condensation is the main effect. PMID:27049846

  7. A hybrid cationic peptide composed of human β-defensin-1 and humanized θ-defensin sequences exhibits salt-resistant antimicrobial activity.

    PubMed

    Olli, Sudar; Nagaraj, Ramakrishnan; Motukupally, Swapna R

    2015-01-01

    We have designed a hybrid peptide by combining sequences of human β-defensin-1 (HBD-1) and θ-defensin, in an attempt to generate a molecule that combines the diversity in structure and biological activity of two different peptides to yield a promising therapeutic candidate. HBD-1 was chosen as it is a natural defensin of humans that is constitutively expressed, but its antibacterial activity is considerably impaired by elevated ionic strength. θ-Defensins are expressed in human bone marrow as a pseudogene and are homologous to rhesus monkey circular minidefensins. Retrocyclins are synthetic human θ-defensins. The cyclic nature of the θ-defensin peptides makes them salt resistant, nonhemolytic, and virtually noncytotoxic in vitro. However, a nonhuman circular molecule developed for clinical use would be less viable than a linear molecule. In this study, we have fused the C-terminal region of HBD-1 to the nonapeptide sequence of a synthetic retrocyclin. Cyclization was achieved by joining the terminal ends of the hybrid peptide by a disulfide bridge. The hybrid peptide with or without the disulfide bridge exhibited enhanced antimicrobial activity against both Gram-negative and Gram-positive bacteria as well as against fungi, including clinical bacterial isolates from eye infections. The peptide retained activity in the presence of NaCl and serum and was nonhemolytic in vitro. Thus, the hybrid peptide generated holds potential as a new class of antibiotics. PMID:25348533

  8. A Hybrid Cationic Peptide Composed of Human β-Defensin-1 and Humanized θ-Defensin Sequences Exhibits Salt-Resistant Antimicrobial Activity

    PubMed Central

    Nagaraj, Ramakrishnan; Motukupally, Swapna R.

    2014-01-01

    We have designed a hybrid peptide by combining sequences of human β-defensin-1 (HBD-1) and θ-defensin, in an attempt to generate a molecule that combines the diversity in structure and biological activity of two different peptides to yield a promising therapeutic candidate. HBD-1 was chosen as it is a natural defensin of humans that is constitutively expressed, but its antibacterial activity is considerably impaired by elevated ionic strength. θ-Defensins are expressed in human bone marrow as a pseudogene and are homologous to rhesus monkey circular minidefensins. Retrocyclins are synthetic human θ-defensins. The cyclic nature of the θ-defensin peptides makes them salt resistant, nonhemolytic, and virtually noncytotoxic in vitro. However, a nonhuman circular molecule developed for clinical use would be less viable than a linear molecule. In this study, we have fused the C-terminal region of HBD-1 to the nonapeptide sequence of a synthetic retrocyclin. Cyclization was achieved by joining the terminal ends of the hybrid peptide by a disulfide bridge. The hybrid peptide with or without the disulfide bridge exhibited enhanced antimicrobial activity against both Gram-negative and Gram-positive bacteria as well as against fungi, including clinical bacterial isolates from eye infections. The peptide retained activity in the presence of NaCl and serum and was nonhemolytic in vitro. Thus, the hybrid peptide generated holds potential as a new class of antibiotics. PMID:25348533

  9. Glucagon-like peptide-2 intracellularly stimulates eNOS phosphorylation and specifically induces submucosal arteriole vasodilation via a sheer stress-independent, local neural mechanism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glucagon-like peptide-2 (GLP-2) is a nutrient-responsive neuropeptide that exerts diverse actions in the gastrointestinal tract, including enhancing mucosal cell survival and proliferation, mucosal blood flow, luminal nutrient uptake, and suppressing gastric motility and secretion. We have shown th...

  10. Conventional Matrices Loaded Onto a Graphene Layer Enhances MALDI-TOF/TOF Signal: Its Application to Improve Detection of Phosphorylated Peptides

    NASA Astrophysics Data System (ADS)

    Rodríguez, Carlos E.; Palacios, Javier; Fajardo, Ignacio; Urdiales, José Luis; Le Guével, Xavier; Lozano, José; Sánchez-Jiménez, Francisca

    2016-02-01

    This is the first study where graphene is used as a MALDI adjuvant in combination with the traditional matrix α-cyano-4-hydroxycinnamic acid (CHCA) to improve the signal intensity of peptide samples. Use of this amended matrix not only leads to increased signals but also to a higher number of peaks detected in complex samples. Additionally, the use of graphene has a stabilizing effect that can also be exploited to improve the detection of easily cleavable molecules.

  11. Discovery of a non-cationic cell penetrating peptide derived from membrane-interacting human proteins and its potential as a protein delivery carrier.

    PubMed

    Young Kim, Hyo; Young Yum, Soo; Jang, Goo; Ahn, Dae-Ro

    2015-01-01

    Cell penetrating peptides (CPPs) are peptides that can be translocated into cells and used as a carrier platform for the intracellular uptake of cargo molecules. Subject to the source of CPP sequences and their positively charged nature, the cytotoxicity and immunogenicity of conventional CPPs needs to be optimized to expand their utility for biomedical applications. In addition to these safety issues, the stability of CPPs needs to be addressed since their positively charged residues are prone to interact with the biological milieu. As an effort to overcome these limitations of the current CPP technology, we isolated CPP candidate sequences and synthesized peptides from twelve isoforms of annexin, a family of membrane-interacting human proteins. The candidate screen returned a CPP rich in hydrophobic residues that showed more efficient cellular uptake than TAT-CPP. We then investigated the uptake mechanism, subcellular localization, and biophysical properties of the newly found CPP, verifying low cytotoxicity, long-term serum stability, and non-immunogenicity. Finally, model proteins conjugated to this peptide were successfully delivered into mammalian cells both in vitro and in vivo, indicating a potential use of the peptide as a carrier for the delivery of macromolecular cargos. PMID:26114640

  12. Middle-Down and Chemical Proteomic Approaches to Reveal Histone H4 Modification Dynamics in Cell Cycle: Label-Free Semi-Quantification of Histone Tail Peptide Modifications Including Phosphorylation and Highly Sensitive Capture of Histone PTM Binding Proteins Using Photo-Reactive Crosslinkers

    PubMed Central

    Yamamoto, Kazuki; Chikaoka, Yoko; Hayashi, Gosuke; Sakamoto, Ryosuke; Yamamoto, Ryuji; Sugiyama, Akira; Kodama, Tatsuhiko; Okamoto, Akimitsu; Kawamura, Takeshi

    2015-01-01

    Mass spectrometric proteomics is an effective approach for identifying and quantifying histone post-translational modifications (PTMs) and their binding proteins, especially in the cases of methylation and acetylation. However, another vital PTM, phosphorylation, tends to be poorly quantified because it is easily lost and inefficiently ionized. In addition, PTM binding proteins for phosphorylation are sometimes resistant to identification because of their variable binding affinities. Here, we present our efforts to improve the sensitivity of detection of histone H4 tail peptide phosphorylated at serine 1 (H4S1ph) and our successful identification of an H4S1ph binder candidate by means of a chemical proteomics approach. Our nanoLC-MS/MS system permitted semi-quantitative label-free analysis of histone H4 PTM dynamics of cell cycle-synchronized HeLa S3 cells, including phosphorylation, methylation, and acetylation. We show that H4S1ph abundance on nascent histone H4 unmethylated at lysine 20 (H4K20me0) peaks from late S-phase to M-phase. We also attempted to characterize effects of phosphorylation at H4S1 on protein–protein interactions. Specially synthesized photoaffinity bait peptides specifically captured 14-3-3 proteins as novel H4S1ph binding partners, whose interaction was otherwise undetectable by conventional peptide pull-down experiments. This is the first report that analyzes dynamics of PTM pattern on the whole histone H4 tail during cell cycle and enables the identification of PTM binders with low affinities using high-resolution mass spectrometry and photo-affinity bait peptides. PMID:26819910

  13. NMR structure of a complex formed by the carboxyl-terminal domain of human RAP74 and a phosphorylated peptide from the central domain of the FCP1 phosphatase.

    PubMed

    Yang, Ao; Abbott, Karen L; Desjardins, Alexandre; Di Lello, Paola; Omichinski, James G; Legault, Pascale

    2009-03-10

    Recycling of RNA polymerase II (RNAPII) requires dephosphorylation of the C-terminal domain (CTD) of the largest subunit of the polymerase. FCP1 enables the recycling of RNAPII via its CTD-specific phosphatase activity, which is stimulated by the RAP74 subunit of the general transcription factor TFIIF. Both the central (centFCP1) and C-terminal (cterFCP1) domains of FCP1 interact independently and specifically with the C-terminal domain of RAP74 (cterRAP74), suggesting that these interactions mediate the stimulatory effect of TFIIF on the CTD phosphatase activity of FCP1. Phosphorylation of FCP1 by casein kinase 2 on residues in its central (T584) and C-terminal (S942 and S944) domains stimulates its binding to RAP74 and its CTD phosphatase activity. To improve our understanding of the FCP1-RAP74 interactions, we previously determined the NMR structure of a complex formed by human cterRAP74 and cterFCP1. We now present the high-resolution NMR structure and thermodynamic characterization by isothermal titration calorimetry of a complex formed by the same cterRAP74 domain and a phosphorylated peptide from the central domain of human FCP1 (centFCP1-PO(4)). Comparison of the cterFCP1-cterRAP74 and centFCP1-PO(4)-cterRAP74 complexes indicates that centFCP1 and cterFCP1 both utilize hydrophobic and acidic residues to recognize the same groove of RAP74, but there are significant differences in the details of their interactions. These differences point to the adaptability of RAP74 to recognize the two regions of FCP1. Our NMR and thermodynamic studies further elucidate the complex molecular mechanism by which TFIIF and FCP1 cooperate for RNAPII recycling. PMID:19215094

  14. Covalent and non-covalent binding in the ion/ion charge inversion of peptide cations with benzene-disulfonic acid anions.

    PubMed

    Stutzman, John R; Luongo, Carl A; McLuckey, Scott A

    2012-06-01

    Protonated angiotensin II and protonated leucine enkephalin-based peptides, which included YGGFL, YGGFLF, YGGFLH, YGGFLK and YGGFLR, were subjected to ion/ion reactions with the doubly deprotonated reagents 4-formyl-1,3-benzenedisulfonic acid (FBDSA) and 1,3-benzenedisulfonic acid (BDSA). The major product of the ion/ion reaction is a negatively charged complex of the peptide and reagent. Following dehydration of [M + FBDSA-H](-) via collisional-induced dissociation (CID), angiotensin II (DRVYIHPF) showed evidence for two product populations, one in which a covalent modification has taken place and one in which an electrostatic modification has occurred (i.e. no covalent bond formation). A series of studies with model systems confirmed that strong non-covalent binding of the FBDSA reagent can occur with subsequent ion trap CID resulting in dehydration unrelated to the adduct. Ion trap CID of the dehydration product can result in cleavage of amide bonds in competition with loss of the FBDSA adduct. This scenario is most likely for electrostatically bound complexes in which the peptide contains both an arginine residue and one or more carboxyl groups. Otherwise, loss of the reagent species from the complex, either as an anion or as a neutral species, is the dominant process for electrostatically bound complexes. The results reported here shed new light on the nature of non-covalent interactions in gas phase complexes of peptide ions that can be used in the rationale design of reagent ions for specific ion/ion reaction applications. PMID:22707160

  15. Phosphorylation site on yeast pyruvate dehydrogenase complex

    SciTech Connect

    Uhlinger, D.J.

    1986-01-01

    The pyruvate dehydrogenase complex was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). Yeast cells were disrupted in a Manton-Gaulin laboratory homogenizer. The pyruvate dehydrogenase complex was purified by fractionation with polyethylene glycol, isoelectric precipitation, ultracentrifugation and chromatography on hydroxylapatite. Final purification of the yeast pyruvate dehydrogenase complex was achieved by cation-exchange high pressure liquid chromatography (HPLC). No endogenous pyruvate dehydrogenase kinase activity was detected during the purification. However, the yeast pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. Tryptic digestion of the /sup 32/P-labeled complex yielded a single phosphopeptide which was purified to homogeniety. The tryptic digest was subjected to chromatography on a C-18 reverse phase HPLC column with a linear gradient of acetonitrile. Radioactive fractions were pooled, concentrated, and subjected to anion-exchange HPLC. The column was developed with a linear gradient of ammonium acetate. Final purification of the phosphopeptide was achieved by chromatography on a C-18 reverse phase HPLC column developed with a linear gradient of acetonitrile. The amino acid sequence of the homogeneous peptide was determined by manual modified Edman degradation.

  16. Peptide Orientation Affects Selectivity in Ion-Exchange Chromatography

    SciTech Connect

    Alpert, Andrew J.; Petritis, Konstantinos; Kangas, Lars J.; Smith, Richard D.; Mechtler, Karl; Mitulovic, Goran; Mohammed, Shabaz; Heck, Albert J.

    2010-06-15

    Here we demonstrate that separation of proteolytic peptides, having the same net charge and one basic residue, is affected by their specific orientation toward the stationary phase in ion-exchange chromatography. In electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with an anion-exchange material, the C-terminus of the peptides is, on average, oriented toward the stationary phase. In cation exchange, the average peptide orientation is the opposite. Data with synthetic peptides, serving as orientation probes, indicate that in tryptic/Lys-C peptides the C-terminal carboxyl group appears to be in a zwitterionic bond with the side chain of the C-terminal Lys/Arg residue. In effect, the side chain is then less basic than the N-terminus, accounting for the specific orientation of tryptic and Lys-C peptides. Analyses of larger sets of peptides, generated from lysates by either Lys-N, Lys-C, or trypsin, reveal that specific peptide orientation affects the ability of harged side chains, such as phosphate residues, to influence retention. Phosphorylated residues that are remote in the sequence from the binding site affect retention less than those that are closer. When a peptide contains multiple charged sites, then orientation is observed to be less rigid and retention tends to be governed by the peptide’s net charge rather than its sequence. These general observations could be of value in confirming a peptide’s identification and, in particular, phosphosite assignments in proteomics analyses. More generally, orientation accounts for the ability of chromatography to separate peptides of the same compositionbut different sequence.

  17. Peptide Orientation Affects Selectivity in Ion-Exchange Chromatography

    SciTech Connect

    Alpert, Andrew J.; Petritis, Konstantinos; Kangas, Lars J.; Smith, R. D.; Mechtler, Karl; Mitulovic, Goran; Mohammed, Shabaz; Heck, Albert J.

    2010-06-15

    Here we demonstrate that separation of proteolytic peptides, having the same net charge and one basic residue, is affected by their specific orientation toward the stationary phase in ion-exchange chromatography. In electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with an anion-exchange material, the C-terminus of the peptides is, on average, oriented toward the stationary phase. In cation exchange, the average peptide orientation is the opposite. Data with synthetic peptides, serving as orientation probes, indicate that in tryptic/ Lys-C peptides the C-terminal carboxyl group appears to be in a zwitterionic bond with the side chain of the C-terminal Lys/Arg residue. In effect, the side chain is then less basic than the N-terminus, accounting for the specific orientation of tryptic and Lys-C peptides. Analyses of larger sets of peptides, generated from lysates by either Lys-N, Lys-C, or trypsin, reveal that specific peptide orientation affects the ability of charged side chains, such as phosphate residues, to influence retention. Phosphorylated residues that are remote in the sequence from the binding site affect retention less than those that are closer. When a peptide contains multiple charged sites, then orientation is observed to be less rigid and retention tends to be governed by the peptide’s net charge rather than its sequence. These general observations could be of value in confirming a peptide’s identification and, in particular, phosphosite assignments in proteomics analyses. More generally, orientation accounts for the ability of chromatography to separate peptides of the same composition but different sequence.

  18. Peptide Orientation Affects Selectivity in Ion-Exchange Chromatography

    PubMed Central

    2010-01-01

    Here we demonstrate that separation of proteolytic peptides, having the same net charge and one basic residue, is affected by their specific orientation toward the stationary phase in ion-exchange chromatography. In electrostatic repulsion−hydrophilic interaction chromatography (ERLIC) with an anion-exchange material, the C-terminus of the peptides is, on average, oriented toward the stationary phase. In cation exchange, the average peptide orientation is the opposite. Data with synthetic peptides, serving as orientation probes, indicate that in tryptic/Lys-C peptides the C-terminal carboxyl group appears to be in a zwitterionic bond with the side chain of the C-terminal Lys/Arg residue. In effect, the side chain is then less basic than the N-terminus, accounting for the specific orientation of tryptic and Lys-C peptides. Analyses of larger sets of peptides, generated from lysates by either Lys-N, Lys-C, or trypsin, reveal that specific peptide orientation affects the ability of charged side chains, such as phosphate residues, to influence retention. Phosphorylated residues that are remote in the sequence from the binding site affect retention less than those that are closer. When a peptide contains multiple charged sites, then orientation is observed to be less rigid and retention tends to be governed by the peptide’s net charge rather than its sequence. These general observations could be of value in confirming a peptide’s identification and, in particular, phosphosite assignments in proteomics analyses. More generally, orientation accounts for the ability of chromatography to separate peptides of the same composition but different sequence. PMID:20481592

  19. Interaction of the non-phosphorylated peptide G7-18NATE with Grb7-SH2 domain requires phosphate for enhanced affinity and specificity.

    PubMed

    Gunzburg, Menachem J; Ambaye, Nigus D; Del Borgo, Mark P; Pero, Stephanie C; Krag, David N; Wilce, Matthew C J; Wilce, Jacqueline A

    2012-01-01

    Src-homology (SH2) domains are an attractive target for the inhibition of specific signalling pathways but pose the challenge of developing a truly specific inhibitor. The G7-18NATE cyclic peptide is reported to specifically inhibit the growth factor receptor bound protein 7 (Grb7) adapter protein, implicated in the progression of several cancer types, via interactions with its SH2 domain. G7-18NATE effectively inhibits the interaction of Grb7 with ErbB3 and focal adhesion kinase in cell lysates and, with the addition of a cell permeability sequence, inhibits the growth and migration of a number of breast cancer cell lines. It is thus a promising lead in the development of therapeutics targeted to Grb7. Here we investigate the degree to which G7-18NATE is specific for the Grb7-SH2 domain compared with closely related SH2 domains including those of Grb10, Grb14, and Grb2 using surface plasmon resonance. We demonstrate that G7-18NATE binds with micromolar binding affinity to Grb7-SH2 domain (K(D)  = 4-6 μm) compared with 50-200 times lower affinity for Grb10, Grb14, and Grb2 but that this specificity depends critically on the presence of phosphate in millimolar concentrations. Other differences in buffer composition, including use of Tris or 2-(N-Morpholino)ethanesulfonic acid or varying the pH, do not impact on the interaction. This suggests that under cellular conditions, G7-18NATE binds with highest affinity to Grb7. In addition, our findings demonstrate that the basis of specificity of G7-18NATE binding to the Grb7-SH2 domain is via other than intrinsic structural features of the protein, representing an unexpected mode of molecular recognition. PMID:22213451

  20. Regulation of formyl peptide receptor binding to rabbit neutrophil plasma membranes. Use of monovalent cations, guanine nucleotides, and bacterial toxins to discriminate among different states of the receptor

    SciTech Connect

    Feltner, D.E.; Marasco, W.A.

    1989-06-01

    The regulation by monovalent cations, guanine nucleotides, and bacterial toxins of (3H)FMLP binding to rabbit neutrophil plasma membranes was studied by using dissociation techniques to identify regulatory effects on separate receptor states. Under conditions of low receptor occupancy (1 nM (3H)FMLP) and in both Na+ and K+ buffers, dissociation is heterogenous, displaying two distinct, statistically significant off rates. (3H)FMLP binding was enhanced by substituting other monovalent cations for Na+. In particular, enhanced binding in the presence of K+ relative to Na+ was caused by additional binding to both rapidly and slowly dissociating receptors. Three receptor dissociation rates, two of which appear to correspond to the two affinity states detected in equilibrium binding studies, were defined by specific GTP and pertussis toxin (PT) treatments. Neither GTP, nor PT or cholera toxins (CT) had an effect on the rate of dissociation of (3H)FMLP from the rapidly dissociating form of the receptor. Both 100 microM GTP and PT treatments increased the percentage of rapidly dissociating receptors, correspondingly decreasing the percentage of slowly dissociating receptors. The observed changes in the rapidly and slowly dissociating receptors after GTP, PT, and CT treatments were caused by an absolute decrease in the amount of binding to the slowly dissociating receptors. However, complete inhibition of slowly dissociating receptor binding by GTP, PT, or both was never observed. Both GTP and PT treatments, but not CT treatment, increased by two-fold the rate of dissociation of 1 nM (3H)FMLP from the slowly dissociating form of the receptor, resulting in a third dissociation rate. Thus, slowly dissociating receptors comprise two different receptor states, a G protein-associated guanine nucleotide and PT-sensitive state and a guanine nucleotide-insensitive state.

  1. Properties of phosphorylated thymidylate synthase.

    PubMed

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Paweł; Rogowska-Wrzesinska, Adelina; Cieśla, Joanna; Zieliński, Zbigniew; Nizioł, Joanna; Jarmuła, Adam; Maj, Piotr; Gołos, Barbara; Wińska, Patrycja; Ostafil, Sylwia; Wałajtys-Rode, Elżbieta; Shugar, David; Rode, Wojciech

    2015-12-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent. PMID:26315778

  2. Observations on the quantitation of the phosphate content of peptides by fast-atom bombardment mass spectrometry.

    PubMed

    Poulter, L; Ang, S G; Williams, D H; Cohen, P

    1987-07-29

    Equimolar mixtures of the phosphorylated and dephosphorylated forms of several peptides have been subjected to fast-atom bombardment mass spectrometry (FABMS), to investigate whether the stoichiometry of phosphorylation can be determined from the relative molecular-ion abundances of the phospho and dephospho derivatives. It is concluded that quantitation can be achieved for peptides with large positive or negative hydrophobicity/hydrophilicity indices (delta F values) where addition of a phosphate group does not alter the distribution of the peptide within the matrix significantly. For peptides with small positive or negative delta F values, phosphopeptides tend to be partially suppressed by their dephosphorylated counterparts. Suppression can be partially or totally overcome by conversion of the peptide to a hydrophobic derivative, and by the selection of an appropriate matrix. Alternatively, addition of a very strong acid, perchloric acid, can even reverse the original suppression effect. This last effect is believed to be due to the increased ionic strength in the matrix, which forces a relatively hydrophilic analyte to the matrix surface; and the ability of such a phosphorylated analyte to form a more stable gas-phase cation. PMID:3038197

  3. Emergence of Daptomycin Resistance in Daptomycin-Naïve Rabbits with Methicillin-Resistant Staphylococcus aureus Prosthetic Joint Infection Is Associated with Resistance to Host Defense Cationic Peptides and mprF Polymorphisms

    PubMed Central

    Mishra, Nagendra N.; Yang, Soo-Jin; Chen, Liang; Muller, Claudette; Saleh-Mghir, Azzam; Kuhn, Sebastian; Peschel, Andreas; Yeaman, Michael R.; Nast, Cynthia C.; Kreiswirth, Barry N.; Crémieux, Anne-Claude; Bayer, Arnold S.

    2013-01-01

    Background Previous studies of both clinically-derived and in vitro passage-derived daptomycin–resistant (DAP-R) Staphylococcus aureus strains demonstrated the coincident emergence of increased DAP MICs and resistance to host defense cationic peptides (HDP-R). Methods In the present investigation, we studied a parental DAP-susceptible (DAP-S) methicillin-resistant Staphylococcus aureus (MRSA) strain and three isogenic variants with increased DAP MICs which were isolated from both DAP-treated and DAP-untreated rabbits with prosthetic joint infections. These strains were compared for: in vitro susceptibility to distinct HDPs differing in size, structure, and origin; i.e.; thrombin-induced platelet microbicidal proteins [tPMPs] and human neutrophil peptide-1 [hNP-1]; cell membrane (CM) phospholipid and fatty acid content; CM order; envelope surface charge; cell wall thickness; and mprF single nucleotide polymorphisms (SNPs) and expression profiles. Results In comparison with the parental strain, both DAP-exposed and DAP-naive strains exhibited: (i) significantly reduced susceptibility to each HDP (P<0.05); (ii) thicker cell walls (P<0.05); (iii) increased synthesis of CM lysyl-phosphatidylglycerol (L-PG); (iv) reduced content of CM phosphatidylglycerol (PG); and (v) SNPs within the mprF locus No significant differences were observed between parental or variant strains in outer CM content of L-PG, CM fluidity, CM fatty acid contents, surface charge, mprF expression profiles or MprF protein content. An isolate which underwent identical in vivo passage, but without evolving increased DAP MICs, retained parental phenotypes and genotype. Conclusions These results suggest: i) DAP MIC increases may occur in the absence of DAP exposures in vivo and may be triggered by organism exposure to endogenous HDPs: and ii) gain-in-function SNPs in mprF may contribute to such HDP-DAP cross-resistance phenotypes, although the mechanism of this relationship remains to be defined. PMID

  4. Azithromycin Synergizes with Cationic Antimicrobial Peptides to Exert Bactericidal and Therapeutic Activity Against Highly Multidrug-Resistant Gram-Negative Bacterial Pathogens

    PubMed Central

    Lin, Leo; Nonejuie, Poochit; Munguia, Jason; Hollands, Andrew; Olson, Joshua; Dam, Quang; Kumaraswamy, Monika; Rivera, Heriberto; Corriden, Ross; Rohde, Manfred; Hensler, Mary E.; Burkart, Michael D.; Pogliano, Joe; Sakoulas, George; Nizet, Victor

    2015-01-01

    Antibiotic resistance poses an increasingly grave threat to the public health. Of pressing concern, rapid spread of carbapenem-resistance among multidrug-resistant (MDR) Gram-negative rods (GNR) is associated with few treatment options and high mortality rates. Current antibiotic susceptibility testing guiding patient management is performed in a standardized manner, identifying minimum inhibitory concentrations (MIC) in bacteriologic media, but ignoring host immune factors. Lacking activity in standard MIC testing, azithromycin (AZM), the most commonly prescribed antibiotic in the U.S., is never recommended for MDR GNR infection. Here we report a potent bactericidal action of AZM against MDR carbapenem-resistant isolates of Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. This pharmaceutical activity is associated with enhanced AZM cell penetration in eukaryotic tissue culture media and striking multi-log-fold synergies with host cathelicidin antimicrobial peptide LL-37 or the last line antibiotic colistin. Finally, AZM monotherapy exerts clear therapeutic effects in murine models of MDR GNR infection. Our results suggest that AZM, currently ignored as a treatment option, could benefit patients with MDR GNR infections, especially in combination with colistin. PMID:26288841

  5. Azithromycin Synergizes with Cationic Antimicrobial Peptides to Exert Bactericidal and Therapeutic Activity Against Highly Multidrug-Resistant Gram-Negative Bacterial Pathogens.

    PubMed

    Lin, Leo; Nonejuie, Poochit; Munguia, Jason; Hollands, Andrew; Olson, Joshua; Dam, Quang; Kumaraswamy, Monika; Rivera, Heriberto; Corriden, Ross; Rohde, Manfred; Hensler, Mary E; Burkart, Michael D; Pogliano, Joe; Sakoulas, George; Nizet, Victor

    2015-07-01

    Antibiotic resistance poses an increasingly grave threat to the public health. Of pressing concern, rapid spread of carbapenem-resistance among multidrug-resistant (MDR) Gram-negative rods (GNR) is associated with few treatment options and high mortality rates. Current antibiotic susceptibility testing guiding patient management is performed in a standardized manner, identifying minimum inhibitory concentrations (MIC) in bacteriologic media, but ignoring host immune factors. Lacking activity in standard MIC testing, azithromycin (AZM), the most commonly prescribed antibiotic in the U.S., is never recommended for MDR GNR infection. Here we report a potent bactericidal action of AZM against MDR carbapenem-resistant isolates of Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. This pharmaceutical activity is associated with enhanced AZM cell penetration in eukaryotic tissue culture media and striking multi-log-fold synergies with host cathelicidin antimicrobial peptide LL-37 or the last line antibiotic colistin. Finally, AZM monotherapy exerts clear therapeutic effects in murine models of MDR GNR infection. Our results suggest that AZM, currently ignored as a treatment option, could benefit patients with MDR GNR infections, especially in combination with colistin. PMID:26288841

  6. Toward a Rational Design of Highly Folded Peptide Cation Conformations. 3D Gas-Phase Ion Structures and Ion Mobility Characterization

    NASA Astrophysics Data System (ADS)

    Pepin, Robert; Laszlo, Kenneth J.; Marek, Aleš; Peng, Bo; Bush, Matthew F.; Lavanant, Helène; Afonso, Carlos; Tureček, František

    2016-07-01

    Heptapeptide ions containing combinations of polar Lys, Arg, and Asp residues with non-polar Leu, Pro, Ala, and Gly residues were designed to study polar effects on gas-phase ion conformations. Doubly and triply charged ions were studied by ion mobility mass spectrometry and electron structure theory using correlated ab initio and density functional theory methods and found to exhibit tightly folded 3D structures in the gas phase. Manipulation of the basic residue positions in LKGPADR, LRGPADK, KLGPADR, and RLGPADK resulted in only minor changes in the ion collision cross sections in helium. Replacement of the Pro residue with Leu resulted in only marginally larger collision cross sections for the doubly and triply charged ions. Disruption of zwitterionic interactions in doubly charged ions was performed by converting the C-terminal and Asp carboxyl groups to methyl esters. This resulted in very minor changes in the collision cross sections of doubly charged ions and even slightly diminished collision cross sections in most triply charged ions. The experimental collision cross sections were related to those calculated for structures of lowest free energy ion conformers that were obtained by extensive search of the conformational space and fully optimized by density functional theory calculations. The predominant factors that affected ion structures and collision cross sections were due to attractive hydrogen bonding interactions and internal solvation of the charged groups that overcompensated their Coulomb repulsion. Structure features typically assigned to the Pro residue and zwitterionic COO-charged group interactions were only secondary in affecting the structures and collision cross sections of these gas-phase peptide ions.

  7. Antimicrobial Peptides in 2014

    PubMed Central

    Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

    2015-01-01

    This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms. PMID:25806720

  8. Antimicrobial peptides in 2014.

    PubMed

    Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

    2015-01-01

    This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms. PMID:25806720

  9. Cationic lipid-mediated nucleic acid delivery: beyond being cationic.

    PubMed

    Rao, N Madhusudhana

    2010-03-01

    Realization of the potential of nucleic acids as drugs is intricately linked to their in vivo delivery. Cationic lipids demonstrated tremendous potential as safe, efficient and scalable in vitro carriers of nucleic acids. For in vivo delivery of nucleic acids, the extant two component liposomal preparations consisting of cationic lipids and nucleic acids have been largely found to be insufficient. Being a soft matter, liposomes readily respond to many physiological variables leading to complex component and morphological changes, thus confounding the efforts in a priori identification of a "competent" formulation. In the recent past many chemical moieties that provide advantage in facing the challenges of barriers in vivo, were incorporated into cationic lipids to improve the transfection efficiency. The cationic lipids, essential for DNA condensation and protection, definitely require additional components to be efficient in vivo. In addition, formulations of cationic lipid carriers with non-lipidic components, mainly peptides, have demonstrated success in in vivo transfection. The present review describes some recent successes of in vivo nucleic acid delivery by cationic lipids. PMID:20060819

  10. [Evaluation of peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method in the identifi cation of Candida species isolated from blood cultures].

    PubMed

    Aydemir, Gonca; Koç, Ayşe Nedret; Atalay, Mustafa Altay

    2016-04-01

    In recent years, increased number of patients who are hospitalized in intensive care units, received immunosuppressive therapy and treated with broad-spectrum antibiotics that can lead an increase in the incidence of systemic candidiasis. In these patients, the most common clinical manifestation is candidemia. Since the identification of Candida species isolated from blood cultures is time consuming by conventional (morphological and biochemical) methods, rapid, reliable and accurate methods are needed. For this purpose novel systems have been developed to identify the agent directly. The aim of this study was to evaluate the peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method for the identification of Candida species by comparing with the conventional methods. A total of 50 patients who were admitted to Erciyes University Medical Faculty Hospital clinics and followed with prediagnosis of systemic fungal infections whose blood cultures were positive for the yeasts between July 2011 and July 2012 were included in the study. The conventional identification of Candida isolates was performed by considering macroscopic and microscopic morphology, germ tube test, cycloheximide sensitivity, urease activity and carbohydrate assimilation patterns with API 20C AUX (bioMerieux, France) test. PNA FISH method was conducted by the use of a commercial kit namely Yeast Traffic Light(®) PNA FISH (AdvanDx, USA). According to morphological and biochemical characteristics (conventional methods), 19 (38%) out of 50 Candida isolates were identified as C.albicans, 12 (24%) as C.glabrata, five (10%) as C.parapsilosis, five (10%) as C.kefyr, four (8%) as C.krusei, two (4%) as C.guilliermondii, two (4%) as C.tropicalis and one (2%) as C.lusitaniae. On the other hand, 24 (48%) of the isolates were identified as C.albicans/C.parapsilosis (with green fluorescence), 16 (32%) as C.glabrata/C.krusei (with red fluorescence) and one (%2) as C.tropicalis (with yellow

  11. Antibacterial properties of cationic steroid antibiotics.

    PubMed

    Savage, Paul B; Li, Chunhong; Taotafa, Uale; Ding, Bangwei; Guan, Qunying

    2002-11-19

    Cationic steroid antibiotics have been developed that display broad-spectrum antibacterial activity. These compounds are comprised of steroids appended with amine groups arranged to yield facially amphiphilic morphology. Examples of these antibiotics are highly bactericidal, while related compounds effectively permeabilize the outer membranes of Gram-negative bacteria sensitizing these organisms to hydrophobic antibiotics. Cationic steroid antibiotics exhibit various levels of eukaryote vs. prokaryote cell selectivity, and cell selectivity can be increased via charge recognition of prokaryotic cells. Studies of the mechanism of action of these antibiotics suggest that they share mechanistic aspects with cationic peptide antibiotics. PMID:12445638

  12. Mapping of phosphorylation sites in polyomavirus large T antigen

    SciTech Connect

    Hassauer, M.; Scheidtmann, K.H.; Walter, G.

    1986-06-01

    The phosphorylation sites of polyomavirus large T antigen from infected or transformed cells were investigated. Tryptic digestion of large T antigen from infected, /sup 32/P/sub i/-labeled cells revealed seven major phosphopeptides. Five of these were phosphorylated only at serine residues, and two were phosphorylated at serine and threonine residues. The overall ratio of phosphoserine to phosphothreonine was 6:1. The transformed cell line B4 expressed two polyomavirus-specific phosphoproteins: large T antigen, which was only weakly phosphorylated, and a truncated form of large T antigen of 34,000 molecular weight which was heavily phosphorylated. Both showed phosphorylation patterns similar to that of large T antigen from infected cells. Peptide analyses of large T antigens encoded by the deletion mutants dl8 and dl23 or of specific fragments of wild-type large T antigen indicated that the phosphorylation sites are located in an amino-terminal region upstream of residue 194. The amino acid composition of the phosphopeptides as revealed by differential labeling with various amino acids indicated that several phosphopeptides contain overlapping sequences and that all phosphorylation sites are located in four tryptic peptides derived from a region between Met71 and Arg191. Two of the potential phosphorylation sites were identified as Ser81 and Thr187. The possible role of this modification of large T antigen is discussed.

  13. Fibronectin phosphorylation by ecto-protein kinase

    SciTech Connect

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru )

    1988-12-01

    The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with ({gamma}-{sup 32})ATP for 10 min at 37{degree}C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with ({gamma}-{sup 32}P)ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation.

  14. Therapeutic utility of antibacterial peptides in wound healing.

    PubMed

    Otvos, Laszlo; Ostorhazi, Eszter

    2015-07-01

    Cationic antimicrobial peptides were first thought to fight infection in animal models by disintegrating bacterial peptides and later by inhibiting bacteria-specific intracellular processes. However, ever increasing evidences indicate that cationic peptides accumulate around and modulate the immune system both systemically and in cutaneous and mucosal surfaces where injuries and infections occur. Native and designer antibacterial peptides as well as cationic peptides, never considered as antibiotics, promote wound healing at every step of cutaneous tissue regeneration. This article provides an introductory list of examples of how cationic peptides are involved in immunostimulation and epithelial tissue repair, eliminating wound infections and promoting wound healing in potential therapeutic utility in sight. Although a few antimicrobial peptides reached the Phase II clinical trial stage, toxicity concerns limit the potential administration routes. Resistance induction to both microbiology actions and the integrity of the innate immune system has to be carefully monitored. PMID:25835521

  15. Phosphorylation of serine residues affects the conformation of the calmodulin binding domain of human protein 4.1.

    PubMed

    Vetter, S W; Leclerc, E

    2001-08-01

    We have previously characterized the calcium-dependent calmodulin (CaM)-binding domain (Ser76-Ser92) of the 135-kDa human protein 4.1 isoform using fluorescence spectroscopy and chemically synthesized nonphosphorylated or serine phosphorylated peptides [Leclerc, E. & Vetter, S. (1998) Eur. J. Biochem. 258, 567-671]. Here we demonstrate that phosphorylation of two serine residues within the 17-residue peptide alters their ability to adopt alpha helical conformation in a position-dependent manner. The helical content of the peptides was determined by CD-spectroscopy and found to increase from 36 to 45% for the Ser80 phosphorylated peptide and reduce to 28% for the Ser84 phosphorylated peptide; the di-phosphorylated peptide showed 32% helical content. Based on secondary structure prediction methods we propose that initial helix formation involves the central residues Leu82-Phe86. The ability of the peptides to adopt alpha helical conformations did not correlate with the observed binding affinities to CaM. We suggest that the reduced CaM-binding affinities observed for the phosphorylated peptides are more likely to be the result of unfavorable sterical and electrostatic interactions introduced into the CaM peptide-binding interface by the phosphate groups, rather than being due to the effect of phosphorylation on the secondary structure of the peptides. PMID:11488924

  16. Kinetics of the inhibition of calcium/calmodulin-dependent protein kinase II by pea protein-derived peptides.

    PubMed

    Li, Huan; Aluko, Rotimi E

    2005-11-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) catalyzes the phosphorylation of various cellular proteins and excessive activities have been implicated in the pathogenesis of various chronic diseases. We hypothesized that positively charged peptides can be produced through enzymatic hydrolysis of pea proteins; such peptides could then bind to negatively charged calmodulin (CaM) at a physiological pH level and inhibit CaMKII activity. Pea protein isolate was hydrolyzed with an alkaline protease (alcalase) and filtered through a 1000-mol wt cutoff membrane. The permeate, which contained low-molecular weight peptides, was used to isolate cationic peptides on an SP-Sepharose column by ion exchange chromatography. Separation of the permeate on the SP-Sepharose column yielded two fractions with net positive charges that were subsequently used for enzyme inhibition studies. Fraction I eluted earlier from the column and contained lower contents of lysine and arginine than Fraction II, which eluted later. Results show that both peptide fractions inhibited CaMKII activity mostly in a competitive manner, although kinetic data suggested that inhibition by Fraction II may be of the mixed type. Kinetic analysis (K(m) and K(i)) showed that affinity of peptides in Fraction II for CaM was more than that in Fraction I, which was directly correlated with the higher inhibitory properties of Fraction II against CaMKII. The results suggest that it may be possible to use pea protein-derived cationic peptides to modulate CaMKII activities. PMID:16111873

  17. Phosphorylation of a neuronal-specific beta-tubulin isotype

    SciTech Connect

    Diaz-Nido, J.; Serrano, L.; Lopez-Otin, C.; Vandekerckhove, J.; Avila, J. )

    1990-08-15

    Adult rats were intracraneally injected with ({sup 32}P) phosphate and brain microtubules isolated. The electrophoretically purified, in vivo phospholabeled, beta-tubulin was digested with the V8-protease and the labeled peptide purified by reversed-phase liquid chromatography. Its amino acid sequence corresponds to the COOH-terminal sequence of a minor neuronal beta 3-tubulin isoform from chicken and human. The phosphorylation site was at serine 444. A synthetic peptide with sequence EMYEDDEEESESQGPK, corresponding to that of the COOH terminus of beta 3-tubulin, was efficiently phosphorylated in vitro by casein kinase II at the same serine 444. The functional meaning of tubulin phosphorylation is still unclear. However, the modification of the protein takes place after microtubule assembly, and phosphorylated tubulin is mainly present in the assembled microtubule protein fraction.

  18. An isotope labeling strategy for quantifying the degree of phosphorylation at multiple sites in proteins.

    PubMed

    Hegeman, Adrian D; Harms, Amy C; Sussman, Michael R; Bunner, Anne E; Harper, Jeffrey F

    2004-05-01

    A procedure for determining the extent of phosphorylation at individual sites of multiply phosphorylated proteins was developed and applied to two polyphosphorylated proteins. The protocol, using simple chemical (Fischer methyl-esterification) and enzymatic (phosphatase) modification steps and an accessible isotopic labeling reagent (methyl alcohol-d(4)), is described in detail. Site-specific phosphorylation stoichiometries are derived from the comparison of chemically identical but isotopically distinct peptide species analyzed by microspray liquid chromatography-mass spectrometry (microLC-MS) using a Micromass Q-TOF2 mass spectrometer. Ten phosphorylation sites were unambiguously identified in tryptic digests of both proteins, and phosphorylation stoichiometries were determined for eight of the ten sites using the isotope-coded strategy. The extent of phosphorylation was also estimated from the mass spectral peak areas for the phosphorylated and unmodified peptides, and these estimates, when compared with stoichiometries determined using the isotope-coded technique, differed only marginally (within approximately 20%). PMID:15121193

  19. Phosphorylation and RLK signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant genomes encode hundreds of receptor-like kinases (RLKs) with an organization of functional domains similar to that of animal receptor kinases. Ligand-dependent phosphorylation has now been demonstrated for several plant RLKs and identification of specific phosphorylation sites followed by thei...

  20. Investigating quantitation of phosphorylation using MALDI-TOF mass spectrometry

    PubMed Central

    Parker, Laurie; Engel-Hall, Aaron; Drew, Kevin; Steinhardt, George; Helseth, Donald L.; Jabon, David; McMurry, Timothy; Angulo, David S.; Kron, Stephen J.

    2010-01-01

    Despite advances in methods and instrumentation for analysis of phosphopeptides using mass spectrometry, it is still difficult to quantify the extent of phosphorylation of a substrate due to physiochemical differences between unphosphorylated and phosphorylated peptides. Here we report experiments to investigate those differences using MALDI-TOF mass spectrometry for a set of synthetic peptides by creating calibration curves of known input ratios of peptides/phosphopeptides and analyzing their resulting signal intensity ratios. These calibration curves reveal subtleties in sequence-dependent differences for relative desorption/ionization efficiencies that cannot be seen from single-point calibrations. We found that the behaviors were reproducible with a variability of 5–10% for observed phosphopeptide signal. Although these data allow us to begin addressing the issues related to modeling these properties and predicting relative signal strengths for other peptide sequences, it is clear this behavior is highly complex and needs to be further explored. PMID:18064576

  1. Antimicrobial peptides: premises and promises.

    PubMed

    Reddy, K V R; Yedery, R D; Aranha, C

    2004-12-01

    Antimicrobial peptides (AMPs) are an important component of the natural defences of most living organisms against invading pathogens. These are relatively small (< 10kDa), cationic and amphipathic peptides of variable length, sequence and structure. During the past two decades several AMPs have been isolated from a wide variety of animals, both vertebrates and invertebrates, and plants as well as from bacteria and fungi. Most of these peptides are obtained from different sources like macrophages, neutrophils, epithelial cells, haemocytes, fat body, reproductive tract, etc. These peptides exhibit broad-spectrum activity against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, protozoa, yeast, fungi and viruses. A few peptides have also been found to be cytotoxic to sperm and tumour cells. AMPs are classified based on the three dimensional structural studies carried out with the help of NMR. The peptides are broadly classified into five major groups namely (a) peptides that form alpha-helical structures, (b) peptides rich in cysteine residues, (c) peptides that form beta-sheet, (d) peptides rich in regular amino acids namely histatin, arginine and proline and (e) peptides composed of rare and modified amino acids. Most of these peptides are believed to act by disrupting the plasma membrane leading to the lysis of the cell. AMPs have been found to be excellent candidates for developing novel antimicrobial agents and a few of these peptides show antimicrobial activity against pathogens causing sexually transmitted infection (STI), including HIV/HSV. Peptides, namely magainin and nisin have been shown to demonstrate contraceptive properties in vitro and in vivo. A few peptides have already entered clinical trials for the treatment of impetigo, diabetic foot ulcers and gastric helicobacter infections. In this review, we discuss the source, structures and mode of action with special reference to therapeutic considerations of various AMPs

  2. Altered protein phosphorylation as a resource for potential AD biomarkers.

    PubMed

    Henriques, Ana Gabriela; Müller, Thorsten; Oliveira, Joana Machado; Cova, Marta; da Cruz E Silva, Cristóvão B; da Cruz E Silva, Odete A B

    2016-01-01

    The amyloidogenic peptide, Aβ, provokes a series of events affecting distinct cellular pathways regulated by protein phosphorylation. Aβ inhibits protein phosphatases in a dose-dependent manner, thus it is expected that the phosphorylation state of specific proteins would be altered in response to Aβ. In fact several Alzheimer's disease related proteins, such as APP and TAU, exhibit pathology associated hyperphosphorylated states. A systems biology approach was adopted and the phosphoproteome, of primary cortical neuronal cells exposed to Aβ, was evaluated. Phosphorylated proteins were recovered and those whose recovery increased or decreased, upon Aβ exposure across experimental sets, were identified. Significant differences were evident for 141 proteins and investigation of their interactors revealed key protein clusters responsive to Aβ treatment. Of these, 73 phosphorylated proteins increased and 68 decreased upon Aβ addition. These phosphorylated proteins represent an important resource of potential AD phospho biomarkers that should be further pursued. PMID:27466139

  3. Altered protein phosphorylation as a resource for potential AD biomarkers

    PubMed Central

    Henriques, Ana Gabriela; Müller, Thorsten; Oliveira, Joana Machado; Cova, Marta; da Cruz e Silva, Cristóvão B.; da Cruz e Silva, Odete A. B.

    2016-01-01

    The amyloidogenic peptide, Aβ, provokes a series of events affecting distinct cellular pathways regulated by protein phosphorylation. Aβ inhibits protein phosphatases in a dose-dependent manner, thus it is expected that the phosphorylation state of specific proteins would be altered in response to Aβ. In fact several Alzheimer’s disease related proteins, such as APP and TAU, exhibit pathology associated hyperphosphorylated states. A systems biology approach was adopted and the phosphoproteome, of primary cortical neuronal cells exposed to Aβ, was evaluated. Phosphorylated proteins were recovered and those whose recovery increased or decreased, upon Aβ exposure across experimental sets, were identified. Significant differences were evident for 141 proteins and investigation of their interactors revealed key protein clusters responsive to Aβ treatment. Of these, 73 phosphorylated proteins increased and 68 decreased upon Aβ addition. These phosphorylated proteins represent an important resource of potential AD phospho biomarkers that should be further pursued. PMID:27466139

  4. Protein phosphorylation in isolated human adipocytes - Adrenergic control of the phosphorylation of hormone-sensitive lipase

    SciTech Connect

    Smiley, R.M. Columbia Univ College of Physicians and Surgeons, New York, NY ); Paul, S.; Browning, M.D.; Leibel, R.L.; Hirsch, J. )

    1990-01-01

    The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with {sup 32}PO{sub 4} in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the {beta}-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (M{sub r} 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the {alpha}-2 agonist clonidine. Epinephrine, a combined {alpha} and {beta} agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the {alpha}-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes.

  5. Toxins and antimicrobial peptides: interactions with membranes

    NASA Astrophysics Data System (ADS)

    Schlamadinger, Diana E.; Gable, Jonathan E.; Kim, Judy E.

    2009-08-01

    The innate immunity to pathogenic invasion of organisms in the plant and animal kingdoms relies upon cationic antimicrobial peptides (AMPs) as the first line of defense. In addition to these natural peptide antibiotics, similar cationic peptides, such as the bee venom toxin melittin, act as nonspecific toxins. Molecular details of AMP and peptide toxin action are not known, but the universal function of these peptides to disrupt cell membranes of pathogenic bacteria (AMPs) or a diverse set of eukaryotes and prokaryotes (melittin) is widely accepted. Here, we have utilized spectroscopic techniques to elucidate peptide-membrane interactions of alpha-helical human and mouse AMPs of the cathelicidin family as well as the peptide toxin melittin. The activity of these natural peptides and their engineered analogs was studied on eukaryotic and prokaryotic membrane mimics consisting of <200-nm bilayer vesicles composed of anionic and neutral lipids as well as cholesterol. Vesicle disruption, or peptide potency, was monitored with a sensitive fluorescence leakage assay. Detailed molecular information on peptidemembrane interactions and peptide structure was further gained through vibrational spectroscopy combined with circular dichroism. Finally, steady-state fluorescence experiments yielded insight into the local environment of native or engineered tryptophan residues in melittin and human cathelicidin embedded in bilayer vesicles. Collectively, our results provide clues to the functional structures of the engineered and toxic peptides and may impact the design of synthetic antibiotic peptides that can be used against the growing number of antibiotic-resistant pathogens.

  6. Identification and quantification of the phosphorylated ovalbumin by high resolution mass spectrometry under dry-heating treatment.

    PubMed

    Wang, Hui; Tu, Zong-Cai; Liu, Guang-Xian; Zhang, Lu; Chen, Yuan

    2016-11-01

    The specific phosphorylation sites and degree of phosphorylation (DP) at each site are directly related to protein's structure and functional properties. Thus, characterizing the introduced phosphate groups is of great importance. This study was to monitor the phosphorylation sites, DP and the number of phosphorylation sites in P-Oval achieved by dry heating in the presence of pyrophosphate for 1, 2 and 5days by using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Two phosphorylation sites were found in natural ovalbumin, but the number of phosphorylation sites increased to 8, 8 and 10 after dry-heating phosphorylation for 1, 2 and 5days, respectively. In addition, dual-phosphorylated peptides were detected for samples without extensive heating. The phosphorylation sites were found to be mainly on Ser residues, which could be the preferred phosphorylation site for dry heating in the presence of pyrophosphate. PMID:27211632

  7. In vivo phosphorylation of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP): CNP in brain myelin is phosphorylated by forskolin- and phorbol ester-sensitive protein kinases.

    PubMed

    Agrawal, H C; Sprinkle, T J; Agrawal, D

    1994-06-01

    2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was phosphorylated in vivo, in brain slices and in a cell free system. Phosphoamino acid analysis of immunoprecipitated CNP labeled in vivo and in brain slices revealed phosphorylation of phosphoserine (94%) and phosphothreonine (5%) residues. Phosphorylation of CNP increased by 3-fold after brain slices were incubated with forskolin. Similarly, incubation of isolated myelin with [gamma-32]ATP with cAMP (5 microM) and cAMP (5 microM)+catalytic unit of cAMP dependent protein kinase dramatically increased CNP2 phosphorylation by 4- and 6-fold, respectively. It is feasible that CNP2 was predominantly phosphorylated on serine and/or threonine residues of the amino terminal peptide of CNP2, and this phosphorylation was catalyzed by protein kinase A. Phosphorylation of CNP1 and CNP2 increased 2-fold by incubating brain slices with phorbol ester. Forskolin and phorbol ester increased the phosphorylation of single, but distinct, CNP peptides. We present the first biochemical evidence that CNP2, on a protein mass basis, is far more heavily phosphorylated than CNP1, suggesting there are more phosphorylation sites on CNP2 than CNP1 and that at least one site is located on the 20-amino acid terminus of CNP2 and that it is likely a PKA site. PMID:8065530

  8. Mining Conditional Phosphorylation Motifs.

    PubMed

    Liu, Xiaoqing; Wu, Jun; Gong, Haipeng; Deng, Shengchun; He, Zengyou

    2014-01-01

    Phosphorylation motifs represent position-specific amino acid patterns around the phosphorylation sites in the set of phosphopeptides. Several algorithms have been proposed to uncover phosphorylation motifs, whereas the problem of efficiently discovering a set of significant motifs with sufficiently high coverage and non-redundancy still remains unsolved. Here we present a novel notion called conditional phosphorylation motifs. Through this new concept, the motifs whose over-expressiveness mainly benefits from its constituting parts can be filtered out effectively. To discover conditional phosphorylation motifs, we propose an algorithm called C-Motif for a non-redundant identification of significant phosphorylation motifs. C-Motif is implemented under the Apriori framework, and it tests the statistical significance together with the frequency of candidate motifs in a single stage. Experiments demonstrate that C-Motif outperforms some current algorithms such as MMFPh and Motif-All in terms of coverage and non-redundancy of the results and efficiency of the execution. The source code of C-Motif is available at: https://sourceforge. net/projects/cmotif/. PMID:26356863

  9. Peptide Fragmentation by Corona Discharge Induced Electrochemical Ionization

    PubMed Central

    Lloyd, John R.; Hess, Sonja

    2010-01-01

    Fundamental studies have greatly improved our understanding of electrospray, including the underlying electrochemical reactions. Generally regarded as disadvantageous, we have recently shown that corona discharge (CD) can be used as an effective method to create a radical cation species [M]+•, thus optimizing the electrochemical reactions that occur on the surface of the stainless steel (SS) electrospray capillary tip. This technique is known as CD initiated electrochemical ionization (CD-ECI). Here, we report on the fundamental studies using CD-ECI to induce analytically useful in-source fragmentation of a range of molecules that complex transition metals. Compounds that have been selectively fragmented using CD-ECI include enolate forming phenylglycine containing peptides, glycopeptides, nucleosides and phosphopeptides. Collision induced dissociation (CID) or other activation techniques were not necessary for CD-ECI fragmentation. A four step mechanism was proposed: 1. Complexation using either Fe in the SS capillary tip material or Cu(II) as an offline complexation reagent; 2. Electrochemical oxidation of the complexed metal and thus formation of a radical cation (e.g.; Fe - e− → Fe +•); 3. Radical fragmentation of the complexed compound. 4. Electrospray ionization of the fragmented neutrals. Fragmentation patterns resembling b- and y-type ions were observed and allowed the localization of the phosphorylation sites. PMID:20869880

  10. Peptide fragmentation by corona discharge induced electrochemical ionization.

    PubMed

    Lloyd, John R; Hess, Sonja

    2010-12-01

    Fundamental studies have greatly improved our understanding of electrospray, including the underlying electrochemical reactions. Generally regarded as disadvantageous, we have recently shown that corona discharge (CD) can be used as an effective method to create a radical cation species [M](+·), thus optimizing the electrochemical reactions that occur on the surface of the stainless steel (SS) electrospray capillary tip. This technique is known as CD initiated electrochemical ionization (CD-ECI). Here, we report on the fundamental studies using CD-ECI to induce analytically useful in-source fragmentation of a range of molecules that complex transition metals. Compounds that have been selectively fragmented using CD-ECI include enolate forming phenylglycine containing peptides, glycopeptides, nucleosides, and phosphopeptides. Collision induced dissociation (CID) or other activation techniques were not necessary for CD-ECI fragmentation. A four step mechanism was proposed: (1) complexation using either Fe in the SS capillary tip material or Cu(II) as an offline complexation reagent; (2) electrochemical oxidation of the complexed metal and thus formation of a radical cation (e.g.; Fe - e(-) → Fe(+·)); (3) radical fragmentation of the complexed compound; (4) electrospray ionization of the fragmented neutrals. Fragmentation patterns resembling b- and y-type ions were observed and allowed the localization of the phosphorylation sites. PMID:20869880

  11. Identification of extracellularly phosphorylated membrane proteins.

    PubMed

    Burghoff, Sandra; Willberg, Wibke; Schrader, Jürgen

    2015-10-01

    Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling. PMID:26152529

  12. Molecular Dynamical Study on Ion Channeling through Peptide Nanotube

    NASA Astrophysics Data System (ADS)

    Sumiya, Norihito; Igami, Daiki; Takeda, Kyozaburo

    2011-12-01

    We theoretically study the possibility of ion channeling through peptide nanotubes (PNTs). After designing the minimal peptide nanorings (PNRs) and their aggregated form (peptide nanotubes, PNT) computationally, we carry out molecular dynamics (MD) calculations for cation channeling. The present MD calculations show that cation channeling through PNTs occurs. Furthermore, inter-ring hydrogen bonds (HBs) survive and maintain the tubular form of PNTs during cation channeling. We introduce mobility such that cation channeling can be evaluated quantitatively. As the ionic radius of the cation becomes smaller, the effective relaxation time τ becomes larger. Accordingly, mobilities of 10-2˜10-3[cm2/volt/sec] are calculated. In contrast, when an anion (F-) passes through the PNT, the inter-ring HBs are broken, thus inducing breakdown of the peptide backbone. Consequently, H atoms from the broken HBs surround the channeling anion (F-) and halt its motion.

  13. Screening for protein phosphorylation using nanoscale reactions on microdroplet arrays.

    PubMed

    Küster, Simon K; Pabst, Martin; Zenobi, Renato; Dittrich, Petra S

    2015-01-26

    We present a novel and straightforward screening method to detect protein phosphorylations in complex protein mixtures. A proteolytic digest is separated by a conventional nanoscale liquid chromatography (nano-LC) separation and the eluate is immediately compartmentalized into microdroplets, which are spotted on a microarray MALDI plate. Subsequently, the enzyme alkaline phosphatase is applied to every second microarray spot to remove the phosphate groups from phosphorylated peptides, which results in a mass shift of n×-80 Da. The MALDI-MS scan of the microarray is then evaluated by a software algorithm to automatically identify the phosphorylated peptides by exploiting the characteristic chromatographic peak profile induced by the phosphatase treatment. This screening method does not require extensive MS/MS experiments or peak list evaluation and can be easily extended to other enzymatic or chemical reactions. PMID:25504774

  14. Phosphorylation of bovine rod photoreceptor cyclic GMP phosphodiesterase.

    PubMed Central

    Udovichenko, I P; Cunnick, J; Gonzales, K; Takemoto, D J

    1993-01-01

    The cyclic GMP phosphodiesterase (PDE) of retinal rods plays a key role in phototransduction and consists of two catalytic subunits (PDE alpha and PDE beta) and two identical inhibitory subunits (PDE gamma). Here we report that PDE alpha and PDE gamma are phosphorylated by protein kinase(s) C (PKC) from brain and rod outer segments (ROS). These same two types of PKC also phosphorylate PDE alpha in trypsin-activated PDE (without PDE gamma). In contrast, cyclic-AMP-dependent protein kinase catalytic subunit phosphorylates both PDE alpha and PDE beta, but not PDE gamma. This kinase does not phosphorylate trypsin-activated PDE. The synthetic peptides AKVISNLLGPREAAV (PDE alpha 30-44) and KQRQTRQFKSKPPKK (PDE gamma 31-45) inhibited phosphorylation of PDE by PKC from ROS. These data suggest that sites (at least one for each subunit) for phosphorylation of PDE by PKC are localized in these corresponding regions of PDE alpha and PDE gamma. Isoenzyme-specific PKC antibodies against peptides unique to the alpha, beta, gamma, delta, epsilon and zeta isoforms of protein kinase C were used to show that a major form of PKC in ROS is PKC alpha. However, other minor forms were also present. Images Figure 1 Figure 4 Figure 6 Figure 7 PMID:8216238

  15. The cubyl cation rearrangements.

    PubMed

    Jalife, Said; Mondal, Sukanta; Cabellos, Jose Luis; Martinez-Guajardo, Gerardo; Fernandez-Herrera, Maria A; Merino, Gabriel

    2016-02-25

    Born-Oppenheimer molecular dynamics simulations and high-level ab initio computations predict that the cage-opening rearrangement of the cubyl cation to the 7H(+)-pentalenyl cation is feasible in the gas phase. The rate-determining step is the formation of the cuneyl cation with an activation barrier of 25.3 kcal mol(-1) at the CCSD(T)/def2-TZVP//MP2/def2-TZVP level. Thus, the cubyl cation is kinetically stable enough to be formed and trapped at moderate temperatures, but it may be rearranged at higher temperatures. PMID:26880646

  16. Comprehensive analysis of phosphorylated proteins of Escherichia coli ribosomes.

    PubMed

    Soung, George Y; Miller, Jennifer L; Koc, Hasan; Koc, Emine C

    2009-07-01

    Phosphorylation of bacterial ribosomal proteins has been known for decades; however, there is still very limited information available on specific locations of the phosphorylation sites in ribosomal proteins and the role they might play in protein synthesis. In this study, we have mapped the specific phosphorylation sites in 24 Escherichia coli ribosomal proteins by tandem mass spectrometry. Detection of phosphorylation was achieved by either phosphorylation specific visualization techniques, ProQ staining, and antibodies for phospho-Ser, Thr, and Tyr; or by mass spectrometry equipped with a capability to detect addition and loss of the phosphate moiety. Enrichment by immobilized metal affinity and/or strong cation exchange chromatography was used to improve the success of detection of the low abundance phosphopeptides. We found the small subunit (30S) proteins S3, S4, S5, S7, S11, S12, S13, S18, and S21 and the large subunit (50S) proteins L1, L2, L3, L5, L6, L7/L12, L13, L14, L16, L18, L19, L21, L22, L28, and L31 to be phosphorylated at one or more residues. Potential roles for each specific site in ribosome function were deduced through careful evaluation of the given phosphorylation sites in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins. PMID:19469554

  17. Structural characterization of a neuroblast-specific phosphorylated region of MARCKS.

    PubMed

    Tinoco, Luzineide W; Fraga, Jully L; Anobom, Cristiane D; Zolessi, Flavio R; Obal, Gonzalo; Toledo, Andrea; Pritsch, Otto; Arruti, Cristina

    2014-04-01

    MARCKS (Myristoylated Alanine-Rich C Kinase substrate) is a natively unfolded protein that interacts with actin, Ca(2+)-Calmodulin, and some plasma membrane lipids. Such interactions occur at a highly conserved region that is specifically phosphorylated by PKC: the Effector Domain. There are two other conserved domains, MH1 (including a myristoylation site) and MH2, also located in the amino terminal region and whose structure and putative protein binding capabilities are currently unknown. MH2 sequence contains a serine that we described as being phosphorylated only in differentiating neurons (S25 in chick). Here, Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy were used to characterize the phosphorylated and unphosphorylated forms of a peptide with the MARCKS sequence surrounding S25. The peptide phosphorylated at this residue is recognized by monoclonal antibody 3C3 (mAb 3C3). CD and NMR data indicated that S25 phosphorylation does not cause extensive modifications in the peptide structure. However, the sharper lines, the absence of multiple spin systems and relaxation dispersion data observed for the phosphorylated peptide suggested a more ordered structure. Surface Plasmon Resonance was employed to compare the binding properties of mAb 3C3 to MARCKS protein and peptide. SPR showed that mAb 3C3 binds to the whole protein and the peptide with a similar affinity, albeit different kinetics. The slightly ordered structure of the phosphorylated peptide might be at the origin of its ability to interact with mAb 3C3 antibody, but this binding did not noticeably modify the peptide structure. PMID:24590112

  18. A potent and highly selective peptide substrate for protein kinase C assay.

    PubMed Central

    Toomik, R; Ek, P

    1997-01-01

    Protein kinases exhibit substrate specificities that are often primarily determined by the amino acids around the phosphorylation sites. Peptides corresponding to protein kinase C phosphorylation sites in several different proteins were synthesized on SPOTs membrane which has recently been found to be applicable for studies of protein kinase specificity. After phosphorylation with protein kinase C, we chose the best phosphorylated peptides for the investigation of the importance of amino acids immediately adjacent to the phosphorylation site. The selectivity of the best protein kinase C substrates from this study was analysed with protein kinases A, CK1 and CK2. According to these tests, the most favourable characteristics of SPOTs-membrane-associated peptides were demonstrated by peptide KRAKRKTAKKR. Kinetic analysis of peptide phosphorylation with protein kinase C revealed an apparent Km of 0.49 +/- 0.13 microM and Vmax of 10.0 +/- 0.5 nmol/min per mg with soluble peptide KRAKRKTAKKR. In addition, we assayed several other soluble peptides commonly used as protein kinase C substrates. Peptide KRAKRKTAKKR showed the lowest Km and the highest Vmax/Km value in comparison with peptides FKKSFKL, pEKRPSQRSKYL and KRAKRKTTKKR. Furthermore, of the peptides tested, KRAKRKTAKKR was the most selective substrate for protein kinase C. The favourable kinetic parameters combined with the selectivity should make the KRAKRKTAKKR peptide useful as a substrate for protein kinase C in the assays of both purified enzyme and in crude cell extracts. PMID:9065763

  19. Oxidation of an Adjacent Methionine Residue Inhibits Regulatory Seryl-phosphorylation of Pyruvate Dehydrogenase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A Met residue is located adjacent to phosphorylation site 1 in the sequences of mitochondrial pyruvate dehydrogenase E1alpha subunits. When synthetic peptides including site 1 were treated with Hydrogen peroxide, the Met residue was oxidized to methionine sulfoxide (MetSO), and the peptides were no...

  20. Microfluidic IEF technique for sequential phosphorylation analysis of protein kinases

    NASA Astrophysics Data System (ADS)

    Choi, Nakchul; Song, Simon; Choi, Hoseok; Lim, Bu-Taek; Kim, Young-Pil

    2015-11-01

    Sequential phosphorylation of protein kinases play the important role in signal transduction, protein regulation, and metabolism in living cells. The analysis of these phosphorylation cascades will provide new insights into their physiological functions in many biological functions. Unfortunately, the existing methods are limited to analyze the cascade activity. Therefore, we suggest a microfluidic isoelectric focusing technique (μIEF) for the analysis of the cascade activity. Using the technique, we show that the sequential phosphorylation of a peptide by two different kinases can be successfully detected on a microfluidic chip. In addition, the inhibition assay for kinase activity and the analysis on a real sample have also been conducted. The results indicate that μIEF is an excellent means for studies on phosphorylation cascade activity.

  1. Assessment of RNA carrier function in peptide amphiphiles derived from the HIV fusion peptide.

    PubMed

    Pratumyot, Yaowalak; Torres, Oscar B; Bong, Dennis

    2016-05-01

    A small library of amphiphilic peptides has been evaluated for duplex RNA carrier function into A549 cells. We studied peptides in which a C-terminal 7-residue cationic domain is attached to a neutral/hydrophobic 23-residue domain that is based on the viral fusion peptide of HIV. We also examined peptides in which the cationic charge was evenly distributed throughout the peptide. Strikingly, subtle sequence variations in the hydrophobic domain that do not alter net hydrophobicity result in wide variation in RNA uptake. Additionally, cyclic cystine variants are much less active as RNA carriers than their open-chain cysteine analogs. With regard to electrostatic effects, we find that lysine is less effective than arginine in facilitating uptake, and that even distribution of cationic residues throughout the peptide sequence results in especially effective RNA carrier function. Overall, minor changes in peptide hydrophobicity, flexibility and charge distribution can significantly alter carrier function. We hypothesize this is due to altered properties of the peptide-RNA assembly rather than peptide secondary structure. PMID:26988874

  2. Isolation of regulatory-competent, phosphorylated cytochrome C oxidase.

    PubMed

    Lee, Icksoo; Salomon, Arthur R; Yu, Kebing; Samavati, Lobelia; Pecina, Petr; Pecinova, Alena; Hüttemann, Maik

    2009-01-01

    The role of posttranslational modifications, specifically reversible phosphorylation as a regulatory mechanism operating in the mitochondria, is a novel research direction. The mitochondrial oxidative phosphorylation system is a particularly interesting unit because it is responsible for the production of the vast majority of cellular energy in addition to free radicals, two factors that are aberrant in numerous human diseases and that may be influenced by reversible phosphorylation of the oxidative phosphorylation complexes. We here describe a detailed protocol for the isolation of mammalian liver and heart mitochondria and subsequently cytochrome c oxidase (CcO) under conditions maintaining the physiological phosphorylation state. The protocol employs the use of activated vanadate, an unspecific tyrosine phosphatase inhibitor, fluoride, an unspecific serine/threonine phosphatase inhibitor, and EGTA, a calcium chelator to prevent the activation of calcium-dependent protein phosphatases. CcO purified without manipulation of signaling pathways shows strong tyrosine phosphorylation on subunits II and IV, whereas tyrosine phosphorylation of subunit I can be induced by the cAMP- and TNFalpha-dependent pathways in liver. Using our protocol on cow liver tissue we further show the identification of a new phosphorylation site on CcO subunit IV tyrosine 11 of the mature protein (corresponding to tyrosine 33 of the precursor peptide) via immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS). This phosphorylation site is located close to the ATP and ADP binding site, which adjusts CcO activity to cellular energy demand, and we propose that phosphorylation of tyrosine 11 enables allosteric regulation. PMID:19426869

  3. Turning peptides in comb silicone polymers.

    PubMed

    Jebors, Said; Pinese, Coline; Nottelet, Benjamin; Parra, Karine; Amblard, Muriel; Mehdi, Ahmad; Martinez, Jean; Subra, Gilles

    2015-03-01

    We have recently reported on a new class of silicone-peptide' biopolymers obtained by polymerization of di-functionalized chlorodimethylsilyl hybrid peptides. Herein, we describe a related strategy based on dichloromethylsilane-derived peptides, which yield novel polymers with a polysiloxane backbone, comparable with a silicone-bearing pendent peptide chains. Interestingly, polymerization is chemoselective toward amino acids side-chains and proceeds in a single step in very mild conditions (neutral pH, water, and room temperature). As potential application, a cationic sequence was polymerized and used for antibacterial coating. PMID:25688748

  4. Antimicrobial peptides

    PubMed Central

    2014-01-01

    With increasing antibiotics resistance, there is an urgent need for novel infection therapeutics. Since antimicrobial peptides provide opportunities for this, identification and optimization of such peptides have attracted much interest during recent years. Here, a brief overview of antimicrobial peptides is provided, with focus placed on how selected hydrophobic modifications of antimicrobial peptides can be employed to combat also more demanding pathogens, including multi-resistant strains, without conferring unacceptable toxicity. PMID:24758244

  5. Struvite and prebiotic phosphorylation.

    NASA Technical Reports Server (NTRS)

    Handschuh, G. J.; Orgel, L. E.

    1973-01-01

    Struvite rather than apatite or amorphous calcium phosphate is precipitated when phosphate is added to seawater containing more than 0.01M NH4+ ions. Struvite may have precipitated from evaporating seawater on the primitive earth, and may have been important for prebiotic phosphorylation.

  6. Peptide-nucleotide microdroplets as a step towards a membrane-free protocell model

    NASA Astrophysics Data System (ADS)

    Koga, Shogo; Williams, David S.; Perriman, Adam W.; Mann, Stephen

    2011-09-01

    Although phospholipid bilayers are ubiquitous in modern cells, their impermeability, lack of dynamic properties, and synthetic complexity are difficult to reconcile with plausible pathways of proto-metabolism, growth and division. Here, we present an alternative membrane-free model, which demonstrates that low-molecular-weight mononucleotides and simple cationic peptides spontaneously accumulate in water into microdroplets that are stable to changes in temperature and salt concentration, undergo pH-induced cycles of growth and decay, and promote α-helical peptide secondary structure. Moreover, the microdroplets selectively sequester porphyrins, inorganic nanoparticles and enzymes to generate supramolecular stacked arrays of light-harvesting molecules, nanoparticle-mediated oxidase activity, and enhanced rates of glucose phosphorylation, respectively. Taken together, our results suggest that peptide-nucleotide microdroplets can be considered as a new type of protocell model that could be used to develop novel bioreactors, primitive artificial cells and plausible pathways to prebiotic organization before the emergence of lipid-based compartmentalization on the early Earth.

  7. Peptide-nucleotide microdroplets as a step towards a membrane-free protocell model.

    PubMed

    Koga, Shogo; Williams, David S; Perriman, Adam W; Mann, Stephen

    2011-09-01

    Although phospholipid bilayers are ubiquitous in modern cells, their impermeability, lack of dynamic properties, and synthetic complexity are difficult to reconcile with plausible pathways of proto-metabolism, growth and division. Here, we present an alternative membrane-free model, which demonstrates that low-molecular-weight mononucleotides and simple cationic peptides spontaneously accumulate in water into microdroplets that are stable to changes in temperature and salt concentration, undergo pH-induced cycles of growth and decay, and promote α-helical peptide secondary structure. Moreover, the microdroplets selectively sequester porphyrins, inorganic nanoparticles and enzymes to generate supramolecular stacked arrays of light-harvesting molecules, nanoparticle-mediated oxidase activity, and enhanced rates of glucose phosphorylation, respectively. Taken together, our results suggest that peptide-nucleotide microdroplets can be considered as a new type of protocell model that could be used to develop novel bioreactors, primitive artificial cells and plausible pathways to prebiotic organization before the emergence of lipid-based compartmentalization on the early Earth. PMID:21860462

  8. Phosphorylation-dephosphorylation of yeast pyruvate dehydrogenase

    SciTech Connect

    Uhlinger, D.J.; Reed, L.J.

    1986-05-01

    Pyruvate dehydrogenase complex (PDC) was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). No pyruvate dehydrogenase (PDH) kinase activity was detected at any stage of the purification. However, the purified PDC was phosphorylated and inactivated by purified PDH kinase from bovine kidney mitochondria, Mg/sup 2 +/, and (..gamma..-/sup 32/P)ATP. The protein-bound radioactivity was localized in the PDH ..cap alpha.. subunit. The phosphorylated, inactivated PDC was dephosphorylated and reactivated with purified bovine PDH phosphatase, Mg/sup 2 +/, and Ca/sup 2 +/. From a tryptic digest of phosphorylated yeast PDC a radioactive peptide was isolated by anion and reverse phase HPLC. The sequence of this tetradecapeptide is Tyr-Gly-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Thr-Tyr-Arg. This sequence is very similar to the sequence of a tryptic phosphopeptide derived from the ..cap alpha.. subunit of bovine kidney and heart PDH: Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser-Tyr-Arg.

  9. A spatiotemporal characterization of the effect of p53 phosphorylation on its interaction with MDM2

    PubMed Central

    ElSawy, Karim M; Sim, Adelene; Lane, David P; Verma, Chandra S; Caves, Leo SD

    2015-01-01

    The interaction of p53 and MDM2 is modulated by the phosphorylation of p53. This mechanism is key to activating p53, yet its molecular determinants are not fully understood. To study the spatiotemporal characteristics of this molecular process we carried out Brownian dynamics simulations of the interactions of the MDM2 protein with a p53 peptide in its wild type state and when phosphorylated at Thr18 (pThr18) and Ser20 (pSer20). We found that p53 phosphorylation results in concerted changes in the topology of the interaction landscape in the diffusively bound encounter complex domain. These changes hinder phosphorylated p53 peptides from binding to MDM2 well before reaching the binding site. The underlying mechanism appears to involve shift of the peptide away from the vicinity of the MDM2 protein, peptide reorientation, and reduction in peptide residence time relative to wild-type p53 peptide. pThr18 and pSr20 p53 peptides experience reduction in residence times by factors of 13.6 and 37.5 respectively relative to the wild-type p53 peptide, indicating a greater role for Ser20 phosphorylation in abrogating p53 MDM2 interactions. These detailed insights into the effect of phosphorylation on molecular interactions are not available from conventional experimental and theoretical approaches and open up new avenues that incorporate molecular interaction dynamics, for stabilizing p53 against MDM2, which is a major focus of anticancer drug lead development. PMID:25584963

  10. Protein kinase CK2 phosphorylates Hsp105 alpha at Ser509 and modulates its function.

    PubMed Central

    Ishihara, Keiichi; Yamagishi, Nobuyuki; Hatayama, Takumi

    2003-01-01

    The 105 kDa heat-shock protein (Hsp) Hsp105 alpha is a mammalian stress protein that belongs to the HSP105/HSP110 family. We have shown previously that Hsp105 alpha exists as non-phosphorylated and phosphorylated forms in vivo, and is phosphorylated by protein kinase CK2 (CK2) in vitro. In this study, to elucidate the role of phosphorylation of Hsp105 alpha, we first analysed the site of phosphorylation of Hsp105 alpha by CK2. Peptide mapping analysis of Hsp105 alpha phosphorylated by CK2 and in vitro phosphorylation experiments using various deletion and substitution mutants of Hsp105 alpha revealed that Hsp105 alpha is phosphorylated at Ser(509) in the beta-sheet domain. Furthermore, Ser(509) in Hsp105 alpha was also phosphorylated in mammalian COS-7 cells, although other sites were phosphorylated as well. Next, we examined the effects of phosphorylation of Hsp105 alpha on its functions using CK2-phosphorylated Hsp105 alpha. Interestingly, Hsp105 alpha suppressed 70 kDa heat-shock cognate protein (Hsc70)-mediated protein folding, whereas the phosphorylation of Hsp105 alpha at Ser(509) abolished the inhibitory activity of Hsp105 alpha in vitro. In accordance with these findings, wild-type Hsp105 alpha, which was thought to be phosphorylated in vivo, had no effect on Hsp70-mediated refolding of heat-denatured luciferase, whereas a non-phosphorylatable mutant of Hsp105 alpha suppressed the Hsp70-mediated refolding of heat-denatured luciferase in mammalian cells. Thus it was suggested that CK2 phosphorylates Hsp105 alpha at Ser(509) and modulates the function of Hsp105 alpha. The regulation of Hsp105 alpha function by phosphorylation may play an important role in a variety of cellular events. PMID:12558502

  11. Purification and sequencing of radish seed calmodulin antagonists phosphorylated by calcium-dependent protein kinase.

    PubMed Central

    Polya, G M; Chandra, S; Condron, R

    1993-01-01

    A family of radish (Raphanus sativus) calmodulin antagonists (RCAs) was purified from seeds by extraction, centrifugation, batch-wise elution from carboxymethyl-cellulose, and high performance liquid chromatography (HPLC) on an SP5PW cation-exchange column. This RCA fraction was further resolved into three calmodulin antagonist polypeptides (RCA1, RCA2, and RCA3) by denaturation in the presence of guanidinium HCl and mercaptoethanol and subsequent reverse-phase HPLC on a C8 column eluted with an acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. The RCA preparation, RCA1, RCA2, RCA3, and other radish seed proteins are phosphorylated by wheat embryo Ca(2+)-dependent protein kinase (CDPK). The RCA preparation contains other CDPK substrates in addition to RCA1, RCA2, and RCA3. The RCA preparation, RCA1, RCA2, and RCA3 inhibit chicken gizzard calmodulin-dependent myosin light chain kinase assayed with a myosin-light chain-based synthetic peptide substrate (fifty percent inhibitory concentrations of RCA2 and RCA3 are about 7 and 2 microM, respectively). N-terminal sequencing by sequential Edman degradation of RCA1, RCA2, and RCA3 revealed sequences having a high homology with the small subunit of the storage protein napin from Brassica napus and with related proteins. The deduced amino acid sequences of RCA1, RCA2, RCA3, and RCA3' (a subform of RCA3) have agreement with average molecular masses from electrospray mass spectrometry of 4537, 4543, 4532, and 4560 kD, respectively. The only sites for serine phosphorylation are near or at the C termini and hence adjacent to the sites of proteolytic precursor cleavage. PMID:8278508

  12. Use of an electrodialytic reactor for the simultaneous β-lactoglobulin enzymatic hydrolysis and fractionation of generated bioactive peptides.

    PubMed

    Doyen, Alain; Husson, Eric; Bazinet, Laurent

    2013-02-15

    The enzymatic hydrolysis of β-lactoglobulin and the fractionation of peptides were performed in one step in an electrodialysis cell with ultrafiltration membranes stacked. After 240 min of treatment, 15 anionic and 4 cationic peptides were detected in the anionic and cationic peptide recovery compartments. Amongst these 15 anionic peptides, 2 hypocholesterolemic, 3 antihypertensive and 1 antibacterial peptides were recovered and concentrated with migration rates ranging from 5.5% and 81.7%. Amongst the 4 cationic peptides, the peptide sequence ALPMHIR, identified as lactokinin and known to exert an important antihypertensive effect, was recovered with an estimated 66% migration rate. To our knowledge, it was the first attempt to perform hydrolysis under an electric field and to simultaneously separate anionic and cationic peptides produced. PMID:23194514

  13. Orientation preferences of backbone secondary amide functional groups in peptide nucleic acid complexes: quantum chemical calculations reveal an intrinsic preference of cationic D-amino acid-based chiral PNA analogues for the P-form.

    PubMed

    Topham, Christopher M; Smith, Jeremy C

    2007-02-01

    Geometric descriptions of nonideal interresidue hydrogen bonding and backbone-base water bridging in the minor groove are established in terms of polyamide backbone carbonyl group orientation from analyses of residue junction conformers in experimentally determined peptide nucleic acid (PNA) complexes. Two types of interresidue hydrogen bonding are identified in PNA conformers in heteroduplexes with nucleic acids that adopt A-like basepair stacking. Quantum chemical calculations on the binding of a water molecule to an O2 base atom in glycine-based PNA thymine dimers indicate that junctions modeled with P-form backbone conformations are lower in energy than a dimer comprising the predominant conformation observed in A-like helices. It is further shown in model systems that PNA analogs based on D-lysine are better able to preorganize in a conformation exclusive to P-form helices than is glycine-based PNA. An intrinsic preference for this conformation is also exhibited by positively charged chiral PNA dimers carrying 3-amino-D-alanine or 4-aza-D-leucine residue units that provide for additional rigidity by side-chain hydrogen bonding to the backbone carbonyl oxygen. Structural modifications stabilizing P-form helices may obviate the need for large heterocycles to target DNA pyrimidine bases via PNA.DNA-PNA triplex formation. Quantum chemical modeling methods are used to propose candidate PNA Hoogsteen strand designs. PMID:17071666

  14. Correlation of Multiple Peptide Mass Spectra for Phosphoprotein Identification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    When collision induced dissociation is used to fragment phosphorylated peptides during tandem mass spectrometry (MS2), an ion exhibiting the neutral loss of phosphoric acid can be the major product. The neutral loss ion can then be fragmented during MS3 for additional resolution of the peptide sequ...

  15. ELISA measurement of specific antibodies to phosphorylated tau in intravenous immunoglobulin products.

    PubMed

    Loeffler, David A; Klaver, Andrea C; Coffey, Mary P

    2015-10-01

    The therapeutic effects of intravenous immunoglobulin (IVIG) products were recently studied in Alzheimer's disease (AD) patients. Pilot studies produced encouraging results but phase II and III trials gave disappointing results; a further study is in progress. IVIG products contain antibodies to tau protein, the main component of neurofibrillary tangles (NFTs). The tau used to detect IVIG's anti-tau antibodies in previous studies was non-phosphorylated recombinant human tau-441, but NFT-associated tau is extensively phosphorylated. The objective of this study was to determine if various IVIG products contain specific antibodies to phosphorylated tau (anti-pTau antibodies). ELISAs were used to evaluate binding of six IVIG products to a 12 amino acid peptide, tau 196-207, which was phosphorylated ("pTau peptide") or non-phosphorylated ("non-pTau peptide") at Serine-199 and Serine-202. Both amino acid residues are phosphorylated in AD NFTs. Each IVIG's "anti-pTau antibody ratio" was calculated by dividing its binding to the pTau peptide by its binding to the non-pTau peptide. Seven experiments were performed and data were pooled, with each experiment contributing one data point from each IVIG product. Mean anti-pTau antibody ratios greater than 1.0, suggesting specific antibodies to phosphorylated tau, were found for three IVIG products. Because administration of antibodies to phosphorylated tau has been found to reduce tau-associated pathology in transgenic mouse models of tauopathy, increasing the levels of anti-pTau antibodies, together with other selected antibodies such as anti-Aβ, in IVIG might increase its ability to slow AD's progression. PMID:26330100

  16. Differences in the sites of phosphorylation of the insulin receptor in vivo and in vitro

    SciTech Connect

    White, M.F.; Takayama, S.; Kahn, C.R.

    1985-08-05

    Phosphorylation of the insulin receptor was studied in intact well differentiated hepatoma cells (Fao) and in a solubilized and partially purified receptor preparation obtained from these cells by affinity chromatography on wheat germ agglutinin agarose. Tryptic peptides containing the phosphorylation sites of the beta-subunit of the insulin receptor were analyzed by reverse-phase high performance liquid chromatography. Phosphoamino acid content of these peptides was determined by acid hydrolysis and high voltage electrophoresis. Separation of the phosphopeptides from unstimulated Fao cells revealed one major and two minor phosphoserine-containing peptides and a single minor phosphothreonine-containing peptide. Insulin (10(-7) M) increased the phosphorylation of the beta-subunit of the insulin receptor 3- to 4-fold in the intact Fao cell. After insulin stimulation, two phosphotyrosine-containing peptides were identified. Tyrosine phosphorylation reached a steady state within 20 s after the addition of insulin and remained nearly constant for 1 h. Under our experimental conditions, no significant change in the amount of (TSP)phosphoserine or (TSP)phosphothreonine associated with the beta-subunit was found during the initial response of cells to insulin. When the insulin receptor was extracted from the Fao cells and incubated in vitro with (gamma-TSP)ATP and MnS , very little phosphorylation occurred in the absence of insulin.

  17. Tonoplast-Bound Protein Kinase Phosphorylates Tonoplast Intrinsic Protein 1

    PubMed Central

    Johnson, Kenneth D.; Chrispeels, Maarten J.

    1992-01-01

    Tonoplast intrinsic protein (TIP) is a member of a family of putative membrane channels found in bacteria, animals, and plants. Plants have seed-specific, vegetative/reproductive organ-specific, and water-stress-induced forms of TIP. Here, we report that the seed-specific TIP is a phosphoprotein whose phosphorylation can be monitored in vivo by allowing bean cotyledons to take up [32P]orthophosphate and in vitro by incubating purified tonoplasts with γ-labeled [32P]ATP. Characterization of the in vitro phosphorylation of TIP indicates that a membrane-bound protein kinase phosphorylates TIP in a Ca2+-dependent manner. The capacity of the isolated tonoplast membranes to phosphorylate TIP declined markedly during seed germination, and this decline occurred well before the development-mediated decrease in TIP occurs. Phosphoamino acid analysis of purified, radiolabeled TIP showed that serine is the major, if not only, phosphorylated residue, and cyanogen bromide cleavage yielded a single radioactive peptide peak on a reverse-phase high-performance liquid chromatogram. Estimation of the molecular mass of the cyanogen bromide phosphopeptide by laser desorption mass spectroscopy led to its identification as the hydrophilic N-terminal domain of TIP. The putative phosphate-accepting serine residue occurs in a consensus phosphorylation site for serine/threonine protein kinases. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:16653198

  18. Phospho-oligosaccharide dependent phosphorylation of ATP citrate lyase.

    PubMed

    Puerta, J; Mato, J M; Alemany, S

    1990-01-01

    The effect of insulin on ATP citrate lyase phosphorylation has been shown to be mimicked by a phospho-oligosaccharide in intact adipocytes. We demonstrate that the addition of phospho-oligosaccharide to intact adipocytes enhances the phosphorylation of ATP citrate lyase in the same tryptic peptide as insulin does. The addition of phospho-oligosaccharide to an adipocyte extract also results in an increase in ATP citrate lyase phosphorylation but in a different site than that observed in intact cells. The phospho-oligosaccharide-dependent incorporation of phosphate into ATP citrate lyase in intact cells is resistant to isopropanol and acetic acid, but the phosphoenzyme phosphorylated in cell extracts is acid labile. In cell extracts, the addition of phospho-oligosaccharide markedly inhibits ATP hydrolysis, which may explain the effect of this molecule on ATP citrate lyase phosphorylation in broken cells. These results support the hypothesis that this phospho-oligosaccharide mediates some of the effects of insulin on protein phosphorylation. They also indicate that caution should be exercised in interpreting the results obtained by adding phospho-oligosaccharide to broken cell preparations. PMID:2119547

  19. Multisite phosphorylation of spinach leaf sucrose-phosphate synthase

    SciTech Connect

    Huber, J.L.; Huber, S.C. )

    1990-05-01

    Spinach leaf sucrose-phosphate synthase is phosphorylated both in vivo and in vitro on serine residues. Phosphorylation of SPS in vivo yields twelve major phosphopeptides after a tryptic digest and two dimensional mapping. The in vivo labeling of three of these SPS P-peptides is reduced in illuminated leaves where the extracted enzyme is activated relative to that of dark leaves. Two of these inhibitory sites are phosphorylated as well when SPS is inactivated in vitro using ({sup 32}P)ATP. In vivo phosphorylation of two other sites is enhanced during mannose feeding of the leaves (in light or dark) which produces the highest activation state of SPS. Overall, the results confirm that light-dark regulation of SPS activity occurs as a result of regulatory seryl-phosphorylation and involves a balance between phosphorylation of sites which inhibit or stimulate activity. Regulation of the SPS protein kinase that inhibits activity is relatively unaffected by phosphate but inhibited by G1c 6-P (IC{sub 50}{approx}5 mM), which may explain the control of SPS activation state by light-dark signals.

  20. Protein Phosphorylation during Coconut Zygotic Embryo Development1

    PubMed Central

    Islas-Flores, Ignacio; Oropeza, Carlos; Hernández-Sotomayor, S.M. Teresa

    1998-01-01

    Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [γ-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species. PMID:9733545

  1. Characterization of Bidentate Phosphoryl Compounds on Soil Particulates using SIMS

    SciTech Connect

    Gary S. Groenewold; Gary L. Gresham; Recep Avci; Muhammedin Deliorman

    2009-03-01

    The presence of organic compounds as surface contaminants on particles can provide valuable data about the particles environment, but identification can be analytically challenging. This is true particularly for compounds that have the potential for strong surface binding, such as compounds capable of multidentate attachment. Direct analysis using time-of-flight secondary ion mass spectrometry was evaluated for characterization of soil particles contaminated with low concentrations of two bidentate organophosphoryl compounds, diphenyl-N,N-di-n-butylcarbamoylmethylphosphine oxide and tetraphenylmethylene diphosphine dioxide. Molecular ions were formed by cationization with H+ and alkali elements Na+ and K+ that are indigenous to the particle surface chemistry. Spectra generated from a contaminated calcareous soil were dominated by K+-containing ions, whereas spectra from a sandy loam had more abundant Na+-species. Cation-bound dimers were also formed which favored incorporation of K+, and a unique aluminosilicate-phosphoryl conjugate cation was also formed when the diphosphoryl ligand was present on the surface. The phosphoryl ligands also underwent fragmentation reactions, the course of which varied depending on the cation that was bound. Minimum detectable surface concentrations were evaluated and were in the 0.04-0.2 monolayer range, depending on the compound and soil particle matrix they was bound to. The ion signature was detected on soil particle surfaces for time periods exceeding six months, suggesting that the characterization approach could be used for environmental exposure history at times well beyond initial exposure.

  2. Synaptic plasticity and phosphorylation

    PubMed Central

    Lee, Hey-Kyoung

    2009-01-01

    A number of neuronal functions, including synaptic plasticity, depend on proper regulation of synaptic proteins, many of which can be rapidly regulated by phosphorylation. Neuronal activity controls the function of these synaptic proteins by exquisitely regulating the balance of various protein kinase and protein phosphatase activity. Recent understanding of synaptic plasticity mechanisms underscores important roles that these synaptic phosphoproteins play in regulating both pre- and post-synaptic functions. This review will focus on key postsynaptic phosphoproteins that have been implicated to play a role in synaptic plasticity. PMID:16904750

  3. Determination of GPCR Phosphorylation Status: Establishing a Phosphorylation Barcode.

    PubMed

    Prihandoko, Rudi; Bradley, Sophie J; Tobin, Andrew B; Butcher, Adrian J

    2015-01-01

    G protein-coupled receptors (GPCRs) are rapidly phosphorylated following agonist occupation in a process that mediates receptor uncoupling from its cognate G protein, a process referred to as desensitization. In addition, this process provides a mechanism by which receptors can engage with arrestin adaptor molecules and couple to downstream signaling pathways. The importance of this regulatory process has been highlighted recently by the understanding that ligands can direct receptor signaling along one pathway in preference to another, the phenomenon of signaling bias that is partly mediated by the phosphorylation status or phosphorylation barcode of the receptor. Methods to determine the phosphorylation status of a GPCR in vitro and in vivo are necessary to understand not only the physiological mechanisms involved in GPCR signaling, but also to fully examine the signaling properties of GPCR ligands. This unit describes detailed methods for determining the overall phosphorylation pattern on a receptor (the phosphorylation barcode), as well as mass spectrometry approaches that can define the precise sites that become phosphorylated. These techniques, coupled with the generation and characterization of receptor phosphorylation-specific antibodies, provide a full palate of techniques necessary to determine the phosphorylation status of any given GPCR subtype. PMID:26344213

  4. Uncouplers of oxidative phosphorylation.

    PubMed

    Terada, H

    1990-07-01

    Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling between the electron transport and phosphorylation reactions and thus inhibit ATP synthesis without affecting the respiratory chain and ATP synthase (H(+)-ATPase). Miscellaneous compounds are known to be uncouplers, but weakly acidic uncouplers are representative because they show very potent activities. The most potent uncouplers discovered so far are the hindered phenol SF 6847, and hydrophobic salicylanilide S-13, which are active in vitro at concentrations in the 10 nM range. For induction of uncoupling, an acid dissociable group, bulky hydrophobic moiety and strong electron-withdrawing group are required. Weakly acidic uncouplers are considered to produce uncoupling by their protonophoric action in the H(+)-impermeable mitochondrial membrane. For exerting these effects, the stability of the respective uncoupler anions in the hydrophobic membrane is very important. High stability is achieved by delocalization of the polar ionic charge through uncoupler (chemical)-specific mechanisms. Such an action of weakly acidic uncouplers is characteristic of the highly efficient membrane targeting action of a nonsite-specific type of bioactive compound. PMID:2176586

  5. Uncouplers of oxidative phosphorylation.

    PubMed Central

    Terada, H

    1990-01-01

    Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling between the electron transport and phosphorylation reactions and thus inhibit ATP synthesis without affecting the respiratory chain and ATP synthase (H(+)-ATPase). Miscellaneous compounds are known to be uncouplers, but weakly acidic uncouplers are representative because they show very potent activities. The most potent uncouplers discovered so far are the hindered phenol SF 6847, and hydrophobic salicylanilide S-13, which are active in vitro at concentrations in the 10 nM range. For induction of uncoupling, an acid dissociable group, bulky hydrophobic moiety and strong electron-withdrawing group are required. Weakly acidic uncouplers are considered to produce uncoupling by their protonophoric action in the H(+)-impermeable mitochondrial membrane. For exerting these effects, the stability of the respective uncoupler anions in the hydrophobic membrane is very important. High stability is achieved by delocalization of the polar ionic charge through uncoupler (chemical)-specific mechanisms. Such an action of weakly acidic uncouplers is characteristic of the highly efficient membrane targeting action of a nonsite-specific type of bioactive compound. PMID:2176586

  6. Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase

    NASA Technical Reports Server (NTRS)

    Bachmann, M.; Shiraishi, N.; Campbell, W. H.; Yoo, B. C.; Harmon, A. C.; Huber, S. C.; Davies, E. (Principal Investigator)

    1996-01-01

    Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.

  7. Latarcins: versatile spider venom peptides.

    PubMed

    Dubovskii, Peter V; Vassilevski, Alexander A; Kozlov, Sergey A; Feofanov, Alexey V; Grishin, Eugene V; Efremov, Roman G

    2015-12-01

    Arthropod venoms feature the presence of cytolytic peptides believed to act synergetically with neurotoxins to paralyze prey or deter aggressors. Many of them are linear, i.e., lack disulfide bonds. When isolated from the venom, or obtained by other means, these peptides exhibit common properties. They are cationic; being mostly disordered in aqueous solution, assume amphiphilic α-helical structure in contact with lipid membranes; and exhibit general cytotoxicity, including antifungal, antimicrobial, hemolytic, and anticancer activities. To suit the pharmacological needs, the activity spectrum of these peptides should be modified by rational engineering. As an example, we provide a detailed review on latarcins (Ltc), linear cytolytic peptides from Lachesana tarabaevi spider venom. Diverse experimental and computational techniques were used to investigate the spatial structure of Ltc in membrane-mimicking environments and their effects on model lipid bilayers. The antibacterial activity of Ltc was studied against a panel of Gram-negative and Gram-positive bacteria. In addition, the action of Ltc on erythrocytes and cancer cells was investigated in detail with confocal laser scanning microscopy. In the present review, we give a critical account of the progress in the research of Ltc. We explore the relationship between Ltc structure and their biological activity and derive molecular characteristics, which can be used for optimization of other linear peptides. Current applications of Ltc and prospective use of similar membrane-active peptides are outlined. PMID:26286896

  8. Protein kinase C catalyses the phosphorylation and activation of rat liver phospholipid methyltransferase.

    PubMed Central

    Villalba, M; Pajares, M A; Renart, M F; Mato, J M

    1987-01-01

    When a partially purified rat liver phospholipid methyltransferase is incubated with [gamma-32P]ATP and rat brain protein kinase C, phospholipid methyltransferase (Mr 50,000, pI 4.75) becomes phosphorylated. Phosphorylation of the enzyme showed Ca2+/lipid-dependency. Protein kinase C-dependent phosphorylation of phospholipid methyltransferase was accompanied by an approx. 2-fold activation of the enzyme activity. Activity changes and enzyme phosphorylation showed the same time course. Activation of the enzyme also showed Ca2+/lipid-dependency. Protein kinase C mediates phosphorylation of predominantly serine residues of the methyltransferase. One major peak of phosphorylation was identified by analysis of tryptic phosphopeptides by isoelectrofocusing. This peak (pI 5.2) differs from that phosphorylated by the cyclic AMP-dependent protein kinase (pI 7.2), demonstrating the specificity of phosphorylation of protein kinase C. Tryptic-peptide mapping by h.p.l.c. of the methyltransferase phosphorylated by protein kinase C revealed one major peak of radioactivity, which could be resolved into two labelled phosphopeptides by t.l.c. The significance of protein kinase C-mediated phosphorylation of phospholipid methyltransferase is discussed. Images Fig. 1. Fig. 4. PMID:3593229

  9. Interrogation of MDM2 phosphorylation in p53 activation using native chemical ligation: the functional role of Ser17 phosphorylation in MDM2 reexamined

    PubMed Central

    Zhan, Changyou; Varney, Kristen; Yuan, Weirong; Zhao, Le; Lu, Wuyuan

    2012-01-01

    The E3 ubiquitin ligase MDM2 functions as a crucial negative regulator of the p53 tumor suppressor protein by antagonizing p53 transactivation activity and targeting p53 for degradation. Cellular stress activates p53 by alleviating MDM2-mediated functional inhibition, even though the molecular mechanisms of stress-induced p53 activation still remain poorly understood. Two opposing models have been proposed to describe the functional and structural role in p53 activation of Ser17 phosphorylation in the N-terminal “lid” (residues 1–24) of MDM2. Using the native chemical ligation technique, we synthesized the p53-binding domain (1–109)MDM2 and its Ser17-phosphorylated analog (1–109)MDM2 pS17 as well as (1–109)MDM2 S17D and (25–109)MDM2, and comparatively characterized their interactions with a panel of p53-derived peptide ligands using surface plasmon resonance, fluorescence polarization, and NMR and CD spectroscopic techniques. We found that the lid is partially structured in apo-MDM2 and occludes p53 peptide binding in a ligand size-dependent manner. Binding of (1–109)MDM2 by the (15–29)p53 peptide fully displaces the lid and renders it completely disordered in the peptide-protein complex. Importantly, neither Ser17 phosphorylation nor the phospho-mimetic mutation S17D has any functional impact on p53 peptide binding to MDM2. Although Ser17 phosphorylation or its mutation to Asp contributes marginally to the stability of the lid conformation in apo-MDM2, neither modification stabilizes apo-MDM2 globally or the displaced lid locally. Our findings demonstrate that Ser17 phosphorylation is functionally neutral with respect to p53 binding, suggesting that MDM2 phosphorylation at a single site is unlikely to play a dominant role in stress-induced p53 activation. PMID:22444248

  10. Protein phosphorylation in stomatal movement

    PubMed Central

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  11. Phosphorylation site prediction in plants.

    PubMed

    Yao, Qiuming; Schulze, Waltraud X; Xu, Dong

    2015-01-01

    Protein phosphorylation events on serine, threonine, and tyrosine residues are the most pervasive protein covalent bond modifications in plant signaling. Both low and high throughput studies reveal the importance of phosphorylation in plant molecular biology. Although becoming more and more common, the proteome-wide screening on phosphorylation by experiments remains time consuming and costly. Therefore, in silico prediction methods are proposed as a complementary analysis tool to enhance the phosphorylation site identification, develop biological hypothesis, or help experimental design. These methods build statistical models based on the experimental data, and they do not have some of the technical-specific bias, which may have advantage in proteome-wide analysis. More importantly computational methods are very fast and cheap to run, which makes large-scale phosphorylation identifications very practical for any types of biological study. Thus, the phosphorylation prediction tools become more and more popular. In this chapter, we will focus on plant specific phosphorylation site prediction tools, with essential illustration of technical details and application guidelines. We will use Musite, PhosPhAt and PlantPhos as the representative tools. We will present the results on the prediction of the Arabidopsis protein phosphorylation events to give users a general idea of the performance range of the three tools, together with their strengths and limitations. We believe these prediction tools will contribute more and more to the plant phosphorylation research community. PMID:25930706

  12. Protein phosphorylation in stomatal movement.

    PubMed

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  13. Factors Affecting Peptide Interactions with Surface-Bound Microgels.

    PubMed

    Nyström, Lina; Nordström, Randi; Bramhill, Jane; Saunders, Brian R; Álvarez-Asencio, Rubén; Rutland, Mark W; Malmsten, Martin

    2016-02-01

    Effects of electrostatics and peptide size on peptide interactions with surface-bound microgels were investigated with ellipsometry, confocal microscopy, and atomic force microscopy (AFM). Results show that binding of cationic poly-L-lysine (pLys) to anionic, covalently immobilized, poly(ethyl acrylate-co-methacrylic acid) microgels increased with increasing peptide net charge and microgel charge density. Furthermore, peptide release was facilitated by decreasing either microgel or peptide charge density. Analogously, increasing ionic strength facilitated peptide release for short peptides. As a result of peptide binding, the surface-bound microgels displayed pronounced deswelling and increased mechanical rigidity, the latter quantified by quantitative nanomechanical mapping. While short pLys was found to penetrate the entire microgel network and to result in almost complete charge neutralization, larger peptides were partially excluded from the microgel network, forming an outer peptide layer on the microgels. As a result of this difference, microgel flattening was more influenced by the lower Mw peptide than the higher. Peptide-induced deswelling was found to be lower for higher Mw pLys, the latter effect not observed for the corresponding microgels in the dispersed state. While the effects of electrostatics on peptide loading and release were similar to those observed for dispersed microgels, there were thus considerable effects of the underlying surface on peptide-induced microgel deswelling, which need to be considered in the design of surface-bound microgels as carriers of peptide loads, for example, in drug delivery or in functionalized biomaterials. PMID:26750986

  14. Sequential and competitive adsorption of peptides at pendant PEO layers.

    PubMed

    Wu, Xiangming; Ryder, Matthew P; McGuire, Joseph; Snider, Joshua L; Schilke, Karl F

    2015-06-01

    Earlier work provided direction for development of responsive drug delivery systems based on modulation of the structure, amphiphilicity, and surface density of bioactive peptides entrapped within pendant polyethylene oxide (PEO) brush layers. In this work, we describe the sequential and competitive adsorption behavior of such peptides at pendant PEO layers. Three cationic peptides were used for this purpose: the arginine-rich, amphiphilic peptide WLBU2, a peptide chemically identical to WLBU2 but of scrambled sequence (S-WLBU2), and the non-amphiphilic peptide poly-L-arginine (PLR). Optical waveguide lightmode spectroscopy (OWLS) was used to quantify the rate and extent of peptide adsorption and elution at surfaces coated with PEO. UV spectroscopy and time-of-flight secondary ion mass spectrometry (TOF-SIMS) were used to quantify the extent of peptide exchange during the course of sequential and competitive adsorption. Circular dichroism (CD) was used to evaluate conformational changes after adsorption of peptide mixtures at PEO-coated silica nanoparticles. Results indicated that amphiphilic peptides are able to displace adsorbed, non-amphiphilic peptides in PEO layers, while non-amphiphilic peptides were not able to displace more amphiphilic peptides. In addition, peptides of greater amphiphilicity dominated the adsorption at the PEO layer from mixtures with less amphiphilic or non-amphiphilic peptides. PMID:25909181

  15. Phosphorylated. beta. -dicarbonyl compounds

    SciTech Connect

    Liorber, B.G.; Tarzivolova, T.A.; Pavlov, V.A.; Zykova, T.V.; Kisilev, V.V.; Tumasheva, N.A.; Slizkii, A.Yu.; Shagvaleev, F.S.

    1987-08-20

    The reaction of trialkyl phosphites with alkyl malonyl chlorides leads to alkyl 3-dialkoxyphosphoryl-3-oxopropionates, which exist in the stable E-enol form. Depending on the basicities of the bases, the reactions of alkyl 3-dialkoxyphosphoryl-3-oxopropionates with nitrogen bases proceed with retention of the C-P bond and the formation of phosphorylated azomethine derivatives or with cleavage of the C-P bond and the liberation of nitrogen-containing derivatives of malonic acid. The /sup 1/H, /sup 13/C, and /sup 13/P NMR spectra were recorded with a Bruker WP-80 NMR spectrometer. The chemical shifts of the protons and carbon atoms are presented relative to tetramethylsilane (TMS). The chemical shifts of the /sup 31/P nuclei were determined relative to H/sub 3/PO/sub 4/.

  16. Integrin β3 Crosstalk with VEGFR Accommodating Tyrosine Phosphorylation as a Regulatory Switch

    PubMed Central

    Malinin, Nikolay L.; Deshmukh, Lalit; Meller, Julia; Mahabeleshwar, Ganapati H.; Weber, Malory E.; Kerr, Bethany A.; Vinogradova, Olga; Byzova, Tatiana V.

    2012-01-01

    Integrins mediate cell adhesion, migration, and survival by connecting intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the importance of the interaction between β3 integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. Here we present in vitro evidence of the direct association between the cytoplasmic tails (CTs) of β3 and VEGFR2. Specifically, the membrane-proximal motif around 801YLSI in VEGFR2 mediates its binding to non-phosphorylated β3CT, accommodating an α-helical turn in integrin bound conformation. We also show that Y747 phosphorylation of β3 enhances the above interaction. To demonstrate the importance of β3 phosphorylation in endothelial cell functions, we synthesized β3CT-mimicking Y747 phosphorylated and unphosphorylated membrane permeable peptides. We show that a peptide containing phospho-Y747 but not F747 significantly inhibits VEGF-induced signaling and angiogenesis. Moreover, phospho-Y747 peptide exhibits inhibitory effect only in WT but not in β3 integrin knock-out or β3 integrin knock-in cells expressing β3 with two tyrosines substituted for phenylalanines, demonstrating its specificity. Importantly, these peptides have no effect on fibroblast growth factor receptor signaling. Collectively these data provide novel mechanistic insights into phosphorylation dependent cross-talk between integrin and VEGFR2. PMID:22363548

  17. Phosphorylation of vaccinia virus core proteins during transcription in vitro.

    PubMed Central

    Moussatche, N; Keller, S J

    1991-01-01

    The phosphorylation of vaccinia virus core proteins has been studied in vitro during viral transcription. The incorporation of [gamma-32P]ATP into protein is linear for the first 2 min of the reaction, whereas incorporation of [3H]UTP into RNA lags for 1 to 2 min before linear synthesis. At least 12 different proteins are phosphorylated on autoradiograms of acrylamide gels, and the majority of label is associated with low-molecular-weight proteins. If the transcription reaction is reduced by dropping the pH to 7 from its optimal of 8.5, two proteins (70 and 80 kDa) are no longer phosphorylated. RNA isolated from the pH 7 transcription reaction hybridized primarily to the vaccinia virus HindIII DNA fragments D to F, whereas the transcripts synthesized at pH 8.5 hybridized to almost all of the HindIII-digested vaccinia virus DNA fragments. The differences between the pH 7.0 and 8.5 transcription reactions in phosphorylation and transcription could be eliminated by preincubating the viral cores with 2 mM ATP. In sum, the results suggest that the phosphorylation of the 70- and 80-kDa peptides may contribute to the regulation of early transcription. Images PMID:2016772

  18. Peptide identification

    DOEpatents

    Jarman, Kristin H [Richland, WA; Cannon, William R [Richland, WA; Jarman, Kenneth D [Richland, WA; Heredia-Langner, Alejandro [Richland, WA

    2011-07-12

    Peptides are identified from a list of candidates using collision-induced dissociation tandem mass spectrometry data. A probabilistic model for the occurrence of spectral peaks corresponding to frequently observed partial peptide fragment ions is applied. As part of the identification procedure, a probability score is produced that indicates the likelihood of any given candidate being the correct match. The statistical significance of the score is known without necessarily having reference to the actual identity of the peptide. In one form of the invention, a genetic algorithm is applied to candidate peptides using an objective function that takes into account the number of shifted peaks appearing in the candidate spectrum relative to the test spectrum.

  19. Pulling peptides across nanochannels: resolving peptide binding and translocation through the hetero-oligomeric channel from Nocardia farcinica.

    PubMed

    Singh, Pratik Raj; Bárcena-Uribarri, Iván; Modi, Niraj; Kleinekathöfer, Ulrich; Benz, Roland; Winterhalter, Mathias; Mahendran, Kozhinjampara R

    2012-12-21

    We investigated translocation of cationic peptides through nanochannels derived from the Gram-positive bacterium Nocardia farcinica at the single-molecule level. The two subunits NfpA and NfpB form a hetero-oligomeric cation selective channel. On the basis of amino acid comparison we performed homology modeling and obtained a channel structurally related to MspA of Mycobacterium smegmatis. The quantitative single-molecule measurements provide an insight into transport processes of solutes through nanochannels. High-resolution ion conductance measurements in the presence of peptides of different charge and length revealed the kinetics of peptide binding. The observed asymmetry in peptide binding kinetics indicated a unidirectional channel insertion in the lipid bilayer. In the case of cationic peptides, the external voltage acts as a driving force that promotes the interaction of the peptide with the channel surface. At low voltage, the peptide just binds to the channel, whereas at higher voltage, the force is strong enough to pull the peptide across the channel. This allows distinguishing quantitatively between peptide binding and translocation through the channel. PMID:23121560

  20. The Natively Disordered Loop of Bcl-2 Undergoes Phosphorylation-Dependent Conformational Change and Interacts with Pin1

    PubMed Central

    Kang, CongBao; Bharatham, Nagakumar; Chia, Joel; Mu, Yuguang; Baek, Kwanghee; Yoon, Ho Sup

    2012-01-01

    Bcl-2 plays a central role in the regulation of apoptosis. Structural studies of Bcl-2 revealed the presence of a flexible and natively disordered loop that bridges the Bcl-2 homology motifs, BH3 and BH4. This loop is phosphorylated on multiple sites in response to a variety of external stimuli, including the microtubule-targeting drugs, paclitaxel and colchicine. Currently, the underlying molecular mechanism of Bcl-2 phosphorylation and its biological significance remain elusive. In this study, we investigated the molecular characteristics of this anti-apoptotic protein. To this end, we generated synthetic peptides derived from the Bcl-2 loop, and multiple Bcl-2 loop truncation mutants that include the phosphorylation sites. Our results demonstrate that S87 in the flexible loop of Bcl-2 is the primary phosphorylation site for JNK and ERK2, suggesting some sequence or structural specificity for the phosphorylation by these kinases. Our NMR studies and molecular dynamics simulation studies support indicate that phosphorylation of S87 induces a conformational change in the peptide. Finally, we show that the phosphorylated peptides of the Bcl-2 loop can bind Pin1, further substantiating the phosphorylation-mediated conformation change of Bcl-2. PMID:23272207

  1. HPLC analysis and purification of peptides.

    PubMed

    Mant, Colin T; Chen, Yuxin; Yan, Zhe; Popa, Traian V; Kovacs, James M; Mills, Janine B; Tripet, Brian P; Hodges, Robert S

    2007-01-01

    High-performance liquid chromatography (HPLC) has proved extremely versatile over the past 25 yr for the isolation and purification of peptides varying widely in their sources, quantity and complexity. This article covers the major modes of HPLC utilized for peptides (size-exclusion, ion-exchange, and reversed-phase), as well as demonstrating the potential of a novel mixed-mode hydrophilic interaction/cation-exchange approach developed in this laboratory. In addition to the value of these HPLC modes for peptide separations, the value of various HPLC techniques for structural characterization of peptides and proteins will be addressed, e.g., assessment of oligomerization state of peptides/proteins by size-exclusion chromatography and monitoring the hydrophilicity/hydrophobicity of amphipathic alpha-helical peptides, a vital precursor for the development of novel antimicrobial peptides. The value of capillary electrophoresis for peptide separations is also demonstrated. Preparative reversed-phase chromatography purification protocols for sample loads of up to 200 mg on analytical columns and instrumentation are introduced for both peptides and recombinant proteins. PMID:18604941

  2. A method for the 32P labeling of peptides or peptide nucleic acid oligomers

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1998-01-01

    A novel approach to the radioactive labeling of peptides and PNA oligomers is described. It is based on the conjugation of a deoxynucleoside 3'-phosphate with the terminal amine of the substrate, followed by phosphorylation of the 5'-hydroxyl group of the nucleotide using T4 polynucleotide kinase and [gamma-32P]ATP.

  3. Phosphorylation of the tumor suppressor CYLD by the breast cancer oncogene IKKε promotes cell transformation

    PubMed Central

    Hutti, Jessica E.; Shen, Rhine R.; Abbott, Derek W.; Zhou, Alicia Y.; Sprott, Kam M.; Asara, John M.; Hahn, William C.; Cantley, Lewis C.

    2009-01-01

    Summary The non-canonical IKK family member IKKε is essential for regulating anti-viral signaling pathways and is a recently-discovered breast cancer oncoprotein. Although several IKKε targets have been described, direct IKKε substrates necessary for regulating cell transformation have not been identified. Here, we performed a screen for putative IKKε substrates using an unbiased proteomic and bioinformatic approach. Using a positional scanning peptide library assay we determined the optimal phosphorylation motif for IKKε and used bioinformatic approaches to predict IKKε substrates. Of these potential substrates, serine 418 of the tumor suppressor CYLD was identified as a likely site of IKKε phosphorylation. We confirmed that CYLD is directly phosphorylated by IKKε, and that IKKε phosphorylates serine 418 in vivo. Phosphorylation of CYLD at serine 418 decreases its deubiquitinase activity and is necessary for IKKε-driven transformation. Together, these observations define IKKε and CYLD as an oncogene-tumor suppressor network that participates in tumorigenesis. PMID:19481526

  4. Tumor-promoting phorbol ester stimulates tyrosine phosphorylation in U-937 monocytes.

    PubMed Central

    Grunberger, G; Zick, Y; Taylor, S I; Gorden, P

    1984-01-01

    Solubilized lectin-purified extracts from human monocyte-like cells (U-937) and freshly isolated human mononuclear cells preincubated in the presence of phorbol 12-myristate 13-acetate (PMA) stimulated phosphorylation of synthetic tyrosine-containing polymers and of casein. Tyrosine phosphorylation was confirmed by phospho amino acid analysis. PMA stimulated phosphorylation of exogenous substrates in a time- and concentration-dependent manner. This phosphorylation reaction did not require addition of phospholipid, diolein, or calcium. Biologically inactive phorbol compounds did not stimulate phosphorylation in this system. In addition, PMA enhanced phosphorylation of a Mr approximately equal to 140,000 protein as well as several other endogenous proteins in the U-937 extracts. PMA treatment stimulated predominantly phosphorylation on tyrosine residues of the Mr 140,000 protein. Tyrosine phosphorylation, typical of growth-promoting peptides such as insulin or epidermal growth factor, is believed to play a role in regulating normal and disordered cellular growth and proliferation. The demonstration of PMA-stimulated tyrosine phosphorylation might provide a clue to the mechanism of cellular differentiation and proliferation induced by the tumor promoter. Images PMID:6201862

  5. Serine phosphorylation of CAPA pyrokinin in cockroaches-a taxon-specific posttranslational modification.

    PubMed

    Sturm, Sebastian; Predel, Reinhard

    2014-07-01

    In insects, posttranslational modifications of neuropeptides are largely restricted to C- and N-terminal amino acids. The most common modifications, N-terminal pyroglutamate formation and C-terminal α-amidation, may prevent a fast degradation of these messenger molecules. This is particularly important for peptide hormones. Other common posttranslational modifications of proteins such as glycosylation and phosphorylation seem to be very rare in insect neuropeptides. To check this assumption, we used a computer algorithm to search an extensive data set of MALDI-TOF mass spectra from cockroach tissues for ion signal patterns indicating peptide phosphorylation. The results verify that phosphorylation is indeed very rare. However, a candidate was found and experimentally verified as phosphorylated CAPA pyrokinin (GGGGpSGETSGMWFGPRL-NH2) in the cockroach Lamproblatta albipalpus (Blattidae, Lamproblattinae). Tandem mass spectrometry revealed the phosphorylation site as Ser(5). Phosphorylated CAPA pyrokinin was then also detected in most other cockroach lineages (e.g. Blaberidae, Polyphagidae) but not in closely related blattid species such as Periplaneta americana. This is remarkable since the sequence of CAPA pyrokinin is identical in Lamproblatta and Periplaneta. A consensus sequence of CAPA pyrokinins of cockroaches revealed a conserved motif that suggests phosphorylation by a Four-jointed/FAM20C related kinase. PMID:24793144

  6. Phosphorylation and Assembly of Glutamate Receptors after Brain Ischemia

    PubMed Central

    Zhang, Fan; Guo, Ailan; Liu, Chunli; Comb, Micheal; Hu, Bingren

    2012-01-01

    Background and Purpose Over-assembly of synaptic glutamate receptors leads to excitotoxicity. The goal of this study is to investigate phosphorylation and assembly of AMPA and NMDA receptors after brain ischemia with reperfusion (I/R). Methods Rats were subjected to 15 min of global ischemia followed by 0.5, 4, and 24 h of reperfusion. Phosphotyrosine (Ptyr) peptides of glutamate receptors in synaptosomal fraction after I/R were identified and quantified by state-of-the-art immuno-affinity purification of Ptyr peptides followed by LC-MS/MS analysis (IAP-LC/MS/MS). Glutamate receptor phosphorylation and synaptic assembly after I/R were studied by biochemical methods. Results Numerous Ptyr sites of AMPA and NMDA were upregulated by about 2- to 37-fold after I/R. A core glutamate receptor kinase, Src kinase, was significantly activated. GluR2/3 and NR2A/B were rapidly clustered from extrasynaptic to synaptic membrane fractions after I/R. GluR2/3 was then translocated into the intracellular pool, whereas NR2A/B remained in the synaptic fraction for as long as 24 h. Consistently, trafficking-related phosphorylation of GluR2/3-S880 was significantly but transiently upregulated, whereas NR2A/B-Y1246 and -Y1472 were significantly and persistently upregulated after I/R. Conclusions Phosphorylation of glutamate receptors at synapses may lead to over-assembly of glutamate receptors, probably via activation of Src family kinases, after I/R. This study provides “global” proteomic information about glutamate receptor tyrosine phosphorylation after brain ischemia. PMID:23212166

  7. Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.

    PubMed

    Chen, Chongguang; Chiu, Yi-Ting; Wu, Wenman; Huang, Peng; Mann, Anika; Schulz, Stefan; Liu-Chen, Lee-Yuan

    2016-02-15

    Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated

  8. An Extensive Survey of Tyrosine Phosphorylation Revealing New Sites in Human Mammary Epithelial Cells

    SciTech Connect

    Heibeck, Tyler H.; Ding, Shi-Jian; Opresko, Lee K.; Zhao, Rui; Schepmoes, Athena A.; Yang, Feng; Tolmachev, Aleksey V.; Monroe, Matthew E.; Camp, David G.; Smith, Richard D.; Wiley, H. S.; Qian, Weijun

    2009-08-01

    Protein tyrosine phosphorylation is a central regulatory mechanism in cell signaling. To extensively characterize the site-specific tyrosine phosphorylation in human cells, we present here a global survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell (HMEC) line by applying anti-phosphotyrosine (pTyr) peptide immunoaffinity purification (IP) coupled with high sensitivity LC-MS/MS. A total of 481 tyrosine phosphorylation sites (covered by 716 unique peptides) from 285 proteins were confidently identified in HMEC following the analysis of both the basal condition and an acute stimulated condition with epidermal growth factor (EGF). The estimated false discovery rate is 1.0% as measured by comparison against a scrambled database search. Comparison of these data to the literature showed significant agreement in site matches. Additionally 281 sites were not previously observed in HMEC culture were found. Twenty-nine of these sites have not been reported in any human cell or tissue system. The global profiling also allowed us to examine the phosphorylation stoichiometry differences based on spectral count information. Comparison of the data to a previous global proteome profiling study illustrates that most of the highly phoshorylated proteins are of relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed for many of the identified proteins, suggesting potentially more important functional roles for those highly phosphorylated pTyr sites within a given protein. By mapping to major signaling networks such as EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which should allow us to select interesting targeted involved in a given pathway for more directed studies. This extensive HMEC tyrosine phosphorylation dataset represents an important database

  9. Structural basis of how stress-induced MDMX phosphorylation activates p53.

    PubMed

    Chen, X; Gohain, N; Zhan, C; Lu, W-Y; Pazgier, M; Lu, W

    2016-04-14

    The tumor-suppressor protein p53 is tightly controlled in normal cells by its two negative regulators--the E3 ubiquitin ligase MDM2 and its homolog MDMX. Under stressed conditions such as DNA damage, p53 escapes MDM2- and MDMX-mediated functional inhibition and degradation, acting to prevent damaged cells from proliferating through induction of cell cycle arrest, DNA repair, senescence or apoptosis. Ample evidence suggests that stress signals induce phosphorylation of MDM2 and MDMX, leading to p53 activation. However, the structural basis of stress-induced p53 activation remains poorly understood because of the paucity of technical means to produce site-specifically phosphorylated MDM2 and MDMX proteins for biochemical and biophysical studies. Herein, we report total chemical synthesis, via native chemical ligation, and functional characterization of (24-108)MDMX and its Tyr99-phosphorylated analog with respect to their ability to interact with a panel of p53-derived peptide ligands and PMI, a p53-mimicking but more potent peptide antagonist of MDMX, using FP and surface plasmon resonance techniques. Phosphorylation of MDMX at Tyr99 weakens peptide binding by approximately two orders of magnitude. Comparative X-ray crystallographic analyses of MDMX and of pTyr99 MDMX in complex with PMI as well as modeling studies reveal that the phosphate group of pTyr99 imposes extensive steric clashes with the C-terminus of PMI or p53 peptide and induces a significant lateral shift of the peptide ligand, contributing to the dramatic decrease in the binding affinity of MDMX for p53. Because DNA damage activates c-Abl tyrosine kinase that phosphorylates MDMX at Tyr99, our findings afford a rare glimpse at the structural level of how stress-induced MDMX phosphorylation dislodges p53 from the inhibitory complex and activates it in response to DNA damage. PMID:26148237

  10. A phosphotyrosine switch regulates organic cation transporters

    PubMed Central

    Sprowl, Jason A.; Ong, Su Sien; Gibson, Alice A.; Hu, Shuiying; Du, Guoqing; Lin, Wenwei; Li, Lie; Bharill, Shashank; Ness, Rachel A.; Stecula, Adrian; Offer, Steven M.; Diasio, Robert B.; Nies, Anne T.; Schwab, Matthias; Cavaletti, Guido; Schlatter, Eberhard; Ciarimboli, Giuliano; Schellens, Jan H. M.; Isacoff, Ehud Y.; Sali, Andrej; Chen, Taosheng; Baker, Sharyn D.; Sparreboom, Alex; Pabla, Navjotsingh

    2016-01-01

    Membrane transporters are key determinants of therapeutic outcomes. They regulate systemic and cellular drug levels influencing efficacy as well as toxicities. Here we report a unique phosphorylation-dependent interaction between drug transporters and tyrosine kinase inhibitors (TKIs), which has uncovered widespread phosphotyrosine-mediated regulation of drug transporters. We initially found that organic cation transporters (OCTs), uptake carriers of metformin and oxaliplatin, were inhibited by several clinically used TKIs. Mechanistic studies showed that these TKIs inhibit the Src family kinase Yes1, which was found to be essential for OCT2 tyrosine phosphorylation and function. Yes1 inhibition in vivo diminished OCT2 activity, significantly mitigating oxaliplatin-induced acute sensory neuropathy. Along with OCT2, other SLC-family drug transporters are potentially part of an extensive ‘transporter-phosphoproteome' with unique susceptibility to TKIs. On the basis of these findings we propose that TKIs, an important and rapidly expanding class of therapeutics, can functionally modulate pharmacologically important proteins by inhibiting protein kinases essential for their post-translational regulation. PMID:26979622

  11. TARP phosphorylation regulates synaptic AMPA receptors through lipid bilayers

    PubMed Central

    Sumioka, Akio; Yan, Dan; Tomita, Susumu

    2010-01-01

    Summary Neurons use neurotransmitters to communicate across synapses, constructing neural circuits in the brain. AMPA-type glutamate receptors are the predominant excitatory neurotransmitter receptors mediating fast synaptic transmission. AMPA receptors localize at synapses by forming protein complexes with transmembrane AMPA receptor regulatory proteins (TARPs) and PSD-95-like MAGUKs. Among the three classes of ionotropic glutamate receptors (AMPA-, NMDA, kainate-type), AMPA receptor activity is most regulatable by neuronal activity to adjust synaptic strength. Here, we mutated the prototypical TARP, stargazin, and found that TARP phosphorylation regulates synaptic AMPA receptor activity in vivo. We also found that stargazin interacts with negatively-charged lipid bilayers in its phosphorylation dependent manner, and that the lipid interaction inhibited stargazin binding to PSD-95. Cationic lipids dissociated stargazin from lipid bilayers and enhanced synaptic AMPA receptor activity in a stargazin phosphorylation-dependent manner. Thus, TARP phosphorylation plays a critical role in regulating AMPA receptor-mediated synaptic transmission via a lipid bilayer interaction. PMID:20547132

  12. Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition

    PubMed Central

    Chang, Ching Ching; Few, Ling Ling; Konrad, Manfred; See Too, Wei Cun

    2016-01-01

    Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis. PMID:27149373

  13. Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry.

    PubMed Central

    Neville, D. C.; Rozanas, C. R.; Price, E. M.; Gruis, D. B.; Verkman, A. S.; Townsend, R. R.

    1997-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an apical membrane Cl- channel regulated by protein phosphorylation. To identify cAMP-dependent protein kinase (PKA)-phosphorylated residues in full-length CFTR, immobilized metal-ion affinity chromatography (IMAC) was used to selectively purify phosphopeptides. The greater specificity of iron-loaded (Fe3+) nitrilotriacetic (NTA). Sepharose compared to iminodiacetic acid (IDA) metal-chelating matrices was demonstrated using a PKA-phosphorylated recombinant NBD1-R protein from CFTR. Fe(3+)-loaded NTA Sepharose preferentially bound phosphopeptides, whereas acidic and poly-His-containing peptides were co-purified using the conventional IDA matrices. IMAC using NTA Sepharose enabled the selective recovery of phosphopeptides and identification of phosphorylated residues from a complex proteolytic digest. Phosphopeptides from PKA-phosphorylated full-length CFTR, generated in Hi5 insect cells using a baculovirus expression system, were purified using NTA Sepharose. Phosphopeptides were identified using matrix-assisted laser desorption mass spectrometry (MALDI/MS) with post-source decay (PSD) analysis and collision-induced dissociation (CID) experiments. Phosphorylated peptides were identified by mass and by the metastable loss of HPO3 and H3PO4 from the parent ions. Peptide sequence and phosphorylation at CFTR residues 660Ser, 737Ser, and 795Ser were confirmed using MALDI/PSD analysis. Peptide sequences and phosphorylation at CFTR residues 700Ser, 712Ser, 768Ser, and 813Ser were deduced from peptide mass, metastable fragment ion formation, and PKA consensus sequences. Peptide sequence and phosphorylation at residue 753Ser was confirmed using MALDI/CID analysis. This is the first report of phosphorylation of 753Ser in full-length CFTR. PMID:9385646

  14. Oxidative and Photosynthetic Phosphorylation Mechanisms

    ERIC Educational Resources Information Center

    Wang, Jui H.

    1970-01-01

    Proposes a molecular mechanism for the coupling of phosphorylation to electron transport in both mitochondria and chloroplasts. Justifies the proposed reaction schemes in terms of thermodynamics and biochemical data. Suggests how areobic respiration could have evolved. (EB)

  15. Hydrophobic peptide auxotrophy in Salmonella typhimurium.

    PubMed Central

    Brãnes, L V; Somers, J M; Kay, W W

    1981-01-01

    The growth of a pleiotropic membrane mutant of Salmonella typhimurium with modified lipopolysaccharide composition was found to be strictly dependent on the peptone component of complex media. Nutritional Shiftdown into minimal media allowed growth for three to four generations. Of 20 commercial peptones, only enzymatic digests supported growth to varying degrees. Neither trace cations, amino acids, vitamins, carbohydrates, lipids, glutathione, polyamines, carbodimides, nor synthetic peptides stimulated growth; however, cells still metabolized carbohydrates, and amino acid transport systems were shown to be functional. A tryptic digest of casein was fractionated into four electrophoretically different peptide fractions of 1,000 to 1,200 molecular weight which supported growth to varying degrees. The best of these was further fractionated to two highly hydrophopic peptides. N-terminal modifications eliminated biological activity. Fluorescein-conjugated goat antibody to rabbit immunoglobulin G was used as a probe to detect antipeptide antibody-peptide complexes on membrane preparations. Cells grown on peptone distributed the peptide into both inner and outer membranes. The peptide could be removed with chaotropic agents, and cells had to be pregrown in peptone-containing media to bind the hydrophobic peptide. The gene (hyp) responsible for peptide auxotrophy was mapped at 44 to 45 units by conjugation. Images PMID:7024254

  16. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding

    PubMed Central

    Ting, Pamela Y.; Johnson, Christian W.; Fang, Cong; Cao, Xiaoqing; Graeber, Thomas G.; Mattos, Carla; Colicelli, John

    2015-01-01

    RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr137. Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr137 phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr137 is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRASY137F and HRASY137E revealed conformation changes radiating from the mutated residue. Although consistent with Tyr137 participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr137 phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRASG12V with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr137 allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling.—Ting, P. Y., Johnson, C. W., Fang, C., Cao, X., Graeber, T. G., Mattos, C., Colicelli, J. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding. PMID:25999467

  17. The self-assembly ability of First microtubule-binding repeat from tau and its modulation by phosphorylation

    SciTech Connect

    Zhou Lianxiu; Zeng Zhiyang; Du Jintang; Zhao Yufen; Li Yanmei . E-mail: liym@mail.tsinghua.edu.cn

    2006-09-22

    Aggregation of abnormally phosphorylated tau in the form of tangs of paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease (AD) and other tauopathies. It is of fundamental importance to study the mechanism of PHF formation and its modulation by phosphorylation. In this work, we have focused on First microtubule-binding repeat of tau encompassing an abnormal phosphorylation site Ser{sup 262}. The assembly propensities of this repeat and its corresponding phosphorylated form were investigated by turbidity and electron microscopy. Additionally, conformation of the two peptides is also analyzed through circular dichroism (CD) and NMR spectroscopy. Our results reveal that both of them are capable of self-assembly and phosphorylation at Ser{sup 262} could speed up the process of assembly. A possible mechanism of PHF formation is proposed and enhancing effect of phosphorylation on assembly provides an explanation to its toxicity in Alzheimer's disease.

  18. Simultaneous Identification of Tyrosine Phosphorylation and Sulfation Sites Utilizing Tyrosine-Specific Bromination

    NASA Astrophysics Data System (ADS)

    Kim, Jong-Seo; Song, Si-Uk; Kim, Hie-Joon

    2011-11-01

    Tyrosine phosphorylation and sulfation play many key roles in the cell. Isobaric phosphotyrosine and sulfotyrosine residues in peptides were determined by mass spectrometry using phosphatase or sulfatase to remove the phosphate or the sulfate group. Unique Br signature was introduced to the resulting tyrosine residues by incubation with 32% HBr at -20 °C for 20 min. MS/MS analysis of the brominated peptide enabled unambiguous determination of the phosphotyrosine and the sulfotyrosine sites. When phosphotyrosine and sulfotyrosine as well as free tyrosine were present in the same peptide, they could be determined simultaneously using either phosphatase or sulfatase following acetylation of the free tyrosine.

  19. MLKL forms cation channels

    PubMed Central

    Xia, Bingqing; Fang, Sui; Chen, Xueqin; Hu, Hong; Chen, Peiyuan; Wang, Huayi; Gao, Zhaobing

    2016-01-01

    The mixed lineage kinase domain-like (MLKL) protein is a key factor in tumor necrosis factor-induced necroptosis. Recent studies on necroptosis execution revealed a commitment role of MLKL in membrane disruption. However, our knowledge of how MLKL functions on membrane remains very limited. Here we demonstrate that MLKL forms cation channels that are permeable preferentially to Mg2+ rather than Ca2+ in the presence of Na+ and K+. Moreover, the N-terminal domain containing six helices (H1-H6) is sufficient to form channels. Using the substituted cysteine accessibility method, we further determine that helix H1, H2, H3, H5 and H6 are transmembrane segments, while H4 is located in the cytoplasm. Finally, MLKL-induced membrane depolarization and cell death exhibit a positive correlation to its channel activity. The Mg2+-preferred permeability and five transmembrane segment topology distinguish MLKL from previously identified Mg2+-permeable channels and thus establish MLKL as a novel class of cation channels. PMID:27033670

  20. Peptide library approach to uncover phosphomimetic inhibitors of the BRCA1 C-terminal domain.

    PubMed

    White, E Railey; Sun, Luxin; Ma, Zhong; Beckta, Jason M; Danzig, Brittany A; Hacker, David E; Huie, Melissa; Williams, David C; Edwards, Ross A; Valerie, Kristoffer; Glover, J N Mark; Hartman, Matthew C T

    2015-05-15

    Many intracellular protein-protein interactions are mediated by the phosphorylation of serine, and phosphoserine-containing peptides can inhibit these interactions. However, hydrolysis of the phosphate by phosphatases, and the poor cell permeability associated with phosphorylated peptides has limited their utility in cellular and in vivo contexts. Compounding the problem, strategies to replace phosphoserine in peptide inhibitors with easily accessible mimetics (such as Glu or Asp) routinely fail. Here, we present an in vitro selection strategy for replacement of phosphoserine. Using mRNA display, we created a 10 trillion member structurally diverse unnatural peptide library. From this library, we found a peptide that specifically binds to the C-terminal domain (BRCT)2 of breast cancer associated protein 1 (BRCA1) with an affinity comparable to phosphorylated peptides. A crystal structure of the peptide bound reveals that the pSer-x-x-Phe motif normally found in BRCA1 (BRCT)2 binding partners is replaced by a Glu-x-x-4-fluoroPhe and that the peptide picks up additional contacts on the protein surface not observed in cognate phosphopeptide binding. Expression of the peptide in human cells led to defects in DNA repair by homologous recombination, a process BRCA1 is known to coordinate. Overall, this work validates a new in vitro selection approach for the development of inhibitors of protein-protein interactions mediated by serine phosphorylation. PMID:25654734

  1. Peptide Library Approach to Uncover Phosphomimetic Inhibitors of the BRCA1 C-Terminal Domain

    PubMed Central

    White, E. Railey; Sun, Luxin; Ma, Zhong; Beckta, Jason M.; Danzig, Brittany A.; Hacker, David E.; Huie, Melissa; Williams, David C.; Edwards, Ross A.; Valerie, Kristoffer; Mark Glover, J. N.; Hartman, Matthew C. T.

    2015-01-01

    Many intracellular protein–protein interactions are mediated by the phosphorylation of serine, and phosphoserine-containing peptides can inhibit these interactions. However, hydrolysis of the phosphate by phosphatases, and the poor cell permeability associated with phosphorylated peptides has limited their utility in cellular and in vivo contexts. Compounding the problem, strategies to replace phosphoserine in peptide inhibitors with easily accessible mimetics (such as Glu or Asp) routinely fail. Here, we present an in vitro selection strategy for replacement of phosphoserine. Using mRNA display, we created a 10 trillion member structurally diverse unnatural peptide library. From this library, we found a peptide that specifically binds to the C-terminal domain (BRCT)2 of breast cancer associated protein 1 (BRCA1) with an affinity comparable to phosphorylated peptides. A crystal structure of the peptide bound reveals that the pSer-x-x-Phe motif normally found in BRCA1 (BRCT)2 binding partners is replaced by a Glu-x-x-4-fluoroPhe and that the peptide picks up additional contacts on the protein surface not observed in cognate phosphopeptide binding. Expression of the peptide in human cells led to defects in DNA repair by homologous recombination, a process BRCA1 is known to coordinate. Overall, this work validates a new in vitro selection approach for the development of inhibitors of protein–protein interactions mediated by serine phosphorylation. PMID:25654734

  2. Phosphorylation sites of the B2 chain of bovine alpha-crystallin

    SciTech Connect

    Chiesa, R.; Gawinowicz-Kolks, M.A.; Kleiman, N.J.; Spector, A.

    1987-05-14

    The B2 chain of bovine lens alpha-crystallin is phosphorylated in a cAMP-dependent reaction. By analysis of /sup 32/P-labelled chymotryptic peptides isolated from alpha-crystallin obtained from lenses labelled in organ culture, two phosphorylated B2 chain fragments were found. Sequence analysis of the fragments gave the following results: Arg-Ala-Pro-Ser-Trp-Ile-Asp-Thr-Gly-Leu and Ser-Leu-Ser-Pro-Phe corresponding to residues 56 to 65 and 43 to 47, respectively. It is established by this work that B1 is a phosphorylated post-translational product of B2. Both the A2 and B2 chains of alpha-crystallin are phosphorylated at a similar site with the sequence Arg-(X)-Pro-Ser. This is an unusual site for cAMP-phosphorylation since the phosphorylated serine is preceded by a proline residue. It may also be of significance that the other B2 chain phosphorylation site even more radically differs from previously reported cAMP-dependent phosphorylation sites.

  3. Conformational selection in the recognition of phosphorylated substrates by the catalytic domain of human Pin1.

    PubMed

    Velazquez, Hector A; Hamelberg, Donald

    2011-11-01

    Post-translational phosphorylation and the related conformational changes in signaling proteins are responsible for regulating a wide range of subcellular processes. Human Pin1 is central to many of these cell signaling pathways in normal and aberrant subcellular processes, catalyzing cis-trans isomerization of the peptide ω-bond in phosphorylated serine/threonine-proline motifs in many proteins. Pin1 has therefore been identified as a possible drug target in many diseases, including cancer and Alzheimer's. The effects of phosphorylation on Pin1 substrates, and the atomistic basis for Pin1 recognition and catalysis, are not well understood. Here, we determine the conformational consequences of phosphorylation on Pin1 substrate analogues and the mechanism of recognition by the catalytic domain of Pin1 using all-atom molecular dynamics simulations. We show that phosphorylation induces backbone conformational changes on the peptide substrate analogues. We also show that Pin1 recognizes specific conformations of its substrate by conformational selection. Furthermore, dynamical correlated motions in the free Pin1 enzyme are present in the enzyme of the enzyme-substrate complex when the substrate is in the transition state configuration, suggesting that these motions play significant roles during catalytic turnover. These results provide a detailed atomistic picture of the mechanism of Pin1 recognition that can be exploited for drug design purposes and further our understanding of the synergistic complexities of post-translational phosphorylation and cis-trans isomerization. PMID:21967280

  4. Functional phosphoproteomic profiling of phosphorylation sites in membrane fractions of salt-stressed Arabidopsis thaliana

    PubMed Central

    2009-01-01

    Background Under conditions of salt stress, plants respond by initiating phosphorylation cascades. Many key phosphorylation events occur at the membrane. However, to date only limited sites have been identified that are phosphorylated in response to salt stress in plants. Results Membrane fractions from three-day and 200 mM salt-treated Arabidopsis suspension plants were isolated, followed by protease shaving and enrichment using Zirconium ion-charged magnetic beads, and tandem mass spectrometry analyses. From this isolation, 18 phosphorylation sites from 15 Arabidopsis proteins were identified. A unique phosphorylation site in 14-3-3-interacting protein AHA1 was predominately identified in 200 mM salt-treated plants. We also identified some phosphorylation sites in aquaporins. A doubly phosphorylated peptide of PIP2;1 as well as a phosphopeptide containing a single phosphorylation site (Ser-283) and a phosphopeptide containing another site (Ser-286) of aquaporin PIP2;4 were identified respectively. These two sites appeared to be novel of which were not reported before. In addition, quantitative analyses of protein phosphorylation with either label-free or stable-isotope labeling were also employed in this study. The results indicated that level of phosphopeptides on five membrane proteins such as AHA1, STP1, Patellin-2, probable inactive receptor kinase (At3g02880), and probable purine permease 18 showed at least two-fold increase in comparison to control in response to 200 mM salt-stress. Conclusion In this study, we successfully identified novel salt stress-responsive protein phosphorylation sites from membrane isolates of abiotic-stressed plants by membrane shaving followed by Zr4+-IMAC enrichment. The identified phosphorylation sites can be important in the salt stress response in plants. PMID:19900291

  5. OGlcNAcylation and Phosphorylation Have Opposing Structural Effects in tau: Phosphothreonine Induces Particular Conformational Order

    PubMed Central

    2015-01-01

    Phosphorylation and OGlcNAcylation are dynamic intracellular protein post-translational modifications that frequently are alternatively observed on the same serine and threonine residues. Phosphorylation and OGlcNAcylation commonly occur in natively disordered regions of proteins, and often have opposing functional effects. In the microtubule-associated protein tau, hyperphosphorylation is associated with protein misfolding and aggregation as the neurofibrillary tangles of Alzheimer’s disease, whereas OGlcNAcylation stabilizes the soluble form of tau. A series of peptides derived from the proline-rich domain (residues 174–251) of tau was synthesized, with free Ser/Thr hydroxyls, phosphorylated Ser/Thr (pSer/pThr), OGlcNAcylated Ser/Thr, and diethylphosphorylated Ser/Thr. Phosphorylation and OGlcNAcylation were found by CD and NMR to have opposing structural effects on polyproline helix (PPII) formation, with phosphorylation favoring PPII, OGlcNAcylation opposing PPII, and the free hydroxyls intermediate in structure, and with phosphorylation structural effects greater than OGlcNAcylation. For tau196–209, phosphorylation and OGlcNAcylation had similar structural effects, opposing a nascent α-helix. Phosphomimic Glu exhibited PPII-favoring structural effects. Structural changes due to Thr phosphorylation were greater than those of Ser phosphorylation or Glu, with particular conformational restriction as the dianion, with mean 3JαN = 3.5 Hz (pThr) versus 5.4 Hz (pSer), compared to 7.2, 6.8, and 6.2 Hz for Thr, Ser, and Glu, respectively, values that correlate with the backbone torsion angle ϕ. Dianionic phosphothreonine induced strong phosphothreonine amide protection and downfield amide chemical shifts (δmean = 9.63 ppm), consistent with formation of a stable phosphate-amide hydrogen bond. These data suggest potentially greater structural importance of threonine phosphorylation than serine phosphorylation due to larger induced structural effects. PMID

  6. Protein phosphorylation in chloroplasts - a survey of phosphorylation targets.

    PubMed

    Baginsky, Sacha

    2016-06-01

    The development of new software tools, improved mass spectrometry equipment, a suite of optimized scan types, and better-quality phosphopeptide affinity capture have paved the way for an explosion of mass spectrometry data on phosphopeptides. Because phosphoproteomics achieves good sensitivity, most studies use complete cell extracts for phosphopeptide enrichment and identification without prior enrichment of proteins or subcellular compartments. As a consequence, the phosphoproteome of cell organelles often comes as a by-product from large-scale studies and is commonly assembled from these in meta-analyses. This review aims at providing some guidance on the limitations of meta-analyses that combine data from analyses with different scopes, reports on the current status of knowledge on chloroplast phosphorylation targets, provides initial insights into phosphorylation site conservation in different plant species, and highlights emerging information on the integration of gene expression with metabolism and photosynthesis by means of protein phosphorylation. PMID:26969742

  7. Cationic Antimicrobial Polymers and Their Assemblies

    PubMed Central

    Carmona-Ribeiro, Ana Maria; de Melo Carrasco, Letícia Dias

    2013-01-01

    Cationic compounds are promising candidates for development of antimicrobial agents. Positive charges attached to surfaces, particles, polymers, peptides or bilayers have been used as antimicrobial agents by themselves or in sophisticated formulations. The main positively charged moieties in these natural or synthetic structures are quaternary ammonium groups, resulting in quaternary ammonium compounds (QACs). The advantage of amphiphilic cationic polymers when compared to small amphiphilic molecules is their enhanced microbicidal activity. Besides, many of these polymeric structures also show low toxicity to human cells; a major requirement for biomedical applications. Determination of the specific elements in polymers, which affect their antimicrobial activity, has been previously difficult due to broad molecular weight distributions and random sequences characteristic of radical polymerization. With the advances in polymerization control, selection of well defined polymers and structures are allowing greater insight into their structure-antimicrobial activity relationship. On the other hand, antimicrobial polymers grafted or self-assembled to inert or non inert vehicles can yield hybrid antimicrobial nanostructures or films, which can act as antimicrobials by themselves or deliver bioactive molecules for a variety of applications, such as wound dressing, photodynamic antimicrobial therapy, food packing and preservation and antifouling applications. PMID:23665898

  8. Cell Penetrating Peptide-Mediated Caveolae-Dependent Activation of Lung Endothelial Nitric Oxide Synthase.

    PubMed

    Hutchinson, Tarun E; Hu, Hanbo; Patel, Jawaharlal M

    2016-01-01

    Cell penetrating peptides can be used as therapeutic agents via modulation of selective cell functions. Nitric oxide (NO) generated by vascular endothelial NO synthase (eNOS) plays a critical role in the NO/ cyclic guanosine 5'-monophosphate (cGMP)-mediated pulmonary vascular function. Here we examined whether internalization of a fifteen amino acid (KRFNSISCSSWRRKR) synthetic peptide (P3) enhances the catalytic activity of eNOS via caveolae/eNOS dissociation leading to NO release and increased cGMP production in pulmonary artery endothelial cells (EC). ECs were treated with varying concentrations of P3 and used to monitor internalization, isolation of caveolae-enriched fraction, the catalytic activity of eNOS, NO/cGMP production, and intracellular Ca(2+) release. Confocal images show timedependent internalization of P3 in EC. Treatment of EC with P3, but not scrambled P3, increased the catalytic activity of eNOS in a dose-dependent manner without change in eNOS expression or phosphorylation. Treatment of EC with P3 stimulated intracellular Ca(2+) release, increased the catalytic activity of phospatidylinsositide 3 kinase (PI3K) and resulted in eNOS/caveolae-1 (Cav-1) dissociation leading to translocation of eNOS to intracellular compartment in EC. P3- mediated activation of eNOS was abolished by intracellular Ca(2+) chelator 1,2-bis(2-aminophenooxy)ethane-N,N,N',N'- tertraacetic acid-AM (BAPTA-AM), PI3K inhibition, or by siRNA-mediated Cav-1 suppression. These results demonstrate that exogenous peptide consisting of cationic amino acids can internalize and enhance the catalytic activity of eNOS via modulation of caveolar signaling and intracellular Ca(2+) release in EC. PMID:26655728

  9. Inhibition of CFTR channels by a peptide toxin of scorpion venom.

    PubMed

    Fuller, Matthew D; Zhang, Zhi-Ren; Cui, Guiying; Kubanek, Julia; McCarty, Nael A

    2004-11-01

    Peptide toxins have been valuable probes in efforts to identify amino acid residues that line the permeation pathway of cation-selective channels. However, no peptide toxins have been identified that interact with known anion-selective channels such as the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR channels are expressed in epithelial cells and are associated with several genetic disorders, including cystic fibrosis and polycystic kidney disease. Several organic inhibitors have been used to investigate the structure of the Cl- permeation pathway in CFTR. However, investigations of the wider cytoplasmic vestibule have been hindered by the lack of a high-affinity blocker that interacts with residues in this area. In this study we show that venom of the scorpion Leiurus quinquestriatus hebraeus reversibly inhibits CFTR, in a voltage-independent manner, by decreasing single-channel mean burst duration and open probability only when applied to the cytoplasmic surface of phosphorylated channels. Venom was able to decrease burst duration and open probability even when CFTR channels were locked open by treatment with either vanadate or adenosine 5'-(beta,gamma-imido)triphosphate, and block was strengthened on reduction of extracellular Cl- concentration, suggesting inhibition by a pore-block mechanism. Venom had no effect on ATP-dependent macroscopic opening rate in channels studied by inside-out macropatches. Interestingly, the inhibitory activity was abolished by proteinase treatment. We conclude that a peptide toxin contained in the scorpion venom inhibits CFTR channels by a pore-block mechanism; these experiments provide the first step toward isolation of the active component, which would be highly valuable as a probe for CFTR structure and function. PMID:15240343

  10. Interphase phosphorylation of lamin A.

    PubMed

    Kochin, Vitaly; Shimi, Takeshi; Torvaldson, Elin; Adam, Stephen A; Goldman, Anne; Pack, Chan-Gi; Melo-Cardenas, Johanna; Imanishi, Susumu Y; Goldman, Robert D; Eriksson, John E

    2014-06-15

    Nuclear lamins form the major structural elements that comprise the nuclear lamina. Loss of nuclear structural integrity has been implicated as a key factor in the lamin A/C gene mutations that cause laminopathies, whereas the normal regulation of lamin A assembly and organization in interphase cells is still undefined. We assumed phosphorylation to be a major determinant, identifying 20 prime interphase phosphorylation sites, of which eight were high-turnover sites. We examined the roles of these latter sites by site-directed mutagenesis, followed by detailed microscopic analysis - including fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and nuclear extraction techniques. The results reveal three phosphorylation regions, each with dominant sites, together controlling lamin A structure and dynamics. Interestingly, two of these interphase sites are hyper-phosphorylated in mitotic cells and one of these sites is within the sequence that is missing in progerin of the Hutchinson-Gilford progeria syndrome. We present a model where different phosphorylation combinations yield markedly different effects on the assembly, subunit turnover and the mobility of lamin A between, and within, the lamina, the nucleoplasm and the cytoplasm of interphase cells. PMID:24741066

  11. Ligand-induced IFN gamma receptor tyrosine phosphorylation couples the receptor to its signal transduction system (p91).

    PubMed Central

    Greenlund, A C; Farrar, M A; Viviano, B L; Schreiber, R D

    1994-01-01

    Herein we report that interferon-gamma (IFN gamma) induces the rapid and reversible tyrosine phosphorylation of the IFN gamma receptor. Using a panel of receptor intracellular domain mutants, we show that a membrane-proximal LPKS sequence (residues 266-269) is required for ligand-induced tyrosine kinase activation and/or kinase-receptor association and biological responsiveness, and a functionally critical membrane-distal tyrosine residue (Y440) is a target of the activated enzyme. The biological significance of Y440 phosphorylation was demonstrated by showing that a receptor-derived nonapeptide corresponding to receptor residues 436-444 and containing phosphorylated Y440 bound specifically to p91, blocked p91 phosphorylation and inhibited the generation of an active p91-containing transcription factor complex. In contrast, nonphosphorylated wild-type, phosphorylated mutant, or phosphorylated irrelevant peptides did not. Moreover, the phosphorylated Y440-containing peptide did not interact with a related but distinct latent transcription factor (p113) which is activatible by IFN alpha but not IFN gamma. These results thus document the specific and inducible association of p91 with the phosphorylated IFN gamma receptor and thereby elucidate the mechanism by which ligand couples the IFN gamma receptor to its signal transduction system. Images PMID:8156998

  12. Role of amphipathicity and hydrophobicity in the balance between hemolysis and peptide-membrane interactions of three related antimicrobial peptides.

    PubMed

    Hollmann, Axel; Martínez, Melina; Noguera, Martín E; Augusto, Marcelo T; Disalvo, Anibal; Santos, Nuno C; Semorile, Liliana; Maffía, Paulo C

    2016-05-01

    Cationic antimicrobial peptides (CAMPs) represent important self defense molecules in many organisms, including humans. These peptides have a broad spectrum of activities, killing or neutralizing many Gram-negative and Gram-positive bacteria. The emergence of multidrug resistant microbes has stimulated research on the development of alternative antibiotics. In the search for new antibiotics, cationic antimicrobial peptides (CAMPs) offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to lysis of microbial membranes and eventually cell death. In particular, the group of linear α-helical cationic peptides has attracted increasing interest from clinical as well as basic research during the last decade. In this work, we studied the biophysical and microbiological characteristics of three new designed CAMPs. We modified a previously studied CAMP sequence, in order to increase or diminish the hydrophobic face, changing the position of two lysines or replacing three leucines, respectively. These mutations modified the hydrophobic moment of the resulting peptides and allowed us to study the importance of this parameter in the membrane interactions of the peptides. The structural properties of the peptides were also correlated with their membrane-disruptive abilities, antimicrobial activities and hemolysis of human red blood cells. PMID:26896660

  13. Cationic polymers and their self-assembly for antibacterial applications.

    PubMed

    Deka, Smriti Rekha; Sharma, Ashwani Kumar; Kumar, Pradee

    2015-01-01

    The present article focuses on the amphiphilic cationic polymers as antibacterial agents. These polymers undergo self-assembly in aqueous conditions and impart biological activity by efficiently interacting with the bacterial cell wall, hence, used in preparing chemical disinfectants and biocides. Both cationic charge as well as hydrophobic segments facilitate interactions with the bacterial cell surface and initiate its disruption. The perturbation in transmembrane potential causes leakage of cytosolic contents followed by cell death. Out of two categories of macromolecules, peptide oligomers and cationic polymers, which have extensively been used as antibacterials, we have elaborated on the current advances made in the area of cationic polymer-based (naturally occurring and commonly employed synthetic polymers and their modified analogs) antibacterial agents. The development of polymer-based antibacterials has helped in addressing challenges posed by the drug-resistant bacterial infections. These polymers provide a new platform to combat such infections in the most efficient manner. This review presents concise discussion on the amphiphilic cationic polymers and their modified analogs having low hemolytic activity and excellent antibacterial activity against array of fungi, bacteria and other microorganisms. PMID:25858132

  14. Fe65 Is Phosphorylated on Ser289 after UV-Induced DNA Damage

    PubMed Central

    Langlands, Hannah; Blain, Peter G.; Jowsey, Paul A.

    2016-01-01

    Fe65 undergoes a phosphatase-sensitive gel mobility shift after DNA damage, consistent with protein phosphorylation. A recent study identified Ser228 as a specific site of phosphorylation, targeted by the ATM and ATR protein kinases, with phosphorylation inhibiting the Fe65-dependent transcriptional activity of the amyloid precursor protein (APP). The direct binding of Fe65 to APP not only regulates target gene expression, but also contributes to secretase-mediated processing of APP, producing cytoactive proteolytic fragments including the APP intracellular domain (AICD) and cytotoxic amyloid β (Aβ) peptides. Given that the accumulation of Aβ peptides in neural plaques is a pathological feature of Alzheimer’s disease (AD), it is essential to understand the mechanisms controlling Aβ production. This will aid in the development of potential therapeutic agents that act to limit the deleterious production of Aβ peptides. The Fe65-APP complex has transcriptional activity and the complex is regulated by multiple post-translational modifications and other protein binding partners. In the present study, we have identified Ser289 as a novel site of UV-induced phosphorylation. Interestingly, this phosphorylation was mediated by ATM, rather than ATR, and occurred independently of APP. Neither phosphorylation nor mutation of Ser289 affected the Fe65-APP interaction, though this was markedly decreased after UV treatment, with a concomitant decrease in the protein levels of APP in cells. Using mutagenesis, we demonstrated that Fe65 Ser289 phosphorylation did not affect the transcriptional activity of the Fe65-APP complex, in contrast to the previously described Ser228 site. PMID:27176072

  15. Antimicrobial peptides.

    PubMed

    Zhang, Ling-Juan; Gallo, Richard L

    2016-01-11

    Antimicrobial peptides and proteins (AMPs) are a diverse class of naturally occurring molecules that are produced as a first line of defense by all multicellular organisms. These proteins can have broad activity to directly kill bacteria, yeasts, fungi, viruses and even cancer cells. Insects and plants primarily deploy AMPs as an antibiotic to protect against potential pathogenic microbes, but microbes also produce AMPs to defend their environmental niche. In higher eukaryotic organisms, AMPs can also be referred to as 'host defense peptides', emphasizing their additional immunomodulatory activities. These activities are diverse, specific to the type of AMP, and include a variety of cytokine and growth factor-like effects that are relevant to normal immune homeostasis. In some instances, the inappropriate expression of AMPs can also induce autoimmune diseases, thus further highlighting the importance of understanding these molecules and their complex activities. This Primer will provide an update of our current understanding of AMPs. PMID:26766224

  16. [Antimicrobial peptide in dentisty. Literature review].

    PubMed

    Sato, F Simain; Rompen, E; Heinen, E

    2009-12-01

    The use of antimicrobial substances has contributed to the development of multiple antimicrobial resistances (1), challenging the pharmaceutical industry to develop with new, innovative, and effective molecules. Discovered around 1980, molecules called natural antimicrobial peptides (AMPs) appear to hold great potential for the treatment of infections. These cationic peptides are able to stop the bacterial development and to control infections. The purpose of this review is to help improve the understanding of the way AMPs operate in the context of the development of new cures against viruses, bacteria, and mushrooms found in the human body in general and in the oral cavity in particular. PMID:20143750

  17. Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants

    PubMed Central

    Bekešová, Slávka; Komis, George; Křenek, Pavel; Vyplelová, Petra; Ovečka, Miroslav; Luptovčiak, Ivan; Illés, Peter; Kuchařová, Anna; Šamaj, Jozef

    2015-01-01

    The aim of the present study is to rationalize acrylamide pendant Phos-Tag™ in-gel discrimination of phosphorylated and non-phosphorylated plant protein species with standard immunoblot analysis, and optimize sample preparation, efficient electrophoretic separation and transfer. We tested variants of the method including extraction buffers suitable for preservation of phosphorylated protein species in crude extracts from plants and we addressed the importance of the cation (Mn2+ or Zn2+) used in the gel recipe for efficient transfer to PVDF membranes for further immunoblot analysis. We demonstrate the monitoring of Medicago sativa stress-induced mitogen activated protein kinase (SIMK) in stress-treated wild type plants and transgenic SIMKK RNAi line. We further show the hyperosmotically-induced phosphorylation of the previously uncharacterized HvMPK4 of barley. The method is validated using inducible phosphorylation of barley and wheat α-tubulin and of Arabidopsis MPK6. Acrylamide pendant Phos-Tag™offers a flexible tool for studying protein phosphorylation in crops and Arabidopsis circumventing radioactive labeling and the use of phosphorylation specific antibodies. PMID:26029234

  18. Cationic Noncovalent Interactions: Energetics and Periodic Trends.

    PubMed

    Rodgers, M T; Armentrout, P B

    2016-05-11

    In this review, noncovalent interactions of ions with neutral molecules are discussed. After defining the scope of the article, which excludes anionic and most protonated systems, methods associated with measuring thermodynamic information for such systems are briefly recounted. An extensive set of tables detailing available thermodynamic information for the noncovalent interactions of metal cations with a host of ligands is provided. Ligands include small molecules (H2, NH3, CO, CS, H2O, CH3CN, and others), organic ligands (O- and N-donors, crown ethers and related molecules, MALDI matrix molecules), π-ligands (alkenes, alkynes, benzene, and substituted benzenes), miscellaneous inorganic ligands, and biological systems (amino acids, peptides, sugars, nucleobases, nucleosides, and nucleotides). Hydration of metalated biological systems is also included along with selected proton-based systems: 18-crown-6 polyether with protonated peptides and base-pairing energies of nucleobases. In all cases, the literature thermochemistry is evaluated and, in many cases, reanchored or adjusted to 0 K bond dissociation energies. Trends in these values are discussed and related to a variety of simple molecular concepts. PMID:26953819

  19. Peptide separation in hydrophilic interaction capillary electrochromatography.

    PubMed

    Fu, Hongjing; Jin, Wenhai; Xiao, Hua; Huang, Haiwei; Zou, Hanfa

    2003-06-01

    Separation of small peptides by hydrophilic interaction capillary electrochromatography (HI-CEC) has been investigated. The negative surface charge of a hydrophilic, strong-cation-exchange stationary phase (PolySULFOETHYL A) provided a substantial cathodic electroosmotic flow (EOF). The influence of acetonitrile content, ionic strength, mobile phase pH as well as applied voltage on the migration of the peptides was studied. Possible retention mechanisms of the peptides in HI-CEC were discussed. It was found that hydrophilic interaction between the solutes and the stationary phase played a major role in this system, especially when mobile phases with high acetonitrile content were used. However, an ion-exchange mechanism and electrophoretic mobility also affect the migration of the peptides in HI-CEC. Elution order and selectivity was proved to be different in HI-CEC and capillary zone electrophoresis (CZE), thus revealing the potential of HI-CEC as a complementary technique to CZE for the separation of peptides. Efficiency and selectivity of HI-CEC for the separation of peptides were demonstrated by baseline separating nine peptides in 6 min. PMID:12858379

  20. Synthetic Multivalent Antifungal Peptides Effective against Fungi

    PubMed Central

    Li, Jianguo; Nandhakumar, Muruganantham; Aung, Thet Tun; Goh, Eunice; Chang, Jamie Ya Ting; Saraswathi, Padhmanaban; Tang, Charles; Safie, Siti Radiah Binte; Lin, Lim Yih; Riezman, Howard; Lei, Zhou; Verma, Chandra S.; Beuerman, Roger W.

    2014-01-01

    Taking advantage of the cluster effect observed in multivalent peptides, this work describes antifungal activity and possible mechanism of action of tetravalent peptide (B4010) which carries 4 copies of the sequence RGRKVVRR through a branched lysine core. B4010 displayed better antifungal properties than natamycin and amphotericin B. The peptide retained significant activity in the presence of monovalent/divalent cations, trypsin and serum and tear fluid. Moreover, B4010 is non-haemolytic and non-toxic to mice by intraperitoneal (200 mg/kg) or intravenous (100 mg/kg) routes. S. cerevisiae mutant strains with altered membrane sterol structures and composition showed hyper senstivity to B4010. The peptide had no affinity for cell wall polysaccharides and caused rapid dissipation of membrane potential and release of vital ions and ATP when treated with C. albicans. We demonstrate that additives which alter the membrane potential or membrane rigidity protect C. albicans from B4010-induced lethality. Calcein release assay and molecular dynamics simulations showed that the peptide preferentially binds to mixed bilayer containing ergosterol over phophotidylcholine-cholesterol bilayers. The studies further suggested that the first arginine is important for mediating peptide-bilayer interactions. Replacing the first arginine led to a 2–4 fold decrease in antifungal activities and reduced membrane disruption properties. The combined in silico and in vitro approach should facilitate rational design of new tetravalent antifungal peptides. PMID:24498363

  1. Real-time monitoring of phosphorylation kinetics with self-assembled nano-oscillators.

    PubMed

    Fang, Yimin; Chen, Shan; Wang, Wei; Shan, Xiaonan; Tao, Nongjian

    2015-02-16

    Phosphorylation is a post-translational modification that is involved in many basic cellular processes and diseases, but is difficult to detect in real time with existing technologies. A label-free detection of phosphorylation is reported in real time with self-assembled nano-oscillators. Each nano-oscillator consists of a gold nanoparticle tethered to a gold surface with a molecular linker. When the nanoparticle is charged, the nano-oscillator can be driven into oscillation with an electric field and detected with a plasmonic imaging approach. The nano-oscillators measure charge change associated with phosphorylation of peptides attached onto a single nanoparticle, allowing us to study the dynamic process of phosphorylation in real time without antibodies down to a few molecules, from which Michaelis and catalytic rate constants are determined. PMID:25583693

  2. A Cholesterol-Based Allostery Model of T Cell Receptor Phosphorylation.

    PubMed

    Swamy, Mahima; Beck-Garcia, Katharina; Beck-Garcia, Esmeralda; Hartl, Frederike A; Morath, Anna; Yousefi, O Sascha; Dopfer, Elaine Pashupati; Molnár, Eszter; Schulze, Anna K; Blanco, Raquel; Borroto, Aldo; Martín-Blanco, Nadia; Alarcon, Balbino; Höfer, Thomas; Minguet, Susana; Schamel, Wolfgang W A

    2016-05-17

    Signaling through the T cell receptor (TCR) controls adaptive immune responses. Antigen binding to TCRαβ transmits signals through the plasma membrane to induce phosphorylation of the CD3 cytoplasmic tails by incompletely understood mechanisms. Here we show that cholesterol bound to the TCRβ transmembrane region keeps the TCR in a resting, inactive conformation that cannot be phosphorylated by active kinases. Only TCRs that spontaneously detached from cholesterol could switch to the active conformation (termed primed TCRs) and then be phosphorylated. Indeed, by modulating cholesterol binding genetically or enzymatically, we could switch the TCR between the resting and primed states. The active conformation was stabilized by binding to peptide-MHC, which thus controlled TCR signaling. These data are explained by a model of reciprocal allosteric regulation of TCR phosphorylation by cholesterol and ligand binding. Our results provide both a molecular mechanism and a conceptual framework for how lipid-receptor interactions regulate signal transduction. PMID:27192576

  3. Peptide arrays for screening cancer specific peptides.

    PubMed

    Ahmed, Sahar; Mathews, Anu Stella; Byeon, Nara; Lavasanifar, Afsaneh; Kaur, Kamaljit

    2010-09-15

    In this paper, we describe a novel method to screen peptides for specific recognition by cancer cells. Seventy peptides were synthesized on a cellulose membrane in an array format, and a direct method to study the peptide-whole cell interaction was developed. The relative binding affinity of the cells for different peptides with respect to a lead 12-mer p160 peptide, identified by phage display, was evaluated using the CyQUANT fluorescence of the bound cells. Screening allowed identification of at least five new peptides that displayed higher affinity (up to 3-fold) for MDA-MB-435 and MCF-7 human cancer cells compared to the p160 peptide. These peptides showed very little binding to the control (noncancerous) human umbilical vein endothelial cells (HUVECs). Three of these peptides were synthesized separately and labeled with fluorescein isothiocyanate (FITC) to study their uptake and interaction with the cancer and control cells using confocal laser scanning microscopy and flow cytometry. The results confirmed the high and specific affinity of an 11-mer peptide 11 (RGDPAYQGRFL) and a 10-mer peptide 18 (WXEAAYQRFL) for the cancer cells versus HUVECs. Peptide 11 binds different receptors on target cancer cells as its sequence contains multiple recognition motifs, whereas peptide 18 binds mainly to the putative p160 receptor. The peptide array-whole cell binding assay reported here is a complementary method to phage display for further screening and optimization of cancer targeting peptides for cancer therapy and diagnosis. PMID:20799711

  4. Survey of small antifungal peptides with chemotherapeutic potential.

    PubMed

    Desbois, Andrew P; Tschörner, David; Coote, Peter J

    2011-08-01

    Many cationic peptides with antimicrobial properties have been isolated from bacteria, fungi, plants, and animals. These peptides vary in molecular size, potency and spectra of activities. This report surveyed the literature to highlight the peptides that have antifungal activity and greatest potential for development as new therapeutic agents. Thus, to be included in the evaluation, each peptide had to fulfil the following criteria: (i) potent antifungal activity, (ii) no, or minimal, mammalian cell toxicity, (iii) of ≤25 amino acids in length, which minimises the costs of synthesis, reduces immunogenicity and enhances bioavailability and stability in vivo, (iv) minimal post-translational modifications (also reduces the production costs). The ~80 peptides that satisfied these criteria are discussed with respect to their structures, mechanisms of antimicrobial action and in vitro and in vivo toxicities. Certainly, some of these small peptides warrant further study and have potential for future exploitation as new antifungal agents. PMID:21470150

  5. Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

    SciTech Connect

    Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

    1986-05-01

    Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca/sup 2 +/ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting /sup 32/P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated /sup 32/P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor.

  6. Mass spectral study of hybrid peptides derived from (R)-aminoxy ester and [beta]-amino acids: The influence of aminoxy peptide bond (CO-NH-O) on peptide fragmentation under electrospray ionization conditions

    NASA Astrophysics Data System (ADS)

    Ramesh, V.; Ramesh, M.; Srinivas, R.; Sharma, G. V. M.; Manohar, V.

    2009-04-01

    A new class of Boc-protected aminoxy hybrid peptides containing repeats of [beta]-hAla-(R)-Ama-, and [beta]-Caa-(R)-Ama- ([beta]-hAla = [beta]3-(S)-hAlanine, (R)-Ama = (R)-aminoxy ester, and [beta]-Caa = (R)-C-linked carbo-[beta]3-amino acid) have been studied by electrospray ionization (ESI) ion-trap and quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) of their protonated, cationized, and negative ions. MS3 CID of protonated aminoxy peptides of [beta]-hAla-(R)-Ama- yield intense [beta]-amino acid characteristic retro-Mannich fragmentation. The bn+ and [bn-methyl imine]+ (n = 3, 5) ions formed by cleavage of aminoxy peptide bond (CO-NH-O) are more intense than bn+ (n = 2, 4) formed by that of peptide bond (CO-NH-C) cleavage. Another characteristic ion observed is due to loss of H3NO from yn+ ions. The cationized (Li+, and Na+) peptides dissociate differently compared to protonated peptides. Intense cationized cn and zn ions are formed due to the cleavage of N-O bond. The deprotonated peptides also show abundant cn- and zn- ions (n = 1, 3, 5) and do not form any yn- ions. All these results clearly indicate the influence of aminoxy peptide bond on fragmentation of these hybrid peptides.

  7. SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

    SciTech Connect

    JOHN C WALKER

    2011-11-01

    Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

  8. Autophagy proteins regulate ERK phosphorylation

    PubMed Central

    Martinez-Lopez, Nuria; Athonvarangkul, Diana; Mishall, Priti; Sahu, Srabani; Singh, Rajat

    2013-01-01

    Autophagy is a conserved pathway that maintains cellular quality control. Extracellular signal-regulated kinase (ERK) controls various aspects of cell physiology including proliferation. Multiple signalling cascades, including ERK, have been shown to regulate autophagy, however whether autophagy proteins (ATG) regulate cell signalling is unknown. Here we show that growth factor exposure increases the interaction of ERK cascade components with ATG proteins in the cytosol and nucleus. ERK and its upstream kinase MEK localize to the extra-luminal face of autophagosomes. ERK2 interacts with ATG proteins via its substrate-binding domains. Deleting Atg7 or Atg5 or blocking LC3 lipidation or ATG5–ATG12 conjugation decreases ERK phosphorylation. Conversely, increasing LC3-II availability by silencing the cysteine protease ATG4B or acute trehalose exposure increases ERK phosphorylation. Decreased ERK phosphorylation in Atg5−/− cells does not occur from overactive phosphatases. Our findings thus reveal an unconventional function of ATG proteins as cellular scaffolds in the regulation of ERK phosphorylation. PMID:24240988

  9. Complete topographical distribution of both the in vivo and in vitro phosphorylation sites of bone sialoprotein and their biological implications.

    PubMed

    Salih, Erdjan; Flückiger, Rudolf

    2004-05-01

    Bone sialoprotein (BSP) is a multifunctional, highly phosphorylated, and glycosylated protein with key roles in biomineralization and tissue remodeling. This work identifies the complete topographical distribution and precise location of both the in vitro and in vivo phosphorylation sites of bovine BSP by a combination of state-of-the-art techniques and approaches. In vitro phosphorylation of native and deglycosylated BSPs by casein kinase II identified seven phosphorylation sites by solid-phase N-terminal peptide sequencing that were within peptides 12-22 (LEDS(P)EENGVFK), 42-62 (FAVQSSSDSS(P)EENGNGDS(P)S(P)EE), 80-91 (EDS(P)DENEDEES(P)E), and 135-145 (EDES(P)DEEEEEE). The in vivo phosphorylation regions and sites were identified by use of a novel thiol reagent, 1-S-mono[(14)C]carboxymethyldithiothreitol. This approach identified all of the phosphopeptides defined by in vitro phosphorylation, but two additional phosphopeptides were defined at residues, 250-264 (DNGYEIYES(P)ENGDPR), and 282-289 (GYDS(P)YDGQ). Furthermore, use of native BSP and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified several of the above peptides, including an additional phosphopeptide at residues 125-130 (AGAT(P)GK) that was not defined in either of the in vitro and in vivo studies described above. Overall, 7 in vitro and 11 in vivo phosphorylation sites were identified unequivocally, with natural variation in the quantitative extent of phosphorylation at each in vivo phosphorylation site. PMID:15004024

  10. Mimicking phosphorylation of alphaB-crystallin affects its chaperone activity.

    PubMed

    Ecroyd, Heath; Meehan, Sarah; Horwitz, Joseph; Aquilina, J Andrew; Benesch, Justin L P; Robinson, Carol V; Macphee, Cait E; Carver, John A

    2007-01-01

    AlphaB-crystallin is a member of the sHsp (small heat-shock protein) family that prevents misfolded target proteins from aggregating and precipitating. Phosphorylation at three serine residues (Ser19, Ser45 and Ser59) is a major post-translational modification that occurs to alphaB-crystallin. In the present study, we produced recombinant proteins designed to mimic phosphorylation of alphaB-crystallin by incorporating a negative charge at these sites. We employed these mimics to undertake a mechanistic and structural investigation of the effect of phosphorylation on the chaperone activity of alphaB-crystallin to protect against two types of protein misfolding, i.e. amorphous aggregation and amyloid fibril assembly. We show that mimicking phosphorylation of alphaB-crystallin results in more efficient chaperone activity against both heat-induced and reduction-induced amorphous aggregation of target proteins. Mimick-ing phosphorylation increased the chaperone activity of alphaB-crystallin against one amyloid-forming target protein (kappa-casein), but decreased it against another (ccbeta-Trp peptide). We observed that both target protein identity and solution (buffer) conditions are critical factors in determining the relative chaperone ability of wild-type and phosphorylated alphaB-crystallins. The present study provides evidence for the regulation of the chaperone activity of alphaB-crystallin by phosphorylation and indicates that this may play an important role in alleviating the pathogenic effects associated with protein conformational diseases. PMID:16928191

  11. Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation.

    PubMed Central

    Johansson, I; Karlsson, M; Shukla, V K; Chrispeels, M J; Larsson, C; Kjellbom, P

    1998-01-01

    PM28A is a major intrinsic protein of the spinach leaf plasma membrane and the major phosphoprotein. Phosphorylation of PM28A is dependent in vivo on the apoplastic water potential and in vitro on submicromolar concentrations of Ca2+. Here, we demonstrate that PM28A is an aquaporin and that its water channel activity is regulated by phosphorylation. Wild-type and mutant forms of PM28A, in which putative phosphorylation sites had been knocked out, were expressed in Xenopus oocytes, and the resulting increase in osmotic water permeability was measured in the presence or absence of an inhibitor of protein kinases (K252a) or of an inhibitor of protein phosphatases (okadaic acid). The results indicate that the water channel activity of PM28A is regulated by phosphorylation of two serine residues, Ser-115 in the first cytoplasmic loop and Ser-274 in the C-terminal region. Labeling of spinach leaves with 32P-orthophosphate and subsequent sequencing of PM28A-derived peptides demonstrated that Ser-274 is phosphorylated in vivo, whereas phosphorylation of Ser-115, a residue conserved among all plant plasma membrane aquaporins, could not be demonstrated. This identifies Ser-274 of PM28A as the amino acid residue being phosphorylated in vivo in response to increasing apoplastic water potential and dephosphorylated in response to decreasing water potential. Taken together, our results suggest an active role for PM28A in maintaining cellular water balance. PMID:9501117

  12. Phosphorylation of Human CTP Synthetase 1 by Protein Kinase A: IDENTIFICATION OF Thr455 AS A MAJOR SITE OF PHOSPHORYLATION*

    PubMed Central

    Choi, Mal-Gi; Carman, George M.

    2007-01-01

    CTP synthetase is an essential enzyme that generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work, we examined the phosphorylation of the human CTPS1-encoded CTP synthetase 1 by protein kinase A. CTP synthetase 1 was expressed and purified from a Saccharomyces cerevisiae ura7Δ ura8Δ double mutant that lacks CTP synthetase activity. Using purified CTP synthetase 1 as a substrate, protein kinase A activity was time- and dose-dependent. The phosphorylation, which primarily occurred on a threonine residue, was accompanied by a 50% decrease in CTP synthetase 1 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr455 was a substrate for protein kinase A. A Thr455 to Ala (T455A) mutation in CTP synthetase 1 was constructed by site-directed mutagenesis and was expressed and purified from the S. cerevisiae ura7Δ ura8Δ mutant. The T455A mutation caused a 78% decrease in protein kinase A phosphorylation, and the loss of the phosphothreonine residue and a major phosphopeptide that were present in the purified wild type enzyme phosphorylated by protein kinase A. The CTP synthetase 1 activity of the T455A mutant enzyme was 2-fold higher than the wild type enzyme. In addition, the T455A mutation caused a 44% decrease in the amount of human CTP synthetase 1 that was phosphorylated in S. cerevisiae cells, and this was accompanied by a 2.5-fold increase in the cellular concentration of CTP and a 1.5-fold increase in the choline-dependent synthesis of phosphatidylcholine. PMID:17189248

  13. Global Analysis of Human Nonreceptor Tyrosine Kinase Specificity Using High-Density Peptide Microarrays

    PubMed Central

    2015-01-01

    Protein kinases phosphorylate substrates in the context of specific phosphorylation site sequence motifs. The knowledge of the specific sequences that are recognized by kinases is useful for mapping sites of phosphorylation in protein substrates and facilitates the generation of model substrates to monitor kinase activity. Here, we have adapted a positional scanning peptide library method to a microarray format that is suitable for the rapid determination of phosphorylation site motifs for tyrosine kinases. Peptide mixtures were immobilized on glass slides through a layer of a tyrosine-free Y33F mutant avidin to facilitate the analysis of phosphorylation by radiolabel assay. A microarray analysis provided qualitatively similar results in comparison with the solution phase peptide library “macroarray” method. However, much smaller quantities of kinases were required to phosphorylate peptides on the microarrays, which thus enabled a proteome scale analysis of kinase specificity. We illustrated this capability by microarray profiling more than 80% of the human nonreceptor tyrosine kinases (NRTKs). Microarray results were used to generate a universal NRTK substrate set of 11 consensus peptides for in vitro kinase assays. Several substrates were highly specific for their cognate kinases, which should facilitate their incorporation into kinase-selective biosensors. PMID:25164267

  14. Phosphorylation in halobacterial signal transduction.

    PubMed Central

    Rudolph, J; Tolliday, N; Schmitt, C; Schuster, S C; Oesterhelt, D

    1995-01-01

    Regulated phosphorylation of proteins has been shown to be a hallmark of signal transduction mechanisms in both Eubacteria and Eukarya. Here we demonstrate that phosphorylation and dephosphorylation are also the underlying mechanism of chemo- and phototactic signal transduction in Archaea, the third branch of the living world. Cloning and sequencing of the region upstream of the cheA gene, known to be required for chemo- and phototaxis in Halobacterium salinarium, has identified cheY and cheB analogs which appear to form part of an operon which also includes cheA and the following open reading frame of 585 nucleotides. The CheY and CheB proteins have 31.3 and 37.5% sequence identity compared with the known signal transduction proteins CheY and CheB from Escherichia coli, respectively. The biochemical activities of both CheA and CheY were investigated following their expression in E.coli, isolation and renaturation. Wild-type CheA could be phosphorylated in a time-dependent manner in the presence of [gamma-32P]ATP and Mg2+, whereas the mutant CheA(H44Q) remained unlabeled. Phosphorylated CheA was dephosphorylated rapidly by the addition of wild-type CheY. The mutant CheY(D53A) had no effect on phosphorylated CheA. The mechanism of chemo- and phototactic signal transduction in the Archaeon H.salinarium, therefore, is similar to the two-component signaling system known from chemotaxis in the eubacterium E.coli. Images PMID:7556066

  15. Dissecting the cation-cation interaction between two uranyl units.

    PubMed

    Tecmer, Paweł; Hong, Sung W; Boguslawski, Katharina

    2016-07-21

    We present a state-of-the-art computational study of the uranyl(vi) and uranyl(v) cation-cation interactions (dications) in aqueous solution. Reliable electronic structures of two interacting uranyl(vi) and uranyl(v) subunits as well as those of the uranyl(vi) and uranyl(v) clusters are presented for the first time. Our theoretical study elucidates the impact of cation-cation interactions on changes in the molecular structure as well as changes in vibrational and UV-Vis spectra of the bare uranyl(vi) and uranyl(v) moieties for different total spin-states and total charges of the dications. PMID:27335229

  16. Peptides Used in the Delivery of Small Noncoding RNA

    PubMed Central

    2015-01-01

    RNA interference (RNAi) is an endogenous process in which small noncoding RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), post-transcriptionally regulate gene expressions. In general, siRNA and miRNA/miRNA mimics are similar in nature and activity except their origin and specificity. Although both siRNAs and miRNAs have been extensively studied as novel therapeutics for a wide range of diseases, the large molecular weight, anionic surface charges, instability in blood circulation, and intracellular trafficking to the RISC after cellular uptake have hindered the translation of these RNAs from bench to clinic. As a result, a great variety of delivery systems have been investigated for safe and effective delivery of small noncoding RNAs. Among these systems, peptides, especially cationic peptides, have emerged as a promising type of carrier due to their inherent ability to condense negatively charged RNAs, ease of synthesis, controllable size, and tunable structure. In this review, we will focus on three major types of cationic peptides, including poly(l-lysine) (PLL), protamine, and cell penetrating peptides (CPP), as well as peptide targeting ligands that have been extensively used in RNA delivery. The delivery strategies, applications, and limitations of these cationic peptides in siRNA/miRNA delivery will be discussed. PMID:25157701

  17. The cation-π interaction.

    PubMed

    Dougherty, Dennis A

    2013-04-16

    The chemistry community now recognizes the cation-π interaction as a major force for molecular recognition, joining the hydrophobic effect, the hydrogen bond, and the ion pair in determining macromolecular structure and drug-receptor interactions. This Account provides the author's perspective on the intellectual origins and fundamental nature of the cation-π interaction. Early studies on cyclophanes established that water-soluble, cationic molecules would forego aqueous solvation to enter a hydrophobic cavity if that cavity was lined with π systems. Important gas phase studies established the fundamental nature of the cation-π interaction. The strength of the cation-π interaction (Li(+) binds to benzene with 38 kcal/mol of binding energy; NH4(+) with 19 kcal/mol) distinguishes it from the weaker polar-π interactions observed in the benzene dimer or water-benzene complexes. In addition to the substantial intrinsic strength of the cation-π interaction in gas phase studies, the cation-π interaction remains energetically significant in aqueous media and under biological conditions. Many studies have shown that cation-π interactions can enhance binding energies by 2-5 kcal/mol, making them competitive with hydrogen bonds and ion pairs in drug-receptor and protein-protein interactions. As with other noncovalent interactions involving aromatic systems, the cation-π interaction includes a substantial electrostatic component. The six (four) C(δ-)-H(δ+) bond dipoles of a molecule like benzene (ethylene) combine to produce a region of negative electrostatic potential on the face of the π system. Simple electrostatics facilitate a natural attraction of cations to the surface. The trend for (gas phase) binding energies is Li(+) > Na(+) > K(+) > Rb(+): as the ion gets larger the charge is dispersed over a larger sphere and binding interactions weaken, a classical electrostatic effect. On other hand, polarizability does not define these interactions. Cyclohexane is

  18. The Cation-π Interaction

    PubMed Central

    DOUGHERTY, DENNIS A.

    2014-01-01

    CONSPECTUS The chemistry community now recognizes the cation-π interaction as a major force for molecular recognition, joining the hydrophobic effect, the hydrogen bond, and the ion pair in determining macromolecular structure and drug-receptor interactions. This Account provides the author’s perspective on the intellectual origins and fundamental nature of the cation-π interaction. Early studies on cyclophanes established that water-soluble, cationic molecules would forgo aqueous solvation to enter a hydrophobic cavity if that cavity was lined with π systems. Important gas phase studies established the fundamental nature of the cation-π interaction. The strength of the cation-π interaction – Li+ binds to benzene with 38 kcal/mol of binding energy; NH4+ with 19 kcal/mol– distinguishes it from the weaker polar-π interactions observed in the benzene dimer or water-benzene complexes. In addition to the substantial intrinsic strength of the cation-π interaction in gas phase studies, the cation-π interaction remains energetically significant in aqueous media and under biological conditions. Many studies have shown that cation-π interactions can enhance binding energies by 2 – 5 kcal/mol, making them competitive with hydrogen bonds and ion pairs in drug-receptor and protein-protein interactions. As with other noncovalent interactions involving aromatic systems, the cation-π interaction includes a substantial electrostatic component. The six (four) Cδ−–Hδ+ bond dipoles of a molecule like benzene (ethylene) combine to produce a region of negative electrostatic potential on the face of the π system. Simple electrostatics facilitate a natural attraction of cations to the surface. The trend for (gas phase) binding energies is Li+>Na+>K+>Rb+: as the ion gets larger the charge is dispersed over a larger sphere and binding interactions weaken, a classical electrostatic effect. On other hand, polarizability does not define these interactions. Cyclohexane

  19. C-Peptide Test

    MedlinePlus

    ... C-peptide is a useful marker of insulin production. The following are some purposes of C-peptide ... it nearly impossible to directly evaluate endogenous insulin production. In these cases, C-peptide measurement is a ...

  20. Overcharging in Biological Systems: Reversal of Electrophoretic Mobility of Aqueous Polyaspartate by Multivalent Cations

    NASA Astrophysics Data System (ADS)

    Kubíčková, Anna; Křížek, Tomáš; Coufal, Pavel; Vazdar, Mario; Wernersson, Erik; Heyda, Jan; Jungwirth, Pavel

    2012-05-01

    Charge reversal as an extreme case of charge compensation is directly observed by capillary electrophoresis for a negatively charged peptide in aqueous solutions of trivalent cations. Atomistic and coarse-grained simulations provide molecular interpretation of this effect showing that it is largely of electrostatic origin with a minor contribution of chemical specificity of the salt ions.

  1. Cell-penetrating recombinant peptides for potential use in agricultural pest control applications

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several important areas of interest intersect in a class of peptides characterized by their highly cationic and partly hydrophobic structure. These molecules have been called cell-penetrating peptides (CPPs) because they possess the ability to translocate across cell membranes. This ability makes ...

  2. Phosphorylation of the insulin receptor in cultured hepatoma cells and a solubilized system

    SciTech Connect

    Kasuga, M.; White, M.F.; Kahn, C.R.

    1985-01-01

    Methods are described which have been used successfully to study insulin receptor autophosphorylation in cultured cells (hepatoma cell line Fao) and detergent solubilized receptor systems. Intact cultured cells were labelled with /sup 32/PO/sub 4//sup 3 -/. Details are given for the solubilization and purification of the insulin receptor and insulin dose-response curves for phosphorylation of the solubilized insulin receptor. Trypsin digestion of a phosphorylated subunit suggests that at least peptides containing sites of /sup 32/P incorporation exist in the receptor molecule.

  3. Self-Assembly of Highly Phosphorylated Silaffins and Their Function in Biosilica Morphogenesis

    NASA Astrophysics Data System (ADS)

    Kröger, Nils; Lorenz, Sonja; Brunner, Eike; Sumper, Manfred

    2002-10-01

    Silaffins are uniquely modified peptides that have been implicated in the biogenesis of diatom biosilica. A method that avoids the harsh anhydrous hydrogen fluoride treatment commonly used to dissolve biosilica allows the extraction of silaffins in their native state. The native silaffins carry further posttranslational modifications in addition to their polyamine moieties. Each serine residue was phosphorylated, and this high level of phosphorylation is essential for biological activity. The zwitterionic structure of native silaffins enables the formation of supramolecular assemblies. Time-resolved analysis of silica morphogenesis in vitro detected a plastic silaffin-silica phase, which may represent a building material for diatom biosilica.

  4. Cellular regulation by protein phosphorylation.

    PubMed

    Fischer, Edmond H

    2013-01-11

    A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca(2+) and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert. PMID:23058924

  5. Antimicrobial Peptides and Their Analogs: Searching for New Potential Therapeutics

    PubMed Central

    Midura-Nowaczek, Krystyna; Markowska, Agnieszka

    2014-01-01

    Antimicrobial peptides (AMPs) are an essential part of innate immunity. These compounds have been considered as potential therapeutics because of their broad-spectrum activities and proven ability to avoid antimicrobial resistance, but their clinical and commercial developments have some limitations, such as susceptibility to proteases and a high cost of peptide production. To overcome these problems, many researchers have tried to develop short active peptides, their modifications and mimics with better properties while retaining their basic features of natural AMPs such as cationic charge and the amphipathic structure. PMID:25374459

  6. Phosphorylation of the C proteins in heterogeneous ribonucleoprotein (hnRNP) particles in HeLa cells: Characterization of in vivo phosphorylation, comparison with in vitro phosphorylation using casein kinase II, and preliminary studies on the effects of phosphorylation on particle structure

    SciTech Connect

    Kleiman, N.J.

    1989-01-01

    Newly formed pre-messenger RNA associates with protein to form heterogeneous ribonucleoprotein (hnRNP) particles. In HeLa cells, hnRNP particles contain six core proteins. Two proteins, termed C{sub 1} and C{sub 2}, are phosphorylated in vitro by casein kinase 11 (CKII). C{sub 1} protein became {sup 32}P-labeled after HeLa cells were incubated with ({sup 32}P)-orthophosphate in vivo (ibid). Because phosphorylation is a ubiquitous regulatory mechanism, C protein phosphorylation was studied in greater detail. C protein phosphorylation in hnRNP particles was investigated in HeLa cells incubated with ({sup 32}P)-orthophosphate in vivo. Immunoblotting in pH 3.5-10 isoelectric focusing (IEF) gels indicated that C proteins focus only at pH 5.0. In pH 4.5-5.5 IEF gels, individually purified C, and 2 proteins resolve into the same four closely spaced, {sup 32}P-labeled bands. A fifth, unlabeled, more basic species was detached when hnRNP particles were purified without NaF. All {sup 32}P-labeled species contained identical amounts of {sup 32}P per unit protein suggesting that charge heterogeneity is not due to differential phosphorylation. Attempts to detect bound carbohydrate were unsuccessful. {sup 32}P-labeled phosphate was readily removed by potato acid phosphatase. E. coli alkaline phosphatase and snake venom phosphodiesterase were ineffective. {sup 32}P-label was found exclusively in phosphoserine. One-dimensional peptide mapping with chymotrypsin and S. aureus protease detected two phosphorylated peptides. C protein phosphorylation was also investigated in vitro. Incubation of hnRNP particles with rabbit liver CKII and {sup 32}P-ATP followed by IEF in pH 4.5-5.5 gels indicated that all four C protein species were {sup 32}P-labeled. {sup 32}P-label was found exclusively in phosphoserine.

  7. Glutathione-based zwitterionic stationary phase for hydrophilic interaction/cation-exchange mixed-mode chromatography.

    PubMed

    Shen, Aijin; Li, Xiuling; Dong, Xuefang; Wei, Jie; Guo, Zhimou; Liang, Xinmiao

    2013-11-01

    As a naturally hydrophilic peptide, glutathione was facilely immobilized onto silica surface to obtain a novel hydrophilic interaction/cation-exchange mixed-mode chromatographic stationary phase (Click TE-GSH) via copper-free "thiol-ene" click chemistry. The resulting material was characterized by solid state (13)C/CP MAS NMR and elemental analysis. The measurement of ζ-potential indicated the cation-exchange characteristics and adjustable surface charge density of Click TE-GSH material. The influence of acetonitrile content and pH value on the retention of ionic compounds was investigated for understanding the chromatographic behaviors. The results demonstrated that Click TE-GSH column could provide both hydrophilic and cation-exchange interaction. Taking advantage of the good hydrophilicity and inherent cation-exchange characteristics of Click TE-GSH material, the resolution of neutral fructosan with high degree of polymerization (DP), basic chitooligosaccharides and strongly acidic carrageenan oligosaccharides was successfully realized in hydrophilic interaction chromatography (HILIC), hydrophilic interaction/cation-exchange mixed-mode chromatography (HILIC/CEX), cation-exchange chromatography (CEX) and electrostatic repulsion/hydrophilic interaction chromatography (ERLIC). On the other hand, the separation of standard peptides varying in hydrophobicity/hydrophilicity and charge was achieved in both CEX and HILIC/CEX mode with high efficiency and distinct selectivity. To further demonstrate the versatility and applicability of Click TE-GSH stationary phase, the separation of a human serum albumin (HSA) tryptic digest was performed in HILIC/CEX mode. Peptides were adequately resolved and up to 86 HSA peptides were identified with sequence coverage of 85%. The results indicated the good potential of Click TE-GSH material in glycomics and proteomics. PMID:24075460

  8. Review: Formation of Peptide Radical Ions Through Dissociative Electron Transfer in Ternary Metal-Ligand-Peptide Complexes

    SciTech Connect

    Chu, Ivan K.; Laskin, Julia

    2011-12-31

    The formation and fragmentation of odd-electron ions of peptides and proteins is of interest to applications in biological mass 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{sup +{sm_bullet}}, radical dications, [M{sup +}H]{sup 2+{sm_bullet}}, radical anions, [M-2H]{sup -{sm_bullet}}. 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 on the role of charge and the 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 such complexes are also important for detailed molecular-level understanding of photosynthesis and respiration in biological systems.

  9. Partial purification of a spinach thylakoid protein kinase that can phosphorylate light-harvesting chlorophyll a/b proteins

    SciTech Connect

    Clark, R.D.; Hind, G.; Bennett, J.

    1985-01-01

    Protein phosphorylation in plant tissues is particularly marked in chloroplasts, protein kinase activity being associated with the outer envelope, the soluble stromal fraction, and the thylakoid membrane. Furthermore, thylakoid-bound activity probably includes several distinct kinases, as suggested by studies of divalent cation specificity and thermal lability carried out with intact thylakoids and by subfractionation of solubilized membranes. Illumination of thylakoids, particularly with red light, promotes the rapid and extensive phosphorylation of the light-harvesting chlorophyll a/b complex (LHCII) on a threonine residue near the amino terminus of the protein. This phosphorylation is thought to be involved in regulating the distribution of absorbed quanta between photosystems II and I and is modulated by the redox state of the thylakoid plastoquinone pool. Neither of the thylakoid kinases reported to date was capable of phosphorylating purified LHCII in vitro or of incorporating phosphate into threonyl residues of exogenous substrates, that some LHCII phosphorylation was catalyzed by a preliminary fraction led workers to suggest that at least one other kinase remained to be isolated. Here, the authors report the solubilization and partial purification of a protein kinase from spinach thylakoids that is capable of phosphorylating LHCII in vitro, and they show that the specific site of phosphorylation is very nearly the same as, if not identical with, the site phosphorylated in organello.

  10. BT cationic peptides: Small peptides that modulate innate immune responses of chicken heterophils and monocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Drug-resistant bacteria pose an enormous threat to public health having developed resistance mechanisms to all existing classes of antibiotics. Fewer novel antibiotics are being developed, so there is an increasing need to identify alternative approaches with less associated resistance. An alternat...

  11. Cation Diffusion in Xenotime

    NASA Astrophysics Data System (ADS)

    Cherniak, D. J.

    2004-05-01

    Xenotime is an important mineral in metamorphic paragenesis, and useful in isotopic dating, garnet-xenotime thermometry, and monazite-xenotime thermometry, so diffusion data for xenotime of cations of geochronological and geochemical importance are of some interest. We report here on diffusion of the rare earth elements Sm, Dy and Yb in synthetic xenotime under dry conditions. The synthetic xenotime was grown via a Na2}CO{3}-MoO_{3 flux method. The source of diffusant for the experiments were REE phosphate powders, with experiments run with sources containing a single REE. Experiments were performed by placing source and xenotime in Pt capsules, and annealing capsules in 1 atm furnaces for times ranging from thirty minutes to a month, at temperatures from 1000 to 1400C. The REE distributions in the xenotime were profiled by Rutherford Backscattering Spectrometry (RBS). The following Arrhenius relations are obtained for diffusion in xenotime, normal to (101): DSm = 1.7x10-4 exp(-442 kJ mol-1/RT) m2}sec{-1 DDy = 3.5x10-7 exp(-365 kJ mol-1/RT) m2}sec{-1 DYb = 7.4x10-7 exp(-371 kJ mol-1/RT) m2}sec{-1. Diffusivities of these REE do not differ greatly in xenotime, in contrast to the findings noted for the REE in zircon (Cherniak et al., 1997), where the LREE diffuse more slowly, and with higher activation energies for diffusion, than the heavier rare earths. In zircon, these differences among diffusion of the rare earths are attributed to the relatively large size of the REE with respect to Zr, for which they substitute in the zircon lattice. With the systematic increase in ionic radius from the heavy to lighter REE, this size mismatch becomes more pronounced and diffusivities of the LREE are as consequence slower. Although xenotime is isostructural with zircon, the REE are more closely matched in size to Y, so in xenotime this effect appears much smaller and the REE diffuse at similar rates. In addition, the process of diffusion in xenotime likely involves simple REE+3

  12. Mycobacterium tuberculosis supports protein tyrosine phosphorylation

    PubMed Central

    Kusebauch, Ulrike; Ortega, Corrie; Ollodart, Anja; Rogers, Richard S.; Sherman, David R.; Moritz, Robert L.; Grundner, Christoph

    2014-01-01

    Reversible protein phosphorylation determines growth and adaptive decisions in Mycobacterium tuberculosis (Mtb). At least 11 two-component systems and 11 Ser/Thr protein kinases (STPKs) mediate phosphorylation on Asp, His, Ser, and Thr. In contrast, protein phosphorylation on Tyr has not been described previously in Mtb. Here, using a combination of phospho-enrichment and highly sensitive mass spectrometry, we show extensive protein Tyr phosphorylation of diverse Mtb proteins, including STPKs. Several STPKs function as dual-specificity kinases that phosphorylate Tyr in cis and in trans, suggesting that dual-specificity kinases have a major role in bacterial phospho-signaling. Mutation of a phosphotyrosine site of the essential STPK PknB reduces its activity in vitro and in live Mtb, indicating that Tyr phosphorylation has a functional role in bacterial growth. These data identify a previously unrecognized phosphorylation system in a human pathogen that claims ∼1.4 million lives every year. PMID:24927537

  13. Hydroxyapatite Growth Inhibition Effect of Pellicle Statherin Peptides.

    PubMed

    Xiao, Y; Karttunen, M; Jalkanen, J; Mussi, M C M; Liao, Y; Grohe, B; Lagugné-Labarthet, F; Siqueira, W L

    2015-08-01

    In our recent studies, we have shown that in vivo-acquired enamel pellicle is a sophisticated biological structure containing a significant portion of naturally occurring salivary peptides. From a functional aspect, the identification of peptides in the acquired enamel pellicle is of interest because many salivary proteins exhibit functional domains that maintain the activities of the native protein. Among the in vivo-acquired enamel pellicle peptides that have been newly identified, 5 peptides are derived from statherin. Here, we assessed the ability of these statherin pellicle peptides to inhibit hydroxyapatite crystal growth. In addition, atomistic molecular dynamics (MD) simulations were performed to better understand the underlying physical mechanisms of hydroxyapatite growth inhibition. A microplate colorimetric assay was used to quantify hydroxyapatite growth. Statherin protein, 5 statherin-derived peptides, and a peptide lacking phosphate at residues 2 and 3 were analyzed. Statherin peptide phosphorylated on residues 2 and 3 indicated a significant inhibitory effect when compared with the 5 other peptides (P < 0.05). MD simulations showed a strong affinity and fast adsorption to hydroxyapatite for phosphopeptides, whereas unphosphorylated peptides interacted weakly with the hydroxyapatite. Our data suggest that the presence of a covalently linked phosphate group (at residues 2 and 3) in statherin peptides modulates the effect of hydroxyapatite growth inhibition. This study provides a mechanism to account for the composition and function of acquired enamel pellicle statherin peptides that will contribute as a base for the development of biologically stable and functional synthetic peptides for therapeutic use against dental caries and/or periodontal disease. PMID:26116492

  14. Cathelicidin peptides as candidates for a novel class of antimicrobials.

    PubMed

    Zanetti, Margherita; Gennaro, Renato; Skerlavaj, Barbara; Tomasinsig, Linda; Circo, Raffaella

    2002-01-01

    Cathelicidin peptides are a numerous group of mammalian cationic antimicrobial peptides. Despite a common evolutionary origin of their genes, peptides display a remarkable variety of sizes, sequences and structures. Their spectra of antimicrobial activity are varied and cover a range of organisms that includes bacteria, fungi and enveloped viruses. In addition, they bind to and neutralize the effects of endotoxin. These features make this family of peptides good candidates in view of a therapeutic use. The most promising ones are currently under evaluation as leads for the development of novel anti-infectives, and synthetic variants are in an advanced stage of development for specific clinical applications. This review focuses on recent studies on the structure and in vitro and in vivo biological activities of these peptides. PMID:11945171

  15. Aberrant protein phosphorylation in Alzheimer disease brain disturbs pro-survival and cell death pathways.

    PubMed

    Perluigi, M; Barone, E; Di Domenico, F; Butterfield, D A

    2016-10-01

    Protein phosphorylation of serine, threonine, and tyrosine residues is one of the most prevalent post-translational modifications fundamental in mediating diverse cellular functions in living cells. Aberrant protein phosphorylation is currently recognized as a critical step in the pathogenesis and progression of Alzheimer disease (AD). Changes in the pattern of protein phosphorylation of different brain regions are suggested to promote AD transition from a presymptomatic to a symptomatic state in response to accumulating amyloid β-peptide (Aβ). Several experimental approaches have been utilized to profile alteration of protein phosphorylation in the brain, including proteomics. Among central pathways regulated by kinases/phosphatases those involved in the activation/inhibition of both pro survival and cell death pathways play a central role in AD pathology. We discuss in detail how aberrant phosphorylation could contribute to dysregulate p53 activity and insulin-mediated signaling. Taken together these results highlight that targeted therapeutic intervention, which can restore phosphorylation homeostasis, either acting on kinases and phosphatases, conceivably may prove to be beneficial to prevent or slow the development and progression of AD. PMID:27425034

  16. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies.

    PubMed

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu

    2016-01-01

    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

  17. The retinoblastoma protein is phosphorylated on multiple sites by human cdc2.

    PubMed Central

    Lees, J A; Buchkovich, K J; Marshak, D R; Anderson, C W; Harlow, E

    1991-01-01

    The retinoblastoma gene product (pRB) is a nuclear phosphoprotein that is thought to play a key role in the negative regulation of cellular proliferation. pRB is phosphorylated in a cell cycle dependent manner, and studies in both actively dividing and differentiated cells suggest that this modification may be essential for cells to progress through the cell cycle. Using tryptic phosphopeptide mapping we have shown that pRB is phosphorylated on multiple serine and threonine residues in vivo and that many of these phosphorylation events can be mimicked in vitro using purified p34cdc2. Using synthetic peptides corresponding to potential cdc2 phosphorylation sites, we have developed a strategy which has allowed the identification of five sites. S249, T252, T373, S807 and S811 are phosphorylated in vivo, and in each case these sites correspond closely to the consensus sequence for phosphorylation by p34cdc2. This and the observation that pRB forms a specific complex with p34cdc2 in vivo suggests that p34cdc2 or a p34cdc2-related protein is a major pRB kinase. Images PMID:1756735

  18. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies

    PubMed Central

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu

    2016-01-01

    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

  19. Multiple biological activities for two peptides derived from the nerve growth factor precursor

    SciTech Connect

    Dicou, Eleni . E-mail: dicou@ipmc.cnrs.fr

    2006-09-01

    ProNGF can be cleaved proteolytically at dibasic residues and liberates two other peptides beside NGF, LIP1 a 29 amino acid (aa) peptide and LIP2 a 38 aa peptide. These peptides were found present in the rat intestine and shown to induce rapid phosphorylation of the Trk receptor in cell lines. The present study describes several novel biological properties for these peptides. They exert an anti-proliferative effect on the mitogenic activity of estrogen and IGF in MCF-7 cells. They protect against in vivo induction of excitotoxic lesions by the glutamatergic analogue ibotenate injected into the developing mouse brain and against in vitro NMDA-induced cell death in primary neuronal cultures. They bind to murine microglial cells and induce phosphorylation of Akt. These results suggest a role for LIP1 and LIP2 in cell survival.

  20. Interaction of Ca2+ and protein phosphorylation in the rabbit platelet release reaction.

    PubMed

    Lyons, R M; Shaw, J O

    1980-02-01

    Ca2+ flux and protein phosphorylation have been implicated as playing an important role in the induction of the platelet release reaction. However, the interactions between Ca2+, protein phosphorylation, and the release reaction have been difficult to study because secretion in human platelets is independent of extracellular Ca2+. Thus, we studied rabbit platelets, which, unlike human platelets, require extracellular Ca2+ for serotonin release to occur. Thrombin, basophil platelet-activating factor (PAF), or ionophore A23187 treatment of intact 32PO43--loaded rabbit platelets resulted in a 200-400% increase in phosphorylation of P7P and P9P, respectively. These peptides were similar in all respects to the peptides phosphorylated in thrombin-treated human platelets. When Ca2+ was replaced in the medium by EGTA, (a) thrombin- and PAF-induced rabbit platelet [3H]serotonin release was inhibited by 60-75%, whereas ionophore-induced release was blocked completely; (b) thrombin-, PAF-, or ionophore-induced P9P phosphorylation was inhibited by 60%; and (c) ionophore-induced P7P phosphorylation was decreased by 60%, whereas that caused by thrombin or PAF was decreased by only 20%. At 0.25-0.5 U/ml of thrombin, phosphorylation preceded [3H]serotonin release with the time for half-maximal release being 26.0 +/- 1.3 s SE (n = 3) and the time for half-maximal phosphorylation being 12.3 +/- 1.3 s SE (n = 3) for P7P and 3.7 +/- 0.17 s SE (n = 3) for P9P. P9P phosphorylation was significantly inhibited (P less than 0.015) by removal by Ca2+ from the medium at a time point before any thrombin- or ionophore-induced serotonin release was detectable. Thus, our data suggest that Ca2+ flux precedes the onset of serotonin secretion and that the rabbit platelet is an appropriate model in which to study the effects of Ca2+ on protein phosphorylation during the platelet release reaction. PMID:6985917

  1. Tobramycin and nebramine as pseudo-oligosaccharide scaffolds for the development of antimicrobial cationic amphiphiles.

    PubMed

    Berkov-Zrihen, Yifat; Herzog, Ido M; Benhamou, Raphael I; Feldman, Mark; Steinbuch, Kfir B; Shaul, Pazit; Lerer, Shachar; Eldar, Avigdor; Fridman, Micha

    2015-03-01

    Antimicrobial cationic amphiphiles derived from aminoglycoside pseudo-oligosaccharide antibiotics interfere with the structure and function of bacterial membranes and offer a promising direction for the development of novel antibiotics. Herein, we report the design and synthesis of cationic amphiphiles derived from the pseudo-trisaccharide aminoglycoside tobramycin and its pseudo-disaccharide segment nebramine. Antimicrobial activity, membrane selectivity, mode of action, and structure-activity relationships were studied. Several cationic amphiphiles showed marked antimicrobial activity, and one amphiphilic nebramine derivative proved effective against all of the tested strains of bacteria; furthermore, against several of the tested strains, this compound was well over an order of magnitude more potent than the parent antibiotic tobramycin, the membrane-targeting antimicrobial peptide mixture gramicidin D, and the cationic lipopeptide polymyxin B, which are in clinical use. PMID:25652188

  2. Synthesis of peptides of Carapax Trionycis and their inhibitory effects on TGF-β1-induced hepatic stellate cells.

    PubMed

    Hu, C L; Peng, X Z; Tang, Y P; Liu, Y W

    2013-12-01

    We previous identified the antifibrotic active ingredients from Carapax Trionycis as two peptides. Here, we synthesized these two peptides (peptide 1 and peptide 2) by a solid phase method and examined their effects on proliferation and activation of cultured hepatic stellate cells (HSC) which are the main ECM (extracellular matrix)-producing cells in fibrosis progression. We demonstrated that peptide 1 and peptide 2 significantly reduced HSC proliferation and activation in a dose dependent manner. Further, peptide 1 and peptide 2 could interfere with TGF-signaling by down-regulating Smad 3 phosphorylation. Thus, these synthetic peptides of Carapax Trionycis could inhibit proliferation and activation of HSC and might be used as a candidate for treatment of liver fibrosis. PMID:24423656

  3. Phosphorylation of ferredoxin and regulation of renal mitochondrial 25-hydroxyvitamin D-1 alpha-hydroxylase activity in vitro.

    PubMed

    Nemani, R; Ghazarian, J G; Moorthy, B; Wongsurawat, N; Strong, R; Armbrecht, H J

    1989-09-15

    The kidney is the principal physiologic site of production of biologically active 1,25-dihydroxyvitamin D. The 25-hydroxyvitamin D-1 alpha-hydroxylase (1-OHase) activity found in renal mitochondria is under tight hormonal control. Parathyroid hormone stimulates the renal conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D in young animals, which is accompanied by dephosphorylation of ferredoxin (Fx), a component of the mitochondrial 1-OHase enzyme complex (Siegel, N., Wongsurawat, N., and Armbrecht, H. J. (1986) J. Biol. Chem. 261, 16998-17003). The present study investigates the capacity of Fx to be phosphorylated in vitro and to modulate the 1-OHase activity of a reconstituted system. Fx was phosphorylated by renal mitochondrial type II protein kinase. Phosphorylation did not alter Fx mobility on sodium dodecyl sulfate gels but did decrease the pI as measured by isoelectric focusing. Amino acid analysis demonstrated that 1 mol of serine and 1 mol of threonine were phosphorylated per mol of Fx. Peptide mapping of phosphorylated Fx was consistent with phosphorylation of serine 88 and threonine 85 or 97. Fx was selectively dephosphorylated by rabbit skeletal muscle protein phosphatase C2 but not C1. Phosphorylation of Fx significantly inhibited the 1-OHase activity of a reconstituted system consisting of Fx reductase, Fx, and renal mitochondrial cytochrome P-450. These findings suggest that phosphorylation/dephosphorylation of Fx may play a role in modulating renal 1,25-dihydroxyvitamin D production. PMID:2768268

  4. Cation Ordering in Layered Nickelates

    NASA Astrophysics Data System (ADS)

    Nelson-Cheeseman, Brittany; Zhou, Hua; Cammarata, Antonio; Hoffman, Jason; Balachandran, Prasanna; Rondinelli, James; Bhattacharya, Anand

    2013-03-01

    The single layer Ruddlesden-Popper nickelates present a model system to understand how the effects of digital dopant cation ordering may affect the properties of 2-dimensional conducting sheets. We investigate the effects of aliovalent A-site cation order on LaSrNiO4 films. Using molecular beam epitaxy, we interleave full layers of SrO and LaO in a series of chemically equivalent films, varying the pattern of SrO and LaO layers relative to the NiO2 layers. Through synchrotron surface x-ray diffraction and Coherant Bragg Rod Analysis (COBRA), we directly investigate the A-site cation order and the resulting atomic displacements for each ordering pattern. We correlate these results with theoretical calculations and transport measurements of the layered nickelate films.

  5. ECD of Tyrosine Phosphorylation in a Triple Quadrupole Mass Spectrometer with a Radio-Frequency-Free Electromagnetostatic Cell

    PubMed Central

    Voinov, Valery G.; Bennett, Samuel E.; Beckman, Joseph S.; Barofsky, Douglas F.

    2014-01-01

    A radio frequency-free electromagnetostatic (EMS) cell devised for electron-capture dissociation (ECD) of ions has been retrofitted into the collision-induced dissociation (CID) section of a triple quadrupole mass spectrometer to enable recording of ECD product-ion mass spectra and simultaneous recording of ECD-CID product-ion mass spectra. This modified instrument can be used to produce easily interpretable ECD and ECD-CID product-ion mass spectra of tyrosine-phosphorylated peptides that cover over 50% of their respective amino-acid sequences and readily identify their respective sites of phosphorylation. ECD fragmentation of doubly protonated, tyrosine-phosphorylated peptides, which was difficult to observe with FT-ICR instruments, occurs efficiently in the EMS cell. PMID:25037842

  6. ECD of Tyrosine Phosphorylation in a Triple Quadrupole Mass Spectrometer with a Radio-Frequency-Free Electromagnetostatic Cell

    NASA Astrophysics Data System (ADS)

    Voinov, Valery G.; Bennett, Samuel E.; Beckman, Joseph S.; Barofsky, Douglas F.

    2014-10-01

    A radio frequency-free electromagnetostatic (EMS) cell devised for electron-capture dissociation (ECD) of ions has been retrofitted into the collision-induced dissociation (CID) section of a triple quadrupole mass spectrometer to enable recording of ECD product-ion mass spectra and simultaneous recording of ECD-CID product-ion mass spectra. This modified instrument can be used to produce easily interpretable ECD and ECD-CID product-ion mass spectra of tyrosine-phosphorylated peptides that cover over 50% of their respective amino-acid sequences and readily identify their respective sites of phosphorylation. ECD fragmentation of doubly protonated, tyrosine-phosphorylated peptides, which was difficult to observe with FT-ICR instruments, occurs efficiently in the EMS cell.

  7. Phosphorylated nano-diamond/ Polyimide Nanocomposites

    NASA Astrophysics Data System (ADS)

    Beyler-Çiǧil, Asli; Çakmakçi, Emrah; Vezir Kahraman, Memet

    2014-08-01

    In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased.

  8. Enhanced binding of RNAP II CTD phosphatase FCP1 to RAP74 following CK2 phosphorylation.

    PubMed

    Abbott, Karen L; Renfrow, Matthew B; Chalmers, Michael J; Nguyen, Bao D; Marshall, Alan G; Legault, Pascale; Omichinski, James G

    2005-03-01

    FCP1 (TFIIF-associated CTD phosphatase) is the first identified CTD-specific phosphatase required to recycle RNA polymerase II (RNAP II). FCP1 activity has been shown to be regulated by the general transcription factors TFIIF (RAP74) and TFIIB, protein kinase CK2 (CK2), and the HIV-1 transcriptional activator Tat. Phosphorylation of FCP1 by CK2 stimulates FCP1 phosphatase activity and enhances binding of RAP74 to FCP1. We have examined consensus CK2 phosphorylation sites (acidic residue n + 3 to serine or threonine residue) located immediately adjacent to both RAP74-binding sites of FCP1. We demonstrate that both of these consensus CK2 sites can be phosphorylated in vitro and that phosphorylation at either CK2 site results in enhanced binding of RAP74 to FCP1. The CK2 site adjacent to the RAP74-binding site in the central domain of FCP1 is phosphorylated at a single threonine site (T584). The CK2 site adjacent to the RAP74-binding site in the carboxyl-terminal domain can be phosphorylated at three successive serine residues (S942-S944), with phosphorylations at S942 and S944 both contributing to enhanced binding to RAP74. With the use of tandem Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR), we demonstrate that the phosphorylation of S942-S944 occurs in a semiordered fashion with the initial phosphorylation occurring at either S942 or S944 followed by a second phosphorylation to yield the S942/S944 diphosphorylated species. Using nuclear magnetic resonance (NMR) spectroscopy, we identify and map chemical shift changes onto the solution structure of the carboxyl-terminal domain of RAP74 (RAP74(436)(-)(517)) on complexation of RAP74(436)(-)(517) with phosphorylated FCP1 peptides. These results provide new functional and structural information on the role of phosphorylation in the recognition of acidic-rich activation domains involved in transcriptional regulation, and bring insights into how CK2 and TFIIF regulate FCP1 function. PMID:15723518

  9. Entrapment enhancement of peptide drugs in niosomes.

    PubMed

    Manosroi, Aranya; Khanrin, Penpan; Werner, Rolf G; Götz, Friedrich; Manosroi, Worapaka; Manosroi, Jiradej

    2010-05-01

    The objective of this study was to enhance the entrapment of various charged peptide drugs [(bacitracin (BCT), insulin and bovine serum albumin (BSA)] in niosomes by modifying the vesicular charge compositions. Cationic, anionic and neutral niosomes were prepared from sorbitan monostearate (Span 60) or polyoxyethylene sorbitan monostearate (Tween 61), cholesterol (CHL), dimethyldioctadecylammonium bromide (DDAB) and/or dicetyl phosphate (DP) in distilled water, by freeze dried empty liposome (FDEL) method. Morphology and vesicular sizes of the vesicles were investigated by optical microscope, TEM, X-ray diffractometry and dynamic light scattering. The entrapment efficiency of the peptides in niosomes was determined by gel electrophoresis and gel documentation. After reconstitution of the empty niosomal powder in phosphate buffer pH 7.0 containing the peptide drugs, they were oligolamellar membrane structure, with the sizes of 40-60 nm, except the neutral niosomes entrapped with insulin and cationic niosomes entrapped with BSA which showed the sizes of 0.1-1.3 microm and 100-150 nm, respectively. The zeta potential values of neutral, cationic and anionic niosomes entrapped with BSA, insulin and BCT were -22.3 +/- 1.52, -30.7 +/- 2.92 and +22.68+/- 1.31 mV, respectively. The entrapment efficiency of BSA, BCT and insulin in neutral niosomes (Tween 61/CHL at 1 : 1 molar ratio) was 72.94, 69.89 and 10.26%, in cationic niosomes (Tween 61/CHL/DDAB at 1 : 1 : 0.05 molar ratio) was 84.54, 32.85 and 87.15% and in anionic niosomes (Tween 61/CHL/DP at 1 : 1 : 0.05 molar ratio) was 50.13, 90.88 and 44.31%, respectively. The highest entrapment efficiency of BSA, BCT and insulin at 72.94, 90.88 and 87.15 was observed in neutral, anionic and cationic niosomes, respectively. The results from this study has suggested the appropriate niosomal formulation to entrap the peptides with different charges and polarity for pharmaceutical application. PMID:20113169

  10. Role of enzyme-peptide substrate backbone hydrogen bonding in determining protein kinase substrate specificities.

    PubMed

    Thomas, N E; Bramson, H N; Miller, W T; Kaiser, E T

    1987-07-14

    As part of a search for peptides that have specificity for selected protein kinases, the possibility that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) recognizes the hydrogen-bonding potential of its peptide substrates was investigated. A-Kinase catalyzes the phosphorylation of five N alpha-methylated and four depsipeptide derivatives of Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) at rates that differ by at least 7 orders of magnitude. These peptide 1 analogues each lack the ability to donate a hydrogen bond at selected positions in the peptide chain. If a particular amide hydrogen of a peptide amide is involved in hydrogen bonding, which is important for enzyme recognition, the prediction is that peptides which contain an ester or a N-methylated bond at that position in peptide 1 will be comparatively poor substrates. In contrast, if a depsipeptide has a reactivity comparable to that of peptide 1 but the analogous N-methylated peptide has a poor reactivity with A-kinase, the result might indicate that the N-methyl group causes unfavorable steric effects. The depsipeptide that lacks a Leu6 amide proton is a good substrate for A-kinase, but the corresponding N-methylated peptide is phosphorylated far less efficiently. This result and others presented in this paper suggest that although enzyme-substrate hydrogen bonding may play some role in A-kinase catalysis of phosphoryl group transfer, other explanations are necessary to account for the relative reactivities of N alpha-methylated and depsi-containing peptide 1 analogues.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3663600

  11. O-GlcNAcylation Antagonizes Phosphorylation of CDH1 (CDC20 Homologue 1).

    PubMed

    Tian, Jie; Geng, Qizhi; Ding, Yuehe; Liao, Ji; Dong, Meng-Qiu; Xu, Xingzhi; Li, Jing

    2016-06-01

    The anaphase promoting complex/cyclosome (APC/C) orchestrates various aspects of the eukaryotic cell cycle. One of its co-activators, Cdh1, is subject to myriad post-translational modifications, such as phosphorylation and ubiquitination. Herein we identify the O-linked N-acetylglucosamine (O-GlcNAc) modification that occurs on Cdh1. Cdh1 is O-GlcNAcylated in cultured cells and mouse brain extracts. Mass spectrometry identifies an O-GlcNAcylated peptide that neighbors a known phosphorylation site. Cell synchronization and mutation studies reveal that O-GlcNAcylation of Cdh1 may antagonize its phosphorylation. Our results thus reveal a pivotal role of O-GlcNAcylation in regulating APC/C activity. PMID:27080259

  12. Insulin rapidly stimulates phosphorylation of a 46-kDa membrane protein on tyrosine residues as well as phosphorylation of several soluble proteins in intact fat cells

    SciTech Connect

    Haering, H.U.; White, M.F.; Machicao, F.; Ermel, B.; Schleicher, E.; Obermaier, B.

    1987-01-01

    It is speculated that the transmission of an insulin signal across the plasma membrane of cells occurs through activation of the tyrosine-specific receptor kinase, autophosphorylation of the receptor, and subsequent phosphorylation of unidentified substrates in the cell. In an attempt to identify possible substrates, the authors labeled intact rat fat cells with (/sup 32/P)orthophosphate and used an antiphosphotyrosine antibody to identify proteins that become phosphorylated on tyrosine residues in an insulin-stimulated way. In the membrane fraction of the fat cells, they found, in addition to the 95-kDa ..beta..-subunit of the receptor, a 46-kDa phosphoprotein that is phosphorylated exclusively on tyrosine residues. This protein is not immunoprecipitated by antibodies against different regions of the insulin receptor and its HPLC tryptic peptide map is different from the tryptic peptide map of the insulin receptor, suggesting that it is not derived from the receptor ..beta..-subunit. Insulin stimulates the tyrosine phosphorylation of the 46-kDa protein within 150 sec in the intact cell 3- to 4-fold in a dose-dependent way at insulin concentrations between 0.5 nM and 100 nM. Insulin (0.5 nM, 100 nM) stimulated within 2 min the /sup 32/P incorporation into a 116-kDa band, a 62 kDa band, and three bands between 45 kDa and 50 kDa 2- to 10-fold. They suggest that the 46-kDa membrane protein and possibly also the soluble proteins are endogenous substrates of the receptor tyrosine kinase in fat cells and that their phosphorylation is an early step in insulin signal transmission.

  13. Starch phosphorylation: insights and perspectives.

    PubMed

    Mahlow, Sebastian; Orzechowski, Sławomir; Fettke, Joerg

    2016-07-01

    During starch metabolism, the phosphorylation of glucosyl residues of starch, to be more precise of amylopectin, is a repeatedly observed process. This phosphorylation is mediated by dikinases, the glucan, water dikinase (GWD) and the phosphoglucan, water dikinase (PWD). The starch-related dikinases utilize ATP as dual phosphate donor transferring the terminal γ-phosphate group to water and the β-phosphate group selectively to either C6 position or C3 position of a glucosyl residue within amylopectin. By the collaborative action of both enzymes, the initiation of a transition of α-glucans from highly ordered, water-insoluble state to a less order state is realized and thus the initial process of starch degradation. Consequently, mutants lacking either GWD or PWD reveal a starch excess phenotype as well as growth retardation. In this review, we focus on the increased knowledge collected over the last years related to enzymatic properties, the precise definition of the substrates, the physiological implications, and discuss ongoing questions. PMID:27147464

  14. Structural and functional characterization of the phosphorylation-dependent interaction between PML and SUMO1.

    PubMed

    Cappadocia, Laurent; Mascle, Xavier H; Bourdeau, Véronique; Tremblay-Belzile, Samuel; Chaker-Margot, Malik; Lussier-Price, Mathieu; Wada, Junya; Sakaguchi, Kazuyasu; Aubry, Muriel; Ferbeyre, Gerardo; Omichinski, James G

    2015-01-01

    PML and several other proteins localizing in PML-nuclear bodies (PML-NB) contain phosphoSIMs (SUMO-interacting motifs), and phosphorylation of this motif plays a key role in their interaction with SUMO family proteins. We examined the role that phosphorylation plays in the binding of the phosphoSIMs of PML and Daxx to SUMO1 at the atomic level. The crystal structures of SUMO1 bound to unphosphorylated and tetraphosphorylated PML-SIM peptides indicate that three phosphoserines directly contact specific positively charged residues of SUMO1. Surprisingly, the crystal structure of SUMO1 bound to a diphosphorylated Daxx-SIM peptide indicate that the hydrophobic residues of the phosphoSIM bind in a manner similar to that seen with PML, but important differences are observed when comparing the phosphorylated residues. Together, the results provide an atomic level description of how specific acetylation patterns within different SUMO family proteins can work together with phosphorylation of phosphoSIM's regions of target proteins to regulate binding specificity. PMID:25497731

  15. Histone tyrosine phosphorylation comes of age

    PubMed Central

    Singh, Rakesh Kumar

    2011-01-01

    Histones were discovered over a century ago and have since been found to be the most extensively post-translationally modified proteins, although tyrosine phosphorylation of histones had remained elusive until recently. The year 2009 proved to be a landmark year for histone tyrosine (Y) phosphorylation as five research groups independently discovered this modification. Three groups describe phosphorylation of Y142 in the variant histone H2A.X, where it may be involved in the cellular decision making process to either undergo DNA repair or apoptosis in response to DNA damage. Further, one group suggests that phosphorylation of histone H3 on Y99 is crucial for its regulated proteolysis in yeast, while another found that Y41 phosphorylation modulates chromatin architecture and oncogenesis in mammalian cells. These pioneering studies provide the initial conceptual framework for further analyses of the diverse roles of tyrosine phosphorylation on different histones, with far reaching implications for human health and disease. PMID:20935492

  16. Prebiotic phosphorylation of nucleosides in formamide

    NASA Technical Reports Server (NTRS)

    Schoffstall, A. M.

    1976-01-01

    Results are presented for an experimental study intended to assess phosphorylation under neither aqueous nor dry thermal conditions. Instead, phosphorylations were attempted in possible nonaqueous prebiotic solvents. Formamide appeared to be the most obvious candidate for phosphorylation studies. Three main classes of phosphorylated products were formed in formamide solution: adenosine monophosphates, cyclic adenosine phosphate, and adenosine diphosphates. Experiments were designed to investigate the extent of phosphorylation of nucleosides in formamide, the relative amounts of nucleoside monophosphate, diphosphates and cyclic phosphate formed and the relative effectiveness of different sources of phosphate as phosphorylating agents in formamide. Reaction variables were temperature, nature of the phosphate or condensed phosphate, nucleoside, concentration of reactants and possible effects of additives. Product identification was based on qualitative and quantitative thin layer chromatography.

  17. Role of flanking sequences and phosphorylation in the recognition of the simian-virus-40 large T-antigen nuclear localization sequences by importin-alpha.

    PubMed Central

    Fontes, Marcos R M; Teh, Trazel; Toth, Gabor; John, Anna; Pavo, Imre; Jans, David A; Kobe, Bostjan

    2003-01-01

    The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser112 in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-alpha (Impalpha), we co-crystallized non-autoinhibited Impalpha with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Impalpha. We confirm the important role of flanking sequences using binding assays. Unexpectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Impalpha. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impalpha, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impalpha. PMID:12852786

  18. Coarse Graining to Investigate Membrane Induced Peptide Folding of Anticancer Peptides

    NASA Astrophysics Data System (ADS)

    Ganesan, Sai; Xu, Hongcheng; Matysiak, Silvina

    Information about membrane induced peptide folding mechanisms using all-atom molecular dynamics simulations is a challenge due to time and length scale issues.We recently developed a low resolution Water Explicit Polarizable PROtein coarse-grained Model by adding oppositely charged dummy particles inside protein backbone beads.These two dummy particles represent a fluctuating dipole,thus introducing structural polarization into the coarse-grained model.With this model,we were able to achieve significant α- β secondary structure content de novo,without any added bias.We extended the model to zwitterionic and anionic lipids,by adding oppositely charged dummy particles inside polar beads, to capture the ability of the head group region to form hydrogen bonds.We use zwitterionic POPC and anionic POPS as our model lipids, and a cationic anticancer peptide,SVS1,as our model peptide.We have characterized the driving forces for SVS1 folding on lipid bilayers with varying anionic and zwitterionic lipid compositions.Based on our results, dipolar interactions between peptide backbone and lipid head groups contribute to stabilize folded conformations.Cooperativity in folding is induced by both intra peptide and membrane-peptide interaction.

  19. Cation-cation interactions and cation exchange in a series of isostructural framework uranyl tungstates

    NASA Astrophysics Data System (ADS)

    Balboni, Enrica; Burns, Peter C.

    2014-05-01

    The isotypical compounds (UO2)3(WO6)(H2O)5 (1), Ag(UO2)3(WO6)(OH)(H2O)3 (2), K(UO2)3(WO6)OH(H2O)4 (3), Rb(UO2)3(WO6)(OH)(H2O)3.5 (4), and Cs(UO2)3(WO6)OH(H2O)3 (5) were synthesized, characterized, and their structures determined. Each crystallizes in space group Cc. (1): a=12.979 (3), b=10.238 (2), c=11.302 (2), β=102.044 (2); (2): a=13.148 (2), b=9.520 (1), c=11.083 (2), β=101.568 (2); (3): a=13.111 (8), b=9.930 (6), c=11.242 (7), β=101.024 (7); (4): a=12.940 (2), b=10.231 (2), c=11.259(2), β=102.205 (2); (5): a=12.983 (3), b=10.191 (3), c=11.263 (4), β=101.661 (4). Compounds 1-5 are a framework of uranyl and tungsten polyhedra containing cation-cation interactions. The framework has three symmetrically distinct U(VI) cations, one tungsten, sixteen to eighteen oxygen atoms, and in 2-5, one monovalent cation. Each atom occupies a general position. Each U(VI) cation is present as a typical (UO2)2+ uranyl ion in an overall pentagonal bipyramidal coordination environment. Each pentagonal bipyramid shares two equatorial edges with two other pentagonal bipyramids, forming a trimer. Trimers are connected into chains by edge-sharing with WO6 octahedra. Chains are linked through cation-cation interactions between two symmetrically independent uranyl ions. This yields a remarkably complex system of intersecting channels that extend along [0 0 1] and [-1 1 0]. The cation exchange properties of 2 and 3 were characterized at room temperature and at 140 °C.

  20. Reinventing cell penetrating peptides using glycosylated methionine sulfonium ion sequences

    DOE PAGESBeta

    Kramer, Jessica R.; Schmidt, Nathan W.; Mayle, Kristine M.; Kamei, Daniel T.; Wong, Gerard C.L.; Deming, Timothy J.

    2015-04-15

    Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess lowmore » cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs.« less

  1. Reinventing Cell Penetrating Peptides Using Glycosylated Methionine Sulfonium Ion Sequences

    PubMed Central

    2015-01-01

    Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess low cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs. PMID:27162954

  2. Reinventing cell penetrating peptides using glycosylated methionine sulfonium ion sequences

    SciTech Connect

    Kramer, Jessica R.; Schmidt, Nathan W.; Mayle, Kristine M.; Kamei, Daniel T.; Wong, Gerard C.L.; Deming, Timothy J.

    2015-04-15

    Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess low cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs.

  3. Phosphorylation and activation of calcineurin by glycogen synthase (casein) kinase-1 and cyclic AMP-dependent protein kinase

    SciTech Connect

    Singh, T.J.; Wang, J.H.

    1986-05-01

    Calcineurin is a phosphoprotein phosphatase that is activated by divalent cations and further stimulated by calmodulin. In this study calcineurin is shown to be a substrate for both glycogen synthase (casein) kinase-1 (CK-1) and cyclic AMP-dependent protein kinase (A-kinase). Either kinase can catalyze the incorporation of 1.0-1.4 mol /sup 32/P/mol calcineurin. Analysis by SDS-PAGE revealed that only the ..cap alpha.. subunit is phosphorylated. Phosphorylation of calcineurin by either kinase leads to its activation. Using p-nitrophenyl phosphate as a substrate the authors observed a 2-3 fold activation of calcineurin by either Mn/sup 2 +/ or Ni/sup 2 +/ (in the presence or absence of calmodulin) after phosphorylation of calcineurin by either CK-1 or A-kinase. In the absence of Mn/sup 2 +/ or Ni/sup 2 +/ phosphorylated calcineurin, like the nonphosphorylated enzyme, showed very little activity. Ni/sup 2 +/ was a more potent activator of phosphorylated calcineurin compared to Mn/sup 2 +/. Higher levels of activation (5-8 fold) of calcineurin by calmodulin was observed when phosphorylated calcineurin was pretreated with Ni/sup 2 +/ before measurement of phosphatase activity. These results indicate that phosphorylation may be an important mechanism by which calcineurin activity is regulated by Ca/sup 2 +/.

  4. [Sugar phosphorylation activities in acetogenic bacteria].

    PubMed

    Jiang, W; Patterson, J A

    1999-12-01

    Seven acetogenic bacteria (Acetitomaculum ruminis, Acetobacterium woodii, Eubacterium limosum as well as isolates A2, A4, A10 and H3HH) were tested for PEP- and ATP-dependent phosphorylation of glucose and 2-deoxyglucose. Although all organisms had detectable phosphorylation activity, substantial variation existed in the rates of both PEP- and ATP-dependent phosphorylation. Isolate Alo had the highest rate of PEP-dependent phosphorylation of 11.62 nmol.L-1.mg-1.min-1. Isolate A10, H3HH as well as E. limosum most likely have a glucose phosphotransferase system(PTS). In contrast, A ruminis, A. woodii and isolate A2, A4 had PEP-dependent glucose phosphorylation rates very similar to control rates, suggesting the lack of PTS activity. The rates of ATP-dependent glucose phosphorylation were higher than PEP-dependent phosphorylation in all organisms surveyed. However, substantial variation existed in the rates of ATP-dependent glucose phosphorylation. The glucose PTS of isolates A10 and H3HH were induced by the presence of extracellular glucose. Moreover, the specific activity of the glucose PTS of both isolates increased as cultures progressed from the early log to late log phase of growth. ATP- and PEP-dependent maltose and sucrose phosphorylation was detected in isolates A10 and H3HH. Although activity was detected in both isolates(A10 and H3HH), the rate of activity varied considerably, depending on the sugar and organism tested. PMID:12555560

  5. An unusual cell penetrating peptide identified using a plasmid display-based functional selection platform

    PubMed Central

    Gao, Shan; Simon, Melissa J.; Hue, Christopher D.; Morrison, Barclay; Banta, Scott

    2011-01-01

    Cell penetrating peptides (CPPs) have tremendous potential for use in gene and drug delivery applications. The selection of new CPPs with desired capabilities from randomized peptide libraries is challenging, since the CPP phenotype is a complex selection target. Here we report the discovery of an unusual new CPP from a randomized peptide library using a functional selection system based on plasmid display (PD). After four rounds of screening of a 14-mer peptide library over PC12 cells, several peptides were identified and tested for their ability to deliver the green fluorescent protein (GFP). One peptide (SG3) exhibited a cell penetrating phenotype, however unlike other well-known CPPs such as TAT or Penetratin, the newly identified peptide was not highly cationic. The PD protocol necessitated the addition of a cationic lipid (Lipofectamine2000), and in the presence of this compound, the SG3 peptide significantly outperformed the well-known TAT CPP in the delivery of GFP to PC12 cells and primary astrocytes. When the SG3 peptide was fused to the pro-apoptotic BH3 peptide from the Bak protein, significant cell death was induced in cultured primary astrocytes, indicating relevant, intracellular delivery of a functional cargo. The PD platform is a useful method for identifying functional new CPPs from randomized libraries with unique delivery capabilities. PMID:21291271

  6. Endosomolytic Nano-Polyplex Platform Technology for Cytosolic Peptide Delivery To Inhibit Pathological Vasoconstriction

    PubMed Central

    2016-01-01

    A platform technology has been developed and tested for delivery of intracellular-acting peptides through electrostatically complexed nanoparticles, or nano-polyplexes, formulated from an anionic endosomolytic polymer and cationic therapeutic peptides. This delivery platform has been initially tested and optimized for delivery of two unique vasoactive peptides, a phosphomimetic of heat shock protein 20 and an inhibitor of MAPKAP kinase II, to prevent pathological vasoconstriction (i.e., vasospasm) in human vascular tissue. These peptides inhibit vasoconstriction and promote vasorelaxation by modulating actin dynamics in vascular smooth muscle cells. Formulating these peptides into nano-polyplexes significantly enhances peptide uptake and retention, facilitates cytosolic delivery through a pH-dependent endosomal escape mechanism, and enhances peptide bioactivity in vitro as measured by inhibition of F-actin stress fiber formation. In comparison to treatment with the free peptides, which were endowed with cell-penetrating sequences, the nano-polyplexes significantly increased vasorelaxation, inhibited vasoconstriction, and decreased F-actin formation in the human saphenous vein ex vivo. These results suggest that these formulations have significant potential for treatment of conditions such as cerebral vasospasm following subarachnoid hemorrhage. Furthermore, because many therapeutic peptides include cationic cell-penetrating segments, this simple and modular platform technology may have broad applicability as a cost-effective approach for enhancing the efficacy of cytosolically active peptides. PMID:26004140

  7. The role of antimicrobial peptides in animal defenses

    NASA Astrophysics Data System (ADS)

    Hancock, Robert E. W.; Scott, Monisha G.

    2000-08-01

    It is becoming clear that the cationic antimicrobial peptides are an important component of the innate defenses of all species of life. Such peptides can be constitutively expressed or induced by bacteria or their products. The best peptides have good activities vs. a broad range of bacterial strains, including antibiotic-resistant isolates. They kill very rapidly, do not easily select resistant mutants, are synergistic with conventional antibiotics, other peptides, and lysozyme, and are able to kill bacteria in animal models. It is known that bacterial infections, especially when treated with antibiotics, can lead to the release of bacterial products such as lipopolysaccharide (LPS) and lipoteichoic acid, resulting in potentially lethal sepsis. In contrast to antibiotics, the peptides actually prevent cytokine induction by bacterial products in tissue culture and human blood, and they block the onset of sepsis in mouse models of endotoxemia. Consistent with this, transcriptional gene array experiments using a macrophage cell line demonstrated that a model peptide, CEMA, blocks the expression of many genes whose transcription was induced by LPS. The peptides do this in part by blocking LPS interaction with the serum protein LBP. In addition, CEMA itself has a direct effect on macrophage gene expression. Because cationic antimicrobial peptides are induced by LPS and are able to dampen the septic response of animal cells to LPS, we propose that, in addition to their role in direct and lysozyme-assisted killing of microbes, they have a role in feedback regulation of cytokine responses. We are currently developing variant peptides as therapeutics against antibiotic-resistant infections.

  8. A Sliding Docking Interaction Is Essential for Sequential and Processive Phosphorylation of an SR Protein by SRPK1

    SciTech Connect

    Ngo, Jacky Chi Ki; Giang, Kayla; Chakrabarti, Sutapa; Ma, Chen-Ting; Huynh, Nhat; Hagopian, Jonathan C.; Dorrestein, Pieter C.; Fu, Xiang-Dong; Adams, Joseph A.; Ghosh, Gourisankar

    2008-09-17

    The 2.9 {angstrom} crystal structure of the core SRPK1:ASF/SF2 complex reveals that the N-terminal half of the basic RS domain of ASF/SF2, which is destined to be phosphorylated, is bound to an acidic docking groove of SRPK1 distal to the active site. Phosphorylation of ASF/SF2 at a single site in the C-terminal end of the RS domain generates a primed phosphoserine that binds to a basic site in the kinase. Biochemical experiments support a directional sliding of the RS peptide through the docking groove to the active site during phosphorylation, which ends with the unfolding of a {beta} strand of the RRM domain and binding of the unfolded region to the docking groove. We further suggest that the priming of the first serine facilitates directional substrate translocation and efficient phosphorylation.

  9. Competitive binding of antagonistic peptides fine-tunes stomatal patterning

    PubMed Central

    Lee, Jin Suk; Hnilova, Marketa; Maes, Michal; Lin, Ya-Chen Lisa; Putarjunan, Aarthi; Han, Soon-Ki; Avila, Julian; U.Torii, Keiko

    2015-01-01

    During development, cells interpret complex, often conflicting signals to make optimal decisions. Plant stomata, the cellular interface between a plant and the atmosphere, develop according to positional cues including a family of secreted peptides, EPIDERMAL PATTERNING FACTORS (EPFs). How these signaling peptides orchestrate pattern formation at a molecular level remains unclear. Here we report that Stomagen/EPF-LIKE9 peptide, which promotes stomatal development, requires ERECTA (ER)-family receptor kinases and interferes with the inhibition of stomatal development by the EPF2-ER module. Both EPF2 and Stomagen directly bind to ER and its co-receptor TOO MANY MOUTHS. Stomagen peptide competitively replaced EPF2 binding to ER. Furthermore, application of EPF2, but not Stomagen, elicited rapid phosphorylation of downstream signaling components in vivo. Our findings demonstrate how a plant receptor agonist and antagonist define inhibitory and inductive cues to fine-tune tissue patterning on the plant epidermis. PMID:26083750

  10. Antimicrobial Lactoferrin Peptides: The Hidden Players in the Protective Function of a Multifunctional Protein

    PubMed Central

    Sinha, Mau; Kaushik, Sanket; Kaur, Punit; Singh, Tej P.

    2013-01-01

    Lactoferrin is a multifunctional, iron-binding glycoprotein which displays a wide array of modes of action to execute its primary antimicrobial function. It contains various antimicrobial peptides which are released upon its hydrolysis by proteases. These peptides display a similarity with the antimicrobial cationic peptides found in nature. In the current scenario of increasing resistance to antibiotics, there is a need for the discovery of novel antimicrobial drugs. In this context, the structural and functional perspectives on some of the antimicrobial peptides found in N-lobe of lactoferrin have been reviewed. This paper provides the comparison of lactoferrin peptides with other antimicrobial peptides found in nature as well as interspecies comparison of the structural properties of these peptides within the native lactoferrin. PMID:23554820

  11. Phosphorylation of human link proteins

    SciTech Connect

    Oester, D.A.; Caterson, B.; Schwartz, E.R.

    1986-06-13

    Three link proteins of 48, 44 and 40 kDa were purified from human articular cartilage and identified with monoclonal anti-link protein antibody 8-A-4. Two sets of lower molecular weight proteins of 30-31 kDa and 24-26 kDa also contained link protein epitopes recognized by the monoclonal antibody and were most likely degradative products of the intact link proteins. The link proteins of 48 and 40 kDa were identified as phosphoproteins while the 44 kDa link protein did not contain /sup 32/P. The phosphorylated 48 and 40 kDa link proteins contained approximately 2 moles PO/sub 4//mole link protein.

  12. Thrombin Ca(2+)-dependently stimulates protein tyrosine phosphorylation in BC3H1 muscle cells.

    PubMed Central

    Offermanns, S; Bombien, E; Schultz, G

    1993-01-01

    The proteinase thrombin, known to act via heptahelical G-protein-coupled receptors, is a mitogenic agent for different cell types, including the mouse muscle cell line BC3H1. In this study, the effect of thrombin on tyrosine phosphorylation was examined using anti-phosphotyrosine antibodies. Thrombin was found to induce phosphorylation of 65-70 and 110-120 kDa proteins in BC3H1 cells. The effect of thrombin was concentration-dependent, being half-maximal and maximal at concentrations of 0.03 and 1 unit/ml respectively. The thrombin-induced increase in phosphorylation was rapid (< or = 10 s) and transient, with a peak response after about 1-2 min. The effect of thrombin could be mimicked by the thrombin receptor agonist peptide SFLLRN-NH2. Preincubation of cells with pertussis toxin (PT) had no effect on thrombin-induced tyrosine phosphorylation. Epidermal growth factor, platelet-derived growth factor and insulin stimulated tyrosine phosphorylation of different proteins, among which were 65-70 and 110-120 kDa proteins. The phorbol ester 12-myristate 13-acetate (PMA) as well as the Ca2+ ionophore A23187 both stimulated tyrosine phosphorylation of proteins identical to those phosphorylated by thrombin, suggesting that activation of protein kinase C (PKC) and elevation of the cytosolic Ca2+ concentration alone are sufficient to induce tyrosine phosphorylation. However, calphostin C and other PKC inhibitors, which completely inhibited tyrosine phosphorylation induced by PMA, had no influence on the effect of thrombin, whereas loading of cells with the intracellular Ca2+ chelator bis-(O-aminophenoxy)ethane-NNN'N'-tetra-acetic acid totally blocked thrombin-stimulated tyrosine phosphorylation. Thus tyrosine phosphorylation stimulated by thrombin is an early PT-insensitive cellular response which is either directly mediated by elevation of cytosolic Ca2+ concentration or by a presently unknown mechanism that requires an elevated cytosolic Ca2+ concentration. Images Figure 1

  13. Brain natriutetic peptide test

    MedlinePlus

    ... medlineplus.gov/ency/article/007509.htm Brain natriuretic peptide test To use the sharing features on this page, please enable JavaScript. Brain natriuretic peptide (BNP) test is a blood test that measures ...

  14. Vasoactive intestinal peptide test

    MedlinePlus

    ... medlineplus.gov/ency/article/003508.htm Vasoactive intestinal peptide test To use the sharing features on this page, please enable JavaScript. Vasoactive intestinal peptide (VIP) is a test that measures the amount ...

  15. Influence of 63Ser phosphorylation and dephosphorylation on the structure of the stathmin helical nucleation sequence: a molecular dynamics study.

    PubMed

    Missimer, John H; Steinmetz, Michel O; van Gunsteren, Wilfred F; Dolenc, Jožica

    2012-10-23

    Phosphorylation is an important mechanism regulating protein-protein interactions involving intrinsically disordered protein regions. Stathmin, an archetypical example of an intrinsically disordered protein, is a key regulator of microtubule dynamics in which phosphorylation of 63Ser within the helical nucleation sequence strongly down-regulates the tubulin binding and microtubule destabilizing activities of the protein. Experimental studies on a peptide encompassing the 19-residue helical nucleation sequence of stathmin (residues 55-73) indicate that phosphorylation of 63Ser destabilizes the peptide's secondary structure by disrupting the salt bridges supporting its helical conformation. In order to investigate this hypothesis at atomic resolution, we performed molecular dynamics simulations of nonphosphorylated and phosphorylated stathmin-[55-73] at room temperature and pressure, neutral pH, and explicit solvation using the recently released GROMOS force field 54A7. In the simulations of nonphosphorylated stathmin-[55-73] emerged salt bridges associated with helical configurations. In the simulations of 63Ser phosphorylated stathmin-[55-73] these configurations dispersed and were replaced by a proliferation of salt bridges yielding disordered configurations. The transformation of the salt bridges was accompanied by emergence of numerous interactions between main and side chains, involving notably the oxygen atoms of the phosphorylated 63Ser. The loss of helical structure induced by phosphorylation is reversible, however, as a final simulation showed. The results extend the hypothesis of salt bridge derangement suggested by experimental observations of the stathmin nucleation sequence, providing new insights into regulation of intrinsically disordered protein systems mediated by phosphorylation. PMID:22978582

  16. Halogenated silanes, radicals, and cations

    NASA Astrophysics Data System (ADS)

    Wang, Liming; He, Yi-Liang

    2008-09-01

    Quantum chemistry study has been carried out on the structure and energetics of halogenated silanes, radicals, and cations (SiHxXy0,+1, X = F, Cl, Br; x + y = 1-4). The geometries are optimized at B3LYP/6-31+G(2df,p) level. The adiabatic ionization energiess (IEas), relative energetics of cations, proton affinities (PAs) of silanes, and the enthalpies of formation are predicted using G3(CC) model chemistry. Non-classical ion complex structures are found for hydrogenated cations and transition states connecting classical and non-classical structures are also located. The most stable cations for silylene and silyl radicals have their classical divalent and trivalent structures, and those for silanes have non-classical structures except for SiH3Br+ and SiH2Br2+. The non-classical structures for halosilane cations imply difficulty in experimentally measurement of the adiabatic ionization energies using photoionization or photoelectron studies. For SiH3X, SiH2X2, and SiHX3, the G3(CC) adiabatic IEas to classical ionic structures closest to their neutrals agree better with the photoelectron spectroscopic measurements. The transition states between classical and non-classical structures also hamper the photoionization determination of the appearance energies for silylene cations from silanes. The G3(CC) results for SiHx0,+1 agree excellently with the photoionization mass spectrometric study, and the results for fluorinated and chlorinated species also agree with the previous theoretical predictions at correlation levels from BAC-MP4 to CCSD(T)/CBS. The predicted enthalpy differences between SiH2Cl+, SiHCl2+, and SiCl3+ are also in accordance with previous kinetics study. The G3(CC) results show large discrepancies to the collision-induced charge transfer and/or dissociation reactions involving SiFx+ and SiClx+ ions, for which the G3(CC) enthalpies of formation are also significantly differed from the previous theoretical predictions, especially on SiFx+ (x = 2-4). The G3

  17. [SYNTHETIC PEPTIDE VACCINES].

    PubMed

    Sergeyev, O V; Barinsky, I F

    2016-01-01

    An update on the development and trials of synthetic peptide vaccines is reviewed. The review considers the successful examples of specific protection as a result of immunization with synthetic peptides using various protocols. The importance of conformation for the immunogenicity of the peptide is pointed out. An alternative strategy of the protection of the organism against the infection using synthetic peptides is suggested. PMID:27145593

  18. Modular evolution of phosphorylation-based signalling systems.

    PubMed

    Jin, Jing; Pawson, Tony

    2012-09-19

    Phosphorylation sites are formed by protein kinases ('writers'), frequently exert their effects following recognition by phospho-binding proteins ('readers') and are removed by protein phosphatases ('erasers'). This writer-reader-eraser toolkit allows phosphorylation events to control a broad range of regulatory processes, and has been pivotal in the evolution of new functions required for the development of multi-cellular animals. The proteins that comprise this system of protein kinases, phospho-binding targets and phosphatases are typically modular in organization, in the sense that they are composed of multiple globular domains and smaller peptide motifs with binding or catalytic properties. The linkage of these binding and catalytic modules in new ways through genetic recombination, and the selection of particular domain combinations, has promoted the evolution of novel, biologically useful processes. Conversely, the joining of domains in aberrant combinations can subvert cell signalling and be causative in diseases such as cancer. Major inventions such as phosphotyrosine (pTyr)-mediated signalling that flourished in the first multi-cellular animals and their immediate predecessors resulted from stepwise evolutionary progression. This involved changes in the binding properties of interaction domains such as SH2 and their linkage to new domain types, and alterations in the catalytic specificities of kinases and phosphatases. This review will focus on the modular aspects of signalling networks and the mechanism by which they may have evolved. PMID:22889906

  19. Sensitive Targeted Quantification of ERK Phosphorylation Dynamics and Stoichiometry in Human Cells without Affinity Enrichment

    DOE PAGESBeta

    Shi, Tujin; Gao, Yuqian; Gaffrey, Matthew J.; Nicora, Carrie D.; Fillmore, Thomas L.; Chrisler, William B.; Gritsenko, Marina A.; Wu, Chaochao; He, Jintang; Bloodsworth, Kent J.; et al

    2014-12-17

    Mass spectrometry-based targeted quantification is a promising technology for site-specific quantification of posttranslational modifications (PTMs). However, a major constraint of most targeted MS approaches is the limited sensitivity for quantifying low-abundance PTMs, requiring the use of affinity reagents to enrich specific PTMs. Herein, we demonstrate the direct site-specific quantification of ERK phosphorylation isoforms (pT, pY, pTpY) and their relative stoichiometries using a highly sensitive targeted MS approach termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM). PRISM provides effective enrichment of target peptides within a given fraction from complex biological matrix with minimal sample losses, followed by selected reactionmore » monitoring (SRM) quantification. The PRISM-SRM approach enabled direct quantification of ERK phosphorylation in human mammary epithelial cells (HMEC) from as little as 25 µg tryptic peptides from whole cell lysates. Compared to immobilized metal-ion affinity chromatography, PRISM provided >10-fold improvement in signal intensities, presumably due to the better peptide recovery of PRISM for handling small size samples. This approach was applied to quantify ERK phosphorylation dynamics in HMEC treated by different doses of EGF at both the peak activation (10 min) and steady state (2 h). At 10 min, the maximal ERK activation was observed with 0.3 ng/mL dose, whereas the maximal steady state level of ERK activation at 2 h was at 3 ng/ml dose, corresponding to 1200 and 9000 occupied receptors, respectively. At 10 min, the maximally activated pTpY isoform represented ~40% of total ERK, falling to less than 10% at 2 h. The time course and dose-response profiles of individual phosphorylated ERK isoforms indicated that singly phosphorylated pT-ERK never increases significantly, while the increase of pY-ERK paralleled that of pTpY-ERK. This data supports for a processive, rather than

  20. Sensitive Targeted Quantification of ERK Phosphorylation Dynamics and Stoichiometry in Human Cells without Affinity Enrichment

    SciTech Connect

    Shi, Tujin; Gao, Yuqian; Gaffrey, Matthew J.; Nicora, Carrie D.; Fillmore, Thomas L.; Chrisler, William B.; Gritsenko, Marina A.; Wu, Chaochao; He, Jintang; Bloodsworth, Kent J.; Zhao, Rui; Camp II, David G.; Liu, Tao; Rodland, Karin D.; Smith, Richard D.; Wiley, H. Steven; Qian, Weijun

    2014-12-17

    Mass spectrometry-based targeted quantification is a promising technology for site-specific quantification of posttranslational modifications (PTMs). However, a major constraint of most targeted MS approaches is the limited sensitivity for quantifying low-abundance PTMs, requiring the use of affinity reagents to enrich specific PTMs. Herein, we demonstrate the direct site-specific quantification of ERK phosphorylation isoforms (pT, pY, pTpY) and their relative stoichiometries using a highly sensitive targeted MS approach termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM). PRISM provides effective enrichment of target peptides within a given fraction from complex biological matrix with minimal sample losses, followed by selected reaction monitoring (SRM) quantification. The PRISM-SRM approach enabled direct quantification of ERK phosphorylation in human mammary epithelial cells (HMEC) from as little as 25 µg tryptic peptides from whole cell lysates. Compared to immobilized metal-ion affinity chromatography, PRISM provided >10-fold improvement in signal intensities, presumably due to the better peptide recovery of PRISM for handling small size samples. This approach was applied to quantify ERK phosphorylation dynamics in HMEC treated by different doses of EGF at both the peak activation (10 min) and steady state (2 h). At 10 min, the maximal ERK activation was observed with 0.3 ng/mL dose, whereas the maximal steady state level of ERK activation at 2 h was at 3 ng/ml dose, corresponding to 1200 and 9000 occupied receptors, respectively. At 10 min, the maximally activated pTpY isoform represented ~40% of total ERK, falling to less than 10% at 2 h. The time course and dose-response profiles of individual phosphorylated ERK isoforms indicated that singly phosphorylated pT-ERK never increases significantly, while the increase of pY-ERK paralleled that of pTpY-ERK. This data supports for a processive, rather than