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Sample records for phosphorylated peptide cations

  1. Formation and Dissociation of Phosphorylated Peptide Radical Cations

    NASA Astrophysics Data System (ADS)

    Kong, Ricky P. W.; Quan, Quan; Hao, Qiang; Lai, Cheuk-Kuen; Siu, Chi-Kit; Chu, Ivan K.

    2012-12-01

    In this study, we generated phosphoserine- and phosphothreonine-containing peptide radical cations through low-energy collision-induced dissociation (CID) of the ternary metal-ligand phosphorylated peptide complexes [CuII(terpy) p M]·2+ and [CoIII(salen) p M]·+ [ p M: phosphorylated angiotensin III derivative; terpy: 2,2':6',2''-terpyridine; salen: N, N '-ethylenebis(salicylideneiminato)]. Subsequent CID of the phosphorylated peptide radical cations ( p M·+) revealed fascinating gas-phase radical chemistry, yielding (1) charge-directed b- and y-type product ions, (2) radical-driven product ions through cleavages of peptide backbones and side chains, and (3) different degrees of formation of [M - H3PO4]·+ species through phosphate ester bond cleavage. The CID spectra of the p M·+ species and their non-phosphorylated analogues featured fragment ions of similar sequence, suggesting that the phosphoryl group did not play a significant role in the fragmentation of the peptide backbone or side chain. The extent of neutral H3PO4 loss was influenced by the peptide sequence and the initial sites of the charge and radical. A preliminary density functional theory study, at the B3LYP 6-311++G(d,p) level of theory, of the neutral loss of H3PO4 from a prototypical model— N-acetylphosphorylserine methylamide—revealed several factors governing the elimination of neutral phosphoryl groups through charge- and radical-induced mechanisms.

  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.

  3. Mechanisms of fragmentation of cationic peptide ions

    NASA Astrophysics Data System (ADS)

    Zhao, Hong; Adams, Jeanette

    1993-06-01

    Fragmentation mechanisms for formation of several commonly occurring product ions in high-energy collision-induced induced decomposition spectra of either (M + Cat2+ - H)+ ions of peptides cationized with alkaline earth metal ions, (M + Ca+)+ ions cationized with alkali metal ions, or (M + H)+ ions are evaluated by using deuterium-labelled peptides. The different sources of hydrogen transferred in the reactions are identified. Our study supports some previously proposed mechanisms but also provides evidence for others.

  4. Fragmentation of phosphorylated and singly charged peptide ions via interaction with metastable atoms

    PubMed Central

    Berkout, Vadym D.; Doroshenko, Vladimir M.

    2008-01-01

    Fragmentation of phosphorylated peptide ions via interaction with electronically excited metastable argon atoms was studied in a linear trap – time-of-flight mass spectrometer. Doubly charged ions of phosphorylated peptides from an Enolase digest were produced by electrospray ionization and subjected to a metastable atom beam in the linear trap. The metastable argon atoms were generated using a glow-discharge source. An intensive series of c- and z- ions were observed in all cases, with the phosphorylation group intact. The formation of molecular radical cations with reduced charge indicated that an electron transfer from a highly excited metastable state of argon to the peptide cation occurred. Additionally, singly charged Bradykinin, Substance P and Fibrinopeptide A molecular ions were fragmented via interaction with electronically excited metastable helium atoms. The fragmentation mechanism was different in this case and involved Penning ionization. PMID:19956340

  5. Fragmentation of phosphorylated and singly charged peptide ions via interaction with metastable atoms.

    PubMed

    Berkout, Vadym D; Doroshenko, Vladimir M

    2008-12-01

    Fragmentation of phosphorylated peptide ions via interaction with electronically excited metastable argon atoms was studied in a linear trap - time-of-flight mass spectrometer. Doubly charged ions of phosphorylated peptides from an Enolase digest were produced by electrospray ionization and subjected to a metastable atom beam in the linear trap. The metastable argon atoms were generated using a glow-discharge source. An intensive series of c- and z- ions were observed in all cases, with the phosphorylation group intact. The formation of molecular radical cations with reduced charge indicated that an electron transfer from a highly excited metastable state of argon to the peptide cation occurred. Additionally, singly charged Bradykinin, Substance P and Fibrinopeptide A molecular ions were fragmented via interaction with electronically excited metastable helium atoms. The fragmentation mechanism was different in this case and involved Penning ionization.

  6. Cationic Hydrophobic Peptides with Antimicrobial Activity

    PubMed Central

    Stark, Margareta; Liu, Li-Ping; Deber, Charles M.

    2002-01-01

    The MICs of cationic, hydrophobic peptides of the prototypic sequence KKAAAXAAAAAXAAWAAXAAAKKKK-amide (where X is one of the 20 commonly occurring amino acids) are in a low micromolar range for a panel of gram-negative and gram-positive bacteria, with no or low hemolytic activity against human and rabbit erythrocytes. The peptides are active only when the average segmental hydrophobicity of the 19-residue core is above an experimentally determined threshold value (where X is Phe, Trp, Leu, Ile, Met, Val, Cys, or Ala). Antimicrobial activity could be increased by using peptides that were truncated from the prototype length to 11 core residues, with X being Phe and with 6 Lys residues grouped at the N terminus. We propose a mechanism for the interaction between these peptides and bacterial membranes similar to the “carpet model,” wherein the Lys residues interact with the anionic phospholipid head groups in the bacterial membrane surface and the hydrophobic core portion of the peptide is then able to interact with the lipid bilayer, causing disruption of the bacterial membrane. PMID:12384369

  7. BT cationic peptides: small peptides that modulate innate immune responses of chicken heterophils and monocytes.

    PubMed

    Kogut, Michael H; Genovese, Kenneth J; He, Haiqi; Swaggerty, Christina L; Jiang, Yi Wei

    2012-01-15

    Neonatal poultry exhibit a transient susceptibility to infectious diseases during the first week of life that stems from inefficient host defense mechanisms. Yet, the initial host immune response to pathogens is a critical determinant of disease resistance and susceptibility. With this context in mind, novel ways to stimulate or modulate the hosts' natural immune response is emerging as an important area of interest for food animal producers including the poultry industry. Specifically, we have been investigating new modulation strategies tailored around the selective stimulation of the host's immune system, and particularly rapid acting innate immunity, as an alternative to direct targeting of microbial pathogens. One such approach that we have been investigating is the use of a group of cationic peptides produced by a Gram-positive soil bacterium, Brevibacillus texasporus (BT peptides). We have previously shown that, provided as a feed additive, BT peptides significantly induced a concentration-dependent protection against cecal colonization and extraintestinal colonization by Salmonella enterica serovar Enteritidis (SE). This protection is not the result of direct antibacterial activity of the BT peptides on the SE since the concentrations used were below the minimum inhibitory concentration for SE. We also found that BT are not absorbed in the intestine, but still induce a significant up-regulation in the functional efficiency of peripheral blood heterophils and monocytes. The mechanisms of this immune modulation are unknown. Here, using in vitro models for measuring: (1) leukocyte oxidative burst, (2) changes in leukocyte cytokine and chemokines gene expression profiles, and (3) phosphorylation of the mitogen activated protein kinases (MAPKs) in leukocytes, we evaluated the role of BT peptides as priming mediators for heterophil and monocyte responses at the level of cell function, gene transcription/expression, and cell phosphorylation following stimulation

  8. 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.

  9. Antiendotoxin activity of cationic peptide antimicrobial agents.

    PubMed

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

    1996-12-01

    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.

  10. 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

  11. Cationic antimicrobial peptides in penaeid shrimp.

    PubMed

    Tassanakajon, Anchalee; Amparyup, Piti; Somboonwiwat, Kunlaya; Supungul, Premruethai

    2011-08-01

    Penaeid shrimp aquaculture has been consistently affected worldwide by devastating diseases that cause a severe loss in production. To fight a variety of harmful microbes in the surrounding environment, particularly at high densities (of which intensive farming represents an extreme example), shrimps have evolved and use a diverse array of antimicrobial peptides (AMPs) as part of an important first-line response of the host defense system. Cationic AMPs in penaeid shrimps composed of penaeidins, crustins, and anti-lipopolysaccharide factors are comprised of multiple classes or isoforms and possess antibacterial and antifungal activities against different strains of bacteria and fungi. Shrimp AMPs are primarily expressed in circulating hemocytes, which is the main site of the immune response, and hemocytes expressing AMPs probably migrate to infection sites to fight against pathogen invasion. Indeed, most AMPs are produced as early as the nauplii developmental stage to protect shrimp larvae from infections. In this review, we discuss the sequence diversity, expression, gene structure, and antimicrobial activities of cationic AMPs in penaeid shrimps. The information available on antimicrobial activities indicates that these shrimp AMPs have potential therapeutic applications in the control of disease problems in aquaculture.

  12. The role of the VQIVYK peptide in tau protein phosphorylation.

    PubMed

    Perez, Mar; Santa-María, Ismael; Tortosa, Elena; Cuadros, Raquel; Del Valle, Mercedes; Hernández, Felix; Moreno, Francisco J; Avila, Jesús

    2007-11-01

    Although it remains unclear whether they are related to one another, tau aggregation and phosphorylation are the main pathological hallmarks of the neuronal disorders known as tauopathies. The capacity to aggregate is impaired in a variant of the tau 3R isoform that lacks residues 306-311 (nomenclature for the largest CNS tau isoform) and hence, we have taken advantage of this feature to study how phosphorylation and aggregation may be related as well as the role of this six amino acid peptide (VQIVYK). Through these analyses, we found that the phosphorylation of the tau variant was higher than that of the complete tau protein and that not only the deletion of these residues, but also the interaction of these residues, in tau 3R, with thioflavin-S augmented tau phosphorylation by glycogen synthase kinase 3. In addition, the binding of the peptide containing the residues 306-311 to the whole tau protein provoked an increase in tau phosphorylation. This observation could be physiologically relevant as may suggest that tau-tau interactions, through those residues, facilitate tau phosphorylation. In summary, our data indicate that deletion of residues VQIVYK, in tau protein produces an increase in tau phosphorylation, without tau aggregation, because the VQIVYK peptide, that favors aggregation, is missing. On the other hand, when the whole tau protein interacts with thioflavin-S or the peptide VQIVYK, an increase in both aggregation and phosphorylation occurs.

  13. Phosphorescent sensor for phosphorylated peptides based on an iridium complex.

    PubMed

    Kang, Jung Hyun; Kim, Hee Jin; Kwon, Tae-Hyuk; Hong, Jong-In

    2014-07-03

    A bis[(4,6-difluorophenyl)pyridinato-N,C(2')]iridium(III) picolinate (FIrpic) derivative coupled with bis(Zn(2+)-dipicolylamine) (ZnDPA) was developed as a sensor (1) for phosphorylated peptides, which are related to many cellular mechanisms. As a control, a fluorescent sensor (2) based on anthracene coupled to ZnDPA was also prepared. When the total negative charge on the phosphorylated peptides was changed to -2, -4, and -6, the emission intensity of sensor 1 gradually increased by factors of up to 7, 11, and 16, respectively. In contrast, there was little change in the emission intensity of sensor 1 upon the addition of a neutral phosphorylated peptide, non-phosphorylated peptides, or various anions such as CO3(2-), NO3(-), SO4(2-), phosphate, azide, and pyrophosphate. Furthermore, sensor 1 could be used to visually discriminate between phosphorylated peptides and adenosine triphosphate in aqueous solution under a UV-vis lamp, unlike fluorescent sensor 2. This enhanced luminance of phosphorescent sensor 1 upon binding to a phosphorylated peptide is attributed to a reduction in the repulsion between the Zn(2+) ions due to the phenoxy anion, its strong metal-to-ligand charge transfer character, and a reduction in self-quenching.

  14. Functions of Cationic Host Defense Peptides in Immunity

    PubMed Central

    Hemshekhar, Mahadevappa; Anaparti, Vidyanand; Mookherjee, Neeloffer

    2016-01-01

    Cationic host defense peptides are a widely distributed family of immunomodulatory molecules with antimicrobial properties. The biological functions of these peptides include the ability to influence innate and adaptive immunity for efficient resolution of infections and simultaneous modulation of inflammatory responses. This unique dual bioactivity of controlling infections and inflammation has gained substantial attention in the last three decades and consequent interest in the development of these peptide mimics as immunomodulatory therapeutic candidates. In this review, we summarize the current literature on the wide range of functions of cationic host defense peptides in the context of the mammalian immune system. PMID:27384571

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    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 IICBGlc, 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.

  16. The dynamic action mechanism of small cationic antimicrobial peptides.

    PubMed

    Lopez Cascales, J J; Garro, A; Porasso, R D; Enriz, R D

    2014-10-21

    Antimicrobial peptides form part of the immune system as protection against the action of external pathogens. The differences that exist between mammalian and microbial cell membrane architectures are key aspects of the ability of these peptides to discriminate between pathogens and host cells. Given that the pathogen membrane is the non-specific target of these cationic peptides, different molecular mechanisms have been suggested to describe the rules that permit them to distinguish between pathogens and mammalian cells. In this context, and setting aside the old fashion idea that cationic peptides act through one mechanism alone, this work will provide insight into the molecular action mechanism of small antimicrobial peptides, based on molecular dynamics simulations of phospholipid bilayers that mimic different cell membrane architectures. After measuring different properties of these lipid bilayers, in the absence and presence of peptides, a four-step action mechanism was suggested on the basis of the formation of phospholipid rafts induced by the presence of these cationic peptides. Thus, this work shows how differences in the bending modulus (k(b)) of these lipid rafts and differences in the free energy profiles (ΔG(z)) associated with the insertion of these peptides into these lipid rafts are key aspects for explaining the action mechanism of these cationic peptides at the molecular level.

  17. Cationic Antimicrobial Peptide Resistance in Neisseria meningitidis

    PubMed Central

    Tzeng, Yih-Ling; Ambrose, Karita D.; Zughaier, Susu; Zhou, Xiaoliu; Miller, Yoon K.; Shafer, William M.; Stephens, David S.

    2005-01-01

    Cationic antimicrobial peptides (CAMPs) are important components of the innate host defense system against microbial infections and microbial products. However, the human pathogen Neisseria meningitidis is intrinsically highly resistant to CAMPs, such as polymyxin B (PxB) (MIC ≥ 512 μg/ml). To ascertain the mechanisms by which meningococci resist PxB, mutants that displayed increased sensitivity (≥4-fold) to PxB were identified from a library of mariner transposon mutants generated in a meningococcal strain, NMB. Surprisingly, more than half of the initial PxB-sensitive mutants had insertions within the mtrCDE operon, which encodes proteins forming a multidrug efflux pump. Additional PxB-sensitive mariner mutants were identified from a second round of transposon mutagenesis performed in an mtr efflux pump-deficient background. Further, a mutation in lptA, the phosphoethanolamine (PEA) transferase responsible for modification of the lipid A head groups, was identified to cause the highest sensitivity to PxB. Mutations within the mtrD or lptA genes also increased meningococcal susceptibility to two structurally unrelated CAMPs, human LL-37 and protegrin-1. Consistently, PxB neutralized inflammatory responses elicited by the lptA mutant lipooligosaccharide more efficiently than those induced by wild-type lipooligosaccharide. mariner mutants with increased resistance to PxB were also identified in NMB background and found to contain insertions within the pilMNOPQ operon involved in pilin biogenesis. Taken together, these data indicated that meningococci utilize multiple mechanisms including the action of the MtrC-MtrD-MtrE efflux pump and lipid A modification as well as the type IV pilin secretion system to modulate levels of CAMP resistance. The modification of meningococcal lipid A head groups with PEA also prevents neutralization of the biological effects of endotoxin by CAMP. PMID:16030233

  18. Doubling down on phosphorylation as a variable peptide modification.

    PubMed

    Cooper, Bret

    2016-09-01

    Some mass spectrometrists believe that searching for variable PTMs like phosphorylation of serine or threonine when using database-search algorithms to interpret peptide tandem mass spectra will increase false-positive matching. The basis for this is the premise that the algorithm compares a spectrum to both a nonphosphorylated peptide candidate and a phosphorylated candidate, which is double the number of candidates compared to a search with no possible phosphorylation. Hence, if the search space doubles, false-positive matching could increase accordingly as the algorithm considers more candidates to which false matches could be made. In this study, it is shown that the search for variable phosphoserine and phosphothreonine modifications does not always double the search space or unduly impinge upon the FDR. A breakdown of how one popular database-search algorithm deals with variable phosphorylation is presented.

  19. Preorganized Peptide Scaffolds as Mimics of Phosphorylated Proteins Binding Sites with a High Affinity for Uranyl.

    PubMed

    Starck, Matthieu; Sisommay, Nathalie; Laporte, Fanny A; Oros, Stéphane; Lebrun, Colette; Delangle, Pascale

    2015-12-07

    Cyclic peptides with two phosphoserines and two glutamic acids were developed to mimic high-affinity binding sites for uranyl found in proteins such as osteopontin, which is believed to be a privileged target of this ion in vivo. These peptides adopt a β-sheet structure that allows the coordination of the latter amino acid side chains in the equatorial plane of the dioxo uranyl cation. Complementary spectroscopic and analytical methods revealed that these cyclic peptides are efficient uranyl chelating peptides with a large contribution from the phosphorylated residues. The conditional affinity constants were measured by following fluorescence tryptophan quenching and are larger than 10(10) at physiological pH. These compounds are therefore promising models for understanding uranyl chelation by proteins, which is relevant to this actinide ion toxicity.

  20. 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

  1. Quality control of cationic cell-penetrating peptides.

    PubMed

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

    2016-01-05

    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.

  2. Inhibition of peptide aggregation by means of enzymatic phosphorylation

    PubMed Central

    Folmert, Kristin; Broncel, Malgorzata; v. Berlepsch, Hans; Ullrich, Christopher Hans; Siegert, Mary-Ann

    2016-01-01

    As is the case in numerous natural processes, enzymatic phosphorylation can be used in the laboratory to influence the conformational populations of proteins. In nature, this information is used for signal transduction or energy transfer, but has also been shown to play an important role in many diseases like tauopathies or diabetes. With the goal of determining the effect of phosphorylation on amyloid fibril formation, we designed a model peptide which combines structural characteristics of α-helical coiled-coils and β-sheets in one sequence. This peptide undergoes a conformational transition from soluble structures into insoluble amyloid fibrils over time and under physiological conditions and contains a recognition motif for PKA (cAMP-dependent protein kinase) that enables enzymatic phosphorylation. We have analyzed the pathway of amyloid formation and the influence of enzymatic phosphorylation on the different states along the conformational transition from random-coil to β-sheet-rich oligomers to protofilaments and on to insoluble amyloid fibrils, and we found a remarkable directing effect from β-sheet-rich structures to unfolded structures in the initial growth phase, in which small oligomers and protofilaments prevail if the peptide is phosphorylated. PMID:28144314

  3. 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...

  4. Alternative mechanisms of action of cationic antimicrobial peptides on bacteria.

    PubMed

    Hale, John D F; Hancock, Robert E W

    2007-12-01

    Cationic antimicrobial peptides are a novel type of antibiotic offering much potential in the treatment of microbial-related diseases. They offer many advantages for commercial development, including a broad spectrum of action and modest size. However, despite the identification or synthetic production of thousands of such peptides, the mode of action remains elusive, except for a few examples. While the dogma for the mechanism of action of antimicrobial peptides against bacteria is believed to be through pore formation or membrane barrier disruption, some peptides clearly act differently and other intracellular target sites have been identified. This article presents an updated review of how cationic antimicrobial peptides are able to affect bacterial killing, with a focus on internal targets.

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

    PubMed Central

    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 IICBGlc, 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

  6. S. Typhimurium strategies to resist killing by cationic antimicrobial peptides.

    PubMed

    Matamouros, Susana; Miller, Samuel I

    2015-11-01

    S. Typhimurium is a broad host range Gram-negative pathogen that must evade killing by host innate immune systems to colonize, replicate, cause disease, and be transmitted to other hosts. A major pathogenic strategy of Salmonellae is entrance, survival, and replication within eukaryotic cell phagocytic vacuoles. These phagocytic vacuoles and gastrointestinal mucosal surfaces contain multiple cationic antimicrobial peptides (CAMPs) which control invading bacteria. S. Typhimurium possesses several key mechanisms to resist killing by CAMPs which involve sensing CAMPs and membrane damage to activate signaling cascades that result in remodeling of the bacterial envelope to reduce its overall negative charge with an increase in hydrophobicity to decrease binding and effectiveness of CAMPs. Moreover Salmonellae have additional mechanisms to resist killing by CAMPs including an outer membrane protease which targets cationic peptides at the surface, and specific efflux pumps which protect the inner membrane from damage. This article is part of a Special Issue entitled: Bacterial Resistance to Antimicrobial Peptides.

  7. Determinants of recombinant production of antimicrobial cationic peptides and creation of peptide variants in bacteria.

    PubMed

    Zhang, L; Falla, T; Wu, M; Fidai, S; Burian, J; Kay, W; Hancock, R E

    1998-06-29

    Cationic peptides possessing antibacterial activity are virtually ubiquitous in nature, and offer exciting prospects as new therapeutic agents. We had previously demonstrated that such peptides could be produced by fusion protein technology in bacteria and several carrier proteins had been tested as fusion partners including glutathione-S-transferase, S. aureus protein A, IgG binding protein and P. aeruginosa outer membrane protein OprF. However these fusion partners, while successfully employed in peptide expression, were not optimized for high level production of cationic peptides (Piers, K., Brow, M. L., and Hancock, R. E. W. 1993, Gene 137, 7-13). In this paper we took advantage of a small replication protein RepA from E. coli and used its truncated version to construct fusion partners. The minimal elements required for high level expression of cationic peptide were defined as a DNA sequence encoding a fusion protein comprising, from the N-terminus, a 68 amino acid carrier region, an anionic prepro domain, a single methionine and the peptide of interest. The 68 amino acid carrier region was a block of three polypeptides consisting of a truncated RepA, a synthetic cellulose binding domain and a hexa histidine domain. The improved system showed high level expression and simplified downstream purification. The active peptide could be yielded by CNBr cleavage of the fusion protein. This novel vector was used to express three classes of cationic peptides including the alpha-helical peptide CEMA, the looped peptide bactenecin and the extended peptide indolicidin. In addition, mutagenesis of the peptide gene to produce peptide variants of CEMA and indolicidin using the improved vector system was shown to be successful.

  8. Novel cationic lipophilic peptides for oligodeoxynucleotide delivery.

    PubMed

    Chan, Enoch; Amon, Michael; Marano, Robert J; Wimmer, Norbert; Kearns, Philip S; Manolios, Nicholas; Rakoczy, P Elizabeth; Toth, Istvan

    2007-06-15

    In search of new oligodeoxynucleotide (ODN) delivery agents, we evaluated novel peptides derived from core peptide H-GLRILLLKV-OH (CP). CP is a fragment designed from the T-cell antigen receptor (TCR) alpha-chain transmembrane sequence. CP was able to enter cells including T-cells and inhibited interleukin-2 (IL-2) production. To examine the effect of increased lipophilicity on cellular uptake and activity of CP, a lipoamino acid (2-aminododecanoic acid) was incorporated into peptide CP resulting in 2-aminodecanoyl-CP (LP). The toxicity of CP and LP was assessed by measuring the haemolytic activity. Neither compound caused any haemolysis of red blood cells. We have also compared the biological activities of the CP and LP. Using a T-cell antigen presentation assay, the more lipophilic LP caused greater inhibition of IL-2 production than the parent CP in the antigen stimulated T-cells. The LP also showed increased permeability than CP in the Caco-2 cell assay. We utilised the enhanced cell permeability property of LP in oligodeoxynucleotide ODN1 delivery. Isothermal titration calorimetry (ITC) suggested that CP and LP complex with ODN1 in a 12:1 (CP:ODN1) and 15:1 (LP:ODN1) ratio. These complexes were then transfected into human retinal pigment epithelial cells. The level of transfection was measured by the decreased production of the protein human vascular endothelial growth factor (hVEGF). The results revealed greater transfection efficiency for both CP and LP (47%, 55% more inhibition) compared to commercially available transfection agent cytofectin GSV. These results suggested that the CP and particularly its lipophilic analogue LP have the potential to be used as oligodeoxynucleotide delivery systems.

  9. Synthesis and characterization of histidine-phosphorylated peptides.

    PubMed Central

    Medzihradszky, K. F.; Phillipps, N. J.; Senderowicz, L.; Wang, P.; Turck, C. W.

    1997-01-01

    Posttranslational phosphorylation of proteins is an important event in many cellular processes. Whereas phosphoesters of serine, threonine, and tyrosine have been studied extensively, only limited information is available for other amino acids modified by a phosphate group. The formation of phosphohistidine residues in proteins was discovered originally in prokaryotic organisms, but also has been found recently in eukaryotic cells. We describe methods for the synthesis and analysis of phosphohistidine-containing peptides, a prerequisite for the investigation of the role of this posttranslational modification in cellular processes. PMID:9232641

  10. Selective phosphorylation of cationic polypeptide aggregated with phosphatidylserine/diacylglycerol/Ca2+/detergent mixed micelles by Ca(2+)-independent but not Ca(2+)-dependent protein kinase C isozymes.

    PubMed

    Mahoney, C W; Huang, K P

    1995-03-14

    Mixed micelles containing Nonidet P40 (NP-40) (829 microM or 4.8 mM), phosphatidylserine (PS) (14.5 or 8 mol%), and 1,2-diacylglycerol (DG) (0.5 or 1 mol%) when preincubated with protein kinase C (PKC) assay mixture containing cationic substrate and CaCl2 (400 microM) formed aggregates in a time-, temperature-, and substrate concentration-dependent manner with a t1/2 approximately 3-12 min (22 degrees C). Concomitant with the formation of these aggregates there was a substantial loss of substrate phosphorylation catalyzed by the Ca(2+)-dependent PKC alpha, beta, and gamma but not the Ca(2+)-independent PKC, delta and epsilon. All cationic PKC substrates tested, neurogranin peptide analog, neurogranin, and histone III-S, formed aggregates with PS/DG/NP-40/Ca2+ mixed micelles in a time-dependent fashion. The poly(cationic-anionic) PKC substrate protamine sulfate also forms aggregates with the mixed micelles in the presence of Ca2+, but without affecting the substrate phosphorylation by the kinase. Under similar conditions, but at 4 degrees C, neither aggregation nor loss of cationic substrate phosphorylation was observed. Another nonionic detergent, octyl glucoside, behaved similarly to NP-40. Phosphatidylinositol (PI) and phosphatidylglycerol like PS, were effective in forming aggregates with NP-40/cationic polypeptide/DG/Ca2+ as monitored by light scattering, yet without affecting substrate phosphorylation. Phosphorylation of cationic substrates by M-kinase, derived from trypsinized PKC beta, was also greatly diminished by the aggregation. In contrast, [3H]phorbol 12,13-dibutyrate binding to PKC beta was unaffected. Formation of the aggregates that were selectively utilized by the Ca(2+)-independent PKCs was dependent on the ratio of cationic substrate to the number of mixed micelles.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Synthetic cationic amphiphilic α-helical peptides as antimicrobial agents.

    PubMed

    Wiradharma, Nikken; Khoe, Ulung; Hauser, Charlotte A E; Seow, See Voon; Zhang, Shuguang; Yang, Yi-Yan

    2011-03-01

    Antimicrobial peptides (AMPs) secreted by the innate immune system are prevalent as the effective first-line of defense to overcome recurring microbial invasions. They have been widely accepted as the blueprints for the development of new antimicrobial agents for the treatment of drug resistant infections. However, there is also a growing concern that AMPs with a sequence that is too close to the host organism's AMP may inevitably compromise its own natural defense. In this study, we design a series of synthetic (non-natural) short α-helical AMPs to expand the arsenal of the AMP families and to gain further insights on their antimicrobial activities. These cationic and amphiphilic peptides have a general sequence of (XXYY)(n) (X: hydrophobic residue, Y: cationic residue, and n: the number of repeat units), and are designed to mimic the folding behavior of the naturally-occurring α-helical AMPs. The synthetic α-helical AMPs with 3 repeat units, (FFRR)(3), (LLRR)(3), and (LLKK)(3), are found to be more selective towards microbial cells than rat red blood cells, with minimum inhibitory concentration (MIC) values that are more than 10 times lower than their 50% hemolytic concentrations (HC(50)). They are effective against Gram-positive B. subtilis and yeast C. albicans; and the studies using scanning electron microscopy (SEM) have elucidated that these peptides possess membrane-lytic activities against microbial cells. Furthermore, non-specific immune stimulation assays of a typical peptide shows negligible IFN-α, IFN-γ, and TNF-α inductions in human peripheral blood mononuclear cells, which implies additional safety aspects of the peptide for both systemic and topical use. Therefore, the peptides designed in this study can be promising antimicrobial agents against the frequently-encountered Gram-positive bacteria- or yeast-induced infections.

  12. 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

  13. An FPGA Implementation to Detect Selective Cationic Antibacterial Peptides

    PubMed Central

    Polanco González, Carlos; Nuño Maganda, Marco Aurelio; Arias-Estrada, Miguel; del Rio, Gabriel

    2011-01-01

    Exhaustive prediction of physicochemical properties of peptide sequences is used in different areas of biological research. One example is the identification of selective cationic antibacterial peptides (SCAPs), which may be used in the treatment of different diseases. Due to the discrete nature of peptide sequences, the physicochemical properties calculation is considered a high-performance computing problem. A competitive solution for this class of problems is to embed algorithms into dedicated hardware. In the present work we present the adaptation, design and implementation of an algorithm for SCAPs prediction into a Field Programmable Gate Array (FPGA) platform. Four physicochemical properties codes useful in the identification of peptide sequences with potential selective antibacterial activity were implemented into an FPGA board. The speed-up gained in a single-copy implementation was up to 108 times compared with a single Intel processor cycle for cycle. The inherent scalability of our design allows for replication of this code into multiple FPGA cards and consequently improvements in speed are possible. Our results show the first embedded SCAPs prediction solution described and constitutes the grounds to efficiently perform the exhaustive analysis of the sequence-physicochemical properties relationship of peptides. PMID:21738652

  14. An FPGA implementation to detect selective cationic antibacterial peptides.

    PubMed

    Polanco González, Carlos; Nuño Maganda, Marco Aurelio; Arias-Estrada, Miguel; del Rio, Gabriel

    2011-01-01

    Exhaustive prediction of physicochemical properties of peptide sequences is used in different areas of biological research. One example is the identification of selective cationic antibacterial peptides (SCAPs), which may be used in the treatment of different diseases. Due to the discrete nature of peptide sequences, the physicochemical properties calculation is considered a high-performance computing problem. A competitive solution for this class of problems is to embed algorithms into dedicated hardware. In the present work we present the adaptation, design and implementation of an algorithm for SCAPs prediction into a Field Programmable Gate Array (FPGA) platform. Four physicochemical properties codes useful in the identification of peptide sequences with potential selective antibacterial activity were implemented into an FPGA board. The speed-up gained in a single-copy implementation was up to 108 times compared with a single Intel processor cycle for cycle. The inherent scalability of our design allows for replication of this code into multiple FPGA cards and consequently improvements in speed are possible. Our results show the first embedded SCAPs prediction solution described and constitutes the grounds to efficiently perform the exhaustive analysis of the sequence-physicochemical properties relationship of peptides.

  15. Bivalent cation binding effect on formation of the peptide bond

    NASA Astrophysics Data System (ADS)

    Remko, Milan; Rode, Bernd Michael

    2000-01-01

    The reactions between formic acid (or glycine) and ammonia, without and with Mg 2+, Ni 2+ and Cu 2+ cations as catalysts, have been studied as model reactions for peptide bond formation using the Becke3LYP functional and 6-311+G(d,p) basis set of DFT theory. Enthalpies and free energies for the stationary points of each reaction have been calculated to determine the thermodynamics of reactions investigated. A substantial decrease in reaction enthalpies and free energies was found for formic acid-ammonia and glycine-ammonia reactions catalysed by Mg 2+, Ni 2+ and Cu 2+ ions compared with those of the uncatalysed amide bond formation. The catalytic effect of the transition metal ions Ni 2+ and Cu 2+ is of similar strength and more pronounced than that of the Mg 2+ cation.

  16. 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.

  17. Atomic force microscopy characterization of kinase-mediated phosphorylation of a peptide monolayer

    PubMed Central

    Zhuravel, Roman; Amit, Einav; Elbaz, Shir; Rotem, Dvir; Chen, Yu-Ju; Friedler, Assaf; Yitzchaik, Shlomo; Porath, Danny

    2016-01-01

    We describe the detailed microscopic changes in a peptide monolayer following kinase-mediated phosphorylation. A reversible electrochemical transformation was observed using square wave voltammetry (SWV) in the reversible cycle of peptide phosphorylation by ERK2 followed by dephosphorylation by alkaline phosphatase. A newly developed method for analyzing local roughness, measured by atomic force microscope (AFM), showed a bimodal distribution. This may indicate either a hole-formation mechanism and/or regions on the surface in which the peptide changed its conformation upon phosphorylation, resulting in increased roughness and current. Our results provide the mechanistic basis for developing biosensors for detecting kinase-mediated phosphorylation in disease. PMID:27841355

  18. Atomic force microscopy characterization of kinase-mediated phosphorylation of a peptide monolayer

    NASA Astrophysics Data System (ADS)

    Zhuravel, Roman; Amit, Einav; Elbaz, Shir; Rotem, Dvir; Chen, Yu-Ju; Friedler, Assaf; Yitzchaik, Shlomo; Porath, Danny

    2016-11-01

    We describe the detailed microscopic changes in a peptide monolayer following kinase-mediated phosphorylation. A reversible electrochemical transformation was observed using square wave voltammetry (SWV) in the reversible cycle of peptide phosphorylation by ERK2 followed by dephosphorylation by alkaline phosphatase. A newly developed method for analyzing local roughness, measured by atomic force microscope (AFM), showed a bimodal distribution. This may indicate either a hole-formation mechanism and/or regions on the surface in which the peptide changed its conformation upon phosphorylation, resulting in increased roughness and current. Our results provide the mechanistic basis for developing biosensors for detecting kinase-mediated phosphorylation in disease.

  19. Charge transfer dissociation (CTD) mass spectrometry of peptide cations using kiloelectronvolt helium cations.

    PubMed

    Hoffmann, William D; Jackson, Glen P

    2014-11-01

    A kiloelectronvolt beam of helium ions is used to ionize and fragment precursor peptide ions starting in the 1+ charge state. The electron affinity of helium cations (24.6 eV) exceeds the ionization potential of protonated peptides and can therefore be used to abstract an electron from--or charge exchange with--the isolated precursor ions. Kiloelectronvolt energies are used, (1) to overcome the Coulombic repulsion barrier between the cationic reactants, (2) to overcome ion-defocussing effects in the ion trap, and (3) to provide additional activation energy. Charge transfer dissociation (CTD) of the [M+H](+) precursor of Substance P gives product ions such as [M+H](2+•) and a dominant series of a ions in both the 1+ and 2+ charge states. These observations, along with the less-abundant a + 1 ions, are consistent with ultraviolet photodissociation (UVPD) results of others and indicate that C-C(α) cleavages are possible through charge exchange with helium ions. Although the efficiencies and timescale of CTD are not yet suitable for on-line chromatography, this new approach to ion activation provides an additional potential tool for the interrogation of gas phase ions.

  20. Cationic Antimicrobial Peptide Resistance Mechanisms of Streptococcal Pathogens

    PubMed Central

    LaRock, Christopher N.; Nizet, Victor

    2015-01-01

    Cationic antimicrobial peptides (CAMPs) are critical front line contributors to host defense against invasive bacterial infection. These immune factors have direct killing activity toward microbes, but many pathogens are able to resist their effects. Group A Streptococcus, group B Streptococcus and Streptococcus pneumoniae are among the most common pathogens of humans and display a variety of phenotypic adaptations to resist CAMPs. Common themes of CAMP resistance mechanisms among the pathogenic streptococci are repulsion, sequestration, export, and destruction. Each pathogen has a different array of CAMP-resistant mechanisms, with invasive disease potential reflecting the utilization of several mechanisms that may act in synergy. Here we discuss recent progress in identifying the sources of CAMP resistance in the medically important Streptococcus genus. Further study of these mechanisms can contribute to our understanding of streptococcal pathogenesis, and may provide new therapeutic targets for therapy and disease prevention. PMID:25701232

  1. Cationic antimicrobial peptides disrupt the Streptococcus pyogenes ExPortal.

    PubMed

    Vega, Luis Alberto; Caparon, Michael G

    2012-09-01

    Although they possess a well-characterized ability to porate the bacterial membrane, emerging research suggests that cationic antimicrobial peptides (CAPs) can influence pathogen behaviour at levels that are sublethal. In this study, we investigated the interaction of polymyxin B and human neutrophil peptide (HNP-1) with the human pathogen Streptococcus pyogenes. At sublethal 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.

  2. Adsorption of cationic peptides to solid surfaces of glass and plastic.

    PubMed

    Kristensen, Kasper; Henriksen, Jonas R; Andresen, Thomas L

    2015-01-01

    Cationic membrane-active peptides have been studied for years in the hope of developing them into novel types of therapeutics. In this article, we investigate an effect that might have significant experimental implications for investigators who wish to study these peptides, namely, that the peptides adsorb to solid surfaces of glass and plastic. Specifically, we use analytical HPLC to systematically quantify the adsorption of the three cationic membrane-active peptides mastoparan X, melittin, and magainin 2 to the walls of commonly used glass and plastic sample containers. Our results show that, at typical experimental peptide concentrations, 90% or more of the peptides might be lost from solution due to rapid adsorption to the walls of the sample containers. Thus, our results emphasize that investigators should always keep these adsorption effects in mind when designing and interpreting experiments on cationic membrane-active peptides. We conclude the article by discussing different strategies for reducing the experimental impact of these adsorption effects.

  3. Studies on cleavage of DNA by N-phosphoryl branched peptides.

    PubMed

    Feng, Yuping; Cao, Shengli; Xiao, Anshan; Xie, Wenjun; Li, Yanmei; Zhao, Yufen

    2006-06-01

    It was found that Nalpha,Nepsilon-di[N-(O,O-diisopropyl)phosphoryl-L-leucy]-L-lysyl-methyl ester (1) and Nalpha,Nepsilon-di[N-(O,O-diisopropyl)phosphoryl-L-phenylalanyl]-L-lysyl-methyl ester (2) could cleave supercoiled DNA such as PUC19 efficiently in 40 mM Britton-Robinson buffer. The cleavage activities for both were investigated by agarose gel electrophoresis. The T4 ligase experiments implied that the cleavage of DNA occurs via a hydrolytic path. The results showed that the cleavage reaction of DNA is dependent on the value of pH and ionic strength in the solution. DNA cleavage is more efficient by N-phosphoryl branched peptide 2 than by N-phosphoryl branched peptide 1. The experiments also show that hydrolysis of DNA by N-phosphoryl branched peptide 1 was accelerated in the presence of Mg2+ or Zn2+ ions. The interactions of DNA with N-phosphoryl branched peptides were also characterized by melting temperature measurements and circular dichroism (CD) techniques. On the basis of experimental data, the possible mechanism of interactions between DNA with N-phosphoryl branched peptides was discussed.

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

    PubMed

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

    2010-12-01

    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.

  5. Roles of Hydrophobicity and Charge Distribution of Cationic Antimicrobial Peptides in Peptide-Membrane Interactions*

    PubMed Central

    Yin, Lois M.; Edwards, Michelle A.; Li, Jessica; Yip, Christopher M.; Deber, Charles M.

    2012-01-01

    Cationic antimicrobial peptides (CAPs) occur as important innate immunity agents in many organisms, including humans, and offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to membrane lysis and eventually cell death. In this work, we studied the biophysical and microbiological characteristics of designed CAPs varying in hydrophobicity levels and charge distributions by a variety of biophysical and biochemical approaches, including in-tandem atomic force microscopy, attenuated total reflection-FTIR, CD spectroscopy, and SDS-PAGE. Peptide structural properties were correlated with their membrane-disruptive abilities and antimicrobial activities. In bacterial lipid model membranes, a time-dependent increase in aggregated β-strand-type structure in CAPs with relatively high hydrophobicity (such as KKKKKKALFALWLAFLA-NH2) was essentially absent in CAPs with lower hydrophobicity (such as KKKKKKAAFAAWAAFAA-NH2). Redistribution of positive charges by placing three Lys residues at both termini while maintaining identical sequences minimized self-aggregation above the dimer level. Peptides containing four Leu residues were destructive to mammalian model membranes, whereas those with corresponding Ala residues were not. This finding was mirrored in hemolysis studies in human erythrocytes, where Ala-only peptides displayed virtually no hemolysis up to 320 μm, but the four-Leu peptides induced 40–80% hemolysis at the same concentration range. All peptides studied displayed strong antimicrobial activity against Pseudomonas aeruginosa (minimum inhibitory concentrations of 4–32 μm). The overall findings suggest optimum routes to balancing peptide hydrophobicity and charge distribution that allow efficient penetration and disruption of the bacterial membranes without damage to mammalian (host) membranes. PMID:22253439

  6. Detection of tyrosine phosphorylated peptides via skimmer collision-induced dissociation/ion trap mass spectrometry.

    PubMed

    Zolodz, Melissa D; Wood, Karl V

    2003-03-01

    Phosphorylation of proteins is an important post-translational protein modification in cellular response to environmental change and occurs in both prokaryotes and eukaryotes. Identification of the amino acid on individual proteins that become phosphorylated in response to extracellular stimulus is essential for understanding the mechanisms involved in the intracellular signals that these modifications facilitate. Most protein kinases catalyze the phosphorylation of proteins on serine, threonine or tyrosine. Although tyrosine phosphorylation is often the least abundant of the three major phosphorylation sites, it is important owing to its role in signal pathways. Currently available methods for the identification of phosphorylation sites can often miss low levels of tyrosine phosphorylations. This paper describes a method for the identification of phosphotyrosine-containing peptides using electrospray ionization on an ion trap mass spectrometer. Skimmer-activated collision-induced dissociation (CID) was used to generate the phosphotyrosine immonium ion at m/z 216. This method is gentle enough that the protonated molecule of the intact peptide is still observed. In-trap CID was employed for the verification of the phosphotyrosine immonium ion. Using this technique, low levels of phosphotyrosine-containing peptides can be identified from peptide mixtures separated by nanoflow micro liquid chromatography/mass spectrometry.

  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.

    2016-05-01

    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.

  8. How Cancer Cells Become Resistant to Cationic Lytic Peptides: It's the Sugar!

    PubMed

    Pierce, Joshua G

    2017-02-16

    In this issue of Cell Chemical Biology, Ishikawa et al. (2017) demonstrate that the loss of cell-surface anionic saccharides can impart resistance toward anticancer peptides. This study provides the first insight into potential resistance mechanisms toward cationic lytic peptides and highlights the important, yet previously unappreciated, role cell-surface glycans can play in cellular resistance mechanisms.

  9. PhosCalc: A tool for evaluating the sites of peptide phosphorylation from Mass Spectrometer data

    PubMed Central

    MacLean, Daniel; Burrell, Michael A; Studholme, David J; Jones, Alexandra ME

    2008-01-01

    Background We have created a software implementation of a published and verified method for assigning probabilities to potential phosphorylation sites on peptides using mass spectrometric data. Our tool, named PhosCalc, determines the number of possible phosphorylation sites and calculates the theoretical masses for the b and y fragment ions of a user-provided peptide sequence. A corresponding user-provided mass spectrum is examined to determine which putative b and y ions have support in the spectrum and a probability score is calculated for each combination of phosphorylation sites. Findings We test the implementation using spectra of phosphopeptides from bovine beta-casein and we compare the results from the implementation to those from manually curated and verified phosphopeptides from our own experiments. We find that the PhosCalc scores are capable of helping a user to identify phosphorylated sites and can remove a bottleneck in high throughput proteomics analyses. Conclusion PhosCalc is available as a web-based interface for examining up to 100 peptides and as a downloadable tool for examining larger numbers of peptides. PhosCalc can be used to speed up identification of phosphorylation sites and can be easily integrated into data handling pipelines making it a very useful tool for those involved in phosphoproteomic research. PMID:18710483

  10. Antimicrobial activity of de novo designed cationic peptides against multi-resistant clinical isolates.

    PubMed

    Faccone, Diego; Veliz, Omar; Corso, Alejandra; Noguera, Martin; Martínez, Melina; Payes, Cristian; Semorile, Liliana; Maffía, Paulo Cesar

    2014-01-01

    Antibiotic resistance is one of the main problems concerning public health or clinical practice. Antimicrobial peptides appear as good candidates for the development of new therapeutic drugs. In this study we de novo designed a group of cationic antimicrobial peptides, analyzed its physicochemical properties, including its structure by circular dichroism and studied its antimicrobial properties against a panel of clinical isolates expressing different mechanisms of resistance. Three cationic alpha helical peptides exhibited antimicrobial activity comparable to, or even better than the comparator omiganan (MBI-226).

  11. Sequential phosphorylation analysis using dye-tethered peptides and microfluidic isoelectric focusing electrophoresis.

    PubMed

    Choi, Hoseok; Choi, Nakchul; Lim, Butaek; Kim, Tae-Wuk; Song, Simon; Kim, Young-Pil

    2015-11-15

    We report a simple method for analyzing sequential phosphorylation by protein kinases using fluorescent peptide substrates and microfluidic isoelectric focusing (μIEF) electrophoresis. When a dye-labeled peptide substrate was sequentially phosphorylated by two consecutive protein kinases (mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3)), its differently phosphorylated forms were easily separated and visualized by fluorescent focusing zones in the μIEF channel based on a change in the isoelectric point (pI) by phosphorylation. As a result, ratiometric and quantitative analysis of the fluorescent focusing regions shifted by phosphorylation enabled the analysis of phosphorylation efficiency and the relevant inhibition of protein kinases (MAPK and GSK3) with high simplicity and selectivity. Furthermore, the GSK3 activity in the cell lysates was elucidated by μIEF electrophoresis in combination with immunoprecipitation. Our results suggest that this method has great potential for analyzing the sequential phosphorylation of multiple protein kinases that are implicated in cellular signaling pathways.

  12. Antimicrobial activity and interactions of cationic peptides derived from Galleria mellonella cecropin D-like peptide with model membranes.

    PubMed

    Oñate-Garzón, José; Manrique-Moreno, Marcela; Trier, Steven; Leidy, Chad; Torres, Rodrigo; Patiño, Edwin

    2017-03-01

    Antimicrobial peptides are effector molecules of the innate immune system against invading pathogens. The cationic charge in their structures has a strong correlation with antimicrobial activity, being responsible for the initial electrostatic interaction between peptides and the anionic microbial surface. This paper contains evidence that charge modification in the neutral peptide Gm cecropin D-like (WT) improved the antimicrobial activity of the modified peptides. Two cationic peptides derived from WT sequence named as ΔM1 and ΔM2, with net charge of +5 and +9, respectively, showed at least an eightfold increase in their antimicrobial activity in comparison to WT. The mechanism of action of these peptides was investigated using small unilamellar vesicles (SUVs) as model membranes. To study permeabilization effects of the peptides on cell membranes, entrapped calcein liposomes were used and the results showed that all peptides induced calcein release from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) SUVs, whereas in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), POPC/POPG and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/POPG SUVs, only ΔM1 and ΔM2 induced a notable permeabilization. In addition, interactions of these peptides with phospholipids at the level of the glycerol backbone and hydrophobic domain were studied through observed changes in generalized polarization and fluorescence anisotropy using probes such as Laurdan and DPH, respectively. The results suggest that peptides slightly ordered the bilayer structure at the level of glycerol backbone and on the hydrophobic core in 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) SUVs, whereas in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/DMPG SUVs, only ΔM1 and ΔM2 peptides increased the order of bilayers. Thus, peptides would be inducing clustering of phospholipids creating phospholipid domains with a higher phase transition temperature.

  13. 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

  14. Detection of selective cationic amphipatic antibacterial peptides by Hidden Markov models.

    PubMed

    Polanco, Carlos; Samaniego, Jose L

    2009-01-01

    Antibacterial peptides are researched mainly for the potential benefit they have in a variety of socially relevant diseases, used by the host to protect itself from different types of pathogenic bacteria. We used the mathematical-computational method known as Hidden Markov models (HMMs) in targeting a subset of antibacterial peptides named Selective Cationic Amphipatic Antibacterial Peptides (SCAAPs). The main difference in the implementation of HMMs was focused on the detection of SCAAP using principally five physical-chemical properties for each candidate SCAAPs, instead of using the statistical information about the amino acids which form a peptide. By this method a cluster of antibacterial peptides was detected and as a result the following were found: 9 SCAAPs, 6 synthetic antibacterial peptides that belong to a subregion of Cecropin A and Magainin 2, and 19 peptides from the Cecropin A family. A scoring function was developed using HMMs as its core, uniquely employing information accessible from the databases.

  15. Delivery of siRNA using ternary complexes containing branched cationic peptides: the role of peptide sequence, branching and targeting.

    PubMed

    Kudsiova, Laila; Welser, Katharina; Campbell, Frederick; Mohammadi, Atefeh; Dawson, Natalie; Cui, Lili; Hailes, Helen C; Lawrence, M Jayne; Tabor, Alethea B

    2016-03-01

    Ternary nanocomplexes, composed of bifunctional cationic peptides, lipids and siRNA, as delivery vehicles for siRNA have been investigated. The study is the first to determine the optimal sequence and architecture of the bifunctional cationic peptide used for siRNA packaging and delivery using lipopolyplexes. Specifically three series of cationic peptides of differing sequence, degrees of branching and cell-targeting sequences were co-formulated with siRNA and vesicles prepared from a 1 : 1 molar ratio of the cationic lipid DOTMA and the helper lipid, DOPE. The level of siRNA knockdown achieved in the human alveolar cell line, A549-luc cells, in both reduced serum and in serum supplemented media was evaluated, and the results correlated to the nanocomplex structure (established using a range of physico-chemical tools, namely small angle neutron scattering, transmission electron microscopy, dynamic light scattering and zeta potential measurement); the conformational properties of each component (circular dichroism); the degree of protection of the siRNA in the lipopolyplex (using gel shift assays) and to the cellular uptake, localisation and toxicity of the nanocomplexes (confocal microscopy). Although the size, charge, structure and stability of the various lipopolyplexes were broadly similar, it was clear that lipopolyplexes formulated from branched peptides containing His-Lys sequences perform best as siRNA delivery agents in serum, with protection of the siRNA in serum balanced against efficient release of the siRNA into the cytoplasm of the cell.

  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. Discovery of a 9-mer Cationic Peptide (LTX-315) as a Potential First in Class Oncolytic Peptide.

    PubMed

    Haug, Bengt Erik; Camilio, Ketil André; Eliassen, Liv Tone; Stensen, Wenche; Svendsen, John Sigurd; Berg, Kristel; Mortensen, Bjarte; Serin, Guillaume; Mirjolet, Jean-Francois; Bichat, Francis; Rekdal, Øystein

    2016-04-14

    Oncolytic immunotherapies represent a new promising strategy in the treatment of cancer. In our efforts to develop oncolytic peptides, we identified a series of chemically modified 9-mer cationic peptides that were highly effective against both drug-resistant and drug-sensitive cancer cells and with lower toxicity toward normal cells. Among these peptides, LTX-315 displayed superior anticancer activity and was selected as a lead candidate. This peptide showed relative high plasma protein binding abilities and a human plasma half-life of 160 min, resulting in formation of nontoxic metabolites. In addition, the lead candidate demonstrated relatively low ability to inhibit CYP450 enzymes. Collectively these data indicated that this peptide has potential to be developed as a new anticancer agent for intratumoral administration and is currently being evaluated in a phase I/IIa study.

  19. Salt-resistant alpha-helical cationic antimicrobial peptides.

    PubMed

    Friedrich, C; Scott, M G; Karunaratne, N; Yan, H; Hancock, R E

    1999-07-01

    Analogues based on the insect cecropin-bee melittin hybrid peptide (CEME) were studied and analyzed for activity and salt resistance. The new variants were designed to have an increase in amphipathic alpha-helical content (CP29 and CP26) and in overall positive charge (CP26). The alpha-helicity of these peptides was demonstrated by circular dichroism spectroscopy in the presence of liposomes. CP29 was shown to have activity against gram-negative bacteria that was similar to or better than those of the parent peptides, and CP26 had similar activity. CP29 had cytoplasmic membrane permeabilization activity, as assessed by the unmasking of cytoplasmic beta-galactosidase, similar to that of CEME and its more positively charged derivative named CEMA, whereas CP26 was substantially less effective. The activity of the peptides was not greatly attenuated by an uncoupler of membrane potential, carbonyl cyanide-m-chlorophenylhydrazone. The tryptophan residue in position 2 was shown to be necessary for interaction with cell membranes, as demonstrated by a complete lack of activity in the peptide CP208. Peptides CP29, CEME, and CEMA were resistant to antagonism by 0.1 to 0.3 M NaCl; however, CP26 was resistant to antagonism only by up to 160 mM NaCl. The peptides were generally more antagonized by 3 and 5 mM Mg2+ and by the polyanion alginate. It appeared that the positively charged C terminus in CP26 altered its ability to permeabilize the cytoplasmic membrane of Escherichia coli, although CP26 maintained its ability to kill gram-negative bacteria. These peptides are potential candidates for future therapeutic drugs.

  20. 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.

  1. 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.

  2. 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

  3. High Specific Selectivity and Membrane-Active Mechanism of Synthetic Cationic Hybrid Antimicrobial Peptides Based on the Peptide FV7.

    PubMed

    Tan, Tingting; Wu, Di; Li, Weizhong; Zheng, Xin; Li, Weifen; Shan, Anshan

    2017-02-06

    Hybrid peptides integrating different functional domains of peptides have many advantages, such as remarkable antimicrobial activity, lower hemolysis and ideal cell selectivity, compared with natural antimicrobial peptides. FV7 (FRIRVRV-NH₂), a consensus amphiphilic sequence was identified as being analogous to host defense peptides. In this study, we designed a series of hybrid peptides FV7-LL-37 (17-29) (FV-LL), FV7-magainin 2 (9-21) (FV-MA) and FV7-cecropin A (1-8) (FV-CE) by combining the FV7 sequence with the small functional sequences LL-37 (17-29) (LL), magainin 2 (9-21) (MA) and cecropin A (1-8) (CE) which all come from well-described natural peptides. The results demonstrated that the synthetic hybrid peptides, in particular FV-LL, had potent antibacterial activities over a wide range of Gram-negative and Gram-positive bacteria with lower hemolytic activity than other peptides. Furthermore, fluorescent spectroscopy indicated that the hybrid peptide FV-LL exhibited marked membrane destruction by inducing outer and inner bacterial membrane permeabilization, while scanning electron microscopy (SEM) and transmission electron microscopy (TEM) demonstrated that FV-LL damaged membrane integrity by disrupting the bacterial membrane. Inhibiting biofilm formation assays also showed that FV-LL had similar anti-biofilm activity compared with the functional peptide sequence FV7. Synthetic cationic hybrid peptides based on FV7 could provide new models for combining different functional domains and demonstrate effective avenues to screen for novel antimicrobial agents.

  4. High Specific Selectivity and Membrane-Active Mechanism of Synthetic Cationic Hybrid Antimicrobial Peptides Based on the Peptide FV7

    PubMed Central

    Tan, Tingting; Wu, Di; Li, Weizhong; Zheng, Xin; Li, Weifen; Shan, Anshan

    2017-01-01

    Hybrid peptides integrating different functional domains of peptides have many advantages, such as remarkable antimicrobial activity, lower hemolysis and ideal cell selectivity, compared with natural antimicrobial peptides. FV7 (FRIRVRV-NH2), a consensus amphiphilic sequence was identified as being analogous to host defense peptides. In this study, we designed a series of hybrid peptides FV7-LL-37 (17–29) (FV-LL), FV7-magainin 2 (9–21) (FV-MA) and FV7-cecropin A (1–8) (FV-CE) by combining the FV7 sequence with the small functional sequences LL-37 (17–29) (LL), magainin 2 (9–21) (MA) and cecropin A (1–8) (CE) which all come from well-described natural peptides. The results demonstrated that the synthetic hybrid peptides, in particular FV-LL, had potent antibacterial activities over a wide range of Gram-negative and Gram-positive bacteria with lower hemolytic activity than other peptides. Furthermore, fluorescent spectroscopy indicated that the hybrid peptide FV-LL exhibited marked membrane destruction by inducing outer and inner bacterial membrane permeabilization, while scanning electron microscopy (SEM) and transmission electron microscopy (TEM) demonstrated that FV-LL damaged membrane integrity by disrupting the bacterial membrane. Inhibiting biofilm formation assays also showed that FV-LL had similar anti-biofilm activity compared with the functional peptide sequence FV7. Synthetic cationic hybrid peptides based on FV7 could provide new models for combining different functional domains and demonstrate effective avenues to screen for novel antimicrobial agents. PMID:28178190

  5. 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

  6. Fragmentation of alpha-Radical Cations of Arginine-Containing Peptides

    SciTech Connect

    Laskin, Julia; Yang, Zhibo; Ng, Dominic C.; Chu, Ivan K.

    2010-04-01

    Fragmentation pathways of peptide radical cations, M+, with well-defined initial location of the radical site were explored using collision-induced dissociation (CID) experiments. Peptide radical cations were produced by gas-phase fragmentation of CoIII(salen)-peptide complexes [salen = N,N´-ethylenebis (salicylideneaminato)]. Subsequent hydrogen abstraction from the -carbon of the side chain followed by Ca-C bond cleavage results in the loss of a neutral side chain and formation of an a-radical cation with the radical site localized on the a-carbon of the backbone. Similar CID spectra dominated by radical-driven dissociation products were obtained for a number of a-radicals when the basic arginine side chain was present in the sequence. In contrast, proton-driven fragmentation dominates CID spectra of a-radicals produced via the loss of the arginine side chain. Our results suggest that in most cases radical migration precedes fragmentation of large peptide radical cations.

  7. Systematic studies of the mass spectrometric properties of alkaline earth metal cationized amino acids and peptides

    NASA Astrophysics Data System (ADS)

    Küjckelmann, Ulrich; Müller, Dietrich; Weber, Carsten

    1997-07-01

    The results of a systematic study of the gas phase interactions of α-amino acids and peptides (4-15 amino acids) with alkaline earth metals, observed with mass spectrometric techniques, are presented. Furthermore, a model for the cationization with calcium at the C-terminal amino acid arginine in rotaviral polypeptides is presented.

  8. Design and Synthesis of a Novel Cationic Peptide with Potent and Broad-Spectrum Antimicrobial Activity

    PubMed Central

    Liu, Wen-Ping; Chen, Ya-Hui; Ming, Xin; Kong, Yi

    2015-01-01

    Antibacterial and antifungal peptides have increasingly been used to combat the antibiotic-resistant microbes in recent years. KW-13, a novel cationic α-helical antibacterial peptide consisting of 13 amino acid residues, was designed and chemically synthesized. The peptide has a net charge of +6 with a total hydrophobic ratio of 38%. The antibacterial experiments revealed that KW-13 strongly inhibited the growth of human pathogenic bacteria with minimal inhibitory concentrations of 4 and 16 μg/mL for Staphylococcus epidermidis and Staphylococcus aureus, respectively, while the hemolytic assay showed that this peptide did not destroy human red blood cells in vitro. Scanning electron microscopy imaging of Escherichia coli confirmed that KW-13 can damage the membrane of bacterial cells. Thus, this peptide could be a potential candidate for the treatment of infectious diseases. PMID:26688811

  9. Interaction of cationic peptides with lipoteichoic acid and gram-positive bacteria.

    PubMed

    Scott, M G; Gold, M R; Hancock, R E

    1999-12-01

    Compounds with antiendotoxin properties have been extensively studied for their potential as therapeutic agents for sepsis attributable to gram-negative bacteria. However, with the increasing incidence of gram-positive sepsis, there is interest in identifying compounds with a broad spectrum of action against both gram-positive and gram-negative bacteria. A series of synthetic alpha-helical cationic peptides related to bee melittin and silk moth cecropin have previously been shown to bind lipopolysaccharide (LPS) with high affinity, inhibit LPS-induced tumor necrosis factor alpha (TNF-alpha) production in vitro and in vivo, and kill gram-negative bacteria. In this study, we analyzed whether these peptides were active against gram-positive bacteria; whether they could bind to lipoteichoic acid (LTA), the major proinflammatory structure on gram-positive bacteria; and whether they could block the ability of LTA to promote the release of cytokines by the RAW 264.7 murine macrophage cell line. We found that the cationic peptides demonstrated moderate growth-inhibitory activity toward gram-positive bacteria. In addition, the peptides bound LTA with high affinity. This correlated with the ability of the peptides to block LTA-induced production of TNF and interleukin-6 by RAW 264.7 cells but did not correlate with their ability to kill the bacteria. The peptides also effectively inhibited LTA-induced TNF production in a whole human blood assay. The peptides were also able to partly block the ability of heat-killed Staphylococcus aureus, as well as soluble products of live S. aureus, to stimulate cytokine production by macrophages. Our results indicate that these cationic peptides may be useful to prevent sepsis and inflammation caused by both gram-negative and gram-positive bacteria.

  10. Cationic activation of galactosyltransferase from rat mammary Golgi membranes by polyamines and by basic peptides and proteins.

    PubMed Central

    Navaratnam, N; Virk, S S; Ward, S; Kuhn, N J

    1986-01-01

    Galactosyltransferase (EC 2.4.1.22) requires bivalent metal ions for its activity. However, preparations of this enzyme solubilized from Golgi membranes of lactating rat mammary gland were shown to be activated not only by Mn2+, Ca2+ and Mg2+, but also by spermine, spermidine, lysyl-lysine, ethylenediamine and other diaminoalkanes, and by a range of basic proteins and peptides, including clupeine, histone, polylysine, ribonuclease, pancreatic trypsin inhibitor, cytochrome c, melittin, avidin and myelin basic protein. Both N-acetyl-lactosamine synthetase and lactose synthetase activities were enhanced. A basic protein fraction was isolated from bovine milk and shown to activate galactosyltransferase at low concentrations. The polyanions ATP, casein, chondroitin sulphate and heparin reversed the activation of galactosyltransferase by several of the above substances. Galactosyltransferase, assayed as a lactose synthetase, showed a 10-fold greater affinity for glucose when Mn2+ ions were replaced by clupeine or by ribonuclease as cationic activator. Evidence was obtained for the presence of an endogenous cationic activator in solubilized Golgi membrane preparations which evoked a similar low apparent Km,glucose. The findings are discussed in the light of cationic activations of glycosyltransferases generally, of the porous nature of the Golgi membrane, and of the unlikelihood of bivalent metal ions being the physiological activators of galactosyltransferase. It is suggested that the natural cationic activator of lactose synthetase may be a secretory protein acting in a manner analogous to the enzyme's activation by alpha-lactalbumin. A scheme is proposed for the two-stage synthesis of lactose and phosphorylation of casein within the cell, to accommodate the apparent incompatibility of these two processes. PMID:3101666

  11. Sequential elution of multiply and singly phosphorylated peptides with polar-copolymerized mixed-mode RP18/SCX material.

    PubMed

    Li, Xiuling; Guo, Zhimou; Sheng, Qianying; Xue, Xingya; Liang, Xinmiao

    2012-06-21

    Novel polar-copolymerized mixed-mode RP18/SCX material was developed for feasible phosphopeptide enrichment, in which multiply and singly phosphorylated peptides could be sequentially eluted and separated with high selectivity.

  12. Specific on-plate enrichment of phosphorylated peptides for direct MALDI-TOF MS analysis.

    PubMed

    Qiao, Liang; Roussel, Christophe; Wan, Jingjing; Yang, Pengyuan; Girault, Hubert H; Liu, Baohong

    2007-12-01

    An on-plate specific enrichment method is presented for the direct analysis of peptides phosphorylation. An array of sintered TiO 2 nanoparticle spots was prepared on a stainless steel plate to provide porous substrate with a very large specific surface and durable functions. These spots were used to selectively capture phosphorylated peptides from peptide mixtures, and the immobilized phosphopeptides could then be analyzed directly by MALDI MS after washing away the nonphosphorylated peptides. beta-Casein and protein mixtures were employed as model samples to investigate the selection efficiency. In this strategy, the steps of phosphopeptide capture, purification, and subsequent mass spectrometry analysis are all successfully accomplished on a single target plate, which greatly reduces sample loss and simplifies analytical procedures. The low detection limit, small sample size, and rapid selective entrapment show that this on-plate strategy is promising for online enrichment of phosphopeptides, which is essential for the analysis of minute amount of samples in high-throughput proteome research.

  13. Assigning structures to gas-phase peptide cations and cation-radicals. An infrared multiphoton dissociation, ion mobility, electron transfer, and computational study of a histidine peptide ion.

    PubMed

    Moss, Christopher L; Chamot-Rooke, Julia; Nicol, Edith; Brown, Jeffery; Campuzano, Iain; Richardson, Keith; Williams, Jonathan P; Bush, Matthew F; Bythell, Benjamin; Paizs, Bela; Turecek, Frantisek

    2012-03-15

    Infrared multiphoton dissociation (IRMPD) spectroscopy, using a free-electron laser, and ion mobility measurements, using both drift-cell and traveling-wave instruments, were used to investigate the structure of gas-phase peptide (AAHAL + 2H)(2+) ions produced by electrospray ionization. The experimental data from the IRMPD spectra and collisional cross section (Ω) measurements were consistent with the respective infrared spectra and Ω calculated for the lowest-energy peptide ion conformer obtained by extensive molecular dynamics searches and combined density functional theory and ab initio geometry optimizations and energy calculations. Traveling-wave ion mobility measurements were employed to obtain the Ω of charge-reduced peptide cation-radicals, (AAHAL + 2H)(+●), and the c(3), c(4), z(3), and z(4) fragments from electron-transfer dissociation (ETD) of (AAHAL + 2H)(2+). The experimental Ω for the ETD charge-reduced and fragment ions were consistent with the values calculated for fully optimized ion structures and indicated that the ions retained specific hydrogen bonding motifs from the precursor ion. In particular, the Ω for the doubly protonated ions and charge-reduced cation-radicals were nearly identical, indicating negligible unfolding and small secondary structure changes upon electron transfer. The experimental Ω for the (AAHAL + 2H)(+●) cation-radicals were compatible with both zwitterionic and histidine radical structures formed by electron attachment to different sites in the precursor ion, but did not allow their distinction. The best agreement with the experimental Ω was found for ion structures fully optimized with M06-2X/6-31+G(d,p) and using both projection approximation and trajectory methods to calculate the theoretical Ω values.

  14. Infrared multiphoton dissociation of peptide cations in a dual pressure linear ion trap mass spectrometer.

    PubMed

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

    2009-10-01

    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 x 10(-3) Torr and the second a low pressure cell operated at nominally 3 x 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 y(1) fragments and immonium ions which are not typically observed by ion trap collision induced dissociation (CID). Photodissociation efficiencies of approximately 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.

  15. 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

  16. Interactions of Cationic Peptides and Ions with Negatively Charged Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Taheri-Araghi, Sattar

    In this thesis we study the interactions of ions and cationic peptides with a negatively charged lipid bilayer in an ionic solution where the electrostatic interactions are screened. We first examine the problem of charge renormalization and inversion of a highly charged bilayer with low dielectric constant. To be specific, we consider an asymmetrically charged lipid bilayer, in which only one layer is negatively charged. In particular, we study how dielectric discontinuities and charge correlations among lipid charges and condensed counterions influence the effective charge of the surface. When counterions are monovalent, e. g. , Na+, our mean-field approach implies that dielectric discontinuities can enhance counterion condensation. A simple scaling picture shows how the effects of dielectric discontinuities and surface-charge distributions are intertwined: Dielectric discontinuities diminish condensation if the backbone charge is uniformly smeared out while counterions are localized in space; they can, however, enhance condensation when the backbone charge is discrete. In the presence of asymmetric salts such as CaCl2, we find that the correlation effect, treated at the Gaussian level, is more pronounced when the surface has a lower dielectric constant, inverting the sign of the charge at a smaller value of Ca2+ concentration. In the last chapter we study binding of cationic peptides onto a lipid-bilayer membrane. The peptide not only interacts electrostatically with anionic lipids, rearranging their spatial distributions, but it can also insert hydrophobically into the membrane, expanding the area of its binding layer (i. e. , the outer layer). We examine how peptide charges and peptide insertion (thus area expansion) are intertwined. Our results show that, depending on the bilayer's surface charge density and peptide hydrophobicity, there is an optimal peptide charge yielding the maximum peptide penetration. Our results shed light on the physics behind the

  17. Is Dissociation of Peptide Radical Cations an Ergodic Process?

    SciTech Connect

    Laskin, Julia; Futrell, Jean H.; Chu, Ivan K.

    2007-08-08

    Achieving a fundamental understanding of the mechanism of unimolecular dissociation of internally excited complex molecules is one of the most important challenges in modern mass spectrometry. One of the central questions is whether the dissociation of large molecules is properly described by statistical theories—RRKM/QET or Phase Space Theories —that have proved to be remarkably successful both for small molecules and a number of small and medium size peptides. The concept question is whether the ergodic assumption that the internal excitation of the ion is randomly redistributed among the vibrational degrees of freedom prior to fragmentation is satisfied for large molecules. The validity of the ergodic hypothesis for dissociation of gas-phase biomolecules has been recently reviewed and will be only briefly discussed here.

  18. Cationic host defense peptides; novel antimicrobial therapeutics against Category A pathogens and emerging infections.

    PubMed

    Findlay, Fern; Proudfoot, Lorna; Stevens, Craig; Barlow, Peter G

    2016-01-01

    Cationic Host Defense Peptides (HDP, also known as antimicrobial peptides) are crucial components of the innate immune system and possess broad-spectrum antibacterial, antiviral, and immunomodulatory activities. They can contribute to the rapid clearance of biological agents through direct killing of the organisms, inhibition of pro-inflammatory mediators such as lipopolysaccharide, and by modulating the inflammatory response to infection. Category A biological agents and materials, as classified by the United States National Institutes for Health, the US Centers for Disease Control and Prevention, and the US Department of Homeland Security, carry the most severe threat in terms of human health, transmissibility, and preparedness. As such, there is a pressing need for novel frontline approaches for prevention and treatment of diseases caused by these organisms, and exploiting the broad antimicrobial activity exhibited by cationic host defense peptides represents an exciting priority area for clinical research. This review will summarize what is known about the antimicrobial and antiviral effects of the two main families of cationic host defense peptides, cathelicidins, and defensins in the context of Category A biological agents which include, but are not limited to; anthrax (Bacillus anthracis), plague (Yersinia pestis), smallpox (Variola major), tularemia (Francisella tularensis). In addition, we highlight priority areas, particularly emerging viral infections, where more extensive research is urgently required.

  19. 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.

  20. CL22 - a novel cationic peptide for efficient transfection of mammalian cells.

    PubMed

    Haines, A M; Irvine, A S; Mountain, A; Charlesworth, J; Farrow, N A; Husain, R D; Hyde, H; Ketteringham, H; McDermott, R H; Mulcahy, A F; Mustoe, T L; Reid, S C; Rouquette, M; Shaw, J C; Thatcher, D R; Welsh, J H; Williams, D E; Zauner, W; Phillips, R O

    2001-01-01

    Condensing peptide-DNA complexes have great potential as nonviral agents for gene delivery. To date, however, such complexes have given transfection activities greatly inferior to adenovirus and somewhat inferior to cationic lipid-DNA complexes, even for cell lines and primary cells in vitro. We report here the identification of a novel condensing peptide, CL22, which forms DNA complexes that efficiently transfect many cell lines, as well as primary dendritic and endothelial cells. We report studies with sequence and structure variants that define some properties of the peptide that contribute to efficient transfection. We demonstrate that the superior transfection activity of CL22 compared with other DNA condensing peptides is conferred at a step after uptake of the complexes into cells. We show that CL22-DNA complexes have transfection activity that is at least equivalent to the best available nonviral agents.

  1. Synthesis and biological activity of lipophilic analogs of the cationic antimicrobial active peptide anoplin.

    PubMed

    Chionis, Kostas; Krikorian, Dimitrios; Koukkou, Anna-Irini; Sakarellos-Daitsiotis, Maria; Panou-Pomonis, Eugenia

    2016-11-01

    Anoplin is a short natural cationic antimicrobial peptide which is derived from the venom sac of the solitary wasp, Anoplius samariensis. Due to its short sequence G(1) LLKR(5) IKT(8) LL-NH2 , it is ideal for research tests. In this study, novel analogs of anoplin were prepared and examined for their antimicrobial, hemolytic activity, and proteolytic stability. Specific substitutions were introduced in amino acids Gly(1) , Arg(5) , and Thr(8) and lipophilic groups with different lengths in the N-terminus in order to investigate how these modifications affect their antimicrobial activity. These cationic analogs exhibited higher antimicrobial activity than the native peptide; they are also nontoxic at their minimum inhibitory concentration (MIC) values and resistant to enzymatic degradation. The substituted peptide GLLKF(5) IKK(8) LL-NH2 exhibited high activity against Gram-negative bacterium Zymomonas mobilis (MIC = 7 µg/ml), and the insertion of octanoic, decanoic, and dodecanoic acid residues in its N-terminus increased the antimicrobial activity against Gram-positive and Gram-negative bacteria (MIC = 5 µg/ml). The conformational characteristics of the peptide analogs were studied by circular dichroism. Structure activity studies revealed that the substitution of specific amino acids and the incorporation of lipophilic groups enhanced the amphipathic α-helical conformation inducing better antimicrobial effects. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  2. 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.

  3. 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.

  4. Early phosphorylation events following the treatment of Swiss 3T3 cells with bombesin and the mammalian bombesin-related peptide, gastrin-releasing peptide.

    PubMed Central

    Isacke, C M; Meisenhelder, J; Brown, K D; Gould, K L; Gould, S J; Hunter, T

    1986-01-01

    Bombesin and the related mammalian peptides, such as gastrin-releasing peptide (GRP), are potent mitogens for some fibroblast cell lines. Here we have examined the bombesin- and GRP-mediated changes in the phosphorylation of proteins in Swiss 3T3 cells and compared these to the events observed after platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and tumor promoter treatment. In agreement with previous reports, bombesin, GRP and PDGF, but not EGF, increased the activity of protein kinase C. This was assayed by an inhibition of [125I]EGF binding, stimulation in phosphorylation of pp60c-src on serine 12 and stimulation in phosphorylation of a group of 80 kd proteins. The different phosphorylated forms of the 80 kd proteins were examined by tryptic peptide mapping and shown to contain multiple phosphorylation sites. An investigation of the tyrosine phosphorylation events following mitogen treatment revealed a significant difference between PDGF and the bombesin peptides. PDGF treatment caused a marked increase in total cellular phosphotyrosine levels, and tyrosine phosphorylation both of known substrates and its own receptor. In contrast, bombesin and GRP treatments resulted in only a weak or undetectable increase in tyrosine phosphorylation of total cellular protein or known substrates. In this respect bombesin and GRP were more similar to EGF. The fact that the bombesin peptides do not induce a phosphorylation response identical with either PDGF or EGF suggests that there is not a single common signal pathway which is activated by all these mitogens. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:2431903

  5. A lesson from Bombinins H, mildly cationic diastereomeric antimicrobial peptides from Bombina skin.

    PubMed

    Mangoni, Maria Luisa

    2013-12-01

    Gene-encoded peptide antibiotics represent fascinating molecules for the development of new antimicrobials with a new mode of action: and one of the richest sources is amphibian skin. In particular, the skin of the fire-bellied toad Bombina genus contains mildly cationic antimicrobial peptides (AMPs), named bombinins H, with attractive properties. Indeed, some members of this peptide family coexist in skin secretions as isomers in which a single D-amino acid (alloisoleucine or leucine) is incorporated as a result of a post-translational modification of the respective gene-encoded Lamino acid. Here, a brief overview of the genes coding for these peptides, their spectrum of antimicrobial activities, mechanism of action and interactions with biological or model membranes is reported. Remarkably, a single D-amino acid substitution represents a unique approach developed by Nature not only to modulate the peptide stability in vivo, but also to confer the all-L peptide and its diastereomer distinctive biological features. Overall, such findings should assist in the generation of new peptide-based anti-infective agents, which are urgently needed because of the growing emergence of microbial strains resistant to conventional antimicrobials.

  6. 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.

  7. 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

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

    PubMed

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

    2015-12-07

    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.

  9. 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.

  10. pH Dependence of microbe sterilization by cationic antimicrobial peptides.

    PubMed

    Walkenhorst, William F; Klein, J Wolfgang; Vo, Phuong; Wimley, William C

    2013-07-01

    We recently described a family of cationic antimicrobial peptides (CAMPs) selected from a combinatorial library that exhibited potent, broad-spectrum activity at neutral pH and low ionic strength. To further delimit the utility and activity profiles of these peptides, we investigated the effects of solution conditions, such as pH and ionic strength, on the efficacy of the peptide antimicrobials against a panel of microorganisms. Peptide minimum sterilizing concentrations (MSCs) varied linearly with pH for each subtype within our family of CAMPs for all organisms tested. The peptides were much less effective against Gram-negative bacteria at high pH, consistent with a decrease in net positive charge on the peptides. A similar trend was observed for the fungus Candida albicans. Surprisingly, the opposite pH trend was observed with the Gram-positive Staphylococcus aureus. In addition, an additive ionic strength effect was observed with increasing buffer strengths at identical pH values. The extreme difference in the observed pH behavior between Gram-negative and Gram-positive organisms is attributed to the presence of native charged molecules in the much thicker peptidoglycan layer of the Gram-positive organism. The novel species-specific effects of pH observed here have important implications for applications using CAMPs and for the design of novel CAMPs.

  11. 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.

  12. Accurate determination of peptide phosphorylation stoichiometry via automated diagonal capillary electrophoresis coupled with mass spectrometry: proof of principle.

    PubMed

    Mou, Si; Sun, Liangliang; Dovichi, Norman J

    2013-11-19

    While reversible protein phosphorylation plays an important role in many cellular processes, simple and reliable measurement of the stoichiometry of phosphorylation can be challenging. This measurement is confounded by differences in the ionization efficiency of phosphorylated and unphosphorylated sites during analysis by mass spectrometry. Here, we demonstrate diagonal capillary electrophoresis-mass spectrometry for the accurate determination of this stoichiometry. Diagonal capillary electrophoresis is a two-dimensional separation method that incorporates an immobilized alkaline phosphatase microreactor at the distal end of the first capillary and employs identical electrophoretic separation modes in both dimensions. The first dimension is used to separate a mixture of the phosphorylated and unphosphorylated forms of a peptide. Fractions are parked in the reactor where they undergo complete dephosphorylation. The products are then periodically transferred to the second capillary and analyzed by mass spectrometry (MS). Because the phosphorylated and unphosphorylated forms differ in charge, they are well resolved in the first dimension separation. Because the unphosphorylated and dephosphorylated peptides are identical, there is no bias in ionization efficiency, and phosphorylation stoichiometry can be determined by the ratio of the signal of the two forms. A calibration curve was generated from mixtures of a phosphorylated standard peptide and its unphosphorylated form, prepared in a bovine serum albumin tryptic digest. This proof of principle experiment demonstrated a linear response across nearly 2 orders of magnitude in stoichiometry.

  13. Tailored-waveform collisional activation of peptide ion electron transfer survivor ions in cation transmission mode ion/ion reaction experiments.

    PubMed

    Han, Hongling; Londry, Frank A; Erickson, David E; McLuckey, Scott A

    2009-04-01

    Broadband resonance excitation via a tailored waveform in a high pressure collision cell (Q2) on a hybrid quadrupole/time-of-flight (QqTOF) tandem mass spectrometer has been implemented for cation transmission mode electron transfer ion/ion reactions of tryptic polypeptides. The frequency components in the broadband waveform were defined to excite the first generation intact electron transfer products for relatively large tryptic peptides. The optimum amplitude of the arbitrary waveform applied has been determined empirically to be 3.0 V(p-p), which is effective for relatively high mass-to-charge (m/z) ratio precursor ions with little elimination of sequence information for low m/z ions. The application of broadband activation during the transmission mode ion/ion reaction obviates frequency and amplitude tuning normally associated with ion trap collision induced dissociation (CID). This approach has been demonstrated with triply and doubly charged tryptic peptides with and without post-translational modifications. Enhanced structural information was achieved by production of a larger number of informative c- and z-type fragments using the tailored waveform on unmodified and modified (phosphorylated and glycosylated) peptides when the first generation intact electron transfer products fell into the defined frequency range. This approach can be applied to a wide range of tryptic peptide ions, making it attractive as a rapid and general approach for ETD LC-MS/MS of tryptic peptides in a QqTOF instrument.

  14. Tailored-waveform Collisional Activation of Peptide Ion Electron Transfer Survivor Ions in Cation Transmission Mode Ion/Ion Reaction Experiments

    PubMed Central

    Han, Hongling; Londry, Frank A.; Erickson, David E.; McLuckey, Scott A.

    2010-01-01

    SUMMARY Broad-band resonance excitation via a tailored waveform in a high pressure collision cell (Q2) on a hybrid quadrupole/time-of-flight (QqTOF) tandem mass spectrometer has been implemented for cation transmission mode electron transfer ion/ion reactions of tryptic polypeptides. The frequency components in the broadband waveform were defined to excite the first generation intact electron transfer products for relatively large tryptic peptides. The optimum amplitude of the arbitrary waveform applied has been determined empirically to be 3.0 Vp-p, which is effective for relatively high mass-to-charge (m/z) ratio precursor ions with little elimination of sequence information for low m/z ions. The application of broadband activation during the transmission mode ion/ion reaction obviates frequency and amplitude tuning normally associated with ion trap collision induced dissociation (CID). This approach has been demonstrated with triply and doubly charged tryptic peptides with and without post-translational modifications. Enhanced structural information was achieved by production of a larger number of informative c- and z-type fragments using the tailored waveform on unmodified and modified (phosphorylated and glycosylated) peptides when the first generation intact electron transfer products fell into the defined frequency range. This approach can be applied to a wide range of tryptic peptide ions, making it attractive as a rapid and general approach for ETD LC-MS/MS of tryptic peptides in a QqTOF instrument. PMID:19305916

  15. Nanoprobing of the effect of Cu2+ cations on misfolding, interaction and aggregation of amyloid β peptide

    PubMed Central

    Lv, Zhengjian; Condron, Margaret M.; Teplow, David B.; Lyubchenko, Yuri L.

    2012-01-01

    Misfolding and aggregation of the amyloid β-protein (Aβ) are hallmarks of Alzheimer’s disease. Both processes are dependent on the environmental conditions, including the presence of divalent cations, such as Cu2+. Cu2+ cations regulate early stages of Aβ aggregation, but the molecular mechanism of Cu2+ regulation is unknown. In this study we applied single molecule AFM force spectroscopy to elucidate the role of Cu2+ cations on interpeptide interactions. By immobilizing one of two interacting Aβ42 molecules on a mica surface and tethering the counterpart molecule onto the tip, we were able to probe the interpeptide interactions in the presence and absence of Cu2+ cations at pH 7.4, 6.8, 6.0, 5.0, and 4.0. The results show that the presence of Cu2+ cations change the pattern of Aβ interactions for pH values between pH 7.4 and pH 5.0. Under these conditions, Cu2+ cations induce Aβ42 peptide structural changes resulting in N–termini interactions within the dimers. Cu2+ cations also stabilize the dimers. No effects of Cu2+ cations on Aβ–Aβ interactions were observed at pH 4.0, suggesting that peptide protonation changes the peptide-cation interaction. The effect of Cu2+ cations on later stages of Aβ aggregation was studied by AFM topographic images. The results demonstrate that substoichiometric Cu2+ cations accelerate the formation of fibrils at pH 7.4 and 5.0, whereas no effect of Cu2+ cations was observed at pH 4.0. Taken together, the combined AFM force spectroscopy and imaging analyses demonstrate that Cu2+ cations promote both the initial and the elongation stages of Aβ aggregation, but protein protonation diminishes the effect of Cu2+. PMID:23143330

  16. Enhanced in-cell folding of reversibly cationized transcription factor using amphipathic peptide.

    PubMed

    Futami, Midori; Nakano, Tomoki; Yasunaga, Mayu; Makihara, Masahiro; Asama, Takashi; Hagihara, Yoshihisa; Nakajima, Yoshihiro; Futami, Junichiro

    2017-04-01

    The intracellular delivery of functionally active transcription factor proteins is emerging as a promising technique for artificial regulation of cellular functions. However, in addition to the cell membrane, which acts as a barrier to macromolecules, the aggregation-favored properties of structurally flexible transcription factor proteins limit the application of this method. In-cell folding technique can be used to overcome these issues. This technique solubilizes denatured protein by reversible alkyl-disulfide cationization (S-cationization), and simultaneously endows efficient intracellular delivery and folding to the biologically active conformation in the reducing environment of the cytosol. Because cationized protein is internalized into cells by adsorption-mediated endocytosis, endosomal escape is crucial for this technique. In this study, we utilized a sensitive luciferase reporter gene assay to quantitatively evaluate in-cell folding of the artificial transcription factor GAL4-VP16. Although the cationic moiety of S-cationized protein was slightly affected, co-transduction of amphipathic peptide Endo-PORTER dramatically improved in-cell folding efficiency. Live cell imaging of fluorescent-labeled GAL4-VP16 revealed that some of the proteins diffused into the cytosol and nucleus through co-transduction with Endo-PORTER. Real-time monitoring of light output of luciferase revealed the kinetics of in-cell folding, supporting that endosomal-release assisted by Endo-PORTER was stimulated by endosome acidification. Because this method can transduce proteins uniformly and repeatedly into living cells, S-cationized transcription factor proteins are widely applicable for the artificial regulation of cellular functions.

  17. Conformations of Cationized Peptides. Determination of Ligand Binding Geometries by Irmpd Spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2009-06-01

    Spectroscopic study of the conformations of metalated amino acids has mapped out in some detail the preferences for canonical (charge solvated) versus zwitterionic (salt bridge) conformations. Corresponding studies of larger peptides are now possible. Here are described results for several singly and doubly charged metal ions with dipeptides and tripeptides. Factors including ion charge, size of cation, and side chain identity and sequence are found to be conformational determinants. IRMPD spectra of the ions were acquired by irradiating the cell with infrared light from the FELIX free electron laser at wavelengths in the approximate range 500 to 1900 cm^{-1}.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. New cationic antimicrobial peptide screened from boiled-dried anchovies by immobilized bacterial membrane liposome chromatography.

    PubMed

    Tang, Wenting; Zhang, Hui; Wang, Li; Qian, Haifeng

    2014-02-19

    An efficient immobilized bacterial membrane liposome chromatography method was used to screen potential antimicrobial peptides from boiled-dried anchovies. A novel cationic antimicrobial peptide (Apep10) was successfully isolated by one-step chromatography. The sequence of Apep10 was identified as GLARCLAGTL by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry (MALDI-Q-TOF MS). The antimicrobial activity assessment indicated that Apep10 inhibited the growth of the reference bacteria (Escherichia coli, Shigella dysenteriae, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Bacillus subtilis, and Streptococcus pneumoniae), with minimal inhibitory concentration (MIC) values ranging from 8 to 64 μg/mL. Almost no cytotoxicity against mouse erythrocytes was observed at concentrations below 20 μg/mL. Nucleotide leakage induced by Apep10 showed that the peptide exhibited permeable activity on the cytoplasmic membrane. Alterations in morphology were observed by scanning electronic microscopy (SEM). Membrane disruption was confirmed by confocal laser scanning microscopy (CLSM) with propidium iodide (PI). The results demonstrate that immobilized bacterial membrane liposome chromatography is a straightforward technique for screening unknown antimicrobial peptides with cell-membrane-interacting activities from boiled-dried anchovies.

  4. 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.

  5. Relative spatial positions of tryptophan and cationic residues in helical membrane-active peptides determine their cytotoxicity.

    PubMed

    Rekdal, Øystein; Haug, Bengt Erik; Kalaaji, Manar; Hunter, Howard N; Lindin, Inger; Israelsson, Ingrid; Solstad, Terese; Yang, Nannan; Brandl, Martin; Mantzilas, Dimitrios; Vogel, Hans J

    2012-01-02

    The cytotoxic activity of 10 analogs of the idealized amphipathic helical 21-mer peptide (KAAKKAA)3, where three of the Ala residues at different positions have been replaced with Trp residues, has been investigated. The peptide's cytotoxic activity was found to be markedly dependent upon the position of the Trp residues within the hydrophobic sector of an idealized α-helix. The peptides with Trp residues located opposite the cationic sector displayed no antitumor activity, whereas those peptides with two or three Trp residues located adjacent to the cationic sector exhibited high cytotoxic activity when tested against three different cancer cell lines. Dye release experiments revealed that in contrast to the peptides with Trp residues located opposite the cationic sector, the peptides with Trp residues located adjacent to the cationic sector induced a strong permeabilizing activity from liposomes composed of a mixture of zwitterionic phosphatidylcholine and negatively charged phosphatidylserine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS)) (2:1) but not from liposomes composed of zwitterionic phosphatidylcholine, POPC. Fluorescence blue shift and quenching experiments revealed that Trp residues inserted deeper into the hydrophobic environment of POPC/POPS liposomes for peptides with high cytotoxic activity. Through circular dichroism studies, a correlation between the cytotoxic activity and the α-helical propensity was established. Structural studies of one inactive and two active peptides in the presence of micelles using NMR spectroscopy showed that only the active peptides adopted highly coiled to helical structures when bound to a membrane surface.

  6. 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.

  7. 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

  8. 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

  9. Comparison of CID, ETD and metastable atom-activated dissociation (MAD) of doubly and triply charged phosphorylated tau peptides.

    PubMed

    Cook, Shannon L; Zimmermann, Carolyn M; Singer, David; Fedorova, Maria; Hoffmann, Ralf; Jackson, Glen P

    2012-06-01

    The fragmentation behavior of the 2+ and 3+ charge states of eleven different phosphorylated tau peptides was studied using collision-induced dissociation (CID), electron transfer dissociation (ETD) and metastable atom-activated dissociation (MAD). The synthetic peptides studied contain up to two known phosphorylation sites on serine or threonine residues, at least two basic residues, and between four and eight potential sites of phosphorylation. CID produced mainly b-/y-type ions with abundant neutral losses of the phosphorylation modification. ETD produced c-/z-type ions in highest abundance but also showed numerous y-type ions at a frequency about 50% that of the z-type ions. The major peaks observed in the ETD spectra correspond to the charge-reduced product ions and small neutral losses from the charge-reduced peaks. ETD of the 2+ charge state of each peptide generally produced fewer backbone cleavages than the 3+ charge state, consistent with previous reports. Regardless of charge state, MAD achieved more extensive backbone cleavage than CID or ETD, while retaining the modification(s) in most cases. In all but one case, unambiguous modification site determination was achieved with MAD. MAD produced 15-20% better sequence coverage than CID and ETD for both the 2+ and 3+ charge states and very different fragmentation products indicating that the mechanism of fragmentation in MAD is unique and complementary to CID and ETD.

  10. Strategies and solid-phase formats for the analysis of protein and peptide phosphorylation employing a novel fluorescent phosphorylation sensor dye.

    PubMed

    Martin, Karen; Steinberg, Thomas H; Goodman, Terrie; Schulenberg, Birte; Kilgore, Jason A; Gee, Kyle R; Beechem, Joseph M; Patton, Wayne F

    2003-06-01

    Protein kinases represent one of the largest families of regulatory enzymes, with more than 2,000 of them being encoded for by the human genome. Many cellular processes are regulated by the reversible phosphorylation of proteins and upwards of 30% of the proteins comprising the eukaryotic proteome are likely to be phosphorylated at some point during their existence. In the past, analysis of global protein phosphorylation has been accomplished through radiolabelling of samples with inorganic (32P or [gamma-32)P] ATP. The approach is limited to specimens amenable to radiolabelling and poses certain safety and disposal problems. Alternatively, immunodetection with antibodies to the common phosphoamino acids may be employed, but the antibodies are relatively expensive and exhibit limited specificity and a certain degree of cross-reactivity. Pro-Q Diamond dye is a new fluorescent phosphosensor technology suitable for the detection of phosphoserine-, phosphothreonine- and phosphotyrosine-containing proteins directly in isoelectric focusing gels, SDS-polyacrylamide gels and two-dimensional gels. Additionally, the technology is appropriate for the detection of phosphoproteins or phosphopeptides arrayed on protein chips or affixed to beads. Dye-stained proteins and peptides can be excited with a laser-based light source of 532 or 543 nm or with a xenon-arc lamp-based system equipped with appropriate band pass filters. Alternatively, ultraviolet light of about 302 nm may be employed, providing that sufficiently long exposure times are used to collect the fluorescence signal. Pro-Q Diamond dye emits maximally at approximately 580 nm. The fluorescence-based detection technology is easy to conduct, cost effective and allows rapid large-scale screening of protein and peptide phosphorylation in a variety of solid-phase assay formats.

  11. Effects of cationic antimicrobial peptides on liquid-preserved boar spermatozoa.

    PubMed

    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.

  12. 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...

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

    PubMed Central

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

    2016-01-01

    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

  14. 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

  15. When cationic cell-penetrating peptides meet hydrocarbons to enhance in-cell cargo delivery.

    PubMed

    Di Pisa, Margherita; Chassaing, Gérard; Swiecicki, Jean-Marie

    2015-05-01

    Cell-penetrating peptides (CPPs) are short sequences often rich in cationic residues with the remarkable ability to cross cell membranes. In the past 20 years, CPPs have gained wide interest and have found numerous applications in the delivery of bioactive cargoes to the cytosol and even the nucleus of living cells. The covalent or non-covalent addition of hydrocarbon moieties to cationic CPPs alters the hydrophobicity/hydrophilicity balance in their sequence. Such perturbation dramatically influences their interaction with the cell membrane, might induce self-assembling properties and modifies their intracellular trafficking. In particular, the introduction of lipophilic moieties changes the subcellular distribution of CPPs and might result in a dramatically increase of the internalization yield of the co-transported cargoes. Herein, we offer an overview of different aspects of the recent findings concerning the properties of CPPs covalently or non-covalently associated to hydrocarbons. We will focus on the impact of the hydrocarbon moieties on the delivery of various cargoes, either covalently or non-covalently bound to the modified CPPs. We will also provide some key elements to rationalize the influence of the hydrocarbons moieties on the cellular uptake. Furthermore, the recent in vitro and in vivo successful applications of acylated CPPs will be summarized to provide a broad view of the versatility of these modified CPPs as small-molecules and oligonucleotides vectors.

  16. The role of the position of the basic residue in the generation and fragmentation of peptide radical cations

    NASA Astrophysics Data System (ADS)

    Wee, Sheena; O'Hair, Richard A. J.; McFadyen, W. David

    2006-03-01

    Using simple di- and tripeptides GX, GGX, GXG, XG and XGG, the influence of the position of the basic residue, X (X = R, K and H), on the formation of peptide radical cations (M+) from [CuII(tpy)M]2+ complexes (where tpy = 2,2':6',2''-terpyridine) was probed. It was found that M+ is formed with greatest abundance when the basic residue is at the C-terminus. For arginine containing peptides, this may be due to further fragmentation of GRG+, RG+ and RGG+ at the MS2 stage. For lysine and histidine containing peptides, when the basic residue is not located at the C-terminus, competing fragmentation pathways that lead to peptide backbone cleavage are more facile than M+ formation. In order to gain some insights into the binding modes of these peptides to [CuII(tpy)]2+, the formation and fragmentation of copper(II) complexes of tripeptides protected as their carboxy methyl/ethyl esters (M-OR', R' = Me/Et) were also probed. The products of the competing fragmentation pathways of [CuII(tpy)M]2+, as well as the formation and fragmentation of [CuII(tpy)(M-OR')]2+, suggest that the unprotected peptides, M, mainly bind as zwitterions to [CuII(tpy)]2+. The fragmentation reactions of the radical cations (M+) were also studied. Radical driven side chain fragmentation reactions of M+ are dependent on both the position of the residue as well as the identity of other residues present in the peptide radical cations. GR and RG, which undergo rearrangement to form a mixed anhydride in their protonated forms, do not undergo the same rearrangement in their radical cation forms.

  17. 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...

  18. 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.

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

    PubMed

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

    2014-01-01

    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.

  20. Counterion condensation in short cationic peptides: limiting mobilities beyond the Onsager-Fuoss theory.

    PubMed

    Wernersson, Erik; Heyda, Jan; Kubíčková, Anna; Křížek, Tomáš; Coufal, Pavel; Jungwirth, Pavel

    2012-03-01

    We investigated the effect of the background electrolyte (BGE) anions on the electrophoretic mobilities of the cationic amino acids arginine and lysine and the polycationic peptides tetraarginine, tetralysine, nonaarginine, and nonalysine. BGEs composed of sodium chloride, sodium propane-1,3-disulfonate, and sodium sulfate were used. For the amino acids, determination of the limiting mobility by extrapolation, using the Onsager-Fuoss (OF) theory expression, yielded consistent estimates. For the peptides, however, the estimates of the limiting mobilities were found to spuriously depend on the BGE salt. This paradox was resolved using molecular modeling. Simulations, on all-atom as well as coarse-grained levels, show that significant counterion condensation, an effect not accounted for in OF theory, occurs for the tetra- and nonapeptides, even for low BGE concentrations. Including this effect in the quantitative estimation of the BGE effect on mobility removed the discrepancy between the estimated limiting mobilities in different salts. The counterion condensation was found to be mainly due to electrostatic interactions, with specific ion effects playing a secondary role. Therefore, the conclusions are likely to be generalizable to other analytes with a similar density of charged groups and OF theory is expected to fail in a predictable way for such analytes.

  1. Reagent Cluster Anions for Multiple Gas-phase Covalent Modifications of Peptide and Protein Cations

    PubMed Central

    Prentice, Boone M.; Stutzman, John R.; McLuckey, Scott A.

    2013-01-01

    Multiple gas phase ion/ion covalent modifications of peptide and protein ions are demonstrated here using cluster-type reagent anions of N-hydroxysulfosuccinimide acetate (sulfo-NHS acetate) and 2-formyl-benzenesulfonic acid (FBMSA). These reagents are used here to selectively modify unprotonated primary amine functionalities of peptides and proteins. Multiple reactive reagent molecules can be present in a single cluster ion, which allows for multiple covalent modifications to be achieved in a single ion/ion encounter and at the ‘cost’ of only a single analyte charge. Multiple derivatizations are demonstrated when the number of available reactive sites on the analyte cation exceeds the number of reagent molecules in the anionic cluster (e.g., data shown here for reactions between the polypeptide [K10+3H]3+ and the reagent cluster [5R5Na-Na]−). This type of gas phase ion chemistry is also applicable to whole protein ions. Here, ubiquitin was successfully modified using an FBMSA cluster anions which, upon collisional activation, produced fragment ions with various numbers of modifications. Data for the pentamer cluster are included here as illustrative of the results obtained for the clusters comprised of 2–6 reagent molecules. PMID:23702708

  2. Lipopolysaccharide Phosphorylation by the WaaY Kinase Affects the Susceptibility of Escherichia coli to the Human Antimicrobial Peptide LL-37*

    PubMed Central

    Bociek, Karol; Ferluga, Sara; Mardirossian, Mario; Benincasa, Monica; Tossi, Alessandro; Gennaro, Renato; Scocchi, Marco

    2015-01-01

    The human cathelicidin LL-37 is a multifunctional host defense peptide with immunomodulatory and antimicrobial roles. It kills bacteria primarily by altering membrane barrier properties, although the exact sequence of events leading to cell lysis has not yet been completely elucidated. Random insertion mutagenesis allowed isolation of Escherichia coli mutants with altered susceptibility to LL-37, pointing to factors potentially relevant to its activity. Among these, inactivation of the waaY gene, encoding a kinase responsible for heptose II phosphorylation in the LPS inner core, leads to a phenotype with decreased susceptibility to LL-37, stemming from a reduced amount of peptide binding to the surface of the cells, and a diminished capacity to lyse membranes. This points to a specific role of the LPS inner core in guiding LL-37 to the surface of Gram-negative bacteria. Although electrostatic interactions are clearly relevant, the susceptibility of the waaY mutant to other cationic helical cathelicidins was unaffected, indicating that particular structural features or LL-37 play a role in this interaction. PMID:26100635

  3. Cationic antimicrobial peptide killing of African trypanosomes and Sodalis glossinidius, a bacterial symbiont of the insect vector of sleeping sickness.

    PubMed

    Haines, Lee R; Hancock, Robert E W; Pearson, Terry W

    2003-01-01

    Nine biochemically distinct cationic antimicrobial peptides were tested in vitro for their effects on bloodstream forms and procyclic (insect) forms of African trypanosomes, the protozoan parasites that cause African sleeping sickness in humans and trypanosomiasis in domestic animals. At low concentrations, one peptide completely inhibited growth of bloodstream forms, one inhibited procyclic forms, and five inhibited both trypanosome life cycle stages. The peptides were also tested on Sodalis glossinidius, a bacterial symbiont of tsetse flies. S. glossinidius was highly resistant to seven of the nine peptides, including both that specifically inhibited either bloodstream or procyclic forms and three of the five that inhibited both trypanosome life cycle stages. The results indicate that several of these peptides may be ideal candidates for therapy of trypanosome infected mammals or for transgenic expression in S. glossinidius as a strategy for inhibiting trypanosome survival, development, and maturation in tsetse and interference with transmission of African sleeping sickness.

  4. A weak cation-exchange monolith as stationary phase for the separation of peptide diastereomers by CEC.

    PubMed

    Ludewig, Ronny; Nietzsche, Sandor; Scriba, Gerhard K E

    2011-01-01

    A CEC weak cation-exchange monolith has been prepared by in situ polymerization of acrylamide, methylenebisacrylamide and 4-acrylamidobutyric acid in a decanol-dimethylsulfoxide mixture as porogen. The columns were evaluated by SEM and characterized with regard to the separation of diastereomers and α/β-isomers of aspartyl peptides. Column preparation was reproducible as evidenced by comparison of the analyte retention times of several columns prepared simultaneously. Analyte separation was achieved using mobile phases consisting of acidic phosphate buffer and ACN. Under these conditions the peptides migrated due to their electrophoretic mobility but the EOF also contributed as driving force as a function of the pH of the mobile phase due to increasing dissociation of the carboxyl groups of the polymer. Raising the pH of the mobile phase also resulted in deprotonation of the peptides reducing analyte mobility. Due to these mechanisms each pair of diastereomeric peptides displayed the highest resolution at a different pH of the buffer component of the mobile phase. Comparing the weak-cation exchange monolith to an RP monolith and a strong cation-exchange monolith different elution order of some peptide diastereomers was observed, clearly illustrating that interactions with the stationary phase contribute to the CEC separations.

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

    PubMed

    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.

  6. Stable isotope N-phosphorylation labeling for Peptide de novo sequencing and protein quantification based on organic phosphorus chemistry.

    PubMed

    Gao, Xiang; Wu, Hanzhi; Lee, Kim-Chung; Liu, Hongxia; Zhao, Yufen; Cai, Zongwei; Jiang, Yuyang

    2012-12-04

    In this paper, we describe the development of a novel stable isotope N-phosphorylation labeling (SIPL) strategy for peptide de novo sequencing and protein quantification based on organic phosphorus chemistry. The labeling reaction could be performed easily and completed within 40 min in a one-pot reaction without additional cleanup procedures. It was found that N-phosphorylation labeling reagents were activated in situ to form labeling intermediates with high reactivity targeting on N-terminus and ε-amino groups of lysine under mild reaction conditions. The introduction of N-terminal-labeled phosphoryl group not only improved the ionization efficiency of peptides and increased the protein sequence coverage for peptide mass fingerprints but also greatly enhanced the intensities of b ions, suppressed the internal fragments, and reduced the complexity of the tandem mass spectrometry (MS/MS) fragmentation patterns of peptides. By using nano liquid chromatography chip/time-of-flight mass spectrometry (nano LC-chip/TOF MS) for the protein quantification, the obtained results showed excellent correlation of the measured ratios to theoretical ratios with relative errors ranging from 0.5% to 6.7% and relative standard deviation of less than 10.6%, indicating that the developed method was reproducible and precise. The isotope effect was negligible because of the deuterium atoms were placed adjacent to the neutral phosphoryl group with high electrophilicity and moderately small size. Moreover, the SIPL approach used inexpensive reagents and was amenable to samples from various sources, including cell culture, biological fluids, and tissues. The method development based on organic phosphorus chemistry offered a new approach for quantitative proteomics by using novel stable isotope labeling reagents.

  7. Zymogen activation confers thermodynamic stability on a key peptide bond and protects human cationic trypsin from degradation.

    PubMed

    Szabó, András; Radisky, Evette S; Sahin-Tóth, Miklós

    2014-02-21

    Human cationic trypsinogen, precursor of the digestive enzyme trypsin, can be rapidly degraded to protect the pancreas when pathological conditions threaten, while trypsin itself is impressively resistant to degradation. For either form, degradation is controlled by two necessary initial proteolytic events: cleavage of the Leu81-Glu82 peptide bond by chymotrypsin C (CTRC) and cleavage of the Arg122-Val123 peptide bond by trypsin. Here we demonstrate that the Leu81-Glu82 peptide bond of human cationic trypsin, but not trypsinogen, is thermodynamically stable, such that cleavage by CTRC leads to an equilibrium mixture containing 10% cleaved and 90% uncleaved trypsin. When cleaved trypsin was incubated with CTRC, the Leu81-Glu82 peptide bond was re-synthesized to establish the same equilibrium. The thermodynamic stability of the scissile peptide bond was not dependent on CTRC or Leu-81, as re-synthesis was also accomplished by other proteases acting on mutated cationic trypsin. The Leu81-Glu82 peptide bond is located within a calcium binding loop, and thermodynamic stability of the bond was strictly dependent on calcium and on the calcium-coordinated residue Glu-85. Trypsinolytic cleavage of the Arg122-Val123 site was also delayed in trypsin relative to trypsinogen in a calcium-dependent manner, but for this bond cleavage was modulated by kinetic rather than thermodynamic control. Our results reveal that the trypsinogen to trypsin conformational switch modulates cleavage susceptibility of nick sites by altering both the thermodynamics and kinetics of cleavage to protect human cationic trypsin from premature degradation.

  8. Molecular Dynamics Simulation of Tau Peptides for the Investigation of Conformational Changes Induced by Specific Phosphorylation Patterns.

    PubMed

    Gandhi, Neha S; Kukic, Predrag; Lippens, Guy; Mancera, Ricardo L

    2017-01-01

    The Tau protein plays an important role due to its biomolecular interactions in neurodegenerative diseases. The lack of stable structure and various posttranslational modifications such as phosphorylation at various sites in the Tau protein pose a challenge for many experimental methods that are traditionally used to study protein folding and aggregation. Atomistic molecular dynamics (MD) simulations can help around deciphering relationship between phosphorylation and various intermediate and stable conformations of the Tau protein which occur on longer timescales. This chapter outlines protocols for the preparation, execution, and analysis of all-atom MD simulations of a 21-amino acid-long phosphorylated Tau peptide with the aim of generating biologically relevant structural and dynamic information. The simulations are done in explicit solvent and starting from nearly extended configurations of the peptide. The scaled MD method implemented in AMBER14 was chosen to achieve enhanced conformational sampling in addition to a conventional MD approach, thereby allowing the characterization of folding for such an intrinsically disordered peptide at 293 K. Emphasis is placed on the analysis of the simulation trajectories to establish correlations with NMR data (i.e., chemical shifts and NOEs). Finally, in-depth discussions are provided for commonly encountered problems.

  9. A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C.

    PubMed

    Luangwedchakarn, Voravich; Day, Noorbibi K; Hitchcock, Remi; Brown, Pam G; Lerner, Danica L; Rucker, Rajivi P; Cianciolo, George J; Good, Robert A; Haraguchi, Soichi

    2003-05-01

    CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.

  10. Downstream signaling molecules bind to different phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) peptides of the high affinity IgE receptor.

    PubMed

    Kimura, T; Kihara, H; Bhattacharyya, S; Sakamoto, H; Appella, E; Siraganian, R P

    1996-11-01

    The cytoplasmic tails of both the beta and gamma subunits of the high affinity IgE receptor (FcepsilonRI) contain a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). This motif plays a critical role in receptor-mediated signal transduction. Synthetic peptides based on the ITAM sequences of the beta and gamma subunits of FcepsilonRI were used to investigate which proteins associate with these motifs. Tyrosine-phosphorylated beta and gamma ITAM peptides immobilized on beads precipitated Syk, Lyn, Shc, Grb2, and phospholipase C-gamma1 from lysates of rat basophilic leukemia RBL-2H3 cells. Syk was precipitated predominantly by the tyrosine-diphosphorylated gamma ITAM peptide, but much less by the diphosphorylated beta ITAM peptide or by the monophosphorylated peptides. Phospholipase C-gamma1, Shc, and Grb2 were precipitated only by the diphosphorylated beta ITAM peptide. Non-phosphorylated ITAM peptides did not precipitate these proteins. In membrane binding assays, fusion proteins containing the Src homology 2 domains of phospholipase C-gamma1, Shc, Syk, and Lyn directly bound the tyrosine-phosphorylated ITAM peptides. Although the ITAM sequences of the beta and gamma subunits of FcepsilonRI are similar, once they are tyrosine-phosphorylated they preferentially bind different downstream signaling molecules. Tyrosine phosphorylation of the ITAM of the gamma subunit recruits and activates Syk, whereas the beta subunit may be important for the Ras signaling pathway.

  11. Structure and further fragmentation of significant [a3 + Na - H]+ ions from sodium-cationized peptides.

    PubMed

    Wang, Huixin; Wang, Bing; Wei, Zhonglin; Zhang, Hao; Guo, Xinhua

    2015-01-01

    A good understanding of gas-phase fragmentation chemistry of peptides is important for accurate protein identification. Additional product ions obtained by sodiated peptides can provide useful sequence information supplementary to protonated peptides and improve protein identification. In this work, we first demonstrate that the sodiated a3 ions are abundant in the tandem mass spectra of sodium-cationized peptides although observations of a3 ions have rarely been reported in protonated peptides. Quantum chemical calculations combined with tandem mass spectrometry are used to investigate this phenomenon by using a model tetrapeptide GGAG. Our results reveal that the most stable [a3 + Na - H](+) ion is present as a bidentate linear structure in which the sodium cation coordinates to the two backbone carbonyl oxygen atoms. Due to structural inflexibility, further fragmentation of the [a3 + Na - H](+) ion needs to overcome several relatively high energetic barriers to form [b2 + Na - H](+) ion with a diketopiperazine structure. As a result, low abundance of [b2 + Na - H](+) ion is detected at relatively high collision energy. In addition, our computational data also indicate that the common oxazolone pathway to generate [b2 + Na - H](+) from the [a3 + Na - H](+) ion is unlikely. The present work provides a mechanistic insight into how a sodium ion affects the fragmentation behaviors of peptides.

  12. 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.

  13. An alpha-helical cationic antimicrobial peptide selectively modulates macrophage responses to lipopolysaccharide and directly alters macrophage gene expression.

    PubMed

    Scott, M G; Rosenberger, C M; Gold, M R; Finlay, B B; Hancock, R E

    2000-09-15

    Certain cationic antimicrobial peptides block the binding of LPS to LPS-binding protein and reduce the ability of LPS to induce the production of inflammatory mediators by macrophages. To gain a more complete understanding of how LPS activates macrophages and how cationic peptides influence this process, we have used gene array technology to profile gene expression patterns in macrophages treated with LPS in the presence or the absence of the insect-derived cationic antimicrobial peptide CEMA (cecropin-melittin hybrid). We found that CEMA selectively blocked LPS-induced gene expression in the RAW 264.7 macrophage cell line. The ability of LPS to induce the expression of >40 genes was strongly inhibited by CEMA, while LPS-induced expression of another 16 genes was relatively unaffected. In addition, CEMA itself induced the expression of a distinct set of 35 genes, including genes involved in cell adhesion and apoptosis. Thus, CEMA, a synthetic alpha-helical peptide, selectively modulates the transcriptional response of macrophages to LPS and can alter gene expression in macrophages.

  14. The Effect of the Secondary Structure on Dissociation of Peptide Radical Cations: Fragmentation of Angiotensin III and Its Analogues

    SciTech Connect

    Yang, Zhibo; Lam, Corey; Chu, Ivan K.; Laskin, Julia

    2008-09-28

    Fragmentation of protonated RVYIHPF and RVYIHPF-OMe and the corresponding radical cations was studied using time- and collision energy-resolved surface-induced dissociation (SID) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially equipped to perform SID experiments. Peptide radical cations were produced by gas-phase fragmentation of CoIII(salen)-peptide complexes. Both the energetics and mechanisms of dissociation of even-electron and odd-electron angiotensin III ions are quite different. Protonated molecules are much more stable towards fragmentation than the corresponding radical cations. RRKM modeling of the experimental data suggests that this stability is largely attributed to differences in threshold energies for dissociation while activation entropies are very similar. Detailed analysis of the experimental data obtained for radical cations demonstrated the presence of two distinct structures separated by a high free-energy barrier. The two families of structures were ascribed to the canonical and zwitterionic forms of the radical cations produced in our experiments.

  15. A cationic amphiphilic peptide ABP-CM4 exhibits selective cytotoxicity against leukemia cells.

    PubMed

    Chen, Yu Qing; Min, Cui; Sang, Ming; Han, Yang Yang; Ma, Xiao; Xue, Xiao Qing; Zhang, Shuang Quan

    2010-08-01

    Some cationic antibacterial peptides exhibit a broad spectrum of cytotoxic activity against cancer cells, which could provide a new class of anticancer drugs. In the present study, the anticancer activity of ABP-CM4, an antibacterial peptide from Bombyx mori, against leukemic cell lines THP-1, K562 and U937 was evaluated, and the cytotoxicity compared with the effects on non-cancerous mammalian cells, including peripheral blood mononuclear cells (PBMCs), HEK-293 and erythrocytes. ABP-CM4 reduced the number of viable cells of the leukemic cell lines after exposure for 24h. The reduction was concentration dependent, and the IC50 values ranged from 14 to 18 microM. Conversely, ABP-CM4, even at 120 microM, exhibited no cytotoxicity toward HEK-293 or PBMCs, indicating that there was no significant effect on these two types of non-cancer cells. ABP-CM4 at a concentration of 200 microM had no hemolytic activity on mammalian erythrocytes. Together, these results suggested a selective cytotoxicity in leukemia cells. Flow cytometry demonstrated that the binding activity of ABP-CM4 to leukemia cells was much higher than that to HEK-293 or PBMCs, and there was almost no binding to erythrocytes. FITC-labeled ABP-CM4 molecules were examined under a confocal microscope and found to be concentrated at the surface of leukemia cells and changes of the cell membrane were determined by a cell permeability assay, which led us to the conclusion that ABP-CM4 could act at the cell membrane for its anticancer activity on leukemia cells. Collectively, our results indicated that ABP-CM4 has the potential for development as a novel antileukemic agent.

  16. Anti-plasmodial action of de novo-designed, cationic, lysine-branched, amphipathic, helical peptides

    PubMed Central

    2012-01-01

    Background A lack of vaccine and rampant drug resistance demands new anti-malarials. Methods In vitro blood stage anti-plasmodial properties of several de novo-designed, chemically synthesized, cationic, amphipathic, helical, antibiotic peptides were examined against Plasmodium falciparum using SYBR Green assay. Mechanistic details of anti-plasmodial action were examined by optical/fluorescence microscopy and FACS analysis. Results Unlike the monomeric decapeptides {(Ac-GXRKXHKXWA-NH2) (X = F,ΔF) (Fm, ΔFm IC50 >100 μM)}, the lysine-branched,dimeric versions showed far greater potency {IC50 (μM) Fd 1.5 , ΔFd 1.39}. The more helical and proteolytically stable ΔFd was studied for mechanistic details. ΔFq, a K-K2 dendrimer of ΔFm and (ΔFm)2 a linear dimer of ΔFm showed IC50 (μM) of 0.25 and 2.4 respectively. The healthy/infected red cell selectivity indices were >35 (ΔFd), >20 (ΔFm)2 and 10 (ΔFq). FITC-ΔFd showed rapid and selective accumulation in parasitized red cells. Overlaying DAPI and FITC florescence suggested that ΔFd binds DNA. Trophozoites and schizonts incubated with ΔFd (2.5 μM) egressed anomalously and Band-3 immunostaining revealed them not to be associated with RBC membrane. Prematurely egressed merozoites from peptide-treated cultures were found to be invasion incompetent. Conclusion Good selectivity (>35), good resistance index (1.1) and low cytotoxicity indicate the promise of ΔFd against malaria. PMID:22853877

  17. Unexpectedly Enhanced Solubility of Aromatic Amino Acids and Peptides in an Aqueous Solution of Divalent Transition-Metal Cations

    NASA Astrophysics Data System (ADS)

    Shi, Guosheng; Dang, Yaru; Pan, Tingting; Liu, Xing; Liu, Hui; Li, Shaoxian; Zhang, Lijuan; Zhao, Hongwei; Li, Shaoping; Han, Jiaguang; Tai, Renzhong; Zhu, Yiming; Li, Jichen; Ji, Qing; Mole, R. A.; Yu, Dehong; Fang, Haiping

    2016-12-01

    We experimentally observed considerable solubility of tryptophan (Trp) in a CuCl2 aqueous solution, which could reach 2-5 times the solubility of Trp in pure water. Theoretical studies show that the strong cation-π interaction between Cu2 + and the aromatic ring in Trp modifies the electronic distribution of the aromatic ring to enhance significantly the water affinity of Trp. Similar solubility enhancement has also been observed for other divalent transition-metal cations (e.g., Zn2 + and Ni2 + ), another aromatic amino acid (phenylalanine), and three aromatic peptides (Trp-Phe, Phe-Phe, and Trp-Ala-Phe).

  18. 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

  19. Cationic peptides combined with betalactams reduce mortality from peritonitis in experimental rat model.

    PubMed

    Ghiselli, Roberto; Giacometti, Andrea; Cirioni, Oscar; Mocchegiani, Federico; Viticchi, Claudio; Scalise, Giorgio; Saba, Vittorio

    2002-11-01

    The efficacy of cationic peptides combined with betalactams was investigated in a peritonitis rat model. Intraabdominal sepsis was induced in adult Wistar rats via cecal ligation and single puncture. The study included eight drug-treated groups: each of them received intravenous polymyxin-E (1 mg/kg), buforin II (1 mg/kg), imipenem (20 mg/kg), amoxicillin-clavulanate (50 mg/kg), polymyxin-E (1 mg/kg) plus imipenem (20 mg/kg), or amoxicillin-clavulanate (50 mg/kg), and buforin II (1 mg/kg) plus imipenem (20 mg/kg), or amoxicillin-clavulanate (50 mg/kg). The study included an untreated control group that received intravenous isotonic sodium chloride solution. All compounds significantly reduced the lethality and the number of bacteria in abdominal fluid compared with saline treatment. Among compounds, imipenem showed the highest antimicrobial activity, while buforin II produced the highest reduction in plasma endotoxin and TNF-alpha levels. Overall, buforin II and imipenem association were the most effective therapeutic approach. Data presented here suggest the potential advantages of combining antimicrobial agents and compounds able to neutralize the biological effect of the endotoxin.

  20. 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

  1. Evaluating Kinase ATP Uptake and Tyrosine Phosphorylation using Multiplexed Quantification of Chemically Labeled and Post-Translationally Modified Peptides

    PubMed Central

    Fang, Bin; Hoffman, Melissa A.; Mirza, Abu-Sayeef; Mishall, Katie M.; Li, Jiannong; Peterman, Scott M.; Smalley, Keiran S. M.; Shain, Kenneth H.; Weinberger, Paul M.; Wu, Jie; Rix, Uwe; Haura, Eric B.; Koomen, John M.

    2015-01-01

    Cancer biologists and other healthcare researchers face an increasing challenge in addressing the molecular complexity of disease. Biomarker measurement tools and techniques now contribute to both basic science and translational research. In particular, liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for multiplexed measurements of protein biomarkers has emerged as a versatile tool for systems biology. Assays can be developed for specific peptides that report on protein expression, mutation, or post-translational modification; discovery proteomics data rapidly translated into multiplexed quantitative approaches. Complementary advances in affinity purification enrich classes of enzymes or peptides representing post-translationally modified or chemically labeled substrates. Here, we illustrate the process for the relative quantification of hundreds of peptides in a single LC-MRM experiment. Desthiobiotinylated peptides produced by activity-based protein profiling (ABPP) using ATP probes and tyrosine-phosphorylated peptides are used as examples. These targeted quantification panels can be applied to further understand the biology of human disease. PMID:25782629

  2. 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.

  3. Salivary mucins inhibit antibacterial activity of the cathelicidin-derived LL-37 peptide but not the cationic steroid CSA-13

    PubMed Central

    Bucki, Robert; Namiot, Dorota B.; Namiot, Zbigniew; Savage, Paul B.; Janmey, Paul A.

    2008-01-01

    Objectives Cationic antimicrobial peptides (CAPs) are the effector molecules of innate immunity, similar in potency to classic antibiotics that function in the first-line of defence against infectious agents. The purpose of this study was to investigate the effects of negatively charged mucins on the antibacterial activity of the positively charged cathelicidin LL-37 peptide, its synthetic analogue WLBU2 and the antimicrobial cationic steroid CSA-13. Methods Mucin, DNA, F-actin and hCAP-18/LL-37 in saliva samples were evaluated by microscopy or immunoblotting. Bacterial killing assays and determination of MICs were used to determine bactericidal activity. Binding of rhodamine-B-labelled LL-37 peptide to mucin was fluorimetrically assessed. Results Microscopic evaluation of saliva after addition of rhodamine-B-labelled LL-37 showed localization similar to that observed after the addition of a specific mucin-binding lectin. Immunoblotting confirmed the presence of hCAP-18/LL-37 in saliva samples and LL-37 peptide bound to isolated submaxillary gland mucin-coated plates. Mucin/LL-37 binding was partially prevented by treatment of mucin with neuraminidase, indicating involvement of sialic acid moieties. Decreased LL-37 and WLBU2 antibacterial activity was observed in the presence of mucin or dialysed human saliva, whereas CSA-13 antibacterial activity was significantly resistant to inhibition by mucins. Conclusions This study shows that the antibacterial LL-37 peptide and its synthetic analogue WLBU2 are inhibited by salivary mucin and that the cationic steroid CSA-13 retains most of its function in the presence of an equal amount of mucin or saliva. PMID:18456648

  4. Formation of AAB-Type Collagen Heterotrimers from Designed Cationic and Aromatic Collagen-Mimetic Peptides: Evaluation of the C-Terminal Cation-π Interactions.

    PubMed

    Chiang, Chu-Harn; Fu, Yi-Hsuan; Horng, Jia-Cherng

    2017-03-13

    Most of natural collagens are heterotrimers composed of two (AAB) or three (ABC) different peptide chains, and thus heterotrimeric constructs are preferable to mimic natural collagens. Exploring the forces to assemble synthetic collagen-mimetic peptides (CMPs) into heterotrimers has been an attractive topic in preparing collagen-related biomaterials. Here we designed and synthesized two cationic CMPs (CR and CK) in which multiple Arg or Lys residues are installed in their C-terminal region, and one aromatic CMP (CF) whose C-terminal end contains multiple Phe residues. Circular dichroism and NMR spectroscopy showed that AAB-type heterotrimers could form in both CR-CF and CK-CF mixtures, suggesting that the C-terminal cation-π interactions between cationic and aromatic residues could serve as a nucleation force and substantially promote the folding of heterotrimers. In particular, only one major heterotrimeric fold was found in each mixture. For CR-CF mixtures, either the heterotrimer with two CR chains and one CF chain or that with one CR chain and two CF chains could form, depending on the molar ratios of CR to CF in solution. By contrast, in CK-CF mixtures only the heterotrimer consisting of two CK chains and one CF chain was found in solution even increasing the ratio of CF, implying that the heterotrimer composed of one CK chain and two CF chains is highly unstable. Additionally, differential scanning calorimetry analysis showed that the folding of these heterotrimers is governed by entropic effects. Together, our results provide a new design to prepare AAB-type collagen heterotrimers and reveal new insights into their folding thermodynamics.

  5. 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.

  6. Primary structure of three cationic peptides from porcine neutrophils. Sequence determination by the combined usage of electrospray ionization mass spectrometry and Edman degradation.

    PubMed

    Mirgorodskaya, O A; Shevchenko, A A; Abdalla, K O; Chernushevich, I V; Egorov, T A; Musoliamov, A X; Kokryakov, V N; Shamova, O V

    1993-09-20

    The primary structure of three major cationic peptides from porcine neutrophils has been determined. The sequencing was made by the combined use of electrospray ionization mass spectrometry and Edman degradation. The determined sequences unambiguously show that these peptides can not be considered as defensins.

  7. 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.

  8. 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.

  9. Identification of proteolytic activities in ROS 17/2.8 cell lysates which cleave peptide substrates for protein kinase C-mediated phosphorylation.

    PubMed

    Guidon, P T; Harrison, P

    1996-04-01

    We have observed two proteolytic activities in cell lysates from the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation, and other peptides containing similar sequences. Both activities are inhibited by Pefabloc, a serine protease inhibitor, while one of the activities is inhibited by either EDTA or aprotinin. The protease inhibitors pepstatin, bestatin, E-64, leupeptin and phosphoramidon do not block either of these proteolytic activities.

  10. Development of helix-stabilized cell-penetrating peptides containing cationic α,α-disubstituted amino acids as helical promoters.

    PubMed

    Yamashita, Hiroko; Misawa, Takashi; Oba, Makoto; Tanaka, Masakazu; Naito, Mikihiko; Kurihara, Masaaki; Demizu, Yosuke

    2017-03-15

    Cell-penetrating peptides (CPP) have attracted many scientists' attention as intracellular delivery tools due to their high cargo molecule transportation efficiency and low cytotoxicity. Therefore, in many research fields CPP, such as HIV-Tat and oligoarginine (Rn), are used to deliver hydrophilic drugs and biomolecules, including proteins, DNA, and RNA. We designed four types of CPP that contained cationic α,α-disubstituted amino acids (Api(C2Gu) and Api(C4Gu)) as helical promoters; i.e., 1-4 [FAM-β-Ala-(l-Arg-l-Arg-Xaa)3-(Gly)3-NH2 (1: Xaa=Api(C2Gu), 2: Xaa=Api(C4Gu)), 3: FAM-β-Ala-(l-Arg)8-Api(C2Gu)-(Gly)3-NH2, and 4: FAM-β-Ala-(l-Arg)5-Api(C2Gu)-(l-Arg)2-Api(C2Gu)-(Gly)3-NH2], and investigated their preferred secondary structures and cell membrane-penetrating ability. As a result, we found that the permeation efficiency of the CPP was affected by the number of helical promoters in their sequences. Specially, peptide 1, which contained three Api(C2Gu) residues, formed a stable helical structure and passed through the cell membrane more efficiently than the other peptides. Moreover, it was demonstrated that the spatial arrangement of the peptides' side chains also influenced their permeability and the helical stabilization of their main chains.

  11. 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.

  12. Mechanical Dilution of Beta-amyloid Peptide and Phosphorylated Tau Protein in Alzheimer's Disease: Too Simple to be True?

    PubMed Central

    2017-01-01

    The neuropathology of Alzheimer's disease (AD) is characterized by the widespread accumulation of neuritic plaques and neurofibrillary tangles composed of deposits of beta-amyloid peptide (Aβ) and abnormally phosphorylated tau protein (phospho-tau) respectively. Considerable effort has been expended to identify methods to retard the deposition of these proteins or to enhance their clearance. It is strikingly surprising that until now, very few researchers have attempted to remove these proteins using mechanical procedures. In this article, we start by showing the rationale of mechanical dilution of cerebrospinal fluid (CSF) as a therapeutic approach in AD. Then, we present models of implantable systems allowing mechanical dilution of CSF by means of CSF replacement and CSF filtration (liquorpheresis). We conclude that even though this approach seems simplistic, it is feasible and deserves exploration.

  13. Charge Transfer Dissociation (CTD) Mass Spectrometry of Peptide Cations: Study of Charge State Effects and Side-Chain Losses

    NASA Astrophysics Data System (ADS)

    Li, Pengfei; Jackson, Glen P.

    2017-01-01

    1+, 2+, and 3+ precursors of substance P and bradykinin were subjected to helium cation irradiation in a 3D ion trap mass spectrometer. Charge exchange with the helium cations produces a variety of fragment ions, the number and type of which are dependent on the charge state of the precursor ions. For 1+ peptide precursors, fragmentation is generally restricted to C-CO backbone bonds (a and x ions), whereas for 2+ and 3+ peptide precursors, all three backbone bonds (C-CO, C-N, and N-Cα) are cleaved. The type of backbone bond cleavage is indicative of possible dissociation channels involved in CTD process, including high-energy, kinetic-based, and ETD-like pathways. In addition to backbone cleavages, amino acid side-chain cleavages are observed in CTD, which are consistent with other high-energy and radical-mediated techniques. The unique dissociation pattern and supplementary information available from side-chain cleavages make CTD a potentially useful activation method for the structural study of gas-phase biomolecules.

  14. Polymer monoliths with low hydrophobicity for strong cation-exchange capillary liquid chromatography of peptides and proteins.

    PubMed

    Gu, Binghe; Li, Yun; Lee, Milton L

    2007-08-01

    Two polymer monoliths were designed and synthesized from commercially available monomers with an attempt to decrease hydrophobicity for strong cation-exchange chromatography. One was prepared from the copolymerization of sulfoethyl methacrylate and poly(ethylene glycol) diacrylate, and the other was synthesized from vinylsulfonic acid and poly(ethylene glycol) diacrylate. Both of the monoliths were synthesized inside 75-microm i.d., UV-transparent fused-silica capillaries by photopolymerization. The hydrophobicities of the two monoliths were systematically evaluated using standard synthetic undecapeptides under ion-exchange conditions and propyl paraben under reversed-phase conditions. The poly(sulfoethyl methacrylate) monolith demonstrated similar hydrophobicity as a monolith prepared from copolymerization of 2-acrylamido-2-methyl-1-propanesulfonic acid and poly(ethylene glycol) diacrylate, and 40% acetonitrile was required to suppress any hydrophobic interactions with peptides under ion-exchange conditions. However, with the use of vinylsulfonic acid as the functional monomer, a monolith with very low hydrophobicity was obtained, making it suitable for strong cation-exchange liquid chromatography of both peptides and proteins. It was found that monolith hydrophobicity could be adjusted by selection of monomers that differ in hydrocarbon content and type of vinyl group. Finally, excellent separations of model protein standards and high-density lipoproteins were achieved using the poly(vinylsulfonic acid) monolith. Five subclasses of high-density lipoproteins were resolved using a simple linear NaCl gradient.

  15. 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

    2016-01-01

    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.

  16. Similarities and differences of copper and zinc cations binding to biologically relevant peptides studied by vibrational spectroscopies.

    PubMed

    Schirer, Alicia; El Khoury, Youssef; Faller, Peter; Hellwig, Petra

    2017-03-20

    GHK and DAHK are biological peptides that bind both copper and zinc cations. Here we used infrared and Raman spectroscopies to study the coordination modes of both copper and zinc ions, at pH 6.8 and 8.9, correlating the data with the crystal structures that are only available for the copper-bound form. We found that Cu(II) binds to deprotonated backbone (amidate), the N-terminus and N(π) of the histidine side chain, in both GHK and DAHK, at pH 6.8 and 8.9. The data for the coordination of zinc at pH 6.8 points to two conformers including both nitrogens of a histidine residue. At pH 8.9, vibrational spectra of the ZnGHK complexes show that equilibria between monomers, oligomers exist, where deprotonated histidine residues as well as deprotonated amide nitrogen are involved in the coordination. A common feature is found: zinc cations coordinate to N(τ) and/or N(π) of the His leading to the formation of GHK and DAHK multimers. In contrast, Cu(II) binds His via N(π) regardless of the peptide, in a pH-independent manner.

  17. 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.

  18. Binding Interactions of Bacterial Lipopolysaccharide and the Cationic Amphiphilic Peptides Polymyxin B and WLBU2

    PubMed Central

    Ryder, Matthew P.; Wu, Xiangming; McKelvey, GregR.; McGuire, Joseph; Schilke, Karl F.

    2014-01-01

    Passage of blood through a sorbent device for removal of bacteria and endotoxin by specific binding with immobilized, membrane-active, bactericidal peptides holds promise for treating severe blood infections. Peptide insertion in the target membrane and rapid/strong binding is desirable, while membrane disruption and release of degradation products to the circulating blood is not. Here we describe interactions between bacterial endotoxin (lipopolysaccharide, LPS) and the membrane-active, bactericidal peptides WLBU2 and polymyxin B (PmB). Analysis of the interfacial behavior of mixtures of LPS and peptide using air-water interfacial tensiometry and optical waveguide lightmode spectroscopy strongly suggests insertion of intact LPS vesicles by the peptide WLBU2 without vesicle destabilization. In contrast, dynamic light scattering (DLS) studies show that LPS vesicles appear to undergo peptide-induced destabilization in the presence of PmB. Circular dichroism spectra further confirm that WLBU2, which shows disordered structure in aqueous solution and substantially helical structure in membrane-mimetic environments, is stably located within the LPS membrane in peptide-vesicle mixtures. We therefore expect that presentation of WLBU2 at an interface, if tethered in a fashion which preserves its mobility and solvent accessibility, will enable the capture of bacteria and endotoxin without promoting reintroduction of endotoxin to the circulating blood, thus minimizing adverse clinical outcomes. On the other hand, our results suggest no such favorable outcome of LPS interactions with polymyxin B. PMID:24905681

  19. Differential binding of L- vs. D-isomers of cationic antimicrobial peptides to the biofilm exopolysaccharide alginate.

    PubMed

    Yin, Lois M; Lee, Soyoung; Mak, Jacky S W; Helmy, Amr S; Deber, Charles M

    2013-08-01

    Alginate is a biofilm exopolysaccharide secreted by the opportunistic pathogen Pseudomonas aeruginosa that acts to prevent the diffusion of antibiotics toward the bacterial cell membrane. Cationic antimicrobial peptides (CAPs) have been increasingly recognized as a viable alternative for prospective antimicrobial agents. The D-isomer chiral counterparts of active L-isomer CAPs tend to show slightly greater antimicrobial activities because bacteria lack proteases to hydrolyze the unnatural D-isomers. Using an enantiomeric pair of synthetic CAPs designed in our laboratory (L-4Leu in the sequence KKKKKKALFALWLAFLA-NH2 and its D-analog D-4Leu), we studied the binding and interactions of Lvs. D-isomers of CAPs with alginate using circular dichroism and Raman spectroscopic techniques. We found that the peptide D-4Leu underwent a more rapid structural transition over time from an initial alginate-induced α-helical conformation to a less soluble β-sheet conformation than L-4Leu, indicating that the D-isomer of this peptide has a relatively greater affinity for alginate. Through Raman spectroscopy it was observed that Raman modes at 1297 cm-1 and 1453 cm-1 wavenumbers were found to differ between the spectra obtained from the insoluble complexes formed between L-4Leu vs. D-4Leu and alginate. These modes were tentatively assigned to CH, and CH3 deformation modes, respectively. Our findings reveal previously undetected subtleties in the binding of this diastereomeric pair of peptides in the microenvironment of a biofilm exopolysaccharide, and provide guidelines for future development of antimicrobial peptides.

  20. Bovine and human cathelicidin cationic host defense peptides similarly suppress transcriptional responses to bacterial lipopolysaccharide.

    PubMed

    Mookherjee, Neeloffer; Wilson, Heather L; Doria, Silvana; Popowych, Yurij; Falsafi, Reza; Yu, Jie Jessie; Li, Yuexin; Veatch, Sarah; Roche, Fiona M; Brown, Kelly L; Brinkman, Fiona S L; Hokamp, Karsten; Potter, Andy; Babiuk, Lorne A; Griebel, Philip J; Hancock, Robert E W

    2006-12-01

    Genomic approaches can be exploited to expose the complexities and conservation of biological systems such as the immune network across various mammalian species. In this study, temporal transcriptional expression profiles were analyzed in human and bovine monocytic cells in response to the TLR-4 agonist, LPS, in the presence or absence of their respective host defense peptides. The cathelicidin peptides, human LL-37 and bovine myeloid antimicrobial peptide-27 (BMAP-27), are homologs, yet they have diverged notably in terms of sequence similarity. In spite of their low sequence similarities, both of these cathelicidin peptides demonstrated potent, antiendotoxin activity in monocytic cells at low, physiologically relevant concentrations. Microarray studies indicated that 10 ng/ml LPS led to the up-regulation of 125 genes in human monocytes, 106 of which were suppressed in the presence of 5 mug/ml of the human peptide LL-37. To confirm and extend these data, temporal transcriptional responses to LPS were assessed in the presence or absence of the species-specific host defense peptides by quantitative real-time PCR. The transcriptional trends of 20 LPS-induced genes were analyzed in bovine and human monocytic cells. These studies demonstrated conserved trends of gene responses in that both peptides were able to profoundly suppress many LPS-induced genes. Consistent with this, the human and bovine peptides suppressed LPS-induced translocation of NF-kappaB subunits p50 and p65 into the nucleus of monocytic cells. However, there were also distinct differences in responses to LPS and the peptides; for example, treatment with 5 mug/ml BMAP-27 alone tended to influence gene expression (RELA, TNF-alpha-induced protein 2, MAPK phosphatase 1/dual specificity phosphatase 1, IkappaBkappaB, NFkappaBIL1, TNF receptor-associated factor 2) to a greater extent than did the same amount of human LL-37. We hypothesize that the immunomodulatory effects of the species-specific host

  1. Electrospray tandem mass spectrometry of alkali-cationized BocN-carbo-alpha,beta- and -beta,alpha-peptides: Differentiation of positional isomers.

    PubMed

    Reddy, P Nagi; Srikanth, R; Srinivas, R; Sharma, V U M; Sharma, G V M; Nagendar, P

    2006-01-01

    Dissociation pathways of a series of alkali-cationized hybrid peptides, viz., Boc-alpha,beta- and -beta,alpha-carbopeptides, synthesized from C-linked carbo-beta3-amino acids [Caa (S)] and alpha-alanine (L-Ala), have been investigated by electrospray ionization tandem mass spectrometry. The positional isomers (six pairs) of the cationized alpha,beta- and beta,alpha-peptides can be differentiated by the collision-induced dissociation (CID) spectra of their [M + Cat-Boc + H]+ ions which give characteristic series of alkali-cationized C- (x(n)+, y(n)+, z(n)+) and N-terminal (a(n)+, b(n)+, c(n)+) ions. Another noteworthy difference is cationized beta,alpha-peptides eliminate a molecule of ammonia whereas this pathway is absent for alpha,beta-peptides. This is useful for identifying the presence of a beta-amino acid at the N-terminus. The CID spectra of [M + Cat-Boc + H]+ ions of these peptide acids show abundant rearrangement [b(n) + 17 + Cat]+ (n = 1 to n-1) ions which is diagnostic for distinguishing between alpha- and beta-amino acid at the C-terminus. MS(n) experiments of [b(n) + Li-H]+ ions from these hybrid peptides showed the loss of CO and 72 u giving rise to [a(n) + Li-H]+ and cationized nitrile product ions which render support to earlier proposals that b(n)+ or [b(n) + Cat-H]+ ions have protonated or cationized oxazolinone structures, respectively.

  2. 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

  3. Iron Oxide Nanoparticles Coated with a Phosphorothioate Oligonucleotide and a Cationic Peptide: Exploring Four Different Ways of Surface Functionalization

    PubMed Central

    Geinguenaud, Frédéric; Banissi, Claire; Carpentier, Antoine F.; Motte, Laurence

    2015-01-01

    The superparamagnetic iron oxide nanoparticles (SPIONs) have great potential in therapeutic and diagnostic applications. Due to their superparamagnetic behavior, they are used clinically as a Magnetic Resonance Imaging (MRI) contrast agent. Iron oxide nanoparticles are also recognized todays as smart drug-delivery systems. However, to increase their specificity, it is essential to functionalize them with a molecule that effectively targets a specific area of the body. Among the molecules that can fulfill this role, peptides are excellent candidates. Oligonucleotides are recognized as potential drugs for various diseases but suffer from poor uptake and intracellular degradation. In this work, we explore four different strategies, based on the electrostatic interactions between the different partners, to functionalize the surface of SPIONs with a phosphorothioate oligonucleotide (ODN) and a cationic peptide labeled with a fluorophore. The internalization of the nanoparticles has been evaluated in vitro on RAW 264.7 cells. Among these strategies, the “«one-step assembly»”, i.e., the direct complexation of oligonucleotides and peptides on iron oxide nanoparticles, provides the best way of coating for the internalization of the nanocomplexes.

  4. Near-UV Photodissociation of Tryptic Peptide Cation Radicals. Scope and Effects of Amino Acid Residues and Radical Sites

    NASA Astrophysics Data System (ADS)

    Nguyen, Huong T. H.; Tureček, František

    2017-02-01

    Peptide cation-radical fragment ions of the z-type, [●AXAR+], [●AXAK+], and [●XAR+], where X = A, C, D, E, F, G, H, K, L, M, N, P, Y, and W, were generated by electron transfer dissociation of peptide dications and investigated by MS3-near-ultraviolet photodissociation (UVPD) at 355 nm. Laser-pulse dependence measurements indicated that the ion populations were homogeneous for most X residues except phenylalanine. UVPD resulted in dissociations of backbone CO-NH bonds that were accompanied by hydrogen atom transfer, producing fragment ions of the [yn]+ type. Compared with collision-induced dissociation, UVPD yielded less side-chain dissociations even for residues that are sensitive to radical-induced side-chain bond cleavages. The backbone dissociations are triggered by transitions to second (B) excited electronic states in the peptide ion R-CH●-CONH- chromophores that are resonant with the 355-nm photon energy. Electron promotion increases the polarity of the B excited states, R-CH+-C●(O-)NH-, and steers the reaction to proceed by transfer of protons from proximate acidic Cα and amide nitrogen positions.

  5. 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.

  6. Systematic evaluation of alternating CID and ETD fragmentation for phosphorylated peptides.

    PubMed

    Kim, Min-Sik; Zhong, Jun; Kandasamy, Kumaran; Delanghe, Bernard; Pandey, Akhilesh

    2011-06-01

    CID has become a routine method for fragmentation of peptides in shotgun proteomics, whereas electron transfer dissociation (ETD) has been described as a preferred method for peptides carrying labile PTMs. Though both of these fragmentation techniques have their obvious advantages, they also have their own drawbacks. By combining data from CID and ETD fragmentation, some of these disadvantages can potentially be overcome because of the complementarity of fragment ions produced. To evaluate alternating CID and ETD fragmentation, we analyzed a complex mixture of phosphopeptides on an LTQ-Orbitrap mass spectrometer. When the CID and ETD-derived spectra were searched separately, we observed 2504, 491, 2584, and 3249 phosphopeptide-spectrum matches from CID alone, ETD alone, decision tree-based CID/ETD, and alternating CID and ETD, respectively. Combining CID and ETD spectra prior to database searching should, intuitively, be superior to either method alone. However, when spectra from the alternating CID and ETD method were merged prior to database searching, we observed a reduction in the number of phosphopeptide-spectrum matches. The poorer identification rates observed after merging CID and ETD spectra are a reflection of a lack of optimized search algorithms for carrying out such searches and perhaps inherent weaknesses of this approach. Thus, although alternating CID and ETD experiments for phosphopeptide identification are desirable for increasing the confidence of identifications, merging spectra prior to database search has to be carefully evaluated further in the context of the various algorithms before adopting it as a routine strategy.

  7. De novo designed cyclic cationic peptides as inhibitors of plant pathogenic bacteria.

    PubMed

    Monroc, Sylvie; Badosa, Esther; Feliu, Lidia; Planas, Marta; Montesinos, Emili; Bardají, Eduard

    2006-11-01

    Head-to-tail cyclic peptides of 4-10 residues consisting of alternating hydrophilic (Lys) and hydrophobic (Leu and Phe) amino acids were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Xanthomonas vesicatoria and Pseudomonas syringae. The antibacterial activity, evaluated as the minimal inhibitory concentration (MIC), the cytotoxicity against human red blood cells and stability towards protease degradation were determined. The influence of cyclization, ring size, and replacement of l-Phe with d-Phe on antibacterial and hemolytic activities was studied and correlated with the degree of structuring and hydrophobicity. Our results showed that linear peptides were inactive against the three bacteria tested. Cyclic peptides were active only toward X. vesicatoria and P. syringae, being c(KLKLKFKLKQ) (BPC10L) the most active peptide with MIC values of 6.25 and 12.5 microM, respectively. The improved antibacterial activity of cyclic peptides compared to their linear counterparts was associated to an increase of the hydrophobicity, represented as RP-HPLC retention time (t(R)), and secondary structure content which are related to an enhanced amphipathicity. A decrease of antibacterial and hemolytic activities was observed when a d-Phe was introduced into the cyclic sequences, which was attributed to their low amphipathicity as shown by their low secondary structure content and low t(R). The small size, simple structure, bactericidal effect, and stability to protease degradation of the best peptides make them potential candidates for the development of effective antibacterial agents for use in plant protection.

  8. Peptide IDR-1002 Inhibits NF-κB Nuclear Translocation by Inhibition of IκBα Degradation and Activates p38/ERK1/2–MSK1-Dependent CREB Phosphorylation in Macrophages Stimulated with Lipopolysaccharide

    PubMed Central

    Huante-Mendoza, Alejandro; Silva-García, Octavio; Oviedo-Boyso, Javier; Hancock, Robert E. W.; Baizabal-Aguirre, Víctor M.

    2016-01-01

    The inflammatory response is a critical molecular defense mechanism of the innate immune system that mediates the elimination of disease-causing bacteria. Repair of the damaged tissue, and the reestablishment of homeostasis, must be accomplished after elimination of the pathogen. The innate defense regulators (IDRs) are short cationic peptides that mimic natural host defense peptides and are effective in eliminating pathogens by enhancing the activity of the immune system while controlling the inflammatory response. Although the role of different IDRs as modulators of inflammation has been reported, there have been only limited studies of the signaling molecules regulated by this type of peptide. The present study investigated the effect of IDR-1002 on nuclear factor κB (NF-κB) and cAMP-response element-binding protein (CREB) transcription factors that are responsible for triggering and controlling inflammation, respectively, in macrophages. We found that TNF-α and COX-2 expression, IκBα phosphorylation, and NF-κB nuclear translocation were strongly inhibited in macrophages pre-incubated with IDR-1002 and then stimulated with lipopolysaccharide (LPS). IDR-1002 also increased CREB phosphorylation at Ser133 via activation of the p38/ERK1/2–MSK1 signaling pathways without detectable expression of the cytokines IL-4, IL-10, and IL-13 involved is suppressing inflammation or alternative activation. Transcriptional activation of NF-κB and CREB is known to require interaction with the transcriptional coactivator CREB-binding protein (CBP). To test for CBP–NF-κB and CBP–CREB complex formation, we performed co-immunoprecipitation assays. These assays showed that IDR-1002 inhibited the interaction between CBP and NF-κB in macrophages stimulated with LPS, which might explain the inhibition of TNF-α and COX-2 expression. Furthermore, the complex between CBP and CREB in macrophages stimulated with IDR-1002 was also inhibited, which might explain why IDR-1002 did

  9. Interaction of divalent cations with peptide fragments from Parkinson's disease genes.

    PubMed

    Remelli, Maurizio; Peana, Massimiliano; Medici, Serenella; Delogu, Lucia Gemma; Zoroddu, Maria Antonietta

    2013-05-07

    Protected Ac-PDEKHEL-NH(2) (PK9-H) and Ac-FCGDGANDCG-NH(2) (PK9-C) peptide fragments corresponding to sequences from residues 1165 to 1171 and 1184 to 1193, respectively, in the Park9 encoded protein from Parkinson's disease gene were tested for their protonation and complex formation capabilities with Cu(II), Zn(II) and Mn(II) ions by potentiometric and UV-Vis measurements. The effects of peptide titration with the metal ions have been followed by mono- and bi-dimensional NMR spectroscopy in order to support the potentiometric results and to understand the details of metal binding. Only mononuclear complexes have been evidenced for all the checked metal ions with PK9-H peptide. Mononuclear and bis-complexes with PK9-C peptide have been evidenced with Cu(II) and Zn(II) metal ions. From the dissociation-constants and pM values obtained for the binary competition diagrams for the systems containing Cu(II), Zn(II) or Mn(II) and the two ligands, the Cu(II) ion is able to bind more efficiently than Zn(II) and Mn(II) metal ions to both ligands.

  10. Interaction of the cationic peptide bactenecin with mixed phospholipid monolayers at the air-water interface.

    PubMed

    López-Oyama, Ana B; Taboada, Pablo; Burboa, María G; Rodríguez, Ezequiel; Mosquera, Víctor; Valdez, Miguel A

    2011-07-01

    The initial mechanism by which antimicrobial peptides target microbes occurs via electrostatic interactions; however, the mechanism is not well understood. We investigate the interaction of the antimicrobial peptide bactenecin with a 50:50 w:w% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG) phospholipid mixture at the air-water interface with different NaCl concentrations (0.01, 0.05, 0.1, 0.5 M) in the subphase. A larger shift of DPPC:DMPG isotherms was obtained for 0.1 M salt concentration at lower and higher pressures, demonstrating the influence of the negative charge of DMPG molecules and the screening of the electrostatic interaction by the salt concentration. Raman spectroscopy of monolayers demonstrated the presence of cysteine-cysteine bridges in bactenecin loops. The peptide adsorption in DPPC:DMPG monolayers observed by AFM images suggests a self-assembled aggregation process, starting with filament-like networks. Domains similar to carpets were formed and pore structures were obtained after a critical peptide concentration, according to the carpet model.

  11. Targeting FOSB with a cationic antimicrobial peptide, TP4, for treatment of triple-negative breast cancer

    PubMed Central

    Ting, Chen-Hung; Chen, Yi-Chun; Wu, Chang-Jer; Chen, Jyh-Yih

    2016-01-01

    Triple-negative breast cancer (TNBC) currently lacks a suitable therapeutic candidate and is thus difficult to treat. Here, we report that a cationic antimicrobial peptide (CAP), tilapia piscidin 4 (TP4), which was derived from Nile tilapia (Oreochromis niloticus), is selectively toxic to TNBC. TP4 acts by inducing an AP-1 protein called FOSB, the expression of which is negatively associated with the pathological grade of TNBC. We show that TP4 is bound to the mitochondria where it disrupts calcium homeostasis and activates FOSB. FOSB overexpression results in TNBC cell death, whereas inhibition of calcium signaling eliminates FOSB induction and blocks TP4-induced TNBC cell death. Both TP4 and anthracyclines strongly induced FOSB, particularly in TNBC, indicating that FOSB may be suitable as a biomarker of drug responses. This study thus provides a novel therapeutic approach toward TNBC through FOSB induction. PMID:27248170

  12. Unblocking the sink: improved CID-based analysis of phosphorylated peptides by enzymatic removal of the basic C-terminal residue.

    PubMed

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

    2014-02-01

    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.

  13. Mechanistic examination of Cβ-Cγ bond cleavages of tryptophan residues during dissociations of molecular peptide radical cations.

    PubMed

    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 [G(n)W](•+) (n = 2-4) and [GXW](•+) (X = C, S, L, F, Y, Q) species. The C(β)-C(γ) bond cleavage of a C-terminal decarboxylated tryptophan residue ([M - CO(2)](•+)) can generate [M - CO(2) - 116](+), [M - CO(2) - 117](•+), and [1H-indole](•+) (m/z 117) species as possible product ions. Competition between the formation of [M - CO(2) - 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 C(β)-C(γ) bond cleavage, can efficiently isomerize to form a more stable π-radical isomer (ion 9) as supported by Rice-Ramsperger-Kassel-Marcus (RRKM) modeling. The C(β)-C(γ) bond cleavage of a tryptophan residue also can occur directly from peptide radical cations containing a basic residue. CID of [WG(n)R](•+) (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 C(β)-C(γ) bond cleavage to prevail over the charge-directed one. DFT calculations predicted that 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.

  14. 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.

  15. Effect of ester chemical structure and peptide bond conformation in fragmentation pathways of differently metal cationized cyclodepsipeptides.

    PubMed

    Banerjee, Raja; Sudarslal, S; Ranganayaki, R S; Raghothama, S

    2011-09-21

    Fragmentation behavior of two classes of cyclodepsipeptides, isariins and isaridins, obtained from the fungus Isaria, was investigated in the presence of different metal ions using multistage tandem mass spectrometry (MS(n)) with collision induced dissociation (CID) and validated by NMR spectroscopy. During MS(n) process, both protonated and metal-cationized isariins generated product ions belonging to the identical 'b-ion' series, exhibiting initial backbone cleavage explicitly at the β-ester bond. Fragmentation behavior for the protonated and metal-cationized acyclic methyl ester derivative of isariins was very similar. On the contrary, isaridins during fragmentation produced ions belonging to the 'b' or/and the 'y' ion series depending on the nature of interacting metal ions, due to initial backbone cleavages at the α-ester linkage or/and at a specific amide linkage. Interestingly, independent of the nature of the interacting metal ions, the product ions formed from the acyclic methyl ester derivative of isaridins belonged only to the 'y-type'. Complementary NMR data showed that, while all metal ions were located around the β-ester group of isariins, the metal ion interacting sites varied across the backbone for isaridins. Combined MS and NMR data suggest that the different behavior in sequence specific charge-driven fragmentation of isariins and isaridins is predetermined because of the constituent β-hydroxy acid residue in isariins and the cis peptide bond in isaridins.

  16. 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.

  17. Influence of a 4-aminomethylbenzoic acid residue on competitive fragmentation pathways during collision-induced dissociation of metal-cationized peptides.

    PubMed

    Osburn, Sandra; Ochola, Sila; Talaty, Erach; Van Stipdonk, Michael

    2007-01-01

    Formation of [bn+17+cat]+ is a prominent collision-induced dissociation (CID) pathway for Li+- and Na+-cationized peptides. Dissociation of protonated and Ag+-cationized peptides instead favors formation of the rival bn+/[bn-1+cat]+ species. In this study the influence of a 4-aminomethylbenzoic acid (4AMBz) residue on the relative intensities of [b(3)-1+cat]+ and [b(3)+17+cat]+ fragment ions was investigated using several model tetrapeptides including those with the general formula A(4AMBz)AX and A(4AMBz)GX (where X=G, A, V). For Li+- and Na+-cationized versions of the peptides there was a significant increase in the intensity of [b(3)-1+cat]+ for the peptides that contain the 4AMBz residue, and in some cases the complete elimination of the [b(3)+17+cat]+ pathway. The influence of the 4AMBz residue may be attributed to the fact that [b(3)-1+cat]+ would be a highly conjugated species containing an aromatic ring substituent. Comparison of CID profiles generated from Na+-cationized AAGV and A(4AMBz)GV suggests an apparent decrease in the critical energy for generation of [b(3)-1+Na]+ relative to that of [b(3)+17+Na]+ when the aromatic amino acid occupies a position such that it leads to the formation of the highly conjugated oxazolinone, thus leading to an increase in formation rate for the former compared to the latter.

  18. Cationic bactericidal peptide 1018 does not specifically target the stringent response alarmone (p)ppGpp

    PubMed Central

    Andresen, Liis; Tenson, Tanel; Hauryliuk, Vasili

    2016-01-01

    The bacterial stringent response is a key regulator of bacterial virulence, biofilm formation and antibiotic tolerance, and is a promising target for the development of new antibacterial compounds. The intracellular nucleotide (p)ppGpp acts as a messenger orchestrating the stringent response. A synthetic peptide 1018 was recently proposed to specifically disrupt biofilms by inhibiting the stringent response via direct interaction with (p)ppGpp (de la Fuente-Núñez et al. (2014) PLoS Pathogens). We have interrogated the specificity of the proposed molecular mechanism. When inhibition of Pseudomonas aeruginosa planktonic and biofilm growth is tested simultaneously in the same assay, peptides 1018 and the control peptide 8101 generated by an inversion of the amino acid sequence of 1018 are equally potent, and, importantly, do not display a preferential activity against biofilm. 1018 inhibits planktonic growth of Escherichia coli equally efficiently either when the alleged target, (p)ppGpp, is essential (MOPS media lacking amino acid L-valine), or dispensable for growth (MOPS media supplemented with L-valine). Genetic disruption of the genes relA and spoT responsible for (p)ppGpp synthesis moderately sensitizes – rather than protects – E. coli to 1018. We suggest that the antimicrobial activity of 1018 does not rely on specific recognition of the stringent response messenger (p)ppGpp. PMID:27819280

  19. Transgenic Brassica juncea plants expressing MsrA1, a synthetic cationic antimicrobial peptide, exhibit resistance to fungal phytopathogens.

    PubMed

    Rustagi, Anjana; Kumar, Deepak; Shekhar, Shashi; Yusuf, Mohd Aslam; Misra, Santosh; Sarin, Neera Bhalla

    2014-06-01

    Cationic antimicrobial peptides (CAPs) have shown potential against broad spectrum of phytopathogens. Synthetic versions with desirable properties have been modeled on these natural peptides. MsrA1 is a synthetic chimera of cecropin A and melittin CAPs with antimicrobial properties. We generated transgenic Brassica juncea plants expressing the msrA1 gene aimed at conferring fungal resistance. Five independent transgenic lines were evaluated for resistance to Alternaria brassicae and Sclerotinia sclerotiorum, two of the most devastating pathogens of B. juncea crops. In vitro assays showed inhibition by MsrA1 of Alternaria hyphae growth by 44-62 %. As assessed by the number and size of lesions and time taken for complete leaf necrosis, the Alternaria infection was delayed and restricted in the transgenic plants with the protection varying from 69 to 85 % in different transgenic lines. In case of S. sclerotiorum infection, the lesions were more severe and spread profusely in untransformed control compared with transgenic plants. The sclerotia formed in the stem of untransformed control plants were significantly more in number and larger in size than those present in the transgenic plants where disease protection of 56-71.5 % was obtained. We discuss the potential of engineering broad spectrum biotic stress tolerance by transgenic expression of CAPs in crop plants.

  20. Real-time monitoring of calcium carbonate and cationic peptide deposition on carboxylate-SAM using a microfluidic SAW biosensor

    PubMed Central

    Pohl, Anna

    2014-01-01

    Summary A microfluidic biosensor with surface acoustic wave technology was used in this study to monitor the interaction of calcium carbonate with standard carboxylate self-assembled monolayer sensor chips. Different fluids, with and without biomolecular components, were investigated. The pH-dependent surface interactions of two bio-inspired cationic peptides, AS8 and ES9, which are similar to an extracellular domain of the chitin synthase involved in mollusc shell formation, were also investigated in a biological buffer system. A range of experimental conditions are described that are suitable to study non-covalent molecular interactions in the presence of ionic substances, such as, mineral precursors below the solubility equilibrium. The peptide ES9, equal to the mollusc chitin synthase epitope, is less sensitive to changes in pH than its counterpart AS8 with a penta-lysine core, which lacks the flanking acidic residues. This study demonstrates the extraordinary potential of microfluidic surface acoustic wave biosensors to significantly expand our experimental capabilities for studying the principles underlying biomineralization in vitro. PMID:25383294

  1. 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

  2. Cationic Peptides and Peptidomimetics Bind Glycosaminoglycans as Potential Sema3A Pathway Inhibitors.

    PubMed

    Corredor, Miriam; Bonet, Roman; Moure, Alejandra; Domingo, Cecilia; Bujons, Jordi; Alfonso, Ignacio; Pérez, Yolanda; Messeguer, Àngel

    2016-03-29

    Semaphorin3A (Sema3A) is a vertebrate-secreted protein that was initially characterized as a repulsive-guidance cue. Semaphorins have crucial roles in several diseases; therefore, the development of Sema3A inhibitors is of therapeutic interest. Sema3A interacts with glycosaminoglycans (GAGs), presumably through its C-terminal basic region. We used different biophysical techniques (i.e., NMR, surface plasmon resonance, isothermal titration calorimetry, fluorescence, and UV-visible spectroscopy) to characterize the binding of two Sema3A C-terminus-derived basic peptides (FS2 and NFS3) to heparin and chondroitin sulfate A. We found that these peptides bind to both GAGs with affinities in the low-micromolar range. On the other hand, a peptoid named SICHI (semaphorin-induced chemorepulsion inhibitor), which is positively charged at physiological pH, was first identified by our group as being able to block Sema3A chemorepulsion and growth-cone collapse in axons at the extracellular level. To elucidate the direct target for the reported SICHI inhibitory effect in the Sema3A signaling pathway, we looked first to the protein-protein interaction between secreted Sema3A and the Nrp1 receptor. However, our results show that SICHI does not bind directly to the Sema3A sema domain or to Nrp1 extracellular domains. We evaluated a new, to our knowledge, hypothesis, according to which SICHI binds to GAGs, thereby perturbing the Sema3A-GAG interaction. By using the above-mentioned techniques, we observed that SICHI binds to GAGs and competes with Sema3A C-terminus-derived basic peptides for binding to GAGs. These data support the ability of SICHI to block the biologically relevant interaction between Sema3A and GAGs, thus revealing SICHI as a new, to our knowledge, class of inhibitors that target the GAG-protein interaction.

  3. A lack of synergy between membrane-permeabilizing cationic antimicrobial peptides and conventional antibiotics.

    PubMed

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

    2015-01-01

    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.

  4. Chemical phosphorylation of histidine-containing peptides based on the sequence of histone H4 and their dephosphorylation by protein histidine phosphatase.

    PubMed

    Attwood, Paul V; Ludwig, Katrin; Bergander, Klaus; Besant, Paul G; Adina-Zada, Abdussalam; Krieglstein, Josef; Klumpp, Susanne

    2010-01-01

    Using peptides based on the amino acid sequences surrounding the two histidine residues in histone H4, we have investigated the kinetics of the phosphorylation and dephosphorylation reactions of their histidine residues, when reacted with potassium phosphoramidate, by (1)H NMR. We have been able to estimate rate constants for the reactions and have shown that there are differences in the kinetics between the two peptides. The kinetics of hydrolysis of phosphoramidate was measured by (31)P NMR and protein histidine phosphatase (PHP) was shown to catalyse the reaction. We have shown that the dephosphorylation of the phosphohistidine of the phosphopeptides is catalysed by PHP. In terms of substrate specificity, there is a small preference for 1-phosphohistidine compared to 3-phosphohistidine, although the rate accelerations for hydrolysis induced by the enzyme were 1100- and 33,333-fold, respectively. The kinetics of both the phosphorylation and dephosphorylation reactions depend on the amino acid sequence surrounding the histidine. PHP shows greater substrate specificity for the peptide whose sequence is similar to that around histidine 18 of histone H4. PHP was unable to catalyse the dephosphorylation of histone H4 that had been phosphorylated with a histone H4 histidine kinase.

  5. Size-exclusive magnetic graphene/mesoporous silica composites with titanium(IV)-immobilized pore walls for selective enrichment of endogenous phosphorylated peptides.

    PubMed

    Sun, Nianrong; Deng, Chunhui; Li, Yan; Zhang, Xiangmin

    2014-07-23

    Developing an effective separation method is necessary for identifying low-abundant endogenous phosphorylated peptides with the removal of proteins. In this work, we prepared size-exclusive magnetic graphene/mesoporous silica composites with titanium(IV)-immobilized pore walls (denoted as Ti(4+)-MGMSs) for capturing endogenous phosphorylated peptides for mass spectrometry analysis. The introduction of hydrophilic polydopamine simplified the synthetic process of Ti(4+)-MGMSs, and the ordered mesoporous channels are beneficial to the trapping of endogenous phosphopeptides while large-size proteins are excluded. Furthermore, the magnetic performance greatly simplifies the entire process of enrichment. With all of the advances, the novel Ti(4+)-MGMSs present high enrichment efficiency either from the low concentration of β-casein tryptic digest (0.5 fmol/μL) or the mixture of β-casein tryptic digest and α-casein (or plus bovine serum albumin, with a mass ratio up to 1:500). Besides, Ti(4+)-MGMSs have also been successfully applied to enrich endogenous phosphorylated peptides from human serum and human saliva.

  6. Effects of Glycine-Extended and Serine13-Phosphorylated Forms of Peptide YY on Food Intake in Rats

    PubMed Central

    Reidelberger, Roger; Haver, Alvin; Chelikani, Prasanth; Keire, David A.; Reeve, Joseph R.

    2011-01-01

    The gut hormone peptide YY(3–36)-amide [PYY(3–36)-NH2] is significantly more potent than PYY(1–36)-NH2 in reducing food intake in rats and humans. Other Gly-extended and Ser13-phosphorylated PYY forms have been detected or predicted based upon known cellular processes of PYY synthesis and modification. Here we compared the effects of 3-h IV infusion of PYY(1–36)-NH2, PYY(3–36)-NH2, PYY(1–36)-Gly-OH, PYY(3–36)-Gly-OH, Ser13(PO3)-PYY(1–36)-NH2, Ser13(PO3)-PYY(3–36)-NH2, Ser13(PO3)-PYY(1–36)-Gly-OH, and Ser13(PO3)-PYY(3–36)-Gly-OH during the early dark period on food intake in freely-feeding rats. PYY(3–36)-NH2 and Ser13(PO3)-PYY(3–36)-NH2 reduced food intake similarly at 50 pmol/kg/min, while only PYY(3–36)-NH2 reduced food intake at 15 pmol/kg/min. PYY(1–36)-NH2 and Ser13(PO3)-PYY(1–36)-NH2 reduced food intake similarly at 50 and 150 pmol/kg/min. In contrast, PYY(1–36)-Gly-OH, PYY(3–36)-Gly-OH, Ser13(PO3)-PYY(3–36)-Gly-OH, and Ser13(PO3)-PYY(1–36)-Gly-OH had no effect on food intake at doses of 50 or 150 pmol/kg/min. Taken together, these results indicate that i) PYY(3–36)-NH2 is significantly more potent than PYY(1–36)-NH2 in reducing food intake, ii) Gly-extended forms of PYY are significantly less potent than non-extended forms, and iii) Ser13-phosphorylation of PYY(3–36)-NH2 decreases the anorexigenic potency PYY(3–36)-NH2, but not PYY(1–36)-NH2. Thus, PYY(3–36)-NH2 appears to be the most potent PYY form for reducing food intake in rats. PMID:21262301

  7. Oral administration of L-mR18L, a single domain cationic amphipathic helical peptide, inhibits lesion formation in ApoE null mice.

    PubMed

    Handattu, Shaila P; Datta, Geeta; Epand, Richard M; Epand, Raquel F; Palgunachari, Mayakonda N; Mishra, Vinod K; Monroe, Candyce E; Keenum, Tamara D; Chaddha, Manjula; Anantharamaiah, G M; Garber, David W

    2010-12-01

    We have shown that Ac-hE18A-NH₂, a dual-domain cationic apolipoprotein-mimetic peptide, reduces plasma cholesterol levels in dyslipidemic mice. Two single-domain cationic peptides based on the lytic class L peptide 18L were developed to test the hypothesis that a single-domain cationic amphipathic peptide can reduce atherosclerosis in apolipoprotein (apo)E null mice when orally administered. To incorporate anti-inflammatory properties, aromatic residues were clustered in the nonpolar face similar to peptide 4F, resulting in modified 18L (m18L). To reduce lytic properties, the Lys residues of 18L were replaced with Arg with the resulting peptide called modified R18L (mR18L). Biophysical studies showed that mR18L had stronger interactions with lipids than did m18L. Peptide mR18L was also more effective than m18L in promoting LDL uptake by HepG2 cells. ApoE null mice received normal chow or chow containing m18L or mR18L for six weeks. A significant reduction in plasma cholesterol and aortic sinus lesion area was seen only in the mR18L group. Plasma from mice administered mR18L, unlike those from the control and m18L groups, did not enhance monocyte adhesion to endothelial cells. Thus oral administration of mR18L reduces plasma cholesterol and lesion formation and inhibits monocyte adhesion.

  8. The Staphylococcus aureus Two-Component Regulatory System, GraRS, Senses and Confers Resistance to Selected Cationic Antimicrobial Peptides

    PubMed Central

    Yang, Soo-Jin; Mishra, Nagendra N.; Meehl, Michael; Ledala, Nagender; Yeaman, Michael R.; Xiong, Yan Q.; Cheung, Ambrose L.

    2012-01-01

    The two-component regulatory system, GraRS, appears to be involved in staphylococcal responses to cationic antimicrobial peptides (CAPs). However, the mechanism(s) by which GraRS is induced, regulated, and modulated remain undefined. In this study, we used two well-characterized MRSA strains (Mu50 and COL) and their respective mutants of graR and vraG (encoding the ABC transporter-dependent efflux pump immediately downstream of graRS), and show that (i) the expression of two key determinants of net positive surface charge (mprF and dlt) is dependent on the cotranscription of both graR and vraG, (ii) reduced expression of mprF and dlt in graR mutants was phenotypically associated with reduced surface-positive charge, (iii) this net reduction in surface-positive charge in graR and vraG mutants, in turn, correlated with enhanced killing by a range of CAPs of diverse structure and origin, including those from mammalian platelets (tPMPs) and neutrophils (hNP-1) and from bacteria (polymyxin B), and (iv) the synthesis and translocation of membrane lysyl-phosphatidylglycerol (an mprF-dependent function) was substantially lower in graR and vraG mutants than in parental strains. Importantly, the inducibility of mprF and dlt transcription via the graRS-vraFG pathway was selective, with induction by sublethal exposure to the CAPs, RP-1 (platelets), and polymyxin B, but not by other cationic molecules (hNP-1, vancomycin, gentamicin, or calcium-daptomycin). Although graR regulates expression of vraG, the expression of graR was codependent on an intact downstream vraG locus. Collectively, these data support an important role of the graRS and vraFG loci in the sensing of and response to specific CAPs involved in innate host defenses. PMID:21986630

  9. CIGB-300, a novel proapoptotic peptide that impairs the CK2 phosphorylation and exhibits anticancer properties both in vitro and in vivo.

    PubMed

    Perea, Silvio E; Reyes, Osvaldo; Baladron, Idania; Perera, Yasser; Farina, Hernán; Gil, Jeovanis; Rodriguez, Arielis; Bacardi, Dania; Marcelo, Jose L; Cosme, Karelia; Cruz, Marisol; Valenzuela, Carmen; López-Saura, Pedro A; Puchades, Yaquelin; Serrano, Joem M; Mendoza, Osmani; Castellanos, Lila; Sanchez, Aniel; Betancourt, Lazaro; Besada, Vladimir; Silva, Ricardo; López, Ernesto; Falcón, Viviana; Hernández, Ignacio; Solares, Margarita; Santana, Agueda; Díaz, Alina; Ramos, Thelvia; López, Carlos; Ariosa, Juan; González, Luis J; Garay, Hilda; Gómez, Daniel; Gómez, Roberto; Alonso, Daniel F; Sigman, Hugo; Herrera, Luis; Acevedo, Boris

    2008-09-01

    Protein Kinase CK2 is a serine-threonine kinase frequently deregulated in many human tumors. Here, we hypothesized that a peptide binder to the CK2 phosphoacceptor site could exhibit anticancer properties in vitro, in tumor animal models, and in cancer patients. By screening a random cyclic peptide phage display library, we identified the CIGB-300 (formerly P15-Tat), a cyclic peptide which abrogates the CK2 phosphorylation by blocking recombinant substrates in vitro. Interestingly, synthetic CIGB-300 led to a dose-dependent antiproliferative effect in a variety of tumor cell lines and induced apoptosis as evidenced by rapid caspase activation. Importantly, CIGB-300 elicited significant antitumor effect both by local and systemic administration in murine syngenic tumors and human tumors xenografted in nude mice. Finally, we performed a First-in-Man trial with CIGB 300 in patients with cervical malignancies. The peptide was found to be safe and well tolerated in the dose range studied. Likewise, signs of clinical benefit were clearly identified after the CIGB-300 treatment as evidenced by significant decrease of the tumor lesion area and histological examination. Our results provide an early proof-of-principle of clinical benefit by using an anti-CK2 approach in cancer. Furthermore, this is the first clinical trial where an investigational drug has been used to target the CK2 phosphorylation domain.

  10. 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.

  11. 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

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

    PubMed

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

    2015-10-01

    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.

  13. 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

    2015-10-02

    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.

  14. Where Does the Electron Go? Stable and Metastable Peptide Cation Radicals Formed by Electron Transfer

    NASA Astrophysics Data System (ADS)

    Pepin, Robert; Layton, Erik D.; Liu, Yang; Afonso, Carlos; Tureček, František

    2017-01-01

    Electron transfer to doubly and triply charged heptapeptide ions containing polar residues Arg, Lys, and Asp in combination with nonpolar Gly, Ala, and Pro or Leu generates stable and metastable charge-reduced ions, (M + 2H)+●, in addition to standard electron-transfer dissociation (ETD) fragment ions. The metastable (M + 2H)+● ions spontaneously dissociate upon resonant ejection from the linear ion trap, giving irregularly shaped peaks with offset m/ z values. The fractions of stable and metastable (M + 2H)+● ions and their mass shifts depend on the presence of Pro-4 and Leu-4 residues in the peptides, with the Pro-4 sequences giving larger fractions of the stable ions while showing smaller mass shifts for the metastables. Conversion of the Asp and C-terminal carboxyl groups to methyl esters further lowers the charge-reduced ion stability. Collisional activation and photodissociation at 355 nm of mass-selected (M + 2H)+● results in different dissociations that give sequence specific MS3 spectra. With a single exception of charge-reduced (LKGLADR + 2H)+●, the MS3 spectra do not produce ETD sequence fragments of the c and z type. Hence, these (M + 2H)+● ions are covalent radicals, not ion-molecule complexes, undergoing dramatically different dissociations in the ground and excited electronic states. The increased stability of the Pro-4 containing (M + 2H)+● ions is attributed to radicals formed by opening of the Pro ring and undergoing further stabilization by hydrogen atom migrations. UV-VIS photodissociation action spectroscopy and time-dependent density functional theory calculations are used in a case in point study of the stable (LKGPADR + 2H)+● ion produced by ETD. In contrast to singly-reduced peptide ions, doubly reduced (M + 3H)+ ions are stable only when formed from the Pro-4 precursors and show all characteristics of even electron ions regarding no photon absorption at 355 nm or ion-molecule reactions, and exhibiting proton driven

  15. Structure-activity relationships for the beta-hairpin cationic antimicrobial peptide polyphemusin I.

    PubMed

    Powers, Jon-Paul S; Rozek, Annett; Hancock, Robert E W

    2004-05-06

    The solution structure of polyphemusin I was determined using (1)H-NMR spectroscopy. Polyphemusin I was found to be an amphipathic, beta-hairpin connected by a type I' beta-turn. The 17 low-energy structures aligned very well over the beta-sheet region while both termini were poorly defined due in part to a hinge-like region centred in the molecule about arginine residues 6 and 16. Conversely, a linear analogue, PM1-S, with all cysteines simultaneously replaced with serine was found to be dynamic in nature, and a lack of medium and long-range NOEs indicated that this molecule displayed no favoured conformation. Circular dichroism (CD) spectroscopy confirmed that in solution, 50% trifluoroethanol (TFE) and in the presence of liposomes, PM1-S remained unstructured. The antimicrobial activity of PM1-S was found to be 4- to 16-fold less than that of polyphemusin I and corresponded with a 4-fold reduction in bacterial membrane depolarization. Both peptides were able to associate with lipid bilayers in a similar fashion; however, PM1-S was completely unable to translocate model membranes while polyphemusin I retained this activity. It was concluded that the disulfide-constrained, beta-sheet structure of polyphemusin I is required for maximum antimicrobial activity. Disruption of this structure results in reduced antimicrobial activity and completely abolishes membrane translocation indicating that the linear PM1-S acts through a different antimicrobial mechanism.

  16. Serine effects on collision-induced dissociation and photodissociation of peptide cation radicals of the z(+•) -type.

    PubMed

    Nguyen, Huong T H; Shaffer, Christopher J; Ledvina, Aaron R; Coon, Joshua J; Tureček, František

    2015-02-15

    The serine residue displays specific effects on the dissociations of peptide fragment cation-radicals of the z(+•) type which are produced by electron transfer dissociation. Energy-resolved collision-induced dissociation (ER-CID), time-resolved infrared multiphoton dissociation (TR-IRMPD), and single-photon UV photodissociation at 355 nm revealed several competitive dissociation pathways consisting of loss of OH radical, water, and backbone cleavages occurring at N-terminal and C-terminal positions relative to the serine residue. The activation modes using slow-heating and UV photon absorption resulted in different relative intensities of fragment ions. This indicated that the dissociations proceeded through several channels with different energy-dependent kinetics. The experimental data were interpreted with the help of electron structure calculations that provided fully optimized structures and relative energies for cis and trans amide isomers of the z4(+•) ions as well as isomerization, dissociation, and transition state energies. UV photon absorption by the z4(+•) ions was due to Cα-radical amide groups created by ETD that provided a new chromophore absorbing at 355 nm.

  17. Serine effects on collision-induced dissociation and photodissociation of peptide cation radicals of the z+•-type

    PubMed Central

    Nguyen, Huong T. H.; Shaffer, Christopher J.; Ledvina, Aaron R.; Coon, Joshua J.

    2014-01-01

    The serine residue displays specific effects on the dissociations of peptide fragment cation-radicals of the z+• type which are produced by electron transfer dissociation. Energy-resolved collision-induced dissociation (ER-CID), time-resolved infrared multiphoton dissociation (TR-IRMPD), and single-photon UV photodissociation at 355 nm revealed several competitive dissociation pathways consisting of loss of OH radical, water, and backbone cleavages occurring at N-terminal and C-terminal positions relative to the serine residue. The activation modes using slow-heating and UV photon absorption resulted in different relative intensities of fragment ions. This indicated that the dissociations proceeded through several channels with different energy-dependent kinetics. The experimental data were interpreted with the help of electron structure calculations that provided fully optimized structures and relative energies for cis and trans amide isomers of the z4+• ions as well as isomerization, dissociation, and transition state energies. UV photon absorption by the z4+• ions was due to Cα-radical amide groups created by ETD that provided a new chromophore absorbing at 355 nm. PMID:26005367

  18. Dual antifungal properties of cationic antimicrobial peptides polybia-MPI: membrane integrity disruption and inhibition of biofilm formation.

    PubMed

    Wang, Kairong; Yan, Jiexi; Dang, Wen; Xie, Junqiu; Yan, Bo; Yan, Wenjin; Sun, Mengyang; Zhang, Bangzhi; Ma, Mingxia; Zhao, Yanyan; Jia, Fengjing; Zhu, Ranran; Chen, Wei; Wang, Rui

    2014-06-01

    With the increasing emergence of resistant fungi, the discovery and development of novel antifungal therapeutics were urgently needed. Compared with conventional antibiotics, the limited propensity of AMPs to induce resistance in pathogens has attracted great interest. In the present study, the antifungal activity and its mechanism-of-action of polybia-MPI, a cationic peptide from the venom of Social wasp Polybia Paulista was investigated. We demonstrated that polybia-MPI could potently inhibit the growth of Candida albicans (C. albicans) and Candida glabrata (C. glabrata). The 50% inhibitory concentrations (IC50) of Polybia-MPI against cancer cells were much higher than the MICs against the tested C. albicans and C. glabrata cells, indicating that polybia-MPI had high selectivity between the fungal and mammalian cells. Our results also indicated that membrane disturbance mechanism was involved in the antifungal activity. Furthermore, polybia-MPI could inhibit the bio film forming of C. glabrata, which was frequently associated with clinically significant biofilm. These results suggest that polybia-MPI has great advantages in the development of antifungal agents.

  19. Assessment of SYBR green I dye-based fluorescence assay for screening antimalarial activity of cationic peptides and DNA intercalating agents.

    PubMed

    Bhatia, Rakesh; Gautam, Ankur; Gautam, Shailendra K; Mehta, Divya; Kumar, Vinod; Raghava, Gajendra P S; Varshney, Grish C

    2015-05-01

    The SYBR green I (SG) dye-based fluorescence assay for screening antimalarial compounds is based on direct quantitation of parasite DNA. We show that DNA-interacting cationic cell-penetrating peptides (CPPs) and intercalating agents compete with SG dye to bind to DNA. Therefore, readouts of this assay, unlike those of the [(3)H]hypoxanthine incorporation assay, for the antimalarial activity of the above DNA binding agents may be erroneous. In the case of CPPs, false readouts can be improved by the removal of excess peptides.

  20. Design of Highly Stabilized β-Hairpin Peptides through Cation-π interactions of Lysine and N-Methyl Lysine with an Aromatic Pocket‡

    PubMed Central

    Riemen, Alexander J.; Waters, Marcey L.

    2009-01-01

    Two tryptophan residues were incorporated on one face of a β-hairpin peptide to form an aromatic pocket that interacts with a lysine or N-methylated lysine via cation-π interactions. The two tryptophan residues were found to pack against the lysine side chain forming an aromatic pocket similar to those observed in trimethylated lysine receptor proteins. Thermal analysis of methylated lysine variant hairpin peptides revealed an increase in thermal stability as the degree of methylation was increased resulting in the most thermally stable β-hairpin reported to date. PMID:19191524

  1. Phosphoryl Transfer Reaction Snapshots in Crystals

    PubMed Central

    Gerlits, Oksana; Tian, Jianhui; Das, Amit; Langan, Paul; Heller, William T.; Kovalevsky, Andrey

    2015-01-01

    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. 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. PMID:25925954

  2. Pleurocidin-family cationic antimicrobial peptides are cytolytic for breast carcinoma cells and prevent growth of tumor xenografts

    PubMed Central

    2011-01-01

    Introduction Cationic antimicrobial peptides (CAPs) defend against microbial pathogens; however, certain CAPs also exhibit anticancer activity. The purpose of this investigation was to determine the effect of the pleurocidin-family CAPs, NRC-03 and NRC-07, on breast cancer cells. Methods MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) and acid phosphatase cell-viability assays were used to assess NRC-03- and NRC-07-mediated killing of breast carcinoma cells. Erythrocyte lysis was determined with hemolysis assay. NRC-03 and NRC-07 binding to breast cancer cells and normal fibroblasts was assessed with fluorescence microscopy by using biotinylated-NRC-03 and -NRC-07. Lactate dehydrogenase-release assays and scanning electron microscopy were used to evaluate the effect of NRC-03 and NRC-07 on the cell membrane. Flow-cytometric analysis of 3,3'-dihexyloxacarbocyanine iodide- and dihydroethidium-stained breast cancer cells was used to evaluate the effects of NRC-03 and NRC-07 on mitochondrial membrane integrity and reactive oxygen species (ROS) production, respectively. Tumoricidal activity of NRC-03 and NRC-07 was evaluated in NOD SCID mice bearing breast cancer xenografts. Results NRC-03 and NRC-07 killed breast cancer cells, including drug-resistant variants, and human mammary epithelial cells but showed little or no lysis of human dermal fibroblasts, umbilical vein endothelial cells, or erythrocytes. Sublethal doses of NRC-03 and, to a lesser extent, NRC-07 significantly reduced the median effective concentration (EC50) of cisplatin for breast cancer cells. NRC-03 and NRC-07 bound to breast cancer cells but not fibroblasts, suggesting that killing required peptide binding to target cells. NRC-03- and NRC-07-mediated killing of breast cancer cells correlated with expression of several different anionic cell-surface molecules, suggesting that NRC-03 and NRC-07 bind to a variety of negatively-charged cell-surface molecules. NRC-03 and NRC-07 also

  3. Anticancer peptide CIGB-300 binds to nucleophosmin/B23, impairs its CK2-mediated phosphorylation, and leads to apoptosis through its nucleolar disassembly activity.

    PubMed

    Perera, Yasser; Farina, Hernán G; Gil, Jeovanis; Rodriguez, Arielis; Benavent, Fernando; Castellanos, Lila; Gómez, Roberto E; Acevedo, Boris E; Alonso, Daniel F; Perea, Silvio E

    2009-05-01

    CIGB-300, formerly known as P15-tat, is a proapoptotic peptide with established antiproliferative activity in vitro and antitumoral activity in vivo. This hypothesis-driven peptide was initially selected for its ability to impair the in vitro CK2-mediated phosphorylation in one of its substrates through direct binding to the conserved acidic phosphoaceptor domain. However, the actual in vivo target(s) on human cancer cells among the hundreds of CK2 substrates as well as the subsequent events that lead to apoptosis on tumor cells remains to be determined. In this work, we identified the multifunctional oncoprotein nucleophosmin/B23 as a major target for CIGB-300. In vivo, the CIGB-300-B23 interaction was shown by pull-down experiments and confirmed by the early in situ colocalization of both molecules in the cell nucleolus. Moreover, CIGB-300 inhibits the CK2-mediated phosphorylation of B23 in a dose-dependent fashion both in vitro and in vivo as shown using the recombinant GST fusion protein and the metabolic labeling approach, respectively. Such phosphorylation impairment was correlated with the ability of CIGB-300 to induce nucleolar disassembly as documented by the use of established markers for nucleolar structure. Finally, we showed that such a sequence of events leads to the rapid and massive onset of apoptosis both at the molecular and cellular levels. Collectively, these findings provide important clues by which the CIGB-300 peptide exerts its proapoptotic effect on tumor cells and highlights the suitability of the B23/CK2 pathway for cancer-targeted therapy.

  4. Improvement of outer membrane-permeabilizing and lipopolysaccharide-binding activities of an antimicrobial cationic peptide by C-terminal modification.

    PubMed

    Piers, K L; Brown, M H; Hancock, R E

    1994-10-01

    Antimicrobial cationic peptides have been discovered in many different organisms and often possess a broad range of activity. In this study, we investigated the mechanisms of actions of melittin and two synthetic peptides, CEME (a cecropin-melittin hybrid) and CEMA, against gram-negative bacteria. CEMA was produced by recombinant DNA procedures and is an analog of CEME with a modified C terminus resulting in two additional positive charges. All three peptides showed good antimicrobial activity against four different gram-negative bacteria, but only CEMA was able to somewhat augment the activity of some conventional antibiotics in synergy studies. Studies using the bacteria Pseudomonas aeruginosa and Enterobacter cloacae showed that the peptides all possessed the ability to permeabilize bacterial outer membranes to the hydrophobic fluorophor 1-N-phenylnaphthylamine and the protein lysozyme, with CEMA being the most active. CEMA also had the strongest relative binding affinity for bacterial endotoxin (lipopolysaccharide). These data collectively indicated that these peptides all cross the outer membrane by the self-promoted uptake pathway and that CEMA is the peptide most effective at accessing this pathway.

  5. Chemical and biological characterization of four new linear cationic α-helical peptides from the venoms of two solitary eumenine wasps.

    PubMed

    Rangel, Marisa; Cabrera, Marcia Perez dos Santos; Kazuma, Kohei; Ando, Kenji; Wang, Xiaoyu; Kato, Manabu; Nihei, Ken-ichi; Hirata, Izaura Yoshico; Cross, Tyra J; Garcia, Angélica Nunes; Faquim-Mauro, Eliana L; Franzolin, Marcia Regina; Fuchino, Hiroyuki; Mori-Yasumoto, Kanami; Sekita, Setsuko; Kadowaki, Makoto; Satake, Motoyoshi; Konno, Katsuhiro

    2011-06-01

    Four novel peptides were isolated from the venoms of the solitary eumenine wasps Eumenes rubrofemoratus and Eumenes fraterculus. Their sequences were determined by MALDI-TOF/TOF (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) analysis, Edman degradation and solid-phase synthesis. Two of them, eumenitin-R (LNLKGLIKKVASLLN) and eumenitin-F (LNLKGLFKKVASLLT), are highly homologous to eumenitin, an antimicrobial peptide from a solitary eumenine wasp, whereas the other two, EMP-ER (FDIMGLIKKVAGAL-NH(2)) and EMP-EF (FDVMGIIKKIAGAL-NH(2)), are similar to eumenine mastoparan-AF (EMP-AF), a mast cell degranulating peptide from a solitary eumenine wasp. These sequences have the characteristic features of linear cationic cytolytic peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, they can be predicted to adopt an amphipathic α-helix secondary structure. In fact, the CD (circular dichroism) spectra of these peptides showed significant α-helical conformation content in the presence of TFE (trifluoroethanol), SDS (sodium dodecylsulfate) and asolectin vesicles. In the biological evaluation, all the peptides exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity.

  6. 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

  7. Competitive Hydrogen Atom Migrations Accompanying Cascade Dissociations of Peptide Cation-Radicals of the z(+•) Type.

    PubMed

    Ledvina, Aaron R; Coon, Joshua J; Tureček, František

    2015-02-01

    We report a combined experimental and computational study of energy-resolved collision-induced dissociation (ER-CID) and time-resolved infrared multiphoton dissociation (TR-IRMPD) of z4 ions prepared by electron transfer dissociation of peptide (Ala-Ala-Asn-Ala-Arg + 2H)(2+) ions. The z4 cation-radicals, (•)ANAR(+), undergo competitive dissociations by backbone cleavage and loss of a CONH2 radical from the Asn side chain. The backbone cleavage proceeds by radical-assisted dissociation of the Asn Cα-CO bond, forming an x2 ion intermediate which rapidly dissociates by HNCO elimination to yield a stable z2 fragment ion, (•)AR(+). The ER-CID and TR-IRMPD data were consistent with the consecutive nature of the backbone dissociation but showed different branching ratios for the two major fragmentations. The ER-CID data showed branching ratios 0.6-1.0 for the side-chain and backbone cleavages whereas the TR-IRMPD data showed an earlier onset for the latter dissociation. Computational analysis of the potential energy surface with density functional theory and ab initio calculations was carried out to provide structures and energies for the reactant ions as well as several intermediates, products, and transition states. Dissociation pathways for cis and trans amide conformers were distinguished and their energies were evaluated. The threshold dissociation energies for the backbone and side-chain dissociations were similar in accordance with the experimental ER-CID branching ratio. The TR-IRMPD data were interpreted by different absorbances of intermediates produced by hydrogen atom migrations along the dissociation pathways.

  8. Competitive Hydrogen Atom Migrations Accompanying Cascade Dissociations of Peptide Cation-Radicals of the z+• Type

    PubMed Central

    Ledvina, Aaron R.; Coon, Joshua J.

    2014-01-01

    We report a combined experimental and computational study of energy-resolved collision-induced dissociation (ER-CID) and time-resolved infrared multiphoton dissociation (TR-IRMPD) of z4 ions prepared by electron transfer dissociation of peptide (Ala-Ala-Asn-Ala-Arg + 2H)2+ ions. The z4 cation-radicals, •ANAR+, undergo competitive dissociations by backbone cleavage and loss of a CONH2 radical from the Asn side chain. The backbone cleavage proceeds by radical-assisted dissociation of the Asn Cα—CO bond, forming an x2 ion intermediate which rapidly dissociates by HNCO elimination to yield a stable z2 fragment ion, •AR+. The ER-CID and TR-IRMPD data were consistent with the consecutive nature of the backbone dissociation but showed different branching ratios for the two major fragmentations. The ER-CID data showed branching ratios 0.6-1.0 for the side-chain and backbone cleavages whereas the TR-IRMPD data showed an earlier onset for the latter dissociation. Computational analysis of the potential energy surface with density functional theory and ab initio calculations was carried out to provide structures and energies for the reactant ions as well as several intermediates, products, and transition states. Dissociation pathways for cis and trans amide conformers were distinguished and their energies were evaluated. The threshold dissociation energies for the backbone and side-chain dissociations were similar in accordance with the experimental ER-CID branching ratio. The TR-IRMPD data were interpreted by different absorbances of intermediates produced by hydrogen atom migrations along the dissociation pathways. PMID:25844055

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

    PubMed Central

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

    2016-01-01

    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

  10. 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

  11. 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

    2016-01-05

    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.

  12. 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

  13. Rational Design of Engineered Cationic Antimicrobial Peptides Consisting Exclusively of Arginine and Tryptophan, and Their Activity against Multidrug-Resistant Pathogens

    PubMed Central

    Deslouches, Berthony; Steckbeck, Jonathan D.; Craigo, Jodi K.; Doi, Yohei; Mietzner, Timothy A.

    2013-01-01

    The emergence of multidrug-resistant (MDR) pathogens underscores the need for new antimicrobial agents to overcome the resistance mechanisms of these organisms. Cationic antimicrobial peptides (CAPs) provide a potential source of new antimicrobial therapeutics. We previously characterized a lytic base unit (LBU) series of engineered CAPs (eCAPs) of 12 to 48 residues demonstrating maximum antibacterial selectivity at 24 residues. Further, Trp substitution in LBU sequences increased activity against both P. aeruginosa and S. aureus under challenging conditions (e.g., saline, divalent cations, and serum). Based on these findings, we hypothesized that the optimal length and, therefore, the cost for maximum eCAP activity under physiologically relevant conditions could be significantly reduced using only Arg and Trp arranged to form idealized amphipathic helices. Hence, we developed a novel peptide series, composed only of Arg and Trp, in a sequence predicted and verified by circular dichroism to fold into optimized amphipathic helices. The most effective antimicrobial activity was achieved at 12 residues in length (WR12) against a panel of both Gram-negative and Gram-positive clinical isolates, including extensively drug-resistant strains, in saline and broth culture and at various pH values. The results demonstrate that the rational design of CAPs can lead to a significant reduction in the length and the number of amino acids used in peptide design to achieve optimal potency and selectivity against specific pathogens. PMID:23507278

  14. Electrospray ionization tandem mass spectrometric study of protonated and alkali- cationized α/ε-hybrid peptides: differentiation of a pair of dipeptide positional isomers.

    PubMed

    Ramesh Babu, A; Raju, G; Purna Chander, C; Shoban Babu, B; Srinivas, R; Sharma, G V M

    2016-01-01

    A new class of Boc-N-protected hybrid peptides derived from L- Ala and ε(6)-Caa (L-Ala = L-Alanine, Caa = C-linked carboamino acid derived from D-xylose) have been studied by positive ion electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). MS(n) spectra of protonated and alkali-cationized hybrid peptides produce characteristic fragmentation involving the peptide backbone, the tert-butyloxycarbonyl (Boc) group, and the side chain. The dipeptide positional isomers are differentiated by the collision-induced dissociation (CID) of the protonated and alkali-cationized peptides. The CID of [M + H](+) ion of Boc-NH-L-Ala-ε-Caa- OCH3 (1) shows a prominent [M + H - C4H8](+) ion, which is totally absent for its positional isomer Boc-NH-ε-Caa-L-Ala-OCH3 (6), which instead shows significant loss of t-butanol. The formation of the [M + Cat - C4H8](+) ion is totally absent and [M + Cat - Boc + H](+) is prominent in the CID of the [M + Cat](+) ion of Boc-NH-L-Ala-ε-Caa- OCH3 (1), whereas the former is highly abundant and the latter is of low abundance for its positional isomer Boc-NH-ε-Caa-L-Ala-OCH3 (6). It is observed that 'b' ions are abundant when oxazolone structures are formed through a five-membered cyclic transition state in tetra-, penta-, and hexapeptides and the cyclization process for larger 'b' ions led to an insignificant abundance. However, the significant 'b' ion is formed in ε,α-dipeptide, which may have a seven-membered substituted 2-oxoazepanium ion structure. The MS(n) spectra of [M + Cat - Boc + H](+) ions of these peptides are found to be significantly different to those of [M + H - Boc + H](+) ions. The CID spectra of [M + Cat - Boc + H](+) ions of peptide acids containing L-Ala at the C-terminus show an abundant N-terminal rearrangement ion, [b(n) + 17 + Cat](+), which is absent for the peptide acids containing ε-Caa at the C-terminus. Thus, the results of these hybrid peptides

  15. Electrochemical genosensor based on peptide nucleic acid-mediated PCR and asymmetric PCR techniques: Electrostatic interactions with a metal cation.

    PubMed

    Kerman, Kagan; Vestergaard, Mun'delanji; Nagatani, Naoki; Takamura, Yuzuru; Tamiya, Eiichi

    2006-04-01

    The unique structure of peptide nucleic acids (PNAs), linking the N-(2-aminoethyl)glycine units that create a neutral backbone, and prevent it from acting as a primer for DNA polymerase, has been utilized in an electrochemical biosensor scheme for simple and sensitive detection of hybridization. When the PNA is targeted against a single-nucleotide polymorphism (SNP) or wild-type site on the gene, PNA-mediated polymerase chain reaction (PCR) clamping method effectively blocks the formation of a PCR product. In our report, PNA probe for PCR clamping was targeted against the wild-type site of alcohol dehydrogenase. The electrostatic interactions between the negatively charged DNA and neutral PNA molecules with redox-active metal cation cobalt(III)hexamine ([Co(NH3)6]3+) were monitored using differential pulse voltammetry. The electrostatic binding of [Co(NH3)6]3+ to DNA provided the basis for the discrimination against PNA/PNA, PNA/DNA, and DNA/DNA hybrid molecules. We have optimized the experimental conditions, such as probe concentration, [Co(NH3)6]3+ concentration, accumulation time for [Co(NH3)6]3+, and target concentration. A new pretreatment method has also been employed to allow fast and simple detection of hybridization reaction between the PCR amplicon and the probe on glassy carbon electrode (GCE) surface. This method was based on the application of a high-temperature treatment (95 degrees C, 5 min), followed by a 1-min incubation in the presence of DNA primers. The excess concentration of DNA primers prevented the rehybridization of the denatured strands, while enabling the target gene sequence to bind with the immobilized probe. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism in standard Roundup Ready soybean samples. The amplicons of asymmetric PCR, which were predominantly single-stranded DNA as a result of unequal primer concentration, hybridized with the DNA probe on the sensor surface efficiently. The

  16. Are the Radical Centers in Peptide Radical Cations Mobile? The Generation, Tautomerism, and Dissociation of Isomeric α-Carbon-Centered Triglycine Radical Cations in the Gas Phase

    SciTech Connect

    Chu, Ivan K.; Zhao, Junfang; Xu, Minjie; Siu, Shiu On; Hopkinson, Alan C.; Siu , K W Michael

    2008-05-31

    The mobility of the radical center in three isomeric triglycine radical cationss[G•GG]+, [GG•G]+, and [GGG•]+shas been investigated theoretically via density functional theory (DFT) and experimentally via tandem mass spectrometry. These radical cations were generated by collision-induced dissociations (CIDs) of Cu(II)-containing ternary complexes that contain the tripeptides YGG, GYG, and GGY, respectively (G and Y are the glycine and tyrosine residues, respectively). Dissociative electron transfer within the complexes led to observation of [Y•GG]+, [GY•G]+, and [GGY•]+; CID resulted in cleavage of the tyrosine side chain as p-quinomethide, yielding [G•GG]+, [GG•G]+, and [GGG•]+, respectively. Interconversions between these isomeric triglycine radical cations have relatively high barriers (g44.7 kcal/mol), in support of the thesis that isomerically pure [G•GG]+, [GG•G]+, and [GGG•]+ can be experimentally produced. This is to be contrasted with barriers < 17 kcal/mol that were encountered in the tautomerism of protonated triglycine [Rodriquez C. F. et al. J. Am. Chem. Soc. 2001, 123, 3006-3012]. The CID spectra of [G•GG]+, [GG•G]+, and [GGG•]+ were substantially different, providing experimental proof that initially these ions have distinct structures. DFT calculations showed that direct dissociations are competitive with interconversions followed by dissociation.

  17. The tetra-aspartate motif in the activation peptide of human cationic trypsinogen is essential for autoactivation control but not for enteropeptidase recognition.

    PubMed

    Nemoda, Zsófia; Sahin-Tóth, Miklós

    2005-08-19

    The activation peptide of vertebrate trypsinogens contains a highly conserved tetra-aspartate sequence (Asp(19-22) in humans) preceding the Lys-Ile scissile bond. A large body of research has defined the primary role of this acidic motif as a specific recognition site for enteropeptidase, the physiological activator of trypsinogen. In addition, the acidic stretch was shown to contribute to the suppression of autoactivation. In the present study, we determined the relative importance of these two activation peptide functions in human cationic trypsinogen. Individual Ala replacements of Asp(19-22) had minimal or no effect on trypsinogen activation catalyzed by human enteropeptidase. Strikingly, a tetra-Ala(19-22) trypsinogen mutant devoid of acidic residues in the activation peptide was still a highly specific substrate for human, but not for bovine, enteropeptidase. In contrast, an intact Asp(19-22) motif was critical for autoactivation control. Thus, single Ala mutations of Asp(19), Asp(20) and Asp(21) resulted in 2-3-fold increased autoactivation, whereas the Asp(22) --> Ala mutant autoactivated at a 66-fold increased rate. These effects were multiplicative in the tri-Ala(19-21) and tetra-Ala(19-22) mutants. Structural modeling revealed that the conserved hydrophobic S2 subsite of trypsin and the unique Asp(218), which forms part of the S3-S4 subsite, participate in distinct inhibitory interactions with the activation peptide. Finally, mutagenesis studies confirmed the significance of the negative charge of Asp(218) in autoactivation control. The results demonstrate that in human cationic trypsinogen the Asp(19-22) motif per se is not required for enteropeptidase recognition, whereas it is essential for maximal suppression of autoactivation. The evolutionary selection of Asp(218), which is absent in the large majority of vertebrate trypsins, provides an additional mechanism of autoactivation control in the human pancreas.

  18. 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

  19. 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

  20. Modulation of calcium oxalate dihydrate growth by selective crystal-face binding of phosphorylated osteopontin and polyaspartate peptide showing occlusion by sectoral (compositional) zoning.

    PubMed

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

    2009-08-28

    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-Asp(86-93)). 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-Asp(86-93) dose-dependently elongated crystal morphology along the {110} faces in a manner similar to OPN. In crystal growth studies using fluorescently tagged poly-Asp(86-93) 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-Asp(86-93) 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-Asp(86-93) 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-Asp(86-93) 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).

  1. The requirement of ammonium or other cations linked with p-cresol sulfate for cross-reactivity with a peptide of myelin basic protein.

    PubMed

    Jackson, Patricia L; Cao, Ligong; Blalock, J Edwin; Whitaker, John N

    2003-10-15

    Urinary myelin basic protein-like material (MBPLM), so designated because of its immunoreactivity with a polyclonal antibody directed against a cryptic epitope located in residues 83-89 of myelin basic protein (MBP), exists in humans normally but increases in concentration in patients with multiple sclerosis who have progressive disease. Given its possible role in reflecting events of neural tissue destruction occurring in multiple sclerosis, urinary MBPLM is a candidate surrogate marker for this phase of the disease. Previously, it has been demonstrated that p-cresol sulfate (PCS) is the dominant component of MBPLM; however, another component(s) was essential in enabling p-cresol sulfate to have molecular mimicry with MBP peptide 83-89 detected by immunoreactivity. In the present investigation, this remaining component(s) was characterized by a combination of high performance size exclusion chromatography followed by nuclear magnetic resonance spectroscopy and shown to be ammonium. The monovalent cation ammonium could be substituted in vitro by several different monovalent and divalent cations, most notably zinc, in restoring to deprotonated p-cresol sulfate its immunoreactivity as MBPLM. These findings indicate the basis for the unexpected molecular mimicry between an epitope of an encephalitogenic protein and a complex containing a small organic molecule, p-cresol sulfate. Furthermore, the reaction of either ammonium or other cations with p-cresol sulfate may represent an in vivo process directly related to damage of axonal membranes.

  2. Investigation of the antimicrobial activities of Snakin-Z, a new cationic peptide derived from Zizyphus jujuba fruits.

    PubMed

    Daneshmand, Fatemeh; Zare-Zardini, Hadi; Ebrahimi, Leila

    2013-01-01

    Snakin-Z is a novel antimicrobial peptide (AMP) that is identified from the fruit of Zizyphus jujuba. This peptide is composed of 31 amino acids which is determined with the sequence of CARLNCVPKGTSGNTETCPCYASLHSCRKYG and molecular weight of 3318.82 Da. Snakin-Z is not identical to any AMP in the peptide database. According to this study, Snakin-Z potentially has antimicrobial property against bacteria and fungi. Minimal inhibitory concentration (MIC) value of this peptide is suitable for antimicrobial activity. We assessed that Snakin-Z could affect Phomopsis azadirachtae with the MIC value of 7.65 μg/mL and vice versa Staphylococcus aureus with the MIC value of 28.8 μg/mL. Interestingly, human red blood cells also showed good tolerance to the Snakin-Z. On the basis of this study, Snakin-Z can be an appropriate candidate for therapeutic applications in the future due to its antimicrobial property.

  3. Plantaricin A, a cationic peptide produced by Lactobacillus plantarum, permeabilizes eukaryotic cell membranes by a mechanism dependent on negative surface charge linked to glycosylated membrane proteins.

    PubMed

    Sand, Sverre L; Nissen-Meyer, Jon; Sand, Olav; Haug, Trude M

    2013-02-01

    Lactobacillus plantarum C11 releases plantaricin A (PlnA), a cationic peptide pheromone that has a membrane-permeabilizing, antimicrobial effect. We have previously shown that PlnA may also permeabilize eukaryotic cells, with a potency that differs between cell types. It is generally assumed that cationic antimicrobial peptides exert their effects through electrostatic attraction to negatively charged phospholipids in the membrane. The aim of the present study was to investigate if removal of the negative charge linked to glycosylated proteins at the cell surface reduces the permeabilizing potency of PlnA. The effects of PlnA were tested on clonal rat anterior pituitary cells (GH(4) cells) using patch clamp and microfluorometric techniques. In physiological extracellular solution, GH(4) cells are highly sensitive to PlnA, but the sensitivity was dramatically reduced in solutions that partly neutralize the negative surface charge of the cells, in agreement with the notion that electrostatic interactions are probably important for the PlnA effects. Trypsination of cells prior to PlnA exposure also rendered the cells less sensitive to the peptide, suggesting that negative charges linked to membrane proteins are involved in the permeabilizing action. Finally, pre-exposure of cells to a mixture of enzymes that split carbohydrate residues from the backbone of glycosylated proteins also impeded the PlnA-induced membrane permeabilization. We conclude that electrostatic attraction between PlnA and glycosylated membrane proteins is probably an essential first step before PlnA can interact with membrane phospholipids. Deviating glycosylation patterns may contribute to the variation in PlnA sensitivity of different cell types, including cancerous cells and their normal counterparts.

  4. Fragmentation of peptide radical cations containing a tyrosine or tryptophan residue: structural features that favor formation of [x(n-1) + H]˙⁺ and [z(n-1) + H]˙⁺ ions.

    PubMed

    Mädler, Stefanie; Lau, Justin Kai-Chi; Williams, Declan; Wang, Yating; Saminathan, Irine S; Zhao, Junfang; Siu, K W Michael; Hopkinson, Alan C

    2014-06-12

    Peptide radical cations A(n)Y(•+) (where n = 3, 4, or 5) and A5W(•+) have been generated by collision-induced dissociation (CID) of [Cu(II)(tpy)(peptide)](•2+) complexes. Apart from the charge-driven fragmentation at the N-Cα bond of the hetero residue producing either [c + 2H](+) or [z - H](•+) ions and radical-driven fragmentation at the Cα-C bond to give a(+) ions, unusual product ions [x + H](•+) and [z + H](•+) are abundant in the CID spectra of the peptides with the hetero residue in the second or third position of the chain. The formation of these ions requires that both the charge and radical be located on the peptide backbone. Energy-resolved spectra established that the [z + H](•+) ion can be produced either directly from the peptide radical cation or via the fragment ion [x + H](•+). Additionally, backbone dissociation by loss of the C-terminal amino acid giving [b(n-1) - H](•+) increases in abundance with the length of the peptides. Mechanisms by which peptide radical cations dissociate have been modeled using density functional theory (B3LYP/6-31++G** level) on tetrapeptides AYAG(•+), AAYG(•+), and AWAG(•+).

  5. Sulphonic acid strong cation-exchange restricted access columns in sample cleanup for profiling of endogenous peptides in multidimensional liquid chromatography. Structure and function of strong cation-exchange restricted access materials.

    PubMed

    Machtejevas, E; Denoyel, R; Meneses, J M; Kudirkaite, V; Grimes, B A; Lubda, D; Unger, K K

    2006-08-04

    In this work, the pore structural parameters and size exclusion properties of LiChrospher strong cation-exchange and reverse phase restricted access materials (RAM) are analysed. The molecular weight size exclusion limit for polystyrenes was found to be about 17.7 kDa, while for standard proteins, the molecular weight size exclusion limit was higher, at approximately 25 kDa. The average pore diameter on a volume basis calculated from the pore network model changes from 8.5 nm (native LiChrospher) to 8.6 nm (diol derivative) to 8.2 nm (sulphonic acid derivative) to 6.9 nm (n-octadecyl derivative). Additional characterisations were performed on restricted access materials with nitrogen sorption at 77 K, water adsorption at 25 degrees C, intrusion-extrusion of water (in order to evaluate the hydrophobic properties of the pores of the hydrophobic RAM), and zeta potential measurements by microelectrophoresis. For peptide analysis out of the biofluids, the strong cation-exchange functionality seems to be particularly suitable mainly because of the high loadability of the strong cation-exchange restricted access material (SCX-RAM) and the fact that one can work under non-denaturing conditions to perform effective chromatographic separations. For bacitracin, the dynamic capacity of the SCX-RAM columns does not reach its maximum value in the analysed range. For lysozyme, the dynamic capacity reaches a value of 0.08 mg/ml of column volume before column is overloaded. Additionally, the proper column operating conditions that lead to the total effective working time of the RAM column to be equal to approximately 500 injections (depending on the type of sample), is comprehensively described. The SCX-RAM column was used in the same system analysing urine samples for the period of 1 month (approximately 150 injections) with run-to-run reproducibility below 5% RSD and below 10% RSD for the relative fractions.

  6. 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

    Gerlits, Oksana; Waltman, Mary Jo; Taylor, Susan; Langan, Paul; Kovalevsky, Andrey

    2013-05-28

    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, Mg(2+), Ca(2+), Sr(2+), and Ba(2+) 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 Mg(2+), 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.

  7. Cascade Dissociations of Peptide Cation-Radicals. Part 1. Scope and Effects of Amino Acid Residues in Penta-, Nona- and Decapeptides

    PubMed Central

    Chung, Thomas W.; Hui, Renjie; Ledvina, Aaron; Coon, Joshua J.

    2013-01-01

    Amino acid residue-specific backbone and side-chain dissociations of peptide z ions in MS3 spectra were elucidated for over 40 pentapeptides with arginine C-terminated sequences of the AAXAR and AAHXR type, nonapeptides of the AAHAAXYAR and AAHAXAYAR type, and AAHAAXYAAR decapeptides. Peptide zn ions containing amino acid residues with readily transferrable benzylic or tertiary β-hydrogen atoms (Phe, Tyr, His, Trp, Val) underwent facile backbone cleavages to form dominant zn-2 or zn-3 ions. These backbone cleavages are thought to be triggered by a side-chain β-hydrogen atom transfer to the z ion Cα radical site followed by homolytic dissociation of the adjacent Cα—CO bond, forming zn-2 + HNCO cation-radicals that spontaneously dissociate by loss of HNCO. Amino acid residues that do not have readily transferrable β-hydrogen atoms (Gly, Ala) do not undergo the zn → zn-2 dissociations. The backbone cleavages compete with side-chain dissociations in z ions containing Asp and Asn residues. Side-chain dissociations are thought to be triggered by α-hydrogen atom transfers that activate the Cβ—Cγ or Cβ—heteroatom bonds for dissociations that dominate the MS3 spectra of z ions from peptides containing Leu, Cys, Lys, Met, Ser, Arg, Glu and Gln residues. The Lys, Arg, Gln, and Glu residues also participate in γ-hydrogen atom transfers that trigger other side-chain dissociations. PMID:22669761

  8. Strong cation exchange chromatography in analysis of posttranslational modifications: innovations and perspectives.

    PubMed

    Edelmann, Mariola J

    2011-01-01

    Strong cation exchange (SCX) chromatography has been utilized as an excellent separation technique that can be combined with reversed-phase (RP) chromatography, which is frequently used in peptide mass spectrometry. Although SCX is valuable as the second component of such two-dimensional separation methods, its application goes far beyond efficient fractionation of complex peptide mixtures. Here I describe how SCX facilitates mapping of the protein posttranslational modifications (PTMs), specifically phosphorylation and N-terminal acetylation. The SCX chromatography has been mainly used for enrichment of these two PTMs, but it might also be beneficial for high-throughput analysis of other modifications that alter the net charge of a peptide.

  9. Enhancement of the direct antimicrobial activity of Lysep3 against Escherichia coli by inserting cationic peptides into its C terminus.

    PubMed

    Ma, Qiang; Guo, Zhimin; Gao, Chencheng; Zhu, Rining; Wang, Shuang; Yu, Ling; Qin, Wanhai; Xia, Xiaojing; Gu, Jingmin; Yan, Guangmou; Lei, Liancheng

    2017-03-01

    Phage lysins are considered promising antimicrobials against resistant bacterial infections. Some lysins have been reported for the prevention and treatment of Gram-positive bacterial infection. Gram-negative bacterial phage lysins, however, can only destroy the bacterial cell wall from inside because of the obstruction of the bacterial outer membrane that prevents direct hydrolysis of the bacterial wall peptidoglycan from the outside, severely restricting the development of lysins against Gram-negative bacteria. In this study, genetic engineering techniques were used to fuse a 5 cationic amino acid polypeptide (KRKRK), a 10 cationic amino acid polypeptide (KRKRKRKRKR), a 15 cationic amino acid polypeptide (KRKRKRKRKRKRKRK), and a polypeptide including both cationic and hydrophobic amino acids (KRKRKFFVAIIP) to the C-terminus of the Escherichia coli phage lysin Lysep3 to obtain four fusion lysins (5aa, 10aa, 15aa, Mix). The bactericidal effects of those four lysins on E. coli were then compared in vitro. Our results showed that the fusion of hydrophobic and positively charged amino acids, Mix, can kill E. coli effectively; the fusion of positively charged amino acids alone at the C-terminus (5aa, 10aa, 15aa) also showed bactericidal activity against E. coli from the outside, with the bactericidal activity gradually increasing with the positive charge at the C-terminus of the lysin. Collectively, improving the positive charge at the C-terminus of E. coli bacteriophage lysin Lysep3 increases its bactericidal ability from outside E. coli, providing a new practical method for the development of anti-Gram-negative bacterial lysins.

  10. Identification of Legionella pneumophila rcp, a pagP-like gene that confers resistance to cationic antimicrobial peptides and promotes intracellular infection.

    PubMed

    Robey, M; O'Connell, W; Cianciotto, N P

    2001-07-01

    In the course of characterizing a locus involved in heme utilization, we identified a Legionella pneumophila gene predicted to encode a protein with homology to the product of the Salmonella enterica serovar Typhimurium pagP gene. In Salmonella, pagP increases resistance to the bactericidal effects of cationic antimicrobial peptides (CAMPs). Mutants with insertions in the L. pneumophila pagP-like gene were generated and showed decreased resistance to different structural classes of CAMPs compared to the wild type; hence, this gene was designated rcp for resistance to cationic antimicrobial peptides. Furthermore, Legionella CAMP resistance was induced by growth in low-magnesium medium. To determine whether rcp had any role in intracellular survival, mutants were tested in the two most relevant host cells for Legionnaires' disease, i.e., amoebae and macrophages. These mutants exhibited a 1,000-fold-decreased recovery during a Hartmannella vermiformis coculture. Complementation of the infectivity defect could be achieved by introduction of a plasmid containing the intact rcp gene. Mutations in rcp consistently reduced both the numbers of bacteria recovered during intracellular infection and their cytopathic capacity for U937 macrophages. The rcp mutant was also more defective for lung colonization of A/J mice. Growth of rcp mutants in buffered yeast extract broth was identical to that of the wild type, indicating that the observed differences in numbers of bacteria recovered from host cells were not due to a generalized growth defect. However, in low-Mg(2+) medium, the rcp mutant was impaired in stationary-phase survival. This is the first demonstration of a pagP-like gene, involved in resistance to CAMPs, being required for intracellular infection and virulence.

  11. Statins reduce amyloid β-peptide production by modulating amyloid precursor protein maturation and phosphorylation through a cholesterol-independent mechanism in cultured neurons.

    PubMed

    Hosaka, Ai; Araki, Wataru; Oda, Akiko; Tomidokoro, Yasushi; Tamaoka, Akira

    2013-03-01

    Statins, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, have been reported to attenuate amyloid-β peptide (Aβ) production in various cellular models. However, the mechanisms by which statins affect neuronal Aβ production have not yet been clarified. Here, we investigated this issue in rat primary cortical neurons using two statins, pitavastatin (PV) and atorvastatin (AV). Treatment of neurons with 0.2-2.5 μM PV or AV for 4 days induced a concentration- and time-dependent reduction in the secretion of both Aβ40 and Aβ42. Moreover, Western blot analyses of cell lysates showed that treatment with PV or AV significantly reduced expression levels of the mature form of amyloid precursor protein (APP) and Thr668-phosphorylated APP (P-APP), but not immature form of APP; the decreases in P-APP levels were more notable than those of mature APP levels. The statin treatment did not alter expression of BACE1 (β-site APP-cleaving enzyme 1) or γ-secretase complex proteins (presenilin 1, nicastrin, APH-1, and PEN-2). In neurons overexpressing APP via recombinant adenoviruses, PV or AV similarly reduced Aβ secretion and the levels of mature APP and P-APP. Statins also markedly reduced cellular cholesterol content in neurons in a concentration-dependent manner. Co-treatment with mevalonate reversed the statin-induced decreases in Aβ secretion and mature APP and P-APP levels, whereas co-treatment with cholesterol did not, despite recovery of cellular cholesterol levels. Finally, cell-surface biotinylation experiments revealed that both statins significantly reduced the levels of cell-surface P-APP without changing those of cell surface mature APP. These results suggest that statins reduce Aβ production by selectively modulating APP maturation and phosphorylation through a mechanism independent of cholesterol reduction in cultured neurons.

  12. Sustained release of hepatocyte growth factor by cationic self-assembling peptide/heparin hybrid hydrogel improves β-cell survival and function through modulating inflammatory response

    PubMed Central

    Liu, Shuyun; Zhang, Lanlan; Cheng, Jingqiu; Lu, Yanrong; Liu, Jingping

    2016-01-01

    Inflammatory response is a major cause of grafts dysfunction in islet transplantation. Hepatocyte growth factor (HGF) had shown anti-inflammatory activity in multiple diseases. In this study, we aim to deliver HGF by self-assembling peptide/heparin (SAP/Hep) hybrid gel to protect β-cell from inflammatory injury. The morphological and slow release properties of SAPs were analyzed. Rat INS-1 β-cell line was treated with tumor necrosis factor α in vitro and transplanted into rat kidney capsule in vivo, and the viability, apoptosis, function, and inflammation of β-cells were evaluated. Cationic KLD1R and KLD2R self-assembled to nanofiber hydrogel, which showed higher binding affinity for Hep and HGF because of electrostatic interaction. Slow release of HGF from cationic SAP/Hep gel is a two-step mechanism involving binding affinity with Hep and molecular diffusion. In vitro and in vivo results showed that HGF-loaded KLD2R/Hep gel promoted β-cell survival and insulin secretion, and inhibited cell apoptosis, cytokine release, T-cell infiltration, and activation of NFκB/p38 MAPK pathways in β-cells. This study suggested that SAP/Hep gel is a promising carrier for local delivery of bioactive proteins in islet transplantation. PMID:27729786

  13. Synthetic long peptide-based vaccine formulations for induction of cell mediated immunity: A comparative study of cationic liposomes and PLGA nanoparticles.

    PubMed

    Varypataki, Eleni Maria; Silva, Ana Luisa; Barnier-Quer, Christophe; Collin, Nicolas; Ossendorp, Ferry; Jiskoot, Wim

    2016-03-28

    Nanoparticulate formulations for synthetic long peptide (SLP)-cancer vaccines as alternative to clinically used Montanide ISA 51- and squalene-based emulsions are investigated in this study. SLPs were loaded into TLR ligand-adjuvanted cationic liposomes and PLGA nanoparticles (NPs) to potentially induce cell-mediated immune responses. The liposomal and PLGA NP formulations were successfully loaded with up to four different compounds and were able to enhance antigen uptake by dendritic cells (DCs) and subsequent activation of T cells in vitro. Subcutaneous vaccination of mice with the different formulations showed that the SLP-loaded cationic liposomes were the most efficient for the induction of functional antigen-T cells in vivo, followed by PLGA NPs which were as potent as or even more than the Montanide and squalene emulsions. Moreover, after transfer of antigen-specific target cells in immunized mice, liposomes induced the highest in vivo killing capacity. These findings, considering also the inadequate safety profile of the currently clinically used adjuvant Montanide ISA-51, make these two particulate, biodegradable delivery systems promising candidates as delivery platforms for SLP-based immunotherapy of cancer.

  14. 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.

  15. Development of a Novel Biosensor Using Cationic Antimicrobial Peptide and Nickel Phthalocyanine Ultrathin Films for Electrochemical Detection of Dopamine

    PubMed Central

    Zampa, Maysa F.; Araújo, Inês Maria de S.; dos Santos Júnior, José Ribeiro; Zucolotto, Valtencir; Leite, José Roberto de S. A.; Eiras, Carla

    2012-01-01

    The antimicrobial peptide dermaseptin 01 (DS 01), from the skin secretion of Phyllomedusa hypochondrialis frogs, was immobilized in nanostructured layered films in conjunction with nickel tetrasulfonated phthalocyanines (NiTsPc), widely used in electronic devices, using layer-by-layer technique. The films were used as a biosensor to detect the presence of dopamine (DA), a neurotransmitter associated with diseases such as Alzheimer's and Parkinson's, with detection limits in the order of 10−6 mol L−1. The use of DS 01 in LbL film generated selectivity in the detection of DA despite the presence of ascorbic acid found in biological fluids. This work is the first to report that the antimicrobial peptide and NiTsPc LbL film exhibits electroanalytical activity to DA oxidation. The selectivity in the detection of DA is a fundamental aspect for the development of electrochemical sensors with potential applications in the biomedical and pharmaceutical industries. PMID:22287966

  16. Development of a novel biosensor using cationic antimicrobial Peptide and nickel phthalocyanine ultrathin films for electrochemical detection of dopamine.

    PubMed

    Zampa, Maysa F; Araújo, Inês Maria de S; Dos Santos Júnior, José Ribeiro; Zucolotto, Valtencir; Leite, José Roberto de S A; Eiras, Carla

    2012-01-01

    The antimicrobial peptide dermaseptin 01 (DS 01), from the skin secretion of Phyllomedusa hypochondrialis frogs, was immobilized in nanostructured layered films in conjunction with nickel tetrasulfonated phthalocyanines (NiTsPc), widely used in electronic devices, using layer-by-layer technique. The films were used as a biosensor to detect the presence of dopamine (DA), a neurotransmitter associated with diseases such as Alzheimer's and Parkinson's, with detection limits in the order of 10(-6) mol L(-1). The use of DS 01 in LbL film generated selectivity in the detection of DA despite the presence of ascorbic acid found in biological fluids. This work is the first to report that the antimicrobial peptide and NiTsPc LbL film exhibits electroanalytical activity to DA oxidation. The selectivity in the detection of DA is a fundamental aspect for the development of electrochemical sensors with potential applications in the biomedical and pharmaceutical industries.

  17. Influence of ionization on the conformational preferences of peptide models. Ramachandran surfaces of N-formyl-glycine amide and N-formyl-alanine amide radical cations.

    PubMed

    Gil, Adrià; Sodupe, Mariona; Bertran, Juan

    2009-09-01

    Ramachandran maps of neutral and ionized HCO-Gly-NH2 and HCO-Ala-NH2 peptide models have been built at the B3LYP/6-31++G(d,p) level of calculation. Direct optimizations using B3LYP and the recently developed MPWB1K functional have also been carried out, as well as single-point calculations at the CCSD(T) level of theory with the 6-311++G(2df,2p) basis set. Results indicate that for both peptide models ionization can cause drastic changes in the shape of the PES in such a way that highly disallowed regions in neutral PES become low-energy regions in the radical cation surface. The structures localized in such regions, epsilonL+* and epsilonD+* are highly stabilized due to the formation of 2-centre-3-electron interactions between the two carbonyl oxygens. Inclusion of solvent effects by the conductor-like polarizable continuum model (CPCM) shows that the solute-solvent interaction energy plays an important role in determining the stability order.

  18. D-Alanylation of Lipoteichoic Acids Confers Resistance to Cationic Peptides in Group B Streptococcus by Increasing the Cell Wall Density

    PubMed Central

    Saar-Dover, Ron; Bitler, Arkadi; Nezer, Ravit; Shmuel-Galia, Liraz; Firon, Arnaud; Shimoni, Eyal; Trieu-Cuot, Patrick; Shai, Yechiel

    2012-01-01

    Cationic antimicrobial peptides (CAMPs) serve as the first line of defense of the innate immune system against invading microbial pathogens. Gram-positive bacteria can resist CAMPs by modifying their anionic teichoic acids (TAs) with D-alanine, but the exact mechanism of resistance is not fully understood. Here, we utilized various functional and biophysical approaches to investigate the interactions of the human pathogen Group B Streptococcus (GBS) with a series of CAMPs having different properties. The data reveal that: (i) D-alanylation of lipoteichoic acids (LTAs) enhance GBS resistance only to a subset of CAMPs and there is a direct correlation between resistance and CAMPs length and charge density; (ii) resistance due to reduced anionic charge of LTAs is not attributed to decreased amounts of bound peptides to the bacteria; and (iii) D-alanylation most probably alters the conformation of LTAs which results in increasing the cell wall density, as seen by Transmission Electron Microscopy, and reduces the penetration of CAMPs through the cell wall. Furthermore, Atomic Force Microscopy reveals increased surface rigidity of the cell wall of the wild-type GBS strain to more than 20-fold that of the dltA mutant. We propose that D-alanylation of LTAs confers protection against linear CAMPs mainly by decreasing the flexibility and permeability of the cell wall, rather than by reducing the electrostatic interactions of the peptide with the cell surface. Overall, our findings uncover an important protective role of the cell wall against CAMPs and extend our understanding of mechanisms of bacterial resistance. PMID:22969424

  19. Cascade dissociations of peptide cation-radicals. Part 1. Scope and effects of amino acid residues in penta-, nona-, and decapeptides.

    PubMed

    Chung, Thomas W; Hui, Renjie; Ledvina, Aaron; Coon, Joshua J; Tureček, Frantisek

    2012-08-01

    Amino acid residue-specific backbone and side-chain dissociations of peptide z ions in MS(3) spectra were elucidated for over 40 pentapeptides with arginine C-terminated sequences of the AAXAR and AAHXR type, nonapeptides of the AAHAAXX"AR and AAHAXAX"AR type, and AAHAAXX"AAR decapeptides. Peptide z(n) ions containing amino acid residues with readily transferrable benzylic or tertiary β-hydrogen atoms (Phe, Tyr, His, Trp, Val) underwent facile backbone cleavages to form dominant z(n-2) or z(n-3) ions. These backbone cleavages are thought to be triggered by a side-chain β-hydrogen atom transfer to the z ion C(α) radical site followed by homolytic dissociation of the adjacent C(α)-CO bond, forming x(n-2) cation-radicals that spontaneously dissociate by loss of HNCO. Amino acid residues that do not have readily transferrable β-hydrogen atoms (Gly, Ala) do not undergo the z(n) → z(n-2) dissociations. The backbone cleavages compete with side-chain dissociations in z ions containing Asp and Asn residues. Side-chain dissociations are thought to be triggered by α-hydrogen atom transfers that activate the C(β)-C(γ) or C(β)-heteroatom bonds for dissociations that dominate the MS(3) spectra of z ions from peptides containing Leu, Cys, Lys, Met, Ser, Arg, Glu, and Gln residues. The Lys, Arg, Gln, and Glu residues also participate in γ-hydrogen atom transfers that trigger other side-chain dissociations.

  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. Lipid complexes with cationic peptides and OAKs; their role in antimicrobial action and in the delivery of antimicrobial agents.

    PubMed

    Epand, Raquel F; Mor, Amram; Epand, Richard M

    2011-07-01

    Antimicrobial agents are toxic to bacteria by a variety of mechanisms. One mechanism that is very dependent on the lipid composition of the bacterial membrane is the clustering of anionic lipid by cationic antimicrobial agents. Certain species of oligo-acyl-lysine (OAK) antimicrobial agents are particularly effective in clustering anionic lipids in mixtures mimicking the composition of bacterial membranes. The clustering of anionic lipids by certain cationic antimicrobial agents contributes to the anti-bacterial action of these agents. Bacterial membrane lipids are a determining factor, resulting in some species of bacteria being more susceptible than others. In addition, lipids can be used to increase the effectiveness of antimicrobial agents when administered in vivo. Therefore, we review some of the structures in which lipid mixtures can assemble, to more effectively be utilized as antimicrobial delivery systems. We describe in more detail the complexes formed between mixtures of lipids mimicking bacterial membranes and an OAK and their usefulness in synergizing with antibiotics to overcome bacterial multidrug resistance.

  2. Cationic Antimicrobial Peptides and Biogenic Silver Nanoparticles Kill Mycobacteria without Eliciting DNA Damage and Cytotoxicity in Mouse Macrophages

    PubMed Central

    Mohanty, Soumitra; Jena, Prajna; Mehta, Ranjit; Pati, Rashmirekha; Banerjee, Birendranath; Patil, Satish

    2013-01-01

    With the emergence of multidrug-resistant mycobacterial strains, better therapeutic strategies are required for the successful treatment of the infection. Although antimicrobial peptides (AMPs) and silver nanoparticles (AgNPs) are becoming one of the popular antibacterial agents, their antimycobacterial potential is not fully evaluated. In this study, we synthesized biogenic-silver nanoparticles using bacterial, fungal, and plant biomasses and analyzed their antibacterial activities in combination with AMPs against mycobacteria. Mycobacterium smegmatis was found to be more susceptible to AgNPs compared to M. marinum. We found that NK-2 showed enhanced killing effect with NP-1 and NP-2 biogenic nanoparticles at a 0.5-ppm concentration, whereas LLKKK-18 showed antibacterial activity only with NP-2 at 0.5-ppm dose against M. smegmatis. In case of M. marinum NK-2 did not show any additive activity with NP-1 and NP-2 and LLKKK-18 alone completely inhibited the bacterial growth. Both NP-1 and NP-2 also showed increased killing of M. smegmatis in combination with the antituberculosis drug rifampin. The sizes and shapes of the AgNPs were determined by transmission electron microscopy and dynamic light scattering. AgNPs showed no cytotoxic or DNA damage effects on macrophages at the mycobactericidal dose, whereas treatment with higher doses of AgNPs caused toxicity and micronuclei formation in cytokinesis blocked cells. Macrophages actively endocytosed fluorescein isothiocyanate-labeled AgNPs resulting in nitric oxide independent intracellular killing of M. smegmatis. Apoptosis and cell cycle studies showed that treatment with higher dose of AgNPs arrested macrophages at the G1-phase. In summary, our data suggest the combined effect of biogenic-AgNPs and antimicrobial peptides as a promising antimycobacterial template. PMID:23689720

  3. Carrier-mediated intestinal absorption of valacyclovir, the L-valyl ester prodrug of acyclovir: 1. Interactions with peptides, organic anions and organic cations in rats.

    PubMed

    Sinko, P J; Balimane, P V

    1998-05-01

    The mechanism of intestinal transport of valacyclovir (VACV), the L-valyl ester prodrug of acyclovir, was investigated in rats using an in situ intestinal perfusion technique. VACV demonstrates an oral bioavailability that is three to five time greater than acyclovir, concentration dependent, and saturable in humans. Homogenate and perfused buffer stability results demonstrated that VACV was increasingly unstable with increasing pH. VACV was converted to ACV in a concentration dependent manner during a single pass through the intestinal segment. Perfusions were performed at 37 degrees C, pH 6.5, and under iso-osmotic conditions (290 +/- 10 mOsm L-1). Intestinal outlet concentrations were corrected for VACV that was converted to ACV during the perfusion. The effective dimensionless intestinal permeability (P*e) of VACV was concentration dependent, saturable (intrinsic Km = 1.2 +/- 0.7 mM), and significantly reduced (p < 0.05) in the presence of peptide analogues (amoxicillin, ampicillin, cefadroxil, and cephradine), by the organic anion, p-amino hippuric acid and by the organic cation quinine. VACV transport was not inhibited by classical nucleoside competitive substrates or inhibitors or by valine. These results suggest that H(+)-oligopeptide, H(+)-organic cation, and organic anion transporters are involved in the small intestinal uptake of VACV. The permeability of VACV in the colon was very low, indicating that VACV is predominantly absorbed from the small intestine. VACV P*e was not altered in the presence of glucose-induced convective fluid flow, suggesting that carrier-mediated, transcellular uptake is the predominant absorption pathway of VACV in rat small intestine. Based on these results, the oral bioavailability of VACV appears to be significantly influenced by the preabsorptive conversion of VACV to the poorly absorbed ACV, by the involvement of multiple transporters in VACV small-intestinal uptake, and by the low permeability of VACV in the colon.

  4. 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.

  5. 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

  6. Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease

    PubMed Central

    Muratovska, Aleksandra; Lightowlers, Robert N.; Taylor, Robert W.; Turnbull, Douglass M.; Smith, Robin A. J.; Wilce, Jacqueline A.; Martin, Stephen W.; Murphy, Michael P.

    2001-01-01

    The selective manipulation of mitochondrial DNA (mtDNA) replication and expression within mammalian cells has proven difficult. One promising approach is to use peptide nucleic acid (PNA) oligomers, nucleic acid analogues that bind selectively to complementary DNA or RNA sequences inhibiting replication and translation. However, the potential of PNAs is restricted by the difficulties of delivering them to mitochondria within cells. To overcome this problem we conjugated a PNA 11mer to a lipophilic phosphonium cation. Such cations are taken up by mitochondria through the lipid bilayer driven by the membrane potential across the inner membrane. As anticipated, phosphonium–PNA (ph–PNA) conjugates of 3.4–4 kDa were imported into both isolated mitochondria and mitochondria within human cells in culture. This was confirmed by using an ion-selective electrode to measure uptake of the ph–PNA conjugates; by cell fractionation in conjunction with immunoblotting; by confocal microscopy; by immunogold-electron microscopy; and by crosslinking ph–PNA conjugates to mitochondrial matrix proteins. In all cases dissipating the mitochondrial membrane potential with an uncoupler prevented ph–PNA uptake. The ph–PNA conjugate selectively inhibited the in vitro replication of DNA containing the A8344G point mutation that causes the human mtDNA disease ‘myoclonic epilepsy and ragged red fibres’ (MERRF) but not the wild-type sequence that differs at a single nucleotide position. Therefore these modified PNA oligomers retain their selective binding to DNA and the lipophilic cation delivers them to mitochondria within cells. When MERRF cells were incubated with the ph–PNA conjugate the ratio of MERRF to wild-type mtDNA was unaffected, even though the ph–PNA content of the mitochondria was sufficient to inhibit MERRF mtDNA replication in a cell-free system. This unexpected finding suggests that nucleic acid derivatives cannot bind their complementary sequences during mt

  7. Cationic osteogenic peptide P15-CSP coatings promote 3-D osteogenesis in poly(epsilon-caprolactone) scaffolds of distinct pore size.

    PubMed

    Li, Xian; Mehr, Nima Ghavidel; Guzmán-Morales, Jessica; Favis, Basil D; De Crescenzo, Gregory; Yakandawala, Nanda; Hoemann, Caroline D

    2017-04-05

    P15-CSP is a biomimetic cationic fusion peptide that stimulates osteogenesis and inhibits bacterial biofilm formation when coated on 2-D surfaces. This study tested the hypothesis that P15-CSP coatings enhance 3-D osteogenesis in a porous but otherwise hydrophobic poly-(ε-caprolactone) (PCL) scaffold. Scaffolds of 84 µm and 141 µm average pore size were coated or not with Layer-by-Layer polyelectrolytes followed by P15-CSP, seeded with adult primary human mesenchymal stem cells (MSCs), and cultured 10 days in proliferation medium, then 21 days in osteogenic medium. Atomic analyses showed that P15-CSP was successfully captured by LbL. After 2 days of culture, MSCs adhered and spread more on P15-CSP coated pores than PCL-only. At day 10, all constructs contained non-mineralized tissue. At day 31, all constructs became enveloped in a "skin" of tissue that, like 2-D cultures, underwent sporadic mineralization in areas of high cell density that extended into some 141 µm edge pores. By quantitative histomorphometry, 2.5-fold more tissue and biomineral accumulated in edge pores versus inner pores. P15-CSP specifically promoted tissue-scaffold integration, 4-fold higher overall biomineralization, and more mineral deposits in the outer 84 µm and inner 141 µm pores than PCL-only (p<0.05). 3-D Micro-CT revealed asymmetric mineral deposition consistent with histological calcium staining. This study provides proof-of-concept that P15-CSP coatings are osteoconductive in PCL pore surfaces with 3-D topography. Biomineralization deeper than 150 µm from the scaffold edge was optimally attained with the larger 141 µm peptide-coated pores. This article is protected by copyright. All rights reserved.

  8. BONE MARROW EXPRESSED ANTIMICROBIAL CATIONIC PEPTIDE LL-37 ENHANCES RESPONSIVENESS OF HEMATOPOIETIC STEM PROGENITOR CELLS TO AN SDF-1 GRADIENT AND ACCELERATES THEIR ENGRAFTMENT AFTER TRANSPLANTATION

    PubMed Central

    Wu, Wan; Kim, Chi Hwa; Liu, Rui; Kucia, Magda; Greco, Nicholas; Ratajczak, Janina; Laughlin, Mary L.; Ratajczak, Mariusz Z.

    2011-01-01

    We report that the bone marrow stroma-released LL-37, a member of the cathelicidin family of antimicrobial peptides, primes/increases responsiveness of murine and human hematopoietic stem/progenitor cells (HSPCs) to an α-chemokine stromal-derived factor-1 (SDF-1) gradient. Accordingly, LL-37 is upregulated in irradiated BM cells and enhances the chemotactic responsiveness of hematopoietic progenitors from all lineages to a low physiological SDF-1 gradient as well as increases their i) adhesiveness, ii) SDF-1-mediated actin polymerization, and iii) MAPKp42/44 phosphorylation. Mice transplanted with bone marrow (BM) cells ex vivo primed by LL-37 showed accelerated recovery of platelet and neutrophil counts by ~3–5 days compared to mice transplanted with unprimed control cells. These priming effects were not mediated by LL-37 binding to its receptor and depended instead on incorporation of the CXCR4 receptor into membrane lipid rafts. We propose that LL-37, which has primarily antimicrobial functions and is harmless to mammalian cells, could be clinically applied to accelerate engraftment as ex vivo priming agent for transplanted human HSPCs. This novel approach would be particularly important in cord blood transplantations, where the number of HSCs available is usually limited. PMID:21931324

  9. 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.

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

    DOE PAGES

    Das, Amit; Gerlits, Oksana O.; Heller, William T.; ...

    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

  11. 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.

  12. Phosphoryl Transfer Reaction Snapshots in Crystals: INSIGHTS INTO THE MECHANISM OF PROTEIN KINASE A CATALYTIC SUBUNIT.

    PubMed

    Gerlits, Oksana; Tian, Jianhui; Das, Amit; Langan, Paul; Heller, William T; Kovalevsky, Andrey

    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 Ca(2+) 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 Ca(2+) cations with Mg(2+) ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. 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.

  13. Cell Surface Expression of the Major Amyloid-β Peptide (Aβ)-degrading Enzyme, Neprilysin, Depends on Phosphorylation by Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK) and Dephosphorylation by Protein Phosphatase 1a*

    PubMed Central

    Kakiya, Naomasa; Saito, Takashi; Nilsson, Per; Matsuba, Yukio; Tsubuki, Satoshi; Takei, Nobuyuki; Nawa, Hiroyuki; Saido, Takaomi C.

    2012-01-01

    Neprilysin is one of the major amyloid-β peptide (Aβ)-degrading enzymes, the expression of which declines in the brain during aging. The decrease in neprilysin leads to a metabolic Aβ imbalance, which can induce the amyloidosis underlying Alzheimer disease. Pharmacological activation of neprilysin during aging therefore represents a potential strategy to prevent the development of Alzheimer disease. However, the regulatory mechanisms mediating neprilysin activity in the brain remain unclear. To address this issue, we screened for pharmacological regulators of neprilysin activity and found that the neurotrophic factors brain-derived neurotrophic factor, nerve growth factor, and neurotrophins 3 and 4 reduce cell surface neprilysin activity. This decrease was mediated by MEK/ERK signaling, which enhanced phosphorylation at serine 6 in the neprilysin intracellular domain (S6-NEP-ICD). Increased phosphorylation of S6-NEP-ICD in primary neurons reduced the levels of cell surface neprilysin and led to a subsequent increase in extracellular Aβ levels. Furthermore, a specific inhibitor of protein phosphatase-1a, tautomycetin, induced extensive phosphorylation of the S6-NEP-ICD, resulting in reduced cell surface neprilysin activity. In contrast, activation of protein phosphatase-1a increased cell surface neprilysin activity and lowered Aβ levels. Taken together, these results indicate that the phosphorylation status of S6-NEP-ICD influences the localization of neprilysin and affects extracellular Aβ levels. Therefore, maintaining S6-NEP-ICD in a dephosphorylated state, either by inhibition of protein kinases involved in its phosphorylation or by activation of phosphatases catalyzing its dephosphorylation, may represent a new approach to prevent reduction of cell surface neprilysin activity during aging and to maintain physiological levels of Aβ in the brain. PMID:22767595

  14. Different effects of transcriptional regulators MarA, SoxS and Rob on susceptibility of Escherichia coli to cationic antimicrobial peptides (CAMPs): Rob-dependent CAMP induction of the marRAB operon.

    PubMed

    Warner, Douglas M; Levy, Stuart B

    2010-02-01

    Cationic antimicrobial peptides (CAMPs), a component of the mammalian immune system, protect the host from bacterial infections. The roles of the Escherichia coli transcriptional regulators MarA, SoxS and Rob in susceptibility to these peptides were examined. Overexpression of marA, either in an antibiotic-resistant marR mutant or from a plasmid, decreased bacterial susceptibility to CAMPs. Overexpression of the soxS gene from a plasmid, which decreased susceptibility to antibiotics, unexpectedly caused no decrease in CAMP susceptibility; instead it produced increased susceptibility to different CAMPs. Deletion or overexpression of rob had little effect on CAMP susceptibility. The marRAB operon was upregulated when E. coli was incubated in sublethal amounts of CAMPs polymyxin B, LL-37 or human beta-defensin-1; however, this upregulation required Rob. Deletion of acrAB increased bacterial susceptibility to polymyxin B, LL-37 and human beta-defensin-1 peptides. Deletion of tolC yielded an even greater increase in susceptibility to these peptides and also led to increased susceptibility to human alpha-defensin-2. Inhibition of cellular proton-motive force increased peptide susceptibility for wild-type and acrAB deletion strains; however, it decreased susceptibility of tolC mutants. These findings demonstrate that CAMPs are both inducers of marA-mediated drug resistance through interaction with Rob and also substrates for efflux in E. coli. The three related transcriptional regulators show different effects on bacterial cell susceptibility to CAMPs.

  15. 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

  16. 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.

  17. The Outer Membrane of Brucella ovis Shows Increased Permeability to Hydrophobic Probes and Is More Susceptible to Cationic Peptides than Are the Outer Membranes of Mutant Rough Brucella abortus Strains

    PubMed Central

    Freer, Enrique; Pizarro-Cerdá, Javier; Weintraub, Andrej; Bengoechea, José-Antonio; Moriyón, Ignacio; Hultenby, Kjell; Gorvel, Jean-Pierre; Moreno, Edgardo

    1999-01-01

    The permeability of the outer membrane (OM) to hydrophobic probes and its susceptibility to bactericidal cationic peptides were investigated for natural rough Brucella ovis and for mutant rough Brucella abortus strains. The OM of B. ovis displayed an abrupt and faster kinetic profile than rough B. abortus during the uptake of the hydrophobic probe N-phenyl-naphthylamine. B. ovis was more sensitive than rough B. abortus to the action of cationic peptides. Bactenecins 5 and 7 induced morphological alterations on the OMs of both rough Brucella strains. B. ovis lipopolysaccharide (LPS) captured considerably more polymyxin B than LPSs from both rough and smooth B. abortus strains. Polymyxin B, poly-l-lysine, and poly-l-ornithine produced a thick coating on the surfaces of both strains, which was more evident in B. ovis than in rough B. abortus. The distinct functional properties of the OMs of these two rough strains correlate with some structural differences of their OMs and with their different biological behaviors in animals and culture cells. PMID:10531286

  18. Effective induction of anti-tumor immunity using p5 HER-2/neu derived peptide encapsulated in fusogenic DOTAP cationic liposomes co-administrated with CpG-ODN.

    PubMed

    Mansourian, Mercedeh; Badiee, Ali; Jalali, Seyed Amir; Shariat, Sheida; Yazdani, Mona; Amin, Mohamdreza; Jaafari, Mahmoud Reza

    2014-11-01

    Cationic liposomes have been used as efficient antigen delivery systems for cancer vaccination. The current study has investigated whether the incorporation of the helper-fusogenic lipid dioleoylphosphatidylethanolamine (DOPE) in cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)-cholesterol enhances the cytosolic delivery of p5 HER-2/neu derived peptide (p5) and promotes cytotoxic T lymphocytes (CTL) response. The p5, which is a very hydrophobic peptide, was encapsulated into liposomes by using three different methods and characterized for their colloidal properties. A chaotropic loading method using 7 M urea provided the highest encapsulation yields. Mice were first immunized with encapsulated p5 in liposomes composed of either DOTAP-cholesterol or DOTAP-cholesterol-DOPE, alone or co-administered with CpG-ODN, as an immunoadjuvant, then, inoculated with a subcutaneous injection of TUBO tumor cells. Results obtained from enzyme-linked immunospot, cytotoxicity and intracellular cytokine assays as well as tumor sizes and animal survival analysis demonstrated that p5 encapsulated in DOTAP-cholesterol-DOPE liposomes co-administered with CpG-ODN greatly enhanced the cytotoxic T lymphocytes response and highly inhibited the tumor progression. The outperformance of DOTAP-cholesterol-DOPE liposomes+CpG-ODN was found to be attributed to its capability in induction of both CD8+ and CD4+ responses. This formulation could be a suitable vaccine candidate against Her2 positive cancers and merits further investigations.

  19. 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.

  20. 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...

  1. Large-Scale Phosphoproteomics Analysis of Whole Saliva Reveals a Distinct Phosphorylation Pattern

    PubMed Central

    Stone, Matthew D.; Chen, Xiaobing; McGowan, Thomas; Bandhakavi, Sricharan; Cheng, Bin; Rhodus, Nelson L.; Griffin, Timothy J.

    2011-01-01

    In-depth knowledge of bodily fluid phosphoproteomes, such as whole saliva, is limited. To better understand the whole saliva phosphoproteome, we generated a large-scale catalog of phosphorylated proteins. To circumvent the wide dynamic range of phosphoprotein abundance in whole saliva, we combined dynamic range compression using hexapeptide beads, strong cation exchange HPLC peptide fractionation, and immobilized metal affinity chromatography prior to mass spectrometry. In total, 217 unique phosphopeptides sites were identified representing 85 distinct phosphoproteins at 2.3% global FDR. From these peptides, 129 distinct phosphorylation sites were identified of which 57 were previously known, but only 11 of which had been previously identified in whole saliva. Cellular localization analysis revealed salivary phosphoproteins had a distribution similar to all known salivary proteins, but with less relative representation in “extracellular” and “plasma membrane” categories compared to salivary glycoproteins. Sequence alignment showed that phosphorylation occurred at acidic-directed kinase, proline-directed, and basophilic motifs. This differs from plasma phosphoproteins, which predominantly occur at Golgi casein kinase recognized sequences. Collectively, these results suggest diverse functions for salivary phosphoproteins and multiple kinases involved in their processing and secretion. In all, this study should lay groundwork for future elucidation of the functions of salivary protein phosphorylation. PMID:21299198

  2. Probing the role of metal cations on the aggregation behavior of amyloid β-peptide at a single molecule level by AFM

    NASA Astrophysics Data System (ADS)

    Xie, Yang; Wang, Jianhua; Liu, Chundong

    2016-09-01

    With the development of nanotechnology, understanding of intermolecular interactions on a single molecule level by atomic force spectroscopy (AFM) has played an important role in molecular biology and biomedical science. In recent years, some research suggested that the presence of metal cations is an important regulator in the processes of misfolding and aggregation of the amyloid β-protein (Aβ), which may be an important etiological factor of Alzheimer's disease. However, the knowledge on the principle of interactions between Aβ and metal cations at the single molecule level is still poor understood. In this paper, the amyloid β-protein (Aβ) was fabricated on substrate of mixed thiol-modified gold nanoparticles using self-assembled monolayer method and the adhesion force in the longitudinal direction between metal cations and Aβ42 were investigated by AFM. The role of metal ions on Aβ aggregation is discussed from the perspective of single molecular force. The force results showed that the specific adhesion force F i and the nonspecific force F 0 between a single Aβ-Aβ pair in control experiment were calculated as 42 ± 3 and 80 pN, respectively. However, F i between a single Aβ-Aβ pair in the presence of Cu2+, Zn2+, Ca2+ and Al3+ increased dramatically to 84 ± 6, 89 ± 3, 73 ± 5, 95 ± 5 pN successively, which indicated that unbinding between Aβ proteins is accelerated in the presence of metal cations. What is more, the imaging results showed that substoichiometric copper cations accelerate the formation of fibrils within 3 days. The combined atomic force spectroscopy and imaging analysis indicate that metal cations play a role in promoting the aggregating behavior of Aβ42.

  3. 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.

  4. Sequence-selective recognition of double-stranded RNA and enhanced cellular uptake of cationic nucleobase and backbone-modified peptide nucleic acids.

    PubMed

    Hnedzko, Dziyana; McGee, Dennis W; Karamitas, Yannis A; Rozners, Eriks

    2017-01-01

    Sequence-selective recognition of complex RNAs in live cells could find broad applications in biology, biomedical research, and biotechnology. However, specific recognition of structured RNA is challenging, and generally applicable and effective methods are lacking. Recently, we found that peptide nucleic acids (PNAs) were unusually well-suited ligands for recognition of double-stranded RNAs. Herein, we report that 2-aminopyridine (M) modified PNAs and their conjugates with lysine and arginine tripeptides form strong (Ka = 9.4 to 17 × 10(7) M(-1)) and sequence-selective triple helices with RNA hairpins at physiological pH and salt concentration. The affinity of PNA-peptide conjugates for the matched RNA hairpins was unusually high compared to the much lower affinity for DNA hairpins of the same sequence (Ka = 0.05 to 1.1 × 10(7) M(-1)). The binding of double-stranded RNA by M-modified PNA-peptide conjugates was a relatively fast process (kon = 2.9 × 10(4) M(-1) sec(-1)) compared to the notoriously slow triple helix formation by oligodeoxynucleotides (kon ∼ 10(3) M(-1) sec(-1)). M-modified PNA-peptide conjugates were not cytotoxic and were efficiently delivered in the cytosol of HEK293 cells at 10 µM. Surprisingly, M-modified PNAs without peptide conjugation were also taken up by HEK293 cells, which, to the best of our knowledge, is the first example of heterocyclic base modification that enhances the cellular uptake of PNA. Our results suggest that M-modified PNA-peptide conjugates are promising probes for sequence-selective recognition of double-stranded RNA in live cells and other biological systems.

  5. FT-IR analysis of phosphorylated protein

    NASA Astrophysics Data System (ADS)

    Ishii, Katsunori; Yoshihashi, Sachiko S.; Chihara, Kunihiro; Awazu, Kunio

    2004-09-01

    Phosphorylation and dephosphorylation, which are the most remarkable posttranslational modifications, are considered to be important chemical reactions that control the activation of proteins. We examine the phosphorylation analysis method by measuring the infrared absorption peak of phosphate group that observed at about 1070cm-1 (9.4μm) with Fourier Transform Infrared Spectrometer (FT-IR). This study indicates that it is possible to identify a phosphorylation by measuring the infrared absorption peak of phosphate group observed at about 1070 cm-1 with FT-IR method. As long as target peptides have the same amino acid sequence, it is possible to identify the phosphorylated sites (threonine, serine and tyrosine).

  6. 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.

  7. Antimicrobial Activity of Cationic Antimicrobial Peptides against Gram-Positives: Current Progress Made in Understanding the Mode of Action and the Response of Bacteria

    PubMed Central

    Omardien, Soraya; Brul, Stanley; Zaat, Sebastian A. J.

    2016-01-01

    Antimicrobial peptides (AMPs) have been proposed as a novel class of antimicrobials that could aid the fight against antibiotic resistant bacteria. The mode of action of AMPs as acting on the bacterial cytoplasmic membrane has often been presented as an enigma and there are doubts whether the membrane is the sole target of AMPs. Progress has been made in clarifying the possible targets of these peptides, which is reported in this review with as focus gram-positive vegetative cells and spores. Numerical estimates are discussed to evaluate the possibility that targets, other than the membrane, could play a role in susceptibility to AMPs. Concerns about possible resistance that bacteria might develop to AMPs are addressed. Proteomics, transcriptomics, and other molecular techniques are reviewed in the context of explaining the response of bacteria to the presence of AMPs and to predict what resistance strategies might be. Emergent mechanisms are cell envelope stress responses as well as enzymes able to degrade and/or specifically bind (and thus inactivate) AMPs. Further studies are needed to address the broadness of the AMP resistance and stress responses observed. PMID:27790614

  8. 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.

  9. 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.

  10. High yield recombinant production of a self-assembling polycationic peptide for silica biomineralization.

    PubMed

    Zerfaß, Christian; Braukmann, Sandra; Nietzsche, Sandor; Hobe, Stephan; Paulsen, Harald

    2015-04-01

    We report the recombinant bacterial expression and purification at high yields of a polycationic oligopeptide, P5S3. The sequence of P5S3 was inspired by a diatom silaffin, a silica precipitating peptide. Like its native model, P5S3 exhibits silica biomineralizing activity, but furthermore has unusual self-assembling properties. P5S3 is efficiently expressed in Escherichia coli as fusion with ketosteroid isomerase (KSI), which causes deposition in inclusion bodies. After breaking the fusion by cyanogen bromide reaction, P5S3 was purified by cation exchange chromatography, taking advantage of the exceptionally high content of basic amino acids. The numerous cationic charges do not prevent, but may even promote counterion-independent self-assembly which in turn leads to silica precipitation. Enzymatic phosphorylation, a common modification in native silica biomineralizing peptides, can be used to modify the precipitation activity.

  11. 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

  12. Hypocretin/Orexin Peptides Excite Rat Neuroendocrine Dopamine Neurons through Orexin 2 Receptor-Mediated Activation of a Mixed Cation Current

    PubMed Central

    Lyons, David J.; Hellysaz, Arash; Ammari, Rachida; Broberger, Christian

    2017-01-01

    Hypocretin/Orexin (H/O) neurons of the lateral hypothalamus are compelling modulator candidates for the chronobiology of neuroendocrine output and, as a consequence, hormone release from the anterior pituitary. Here we investigate the effects of H/O peptides upon tuberoinfundibular dopamine (TIDA) neurons – cells which control, via inhibition, the pituitary secretion of prolactin. In whole cell recordings performed in male rat hypothalamic slices, application of H/O-A, as well as H/O-B, excited oscillating TIDA neurons, inducing a reversible depolarising switch from phasic to tonic discharge. The H/O-induced inward current underpinning this effect was post-synaptic (as it endured in the presence of tetrodotoxin), appeared to be carried by a Na+-dependent transient receptor potential-like channel (as it was blocked by 2-APB and was diminished by removal of extracellular Na+), and was a consequence of OX2R receptor activation (as it was blocked by the OX2R receptor antagonist TCS OX2 29, but not the OX1R receptor antagonist SB 334867). Application of the hormone, melatonin, failed to alter TIDA membrane potential or oscillatory activity. This first description of the electrophysiological effects of H/Os upon the TIDA network identifies cellular mechanisms that may contribute to the circadian rhythmicity of prolactin secretion. PMID:28145492

  13. 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

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

    PubMed

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

    2016-10-01

    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. Graphical Abstract ᅟ.

  15. The Lipopolysaccharide of Brucella abortus BvrS/BvrR Mutants Contains Lipid A Modifications and Has Higher Affinity for Bactericidal Cationic Peptides

    PubMed Central

    Manterola, Lorea; Moriyón, Ignacio; Moreno, Edgardo; Sola-Landa, Alberto; Weiss, David S.; Koch, Michel H. J.; Howe, Jörg; Brandenburg, Klaus; López-Goñi, Ignacio

    2005-01-01

    The two-component BvrS/BvrR system is essential for Brucella abortus virulence. It was shown previously that its dysfunction abrogates expression of some major outer membrane proteins and increases bactericidal peptide sensitivity. Here, we report that BvrS/BvrR mutants have increased surface hydrophobicity and susceptibility to killing by nonimmune serum. The bvrS and bvrR mutant lipopolysaccharides (LPSs) bound more polymyxin B, chimeras constructed with bvrS mutant cells and parental LPS showed augmented polymyxin B resistance, and, conversely, parental cells and bvrS mutant LPS chimeras were more sensitive and displayed polymyxin B-characteristic outer membrane lesions, implicating LPS as being responsible for the phenotype of the BvrS/BvrR mutants. No qualitative or quantitative changes were detected in other envelope and outer membrane components examined: periplasmic β(1-2) glucans, native hapten polysaccharide, and phospholipids. The LPS of the mutants was similar to parental LPS in O-polysaccharide polymerization and fine structure but showed both increased underacylated lipid A species and higher acyl-chain fluidity that correlated with polymyxin B binding. These lipid A changes did not alter LPS cytokine induction, showing that in contrast to other gram-negative pathogens, recognition by innate immune receptors is not decreased by these changes in LPS structure. Transcription of Brucella genes required for incorporating long acyl chains into lipid A (acpXL and lpxXL) or implicated in lipid A acylation control (bacA) was not affected. We propose that in Brucella the outer membrane homeostasis depends on the functioning of BvrS/BvrR. Accordingly, disruption of BvrS/BvrR damages the outer membrane, thus contributing to the severe attenuation manifested by bvrS and bvrR mutants. PMID:16077108

  16. The lipopolysaccharide of Brucella abortus BvrS/BvrR mutants contains lipid A modifications and has higher affinity for bactericidal cationic peptides.

    PubMed

    Manterola, Lorea; Moriyón, Ignacio; Moreno, Edgardo; Sola-Landa, Alberto; Weiss, David S; Koch, Michel H J; Howe, Jörg; Brandenburg, Klaus; López-Goñi, Ignacio

    2005-08-01

    The two-component BvrS/BvrR system is essential for Brucella abortus virulence. It was shown previously that its dysfunction abrogates expression of some major outer membrane proteins and increases bactericidal peptide sensitivity. Here, we report that BvrS/BvrR mutants have increased surface hydrophobicity and susceptibility to killing by nonimmune serum. The bvrS and bvrR mutant lipopolysaccharides (LPSs) bound more polymyxin B, chimeras constructed with bvrS mutant cells and parental LPS showed augmented polymyxin B resistance, and, conversely, parental cells and bvrS mutant LPS chimeras were more sensitive and displayed polymyxin B-characteristic outer membrane lesions, implicating LPS as being responsible for the phenotype of the BvrS/BvrR mutants. No qualitative or quantitative changes were detected in other envelope and outer membrane components examined: periplasmic beta(1-2) glucans, native hapten polysaccharide, and phospholipids. The LPS of the mutants was similar to parental LPS in O-polysaccharide polymerization and fine structure but showed both increased underacylated lipid A species and higher acyl-chain fluidity that correlated with polymyxin B binding. These lipid A changes did not alter LPS cytokine induction, showing that in contrast to other gram-negative pathogens, recognition by innate immune receptors is not decreased by these changes in LPS structure. Transcription of Brucella genes required for incorporating long acyl chains into lipid A (acpXL and lpxXL) or implicated in lipid A acylation control (bacA) was not affected. We propose that in Brucella the outer membrane homeostasis depends on the functioning of BvrS/BvrR. Accordingly, disruption of BvrS/BvrR damages the outer membrane, thus contributing to the severe attenuation manifested by bvrS and bvrR mutants.

  17. 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

    2016-10-01

    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.

  18. Cationic PEGylated liposomes incorporating an antimicrobial peptide tilapia hepcidin 2-3: an adjuvant of epirubicin to overcome multidrug resistance in cervical cancer cells.

    PubMed

    Juang, Vivian; Lee, Hsin-Pin; Lin, Anya Maan-Yuh; Lo, Yu-Li

    Antimicrobial peptides (AMPs) have been recently evaluated as a new generation of adjuvants in cancer chemotherapy. In this study, we designed PEGylated liposomes encapsulating epirubicin as an antineoplastic agent and tilapia hepcidin 2-3, an AMP, as a multidrug resistance (MDR) transporter suppressor and an apoptosis/autophagy modulator in human cervical cancer HeLa cells. Cotreatment of HeLa cells with PEGylated liposomal formulation of epirubicin and hepcidin 2-3 significantly increased the cytotoxicity of epirubicin. The liposomal formulations of epirubicin and/or hepcidin 2-3 were found to noticeably escalate the intracellular H2O2 and O2(-) levels of cancer cells. Furthermore, these treatments considerably reduced the mRNA expressions of MDR protein 1, MDR-associated protein (MRP) 1, and MRP2. The addition of hepcidin 2-3 in liposomes was shown to markedly enhance the intracellular epirubicin uptake and mainly localized into the nucleus. Moreover, this formulation was also found to trigger apoptosis and autophagy in HeLa cells, as validated by significant increases in the expressions of cleaved poly ADP ribose polymerase, caspase-3, caspase-9, and light chain 3 (LC3)-II, as well as a decrease in mitochondrial membrane potential. The apoptosis induction was also confirmed by the rise in sub-G1 phase of cell cycle assay and apoptosis percentage of annexin V/propidium iodide assay. We found that liposomal epirubicin and hepcidin 2-3 augmented the accumulation of GFP-LC3 puncta as amplified by chloroquine, implying the involvement of autophagy. Interestingly, the partial inhibition of necroptosis and the epithelial-mesenchymal transition by this combination was also verified. Altogether, our results provide evidence that coincubation with PEGylated liposomes of hepcidin 2-3 and epirubicin caused programmed cell death in cervical cancer cells through modulation of multiple signaling pathways, including MDR transporters, apoptosis, autophagy, and/or necroptosis

  19. Cationic PEGylated liposomes incorporating an antimicrobial peptide tilapia hepcidin 2–3: an adjuvant of epirubicin to overcome multidrug resistance in cervical cancer cells

    PubMed Central

    Juang, Vivian; Lee, Hsin-Pin; Lin, Anya Maan-Yuh; Lo, Yu-Li

    2016-01-01

    Antimicrobial peptides (AMPs) have been recently evaluated as a new generation of adjuvants in cancer chemotherapy. In this study, we designed PEGylated liposomes encapsulating epirubicin as an antineoplastic agent and tilapia hepcidin 2–3, an AMP, as a multidrug resistance (MDR) transporter suppressor and an apoptosis/autophagy modulator in human cervical cancer HeLa cells. Cotreatment of HeLa cells with PEGylated liposomal formulation of epirubicin and hepcidin 2–3 significantly increased the cytotoxicity of epirubicin. The liposomal formulations of epirubicin and/or hepcidin 2–3 were found to noticeably escalate the intracellular H2O2 and O2− levels of cancer cells. Furthermore, these treatments considerably reduced the mRNA expressions of MDR protein 1, MDR-associated protein (MRP) 1, and MRP2. The addition of hepcidin 2–3 in liposomes was shown to markedly enhance the intracellular epirubicin uptake and mainly localized into the nucleus. Moreover, this formulation was also found to trigger apoptosis and autophagy in HeLa cells, as validated by significant increases in the expressions of cleaved poly ADP ribose polymerase, caspase-3, caspase-9, and light chain 3 (LC3)-II, as well as a decrease in mitochondrial membrane potential. The apoptosis induction was also confirmed by the rise in sub-G1 phase of cell cycle assay and apoptosis percentage of annexin V/propidium iodide assay. We found that liposomal epirubicin and hepcidin 2–3 augmented the accumulation of GFP-LC3 puncta as amplified by chloroquine, implying the involvement of autophagy. Interestingly, the partial inhibition of necroptosis and the epithelial–mesenchymal transition by this combination was also verified. Altogether, our results provide evidence that coincubation with PEGylated liposomes of hepcidin 2–3 and epirubicin caused programmed cell death in cervical cancer cells through modulation of multiple signaling pathways, including MDR transporters, apoptosis, autophagy, and

  20. Biological activities of lipopolysaccharides of Proteus spp. and their interactions with polymyxin B and an 18-kDa cationic antimicrobial protein (CAP18)-derived peptide.

    PubMed

    St Swierzko, A; Kirikae, T; Kirikae, F; Hirata, M; Cedzynski, M; Ziolkowski, A; Hirai, Y; Kusumoto, S; Yokochi, T; Nakano, M

    2000-02-01

    The saccharide constituents of lipopolysaccharides (LPS) of Proteus spp. vary with the strain and contain unique components about which little is known. The biological activities of LPS and lipid A from S- and R-forms of 10 Proteus strains were examined. LPS from all S-form Proteus strains was lethal to D-(+)-galactosamine (GalN)-loaded, LPS-responsive, C3H/HeN mice, but not to LPS-hypo-responsive C3H/HeJ mice. P. vulgaris 025 LPS evoked strong anaphylactoid reactions in N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP)-primed C3H/HeJ mice. LPS from S- and R-form Proteus strains induced production of nitric oxide (NO) and tumour necrosis factor (TNF) by macrophages isolated from C3H/HeN but not C3H/HeJ mice. Lipid A from Proteus strains also induced NO and TNF production, although lipid A was less potent than LPS. The effects of LPS were mainly dependent on CD14; LPS-induced NO and TNF production in CD14+ J774.1 cells was significantly greater than in CD14-J7.DEF.3 cells. All LPS from Proteus strains, and especially from P. vulgaris 025, exhibited higher anti-complementary activity than LPS from Escherichia coli or Pseudomonas aeruginosa. Polymyxin B inactivated proteus LPS in a dose-dependent manner, but these LPS preparations were more resistant to polymyxin B than E. coli LPS. CAP18(109-135), a granulocyte-derived peptide, inhibited proteus LPS endotoxicity only when the LPS:CAP18(109-135) ratio was appropriate, which suggests that CAP18(109-135) acts through a different mechanism than polymyxin B. The results indicate that LPS from Proteus spp. are potently endotoxic, but that the toxicity is different from that of LPS from E. coli or Salmonella spp. and even varies among different Proteus strains. The variation in biological activities among proteus LPS may be due to unique components within the respective LPS.

  1. [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

  2. 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

  3. 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.

  4. Chlamydia trachomatis tarp is phosphorylated by src family tyrosine kinases.

    PubMed

    Jewett, Travis J; Dooley, Cheryl A; Mead, David J; Hackstadt, Ted

    2008-06-27

    The translocated actin recruiting phosphoprotein (Tarp) is injected into the cytosol shortly after Chlamydia trachomatis attachment to a target cell and subsequently phosphorylated by an unidentified tyrosine kinase. A role for Tarp phosphorylation in bacterial entry is unknown. In this study, recombinant C. trachomatis Tarp was employed to identify the host cell kinase(s) required for phosphorylation. Each tyrosine rich repeat of L2 Tarp harbors a sequence similar to a Src and Abl kinase consensus target. Furthermore, purified p60-src, Yes, Fyn, and Abl kinases were able to phosphorylate Tarp. Mutagenesis of potential tyrosines within a single tyrosine rich repeat peptide indicated that both Src and Abl kinases phosphorylate the same residues suggesting that C. trachomatis Tarp may serve as a substrate for multiple host cell kinases. Surprisingly, chemical inhibition of Src and Abl kinases prevented Tarp phosphorylation in culture and had no measurable effect on bacterial entry into host cells.

  5. [Phosphorylation of tau protein].

    PubMed

    Uchida, T; Ishiguro, K

    1990-05-01

    In aged human brain and particularly in Alzheimer's disease brain, paired helical filaments (PHFs) accumulate in the neuronal cell. Recently, it has been found that the highly phosphorylated tau protein, one of the microtubule-associated proteins (MAPs), is a component of PHF. The authors attempted to clarify the mechanism underlying the accumulation of PHF from the following two aspects; 1) What is the mechanism of phosphorylation of tau protein? 2) Is the highly phosphorylated tau protein capable of forming PHFs? From rat or bovine microtubule proteins we partially purified and characterized a novel protein kinase that specifically phosphorylated tau and MAP2 among many proteins in the brain extract, and which formed a PHF epitope on the phosphorylated human tau. This enzyme was one of the protein serine/threonine kinases and was independent of known second messengers. The phosphorylation of tau by this enzyme was stimulated by tubulin under the condition of microtubule formation, suggesting that the phosphorylation of tau could occur concomitantly with microtubule formation in the brain. Since this kinase was usually bound to tau but not directly to tubulin, the enzyme was associated with microtubules through tau. From these properties related to tau, this kinase is designated as tau protein kinase. The tau that been phosphorylated with this kinase using [gamma-32P]ATP as a phosphate donor, was digested by endoprotinase Lys-C to produce three labeled fragments, K1, K2 and K3. These three fragments were sequenced and the phosphorylation sites on tau by this kinase were identified. The K2 fragment overlapped with the tau-1 site known to be one of the phosphorylation site in PHF. This result strengthens the possibility that tau protein phosphorylated by tau protein kinase is incorporated into PHF. Tubulin binding sites on tau were located between K1 and K3 fragments, while K2 fragment was located in the neighboring to N-terminus of K1. No phosphorylated sites were

  6. Focused fluorescent probe library for metal cations and biological anions.

    PubMed

    Rhee, Hyun-Woo; Lee, Sang Wook; Lee, Jun-Seok; Chang, Young-Tae; Hong, Jong-In

    2013-09-09

    A focused fluorescent probe library for metal cations was developed by combining metal chelators and picolinium/quinolinium moieties as combinatorial blocks connected through a styryl group. Furthermore, metal complexes derived from metal chelators having high binding affinities for metal cations were used to construct a focused probe library for phosphorylated biomolecules. More than 250 fluorescent probes were screened for identifying an ultraselective probe for dTTP.

  7. Substrate phosphorylation affects degradation and interaction to endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme.

    PubMed

    Machado, M F M; Cunha, F M; Berti, D A; Heimann, A S; Klitzke, C F; Rioli, V; Oliveira, V; Ferro, E S

    2006-01-13

    Recent findings from our laboratory suggest that intracellular peptides containing putative post-translational modification sites (i.e., phosphorylation) could regulate specific protein interactions. Here, we extend our previous observations showing that peptide phosphorylation changes the kinetic parameters of structurally related endopeptidase EP24.15 (EC 3.4.24.15), neurolysin (EC 3.4.24.16), and angiotensin-converting enzyme (EC 3.4.15.1). Phosphorylation of peptides that are degraded by these enzymes leads to reduced degradation, whereas phosphorylation of peptides that interacted as competitive inhibitors of these enzymes alters only the K(i)'s. These data suggest that substrate phosphorylation could be one of the mechanisms whereby some intracellular peptides would escape degradation and could be regulating protein interactions within cells.

  8. 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.

  9. 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

  10. 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

    2016-07-28

    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.

  11. Mass spectrometric phosphoproteome analysis of HIV-infected brain reveals novel phosphorylation sites and differential phosphorylation patterns

    PubMed Central

    Uzasci, Lerna; Auh, Sungyoung; Cotter, Robert J.; Nath, Avindra

    2016-01-01

    Purpose To map the phosphoproteome and identify changes in the phosphorylation patterns in the HIV-infected and uninfected brain using high-resolution mass spectrometry. Experimental Design Parietal cortex from brain of individuals with and without HIV infection were lysed and trypsinized. The peptides were labeled with iTRAQ reagents, combined, phospho-enriched by titanium dioxide chromatography, and analyzed by LC-MS/MS with high-resolution. Results Our phosphoproteomic workflow resulted in the identification of 112 phosphorylated proteins and 17 novel phosphorylation sites in all the samples that were analyzed. The phosphopeptide sequences were searched for kinase substrate motifs which revealed potential kinases involved in important signaling pathways. The site-specific phosphopeptide quantification showed that peptides from neurofilament medium polypeptide, myelin basic protein, and 2′–3′-cyclic nucleotide-3′ phosphodiesterase have relatively higher phosphorylation levels during HIV infection. Clinical Relevance This study has enriched the global phosphoproteome knowledge of the human brain by detecting novel phosphorylation sites on neuronal proteins and identifying differentially phosphorylated brain proteins during HIV infection. Kinases that lead to unusual phosphorylations could be therapeutic targets for the treatment of HIV-associated neurocognitive disorders (HAND). PMID:26033855

  12. 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.

  13. Altered localization and cytoplasmic domain-binding properties of tyrosine-phosphorylated beta 1 integrin

    PubMed Central

    1994-01-01

    We describe a novel approach to study tyrosine-phosphorylated (PY) integrins in cells transformed by virally encoded tyrosine kinases. We have synthesized a peptide (PY beta 1 peptide) that represents a portion of the cytoplasmic domain of the beta 1 integrin subunit and is phosphorylated on the tyrosine residue known to be the target of oncogenic tyrosine kinases. Antibodies prepared against the PY beta 1 peptide, after removal of cross-reacting antibodies by absorption and affinity purification, recognized the PY beta 1 peptide and the tyrosine-phosphorylated form of the intact beta 1 subunit, but did not bind the nonphosphorylated beta 1 peptide, the nonphosphorylated beta 1 subunit or other unrelated tyrosine-phosphorylated proteins. The anti- PY beta 1 antibodies labeled the podosomes of Rous sarcoma virus- transformed fibroblasts, but did not detectably stain nontransformed fibroblasts. The localization of the tyrosine phosphorylated beta 1 subunits appeared distinct from that of the beta 1 subunit. Adhesion plaques were stained by the anti-beta 1 subunit antibodies in Rous sarcoma virus-transformed fibroblasts plated on fibronectin, whereas neither podosomes nor adhesion plaques were labeled on vitronectin or on uncoated plates. Anti-phosphotyrosine antibodies labeled podosomes, adhesion plaques and cell-cell boundaries regardless of the substratum. One of the SH2 domains of the p85 subunit of phosphatidylinositol-3- kinase bound to the PY beta 1 peptide, but not to the non- phosphorylated beta 1 cytoplasmic peptide. Other SH2 domains did not bind to the PY beta 1 peptide. These results show that the phosphorylated form of the beta 1 integrin subunit is detected in a different subcellular localization than the nonphosphorylated form and suggest that the phosphorylation on tyrosine of the beta 1 subunit cytoplasmic domain may affect cellular signaling pathways. PMID:7520449

  14. Phosphorylation: Implications in Cancer.

    PubMed

    Singh, Vishakha; Ram, Mahendra; Kumar, Rajesh; Prasad, Raju; Roy, Birendra Kumar; Singh, Kaushal Kumar

    2017-02-01

    Post translational modifications (PTMs) are involved in variety of cellular activities and phosphorylation is one of the most extensively studied PTM, which regulates a number of cellular functions like cell growth, differentiation, apoptosis and cell signaling in healthy condition. However, alterations in phosphorylation pathways result in serious outcomes in the form of diseases, especially cancer. Many signalling pathways including Tyrosine kinase, MAP kinase, Cadherin-catenin complex, Cyclin-dependent kinase etc. are major players of the cell cycle and deregulation in their phosphorylation-dephosphorylation cascade has been shown to be manifested in the form of various types of cancers. Tyrosine kinase family encompasses the greatest number of oncoproteins. MAPK cascade has an importance role in cancer growth and progression. Bcl-2 family proteins serve either proapoptotic or antiapoptotic function. Cadherin-catenin complex regulates cell adhesion properties and cyclins are the key regulators of cell cycle. Altered phosphorylations in any of the above pathways are strongly associated with cancer, at the same time they serve as the potential tergets for drug development against cancer. Drugs targeting tyrosine kinase are potent anticancer drugs. Inhibitors of MEK, PI3K and ERK signalling pathways are undergoing clinical trials. Thus, drugs targeting phosphorylation pathways represent a promising area for cancer therapy.

  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.

  16. 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.

  17. Comprehensive Analysis of Phosphorylated Proteins of E. coli Ribosomes

    PubMed Central

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

    2009-01-01

    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 twenty-four E. coli ribosomal proteins by tandem mass spectrometry. Specific 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 the 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, 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 site of the phosphorylation in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins. PMID:19469554

  18. Phosphorylation sites in human erythrocyte band 3 protein.

    PubMed

    Yannoukakos, D; Vasseur, C; Piau, J P; Wajcman, H; Bursaux, E

    1991-01-30

    The human red cell anion-exchanger, band 3 protein, is one of the main phosphorylated proteins of the erythrocyte membrane. Previous studies from this laboratory have shown that ATP-depletion of the red blood cell decreased the anion-exchange rate, suggesting that band 3 protein phosphorylation could be involved in the regulation of anion transport function (Bursaux et al. (1984) Biochim. Biophys. Acta 777, 253-260). Phosphorylation occurs mainly on the cytoplasmic domain of the protein and the major site of phosphorylation was assigned to tyrosine-8 (Dekowski et al. (1983) J. Biol. Chem. 258, 2750-2753). This site being very far from the integral, anion-exchanger domain, the aim of the present study was to determine whether phosphorylation sites exist in the integral domain. The phosphorylation reaction was carried out on isolated membranes in the presence of [gamma-32P]ATP and phosphorylated band 3 protein was then isolated. Both the cytoplasmic and the membrane spanning domains were purified. The predominant phosphorylation sites were found on the cytoplasmic domain. RP-HPLC analyses of the tryptic peptides of whole band 3 protein, and of the isolated cytoplasmic and membrane-spanning domains allowed for the precise localization of the phosphorylated residues. 80% of the label was found in the N-terminal tryptic peptide (T-1), (residues 1-56). In this region, all the residues susceptible to phosphorylation were labeled but in varying proportion. Under our conditions, the most active membrane kinase was a tyrosine kinase, activated preferentially by Mn2+ but also by Mg2+. Tyrosine-8 was the main phosphate acceptor residue (50-70%) of the protein, tyrosine-21 and tyrosine-46 residues were also phosphorylated but to a much lesser extent. The main targets of membrane casein kinase, preferentially activated by Mg2+, were serine-29, serine-50, and threonine(s)-39, -42, -44, -48, -49, -54 residue(s) located in the T-1 peptide. A tyrosine phosphatase activity was

  19. Phosphorylation regulates mycobacterial proteasome.

    PubMed

    Anandan, Tripti; Han, Jaeil; Baun, Heather; Nyayapathy, Seeta; Brown, Jacob T; Dial, Rebekah L; Moltalvo, Juan A; Kim, Min-Seon; Yang, Seung Hwan; Ronning, Donald R; Husson, Robert N; Suh, Joowon; Kang, Choong-Min

    2014-09-01

    Mycobacterium tuberculosis possesses a proteasome system that is required for the microbe to resist elimination by the host immune system. Despite the importance of the proteasome in the pathogenesis of tuberculosis, the molecular mechanisms by which proteasome activity is controlled remain largely unknown. Here, we demonstrate that the α-subunit (PrcA) of the M. tuberculosis proteasome is phosphorylated by the PknB kinase at three threonine residues (T84, T202, and T178) in a sequential manner. Furthermore, the proteasome with phosphorylated PrcA enhances the degradation of Ino1, a known proteasomal substrate, suggesting that PknB regulates the proteolytic activity of the proteasome. Previous studies showed that depletion of the proteasome and the proteasome-associated proteins decreases resistance to reactive nitrogen intermediates (RNIs) but increases resistance to hydrogen peroxide (H2O2). Here we show that PknA phosphorylation of unprocessed proteasome β-subunit (pre-PrcB) and α-subunit reduces the assembly of the proteasome complex and thereby enhances the mycobacterial resistance to H2O2 and that H2O2 stress diminishes the formation of the proteasome complex in a PknA-dependent manner. These findings indicate that phosphorylation of the M. tuberculosis proteasome not only modulates proteolytic activity of the proteasome, but also affects the proteasome complex formation contributing to the survival of M. tuberculosis under oxidative stress conditions.

  20. 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.

  1. Protein phosphorylation and photorespiration.

    PubMed

    Hodges, M; Jossier, M; Boex-Fontvieille, E; Tcherkez, G

    2013-07-01

    Photorespiration allows the recycling of carbon atoms of 2-phosphoglycolate produced by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenase activity, as well as the removal of potentially toxic metabolites. The photorespiratory pathway takes place in the light, encompasses four cellular compartments and interacts with several other metabolic pathways and functions. Therefore, the regulation of this cycle is probably of paramount importance to plant metabolism, however, our current knowledge is poor. To rapidly respond to changing conditions, proteins undergo a number of different post-translational modifications that include acetylation, methylation and ubiquitylation, but protein phosphorylation is probably the most common. The reversible covalent addition of a phosphate group to a specific amino acid residue allows the modulation of protein function, such as activity, subcellular localisation, capacity to interact with other proteins and stability. Recent data indicate that many photorespiratory enzymes can be phosphorylated, and thus it seems that the photorespiratory cycle is, in part, regulated by protein phosphorylation. In this review, the known phosphorylation sites of each Arabidopsis thaliana photorespiratory enzyme and several photorespiratory-associated proteins are described and discussed. A brief account of phosphoproteomic protocols is also given since the published data compiled in this review are the fruit of this approach.

  2. Phosphorylation of erythrocyte membrane liberates calcium

    SciTech Connect

    Chauhan, V.P.S.; Brockerhoff, H.

    1986-05-01

    Phosphorylation of permeabilized erythrocyte ghost membranes with ATP results in an increase free calcium level as measured with the help of Ca/sup 2 +/ electrode and /sup 45/Ca. This effect could not be observed in the presence of p/sup -/ chloromercuric benzoate, an inhibitor of kinases. The rise in the free calcium due to phosphorylation of the membrane was accompanied by a decrease in the level of phosphatidylinositol (PI) and an increase in phosphatidylinositolmonophosphate (PIP) and phosphatidylinositolbisphosphate (PIP/sub 2/). These results support the proposal that an inositol shuttle, PI in equilibrium PIP in equilibrium PIP/sub 2/, operates to maintain the intracellular calcium concentration. The cation is believed to be sequestered in a cage formed by the head groups of two acidic phospholipid molecules, e.g., phosphatidylserine and phosphatidylinositol, with the participation of both PO and fatty acid ester CO groups. When the inositol group of such a cage is phosphorylated, inter-headgroup hydrogen bonding between the lipids is broken. As a result the cage opens and calcium is released.

  3. 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.

  4. Identification of a phosphorylation site in the hinge region of the human progesterone receptor and additional amino-terminal phosphorylation sites.

    PubMed

    Knotts, T A; Orkiszewski, R S; Cook, R G; Edwards, D P; Weigel, N L

    2001-03-16

    We have previously reported the identification of seven in vivo phosphorylation sites in the amino-terminal region of the human progesterone receptor (PR). From our previous in vivo studies, it was evident that several phosphopeptides remained unidentified. In particular, we wished to determine whether human PR contains a phosphorylation site in the hinge region, as do other steroid receptors including chicken PR, human androgen receptor, and mouse estrogen receptor. Previously, problematic trypsin cleavage sites hampered our ability to detect phosphorylation sites in large incomplete tryptic peptides. Using a combination of mass spectrometry and in vitro phosphorylation, we have identified six previously unidentified phosphorylation sites in human PR. Using nanoelectrospray ionization mass spectrometry, we have identified two new in vivo phosphorylation sites, Ser(20) and Ser(676), in baculovirus-expressed human PR. Ser(676) is analogous to the hinge site identified in other steroid receptors. Additionally, precursor ion scans identified another phosphopeptide that contains Ser(130)-Pro(131), a likely candidate for phosphorylation. In vitro phosphorylation of PR with Cdk2 has revealed five additional in vitro Cdk2 phosphorylation sites: Ser(25), Ser(213), Thr(430), Ser(554), and Ser(676). At least two of these, Ser(213) and Ser(676), are authentic in vivo sites. We confirmed the presence of the Cdk2-phosphorylated peptide containing Ser(213) in PR from in vivo labeled T47D cells, indicating that this is an in vivo site. Our combined studies indicate that most, if not all, of the Ser-Pro motifs in human PR are sites for phosphorylation. Taken together, these data indicate that the phosphorylation of PR is highly complex, with at least 14 phosphorylation sites.

  5. 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.

  6. Uniformly cationized protein efficiently reaches the cytosol of mammalian cells.

    PubMed

    Futami, Midori; Watanabe, Yasuyoshi; Asama, Takashi; Murata, Hitoshi; Tada, Hiroko; Kosaka, Megumi; Yamada, Hidenori; Futami, Junichiro

    2012-10-17

    Protein cationization techniques are powerful protein transduction methods for mammalian cells. As we demonstrated previously, cationized proteins with limited conjugation to polyethylenimine have excellent ability to enter into cells by adsorption-mediated endocytosis [Futami, J., et al. (2005) J. Biosci. Bioeng. 99, 95-103]. In this study, we show that proteins with extensive and uniform cationization covering the protein surface reach the cytoplasm and nucleus more effectively than proteins with limited cationic polymers or proteins that are fused to cationic peptides. Although extensive modification of carboxylates results in loss of protein function, chicken avidin retains biotin-binding ability even after extensive amidation of carboxylates. Using this cationized avidin carrier system, the protein transduction ability of variously cationized avidins was investigated using biotinylated protein as a probe. The results revealed that cationized avidins bind rapidly to the cell surface followed by endocytotic uptake. Small amounts of uniformly cationized avidin showed direct penetration into the cytoplasm within a 15 min incubation. This penetration route seemed to be energy dependent and functioned under cellular physiological conditions. A biotinylated exogenous transcription factor protein that penetrated cells was demonstrated to induce target gene expression in living cells.

  7. A Proteomic Approach to Identify Phosphorylation-Dependent Targets of BRCT Domains

    DTIC Science & Technology

    2007-03-01

    AD_________________ Award Number: W81XWH-05-1-0233 TITLE: A Proteomic Approach to...COVERED (From - To) 1 Mar 2006 – 28 Feb 2007 5a. CONTRACT NUMBER A Proteomic Approach to Identify Phosphorylation-Dependent Targets of BRCT...this award) BRCT domain, peptide library, OPAL, peptide array, proteomics , genome wide, signal transduction pathways, androgen receptor 16. SECURITY

  8. Regulation of phenylalanine hydroxylase: conformational changes upon phosphorylation detected by H/D exchange and mass spectrometry.

    PubMed

    Li, Jun; Fitzpatrick, Paul F

    2013-07-15

    The enzyme phenylalanine hydroxylase catalyzes the hydroxylation of excess phenylalanine in the liver to tyrosine. The enzyme is regulated allosterically by phenylalanine and by phosphorylation of Ser16. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into any structural change upon phosphorylation. Peptides in both the catalytic and regulatory domains show increased deuterium incorporation into the phosphorylated protein. Deuterium is incorporated into fewer peptides than when the enzyme is activated by phenylalanine, and the incorporation is slower. This establishes that the conformational change upon phosphorylation of phenylalanine hydroxylase is different from and less extensive than that upon phenylalanine activation.

  9. Click conjugation of a binuclear terbium(III) complex for real-time detection of tyrosine phosphorylation.

    PubMed

    Akiba, Hiroki; Sumaoka, Jun; Tsumoto, Kouhei; Komiyama, Makoto

    2015-04-07

    Phosphorylation of proteins is closely associated with various diseases, and, therefore, its detection is vitally important in molecular biology and drug discovery. Previously, we developed a binuclear Tb(III) complex, which emits notable luminescence only in the presence of phosphotyrosine. In this study, we conjugated a newly synthesized binuclear Tb(III) complex to substrate peptides by using click chemistry. Using these conjugates, we were able to detect tyrosine phosphorylation in real time. These conjugates were superior to nonconjugated Tb(III) complexes for the detection of tyrosine phosphorylation, especially when the substrate peptides used were positively charged. Luminescence intensity upon phosphorylation was enhanced 10-fold, making the luminescence intensity of this system one of the largest among lanthanide luminescence-based systems. We also determined Michaelis-Menten parameters for the phosphorylation of various kinase/peptide combinations and quantitatively analyzed the effects of mutations in the peptide substrates. Furthermore, we successfully monitored the inhibition of enzymatic phosphorylation by inhibitors in real time. Advantageously, this system detects only the phosphorylation of tyrosine (phosphorylated serine and threonine are virtually silent) and is applicable to versatile peptide substrates. Our study thus demonstrates the applicability of this system for the analysis of kinase activity, which could lead to drug discovery.

  10. 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.

  11. Peptide mass fingerprinting.

    PubMed

    Thiede, Bernd; Höhenwarter, Wolfgang; Krah, Alexander; Mattow, Jens; Schmid, Monika; Schmidt, Frank; Jungblut, Peter R

    2005-03-01

    Peptide mass fingerprinting by MALDI-MS and sequencing by tandem mass spectrometry have evolved into the major methods for identification of proteins following separation by two-dimensional gel electrophoresis, SDS-PAGE or liquid chromatography. One main technological goal of proteome analyses beside high sensitivity and automation was the comprehensive analysis of proteins. Therefore, the protein species level with the essential information on co- and post-translational modifications must be achieved. The power of peptide mass fingerprinting for protein identification was described here, as exemplified by the identification of protein species with high molecular masses (spectrin alpha and beta), low molecular masses (elongation factor EF-TU fragments), splice variants (alpha A crystallin), aggregates with disulfide bridges (alkylhydroperoxide reductase), and phosphorylated proteins (heat shock protein 27). Helpful tools for these analyses were the use of the minimal protein identifier concept and the software program MS-Screener to remove mass peaks assignable to contaminants and neighbor spots.

  12. 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

  13. 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

  14. Novel imidazolium salt--peptide conjugates and their antimicrobial activity.

    PubMed

    Reinhardt, A; Horn, M; Schmauck, J Pieper Gen; Bröhl, A; Giernoth, R; Oelkrug, C; Schubert, A; Neundorf, I

    2014-12-17

    Our study presents innovative research dealing with the synthesis and biological evaluation of conjugates out of antimicrobial peptides (AMPs) and imidazolium cations that are derived from ionic liquids. AMPs are considered as promising alternatives to common antibiotics due to their different activity mechanisms. Antibacterial effects have also been described for ionic liquids bearing imidazolium cations . Besides single coupling of carboxy-functionalized imidazolium cations to the peptide N-terminal we also developed conjugates bearing multiple copies of imidazolium cations. The combination of both compounds resulted in synergistic effects that were most pronounced when more imidazolium cations were attached to the peptides. In addition, antibacterial activity even in drug-resistant bacterial strains could be observed. Moreover, the novel compounds showed good selectivity only against bacterial cells, an observation that was further proven by lipid interaction studies using giant unilamellar vesicles.

  15. 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.

  16. 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

    SciTech Connect

    Smith, Jeremy C; Topham, Christopher

    2007-01-01

    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 base pair 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 PNADNA-PNA triplex formation. Quantum chemical modeling methods are used to propose candidate PNA Hoogsteen strand designs.

  17. Determination of GPCR Phosphorylation Status: Establishing a Phosphorylation Barcode.

    PubMed

    Prihandoko, Rudi; Bradley, Sophie J; Tobin, Andrew B; Butcher, Adrian J

    2015-06-01

    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.

  18. 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.

  19. 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.

  20. Phosphorylation by casein kinase II affects the interaction of caldesmon with smooth muscle myosin and tropomyosin.

    PubMed Central

    Bogatcheva, N V; Vorotnikov, A V; Birukov, K G; Shirinsky, V P; Gusev, N B

    1993-01-01

    Smooth muscle caldesmon was phosphorylated by casein kinase II, and the effects of phosphorylation on the interaction of caldesmon and its chymotryptic peptides with myosin and tropomyosin were investigated. The N-terminal chymotryptic peptide of caldesmon of molecular mass 27 kDa interacted with myosin. Phosphorylation of Ser-73 catalysed by casein kinase II resulted in a 2-fold decrease in the affinity of the native caldesmon (or its 27 kDa N-terminal peptide) for smooth muscle myosin. At low ionic strength, caldesmon and its N-terminal peptides of molecular masses 25 and 27 kDa were retarded on a column of immobilized tropomyosin. Phosphorylation of Ser-73 led to a 2-4-fold decrease in the affinity of caldesmon (or its N-terminal peptides) for tropomyosin. Thus phosphorylation of Ser-73 catalysed by casein kinase II affects the interaction of caldesmon with both smooth muscle myosin and tropomyosin. Images Figure 1 Figure 2 Figure 3 PMID:8452532

  1. Hypo-phosphorylation of salivary peptidome as a clue to the molecular pathogenesis of autism spectrum disorders.

    PubMed

    Castagnola, Massimo; Messana, Irene; Inzitari, Rosanna; Fanali, Chiara; Cabras, Tiziana; Morelli, Alessandra; Pecoraro, Anna Maria; Neri, Giovanni; Torrioli, Maria Giulia; Gurrieri, Fiorella

    2008-12-01

    RP-HPLC-ESI-MS profile of naturally occurring salivary peptides of subjects with autistic spectrum disorder [ASD; N = 27:12 with diagnosis of autism, 1 with diagnosis of Asperger, 14 with diagnosis of pervasive developmental disorders not otherwise specified (PDD-NOS)] was compared to that of age-matched controls with the goal of identifying differences that could turn out to become hallmarks of at least a subgroup of ASD individuals. Phosphorylation level of four specific salivary phospho-peptides, namely statherin, histatin 1 (both, p < 0.0001) and acidic proline-rich proteins (both entire and truncated isoforms) (p < 0.005) was found significantly lower in autistic patients, with hypo-phosphorylation of at least one peptide observed in 18 ASD subjects (66%). Developmental scale assessment (Griffith or WISC-R) carried out on 14 ASD subjects highlighted a normal to borderline cognitive development in 10 of them, all included in the hypo-phosphorylated group. Phosphorylation of salivary peptides involves a Golgi casein kinase common to many organs and tissues, CNS included, whose expression seems to be synchronized during fetal development. Hypo-phosphorylation of salivary peptides suggests potential asynchronies in the phosphorylation of other secretory proteins, which could be relevant in CNS development either during embryonic development or in early infancy. These results suggest that analysis of salivary phospho-peptides might help to discriminate a considerable subgroup of ASD patients.

  2. 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

  3. 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.

  4. Protein tyrosine phosphorylation in streptomycetes.

    PubMed

    Waters, B; Vujaklija, D; Gold, M R; Davies, J

    1994-07-01

    Using phosphotyrosine-specific antibodies, we demonstrate that in several Streptomyces spp. a variety of proteins are phosphorylated on tyrosine residues. Tyrosine phosphorylation was found in a number of Streptomyces species including Streptomyces lividans, Streptomyces hygroscopicus and Streptomyces lavendulae. Each species exhibited a unique pattern of protein tyrosine phosphorylation. Moreover, the patterns of tyrosine phosphorylation varied during the growth phase and were also influenced by culture conditions. We suggest that metabolic shifts during the complex growth cycle of these filamentous bacteria, and possibly secondary metabolic pathways, may be controlled by the action of protein tyrosine kinases and phosphatases, as has been demonstrated in signal transduction pathways in eukaryotic organisms.

  5. Selective disulfide bond cleavage in gold(I) cationized polypeptide ions formed via gas-phase ion/ion cation switching.

    PubMed

    Gunawardena, Harsha P; O'Hair, Richard A J; McLuckey, Scott A

    2006-09-01

    Gaseous multiply protonated disulfide-linked peptides have been subjected to reactions with AuCl2(-) ions to explore the possibility of effecting cation switching of Au+ for two protons and to determine whether cationization by Au+ ions affords selective dissociation of disulfide linkages. The incorporation of Au+ into several model disulfide-linked peptides proved to be straightforward. The primary ion/ion reaction channels were proton transfer, which does not lead to Au+ incorporation, and attachment of AuCl2(-) ions to the polypeptide cation, which does incorporate Au+. Fragmentation of the attachment product, the extent of which varied with peptide and charge state, led to losses of one or more molecules of HCl and, to some extent, cleavage of polypeptides at the disulfide linkage into its two constituent chains. Collisional activation of the intact metal-ion-incorporated peptides showed cleavage of the disulfide linkage to be a major, and in some cases exclusive, process. Cations with protons as the only cationizing agents showed only small contributions from cleavage of the disulfide linkage. These results indicate that Au+ incorporation into a disulfide-linked polypeptide ion is a promising way to effect selective dissociation of disulfide bonds. Cation switching via ion/ion reactions is a convenient means for incorporating gold and is attractive because it avoids the requirement of adding metal salts to the analyte solution.

  6. Phosphorylation of mammalian initiation factor eIF-4B

    SciTech Connect

    Duncan, R.F.; Milburn, S.C.; Cooper, R.; Gould, K.; Hunter, T.; Hershey, J.W.B.

    1987-05-01

    The phosphorylation of initiation factors appears to be an important mechanism for regulating the rate of translation in mammalian cells. eIF-4B (80 kDa) purified from HeLa cells exhibits a complex array of 8 to 12 spots when analyzed by 2-dimensional isoelectric focusing - SDS polyacrylamide gel electrophoresis. A similar array of eIF-4B spots is seen when total lysate proteins are analyzed by immunoblotting with anti-eIF-4B antiserum or with antibodies affinity-purified from the most basic eIF-4B spot. The multiple forms of eIF-4B are due to phosphorylation, since all but the most basic spot are labeled with (/sup 32/P)phosphate in vivo and the action of alkaline phosphatase in vitro reduces the array to only two spots. Tryptic peptide maps of phosphopeptides from each of the various isoelectric variants of eIF-4B show a similar complexity, suggesting that a number of different sites are phosphorylated in a random order. When serum-deprived HeLa cells are treated with phorbol ester, both the protein synthesis rate and the extent of eIF-4B phosphorylation increase, suggesting that C kinase may be a regulator of translation. Purified C kinase phosphorylates a number of pure initiation factors in vitro, but eIF-4B is the strongest target protein. When pure eIF-4B is treated, the entire mass of eIF-4B is shifted to the most acidic spots, indicating very strong phosphorylation. Attempts are being made to detect differences in the in vitro activities of the non-phosphorylated and highly phosphorylated forms.

  7. Hydrogen-deuterium exchange in imidazole as a tool for studying histidine phosphorylation.

    PubMed

    Cebo, Małgorzata; Kielmas, Martyna; Adamczyk, Justyna; Cebrat, Marek; Szewczuk, Zbigniew; Stefanowicz, Piotr

    2014-12-01

    Isotope exchange at the histidine C2 atom of imidazole in D2O solution is well known to occur at a significantly slower rate than the exchange of amide protons. Analysis of the kinetics of this isotope-exchange reaction is proposed herein as a method of detecting histidine phosphorylation. This modification of His-containing peptides is challenging to pinpoint because of its instability under acidic conditions as well as during CID-MS analysis. In this work, we investigated the effect of phosphorylation of the histidine side chain in peptides on deuterium-hydrogen exchange (DHX) in the imidazole. The results demonstrate that phosphorylation dramatically slows the rate of the DHX reaction. This phenomenon can be applied to detect phosphorylation of peptides at the histidine residue (e.g., in enzymatic digests). We also found that the influence of the peptide sequence on the exchange kinetics is relatively small. A CID fragmentation experiment revealed that there was no detectable hydrogen scrambling in peptides deuterated at C2 of the imidazole ring. Therefore, MS/MS can be used to directly identify the locations of deuterium ions incorporated into peptides containing multiple histidine moieties.

  8. Lipopolysaccharide Neutralization by Cationic-Amphiphilic Polymers through Pseudoaggregate Formation.

    PubMed

    Uppu, Divakara S S M; Haldar, Jayanta

    2016-03-14

    Synthetic polymers incorporating the cationic charge and hydrophobicity to mimic the function of antimicrobial peptides (AMPs) have been developed. These cationic-amphiphilic polymers bind to bacterial membranes that generally contain negatively charged phospholipids and cause membrane disintegration resulting in cell death; however, cationic-amphiphilic antibacterial polymers with endotoxin neutralization properties, to the best of our knowledge, have not been reported. Bacterial endotoxins such as lipopolysaccharide (LPS) cause sepsis that is responsible for a great amount of mortality worldwide. These cationic-amphiphilic polymers can also bind to negatively charged and hydrophobic LPS and cause detoxification. Hence, we envisaged that cationic-amphiphilic polymers can have both antibacterial as well as LPS binding properties. Here we report synthetic amphiphilic polymers with both antibacterial as well as endotoxin neutralizing properties. Levels of proinflammatory cytokines in human monocytes caused by LPS stimulation were inhibited by >80% when coincubated with these polymers. These reductions were found to be dependent on concentration and, more importantly, on the side-chain chemical structure due to variations in the hydrophobicity profiles of these polymers. These cationic-amphiphilic polymers bind and cause LPS neutralization and detoxification. Investigations of polymer interaction with LPS using fluorescence spectroscopy and dynamic light scattering (DLS) showed that these polymers bind but neither dissociate nor promote LPS aggregation. We show that polymer binding to LPS leads to sort of a pseudoaggregate formation resulting in LPS neutralization/detoxification. These findings provide an unusual mechanism of LPS neutralization using novel synthetic cationic-amphiphilic polymers.

  9. 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...

  10. An Extensive Survey of Tyrosine Phosphorylation Revealing New Sites in Human Mammary Epithelial Cells

    PubMed Central

    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. Steven; Qian, Wei-Jun

    2010-01-01

    Protein tyrosine phosphorylation represents a central regulatory mechanism in cell signaling. Here we present an extensive survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell line by applying anti-phosphotyrosine peptide immunoaffinity purification coupled with high sensitivity capillary liquid chromatography tandem mass spectrometry. 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 acute stimulation with epidermal growth factor (EGF). The estimated false discovery rate was 1.0% as determined by searching against a scrambled database. Comparison of these data with existing literature showed significant agreement for previously reported sites. However, we observed 281 sites that were not previously reported for HMEC cultures and 29 of which have not been reported for any human cell or tissue system. The analysis showed that the majority of highly phosphorylated proteins were relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed, raising the possibility of more important functional roles for such highly phosphorylated pTyr sites. By mapping to major signaling networks, such as the EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which provides interesting targets for future hypothesis-driven and targeted quantitative studies involving tyrosine phosphorylation in HMEC or other human systems. PMID:19534553

  11. 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.

  12. Channel modulation by tyrosine phosphorylation in an identified leech neuron.

    PubMed Central

    Aniksztejn, L; Catarsi, S; Drapeau, P

    1997-01-01

    1. We have examined the effects of tyrosine phosphorylation on a spontaneously active cation channel that also participates in the modulation of pressure-sensitive (P) neurons in the leech. Cation channel activity in cell-attached or isolated, inside-out membrane patches from P cells in culture was monitored before and after treatments that altered the level of tyrosine phosphorylation. 2. In cell-attached recordings from intact P cells, bath application of genistein, an inhibitor of tyrosine kinases, resulted in a 6.6 +/- 2.6-fold increase in channel activity with no change in the mean open time or amplitude. Daidzein, an inactive form of genistein, was without effect. Addition of pervanadate, a membrane-permeant inhibitor of tyrosine phosphatases, had no effect on its own and blocked the effect of subsequent addition of genistein. 3. In inside-out P cell membrane patch recordings, exposure to a catalytically active fragment of a tyrosine phosphatase resulted in a 10.3 +/- 3.6-fold increase in channel activity with no change in the mean open time or amplitude. Orthovanadate had no effect on channel activity and, when added with the phosphatase, prevented the increase in activity. 4. Our results demonstrate that the basal activity of cation channels is increased by tyrosine dephosphorylation, suggesting a constitutive modulation of channel activity under resting conditions. PMID:9023773

  13. N-->S phosphoryl migration in phosphoryl glutathion.

    PubMed

    Yang, H J; Liu, J; Zhao, Y F

    1993-07-01

    It was found that in the case of N-(diisopropylphosphoryl) glutathion (reduced form), 2, N-->S phosphoryl migration took place, but not for N,N-bis(diisopropylphosphoryl) glutathion (oxidized form) or N-diisopropylphosphoryl cysteine. These results were deduced by 31P-NMR tracing experiments. It was shown that phosphoryl migration was catalyzed by an intramolecular carboxyl group, and a mechanism involving a mixed carboxyl-phosphoric anhydride was proposed. A competitive reaction between the amino and thiol group toward diisopropyl phosphite indicated that the phospho-thiol derived from N-(diisopropylphosphoryl) glutathion (reduced form), 2, did not result from direct phosphorylation of the thiol group. N,S-Bis(diisopropylphosphoryl) glutathion provides an authentic sample to confirm the migrated phosphoryl thiol product.

  14. Structure-function relationships of antimicrobial peptides.

    PubMed

    Hwang, P M; Vogel, H J

    1998-01-01

    Antimicrobial peptides are ubiquitously produced throughout nature. Many of these relatively short peptides (6-50 residues) are lethal towards bacteria and fungi, yet they display minimal toxicity towards mammalian cells. All of the peptides are highly cationic and hydrophobic. It is widely believed that they act through nonspecific binding to biological membranes, even though the exact nature of these interactions is presently unclear. High-resolution nuclear magnetic resonance (NMR) has contributed greatly to knowledge in this field, providing insight about peptide structure in aqueous solution, in organic cosolvents, and in micellar systems. Solid-state NMR can provide additional information about peptide-membrane binding. Here we review our current knowledge about the structure of antimicrobial peptides. We also discuss studies pertaining to the mechanism of action. Despite the different three-dimensional structural motifs of the various classes, they all have similar amphiphilic surfaces that are well-suited for membrane binding. Many antimicrobial peptides bind in a membrane-parallel orientation, interacting only with one face of the bilayer. This may be sufficient for antimicrobial action. At higher concentrations, peptides and phospholipids translocate to form multimeric transmembrane channels that seem to contribute to the peptide's hemolytic activity. An understanding of the key features of the secondary and tertiary structures of the antimicrobial peptides and their effects on bactericidal and hemolytic activity can aid the rational design of improved analogs for clinical use.

  15. 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

  16. A phosphotyrosine switch regulates organic cation transporters.

    PubMed

    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

    2016-03-16

    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.

  17. Identification of kinases phosphorylating 13 sites in the nuclear, DNA-binding protein NUCKS.

    PubMed

    Grundt, Kirsten; Thiede, Bernd; Østvold, Anne Carine

    2017-03-01

    NUCKS is a vertebrate specific, nuclear and DNA-binding phospho protein. The protein is highly expressed in rapidly dividing cells, and is overexpressed in a number of cancer tissues. The phosphorylation of NUCKS is cell cycle and DNA-damage regulated, but little is known about the responsible kinases. By utilizing in vitro and in vivo phosphorylation assays using isolated NUCKS as well as synthetic NUCKS-derived peptides in combination with mass spectrometry, phosphopeptide mapping, phosphphoamino acid analyses, phosphospecific antibodies and the use of specific kinase inhibitors, we found that NUCKS is phosphorylated on 11 sites by CK2. At least 7 of the CK2 sites are phosphorylated in vivo. We also found that NUCKS is phosphorylated on two sites by ATM kinase and DNA-PK in vitro, and is phosphorylated in vivo by ATM kinase in γ-irradiated cells. All together, we identified three kinases phosphorylating 13 out of 39 in vivo phosphorylated sites in mammalian NUCKS. The identification of CK2 and PIKK kinases as kinases phosphorylating NUCKS in vivo provide further evidence for the involvement of NUCKS in cell cycle control and DNA repair.

  18. 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.

  19. TARP phosphorylation regulates synaptic AMPA receptors through lipid bilayers.

    PubMed

    Sumioka, Akio; Yan, Dan; Tomita, Susumu

    2010-06-10

    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 membrane-associated guanylate kinases. Among the three classes of ionotropic glutamate receptors (AMPA, NMDA, and 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 a 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.

  20. 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

  1. 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)

  2. Engineering short peptide sequences for uranyl binding.

    PubMed

    Lebrun, Colette; Starck, Matthieu; Gathu, Vicky; Chenavier, Yves; Delangle, Pascale

    2014-12-08

    Peptides are interesting tools to rationalize uranyl-protein interactions, which are relevant to uranium toxicity in vivo. Structured cyclic peptide scaffolds were chosen as promising candidates to coordinate uranyl thanks to four amino acid side chains pre-oriented towards the dioxo cation equatorial plane. The binding of uranyl by a series of decapeptides has been investigated with complementary analytical and spectroscopic methods to determine the key parameters for the formation of stable uranyl-peptide complexes. The molar ellipticity of the uranyl complex at 195 nm is directly correlated to its stability, which demonstrates that the β-sheet structure is optimal for high stability in the peptide series. Cyclodecapeptides with four glutamate residues exhibit the highest affinities for uranyl with log KC =8.0-8.4 and, therefore, appear as good starting points for the design of high-affinity uranyl-chelating peptides.

  3. Cation diffusion in titanomagnetites

    NASA Astrophysics Data System (ADS)

    Aragon, R.; McCallister, R. H.; Harrison, H. R.

    1984-02-01

    Interdiffusion couple experiments were performed with titanomagnetite single crystals at 1,000°C, 1,100° C and 1,200° C in various buffered atmospheres. The dependence of the interdiffusion coefficient on oxygen fugacity, composition and temperature was interpreted in terms of point defect structure. Estimates of the cation tracer diffusivities indicate that Fe migrates via a point defect mechanism, involving mixed tetrahedral-octahedral site jumps, with an activation energy of 33 Kcal/mole; whereas Ti migration is one to two orders of magnitude slower, is restricted to octahedral sites and has an activation energy of 60 Kcal/mole.

  4. De Novo Design of Potent Antimicrobial Peptides

    PubMed Central

    Frecer, V.; Ho, B.; Ding, J. L.

    2004-01-01

    Lipopolysaccharide (LPS), shed by gram-negative bacteria during infection and antimicrobial therapy, may lead to lethal endotoxic shock syndrome. A rational design strategy based on the presumed mechanism of antibacterial effect was adopted to design cationic antimicrobial peptides capable of binding to LPS through tandemly repeated sequences of alternating cationic and nonpolar residues. The peptides were designed to achieve enhanced antimicrobial potency due to initial bacterial membrane binding with a reduced risk of endotoxic shock. The peptides designed displayed binding affinities to LPS and lipid A (LA) in the low micromolar range and by molecular modeling were predicted to form amphipathic β-hairpin-like structures when they bind to LPS or LA. They also exhibited strong effects against gram-negative bacteria, with MICs in the nanomolar range, and low cytotoxic and hemolytic activities at concentrations significantly exceeding their MICs. Quantitative structure-activity relationship (QSAR) analysis of peptide sequences and their antimicrobial, cytotoxic, and hemolytic activities revealed that site-directed substitutions of residues in the hydrophobic face of the amphipathic peptides with less lipophilic residues selectively decrease the hemolytic effect without significantly affecting the antimicrobial or cytotoxic activity. On the other hand, the antimicrobial effect can be enhanced by substitutions in the polar face with more polar residues, which increase the amphipathicity of the peptide. On the basis of the QSARs, new analogs that have strong antimicrobial effects but that lack hemolytic activity can be proposed. The findings highlight the importance of peptide amphipathicity and allow a rational method that can be used to dissociate the antimicrobial and hemolytic effects of cationic peptides, which have potent antimicrobial properties, to be proposed. PMID:15328096

  5. Phosphorylation regulates human OCT4.

    PubMed

    Brumbaugh, Justin; Hou, Zhonggang; Russell, Jason D; Howden, Sara E; Yu, Pengzhi; Ledvina, Aaron R; Coon, Joshua J; Thomson, James A

    2012-05-08

    The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography-MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency.

  6. Phosphorylation regulates human OCT4

    PubMed Central

    Brumbaugh, Justin; Russell, Jason D.; Howden, Sara E.; Yu, Pengzhi; Ledvina, Aaron R.; Coon, Joshua J.; Thomson, James A.

    2012-01-01

    The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography–MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency. PMID:22474382

  7. 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.

  8. 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.

  9. 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

  10. Musite, a Tool for Global Prediction of General and Kinase-specific Phosphorylation Sites*

    PubMed Central

    Gao, Jianjiong; Thelen, Jay J.; Dunker, A. Keith; Xu, Dong

    2010-01-01

    Reversible protein phosphorylation is one of the most pervasive post-translational modifications, regulating diverse cellular processes in various organisms. High throughput experimental studies using mass spectrometry have identified many phosphorylation sites, primarily from eukaryotes. However, the vast majority of phosphorylation sites remain undiscovered, even in well studied systems. Because mass spectrometry-based experimental approaches for identifying phosphorylation events are costly, time-consuming, and biased toward abundant proteins and proteotypic peptides, in silico prediction of phosphorylation sites is potentially a useful alternative strategy for whole proteome annotation. Because of various limitations, current phosphorylation site prediction tools were not well designed for comprehensive assessment of proteomes. Here, we present a novel software tool, Musite, specifically designed for large scale predictions of both general and kinase-specific phosphorylation sites. We collected phosphoproteomics data in multiple organisms from several reliable sources and used them to train prediction models by a comprehensive machine-learning approach that integrates local sequence similarities to known phosphorylation sites, protein disorder scores, and amino acid frequencies. Application of Musite on several proteomes yielded tens of thousands of phosphorylation site predictions at a high stringency level. Cross-validation tests show that Musite achieves some improvement over existing tools in predicting general phosphorylation sites, and it is at least comparable with those for predicting kinase-specific phosphorylation sites. In Musite V1.0, we have trained general prediction models for six organisms and kinase-specific prediction models for 13 kinases or kinase families. Although the current pretrained models were not correlated with any particular cellular conditions, Musite provides a unique functionality for training customized prediction models

  11. Antimicrobial peptides activate the Rcs regulon through the outer membrane lipoprotein RcsF.

    PubMed

    Farris, Carol; Sanowar, Sarah; Bader, Martin W; Pfuetzner, Richard; Miller, Samuel I

    2010-10-01

    Salmonella enterica species are exposed to envelope stresses due to their environmental and infectious lifestyles. Such stresses include amphipathic cationic antimicrobial peptides (CAMPs), and resistance to these peptides is an important property for microbial virulence for animals. Bacterial mechanisms used to sense and respond to CAMP-induced envelope stress include the RcsFCDB phosphorelay, which contributes to survival from polymyxin B exposure. The Rcs phosphorelay includes two inner membrane (IM) proteins, RcsC and RcsD; the response regulator RcsB; the accessory coregulator RcsA; and an outer membrane bound lipoprotein, RcsF. Transcriptional activation of the Rcs regulon occurred within minutes of exposure to CAMP and during the first detectable signs of CAMP-induced membrane disorder. Rcs transcriptional activation by CAMPs required RcsF and preservation of its two internal disulfide linkages. The rerouting of RcsF to the inner membrane or its synthesis as an unanchored periplasmic protein resulted in constitutive activation of the Rcs regulon and RcsCD-dependent phosphorylation. These findings suggest that RcsFCDB activation in response to CAMP-induced membrane disorder is a result of a change in structure or availability of RcsF to the IM signaling constituents of the Rcs phosphorelay.

  12. Application of capillary isotachophoresis in peptide analysis.

    PubMed

    Kasicka, V; Prusík, Z

    1991-09-13

    This paper gives a broad and detailed review of the applications of one of the modern high-performance electromigration separation techniques--capillary isotachophoresis (ITP)--in peptide analysis. Examples are presented of the utilization of capillary ITP for peptide analysis in the fields of chemistry, general and clinical biochemistry, biology, biotechnology, pharmacy and the food industry. The complete composition of all the electrolyte systems used for peptide ITP analyses in both cationic and anionic techniques is given in tabular form. According to the purpose of analysis the applications are divided into several sections: model studies, determination of physico-chemical characteristics, purity control of both intermediate and final peptide preparations, including the determination of low-molecular-mass ionogenic admixtures, and the analysis of peptides in biological fluids and tissue extracts. In addition to the main applications the theoretical and methodological aspects of peptide ITP analysis are discussed. The basic electromigration properties of peptides (their polyampholyte character, effective and absolute mobilities, acid-base equilibria) are explained and the selection of parameters for peptide ITP analysis is described in detail. The advantages and disadvantages of ITP compared with other electrophoretic and chromatographic methods used for peptide analysis are discussed.

  13. 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.

  14. Contributions of cation-π interactions to the collagen triple helix stability.

    PubMed

    Chen, Chia-Ching; Hsu, Wei; Hwang, Kuo-Chu; Hwu, Jih Ru; Lin, Chun-Cheng; Horng, Jia-Cherng

    2011-04-01

    Cation-π interactions are found to be an important noncovalent force in proteins. Collagen is a right-handed triple helix composed of three left-handed PPII helices, in which (X-Y-Gly) repeats dominate in the sequence. Molecular modeling indicates that cation-π interactions could be formed between the X and Y positions in adjacent collagen strands. Here, we used a host-guest peptide system: (Pro-Hyp-Gly)(3)-(Pro-Y-Gly-X-Hyp-Gly)-(Pro-Hyp-Gly)(3), where X is an aromatic residue and Y is a cationic residue, to study the cation-π interaction in the collagen triple helix. Circular dichroism (CD) measurements and Tm data analysis show that the cation-π interactions involving Arg have a larger contribution to the conformational stability than do those involving Lys, and Trp forms a weaker cation-π interaction with cationic residues than expected as a result of steric effects. The results also show that the formation of cation-π interactions between Arg and Phe depends on their relative positions in the strand. Moreover, the fluorinated and methylated Phe substitutions show that an electron-withdrawing or electron-donating substituent on the aromatic ring can modulate its π-electron density and the cation-π interaction in collagen. Our data demonstrate that the cation-π interaction could play an important role in stabilizing the collagen triple helix.

  15. Chemical ionization of neutral peptides produced by matrix-assisted laser desorption

    NASA Astrophysics Data System (ADS)

    Belov, Mikhail E.; Myatt, Christopher P.; Derrick, Peter J.

    1998-03-01

    The cationization in the gas phase of neutral peptides formed under the conditions of matrix-assisted laser desorption (MALD) has been demonstrated. The beam of neutral peptides formed by MALD using one laser was intercepted several hundred micrometers above the sample surface by a beam of cations produced using a second laser. The intensity of cationized neutral gramicidin S formed in this way was comparable to the ion signal produced by conventional matrix-assisted laser desorption/ionization (MALDI). The threshold fluences for MALD of neutral peptides have been shown to be lower than those for formation of ions from the same samples by MALDI.

  16. Cell volume-regulated cation channels.

    PubMed

    Wehner, Frank

    2006-01-01

    perception of a variety of different stimuli including mechanical and (hypo-) osmotic stress. As a peculiarity, phospholemman (PLM, a 72 AA peptide also employed in ion transport regulation) appears to be activated under, both, hypertonic and hypotonic conditions, preferentially operating as a cation and anion channel, respectively.

  17. 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

  18. The effect of dexamethasone/cell-penetrating peptide nanoparticles on gene delivery for inner ear therapy

    PubMed Central

    Yoon, Ji Young; Yang, Keum-Jin; Park, Shi-Nae; Kim, Dong-Kee; Kim, Jong-Duk

    2016-01-01

    Dexamethasone (Dex)-loaded PHEA-g-C18-Arg8 (PCA) nanoparticles (PCA/Dex) were developed for the delivery of genes to determine the synergistic effect of Dex on gene expression. The cationic PCA nanoparticles were self-assembled to create cationic micelles containing an octadecylamine (C18) core with Dex and an arginine 8 (Arg8) peptide shell for electrostatic complexation with nucleic acids (connexin 26 [Cx26] siRNA, green fluorescent protein [GFP] DNA or brain-derived neurotrophic factor [BDNF] pDNA). The PCA/Dex nanoparticles conjugated with Arg8, a cell-penetrating peptide that enhances permeability through a round window membrane in the inner ear for gene delivery, exhibited high uptake efficiency in HEI-OC1 cells. This potential carrier co-delivering Dex and the gene into inner ear cells has a diameter of 120–140 nm and a zeta potential of 20–25 mV. Different types of genes were complexed with the Dex-loaded PCA nanoparticle (PCA/Dex/gene) for gene expression to induce additional anti-inflammatory effects. PCA/Dex showed mildly increased expression of GFP and lower mRNA expression of inflammatory cytokines (IL1b, IL12, and INFr) than did Dex-free PCA nanoparticles and Lipofectamine® reagent in HEI-OC1 cells. In addition, after loading Cx26 siRNA onto the surface of PCA/Dex, Cx26 gene expression was downregulated according to real-time polymerase chain reaction for 24 h, compared with that using Lipofectamine reagent. After loading BDNF DNA into PCA/Dex, increased expression of BDNF was observed for 30 h, and its signaling pathway resulted in an increase in phosphorylation of Akt, observed by Western blotting. Thus, Dex within PCA/Dex/gene nanoparticles created an anti-inflammatory effect and enhanced gene expression. PMID:27895484

  19. 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.

  20. Liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometric characterization of protein kinase C phosphorylation.

    PubMed

    Chalmers, Michael J; Quinn, John P; Blakney, Greg T; Emmett, Mark R; Mischak, Harold; Gaskell, Simon J; Marshall, Alan G

    2003-01-01

    A vented column, capillary liquid chromatography (LC) microelectrospray ionization (ESI) Fourier transform ion cyclotron resonance (FT-ICR (9.4 T)) mass spectrometry (MS) approach to phosphopeptide identification is described. A dual-ESI source capable of rapid (approximately 200 ms) switching between two independently controlled ESI emitters was constructed. The dual-ESI source, combined with external ion accumulation in a linear octopole ion trap, allowed for internal calibration of every mass spectrum during LC. LC ESI FT-ICR positive-ion MS of protein kinase C (PKC) revealed four previously unidentified phosphorylated peptides (one within PKC(alpha), one within PKC(delta), and two within PKC(zeta)). Internal calibration improved the mass accuracy for LC MS spectra from an absolute mean (47 peptide ions) of 11.5 ppm to 1.5 ppm. Five additional (out of eight known) activating sites of PKC phosphorylation, not detected in positive-ion experiments, were observed by subsequent negative-ion direct infusion nanoelectrospray. Extension of the method to enable infrared multiphoton dissociation of all ions in the ICR cell prior to every other mass measurement revealed the diagnostic neutral loss of H3PO4 from phosphorylated peptide ions. The combination of accurate-mass MS and MS/MS offers a powerful new tool for identifying the presence and site(s) of phosphorylation in peptides, without the need for additional wet chemical derivatization.

  1. Regulation of casein kinase 2 by phosphorylation/dephosphorylation.

    PubMed Central

    Agostinis, P; Goris, J; Pinna, L A; Merlevede, W

    1987-01-01

    The effects of various polycation-stimulated (PCS) phosphatases and of the active catalytic subunit of the ATPMg-dependent (AMDc) protein phosphatase on the activity of casein kinase 2 (CK-2) were investigated by using the synthetic peptide substrate Ser-Glu-Glu-Glu-Glu-Glu, whose phosphorylated derivative is entirely insensitive to these protein phosphatases. Previous dephosphorylation of native CK-2 enhances its specific activity 2-3-fold. Such an effect, accounted for by an increase in Vmax, is more readily promoted by the PCS phosphatases than by the AMDc phosphatase. The phosphate incorporated by autophosphorylation could not be removed by the protein phosphatases, suggesting the involvement of phosphorylation site(s) other than the one(s) affected by intramolecular autophosphorylation. The activation of CK-2 by the phosphatase pretreatment is neutralized during the kinase assay; the mechanism of this phenomenon, which is highly dependent on the kinase concentration, is discussed. Images Fig. 4. PMID:2829841

  2. Regulation of casein kinase 2 by phosphorylation/dephosphorylation.

    PubMed

    Agostinis, P; Goris, J; Pinna, L A; Merlevede, W

    1987-12-15

    The effects of various polycation-stimulated (PCS) phosphatases and of the active catalytic subunit of the ATPMg-dependent (AMDc) protein phosphatase on the activity of casein kinase 2 (CK-2) were investigated by using the synthetic peptide substrate Ser-Glu-Glu-Glu-Glu-Glu, whose phosphorylated derivative is entirely insensitive to these protein phosphatases. Previous dephosphorylation of native CK-2 enhances its specific activity 2-3-fold. Such an effect, accounted for by an increase in Vmax, is more readily promoted by the PCS phosphatases than by the AMDc phosphatase. The phosphate incorporated by autophosphorylation could not be removed by the protein phosphatases, suggesting the involvement of phosphorylation site(s) other than the one(s) affected by intramolecular autophosphorylation. The activation of CK-2 by the phosphatase pretreatment is neutralized during the kinase assay; the mechanism of this phenomenon, which is highly dependent on the kinase concentration, is discussed.

  3. 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

  4. 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.

  5. Protein Phosphorylation in Amyloplasts Regulates Starch Branching Enzyme Activity and Protein–Protein Interactions

    PubMed Central

    Tetlow, Ian J.; Wait, Robin; Lu, Zhenxiao; Akkasaeng, Rut; Bowsher, Caroline G.; Esposito, Sergio; Kosar-Hashemi, Behjat; Morell, Matthew K.; Emes, Michael J.

    2004-01-01

    Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with γ-32P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated 32P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule–associated phosphoproteins after incubation of intact amyloplasts with γ-32P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein

  6. Characterization of a possible uptake mechanism of selective antibacterial peptides.

    PubMed

    Polanco, Carlos; Samaniego, José Lino; Castañón-González, Jorge Alberto; Buhse, Thomas; Sordo, Marili Leopold

    2013-01-01

    Selective antibacterial peptides containing less than 30 amino acid residues, cationic, with amphipathic properties, have been the subject of several studies due to their active participation and beneficial effects in strengthening the immune system of all living organisms. This manuscript reports the results of a comparison between the group of selective antibacterial peptides and another group called "cell penetrating peptides". An important number of the selective antibacterial peptides are cell penetrating peptides, suggesting that their toxicity is related to their uptake mechanism. The verification of this observation also includes the adaptation of a method previously published, called Polarity index, which reproduces and confirms the action of this new set of peptides. The efficiency of this method was verified based on four different databases, yielding a high score. The verification was based exclusively on the peptides already reported in the databases which have been experimentally verified.

  7. Cationic liposomes as vaccine adjuvants.

    PubMed

    Christensen, Dennis; Korsholm, Karen Smith; Andersen, Peter; Agger, Else Marie

    2011-04-01

    The application of cationic liposomes as vaccine delivery systems and adjuvants has been investigated extensively over the last few decades. However, cationic liposomes are, in general, not sufficiently immunostimulatory, which is why the combination of liposomes with immunostimulating ligands has arisen as a strategy in the development of novel adjuvant systems. Within the last 5 years, two novel adjuvant systems based on cationic liposomes incorporating Toll-like receptor or non-Toll-like receptor immunostimulating ligands have progressed from preclinical testing in smaller animal species to clinical testing in humans. The immune responses that these clinical candidates induce are primarily of the Th1 type for which there is a profound unmet need. Furthermore, a number of new cationic liposome-forming surfactants with notable immunostimulatory properties have been discovered. In this article we review the recent progress on the application of cationic liposomes as vaccine delivery systems/adjuvants.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. Phosphorylation and dephosphorylation of spectrin.

    PubMed

    Fairbanks, G; Avruch, J; Dino, J E; Patel, V P

    1978-01-01

    The phosphorylation of spectrin polypeptide 2 is thought to be involved in the metabolically dependent regulation of red cell shape and deformability. Spectrin phosphorylation is not affected by cAMP. The reaction in isolated membranes resembles the cAMP-independent, salt-stimulated phosphorylation of an exogenous substrate, casein, by enzyme(s) present both in isolated membranes and cytoplasmic extracts. Spectrin kinase is selectively eluted from membranes by 0.5 M NaCl and co-fractionates with eluted casein kinase. Phosphorylation of band 3 in the membrane is inhibited by salt, but the band 3 kinase is otherwise indistinguishable operationally from spectrin kinase. The membrane-bound casein (spectrin) kinase is not eluted efficiently with spectrin at low ionic strength; about 80% of the activity is apparently bound at sites (perhaps on or near band 3) other than spectrin. Partitioning of casein kinase between cytoplasm and membrane is metabolically dependent; the proportion of casein kinase on the membrane can range from 25% to 75%, but for fresh cells is normally about 40%. Dephosphorylation of phosphorylated spectrin has not been studied intensively. Slow release of 32Pi from [32P] spectrin on the membrane can be demonstrated, but phosphatase activity measured against solubilized [32P] spectrin is concentrated in the cytoplasm. The crude cytoplasmic phosphospectrin phosphatase is inhibited by various anions--notably, ATP and 2,3-DPG at physiological concentrations. Regulation of spectrin phosphorylation in intact cells has not been studied. We speculate that spectrin phosphorylation state may be regulated 1) by metabolic intermediates and other internal chemical signals that modulate kinase and phosphatase activities per se or determine their intracellular localization and 2) by membrane deformation that alters enzyme-spectrin interaction locally. Progress in the isolation and characterization of spectrin kinase and phosphospectrin phosphatase should lead to

  13. A cumulative experience examining the effect of natural and synthetic antimicrobial peptides vs. Chlamydia trachomatis.

    PubMed

    Yasin, B; Pang, M; Wagar, E A

    2004-08-01

    We tested the activity of 48 structurally diverse antimicrobial peptides against Chlamydia trachomatis, serovar L2. The peptides' activity against C. trachomatis, serovar L2 was measured in 48-h McCoy cell shell vial assays. Peptides of 16-20 amino acids were more active than larger peptides, such as defensins. Beta-sheet protegrins, as well as alpha-helical peptides such as novispirin (G-10) were equally active. Enantiomers were as active as native structures. Moderate-sized circular mini-defensins were less effective against C. trachomatis. Moderate-sized cationic peptides may be useful in microbicide preparations designed to prevent chlamydial infection.

  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. Calcium/calmodulin-dependent phosphorylation of vimentin in rat sertoli cells.

    PubMed Central

    Spruill, W A; Zysk, J R; Tres, L L; Kierszenbaum, A L

    1983-01-01

    Ca2+-dependent protein phosphorylation and the role of calmodulin in this process was investigated in subcellular fractions of primary cultures of rat Sertoli cells. Significant Ca2+/calmodulin-dependent protein phosphorylation in Sertoli cells was restricted to the cytosol fraction. The calmodulin dependence of these effects was confirmed by using the calmodulin inhibitor trifluoperazine. One of the Ca2+/calmodulin-dependent phosphoproteins was identified as the intermediate filament protein vimentin, based on the following criteria: (i) migration pattern in two-dimensional polyacrylamide gels, (ii) Ca2+/calmodulin-dependent phosphorylation of a 58-kilodalton protein present in detergent-insoluble intermediate filament protein extract of Sertoli cells, and (iii) peptide mapping of the phosphoprotein. These data support a role for Ca2+/calmodulin-dependent protein phosphorylation in the modulation of Sertoli cell cytoskeletal components. Images PMID:6572367

  16. Three new antimicrobial peptides from the scorpion Pandinus imperator.

    PubMed

    Zeng, Xian-Chun; Zhou, Lingli; Shi, Wanxia; Luo, Xuesong; Zhang, Lei; Nie, Yao; Wang, Jinwei; Wu, Shifen; Cao, Bin; Cao, Hanjun

    2013-07-01

    Three novel cysteine-free venom peptides, which were referred to as Pantinin-1, Pantinin-2 and Pantinin-3, respectively, have been identified from the scorpion Pandinus imperator by cDNA cloning strategy. The precursor of each peptide consists of a signal peptide, a mature peptide with no disulfide bridges, and an acidic propeptide with a typical processing signal. Each of the three peptides is an α-helical, cationic and amphipathic molecule with 13 or 14 amino acid residues. Their amino acid sequences are homologous to those of some 13-mer antimicrobial peptides isolated from scorpions. Antimicrobial assay showed that all the three peptides possess relatively strong activities against Gram-positive bacteria and a fungus, but have very weak antimicrobial activities against Gram-negative bacteria. Toxicity assay showed that the three peptides exhibit very low or mild hemolytic activities against human red blood cells. It is interesting to see that Pantinin-3 is able to potently inhibit the growth of vancomycin-resistant Enterococcus (VRE) S13, a pathogen that can cause a number of human infections; this suggests that Pantinin-3 has great potential to be applied in the treatment of VRE infections. Our findings gain new insights into the structure/function relationships of the small linear cationic antimicrobial peptides from scorpions, and provide new templates for designing of antimicrobial agents targeting antibiotic-resistant pathogenic bacteria.

  17. Tyrosine phosphorylation of phosphoinositide-dependent kinase 1 by the insulin receptor is necessary for insulin metabolic signaling.

    PubMed

    Fiory, Francesca; Alberobello, Anna Teresa; Miele, Claudia; Oriente, Francesco; Esposito, Iolanda; Corbo, Vincenzo; Ruvo, Menotti; Tizzano, Barbara; Rasmussen, Thomas E; Gammeltoft, Steen; Formisano, Pietro; Beguinot, Francesco

    2005-12-01

    In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR(Delta43)) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR(Delta43) (L6(IRDelta43)) also showed no insulin effect on glucose uptake and glycogen synthase, accompanied by a >80% decrease in insulin induction of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activity and tyrosine phosphorylation and of protein kinase B (PKB) phosphorylation at Thr(308). Insulin induced the phosphatidylinositol 3 kinase-dependent coprecipitation of PDK-1 with wild-type IR (IR(WT)), but not IR(Delta43). Based on overlay blotting, PDK-1 directly bound IR(WT), but not IR(Delta43). Insulin-activated IR(WT), and not IR(Delta43), phosphorylated PDK-1 at tyrosines 9, 373, and 376. The IR C-terminal 43-amino-acid peptide (C-terminal peptide) inhibited in vitro PDK-1 tyrosine phosphorylation by the IR. Tyr-->Phe substitution prevented this inhibitory action. In the L6(hIR) cells, the C-terminal peptide coprecipitated with PDK-1 in an insulin-stimulated fashion. This peptide simultaneously impaired the insulin effect on PDK-1 coprecipitation with IR(WT), on PDK-1 tyrosine phosphorylation, on PKB phosphorylation at Thr(308), and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6(IRDelta43) compared to control cells. In L6 cells expressing IR(WT), the C-terminal peptide also impaired insulin-dependent PDK-1 membrane persistence. Thus, PDK-1 directly binds to the insulin receptor, followed by PDK-1 activation and insulin metabolic effects.

  18. Feedback Inhibition in the PhoQ/PhoP Signaling System by a Membrane Peptide

    PubMed Central

    Lippa, Andrew M.; Goulian, Mark

    2009-01-01

    The PhoQ/PhoP signaling system responds to low magnesium and the presence of certain cationic antimicrobial peptides. It regulates genes important for growth under these conditions, as well as additional genes important for virulence in many gram-negative pathogens. PhoQ is a sensor kinase that phosphorylates and activates the transcription factor PhoP. Since feedback inhibition is a common theme in stress-response circuits, we hypothesized that some members of the PhoP regulon may play such a role in the PhoQ/PhoP pathway. We therefore screened for PhoP-regulated genes that mediate feedback in this system. We found that deletion of mgrB (yobG), which encodes a 47 amino acid peptide, results in a potent increase in PhoP-regulated transcription. In addition, over-expression of mgrB decreased transcription at both high and low concentrations of magnesium. Localization and bacterial two-hybrid studies suggest that MgrB resides in the inner-membrane and interacts directly with PhoQ. We further show that MgrB homologs from Salmonella typhimurium and Yersinia pestis also repress PhoP-regulated transcription in these organisms. In cell regulatory circuits, feedback has been associated with modulating the induction kinetics and/or the cell-to-cell variability in response to stimulus. Interestingly, we found that elimination of MgrB-mediated feedback did not have a significant effect on the kinetics of reporter protein production and did not decrease the variability in expression among cells. Our results indicate MgrB is a broadly conserved membrane peptide that is a critical mediator of negative feedback in the PhoQ/PhoP circuit. This new regulator may function as a point of control that integrates additional input signals to modulate the activity of this important signaling system. PMID:20041203

  19. Strategies for the Gas Phase Modification of Cationized Arginine via Ion/ion Reactions.

    PubMed

    Prentice, Boone M; McGee, William M; Stutzman, John R; McLuckey, Scott A

    2013-11-15

    The gas phase acetylation of cationized arginine residues is demonstrated here using ion/ion reactions with sulfosuccinimidyl acetate (sulfo-NHS acetate) anions. Previous reports have demonstrated the gas phase modification of uncharged primary amine (the N-terminus and ε-amino side chain of lysine) and uncharged guanidine (the arginine side chain) functionalities via sulfo-NHS ester chemistry. Herein, charge-saturated arginine-containing peptides that contain sodium ions as the charge carriers, such as [ac-ARAAARA+2Na](2+), are shown to exhibit strong reactivity towards sulfo-NHS acetate whereas the protonated peptide analogues exhibit no such reactivity. This difference in reactivity is attributed to the lower sodium ion (as compared to proton) affinity of the arginine, which results in increased nucleophilicity of the cationized arginine guanidinium functionality. This increased nucleophilicity improves the arginine residue's reactivity towards sulfo-NHS esters and enhances the gas phase covalent modification pathway. No such dramatic increase in reactivity towards sulfo-NHS acetate has been observed upon sodium cationization of lysine amino acid residues, indicating that this behavior appears to be unique to arginine. The sodium cationization process is demonstrated in the condensed phase by simply spiking sodium chloride into the peptide sample solution and in the gas phase by a peptide-sodium cation exchange process with a sulfo-NHS acetate sodium-bound dimer cluster reagent. This methodology demonstrates several ways by which arginine can be covalently modified in the gas phase even when it is charged. Collisional activation of an acetylated arginine product can result in deguanidination of the residue, generating an ornithine. This gas phase ornithination exhibits similar site-specific fragmentation behavior to that observed with peptides ornithinated in solution and may represent a useful approach for inducing selective peptide cleavages.

  20. Synthetic cation-selective nanotube: Permeant cations chaperoned by anions

    NASA Astrophysics Data System (ADS)

    Hilder, Tamsyn A.; Gordon, Dan; Chung, Shin-Ho

    2011-01-01

    The ability to design ion-selective, synthetic nanotubes which mimic biological ion channels may have significant implications for the future treatment of bacteria, diseases, and as ultrasensitive biosensors. We present the design of a synthetic nanotube made from carbon atoms that selectively allows monovalent cations to move across and rejects all anions. The cation-selective nanotube mimics some of the salient properties of biological ion channels. Before practical nanodevices are successfully fabricated it is vital that proof-of-concept computational studies are performed. With this in mind we use molecular and stochastic dynamics simulations to characterize the dynamics of ion permeation across a single-walled (10, 10), 36 Å long, carbon nanotube terminated with carboxylic acid with an effective radius of 5.08 Å. Although cations encounter a high energy barrier of 7 kT, its height is drastically reduced by a chloride ion in the nanotube. The presence of a chloride ion near the pore entrance thus enables a cation to enter the pore and, once in the pore, it is chaperoned by the resident counterion across the narrow pore. The moment the chaperoned cation transits the pore, the counterion moves back to the entrance to ferry another ion. The synthetic nanotube has a high sodium conductance of 124 pS and shows linear current-voltage and current-concentration profiles. The cation-anion selectivity ratio ranges from 8 to 25, depending on the ionic concentrations in the reservoirs.

  1. 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.

  2. 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.

  3. Peptide Transport through the Blood-Brain Barrier

    DTIC Science & Technology

    1989-01-01

    analogues for treatment of membiry,.ACIH analogues for treatmen of post-traumatic epilepsy), should these peptides be capable of traversing the BBB...thus far. In addition, since cationization does not destroy immunoglobulin G antigenicity, the use of cationized immunoglobulins may be used for... aerosolized insulin. Mixed-meal studies and long-term use in Type I diabetes. N. Engl. J. Med. 312:1078-1084. 12. Pardridge, W.M. (1988): Recent advances in

  4. [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.

  5. Phosphorylation of the herpes simplex virus type 1 tegument protein VP22.

    PubMed

    Elliott, G; O'Reilly, D; O'Hare, P

    1996-12-01

    The herpes simplex virus type 1 tegument protein VP22 is known to be highly phosphorylated during infection. Here we show that two electrophoretic forms of VP22 can be identified in infected cell extracts and that this heterogeneity is accounted for by phosphorylation. Furthermore, the nonphosphorylated form of VP22 appears to be specifically incorporated into virions. We also show that the phosphorylated form of VP22 is the only form detected during transient transfection and as such that VP22 can act as a substrate for a cellular kinase. Phospho-amino acid and phospho-peptide analyses of in vivo labeled VP22 were utilized to demonstrate that the phosphorylation profiles of VP22 synthesized during transfection and infection are the same. In both cases VP22 was modified solely on serine residues located in the N-terminal 120 residues of the protein. Moreover, in vitro phosphorylation was utilized to show that the constitutive cellular kinase, casein kinase II, which has four serine consensus recognition sites at the N-terminus of VP22, phosphorylates VP22 in the same manner as observed in vivo. This kinase also phosphorylates VP22 at the N-terminus in intact capsid-tegument structures. Casein kinase II is therefore likely to be the major kinase of VP22 during infection.

  6. Haemophilus ducreyi LspA proteins are tyrosine phosphorylated by macrophage-encoded protein tyrosine kinases.

    PubMed

    Deng, Kaiping; Mock, Jason R; Greenberg, Steven; van Oers, Nicolai S C; Hansen, Eric J

    2008-10-01

    The LspA proteins (LspA1 and LspA2) of Haemophilus ducreyi are necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines, an event that can be correlated with a reduction in the level of active Src family protein tyrosine kinases (PTKs) in these eukaryotic cells. During studies investigating this inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine phosphorylated after wild-type H. ducreyi cells were incubated with macrophages. LspA proteins in cell-free concentrated H. ducreyi culture supernatant fluid could also be tyrosine phosphorylated by macrophages. This ability to tyrosine phosphorylate the LspA proteins was not limited to immune cell lineages but could be accomplished by both HeLa and COS-7 cells. Kinase inhibitor studies with macrophages demonstrated that the Src family PTKs were required for this tyrosine phosphorylation activity. In silico methods and site-directed mutagenesis were used to identify EPIYG and EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. A total of four tyrosines could be phosphorylated in LspA1, with LspA2 containing eight predicted tyrosine phosphorylation motifs. Purified LspA1 fusion proteins containing either the EPIYG or EPVYA motifs were shown to be phosphorylated by purified Src PTK in vitro. Macrophage lysates could also tyrosine phosphorylate the LspA proteins and an LspA1 fusion protein via a mechanism that was dependent on the presence of both divalent cations and ATP. Several motifs known to interact with or otherwise affect eukaryotic kinases were identified in the LspA proteins.

  7. Haemophilus ducreyi LspA Proteins Are Tyrosine Phosphorylated by Macrophage-Encoded Protein Tyrosine Kinases▿

    PubMed Central

    Deng, Kaiping; Mock, Jason R.; Greenberg, Steven; van Oers, Nicolai S. C.; Hansen, Eric J.

    2008-01-01

    The LspA proteins (LspA1 and LspA2) of Haemophilus ducreyi are necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines, an event that can be correlated with a reduction in the level of active Src family protein tyrosine kinases (PTKs) in these eukaryotic cells. During studies investigating this inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine phosphorylated after wild-type H. ducreyi cells were incubated with macrophages. LspA proteins in cell-free concentrated H. ducreyi culture supernatant fluid could also be tyrosine phosphorylated by macrophages. This ability to tyrosine phosphorylate the LspA proteins was not limited to immune cell lineages but could be accomplished by both HeLa and COS-7 cells. Kinase inhibitor studies with macrophages demonstrated that the Src family PTKs were required for this tyrosine phosphorylation activity. In silico methods and site-directed mutagenesis were used to identify EPIYG and EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. A total of four tyrosines could be phosphorylated in LspA1, with LspA2 containing eight predicted tyrosine phosphorylation motifs. Purified LspA1 fusion proteins containing either the EPIYG or EPVYA motifs were shown to be phosphorylated by purified Src PTK in vitro. Macrophage lysates could also tyrosine phosphorylate the LspA proteins and an LspA1 fusion protein via a mechanism that was dependent on the presence of both divalent cations and ATP. Several motifs known to interact with or otherwise affect eukaryotic kinases were identified in the LspA proteins. PMID:18678665

  8. Inhibition of insulin-stimulated phosphorylation of the intracellular domain of phospholemman decreases insulin-dependent GLUT4 translocation in streptolysin-O-permeabilized adipocytes.

    PubMed Central

    Walaas, O; Horn, R S; Walaas, S I

    1999-01-01

    A variety of studies indicate that protein kinase C might be involved in the insulin signalling cascade leading to translocation of the insulin-regulated glucose transporter GLUT4 from intracellular pools to the plasma membrane. Phospholemman is a plasma-membrane protein kinase C substrate whose phosphorylation is increased by insulin in intact muscle [Walaas, Czernik, Olstad, Sletten and Walaas (1994) Biochem. J. 304, 635-640]. The present study examined whether the inhibition of phospholemman phosphorylation modulates the effects of insulin on GLUT4 translocation. For this purpose, a synthetic peptide derived from the intracellular domain of phospholemman with the phosphorylatable serine residues replaced with alanine residues was prepared. This peptide was found to decrease the protein kinase C-catalysed phosphorylation of a synthetic phospholemman peptide in vitro. When introduced into streptolysin-O-permeabilized adipocytes, the peptide decreased the effects of insulin on both the phosphorylation of phospholemman and the recruitment of GLUT4 to the plasma membrane. Similarly, the internalization of phospholemman antibodies, which also decreased the protein kinase C-mediated phosphorylation of the synthetic phospholemman peptide in vitro, decreased the effect of insulin on GLUT4 translocation in the adipocytes. The results suggest that phosphorylation of the intracellular domain of phospholemman might be involved in modulating the insulin-induced translocation of GLUT4 to the plasma membrane. PMID:10493924

  9. Antimicrobial Peptides

    PubMed Central

    Bahar, Ali Adem; Ren, Dacheng

    2013-01-01

    The rapid increase in drug-resistant infections has presented a serious challenge to antimicrobial therapies. The failure of the most potent antibiotics to kill “superbugs” emphasizes the urgent need to develop other control agents. Here we review the history and new development of antimicrobial peptides (AMPs), a growing class of natural and synthetic peptides with a wide spectrum of targets including viruses, bacteria, fungi, and parasites. We summarize the major types of AMPs, their modes of action, and the common mechanisms of AMP resistance. In addition, we discuss the principles for designing effective AMPs and the potential of using AMPs to control biofilms (multicellular structures of bacteria embedded in extracellular matrixes) and persister cells (dormant phenotypic variants of bacterial cells that are highly tolerant to antibiotics). PMID:24287494

  10. Function of Estrogen Receptor Tryosine Phosphorylation

    DTIC Science & Technology

    1998-07-01

    6219 TITLE: Function of Estrogen Receptor Tryosine Phosphorylation PRINCIPAL INVESTIGATOR: Matthew R. Yudt CONTRACTING ORGANIZATION: University of...Estrogen Receptor Tryosine Phosphorylation ~DAMD17-96-1-6219 6. AUTHOR(S) Matthew R. Yudt 7. PERFORMING ORGANIZATION NAME11S) AND AODRESS(ES...this model, tyrosine 537 (Y537) phosphorylation of one monomer interacts with another tyrosine phosphorylated monomer to constitute an hER dimer

  11. Different modes of endothelial-smooth muscle cell interaction elicit differential β-catenin phosphorylations and endothelial functions.

    PubMed

    Chang, Shun-Fu; Chen, Li-Jing; Lee, Pei-Ling; Lee, Ding-Yu; Chien, Shu; Chiu, Jeng-Jiann

    2014-02-04

    β-Catenin phosphorylation plays important roles in modulating its functions, but the effects of different phosphorylated forms of β-catenin in response to heterocellular interaction are unclear. Here we investigated whether distinct modes of phosphorylation on β-catenin could be triggered through heterocellular interactions between endothelial cells (ECs) and smooth muscle cells (SMCs), and the consequent modulation of EC functions. ECs were cocultured with SMCs to initiate direct contact and paracrine interaction. EC-SMC coculture induced EC β-catenin phosphorylations simultaneously at tyrosine 142 (Tyr142) and serine 45/threonine 41 (Ser45/Thr41) at the cytoplasm/nuclei and the membrane, respectively. Treating ECs with SMC-conditional medium induced β-catenin phosphorylation only at Ser45/Thr41. These findings indicate that different phosphorylation effects of EC-SMC coculture were induced through heterocellular direct contact and paracrine effects, respectively. Using specific blocking peptides, antagonists, and siRNAs, we found that the β-catenin Tyr142-phosphorylation was mediated by connexin 43/Fer and that the β-catenin Ser45/Thr41-phosphorylation was mediated by SMC-released bone morphogenetic proteins through VE-cadherin and bone morphogenetic protein receptor-II/Smad5. Transfecting ECs with β-catenin-Tyr142 or -Ser45 mutants showed that these two phosphorylated forms of β-catenin modulate differential EC function: The Tyr142-phosphorylated β-catenin stimulates vascular cell-adhesion molecule-1 expression to increase EC-monocytic adhesion, but the Ser45/Thr41-phosphorylated β-catenin attenuates VE-cadherin-dependent junction structures to increase EC permeability. Our findings provide new insights into the understanding of regulatory complexities of distinct modes of β-catenin phosphorylations under EC-SMC interactions and suggest that different phosphorylated forms of β-catenin play important roles in modulating vascular pathophysiology

  12. Tyrosine phosphorylation of WW proteins

    PubMed Central

    Reuven, Nina; Shanzer, Matan

    2015-01-01

    A number of key regulatory proteins contain one or two copies of the WW domain known to mediate protein–protein interaction via proline-rich motifs, such as PPxY. The Hippo pathway components take advantage of this module to transduce tumor suppressor signaling. It is becoming evident that tyrosine phosphorylation is a critical regulator of the WW proteins. Here, we review the current knowledge on the involved tyrosine kinases and their roles in regulating the WW proteins. PMID:25627656

  13. In vivo and in vitro phosphorylation of the alpha 7/PRS1 subunit of Saccharomyces cerevisiae 20 S proteasome: in vitro phosphorylation by protein kinase CK2 is absolutely dependent on polylysine.

    PubMed

    Pardo, P S; Murray, P F; Walz, K; Franco, L; Passeron, S

    1998-01-15

    In this paper, we show that the Saccharomyces cerevisiae 20 S proteasome subunit 1 (PRS1), recently renamed as alpha 7, is the main in vivo phosphorylated and in vitro CK2-phosphorylatable proteasome component. In vitro phosphorylation occurs only in the presence of polylysine, a characteristic that distinguishes the yeast proteasome from mammalian ones which are phosphorylated by CK2 in the absence of polylysine. A peptide reproducing the long acidic C-terminal tail of alpha 7/PRS1, where consensus CK2 phosphorylation sites are located, was also phosphorylated by the CK2 holoenzyme in a polylysine-dependent manner, suggesting that this region contains structural features responsible for this particular behavior.

  14. A Highly Scalable Peptide-Based Assay System for Proteomics

    PubMed Central

    Kozlov, Igor A.; Thomsen, Elliot R.; Munchel, Sarah E.; Villegas, Patricia; Capek, Petr; Gower, Austin J.; K. Pond, Stephanie J.; Chudin, Eugene; Chee, Mark S.

    2012-01-01

    We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays. PMID:22701568

  15. 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.

  16. Formyl peptides and ATP stimulate Ca2+ and Na+ inward currents through non-selective cation channels via G-proteins in dibutyryl cyclic AMP-differentiated HL-60 cells. Involvement of Ca2+ and Na+ in the activation of beta-glucuronidase release and superoxide production.

    PubMed

    Krautwurst, D; Seifert, R; Hescheler, J; Schultz, G

    1992-12-15

    In human neutrophils, the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) induces increases in the intracellular free Ca2+ concentration ([Ca2+]i) with subsequent activation of beta-glucuronidase release and superoxide (O2-) production. Results from several laboratories suggest that the increase in [Ca2+]i is due to activation of non-selective cation (NSC) channels. We studied the biophysical characteristics, pharmacological modulation and functional role of NSC channels in dibutyryl cyclic AMP (Bt2cAMP)-differentiated HL-60 cells. fMLP increased [Ca2+]i by release of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular space. fMLP also induced Mn2+ influx. Ca2+ and Mn2+ influxes were inhibited by 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365). Under whole-cell voltage-clamp conditions, fMLP and ATP (a purinoceptor agonist) activated inward currents characterized by a linear current-voltage relationship and a reversal potential near 0 mV. NSC channels were substantially more permeable to Na+ than to Ca2+. SK&F 96365 inhibited fMLP- and ATP-stimulated currents with a half-maximal effect at about 3 microM. Pertussis toxin prevented stimulation by fMLP of NSC currents and reduced ATP-stimulated currents by about 80%. Intracellular application of the stable GDP analogue, guanosine 5'-O-[2-thio]diphosphate, completely blocked stimulation by agonists of NSC currents. In excised inside-out patches, single channel openings with an amplitude of 0.24 pA were observed in the presence of fMLP and the GTP analogue, guanosine 5'-O-[3-thio]triphosphate. The bath solution contained neither Ca2+ nor ATP. The current/voltage relationship was linear with a conductance of 4-5 pS and reversed at about 0 mV. fMLP-induced beta-glucuronidase release and O2- production were substantially reduced by replacement of extracellular CaCl2 or NaCl by ethylenebis(oxyethylenenitrilo)tetra-acetic acid and

  17. Phosphorylation of the human Fhit tumor suppressor on tyrosine 114 in Escherichia coli and unexpected steady state kinetics of the phosphorylated forms.

    PubMed

    Garrison, Preston N; Robinson, Angela K; Pekarsky, Yuri; Croce, Carlo M; Barnes, Larry D

    2005-04-26

    The human tumor suppressor Fhit is a homodimeric histidine triad (HIT) protein of 147 amino acids which has Ap(3)A hydrolase activity. We have recently discovered that Fhit is phosphorylated in vivo and is phosphorylated in vitro by Src kinase [Pekarsky, Y., Garrison, P. N., Palamarchuk, A., Zanesi, N., Aqeilan, R. I., Huebner, K., Barnes, L. D., and Croce, C. M. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 3775-3779]. Now we have coexpressed Fhit with the elk tyrosine kinase in Escherichia coli to generate phosphorylated forms of Fhit. Unphosphorylated Fhit, Fhit phosphorylated on one subunit, and Fhit phosphorylated on both subunits were purified to apparent homogeneity by column chromatography on anion-exchange and gel filtration resins. MALDI-TOF and HPLC-ESI tandem mass spectrometry of intact Fhit and proteolytic peptides of Fhit demonstrated that Fhit is phosphorylated on Y(114) on either one or both subunits. Monophosphorylated Fhit exhibited monophasic kinetics with K(m) and k(cat) values approximately 2- and approximately 7-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic kinetics. One site had K(m) and k(cat) values approximately 2- and approximately 140-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. The second site had a K(m) approximately 60-fold higher and a k(cat) approximately 6-fold lower than the corresponding values for unphosphorylated Fhit. The unexpected kinetic patterns for the phosphorylated forms suggest the system may be enzymologically novel. The decreases in the values of K(m) and k(cat) for the phosphorylated forms in comparison to those of unphosphorylated Fhit favor the formation and lifetime of the Fhit-Ap(3)A complex, which may enhance the tumor suppressor activity of Fhit.

  18. Phosphorylation of tau is regulated by PKN.

    PubMed

    Taniguchi, T; Kawamata, T; Mukai, H; Hasegawa, H; Isagawa, T; Yasuda, M; Hashimoto, T; Terashima, A; Nakai, M; Mori, H; Ono, Y; Tanaka, C

    2001-03-30

    For the phosphorylation state of microtubule-associated protein, tau plays a pivotal role in regulating microtubule networks in neurons. Tau promotes the assembly and stabilization of microtubules. The potential for tau to bind to microtubules is down-regulated after local phosphorylation. When we investigated the effects of PKN activation on tau phosphorylation, we found that PKN triggers disruption of the microtubule array both in vitro and in vivo and predominantly phosphorylates tau in microtubule binding domains (MBDs). PKN has a catalytic domain highly homologous to protein kinase C (PKC), a kinase that phosphorylates Ser-313 (= Ser-324, the number used in this study) in MBDs. Thus, we identified the phosphorylation sites of PKN and PKC subtypes (PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, -zeta, and -lambda) in MBDs. PKN phosphorylates Ser-258, Ser-320, and Ser-352, although all PKC subtypes phosphorylate Ser-258, Ser-293, Ser-324, and Ser-352. There is a PKN-specific phosphorylation site, Ser-320, in MBDs. HIA3, a novel phosphorylation-dependent antibody recognizing phosphorylated tau at Ser-320, showed immunoreactivity in Chinese hamster ovary cells expressing tau and the active form of PKN, but not in Chinese hamster ovary cells expressing tau and the inactive form of PKN. The immunoreactivity for phosphorylated tau at Ser-320 increased in the presence of a phosphatase inhibitor, FK506 treatment, which means that calcineurin (protein phosphatase 2B) may be involved in dephosphorylating tau at Ser-320 site. We also noted that PKN reduces the phosphorylation recognized by the phosphorylation-dependent antibodies AT8, AT180, and AT270 in vivo. Thus PKN serves as a regulator of microtubules by specific phosphorylation of tau, which leads to disruption of tubulin assembly.

  19. Anticancer activities of bovine and human lactoferricin-derived peptides.

    PubMed

    Arias, Mauricio; Hilchie, Ashley L; Haney, Evan F; Bolscher, Jan G M; Hyndman, M Eric; Hancock, Robert E W; Vogel, Hans J

    2017-02-01

    Lactoferrin (LF) is a mammalian host defense glycoprotein with diverse biological activities. Peptides derived from the cationic region of LF possess cytotoxic activity against cancer cells in vitro and in vivo. Bovine lactoferricin (LFcinB), a peptide derived from bovine LF (bLF), exhibits broad-spectrum anticancer activity, while a similar peptide derived from human LF (hLF) is not as active. In this work, several peptides derived from the N-terminal regions of bLF and hLF were studied for their anticancer activities against leukemia and breast-cancer cells, as well as normal peripheral blood mononuclear cells. The cyclized LFcinB-CLICK peptide, which possesses a stable triazole linkage, showed improved anticancer activity, while short peptides hLF11 and bLF10 were not cytotoxic to cancer cells. Interestingly, hLF11 can act as a cell-penetrating peptide; when combined with the antimicrobial core sequence of LFcinB (RRWQWR) through either a Pro or Gly-Gly linker, toxicity to Jurkat cells increased. Together, our work extends the library of LF-derived peptides tested for anticancer activity, and identified new chimeric peptides with high cytotoxicity towards cancerous cells. Additionally, these results support the notion that short cell-penetrating peptides and antimicrobial peptides can be combined to create new adducts with increased potency.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. Small cationic antimicrobial peptidomimetics: emerging candidate for the development of potential anti-infective agents.

    PubMed

    Lohan, Sandeep; Bisht, Gopal Singh

    2013-01-01

    Rapid increase in the emergence and spread of microbes resistant to conventionally used antibiotics has become a major threat to global health care. Antimicrobial peptides (AMPs) are considered as a potential source of novel antibiotics because of their numerous advantages such as broad-spectrum activity, lower tendency to induce resistance, immunomodulatory response and unique mode of action. However, AMPs have several drawbacks such as; susceptibility to protease degradation, toxicity and high costs of manufacturing. Therefore, extensive research efforts are underway to explore the therapeutic potential of these fascinating natural compounds. This review highlights the potential of small cationic antimicrobial peptidomimetics (SCAMPs; M.W. ≅ 700 Da) as new generation antibiotics. In particular, we focused on recently identified small active pharmacophore from bulky templates of native AMPs, β-peptides, and lipopeptides. In addition, various design strategies recently undertaken to improve the physicochemical properties (proteolytic stability & plasma protein binding) of small cationic peptides have also been discussed.

  5. 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.

  6. HF‐Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization

    PubMed Central

    Raz, Richard; Burlina, Fabienne; Ismail, Mohamed; Downward, Julian; Li, Jiejin; Smerdon, Stephen J.; Quibell, Martin; White, Peter D.

    2016-01-01

    Abstract We have developed a convenient method for the direct synthesis of peptide thioesters, versatile intermediates for peptide ligation and cyclic peptide synthesis. The technology uses a modified Boc SPPS strategy that avoids the use of anhydrous HF. Boc in situ neutralization protocols are used in combination with Merrifield hydroxymethyl resin and TFA/TMSBr cleavage. Avoiding HF extends the scope of Boc SPPS to post‐translational modifications that are compatible with the milder cleavage conditions, demonstrated here with the synthesis of the phosphorylated protein CHK2. Peptide thioesters give easy, direct, access to cyclic peptides, illustrated by the synthesis of cyclorasin, a KRAS inhibitor. PMID:27654901

  7. 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 ...

  8. Analysis of the in vivo phosphorylation state of rabbit skeletal muscle glycogen synthase by fast-atom-bombardment mass spectrometry.

    PubMed

    Poulter, L; Ang, S G; Gibson, B W; Williams, D H; Holmes, C F; Caudwell, F B; Pitcher, J; Cohen, P

    1988-08-15

    The in vivo phosphorylation state of glycogen synthase was re-examined by fast-atom-bombardment mass spectrometry and a procedure in which phosphoserine residues are first converted to S-ethylcysteine. In animals injected with the beta-adrenergic antagonist propranolol, the phosphorylation sites in the N-terminal (N) and C-terminal (C) cyanogen bromide peptides were identified as the serine residues at N7, the region C28-C39, C42, C46 and C100. In animals injected with adrenalin, the phosphorylation of N7 increased from 0.6 to 0.8 mol/mol, the region C28-C39 from 0.7 to 1.2 mol/mol and C100 from 0.3 to 0.6 mol/mol. The phosphorylation states of C42 (0.7 mol/mol) and C46 (0.9 mol/mol) were unchanged. In addition, two further serine residues became phosphorylated at positions N10 (0.5 mol/mol) and C87 (0.5 mol/mol), which were not phosphorylated in the absence of adrenalin. Residues N10 and C42 have not been recognized as in vivo sites of phosphorylation previously. The results suggest that N10 is phosphorylated by a novel protein kinase which may be activated by cyclic-AMP-dependent protein kinase. The phosphorylation of C42 is likely to be catalysed by glycogen synthase kinase 3. The protein kinases responsible for phosphorylating N7, the region C28-C39, C46, C87 and C100 in vivo and the molecular mechanisms by which adrenalin inactivates glycogen synthase in vivo are discussed. Residue N3, a major site phosphorylated by casein kinase-I in vitro is not phosphorylated in vivo. This and other evidence indicates that casein kinase-I is not a glycogen synthase kinase in vivo.

  9. Sulfur Pentafluoride is a Preferred Reagent Cation for Negative Electron Transfer Dissociation.

    PubMed

    Rush, Matthew J P; Riley, Nicholas M; Westphall, Michael S; Syka, John E P; Coon, Joshua J

    2017-03-27

    Negative mode proteome analysis offers access to unique portions of the proteome and several acidic post-translational modifications; however, traditional collision-based fragmentation methods fail to reliably provide sequence information for peptide anions. Negative electron transfer dissociation (NETD), on the other hand, can sequence precursor anions in a high-throughput manner. Similar to other ion-ion methods, NETD is most efficient with peptides of higher charge state because of the increased electrostatic interaction between reacting molecules. Here we demonstrate that NETD performance for lower charge state precursors can be improved by altering the reagent cation. Specifically, the recombination energy of the NETD reaction-largely dictated by the ionization energy (IE) of the reagent cation-can affect the extent of fragmentation. We compare the NETD reagent cations of C16H10(●+) (IE = 7.9 eV) and SF5(●+) (IE = 9.6 eV) on a set of standard peptides, concluding that SF5(●+) yields greater sequence ion generation. Subsequent proteome-scale nLC-MS/MS experiments comparing C16H10(●+) and SF5(●+) further supported this outcome: analyses using SF5(●+) yielded 4637 peptide spectral matches (PSMs) and 2900 unique peptides, whereas C16H10(●+) produced 3563 PSMs and 2231 peptides. The substantive gain in identification power with SF5(●+) was largely driven by improved identification of doubly deprotonated precursors, indicating that increased NETD recombination energy can increase product ion yield for low charge density precursors. This work demonstrates that SF5(●+) is a viable, if not favorable, reagent cation for NETD, and provides improved fragmentation over the commonly used fluoranthene reagent. Graphical Abstract ᅟ.

  10. C-Peptide Test

    MedlinePlus

    ... vital for the body to use its main energy source, glucose . Since C-peptide and insulin are produced ... these cases, C-peptide measurement is a useful alternative to testing for insulin. C-peptide measurements can ...

  11. Neuromedin B receptors regulate EGF receptor tyrosine phosphorylation in lung cancer cells

    PubMed Central

    Moody, Terry W.; Berna, Marc J.; Mantey, Samuel; Sancho, Veronica; Ridnour, Lisa; Wink, David A.; Chan, Daniel; Giaccone, Giuseppe; Jensen, Robert T.

    2014-01-01

    Neuromedin B (NMB), a member of the bombesin family of peptides, is an autocrine growth factor for many lung cancer cells. The present study investigated the ability of NMB to cause transactivation of the epidermal growth factor (EGF) receptor in lung cancer cells. By Western blot, addition of NMB or related peptides to NCI-H1299 human non-small cell lung cancer (NSCLC) cells, caused phosphorylation of Tyr1068 of the EGF receptor. The signal was amplified using NCI-H1299 cells stably transected with NMB receptors. The transactivation of the EGF receptor or the tyrosine phosphorylation of ERK caused by NMB-like peptides was inhibited by AG1478 or gefitinib (tyrosine kinase inhibitors) and NMB receptor antagonist PD168368 but not the GRP receptor antagonist, BW2258U89. The transactivation of the EGF receptor caused by NMB-like peptides was inhibited by GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), or transforming growth factor (TGF)α antibody. The transactivation of the EGF receptor and the increase in reactive oxygen species caused by NMB-like peptides was inhibited by N-acetylcysteine (NAC) or Tiron. Gefitinib inhibited the proliferation of NCI-H1299 cells and its sensitivity was increased by the addition of PD168368. The results indicate that the NMB receptor regulates EGF receptor transactivation by a mechanism dependent on Src as well as metalloprotease activation and generation of reactive oxygen species. PMID:20388507

  12. gCap39 is phosphorylated. Stimulation by okadaic acid and preferential association with nuclei.

    PubMed

    Onoda, K; Yin, H L

    1993-02-25

    gCap39 is an actin filament end-capping protein which is a member of the gelsolin family. Unlike gelsolin, gCap39 does not sever actin filaments and is a cytoplasmic as well as nuclear protein. We report here that gCap39 is phosphorylated, while gelsolin is not. gCap39 is phosphorylated on serines and threonines at multiple sites, and phospho-gCap39 is resolved by isofocusing into multiple isoforms which are more acidic than unphosphorylated gCap39. In vitro dephosphorylation eliminates the acidic isoforms. Okadaic acid, a protein phosphatase inhibitor, stimulates gCap39 phosphorylation in vivo. It preferentially increases labeling of several peptides and enhances labeling of phosphothreonines relative to phosphoserines. The phosphorylation state of gCap39 in cells is therefore regulated by a balance between kinases and okadaic acid-sensitive phosphatases, and phosphorylation sites containing threonines appear to be particularly sensitive to the phosphatases. Subcellular fractionation shows that the nuclear fraction contains 17 +/- 5% (n = 3) of total gCap39. Compared with the soluble cytoplasm, nuclear gCap39 has a 1.7 +/- 0.2 (n = 3) fold increase in the amount of 32P label incorporation and a higher ratio of acidic/basic gCap39. We conclude that phospho-gCap39 is preferentially associated with nuclei and suggest that phosphorylation of gCap39 is functionally significant.

  13. Phosphorylation of multifunctional galectins by protein kinases CK1, CK2, and PKA.

    PubMed

    Kübler, Dieter; Seidler, Jörg; André, Sabine; Kumar, Sonu; Schwartz-Albiez, Reinhard; Lehmann, Wolf-Dieter; Gabius, Hans-Joachim

    2014-03-15

    Phosphorylation is known to have a strong impact on protein functions. We analyzed members of the lectin family of multifunctional galectins as targets of the protein kinases CK1, CK2, and PKA. Galectins are potent growth regulators able to bind both glycan and peptide motifs at intra- and extracellular sites. Performing in vitro kinase assays, galectin phosphorylation was detected by phosphoprotein staining and autoradiography. The insertion of phosphoryl groups varied to a large extent depending on the type of kinase applied and the respective galectin substrate. Sites of phosphorylation observed in the recombinant galectins were determined by a strategic combination of phosphopeptide enrichment and nano-ultra-performance liquid chromatography tandem mass spectrometry (nanoUPLC-MS/MS). By in silico modeling, phosphorylation sites were visualized three-dimensionally. Our results reveal galectin-type-specific Ser-/Thr-dependent phosphorylation beyond the known example of galectin-3. These data are the basis for functional studies and also illustrate the analytical sensitivity of the applied methods for further work on human lectins.

  14. 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

  15. 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.

  16. 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.

  17. Cation affinity numbers of Lewis bases.

    PubMed

    Lindner, Christoph; Tandon, Raman; Maryasin, Boris; Larionov, Evgeny; Zipse, Hendrik

    2012-01-01

    Using selected theoretical methods the affinity of a large range of Lewis bases towards model cations has been quantified. The range of model cations includes the methyl cation as the smallest carbon-centered electrophile, the benzhydryl and trityl cations as models for electrophilic substrates encountered in Lewis base-catalyzed synthetic procedures, and the acetyl cation as a substrate model for acyl-transfer reactions. Affinities towards these cationic electrophiles are complemented by data for Lewis-base addition to Michael acceptors as prototypical neutral electrophiles.

  18. An improved method for utilization of peptide substrates for antibody characterization and enzymatic assays.

    PubMed

    Ghosh, Inca; Sun, Luo; Evans, Thomas C; Xu, Ming-Qun

    2004-10-01

    Synthetic peptides have become an important tool in antibody production and enzyme characterization. The small size of peptides, however, has hindered their use in assays systems, such as Western blots, and as immunogens. Here, we present a facile method to improve the properties of peptides for multiple applications by ligating the peptides to intein-generated carrier proteins. The stoichiometric ligation of peptide and carrier achieved by intein-mediated protein ligation (IPL) results in the ligation product migrating as a single band on a SDS-PAGE gel. The carrier proteins, HhaI methylase (M.HhaI) and maltose-binding protein (MBP), were ligated to various peptides; the ligated carrier-peptide products gave sharp, reproducible bands when used as positive controls for antibodies raised against the same peptides during Western blot analysis. We further show that ligation of the peptide antigens to a different thioester-tagged carrier protein, paramyosin, produced immunogens for the production of antisera in rabbits or mice. Furthermore, we demonstrate the generation of a substrate for enzymatic assays by ligating a peptide containing the phosphorylation site for Abl protein tyrosine kinase to a carrier protein. This carrier-peptide protein was used as a kinase substrate that could easily be tested for phosphorylation using a phosphotyrosine antibody in Western blot analysis. These techniques do not require sophisticated equipment, reagents, or skills thereby providing a simple method for research and development.

  19. 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.

  20. Matrix-assisted laser desorption mass spectrometry of gas-phase peptide-metal complexes

    NASA Astrophysics Data System (ADS)

    Hortal, Ana R.; Hurtado, Paola; Martínez-Haya, Bruno

    2008-12-01

    Cation attachment to a model peptide has been investigated in matrix-assisted laser desorption experiments. Angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) is chosen as a system for study, and Cu2+ and K+ salts are used as cationizing agents. Three fundamentally different types of samples are investigated: (1) a crystalline sample of Ang I, metal salt and MALDI matrix, prepared with the conventional dried droplet method; (2) a solvent-free fine powder mixture of the same three compounds, and (3) a solution of the angiotensin and the metal salt in an ionic liquid matrix (a molten organic salt that acts as a MALDI active solvent). Effective protonation and cationization of the peptide are achieved with the three methods. The transition metal systematically provides more efficient cationization than the alkali metal. At sufficiently high concentration of the salt, the attachment of up to four copper cations to the angiotensin is observed in the MALDI spectrum. In contrast, only one K+ cation is efficiently bound to the peptide. For a given salt concentration, the highest degree of cationization is obtained in the laser desorption from the ionic liquid matrix. This is attributed to the efficient transfer of free metal cations to the desorption plume, where the complexation takes place.

  1. Post-translational phosphorylation affects the IgE binding capacity of caseins.

    PubMed

    Bernard, H; Meisel, H; Creminon, C; Wal, J M

    2000-02-11

    IgE response specific to those molecular regions of casein that contain a major phosphorylation site was analyzed using native and modified caseins and derived peptides. This study included (i) the naturally occurring common variants A1 and A from beta- and alphas2-caseins, respectively, which were purified in the native form and then dephosphorylated, (ii) a purified rare variant D of alphas2-casein which lacks one major phosphorylation site, and (iii) the native and dephosphorylated tryptic fragment f(1-25) from beta-casein. Direct and indirect ELISA using sera from patients allergic to milk showed that the IgE response to caseins is affected by modifying or eliminating the major phosphorylation site.

  2. 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

    2016-06-03

    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.

  3. Tyrosine-specific phosphorylation of calmodulin by the insulin receptor kinase purified from human placenta.

    PubMed Central

    Sacks, D B; Fujita-Yamaguchi, Y; Gale, R D; McDonald, J M

    1989-01-01

    It has previously been demonstrated that calmodulin can be phosphorylated in vitro and in vivo by both tyrosine-specific and serine/threonine protein kinase. We demonstrate here that the insulin receptor tyrosine kinase purified from human placenta phosphorylates calmodulin. The highly purified receptors (prepared by insulin-Sepharose chromatography) were 5-10 times more effective in catalysing the phosphorylation of calmodulin than an equal number of partially purified receptors (prepared by wheat-germ agglutinin-Sepharose chromatography). Phosphorylation occurred exclusively on tyrosine residues, up to a maximum of 1 mol [0.90 +/- 0.14 (n = 5)] of phosphate incorporated/mol of calmodulin. Phosphorylation of calmodulin was dependent on the presence of certain basic proteins and divalent cations. Some of these basic proteins, i.e. polylysine, polyarginine, polyornithine, protamine sulphate and histones H1 and H2B, were also able to stimulate the phosphorylation of calmodulin via an insulin-independent activation of the receptor tyrosine kinase. Addition of insulin further increased incorporation of 32P into calmodulin. The magnitude of the effect of insulin was dependent on the concentration and type of basic protein used, ranging from 0.5- to 9.0-fold stimulation. Maximal phosphorylation of calmodulin was obtained at an insulin concentration of 10(-10) M, with half-maximal effect at 10(-11) M. Either Mg2+ or Mn2+ was necessary to obtain phosphorylation, but Mg2+ was far more effective than Mn2+. In contrast, maximal phosphorylation of calmodulin was observed in the absence of Ca2+. Inhibition of phosphorylation was observed as free Ca2+ concentration exceeded 0.1 microM, with almost complete inhibition at 30 microM free Ca2+. The Km for calmodulin was approx. 0.1 microM. To gain further insight into the effects of basic proteins in this system, we examined the binding of calmodulin to the insulin receptor and the polylysine. Calmodulin binds to the insulin

  4. Identification of Phosphorylation-Dependent Binding Partners of Aquaporin-2 Using Protein Mass Spectrometry

    PubMed Central

    Zwang, Nicholas A.; Hoffert, Jason D.; Pisitkun, Trairak; Moeller, Hanne B.; Fenton, Robert A.; Knepper, Mark A.

    2008-01-01

    Vasopressin-mediated control of water permeability in the renal collecting duct occurs in part through regulation of the distribution of aquaporin-2 (AQP2) between the apical plasma membrane and intracellular membrane compartments. Phosphorylation of Ser-256 at AQP2's cytoplasmic COOH-terminus is well-accepted as a critical step for translocation. The aim of this study was to identify binding partners to phosphorylated versus nonphosphorylated forms of the AQP2 COOH-terminus via a targeted comparative proteomic approach. Cytosol from inner medullary collecting ducts isolated from rat kidneys was incubated with “bait” peptides, representing the COOH-terminal AQP2 tail in its nonphosphorylated and phosphorylated forms, to capture differentially-bound proteins prior to LC-MS/MS analysis. Mass spectrometric results were confirmed by immunoblotting. Immunoprecipitation was performed using an AQP2 COOH-terminal antibody combined with immunblotting against the proposed binding partners in order to demonstrate interactions with native AQP2. Our studies confirmed previously identified interactions between AQP2 and hsc70, hsp70−1 and −2, as well as annexin II. These proteins were found to bind less to the Ser-256-phosphorylated AQP2 than to the non-phosphorylated form. In contrast, another heat shock protein, hsp70−5 (BiP/grp78), bound to phosphorylated AQP2 more avidly than to non-phosphorylated AQP2. Immunogold EM studies demonstrated that BiP is present not only in the ER, but in the cytoplasm and apical plasma membrane of rat collecting duct cells. Furthermore, confocal immunofluorescence studies showed partial colocalization of BiP with AQP2 in non-ER compartments. These results suggest that phosphorylation of AQP2 at Ser-256 may regulate AQP2 trafficking in part by mediating differential binding of hsp70 family proteins to the COOH-terminal tail. PMID:19209902

  5. Protein kinase A activation of the surfactant protein B gene is mediated by phosphorylation of thyroid transcription factor 1.

    PubMed

    Yan, C; Whitsett, J A

    1997-07-11

    Thyroid transcription factor 1 (TTF-1) is a homeodomain-containing nuclear transcription factor expressed in epithelial cells of the lung and thyroid. TTF-1 binds to and activates the transcription of genes expressed selectively in the respiratory epithelium including pulmonary surfactant A, B, C and Clara cell secretory protein. Transfection with a plasmid encoding the cyclic AMP-dependent protein kinase (protein kinase A; PKA) catalytic subunit, Cat-beta, stimulated the phosphorylation of a TTF-1-flag fusion protein 6-7-fold in H441 pulmonary adenocarcinoma cells. Recombinant TTF-1 was phosphorylated by purified PKA catalytic subunit in the presence of [gamma-32P]ATP. PKA catalytic subunit family members, Cat-alpha and Cat-beta, markedly enhanced the transcriptional activation of surfactant B gene promoters by TTF-1 in vitro. Peptide mapping was used to identify a PKA phosphorylation site at the NH2 terminus of TTF-1. A 17-amino acid synthetic peptide comprising this site completely inhibited the PKA-dependent phosphorylation of TTF-1 in vitro. A substitution mutation of TTF-1 (Thr9 two head right arrow Ala) abolished phosphorylation by PKA and reduced transactivation of the surfactant B gene promoter. Transfection with a plasmid encoding the cAMP regulatory element binding factor inhibited transcriptional activity of the surfactant protein B gene promoter. Phosphorylation of TTF-1 mediates PKA-dependent activation of surfactant protein B gene transcription.

  6. Novel Antifungal Peptides Produced by Leuconostoc mesenteroides DU15 Effectively Inhibit Growth of Aspergillus niger.

    PubMed

    Muhialdin, Belal J; Hassan, Zaiton; Abu Bakar, Fatimah; Algboory, Hussein L; Saari, Nazamid

    2015-05-01

    The ability of Leuconostoc mesenteroides DU15 to produce antifungal peptides that inhibit growth of Aspergillus niger was evaluated under optimum growth conditions of 30 °C for 48 h. The cell-free supernatant showed inhibitory activity against A. niger. Five novel peptides were isolated with the sequences GPFPL, YVPLF, LLHGVPLP, GPFPLEMTLGPT, and TVYPFPGPL as identified by de novo sequencing using PEAKS 6 software. Peptide LLHGVPLP was the only positively charged (cationic peptides) and peptide GPFPLEMTLGPT negatively charged (anionic), whereas the rest are neutral. The identified peptides had high hydrophobicity ratio and low molecular weights with amino acids sequences ranging from 5 to 12 residues. The mode of action of these peptides is observed under the scanning electron microscope and is due to cell lysis of fungi. This work reveals the potential of peptides from L. mesenteroides DU15 as natural antifungal preservatives in inhibiting the growth of A. niger that is implicated to the spoilage during storage.

  7. 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.

    PubMed Central

    Häring, 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, we labeled intact rat fat cells with [32P]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, we 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. The insulin effect starts after 30 sec, is maximal at 150 sec, and declines to almost basal values by 5 min. Furthermore, the antiphosphotyrosine antibody precipitated at least five proteins in the soluble fraction of the fat cell. Insulin (0.5 nM, 100 nM) stimulated within 2 min the 32P incorporation into a 116-kDa band, a 62-kDa band, and three bands between 45 kDa and 50 kDa 2- to 10-fold. We 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. Images PMID:3540953

  8. Regulation of phosphorylation of nicotinic acetylcholine receptors in mouse BC3H1 myocytes

    SciTech Connect

    Smith, M.M.; Merlie, J.P.; Lawrence, J.C. Jr.

    1987-09-01

    By using /sup 32/P-labeling methods and performing immunoprecipitations with specific antibodies, the authors have found that three subunits of the nicotinic acetylcholine receptor and phosphorylated in mouse skeletal muscle cells. In nonstimulated cells, the molar ratios of phosphate estimated in ..cap alpha.., ..beta.., and delta subunits were 0.02, 0.05, and 0.5, respectively. All three subunits contained predominantly phosphoserine with some phosphothreonine; the ..beta.., subunit also contained phosphotyrosine. Incubating cells with agents that stimulate cAMP-dependent pathways (isoproterenol, forskolin, 8-Br-cAMP) increased the phosphorylation of the delta subunit by 50%, but phosphate labeling of the ..beta.. subunit was depressed by a third. In contrast, when cells were incubated with the divalent cation ionophores A-23187 or ionomycin, phosphorylation of both the delta and ..beta.. subunits increased. The results indicate that acetylcholine receptors are phosphorylated to significant levels in skeletal muscle cells and that cAMP-dependent and Ca/sup 2 +/-dependent pathways exist for controlling the phosphorylation state of the receptor subunits.

  9. Beta-endorphin chimeric peptides: Transport through the blood-brain barrier in vivo and cleavage of disulfide linkage by brain

    SciTech Connect

    Pardridge, W.M.; Triguero, D.; Buciak, J.L. )

    1990-02-01

    Water soluble peptides are normally not transported through the blood-brain barrier (BBB). Chimeric peptides may be transportable through the BBB and are formed by the covalent coupling of a nontransportable peptide to a transportable peptide vector, e.g. cationized albumin, using disulfide-based coupling reagents such as N-succinimidyl 3-(2-pyridyldithio(propionate)) (SPDP). The transcytosis of peptide into brain parenchyma, as opposed to vascular sequestration of blood-borne peptide, was quantified using an internal carotid artery perfusion/capillary depletion method. It is shown that (125I)beta-endorphin is not transported through the BBB, but is rapidly cleaved to free (125I) tyrosine via capillary peptidase. Therefore, chimeric peptide was prepared using (125I) (D-Ala2)beta-endorphin (DABE), owing to the resistance of this analogue to peptidase degradation. The (125I) DABE-cationized albumin chimeric peptide is shown to enter brain parenchyma at a rate comparable to that reported previously for unconjugated cationized albumin. When the (125I) DABE-cationized albumin chimeric peptide was incubated with rat brain homogenate at 37 C, the free (125I) DABE was liberated from the cationized albumin conjugate prior to its subsequent degradation into free (125I) tyrosine. Approximately 50% of the chimeric peptide was cleaved within 60 sec of incubation at 37 C. These studies demonstrate that (1) (125I)beta-endorphin is not transported through the BBB in its unconjugated form, (2) a (125I) DABE-cationized albumin chimeric peptide is transported through the BBB into brain parenchyma at a rate comparable to the unconjugated cationized albumin, and (3) brain contains the necessary disulfide reductases for rapid cleavage of the chimeric peptide into free beta-endorphin and this cleavage occurs before degradation of the (125I) DABE into (125I) tyrosine.

  10. Alterations of molecular and behavioral responses to cocaine by selective inhibition of Elk-1 phosphorylation.

    PubMed

    Besnard, Antoine; Bouveyron, Nicolas; Kappes, Vincent; Pascoli, Vincent; Pagès, Christiane; Heck, Nicolas; Vanhoutte, Peter; Caboche, Jocelyne

    2011-10-05

    Activation of the extracellular signal-regulated kinase (ERK) signaling pathway in the striatum is crucial for molecular adaptations and long-term behavioral alterations induced by cocaine. In response to cocaine, ERK controls the phosphorylation levels of both mitogen and stress-activated protein kinase 1 (MSK-1), a nuclear kinase involved in histone H3 (Ser10) and cAMP response element binding protein phosphorylation, and Elk-1, a transcription factor involved in serum response element (SRE)-driven gene regulations. We recently characterized the phenotype of msk-1 knock-out mice in response to cocaine. Herein, we wanted to address the role of Elk-1 phosphorylation in cocaine-induced molecular, morphological, and behavioral responses. We used a cell-penetrating peptide, named TAT-DEF-Elk-1 (TDE), which corresponds to the DEF docking domain of Elk-1 toward ERK and inhibits Elk-1 phosphorylation induced by ERKs without modifying ERK or MSK-1 in vitro. The peptide was injected in vivo before cocaine administration in mice. Immunocytochemical, molecular, morphological, and behavioral studies were performed. The TDE inhibited Elk-1 and H3 (Ser10) phosphorylation induced by cocaine, sparing ERK and MSK-1 activation. Consequently, TDE altered cocaine-induced regulation of genes bearing SRE site(s) in their promoters, including c-fos, zif268, ΔFosB, and arc/arg3.1 (activity-regulated cytoskeleton-associated protein). In a chronic cocaine administration paradigm, TDE reversed cocaine-induced increase in dendritic spine density. Finally, the TDE delayed the establishment of cocaine-induced psychomotor sensitization and conditioned-place preference. We conclude that Elk-1 phosphorylation downstream from ERK is a key molecular event involved in long-term neuronal and behavioral adaptations to cocaine.

  11. Phosphorylation of Ser8 promotes zinc-induced dimerization of the amyloid-β metal-binding domain.

    PubMed

    Kulikova, Alexandra A; Tsvetkov, Philipp O; Indeykina, Maria I; Popov, Igor A; Zhokhov, Sergey S; Golovin, Andrey V; Polshakov, Vladimir I; Kozin, Sergey A; Nudler, Evgeny; Makarov, Alexander A

    2014-10-01

    Zinc-induced aggregation of the amyloid-β peptide (Aβ) is a hallmark molecular feature of Alzheimer's disease (AD). Recently it was shown that phosphorylation of Aβ at Ser8 promotes the formation of toxic aggregates. In this work, we have studied the impact of Ser8 phosphorylation on the mode of zinc interaction with the Aβ metal-binding domain 1-16 using isothermal titration calorimetry, electrospray ionization mass spectrometry and NMR spectroscopy. We have discovered a novel zinc binding site ((6)HDpS(8)) in the phosphorylated peptide, in which the zinc ion is coordinated by the imidazole ring of His6, the phosphate group attached to Ser8 and a backbone carbonyl group of His6 or Asp7. Interaction of the zinc ion with this site involves His6, thereby withdrawing it from the interaction pattern observed in the non-modified peptide. This event was found to stimulate dimerization of peptide chains through the (11)EVHH(14) site, where the zinc ion is coordinated by the two pairs of Glu11 and His14 in the two peptide subunits. The proposed molecular mechanism of zinc-induced dimerization could contribute to the understanding of initiation of pathological Aβ aggregation, and the (11)EVHH(14) tetrapeptide can be considered as a promising drug target for the prevention of amyloidogenesis.

  12. [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.

  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.

  14. Heavy metal cations permeate the TRPV6 epithelial cation channel.

    PubMed

    Kovacs, Gergely; Danko, Tamas; Bergeron, Marc J; Balazs, Bernadett; Suzuki, Yoshiro; Zsembery, Akos; Hediger, Matthias A

    2011-01-01

    TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. This calcium-selective channel is highly expressed in the duodenum and the placenta, being responsible for calcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not only permeable to calcium but also to other divalent cations in epithelial tissues. In this study, we tested whether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basal intracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6 increased the permeability for Ca(2+), Ba(2+), Sr(2+), Mn(2+), Zn(2+), Cd(2+), and interestingly also for La(3+) and Gd(3+). These results were confirmed using the patch clamp technique. (45)Ca uptake experiments showed that cadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calcium influx at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium transport but also in the transport of other divalent cations, including heavy metal ions, which may have toxicological implications.

  15. Determinants of chymotrypsin C cleavage specificity in the calcium binding loop of human cationic trypsinogen

    PubMed Central

    Szabó, András; Sahin-Tóth, Miklós

    2013-01-01

    SUMMARY The pancreatic serine protease chymotrypsin C (CTRC) cleaves the Leu81-Glu82 peptide bond in the calcium binding loop of human cationic trypsinogen and thereby promotes its degradation. This serves as a protective mechanism against ectopic trypsinogen activation in the pancreas. In the present study we demonstrate that cleavage of the Leu81-Glu82 peptide bond by CTRC is highly specific and other human pancreatic chymotrypsins (CTRB1, CTRB2, CTRL1) and elastases (ELA2A, ELA3A, ELA3B) do not catalyze this reaction. To elucidate the mechanistic basis for CTRC specificity, we surveyed the primary (P1) cleavage preference of these pancreatic proteases on peptide substrates. We found that CTRC cleaved after a P1 Leu with at least 10-fold higher catalytic efficiency than other enzymes tested. To assess extended subsite interactions, we introduced Ala-mutations into human cationic trypsinogen at the P3, P1' P3' and P4' amino-acid positions, where P1-P1' corresponds to Leu81-Glu82. Interestingly, CTRC mediated cleavage was stimulated 3-fold by mutation E82A and unaffected by mutations E79A and N84A, but all three mutations compromised specificity and resulted in increased cleavage by ELA2A. Mutation E85A decreased CTRC cleavage by 2-fold. Remarkably, other chymotrypsins and elastases did not cleave human cationic trypsinogen even with the L81F or L81A mutations, which introduced favorable P1 residues for these enzymes. We conclude that specific cleavage of the Leu81-Glu82 peptide bond in human cationic trypsinogen by CTRC is primarily determined by its distinctively high activity on leucyl peptide bonds, whereas the P1' Glu82, P3' Asn84 and P4' Glu85 residues serve as additional specificity determinants. PMID:23035638

  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. An insulin-sensitive cytosolic protein kinase accounts for the regulation of ATP citrate-lyase phosphorylation.

    PubMed Central

    Yu, K T; Benjamin, W B; Ramakrishna, S; Khalaf, N; Czech, M P

    1990-01-01

    Purified rat liver ATP citrate-lyase is phosphorylated on serine residues by an insulin-stimulated cytosolic kinase activity partially purified from rat adipocytes [Yu, Khalaf & Czech (1987) J. Biol. Chem. 262, 16677-16685]. The Km for lyase phosphorylation by this hormone-sensitive kinase activity is approx. 3 microM. Two-dimensional tryptic-peptide mapping of the 32P-labelled lyase reveals that the kinase-catalysed phosphorylation occurs primarily on a specific peptide. In intact 32P-labelled adipocytes, insulin enhances the serine phosphorylation of ATP citrate-lyase by 2-3-fold. Tryptic digestion of the 32P-labelled lyase immunopurified from insulin-treated adipocytes also yields one major phosphopeptide. 32P-labelled lyase tryptic peptides derived from labelling experiments in vitro and in vivo exhibit identical electrophoretic and chromatographic migration profiles. Furthermore, radio-sequencing of the phosphopeptide from lyase 32P-labelled in vitro indicates that serine-3 from the N-terminus is phosphorylated by the insulin-stimulated cytosolic kinase, in agreement with previous studies on the position of the phosphoserine residue in ATP citrate-lyase isolated from insulin-treated cells. Taken together, the similarity in site-specific phosphorylation of ATP citrate-lyase from insulin-treated adipocytes to that catalysed by the hormone-activated cytosolic kinase in vitro strongly suggests that this kinase mediates insulin action on lyase phosphorylation in intact cells. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2114095

  18. Cationic electrodepositable coating composition comprising lignin

    DOEpatents

    Fenn, David; Bowman, Mark P; Zawacky, Steven R; Van Buskirk, Ellor J; Kamarchik, Peter

    2013-07-30

    A cationic electrodepositable coating composition is disclosed. The present invention in directed to a cationic electrodepositable coating composition comprising a lignin-containing cationic salt resin, that comprises (A) the reaction product of: lignin, an amine, and a carbonyl compound; (B) the reaction product of lignin, epichlorohydrin, and an amine; or (C) combinations thereof.

  19. Method for loading lipsomes with ionizable phosphorylated hydrophobic compounds, pharmaceutical preparations and a method for administering the preparations

    DOEpatents

    Mehlhorn, R.J.

    1998-10-27

    A method of entrapping ionizable compounds, preferably phosphorylated hydrophobic compounds, into liposomes having transmembrane gradients is disclosed. The procedures involve forming liposomes in an acidic medium or a basic medium, adding to the acidic medium a cationic compound or to the basic medium an anionic compound and then adding a base to the cationic-containing medium or an acid to the anionic-containing medium, thereby inducing the ionizable compound into the liposomes` internal aqueous phase. The compound-entrapped liposomes prepared in accordance with the disclosed methods may be used as pharmaceutical preparations. Methods of administering such pharmaceutical preparations are also disclosed. 2 figs.

  20. Method for loading lipsomes with ionizable phosphorylated hydrophobic compounds, pharmaceutical preparations and a method for administering the preparations

    DOEpatents

    Mehlhorn, Rolf Joachim

    1998-10-27

    A method of entrapping ionizable compounds, preferably phosphorylated hydrophobic compounds, into liposomes having transmembrane gradients is disclosed. The procedures involve forming liposomes in an acidic medium or a basic medium, adding to the acidic medium a cationic compound or to the basic medium an anionic compound and then adding a base to the cationic-containing medium or an acid to the anionic-containing medium, thereby inducing the ionizable compound into the liposomes' internal aqueous phase. The compound-entrapped liposomes prepared in accordance with the disclosed methods may be used as pharmaceutical preparations. Methods of administering such pharmaceutical preparations are also disclosed.

  1. Homo sapiens dullard protein phosphatase shows a preference for the insulin-dependent phosphorylation site of lipin1.

    PubMed

    Wu, Rui; Garland, Megan; Dunaway-Mariano, Debra; Allen, Karen N

    2011-04-19

    Human lipin1 catalyzes the highly regulated conversion of phosphatidic acids to diacylglycerides. Lipin's cellular location, protein partners, and biological function are directed by phosphorylation-dephosphorylation events catalyzed by the phosphoserine phosphatase dullard. To define the determinants of dullard substrate recognition and catalysis, and hence, lipin regulation, steady-state kinetic analysis was performed on phosphoserine-bearing nonapeptides based on the phosphorylation sites of lipin. The results demonstrate that dullard shows specificity for the peptide corresponding to the insulin-dependent phosphorylation site (Ser106) of lipin with a k(cat)/K(m) of 2.9 × 10(4) M(-1) s(-1). These results are consistent with a coil-loop structure for the insulin-dependent phosphorylation site on human lipin1 and make unlikely the requirement for an adaptor protein to confer activity such as that proposed for the yeast homologue.

  2. Reinventing cell penetrating peptides using glycosylated methionine sulfonium ion sequences

    DOE PAGES

    Kramer, Jessica R.; Schmidt, Nathan W.; Mayle, Kristine M.; ...

    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

  3. 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.

  4. 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

  5. Green synthesis of peptide-templated gold nanoclusters as novel fluorescence probes for detecting protein kinase activity.

    PubMed

    Song, Wei; Liang, Ru-Ping; Wang, Ying; Zhang, Li; Qiu, Jian-Ding

    2015-06-21

    A green method was employed for synthesizing peptide-templated nanoclusters without requiring strong reducing agents. Using synthetic peptide-gold nanoclusters as fluorescence probes, a novel assay for detecting protein kinase is developed based on phosphorylation against carboxypeptidase Y digestion.

  6. Identification of phosphorylated butyrylcholinesterase in human plasma using immunoaffinity purification and mass spectrometry

    SciTech Connect

    Aryal, Uma K.; Lin, Chiann Tso; Kim, Jong Seo; Heibeck, Tyler H.; Wang, Jun; Qian, Weijun; Lin, Yuehe

    2012-04-20

    Paraoxon (diethyl 4-nitrophenyl phosphate) is an active metabolite of the common insecticide parathion and is acutely toxic due to the inhibition of cholinesterase (ChE) activity in the nervous systems. The Inhibition of butyrylcholinesterase (BChE) activity by paraoxon is due to the formation of phosphorylated BChE adduct, and the detection of the phosphorylated BChE adduct in human plasma can serve as an exposure biomarker of organophosphate pesticides and nerve agents. In this study, we performed immunoaffinity purification and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for identifying phosphorylated BChE in human plasma treated by paraoxon. BChE was captured by biotinylated anti-BChE polyclonal antibodies conjugated to streptavidin magnetic beads. Western blot analysis showed that the antibody was effective to recognize both native and modified BChE with high specificity. The exact phosphorylation site of BChE was confirmed on Serine 198 by MS/MS with a 108 Da modification mass and accurately measured parent ion masses. The phosphorylated BChE peptide was also successfully detected in the immunoaffinity purified sample from paraoxon treated human plasma. Thus, immunoaffinity purification combined with mass spectrometry represents a viable approach for the detection of paraoxon-modified BChE and other forms of modified BChE as exposure biomarkers of organophosphates and nerve agents.

  7. Microgravity alters protein phosphorylation changes during initiation of sea urchin sperm motility

    NASA Technical Reports Server (NTRS)

    Tash, J. S.; Bracho, G. E.

    1999-01-01

    European Space Agency (ESA) studies demonstrated that bull sperm swim with higher velocity in microgravity (microG) than at 1 G. Coupling between protein phosphorylation and sperm motility during activation in microG and at 1 G was examined in the ESA Biorack on two space shuttle missions. Immotile sperm were activated to swim (86-90% motility) at launch +20 h by dilution into artificial seawater (ASW). Parallel ground controls were performed 2 h after the flight experiment. Activation after 0, 30, and 60 s was terminated with electrophoresis sample buffer and samples analyzed for phosphoamino acids by Western blotting. Phosphorylation of a 130-kDa phosphothreonine-containing protein (FP130) occurred three to four times faster in microG than at 1 G. A 32-kDa phosphoserine-containing protein was significantly stimulated at 30 s but returned to 1 G control levels at 60 s. The rate of FP130 phosphorylation in microG was attenuated by D2O, suggesting that changes in water properties participate in altering signal transduction. Changes in FP130 phosphorylation triggered by the egg peptide speract were delayed in microG. These results demonstrate that previously observed effects of microG on sperm motility are coupled to changes in phosphorylation of specific flagellar proteins and that early events of sperm activation and fertilization are altered in microG.

  8. Follicle-stimulating hormone-dependent phosphorylation of vimentin in cultures of rat Sertoli cells.

    PubMed Central

    Spruill, W A; Steiner, A L; Tres, L L; Kierszenbaum, A L

    1983-01-01

    Endogenous protein phosphorylation was investigated in cultured rat Sertoli cells after treatment with follicle-stimulating hormone (FSH) and pharmacological agents that activate cAMP-dependent protein kinases. In intact Sertoli cells, both phosphorylation and dephosphorylation of proteins occurred in response to treatment with these agents. Studies using cell-free preparations suggest that four phosphoproteins phosphorylated by cAMP or the catalytic subunit of cAMP-dependent protein kinase were also phosphorylated in a FSH-dependent manner in intact cells. These data suggest that FSH-dependent phosphorylation in Sertoli cells occurs through activation of a cAMP-dependent protein kinase. A FSH-dependent phosphoprotein with a molecular weight of 58,000 was identified as the intermediate filament protein vimentin, based on its migration in two-dimensional gels and its peptide map. The cellular distribution of vimentin was monitored by immunofluorescence in Sertoli cells after treatment with FSH. Results of this study support a role for intermediate filaments in FSH-dependent events in Sertoli cells. Images PMID:6302679

  9. Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation

    NASA Astrophysics Data System (ADS)

    Karim, Christine B.; Michel Espinoza-Fonseca, L.; James, Zachary M.; Hanse, Eric A.; Gaynes, Jeffrey S.; Thomas, David D.; Kelekar, Ameeta

    2015-09-01

    We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics (MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa’s BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC incorporated within the BH3 domain, revealed equilibrium between ordered and dynamically disordered states. Mcl-1 further restricted the ordered component for non-phosphorylated Noxa, but left the pSer13 Noxa profile unchanged. Microsecond MD simulations indicated that the BH3 domain of unphosphorylated Noxa is housed within a flexible loop connecting two antiparallel β-sheets, flanked by disordered N- and C-termini and Ser13 phosphorylation creates a network of salt-bridges that facilitate the interaction between the N-terminus and the BH3 domain. EPR showed that a spin label inserted near the N-terminus was weakly immobilized in unphosphorylated Noxa, consistent with a solvent-exposed helix/loop, but strongly constrained in pSer13 Noxa, indicating a more ordered peptide backbone, as predicted by MD simulations. Together these studies reveal a novel mechanism by which phosphorylation of a distal serine inhibits a pro-apoptotic BH3 domain and promotes cell survival.

  10. The abnormal phosphorylation of tau protein at Ser-202 in Alzheimer disease recapitulates phosphorylation during development.

    PubMed

    Goedert, M; Jakes, R; Crowther, R A; Six, J; Lübke, U; Vandermeeren, M; Cras, P; Trojanowski, J Q; L