Multimarker Strategy for Heart Failure Prognostication. Value of Neurohormonal Biomarkers: Neprilysin vs NT-proBNP.

PubMed

Bayes-Genis, Antoni; Barallat, Jaume; Galán, Amparo; de Antonio, Marta; Domingo, Mar; Zamora, Elisabet; Gastelurrutia, Paloma; Vila, Joan; Peñafiel, Judith; Gálvez-Montón, Carolina; Lupón, Josep

2015-12-01

Neprilysin breaks down numerous vasoactive peptides. The soluble form of neprilysin, which was recently identified in heart failure, is associated with cardiovascular outcomes. Within a multibiomarker strategy, we directly compared soluble neprilysin and N-terminal pro-B-type natriuretic peptide as risk stratifiers in a real-life cohort of heart failure patients. Soluble neprilysin, N-terminal pro-B-type natriuretic peptide, ST2, and high-sensitivity troponin T levels were measured in 797 consecutive ambulatory heart failure patients followed up for 4.7 years. Comprehensive multivariable analyses and soluble neprilysin vs N-terminal pro-B-type natriuretic peptide head-to-head assessments of performance were performed. A primary composite endpoint included cardiovascular death or heart failure hospitalization. A secondary endpoint explored cardiovascular death alone. Median soluble neprilysin and N-terminal pro-B-type natriuretic peptide concentrations were 0.64ng/mL and 1187 ng/L, respectively. Both biomarkers significantly correlated with age (P<.001) and ST2 (P<.001), but only N-terminal pro-B-type natriuretic peptide significantly correlated with estimated glomerular filtration rate (P<.001), body mass index (P<.001), left ventricular ejection fraction (P=.02) and high-sensitivity troponin T (P<.001). In multivariable Cox regression analyses, soluble neprilysin remained independently associated with the composite endpoint (hazard ratio=1.14; 95% confidence interval, 1.02-1.27; P=.03) and cardiovascular death (hazard ratio=1.15; 95% confidence interval, 1.01-1.31; P=.04), but N-terminal pro-B-type natriuretic peptide did not. The head-to-head soluble neprilysin vs N-terminal pro-B-type natriuretic peptide comparison showed good calibration and similar discrimination and reclassification for both neurohormonal biomarkers, but only soluble neprilysin improved overall goodness-of-fit. When added to a multimarker strategy, soluble neprilysin remained an independent prognosticator, while N-terminal pro-B-type natriuretic peptide lost significance as a risk stratifier in ambulatory patients with heart failure. Both biomarkers performed similarly in head-to-head analyses. Copyright © 2015 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

A peptide N-terminal protection strategy for comprehensive glycoproteome analysis using hydrazide chemistry based method

PubMed Central

Huang, Junfeng; Qin, Hongqiang; Sun, Zhen; Huang, Guang; Mao, Jiawei; Cheng, Kai; Zhang, Zhang; Wan, Hao; Yao, Yating; Dong, Jing; Zhu, Jun; Wang, Fangjun; Ye, Mingliang; Zou, Hanfa

2015-01-01

Enrichment of glycopeptides by hydrazide chemistry (HC) is a popular method for glycoproteomics analysis. However, possible side reactions of peptide backbones during the glycan oxidation in this method have not been comprehensively studied. Here, we developed a proteomics approach to locate such side reactions and found several types of the side reactions that could seriously compromise the performance of glycoproteomics analysis. Particularly, the HC method failed to identify N-terminal Ser/Thr glycopeptides because the oxidation of vicinal amino alcohol on these peptides generates aldehyde groups and after they are covalently coupled to HC beads, these peptides cannot be released by PNGase F for identification. To overcome this drawback, we apply a peptide N-terminal protection strategy in which primary amine groups on peptides are chemically blocked via dimethyl labeling, thus the vicinal amino alcohols on peptide N-termini are eliminated. Our results showed that this strategy successfully prevented the oxidation of peptide N-termini and significantly improved the coverage of glycoproteome. PMID:25959593

A Convenient Approach to Synthesizing Peptide C-Terminal N-Alkyl Amides

PubMed Central

Fang, Wei-Jie; Yakovleva, Tatyana; Aldrich, Jane V.

2014-01-01

Peptide C-terminal N-alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C-terminal N-alkyl amides using the well-known Fukuyama N-alkylation reaction on a standard resin commonly used for the synthesis of peptide C-terminal primary amides, the PAL-PEG-PS (Peptide Amide Linker-polyethylene glycol-polystyrene) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N-alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N-alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N-alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N-alkyl amides. PMID:22252422

Isolation and characterization of a cDNA from Cuphea lanceolata encoding a beta-ketoacyl-ACP reductase.

PubMed

Klein, B; Pawlowski, K; Höricke-Grandpierre, C; Schell, J; Töpfer, R

1992-05-01

A cDNA encoding beta-ketoacyl-ACP reductase (EC 1.1.1.100), an integral part of the fatty acid synthase type II, was cloned from Cuphea lanceolata. This cDNA of 1276 bp codes for a polypeptide of 320 amino acids with 63 N-terminal residues presumably representing a transit peptide and 257 residues corresponding to the mature protein of 27 kDa. The encoded protein shows strong homology with the amino-terminal sequence and two tryptic peptides from avocado mesocarp beta-ketoacyl-ACP reductase, and its total amino acid composition is highly similar to those of the beta-ketoacyl-ACP reductases of avocado and spinach. Amino acid sequence homologies to polyketide synthase, beta-ketoreductases and short-chain alcohol dehydrogenases are discussed. An engineered fusion protein lacking most of the transit peptide, which was produced in Escherichia coli, was isolated and proved to possess beta-ketoacyl-ACP reductase activity. Hybridization studies revealed that in C. lanceolata beta-ketoacyl-ACP reductase is encoded by a small family of at least two genes and that members of this family are expressed in roots, leaves, flowers and seeds.

Quantitative analysis of pyroglutamic acid in peptides.

PubMed

Suzuki, Y; Motoi, H; Sato, K

1999-08-01

A simplified and rapid procedure for the determination of pyroglutamic acid in peptides was developed. The method involves the enzymatic cleavage of an N-terminal pyroglutamate residue using a thermostable pyroglutamate aminopeptidase and isocratic HPLC separation of the resulting enzymatic hydrolysate using a column switching technique. Pyroglutamate aminopeptidase from a thermophilic archaebacteria, Pyrococcus furiosus, cleaves N-terminal pyroglutamic acid residue independent of the molecular weight of the substrate. It cleaves more than 85% of pyroglutamate from peptides whose molecular weight ranges from 362.4 to 4599.4 Da. Thus, a new method is presented that quantitatively estimates N-terminal pyroglutamic acid residue in peptides.

Design of chimeric peptide ligands to galanin receptors and substance P receptors.

PubMed

Langel, U; Land, T; Bartfai, T

1992-06-01

Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (KD(1) less than 0.1 nM and KD(2) approximately 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (KD approximately 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD approximately 1 nM) or its N-terminal fragment galanin-(1-13) (KD approximately 1 microM), which constitutes the N-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (KD = 0.4 microM), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp2], in the galanin portion of the chimeric peptide or substitution of ([Phe7] or [Met11]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

C-terminal peptide extension via gas-phase ion/ion reactions

PubMed Central

Peng, Zhou; McLuckey, Scott A.

2015-01-01

The formation of peptide bonds is of great importance from both a biological standpoint and in routine organic synthesis. Recent work from our group demonstrated the synthesis of peptides in the gas-phase via ion/ion reactions with sulfo-NHS reagents, which resulted in conjugation of individual amino acids or small peptides to the N-terminus of an existing ‘anchor’ peptide. Here, we demonstrate a complementary approach resulting in the C-terminal extension of peptides. Individual amino acids or short peptides can be prepared as reagents by incorporating gas phase-labile protecting groups to the reactive C-terminus and then converting the N-terminal amino groups to the active ketenimine reagent. Gas-phase ion/ion reactions between the anionic reagents and doubly protonated “anchor” peptide cations results in extension of the “anchor” peptide with new amide bond formation at the C-terminus. We have demonstrated that ion/ion reactions can be used as a fast, controlled, and efficient means for C-terminal peptide extension in the gas phase. PMID:26640400

Resin-Bound Crypto-Thioester for Native Chemical Ligation.

PubMed

Naruse, Naoto; Ohkawachi, Kento; Inokuma, Tsubasa; Shigenaga, Akira; Otaka, Akira

2018-04-20

The resin-bound N-sulfanylethylanilide (SEAlide) peptide was found to function as a crypto-thioester peptide. Exposure of the peptide resin to an aqueous solution under neutral conditions in the presence of thiols affords thioesters without accompanying racemization of C-terminal amino acids. Furthermore, the resin-bound SEAlide peptides react with N-terminal cysteinyl peptides in the absence of phosphate salts to afford ligated products, whereas soluble SEAlide peptides do not. This unexpected difference in reactivity of the SEAlide peptides allows for a one-pot/three-fragment ligation using resin-bound and unbound peptides.

Chemical Cleavage of an Asp-Cys Sequence Allows Efficient Production of Recombinant Peptides with an N-Terminal Cysteine Residue.

PubMed

Pane, Katia; Verrillo, Mariavittoria; Avitabile, Angela; Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Di Maro, Antimo; Rega, Camilla; Amoresano, Angela; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

2018-04-18

Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging, drug discovery, diagnostics, and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20-37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20, an antimicrobial peptide from the C-terminus of human thrombin, (C)ApoB L , an antimicrobial peptide from an inner region of human Apolipoprotein B, and (C)p53pAnt, an anticancer peptide containing the C-terminal region of the p53 protein fused to the cell penetrating peptide Penetratin. Cleavage efficiency of Asp-Cys bonds in the four fusion proteins was studied as a function of pH, temperature, and incubation time. In spite of the differences in the amino acid sequence (GTGDCGKY, GTGDCHVA, GSGTDCGSR, SQGSDCGSR) we obtained for all the proteins a cleavage efficiency of about 70-80% after 24 h incubation at 60 °C and pH 2. All the peptides were produced with very good yield (5-16 mg/L of LB cultures), high purity (>96%), and the expected content of free thiol groups (1 mol per mole of peptide). Furthermore, (C)GKY20 was modified with PyMPO-maleimide, a commercially available fluorophore bearing a thiol reactive group, and with 6-hydroxy-2-cyanobenzothiazole, a reagent specific for N-terminal cysteines, with yields of 100% thus demonstrating that our method is very well suited for the production of fully reactive peptides with an N-terminal cysteine residue.

A new pH-responsive peptide tag for protein purification.

PubMed

Nonaka, Takahiro; Tsurui, Noriko; Mannen, Teruhisa; Kikuchi, Yoshimi; Shiraki, Kentaro

2018-06-01

This paper describes a new pH-responsive peptide tag that adds a protein reversible precipitation and redissolution character. This peptide tag is a part of a cell surface protein B (CspB) derived from Corynebacterium glutamicum. Proinsulin that genetically fused with a peptide of N-terminal 6, 17, 50, or 250 amino acid residues of CspB showed that the reversible precipitation and redissolution depended on the pH. The transition occurred within a physiological and narrow pH range. A CspB50 tag comprising 50 amino acid residues of N-terminal CspB was further evaluated as a representative using other pharmaceutical proteins. Below pH 6.8, almost all CspB50-Teriparatide fusion formed an aggregated state. Subsequent addition of alkali turned the cloudy protein solution transparent above pH 7.3, in which almost all the CspB50-Teriparatide fusion redissolved. The CspB50-Bivalirudin fusion showed a similar behavior with slightly different pH range. This tag is offering a new protein purification method based on liquid-solid separation which does not require an affinity ligand. This sharp response around neutral pH is useful as a pH-responsive tag for the purification of unstable proteins at a non-physiological pH. Copyright © 2018 Elsevier Inc. All rights reserved.

Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates.

PubMed

Kleifeld, Oded; Doucet, Alain; Prudova, Anna; auf dem Keller, Ulrich; Gioia, Magda; Kizhakkedathu, Jayachandran N; Overall, Christopher M

2011-09-22

Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria.

N-terminal pro-brain natriuretic peptide and high-sensitivity troponin in the evaluation of acute chest pain of uncertain etiology. A PITAGORAS substudy.

PubMed

Sanchis, Juan; Bardají, Alfredo; Bosch, Xavier; Loma-Osorio, Pablo; Marín, Francisco; Sánchez, Pedro L; Calvo, Francisco; Avanzas, Pablo; Hernández, Carolina; Serrano, Silvia; Carratalá, Arturo; Barrabés, José A

2013-07-01

High-sensitivity troponin assays have improved the diagnosis of acute coronary syndrome in patients presenting with chest pain and normal troponin levels as measured by conventional assays. Our aim was to investigate whether N-terminal pro-brain natriuretic peptide provides additional information to troponin determination in these patients. A total of 398 patients, included in the PITAGORAS study, presenting to the emergency department with chest pain and normal troponin levels as measured by conventional assay in 2 serial samples (on arrival and 6 h to 8h later) were studied. The samples were also analyzed in a central laboratory for high-sensitivity troponin T (both samples) and for N-terminal pro-brain natriuretic peptide (second sample). The endpoints were diagnosis of acute coronary syndrome and the composite endpoint of in-hospital revascularization or a 30-day cardiac event. Acute coronary syndrome was adjudicated to 79 patients (20%) and the composite endpoint to 59 (15%). When the N-terminal pro-brain natriuretic peptide quartile increased, the diagnosis of acute coronary syndrome also increased (12%, 16%, 23% and 29%; P=.01), as did the risk of the composite endpoint (6%, 13%, 16% and 24%; P=.004). N-terminal pro-brain natriuretic peptide elevation (>125ng/L) was associated with both endpoints (relative risk= 2.0; 95% confidence interval, 1.2-3.3; P=.02; relative risk=2.4; 95% confidence interval, 1.4-4.2; P=.004). However, in the multivariable models adjusted by clinical and electrocardiographic data, a predictive value was found for high-sensitivity T troponin but not for N-terminal pro-brain natriuretic peptide. In low-risk patients with chest pain of uncertain etiology evaluated using high-sensitivity T troponin, N-terminal pro-brain natriuretic peptide does not contribute additional predictive value to diagnosis or the prediction of short-term outcomes. Copyright © 2012 Sociedad Española de Cardiología. Published by Elsevier Espana. All rights reserved.

An improved stable isotope N-terminal labeling approach with light/heavy TMPP to automate proteogenomics data validation: dN-TOP.

PubMed

Bertaccini, Diego; Vaca, Sebastian; Carapito, Christine; Arsène-Ploetze, Florence; Van Dorsselaer, Alain; Schaeffer-Reiss, Christine

2013-06-07

In silico gene prediction has proven to be prone to errors, especially regarding precise localization of start codons that spread in subsequent biological studies. Therefore, the high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and especially proteogenomics fields. The trimethoxyphenyl phosphonium (TMPP) labeling approach (N-TOP) is an efficient N-terminomic approach that allows the characterization of both N-terminal and internal peptides in a single experiment. Due to its permanent positive charge, TMPP labeling strongly affects MS/MS fragmentation resulting in unadapted scoring of TMPP-derivatized peptide spectra by classical search engines. This behavior has led to difficulties in validating TMPP-derivatized peptide identifications with usual score filtering and thus to low/underestimated numbers of identified N-termini. We present herein a new strategy (dN-TOP) that overwhelmed the previous limitation allowing a confident and automated N-terminal peptide validation thanks to a combined labeling with light and heavy TMPP reagents. We show how this double labeling allows increasing the number of validated N-terminal peptides. This strategy represents a considerable improvement to the well-established N-TOP method with an enhanced and accelerated data processing making it now fully compatible with high-throughput proteogenomics studies.

Improving cell penetration of helical peptides stabilized by N-terminal crosslinked aspartic acids.

PubMed

Zhao, Hui; Jiang, Yanhong; Tian, Yuan; Yang, Dan; Qin, Xuan; Li, Zigang

2017-01-04

Cell penetration and nucleus translocation efficiency are important for the cellular activities of peptide therapeutics. For helical peptides stabilized by N-terminal crosslinked aspartic acid, correlations between their penetration efficiency/nucleus translocation and physicochemical properties were studied. An increase in hydrophobicity and isoelectric point will promote cellular uptake and nucleus translocation of stabilized helices.

The histone H3 N-terminal tail: a computational analysis of the free energy landscape and kinetics.

PubMed

Zheng, Yuqing; Cui, Qiang

2015-05-28

Histone tails are the short peptide protrusions outside of the nucleosome core particle and they play a critical role in regulating chromatin dynamics and gene activity. A histone H3 N-terminal tail, like other histone tails, can be covalently modified on different residues to activate or repress gene expression. Previous studies have indicated that, despite its intrinsically disordered nature, the histone H3 N-terminal tail has regions of notable secondary structural propensities. To further understand the structure-dynamics-function relationship in this system, we have carried out 75.6 μs long implicit solvent simulations and 29.3 μs long explicit solvent simulations. The extensive samplings allow us to better characterize not only the underlying free energy landscape but also kinetic properties through Markov state models (MSM). Dihedral principal component analysis (dPCA) and locally scaled diffusion map (LSDMap) analysis yield consistent results that indicate an overall flat free energy surface with several shallow basins that correspond to conformations with a high α-helical propensity in two regions of the peptide. Kinetic information extracted from Markov state models reveals rapid transitions between different metastable states with mean first passage times spanning from several hundreds of nanoseconds to hundreds of microseconds. These findings shed light on how the dynamical nature of the histone H3 N-terminal tail is related to its function. The complementary nature of dPCA, LSDMap and MSM for the analysis of biomolecules is also discussed.

Truncated Glucagon-like Peptide-1 and Exendin-4 α-Conotoxin pl14a Peptide Chimeras Maintain Potency and α-Helicity and Reveal Interactions Vital for cAMP Signaling in Vitro*

PubMed Central

Swedberg, Joakim E.; Schroeder, Christina I.; Mitchell, Justin M.; Fairlie, David P.; Edmonds, David J.; Griffith, David A.; Ruggeri, Roger B.; Derksen, David R.; Loria, Paula M.; Price, David A.; Liras, Spiros; Craik, David J.

2016-01-01

Glucagon-like peptide-1 (GLP-1) signaling through the glucagon-like peptide 1 receptor (GLP-1R) is a key regulator of normal glucose metabolism, and exogenous GLP-1R agonist therapy is a promising avenue for the treatment of type 2 diabetes mellitus. To date, the development of therapeutic GLP-1R agonists has focused on producing drugs with an extended serum half-life. This has been achieved by engineering synthetic analogs of GLP-1 or the more stable exogenous GLP-1R agonist exendin-4 (Ex-4). These synthetic peptide hormones share the overall structure of GLP-1 and Ex-4, with a C-terminal helical segment and a flexible N-terminal tail. Although numerous studies have investigated the molecular determinants underpinning GLP-1 and Ex-4 binding and signaling through the GLP-1R, these have primarily focused on the length and composition of the N-terminal tail or on how to modulate the helicity of the full-length peptides. Here, we investigate the effect of C-terminal truncation in GLP-1 and Ex-4 on the cAMP pathway. To ensure helical C-terminal regions in the truncated peptides, we produced a series of chimeric peptides combining the N-terminal portion of GLP-1 or Ex-4 and the C-terminal segment of the helix-promoting peptide α-conotoxin pl14a. The helicity and structures of the chimeric peptides were confirmed using circular dichroism and NMR, respectively. We found no direct correlation between the fractional helicity and potency in signaling via the cAMP pathway. Rather, the most important feature for efficient receptor binding and signaling was the C-terminal helical segment (residues 22–27) directing the binding of Phe22 into a hydrophobic pocket on the GLP-1R. PMID:27226591

Rust fungal effectors mimic host transit peptides to translocate into chloroplasts.

PubMed

Petre, Benjamin; Lorrain, Cécile; Saunders, Diane G O; Win, Joe; Sklenar, Jan; Duplessis, Sébastien; Kamoun, Sophien

2016-04-01

Parasite effector proteins target various host cell compartments to alter host processes and promote infection. How effectors cross membrane-rich interfaces to reach these compartments is a major question in effector biology. Growing evidence suggests that effectors use molecular mimicry to subvert host cell machinery for protein sorting. We recently identified chloroplast-targeted protein 1 (CTP1), a candidate effector from the poplar leaf rust fungus Melampsora larici-populina that carries a predicted transit peptide and accumulates in chloroplasts and mitochondria. Here, we show that the CTP1 transit peptide is necessary and sufficient for accumulation in the stroma of chloroplasts. CTP1 is part of a Melampsora-specific family of polymorphic secreted proteins. Two members of that family, CTP2 and CTP3, also translocate in chloroplasts in an N-terminal signal-dependent manner. CTP1, CTP2 and CTP3 are cleaved when they accumulate in chloroplasts, while they remain intact when they do not translocate into chloroplasts. Our findings reveal that fungi have evolved effector proteins that mimic plant-specific sorting signals to traffic within plant cells. © 2015 John Wiley & Sons Ltd.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berbís, M. Álvaro; André, Sabine; Cañada, F. Javier

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 uniquemore » 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.« less

Structural insights into the backbone-circularized granulocyte colony-stimulating factor containing a short connector.

PubMed

Miyafusa, Takamitsu; Shibuya, Risa; Honda, Shinya

2018-06-02

Backbone circularization is a powerful approach for enhancing the structural stability of polypeptides. Herein, we present the crystal structure of the circularized variant of the granulocyte colony-stimulating factor (G-CSF) in which the terminal helical region was circularized using a short, two-amino acid connector. The structure revealed that the N- and C-termini were indeed connected by a peptide bond. The local structure of the C-terminal region transited from an α helix to 3 10 helix with a bend close to the N-terminal region, indicating that the structural change offset the insufficient length of the connector. This is the first-ever report of a crystal structure of the backbone of a circularized protein. It will facilitate the development of backbone circularization methodology. Copyright © 2018 Elsevier Inc. All rights reserved.

Peptide fragments of a beta-defensin derivative with potent bactericidal activity.

PubMed

Reynolds, Natalie L; De Cecco, Martin; Taylor, Karen; Stanton, Chloe; Kilanowski, Fiona; Kalapothakis, Jason; Seo, Emily; Uhrin, Dusan; Campopiano, Dominic; Govan, John; Macmillan, Derek; Barran, Perdita; Dorin, Julia R

2010-05-01

Beta-defensins are known to be both antimicrobial and able to chemoattract various immune cells. Although the sequences of paralogous genes are not highly conserved, the core defensin structure is retained. Defb14-1C(V) has bactericidal activity similar to that of its parent peptide (murine beta-defensin Defb14) despite all but one of the canonical six cysteines being replaced with alanines. The 23-amino-acid N-terminal half of Defb14-1C(V) is a potent antimicrobial while the C-terminal half is not. Here, we use a library of peptide derivatives to demonstrate that the antimicrobial activity can be localized to a particular region. Overlapping fragments of the N-terminal region were tested for their ability to kill Gram-positive and Gram-negative bacteria. We demonstrate that the most N-terminal fragments (amino acids 1 to 10 and 6 to 17) are potent antimicrobials against Gram-negative bacteria whereas fragments based on sequence more C terminal than amino acid 13 have very poor activity against both Gram-positive and -negative types. We further test a series of N-terminal deletion peptides in both their monomeric and dimeric forms. We find that bactericidal activity is lost against both Gram types as the deletion region increases, with the point at which this occurs varying between bacterial strains. The dimeric form of the peptides is more resistant to the peptide deletions, but this is not due just to increased charge. Our results indicate that the primary sequence, together with structure, is essential in the bactericidal action of this beta-defensin derivative peptide and importantly identifies a short fragment from the peptide that is a potent bactericide.

Peptide Fragments of a β-Defensin Derivative with Potent Bactericidal Activity ▿

PubMed Central

Reynolds, Natalie L.; De Cecco, Martin; Taylor, Karen; Stanton, Chloe; Kilanowski, Fiona; Kalapothakis, Jason; Seo, Emily; Uhrin, Dusan; Campopiano, Dominic; Govan, John; Macmillan, Derek; Barran, Perdita; Dorin, Julia R.

2010-01-01

β-Defensins are known to be both antimicrobial and able to chemoattract various immune cells. Although the sequences of paralogous genes are not highly conserved, the core defensin structure is retained. Defb14-1CV has bactericidal activity similar to that of its parent peptide (murine β-defensin Defb14) despite all but one of the canonical six cysteines being replaced with alanines. The 23-amino-acid N-terminal half of Defb14-1CV is a potent antimicrobial while the C-terminal half is not. Here, we use a library of peptide derivatives to demonstrate that the antimicrobial activity can be localized to a particular region. Overlapping fragments of the N-terminal region were tested for their ability to kill Gram-positive and Gram-negative bacteria. We demonstrate that the most N-terminal fragments (amino acids 1 to 10 and 6 to 17) are potent antimicrobials against Gram-negative bacteria whereas fragments based on sequence more C terminal than amino acid 13 have very poor activity against both Gram-positive and -negative types. We further test a series of N-terminal deletion peptides in both their monomeric and dimeric forms. We find that bactericidal activity is lost against both Gram types as the deletion region increases, with the point at which this occurs varying between bacterial strains. The dimeric form of the peptides is more resistant to the peptide deletions, but this is not due just to increased charge. Our results indicate that the primary sequence, together with structure, is essential in the bactericidal action of this β-defensin derivative peptide and importantly identifies a short fragment from the peptide that is a potent bactericide. PMID:20176896

Structure stability of lytic peptides during their interactions with lipid bilayers.

PubMed

Chen, H M; Lee, C H

2001-10-01

In this work, molecular dynamics simulations were used to examine the consequences of a variety of analogs of cecropin A on lipid bilayers. Analog sequences were constructed by replacing either the N- or C-terminal helix with the other helix in native or reverse sequence order, by making palindromic peptides based on both the N- and C-terminal helices, and by deleting the hinge region. The structure of the peptides was monitored throughout the simulation. The hinge region appeared not to assist in maintaining helical structure but help in motion flexibility. In general, the N-terminal helix of peptides was less stable than the C-terminal one during the interaction with anionic lipid bilayers. Sequences with hydrophobic helices tended to regain helical structure after an initial loss while sequences with amphipathic helices were less able to do this. The results suggests that hydrophobic design peptides have a high structural stability in an anionic membrane and are the candidates for experimental investigation.

C-terminal N-alkylated peptide amides resulting from the linker decomposition of the Rink amide resin: a new cleavage mixture prevents their formation.

PubMed

Stathopoulos, Panagiotis; Papas, Serafim; Tsikaris, Vassilios

2006-03-01

Decomposition of the resin linkers during TFA cleavage of the peptides in the Fmoc strategy leads to alkylation of sensitive amino acids. The C-terminal amide alkylation, reported for the first time, is shown to be a major problem in peptide amides synthesized on the Rink amide resin. This side reaction occurs as a result of the Rink amide linker decomposition under TFA treatment of the peptide resin. The use of 1,3-dimethoxybenzene in a cleavage cocktail prevents almost quantitatively formation of C-terminal N-alkylated peptide amides. Oxidized by-product in the tested Cys- and Met-containing peptides were not observed, even if thiols were not used in the cleavage mixture. Copyright (c) 2005 European Peptide Society and John Wiley & Sons, Ltd.

NMR assignment of a PDZ domain in complex with a HPV51 E6 derived N-terminally pyroglutamic acid modified peptide.

PubMed

Mischo, André; Ohlenschläger, Oliver; Ramachandran, Ramadurai; Görlach, Matthias

2013-04-01

The resonance assignment of an amino-terminal pyroglutamic acid containing peptide derived from the E6 protein of human papillomavirus (HPV) type 51 in complex with PDZ domain 2 of hDlg/SAP-97 is reported. The assignments include (1)H, (13)C and (15)N resonances for the protein and peptide in the complex and all of the peptide's pyroglutamic acid nuclei.

Cathepsin H Functions as an Aminopeptidase in Secretory Vesicles for Production of Enkephalin and Galanin Peptide Neurotransmitters

PubMed Central

Lu, W. Douglas; Funkelstein, Lydiane; Toneff, Thomas; Reinheckel, Thomas; Peters, Christoph; Hook, Vivian

2012-01-01

Peptide neurotransmitters function as key intercellular signaling molecules in the nervous system. These peptides are generated in secretory vesicles from proneuropeptides by proteolytic processing at dibasic residues, followed by removal of N- and/or C-terminal basic residues to form active peptides. Enkephalin biosynthesis from proenkephalin utilizes the cysteine protease cathepsin L and the subtilisin-like prohormone convertase 2 (PC2). Cathepsin L generates peptide intermediates with N-terminal basic residue extensions, which must be removed by an aminopeptidase. In this study, we identified cathepsin H as an aminopeptidase in secretory vesicles that produces (Met)enkephalin (ME) by sequential removal of basic residues from KR-ME and KK-ME, supported by in vivo knockout of the cathepsin H gene. Localization of cathepsin H in secretory vesicles was demonstrated by immunoelectron microscopy and confocal immunofluorescence microscopy. Purified human cathepsin H sequentially removes N-terminal basic residues to generate ME, with peptide products characterized by nano-LC-MS/MS tandem mass spectrometry. Cathepsin H shows highest activities for cleaving N-terminal basic residues (Arg and Lys) among amino acid fluorogenic substrates. Notably, knockout of the cathepsin H gene results in reduction of ME in mouse brain. Cathepsin H deficient mice also show a substantial decrease in galanin peptide neurotransmitter levels in brain. These results illustrate a role for cathepsin H as an aminopeptidase for enkephalin and galanin peptide neurotransmitter production. PMID:22582844

UV Resonance Raman Elucidation of the Terminal and Internal Peptide Bond Conformations of Crystalline and Solution Oligoglycines.

PubMed

Bykov, Sergei V; Asher, Sanford A

2010-11-30

Spectroscopic investigations of macromolecules generally attempt to interpret the measured spectra in terms of the summed contributions of the different molecular fragments. This is the basis of the local mode approximation in vibrational spectroscopy. In the case of resonance Raman spectroscopy independent contributions of molecular fragments require both a local mode-like behavior and the uncoupled electronic transitions. Here we show that the deep UV resonance Raman spectra of aqueous solution phase oligoglycines show independent peptide bond molecular fragment contributions indicating that peptide bonds electronic transitions and vibrational modes are uncoupled. We utilize this result to separately determine the conformational distributions of the internal and penultimate peptide bonds of oligoglycines. Our data indicate that in aqueous solution the oligoglycine terminal residues populate conformations similar to those found in crystals (3(1)-helices and β-strands), but with a broader distribution, while the internal peptide bond conformations are centered around the 3(1)-helix Ramachandran angles.

Mimicking of Arginine by Functionalized N(ω)-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples.

PubMed

Keller, Max; Kuhn, Kilian K; Einsiedel, Jürgen; Hübner, Harald; Biselli, Sabrina; Mollereau, Catherine; Wifling, David; Svobodová, Jaroslava; Bernhardt, Günther; Cabrele, Chiara; Vanderheyden, Patrick M L; Gmeiner, Peter; Buschauer, Armin

2016-03-10

Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

Analysis of the differentially expressed low molecular weight peptides in human serum via an N-terminal isotope labeling technique combining nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry.

PubMed

Leng, Jiapeng; Zhu, Dong; Wu, Duojiao; Zhu, Tongyu; Zhao, Ningwei; Guo, Yinlong

2012-11-15

Peptidomics analysis of human serum is challenging due to the low abundance of serum peptides and interference from the complex matrix. This study analyzed the differentially expressed (DE) low molecular weight peptides in human serum integrating a DMPITC-based N-terminal isotope labeling technique with nano-liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry (nano-LC/MALDI-MS). The workflow introduced a [d(6)]-4,6-dimethoxypyrimidine-2-isothiocyanate (DMPITC)-labeled mixture of aliquots from test samples as the internal standard. The spiked [d(0)]-DMPITC-labeled samples were separated by nano-LC then spotted on the MALDI target. Both quantitative and qualitative studies for serum peptides were achieved based on the isotope-labeled peaks. The DMPITC labeling technique combined with nano-LC/MALDI-MS not only minimized the errors in peptide quantitation, but also allowed convenient recognition of the labeled peptides due to the 6 Da mass difference. The data showed that the entire research procedure as well as the subsequent data analysis method were effective, reproducible, and sensitive for the analysis of DE serum peptides. This study successfully established a research model for DE serum peptides using DMPITC-based N-terminal isotope labeling and nano-LC/MALDI-MS. Application of the DMPITC-based N-terminal labeling technique is expected to provide a promising tool for the investigation of peptides in vivo, especially for the analysis of DE peptides under different biological conditions. Copyright © 2012 John Wiley & Sons, Ltd.

Effect of N-Terminal Acylation on the Activity of Myostatin Inhibitory Peptides.

PubMed

Takayama, Kentaro; Nakamura, Akari; Rentier, Cédric; Mino, Yusaku; Asari, Tomo; Saga, Yusuke; Taguchi, Akihiro; Yakushiji, Fumika; Hayashi, Yoshio

2016-04-19

Inhibition of myostatin, which negatively regulates skeletal muscle growth, is a promising strategy for the treatment of muscle atrophic disorders, such as muscular dystrophy, cachexia and sarcopenia. Recently, we identified peptide A (H-WRQNTRYSRIEAIKIQILSKLRL-NH2 ), the 23-amino-acid minimum myostatin inhibitory peptide derived from mouse myostatin prodomain, and highlighted the importance of its N-terminal tryptophan residue for the effective inhibition. In this study, we synthesized a series of acylated peptide derivatives focused on the tryptophan residue to develop potent myostatin inhibitors. As a result of the investigation, a more potent derivative of peptide A was successfully identified in which the N-terminal tryptophan residue is replaced with a 2-naphthyloxyacetyl moiety to give an inhibitory peptide three times (1.19±0.11 μm) more potent than parent peptide A (3.53±0.25 μm). This peptide could prove useful as a new starting point for the development of improved inhibitory peptides. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kong, Rui; Xu, Kai; Zhou, Tongqing

The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. In this paper, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showedmore » that the N-terminal portion of the fusion peptide can be solvent-exposed. Finally, these results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design.« less

Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody

DOE PAGES

Kong, Rui; Xu, Kai; Zhou, Tongqing; ...

2016-05-13

The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. In this paper, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showedmore » that the N-terminal portion of the fusion peptide can be solvent-exposed. Finally, these results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design.« less

Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Dupré, Mathieu; Cantel, Sonia; Martinez, Jean; Enjalbal, Christine

2012-02-01

By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (bn-1 + H2O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H2O and NH3) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment ion in the high mass range of the MS/MS spectra. The mass difference between this signal and the protonated molecular ion corresponds to the mass of the C-terminal residue. It allowed a straightforward identification of the amino acid positioned at this extremity. It must be emphasized that a neutral residue loss can be misattributed to the formation of a ym-1 ion, i.e., to the loss of the N-terminal residue following the a1-ym-1 fragmentation channel. Extreme caution must be adopted when reading the direct sequence ion on the positive ion MS/MS spectra of singly charged peptides not to mix up the attribution of the N- and C-terminal amino acids. Although such peculiar fragmentation behavior is of obvious interest for de novo peptide sequencing, it can also be exploited in proteomics, especially for studies involving digestion protocols carried out with proteolytic enzymes other than trypsin (Lys-N, Glu-C, and Asp-N) that produce arginine-containing peptides.

[Diagnostic values of type III Procollagen N-terminal peptide and combination assay of type III procollagen N-terminal peptide with CEA and CA 19-9 in gastric cancer].

PubMed

Akazawa, S; Harada, A; Futatsuki, K

1984-07-01

It is known that interstitial collagens are initially synthesized as precursors (procollagen), which possess extra peptide segments at both ends of the molecules. The authors attempted to detect the aminoterminal peptide of type III procollagen (type III-N-peptide) and also to measure the carcinoembryonic antigen (CEA) and carbohydrate antigen (CA 19-9) together in sera of patients with gastric cancer. The results showed that: (1) mean serum levels and positive ratios of the type III-N-peptide increased as the clinical stage of the patients with gastric cancer advanced; (2) serum levels of the type III-N-peptide were not correlated either with those of CEA or CA 19-9; (3) positive ratios of type III-N-peptide, CEA and CA 19-9 were 51.7%, 44.8% and 48.3%, respectively: (4) positive ratio in combination of the type III-N-peptide with CEA was 69.3% and that in combination of the type III-N-peptide with CEA and CA 19-9 was 72.4%. These results suggest that type III-N-peptide is available for diagnosis of gastric cancer and, that the combination assay of type III-N-peptide with CEA and CA 19-9 is more effective than a single assay for diagnosis.

Investigation of the Enzymes Involved in Lantibiotic Biosynthesis: Lacticin 481 and Haloduracin

ERIC Educational Resources Information Center

Ihnken, Leigh Anne Furgerson

2009-01-01

Lantibiotics are cyclic peptides that exhibit a range of biological properties, including antimicrobial activity. They are ribosomally-synthesized as linear precursor peptides that consist of two regions, an N-terminal leader peptide and a C-terminal propeptide (or structural) region. The structural region undergoes extensive enzyme-catalyzed…

Human lysozyme possesses novel antimicrobial peptides within its N-terminal domain that target bacterial respiration.

PubMed

Ibrahim, Hisham R; Imazato, Kenta; Ono, Hajime

2011-09-28

Human milk lysozyme is thought to be a key defense factor in protecting the gastrointestinal tract of newborns against bacterial infection. Recently, evidence was found that pepsin, under conditions relevant to the newborn stomach, cleaves chicken lysozyme (cLZ) at specific loops to generate five antimicrobial peptide motifs. This study explores the antimicrobial role of the corresponding peptides of human lysozyme (hLZ), the actual protein in breast milk. Five peptide motifs of hLZ, one helix-loop-helix (HLH), its two helices (H1 and H2), and two helix-sheet motifs, H2-β-strands 1-2 (H2-S12) or H2-β-strands 1-3 (H2-S13), were synthesized and examined for antimicrobial action. The five peptides of hLZ exhibit microbicidal activity to various degrees against several bacterial strains. The HLH peptide and its N-terminal helix (H1) were significantly the most potent bactericidal to Gram-positive and Gram-negative bacteria and the fungus Candida albicans . Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and are able to dissipate the membrane potential-dependent respiration of Gram-positive bacteria. This finding is the first to describe that hLZ possesses multiple antimicrobial peptide motifs within its N-terminal domain, providing insight into new classes of antibiotic peptides with potential use in the treatment of infectious diseases.

N-terminal amphipathic helix as a trigger of hemolytic activity in antimicrobial peptides: a case study in latarcins.

PubMed

Polyansky, Anton A; Vassilevski, Alexander A; Volynsky, Pavel E; Vorontsova, Olga V; Samsonova, Olga V; Egorova, Natalya S; Krylov, Nicolay A; Feofanov, Alexei V; Arseniev, Alexander S; Grishin, Eugene V; Efremov, Roman G

2009-07-21

In silico structural analyses of sets of alpha-helical antimicrobial peptides (AMPs) are performed. Differences between hemolytic and non-hemolytic AMPs are revealed in organization of their N-terminal region. A parameter related to hydrophobicity of the N-terminal part is proposed as a measure of the peptide propensity to exhibit hemolytic and other unwanted cytotoxic activities. Based on the information acquired, a rational approach for selective removal of these properties in AMPs is suggested. A proof of concept is gained through engineering specific mutations that resulted in elimination of the hemolytic activity of AMPs (latarcins) while leaving the beneficial antimicrobial effect intact.

Bromine isotopic signature facilitates de novo sequencing of peptides in free-radical-initiated peptide sequencing (FRIPS) mass spectrometry.

PubMed

Nam, Jungjoo; Kwon, Hyuksu; Jang, Inae; Jeon, Aeran; Moon, Jingyu; Lee, Sun Young; Kang, Dukjin; Han, Sang Yun; Moon, Bongjin; Oh, Han Bin

2015-02-01

We recently showed that free-radical-initiated peptide sequencing mass spectrometry (FRIPS MS) assisted by the remarkable thermochemical stability of (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (TEMPO) is another attractive radical-driven peptide fragmentation MS tool. Facile homolytic cleavage of the bond between the benzylic carbon and the oxygen of the TEMPO moiety in o-TEMPO-Bz-C(O)-peptide and the high reactivity of the benzylic radical species generated in •Bz-C(O)-peptide are key elements leading to extensive radical-driven peptide backbone fragmentation. In the present study, we demonstrate that the incorporation of bromine into the benzene ring, i.e. o-TEMPO-Bz(Br)-C(O)-peptide, allows unambiguous distinction of the N-terminal peptide fragments from the C-terminal fragments through the unique bromine doublet isotopic signature. Furthermore, bromine substitution does not alter the overall radical-driven peptide backbone dissociation pathways of o-TEMPO-Bz-C(O)-peptide. From a practical perspective, the presence of the bromine isotopic signature in the N-terminal peptide fragments in TEMPO-assisted FRIPS MS represents a useful and cost-effective opportunity for de novo peptide sequencing. Copyright © 2015 John Wiley & Sons, Ltd.

Envisaging Structural Insight of a Terminally Protected Proline Dipeptide by Raman Spectroscopy and Density Functional Theory Analyses.

PubMed

Das, Supriya; Pal, Uttam; Chatterjee, Moumita; Pramanik, Sumit Kumar; Banerji, Biswadip; Maiti, Nakul C

2016-12-15

The proline residue in a protein sequence generates constraints to its secondary structure as the associated torsion angles become a part of the heterocyclic ring. It becomes more significant when two consecutive proline residues link via amide linkage and produce additional configurational constraint to a protein's folding and stability. In the current manuscript we have illustrated conformation preference of a novel dipeptide, (R)-tert-butyl 2-((S)-2-(methoxycarbonyl)pyrrolidine-1-carbonyl)pyrrolidine-1-carboxylate. The dipeptide crystallized in the orthorhombic crystalline state and produced rod-shaped macroscopic material. The analysis of the crystal coordinates showed dihedral angles (φ, ψ) of the interlinked amide groups as (+72°, -147°) and the dihedral angles (φ, ψ) produced with the next carbonyl were (-68°, +151°), indicating polyglycine II (PGII) and polyproline II (PPII)-like helix states at the N- and C-terminals, respectively. These two states, PGII and PPII, are mirror image configurations and are expected to produce similar vibration bands from the associated carbonyl groups. However, the unique atomic arrangement in the molecule produces three carbonyl groups and one of them was very specific, being part of the main peptide linkage that connects both the pyrrolidine rings. The carbonyl group in the peptide bond exhibited a Raman vibration frequency at ∼1642 cm -1 and is considered a signatory Raman marker band for the peptide bond linking two heterochiral proline residues. The carbonyl group (t-Boc) at the N-terminal of the peptide showed a characteristic vibration at ∼1685 cm -1 and the C-terminal carbonyl group as a part of the ester showed a vibration signature at a significantly high frequency (1746 cm -1 ). Conformation analyses performed with density functional theory (DFT) calculations depicted that the dipeptide was stabilized in vacuum with dihedral angles (+72°, -154°) and (-72°, +151°) at the N- and C-terminals, respectively. Molecular dynamics (MD) simulation also showed that the peptide conformation having dihedral angles around (+75°, -150°) and (-75°, +150°) at the N- and C-terminals, respectively, was reasonably stable in water. Due to unique absence of the amide N-H, the peptide was ineffective in forming any intramolecular hydrogen bonding. MD investigation, however, revealed an intermolecular hydrogen bonding interaction with the water molecules, leading to its stability in aqueous solution. Metadynamics simulation analysis of the dipeptide in water also supported the PGII-PPII-like conformation at the N- and C-terminals, respectively, as the energetically stable conformation among the other possible combinations of conformations. The possible electronic transitions along with the HOMO-LUMO analysis further depicted the stability of the dipeptide in water and their possible absorption pattern. Time-dependent density functional theory (TDDFT) analysis showed strong negative rotatory strength of the dipeptide around 210 nm in water and acetonitrile, and it could be the source of experimentally observed high-amplitude negative absorption in the circular dichroism (CD) spectra around 200-203 nm. The very weak positive band (signature) in the region at ∼228 nm in CD spectra could also be correlated to the positive rotatory strength at 228 nm observed in ECD. To test the effect of such a dipeptide on a living cell, an MTT assay was performed and the result indicated no cytotoxic effect toward human hepatocellular carcinoma Hep G2 cancer cell lines.

Opposite Electron-Transfer Dissociation and Higher-Energy Collisional Dissociation Fragmentation Characteristics of Proteolytic K/R(X)n and (X)nK/R Peptides Provide Benefits for Peptide Sequencing in Proteomics and Phosphoproteomics.

PubMed

Tsiatsiani, Liana; Giansanti, Piero; Scheltema, Richard A; van den Toorn, Henk; Overall, Christopher M; Altelaar, A F Maarten; Heck, Albert J R

2017-02-03

A key step in shotgun proteomics is the digestion of proteins into peptides amenable for mass spectrometry. Tryptic peptides can be readily sequenced and identified by collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) because the fragmentation rules are well-understood. Here, we investigate LysargiNase, a perfect trypsin mirror protease, because it cleaves equally specific at arginine and lysine residues, albeit at the N-terminal end. LysargiNase peptides are therefore practically tryptic-like in length and sequence except that following ESI, the two protons are now both positioned at the N-terminus. Here, we compare side-by-side the chromatographic separation properties, gas-phase fragmentation characteristics, and (phospho)proteome sequence coverage of tryptic (i.e., (X) n K/R) and LysargiNase (i.e., K/R(X) n ) peptides using primarily electron-transfer dissociation (ETD) and, for comparison, HCD. We find that tryptic and LysargiNase peptides fragment nearly as mirror images. For LysargiNase predominantly N-terminal peptide ions (c-ions (ETD) and b-ions (HCD)) are formed, whereas for trypsin, C-terminal fragment ions dominate (z-ions (ETD) and y-ions (HCD)) in a homologous mixture of complementary ions. Especially during ETD, LysargiNase peptides fragment into low-complexity but information-rich sequence ladders. Trypsin and LysargiNase chart distinct parts of the proteome, and therefore, the combined use of these enzymes will benefit a more in-depth and reliable analysis of (phospho)proteomes.

Utilization of peptide carrier system to improve intestinal absorption: targeting prolidase as a prodrug-converting enzyme

NASA Technical Reports Server (NTRS)

Bai, J. P.; Hu, M.; Subramanian, P.; Mosberg, H. I.; Amidon, G. L.

1992-01-01

The feasibility of targeting prolidase as a peptide prodrug-converting enzyme has been examined. The enzymatic hydrolysis by prolidase of substrates for the peptide transporter L-alpha-methyldopa-pro and several dipeptide analogues without an N-terminal alpha-amino group (phenylpropionylproline, phenylacetylproline, N-benzoylproline, and N-acetylproline) was investigated. The Michaelis-Menten parameters Km and Vmax for L-alpha-methyldopa-pro are 0.09 +/- 0.02 mM and 3.98 +/- 0.25 mumol/min/mg protein, respectively. However, no hydrolysis of the dipeptide analogues without an N-terminal alpha-amino group is observed, suggesting that an N-terminal alpha-amino group is required for prolidase activity. These results demonstrate that prolidase may serve as a prodrug-converting enzyme for the dipeptide-type prodrugs, utilizing the peptide carrier for transport of prodrugs into the mucosal cells and prolidase, a cytosolic enzyme, to release the drug. However, a free alpha-amino group appears to be necessary for prolidase hydrolysis.

Plasmatic levels of N-terminal pro-atrial natriuretic peptide in preeclamptic patients and healthy normotensive pregnant women.

PubMed

Reyna-Villasmil, Eduardo; Mejia-Montilla, Jorly; Reyna-Villasmil, Nadia; Mayner-Tresol, Gabriel; Herrera-Moya, Pedro; Fernández-Ramírez, Andreina; Rondón-Tapía, Marta

2018-05-11

To compare plasma N-terminal pro-atrial natriuretic peptide concentrations in preeclamptic patients and healthy normotensive pregnant women. A cases-controls study was done with 180 patients at Hospital Central Dr. Urquinaona, Maracaibo, Venezuela, that included 90 preeclamptic patients (group A; cases) and 90 healthy normotensive pregnant women selected with the same age and body mass index similar to group A (group B; controls). Blood samples were collected one hour after admission and prior to administration of any medication in group A to determine plasma N-terminal pro-atrial natriuretic peptide and other laboratory parameters. Plasma N-terminal pro-atrial natriuretic peptide concentrations in group A (mean 1.01 [0.26] pg/mL) showed a significant difference when compared with patients in group B (mean 0.55 [0.07] pg/mL; P<.001]. There was no significant correlation with systolic and diastolic blood pressure values in preeclamptic patients (P=ns). A cut-off value of 0.66ng/mL had an area under the curve of 0.93, sensitivity of 87.8%, specificity of 83.3%, a positive predictive value of 84.0% and a negative predictive value of 87.2%, with a diagnostic accuracy of 85.6%. Preeclamptic patients have significantly higher concentrations of plasma N-terminal pro-atrial natriuretic peptide compared with healthy normotensive pregnant women, with high predictive values for diagnosis. Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

Glucagon-Like Peptide-1 Receptor Ligand Interactions: Structural Cross Talk between Ligands and the Extracellular Domain

PubMed Central

West, Graham M.; Willard, Francis S.; Sloop, Kyle W.; Showalter, Aaron D.; Pascal, Bruce D.; Griffin, Patrick R.

2014-01-01

Activation of the glucagon-like peptide-1 receptor (GLP-1R) in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM). Like other class B G protein-coupled receptors (GPCRs), the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX) to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R) were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R) peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R). In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands. PMID:25180755

Tidbits for the synthesis of bis(2-sulfanylethyl)amido (SEA) polystyrene resin, SEA peptides and peptide thioesters.

PubMed

Ollivier, Nathalie; Raibaut, Laurent; Blanpain, Annick; Desmet, Rémi; Dheur, Julien; Mhidia, Reda; Boll, Emmanuelle; Drobecq, Hervé; Pira, Silvain L; Melnyk, Oleg

2014-02-01

Protein total chemical synthesis enables the atom-by-atom control of the protein structure and therefore has a great potential for studying protein function. Native chemical ligation of C-terminal peptide thioesters with N-terminal cysteinyl peptides and related methodologies are central to the field of protein total synthesis. Consequently, methods enabling the facile synthesis of peptide thioesters using Fmoc-SPPS are of great value. Herein, we provide a detailed protocol for the preparation of bis(2-sulfanylethyl)amino polystyrene resin as a starting point for the synthesis of C-terminal bis(2-sulfanylethyl)amido peptides and of peptide thioesters derived from 3-mercaptopropionic acid. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

The unique N-terminal zinc finger of synaptotagmin-like protein 4 reveals FYVE structure.

PubMed

Miyamoto, Kazuhide; Nakatani, Arisa; Saito, Kazuki

2017-12-01

Synaptotagmin-like protein 4 (Slp4), expressed in human platelets, is associated with dense granule release. Slp4 is comprised of the N-terminal zinc finger, Slp homology domain, and C2 domains. We synthesized a compact construct (the Slp4N peptide) corresponding to the Slp4 N-terminal zinc finger. Herein, we have determined the solution structure of the Slp4N peptide by nuclear magnetic resonance (NMR). Furthermore, experimental, chemical modification of Cys residues revealed that the Slp4N peptide binds two zinc atoms to mediate proper folding. NMR data showed that eight Cys residues coordinate zinc atoms in a cross-brace fashion. The Simple Modular Architecture Research Tool database predicted the structure of Slp4N as a RING finger. However, the actual structure of the Slp4N peptide adopts a unique C 4 C 4 -type FYVE fold and is distinct from a RING fold. To create an artificial RING finger (ARF) with specific ubiquitin-conjugating enzyme (E2)-binding capability, cross-brace structures with eight zinc-ligating residues are needed as the scaffold. The cross-brace structure of the Slp4N peptide could be utilized as the scaffold for the design of ARFs. © 2017 The Protein Society.

Membrane interaction of the N-terminal domain of chemokine receptor CXCR1.

PubMed

Haldar, Sourav; Raghuraman, H; Namani, Trishool; Rajarathnam, Krishna; Chattopadhyay, Amitabha

2010-06-01

The N-terminal domain of chemokine receptors constitutes one of the two critical ligand binding sites, and plays important roles by mediating binding affinity, receptor selectivity, and regulating function. In this work, we monitored the organization and dynamics of a 34-mer peptide of the CXC chemokine receptor 1 (CXCR1) N-terminal domain and its interaction with membranes by utilizing a combination of fluorescence-based approaches and surface pressure measurements. Our results show that the CXCR1 N-domain 34-mer peptide binds vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and upon binding, the tryptophan residues of the peptide experience motional restriction and exhibit red edge excitation shift (REES) of 19nm. These results are further supported by increase in fluorescence anisotropy and mean fluorescence lifetime upon membrane binding. These results constitute one of the first reports demonstrating membrane interaction of the N-terminal domain of CXCR1 and gain relevance in the context of the emerging role of cellular membranes in chemokine signaling.

Total chemical synthesis of proteins without HPLC purification† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc01883a Click here for additional data file.

PubMed Central

Loibl, S. F.; Harpaz, Z.; Zitterbart, R.

2016-01-01

The total chemical synthesis of proteins is a tedious and time-consuming endeavour. The typical steps involve solid phase synthesis of peptide thioesters and cysteinyl peptides, native chemical ligation (NCL) in solution, desulfurization or removal of ligation auxiliaries in the case of extended NCL as well as many intermediary and final HPLC purification steps. With an aim to facilitate and improve the throughput of protein synthesis we developed the first method for the rapid chemical total on-resin synthesis of proteins that proceeds without a single HPLC-purification step. The method relies on the combination of three orthogonal protein tags that allow sequential immobilization (via the N-terminal and C-terminal ends), extended native chemical ligation and release reactions. The peptide fragments to be ligated are prepared by conventional solid phase synthesis and used as crude materials in the subsequent steps. An N-terminal His6 unit permits selective immobilization of the full length peptide thioester onto Ni-NTA agarose beads. The C-terminal peptide fragment carries a C-terminal peptide hydrazide and an N-terminal 2-mercapto-2-phenyl-ethyl ligation auxiliary, which serves as a reactivity tag for the full length peptide. As a result, only full length peptides, not truncation products, react in the subsequent on-bead extended NCL. After auxiliary removal the ligation product is liberated into solution upon treatment with mild acid, and is concomitantly captured by an aldehyde-modified resin. This step allows the removal of the most frequently observed by-product in NCL chemistry, i.e. the hydrolysed peptide thioester (which does not contain a C-terminal peptide hydrazide). Finally, the target protein is released with diluted hydrazine or acid. We applied the method in the synthesis of 46 to 126 amino acid long MUC1 proteins comprising 2–6 copies of a 20mer tandem repeat sequence. Only three days were required for the parallel synthesis of 9 MUC1 proteins which were obtained in 8–33% overall yield with 90–98% purity despite the omission of HPLC purification. PMID:28451120

Characterizing the Specificity and Co-operation of Aminopeptidases in the Cytosol and ER During MHC Class I antigen Presentation1

PubMed Central

Hearn, Arron; York, Ian A.; Bishop, Courtney; Rock, Kenneth L.

2010-01-01

Many MHC class I binding peptides are generated as N-extended precursors during protein degradation by the proteasome. These peptides can be subsequently trimmed by aminopeptidases in the cytosol and/or the ER to produce mature epitope. However, the contribution and specificity of each of these subcellular compartments in removing N-terminal amino acids for antigen presentation is not well defined. Here we investigate this issue for antigenic precursors that are expressed in the cytosol. By systematically varying the N-terminal flanking sequences of peptides we show that the amino acids upstream of an epitope precursor are a major determinant of the amount of antigen presentation. In many cases MHC class I binding peptides are produced through sequential trimming in both the cytosol and ER. Trimming of flanking residues in the cytosol contributes most to sequences that are poorly trimmed in the ER. Since N-terminal trimming has different specificity in the cytosol and ER, the cleavage of peptides in both of these compartments serves to broaden the repertoire of sequences that are presented. PMID:20351195

NMR assignments of SPOC domain of the human transcriptional corepressor SHARP in complex with a C-terminal SMRT peptide.

PubMed

Mikami, Suzuka; Kanaba, Teppei; Ito, Yutaka; Mishima, Masaki

2013-10-01

The transcriptional corepressor SMRT/HDAC1-associated repressor protein (SHARP) recruits histone deacetylases. Human SHARP protein is thought to function in processes involving steroid hormone responses and the Notch signaling pathway. SHARP consists of RNA recognition motifs (RRMs) in the N-terminal region and the spen paralog and ortholog C-terminal (SPOC) domain in the C-terminal region. It is known that the SPOC domain binds the LSD motif in the C-terminal tail of corepressors silencing mediator for retinoid and thyroid receptor (SMRT)/nuclear receptor corepressor (NcoR). We are interested in delineating the mechanism by which the SPOC domain recognizes the LSD motif of the C-terminal tail of SMRT/NcoR. To this end, we are investigating the tertiary structure of the SPOC/SMRT peptide using NMR. Herein, we report on the (1)H, (13)C and (15)N resonance assignments of the SPOC domain in complex with a SMRT peptide, which contributes towards a structural understanding of the SPOC/SMRT peptide and its molecular recognition.

Synthesis of antifungal vaccines by conjugation of β-1,2 trimannosides with T-cell peptides and covalent anchoring of neoglycopeptide to tetanus toxoid.

PubMed

Cartmell, Jonathan; Paszkiewicz, Eugenia; Dziadek, Sebastian; Tam, Pui-Hang; Luu, Thanh; Sarkar, Susmita; Lipinski, Tomasz; Bundle, David R

2015-02-11

Selective strategies for the construction of novel three component glycoconjugate vaccines presenting Candida albicans cell wall glycan (β-1,2 mannoside) and polypeptide fragments on a tetanus toxoid carrier are described. The first of two conjugation strategies employed peptides bearing an N-terminal thiopropionyl residue for conjugation to a trisaccharide equipped with an acrylate linker and a C-terminal S-acetyl thioglycolyl moiety for subsequent linking of neoglycopeptide to bromoacetylated tetanus toxoid. Michael addition of acrylate trisaccharides to peptide thiol under mildly basic conditions gave a mixture of N- and C- terminal glyco-peptide thioethers. An adaptation of this strategy coordinated S-acyl protection with anticipated thioester exchange equilibria. This furnished a single chemically defined fully synthetic neoglycopeptide conjugate that could be anchored to a tetanus toxoid carrier and avoids the introduction of exogenous antigenic groups. The second strategy retained the N-terminal thiopropionyl residue but replaced the C-terminal S-acetate functionality with an azido group that allowed efficient, selective formation of neoglycopeptide thioethers and subsequent conjugation of these with propargylated tetanus toxoid, but introduced potentially antigenic triazole linkages. Copyright © 2014 Elsevier Ltd. All rights reserved.

On the terminal homologation of physiologically active peptides as a means of increasing stability in human serum--neurotensin, opiorphin, B27-KK10 epitope, NPY.

PubMed

Seebach, Dieter; Lukaszuk, Aneta; Patora-Komisarska, Krystyna; Podwysocka, Dominika; Gardiner, James; Ebert, Marc-Olivier; Reubi, Jean Claude; Cescato, Renzo; Waser, Beatrice; Gmeiner, Peter; Hübner, Harald; Rougeot, Catherine

2011-05-01

The terminal homologation by CH(2) insertion into the peptides mentioned in the title is described. This involves replacement of the N-terminal amino acid residue by a β(2) - and of the C-terminal amino acid residue by a β(3) -homo-amino acid moiety (β(2) hXaa and β(3) hXaa, resp.; Fig. 1). In this way, the structure of the peptide chain from the N-terminal to the C-terminal stereogenic center is identical, and the modified peptide is protected against cleavage by exopeptidases (Figs. 2 and 3). Neurotensin (NT; 1) and its C-terminal fragment NT(8-13) are ligands of the G-protein-coupled receptors (GPCR) NT1, NT2, NT3, and NT analogs are promising tools to be used in cancer diagnostics and therapy. The affinities of homologated NT analogs, 2b-2e, for NT1 and NT2 receptors were determined by using cell homogenates and tumor tissues (Table 1); in the latter experiments, the affinities for the NT1 receptor are more or less the same as those of NT (0.5-1.3 vs. 0.6 nM). At the same time, one of the homologated NT analogs, 2c, survives in human plasma for 7 days at 37° (Fig. 6). An NMR analysis of NT(8-13) (Tables 2 and 4, and Fig. 8) reveals that this N-terminal NT fragment folds to a turn in CD(3) OH. - In the case of the human analgesic opiorphin (3a), a pentapeptide, and of the HIV-derived B27-KK10 (4a), a decapeptide, terminal homologation (→3b and 4b, resp.) led to a 7- and 70-fold half-life increase in plasma (Fig. 9). With N-terminally homologated NPY, 5c, we were not able to determine serum stability; the peptide consisting of 36 amino acid residues is subject to cleavage by endopetidases. Three of the homologated compounds, 2b, 2c, and 5c, were shown to be agonists (Fig. 7 and 11). A comparison of terminal homologation with other stability-increasing terminal modifications of peptides is performed (Fig. 5), and possible applications of the neurotensin analogs, described herein, are discussed. Copyright © 2011 Verlag Helvetica Chimica Acta AG, Zürich.

Structure-activity analysis of synthetic alpha-thrombin-receptor-activating peptides.

PubMed

Van Obberghen-Schilling, E; Rasmussen, U B; Vouret-Craviari, V; Lentes, K U; Pavirani, A; Pouysségur, J

1993-06-15

alpha-Thrombin stimulates G-protein-coupled effectors leading to secretion and aggregation in human platelets, and to a mitogenic response in CCL39 hamster fibroblasts. alpha-Thrombin receptors can be activated by synthetic peptides corresponding to the receptor sequence starting with serine-42, at the proposed cleavage site. We have previously determined that the agonist domain of receptor-activating peptides resides within the five N-terminal residues [Vouret-Craviari, Van Obberghen-Schilling, Rasmussen, Pavirani, Lecocq and Pouysségur (1992) Mol. Biol. Cell. 3, 95-102], although the 7-residue peptide (SFFLRNP) corresponding to the hamster alpha-thrombin receptor was 10 times more potent than the 5-residue peptide for activation of human platelets. In the present study we have analysed the role of individual amino acids in receptor activation by using a series of modified hexa- or hepta-peptides derived from the human alpha-thrombin-receptor sequence. Cellular events examined here include phospholipase C activation, adenylyl cyclase inhibition and DNA synthesis stimulation in non-transformed CCL39 fibroblasts and a tumorigenic variant of that line (A71 cells). Modification of the peptide sequence had similar functional consequence for each of the assays described, indicating that either a unique receptor or pharmacologically indistinguishable receptor subtypes activate distinct G-protein signalling pathways. Furthermore, we found that: (1) the N-terminal serine can be replaced by small or intermediately sized amino acids (+/- hydroxyl groups) without loss of activity. However, its replacement by an aromatic side-chain or omission of the N-terminal amino group severely reduces activity. (2) An aromatic side-chain on the penultimate N-terminal residue appears to play a critical role since phenylalanine in this position can be substituted by tyrosine without complete loss of activity whereas an alanine in its place is not tolerated. (3) Deletion of the first, second or third N-terminal residue leads to a loss of activity, suggesting that a defined spacing of more than one structural component may be important for ligand-receptor interaction. Finally, we did not observe an antagonistic effect of the inactive peptides on phospholipase C activation or DNA synthesis induced by alpha-thrombin (1 nM) or SFLLRNP (3 microM).

Structure-activity analysis of synthetic alpha-thrombin-receptor-activating peptides.

PubMed Central

Van Obberghen-Schilling, E; Rasmussen, U B; Vouret-Craviari, V; Lentes, K U; Pavirani, A; Pouysségur, J

1993-01-01

alpha-Thrombin stimulates G-protein-coupled effectors leading to secretion and aggregation in human platelets, and to a mitogenic response in CCL39 hamster fibroblasts. alpha-Thrombin receptors can be activated by synthetic peptides corresponding to the receptor sequence starting with serine-42, at the proposed cleavage site. We have previously determined that the agonist domain of receptor-activating peptides resides within the five N-terminal residues [Vouret-Craviari, Van Obberghen-Schilling, Rasmussen, Pavirani, Lecocq and Pouysségur (1992) Mol. Biol. Cell. 3, 95-102], although the 7-residue peptide (SFFLRNP) corresponding to the hamster alpha-thrombin receptor was 10 times more potent than the 5-residue peptide for activation of human platelets. In the present study we have analysed the role of individual amino acids in receptor activation by using a series of modified hexa- or hepta-peptides derived from the human alpha-thrombin-receptor sequence. Cellular events examined here include phospholipase C activation, adenylyl cyclase inhibition and DNA synthesis stimulation in non-transformed CCL39 fibroblasts and a tumorigenic variant of that line (A71 cells). Modification of the peptide sequence had similar functional consequence for each of the assays described, indicating that either a unique receptor or pharmacologically indistinguishable receptor subtypes activate distinct G-protein signalling pathways. Furthermore, we found that: (1) the N-terminal serine can be replaced by small or intermediately sized amino acids (+/- hydroxyl groups) without loss of activity. However, its replacement by an aromatic side-chain or omission of the N-terminal amino group severely reduces activity. (2) An aromatic side-chain on the penultimate N-terminal residue appears to play a critical role since phenylalanine in this position can be substituted by tyrosine without complete loss of activity whereas an alanine in its place is not tolerated. (3) Deletion of the first, second or third N-terminal residue leads to a loss of activity, suggesting that a defined spacing of more than one structural component may be important for ligand-receptor interaction. Finally, we did not observe an antagonistic effect of the inactive peptides on phospholipase C activation or DNA synthesis induced by alpha-thrombin (1 nM) or SFLLRNP (3 microM). PMID:7686363

Resin-assisted Enrichment of N-terminal Peptides for Characterizing Proteolytic Processing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jong Seo; Dai, Ziyu; Aryal, Uma K.

2013-06-17

Proteolytic processing is a ubiquitous, irreversible posttranslational modification that plays an important role in cellular regulation in all living organisms. Herein we report a resin-assisted positive selection method for specifically enriching protein N-terminal peptides to facilitate the characterization of proteolytic processing events by liquid chromatography-tandem mass spectrometry. In this approach, proteins are initially reduced and alkylated and their lysine residues are converted to homoarginines. Then, protein N-termini are selectively converted to reactive thiol groups. We demonstrate that these sequential reactions were achieved with nearly quantitative efficiencies. Thiol-containing N-terminal peptides are then captured (>98% efficiency) by a thiol-affinity resin, a significantmore » improvement over the traditional avidin/biotin enrichment. Application to cell lysates of Aspergillus niger, a filamentous fungus of interest for biomass degradation, enabled the identification of 1672 unique protein N-termini and proteolytic cleavage sites from 690 unique proteins.« less

Structure-activity relationship of HP (2-20) analog peptide: enhanced antimicrobial activity by N-terminal random coil region deletion.

PubMed

Park, Yoonkyung; Park, Seong-Cheol; Park, Hae-Kyun; Shin, Song Yub; Kim, Yangmee; Hahm, Kyung-Soo

2007-01-01

HP (2-20) (AKKVFKRLEKLFSKIQNDK) is a 19-aa antimicrobial peptide derived from N-terminus of Helicobacter pylori Ribosomal protein L1 (RpL1). In the previous study, several analogs with amino acid substitutions were designed to increase or decrease only the net hydrophobicity. In particular, substitutions of Gln(16) and Asp(18) with Trp (Anal 3) for hydrophobic amino acid caused a dramatic increase in antibiotic activity without a hemolytic effect. HP-A3 is a potent antimicrobial peptide that forms, in a hydrophobic medium, an amphipathic structure consisting of an N-terminal random coil region (residues 2-5) and extended C-terminal regular alpha-helical region (residues 6-20). To obtain the short and potent alpha-helical antimicrobial peptide, we synthesized a N-terminal random coil deleted HP-A3 (A3-NT) and examined their antimicrobial activity and mechanism of action. The resulting 15mer peptide showed increased antibacterial and antifungal activity to 2- and 4-fold, respectively, without hemolysis. Confocal fluorescence microscopy studies showed that A3-NT was accumulated in the plasma membrane. Flow cytometric analysis revealed that A3-NT acted in salt- and energy-independent manner. Furthermore, A3-NT causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy. Circular dichroism (CD) analysis revealed that A3-NT showed higher alpha-helical contents than the HP-A3 peptide in 50% TFE solution. Therefore, the cell-lytic efficiency of HP-A3, which depended on the alpha-helical content of peptide, correlated linearly with their antimicrobial potency.

Synthetic procedure for N-Fmoc amino acyl-N-sulfanylethylaniline linker as crypto-peptide thioester precursor with application to native chemical ligation.

PubMed

Sakamoto, Ken; Sato, Kohei; Shigenaga, Akira; Tsuji, Kohei; Tsuda, Shugo; Hibino, Hajime; Nishiuchi, Yuji; Otaka, Akira

2012-08-17

N-sulfanylethylanilide (SEAlide) peptides 1, obtainable using Fmoc-based solid-phase peptide synthesis (Fmoc SPPS), function as crypto-thioesters in native chemical ligation (NCL), yielding a wide variety of peptides/proteins. Their acylating potential with N-terminal cysteinyl peptides 2 can be tuned by the presence or absence of phosphate salts, leading to one-pot/multifragment ligation, operating under kinetically controlled conditions. SEAlide peptides have already been shown to be promising for use in protein synthesis; however, a widely applicable method for the synthesis of N-Fmoc amino acyl-N-sulfanylethylaniline linkers 4, required for the preparation of SEAlide peptides, is unavailable. The present study addresses the development of efficient condensation protocols of 20 naturally occurring amino acid derivatives to the N-sulfanylethylaniline linker 5. N-Fmoc amino acyl aniline linkers 4 of practical use in NCL chemistry, except in the case of the proline- or aspartic acid-containing linker, were successfully synthesized by coupling of POCl(3)- or SOCl(2)-activated Fmoc amino acid derivatives with sodium anilide species 6, without accompanying racemization and loss of side-chain protection. Furthermore, SEAlide peptides 7 possessing various C-terminal amino acids (Gly, His, Phe, Ala, Asn, Ser, Glu, and Val) were shown to be of practical use in NCL chemistry.

Native Chemical Ligation Strategy to Overcome Side Reactions during Fmoc-Based Synthesis of C-Terminal Cysteine-Containing Peptides.

PubMed

Lelièvre, Dominique; Terrier, Victor P; Delmas, Agnès F; Aucagne, Vincent

2016-03-04

The Fmoc-based solid phase synthesis of C-terminal cysteine-containing peptides is problematic, due to side reactions provoked by the pronounced acidity of the Cα proton of cysteine esters. We herein describe a general strategy consisting of the postsynthetic introduction of the C-terminal Cys through a key chemoselective native chemical ligation reaction with N-Hnb-Cys peptide crypto-thioesters. This method was successfully applied to the demanding peptide sequences of two natural products of biological interest, giving remarkably high overall yields compared to that of a state of the art strategy.

Chloroplast-encoded serotonin N-acetyltransferase in the red alga Pyropia yezoensis: gene transition to the nucleus from chloroplasts.

PubMed

Byeon, Yeong; Yool Lee, Hyoung; Choi, Dong-Woog; Back, Kyoungwhan

2015-02-01

Melatonin biosynthesis involves the N-acetylation of arylalkylamines such as serotonin, which is catalysed by serotonin N-acetyltransferase (SNAT), the penultimate enzyme of melatonin biosynthesis in both animals and plants. Here, we report the functional characterization of a putative N-acetyltransferase gene in the chloroplast genome of the alga laver (Pyropia yezoensis, formerly known as Porphyra yezoensis) with homology to the rice SNAT gene. To confirm that the putative Pyropia yezoensis SNAT (PySNAT) gene encodes an SNAT, we cloned the full-length chloroplastidic PySNAT gene by PCR and purified the recombinant PySNAT protein from Escherichia coli. PySNAT was 174 aa and had 50% amino acid identity with cyanobacteria SNAT. Purified recombinant PySNAT showed a peak activity at 55 °C with a K m of 467 µM and V max of 28 nmol min-1 mg(-1) of protein. Unlike other plant SNATs, PySNAT localized to the cytoplasm due to a lack of N-terminal chloroplast transit peptides. Melatonin was present at 0.16ng g(-1) of fresh mass but increased during heat stress. Phylogenetic analysis of the sequence suggested that PySNAT has evolved from the cyanobacteria SNAT gene via endosymbiotic gene transfer. Additionally, the chloroplast transit peptides of plant SNATs were acquired 1500 million years ago, concurrent with the appearance of green algae. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Post-staining electroblotting for efficient and reliable peptide blotting.

PubMed

Lee, Der-Yen; Chang, Geen-Dong

2015-01-01

Post-staining electroblotting has been previously described to transfer Coomassie blue-stained proteins from polyacrylamide gel onto polyvinylidene difluoride (PVDF) membranes. Actually, stained peptides can also be efficiently and reliably transferred. Because of selective staining procedures for peptides and increased retention of stained peptides on the membrane, even peptides with molecular masses less than 2 kDa such as bacitracin and granuliberin R are transferred with satisfactory results. For comparison, post-staining electroblotting is about 16-fold more sensitive than the conventional electroblotting for visualization of insulin on the membrane. Therefore, the peptide blots become practicable and more accessible to further applications, e.g., blot overlay detection or immunoblotting analysis. In addition, the efficiency of peptide transfer is favorable for N-terminal sequence analysis. With this method, peptide blotting can be normalized for further analysis such as blot overlay assay, immunoblotting, and N-terminal sequencing for identification of peptide in crude or partially purified samples.

Peptide mimics of the M13 coat protein transmembrane segment. Retention of helix-helix interaction motifs.

PubMed

Wang, C; Deber, C M

2000-05-26

Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK. The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N-terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation. Using the propensity of the protein to undergo thermally induced alpha-helix --> beta-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24-42). Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics. Molecular dynamics simulations support the tight "two in-two out" dimerization motif for V31A consistent with mutagenesis data. The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis.

Preparation and Analysis of N-Terminal Chemokine Receptor Sulfopeptides Using Tyrosylprotein Sulfotransferase Enzymes.

PubMed

Seibert, Christoph; Sanfiz, Anthony; Sakmar, Thomas P; Veldkamp, Christopher T

2016-01-01

In most chemokine receptors, one or multiple tyrosine residues have been identified within the receptor N-terminal domain that are, at least partially, modified by posttranslational tyrosine sulfation. For example, tyrosine sulfation has been demonstrated for Tyr-3, -10, -14, and -15 of CCR5, for Tyr-3, -14, and -15 of CCR8, and for Tyr-7, -12, and -21 of CXCR4. While there is evidence for several chemokine receptors that tyrosine sulfation is required for optimal interaction with the chemokine ligands, the precise role of tyrosine sulfation for chemokine receptor function remains unclear. Furthermore, the function of the chemokine receptor N-terminal domain in chemokine binding and receptor activation is also not well understood. Sulfotyrosine peptides corresponding to the chemokine receptor N-termini are valuable tools to address these important questions both in structural and functional studies. However, due to the lability of the sulfotyrosine modification, these peptides are difficult to obtain using standard peptide chemistry methods. In this chapter, we provide methods to prepare sulfotyrosine peptides by enzymatic in vitro sulfation of peptides using purified recombinant tyrosylprotein sulfotransferase (TPST) enzymes. In addition, we also discuss alternative approaches for the generation of sulfotyrosine peptides and methods for sulfopeptide analysis. © 2016 Elsevier Inc. All rights reserved.

Preparation and analysis of N-terminal chemokine receptor sulfopeptides using tyrosylprotein sulfotransferase enzymes

PubMed Central

Seibert, Christoph; Sanfiz, Anthony; Sakmar, Thomas P.; Veldkamp, Christopher T.

2016-01-01

In most chemokine receptors, one or multiple tyrosine residues have been identified within the receptor N-terminal domain that are, at least partially, modified by post-translational tyrosine sulfation. For example, tyrosine sulfation has been demonstrated for Tyr-3, -10, -14, and -15 of CCR5, for Tyr-3, -14, and -15 of CCR8 and for Tyr-7, -12, and -21 of CXCR4. While there is evidence for several chemokine receptors that tyrosine sulfation is required for optimal interaction with the chemokine ligands, the precise role of tyrosine sulfation for chemokine receptor function remains unclear. Furthermore, the function of the chemokine receptor N-terminal domain in chemokine binding and receptor activation is also not well understood. Sulfotyrosine peptides corresponding to the chemokine receptor N-termini are valuable tools to address these important questions both in structural and functional studies. However, due to the liability of the sulfotyrosine modification, these peptides are difficult to obtain using standard peptide chemistry methods. In this chapter, we provide methods to prepare sulfotyrosine peptides by enzymatic in vitro sulfation of peptides using purified recombinant tyrosylprotein sulfotransferase (TPST) enzymes. In addition, we also discuss alternative approaches for the generation of sulfotyrosine peptides and methods from sulfopeptide analysis. PMID:26921955

The Specificity of Trimming of MHC Class I-Presented Peptides in the Endoplasmic Reticulum1

PubMed Central

Hearn, Arron; York, Ian A.; Rock, Kenneth L.

2010-01-01

Aminopeptidases in the endoplasmic reticulum (ER) can cleave antigenic peptides and in so doing either create or destroy MHC class I-presented epitopes. However the specificity of this trimming process overall and of the major ER aminopeptidase ERAP1 in particular is not well understood. This issue is important because peptide trimming influences the magnitude and specificity of CD8 T cell responses. By systematically varying the N-terminal flanking sequences of peptides in a cell free biochemical system and in intact cells, we elucidated the specificity of ERAP1 and of ER trimming overall. ERAP1 can cleave after many amino acids on the N-terminus of epitope precursors but does so at markedly different rates. The specificity seen with purified ERAP1 is similar to that observed for trimming and presentation of epitopes in the ER of intact cells. We define N-terminal sequences that are favorable or unfavorable for antigen presentation in ways that are independent from the epitopes core sequence. When databases of known presented peptides were analyzed, the residues that were preferred for the trimming of model peptide precursors were found to be overrepresented in N-terminal flanking sequences of epitopes generally. These data define key determinants in the specificity of antigen processing. PMID:19828632

Upregulation of α7 Nicotinic Receptors by Acetylcholinesterase C-Terminal Peptides

PubMed Central

Bond, Cherie E.; Zimmermann, Martina; Greenfield, Susan A.

2009-01-01

Background The alpha-7 nicotinic acetylcholine receptor (α7-nAChR) is well known as a potent calcium ionophore that, in the brain, has been implicated in excitotoxicity and hence in the underlying mechanisms of neurodegenerative disorders such as Alzheimer's disease. Previous research implied that the activity of this receptor may be modified by exposure to a peptide fragment derived from the C-terminal region of the enzyme acetylcholinesterase. This investigation was undertaken to determine if the functional changes observed could be attributed to peptide binding interaction with the α7-nAChR, or peptide modulation of receptor expression. Methodology/Principal Findings This study provides evidence that two peptides derived from the C-terminus of acetylcholinesterase, not only selectively displace specific bungarotoxin binding at the α7-nAChR, but also alter receptor binding properties for its familiar ligands, including the alternative endogenous agonist choline. Of more long-term significance, these peptides also induce upregulation of α7-nAChR mRNA and protein expression, as well as enhancing receptor trafficking to the plasma membrane. Conclusions/Significance The results reported here demonstrate a hitherto unknown relationship between the α7-nAChR and the non-enzymatic functions of acetylcholinesterase, mediated independently by its C-terminal domain. Such an interaction may prove valuable as a pharmacological tool, prompting new approaches for understanding, and combating, the process of neurodegeneration. PMID:19287501

Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor-Derived Peptides for Regulation of Mast Cell Degranulation.

PubMed

Yang, Yoosoo; Kong, Byoungjae; Jung, Younghoon; Park, Joon-Bum; Oh, Jung-Mi; Hwang, Jaesung; Cho, Jae Youl; Kweon, Dae-Hyuk

2018-01-01

Vesicle-associated V-soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and target membrane-associated T-SNAREs (syntaxin 4 and SNAP-23) assemble into a core trans -SNARE complex that mediates membrane fusion during mast cell degranulation. This complex plays pivotal roles at various stages of exocytosis from the initial priming step to fusion pore opening and expansion, finally resulting in the release of the vesicle contents. In this study, peptides with the sequences of various SNARE motifs were investigated for their potential inhibitory effects against SNARE complex formation and mast cell degranulation. The peptides with the sequences of the N-terminal regions of vesicle-associated membrane protein 2 (VAMP2) and VAMP8 were found to reduce mast cell degranulation by inhibiting SNARE complex formation. The fusion of protein transduction domains to the N-terminal of each peptide enabled the internalization of the fusion peptides into the cells equally as efficiently as cell permeabilization by streptolysin-O without any loss of their inhibitory activities. Distinct subsets of mast cell granules could be selectively regulated by the N-terminal-mimicking peptides derived from VAMP2 and VAMP8, and they effectively decreased the symptoms of atopic dermatitis in mouse models. These results suggest that the cell membrane fusion machinery may represent a therapeutic target for atopic dermatitis.

An optimized transit peptide for effective targeting of diverse foreign proteins into chloroplasts in rice.

PubMed

Shen, Bo-Ran; Zhu, Cheng-Hua; Yao, Zhen; Cui, Li-Li; Zhang, Jian-Jun; Yang, Cheng-Wei; He, Zheng-Hui; Peng, Xin-Xiang

2017-04-11

Various chloroplast transit peptides (CTP) have been used to successfully target some foreign proteins into chloroplasts, but for other proteins these same CTPs have reduced localization efficiencies or fail completely. The underlying cause of the failures remains an open question, and more effective CTPs are needed. In this study, we initially observed that two E.coli enzymes, EcTSR and EcGCL, failed to be targeted into rice chloroplasts by the commonly-used rice rbcS transit peptide (rCTP) and were subsequently degraded. Further analyses revealed that the N-terminal unfolded region of cargo proteins is critical for their localization capability, and that a length of about 20 amino acids is required to attain the maximum localization efficiency. We considered that the unfolded region may alleviate the steric hindrance produced by the cargo protein, by functioning as a spacer to which cytosolic translocators can bind. Based on this inference, an optimized CTP, named RC2, was constructed. Analyses showed that RC2 can more effectively target diverse proteins, including EcTSR and EcGCL, into rice chloroplasts. Collectively, our results provide further insight into the mechanism of CTP-mediated chloroplastic localization, and more importantly, RC2 can be widely applied in future chloroplastic metabolic engineering, particularly for crop plants.

Antifungal properties of durancins isolated from Enterococcus durans A5-11 and of its synthetic fragments.

PubMed

Belguesmia, Y; Choiset, Y; Rabesona, H; Baudy-Floc'h, M; Le Blay, G; Haertlé, T; Chobert, J-M

2013-04-01

The aim of this work was to study the antifungal properties of durancins isolated from Enterococcus durans A5-11 and of their chemically synthesized fragments. Enterococcus durans A5-11 is a lactic acid bacteria strain isolated from traditional Mongolian airag cheese. This strain inhibits the growth of several fungi including Fusarium culmorum, Penicillium roqueforti and Debaryomyces hansenii. It produces two bacteriocins: durancin A5-11a and durancin A5-11b, which have similar antimicrobial properties. The whole durancins A5-11a and A5-11b, as well as their N- and C-terminal fragments were synthesized, and their antifungal properties were studied. C-terminal fragments of both durancins showed stronger antifungal activities than other tested peptides. Treatment of D. hansenii LMSA2.11.003 strain with 2 mmol l(-1) of the synthetic peptides led to the loss of the membrane integrity and to several changes in the ultra-structure of the yeast cells. Chemically synthesized durancins and their synthetic fragments showed different antimicrobial properties from each other. N-terminal peptides show activities against both bacterial and fungal strains tested. C-terminal peptides have specific activities against tested fungal strain and do not show antibacterial activity. However, the C-terminal fragment enhances the activity of the N-terminal fragment in the whole bacteriocins against bacteria. © 2012 The Society for Applied Microbiology.

Symplocin A, a Linear Peptide from the Bahamian Cyanobacterium Symploca sp. Configurational Analysis of N,N-Dimethylamino Acids by Chiral-Phase HPLC of Naphthacyl Esters†

PubMed Central

Molinski, Tadeusz F.; Reynolds, Kirk A.; Morinaka, Brandon I.

2012-01-01

The absolute stereostructures of the components of symplocin A (3), a new N,N-dimethyl-terminated peptide from the Bahamian cyanobacterium, Symploca sp., were assigned from spectroscopic analysis, including MS and 2D NMR and Marfey’s analysis. The complete absolute configuration of symplocin A, including the unexpected D-configurations of the terminal N,N-dimethylisoleucine and valic acid residues, were assigned by chiral-phase HPLC of the corresponding 2-naphthacyl esters, a highly sensitive, complementary strategy for assignment of N-blocked peptide residues where Marfey’s method is ineffectual, or other methods fall short. Symplocin A exhibited potent activity as an inhibitor of cathepsin E (IC50 300 pM). PMID:22360587

Designer Self-Assembling Peptide Nanofiber Scaffolds Containing Link Protein N-Terminal Peptide Induce Chondrogenesis of Rabbit Bone Marrow Stem Cells

PubMed Central

Wang, Baichuan; Sun, Caixia; Shao, Zengwu; Yang, Shuhua; Che, Biao; Wu, Qiang; Liu, Jianxiang

2014-01-01

Designer self-assembling peptide nanofiber hydrogel scaffolds have been considered as promising biomaterials for tissue engineering because of their excellent biocompatibility and biofunctionality. Our previous studies have shown that a novel designer functionalized self-assembling peptide nanofiber hydrogel scaffold (RLN/RADA16, LN-NS) containing N-terminal peptide sequence of link protein (link N) can promote nucleus pulposus cells (NPCs) adhesion and three-dimensional (3D) migration and stimulate biosynthesis of type II collagen and aggrecan by NPCs in vitro. The present study has extended these investigations to determine the effects of this functionalized LN-NS on bone marrow stem cells (BMSCs), a potential cell source for NP regeneration. Although the functionalized LN-NS cannot promote BMSCs proliferation, it significantly promotes BMSCs adhesion compared with that of the pure RADA16 hydrogel scaffold. Moreover, the functionalized LN-NS remarkably stimulates biosynthesis and deposition of type II collagen and aggrecan. These data demonstrate that the functionalized peptide nanofiber hydrogel scaffold containing link N peptide as a potential matrix substrate will be very useful in the NP tissue regeneration. PMID:25243141

Comparison of N-terminal modifications on neurotensin(8-13) analogues correlates peptide stability but not binding affinity with in vivo efficacy.

PubMed

Orwig, Kevin S; Lassetter, McKensie R; Hadden, M Kyle; Dix, Thomas A

2009-04-09

Neurotensin(8-13) and two related analogues were used as model systems to directly compare various N-terminal peptide modifications representing both commonly used and novel capping groups. Each N-terminal modification prevented aminopeptidase cleavage but surprisingly differed in its ability to inhibit cleavage at other sites, a phenomenon attributed to long-range conformational effects. None of the capping groups were inherently detrimental to human neurotensin receptor 1 (hNTR1) binding affinity or receptor agonism. Although the most stable peptides exhibited the lowest binding affinities and were the least potent receptor agonists, they produced the largest in vivo effects. Of the parameters studied only stability significantly correlated with in vivo efficacy, demonstrating that a reduction in binding affinity at NTR1 can be countered by increased in vivo stability.

Abundant pyroglutamate-modified ABri and ADan peptides in extracellular and vascular amyloid deposits in familial British and Danish dementias.

PubMed

Saul, Anika; Lashley, Tammaryn; Revesz, Tamas; Holton, Janice; Ghiso, Jorge A; Coomaraswamy, Janaky; Wirths, Oliver

2013-05-01

Familial British and familial Danish dementia (FDD) are progressive neurodegenerative disorders characterized by cerebral deposition of the amyloidogenic peptides ABri and ADan, respectively. These amyloid peptides start with an N-terminal glutamate residue, which can be posttranslationally converted into a pyroglutamate (pGlu) modified form, a mechanism which has been extensively described to be relevant for amyloid-beta (Aβ) peptides in Alzheimer's disease. Like pGlu-Aβ peptides, pGlu-ABri peptides have an increased aggregation propensity and show higher toxicity on human neuroblastoma cells as their nonmodified counterparts. We have generated novel N-terminal specific antibodies detecting the pGlu-modified forms of ABri and ADan peptides. With these antibodies we were able to identify abundant extracellular amyloid plaques, vascular, and parenchymal deposits in human familial British dementia and FDD brain tissue, and in a mouse model for FDD. Double-stainings using C-terminal specific antibodies in human samples revealed that highly aggregated pGlu-ABri and pGlu-ADan peptides are mainly present in plaque cores and central vascular deposits, leading to the assumption that these peptides have seeding properties. Furthermore, in an FDD-mouse model ADan peptides were detected in presynaptic terminals of the hippocampus where they might contribute to impaired synaptic transmission. These similarities of ABri and ADan to Aβ in Alzheimer's disease suggest that the posttranslational pGlu-modification of amyloid peptides might represent a general pathological mechanism leading to increased aggregation and toxicity in these forms of degenerative dementias. Copyright © 2013 Elsevier Inc. All rights reserved.

Abundant pyroglutamate-modified ABri and ADan peptides in extracellular and vascular amyloid deposits in familial British and Danish dementias

PubMed Central

Saul, Anika; Lashley, Tammaryn; Revesz, Tamas; Holton, Janice; Ghiso, Jorge A.; Coomaraswamy, Janaky; Wirths, Oliver

2013-01-01

Familial British (FBD) and familial Danish dementia (FDD) are progressive neurodegenerative disorders characterized by cerebral deposition of the amyloidogenic peptides ABri and ADan. These amyloid peptides start with an N-terminal glutamate residue, which can be posttranslationally converted into a pyroglutamate (pGlu-) modified form, a mechanism which has been extensively described to be relevant for Aβ peptides in Alzheimer’s disease (AD). Like pGlu-Aβ peptides, pGlu-ABri peptides have an increased aggregation propensity and show higher toxicity on human neuroblastoma cells as their non-modified counterparts. We have generated novel N-terminal specific antibodies detecting the pGlu-modified forms of ABri and ADan peptides. With these antibodies we were able to identify abundant extracellular amyloid plaques, vascular and parenchymal deposits in human FBD and FDD brain tissue, as well as in a mouse model for FDD. Double-stainings using C-terminal specific antibodies in human samples revealed that highly aggregated pGlu-ABri and pGlu-ADan peptides are mainly present in plaque cores and central vascular deposits, leading to the assumption that these peptides have seeding properties. Furthermore, in an FDD-mouse model ADan peptides were detected in pre-synaptic terminals of the hippocampus where they might contribute to impaired synaptic transmission. These similarities of ABri and ADan to Aβ in AD suggest that the posttranslational pGlu-modification of amyloid peptides might represent a general pathological mechanism leading to increased aggregation and toxicity in these forms of degenerative dementias. PMID:23261769

Comparable increase of B-type natriuretic peptide and amino-terminal pro-B-type natriuretic peptide levels in patients with severe sepsis, septic shock, and acute heart failure.

PubMed

Rudiger, Alain; Gasser, Stefan; Fischler, Manuel; Hornemann, Thorsten; von Eckardstein, Arnold; Maggiorini, Marco

2006-08-01

B-type natriuretic peptide (BNP) and N-terminal pro-BNP measurements are used for the diagnosis of congestive heart failure (HF). However, the diagnostic value of these tests is unknown under septic conditions. We compared patients with severe sepsis or septic shock and patients with acute HF to unravel the influence of the underlying diagnosis on BNP and N-terminal pro-BNP levels. Prospective, clinical study. Academic medical intensive care unit (ICU). A total of 249 consecutive patients were screened for the diagnosis of sepsis or HF. Sepsis was defined according to published guidelines. HF was diagnosed in the presence of an underlying heart disease and congestive HF, pulmonary edema, or cardiogenic shock. BNP and N-terminal pro-BNP were measured from blood samples that were drawn daily for routine analysis. We identified 24 patients with severe sepsis or septic shock and 51 patients with acute HF. At admission, the median (range) BNP and N-terminal pro-BNP levels were 572 (13-1,300) and 6,526 (198-70,000) ng/L in patients with sepsis and 581 (6-1,300) and 4,300 (126-70,000) ng/L in patients with HF. The natriuretic peptide levels increased during the ICU stay, but the differences between the groups were not significant. Nine patients with sepsis and eight patients with HF were monitored with a pulmonary artery catheter. Mean (sd) pulmonary artery occlusion pressure were 16 (4.2) and 22 (5.3) mm Hg (p = .02), and cardiac indexes were 4.6 (2.8) and 2.2 (0.6) L/min/m (p = .03) in patients with sepsis and HF, respectively. Despite these clear hemodynamic differences BNP and N-terminal pro-BNP levels were not statistically different between the two groups. In patients with severe sepsis or septic shock, BNP and N-terminal pro-BNP values are highly elevated and, despite significant hemodynamic differences, comparable with those found in acute HF patients. It remains to be determined how elevations of natriuretic peptide levels are linked to inflammation and sepsis-associated myocardial dysfunction.

Highly potent antimicrobial peptides from N-terminal membrane-binding region of E. coli MreB.

PubMed

Saikia, Karabi; Sravani, Yalavarthi Durga; Ramakrishnan, Vibin; Chaudhary, Nitin

2017-02-23

Microbial pathogenesis is a serious health concern. The threat escalates as the existing conventional antimicrobials are losing their efficacy against the evolving pathogens. Peptides hold promise to be developed into next-generation antibiotics. Antimicrobial peptides adopt amphipathic structures that could selectively bind to and disrupt the microbial membranes. Interaction of proteins with membranes is central to all living systems and we reasoned that the membrane-binding domains in microbial proteins could be developed into efficient antimicrobials. This is an interesting approach as self-like sequences could elude the microbial strategies of degrading the antimicrobial peptides, one of the mechanisms of showing resistance to antimicrobials. We selected the 9-residue-long membrane-binding region of E. coli MreB protein. The 9-residue peptide (C-terminal amide) and its N-terminal acetylated analog displayed broad-spectrum activity, killing Gram-negative bacteria, Gram-positive bacteria, and fungi. Extension with a tryptophan residue at the N-terminus drastically improved the activity of the peptides with lethal concentrations ≤10 μM against all the organisms tested. The tryptophan-extended peptides caused complete killing of C. albicans as well as gentamicin and methicillin resistant S. aureus at 5 μM concentration. Lipid-binding studies and electron microscopic analyses of the peptide-treated microbes suggest membrane disruption as the mechanism of killing.

The M-T Hook Structure Is Critical for Design of HIV-1 Fusion Inhibitors*

PubMed Central

Chong, Huihui; Yao, Xue; Sun, Jianping; Qiu, Zonglin; Zhang, Meng; Waltersperger, Sandro; Wang, Meitian; Cui, Sheng; He, Yuxian

2012-01-01

CP621-652 is a potent HIV-1 fusion inhibitor peptide derived from the C-terminal heptad repeat of gp41. We recently identified that its N-terminal residues Met-626 and Thr-627 adopt a unique hook-like structure (termed M-T hook) thus stabilizing the interaction of the inhibitor with the deep pocket on the N-terminal heptad repeat. In this study, we further demonstrated that the M-T hook structure is a key determinant of CP621-652 in terms of its thermostability and anti-HIV activity. To directly define the structure and function of the M-T hook, we generated the peptide MT-C34 by incorporating Met-626 and Thr-627 into the N terminus of the C-terminal heptad repeat-derived peptide C34. The high resolution crystal structure (1.9 Å) of MT-C34 complexed by an N-terminal heptad repeat-derived peptide reveals that the M-T hook conformation is well preserved at the N-terminal extreme of the inhibitor. Strikingly, addition of two hook residues could dramatically enhance the binding affinity and thermostability of 6-helix bundle core. Compared with C34, MT-C34 exhibited significantly increased activity to inhibit HIV-1 envelope-mediated cell fusion (6.6-fold), virus entry (4.5-fold), and replication (6-fold). Mechanistically, MT-C34 had a 10.5-fold higher increase than C34 in blocking 6-helix bundle formation. We further showed that MT-C34 possessed higher potency against T20 (Enfuvirtide, Fuzeon)-resistant HIV-1 variants. Therefore, this study provides convincing data for our proposed concept that the M-T hook structure is critical for designing HIV-1 fusion inhibitors. PMID:22879603

Efficient peptide ligation between allyl-protected Asp and Cys followed by palladium-mediated deprotection.

PubMed

Kamo, Naoki; Hayashi, Gosuke; Okamoto, Akimitsu

2018-04-24

An efficient method for peptide ligation between C-terminal Asp(OAllyl) and N-terminal Cys has been developed. Peptide ligation and removal of the allyl group at the Asp carboxylate side chain proceeded in one pot by adding a small amount of Pd/TPPTS complex. Based on this efficient synthetic method, PEP-19 (61 amino acids), which is highly expressed in Purkinje cells, was synthesized.

Border control: selectivity of chloroplast protein import and regulation at the TOC-complex

PubMed Central

Demarsy, Emilie; Lakshmanan, Ashok M.; Kessler, Felix

2014-01-01

Plants have evolved complex and sophisticated molecular mechanisms to regulate their development and adapt to their surrounding environment. Particularly the development of their specific organelles, chloroplasts and other plastid-types, is finely tuned in accordance with the metabolic needs of the cell. The normal development and functioning of plastids require import of particular subsets of nuclear encoded proteins. Most preproteins contain a cleavable sequence at their N terminal (transit peptide) serving as a signal for targeting to the organelle and recognition by the translocation machinery TOC–TIC (translocon of outer membrane complex–translocon of inner membrane complex) spanning the dual membrane envelope. The plastid proteome needs constant remodeling in response to developmental and environmental factors. Therefore selective regulation of preprotein import plays a crucial role in plant development. In this review we describe the diversity of transit peptides and TOC receptor complexes, and summarize the current knowledge and potential directions for future research concerning regulation of the different Toc isoforms. PMID:25278954

Border control: selectivity of chloroplast protein import and regulation at the TOC-complex.

PubMed

Demarsy, Emilie; Lakshmanan, Ashok M; Kessler, Felix

2014-01-01

Plants have evolved complex and sophisticated molecular mechanisms to regulate their development and adapt to their surrounding environment. Particularly the development of their specific organelles, chloroplasts and other plastid-types, is finely tuned in accordance with the metabolic needs of the cell. The normal development and functioning of plastids require import of particular subsets of nuclear encoded proteins. Most preproteins contain a cleavable sequence at their N terminal (transit peptide) serving as a signal for targeting to the organelle and recognition by the translocation machinery TOC-TIC (translocon of outer membrane complex-translocon of inner membrane complex) spanning the dual membrane envelope. The plastid proteome needs constant remodeling in response to developmental and environmental factors. Therefore selective regulation of preprotein import plays a crucial role in plant development. In this review we describe the diversity of transit peptides and TOC receptor complexes, and summarize the current knowledge and potential directions for future research concerning regulation of the different Toc isoforms.

Paramyxovirus F1 protein has two fusion peptides: implications for the mechanism of membrane fusion.

PubMed

Peisajovich, S G; Samuel, O; Shai, Y

2000-03-10

Viral fusion proteins contain a highly hydrophobic segment, named the fusion peptide, which is thought to be responsible for the merging of the cellular and viral membranes. Paramyxoviruses are believed to contain a single fusion peptide at the N terminus of the F1 protein. However, here we identified an additional internal segment in the Sendai virus F1 protein (amino acids 214-226) highly homologous to the fusion peptides of HIV-1 and RSV. A synthetic peptide, which includes this region, was found to induce membrane fusion of large unilamellar vesicles, at concentrations where the known N-terminal fusion peptide is not effective. A scrambled peptide as well as several peptides from other regions of the F1 protein, which strongly bind to membranes, are not fusogenic. The functional and structural characterization of this active segment suggest that the F1 protein has an additional internal fusion peptide that could participate in the actual fusion event. The presence of homologous regions in other members of the same family suggests that the concerted action of two fusion peptides, one N-terminal and the other internal, is a general feature of paramyxoviruses. Copyright 2000 Academic Press.

Antimicrobial and Immunomodulatory Activities of PR-39 Derived Peptides

PubMed Central

Veldhuizen, Edwin J. A.; Schneider, Viktoria A. F.; Agustiandari, Herfita; van Dijk, Albert; Tjeerdsma-van Bokhoven, Johanna L. M.; Bikker, Floris J.; Haagsman, Henk P.

2014-01-01

The porcine cathelicidin PR-39 is a host defence peptide that plays a pivotal role in the innate immune defence of the pig against infections. Besides direct antimicrobial activity, it is involved in immunomodulation, wound healing and several other biological processes. In this study, the antimicrobial- and immunomodulatory activity of PR-39, and N- and C-terminal derivatives of PR-39 were tested. PR-39 exhibited an unexpected broad antimicrobial spectrum including several Gram positive strains such as Bacillus globigii and Enterococcus faecalis. Of organisms tested, only Staphylococcus aureus was insensitive to PR-39. Truncation of PR-39 down to 15 (N-terminal) amino acids did not lead to major loss of activity, while peptides corresponding to the C-terminal part of PR-39 were hampered in their antimicrobial activity. However, shorter peptides were all much more sensitive to inhibition by salt. Active peptides induced ATP leakage and loss of membrane potential in Bacillus globigii and Escherichia coli, indicating a lytic mechanism of action for these peptides. Finally, only the mature peptide was able to induce IL-8 production in porcine macrophages, but some shorter peptides also had an effect on TNF-α production showing differential regulation of cytokine induction by PR-39 derived peptides. None of the active peptides showed high cytotoxicity highlighting the potential of these peptides for use as an alternative to antibiotics. PMID:24755622

Molecular dynamics simulation of the enterostatin APGPR and VPDPR peptides in water

NASA Astrophysics Data System (ADS)

Trucco, Gabriella; Fornili, Sandro L.

2007-09-01

We report on structural and dynamic properties in water of all the isomers of both peptides related to the trans and cis conformations of the peptide bonds preceding the proline (Pro) residues. Free-energy calculations indicate that the isomers having the Pro closer to the N-terminus (Pro1) in trans and the Pro2 in cis conformations are the most populated. Furthermore, the backbone is more flexible for APGPR than for VPDPR, and its conformation is more stable in the hydrophilic C-terminal moiety than in the hydrophobic N-terminal region.

Identification of C-terminal phosphorylation sites of N-formyl peptide receptor-1 (FPR1) in human blood neutrophils.

PubMed

Maaty, Walid S; Lord, Connie I; Gripentrog, Jeannie M; Riesselman, Marcia; Keren-Aviram, Gal; Liu, Ting; Dratz, Edward A; Bothner, Brian; Jesaitis, Algirdas J

2013-09-20

Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (∼34,000 Mr) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu(312)-Arg(322) and Arg(323)-Lys(350)) and extracellular FPR1 peptide (Ile(191)-Arg(201)) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala(323)-Lys(350) only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr(325), Ser(328), Thr(329), Thr(331), Ser(332), Thr(334), and Thr(339). No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC50 = 33 ± 8 to 74 ± 21 nM. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of FPR1 phosphorylation can be monitored with C-terminal tail FPR1-phosphospecific antibodies.

Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates.

PubMed

Teixeira, Luis Gustavo D; Malavolta, Luciana; Bersanetti, Patrícia A; Schreier, Shirley; Carmona, Adriana K; Nakaie, Clovis R

2015-01-01

Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC0-AngI and TOAC1-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe8-His9 peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr4 fluorescence by the differently positioned TOAC residues corroborated the data obtained by the other spectroscopic techniques. Lastly, we demonstrated the feasibility of monitoring the progress of ACE-catalyzed hydrolysis of TOAC-labeled peptides by following time-dependent changes in their EPR spectra.

Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates

PubMed Central

Teixeira, Luis Gustavo D.; Malavolta, Luciana; Bersanetti, Patrícia A.; Schreier, Shirley; Carmona, Adriana K.; Nakaie, Clovis R.

2015-01-01

Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC0-AngI and TOAC1-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe8-His9 peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr4 fluorescence by the differently positioned TOAC residues corroborated the data obtained by the other spectroscopic techniques. Lastly, we demonstrated the feasibility of monitoring the progress of ACE-catalyzed hydrolysis of TOAC-labeled peptides by following time-dependent changes in their EPR spectra. PMID:26317625

Sensing pH via p-cyanophenylalanine fluorescence: Application to determine peptide pKa and membrane penetration kinetics.

PubMed

Pazos, Ileana M; Ahmed, Ismail A; Berríos, Mariana I León; Gai, Feng

2015-08-15

We expand the spectroscopic utility of a well-known infrared and fluorescence probe, p-cyanophenylalanine, by showing that it can also serve as a pH sensor. This new application is based on the notion that the fluorescence quantum yield of this unnatural amino acid, when placed at or near the N-terminal end of a polypeptide, depends on the protonation status of the N-terminal amino group of the peptide. Using this pH sensor, we are able to determine the N-terminal pKa values of nine tripeptides and also the membrane penetration kinetics of a cell-penetrating peptide. Taken together, these examples demonstrate the applicability of using this unnatural amino acid fluorophore to study pH-dependent biological processes or events that accompany a pH change. Copyright © 2015 Elsevier Inc. All rights reserved.

Peptides mimicking viral proteins of porcine circovirus type 2 were profiled by the spectrum of mouse anti-PCV2 antibodies.

PubMed

Hung, Ling-Chu; Yang, Cheng-Yao; Cheng, Ivan-Chen

2017-05-15

Porcine circovirus 2 (PCV2) is a small, non-enveloped DNA virus causing swine lymphocyte depletion and severe impact on the swine industry. The aim of this study was to evaluate the antigenicity and immunogenicity of specific peptides, and seeking the potential candidate of PCV2 peptide-based vaccine. It's initiating from peptides reacting with PCV2-infected pig sera and peptide-immunized mouse sera. The data showed that the sera from PCV2-infected pigs could react with the N-terminal (C1), middle region (C2), and C-terminal peptide (C3) of PCV2 capsid protein (CP), ORF3 protein (N1), ORF6 protein (N2) and ORF9 protein (N3). This study demonstrated that anti-PCV2 mouse antisera could be generated by specific synthetic peptides (C3 and N2) and recognized PCV2 viral protein. We found that the tertiary or linear form C-terminal sequence (C3) of PCV2 capsid peptide only appeared a local distribution in the nucleus of PCV2-infected PK cells, virus-like particles of PCV2 major appeared a local distribution in the cytoplasm, and ORF 6 protein of PCV2 were shown unusually in cytoplasm. Furthermore, most residues of the C1 and the C3 were presented on the surface of PCV2 CP, in the view of 3-D structure of the CP. Our data demonstrated that PCV2-infected pigs had higher OD 405 value of anti-C3 IgG on Day 1, Month 3 and Month 6 than in Month 1. These pigs had higher anti-C3 IgM level in Month 3 and Month 6 than on Day 1 (P < 0.01). We demonstrated that the key peptide (C3) mimic the C-terminal of PCV2 capsid protein which were capable of inducing antibodies. The specific antibody against the C3 were confirmed as the serological marker in PCV2-infected pigs.

Conformational Dynamics of Titin PEVK Explored with FRET Spectroscopy

PubMed Central

Huber, Tamás; Grama, László; Hetényi, Csaba; Schay, Gusztáv; Fülöp, Lívia; Penke, Botond; Kellermayer, Miklós S.Z.

2012-01-01

The proline-, glutamate-, valine-, and lysine-rich (PEVK) domain of the giant muscle protein titin is thought to be an intrinsically unstructured random-coil segment. Various observations suggest, however, that the domain may not be completely devoid of internal interactions and structural features. To test the validity of random polymer models for PEVK, we determined the mean end-to-end distances of an 11- and a 21-residue synthetic PEVK peptide, calculated from the efficiency of the fluorescence resonance energy transfer (FRET) between an N-terminal intrinsic tryptophan donor and a synthetically added C-terminal IAEDANS acceptor obtained in steady-state and time-resolved experiments. We find that the contour-length scaling of mean end-to-end distance deviates from predictions of a purely statistical polymer chain. Furthermore, the addition of guanidine hydrochloride decreased, whereas the addition of salt increased the FRET efficiency, pointing at the disruption of structure-stabilizing interactions. Increasing temperature between 10 and 50°C increased the normalized FRET efficiency in both peptides but with different trajectories, indicating that their elasticity and conformational stability are different. Simulations suggest that whereas the short PEVK peptide displays an overall random structure, the long PEVK peptide retains residual, loose helical configurations. Transitions in the local structure and dynamics of the PEVK domain may play a role in the modulation of passive muscle mechanics. PMID:23062340

Comparative measurement of N-terminal pro-brain natriuretic peptide and brain natriuretic peptide in ambulatory patients with heart failure.

PubMed

Masson, Serge; Vago, Tarcisio; Baldi, Gabriella; Salio, Monica; De Angelis, Noeleen; Nicolis, Enrico; Maggioni, Aldo P; Latini, Roberto; Norbiato, Guido; Bevilacqua, Maurizio

2002-08-01

It is not clear whether brain natriuretic peptide (BNP) or N-terminal proBNP (NT-proBNP) is superior as a diagnostic and prognostic indicator in cardiac diseases. Here, we compare the clinical correlations of both peptides in a population of 92 ambulatory patients with heart failure, using a well-established immunoradiometric assay (IRMA) for BNP and an automated electrochemiluminescence immunoassay for NT-proBNP. The analytical correlation between the two peptides was satisfactory over a wide range of concentrations (1-686 pM for BNP) with the equation: NT-proBNP = 3.48 x BNP -19 and a correlation coefficient r2=0.94. In addition, the concentration of both peptides increased in a similar fashion according to the severity of the disease New York Heart Association (NYHA) functional class, left ventricular ejection fraction, etiology) and age; for instance, the ratios between median levels measured in NYHA class III vs. class II patients were comparable for BNP (383 vs. 16 pM, ratio 24) and NT-proBNP (1306 vs. 57 pM, ratio 23). We conclude that N-terminal proBNP, as assayed in the present study, correlates equally to BNP with clinical variables in patients with heart failure.

Protein Traffic to the Plasmodium falciparum apicoplast: evidence for a sorting branch point at the Golgi.

PubMed

Heiny, Sabrina R; Pautz, Sabine; Recker, Mario; Przyborski, Jude M

2014-12-01

Plasmodium falciparum, similar to many other apicomplexan parasites, contains an apicoplast, a plastid organelle of secondary endosymbiotic origin. Nuclear-encoded proteins are targeted to the apicoplast by a bipartite topogenic signal consisting of (i) an endoplasmic reticulum (ER)-type N-terminal secretory signal peptide, followed by (ii) a plant-like transit peptide. Although the signals responsible for transport of most proteins to the apicoplast are well described, the route of trafficking from the ER to the outermost apicoplast membrane is still a matter of debate. Current models of trafficking to the apicoplast suggest that proteins destined for this organelle are, on entry into the lumen of the ER, diverted from the default secretory pathway to a specialized vesicular system which carries proteins directly from the ER to the outer apicoplast membrane. Here, we have re-examined this trafficking pathway. By titrating wild-type and mutant apicoplast transit peptides against different ER retrieval sequences and studying protein transport in a brefeldin A-resistant parasite line, we generated data which suggest a direct involvement of the Golgi in traffic of soluble proteins to the P. falciparum apicoplast. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Role of the Simian Virus 5 Fusion Protein N-Terminal Coiled-Coil Domain in Folding and Promotion of Membrane Fusion

PubMed Central

West, Dava S.; Sheehan, Michael S.; Segeleon, Patrick K.; Dutch, Rebecca Ellis

2005-01-01

Formation of a six-helix bundle comprised of three C-terminal heptad repeat regions in antiparallel orientation in the grooves of an N-terminal coiled-coil is critical for promotion of membrane fusion by paramyxovirus fusion (F) proteins. We have examined the effect of mutations in four residues of the N-terminal heptad repeat in the simian virus 5 (SV5) F protein on protein folding, transport, and fusogenic activity. The residues chosen have previously been shown from study of isolated peptides to have differing effects on stability of the N-terminal coiled-coil and six-helix bundle (R. E. Dutch, G. P. Leser, and R. A. Lamb, Virology 254:147-159, 1999). The mutant V154M showed reduced proteolytic cleavage and surface expression, indicating a defect in intracellular transport, though this mutation had no effect when studied in isolated peptides. The mutation I137M, previously shown to lower thermostability of the six-helix bundle, resulted in an F protein which was properly processed and transported to the cell surface but which had reduced fusogenic activity. Finally, mutations at L140M and L161M, previously shown to disrupt α-helix formation of isolated N-1 peptides but not to affect six-helix bundle formation, resulted in F proteins that were properly processed. Interestingly, the L161M mutant showed increased syncytium formation and promoted fusion at lower temperatures than the wild-type F protein. These results indicate that interactions separate from formation of an N-terminal coiled-coil or six-helix bundle are important in the initial folding and transport of the SV5 F protein and that mutations that destabilize the N-terminal coiled-coil can result in stimulation of membrane fusion. PMID:15650180

Detection of diastereomer peptides as the intermediates generating D-amino acids during acid hydrolysis of peptides.

PubMed

Miyamoto, Tetsuya; Sekine, Masae; Ogawa, Tetsuhiro; Hidaka, Makoto; Watanabe, Hidenori; Homma, Hiroshi; Masaki, Haruhiko

2016-11-01

In this study, we investigated whether the amino acid residues within peptides were isomerized (and the peptides converted to diastereomers) during the early stages of acid hydrolysis. We demonstrate that the model dipeptides L-Ala-L-Phe and L-Phe-L-Ala are epimerized to produce the corresponding diastereomers at a very early stage, prior to their acid hydrolytic cleavage to amino acids. Furthermore, the sequence-inverted dipeptides were generated via formation of a diketopiperazine during hydrolytic incubation, and these dipeptides were also epimerized. The proportion of diastereomers increased rapidly during incubation for 0.5-2 h. During acid hydrolysis, C-terminal residues of the model dipeptides were isomerized faster than N-terminal residues, consistent with the observation that the D-amino acid values of the C-terminal residues determined by the 0 h-extrapolating method were larger than those of the N-terminal residues. Thus, the artificial D-amino acid contents determined by the 0 h-extrapolating method appear to be products of the isomerization of amino acid residues during acid hydrolysis.

Influence of Dimerization of Lipopeptide Laur-Orn-Orn-Cys-NH2 and an N-terminal Peptide of Human Lactoferricin on Biological Activity.

PubMed

Kamysz, Elżbieta; Sikorska, Emilia; Dawgul, Małgorzata; Tyszkowski, Rafał; Kamysz, Wojciech

Lactoferrin (LF) is a naturally occurring antimicrobial peptide that is cleaved by pepsin to lactoferricin (LFcin). LFcin has an enhanced antimicrobial activity as compared to that of LF. Recently several hetero- and homodimeric antimicrobial peptides stabilized by a single disulfide bond linking linear polypeptide chains have been discovered. We have demonstrated that the S-S bond heterodimerization of lipopeptide Laur-Orn-Orn-Cys-NH 2 (peptide III) and the synthetic N -terminal peptide of human lactoferricin (peptide I) yields a dimer (peptide V), which is almost as microbiologically active as the more active monomer and at the same time it is much less toxic. Furthermore, it has been found that the S-S bond homodimerization of both peptide I and peptide III did not affect antimicrobial and haemolytic activity of the compounds. The homo- and heterodimerization of peptides I and III resulted in either reduction or loss of antifungal activity. This work suggests that heterodimerization of antimicrobial lipopeptides via intermolecular disulfide bond might be a powerful modification deserving consideration in the design of antimicrobial peptides.

Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence.

PubMed Central

Casal, J I; Langeveld, J P; Cortés, E; Schaaper, W W; van Dijk, E; Vela, C; Kamstrup, S; Meloen, R H

1995-01-01

The N-terminal domain of the major capsid protein VP2 of canine parvovirus was shown to be an excellent target for development of a synthetic peptide vaccine, but detailed information about number of epitopes, optimal length, sequence choice, and site of coupling to the carrier protein was lacking. Therefore, several overlapping peptides based on this N terminus were synthesized to establish conditions for optimal and reproducible induction of neutralizing antibodies in rabbits. The specificity and neutralizing ability of the antibody response for these peptides were determined. Within the N-terminal 23 residues of VP2, two subsites able to induce neutralizing antibodies and which overlapped by only two glycine residues at positions 10 and 11 could be discriminated. The shortest sequence sufficient for neutralization induction was nine residues. Peptides longer than 13 residues consistently induced neutralization, provided that their N termini were located between positions 1 and 11 of VP2. The orientation of the peptides at the carrier protein was also of importance, being more effective when coupled through the N terminus than through the C terminus to keyhole limpet hemocyanin. The results suggest that the presence of amino acid residues 2 to 21 (and probably 3 to 17) of VP2 in a single peptide is preferable for a synthetic peptide vaccine. PMID:7474152

Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence.

PubMed

Casal, J I; Langeveld, J P; Cortés, E; Schaaper, W W; van Dijk, E; Vela, C; Kamstrup, S; Meloen, R H

1995-11-01

The N-terminal domain of the major capsid protein VP2 of canine parvovirus was shown to be an excellent target for development of a synthetic peptide vaccine, but detailed information about number of epitopes, optimal length, sequence choice, and site of coupling to the carrier protein was lacking. Therefore, several overlapping peptides based on this N terminus were synthesized to establish conditions for optimal and reproducible induction of neutralizing antibodies in rabbits. The specificity and neutralizing ability of the antibody response for these peptides were determined. Within the N-terminal 23 residues of VP2, two subsites able to induce neutralizing antibodies and which overlapped by only two glycine residues at positions 10 and 11 could be discriminated. The shortest sequence sufficient for neutralization induction was nine residues. Peptides longer than 13 residues consistently induced neutralization, provided that their N termini were located between positions 1 and 11 of VP2. The orientation of the peptides at the carrier protein was also of importance, being more effective when coupled through the N terminus than through the C terminus to keyhole limpet hemocyanin. The results suggest that the presence of amino acid residues 2 to 21 (and probably 3 to 17) of VP2 in a single peptide is preferable for a synthetic peptide vaccine.

Fischer carbene mediated covalent grafting of a peptide nucleic acid on gold surfaces and IR optical detection of DNA hybridization with a transition metalcarbonyl label

NASA Astrophysics Data System (ADS)

Srivastava, Pratima; Ghasemi, Mahsa; Ray, Namrata; Sarkar, Amitabha; Kocabova, Jana; Lachmanova, Stepanka; Hromadova, Magdalena; Boujday, Souhir; Cauteruccio, Silvia; Thakare, Pramod; Licandro, Emanuela; Fosse, Céline; Salmain, Michèle

2016-11-01

Amine-reactive surfaces comprising N-hydroxysuccinimide ester groups as well as much more unusual Fischer alkoxymetallocarbene groups were generated on gold-coated surfaces via self-assembled monolayers of carboxy- and azido-terminated thiolates, respectively. These functions were further used to immobilize homothymine peptide nucleic acid (PNA) decamer in a covalent fashion involving the primary amine located at its N-terminus. These stepwise processes were monitored by polarization modulation reflection - absorption infrared spectroscopy (PM-RAIRS) that gave useful information on the molecular composition of the organic layers. PNA grafting and hybridization with complementary DNA strand were successfully transduced by quartz crystal microbalance (QCM) measurements. Unfortunately, attempts to transduce the hybridization optically by IR in a label-free fashion were inconclusive. Therefore we undertook to introduce an IR reporter group, namely a transition metalcarbonyl (TMC) entity at the 5‧ terminus of complementary DNA. Evidence for the formation of PNA-DNA heteroduplex was brought by the presence of ν(Ctbnd O) bands in the 2000 cm-1 region of the IR spectrum of the gold surface owing to the metalcarbonyl label.

C-Terminal Protein Characterization by Mass Spectrometry: Isolation of C-Terminal Fragments from Cyanogen Bromide-Cleaved Protein

PubMed Central

Nika, Heinz; Hawke, David H.; Angeletti, Ruth Hogue

2014-01-01

A sample preparation method for protein C-terminal peptide isolation from cyanogen bromide (CNBr) digests has been developed. In this strategy, the analyte was reduced and carboxyamidomethylated, followed by CNBr cleavage in a one-pot reaction scheme. The digest was then adsorbed on ZipTipC18 pipette tips for conjugation of the homoserine lactone-terminated peptides with 2,2′-dithiobis (ethylamine) dihydrochloride, followed by reductive release of 2-aminoethanethiol from the derivatives. The thiol-functionalized internal and N-terminal peptides were scavenged on activated thiol sepharose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were replaced directly on the support, allowing the reactions to proceed at minimal sample loss. By this sequence of solid-phase reactions, the C-terminal peptide could be recognized uniquely in mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-level amounts of a whole, intact model protein. The C-terminal fragments were retrieved selectively and efficiently from the affinity support. The use of covalent chromatography for C-terminal peptide purification enabled recovery of the depleted material for further chemical and/or enzymatic manipulation. The sample preparation method provides for robustness and simplicity of operation and is anticipated to be expanded to gel-separated proteins and in a scaled-up format to high-throughput protein profiling in complex biological mixtures. PMID:24688319

Synthesis of histone proteins by CPE ligation using a recombinant peptide as the C-terminal building block.

PubMed

Kawakami, Toru; Yoshikawa, Ryo; Fujiyoshi, Yuki; Mishima, Yuichi; Hojo, Hironobu; Tajima, Shoji; Suetake, Isao

2015-11-01

The post-translational modification of histones plays an important role in gene expression. We report herein on a method for synthesizing such modified histones by ligating chemically prepared N-terminal peptides and C-terminal recombinant peptide building blocks. Based on their chemical synthesis, core histones can be categorized as two types; histones H2A, H2B and H4 which contain no Cys residues, and histone H3 which contains a Cys residue(s) in the C-terminal region. A combination of native chemical ligation and desulphurization can be simply used to prepare histones without Cys residues. For the synthesis of histone H3, the endogenous Cys residue(s) must be selectively protected, while keeping the N-terminal Cys residue of the C-terminal building block that is introduced for purposes of chemical ligation unprotected. To this end, a phenacyl group was successfully utilized to protect endogenous Cys residue(s), and the recombinant peptide was ligated with a peptide containing a Cys-Pro ester (CPE) sequence as a thioester precursor. Using this approach it was possible to prepare all of the core histones H2A, H2B, H3 and H4 with any modifications. The resulting proteins could then be used to prepare a core histone library of proteins that have been post-translationally modified. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Toxoplasma gondii peptide ligands open the gate of the HLA class I binding groove

PubMed Central

McMurtrey, Curtis; Trolle, Thomas; Sansom, Tiffany; Remesh, Soumya G; Kaever, Thomas; Bardet, Wilfried; Jackson, Kenneth; McLeod, Rima; Sette, Alessandro; Nielsen, Morten; Zajonc, Dirk M; Blader, Ira J; Peters, Bjoern; Hildebrand, William

2016-01-01

HLA class I presentation of pathogen-derived peptide ligands is essential for CD8+ T-cell recognition of Toxoplasma gondii infected cells. Currently, little data exist pertaining to peptides that are presented after T. gondii infection. Herein we purify HLA-A*02:01 complexes from T. gondii infected cells and characterize the peptide ligands using LCMS. We identify 195 T. gondii encoded ligands originating from both secreted and cytoplasmic proteins. Surprisingly, T. gondii ligands are significantly longer than uninfected host ligands, and these longer pathogen-derived peptides maintain a canonical N-terminal binding core yet exhibit a C-terminal extension of 1–30 amino acids. Structural analysis demonstrates that binding of extended peptides opens the HLA class I F’ pocket, allowing the C-terminal extension to protrude through one end of the binding groove. In summary, we demonstrate that unrealized structural flexibility makes MHC class I receptive to parasite-derived ligands that exhibit unique C-terminal peptide extensions. DOI: http://dx.doi.org/10.7554/eLife.12556.001 PMID:26824387

N-terminal pro-B-type natriuretic peptide and the prediction of primary cardiovascular events: results from 15-year follow-up of WOSCOPS

PubMed Central

Welsh, Paul; Doolin, Orla; Willeit, Peter; Packard, Chris; Macfarlane, Peter; Cobbe, Stuart; Gudnason, Vilmundur; Di Angelantonio, Emanuele; Ford, Ian; Sattar, Naveed

2013-01-01

Aims To test whether N-terminal pro-B-type natriuretic peptide (NT-proBNP) was independently associated with, and improved the prediction of, cardiovascular disease (CVD) in a primary prevention cohort. Methods and results In the West of Scotland Coronary Prevention Study (WOSCOPS), a cohort of middle-aged men with hypercholesterolaemia at a moderate risk of CVD, we related the baseline NT-proBNP (geometric mean 28 pg/mL) in 4801 men to the risk of CVD over 15 years during which 1690 experienced CVD events. Taking into account the competing risk of non-CVD death, NT-proBNP was associated with an increased risk of all CVD [HR: 1.17 (95% CI: 1.11–1.23) per standard deviation increase in log NT-proBNP] after adjustment for classical and clinical cardiovascular risk factors plus C-reactive protein. N-terminal pro-B-type natriuretic peptide was more strongly related to the risk of fatal [HR: 1.34 (95% CI: 1.19–1.52)] than non-fatal CVD [HR: 1.17 (95% CI: 1.10–1.24)] (P= 0.022). The addition of NT-proBNP to traditional risk factors improved the C-index (+0.013; P < 0.001). The continuous net reclassification index improved with the addition of NT-proBNP by 19.8% (95% CI: 13.6–25.9%) compared with 9.8% (95% CI: 4.2–15.6%) with the addition of C-reactive protein. N-terminal pro-B-type natriuretic peptide correctly reclassified 14.7% of events, whereas C-reactive protein correctly reclassified 3.4% of events. Results were similar in the 4128 men without evidence of angina, nitrate prescription, minor ECG abnormalities, or prior cerebrovascular disease. Conclusion N-terminal pro-B-type natriuretic peptide predicts CVD events in men without clinical evidence of CHD, angina, or history of stroke, and appears related more strongly to the risk for fatal events. N-terminal pro-B-type natriuretic peptide also provides moderate risk discrimination, in excess of that provided by the measurement of C-reactive protein. Clinical trial registration WOSCOPS was carried out and completed prior to the requirement for clinical trial registration. PMID:22942340

Sequence-specific, nanomolar peptide binding via cucurbit[8]uril-induced folding and inclusion of neighboring side chains.

PubMed

Smith, Lauren C; Leach, David G; Blaylock, Brittney E; Ali, Omar A; Urbach, Adam R

2015-03-18

This paper describes the molecular recognition of the tripeptide Tyr-Leu-Ala by the synthetic receptor cucurbit[8]uril (Q8) in aqueous buffer with nanomolar affinity and exceptional specificity. This combination of characteristics, which also applies to antibodies, is desirable for applications in biochemistry and biotechnology but has eluded supramolecular chemists for decades. Building on prior knowledge that Q8 binds to peptides with N-terminal aromatic residues, a library screen of 105 peptides was designed to test the effects of residues adjacent to N-terminal Trp, Phe, or Tyr. The screen used tetramethylbenzobis(imidazolium) (MBBI) as a fluorescent indicator and resulted in the unexpected discovery that MBBI can serve not only as a turn-off sensor via the simultaneous inclusion of a Trp residue but also as a turn-on sensor via the competitive displacement of MBBI upon binding of Phe- or Tyr-terminated peptides. The unusual fluorescence response of the Tyr series prompted further investigation by (1)H NMR spectroscopy, electrospray ionization mass spectrometry, and isothermal titration calorimetry. From these studies, a novel binding motif was discovered in which only 1 equiv of peptide binds to Q8, and the side chains of both the N-terminal Tyr residue and its immediate neighbor bind within the Q8 cavity. For the peptide Tyr-Leu-Ala, the equilibrium dissociation constant value is 7.2 nM, whereas that of its sequence isomer Tyr-Ala-Leu is 34 μM. The high stability, recyclability, and low cost of Q8 combined with the straightforward incorporation of Tyr-Leu-Ala into recombinant proteins should make this system attractive for the development of biological applications.

N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana *

PubMed Central

Ndah, Elvis; Jonckheere, Veronique

2017-01-01

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes. PMID:28432195

N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana.

PubMed

Willems, Patrick; Ndah, Elvis; Jonckheere, Veronique; Stael, Simon; Sticker, Adriaan; Martens, Lennart; Van Breusegem, Frank; Gevaert, Kris; Van Damme, Petra

2017-06-01

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The localization of a vitamin K-induced modification in an N-terminal fragment of human prothrombin

PubMed Central

Skotland, Tore; Holm, Turid; Østerud, Bjarne; Flengsrud, Ragnar; Prydz, Hans

1974-01-01

1. The N-terminal fragment (PF-I) split off from prothrombin during coagulation was purified to homogeneity from human serum. 2. The apparent molecular weight is 27000±2000 in sodium dodecyl sulphate–polyacrylamide-gel electrophoresis, whereas a value of about 19600 is obtained by calculation based on amino acid and carbohydrate analyses. The N-terminal sequence is an Ala-Asx bond. The fragment contains about 16% carbohydrate, binds phospholipids in the presence of Ca2+ and is adsorbed to BaSO4. The pKa of its BaSO4-binding group(s) is 3.1–3.5. 3. By CNBr cleavage of fragment PF-I two peptides (C-1 and C-2) were obtained with molecular weights of about 5900 (C-2) and 12400 (C-1) on the basis of amino acid and carbohydrate analyses. Only the smaller (N-terminal) peptide is adsorbed to BaSO4 and, since the ability of the whole protein to bind to BaSO4 is known to be absent in samples obtained from patients treated with vitamin K antagonists, this peptide probably contains the site of a modification to the structure of the protein which occurs during biosynthesis and depends on vitamin K. This peptide does not contain hexosamine or sialic acid. ImagesFig. 2. PMID:4219283

Structural basis for the mechanism and substrate specificity of glycocyamine kinase, a phosphagen kinase family member

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Kap; Pullalarevu, Sadhana; Surabian, Karen Talin

2010-03-12

Glycocyamine kinase (GK), a member of the phosphagen kinase family, catalyzes the Mg{sup 2+}-dependent reversible phosphoryl group transfer of the N-phosphoryl group of phosphoglycocyamine to ADP to yield glycocyamine and ATP. This reaction helps to maintain the energy homeostasis of the cell in some multicelullar organisms that encounter high and variable energy turnover. GK from the marine worm Namalycastis sp. is heterodimeric, with two homologous polypeptide chains, {alpha} and {beta}, derived from a common pre-mRNA by mutually exclusive N-terminal alternative exons. The N-terminal exon of GK{beta} encodes a peptide that is different in sequence and is 16 amino acids longermore » than that encoded by the N-terminal exon of GK{alpha}. The crystal structures of recombinant GK{alpha}{beta} and GK{beta}{beta} from Namalycastis sp. were determined at 2.6 and 2.4 {angstrom} resolution, respectively. In addition, the structure of the GK{beta}{beta} was determined at 2.3 {angstrom} resolution in complex with a transition state analogue, Mg{sup 2+}-ADP-NO{sub 3}{sup -}-glycocyamine. Consistent with the sequence homology, the GK subunits adopt the same overall fold as that of other phosphagen kinases of known structure (the homodimeric creatine kinase (CK) and the monomeric arginine kinase (AK)). As with CK, the GK N-termini mediate the dimer interface. In both heterodimeric and homodimeric GK forms, the conformations of the two N-termini are asymmetric, and the asymmetry is different than that reported previously for the homodimeric CKs from several organisms. The entire polypeptide chains of GK{alpha}{beta} are structurally defined, and the longer N-terminus of the {beta} subunit is anchored at the dimer interface. In GK{beta}{beta} the 24 N-terminal residues of one subunit and 11 N-terminal residues of the second subunit are disordered. This observation is consistent with a proposal that the GK{alpha}{beta} amino acids involved in the interface formation were optimized once a heterodimer emerged as the physiological form of the enzyme. As a consequence, the homodimer interface (either solely {alpha} or solely {beta} chains) has been corrupted. In the unbound state, GK exhibits an open conformation analogous to that observed with ligand-free CK or AK. Upon binding the transition state analogue, both subunits of GK undergo the same closure motion that clasps the transition state analogue, in contrast to the transition state analogue complexes of CK, where the corresponding transition state analogue occupies only one subunit, which undergoes domain closure. The active site environments of the GK, CK, and AK at the bound states reveal the structural determinants of substrate specificity. Despite the equivalent binding in both active sites of the GK dimer, the conformational asymmetry of the N-termini is retained. Thus, the coupling between the structural asymmetry and negative cooperativity previously proposed for CK is not supported in the case of GK.« less

A Novel N14Y Mutation in Connexin26 in Keratitis-Ichthyosis-Deafness Syndrome

PubMed Central

Arita, Ken; Akiyama, Masashi; Aizawa, Tomoyasu; Umetsu, Yoshitaka; Segawa, Ikuo; Goto, Maki; Sawamura, Daisuke; Demura, Makoto; Kawano, Keiichi; Shimizu, Hiroshi

2006-01-01

Connexins (Cxs) are transmembranous proteins that connect adjacent cells via channels known as gap junctions. The N-terminal 21 amino acids of Cx26 are located at the cytoplasmic side of the channel pore and are thought to be essential for the regulation of channel selectivity. We have found a novel mutation, N14Y, in the N-terminal domain of Cx26 in a case of keratitis-ichthyosis-deafness syndrome. Reduced gap junctional intercellular communication was observed in the patient’s keratinocytes by the dye transfer assay using scrape-loading methods. The effect of this mutation on molecular structure was investigated using synthetic N-terminal peptides from both wild-type and mutated Cx26. Two-dimensional 1H nuclear magnetic resonance and circular dichroism measurements demonstrated that the secondary structures of these two model peptides are similar to each other. However, several novel nuclear Overhauser effect signals appeared in the N14Y mutant, and the secondary structure of the mutant peptide was more susceptible to induction of 2,2,2-trifluoroethanol than wild type. Thus, it is likely that the N14Y mutation induces a change in local structural flexibility of the N-terminal domain, which is important for exerting the activity of the channel function, resulting in impaired gap junctional intercellular communication. PMID:16877344

Probing Charge Transport through Peptide Bonds.

PubMed

Brisendine, Joseph M; Refaely-Abramson, Sivan; Liu, Zhen-Fei; Cui, Jing; Ng, Fay; Neaton, Jeffrey B; Koder, Ronald L; Venkataraman, Latha

2018-02-15

We measure the conductance of unmodified peptides at the single-molecule level using the scanning tunneling microscope-based break-junction method, utilizing the N-terminal amine group and the C-terminal carboxyl group as gold metal-binding linkers. Our conductance measurements of oligoglycine and oligoalanine backbones do not rely on peptide side-chain linkers. We compare our results with alkanes terminated asymmetrically with an amine group on one end and a carboxyl group on the other to show that peptide bonds decrease the conductance of an otherwise saturated carbon chain. Using a newly developed first-principles approach, we attribute the decrease in conductance to charge localization at the peptide bond, which reduces the energy of the frontier orbitals relative to the Fermi energy and the electronic coupling to the leads, lowering the tunneling probability. Crucially, this manifests as an increase in conductance decay of peptide backbones with increasing length when compared with alkanes.

Substrate specificity of platypus venom L-to-D-peptide isomerase.

PubMed

Bansal, Paramjit S; Torres, Allan M; Crossett, Ben; Wong, Karen K Y; Koh, Jennifer M S; Geraghty, Dominic P; Vandenberg, Jamie I; Kuchel, Philip W

2008-04-04

The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, alpha-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, beta-branched or long side chains with polar terminal groups, viz. Ala, alpha-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L-to-D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.

Even-electron [M-H](+) ions generated by loss of AgH from argentinated peptides with N-terminal imine groups.

PubMed

Plaviak, Alexandra; Osburn, Sandra; Patterson, Khiry; van Stipdonk, Michael J

2016-01-15

Experiments were performed to probe the creation of apparent even-electron, [M-H](+) ions by CID of Ag-cationized peptides with N-terminal imine groups (Schiff bases). Imine-modified peptides were prepared using condensation reactions with aldehydes. Ag(+) -cationized precursors were generated by electrospray ionization (ESI). Tandem mass spectrometry (MS(n) ) and collision-induced dissociation (CID) were performed using a linear ion trap mass spectrometer. Loss of AgH from peptide [M + Ag](+) ions, at the MS/MS stage, creates closed-shell [M-H](+) ions from imine-modified peptides. Isotope labeling unambiguously identifies the imine C-H group as the source of H eliminated in AgH. Subsequent CID of the [M-H](+) ions generated sequence ions that are analogous to those produced from [M + H](+) ions of the imine-modified peptides. Experiments show (a) formation of novel even-electron peptide cations by CID and (b) the extent to which sequence ions (conventional b, a and y ions) are generated from peptides with fixed charge site and thus lacking a conventional mobile proton. Copyright © 2015 John Wiley & Sons, Ltd.

Plasma matrix metalloproteinase-9 better predicts outcome than N-terminal protype-B natriuretic peptide in patients with systolic heart failure and a high prevalence of coronary artery disease.

PubMed

Dini, Frank Lloyd; Buralli, Simona; Bajraktari, Gani; Elezi, Shpend; Duranti, Emiliano; Metelli, Maria Rita; Carpi, Angelo; Taddei, Stefano

2010-05-01

Metalloproteinases have been proposed as biochemical markers of left ventricular (LV) remodeling in systolic heart failure (HF). However, their role in the prognostic stratification of these patients remains controversial. In the present study, we aimed at investigating the value of plasma metalloproteinases-3 and -9 in comparison with N-terminal protype-B natriuretic peptide in patients with systolic HF. One hundred and 27 consecutive patients hospitalized for systolic HF (LV ejection fraction < 45%) were enrolled. Coronary artery disease (CAD) was the aetiology in 67% of the study patients. Plasma metalloproteinases-3 and -9 and N-terminal protype-B natriuretic peptide levels were assessed. A complete echocardiographic and Doppler examination was also performed. Follow-up period was 24-15 months. On univariate analysis, a number of measurements predicted cardiac events in the following order of power: NYHA class >2, LV ejection fraction < 25%, metalloproteinases-9 > 238 ng/ml, mitral E wave deceleration time < 150 ms, N-terminal protype-B natriuretic peptide > 1586 pg/ml and metalloproteinases-3 > 15 ng/ml. However, on multivariate analysis the only independent variables of cardiac events were NYHA class (OR=2.26, p=0.059) and plasma metalloproteinases-9 (OR=2.00, p=0.029). On Kaplan-Meier survival analysis, patients with elevated levels of metalloproteinases-9 exhibited a significantly worse event free-survival at 45 months than those without (21% vs. 54%, log-rank: 13.93, p=0.0002). A worse survival was also observed in patients with elevated N-terminal protype-B natriuretic peptide levels with respect to those without (18% vs. 46%, log-rank: 9.11, p=0.025). Our results demonstrated the value of plasma metalloproteinases-9 levels for prognostication of patients with systolic HF and a high prevalence of CAD. 2009. Published by Elsevier SAS.

Amino acid residue Y196E substitution and C-terminal peptide synergistically alleviate the toxicity of Clostridium perfringens epsilon toxin.

PubMed

Yao, Wenwu; Kang, Lin; Gao, Shan; Zhuang, Xiangjin; Zhang, Tao; Yang, Hao; Ji, Bin; Xin, Wenwen; Wang, Jinglin

2015-06-15

Epsilon toxin (ETX) is produced by Clostridium perfringens type B and D strains, and is the causative agent of a lethal enterotoxemia in livestock animals and possibly in humans. However, many details of ETX structure and activity are not known. Therefore, it is important to clarify the relationship between ETX structure and activity. To explore the effect and mechanism of ETX amino acid residue Y196E substitution and C-terminal peptide on toxicity, four recombinant proteins, rETX (without 13 N-terminal peptides and 23 C-terminal peptides), rETX-C (rETX with 23 C-terminal peptides), rETX(Y196E) (rETX with an amino acid residue substitution at Y196) and rETX(Y196E)-C (rETX-C with a Y196E mutation), were constructed in this study. Both the amino acid residue Y196E substitution and the C-terminal peptide reduce ETX toxicity to a similar extent, and the two factors synergistically alleviate ETX toxicity. In addition, we demonstrated that the C-terminal peptides and Y196E amino acid mutation reduce the toxin toxicity in two different pathways: the C-terminal peptides inhibit the binding activity of toxins to target cells, and the Y196E amino acid mutation slightly inhibits the pore-forming or heptamer-forming process. Interaction between the two factors was not observed in pore-forming or binding assays but toxicity assays, which demonstrated that the relationship between domains of the toxin is more complicated than previously appreciated. However, the exact mechanism of synergistic action is not yet clarified. Copyright © 2015 Elsevier Ltd. All rights reserved.

Purification and characterization of an N alpha-acetyltransferase from Saccharomyces cerevisiae.

PubMed

Lee, F J; Lin, L W; Smith, J A

1988-10-15

N alpha-Acetyltransferase, which catalyzes the transfer of an acetyl group from acetyl coenzyme A to the alpha-NH2 group of proteins and peptides, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 4,600-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, hydroxylapatite, DE52 cellulose, and Affi-Gel blue. The Mr of the native enzyme was estimated to be 180,000 +/- 10,000 by gel filtration chromatography, and the Mr of each subunit was estimated to be 95,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pH optimum near 9.0, and its pI is 4.3 as determined by chromatofocusing on Mono-P. The enzyme catalyzed the transfer of an acetyl group to various synthetic peptides, including human adrenocorticotropic hormone (ACTH) (1-24) and its [Phe2] analogue, yeast alcohol dehydrogenase I (1-24), yeast alcohol dehydrogenase II (1-24), and human superoxide dismutase (1-24). These peptides contain either Ser or Ala as NH2-terminal residues which together with Met are the most commonly acetylated NH2-terminal residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). Yeast enolase, containing a free NH2-terminal Ala residue, is known not to be N alpha-acetylated in vivo (Chin, C. C. Q., Brewer, J. M., and Wold, F. (1981) J. Biol. Chem. 256, 1377-1384), and enolase (1-24), a synthetic peptide mimicking the protein's NH2 terminus, was not acetylated in vitro by yeast acetyltransferase. The enzyme did not catalyze the N alpha-acetylation of other synthetic peptides including ACTH(11-24), ACTH(7-38), ACTH(18-39), human beta-endorphin, yeast superoxide dismutase (1-24). Each of these peptides has an NH2-terminal residue which is rarely acetylated in proteins (Lys, Phe, Arg, Tyr, Val, respectively). Among a series of divalent cations, Cu2+ and Zn2+ were demonstrated to be the most potent inhibitors. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate and N-bromosuccinimide.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Yingying; Triscari, Joseph M.; Tseng, George C.

Data mining was performed on 28 330 unique peptide tandem mass spectra for which sequences were assigned with high confidence. By dividing the spectra into different sets based on structural features and charge states of the corresponding peptides, chemical interactions involved in promoting specific cleavage patterns in gas-phase peptides were characterized. Pairwise fragmentation maps describing cleavages at all Xxx-Zzz residue combinations for b and y ions reveal that the difference in basicity between Arg and Lys results in different dissociation patterns for singly charged Arg- and Lys-ending tryptic peptides. While one dominant protonation form (proton localized) exists for Arg-ending peptides,more » a heterogeneous population of different protonated forms or more facile interconversion of protonated forms (proton partially mobile) exists for Lys-ending peptides. Cleavage C-terminal to acidic residues dominates spectra from peptides that have a localized proton and cleavage N-terminal to Pro dominates those that have a mobile or partially mobile proton. When Pro is absent from peptides that have a mobile or partially mobile proton, cleavage at each peptide bond becomes much more prominent. Whether the above patterns can be found in b ions, y ions, or both depends on the location of the proton holder(s). Enhanced cleavages C-terminal to branched aliphatic residues (Ile, Val, Leu) are observed in both b and y ions from peptides that have a mobile proton, as well as in y ions from peptides that have a partially mobile proton; enhanced cleavages N-terminal to these residues are observed in b ions from peptides that have a partially mobile proton. Statistical tools have been designed to visualize the fragmentation maps and measure the similarity between them. The pairwise cleavage patterns observed expand our knowledge of peptide gas-phase fragmentation behaviors and should be useful in algorithm development that employs improved models to predict fragment ion intensities.« less

A highly sensitive peptide substrate for detecting two Aß-degrading enzymes: neprilysin and insulin-degrading enzyme.

PubMed

Chen, Po-Ting; Liao, Tai-Yan; Hu, Chaur-Jong; Wu, Shu-Ting; Wang, Steven S-S; Chen, Rita P-Y

2010-06-30

Neprilysin has been singled out as the most promising candidate for use in the degradation of Abeta as a therapy for Alzheimer's disease. In this study, a quenched fluorogenic peptide substrate containing the first seven residues of the Abeta peptide plus a C-terminal Cysteine residue was synthesized to detect neprilysin activity. A fluorophore was attached to the C-terminal Cysteine and its fluorescence was quenched by a quencher linked to the N-terminus of the peptide. When this peptide substrate was degraded by an endopeptidase, fluorescence was produced and proved to be a sensitive detection system for endopeptidase activity. Our results showed that this assay system was extremely sensitive to neprilysin and insulin-degrading enzyme, but insensitive, or much less sensitive, to other Abeta-degrading enzymes. As low as 0.1 nM of neprilysin and 0.2 nM of insulin-degrading enzyme can be detected. Copyright 2010 Elsevier B.V. All rights reserved.

Cytoplasmic bacteriophage display system

DOEpatents

Studier, F.W.; Rosenberg, A.H.

1998-06-16

Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest. 1 fig.

Cytoplasmic bacteriophage display system

DOEpatents

Studier, F. William; Rosenberg, Alan H.

1998-06-16

Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest.

Group A Streptococcal vaccine candidate: contribution of epitope to size, antigen presenting cell interaction and immunogenicity.

PubMed

Zaman, Mehfuz; Chandrudu, Saranya; Giddam, Ashwini K; Reiman, Jennifer; Skwarczynski, Mariusz; McPhun, Virginia; Moyle, Peter M; Batzloff, Michael R; Good, Michael F; Toth, Istvan

2014-12-01

Utilize lipopeptide vaccine delivery system to develop a vaccine candidate against Group A Streptococcus. Lipopeptides synthesized by solid-phase peptide synthesis-bearing carboxyl (C)-terminal and amino (N)-terminal Group A Streptococcus peptide epitopes. Nanoparticles formed were evaluated in vivo. Immune responses were induced in mice without additional adjuvant. We demonstrated for the first time that incorporation of the C-terminal epitope significantly enhanced the N-terminal epitope-specific antibody response and correlated with forming smaller nanoparticles. Antigen-presenting cells had increased uptake and maturation by smaller, more immunogenic nanoparticles. Antibodies raised by vaccination recognized isolates. Demonstrated the lipopeptidic nanoparticles to induce an immune response which can be influenced by the combined effect of epitope choice and size.

An N-terminal peptide extension results in efficient expression, but not secretion, of a synthetic horseradish peroxidase gene in transgenic tobacco.

PubMed

Kis, Mihaly; Burbridge, Emma; Brock, Ian W; Heggie, Laura; Dix, Philip J; Kavanagh, Tony A

2004-03-01

Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N-terminal and C-terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence. Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N-terminal or the C-terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV-35S) or the tobacco RUBISCO-SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium-mediated transformation. To study the effects of the N- and C-terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques. Transgenic tobacco plants can exhibit a ten-fold increase in peroxidase activity compared with wild-type tobacco levels, and the majority of this activity is located in the symplast. The N-terminal extension is essential for the production of high levels of recombinant protein, while the C-terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels. There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N-terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency. Plants have been generated with greatly elevated cytosolic peroxidase activity, and smaller increases in apoplastic activity. These will be valuable for exploring the role of these enzymes in stress amelioration and plant development.

Neurospora tryptophan synthase: N-terminal analysis and the sequence of the pyridoxal phosphate active site peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pratt, M.L.; Hsu, P.Y.; DeMoss, J.A.

1986-05-01

Tryptophan synthase (TS), which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein requiring pyridoxal phosphate (B6P) for two of its three distinct enzyme activities. TS from Neurospora has a blocked N-terminal, is a homodimer of 150 KDa and binds one mole of B6P per mole of subunit. The authors shown the N-terminal residue to be acyl-serine. The B6P-active site of holoenzyme was labelled by reduction of the B6P-Schiff base with (/sup 3/H)-NaBH/sub 4/, and resulted in a proportionate loss of activity in the two B6P-requiring reactions. SDS-polyacrylamide gel electrophoresis of CNBr-generated peptides showed the labelled, active sitemore » peptide to be 6 KDa. The sequence of this peptide, purified to apparent homogeneity by a combination of C-18 reversed phase and TSK gel filtration HPLC is: gly-arg-pro-gly-gln-leu-his-lys-ala-glu-arg-leu-thr-glu-tyr-ala-gly-gly-ala-gln-ile-xxx-leu-lys-arg-glu-asp-leu-asn-his-xxx-gly-xxx-his-/sub ***/-ile-asn-asn-ala-leu. Although four residues (xxx, /sub ***/) are unidentified, this peptide is minimally 78% homologous with the corresponding peptide from yeast TS, in which residue (/sub ***/) is the lysine that binds B6P.« less

Chromogranin A promotes peptide hormone sorting to mobile granules in constitutively and regulated secreting cells: role of conserved N- and C-terminal peptides.

PubMed

Montero-Hadjadje, Maité; Elias, Salah; Chevalier, Laurence; Benard, Magalie; Tanguy, Yannick; Turquier, Valérie; Galas, Ludovic; Yon, Laurent; Malagon, Maria M; Driouich, Azeddine; Gasman, Stéphane; Anouar, Youssef

2009-05-01

Chromogranin A (CgA) has been proposed to play a major role in the formation of dense-core secretory granules (DCGs) in neuroendocrine cells. Here, we took advantage of unique features of the frog CgA (fCgA) to assess the role of this granin and its potential functional determinants in hormone sorting during DCG biogenesis. Expression of fCgA in the constitutively secreting COS-7 cells induced the formation of mobile vesicular structures, which contained cotransfected peptide hormones. The fCgA and the hormones coexpressed in the newly formed vesicles could be released in a regulated manner. The N- and C-terminal regions of fCgA, which exhibit remarkable sequence conservation with their mammalian counterparts were found to be essential for the formation of the mobile DCG-like structures in COS-7 cells. Expression of fCgA in the corticotrope AtT20 cells increased pro-opiomelanocortin levels in DCGs, whereas the expression of N- and C-terminal deletion mutants provoked retention of the hormone in the Golgi area. Furthermore, fCgA, but not its truncated forms, promoted pro-opiomelanocortin sorting to the regulated secretory pathway. These data demonstrate that CgA has the intrinsic capacity to induce the formation of mobile secretory granules and to promote the sorting and release of peptide hormones. The conserved terminal peptides are instrumental for these activities of CgA.

Releasing N-glycan from peptide N-terminus by N-terminal succinylation assisted enzymatic deglycosylation.

PubMed

Weng, Yejing; Sui, Zhigang; Jiang, Hao; Shan, Yichu; Chen, Lingfan; Zhang, Shen; Zhang, Lihua; Zhang, Yukui

2015-04-22

Due to the important roles of N-glycoproteins in various biological processes, the global N-glycoproteome analysis has been paid much attention. However, by current strategies for N-glycoproteome profiling, peptides with glycosylated Asn at N-terminus (PGANs), generated by protease digestion, could hardly be identified, due to the poor deglycosylation capacity by enzymes. However, theoretically, PGANs occupy 10% of N-glycopeptides in the typical tryptic digests. Therefore, in this study, we developed a novel strategy to identify PGANs by releasing N-glycans through the N-terminal site-selective succinylation assisted enzymatic deglycosylation. The obtained PGANs information is beneficial to not only achieve the deep coverage analysis of glycoproteomes, but also discover the new biological functions of such modification.

Collision-induced dissociation of diazirine-labeled peptide ions. Evidence for Brønsted-acid assisted elimination of nitrogen.

PubMed

Marek, Aleš; Tureček, František

2014-05-01

Gas-phase dissociations were investigated for several peptide ions containing the Gly-Leu* N-terminal motif where Leu* was a modified norleucine residue containing the photolabile diazirine ring. Collisional activation of gas-phase peptide cations resulted in facile N₂ elimination that competed with backbone dissociations. A free lysine ammonium group can act as a Brønsted acid to facilitate N₂ elimination. This dissociation was accompanied by insertion of a lysine proton in the side chain of the photoleucine residue, as established by deuterium labeling and gas-phase sequencing of the products. Electron structure calculations were used to provide structures and energies of reactants, intermediates, and transition states for Gly-Leu*-Gly-Gly-Lys amide ions that were combined with RRKM calculations of unimolecular rate constants. The calculations indicated that Brønsted acid-catalyzed eliminations were kinetically preferred over direct loss of N₂ from the diazirine ring. Mechanisms are proposed to explain the proton-initiated reactions and discuss the reaction products. The non-catalyzed diazirine ring cleavage and N₂ loss is proposed as a thermometer dissociation for peptide ion dissociations.

Empirical parameterization of a model for predicting peptide helix/coil equilibrium populations.

PubMed Central

Andersen, N. H.; Tong, H.

1997-01-01

A modification of the Lifson-Roig formulation of helix/coil transitions is presented; it (1) incorporates end-capping and coulombic (salt bridges, hydrogen bonding, and side-chain interactions with charged termini and the helix dipole) effects, (2) helix-stabilizing hydrophobic clustering, (3) allows for different inherent termination probabilities of individual residues, and (4) differentiates helix elongation in the first versus subsequent turns of a helix. Each residue is characterized by six parameters governing helix formation. The formulation of the conditional probability of helix initiation and termination that we developed is essentially the same as one presented previously (Shalongo W, Stellwagen, E. 1995. Protein Sci 4:1161-1166) and nearly the mathematical equivalent of the new capping formulation incorporated in the model presented by Rohl et al. (1996. Protein Sci 5:2623-2637). Side-chain/side-chain interactions are, in most cases, incorporated as context dependent modifications of propagation rather than nucleation parameters. An alternative procedure for converting [theta]221 values to experimental fractional helicities () is presented. Tests of the program predictions suggest this method may have some advantages both for designed peptides and for the analysis of secondary structure preferences that could drive the formation of molten-globule intermediates on protein folding pathways. The model predicts the fractional helicity of 385 peptides with a root-mean-square deviation (RMSD) of 0.050 and locates (with precise definition of the termini in many cases) helices in proteins as well as competing methods. The propagation and nucleation parameters were derived from NMR data and from the CD data for a 79 peptide "learning set" for which an excellent fit resulted (RMSD = 0.0295). The current set of parameter corrections for capping boxes, helix dipole interactions, and side-chain/side-chain interactions (coulombic, hydrogen bonding and hydrophobic clustering), although still under development provide a significant improvement in both helix/coil equilibrium prediction for peptides and helix location in protein sequences. This is clearly evident in the rms deviations between CD measures and calculated values of fractional helicity for different classes of peptides before and after applying the corrections: for peptides lacking capping boxes and i/i + 3 and i/i + 4 side-chain/side-chain interactions RMSD = 0.044 (n = 164) versus RMSD = 0.054 (0.172 without the corrections, n = 221) for peptides that required context-dependent corrections of the parameters. If we restrict the analysis to N-acylated peptides with helix stabilizing side-chain/side-chain interactions (including N-capping boxes), the degree to which our corrections account for the stabilizing interaction can be judged from the change in helicity underestimation, (calc-CD): -0.15 +/- 0.10, which is reduced to -0.018 +/- 0.048 (n = 191) upon applying the corrections. PMID:9300492

Production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli

PubMed Central

Handa, Sumit; Spradling, Tyler J.; Dempsey, Daniel R.; Merkler, David J.

2013-01-01

Most mammalian bioactive peptides possess a C-terminal amino acid amide moiety. The presence of the C-terminal amide is a significant impediment to the recombinant production of α-amidated peptides. α-Amidated peptides are produced in vivo by the enzymatic cleavage of a precursor with a C-terminal glycine residue. Peptidylglycine α-hydroxylating monooxygenase catalyzes the key step in the oxidation of the glycine-extended precursors to the α-amidated peptide. Herein, we detail the production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli possessing a N-terminal fusion to thioredoxin (Trx). Trx was fused to hPHMcc to enhance the yield of the resulting 52 kDa protein as a soluble and catalytically active enzyme. The Trx-hPHMcc-His6 fusion was purified to homogeneity and exhibited steady-state kinetic parameters that were similar to purified rat PHMcc. The bacterial production of recombinant hPHMcc will foster efforts to generate α-amidated peptides by the co-expression of hPHMcc and the α-amidated peptide precursors in E. coli or the in vitro amidation of recombinantly expressed α-amidated peptide precursors. PMID:22554821

Identification of key residues for the binding of glucagon to the N-terminal domain of its receptor: an alanine scan and modeling study.

PubMed

Prévost, M; Vertongen, P; Waelbroeck, M

2012-10-01

Glucagon plays an essential role in the glycemia maintenance during fasting, but also aggravates hyperglycemia in diabetic patients. A series of analogues of glucagon were synthesized replacing each amino acid of the C-terminal region (residues 15-29) with alanine. The residues affecting the binding to the glucagon receptor are found to be located on one face of the glucagon helix. Several 3-dimensional models of the N-terminal domain of the glucagon receptor in complex with its ligand peptide were built and used to analyze the peptide-receptor interface in terms of the nature of the peptide residues and the interactions they form with the receptor. The models suggest that glucagon keeps its native helical structure upon binding, and that a large part of the interface formed with the receptor is hydrophobic. We find that in the C-terminal region, F22, V23, M27, and D15 are the most important residues for peptide binding. They bury a large portion of their solvent accessible surface area and make numerous interactions with the receptor mainly of the hydrophobic type. © Georg Thieme Verlag KG Stuttgart · New York.

A Conserved Acidic Motif in the N-Terminal Domain of Nitrate Reductase Is Necessary for the Inactivation of the Enzyme in the Dark by Phosphorylation and 14-3-3 Binding1

PubMed Central

Pigaglio, Emmanuelle; Durand, Nathalie; Meyer, Christian

1999-01-01

It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611–621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II. PMID:9880364

Design and characterization of the anion-sensitive coiled-coil peptide.

PubMed Central

Hoshino, M.; Yumoto, N.; Yoshikawa, S.; Goto, Y.

1997-01-01

As a model for analyzing the role of charge repulsion in proteins and its shielding by the solvent, we designed a peptide of 27 amino acid residues that formed a homodimeric coiled-coil. The interface between the coils consisted of hydrophobic Leu and Val residues, and 10 Lys residues per monomer were incorporated into the positions exposed to solvent. During the preparation of a disulfide-linked dimer in which the two peptides were linked in parallel by the two disulfide bonds located at the N and C terminals, a cyclic monomer with an intramolecular disulfide bond was also obtained. On the basis of CD and 1H-NMR, the conformational stabilities of these isomers and several reference peptides were examined. Whereas all these peptides were unfolded in the absence of salt at pH 4.7 and 20 degrees C, the addition of NaClO4 cooperatively stabilized the alpha-helical conformation. The crosslinking of the peptides by disulfide bonds significantly decreased the midpoint salt concentration of the transition. The 1H-NMR spectra in the presence of NaClO4 suggested that, whereas the disulfide-bonded dimer assumed a native-like conformation, the cyclic monomer assumed a molten globule-like conformation with disordered side chains. However, the cyclic monomer exhibited cooperative transitions against temperature and Gdn-HCl that were only slightly less cooperative than those of the disulfide-bonded parallel dimer. These results indicate that the charge repulsion critically destabilizes the native-like state as well as the molten globule-like state, and that the solvent-dependent charge repulsion may be useful for controlling the conformation of designed peptides. PMID:9232640

Application of synthetic peptides for detection of anti-citrullinated peptide antibodies.

PubMed

Trier, Nicole Hartwig; Holm, Bettina Eide; Slot, Ole; Locht, Henning; Lindegaard, Hanne; Svendsen, Anders; Nielsen, Christoffer Tandrup; Jacobsen, Søren; Theander, Elke; Houen, Gunnar

2016-02-01

Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and represent an important tool for the serological diagnosis of RA. In this study, we describe ACPA reactivity to overlapping citrullinated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-derived peptides and analyze their potential as substrates for ACPA detection by streptavidin capture enzyme-linked immunosorbent assay. Using systematically overlapping peptides, containing a 10 amino acid overlap, labelled with biotin C-terminally or N-terminally, sera from 160 individuals (RA sera (n=60), healthy controls (n=40), systemic lupus erythematosus (n=20), Sjögren's syndrome (n=40)) were screened for antibody reactivity. Antibodies to a panel of five citrullinated EBNA-1 peptides were found in 67% of RA sera, exclusively of the IgG isotype, while 53% of the patient sera reacted with a single peptide, ARGGSRERARGRGRG-Cit-GEKR, accounting for more than half of the ACPA reactivity alone. Moreover, these antibodies were detected in 10% of CCP2-negative RA sera. In addition, 47% of the RA sera reacted with two or three citrullinated EBNA-1 peptides from the selected peptide panel. Furthermore, a negative correlation between the biotin attachment site and the location of citrulline in the peptides was found, i.e. the closer the citrulline was located to biotin, the lower the antibody reactivity. Our data suggest that citrullinated EBNA-1 peptides may be considered a substrate for the detection of ACPAs and that the presence of Epstein-Barr virus may play a role in the induction of these autoantibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

HgNO3 sensitivity of AlGaN/GaN field effect transistors functionalized with phytochelating peptides

NASA Astrophysics Data System (ADS)

Rohrbaugh, Nathaniel; Hernandez-Balderrama, Luis; Kaess, Felix; Kirste, Ronny; Collazo, Ramon; Ivanisevic, Albena

2016-06-01

This study examined the conductance sensitivity of AlGaN/GaN field effect transistors in response to varying Hg/HNO3 solutions. FET surfaces were covalently functionalized with phytochelatin-5 peptides in order to detect Hg in solution. Results showed a resilience of peptide-AlGaN/GaN bonds in the presence of strong HNO3 aliquots, with significant degradation in FET ID signal. However, devices showed strong and varied response to Hg concentrations of 1, 10, 100, and 1000 ppm. The gathered statistically significant results indicate that peptide terminated AlGaN/GaN devices are capable of differentiating between Hg solutions and demonstrate device sensitivity.

The minimum activation peptide from ilvH can activate the catalytic subunit of AHAS from different species.

PubMed

Zhao, Yuefang; Niu, Congwei; Wen, Xin; Xi, Zhen

2013-04-15

Acetohydroxyacid synthases (AHASs), which catalyze the first step in the biosynthesis of branched-chain amino acids, are composed of a catalytic subunit (CSU) and a regulatory subunit (RSU). The CSU harbors the catalytic site, and the RSU is responsible for the activation and feedback regulation of the CSU. Previous results from Chipman and co-workers and our lab have shown that heterologous activation can be achieved among isozymes of Escherichia coli AHAS. It would be interesting to find the minimum peptide of ilvH (the RSU of E. coli AHAS III) that could activate other E. coli CSUs, or even those of ## species. In this paper, C-terminal, N-terminal, and C- and N-terminal truncation mutants of ilvH were constructed. The minimum peptide to activate ilvI (the CSU of E. coli AHAS III) was found to be ΔN 14-ΔC 89. Moreover, this peptide could not only activate its homologous ilvI and heterologous ilvB (CSU of E. coli AHAS I), but also heterologously activate the CSUs of AHAS from Saccharomyces cerevisiae, Arabidopsis thaliana, and Nicotiana plumbaginifolia. However, this peptide totally lost its ability for feedback regulation by valine, thus suggesting different elements for enzymatic activation and feedback regulation. Additionally, the apparent dissociation constant (Kd ) of ΔN 14-ΔC 89 when binding CSUs of different species was found to be 9.3-66.5 μM by using microscale thermophoresis. The ability of this peptide to activate different CSUs does not correlate well with its binding ability (Kd ) to these CSUs, thus implying that key interactions by specific residues is more important than binding ability in promoting enzymatic reactions. The high sequence similarity of the peptide ΔN 14-ΔC 89 to RSUs across species hints that this peptide represents the minimum activation motif in RSU and that it regulates all AHASs. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Amyloid fibril formation of peptides derived from the C-terminus of CETP modulated by lipids

DOE Office of Scientific and Technical Information (OSTI.GOV)

García-González, Victor; Mas-Oliva, Jaime, E-mail: jmas@ifc.unam.mx; División de Investigación, Facultad de Medicina, Universidad Nacional Autónoma de México, 04510 México, DF

2013-04-26

Highlights: •The secondary structure of a C-terminal peptide derived from CETP was studied. •Lipids modulate secondary structure changes of a C-terminal peptide derived from CETP. •Lysophosphatidic acid maintains a functional α-helix and prevents fibril formation. •Transfer of lipids by CETP is related to the presence of an α-helix at its C-end. -- Abstract: Cholesteryl-ester transfer protein (CETP) is a plasmatic protein involved in neutral lipid transfer between lipoproteins. Focusing on the last 12 C-terminus residues we have previously shown that mutation D{sub 470}N promotes a conformational change towards a β-secondary structure. In turn, this modification leads to the formation ofmore » oligomers and fibrillar structures, which cause cytotoxic effects similar to the ones provoked by amyloid peptides. In this study, we evaluated the role of specific lipid arrangements on the structure of peptide helix-Z (D{sub 470}N) through the use of thioflavin T fluorescence, peptide bond absorbance, circular dichroism and electron microscopy. The results indicate that the use of micelles formed with lysophosphatidylcholine and lysophosphatidic acid (LPA) under neutral pH induce a conformational transition of peptide helix-Z containing a β-sheet conformation to a native α-helix structure, therefore avoiding the formation of amyloid fibrils. In contrast, incubation with phosphatidic acid does not change the profile for the β-sheet conformation. When the electrostatic charge at the surface of micelles or vesicles is regulated through the use of lipids such as phospholipid and LPA, minimal changes and the presence of β-structures were recorded. Mixtures with a positive net charge diminished the percentage of β-structure and the amount of amyloid fibrils. Our results suggest that the degree of solvation determined by the presence of a free hydroxyl group on lipids such as LPA is a key condition that can modulate the secondary structure and the consequent formation of amyloid fibrils in the highly flexible C-terminus domain of CETP.« less

Bovine and human lactoferricin peptides: chimeras and new cyclic analogs.

PubMed

Arias, Mauricio; McDonald, Lindsey J; Haney, Evan F; Nazmi, Kamran; Bolscher, Jan G M; Vogel, Hans J

2014-10-01

Lactoferrin (LF) is an important antimicrobial and immune regulatory protein present in neutrophils and most exocrine secretions of mammals. The antimicrobial activity of LF has been related to the presence of an antimicrobial peptide sequence, called lactoferricin (LFcin), located in the N-terminal region of the protein. The antimicrobial activity of bovine LFcin is considerably stronger than the human version. In this work, chimera peptides combining segments of bovine and human LFcin were generated in order to study their antimicrobial activity and mechanism of action. In addition, the relevance of the conserved disulfide bridge and the resulting cyclic structure of both LFcins were analyzed by using "click chemistry" and sortase A-catalyzed cyclization of the peptides. The N-terminal region of bovine LFcin (residues 17-25 of bovine LF) proved to be very important for the antimicrobial activity of the chimera peptides against E. coli, when combined with the C-terminal region of human LFcin. Similarly the cyclic bovine LFcin analogs generated by "click chemistry" and sortase A preserved the antimicrobial activity of the original peptide, showing the significance of these two techniques in the design of cyclic antimicrobial peptides. The mechanism of action of bovine LFcin and its active derived peptides was strongly correlated with membrane leakage in E. coli and up to some extent with the ability to induce vesicle aggregation. This mechanism was also preserved under conditions of high ionic strength (150 mM NaCl) illustrating the importance of these peptides in a more physiologically relevant system.

New features of the delta opioid receptor: conformational properties of deltorphin I analogues.

PubMed

Balboni, G; Marastoni, M; Picone, D; Salvadori, S; Tancredi, T; Temussi, P A; Tomatis, R

1990-06-15

Deltorphin I is an opioid peptide of sequence H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, recently isolated from the skin of Phyllomedusa bicolor. Its enormous selectivity towards the delta opioid receptor and the similarity of the conformation of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-he-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide, prompted the synthesis, biological evaluation and comparative conformational study of four analogs. A 1H-NMR study showed that the conformational preferences of the N-terminal sequences of all peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects in the interaction with the membrane and at the receptor site and of hydrophobicity of the C-terminal part, when structured in a folded conformation.

Antibacterial activity in bovine lactoferrin-derived peptides.

PubMed Central

Hoek, K S; Milne, J M; Grieve, P A; Dionysius, D A; Smith, R

1997-01-01

Several peptides sharing high sequence homology with lactoferricin B (Lf-cin B) were generated from bovine lactoferrin (Lf) with recombinant chymosin. Two peptides were copurified, one identical to Lf-cin B and another differing from Lf-cin B by the inclusion of a C-terminal alanine (lactoferricin). Two other peptides were copurified from chymosin-hydrolyzed Lf, one differing from Lf-cin B by the inclusion of C-terminal alanyl-leucine and the other being a heterodimer linked by a disulfide bond. These peptides were isolated in a single step from chymosin-hydrolyzed Lf by membrane ion-exchange chromatography and were purified by reverse-phase high-pressure liquid chromatography (HPLC). They were characterized by N-terminal Edman sequencing, mass spectrometry, and antibacterial activity determination. Pure lactoferricin, prepared from pepsin-hydrolyzed Lf, was purified by standard chromatography techniques. This peptide was analyzed against a number of gram-positive and gram-negative bacteria before and after reduction of its disulfide bond or cleavage after its single methionine residue and was found to inhibit the growth of all the test bacteria at a concentration of 8 microM or less. Subfragments of lactoferricin were isolated from reduced and cleaved peptide by reverse-phase HPLC. Subfragment 1 (residues 1 to 10) was active against most of the test microorganisms at concentrations of 10 to 50 microM. Subfragment 2 (residues 11 to 26) was active against only a few microorganisms at concentrations up to 100 microM. These antibacterial studies indicate that the activity of lactoferricin is mainly, but not wholly, due to its N-terminal region. PMID:8980754

Convergent Synthesis of N-Linked Glycopeptides via Aminolysis of ω-Asp p-Nitrophenyl Thioesters in Solution.

PubMed

Du, Jing-Jing; Gao, Xiao-Fei; Xin, Ling-Ming; Lei, Ze; Liu, Zheng; Guo, Jun

2016-10-07

An efficient N-linked glycosylation reaction between glycosylamines and p-nitrophenyl thioester peptides has been developed. The reaction conditions are mild and compatible with the C-terminal free carboxylic acid group and the unprotected N-linked sialyloligosaccharide. By means of this convergent strategy, a versatile N-glycopeptide fragment containing an N-terminal Thz and a C-terminal thioester was readily prepared, which is available for the synthesis of long glycopeptides and glycoproteins using the protocol of native chemical ligation.

Protein N- and C-Termini Identification Using Mass Spectrometry and Isotopic Labeling

USDA-ARS?s Scientific Manuscript database

A new method for protein N- and C-terminal analysis using mass spectrometry is introduced. A novel stable isotopic labeling scheme has been developed to identify terminal peptides generated from an enzyme digestion for the determination of both N- and C-termini of the protein. This method works dire...

Highly sensitive detection of influenza virus by boron-doped diamond electrode terminated with sialic acid-mimic peptide.

PubMed

Matsubara, Teruhiko; Ujie, Michiko; Yamamoto, Takashi; Akahori, Miku; Einaga, Yasuaki; Sato, Toshinori

2016-08-09

The progression of influenza varies according to age and the presence of an underlying disease; appropriate treatment is therefore required to prevent severe disease. Anti-influenza therapy, such as with neuraminidase inhibitors, is effective, but diagnosis at an early phase of infection before viral propagation is critical. Here, we show that several dozen plaque-forming units (pfu) of influenza virus (IFV) can be detected using a boron-doped diamond (BDD) electrode terminated with a sialic acid-mimic peptide. The peptide was used instead of the sialyloligosaccharide receptor, which is the common receptor of influenza A and B viruses required during the early phase of infection, to capture IFV particles. The peptide, which was previously identified by phage-display technology, was immobilized by click chemistry on the BDD electrode, which has excellent electrochemical characteristics such as low background current and weak adsorption of biomolecules. Electrochemical impedance spectroscopy revealed that H1N1 and H3N2 IFVs were detectable in the range of 20-500 pfu by using the peptide-terminated BDD electrode. Our results demonstrate that the BDD device integrated with the receptor-mimic peptide has high sensitivity for detection of a low number of virus particles in the early phase of infection.

STUDIES ON THE IMMUNE RESPONSE TO A CHARACTERIZED ANTIGENIC DETERMINANT OF THE TOBACCO MOSAIC VIRUS PROTEIN

PubMed Central

Spitler, Lynn; Benjamini, E.; Young, Janis D.; Kaplan, Harvey; Fudenberg, H. H.

1970-01-01

The following peptides have previously been shown to bind specifically with antibodies to TMVP: (a) An eicosapeptide representing residues 93–112 of TMVP and having the sequence Ileu-Ileu-Glu-Val-Glu-AspNH2-GluNH2-Ala-AspNH2-Pro-Thr-Thr-Ala-Glu-Thr-Leu-Asp-Ala-Thr-Arg. (b) Its C-terminal decapeptide. (c) Its C-terminal pentapeptide. (d) N-octanoyl-C-terminal-tripeptide. (e) (Lys)4-C-terminal-pentapeptide. (f) (Lys)7 C-terminal-pentapeptide. The present communication deals with the investigation of several parameters of the immunological activity of the peptides. The results show that none of the peptides tested were immunogenic in guinea pigs, nor did they stimulate the incorporation of 14C-thymidine by spleen cells derived from TMVP-primed animals. Results also showed that all of the peptides tested could elicit specific delayed and immediate skin reactions in TMVP-sensitized guinea pigs, and furthermore, that the peptides could specifically inhibit the migration of peritoneal exudate cells derived from these animals. The elicitation of delayed skin reactions and the ability to inhibit migration of peritoneal exudate cells were independent of carrier specificity. PMID:5409944

LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells.

USDA-ARS?s Scientific Manuscript database

LysK is a staphylococcal bacteriophage endolysin composed of three domains, an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain (cleaves between D-alanine of the stem peptide and glycine of the cross-bridge peptide) a mid-protein amidase 2 domain (N-ace...

C-terminal sequence of amyloid-resistant type F apolipoprotein A-II inhibits amyloid fibril formation of apolipoprotein A-II in mice

PubMed Central

Sawashita, Jinko; Zhang, Beiru; Hasegawa, Kazuhiro; Mori, Masayuki; Naiki, Hironobu; Kametani, Fuyuki; Higuchi, Keiichi

2015-01-01

In murine senile amyloidosis, misfolded serum apolipoprotein (apo) A-II deposits as amyloid fibrils (AApoAII) in a process associated with aging. Mouse strains carrying type C apoA-II (APOA2C) protein exhibit a high incidence of severe systemic amyloidosis. Previously, we showed that N- and C-terminal sequences of apoA-II protein are critical for polymerization into amyloid fibrils in vitro. Here, we demonstrate that congenic mouse strains carrying type F apoA-II (APOA2F) protein, which contains four amino acid substitutions in the amyloidogenic regions of APOA2C, were absolutely resistant to amyloidosis, even after induction of amyloidosis by injection of AApoAII. In vitro fibril formation tests showed that N- and C-terminal APOA2F peptides did not polymerize into amyloid fibrils. Moreover, a C-terminal APOA2F peptide was a strong inhibitor of nucleation and extension of amyloid fibrils during polymerization. Importantly, after the induction of amyloidosis, we succeeded in suppressing amyloid deposition in senile amyloidosis-susceptible mice by treatment with the C-terminal APOA2F peptide. We suggest that the C-terminal APOA2F peptide might inhibit further extension of amyloid fibrils by blocking the active ends of nuclei (seeds). We present a previously unidentified model system for investigating inhibitory mechanisms against amyloidosis in vivo and in vitro and believe that this system will be useful for the development of novel therapies. PMID:25675489

Anti-senescence and Anti-inflammatory Effects of the C-terminal Moiety of PTHrP Peptides in OA Osteoblasts.

PubMed

Platas, Julia; Guillén, Maria Isabel; Gomar, Francisco; Castejón, Miguel Angel; Esbrit, Pedro; Alcaraz, Maria José

2017-05-01

Osteoarthritis (OA) is characterized by degenerative changes in the whole joint leading to physical disability in the elderly population. This condition is associated with altered bone metabolism in subchondral areas suggesting that therapeutic strategies aimed at modifying bone cell metabolism may be of interest. We have investigated the effects of several parathyroid hormone-related protein (PTHrP)-derived peptides (1-37): (N-terminal), (107-111) and (107-139) (C-terminal) on senescence features induced by inflammatory stress in human OA osteoblasts. Incubation of these primary cells with interleukin(IL)-1β led to an increased expression of senescence markers senescence-associated-β-galactosidase activity, γH2AX foci, p16, p21, p53, and caveolin-1. PTHrP (107-111) and PTHrP (107-139) significantly reduced all these parameters. Both peptides decreased the production of IL-6 and prostaglandin E2 which was the consequence of cyclo-oxygenase-2 downregulation. PTHrP (107-139) also reduced tumor necrosis factor-α release. These anti-inflammatory effects would be related to the reduction of nuclear factor-κB activation by both peptides and activator protein-1 by PTHrP (107-139). The three PTHrP peptides favored osteoblastic function although the C-terminal domain of PTHrP was more efficient than its N-terminal domain. Our data support an anti-senescence and anti-inflammatory role for the C-terminal moiety of PTHrP with potential applications in chronic inflammatory conditions such as OA. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R)

PubMed Central

Koole, Cassandra; Reynolds, Christopher A.; Mobarec, Juan C.; Hick, Caroline; Sexton, Patrick M.; Sakmar, Thomas P.

2017-01-01

The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target in the management of type II diabetes mellitus, with actions including regulation of insulin biosynthesis and secretion, promotion of satiety, and preservation of β-cell mass. Like most class B G protein-coupled receptors (GPCRs), there is limited knowledge linking biological activity of the GLP-1R with the molecular structure of an intact, full-length, and functional receptor·ligand complex. In this study, we have utilized genetic code expansion to site-specifically incorporate the photoactive amino acid p-azido-l-phenylalanine (azF) into N-terminal residues of a full-length functional human GLP-1R in mammalian cells. UV-mediated photolysis of azF was then carried out to induce targeted photocross-linking to determine the proximity of the azido group in the mutant receptor with the peptide exendin-4. Cross-linking data were compared directly with the crystal structure of the isolated N-terminal extracellular domain of the GLP-1R in complex with exendin(9–39), revealing both similarities as well as distinct differences in the mode of interaction. Generation of a molecular model to accommodate the photocross-linking constraints highlights the potential influence of environmental conditions on the conformation of the receptor·peptide complex, including folding dynamics of the peptide and formation of dimeric and higher order oligomeric receptor multimers. These data demonstrate that crystal structures of isolated receptor regions may not give a complete reflection of peptide/receptor interactions and should be combined with additional experimental constraints to reveal peptide/receptor interactions occurring in the dynamic, native, and full-length receptor state. PMID:28283573

Design and activity of novel lactoferrampin analogues against O157:H7 enterohemorrhagic Escherichia coli.

PubMed

Cruz, Jenniffer; Ortiz, Claudia; Guzmán, Fanny; Cárdenas, Constanza; Fernandez-Lafuente, Roberto; Torres, Rodrigo

2014-04-01

Lactoferrampin 265-284 (LFampin 265-284) is a peptide consisting of residues 265-284 of N1-domain of bovine Lactoferrin (LF). This peptide has several cationic groups in the C-terminal lobe, exhibiting an antibacterial activity against a wide range of microorganisms. However, LFampin 265-284 exhibits low antimicrobial activity against the O157:H7 enterohaemorrhagic Escherichia coli (EHEC O157:H7) when compared with Lactoferrin chimera and Lactoferricin. Here, we have designed three analogues of LFampin 265-284 based on the distribution of cationic groups, hydrophobicity, size, and sequence. Analogues were synthesized by solid phase chemistry using Fmoc methodology obtaining peptides with 95% purity. All peptides maintain the ability to adopt helical conformations (checked by circular dichroism spectra and molecular simulations). Some of these analogues exhibited a significant increase in antimicrobial activity by counting colony forming units against EHEC O157:H7 compared to native LFampin 265-284, with MIC of 10 and 40 µM for 264G-D265K and 264G-D265K/S272R, respectively. The incorporation of a GKLI sequence in the N-terminal lobe increased dramatically its antibacterial activity, an effect which has been attributed to the addition of cationic groups in the N-terminal side that may stabilize the helical conformation of the new designed peptides. Copyright © 2013 Wiley Periodicals, Inc.

The N-terminal domain of substance P is required for complete homologous desensitization but not phosphorylation of the rat neurokinin-1 receptor.

PubMed

Vigna, S R

2001-02-01

The agonist activity of substance P (SP) is a function of the C-terminal domain of the peptide. A C-terminal SP fragment (SP(6-11)) and analog (septide) and neurokinin A (NKA; a related tachykinin with a divergent N-terminal amino acid sequence) were found to be full neurokinin-1 receptor (NK-1R) agonists, but were not able to desensitize the receptor maximally as much as SP. Substance P caused 95.6 +/- 0.9% maximal desensitization of the NK-1R whereas SP(6-11), septide, and NKA(only)caused 74 +/- 3.5, 50.6 +/- 8, and 71.5 +/- 4.4% maximal desensitization, respectively (mean +/- SEM; P < 0.001 vs SP). When a series of SP C-terminal fragment peptides were tested for their NK-1R desensitizing activity, it was found that SP(5-11)and SP(6-11)caused significantly less maximal NK-1R desensitization than SP. SP N-terminal fragment peptides had no effect on the ability of SP(6-11)to compete with(3)H-SP binding, generate an IP(3)response, or cause NK-1R desensitization when tested with or without SP(6-11). SP, SP(6-11), septide, and NKA all maximally stimulated 8-9-fold increases in NK-1R phosphorylation. When attached to the C-terminal domain of SP responsible for NK-1R binding and agonism, the N-terminus of SP is responsible for 25-50% of homologous desensitization and this may occur via a mechanism other than NK-1R phosphorylation. Copyright 2001 Harcourt Publishers Ltd.

Cellular targeting and host-specific recognition of cyst nematode CLE proteins

USDA-ARS?s Scientific Manuscript database

Cyst nematodes produce secreted peptide mimics of plant CLAVATA3/ESR (CLE) peptides likely involved in redirecting CLE signaling pathways active in roots to form unique and essential feeding cells. The hallmark structure of plant CLEs, which includes an N-terminal signal peptide, a highly variable d...

ANTI-11[E]-PYROGLUTAMATE-MODIFIED AMYLOID β ANTIBODIES CROSS-REACT WITH OTHER PATHOLOGICAL Aβ SPECIES: RELEVANCE FOR IMMUNOTHERAPY

PubMed Central

Perez-Garmendia, Roxanna; Ibarra-Bracamontes, Vanessa; Vasilevko, Vitaly; Luna-Muñoz, Jose; Mena, Raul; Govezensky, Tzipe; Acero, Gonzalo; Manoutcharian, Karen; Cribbs, David H.; Gevorkian, Goar

2010-01-01

N-truncated/modified forms of amyloid beta (Aß) peptide are found in diffused and dense core plaques in Alzheimer's disease (AD) and Down's syndrome patients as well as animal models of AD, and represent highly desirable therapeutic targets. In the present study we have focused on Ntruncated/modified Aβ peptide bearing amino-terminal pyroglutamate at position 11 (AβN11(pE)). We identified two B-cell epitopes recognized by rabbit anti-AβN11(pE) polyclonal antibodies. Interestingly, rabbit anti-AβN11(pE) polyclonal antibodies bound also to full-length Aβ1-42 and N-truncated/modified AβN3(pE), suggesting that the three peptides may share a common B-cell epitope. Importantly, rabbit anti-AβN11(pE) antibodies bound to naturally occurring Aβ aggregates present in brain samples from AD patients. These results are potentially important for developing novel immunogens for targeting N-truncated/modified Aβ aggregates as well, since the most commonly used immunogens in the majority of vaccine studies have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Aβ, which is absent in N-amino truncated peptides. PMID:20864186

Expression, refolding and crystallizations of the Grb2-like (GADS) C-terminal SH3 domain complexed with a SLP-76 motif peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faravelli, Alessandro; Dimasi, Nazzareno, E-mail: ndimasi@gmail.com

Several crystals of the Grb2-like C-terminal SH3 domain in complex with a motif peptide from the SLP-76 protein were obtained and characterized. The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli,more » refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.« less

The C terminus of the catalytic domain of type A botulinum neurotoxin may facilitate product release from the active site.

PubMed

Mizanur, Rahman M; Frasca, Verna; Swaminathan, Subramanyam; Bavari, Sina; Webb, Robert; Smith, Leonard A; Ahmed, S Ashraf

2013-08-16

Botulinum neurotoxins are the most toxic of all compounds. The toxicity is related to a poor zinc endopeptidase activity located in a 50-kDa domain known as light chain (Lc) of the toxin. The C-terminal tail of Lc is not visible in any of the currently available x-ray structures, and it has no known function but undergoes autocatalytic truncations during purification and storage. By synthesizing C-terminal peptides of various lengths, in this study, we have shown that these peptides competitively inhibit the normal catalytic activity of Lc of serotype A (LcA) and have defined the length of the mature LcA to consist of the first 444 residues. Two catalytically inactive mutants also inhibited LcA activity. Our results suggested that the C terminus of LcA might interact at or near its own active site. By using synthetic C-terminal peptides from LcB, LcC1, LcD, LcE, and LcF and their respective substrate peptides, we have shown that the inhibition of activity is specific only for LcA. Although a potent inhibitor with a Ki of 4.5 μm, the largest of our LcA C-terminal peptides stimulated LcA activity when added at near-stoichiometric concentration to three versions of LcA differing in their C-terminal lengths. The result suggested a product removal role of the LcA C terminus. This suggestion is supported by a weak but specific interaction determined by isothermal titration calorimetry between an LcA C-terminal peptide and N-terminal product from a peptide substrate of LcA. Our results also underscore the importance of using a mature LcA as an inhibitor screening target.

Characterization of the N-terminal segment used by the barley yellow dwarf virus movement protein to promote interaction with the nuclear membrane of host plant cells.

PubMed

Dennison, Sarah Rachel; Harris, Frederick; Brandenburg, Klaus; Phoenix, David Andrew

2007-11-01

The barley yellow dwarf virus movement protein (BYDV-MP) requires its N-terminal sequence to promote the transport of viral RNA into the nuclear compartment of host plant cells. Here, graphical analysis predicts that this sequence would form a membrane interactive amphiphilic alpha-helix. Confirming this prediction, NT1, a peptide homologue of the BYDV-MP N-terminal sequence, was found to be alpha-helical (65%) in the presence of vesicles mimics of the nuclear membrane. The peptide increased the fluidity of these nuclear membrane mimics (rise in wavenumber of circa 0.5-1.0 cm(-1)) and induced surface pressure changes of 2 mN m(-1) in lipid monolayers with corresponding compositions. Taken with isotherm analysis these results suggest that BYDV-MP forms an N-terminal amphiphilic alpha-helix, which partitions into the nuclear membrane primarily through thermodynamically stable associations with the membrane lipid headgroup region. We speculate that these associations may play a role in targeting of the nuclear membrane by BYDM-MP.

Gas-phase structure and fragmentation pathways of singly protonated peptides with N-terminal arginine.

PubMed

Bythell, Benjamin J; Csonka, István P; Suhai, Sándor; Barofsky, Douglas F; Paizs, Béla

2010-11-25

The gas-phase structures and fragmentation pathways of the singly protonated peptide arginylglycylaspartic acid (RGD) are investigated by means of collision-induced-dissociation (CID) and detailed molecular mechanics and density functional theory (DFT) calculations. It is demonstrated that despite the ionizing proton being strongly sequestered at the guanidine group, protonated RGD can easily be fragmented on charge directed fragmentation pathways. This is due to facile mobilization of the C-terminal or aspartic acid COOH protons thereby generating salt-bridge (SB) stabilized structures. These SB intermediates can directly fragment to generate b(2) ions or facilely rearrange to form anhydrides from which both b(2) and b(2)+H(2)O fragments can be formed. The salt-bridge stabilized and anhydride transition structures (TSs) necessary to form b(2) and b(2)+H(2)O are much lower in energy than their traditional charge solvated counterparts. These mechanisms provide compelling evidence of the role of SB and anhydride structures in protonated peptide fragmentation which complements and supports our recent findings for tryptic systems (Bythell, B. J.; Suhai, S.; Somogyi, A.; Paizs, B. J. Am. Chem. Soc. 2009, 131, 14057-14065.). In addition to these findings we also report on the mechanisms for the formation of the b(1) ion, neutral loss (H(2)O, NH(3), guanidine) fragment ions, and the d(3) ion.

60 YEARS OF POMC: N-terminal POMC peptides and adrenal growth.

PubMed

Bicknell, Andrew B

2016-05-01

The peptide hormones contained within the sequence of proopiomelanocortin (POMC) have diverse roles ranging from pigmentation to regulation of adrenal function to control of our appetite. It is generally acknowledged to be the archetypal hormone precursor, and as its biology has been unravelled, so too have many of the basic principles of hormone biosynthesis and processing. This short review focuses on one group of its peptide products, namely, those derived from the N-terminal of POMC and their role in the regulation of adrenal growth. From a historical and a personal perspective, it describes how their role in regulating proliferation of the adrenal cortex was identified and also highlights the key questions that remain to be answered. © 2016 Society for Endocrinology.

Hypochlorous Acid Reacts with the N-Terminal Methionines of Proteins to Give Dehydromethionine, a Potential Biomarker for Neutrophil-Induced Oxidative Stress†

PubMed Central

Beal, Jennifer L.; Foster, Steven B.; Ashby, Michael T.

2009-01-01

Electrophilic halogenating agents, including hypohalous acids and haloamines, oxidize free methionine and the N-terminal methionines of peptides and proteins (e.g., Met-1 of anti-inflammatory peptide 1 and ubiquitin) to produce dehydromethionine (a five-membered isothiazolidinium heterocycle). Amide derivatives of methionine are oxidized to the corresponding sulfoxide derivatives under the same reaction conditions (e.g., Met-3 of anti-inflammatory peptide 1). Other biological oxidants, including hydrogen peroxide and peroxynitrite, also only produce the corresponding sulfoxides. Hypothiocyanite does not react with methionine residues. It is suggested that dehydromethionine may be a useful biomarker for the myeloperoxidase-induced oxidative stress associated with many inflammatory diseases. PMID:19839600

Targeted mass spectrometric analysis of N-terminally truncated isoforms generated via alternative translation initiation.

PubMed

Kobayashi, Ryuji; Patenia, Rebecca; Ashizawa, Satoshi; Vykoukal, Jody

2009-07-21

Alternative translation initiation is a mechanism whereby functionally altered proteins are produced from a single mRNA. Internal initiation of translation generates N-terminally truncated protein isoforms, but such isoforms observed in immunoblot analysis are often overlooked or dismissed as degradation products. We identified an N-terminally truncated isoform of human Dok-1 with N-terminal acetylation as seen in the wild-type. This Dok-1 isoform exhibited distinct perinuclear localization whereas the wild-type protein was distributed throughout the cytoplasm. Targeted analysis of blocked N-terminal peptides provides rapid identification of protein isoforms and could be widely applied for the general evaluation of perplexing immunoblot bands.

Cardiac peptide stability, aprotinin and room temperature: importance for assessing cardiac function in clinical practice.

PubMed

Buckley, M G; Marcus, N J; Yacoub, M H

1999-12-01

Brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP) and N-terminal ANP are good research indices of the severity of heart failure. The stability of these peptides at room temperature has become an important factor in assessing their use as indicators of cardiac function in routine clinical practice. Inhibitors such as aprotinin are routinely added in the blood collection process, but may provide no benefit in sample collection and routine clinical practice. We assessed the stability of BNP, ANP and N-terminal ANP in blood samples collected in either the presence or the absence of the protease inhibitor aprotinin. Blood, either with or without aprotinin, was processed immediately (initial; 0 h) and after blood samples had been left for 3 h, 2 days or 3 days at room temperature. These times were chosen to reflect processing in a hospital outpatient clinic (2-3 h), or when posted from general practice (2-3 days). Initial plasma BNP, ANP and N-terminal ANP levels in the absence of aprotinin were 28.2+/-5.4, 44.2+/-7.9 and 1997+/-608 pg/ml respectively, and were not significantly different from initial values in the presence of aprotinin (29.0+/-5.9, 45.2+/-8.0 and 2009+/-579 pg/ml respectively). After 3 h at room temperature, there was a significant fall in ANP in the absence of aprotinin (36. 7+/-7.9 pg/ml; P<0.005), but not in the presence of aprotinin (41. 2+/-7.6 pg/ml). Both BNP and N-terminal ANP were unchanged in either the absence (BNP, 27.6+/-5.5 pg/ml; N-terminal ANP, 2099+/-613 pg/ml) or the presence (BNP, 29.4+/-5.6 pg/ml; N-terminal ANP, 1988+/-600 pg/ml) of aprotinin. After 2 days at room temperature, ANP had fallen significantly in both the absence (16.9+/-3.4 pg/ml) and the presence (24.0+/-5.0 pg/ml) of aprotinin compared with initial values, and there was a significant difference in ANP levels in the absence and presence of aprotinin (P<0.001). ANP levels had decreased further after 3 days at room temperature, to 11.9+/-3.4 pg/ml (no aprotinin) and 20.3+/-5.0 pg/ml (aprotinin added); these values were significantly different (P=0.002). In contrast, there was no change in the levels of BNP or N-terminal ANP after 2 or 3 days at room temperature, in either the absence or the presence of aprotinin. These studies indicate that aprotinin adds little benefit to the stability of cardiac peptides at room temperature. Blood samples for BNP and N-terminal ANP measurement used as a test of heart function in hospital clinics and by general practitioners in the community could be taken into blood tubes containing only EDTA as anticoagulant and without the additional step of adding the routinely used inhibitor aprotinin.

Evolution in Action: N and C Termini of Subunits in Related T=4 Viruses Exchange Roles as Molecular Switches

PubMed Central

Speir, Jeffrey A.; Taylor, Derek J.; Natarajan, Padmaja; Pringle, Fiona M.; Ball, L. Andrew; Johnson, John E.

2010-01-01

Summary The T=4 tetravirus and T=3 nodavirus capsid proteins undergo closely similar autoproteolysis to produce the N-terminal ß and C-terminal, lipophilic γ polypeptides. The γ peptides and N-termini of ß also act as molecular switches that determine their quasi-equivalent capsid structures. The crystal structure of Providence virus (PrV), only the second of a tetravirus (the first was NωV), reveals conserved folds and cleavage sites, but the protein termini have completely different structures and the opposite functions of those in N⌉V. N-termini of ß form the molecular switch in PrV, while γ peptides have this role in N⌉V. PrV γ peptides instead interact with packaged RNA at the particle 2-folds using a repeating sequence pattern found in only four other RNA or membrane binding proteins. The disposition of peptide termini in PrV is closely related to those in nodaviruses suggesting that PrV may be closer to the primordial T=4 particle than NωV. PMID:20541507

Interaction of MreB-derived antimicrobial peptides with membranes.

PubMed

Saikia, Karabi; Chaudhary, Nitin

2018-03-25

Antimicrobial peptides are critical components of defense systems in living forms. The activity is conferred largely by the selective membrane-permeabilizing ability. In our earlier work, we derived potent antimicrobial peptides from the 9-residue long, N-terminal amphipathic helix of E. coli MreB protein. The peptides display broad-spectrum activity, killing not only Gram-positive and Gram-negative bacteria but opportunistic fungus, Candida albicans as well. These results proved that membrane-binding stretches of bacterial proteins could turn out to be self-harming when applied from outside. Here, we studied the membrane-binding and membrane-perturbing potential of these peptides. Steady-state tryptophan fluorescence studies with tryptophan extended peptides, WMreB 1-9 and its N-terminal acetylated analog, Ac-WMreB 1-9 show preferential binding to negatively-charged liposomes. Both the peptides cause permeabilization of E. coli inner and outer-membranes. Tryptophan-lacking peptides, though permeabilize the outer-membrane efficiently, little permeabilization of the inner-membrane is observed. These data attest membrane-destabilization as the mechanism of rapid bacterial killing. This study is expected to motivate the research in identifying microbes' self-sequences to combat them. Copyright © 2018 Elsevier Inc. All rights reserved.

Structural analysis of the starfish SALMFamide neuropeptides S1 and S2: the N-terminal region of S2 facilitates self-association.

PubMed

Otara, Claire B; Jones, Christopher E; Younan, Nadine D; Viles, John H; Elphick, Maurice R

2014-02-01

The neuropeptides S1 (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide), which share sequence similarity, were discovered in the starfish Asterias rubens and are prototypical members of the SALMFamide family of neuropeptides in echinoderms. SALMFamide neuropeptides act as muscle relaxants and both S1 and S2 cause relaxation of cardiac stomach and tube foot preparations in vitro but S2 is an order of magnitude more potent than S1. Here we investigated a structural basis for this difference in potency using spectroscopic techniques. Circular dichroism spectroscopy showed that S1 does not have a defined structure in aqueous solution and this was supported by 2D nuclear magnetic resonance experiments. In contrast, we found that S2 has a well-defined conformation in aqueous solution. However, the conformation of S2 was concentration dependent, with increasing concentration inducing a transition from an unstructured to a structured conformation. Interestingly, this property of S2 was not observed in an N-terminally truncated analogue of S2 (short S2 or SS2; SFNSGLTFamide). Collectively, the data obtained indicate that the N-terminal region of S2 facilitates peptide self-association at high concentrations, which may have relevance to the biosynthesis and/or bioactivity of S2 in vivo. © 2013.

Growth hormone-releasing hormone stimulates and somatostatin inhibits the release of a novel protein by cultured rat pituitary cells.

PubMed

Tachibana, K; Marquardt, H; Yokoya, S; Friesen, H G

1988-10-01

We have reported that the secretion of at least 17 distinct peptides [including rat (rGH)] GH by cultured rat pituitary cells was stimulated by GH-releasing hormone and inhibited by somatostatin, when analyzed by two-dimensional polyacrylamide gel electrophoresis. Three of these peptides (no. 23, 24, and 25) were not rGH immunoreactive. In order to determine whether these three peptides are fragments, degradation products or posttranscriptionally modified forms of rGH, rGH and peptide no. 23 were characterized structurally. From partial peptide maps of rGH and peptide no. 23 by V8 protease or chymotrypsin, it appeared that these peptides were not related to each other. By N-terminal microsequencing of two-dimensional polyacrylamide gel electrophoresis purified peptide, we have obtained the sequence of 24 N-terminal amino acid residues of peptide no. 23. This sequence has no significant homology with rGH or any other reported protein sequence. Antiserum was generated against a synthetic oligopeptide corresponding to amino acid residues 3-24 of peptide no. 23. The antiserum cross-reacted with peptides no. 23, 24, and 25 upon Western blot analysis. These results indicate that peptide no. 23 has a novel structure unrelated to other pituitary hormones. Since its secretion is influenced by GH-releasing hormone and somatostatin, peptide no. 23 may represent a previously unrecognized structurally unique growth factor.

C-reactive protein and N-terminal prohormone brain natriuretic peptide as biomarkers in acute exacerbations of COPD leading to hospitalizations.

PubMed

Chen, Yu-Wei Roy; Chen, Virginia; Hollander, Zsuzsanna; Leipsic, Jonathon A; Hague, Cameron J; DeMarco, Mari L; FitzGerald, J Mark; McManus, Bruce M; Ng, Raymond T; Sin, Don D

2017-01-01

There are currently no accepted and validated blood tests available for diagnosing acute exacerbations of chronic obstructive pulmonary disease (AECOPD). In this study, we sought to determine the discriminatory power of blood C-reactive protein (CRP) and N-terminal prohormone brain natriuretic peptide (NT-proBNP) in the diagnosis of AECOPD requiring hospitalizations. The study cohort consisted of 468 patients recruited in the COPD Rapid Transition Program who were hospitalized with a primary diagnosis of AECOPD, and 110 stable COPD patients who served as controls. Logistic regression was used to build a classification model to separate AECOPD from convalescent or stable COPD patients. Performance was assessed using an independent validation set of patients who were not included in the discovery set. Serum CRP and whole blood NT-proBNP concentrations were highest at the time of hospitalization and progressively decreased over time. Of the 3 classification models, the one with both CRP and NT-proBNP had the highest AUC in discriminating AECOPD (cross-validated AUC of 0.80). These data were replicated in a validation cohort with an AUC of 0.88. A combination of CRP and NT-proBNP can reasonably discriminate AECOPD requiring hospitalization versus clinical stability and can be used to rapidly diagnose patients requiring hospitalization for AECOPD.

Structural analysis of a functional DIAP1 fragment bound to grim and hid peptides.

PubMed

Wu, J W; Cocina, A E; Chai, J; Hay, B A; Shi, Y

2001-07-01

The inhibitor of apoptosis protein DIAP1 suppresses apoptosis in Drosophila, with the second BIR domain (BIR2) playing an important role. Three proteins, Hid, Grim, and Reaper, promote apoptosis, in part by binding to DIAP1 through their conserved N-terminal sequences. The crystal structures of DIAP1-BIR2 by itself and in complex with the N-terminal peptides from Hid and Grim reveal that these peptides bind a surface groove on DIAP1, with the first four amino acids mimicking the binding of the Smac tetrapeptide to XIAP. The next 3 residues also contribute to binding through hydrophobic interactions. Interestingly, peptide binding induces the formation of an additional alpha helix in DIAP1. Our study reveals the structural conservation and diversity necessary for the binding of IAPs by the Drosophila Hid/Grim/Reaper and the mammalian Smac proteins.

Duck-billed platypus venom peptides induce Ca2+ influx in neuroblastoma cells.

PubMed

Kita, Masaki; Black, David StC; Ohno, Osamu; Yamada, Kaoru; Kigoshi, Hideo; Uemura, Daisuke

2009-12-23

The duck-billed platypus (Ornithorhynchus anatinus) is one of the few venomous Australian mammals. We previously found that its crude venom potently induces Ca(2+) influx in human neuroblastoma IMR-32 cells. Guided by this bioassay, we identified 11 novel peptides, including the heptapeptide H-His-Asp-His-Pro-Asn-Pro-Arg-OH (1). Compounds 1-4 and 5-11 coincided with the 6-9 N-terminal residues of Ornithorhynchus venom C-type natriuretic peptide (OvCNP) and the 132-150 part of OvCNP precursor peptide, respectively. Heptapeptide 1, which is one of the primary components of the venom fluid (approximately 200 ng/microL), induced a significant increase in [Ca(2+)](i) in IMR-32 cells at 75 microM. To the best of our knowledge, this is the first example of the isolation of the N-terminal linear fragments of CNPs in any mammal.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Camarero, J A; Hackel, B J; de Yoreo, J J

C-terminal peptide thioesters are key intermediates for the synthesis/semisynthesis of proteins and for the production of cyclic peptides by native chemical ligation. They can be synthetically prepared by solid-phase peptide synthesis (SPPS) methods or biosynthetically by protein splicing techniques. Until recently, the chemical synthesis of C-terminal a-thioester peptides by SPPS was largely restricted to the Boc/Benzyl methodology because of the poor stability of the thioester bond to the basic conditions employed for the deprotection of the N{sup {alpha}}-Fmoc group. In the present work, we describe a new method for the SPPS of C-terminal thioesters by Fmoc/t-Bu chemistry. This method ismore » based on the use of an aryl hydrazide linker, which is totally stable to the Fmoc-SPPS conditions. Once the peptide synthesis has been completed, activation of the linker can be achieved by mild oxidation. This step transforms the hydrazide group into a highly reactive diazene intermediate which can react with different H-AA-SEt to yield the corresponding {alpha}-thioester peptide in good yields. This method has been successfully used for the generation of different thioester peptides, circular peptides and a fully functional SH3 protein domain.« less

Comprehensive mutational analysis of the M13 major coat protein: improved scaffolds for C-terminal phage display.

PubMed

Held, Heike A; Sidhu, Sachdev S

2004-07-09

A peptide was fused to the C terminus of the M13 bacteriophage major coat protein (P8), and libraries of P8 mutants were screened to select for variants that displayed the peptide with high efficiency. Over 600 variants were sequenced to compile a comprehensive database of P8 sequence diversity compatible with assembly into the wild-type phage coat. The database reveals that, while the alpha-helical P8 molecule was highly tolerant to mutations, certain functional epitopes were required for efficient incorporation. Three hydrophobic epitopes were located approximately equidistantly along the length of the alpha-helix. In addition, a positively charged epitope was required directly opposite the most C-terminal hydrophobic epitope and on the same side as the other two epitopes. Both ends of the protein were highly tolerant to mutations, consistent with the use of P8 as a scaffold for both N and C-terminal phage display. Further rounds of selection were used to enrich for P8 variants that supported higher levels of C-terminal peptide display. The largest improvements in display resulted from mutations around the junction between P8 and the C-terminal linker, and additional mutations in the N-terminal region were selected for further improvements in display. The best P8 variants improved C-terminal display more than 100-fold relative to the wild-type, and these variants could support the simultaneous display of N and C-terminal fusions. These finding provide information on the requirements for filamentous phage coat assembly, and provide improved scaffolds for phage display technology. Copyright 2004 Elsevier Ltd.

Structural Characterization of the N Terminus of IpaC from Shigella flexneri

PubMed Central

Harrington, Amanda T.; Hearn, Patricia D.; Picking, Wendy L.; Barker, Jeffrey R.; Wessel, Andrew; Picking, William D.

2003-01-01

The primary effector for Shigella invasion of epithelial cells is IpaC, which is secreted via a type III secretion system. We recently reported that the IpaC N terminus is required for type III secretion and possibly other functions. In this study, mutagenesis was used to identify an N-terminal secretion signal and to determine the functional importance of the rest of the IpaC N terminus. The 15 N-terminal amino acids target IpaC for secretion by Shigella flexneri, and placing additional amino acids at the N terminus does not interfere with IpaC secretion. Furthermore, amino acid sequences with no relationship to the native IpaC secretion signal can also direct its secretion. Deletions introduced beyond amino acid 20 have no effect on secretion and do not adversely affect IpaC function in vivo until they extend beyond residue 50, at which point invasion function is completely eliminated. Deletions introduced at amino acid 100 and extending toward the N terminus reduce IpaC's invasion function but do not eliminate it until they extend to the N-terminal side of residue 80, indicating that a region from amino acid 50 to 80 is critical for IpaC invasion function. To explore this further, the ability of an IpaC N-terminal peptide to associate in vitro with its translocon partner IpaB and its chaperone IpgC was studied. The N-terminal peptide binds tightly to IpaB, but the IpaC central hydrophobic region also appears to participate in this binding. The N-terminal peptide also associates with the chaperone IpgC and IpaB is competitive for this interaction. Based on additional biophysical data, we propose that a region between amino acids 50 and 80 is required for chaperone binding, and that the IpaB binding domain is located downstream from, and possibly overlapping, this region. From these data, we propose that the secretion signal, chaperone binding region, and IpaB binding domain are located at the IpaC N terminus and are essential for presentation of IpaC to host cells during bacterial entry; however, IpaC effector activity may be located elsewhere. PMID:12595440

[Diagnostic values of serum type III procollagen N-terminal peptide in type IV gastric cancer].

PubMed

Akazawa, S; Fujiki, T; Kanda, Y; Kumai, R; Yoshida, S

1985-04-01

Since increased synthesis of collagen has been demonstrated in tissue of type IV gastric cancer, we attempted to distinguish type IV gastric cancer from other cancers by measuring serum levels of type III procollagen N-terminal peptide (type III-N-peptide). Mean serum levels in type IV gastric cancer patients without metastasis were found to be elevated above normal values and developed a tendency to be higher than those in types I, II and III gastric cancer patients without metastasis. Highly positive ratios were found in patients with liver diseases including hepatoma and colon cancer, biliary tract cancer, and esophageal cancer patients with liver, lung or bone metastasis, but only 2 out of 14 of these cancer patients without such metastasis showed positive serum levels of type III-N-peptide. Positive cases in patients with type IV gastric cancer were obtained not only in the group with clinical stage IV but also in the groups with clinical stages II and III. In addition, high serum levels of type III-N-peptide in patients with type IV gastric cancer were seen not only in the cases with liver, lung or bone metastasis but also in cases with disseminated peritoneal metastasis alone. These results suggest that if the serum level of type III-N-peptide is elevated above normal values, type IV gastric cancer should be suspected after ruling out liver diseases, myelofibrosis and liver, lung or bone metastasis.

Location of alkali metal binding sites in endothelin A selective receptor antagonists, cyclo(D-Trp-D-Asp-Pro-D-Val-Leu) and cyclo(D-Trp-D-Asp-Pro-D-Ile-Leu), from multistep collisionally activated decompositions.

PubMed

Ngoka, L C; Gross, M L

2000-02-01

We previously showed by using mass spectrometry that endothelin A selective receptor antagonists BQ123 and JKC301 form novel coordination compounds with sodium ions. This property may underlie the ability of an ET(A) antagonist to induce net tubular sodium reabsorption in the proximal tubule cells and reverse acute renal failure induced by severe ischemia. We have now defined the metal binding sites on BQ123 and JKC301 by subjecting the metal-containing peptides to multiple stages of collisionally activated decomposition (CAD) in an ion trap mass spectrometer. When submitted to low-energy CAD, the ring opens at the Asp-Pro amide bond. The metal ion, which bonds, inter alia, to the carbonyl oxygen of the proline residue, acts as a fixed charge site, and directs a charge-remote, sequence-specific fragmentation of the ring-opened peptide. Amino acid residues are sequentially cleaved from the C-terminal end, and the terminal aziridinone structure moves one step toward the N-terminus with each C-terminal amino acid residue removed. These observations are the basis of a new method to sequence cyclic peptides. Amino acid residues are observed as sets of three ions, a*(n)PD, b*(n)PD and c*(n)PD where n is the number of amino acid residues in the peptide. Copyright 2000 John Wiley & Sons, Ltd.

Identification of amino acids in the N-terminal SH2 domain of phospholipase C gamma 1 important in the interaction with epidermal growth factor receptor.

PubMed

Gergel, J R; McNamara, D J; Dobrusin, E M; Zhu, G; Saltiel, A R; Miller, W T

1994-12-13

Photoaffinity labeling and site-directed mutagenesis have been used to identify amino acid residues of the phospholipase C gamma 1 (PLC gamma 1) N-terminal SH2 domain involved in recognition of the activated epidermal growth factor receptor (EGFR). The photoactive amino acid p-benzoylphenylalanine (Bpa) was incorporated into phosphotyrosine-containing peptides derived from EGFR autophosphorylation sites Tyr992 and Tyr1068. Irradiation of these labels in the presence of SH2 domains showed cross-linking which was time-dependent and specific; labeling was inhibited with non-Bpa-containing peptides from EGFR in molar excess. The phosphotyrosine residue on the peptides was important for SH2 recognition, as dephosphorylated peptides did not cross-link. Radiolabeled peptides were used to identify sites of cross-linking to the N-terminal SH2 of PLC gamma 1. Bpa peptide-SH2 complexes were digested with trypsin, and radioactive fragments were purified by HPLC and analyzed by Edman sequencing. These experiments showed Arg562 and an additional site in the alpha A-beta B region of the SH2 domain, most likely Glu587, to be labeled by the Tyr992-derived peptide. Similar analysis of the reaction with the Tyr1068-derived photoaffinity label identified Leu653 as the cross-linked site. Mutation of the neighboring residues of Glu587 decreased photo-cross-linking, emphasizing the importance of this region of the molecule for recognition. These results are consistent with evidence from the v-Src crystal structure and implicate the loop spanning residues Gln640-Ser654 of PLC gamma 1 in specific recognition of phosphopeptides.

Convergent synthesis of proteins by kinetically controlled ligation

DOEpatents

Kent, Stephen; Pentelute, Brad; Bang, Duhee; Johnson, Erik; Durek, Thomas

2010-03-09

The present invention concerns methods and compositions for synthesizing a polypeptide using kinetically controlled reactions involving fragments of the polypeptide for a fully convergent process. In more specific embodiments, a ligation involves reacting a first peptide having a protected cysteyl group at its N-terminal and a phenylthioester at its C-terminal with a second peptide having a cysteine residue at its N-termini and a thioester at its C-termini to form a ligation product. Subsequent reactions may involve deprotecting the cysteyl group of the resulting ligation product and/or converting the thioester into a thiophenylester.

Analysis of the Intrinsically Disordered N-Terminus of the DNA Junction-Resolving Enzyme T7 Endonuclease I: Identification of Structure Formed upon DNA Binding

PubMed Central

2016-01-01

The four-way (Holliday) DNA junction of homologous recombination is processed by the symmetrical cleavage of two strands by a nuclease. These junction-resolving enzymes bind to four-way junctions in dimeric form, distorting the structure of the junction in the process. Crystal structures of T7 endonuclease I have been determined as free protein, and the complex with a DNA junction. In neither crystal structure was the N-terminal 16-amino acid peptide visible, yet deletion of this peptide has a marked effect on the resolution process. Here we have investigated the N-terminal peptide by inclusion of spin-label probes at unique sites within this region, studied by electron paramagnetic resonance. Continuous wave experiments show that these labels are mobile in the free protein but become constrained on binding a DNA junction, with the main interaction occurring for residues 7–10 and 12. Distance measurements between equivalent positions within the two peptides of a dimer using PELDOR showed that the intermonomeric distances for residues 2–12 are long and broadly distributed in the free protein but are significantly shortened and become more defined on binding to DNA. These results suggest that the N-terminal peptides become more organized on binding to the DNA junction and nestle into the minor grooves at the branchpoint, consistent with the biochemical data indicating an important role in the resolution process. This study demonstrates the presence of structure within a protein region that cannot be viewed by crystallography. PMID:27387136

Endogenous peptide profile for elucidating biosynthetic processing of the ghrelin precursor.

PubMed

Tsuchiya, Takashi; Iwakura, Hiroshi; Minamino, Naoto; Kangawa, Kenji; Sasaki, Kazuki

2017-09-02

Ghrelin is an orexigenic peptide primarily produced by gastric endocrine cells. The biosynthetic cleavage site of ghrelin has been well documented, but how its downstream region undergoes proteolytic processing remains poorly explored. Here, we provide the first snapshot of endogenous peptides from the ghrelin precursor by profiling the secretopeptidome of cultured mouse ghrelin-producing cells during exocytosis. Mapping of MS/MS sequenced peptides to the precursor highlighted three atypical monobasic processing sites, including the established C-terminus of ghrelin and the N-terminal cleavage site for obestatin, a putative 23-amino-acid C-terminally amidated peptide. However, we found that mouse obestatin does not occur in the form originally reported, but that a different amidation site is used to generate a shorter peptide. These data can be extended to study and characterize the precursor-derived peptides located downstream of ghrelin in different biological contexts. Copyright © 2017 Elsevier Inc. All rights reserved.

The fate of b-ions in the two worlds of collision-induced dissociation.

PubMed

Waldera-Lupa, Daniel M; Stefanski, Anja; Meyer, Helmut E; Stühler, Kai

2013-12-01

Fragment analysis of proteins and peptides by mass spectrometry using collision-induced dissociation (CID) revealed that the pairwise generated N-terminal b- and C-terminal y-ions have different stabilities resulting in underrepresentation of b-ions. Detailed analyses of large-scale spectra databases and synthetic peptides underlined these observations and additionally showed that the fragmentation pattern depends on utilized CID regime. To investigate this underrepresentation further we systematically compared resonant excitation energy and beam-type CID facilitated on different mass spectrometer platforms: (i) quadrupole time-of-flight, (ii) linear ion trap and (iii) three-dimensional ion trap. Detailed analysis of MS/MS data from a standard tryptic protein digest revealed that b-ions are significantly underrepresented on all investigated mass spectrometers. By N-terminal acetylation of tryptic peptides we show for the first time that b-ion cyclization reaction significantly contributes to b-ion underrepresentation even on ion trap instruments and accounts for at most 16% of b-ion loss. © 2013.

Relationship between helix stability and binding affinities: molecular dynamics simulations of Bfl-1/A1-binding pro-apoptotic BH3 peptide helices in explicit solvent.

PubMed

Modi, Vivek; Lama, Dilraj; Sankararamakrishnan, Ramasubbu

2013-01-01

The anti-apoptotic protein Bfl-1, also known as A1, belongs to the Bcl-2 family of proteins and interacts with pro-apoptotic Bcl-2 counterparts to regulate programmed cell death. As demonstrated for other anti-apoptotic Bcl-2 proteins, Bfl-1/A1 has also been shown to be overexpressed in various human cancers and hence they are attractive targets for anticancer drugs. Peptides derived from the BH3 region of pro-apoptotic Bcl-2 proteins have been shown to elicit similar biological response as that of parent proteins. BH3 peptides from different pro-apoptotic proteins have wide range of affinities for Bfl-1/A1. Experimentally determined complex structures show that the hydrophobic side of amphipathic BH3 peptides binds to the hydrophobic groove formed by the α-helical bundle of Bfl-1/A1 protein. Apart from the length and amino acid composition, a BH3 peptide's ability to form a stable helical structure has been suggested to be important for its high binding affinity. Molecular dynamics simulations of three BH3 peptides derived from the pro-apoptotic proteins Bak, Bid, and Bmf were carried out each for a period of at least 100 ns after 2 ns equilibration run. The length of simulated BH3 peptides varied from 22 to 24 residues and their binding affinities for Bfl-1/A1 varied from 1 to 180 nM. Our results show that the hydrophobic residues from the hydrophobic face of BH3 peptides tend to cluster together quickly to avoid being exposed to the solvent. This resulted in either reduction of helix length or complete loss of helical character. Bak and Bid BH3 peptides with high affinities for Bf1-1/A1 have stable helical segments in the N-terminal region. The highly conserved Leu residue lies just outside the helical region at the C-terminal end. Capping interactions arising out of N-cap residues seem to be extremely important to maintain the helical stability. Favorable hydrophilic interactions between residues also give further stability to the helix fragment and at least one of the interacting residues resides within the helical region. Bmf BH3 peptide with a weaker binding affinity for Bmf-1/A1 completely lost its helical character at the end of 100 ns production run and a further 50 ns simulation showed that the Bmf peptide continues to remain in random conformation. The present study clearly establishes a link between a BH3 peptide's ability to form a stable helical segment and its high binding affinity for an anti-apoptotic protein. To further test this hypothesis, we simulated a mutant Bmf peptide for 100 ns in which two residues R129 and H146 were substituted by Asn in silico in the wild-type peptide. Introduction of N-terminal Asn clearly enabled the formation of capping interactions at the N-terminus and resulted in a stable N-terminal helical segment. This demonstrates that the knowledge of interactions that help to maintain stable helical segments in a high-affinity BH3 peptide will help in designing highly specific peptide-based drugs/inhibitors. Such molecules will have the ability to bind a particular anti-apoptotic protein with high affinity.

Protease-catalyzed peptide bond formation: application to synthesis of the COOH-terminal octapeptide of cholecystokinin.

PubMed

Kullmann, W

1982-05-01

This study of protease-catalyzed peptide synthesis reports the preparation of the COOH-terminal octapeptide amide of cholecystokinin. The octapeptide was assembled by chemical condensation of two tetrapeptide segments that had been synthesized through the concerted catalytic reactions of several proteases of different specificities. The resulting octapeptide derivative was subjected to catalytic transfer hydrogenation, followed by sulfation of its tyrosine residue and removal of the N alpha-protecting group. The homogeneous target peptide was obtained after purification via partition chromatography, gel filtration, and ion-exchange chromatography. The synthetic octapeptide stimulated amylase release from pancreatic acinar cells.

Processing of the precursor of protamine P2 in mouse. Peptide mapping and N-terminal sequence analysis of intermediates.

PubMed Central

Carré-Eusèbe, D; Lederer, F; Lê, K H; Elsevier, S M

1991-01-01

Protamine P2, the major basic chromosomal protein of mouse spermatozoa, is synthesized as a precursor almost twice as long as the mature protein, its extra length arising from an N-terminal extension of 44 amino acid residues. This precursor is integrated into chromatin of spermatids, and the extension is processed during chromatin condensation in the haploid cells. We have studied processing in the mouse and have identified two intermediates generated by proteolytic cleavage of the precursor. H.p.l.c. separated protamine P2 from four other spermatid proteins, including the precursor and three proteins known to possess physiological characteristics expected of processing intermediates. Peptide mapping indicated that all of these proteins were structurally similar. Two major proteins were further purified by PAGE, transferred to poly(vinylidene difluoride) membranes and submitted to automated N-terminal sequence analysis. Both sequences were found within the deduced sequence of the precursor extension. The N-terminus of the larger intermediate, PP2C, was Gly-12, whereas the N-terminus of the smaller, PP2D, was His-21. Both processing sites involved a peptide bond in which the carbonyl function was contributed by an acidic amino acid. Images Fig. 1. Fig. 3. Fig. 4. PMID:1854346

The pH-dependent assembly of Chaplin E from Streptomyces coelicolor.

PubMed

Dokouhaki, Mina; Hung, Andrew; Day, Li; Gras, Sally L

2017-05-01

Chaplin E, is one of five self-assembling peptides secreted by Streptomyces coelicolor that assist aerial growth by lowering the surface tension of water. Although the surface activity of a mixture of chaplin peptides has observed to depend on pH, it is unclear how the solvent environment (i.e. pH) influences the structure, assembly and subsequent functionality of these individual peptides. In this study, the conformation and fibril forming propensity of the Chaplin E peptide was assessed as a function of pH using a combination of experimental measurements and molecular dynamics simulations. At an acidic pH of 3.0, Chaplin E retained a random coil structure, whereas at the isoelectric point of 6.7 or a basic pH of 10.0, Chaplin E rapidly formed amyloid fibrils rich in β-sheet structure with high efficiency (>93%). Molecular dynamics simulations indicate the persistence of greater α-helical content at the N-terminus at high pH; this is likely partly due to the lack of electrostatic repulsion between residues His6 and Lys10. Since fibril formation was observed at high but not at low pH, we propose that the presence of an N-terminal α-helix in the monomeric form of Chaplin E is required for aggregation and conversion to β-amyloid fibrils. The pH sensitivity of Chaplin E peptide structure provides a route to control peptide assembly and may be important for the physiological function of this peptide, as a surface active agent in the transition from vegetative to aerial growth and could assist Streptomyces coelicolor in response to environmental fluctuations in pH. Copyright © 2017 Elsevier Inc. All rights reserved.

New insights into the molecular interaction of the C-terminal sequence of CXCL4 with fibroblast growth factor-2.

PubMed

Ragona, Laura; Tomaselli, Simona; Quemener, Cathy; Zetta, Lucia; Bikfalvi, Andreas

2009-04-24

Full-length CXCL4 chemokine and a peptide derived from its carboxyl-terminal domain exhibits significant antiangiogenic and anti-tumor activity in vivo and in vitro by interacting with fibroblast growth factor (FGF). In this study we used NMR spectroscopy to characterize at a molecular level the interactions between CXCL4 (47-70) and FGF-2 identifying the peptide residues mainly involved in the contact area with the growth factor. Altogether NMR data point to a major role of the hydrophobic contributions of the C-terminal region of CXCL4 (47-70) peptide in addition to specific contacts established by the N-terminal region through cysteine side chain. The proposed recognition mode constitutes a rationale for the observed effects of CXCL4 (47-70) on FGF-2 biological activity and lays the basis for developing novel inhibitors of angiogenesis.

Isolation and characterization of the CNBr peptides from the proteolytically derived N-terminal fragment of ovine opsin.

PubMed Central

Brett, M; Findlay, J B

1983-01-01

Ovine rhodopsin may be cleaved in situ by Staphylococcus aureus V8 proteinase into two membrane-bound fragments designated V8-L (27 000 mol.wt.) and V8-S (12 000 mol.wt.). After purification of the proteolysed complex by affinity chromatography in detergent using concanavalin A immobilized on Sepharose 4B, the two polypeptide fragments may be separated by gel-permeation chromatography on Sephadex LH-60. Digestion of the N-terminal-derived V8-L fragment with CNBr in 70% (v/v) trifluoroacetic acid resulted in a peptide mixture that could be fractionated by procedures involving gel-permeation chromatography in organic and aqueous solvents and the use of differential solubility. The complete or partial sequences of all ten peptides are reported. PMID:6224479

Gas-phase conformation-specific photofragmentation of proline-containing peptide ions.

PubMed

Kim, Tae-Young; Valentine, Stephen J; Clemmer, David E; Reilly, James P

2010-08-01

Singly-protonated proline-containing peptides with N-terminal arginine are photodissociated with vacuum ultraviolet (VUV) light in an ESI linear ion trap/orthogonal-TOF (LIT/o-TOF). When proline is the nth residue from the N-terminus, unusual b(n) + 2 and a(n) + 2 ions are observed. Their formation is explained by homolytic cleavage of the C(alpha)-C bond in conjunction with a rearrangement of electrons and an amide hydrogen. The latter is facilitated by a proline-stabilized gas-phase peptide conformation. Copyright 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

C-terminal Lysine-Linked Magainin 2 with Increased Activity Against Multidrug-Resistant Bacteria.

PubMed

Lorenzón, Esteban N; Santos-Filho, Norival A; Ramos, Matheus A S; Bauab, Tais M; Camargo, Ilana L B C; Cilli, Eduardo M

2016-01-01

Due to the growing problem of antibiotic-resistant microorganisms, the development of novel antimicrobial agents is a very important challenge. Dimerization of cationic antimicrobial peptides (cAMPs) is a potential strategy for enhancing antimicrobial activity. Here, we studied the effects of magainin 2 (MG2) dimerization on its structure and biological activity. Lysine and glutamic acid were used to synthesize the C- and N-terminal dimers of MG2, respectively, in order to evaluate the impact of linker position used to obtain the dimers. Both MG2 and its dimeric versions showed a random coil structure in aqueous solution. However, in the presence of a structure-inducing solvent or a membrane mimetic, all peptides acquired helical structure. N-terminal dimerization did not affect the biological activity of the peptide. On the other hand, the C-terminal dimer, (MG2)2K, showed antimicrobial activity 8-16 times higher than that of MG2, and the time required to kill Escherichia coli was lower. The enhanced antimicrobial activity was related to membrane permeabilization. (MG2)2K was also more active against multidrug-resistant bacteria of clinical origin. Overall, the results presented here demonstrate that C-terminal lysine-linked dimerization improve the activity of MG2, and (MG2)2K can be considered as a potential antimicrobial agent.

The N-degradome of Escherichia coli

PubMed Central

Humbard, Matthew A.; Surkov, Serhiy; De Donatis, Gian Marco; Jenkins, Lisa M.; Maurizi, Michael R.

2013-01-01

The N-end rule is a conserved mechanism found in Gram-negative bacteria and eukaryotes for marking proteins to be degraded by ATP-dependent proteases. Specific N-terminal amino acids (N-degrons) are sufficient to target a protein to the degradation machinery. In Escherichia coli, the adaptor ClpS binds an N-degron and delivers the protein to ClpAP for degradation. As ClpS recognizes N-terminal Phe, Trp, Tyr, and Leu, which are not found at the N terminus of proteins translated and processed by the canonical pathway, proteins must be post-translationally modified to expose an N-degron. One modification is catalyzed by Aat, an enzyme that adds leucine or phenylalanine to proteins with N-terminal lysine or arginine; however, such proteins are also not generated by the canonical protein synthesis pathway. Thus, the mechanisms producing N-degrons in proteins and the frequency of their occurrence largely remain a mystery. To address these issues, we used a ClpS affinity column to isolate interacting proteins from E. coli cell lysates under non-denaturing conditions. We identified more than 100 proteins that differentially bound to a column charged with wild-type ClpS and eluted with a peptide bearing an N-degron. Thirty-two of 37 determined N-terminal peptides had N-degrons. Most of the proteins were N-terminally truncated by endoproteases or exopeptidases, and many were further modified by Aat. The identities of the proteins point to possible physiological roles for the N-end rule in cell division, translation, transcription, and DNA replication and reveal widespread proteolytic processing of cellular proteins to generate N-end rule substrates. PMID:23960079

Electron transfer dissociation of synthetic and natural peptides containing lanthionine/methyllanthionine bridges.

PubMed

Dolle, Ashwini B; Jagadeesh, Narasimhappagari; Bhaumik, Suman; Prakash, Sunita; Biswal, Himansu S; Gowd, Konkallu Hanumae

2018-06-15

The modes of cleavage of lanthionine/methyllanthionine bridges under electron transfer dissociation (ETD) were investigated using synthetic and natural lantipeptides. Knowledge of the mass spectrometric fragmentation of lanthionine/methyllanthionine bridges may assist in the development of analytical methods for the rapid discovery of new lantibiotics. The present study strengthens the advantage of ETD in the characterization of posttranslational modifications of peptides and proteins. Synthetic and natural lantipeptides were obtained by desulfurization of peptide disulfides and cyanogen bromide digestion of the lantibiotic nisin, respectively. These peptides were subjected to electrospray ionization collision-induced dissociation tandem mass spectrometry (CID-MS/MS) and ETD-MS/MS using an HCT ultra ETDII ion trap mass spectrometer. MS 3 CID was performed on the desired product ions to prove cleavage of the lanthionine/methyllanthionine bridge during ETD-MS/MS. ETD has advantages over CID in the cleavage of the side chain of lanthionine/methyllanthionine bridges. The cleavage of the N-Cα backbone peptide bond followed by C-terminal side chain of the lanthionine bridge results in formation of c •+ and z + ions. Cleavage at the preceding peptide bond to the C-terminal side chain of lanthionine/methyllanthionine bridges yields specific fragments with the cysteine/methylcysteine thiyl radical and dehydroalanine. ETD successfully cleaves the lanthionine/methyllanthionine bridges of synthetic and natural lantipeptides. Diagnostic fragment ions of ETD cleavage of lanthionine/methyllanthionine bridges are the N-terminal cysteine/methylcysteine thiyl radical and C-terminal dehydroalanine. Detection of the cysteine/methylcysteine thiyl radical and dehydroalanine in combined ETD-CID-MS may be used for the rapid identification of lantipeptide natural products. Copyright © 2018 John Wiley & Sons, Ltd.

Levels of the Novel Glycoprotein Lacritin in Human Tears After Laser Refractive Surgery

DTIC Science & Technology

2013-10-01

13. SUPPLEMENTARY NOTES 14. ABSTRACT Lacritin is a naturally occurring tear protein with antimicrobial activity that is capable of stimulating...Development and validation testing of lacritin assay. Lacritin Peptide , Anti-N-Terminal Anti-lacritin (Pep Lac N-Term) polyclonal antibodies were...generated in rabbits against a synthetic peptide corresponding to the first 19 N-terminus amino acids of mature human lacritin as previously described

Mapping the protein domain structures of the respiratory mucins: a mucin proteome coverage study.

PubMed

Cao, Rui; Wang, T Tiffany; DeMaria, Genevieve; Sheehan, John K; Kesimer, Mehmet

2012-08-03

Mucin genes encode a family of the largest expressed proteins in the human genome. The proteins are highly substituted with O-linked oligosaccharides that greatly restrict access to the peptide backbones. The genomic organization of the N-terminal, O-glycosylated, and C-terminal regions of most of the mucins has been established and is available in the sequence databases. However, much less is known about the fate of their exposed protein regions after translation and secretion, and to date, detailed proteomic studies complementary to the genomic studies are rather limited. Using mucins isolated from cultured human airway epithelial cell secretions, trypsin digestion, and mass spectrometry, we investigated the proteome coverage of the mucins responsible for the maintenance and protection of the airway epithelia. Excluding the heavily glycosylated mucin domains, up to 85% coverage of the N-terminal region of the gel-forming mucins MUC5B and MUC5AC was achieved, and up to 60% of the C-terminal regions were covered, suggesting that more N- and sparsely O-glycosylated regions as well as possible other modifications are available at the C-terminus. All possible peptides from the cysteine-rich regions that interrupt the heavily glycosylated mucin domains were identified. Interestingly, 43 cleavage sites from 10 different domains of MUC5B and MUC5AC were identified, which possessed a non-tryptic cleavage site on the N-terminal end of the peptide, indicating potential exposure to proteolytic and/or "spontaneous cleavages". Some of these non-tryptic cleavages may be important for proper maturation of the molecule, before and/or after secretion. Most of the peptides identified from MUC16 were from the SEA region. Surprisingly, three peptides were clearly identified from its heavily glycosylated regions. Up to 25% coverage of MUC4 was achieved covering seven different domains of the molecule. All peptides from the MUC1 cytoplasmic domain were detected along with the three non-tryptic cleavages in the region. Only one peptide was identified from MUC20, which led us to successful antisera raised against the molecule. Taken together, this report represents our current efforts to dissect the complexities of mucin macromolecules. Identification of regions accessible to proteolysis can help in the design of effective antibodies and points to regions that might be available for mucin-protein interactions and identification of cleavage sites will enable understanding of their pre- and post-secretory processing in normal and disease environments.

The delta-selective opioid peptide dermenkephalin and the mu-selective hybrid peptide dermenkephalin-[1-4]-dermophin-[5-7] display strikingly different conformations despite identical tetrapeptide N-termini. A quantitative 2-D NMR and molecular modeling analysis.

PubMed

Riand, J; Baron, D; Nicolas, P; Benajiba, A; Teng, Y; Naim, M

1999-12-01

The selective recognition of the aminoterminal binding pharmacophore Tyr-D-Xaa-Phe of the opioid heptapeptide dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (DRM)1, and of dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (DREK), by the mu-opioid receptor and delta-opioid receptor, respectively, depends upon the constitution / conformation of the C-terminal tripeptide. The hybrid peptide DREK-[1-4]-DRM-[5-7] is very potent at, and exquisitely selective for the mu-opioid receptor, and differs only from dermenkephalin by its C-terminal tripeptide. Comparison of the structural features of DREK-[1-4]-DRM-[5-7] and dermenkephalin by nmr analysis and molecular modeling revealed striking differences, as well in the trans (Tyr5 - Pro6) isomer (population 75%) than in the cis isomer.. Whereas the folded C-terminal tail of dermenkephalin influenced the tertiary structure of the N-terminal tetrapeptide and placed the Tyr1 and Phe3 aromatic rings in definite orientations that are best suited for the delta-receptor, there were only weak contacts, as shown by NOE data, between the aminoterminal and carboxyterminal parts of the hybrid peptide. This promoted increased flexibility of the whole backbone and relaxed orientations for the side-chains of Tyr1 and Phe3 that are compatible with the mu-receptor but unsuitable for the delta-receptor. The steric hindrance introduced by Pro6 in DREK-[1-4]-DRM-[5-7], plus the absence of large hydrophobic side-chains in positions 5 and 6 may prevent close contacts between the N-terminal and C-terminal domains and reorientation of the main pharmacophoric elements Tyr1 and Phe3.

Spontaneous Isomerization of Peptide Cation Radicals Following Electron Transfer Dissociation Revealed by UV-Vis Photodissociation Action Spectroscopy.

PubMed

Imaoka, Naruaki; Houferak, Camille; Murphy, Megan P; Nguyen, Huong T H; Dang, Andy; Tureček, František

2018-01-16

Peptide cation radicals of the z-type were produced by electron transfer dissociation (ETD) of peptide dications and studied by UV-Vis photodissociation (UVPD) action spectroscopy. Cation radicals containing the Asp (D), Asn (N), Glu (E), and Gln (Q) residues were found to spontaneously isomerize by hydrogen atom migrations upon ETD. Canonical N-terminal [z 4 + H] +● fragment ion-radicals of the R-C ● H-CONH- type, initially formed by N-C α bond cleavage, were found to be minor components of the stable ion fraction. Vibronically broadened UV-Vis absorption spectra were calculated by time-dependent density functional theory for several [ ● DAAR + H] + isomers and used to assign structures to the action spectra. The potential energy surface of [ ● DAAR + H] + isomers was mapped by ab initio and density functional theory calculations that revealed multiple isomerization pathways by hydrogen atom migrations. The transition-state energies for the isomerizations were found to be lower than the dissociation thresholds, accounting for the isomerization in non-dissociating ions. The facile isomerization in [ ● XAAR + H] + ions (X = D, N, E, and Q) was attributed to low-energy intermediates having the radical defect in the side chain that can promote hydrogen migration along backbone C α positions. A similar side-chain mediated mechanism is suggested for the facile intermolecular hydrogen migration between the c- and [z + H] ● -ETD fragments containing Asp, Asn, Glu, and Gln residues. Graphical Abstract ᅟ.

Spontaneous Isomerization of Peptide Cation Radicals Following Electron Transfer Dissociation Revealed by UV-Vis Photodissociation Action Spectroscopy

NASA Astrophysics Data System (ADS)

Imaoka, Naruaki; Houferak, Camille; Murphy, Megan P.; Nguyen, Huong T. H.; Dang, Andy; Tureček, František

2018-01-01

Peptide cation radicals of the z-type were produced by electron transfer dissociation (ETD) of peptide dications and studied by UV-Vis photodissociation (UVPD) action spectroscopy. Cation radicals containing the Asp (D), Asn (N), Glu (E), and Gln (Q) residues were found to spontaneously isomerize by hydrogen atom migrations upon ETD. Canonical N-terminal [z4 + H]+● fragment ion-radicals of the R-C●H-CONH- type, initially formed by N-Cα bond cleavage, were found to be minor components of the stable ion fraction. Vibronically broadened UV-Vis absorption spectra were calculated by time-dependent density functional theory for several [●DAAR + H]+ isomers and used to assign structures to the action spectra. The potential energy surface of [●DAAR + H]+ isomers was mapped by ab initio and density functional theory calculations that revealed multiple isomerization pathways by hydrogen atom migrations. The transition-state energies for the isomerizations were found to be lower than the dissociation thresholds, accounting for the isomerization in non-dissociating ions. The facile isomerization in [●XAAR + H]+ ions (X = D, N, E, and Q) was attributed to low-energy intermediates having the radical defect in the side chain that can promote hydrogen migration along backbone Cα positions. A similar side-chain mediated mechanism is suggested for the facile intermolecular hydrogen migration between the c- and [z + H]●-ETD fragments containing Asp, Asn, Glu, and Gln residues. [Figure not available: see fulltext.

Mechanism of Dual Targeting of the Phytochrome Signaling Component HEMERA/pTAC12 to Plastids and the Nucleus.

PubMed

Nevarez, P Andrew; Qiu, Yongjian; Inoue, Hitoshi; Yoo, Chan Yul; Benfey, Philip N; Schnell, Danny J; Chen, Meng

2017-04-01

HEMERA (HMR) is a nuclear and plastidial dual-targeted protein. While it functions in the nucleus as a transcriptional coactivator in phytochrome signaling to regulate a distinct set of light-responsive, growth-relevant genes, in plastids it is known as pTAC12, which associates with the plastid-encoded RNA polymerase, and is essential for inducing the plastomic photosynthetic genes and initiating chloroplast biogenesis. However, the mechanism of targeting HMR to the nucleus and plastids is still poorly understood. Here, we show that HMR can be directly imported into chloroplasts through a transit peptide residing in the N-terminal 50 amino acids. Upon cleavage of the transit peptide and additional proteolytic processing, mature HMR, which begins from Lys-58, retains its biochemical properties in phytochrome signaling. Unexpectedly, expression of mature HMR failed to rescue not only the plastidial but also the nuclear defects of the hmr mutant. This is because the predicted nuclear localization signals of HMR are nonfunctional, and therefore mature HMR is unable to accumulate in either plastids or the nucleus. Surprisingly, fusing the transit peptide of the small subunit of Rubisco with mature HMR rescues both its plastidial and nuclear localization and functions. These results, combined with the observation that the nuclear form of HMR has the same reduced molecular mass as plastidial HMR, support a retrograde protein translocation mechanism in which HMR is targeted first to plastids, processed to the mature form, and then relocated to the nucleus. © 2017 American Society of Plant Biologists. All Rights Reserved.

Triaspartate: a model system for conformationally flexible DDD motifs in proteins.

PubMed

Duitch, Laura; Toal, Siobhan; Measey, Thomas J; Schweitzer-Stenner, Reinhard

2012-05-03

Understanding the interactions that govern turn formation in the unfolded state of proteins is necessary for a complete picture of the role that these turns play in both normal protein folding and functionally relevant yet disordered linear motifs. It is still unclear, however, whether short peptides can adopt stable turn structures in aqueous environments in the absence of any nonlocal interactions. To explore the effect that nearest-neighbor interactions and the local peptide environment have on the turn-forming capability of individual amino acid residues in short peptides, we combined vibrational (IR, Raman, and VCD), UV-CD, and (1)H NMR spectroscopies in order to probe the conformational ensemble of the central aspartic acid residue of the triaspartate peptide (DDD). The study was motivated by the recently discovered turn propensities of aspartic acid in GDG (Hagarman; et al. Chem.-Eur. J. 2011, 17, 6789). We investigated the DDD peptide under both acidic and neutral conditions in order to elucidate the effect that side-chain protonation has on the conformational propensity of the central aspartic acid residue. Amide I' profiles were analyzed in terms of two-dimensional Gaussian distributions representing conformational subdistributions in Ramachandran space. Interestingly, our results show that while the protonated form of the DDD peptide samples various turn-like conformations similar to GDG, deprotonation of the peptide eliminates this propensity for turns, causing the fully ionized peptide to exclusively sample pPII and β-strand-like structures. To further explore the factors stabilizing these more extended conformations in fully ionized DDD, we analyzed the temperature dependence of both the UV-CD spectrum and the (3)J(H(N),H(α)) coupling constants of the two amide protons (N- and C-terminal) in terms of a simple two-state (pPII-β) thermodynamic model. Thus, we were able to obtain the enthalpic and entropic differences between the pPII and β-strand conformations of the central and C-terminal residue. For the central residue, we obtained ΔH(3) = -12.0 kJ/mol and ΔS(3) = -73.8 J/mol·K, resulting in a much larger room-temperature Gibbs free energy of 10.0 kJ/mol, which effectively locks the C-terminal in a β-like conformation. A comparison of the temperature dependence of the chemical shifts reveals that there is indeed some type of protection of the amide protons from solvent in ionized DDD. This finding and several other lines of evidence suggest that both conformations of ionized DDD are stabilized by hydrogen bonding between the carboxylate groups of the central and C-terminal residue and the respective amide protons. These hydrogen bonds can be expected to be eliminated by side-chain protonation and substituted by hydrogen bonds between the N-terminal amide proton and the C-terminal carbonyl group as well as between the central aspartate side chain and the N-terminal amide proton. Hence, our results are indicative of a pH-induced switch in hydrogen-bonding patterns of aspartic acid motifs.

Plasma Biomarkers Reflecting Profibrotic Processes in Heart Failure With a Preserved Ejection Fraction

PubMed Central

Zile, Michael R.; Jhund, Pardeep S.; Baicu, Catalin F.; Claggett, Brian L.; Pieske, Burkert; Voors, Adriaan A.; Prescott, Margaret F.; Shi, Victor; Lefkowitz, Martin; McMurray, John J.V.; Solomon, Scott D.

2017-01-01

Background Heart failure with preserved ejection fraction is a clinical syndrome that has been associated with changes in the extracellular matrix. The purpose of this study was to determine whether profibrotic biomarkers accurately reflect the presence and severity of disease and underlying pathophysiology and modify response to therapy in patients with heart failure with preserved ejection fraction. Methods and Results Four biomarkers, soluble form of ST2 (an interleukin-1 receptor family member), galectin-3, matrix metalloproteinase-2, and collagen III N-terminal propeptide were measured in the Prospective Comparison of ARNI With ARB on Management of Heart Failure With Preserved Ejection Fraction (PARAMOUNT) trial at baseline, 12 and 36 weeks after randomization to valsartan or LCZ696. We examined the relationship between baseline biomarkers, demographic and echocardiographic characteristics, change in primary (change in N-terminal pro B-type natriuretic peptide) and secondary (change in left atrial volume) end points. The median (interquartile range) value for soluble form of ST2 (33 [24.6–48.1] ng/mL) and galectin 3 (17.8 [14.1–22.8] ng/mL) were higher, and for matrix metalloproteinase-2 (188 [155.5–230.6] ng/mL) lower, than in previously published referent controls; collagen III N-terminal propeptide (5.6 [4.3–6.9] ng/mL) was similar to referent control values. All 4 biomarkers correlated with severity of disease as indicated by N-terminal pro B-type natriuretic peptide, E/E′, and left atrial volume. Baseline biomarkers did not modify the response to LCZ696 for lowering N-terminal pro B-type natriuretic peptide; however, left atrial volume reduction varied by baseline level of soluble form of ST2 and galectin 3; patients with values less than the observed median (<33 ng/mL soluble form of ST2 and <17.8 ng/mL galectin 3) had reduction in left atrial volume, those above median did not. Although LCZ696 reduced N-terminal pro B-type natriuretic peptide, levels of the other 4 biomarkers were not affected over time. Conclusions In patients with heart failure with preserved ejection fraction, biomarkers that reflect collagen homeostasis correlated with the presence and severity of disease and underlying pathophysiology, and may modify the structural response to treatment. PMID:26754625

28-mer Fragment Derived from Enterocin CRL35 Displays an Unexpected Bactericidal Effect on Listeria Cells.

PubMed

Masias, Emilse; Sanches, Paulo R S; Dupuy, Fernando G; Acuna, Leonardo; Bellomio, Augusto; Cilli, Eduardo; Saavedra, Lucila; Minahk, Carlos

2015-01-01

Two shorter peptides derived from enterocin CRL35, a 43-mer bacteriocin, were synthesized i.e. the N-terminal fragment spanning from residues 1 to 15, and a 28-mer fragment that represents the C-terminal of enterocin CRL35, the residues 16 to 43. The separate peptides showed no activity when combined. On one hand, the 28-mer peptide displayed an unpredicted antimicrobial activity. On the other, 15- mer peptide had no consistent anti-Listeria effect. The dissociation constants calculated from experimental data indicated that all peptides could bind at similar extent to the sensitive cells. However, transmembrane electrical potential was not dissipated to the same level by the different peptides; whereas the full-length and the C-terminal 28-mer fragment induced almost full dissipation, 15-mer fragment produced only a slow and incomplete effect. Furthermore, a different interaction of each peptide with membranes was demonstrated based on studies carried out with liposomes, which led us to conclude that activity was related to structure rather than to net positive charges. These results open up the possibility of designing new peptides based on the 28-mer fragment with enhanced activity, which would represent a promising approach for combating Listeria and other pathogens.

Sequence-Independent Cloning and Post-Translational Modification of Repetitive Protein Polymers through Sortase and Sfp-Mediated Enzymatic Ligation.

PubMed

Ott, Wolfgang; Nicolaus, Thomas; Gaub, Hermann E; Nash, Michael A

2016-04-11

Repetitive protein-based polymers are important for many applications in biotechnology and biomaterials development. Here we describe the sequential additive ligation of highly repetitive DNA sequences, their assembly into genes encoding protein-polymers with precisely tunable lengths and compositions, and their end-specific post-translational modification with organic dyes and fluorescent protein domains. Our new Golden Gate-based cloning approach relies on incorporation of only type IIS BsaI restriction enzyme recognition sites using PCR, which allowed us to install ybbR-peptide tags, Sortase c-tags, and cysteine residues onto either end of the repetitive gene polymers without leaving residual cloning scars. The assembled genes were expressed in Escherichia coli and purified using inverse transition cycling (ITC). Characterization by cloud point spectrophotometry, and denaturing polyacrylamide gel electrophoresis with fluorescence detection confirmed successful phosphopantetheinyl transferase (Sfp)-mediated post-translational N-terminal labeling of the protein-polymers with a coenzyme A-647 dye (CoA-647) and simultaneous sortase-mediated C-terminal labeling with a GFP domain containing an N-terminal GG-motif in a one-pot reaction. In a further demonstration, we installed an N-terminal cysteine residue into an elastin-like polypeptide (ELP) that was subsequently conjugated to a single chain poly(ethylene glycol)-maleimide (PEG-maleimide) synthetic polymer, noticeably shifting the ELP cloud point. The ability to straightforwardly assemble repetitive DNA sequences encoding ELPs of precisely tunable length and to post-translationally modify them specifically at the N- and C- termini provides a versatile platform for the design and production of multifunctional smart protein-polymeric materials.

Identity of a peptide domain of human C9 that is bound by the cell-surface complement inhibitor, CD59.

PubMed

Chang, C P; Hüsler, T; Zhao, J; Wiedmer, T; Sims, P J

1994-10-21

The CD59 antigen is a plasma membrane glycoprotein that serves as an inhibitor of the C5b-9 complex of complement. This inhibitory activity appears related to the capacity of CD59 to bind with high affinity to sites that are nascently exposed in the alpha-chain subunit of human C8, as well as within the C9b domain (amino acid residues 245-538) of human C9, during assembly of the C5b-9 complex on the target membrane (Ninomiya, H., and Sims, P. J. (1992) J. Biol. Chem. 267, 13675-13680). The CD59 binding site in C9 was first investigated by N-terminal sequencing of CD59-binding peptides generated by limited digest of the isolated C9b domain. These experiments revealed a 17-kDa fragment (starting at C9 residue Thr-320) that retained affinity for CD59, suggesting the possibility for localizing the CD59 binding site by mapping with small C9-derived peptides. Peptides spanning the entire C9b sequence were expressed in Escherichia coli and then probed with CD59. CD59 bound specifically to all peptides starting N-terminal to C9 residue 359 with C termini extending beyond residue 411. Little to no CD59 binding was observed for various C9-derived peptides that started C-terminal to residue 359 or that were truncated N-terminal to residue 411. Affinity-purified antibody against C9 residues 320-411 inhibited CD59 binding to C9 by > 50% and completely inhibited its binding to the isolated C9b domain. Little to no specific binding of CD59 was detected for peptides restricted to the putative hinge domain within C9b (residues 245-271). These results indicate that a CD59 binding site is located between residues 320 and 411 of the C9 polypeptide and suggest that the affinity of this site is principally determined by residues 359-411.

Physical and mental recovery after conventional aortic valve surgery.

PubMed

Petersen, Johannes; Vettorazzi, Eik; Winter, Lena; Schmied, Wolfram; Kindermann, Ingrid; Schäfers, Hans-Joachim

2016-12-01

Physical and mental recovery are important factors to consider in the treatment of aortic valve disease, and the process of recovery is not well known. We investigated the course of physical and mental recovery directly after conventional aortic valve surgery. In a longitudinal study, 60 patients undergoing elective aortic valve surgery were studied preoperatively and at intervals of 4 weeks after aortic valve surgery. The last measurement was taken 6 months postoperatively. Measurements included the 6-minute walk test and N-terminal pro-B-type natriuretic peptide. Mental recovery was assessed by the Short Form Health Survey and the Hospital Anxiety and Depression Scale. All parameters were compared with published healthy norms. All parameters except for the anxiety score showed a significant decline after the first postoperative measurement at 1 week after aortic valve surgery. The baseline level was restored at 1 to 3 weeks (anxiety, depression, mental quality of life, Borg scale), 4 to 6 weeks (6-minute walk test, physical quality of life), and 9 weeks (N-terminal pro-B-type natriuretic peptide) after the first postoperative week. Significantly better values than preoperatively for the first time were reached at 2 to 3 weeks (anxiety, depression, mental quality of life), 5 weeks (6-minute walk test), 8 weeks (physical quality of life), and 12 weeks (N-terminal pro-B-type natriuretic peptide) after the first postoperative week. At 3 months postoperatively, significant improvements (P < .001) were seen in walk distance (+212 m), dyspnea (-1.11), physical (+12.38) and mental quality of life (+7.71), anxiety (-3.74), and depression (-3.62) compared with the first week postoperatively. At 6 months postoperatively, all parameters were significantly improved compared with preoperative data and, except for the N-terminal pro-B-type natriuretic peptide value, significantly better or equal compared with published healthy norms. After conventional aortic valve surgery, the most pronounced recovery was seen in the first 6 weeks postoperatively. Physical quality of life and N-terminal pro-B-type natriuretic peptide required a prolonged time for a complete recovery. Copyright © 2016 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

Expression, Refolding and Crystallizations of the Grb2-like (GADS) C-Terminal SH3 Domain Complexed with a SLP-76 Motif Peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faravelli,A.; Dimasi, N.

The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli, refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.

Towards Understanding the Tandem Mass Spectra of Protonated Oligopeptides. 2: The Proline Effect in Collision-Induced Dissociation of Protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp)

NASA Astrophysics Data System (ADS)

Bleiholder, Christian; Suhai, Sándor; Harrison, Alex G.; Paizs, Béla

2011-06-01

The product ion spectra of proline-containing peptides are commonly dominated by y n ions generated by cleavage at the N-terminal side of proline residues. This proline effect is investigated in the current work by collision-induced dissociation (CID) of protonated Ala-Ala-Xxx-Pro-Ala (Xxx includes Ala, Ser, Leu, Val, Phe, and Trp) in an electrospray/quadrupole/time-of-flight (QqTOF) mass spectrometer and by quantum chemical calculations on protonated Ala-Ala-Ala-Pro-Ala. The CID spectra of all investigated peptides show a dominant y 2 ion (Pro-Ala sequence). Our computational results show that the proline effect mainly arises from the particularly low threshold energy for the amide bond cleavage N-terminal to the proline residue, and from the high proton affinity of the proline-containing C-terminal fragment produced by this cleavage. These theoretical results are qualitatively supported by the experimentally observed y 2 / b 3 abundance ratios for protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp). In the post-cleavage phase of fragmentation the N-terminal oxazolone fragment with the Ala-Ala-Xxx sequence and Pro-Ala compete for the ionizing proton for these peptides. As the proton affinity of the oxazolone fragment increases, the y 2 / b 3 abundance ratio decreases.

Towards understanding the tandem mass spectra of protonated oligopeptides. 2: The proline effect in collision-induced dissociation of protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp).

PubMed

Bleiholder, Christian; Suhai, Sándor; Harrison, Alex G; Paizs, Béla

2011-06-01

The product ion spectra of proline-containing peptides are commonly dominated by y(n) ions generated by cleavage at the N-terminal side of proline residues. This proline effect is investigated in the current work by collision-induced dissociation (CID) of protonated Ala-Ala-Xxx-Pro-Ala (Xxx includes Ala, Ser, Leu, Val, Phe, and Trp) in an electrospray/quadrupole/time-of-flight (QqTOF) mass spectrometer and by quantum chemical calculations on protonated Ala-Ala-Ala-Pro-Ala. The CID spectra of all investigated peptides show a dominant y(2) ion (Pro-Ala sequence). Our computational results show that the proline effect mainly arises from the particularly low threshold energy for the amide bond cleavage N-terminal to the proline residue, and from the high proton affinity of the proline-containing C-terminal fragment produced by this cleavage. These theoretical results are qualitatively supported by the experimentally observed y(2)/b(3) abundance ratios for protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp). In the post-cleavage phase of fragmentation the N-terminal oxazolone fragment with the Ala-Ala-Xxx sequence and Pro-Ala compete for the ionizing proton for these peptides. As the proton affinity of the oxazolone fragment increases, the y(2)/b(3) abundance ratio decreases.

Mutagenesis of NosM Leader Peptide Reveals Important Elements in Nosiheptide Biosynthesis

PubMed Central

Jin, Liang; Wu, Xuri; Xue, Yanjiu; Jin, Yue; Wang, Shuzhen

2016-01-01

ABSTRACT Nosiheptide, a typical member of the ribosomally synthesized and posttranslationally modified peptides (RiPPs), exhibits potent activity against multidrug-resistant Gram-positive bacterial pathogens. The precursor peptide of nosiheptide (NosM) is comprised of a leader peptide with 37 amino acids and a core peptide containing 13 amino acids. To pinpoint elements in the leader peptide that are essential for nosiheptide biosynthesis, a collection of mutants with unique sequence features, including N- and C-terminal motifs, peptide length, and specific sites in the leader peptide, was generated by mutagenesis in vivo. The effects of various mutants on nosiheptide biosynthesis were evaluated. In addition to the necessity of a conserved motif LEIS box, native length and the N-terminal 12 amino acid residues were indispensable, and single-site substitutions of these 12 amino acid residues resulted in changes ranging from a greater-than-5-fold decrease to a 2-fold increase of nosiheptide production, depending on the sites and substituted residues. Moreover, although the C-terminal motif is not conservative, significant effects of this portion on nosiheptide production were also evident. Taken together, the present results further highlight the importance of the leader peptide in nosiheptide biosynthesis, and provide new insights into the diversity and specificity of leader peptides in the biosynthesis of various RiPPs. IMPORTANCE As a representative thiopeptide, nosiheptide exhibits excellent antibacterial activity. Although the biosynthetic gene cluster and several modification steps have been revealed, the presence and roles of the leader peptide within the precursor peptide of the nosiheptide gene cluster remain elusive. Thus, identification of specific elements in the leader peptide can significantly facilitate the genetic manipulation of the gene cluster for increasing nosiheptide production or generating diverse analogues. Given the complexity of the biosynthetic process, the instability of the leader peptide, and the unavailability of intermediates, cocrystallization of intermediates, leader peptide, and modification enzymes is currently not feasible. Therefore, a mutagenesis approach was used to construct a series of leader peptide mutants to uncover a number of crucial and characteristic elements affecting nosiheptide biosynthesis, which moves a considerable distance toward a thorough understanding of the biosynthetic machinery for thiopeptides. PMID:27913416

Structure of the Paramyxovirus Parainfluenza Virus 5 Nucleoprotein in Complex with an Amino-Terminal Peptide of the Phosphoprotein.

PubMed

Aggarwal, Megha; Leser, George P; Kors, Christopher A; Lamb, Robert A

2018-03-01

Parainfluenza virus 5 (PIV5) belongs to the family Paramyxoviridae , which consists of enveloped viruses with a nonsegmented negative-strand RNA genome encapsidated by the nucleoprotein (N). Paramyxovirus replication is regulated by the phosphoprotein (P) through protein-protein interactions with N and the RNA polymerase (L). The chaperone activity of P is essential to maintain the unassembled RNA-free form of N in order to prevent nonspecific RNA binding and premature N oligomerization. Here, we determined the crystal structure of unassembled PIV5 N in complex with a P peptide (N 0 P) derived from the N terminus of P (P50) at 2.65 Å. The PIV5 N 0 P consists of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a hinge region. The cleft at the hinge region of RNA-bound PIV5 N was previously shown to be an RNA binding site. The N 0 P structure shows that the P peptide binds to the CTD of N and extends toward the RNA binding site to inhibit N oligomerization and, hence, RNA binding. Binding of P peptide also keeps the PIV5 N in the open form. A molecular dynamics (MD) analysis of both the open and closed forms of N shows the flexibility of the CTD and the preference of the N protein to be in an open conformation. The gradual opening of the hinge region, to release the RNA, was also observed. Together, these results advance our knowledge of the conformational swapping of N required for the highly regulated paramyxovirus replication. IMPORTANCE Paramyxovirus replication is regulated by the interaction of P with N and L proteins. Here, we report the crystal structure of unassembled parainfluenza virus 5 (PIV5) N chaperoned with P peptide. Our results provide a detailed understanding of the binding of P to N. The conformational switching of N between closed and open forms during its initial interaction with P, as well as during RNA release, was analyzed. Our data also show the plasticity of the CTD and the importance of domain movement for conformational switching. The results improve our understanding of the mechanism of interchanging N conformations for RNA replication and release. Copyright © 2018 American Society for Microbiology.

Novel activity of angiotensin-converting enzyme. Hydrolysis of cholecystokinin and gastrin analogues with release of the amidated C-terminal dipeptide.

PubMed Central

Dubreuil, P; Fulcrand, P; Rodriguez, M; Fulcrand, H; Laur, J; Martinez, J

1989-01-01

ACE (angiotensin-converting enzyme; peptidyl dipeptidase A; EC 3.4.15.1), cleaves C-terminal dipeptides from active peptides containing a free C-terminus. We investigated the hydrolysis of cholecystokinin-8 [CCK-8; Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] and of various gastrin analogues by purified rabbit lung ACE. Although these peptides are amidated at their C-terminal end, they were metabolized by ACE to several peptide fragments. These fragments were analysed by h.p.l.c., isolated and identified by comparison with synthetic fragments, and by amino acid analysis. The initial and major site of hydrolysis was the penultimate peptide bond, which generated a major product, the C-terminal amidated dipeptide Asp-Phe-NH2. As a secondary cleavage, ACE subsequently released di- or tri-peptides from the C-terminal end of the remaining N-terminal fragments. The cleavage of CCK-8 and gastrin analogues was inhibited by ACE inhibitors (Captopril and EDTA), but not by other enzyme inhibitors (phosphoramidon, thiorphan, bestatin etc.). Hydrolysis of [Leu15]gastrin-(14-17)-peptide [Boc (t-butoxycarbonyl)-Trp-Leu-Asp-Phe-NH2] in the presence of ACE was found to be dependent on the chloride-ion concentration. Km values for the hydrolysis of CCK-8, [Leu15]gastrin-(11-17)-peptide and Boc-[Leu15]gastrin-(14-17)-peptide at an NaCl concentration of 300 mM were respectively 115, 420 and 3280 microM, and the catalytic constants were about 33, 115 and 885 min-1. The kcat/Km for the reactions at 37 degrees C was approx. 0.28 microM-1.min-1, which is approx. 35 times less than that reported for the cleavage of angiotensin I. These results suggest that ACE might be involved in the metabolism in vivo of CCK and gastrin short fragments. PMID:2554881

The Origins of Specificity in the Microcin-Processing Protease TldD/E.

PubMed

Ghilarov, Dmitry; Serebryakova, Marina; Stevenson, Clare E M; Hearnshaw, Stephen J; Volkov, Dmitry S; Maxwell, Anthony; Lawson, David M; Severinov, Konstantin

2017-10-03

TldD and TldE proteins are involved in the biosynthesis of microcin B17 (MccB17), an Escherichia coli thiazole/oxazole-modified peptide toxin targeting DNA gyrase. Using a combination of biochemical and crystallographic methods we show that E. coli TldD and TldE interact to form a heterodimeric metalloprotease. TldD/E cleaves the N-terminal leader sequence from the modified MccB17 precursor peptide, to yield mature antibiotic, while it has no effect on the unmodified peptide. Both proteins are essential for the activity; however, only the TldD subunit forms a novel metal-containing active site within the hollow core of the heterodimer. Peptide substrates are bound in a sequence-independent manner through β sheet interactions with TldD and are likely cleaved via a thermolysin-type mechanism. We suggest that TldD/E acts as a "molecular pencil sharpener": unfolded polypeptides are fed through a narrow channel into the active site and processively truncated through the cleavage of short peptides from the N-terminal end. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Serum levels of natriuretic peptides in children with various types of loading conditions.

PubMed

Eerola, Anneli; Jokinen, Eero; Pihkala, Jaana I

2009-06-01

To evaluate the influence of volume overload of the left (LV) and right ventricle (RV) and pressure overload of LV and restrictive physiology on levels of N-terminal proatriopeptide (ANPN) and N-terminal pro-brain natriuretic peptide (NT-proBNP). We studied 41 children with atrial septal defect (ASD), 35 with patent ductus arteriosus (PDA), 27 with coarctation of the aorta (CoA), 25 with restrictive physiology caused by Mulibrey nanism, and 64 control children. We measured serum concentrations of natriuretic peptides and evaluated ventricular size and function with echocardiography. In patients with ASD, PDA, and Mulibrey nanism, levels of both ANPN and NT-proBNP were higher than in controls but in children with CoA, only ANPN levels were higher. ANPN levels correlated with RV size in ASD and NT-proBNP levels with LV size in PDA. In patients with restriction, NT-proBNP levels correlated negatively with LV size. Correlation between echo measurements and levels of natriuretic peptides varied according to loading condition. Measurement of natriuretic peptide levels provides a supplemental method for non-invasive haemodynamic evaluation of children's heart disease.

Protease-catalyzed peptide bond formation: application to synthesis of the COOH-terminal octapeptide of cholecystokinin.

PubMed Central

Kullmann, W

1982-01-01

This study of protease-catalyzed peptide synthesis reports the preparation of the COOH-terminal octapeptide amide of cholecystokinin. The octapeptide was assembled by chemical condensation of two tetrapeptide segments that had been synthesized through the concerted catalytic reactions of several proteases of different specificities. The resulting octapeptide derivative was subjected to catalytic transfer hydrogenation, followed by sulfation of its tyrosine residue and removal of the N alpha-protecting group. The homogeneous target peptide was obtained after purification via partition chromatography, gel filtration, and ion-exchange chromatography. The synthetic octapeptide stimulated amylase release from pancreatic acinar cells. Images PMID:6283547

Structure of the Paramyxovirus Parainfluenza Virus 5 Nucleoprotein in Complex with an Amino-Terminal Peptide of the Phosphoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aggarwal, Megha; Leser, George P.; Kors, Christopher A.

Parainfluenza virus 5 (PIV5) belongs to the familyParamyxoviridae, which consists of enveloped viruses with a nonsegmented negative-strand RNA genome encapsidated by the nucleoprotein (N). Paramyxovirus replication is regulated by the phosphoprotein (P) through protein-protein interactions with N and the RNA polymerase (L). The chaperone activity of P is essential to maintain the unassembled RNA-free form of N in order to prevent nonspecific RNA binding and premature N oligomerization. Here, we determined the crystal structure of unassembled PIV5 N in complex with a P peptide (N 0P) derived from the N terminus of P (P50) at 2.65 Å. The PIV5 Nmore » 0P consists of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a hinge region. The cleft at the hinge region of RNA-bound PIV5 N was previously shown to be an RNA binding site. The N 0P structure shows that the P peptide binds to the CTD of N and extends toward the RNA binding site to inhibit N oligomerization and, hence, RNA binding. Binding of P peptide also keeps the PIV5 N in the open form. A molecular dynamics (MD) analysis of both the open and closed forms of N shows the flexibility of the CTD and the preference of the N protein to be in an open conformation. The gradual opening of the hinge region, to release the RNA, was also observed. Together, these results advance our knowledge of the conformational swapping of N required for the highly regulated paramyxovirus replication. IMPORTANCEParamyxovirus replication is regulated by the interaction of P with N and L proteins. Here, we report the crystal structure of unassembled parainfluenza virus 5 (PIV5) N chaperoned with P peptide. Our results provide a detailed understanding of the binding of P to N. The conformational switching of N between closed and open forms during its initial interaction with P, as well as during RNA release, was analyzed. Our data also show the plasticity of the CTD and the importance of domain movement for conformational switching. The results improve our understanding of the mechanism of interchanging N conformations for RNA replication and release.« less

Evidence for N- and C-terminal processing of a plant defense-related enzyme: Primary structure of tobacco prepro-β-1,3-glucanase

PubMed Central

Shinshi, H.; Wenzler, H.; Neuhaus, J.-M.; Felix, G.; Hofsteenge, J.; Meins, F.

1988-01-01

Tobacco glucan endo-1,3-β-glucosidase (β-1,3-glucanase; 1,3-β-D-glucan glucanohydrolase; EC 3.2.1.39) exhibits complex hormonal and developmental regulation and is induced when plants are infected with pathogens. We determined the primary structure of this enzyme from the nucleotide sequence of five partial cDNA clones and the amino acid sequence of five peptides covering a total of 70 residues. β-1,3-Glucanase is produced as a 359-residue preproenzyme with an N-terminal hydrophobic signal peptide of 21 residues and a C-terminal extension of 22 residues containing a putative N-glycosylation site. The results of pulse-chase experiments with tunicamycin provide evidence that the first step in processing is loss of the signal peptide and addition of an oligosaccharide side chain. The glycosylated intermediate is further processed with the loss of the oligosaccharide side chain and C-terminal extension to give the mature enzyme. Heterogeneity in the sequences of cDNA clones and of mature protein and in Southern blot analysis of restriction endonuclease fragments indicates that tobacco β-1,3-glucanase is encoded by a small gene family. Two or three members of this family appear to have their evolutionary origin in each of the progenitors of tobacco, Nicotiana sylvestris and Nicotiana tomentosiformis. Images PMID:16593965

Vaccatides: Antifungal Glutamine-Rich Hevein-Like Peptides from Vaccaria hispanica

PubMed Central

Wong, Ka H.; Tan, Wei Liang; Kini, Shruthi G.; Xiao, Tianshu; Serra, Aida; Sze, Sui Kwan; Tam, James P.

2017-01-01

Hevein and hevein-like peptides are disulfide-constrained chitin-binding cysteine-rich peptides. They are divided into three subfamilies, 6C-, 8C-, and 10C-hevein-like peptides, based on the number of cysteine residues. In addition, hevein-like peptides can exist in two forms, short and long. The long C-terminal form found in hevein and 10C-hevein-like peptides contain a C-terminal protein cargo. In contrast, the short form without a protein cargo is found in all three subfamilies. Here, we report the discovery and characterization of two novel glutamine-rich and protein cargo-free 8C-hevein-like peptides, vaccatides vH1 and vH2, from Vaccaria hispanica of the Caryophyllaceae family. Proteomic analyses showed that the vaccatides are 40–41 amino acids in length and contain a chitin-binding domain. NMR determination revealed that vaccatide vH2 displays a highly compact structure with a N-terminal cystine knot and an addition C-terminal disulfide bond. Stability studies showed that this compact structure renders vaccatide vH2 resistant to thermal, chemical and proteolytic degradation. The chitin-binding vH2 was shown to inhibit the mycelium growth of four phyto-pathogenic fungal strains with IC50 values in the micromolar range. Our findings show that vaccatides represent a new family of 8C-hevein-like peptides, which are protein cargo-free and glutamine-rich, characteristics that differentiate them from the prototypic hevein and the 10C-hevein-like peptides. In summary, this study enriches the existing library of hevein-like peptides and provides insight into their molecular diversity in sequence, structure and biosynthesis. Additionally, their highly disulfide-constrained structure could be used as a scaffold for developing metabolically and orally active peptidyl therapeutics. PMID:28680440

Vaccatides: Antifungal Glutamine-Rich Hevein-Like Peptides from Vaccaria hispanica.

PubMed

Wong, Ka H; Tan, Wei Liang; Kini, Shruthi G; Xiao, Tianshu; Serra, Aida; Sze, Sui Kwan; Tam, James P

2017-01-01

Hevein and hevein-like peptides are disulfide-constrained chitin-binding cysteine-rich peptides. They are divided into three subfamilies, 6C-, 8C-, and 10C-hevein-like peptides, based on the number of cysteine residues. In addition, hevein-like peptides can exist in two forms, short and long. The long C-terminal form found in hevein and 10C-hevein-like peptides contain a C-terminal protein cargo. In contrast, the short form without a protein cargo is found in all three subfamilies. Here, we report the discovery and characterization of two novel glutamine-rich and protein cargo-free 8C-hevein-like peptides, vaccatides vH1 and vH2, from Vaccaria hispanica of the Caryophyllaceae family. Proteomic analyses showed that the vaccatides are 40-41 amino acids in length and contain a chitin-binding domain. NMR determination revealed that vaccatide vH2 displays a highly compact structure with a N-terminal cystine knot and an addition C-terminal disulfide bond. Stability studies showed that this compact structure renders vaccatide vH2 resistant to thermal, chemical and proteolytic degradation. The chitin-binding vH2 was shown to inhibit the mycelium growth of four phyto-pathogenic fungal strains with IC 50 values in the micromolar range. Our findings show that vaccatides represent a new family of 8C-hevein-like peptides, which are protein cargo-free and glutamine-rich, characteristics that differentiate them from the prototypic hevein and the 10C-hevein-like peptides. In summary, this study enriches the existing library of hevein-like peptides and provides insight into their molecular diversity in sequence, structure and biosynthesis. Additionally, their highly disulfide-constrained structure could be used as a scaffold for developing metabolically and orally active peptidyl therapeutics.

An intrinsic agonist mechanism for activation of glucagon-like peptide-1 receptor by its extracellular domain

PubMed Central

Yin, Yanting; Zhou, X Edward; Hou, Li; Zhao, Li-Hua; Liu, Bo; Wang, Gaihong; Jiang, Yi; Melcher, Karsten; Xu, H Eric

2016-01-01

The glucagon-like peptide-1 receptor is a class B G protein coupled receptor (GPCR) that plays key roles in glucose metabolism and is a major therapeutic target for diabetes. The classic two-domain model for class B GPCR activation proposes that the apo-state receptor is auto-inhibited by its extracellular domain, which physically interacts with the transmembrane domain. The binding of the C-terminus of the peptide hormone to the extracellular domain allows the N-terminus of the hormone to insert into the transmembrane domain to induce receptor activation. In contrast to this model, here we demonstrate that glucagon-like peptide-1 receptor can be activated by N-terminally truncated glucagon-like peptide-1 or exendin-4 when fused to the receptor, raising the question regarding the role of N-terminal residues of peptide hormone in glucagon-like peptide-1 receptor activation. Mutations of cysteine 347 to lysine or arginine in intracellular loop 3 transform the receptor into a G protein-biased receptor and allow it to be activated by a nonspecific five-residue linker that is completely devoid of exendin-4 or glucagon-like peptide-1 sequence but still requires the presence of an intact extracellular domain. Moreover, the extracellular domain can activate the receptor in trans in the presence of an intact peptide hormone, and specific mutations in three extracellular loops abolished this extracellular domain trans-activation. Together, our data reveal a dominant role of the extracellular domain in glucagon-like peptide-1 receptor activation and support an intrinsic agonist model of the extracellular domain, in which peptide binding switches the receptor from the auto-inhibited state to the auto-activated state by releasing the intrinsic agonist activity of the extracellular domain. PMID:27917297

Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics.

PubMed

Prudova, Anna; auf dem Keller, Ulrich; Butler, Georgina S; Overall, Christopher M

2010-05-01

Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses using iTRAQ-TAILS links gelatinases with new mechanisms of action in angiogenesis and reveals unpredicted restrictions in substrate repertoires for these two very similar proteases.

Multiplex N-terminome Analysis of MMP-2 and MMP-9 Substrate Degradomes by iTRAQ-TAILS Quantitative Proteomics*

PubMed Central

Prudova, Anna; auf dem Keller, Ulrich; Butler, Georgina S.; Overall, Christopher M.

2010-01-01

Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses using iTRAQ-TAILS links gelatinases with new mechanisms of action in angiogenesis and reveals unpredicted restrictions in substrate repertoires for these two very similar proteases. PMID:20305284

Studies on the structures of the Tm, Sj, M1, Can, Sext and Hu blood group antigens.

PubMed

Dahr, W; Knuppertz, G; Beyreuther, K; Moulds, J J; Moulds, M; Wilkinson, S; Capon, C; Fournet, B; Issitt, P D

1991-08-01

The Glycophorins (GPs = sialoglycoproteins) in erythrocyte membranes from various Black individuals, some of which exhibit the M1, Can, Sj, Tm, Sext and/or Hu antigens, and several Caucasian donors, including pooled fetal red cells, were studied. Using agglutination inhibition assays with GP fractions, GP fragments and chemically modified GPs as well as trypsin treatment of intact red cells, the antigens defined by anti-M1, anti-M+M1, anti-Can and anti-Tm sera were found to be located on the N-terminal tryptic peptide (T2, residues 1-31) of the major GP (GP A = MN sialoglycoprotein). Evidence was obtained that the N-terminal amino-acid residue, NeuNAc and/or (a) different sugar residue(s) are involved in the antigens. Amino-acid sequence and composition analyses excluded an amino-acid exchange within the N-terminal region (residues 1-31) of GP A. Carbohydrate analyses revealed the attachment of GlcNAc residues (up to about five, dependent on the strength of the above-mentioned antigens) to O-glycosidically linked oligosaccharides within the N-terminal portion (residues 1-31) of GP A. As judged from the carbohydrate compositions of peptides, the alteration of the O-glycosidic oligosaccharides is associated with a slight increase of the Gal and Fuc contents and a slight decrease of the NeuNAc level. Analyses of small, secondary cyanogen bromide and V8 proteinase peptides from the N-terminal region of GP A from Blacks, Caucasians and Caucasian fetal cells suggest that the variable attachment of small quantities of GlcNAc (about 0.03 to about 0.2 residues per peptide molecule) accounts, at least in part, for the polymorphisms detected by anti-Can and the original anti-Tm (serum Sheerin). Remarkably, the GlcNAc-containing O-glycosidic oligosaccharides occur only in small quantities, or not all at, within the positions 32-61 of GP A and the glycosylated domains of GP B and GP C.(ABSTRACT TRUNCATED AT 400 WORDS)

Conformation and molecular topography of the N-terminal segment of surfactant protein B in structure-promoting environments.

PubMed Central

Gordon, L. M.; Horvath, S.; Longo, M. L.; Zasadzinski, J. A.; Taeusch, H. W.; Faull, K.; Leung, C.; Waring, A. J.

1996-01-01

Although the effects of surfactant protein B (SP-B) on lipid surface activity in vitro and in vivo are well known, the relationship between molecular structure and function is still not fully understood. To further characterize protein structure-activity correlations, we have used physical techniques to study conformation, orientation, and molecular topography of N-terminal SP-B peptides in lipids and structure-promoting environments. Fourier transform infrared (FTIR) and CD measurements of SP-B1-25 (residues 1-25) in methanol, SDS micelles, egg yolk lecithin (EYL) liposomes, and surfactant lipids indicate the peptide has a dominant helical content, with minor turn and disordered components. Polarized FTIR studies of SP-B1-25 indicate the long molecular axis lies at an oblique angle to the surface of lipid films. Truncated peptides were similarly examined to assign more accurately the discrete conformations within the SP-B1-25 sequence. Residues Cys-8-Gly-25 are largely alpha-helix in methanol, whereas the N-terminal segment Phe-1-Cys-8 had turn and helical propensities. Addition of SP-B1-25 spin-labeled at the N-terminal Phe (i.e., SP-B1-25) to SDS, EYL, or surfactant lipids yielded electron spin resonance spectra that reflect peptide bound to lipids, but retaining considerable mobility. The absence of characteristic radical broadening indicates that SP-B1-25 is minimally aggregated when it interacts with these lipids. Further, the high polarity of SP-B1-25 argues that the reporter on Phe-1 resides in the headgroup of the lipid dispersions. The blue-shift in the endogenous fluorescence of Trp-9 near the N-terminus of SP-B1-25 suggests that this residue also lies near the lipid headgroup. A summary model based on the above physical experiments is presented for SP-B1-25 interacting with lipids. PMID:8844855

Interactions of chromogranin A-derived vasostatins and monolayers of phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine.

PubMed

Blois, Anna; Holmsen, Holm; Martino, Guglielmo; Corti, Angelo; Metz-Boutigue, Marie-Hélène; Helle, Karen B

2006-03-15

Vasostatin-I (CgA1-76) is a naturally occurring and biologically active N-terminal peptide derived from chromogranin A (CgA), produced and secreted at high concentrations by neuroendocrine tissues and also from a range of neuroendocrine tumors. This study aims to examine the hypothesis that in the absence of classical protein receptors CgA1-76 may, like its two derived peptides CgA1-40 and CgA47-66, perturb the lipid microenvironment of other membrane receptors, as a basis for the largely inhibitory activities of these CgA peptides. The nature of the interactions between phospholipids and vasostatin-derived fragments was studied in the Langmuir film balance apparatus at 37 degrees C. The synthetic peptides CgA1-40 and CgA47-66 and a recombinant fragment (VS-I) containing vasostatin-I (Ser-Thr-Ala-CgA1-78) were compared for their effects on monolayers of phosphatidylcholine and phosphatidylethanolamine from pig brain and defined species of phosphatidylserine. Marked differences in surface pressure-area isotherms and phase-transition plateaus were apparent with the three classes of phospholipids on VS-I, CgA1-40 and CgA47-66 in physiological buffer or pure water. The results indicate that VS-I and CgA47-66 at 5-10 nM concentrations may engage in electrostatic as well as hydrophobic interactions with membrane-relevant phospholipids at physiological conditions, VS-I in particular enhancing the fluidity of saturated species of phosphatidylserine.

Immunological and biochemical characterization of processing products from the neurotensin/neuromedin N precursor in the rat medullary thyroid carcinoma 6-23 cell line.

PubMed Central

Bidard, J N; de Nadai, F; Rovere, C; Moinier, D; Laur, J; Martinez, J; Cuber, J C; Kitabgi, P

1993-01-01

Neurotensin (NT) and neuromedin N (NN) are two related biologically active peptides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide starting with an N-terminal signal peptide and containing in its C-terminal region one copy each of NT and NN. NN precedes NT and is separated from it by a Lys-Arg sequence. Two other Lys-Arg sequences flank the N-terminus of NN and the C-terminus of NT. A fourth Lys-Arg sequence occurs near the middle of the precursor and is followed by an NN-like sequence. Finally, an Arg-Arg pair is present within the NT moiety. The four Lys-Arg doublets represent putative processing sites in the precursor molecule. The present study was designed to investigate the post-translational processing of the NT/NN precursor in the rat medullary thyroid carcinoma (rMTC) 6-23 cell line, which synthesizes large amounts of NT upon dexamethasone treatment. Five region-specific antisera recognizing the free N- or C-termini of sequences adjacent to the basic doublets were produced, characterized and used for immunoblotting and radioimmunoassay studies in combination with gel filtration, reverse-phase h.p.l.c. and trypsin digestion of rMTC 6-23 cell extracts. Because two of the antigenic sequences, i.e. NN and the NN-like sequence, start with a lysine residue that is essential for recognition by their respective antisera, a micromethod by which trypsin specifically cleaves at arginine residues was developed. The results show that dexamethasone-treated rMTC 6-23 cells produced comparable amounts of NT, NN and a peptide corresponding to a large N-terminal precursor fragment lacking the NN and NT moieties. This large fragment was purified. N-Terminal sequencing revealed that it started at residue Ser23 of the prepro-NT/NN sequence, and thus established the Cys22-Ser23 bond as the cleavage site of the signal peptide. Two other large N-terminal fragments bearing respectively the NN and NT sequences at their C-termini were present in lower amounts. The NN-like sequence was internal to all the large fragments. There was no evidence for the presence of peptides with the NN-like sequence at their N-termini. This shows that, in rMTC 6-23 cells, the precursor is readily processed at the three Lys-Arg doublets that flank and separate the NT and NN sequences. In contrast, the Lys-Arg doublet that precedes the NN-like sequence is not processed in this system.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 3 PMID:8471039

Copper(II) complexes of alloferon 1 with point mutations (H1A) and (H9A) stability structure and biological activity.

PubMed

Matusiak, Agnieszka; Kuczer, Mariola; Czarniewska, Elżbieta; Rosiński, Grzegorz; Kowalik-Jankowska, Teresa

2014-09-01

Mono- and polynuclear copper(II) complexes of the alloferon 1 with point mutations (H1A) A(1)GVSGH(6)GQH(9)GVH(12)G (Allo1A) and (H9A) H(1)GVSGH(6)GQA(9)GVH(12)G (Allo9A) have been studied by potentiometric, UV-visible, CD, EPR spectroscopic and mass spectrometry (MS) methods. To obtain a complete complex speciation different metal-to-ligand molar ratios ranging from 1:1 to 4:1 for Allo1A and to 3:1 for Allo9A were studied. The presence of the His residue in first position of the peptide chain changes the coordination abilities of the Allo9A peptide in comparison to that of the Allo1A. Imidazole-N3 atom of N-terminal His residue of the Allo9A peptide forms stable 6-membered chelate with the terminal amino group. Furthermore, the presence of two additional histidine residues in the Allo9A peptide (H(6),H(12)) leads to the formation of the CuL complex with 4N {NH2,NIm-H(1),NIm-H(6),NIm-H(12)} binding site in wide pH range (5-8). For the Cu(II)-Allo1A system, the results demonstrated that at physiological pH7.4 the predominant complex the CuH-1L consists of the 3N {NH2,N(-),CO,NIm} coordination mode. The inductions of phenoloxidase activity and apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH7.4 were studied. The Allo1A, Allo1K peptides and their copper(II) complexes displayed the lowest hemocytotoxic activity while the most active was the Cu(II)-Allo9A complex formed at pH7.4. The results may suggest that the N-terminal-His(1) and His(6) residues may be more important for their proapoptotic properties in insects than those at positions 9 and 12 in the peptide chain. Copyright © 2014 Elsevier Inc. All rights reserved.

Immunocontraceptive efficacy of synthetic peptides corresponding to major antigenic determinants of chicken riboflavin carrier protein in the female rats.

PubMed

Subramanian, S; Karande, A A; Adiga, P R

2000-09-01

Earlier studies have demonstrated that antibodies directed towards the N-terminal (residues 10-17) and C-terminal (residues 200-207) regions on chicken riboflavin carrier protein (RCP; 219 AA) are effective in pregnancy termination in rodents and sub-human primates. In the present study, the immunocontraceptive potential of three additional immunodominant sequences comprising of residues 33-49, 64 83 and 130-147 (CYA, CED and CGE peptides, respectively) of chicken RCP was investigated. The three antigenic peptides were synthesized by using Fmoc chemistry. Oligoclonal antibodies were generated in rabbits. Bioneutralizing capacity of these peptides was assessed by passive and active immunoneutralization studies. All the three peptides-specific antisera recognized their cognate epitopes on native RCP. When the affinity purified peptide IgG were administered on three consecutive days to pregnant rats (on days 10, 11 and 12), it was observed that the rats injected with CED and CGE-IgG failed to deliver any pups whereas the animals which received CYA IgG delivered normal pups. Active immunization of fertile female rats with CED or CGE peptide conferred protection from pregnancy. These results demonstrate the presence of two additional stretches in chicken RCP which can serve as mini-vaccines.

Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain.

PubMed

Runge, Steffen; Thøgersen, Henning; Madsen, Kjeld; Lau, Jesper; Rudolph, Rainer

2008-04-25

The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B1 of the seven-transmembrane G protein-coupled receptors, and its natural agonist ligand is the peptide hormone glucagon-like peptide-1 (GLP-1). GLP-1 is involved in glucose homeostasis, and activation of GLP-1R in the plasma membrane of pancreatic beta-cells potentiates glucose-dependent insulin secretion. The N-terminal extracellular domain (nGLP-1R) is an important ligand binding domain that binds GLP-1 and the homologous peptide Exendin-4 with differential affinity. Exendin-4 has a C-terminal extension of nine amino acid residues known as the "Trp cage", which is absent in GLP-1. The Trp cage was believed to interact with nGLP-1R and thereby explain the superior affinity of Exendin-4. However, the molecular details that govern ligand binding and specificity of nGLP-1R remain undefined. Here we report the crystal structure of human nGLP-1R in complex with the antagonist Exendin-4(9-39) solved by the multiwavelength anomalous dispersion method to 2.2A resolution. The structure reveals that Exendin-4(9-39) is an amphipathic alpha-helix forming both hydrophobic and hydrophilic interactions with nGLP-1R. The Trp cage of Exendin-4 is not involved in binding to nGLP-1R. The hydrophobic binding site of nGLP-1R is defined by discontinuous segments including primarily a well defined alpha-helix in the N terminus of nGLP-1R and a loop between two antiparallel beta-strands. The structure provides for the first time detailed molecular insight into ligand binding of the human GLP-1 receptor, an established target for treatment of type 2 diabetes.

Influence of specific amino acid side-chains on the antimicrobial activity and structure of bovine lactoferrampin.

PubMed

Haney, Evan F; Nazmi, Kamran; Bolscher, Jan G M; Vogel, Hans J

2012-06-01

Lactoferrin is an 80 kDa iron binding protein found in the secretory fluids of mammals and it plays a major role in host defence. An antimicrobial peptide, lactoferrampin, was identified through sequence analysis of bovine lactoferrin and its antimicrobial activity against a wide range of bacteria and yeast species is well documented. In the present work, the contribution of specific amino acid residues of lactoferrampin was examined to evaluate the role that they play in membrane binding and bilayer disruption. The structures of all the bovine lactoferrampin derivatives were examined with circular dichroism and nuclear magnetic resonance spectroscopy, and their interactions with phospholipids were evaluated with differential scanning calorimetry and isothermal titration calorimetry techniques. From our results it is apparent that the amphipathic N-terminal helix anchors the peptide to membranes with Trp 268 and Phe 278 playing important roles in determining the strength of the interaction and for inducing peptide folding. In addition, the N-terminal helix capping residues (DLI) increase the affinity for negatively charged vesicles and they mediate the depth of membrane insertion. Finally, the unique flexibility in the cationic C-terminal region of bovine lactoferrampin does not appear to be essential for the antimicrobial activity of the peptide.

Localization of the Intracellular Activity Domain of Pasteurella multocida Toxin to the N Terminus

PubMed Central

Wilson, Brenda A.; Ponferrada, Virgilio G.; Vallance, Jefferson E.; Ho, Mengfei

1999-01-01

We have shown that Pasteurella multocida toxin (PMT) directly causes transient activation of Gqα protein that is coupled to phosphatidylinositol-specific phospholipase Cβ1 in Xenopus oocytes (B. A. Wilson, X. Zhu, M. Ho, and L. Lu, J. Biol. Chem. 272:1268–1275, 1997). We found that antibodies directed against an N-terminal peptide of PMT inhibited the toxin-induced response in Xenopus oocytes, but antibodies against a C-terminal peptide did not. To test whether the intracellular activity domain of PMT is localized to the N terminus, we conducted a deletion mutational analysis of the PMT protein, using the Xenopus oocyte system as a means of screening for toxin activity. Using PCR and conventional cloning techniques, we cloned from a toxinogenic strain of P. multocida the entire toxA gene, encoding the 1,285-amino-acid PMT protein, and expressed the recombinant toxin as a His-tagged fusion protein in Escherichia coli. We subsequently generated a series of N-terminal and C-terminal deletion mutants and expressed the His-tagged PMT fragments in E. coli. These proteins were screened for cytotoxic activity on cultured Vero cells and for intracellular activity in the Xenopus oocyte system. Only the full-length protein without the His tag exhibited activity on Vero cells. The full-length PMT and N-terminal fragments containing the first 500 residues elicited responses in oocytes, but the C-terminal 780 amino acid fragment did not. Our results confirm that the intracellular activity domain of PMT is localized to the N-terminal 500 amino acids of the protein and that the C terminus is required for entry into cells. PMID:9864199

Crystal Structure of the Marburg Virus Nucleoprotein Core Domain Chaperoned by a VP35 Peptide Reveals a Conserved Drug Target for Filovirus.

PubMed

Zhu, Tengfei; Song, Hao; Peng, Ruchao; Shi, Yi; Qi, Jianxun; Gao, George F

2017-09-15

Filovirus nucleoprotein (NP), viral protein 35 (VP35), and polymerase L are essential for viral replication and nucleocapsid formation. Here, we identify a 28-residue peptide (NP binding peptide [NPBP]) from Marburg virus (MARV) VP35 through sequence alignment with previously identified Ebola virus (EBOV) NPBP, which bound to the core region (residues 18 to 344) of the N-terminal portion of MARV NP with high affinity. The crystal structure of the MARV NP core/NPBP complex at a resolution of 2.6 Å revealed that NPBP binds to the C-terminal region of the NP core via electrostatic and nonpolar interactions. Further structural analysis revealed that the MARV and EBOV NP cores hold a conserved binding pocket for NPBP, and this pocket could serve as a promising target for the design of universal drugs against filovirus infection. In addition, cross-binding assays confirmed that the NP core of MARV or EBOV can bind the NPBP from the other virus, although with moderately reduced binding affinities that result from termini that are distinct between the MARV and EBOV NPBPs. IMPORTANCE Historically, Marburg virus (MARV) has caused severe disease with up to 90% lethality. Among the viral proteins produced by MARV, NP and VP35 are both multifunctional proteins that are essential for viral replication. In its relative, Ebola virus (EBOV), an N-terminal peptide from VP35 binds to the NP N-terminal region with high affinity. Whether this is a common mechanism among filoviruses is an unsolved question. Here, we present the crystal structure of a complex that consists of the core domain of MARV NP and the NPBP peptide from VP35. As we compared MARV NPBP with EBOV NPBP, several different features at the termini were identified. Although these differences reduce the affinity of the NP core for NPBPs across genera, a conserved pocket in the C-terminal region of the NP core makes cross-species binding possible. Our results expand our knowledge of filovirus NP-VP35 interactions and provide more details for therapeutic intervention. Copyright © 2017 American Society for Microbiology.

Folding and conformational consequences of glycine to alanine replacements at different positions in a collagen model peptide.

PubMed

Bhate, Manjiri; Wang, Xin; Baum, Jean; Brodsky, Barbara

2002-05-21

The collagen model peptide T1-892 includes a C-terminal nucleation domain, (Gly-Pro-Hyp)(4), and an N-terminal (Gly-X-Y)(6) sequence taken from type I collagen. In osteogenesis imperfecta (OI) and other collagen diseases, single base mutations often convert one Gly to a larger residue, and T1-892 homologues modeling such mutations were synthesized with Gly to Ala substitutions in either the (Gly-Pro-Hyp)(4) domain, Gly25Ala, or the (Gly-X-Y)(6) domain, Gly10Ala. CD and NMR studies show the Gly10Ala peptide forms a normal triple-helix at the C-terminal end and propagates from the C- to the N-terminus until the Gly --> Ala substitution is encountered. At this point, triple-helix folding is terminated and cannot be reinitiated, leaving a nonhelical N-terminus. A decreased thermal stability is observed as a result of the shorter length of the triple-helix. In contrast, introduction of the Gly to Ala replacement at position 25, in the nucleation domain, shifts the monomer/trimer equilibrium toward the monomer form. The increased monomer and lower trimer populations are reflected in the dramatic decrease in triple-helix content and stability. Unlike the Ala replacement at position 10, the Ala substitution in the (Gly-Pro-Hyp)(4) region can still be incorporated into a triple-helix, but at a greatly decreased rate of folding, since the original efficient nucleation site is no longer operative. The specific consequences of Gly to Ala replacements in two distinctive sequences in this triple-helical peptide may help clarify the variability in OI clinical severity resulting from mutations at different sites along type I collagen chains.

Free energy landscapes of peptides by enhanced conformational sampling.

PubMed

Nakajima, N; Higo, J; Kidera, A; Nakamura, H

2000-02-11

The free energy landscapes of peptide conformations in water have been observed by the enhanced conformational sampling method, applying the selectively enhanced multicanonical molecular dynamics simulations. The conformations of the peptide dimers, -Gly-Gly-, -Gly-Ala-, -Gly-Ser-, -Ala-Gly-, -Asn-Gly-, -Pro-Gly-, -Pro-Ala-, and -Ala-Ala-, which were all blocked with N-terminal acetyl and C-terminal N-methyl groups, were individually sampled with the explicit TIP3P water molecules. From each simulation trajectory, we obtained the canonical ensemble at 300 K, from which the individual three-dimensional landscape was drawn by the potential of mean force using the three reaction coordinates: the backbone dihedral angle, psi, of the first amino acid, the backbone dihedral angle, phi, of the second amino acid, and the distance between the carbonyl oxygen of the N-terminal acetyl group and the C-terminal amide proton. The most stable state and several meta-stable states correspond to extended conformations and typical beta-turn conformations, and their free energy values were accounted for from the potentials of mean force at the states. In addition, the contributions from the intra-molecular energies of peptides and those from the hydration effects were analyzed. Consequently, the stable beta-turn conformations in the free energy landscape were consistent with the empirically preferred beta-turn types for each amino acid sequence. The thermodynamic values for the hydration effect were decomposed and they correlated well with the empirical values estimated from the solvent accessible surface area of each molecular conformation during the trajectories. The origin of the architecture of protein local fragments was analyzed from the viewpoint of the free energy and its decomposed factors. Copyright 2000 Academic Press.

Inhibiting nucleation of amyloid structure in a huntingtin fragment by targeting α-helix rich oligomeric intermediates

PubMed Central

Mishra, Rakesh; Jayaraman, Murali; Roland, Bartholomew P.; Landrum, Elizabeth; Fullam, Timothy; Kodali, Ravindra; Thakur, Ashwani K.; Arduini, Irene; Wetzel, Ronald

2011-01-01

Although oligomeric intermediates are transiently formed in almost all known amyloid assembly reactions, their mechanistic roles are poorly understood. Recently we demonstrated a critical role for the 17 amino acid N-terminal segment (httNT) of huntingtin (htt) in oligomer-mediated amyloid assembly of htt N-terminal fragments. In this mechanism, the httNT segment forms the α-helix rich core of the oligomers, leaving most or all of each polyglutamine (polyQ) segment disordered and solvent-exposed. Nucleation of amyloid structure occurs within this local high concentration of disordered polyQ. Here we demonstrate the kinetic importance of httNT self-assembly by describing inhibitory httNT-containing peptides that appear to work by targeting nucleation within the oligomer fraction. These molecules inhibit amyloid nucleation by forming mixed oligomers with the httNT domains of polyQ-containing htt N-terminal fragments. In one class of inhibitor, nucleation is passively suppressed due to the reduced local concentration of polyQ within the mixed oligomer. In the other class, nucleation is actively suppressed by a proline-rich polyQ segment covalently attached to httNT. Studies with D-amino acid and scrambled sequence versions of httNT suggest that inhibition activity is strongly linked to the propensity of inhibitory peptides to make amphipathic α-helices. HttNT derivatives with C-terminal cell penetrating peptide segments, also exhibit excellent inhibitory activity. The httNT-based peptides described here, especially those with protease-resistant D-amino acids and/or with cell penetrating sequences, may prove useful as lead therapeutics for inhibiting nucleation of amyloid formation in Huntington’s disease. PMID:22178478

O-linked oligosaccharides on insulin receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collier, E.; Gorden, P.

1991-02-01

The insulin receptor, an integral membrane glycoprotein, is synthesized as a single-chain precursor that is cleaved to produce two mature subunits, both of which contain N-linked oligosaccharide chains and covalently linked fatty acids. We report that the beta-subunit also contains O-linked oligosaccharides. The proreceptor, alpha-subunit, and beta-subunit were labeled with (3H)mannose and (3H)galactose in the presence or absence of an inhibitor of O-linked glycosylation. Tryptic peptides from each component were separated by reverse-phase high-performance liquid chromatography. N- and O-linked oligosaccharide chains were identified on these peptides by specific enzymatic digestions. The proreceptor and alpha-subunit contained only N-linked oligosaccharides, whereas themore » beta-subunit contained both N- and O-linked oligosaccharides. The O-linked oligosaccharide chains were attached to a single tryptic fraction of the beta-subunit, which also contained N-linked chains. This fraction was further localized to the NH2-terminal tryptic peptide of the beta-subunit by specific immunoprecipitation with an anti-peptide antibody with specificity for this region. Binding of insulin and autophosphorylation of the beta-subunit were not dependent on O-linked glycosylation, because cells grown in the presence of the inhibitor exhibited a normal dose response to insulin. Therefore, the insulin receptor contains O-linked oligosaccharides on the NH2-terminal tryptic peptide of the beta-subunit, and these O-linked oligosaccharides are not necessary to the binding or autophosphorylation function of the receptor.« less

Antigen-antibody interaction. The immunodominant region of EDP208 pili.

PubMed

Worobec, E A; Paranchych, W; Parker, J M; Taneja, A K; Hodges, R S

1985-01-25

The EDP208 pilus contains a major antigenic determinant in the N-terminal dodecapeptide, as shown by E. A. Worobec, A. K. Taneja, R. S. Hodges, and W. Paranchych ((1983) J. Bacteriol. 153, 955-961). This peptide was chemically synthesized, coupled to bovine serum albumin with N-hydroxysuccinimidyl p-azido-benzoate, and used in immunoblot and enzyme-linked immunosorbent assays to show it was capable of reacting with anti-EDP208 pilus antibodies. Antibodies raised against the synthetic peptide conjugate were also capable of reacting with whole pili in these assays. To further examine the specific residues responsible for the antigenicity of this site, several peptide analogs were chemically synthesized. The relative affinity of these peptides for anti-EDP208 pilus antibodies was determined by a competitive enzyme-linked immunosorbent assay using the Fab fragment of anti-EDP208 pilus immunoglobulin G. From these results we established that the antigenic region of this peptide was the N-terminal pentapeptide, N-acetyl-Thr-Asp-Leu-Leu-Ala, and the key residues responsible for the antibody-antigen interaction are the N-acetyl-Thr1, Leu3, and Leu4. Hydrophobic interactions involving the methyl of the acetyl group and the leucine side chains make the largest contributions to the antigen-antibody interaction, while a lesser contribution is made by the Thr1 hydroxyl. The side chains of Asp2 and Ala5 contribute only weakly to the stabilization of the antigen-antibody complex.

The signal peptide-like segment of hpaXm is required for its association to the cell wall in transgenic tobacco plants

PubMed Central

Li, Le; Miao, Weiguo; Liu, Wenbo; Zhang, Shujian

2017-01-01

Harpins, encoded by hrp (hypersensitive response and pathogenicity) genes of Gram-negative plant pathogens, are elicitors of hypersensitive response (HR). HpaXm is a novel harpin-like protein described from cotton leaf blight bacteria, Xanthomonas citri subsp. malvacearum—a synonym of X. campestris pv. malvacearum (Smith 1901–1978). A putative signal peptide (1-MNSLNTQIGANSSFL-15) of hpaXm was predicted in the nitroxyl-terminal (N-terminal)by SignalP (SignalP 3.0 server). Here, we explored the function of the N-terminal leader peptide like segment of hpaXm using transgenic tobacco (Nicotiana tabacum cv. Xanthi nc.). Transgenic tobacco lines expressing the full-length hpaXm and the signal peptide-like segment-deleted mutant hpaXmΔLP were developed using transformation mediated by Agrobacterium tumefaciens. The target genes were confirmed integrated into the tobacco genomes and expressed normally. Using immune colloidal-gold detection technique, hpaXm protein was found to be transferred to the cytoplasm, the cell membrane, and organelles such as chloroplasts, mitochondria, and nucleus, as well as the cell wall. However, the deletion mutant hpaXmΔLP expressed in transgenic tobacco was found unable to cross the membrane to reach the cell wall. Additionally, soluble proteins extracted from plants transformed with hpaXm and hpaXmΔLP were bio-active. Defensive micro-HR induced by the transgene expression of hpaXm and hpaXmΔLP were observed on transgenic tobacco leaves. Disease resistance bioassays to tobacco mosaic virus (TMV) showed that tobacco plants transformed with hpaXm and with hpaXmΔLP exhibited enhanced resistance to TMV. In summary, the N-terminal signal peptide-like segment (1–45 bp) in hpaXm sequence is not necessary for transgene expression, bioactivity of hpaXm and resistance to TMV in transgenic tobacco, but is required for the protein to be translocated to the cell wall. PMID:28141855

Circular dichroism study of the carbohydrate-modified opioid peptides

NASA Astrophysics Data System (ADS)

Horvat, Štefica; Otvos, Laszlo; Urge, Laszlo; Horvat, Jaroslav; Čudić, Mare; Varga-Defterdarović, Lidija

1999-09-01

The conformational preferences of enkephalins and the related glycoconjugates in which free or protected carbohydrate moieties were linked to the opioid peptides through an ether, ester or amide bond were investigated by circular dichroism spectroscopy in water, trifluoroethanol and water-trifluoroethanol mixtures. The analysis of the spectra revealed that the conformation of the enkephalin molecule is very sensitive to slight changes in the peptide structure around the C-terminal region. It was found that the type II β-turn structures are populated in N-terminal tetrapeptide enkephalin fragment, while leucine-enkephalin amide feature a type I (III) β-turn structure in solution. Incorporation of the sugar moiety into opioid peptide compound did not significantly influence the overall conformation of the peptide backbone, although minor intensity changes may reflect shifts in the population of the different turn systems. These small structural alterations can be responsible for the receptor-subtype selectivity of the various carbohydrate-modified enkephalin analogs.

The dominant role of side chains in supramolecular double helical organisation in synthetic tripeptides

NASA Astrophysics Data System (ADS)

Sharma, Ankita; Tiwari, Priyanka; Dutt Konar, Anita

2018-06-01

Peptide self-assembled nanostructures have attracted attention recently owing to their promising applications in diversified avenues. To validate the importance of sidechains in supramolecular architectural stabilization, herein this report describes the self-assembly propensities involving weak interactions in a series of model tripeptides Boc-Xaa-Aib-Yaa-OMe I-IV, (where Xaa = 4-F-Phe/NMeSer/Ile & Yaa = Tyr in peptide I-III respectively and Xaa = 4-F-Phe & Yaa = Ile in peptide IV) differing in terminal side chains. The solid state structural analysis reveals that tripeptide (I) displays supramolecular preference for double helical architecture. However, when slight modification has been introduced in the N-terminal side chains disfavour the double helical organisation (Peptide II and III). Indeed the peptides display sheet like ensemble within the framework. Besides replacement of C-terminal Tyr by Ile in peptide I even do not promote the architecture, emphasizing the dominant role of balance of side chains in stabilizing double helical organisation. The CD measurements, concentration dependant studies, NMR titrations and ROESY spectra are well in agreement with the solid state conformational investigation. Moreover the morphological experiments utilizing FE-SEM, support the heterogeneity present in the peptides. Thus this work may not only hold future promise in understanding the structure and function of neurodegenerative diseases but also assist in rational design of protein modification in biologically active peptides.

Structures of the peptide-modifying radical SAM enzyme SuiB elucidate the basis of substrate recognition.

PubMed

Davis, Katherine M; Schramma, Kelsey R; Hansen, William A; Bacik, John P; Khare, Sagar D; Seyedsayamdost, Mohammad R; Ando, Nozomi

2017-09-26

Posttranslational modification of ribosomally synthesized peptides provides an elegant means for the production of biologically active molecules known as RiPPs (ribosomally synthesized and posttranslationally modified peptides). Although the leader sequence of the precursor peptide is often required for turnover, the exact mode of recognition by the modifying enzymes remains unclear for many members of this class of natural products. Here, we have used X-ray crystallography and computational modeling to examine the role of the leader peptide in the biosynthesis of a homolog of streptide, a recently identified peptide natural product with an intramolecular lysine-tryptophan cross-link, which is installed by the radical S -adenosylmethionine (SAM) enzyme, StrB. We present crystal structures of SuiB, a close ortholog of StrB, in various forms, including apo SuiB, SAM-bound SuiB, and a complex of SuiB with SAM and its peptide substrate, SuiA. Although the N-terminal domain of SuiB adopts a typical RRE (RiPP recognition element) motif, which has been implicated in precursor peptide recognition, we observe binding of the leader peptide in the catalytic barrel rather than the N-terminal domain. Computational simulations support a mechanism in which the leader peptide guides posttranslational modification by positioning the cross-linking residues of the precursor peptide within the active site. Together the results shed light onto binding of the precursor peptide and the associated conformational changes needed for the formation of the unique carbon-carbon cross-link in the streptide family of natural products.

The Human Antimicrobial Protein Bactericidal/Permeability-Increasing Protein (BPI) Inhibits the Infectivity of Influenza A Virus

PubMed Central

Pinkenburg, Olaf; Meyer, Torben; Bannert, Norbert; Norley, Steven; Bolte, Kathrin; Czudai-Matwich, Volker; Herold, Susanne; Gessner, André; Schnare, Markus

2016-01-01

In addition to their well-known antibacterial activity some antimicrobial peptides and proteins (AMPs) display also antiviral effects. A 27 aa peptide from the N-terminal part of human bactericidal/permeability-increasing protein (BPI) previously shown to harbour antibacterial activity inhibits the infectivity of multiple Influenza A virus strains (H1N1, H3N2 and H5N1) the causing agent of the Influenza pneumonia. In contrast, the homologous murine BPI-peptide did not show activity against Influenza A virus. In addition human BPI-peptide inhibits the activation of immune cells mediated by Influenza A virus. By changing the human BPI-peptide to the sequence of the mouse homologous peptide the antiviral activity was completely abolished. Furthermore, the human BPI-peptide also inhibited the pathogenicity of the Vesicular Stomatitis Virus but failed to interfere with HIV and measles virus. Electron microscopy indicate that the human BPI-peptide interferes with the virus envelope and at high concentrations was able to destroy the particles completely. PMID:27273104

Identification and Mechanistic Analysis of a Novel Tick-Derived Inhibitor of Thrombin

PubMed Central

Jablonka, Willy; Kotsyfakis, Michalis; Mizurini, Daniella M.; Monteiro, Robson Q.; Lukszo, Jan; Drake, Steven K.; Ribeiro, José M. C.; Andersen, John F.

2015-01-01

A group of peptides from the salivary gland of the tick Hyalomma marginatum rufipes, a vector of Crimean Congo hemorrhagic fever show weak similarity to the madanins, a group of thrombin-inhibitory peptides from a second tick species, Haemaphysalis longicornis. We have evaluated the anti-serine protease activity of one of these H. marginatum peptides that has been given the name hyalomin-1. Hyalomin-1 was found to be a selective inhibitor of thrombin, blocking coagulation of plasma and inhibiting S2238 hydrolysis in a competitive manner with an inhibition constant (Ki) of 12 nM at an ionic strength of 150 mM. It also blocks the thrombin-mediated activation of coagulation factor XI, thrombin-mediated platelet aggregation, and the activation of coagulation factor V by thrombin. Hyalomin-1 is cleaved at a canonical thrombin cleavage site but the cleaved products do not inhibit coagulation. However, the C-terminal cleavage product showed non-competitive inhibition of S2238 hydrolysis. A peptide combining the N-terminal parts of the molecule with the cleavage region did not interact strongly with thrombin, but a 24-residue fragment containing the cleavage region and the C-terminal fragment inhibited the enzyme in a competitive manner and also inhibited coagulation of plasma. These results suggest that the peptide acts by binding to the active site as well as exosite I or the autolysis loop of thrombin. Injection of 2.5 mg/kg of hyalomin-1 increased arterial occlusion time in a mouse model of thrombosis, suggesting this peptide could be a candidate for clinical use as an antithrombotic. PMID:26244557

Development and use of domain-specific antibodies in a characterization of the large subunits of soybean photosystem 1

NASA Technical Reports Server (NTRS)

Henry, R. L.; Takemoto, L. J.; Murphy, J.; Gallegos, G. L.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

The molecular architecture of the soybean photosystem 1 reaction center complex was examined using a combination of surface labeling and immunological methodology on isolated thylakoid membranes. Synthetic peptides (12 to 14 amino acids in length) were prepared which correspond to the N-terminal regions of the 83 and 82.4 kDa subunits of photosystem 1 (the PsaA and PsaB proteins, respectively). Similarly, a synthetic peptide was prepared corresponding to the C-terminal region of the PsaB subunit. These peptides were conjugated to a carrier protein, and were used for the production of polyclonal antibodies in rabbits. The resulting sera could distinguish between the PsaA and PsaB photosystem 1 subunits by Western blot analysis, and could identify appropriate size classes of cyanogen bromide cleavage fragments as predicted from the primary sequences of these two subunits. When soybean thylakoid membranes were surface-labeled with N-hydroxysuccinimidobiotin, several subunits of the complete photosystem 1 lipid/protein complex incorporated label. These included the light harvesting chlorophyll proteins of photosystem 1, and peptides thought to aid in the docking of ferredoxin to the complex during photosynthetic electron transport. However, the PsaA and PsaB subunits showed very little biotinylation. When these subunits were examined for the domains to which biotin did attach, most of the observed label was associated with the N-terminal domain of the PsaA subunit, as identified using a domain-specific polyclonal antisera.

Structure of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser of Aβ-peptide with phospholipase A2 from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution.

PubMed

Mirza, Zeenat; Pillai, Vikram Gopalakrishna; Zhong, Wei-Zhu

2014-03-10

Alzheimer's disease (AD) is one of the most significant social and health burdens of the present century. Plaques formed by extracellular deposits of amyloid β (Aβ) are the prime player of AD's neuropathology. Studies have implicated the varied role of phospholipase A2 (PLA2) in brain where it contributes to neuronal growth and inflammatory response. Overall contour and chemical nature of the substrate-binding channel in the low molecular weight PLA2s are similar. This study involves the reductionist fragment-based approach to understand the structure adopted by N-terminal fragment of Alzheimer's Aβ peptide in its complex with PLA2. In the current communication, we report the structure determined by X-ray crystallography of N-terminal sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser (DAEFRHDS) of Aβ-peptide with a Group I PLA2 purified from venom of Andaman Cobra sub-species Naja naja sagittifera at 2.0 Å resolution (Protein Data Bank (PDB) Code: 3JQ5). This is probably the first attempt to structurally establish interaction between amyloid-β peptide fragment and hydrophobic substrate binding site of PLA2 involving H bond and van der Waals interactions. We speculate that higher affinity between Aβ and PLA2 has the therapeutic potential of decreasing the Aβ-Aβ interaction, thereby reducing the amyloid aggregation and plaque formation in AD.

Identification and characterization of antimicrobial peptides from the skin of the endangered frog Odorrana ishikawae.

PubMed

Iwakoshi-Ukena, Eiko; Ukena, Kazuyoshi; Okimoto, Aiko; Soga, Miyuki; Okada, Genya; Sano, Naomi; Fujii, Tamotsu; Sugawara, Yoshiaki; Sumida, Masayuki

2011-04-01

The endangered anuran species, Odorrana ishikawae, is endemic to only two small Japanese Islands, Amami and Okinawa. To assess the innate immune system in this frog, we investigated antimicrobial peptides in the skin using artificially bred animals. Nine novel antimicrobial peptides containing the C-terminal cyclic heptapeptide domain were isolated on the basis of antimicrobial activity against Escherichia coli. The peptides were members of the esculentin-1 (two peptides), esculentin-2 (one peptide), palustrin-2 (one peptide), brevinin-2 (three peptides) and nigrocin-2 (two peptides) antimicrobial peptide families. They were named esculentin-1ISa, esculentin-1ISb, esculentin-2ISa, palustrin-2ISa, brevinin-2ISa, brevinin-2ISb, brevinin-2ISc, nigrocin-2ISa and nigrocin-2ISb. Peptide primary structures suggest a close relationship with the Asian odorous frogs, Odorrana grahami and Odorrana hosii. These antimicrobial peptides possessed a broad-spectrum of growth inhibition against five microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis and Candida albicans). Nine different cDNAs encoding the precursor proteins were also cloned and showed that the precursor proteins exhibited a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and an antimicrobial peptide at the C-terminus. Copyright © 2010 Elsevier Inc. All rights reserved.

Identification of Cell Adhesive Sequences in the N-terminal Region of the Laminin α2 Chain*

PubMed Central

Hozumi, Kentaro; Ishikawa, Masaya; Hayashi, Takemitsu; Yamada, Yuji; Katagiri, Fumihiko; Kikkawa, Yamato; Nomizu, Motoyoshi

2012-01-01

The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117–128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin β1 and anti-integrin α2β1 antibodies. These results suggest that A2-8 promotes an integrin α2β1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2β1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2β1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2β1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions. PMID:22654118

Long-term stability assessment of AlGaN/GaN field effect transistors modified with peptides: Device characteristics vs. surface properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rohrbaugh, Nathaniel; Bryan, Isaac; Bryan, Zachary

AlGaN/GaN Field Effect Transistors (FETs) are promising biosensing devices. Functionalization of these devices is explored in this study using an in situ approach with phosphoric acid etchant and a phosphonic acid derivative. Devices are terminated on peptides and soaked in water for up to 168 hrs to examine FETs for both device responses and surface chemistry changes. Measurements demonstrated threshold voltage shifting after the functionalization and soaking processes, but demonstrated stable FET behavior throughout. X-ray photoelectron spectroscopy and atomic force microscopy confirmed peptides attachment to device surfaces before and after water soaking. Results of this work point to the stabilitymore » of peptide coated functionalized AlGaN/GaN devices in solution and support further research of these devices as disposable, long term, in situ biosensors.« less

Heart failure, saxagliptin, and diabetes mellitus: observations from the SAVOR-TIMI 53 randomized trial.

PubMed

Scirica, Benjamin M; Braunwald, Eugene; Raz, Itamar; Cavender, Matthew A; Morrow, David A; Jarolim, Petr; Udell, Jacob A; Mosenzon, Ofri; Im, KyungAh; Umez-Eronini, Amarachi A; Pollack, Pia S; Hirshberg, Boaz; Frederich, Robert; Lewis, Basil S; McGuire, Darren K; Davidson, Jaime; Steg, Ph Gabriel; Bhatt, Deepak L

2014-10-28

Diabetes mellitus and heart failure frequently coexist. However, few diabetes mellitus trials have prospectively evaluated and adjudicated heart failure as an end point. A total of 16 492 patients with type 2 diabetes mellitus and a history of, or at risk of, cardiovascular events were randomized to saxagliptin or placebo (mean follow-up, 2.1 years). The primary end point was the composite of cardiovascular death, myocardial infarction, or ischemic stroke. Hospitalization for heart failure was a predefined component of the secondary end point. Baseline N-terminal pro B-type natriuretic peptide was measured in 12 301 patients. More patients treated with saxagliptin (289, 3.5%) were hospitalized for heart failure compared with placebo (228, 2.8%; hazard ratio, 1.27; 95% confidence intercal, 1.07-1.51; P=0.007). Corresponding rates at 12 months were 1.9% versus 1.3% (hazard ratio, 1.46; 95% confidence interval, 1.15-1.88; P=0.002), with no significant difference thereafter (time-varying interaction, P=0.017). Subjects at greatest risk of hospitalization for heart failure had previous heart failure, an estimated glomerular filtration rate ≤60 mL/min, or elevated baseline levels of N-terminal pro B-type natriuretic peptide. There was no evidence of heterogeneity between N-terminal pro B-type natriuretic peptide and saxagliptin (P for interaction=0.46), although the absolute risk excess for heart failure with saxagliptin was greatest in the highest N-terminal pro B-type natriuretic peptide quartile (2.1%). Even in patients at high risk of hospitalization for heart failure, the risk of the primary and secondary end points were similar between treatment groups. In the context of balanced primary and secondary end points, saxagliptin treatment was associated with an increased risk or hospitalization for heart failure. This increase in risk was highest among patients with elevated levels of natriuretic peptides, previous heart failure, or chronic kidney disease. http://www.clinicaltrials.gov. Unique identifier: NCT01107886. © 2014 American Heart Association, Inc.

Structural and pharmacological characteristics of chimeric peptides derived from peptide E and beta-endorphin reveal the crucial role of the C-terminal YGGFL and YKKGE motifs in their analgesic properties.

PubMed

Condamine, Eric; Courchay, Karine; Rego, Jean-Claude Do; Leprince, Jérôme; Mayer, Catherine; Davoust, Daniel; Costentin, Jean; Vaudry, Hubert

2010-05-01

Peptide E (a 25-amino acid peptide derived from proenkephalin A) and beta-endorphin (a 31-amino acid peptide derived from proopiomelanocortin) bind with high affinity to opioid receptors and share structural similarities but induce analgesic effects of very different intensity. Indeed, whereas they possess the same N-terminus Met-enkephalin message sequence linked to a helix by a flexible spacer and a C-terminal part in random coil conformation, in contrast with peptide E, beta-endorphin produces a profound analgesia. To determine the key structural elements explaining this very divergent opioid activity, we have compared the structural and pharmacological characteristics of several chimeric peptides derived from peptide E and beta-endorphin. Structures were obtained under the same experimental conditions using circular dichroism, computational estimation of helical content and/or nuclear magnetic resonance spectroscopy (NMR) and NMR-restrained molecular modeling. The hot-plate and writhing tests were used in mice to evaluate the antinociceptive effects of the peptides. Our results indicate that neither the length nor the physicochemical profile of the spacer plays a fundamental role in analgesia. On the other hand, while the functional importance of the helix cannot be excluded, the last 5 residues in the C-terminal part seem to be crucial for the expression or absence of the analgesic activity of these peptides. These data raise the question of the true function of peptides E in opioidergic systems. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Plasma Biomarkers Reflecting Profibrotic Processes in Heart Failure With a Preserved Ejection Fraction: Data From the Prospective Comparison of ARNI With ARB on Management of Heart Failure With Preserved Ejection Fraction Study.

PubMed

Zile, Michael R; Jhund, Pardeep S; Baicu, Catalin F; Claggett, Brian L; Pieske, Burkert; Voors, Adriaan A; Prescott, Margaret F; Shi, Victor; Lefkowitz, Martin; McMurray, John J V; Solomon, Scott D

2016-01-01

Heart failure with preserved ejection fraction is a clinical syndrome that has been associated with changes in the extracellular matrix. The purpose of this study was to determine whether profibrotic biomarkers accurately reflect the presence and severity of disease and underlying pathophysiology and modify response to therapy in patients with heart failure with preserved ejection fraction. Four biomarkers, soluble form of ST2 (an interleukin-1 receptor family member), galectin-3, matrix metalloproteinase-2, and collagen III N-terminal propeptide were measured in the Prospective Comparison of ARNI With ARB on Management of Heart Failure With Preserved Ejection Fraction (PARAMOUNT) trial at baseline, 12 and 36 weeks after randomization to valsartan or LCZ696. We examined the relationship between baseline biomarkers, demographic and echocardiographic characteristics, change in primary (change in N-terminal pro B-type natriuretic peptide) and secondary (change in left atrial volume) end points. The median (interquartile range) value for soluble form of ST2 (33 [24.6-48.1] ng/mL) and galectin 3 (17.8 [14.1-22.8] ng/mL) were higher, and for matrix metalloproteinase-2 (188 [155.5-230.6] ng/mL) lower, than in previously published referent controls; collagen III N-terminal propeptide (5.6 [4.3-6.9] ng/mL) was similar to referent control values. All 4 biomarkers correlated with severity of disease as indicated by N-terminal pro B-type natriuretic peptide, E/E', and left atrial volume. Baseline biomarkers did not modify the response to LCZ696 for lowering N-terminal pro B-type natriuretic peptide; however, left atrial volume reduction varied by baseline level of soluble form of ST2 and galectin 3; patients with values less than the observed median (<33 ng/mL soluble form of ST2 and <17.8 ng/mL galectin 3) had reduction in left atrial volume, those above median did not. Although LCZ696 reduced N-terminal pro B-type natriuretic peptide, levels of the other 4 biomarkers were not affected over time. In patients with heart failure with preserved ejection fraction, biomarkers that reflect collagen homeostasis correlated with the presence and severity of disease and underlying pathophysiology, and may modify the structural response to treatment. URL: http://www.clinicaltrials.gov. Unique identifier: NCT00887588. © 2016 American Heart Association, Inc.

Male Sterile2 Encodes a Plastid-Localized Fatty Acyl Carrier Protein Reductase Required for Pollen Exine Development in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, W.; Shanklin, J.; Yu, X.-H.

Male Sterile2 (MS2) is predicted to encode a fatty acid reductase required for pollen wall development in Arabidopsis (Arabidopsis thaliana). Transient expression of MS2 in tobacco (Nicotiana benthamiana) leaves resulted in the accumulation of significant levels of C16 and C18 fatty alcohols. Expression of MS2 fused with green fluorescent protein revealed that an amino-terminal transit peptide targets the MS2 to plastids. The plastidial localization of MS2 is biologically important because genetic complementation of MS2 in ms2 homozygous plants was dependent on the presence of its amino-terminal transit peptide or that of the Rubisco small subunit protein amino-terminal transit peptide. Inmore » addition, two domains, NAD(P)H-binding domain and sterile domain, conserved in MS2 and its homologs were also shown to be essential for MS2 function in pollen exine development by genetic complementation testing. Direct biochemical analysis revealed that purified recombinant MS2 enzyme is able to convert palmitoyl-Acyl Carrier Protein to the corresponding C16:0 alcohol with NAD(P)H as the preferred electron donor. Using optimized reaction conditions (i.e. at pH 6.0 and 30 C), MS2 exhibits a K{sub m} for 16:0-Acyl Carrier Protein of 23.3 {+-} 4.0 {mu}m, a V{sub max} of 38.3 {+-} 4.5 nmol mg{sup -1} min{sup -1}, and a catalytic efficiency/K{sub m} of 1,873 m{sup -1} s{sup -1}. Based on the high homology of MS2 to other characterized fatty acid reductases, it was surprising that MS2 showed no activity against palmitoyl- or other acyl-coenzyme A; however, this is consistent with its plastidial localization. In summary, genetic and biochemical evidence demonstrate an MS2-mediated conserved plastidial pathway for the production of fatty alcohols that are essential for pollen wall biosynthesis in Arabidopsis.« less

Analysis of the HLA-DR peptidome from human dendritic cells reveals high affinity repertoires and nonconventional pathways of peptide generation.

PubMed

Ciudad, M Teresa; Sorvillo, Nicoletta; van Alphen, Floris P; Catalán, Diego; Meijer, Alexander B; Voorberg, Jan; Jaraquemada, Dolores

2017-01-01

Dendritic cells (DCs) are the major professional APCs of the immune system; however, their MHC-II-associated peptide repertoires have been hard to analyze, mostly because of their scarce presence in blood and tissues. In vitro matured human monocyte-derived DCs (MoDCs) are widely used as professional APCs in experimental systems. In this work, we have applied mass spectrometry to identify the HLA-DR-associated self-peptide repertoires from small numbers of mature MoDCs (∼5 × 10 6 cells), derived from 7 different donors. Repertoires of 9 different HLA-DR alleles were defined from analysis of 1319 peptides, showing the expected characteristics of MHC-II-associated peptides. Most peptides identified were predicted high binders for their respective allele, formed nested sets, and belonged to endo-lysosomal pathway-degraded proteins. Approximately 20% of the peptides were derived from cytosolic and nuclear proteins, a recurrent finding in HLA-DR peptide repertoires. Of interest, most of these peptides corresponded to single sequences, did not form nested sets, and were located at the C terminus of the parental protein, which suggested alternative processing. Analysis of cleavage patterns for terminal peptides predominantly showed aspartic acid before the cleavage site of both C- and N-terminal peptides and proline immediately after the cleavage site in C-terminal peptides. Proline was also frequent next to the cut sites of internal peptides. These data provide new insights into the Ag processing capabilities of DCs. The relevance of these processing pathways and their contribution to response to infection, tolerance induction, or autoimmunity deserve further analysis. © Society for Leukocyte Biology.

Ranatensin-HL: A Bombesin-Related Tridecapeptide from the Skin Secretion of the Broad-Folded Frog, Hylarana latouchii.

PubMed

Lin, Yan; Chen, Tianbao; Zhou, Mei; Wang, Lei; Su, Songkun; Shaw, Chris

2017-07-04

Bombesin-related peptides are a family of peptides whose prototype was discovered in amphibian skin and which exhibit a wide range of biological activities. Since the initial isolation of bombesin from Bombina bombina skin, diverse forms of bombesin-related peptides have been found in the skins across Anura. In this study, a novel bombesin-related peptide of the ranatensin subfamily, named ranatensin-HL, was structurally-characterised from the skin secretion of the broad-folded frog, Hylarana latouchii , through combination of molecular cloning and mass spectrometric methodologies. It is composed of 13 amino acid residues, pGlu-RAGNQWAIGHFM-NH₂, and resembles an N-terminally extended form of Xenopus neuromedin B. Ranatensin-HL and its C-terminal decapeptide (ranatensin-HL-10) were chemically synthesised and subjected to in vitro smooth muscle assays in which they were found to display moderate stimulatory effects on rat urinary bladder and uterus smooth muscles with EC 50 values in the range of 1-10 nM. The prepro-ranatensin-HL was highly homological to a bombesin-like peptide from Rana catesbeiana at both nucleotide and amino acid levels, which might provide a clue for the taxonomic classification of ranid frogs in the future.

Sialogogic activity in the rat of peptides analogous to [Tyr8]-substance P in which substitutions have been made in the N-terminal amino acids.

PubMed

Higa, K; Gao, C; Motokawa, W; Abe, K

2001-04-01

In order to elucidate the regulatory roles for salivation of amino acids in positions 1-4 of the N-terminal region of [Tyr8]-substance P (SP), the structure-sialogogic activity correlations of various synthetic octa- to undecapeptides replaced in positions 1-4 of [Tyr8]-SP with each of 19 common amino acids, one by one, and with the same sequence of the C-terminal hepatapeptide as that of [Tyr8]-SP, were studied in the submandibular glands of rats after intraperitoneal injection. Each of 19 octa-, nona-, deca- and undecapeptides with replaced amino acids and a penta- to decapeptide with the progressive elimination of the N-terminal portion were newly synthesized by the multipin peptide method. All octa- to undecapeptides replaced with each of 19 common amino acids in positions 1-4 had sialogogic activities. In 19 octa- and decapeptides in which P4 and P2 had been replaced, four and three replacements, respectively, had significantly increased secretory activities. In contrast, in 19 nonapeptides in which K3 had been replaced, none had significantly increased secretory activities. Furthermore, in 19 undecapeptides in which R1 had been replaced, most replacements had significantly increased or equipotent activities for fluid secretion. It is concluded that amino acids in the N-terminal region of various tachykinins may not need to be strictly conserved and that amino acid residues in the N-terminal portion, R1 in particular and P2, may strongly inhibit secretory activity.

Synthetic signal sequences that enable efficient secretory protein production in the yeast Kluyveromyces marxianus.

PubMed

Yarimizu, Tohru; Nakamura, Mikiko; Hoshida, Hisashi; Akada, Rinji

2015-02-14

Targeting of cellular proteins to the extracellular environment is directed by a secretory signal sequence located at the N-terminus of a secretory protein. These signal sequences usually contain an N-terminal basic amino acid followed by a stretch containing hydrophobic residues, although no consensus signal sequence has been identified. In this study, simple modeling of signal sequences was attempted using Gaussia princeps secretory luciferase (GLuc) in the yeast Kluyveromyces marxianus, which allowed comprehensive recombinant gene construction to substitute synthetic signal sequences. Mutational analysis of the GLuc signal sequence revealed that the GLuc hydrophobic peptide length was lower limit for effective secretion and that the N-terminal basic residue was indispensable. Deletion of the 16th Glu caused enhanced levels of secreted protein, suggesting that this hydrophilic residue defined the boundary of a hydrophobic peptide stretch. Consequently, we redesigned this domain as a repeat of a single hydrophobic amino acid between the N-terminal Lys and C-terminal Glu. Stretches consisting of Phe, Leu, Ile, or Met were effective for secretion but the number of residues affected secretory activity. A stretch containing sixteen consecutive methionine residues (M16) showed the highest activity; the M16 sequence was therefore utilized for the secretory production of human leukemia inhibitory factor protein in yeast, resulting in enhanced secreted protein yield. We present a new concept for the provision of secretory signal sequence ability in the yeast K. marxianus, determined by the number of residues of a single hydrophobic residue located between N-terminal basic and C-terminal acidic amino acid boundaries.

Structure determination of a peptide model of the repeated helical domain in Samia cynthia ricini silk fibroin before spinning by a combination of advanced solid-state NMR methods.

PubMed

Nakazawa, Yasumoto; Asakura, Tetsuo

2003-06-18

Fibrous proteins unlike globular proteins, contain repetitive amino acid sequences, giving rise to very regular secondary protein structures. Silk fibroin from a wild silkworm, Samia cynthia ricini, consists of about 100 repeats of alternating polyalanine (poly-Ala) regions of 12-13 residues in length and Gly-rich regions. In this paper, the precise structure of the model peptide, GGAGGGYGGDGG(A)(12)GGAGDGYGAG, which is a typical repeated sequence of the silk fibroin, was determined using a combination of three kinds of solid-state NMR studies; a quantitative use of (13)C CP/MAS NMR chemical shift with conformation-dependent (13)C chemical shift contour plots, 2D spin diffusion (13)C solid-state NMR under off magic angle spinning and rotational echo double resonance. The structure of the model peptide corresponding to the silk fibroin structure before spinning was determined. The torsion angles of the central Ala residue, Ala(19), in the poly-Ala region were determined to be (phi, psi) = (-59 degrees, -48 degrees ) which are values typically associated with alpha-helical structures. However, the torsion angles of the Gly(25) residue adjacent to the C-terminal side of the poly-Ala chain were determined to be (phi, psi) = (-66 degrees, -22 degrees ) and those of Gly(12) and Ala(13) residues at the N-terminal of the poly-Ala chain to be (phi, psi) = (-70 degrees, -30 degrees ). In addition, REDOR experiments indicate that the torsion angles of the two C-terminal Ala residues, Ala(23) and Ala(24), are (phi, psi) = (-66 degrees, -22 degrees ) and those of N-terminal two Ala residues, Ala(13) and Ala(14) are (phi, psi) = (-70 degrees, -30 degrees ). Thus, the local structure of N-terminal and C-terminal residues, and also the neighboring residues of alpha-helical poly-Ala chain in the model peptide is a more strongly wound structure than found in typical alpha-helix structures.

Structure and biological activity of glucagon and glucagon-like peptide from a primitive bony fish, the bowfin (Amia calva).

PubMed

Conlon, J M; Youson, J H; Mommsen, T P

1993-11-01

The bowfin, Amia calva (order Amiiformes) occupies an important position in phylogeny as a surviving representative of a group of primitive ray-finned fishes from which the present-day teleosts may have evolved. Glucagon and glucagon-like peptide (GLP) were isolated from an extract of bowfin pancreas and their primary structures determined. Bowfin glucagon shows only four amino acid substitutions compared with human glucagon, and bowfin glucagon was equipotent and equally effective as human glucagon in stimulation of glycogenolysis in dispersed hepatocytes from a teleost fish, the copper rockfish, Sebastes caurinus. In contrast, bowfin GLP shows 15 amino acid substitutions and three amino acid deletions compared with the corresponding region of human GLP-1-(7-37)-peptide. In particular, the bowfin peptide contains an N-terminal tyrosine residue rather than the N-terminal histidine residue found in all other glucagon-related peptides so far characterized. Bowfin GLP stimulated glycogenolysis in rockfish hepatocytes, but was 3-fold less effective and 23-fold less potent than human GLP-1-(7-37)-peptide. We speculate that selective mutations in the GLP domain of bowfin preproglucagon may be an adaptive response to the previously demonstrated low biological potency of bowfin insulin.

Structure and biological activity of glucagon and glucagon-like peptide from a primitive bony fish, the bowfin (Amia calva).

PubMed Central

Conlon, J M; Youson, J H; Mommsen, T P

1993-01-01

The bowfin, Amia calva (order Amiiformes) occupies an important position in phylogeny as a surviving representative of a group of primitive ray-finned fishes from which the present-day teleosts may have evolved. Glucagon and glucagon-like peptide (GLP) were isolated from an extract of bowfin pancreas and their primary structures determined. Bowfin glucagon shows only four amino acid substitutions compared with human glucagon, and bowfin glucagon was equipotent and equally effective as human glucagon in stimulation of glycogenolysis in dispersed hepatocytes from a teleost fish, the copper rockfish, Sebastes caurinus. In contrast, bowfin GLP shows 15 amino acid substitutions and three amino acid deletions compared with the corresponding region of human GLP-1-(7-37)-peptide. In particular, the bowfin peptide contains an N-terminal tyrosine residue rather than the N-terminal histidine residue found in all other glucagon-related peptides so far characterized. Bowfin GLP stimulated glycogenolysis in rockfish hepatocytes, but was 3-fold less effective and 23-fold less potent than human GLP-1-(7-37)-peptide. We speculate that selective mutations in the GLP domain of bowfin preproglucagon may be an adaptive response to the previously demonstrated low biological potency of bowfin insulin. PMID:8240302

The Human Leukocyte Antigen (HLA)-B27 Peptidome in Vivo, in Spondyloarthritis-susceptible HLA-B27 Transgenic Rats and the Effect of Erap1 Deletion *

PubMed Central

Barnea, Eilon; Melamed Kadosh, Dganit; Haimovich, Yael; Satumtira, Nimman; Dorris, Martha L.; Nguyen, Mylinh T.; Hammer, Robert E.; Tran, Tri M.; Colbert, Robert A.; Taurog, Joel D.

2017-01-01

HLA-B27 is a class I major histocompatibility (MHC-I) allele that confers susceptibility to the rheumatic disease ankylosing spondylitis (AS) by an unknown mechanism. ERAP1 is an aminopeptidase that trims peptides in the endoplasmic reticulum for binding to MHC-I molecules. ERAP1 shows genetic epistasis with HLA-B27 in conferring susceptibility to AS. Male HLA-B27 transgenic rats develop arthritis and serve as an animal model of AS, whereas female B27 transgenic rats remain healthy. We used large scale quantitative mass spectrometry to identify over 15,000 unique HLA-B27 peptide ligands, isolated after immunoaffinity purification of the B27 molecules from the spleens of HLA-B27 transgenic rats. Heterozygous deletion of Erap1, which reduced the Erap1 level to less than half, had no qualitative or quantitative effects on the B27 peptidome. Homozygous deletion of Erap1 affected approximately one-third of the B27 peptidome but left most of the B27 peptidome unchanged, suggesting the possibility that some of the HLA-B27 immunopeptidome is not processed in the presence of Erap1. Deletion of Erap1 was permissive for the AS-like phenotype, increased mean peptide length and increased the frequency of C-terminal hydrophobic residues and of N-terminal Ala, Ser, or Lys. The presence of Erap1 increased the frequency of C-terminal Lys and Arg, of Glu and Asp at intermediate residues, and of N-terminal Gly. Several peptides of potential interest in AS pathogenesis, previously identified in human cell lines, were isolated. However, rats susceptible to arthritis had B27 peptidomes similar to those of non-susceptible rats, and no peptides were found to be uniquely associated with arthritis. Whether specific B27-bound peptides are required for AS pathogenesis remains to be determined. Data are available via ProteomeXchange with identifier PXD005502. PMID:28188227

Crystal Structure of Ribosome-Inactivating Protein Ricin A Chain in Complex with the C-Terminal Peptide of the Ribosomal Stalk Protein P2.

PubMed

Shi, Wei-Wei; Tang, Yun-Sang; Sze, See-Yuen; Zhu, Zhen-Ning; Wong, Kam-Bo; Shaw, Pang-Chui

2016-10-13

Ricin is a type 2 ribosome-inactivating protein (RIP), containing a catalytic A chain and a lectin-like B chain. It inhibits protein synthesis by depurinating the N-glycosidic bond at α-sarcin/ricin loop (SRL) of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation center of the ribosome. Here, we present the 1.6 Å crystal structure of Ricin A chain (RTA) complexed to the C-terminal peptide of the ribosomal stalk protein P2, which plays a crucial role in specific recognition of elongation factors and recruitment of eukaryote-specific RIPs to the ribosomes. Our structure reveals that the C-terminal GFGLFD motif of P2 peptide is inserted into a hydrophobic pocket of RTA, while the interaction assays demonstrate the structurally untraced SDDDM motif of P2 peptide contributes to the interaction with RTA. This interaction mode of RTA and P protein is in contrast to that with trichosanthin (TCS), Shiga-toxin (Stx) and the active form of maize RIP (MOD), implying the flexibility of the P2 peptide-RIP interaction, for the latter to gain access to ribosome.

Cn-AMP2 from green coconut water is an anionic anticancer peptide.

PubMed

Prabhu, Saurabh; Dennison, Sarah R; Mura, Manuela; Lea, Robert W; Snape, Timothy J; Harris, Frederick

2014-12-01

Globally, death due to cancers is likely to rise to over 20 million by 2030, which has created an urgent need for novel approaches to anticancer therapies such as the development of host defence peptides. Cn-AMP2 (TESYFVFSVGM), an anionic host defence peptide from green coconut water of the plant Cocos nucifera, showed anti-proliferative activity against the 1321N1 and U87MG human glioma cell lines with IC50 values of 1.25 and 1.85 mM, respectively. The membrane interactive form of the peptide was found to be an extended conformation, which primarily included β-type structures (levels > 45%) and random coil architecture (levels > 45%). On the basis of these and other data, it is suggested that the short anionic N-terminal sequence (TES) of Cn-AMP2 interacts with positively charged moieties in the cancer cell membrane. Concomitantly, the long hydrophobic C-terminal sequence (YFVFSVGM) of the peptide penetrates the membrane core region, thereby driving the translocation of Cn-AMP2 across the cancer cell membrane to attack intracellular targets and induce anti-proliferative mechanisms. This work is the first to demonstrate that anionic host defence peptides have activity against human glioblastoma, which potentially provides an untapped source of lead compounds for development as novel agents in the treatment of these and other cancers. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Versatile platform for nanotechnology based on circular permutations of chaperonin protein

NASA Technical Reports Server (NTRS)

McMillan, R. Andrew (Inventor); Kagawa, Hiromi (Inventor); Paavola, Chad D. (Inventor); Chan, Suzanne L. (Inventor); Li, Yi-Fen (Inventor); Trent, Jonathan D. (Inventor)

2010-01-01

The present invention provides chaperonin polypeptides which are modified to include N-terminal and C-terminal ends that are relocated from the central pore region to various different positions in the polypeptide which are located on the exterior of the folded modified chaperonin polypeptide. In the modified chaperonin polypeptide, the naturally-occurring N-terminal and C-terminal ends are joined together directly or with an intervening linker peptide sequence. The relocated N-terminal or C-terminal ends can be covalently joined to, or bound with another molecule such as a nucleic acid molecule, a lipid, a carbohydrate, a second polypeptide, or a nanoparticle. The modified chaperonin polypeptides can assemble into double-ringed chaperonin structures. Further, the chaperonin structures can organize into higher order structures such as nanofilaments or nanoarrays which can be used to produce nanodevices and nanocoatings.

The β1a Subunit of the Skeletal DHPR Binds to Skeletal RyR1 and Activates the Channel via Its 35-Residue C-Terminal Tail

PubMed Central

Rebbeck, Robyn T.; Karunasekara, Yamuna; Gallant, Esther M.; Board, Philip G.; Beard, Nicole A.; Casarotto, Marco G.; Dulhunty, Angela F.

2011-01-01

Although it has been suggested that the C-terminal tail of the β1a subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca2+ release in skeletal muscle by interacting with the skeletal ryanodine receptor (RyR1), a direct functional interaction between the two proteins has not been demonstrated previously. Such an interaction is reported here. A peptide with the sequence of the C-terminal 35 residues of β1a bound to RyR1 in affinity chromatography. The full-length β1a subunit and the C-terminal peptide increased [3H]ryanodine binding and RyR1 channel activity with an AC50 of 450–600 pM under optimal conditions. The effect of the peptide was dependent on cytoplasmic Ca2+, ATP, and Mg2+ concentrations. There was no effect of the peptide when channel activity was very low as a result of Mg2+ inhibition or addition of 100 nM Ca2+ (without ATP). Maximum increases were seen with 1–10 μM Ca2+, in the absence of Mg2+ inhibition. A control peptide with the C-terminal 35 residues in a scrambled sequence did not bind to RyR1 or alter [3H]ryanodine binding or channel activity. This high-affinity in vitro functional interaction between the C-terminal 35 residues of the DHPR β1a subunit and RyR1 may support an in vivo function of β1a during voltage-activated Ca2+ release. PMID:21320436

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

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- OCH 3 (1) shows a prominent [M + H - C 4 H 8 ] + ion, which is totally absent for its positional isomer Boc-NH-ε-Caa-L-Ala-OCH 3 (6), which instead shows significant loss of t-butanol. The formation of the [M + Cat - C 4 H 8 ] + 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- OCH 3 (1), whereas the former is highly abundant and the latter is of low abundance for its positional isomer Boc-NH-ε-Caa-L-Ala-OCH 3 (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 provide sequencing information, the structure of the cyclic intermediate involved in the formation of the rearrangement ion, and distinguish a pair of dipeptide positional isomers.

Insulation of the Chicken β-Globin Chromosomal Domain from a Chromatin-Condensing Protein, MENT

PubMed Central

Istomina, Natalia E.; Shushanov, Sain S.; Springhetti, Evelyn M.; Karpov, Vadim L.; A. Krasheninnikov, Igor; Stevens, Kimberly; Zaret, Kenneth S.; Singh, Prim B.; Grigoryev, Sergei A.

2003-01-01

Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken β-globin domain. We observed two sharp transitions of MENT concentration coinciding with the β-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation. PMID:12944473

Diverse Peptide Hormones Affecting Root Growth Identified in the Medicago truncatula Secreted Peptidome.

PubMed

Patel, Neha; Mohd-Radzman, Nadiatul A; Corcilius, Leo; Crossett, Ben; Connolly, Angela; Cordwell, Stuart J; Ivanovici, Ariel; Taylor, Katia; Williams, James; Binos, Steve; Mariani, Michael; Payne, Richard J; Djordjevic, Michael A

2018-01-01

Multigene families encoding diverse secreted peptide hormones play important roles in plant development. A need exists to efficiently elucidate the structures and post-translational-modifications of these difficult-to-isolate peptide hormones in planta so that their biological functions can be determined. A mass spectrometry and bioinformatics approach was developed to comprehensively analyze the secreted peptidome of Medicago hairy root cultures and xylem sap. We identified 759 spectra corresponding to the secreted products of twelve peptide hormones including four CEP ( C -TERMINALLY E NCODED P EPTIDE), two CLE ( CL V3/ E NDOSPERM SURROUNDING REGION RELATED) and six XAP ( X YLEM SAP A SSOCIATED P EPTIDE) peptides. The MtCEP1, MtCEP2, MtCEP5 and MtCEP8 peptides identified differed in post-translational-modifications. Most were hydroxylated at conserved proline residues but some MtCEP1 derivatives were tri-arabinosylated. In addition, many CEP peptides possessed unexpected N - and C -terminal extensions. The pattern of these extensions suggested roles for endo- and exoproteases in CEP peptide maturation. Longer than expected, hydroxylated and homogeneously modified mono- and tri-arabinosylated CEP peptides corresponding to their in vivo structures were chemically synthesized to probe the effect of these post-translational-modifications on function. The ability of CEP peptides to elevate root nodule number was increased by hydroxylation at key positions. MtCEP1 peptides with N -terminal extensions or with tri-arabinosylation modification, however, were unable to impart increased nodulation. The MtCLE5 and MtCLE17 peptides identified were of precise size, and inhibited main root growth and increased lateral root number. Six XAP peptides, each beginning with a conserved DY sulfation motif, were identified including MtXAP1a, MtXAP1b, MtXAP1c, MtXAP3, MtXAP5 and MtXAP7. MtXAP1a and MtXAP5 inhibited lateral root emergence. Transcriptional analyses demonstrated peptide hormone gene expression in the root vasculature and tip. Since hairy roots can be induced on many plants, their corresponding root cultures may represent ideal source materials to efficiently identify diverse peptide hormones in vivo in a broad range of species. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

A peptidases-resistant glycosylated analogue of substance P-(5-11). Specificity towards substance P receptors.

PubMed

Poujade, C; Lavielle, S; Torrens, Y; Beaujouan, J C; Glowinski, J; Marquet, A

1984-09-01

Glycosylated analogues of the C-terminal heptapeptide of substance P either free or blocked on the N-terminal glutamine were synthesized in order to develop a metabolically stable peptide that would have an increased specificity for one type of receptor. Of the analogue described, (N-alpha-Boc-beta-D-Glc-p (1----5) Gln) -Gln-Phe-Phe-Gly-Leu-Met-NH2 is highly resistant to degradation on exposure to rat hypothalamic slices. This glycosylated peptide is about one third as potent as substance P in eliciting contractions of the guinea-pig ileum and is almost devoided of affinity for the 125I-Bolton Hunter-SP specific binding sites on rat brain synaptosomes.

Tyrosinase-catalyzed site-specific immobilization of engineered C-phycocyanin to surface

PubMed Central

Faccio, Greta; Kämpf, Michael M.; Piatti, Chiara; Thöny-Meyer, Linda; Richter, Michael

2014-01-01

Enzymatic crosslinking of proteins is often limited by the steric availability of the target residues, as of tyrosyl side chains in the case of tyrosinase. Carrying an N-terminal peptide-tag containing two tyrosine residues, the fluorescent protein C-phycocyanin HisCPC from Synechocystis sp. PCC6803 was crosslinked to fluorescent high-molecular weight forms with tyrosinase. Crosslinking with tyrosinase in the presence of L-tyrosine produced non fluorescent high-molecular weight products. Incubated in the presence of tyrosinase, HisCPC could also be immobilized to amino-modified polystyrene beads thus conferring a blue fluorescence. Crosslinking and immobilization were site-specific as both processes required the presence of the N-terminal peptide in HisCPC. PMID:24947668

Identification of a maize nucleic acid-binding protein (NBP) belonging to a family of nuclear-encoded chloroplast proteins.

PubMed Central

Cook, W B; Walker, J C

1992-01-01

A cDNA encoding a nuclear-encoded chloroplast nucleic acid-binding protein (NBP) has been isolated from maize. Identified as an in vitro DNA-binding activity, NBP belongs to a family of nuclear-encoded chloroplast proteins which share a common domain structure and are thought to be involved in posttranscriptional regulation of chloroplast gene expression. NBP contains an N-terminal chloroplast transit peptide, a highly acidic domain and a pair of ribonucleoprotein consensus sequence domains. NBP is expressed in a light-dependent, organ-specific manner which is consistent with its involvement in chloroplast biogenesis. The relationship of NBP to the other members of this protein family and their possible regulatory functions are discussed. Images PMID:1346929

Studies on lactoferricin-derived Escherichia coli membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides.

PubMed

Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

2011-06-17

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.

Studies on Lactoferricin-derived Escherichia coli Membrane-active Peptides Reveal Differences in the Mechanism of N-Acylated Versus Nonacylated Peptides*

PubMed Central

Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E.; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

2011-01-01

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of Gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis. PMID:21515687

Opioid and neurokinin activities of substance P fragments and their analogs.

PubMed

Lei, S Z; Lipkowski, A W; Wilcox, G L

1991-02-07

Newly developed substance P (SP) analogs with altered N-terminal sequences which equalize the lipophilicity of the N-terminal and C-terminal elements and of their fusion product were examined using i.t. injection in mice. I.t. injection of either the full length analog or the C-terminal hexapeptide (CP) produced biting and scratching behavior similar to that elicited by SP. SPF was approximately 5-fold and CP 14-fold less potent than native SP. The N-terminal peptide (NP) was inactive by itself but inhibited CP-elicited behavior. Naloxone antagonized this action of NP and shifted the SPF dose-response curve 4-fold to the left. However, naloxone had no effect on the action of CP or on the action of any of the native neurokinins. The results are consistent with the hypothesis that N- and C-terminal analogs of SP can have opioid and SP-like actions, respectively, in the CNS of rodents. Furthermore, analogs of SP which include at least the terminal tetrapeptide retain neurokinin activity.

Peptide Handling by HLA-B27 Subtypes Influences Their Biological Behavior, Association with Ankylosing Spondylitis and Susceptibility to Endoplasmic Reticulum Aminopeptidase 1 (ERAP1)*

PubMed Central

García-Medel, Noel; Sanz-Bravo, Alejandro; Alvarez-Navarro, Carlos; Gómez-Molina, Patricia; Barnea, Eilon; Marcilla, Miguel; Admon, Arie; de Castro, José A. López

2014-01-01

HLA-B27 is strongly associated with ankylosing spondylitis (AS). We analyzed the relationship between structure, peptide specificity, folding, and stability of the seven major HLA-B27 subtypes to determine the role of their constitutive peptidomes in the pathogenicity of this molecule. Identification of large numbers of ligands allowed us to define the differences among subtype-bound peptidomes and to elucidate the peptide features associated with AS and molecular stability. The peptides identified only in AS-associated or high thermostability subtypes with identical A and B pockets were longer and had bulkier and more diverse C-terminal residues than those found only among non-AS-associated/lower-thermostability subtypes. Peptides sequenced from all AS-associated subtypes and not from non-AS-associated ones, thus strictly correlating with disease, were very rare. Residue 116 was critical in determining peptide binding, thermodynamic properties, and folding, thus emerging as a key feature that unified HLA-B27 biology. HLA-B27 ligands were better suited to TAP transport than their N-terminal precursors, and AS-associated subtype ligands were better than those from non-AS-associated subtypes, suggesting a particular capacity of AS-associated subtypes to bind epitopes directly produced in the cytosol. Peptides identified only from AS-associated/high-thermostability subtypes showed a higher frequency of ERAP1-resistant N-terminal residues than ligands found only in non-AS-associated/low-thermostability subtypes, reflecting a more pronounced effect of ERAP1 on the former group. Our results reveal the basis for the relationship between peptide specificity and other features of HLA-B27, provide a unified view of HLA-B27 biology and pathogenicity, and suggest a larger influence of ERAP1 polymorphism on AS-associated than non-AS-associated subtypes. PMID:25187574

Peptide handling by HLA-B27 subtypes influences their biological behavior, association with ankylosing spondylitis and susceptibility to endoplasmic reticulum aminopeptidase 1 (ERAP1).

PubMed

García-Medel, Noel; Sanz-Bravo, Alejandro; Alvarez-Navarro, Carlos; Gómez-Molina, Patricia; Barnea, Eilon; Marcilla, Miguel; Admon, Arie; de Castro, José A López

2014-12-01

HLA-B27 is strongly associated with ankylosing spondylitis (AS). We analyzed the relationship between structure, peptide specificity, folding, and stability of the seven major HLA-B27 subtypes to determine the role of their constitutive peptidomes in the pathogenicity of this molecule. Identification of large numbers of ligands allowed us to define the differences among subtype-bound peptidomes and to elucidate the peptide features associated with AS and molecular stability. The peptides identified only in AS-associated or high thermostability subtypes with identical A and B pockets were longer and had bulkier and more diverse C-terminal residues than those found only among non-AS-associated/lower-thermostability subtypes. Peptides sequenced from all AS-associated subtypes and not from non-AS-associated ones, thus strictly correlating with disease, were very rare. Residue 116 was critical in determining peptide binding, thermodynamic properties, and folding, thus emerging as a key feature that unified HLA-B27 biology. HLA-B27 ligands were better suited to TAP transport than their N-terminal precursors, and AS-associated subtype ligands were better than those from non-AS-associated subtypes, suggesting a particular capacity of AS-associated subtypes to bind epitopes directly produced in the cytosol. Peptides identified only from AS-associated/high-thermostability subtypes showed a higher frequency of ERAP1-resistant N-terminal residues than ligands found only in non-AS-associated/low-thermostability subtypes, reflecting a more pronounced effect of ERAP1 on the former group. Our results reveal the basis for the relationship between peptide specificity and other features of HLA-B27, provide a unified view of HLA-B27 biology and pathogenicity, and suggest a larger influence of ERAP1 polymorphism on AS-associated than non-AS-associated subtypes. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Pyrazole amino acids: hydrogen bonding directed conformations of 3-amino-1H-pyrazole-5-carboxylic acid residue.

PubMed

Kusakiewicz-Dawid, Anna; Porada, Monika; Ochędzan-Siodłak, Wioletta; Broda, Małgorzata A; Bujak, Maciej; Siodłak, Dawid

2017-09-01

A series of model compounds containing 3-amino-1H-pyrazole-5-carboxylic acid residue with N-terminal amide/urethane and C-terminal amide/hydrazide/ester groups were investigated by using NMR, Fourier transform infrared, and single-crystal X-ray diffraction methods, additionally supported by theoretical calculations. The studies demonstrate that the most preferred is the extended conformation with torsion angles ϕ and ψ close to ±180°. The studied 1H-pyrazole with N-terminal amide/urethane and C-terminal amide/hydrazide groups solely adopts this energetically favored conformation confirming rigidity of that structural motif. However, when the C-terminal ester group is present, the second conformation with torsion angles ϕ and ψ close to ±180° and 0°, respectively, is accessible. The conformational equilibrium is observed in NMR and Fourier transform infrared studies in solution in polar environment as well as in the crystal structures of other related compounds. The observed conformational preferences are clearly related to the presence of intramolecular interactions formed within the studied residue. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Alkynyl-Containing Peptides of Marine Origin: A Review

PubMed Central

Chai, Qiu-Ye; Yang, Zhen; Lin, Hou-Wen; Han, Bing-Nan

2016-01-01

Since the 1990s, a number of terminal alkynyl residue-containing cyclic/acyclic peptides have been identified from marine organisms, especially cyanobacteria and marine mollusks. This review has presented 66 peptides, which covers over 90% marine peptides with terminal alkynyl fatty acyl units. In fact, more than 90% of these peptides described in the literature are of cyanobacterial origin. Interestingly, all the linear peptides featured with terminal alkyne were solely discovered from marine cyanobacteria. The objective of this article is to provide an overview on the types, structural characterization of these unusual terminal alkynyl fatty acyl units, as well as the sources and biological functions of their composed peptides. Many of these peptides have a variety of biological activities, including antitumor, antibacterial, antimalarial, etc. Further, we have also discussed the evident biosynthetic origin responsible for formation of terminal alkynes of natural PKS (polyketide synthase)/NRPS (nonribosome peptide synthetase) hybrids. PMID:27886049

Prohormone convertases differentially process pro-neurotensin/neuromedin N in tissues and cell lines.

PubMed

Kitabgi, Patrick

2006-08-01

Neurotensin (NT) is synthesized as part of a larger precursor that also contains neuromedin N (NN), a six-amino acid neurotensin-like peptide. NT and NN are located in the C-terminal region of the precursor (pro-NT/NN) where they are flanked and separated by three Lys-Arg sequences. A fourth dibasic sequence is present in the middle of the precursor. Dibasics are the consensus sites recognized and cleaved by specialized endoproteases that belong to the family of proprotein convertases (PCs). In tissues that express pro-NT/NN, the three C-terminal Lys-Arg sites are differentially processed, whereas the middle dibasic is poorly cleaved. Processing gives rise mainly to NT and NN in the brain, to NT and a large peptide with a C-terminal NN moiety (large NN) in the gut, and to NT, large NN, and a large peptide with a C-terminal NT moiety (large NT) in the adrenals. Recent evidence indicates that PC1, PC2, and PC5-A are the prohormone convertases responsible for the processing patterns observed in the gut, brain, and adrenals, respectively. As NT, NN, large NT, and large NN are all endowed with biological activity, the evidence reviewed in this paper supports the idea that posttranslational processing of pro-NT/NN in tissues may generate biological diversity of pathophysiological relevance.

Neurotensin and neuromedin N are differentially processed from a common precursor by prohormone convertases in tissues and cell lines.

PubMed

Kitabgi, Patrick

2010-01-01

Neurotensin (NT) is synthesized as part of a larger precursor that also contains neuromedin N (NN), a six amino acid NT-like peptide. NT and NN are located in the C-terminal region of the precursor (pro-NT/NN) where they are flanked and separated by three Lys-Arg sequences. A fourth dibasic sequence is present in the middle of the precursor. Dibasics are the consensus sites recognized and cleaved by specialized endoproteases that belong to the family of proprotein convertases (PCs). In tissues that express pro-NT/NN, the three C-terminal Lys-Arg sites are differentially processed, whereas the middle dibasic is poorly cleaved. Processing gives rise mainly to NT and NN in the brain, NT and a large peptide with a C-terminal NN moiety (large NN) in the gut, and NT, large NN, and a large peptide with a C-terminal NT moiety (large NT) in the adrenals. Recent evidence indicates that PC1, PC2, and PC5-A are the prohormone convertases responsible for the processing patterns observed in the gut, brain, and adrenals, respectively. As NT, NN, large NT, and large NN are all endowed with biological activity, the evidence reviewed here supports the idea that posttranslational processing of pro-NT/NN in tissues may generate biological diversity of pathophysiological relevance.

The catalytic chain of human complement subcomponent C1r. Purification and N-terminal amino acid sequences of the major cyanogen bromide-cleavage fragments.

PubMed

Arlaud, G J; Gagnon, J; Porter, R R

1982-01-01

1. The a- and b-chains of reduced and alkylated human complement subcomponent C1r were separated by high-pressure gel-permeation chromatography and isolated in good yield and in pure form. 2. CNBr cleavage of C1r b-chain yielded eight major peptides, which were purified by gel filtration and high-pressure reversed-phase chromatography. As determined from the sum of their amino acid compositions, these peptides accounted for a minimum molecular weight of 28 000, close to the value 29 100 calculated from the whole b-chain. 3. N-Terminal sequence determinations of C1r b-chain and its CNBr-cleavage peptides allowed the identification of about two-thirds of the amino acids of C1r b-chain. From our results, and on the basis of homology with other serine proteinases, an alignment of the eight CNBr-cleavage peptides from C1r b-chain is proposed. 4. The residues forming the 'charge-relay' system of the active site of serine proteinases (His-57, Asp-102 and Ser-195 in the chymotrypsinogen numbering) are found in the corresponding regions of C1r b-chain, and the amino acid sequence around these residues has been determined. 5. The N-terminal sequence of C1r b-chain has been extended to residue 60 and reveals that C1r b-chain lacks the 'histidine loop', a disulphide bond that is present in all other known serine proteinases.

Effects of Hydrophilic Residues and Hydrophobic Length on Flip-Flop Promotion by Transmembrane Peptides.

PubMed

Nakao, Hiroyuki; Hayashi, Chihiro; Ikeda, Keisuke; Saito, Hiroaki; Nagao, Hidemi; Nakano, Minoru

2018-04-19

Peptide-induced phospholipid flip-flop (scrambling) was evaluated using transmembrane model peptides in which the central residue was substituted with various amino acid residues (sequence: Ac-GKK(LA) n XW(LA) n LKKA-CONH 2 ). Peptides with a strongly hydrophilic residue (X = Q, N, or H) had higher scramblase activity than that of other peptides, and the activity was also dependent on the length of the peptides. Peptides with a hydrophobic stretch of 17 residues showed high flip-promotion propensity, whereas those of 21 and 25 residues did not, suggesting that membrane thinning under negative mismatch conditions promotes the flipping. Interestingly, a hydrophobic stretch of 19 residues intensively promoted phospholipid scrambling and membrane leakage. The distinctive characteristics of the peptide were ascribed by long-term molecular dynamics simulation to the arrangement of central glutamine and terminal four lysine residues on the same side of the helix. The combination of simulated and experimental data enables understanding of the mechanisms by which transmembrane helices, and ultimately unidentified scramblases in biomembranes, cause lipid scrambling.

Enhancement of endotoxin neutralization by coupling of a C12-alkyl chain to a lactoferricin-derived peptide

PubMed Central

2004-01-01

Antibacterial peptide acylation, which mimics the structure of the natural lipopeptide polymyxin B, increases antimicrobial and endotoxin-neutralizing activities. The interaction of the lactoferricin-derived peptide LF11 and its N-terminally acylated analogue, lauryl-LF11, with different chemotypes of bacterial lipopolysaccharide (LPS Re, Ra and smooth S form) was investigated by biophysical means and was related to the peptides' biological activities. Both peptides exhibit high antibacterial activity against the three strains of Salmonella enterica differing in the LPS chemotype. Lauryl-LF11 has one order of magnitude higher activity against Re-type, but activity against Ra- and S-type bacteria is comparable with that of LF11. The alkyl derivative peptide lauryl-LF11 shows a much stronger inhibition of the LPS-induced cytokine induction in human mononuclear cells than LF11. Although peptide–LPS interaction is essentially of electrostatic nature, the lauryl-modified peptide displays a strong hydrophobic component. Such a feature might then explain the fact that saturation of the peptide binding takes place at a much lower peptide/LPS ratio for LF11 than for lauryl-LF11, and that an overcompensation of the negative LPS backbone charges is observed for lauryl-LF11. The influence of LF11 on the gel-to-liquid-crystalline phase-transition of LPS is negligible for LPS Re, but clearly fluidizing for LPS Ra. In contrast, lauryl-LF11 causes a cholesterol-like effect in the two chemotypes, fluidizing in the gel and rigidifying of the hydrocarbon chains in the liquid-crystalline phase. Both peptides convert the mixed unilamellar/non-lamellar aggregate structure of lipid A, the ‘endotoxic principle’ of LPS, into a multilamellar one. These data contribute to the understanding of the mechanisms of the peptide-mediated neutralization of endotoxin and effect of lipid modification of peptides. PMID:15344905

The development and application of new crystallization method for tobacco mosaic virus coat protein.

PubMed

Li, Xiangyang; Song, Baoan; Hu, Deyu; Wang, Zhenchao; Zeng, Mengjiao; Yu, Dandan; Chen, Zhuo; Jin, Linhong; Yang, Song

2012-11-21

Although tobacco mosaic virus (TMV) coat protein (CP) has been isolated from virus particles and its crystals have grown in ammonium sulfate buffers for many years, to date, no one has reported on the crystallization of recombinant TMV-CP connecting peptides expressed in E. coli. In the present papers genetically engineered TMV-CP was expressed, into which hexahistidine (His) tags or glutathione-S-transferase (GST) tags were incorporated. Considering that GST-tags are long peptides and His-tags are short peptides, an attempt was made to grow crystals of TMV-CP cleaved GST-tags (WT-TMV-CP32) and TMV-CP incorporated His-tags (WT-His-TMV-CP12) simultaneously in ammonium sulfate buffers and commercial crystallization reagents. It was found that the 20S disk form of WT-TMV-CP32 and WT-His-TMV-CP12 did not form high resolution crystals by using various crystallization buffers and commercial crystallization reagents. Subsequently, a new experimental method was adopted in which a range of truncated TMV-CP was constructed by removing several amino acids from the N- or the C-terminal, and high resolution crystals were grown in ammonium sulfate buffers and commercial crystallization reagents. The new crystallization method was developed and 3.0 Å resolution macromolecular crystal was thereby obtained by removing four amino acids at the C-terminal of His-TMV-CP and connecting six His-tags at the N-terminal of His-TMV-CP (TR-His-TMV-CP19). The Four-layer aggregate disk structure of TR-His-TMV-CP19 was solved. This phenomenon showed that peptides at the C-terminus hindered the growth of high resolution crystals and the peptides interactions at the N-terminus were attributed to the quality of TMV-CP crystals. A 3.0 Å resolution macromolecular crystal of TR-His-TMV-CP19 was obtained and the corresponding structure was solved by removing four amino acids at the C-terminus of TMV-CP and connecting His-tags at the N-terminus of TMV-CP. It indicated that short peptides influenced the resolution of TMV-CP crystals.

A Statistics-based Platform for Quantitative N-terminome Analysis and Identification of Protease Cleavage Products*

PubMed Central

auf dem Keller, Ulrich; Prudova, Anna; Gioia, Magda; Butler, Georgina S.; Overall, Christopher M.

2010-01-01

Terminal amine isotopic labeling of substrates (TAILS), our recently introduced platform for quantitative N-terminome analysis, enables wide dynamic range identification of original mature protein N-termini and protease cleavage products. Modifying TAILS by use of isobaric tag for relative and absolute quantification (iTRAQ)-like labels for quantification together with a robust statistical classifier derived from experimental protease cleavage data, we report reliable and statistically valid identification of proteolytic events in complex biological systems in MS2 mode. The statistical classifier is supported by a novel parameter evaluating ion intensity-dependent quantification confidences of single peptide quantifications, the quantification confidence factor (QCF). Furthermore, the isoform assignment score (IAS) is introduced, a new scoring system for the evaluation of single peptide-to-protein assignments based on high confidence protein identifications in the same sample prior to negative selection enrichment of N-terminal peptides. By these approaches, we identified and validated, in addition to known substrates, low abundance novel bioactive MMP-2 targets including the plasminogen receptor S100A10 (p11) and the proinflammatory cytokine proEMAP/p43 that were previously undescribed. PMID:20305283

Crystal Structure of the Golgi-Associated Human Nα-Acetyltransferase 60 Reveals the Molecular Determinants for Substrate-Specific Acetylation.

PubMed

Støve, Svein Isungset; Magin, Robert S; Foyn, Håvard; Haug, Bengt Erik; Marmorstein, Ronen; Arnesen, Thomas

2016-07-06

N-Terminal acetylation is a common and important protein modification catalyzed by N-terminal acetyltransferases (NATs). Six human NATs (NatA-NatF) contain one catalytic subunit each, Naa10 to Naa60, respectively. In contrast to the ribosome-associated NatA to NatE, NatF/Naa60 specifically associates with Golgi membranes and acetylates transmembrane proteins. To gain insight into the molecular basis for the function of Naa60, we developed an Naa60 bisubstrate CoA-peptide conjugate inhibitor, determined its X-ray structure when bound to CoA and inhibitor, and carried out biochemical experiments. We show that Naa60 adapts an overall fold similar to that of the catalytic subunits of ribosome-associated NATs, but with the addition of two novel elongated loops that play important roles in substrate-specific binding. One of these loops mediates a dimer to monomer transition upon substrate-specific binding. Naa60 employs a catalytic mechanism most similar to Naa50. Collectively, these data reveal the molecular basis for Naa60-specific acetyltransferase activity with implications for its Golgi-specific functions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Stable isotope-assisted NMR characterization of interaction between lipid A and sarcotoxin IA, a cecropin-type antibacterial peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yagi-Utsumi, Maho; Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603; Yamaguchi, Yoshiki

Highlights: ► Recombinant sarcotoxin IA was successfully produced with {sup 13}C- and {sup 15}N-labeling. ► Sarcotoxin IA adopts an N-terminal α-helix upon binding to lipid A-embedding micelles. ► Two lysine residues are involved in lipid A-mediated antibacterial activities. -- Abstract: Sarcotoxin IA is a 39-residue cecropin-type peptide from Sarcophaga peregrina. This peptide exhibits antibacterial activity against Gram-negative bacteria through its interaction with lipid A, a core component of lipopolysaccharides. To acquire detailed structural information on this specific interaction, we performed NMR analysis using bacterially expressed sarcotoxin IA analogs with {sup 13}C- and {sup 15}N-labeling along with lipid A-embedding micelles composedmore » of dodecylphosphocholine. By inspecting the stable isotope-assisted NMR data, we revealed that the N-terminal segment (Leu3–Arg18) of sarcotoxin IA formed an amphiphilic α-helix upon its interaction with the aqueous micelles. Furthermore, chemical shift perturbation data indicated that the amino acid residues displayed on this α-helix were involved in the specific interaction with lipid A. On the basis of these data, we successfully identified Lys4 and Lys5 as key residues in the interaction with lipid A and the consequent antibacterial activity. Therefore, these results provide unique information for designing chemotherapeutics based on antibacterial peptide structures.« less

One new kind of phytohormonal signaling integrator: Up-and-coming GASA family genes.

PubMed

Zhang, Shengchun; Wang, Xiaojing

2017-02-01

GASA proteins are characterized by an N-terminal signal peptide and a C-terminal conserved GASA domain with 12 invariant cysteine residues. Despite being widely distributed among plant species, their functions are not completely elucidated and little is known about their mechanism of action. This review focuses on the current knowledge about the molecular structure, protein subcellular localization and phytohormones responses of this up-and-coming family of peptides. Furthermore, we discussed the roles of GASA proteins in plant growth and development, plant responses to biotic or abiotic stresses and their participation in phytohormonal signaling integration.

Identification of the WW domain-interaction sites in the unstructured N-terminal domain of EBV LMP 2A.

PubMed

Seo, Min-Duk; Park, Sung Jean; Kim, Hyun-Jung; Lee, Bong Jin

2007-01-09

Epstein-Barr virus latency is maintained by the latent membrane protein (LMP) 2A, which mimics the B-cell receptor (BCR) and perturbs BCR signaling. The cytoplasmic N-terminal domain of LMP2A is composed of 119 amino acids. The N-terminal domain of LMP2A (LMP2A NTD) contains two PY motifs (PPPPY) that interact with the WW domains of Nedd4 family ubiquitin-protein ligases. Based on our analysis of NMR data, we found that the LMP2A NTD adopts an overall random-coil structure in its native state. However, the region between residues 60 and 90 was relatively ordered, and seemed to form the hydrophobic core of the LMP2A NTD. This region resides between two PY motifs and is important for WW domain binding. Mapping of the residues involved in the interaction between the LMP2A NTD and WW domains was achieved by chemical shift perturbation, by the addition of WW2 and WW3 peptides. Interestingly, the binding of the WW domains mainly occurred in the hydrophobic core of the LMP2A NTD. In addition, we detected a difference in the binding modes of the two PY motifs against the two WW peptides. The binding of the WW3 peptide caused the resonances of five residues (Tyr(60), Glu(61), Asp(62), Trp(65), and Gly(66)) just behind the N-terminal PY motif of the LMP2A NTD to disappear. A similar result was obtained with WW2 binding. However, near the C-terminal PY motif, the chemical shift perturbation caused by WW2 binding was different from that due to WW3 binding, indicating that the residues near the PY motifs are involved in selective binding of WW domains. The present work represents the first structural study of the LMP2A NTD and provides fundamental structural information about its interaction with ubiquitin-protein ligase.

PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands.

PubMed

Walkup, Ward G; Kennedy, Mary B

2014-06-01

PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

Engineering peptide ligase specificity by proteomic identification of ligation sites.

PubMed

Weeks, Amy M; Wells, James A

2018-01-01

Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme-substrate specificity limits its utility. We developed an approach for comprehensively characterizing peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme-substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins, including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. Our methods provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.

Neurotensin may function as a regulatory peptide in small cell lung cancer.

PubMed

Davis, T P; Crowell, S; McInturff, B; Louis, R; Gillespie, T

1991-01-01

Neurotensin (NT) has been postulated to act as a modulatory agent in the central nervous system. Besides its presence in mammalian brain, NT is produced by small cell carcinoma of the lung (SCLC) and cell lines derived from these tumors. Receptors have also been characterized in some SCLC cell lines leading to the suggestion that NT could regulate the growth of SCLC in an autocrine fashion similar to bombesin/GRP. Previously, we had reported that a 10 nM dose of NT and NT(8-13), but not NT(1-8), elevated cytosolic Ca2+, indicating that SCLC NT receptors may use Ca2+ as a second messenger. Using intact SCLC cells we report that time-course incubations with NT lead to the formation of the amino-terminal fragment NT(1-8) and small amounts of the C-terminal fragment NT(9-13). These fragments are formed by metalloendopeptidase 3.4.24.15 cleaving enzyme at the Arg8-Arg9 bond of NT. Significant levels of soluble 3.4.24.15 (10-17 nmoles/mg Pr-/min) are present in SCLC cell lines. Using the in vitro clonogenic assay we tested the effect of 0.5, 5.0 and 10.0 nM doses of NT, NT(1-8) and NT(8-13) on SCLC clonal growth. NT and the C-terminal fragment NT(8-13) stimulated colony formation whereas the N-terminal fragment did not. In summary, NT may function as a regulatory peptide in SCLC through the formation of peptide fragments.

Elucidation of Ligand-Dependent Modulation of Disorder-Order Transitions in the Oncoprotein MDM2.

PubMed

Bueren-Calabuig, Juan A; Michel, Julien

2015-06-01

Numerous biomolecular interactions involve unstructured protein regions, but how to exploit such interactions to enhance the affinity of a lead molecule in the context of rational drug design remains uncertain. Here clarification was sought for cases where interactions of different ligands with the same disordered protein region yield qualitatively different results. Specifically, conformational ensembles for the disordered lid region of the N-terminal domain of the oncoprotein MDM2 in the presence of different ligands were computed by means of a novel combination of accelerated molecular dynamics, umbrella sampling, and variational free energy profile methodologies. The resulting conformational ensembles for MDM2, free and bound to p53 TAD (17-29) peptide identify lid states compatible with previous NMR measurements. Remarkably, the MDM2 lid region is shown to adopt distinct conformational states in the presence of different small-molecule ligands. Detailed analyses of small-molecule bound ensembles reveal that the ca. 25-fold affinity improvement of the piperidinone family of inhibitors for MDM2 constructs that include the full lid correlates with interactions between ligand hydrophobic groups and the C-terminal lid region that is already partially ordered in apo MDM2. By contrast, Nutlin or benzodiazepinedione inhibitors, that bind with similar affinity to full lid and lid-truncated MDM2 constructs, interact additionally through their solubilizing groups with N-terminal lid residues that are more disordered in apo MDM2.

Sequence dependent N-terminal rearrangement and degradation of peptide nucleic acid (PNA) in aqueous solution

NASA Technical Reports Server (NTRS)

Eriksson, M.; Christensen, L.; Schmidt, J.; Haaima, G.; Orgel, L.; Nielsen, P. E.

1998-01-01

The stability of the PNA (peptide nucleic acid) thymine monomer inverted question markN-[2-(thymin-1-ylacetyl)]-N-(2-aminoaminoethyl)glycine inverted question mark and those of various PNA oligomers (5-8-mers) have been measured at room temperature (20 degrees C) as a function of pH. The thymine monomer undergoes N-acyl transfer rearrangement with a half-life of 34 days at pH 11 as analyzed by 1H NMR; and two reactions, the N-acyl transfer and a sequential degradation, are found by HPLC analysis to occur at measurable rates for the oligomers at pH 9 or above. Dependent on the amino-terminal sequence, half-lives of 350 h to 163 days were found at pH 9. At pH 12 the half-lives ranged from 1.5 h to 21 days. The results are discussed in terms of PNA as a gene therapeutic drug as well as a possible prebiotic genetic material.

Isolation, cloning, and characterization of the 2S albumin: a new allergen from hazelnut.

PubMed

Garino, Cristiano; Zuidmeer, Laurian; Marsh, Justin; Lovegrove, Alison; Morati, Maria; Versteeg, Serge; Schilte, Piet; Shewry, Peter; Arlorio, Marco; van Ree, Ronald

2010-09-01

2S albumins are the major allergens involved in severe food allergy to nuts, seeds, and legumes. We aimed to isolate, clone, and express 2S albumin from hazelnut and determine its allergenicity. 2S albumin from hazelnut extract was purified using size exclusion chromatography and RP-HPLC. After N-terminal sequencing, degenerated and poly-d(T) primers were used to clone the 2S albumin sequence from hazelnut cDNA. After expression in Escherichia coli and affinity purification, IgE reactivity was evaluated by Immunoblot/ImmunoCAP (inhibition) analyses using sera of nut-allergic patients. N-terminal sequencing of a approximately 10 kDa peak from size exclusion chromatography/RP-HPLC gave two sequences highly homologous to pecan 2S albumin, an 11 amino acid (aa) N-terminal and a 10 aa internal peptide. The obtained clone (441 bp) encoded a 147 aa hazelnut 2S albumin consisting of a putative signal peptide (22 aa), a linker peptide (20 aa), and the mature protein sequence (105 aa). The latter was successfully expressed in E. coli. Both recombinant and natural 2S albumin demonstrated similar IgE reactivity in Immunoblot/ImmunoCAP (inhibition) analyses. We confirmed the postulated role of hazelnut 2S albumin as an allergen. The availability of recombinant molecules will allow establishing the importance of hazelnut 2S albumin for hazelnut allergy.

Impacts of the N-terminal fragment analog of human parathyroid hormone on structure, composition and biomechanics of bone.

PubMed

Chunxiao, Wang; Yu, Zhang; Wentao, Liu; Jingjing, Liu; Jiahui, Ye; Qingmei, Chen

2012-12-18

Osteoporosis is a skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, and it is a serious threat to human lives. We previously showed that the N-terminal peptide analog of human parathyroid hormone (Pro-Pro-PTH(1-34)) enhanced plasma calcium concentration. In this paper, we study the impact of PTH N-terminal fragment analog on the structure, component, and mechanical properties of the rat bones. Daily subcutaneous injections of Pro-Pro-hPTH (1-34) induces 26.5-32.8% increase in femur bone mineral density (BMD), 23.0-34.2% decrease the marrow cavity or increase in trabecular bone area. The peptide also increases 16.0-59.5%, 28.8-48.2% and 14.0-17.8% of bone components of calcium, phosphorus and collagen, respectively. In terms of mechanic properties, administration of the peptide elevates the bone rigidity by 45.4-76.6%, decreases the flexibility by 23.0-31.6%, and improves modulus of elasticity by 32.8-63.4%. The results suggest that Pro-Pro-hPTH (1-34) has a positive effect on bone growth and strength, and possesses anti-fracture capability, thus a potential candidate for the application for the treatment of osteoporosis. Copyright © 2012 Elsevier B.V. All rights reserved.

Inward open characterization of EmrD transporter with molecular dynamics simulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Xianwei; Wang, Boxiong, E-mail: boxiong_wang@yahoo.com

EmrD is a member of the multidrug resistance exporter family. Up to now, little is known about the structural dynamics that underline the function of the EmrD protein in inward-facing open state and how the EmrD transits from an occluded state to an inward open state. For the first time the article applied the AT simulation to investigate the membrane transporter protein EmrD, and described the dynamic features of the whole protein, the domain, the helices, and the amino acid residues during an inward-open process from its occluded state. The gradual inward-open process is different from the current model ofmore » rigid-body domain motion in alternating-access mechanism. Simulation results show that the EmrD inward-open conformational fluctuation propagates from a C-terminal domain to an N-terminal domain via the linker region during the transition from its occluded state. The conformational fluctuation of the C-terminal domain is larger than that of the N-terminal domain. In addition, it is observed that the helices exposed to the surrounding membrane show a higher level of flexibility than the other regions, and the protonated E227 plays a key role in the transition from the occluded to the open state. -- Highlights: •This study described the dynamic features of the whole EmrD protein, during an inward-open process from its occluded state. •The EmrD inward-open conformational fluctuation propagates from a C-terminal domain to an N-terminal domain via the linker region during the transition from its occluded state. •The conformational fluctuation of the C-terminal domain is larger than that of the N-terminal domain. •The protonated E227 plays a key role in the transition from the occluded to the open state.« less

Intestinal cell targeting of a stable recombinant Cu-Zn SOD from Cucumis melo fused to a gliadin peptide.

PubMed

Intes, Laurent; Bahut, Muriel; Nicole, Pascal; Couvineau, Alain; Guette, Catherine; Calenda, Alphonse

2012-05-31

The mRNA encoding full length chloroplastic Cu-Zn SOD (superoxide dismutase) of Cucumis melo (Cantaloupe melon) was cloned. This sequence was then used to generate a mature recombinant SOD by deleting the first 64 codons expected to encode a chloroplastic peptide signal. A second hybrid SOD was created by inserting ten codons to encode a gliadin peptide at the N-terminal end of the mature SOD. Taking account of codon bias, both recombinant proteins were successfully expressed and produced in Escherichia coli. Both recombinant SODs display an enzymatic activity of ~5000U mg(-1) and were shown to be stable for at least 4h at 37°C in biological fluids mimicking the conditions of intestinal transit. These recombinant proteins were capable in vitro, albeit at different levels, of reducing ROS-induced-apoptosis of human epithelial cells. They also stimulated production and release in a time-dependent manner of an autologous SOD activity from cells located into jejunum biopsies. Nevertheless, the fused gliadin peptide enable the recombinant Cu-Zn SOD to maintain a sufficiently sustained interaction with the intestinal cells membrane in vivo rather than being eliminated with the flow. According to these observations, the new hybrid Cu-Zn SOD should show promise in applications for managing inflammatory bowel diseases. Copyright © 2012 Elsevier B.V. All rights reserved.

Analysis of the role of tripeptidyl peptidase II in MHC class I antigen presentation in vivo1

PubMed Central

Kawahara, Masahiro; York, Ian A.; Hearn, Arron; Farfan, Diego; Rock, Kenneth L.

2015-01-01

Previous experiments using enzyme inhibitors and RNAi in cell lysates and cultured cells have suggested that tripeptidyl peptidase II (TPPII) plays a role in creating and destroying MHC class I-presented peptides. However, its precise contribution to these processes has been controversial. To elucidate the importance of TPPII in MHC class I antigen presentation, we analyzed TPPII-deficient gene-trapped mice and cell lines from these animals. In these mice, the expression level of TPPII was reduced by >90% compared to wild-type mice. Thymocytes from TPPII gene-trapped mice displayed more MHC class I on the cell surface, suggesting that TPPII normally limits antigen presentation by destroying peptides overall. TPPII gene-trapped mice responded as well as did wild-type mice to four epitopes from lymphocytic choriomeningitis virus (LCMV). The processing and presentation of peptide precursors with long N-terminal extensions in TPPII gene-trapped embryonic fibroblasts was modestly reduced, but in vivo immunization with recombinant lentiviral or vaccinia virus vectors revealed that such peptide precursors induced an equivalent CD8 T cell response in wild type and TPPII-deficient mice. These data indicate while TPPII contributes to the trimming of peptides with very long N-terminal extensions, TPPII is not essential for generating most MHC class I-presented peptides or for stimulating CTL responses to several antigens in vivo. PMID:19841172

Negative Ion In-Source Decay Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Sequencing Acidic Peptides

NASA Astrophysics Data System (ADS)

McMillen, Chelsea L.; Wright, Patience M.; Cassady, Carolyn J.

2016-05-01

Matrix-assisted laser desorption/ionization (MALDI) in-source decay was studied in the negative ion mode on deprotonated peptides to determine its usefulness for obtaining extensive sequence information for acidic peptides. Eight biological acidic peptides, ranging in size from 11 to 33 residues, were studied by negative ion mode ISD (nISD). The matrices 2,5-dihydroxybenzoic acid, 2-aminobenzoic acid, 2-aminobenzamide, 1,5-diaminonaphthalene, 5-amino-1-naphthol, 3-aminoquinoline, and 9-aminoacridine were used with each peptide. Optimal fragmentation was produced with 1,5-diaminonphthalene (DAN), and extensive sequence informative fragmentation was observed for every peptide except hirudin(54-65). Cleavage at the N-Cα bond of the peptide backbone, producing c' and z' ions, was dominant for all peptides. Cleavage of the N-Cα bond N-terminal to proline residues was not observed. The formation of c and z ions is also found in electron transfer dissociation (ETD), electron capture dissociation (ECD), and positive ion mode ISD, which are considered to be radical-driven techniques. Oxidized insulin chain A, which has four highly acidic oxidized cysteine residues, had less extensive fragmentation. This peptide also exhibited the only charged localized fragmentation, with more pronounced product ion formation adjacent to the highly acidic residues. In addition, spectra were obtained by positive ion mode ISD for each protonated peptide; more sequence informative fragmentation was observed via nISD for all peptides. Three of the peptides studied had no product ion formation in ISD, but extensive sequence informative fragmentation was found in their nISD spectra. The results of this study indicate that nISD can be used to readily obtain sequence information for acidic peptides.

Negative Ion In-Source Decay Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Sequencing Acidic Peptides.

PubMed

McMillen, Chelsea L; Wright, Patience M; Cassady, Carolyn J

2016-05-01

Matrix-assisted laser desorption/ionization (MALDI) in-source decay was studied in the negative ion mode on deprotonated peptides to determine its usefulness for obtaining extensive sequence information for acidic peptides. Eight biological acidic peptides, ranging in size from 11 to 33 residues, were studied by negative ion mode ISD (nISD). The matrices 2,5-dihydroxybenzoic acid, 2-aminobenzoic acid, 2-aminobenzamide, 1,5-diaminonaphthalene, 5-amino-1-naphthol, 3-aminoquinoline, and 9-aminoacridine were used with each peptide. Optimal fragmentation was produced with 1,5-diaminonphthalene (DAN), and extensive sequence informative fragmentation was observed for every peptide except hirudin(54-65). Cleavage at the N-Cα bond of the peptide backbone, producing c' and z' ions, was dominant for all peptides. Cleavage of the N-Cα bond N-terminal to proline residues was not observed. The formation of c and z ions is also found in electron transfer dissociation (ETD), electron capture dissociation (ECD), and positive ion mode ISD, which are considered to be radical-driven techniques. Oxidized insulin chain A, which has four highly acidic oxidized cysteine residues, had less extensive fragmentation. This peptide also exhibited the only charged localized fragmentation, with more pronounced product ion formation adjacent to the highly acidic residues. In addition, spectra were obtained by positive ion mode ISD for each protonated peptide; more sequence informative fragmentation was observed via nISD for all peptides. Three of the peptides studied had no product ion formation in ISD, but extensive sequence informative fragmentation was found in their nISD spectra. The results of this study indicate that nISD can be used to readily obtain sequence information for acidic peptides.

The role of the N-terminal tail for the oligomerization, folding and stability of human frataxin☆

PubMed Central

Faraj, Santiago E.; Venturutti, Leandro; Roman, Ernesto A.; Marino-Buslje, Cristina B.; Mignone, Astor; Tosatto, Silvio C.E.; Delfino, José M.; Santos, Javier

2013-01-01

The N-terminal stretch of human frataxin (hFXN) intermediate (residues 42–80) is not conserved throughout evolution and, under defined experimental conditions, behaves as a random-coil. Overexpression of hFXN56–210 in Escherichia coli yields a multimer, whereas the mature form of hFXN (hFXN81–210) is monomeric. Thus, cumulative experimental evidence points to the N-terminal moiety as an essential element for the assembly of a high molecular weight oligomer. The secondary structure propensity of peptide 56–81, the moiety putatively responsible for promoting protein–protein interactions, was also studied. Depending on the environment (TFE or SDS), this peptide adopts α-helical or β-strand structure. In this context, we explored the conformation and stability of hFXN56–210. The biophysical characterization by fluorescence, CD and SEC-FPLC shows that subunits are well folded, sharing similar stability to hFXN90–210. However, controlled proteolysis indicates that the N-terminal stretch is labile in the context of the multimer, whereas the FXN domain (residues 81–210) remains strongly resistant. In addition, guanidine hydrochloride at low concentration disrupts intermolecular interactions, shifting the ensemble toward the monomeric form. The conformational plasticity of the N-terminal tail might impart on hFXN the ability to act as a recognition signal as well as an oligomerization trigger. Understanding the fine-tuning of these activities and their resulting balance will bear direct relevance for ultimately comprehending hFXN function. PMID:23951553

Isobaric Quantification of Cerebrospinal Fluid Amyloid-β Peptides in Alzheimer's Disease: C-Terminal Truncation Relates to Early Measures of Neurodegeneration.

PubMed

Rogeberg, Magnus; Almdahl, Ina Selseth; Wettergreen, Marianne; Nilsson, Lars N G; Fladby, Tormod

2015-11-06

The amyloid beta (Aβ) peptide is the main constituent of the plaques characteristic of Alzheimer's disease (AD). Measurement of Aβ1-42 in cerebrospinal fluid (CSF) is a valuable marker in AD research, where low levels indicate AD. Although the use of immunoassays measuring Aβ1-38 and Aβ1-40 in addition to Aβ1-42 has increased, quantitative assays of other Aβ peptides remain rarely explored. We recently discovered novel Aβ peptides in CSF using antibodies recognizing the Aβ mid-domain region. Here we have developed a method using both Aβ N-terminal and mid-domain antibodies for immunoprecipitation in combination with isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for relative quantification of endogenous Aβ peptides in CSF. The developed method was used in a pilot study to produce Aβ peptide profiles from 38 CSF samples. Statistical comparison between CSF samples from 19 AD patients and 19 cognitively healthy controls revealed no significant differences at group level. A significant correlation was found between several larger C-terminally truncated Aβ peptides and protein biomarkers for neuronal damage, particularly prominent in the control group. Comparison of the isobaric quantification with immunoassays measuring Aβ1-38 or Aβ1-40 showed good correlation (r(2) = 0.84 and 0.85, respectively) between the two analysis methods. The developed method could be used to assess disease-modifying therapies directed at Aβ production or degradation.

Enterocin T, a novel class IIa bacteriocin produced by Enterococcus sp. 812.

PubMed

Chen, Yi-Sheng; Yu, Chi-Rong; Ji, Si-Hua; Liou, Min-Shiuan; Leong, Kun-Hon; Pan, Shwu-Fen; Wu, Hui-Chung; Lin, Yu-Hsuan; Yu, Bi; Yanagida, Fujitoshi

2013-09-01

Enterococcus sp. 812, isolated from fresh broccoli, was previously found to produce a bacteriocin active against a number of Gram-positive bacteria, including Listeria monocytogenes. Bacteriocin activity decreased slightly after autoclaving (121 °C for 15 min), but was inactivated by protease K. Mass spectrometry analysis revealed the bacteriocin mass to be approximately 4,521.34 Da. N-terminal amino acid sequencing yielded a partial sequence, NH2-ATYYGNGVYXDKKKXWVEWGQA, by Edman degradation, which contained the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The obtained partial sequence showed high homology with some enterococcal bacteriocins; however, no identical peptide or protein was found. This peptide was therefore considered to be a novel bacteriocin produced by Enterococcus sp. 812 and was termed enterocin T.

Peptoid-Substituted Hybrid Antimicrobial Peptide Derived from Papiliocin and Magainin 2 with Enhanced Bacterial Selectivity and Anti-inflammatory Activity.

PubMed

Shin, Areum; Lee, Eunjung; Jeon, Dasom; Park, Young-Guen; Bang, Jeong Kyu; Park, Yong-Sun; Shin, Song Yub; Kim, Yangmee

2015-06-30

Antimicrobial peptides (AMPs) are important components of the host innate immune system. Papiliocin is a 37-residue AMP purified from larvae of the swallowtail butterfly Papilio xuthus. Magainin 2 is a 23-residue AMP purified from the skin of the African clawed frog Xenopus laevis. We designed an 18-residue hybrid peptide (PapMA) incorporating N-terminal residues 1-8 of papiliocin and N-terminal residues 4-12 of magainin 2, joined by a proline (Pro) hinge. PapMA showed high antimicrobial activity but was cytotoxic to mammalian cells. To decrease PapMA cytotoxicity, we designed a lysine (Lys) peptoid analogue, PapMA-k, which retained high antimicrobial activity but displayed cytotoxicity lower than that of PapMA. Fluorescent dye leakage experiments and confocal microscopy showed that PapMA targeted bacterial cell membranes whereas PapMA-k penetrated bacterial cell membranes. Nuclear magnetic resonance experiments revealed that PapMA contained an N-terminal α-helix from Lys(3) to Lys(7) and a C-terminal α-helix from Lys(10) to Lys(17), with a Pro(9) hinge between them. PapMA-k also had two α-helical structures in the same region connected with a flexible hinge residue at Nlys(9), which existed in a dynamic equilibrium of cis and trans conformers. Using lipopolysaccharide-stimulated RAW264.7 macrophages, the anti-inflammatory activity of PapMA and PapMA-k was confirmed by inhibition of nitric oxide and inflammatory cytokine production. In addition, treatment with PapMA and PapMA-k decreased the level of ultraviolet irradiation-induced expression of genes encoding matrix metalloproteinase-1 (MMP-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in human keratinocyte HaCaT cells. Thus, PapMA and PapMA-k are potent peptide antibiotics with antimicrobial and anti-inflammatory activity, with PapMA-k displaying enhanced bacterial selectivity.

Coulomb repulsion in short polypeptides.

PubMed

Norouzy, Amir; Assaf, Khaleel I; Zhang, Shuai; Jacob, Maik H; Nau, Werner M

2015-01-08

Coulomb repulsion between like-charged side chains is presently viewed as a major force that impacts the biological activity of intrinsically disordered polypeptides (IDPs) by determining their spatial dimensions. We investigated short synthetic models of IDPs, purely composed of ionizable amino acid residues and therefore expected to display an extreme structural and dynamic response to pH variation. Two synergistic, custom-made, time-resolved fluorescence methods were applied in tandem to study the structure and dynamics of the acidic and basic hexapeptides Asp6, Glu6, Arg6, Lys6, and His6 between pH 1 and 12. (i) End-to-end distances were obtained from the short-distance Förster resonance energy transfer (sdFRET) from N-terminal 5-fluoro-l-tryptophan (FTrp) to C-terminal Dbo. (ii) End-to-end collision rates were obtained for the same peptides from the collision-induced fluorescence quenching (CIFQ) of Dbo by FTrp. Unexpectedly, the very high increase of charge density at elevated pH had no dynamical or conformational consequence in the anionic chains, neither in the absence nor in the presence of salt, in conflict with the common view and in partial conflict with accompanying molecular dynamics simulations. In contrast, the cationic peptides responded to ionization but with surprising patterns that mirrored the rich individual characteristics of each side chain type. The contrasting results had to be interpreted, by considering salt screening experiments, N-terminal acetylation, and simulations, in terms of an interplay of local dielectric constant and peptide-length dependent side chain charge-charge repulsion, side chain functional group solvation, N-terminal and side chain charge-charge repulsion, and side chain-side chain as well as side chain-backbone interactions. The common picture that emerged is that Coulomb repulsion between water-solvated side chains is efficiently quenched in short peptides as long as side chains are not in direct contact with each other or the main chain.

Plasma atrial natriuretic peptide and N-terminal pro B-type natriuretic peptide concentrations in dogs with right-sided congestive heart failure

PubMed Central

KANNO, Nobuyuki; HORI, Yasutomo; HIDAKA, Yuichi; CHIKAZAWA, Seishiro; KANAI, Kazutaka; HOSHI, Fumio; ITOH, Naoyuki

2015-01-01

The clinical utility of plasma natriuretic peptide concentrations in dogs with right-sided congestive heart failure (CHF) remains unclear. We investigated whether plasma levels of atrial natriuretic peptide (ANP) and N-terminal pro B-type natriuretic peptide (NT-proBNP) are useful for assessing the congestive signs of right-sided heart failure in dogs. This retrospective study enrolled 16 healthy dogs and 51 untreated dogs with presence (n=28) or absence (n=23) of right-sided CHF. Medical records of physical examinations, thoracic radiography and echocardiography were reviewed. The plasma concentration of canine ANP was measured with a chemiluminescent enzyme immunoassay. Plasma NT-proBNP concentrations were determined using an enzyme immunoassay. Plasma ANP and NT-proBNP concentrations in dogs with right-sided CHF were significantly higher than in healthy controls and those without right-sided CHF. The plasma NT-proBNP concentration >3,003 pmol/l used to identify right-sided CHF had a sensitivity of 88.5% and specificity of 90.3%. An area under the ROC curve (AUC) was 0.93. The AUC for NT-proBNP was significantly higher than the AUCs for the cardiothoracic ratio, vertebral heart score, ratio of right ventricular end-diastolic internal diameter to body surface area, tricuspid late diastolic flow and ratio of the velocities of tricuspid early to late diastolic flow. These results suggest that plasma ANP and NT-proBNP concentrations increase markedly in dogs with right-sided CHF. Particularly, NT-proBNP is simple and helpful biomarkers to assess the right-sided CHF. PMID:26607133

Using Gas-Phase Guest-Host Chemistry to Probe the Structures of b Ions of Peptides

NASA Astrophysics Data System (ADS)

Somogyi, Árpád; Harrison, Alex G.; Paizs, Béla

2012-12-01

Middle-sized b n ( n ≥ 5) fragments of protonated peptides undergo selective complex formation with ammonia under experimental conditions typically used to probe hydrogen-deuterium exchange in Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Other usual peptide fragments like y, a, a*, etc., and small b n ( n ≤ 4) fragments do not form stable ammonia adducts. We propose that complex formation of b n ions with ammonia is characteristic to macrocyclic isomers of these fragments. Experiments on a protonated cyclic peptide and N-terminal acetylated peptides fully support this hypothesis; the protonated cyclic peptide does form ammonia adducts while linear b n ions of acetylated peptides do not undergo complexation. Density functional theory (DFT) calculations on the proton-bound dimers of all-Ala b 4 , b 5 , and b 7 ions and ammonia indicate that the ionizing proton initially located on the peptide fragment transfers to ammonia upon adduct formation. The ammonium ion is then solvated by N+-H…O H-bonds; this stabilization is much stronger for macrocyclic b n isomers due to the stable cage-like structure formed and entropy effects. The present study demonstrates that gas-phase guest-host chemistry can be used to selectively probe structural features (i.e., macrocyclic or linear) of fragments of protonated peptides. Stable ammonia adducts of b 9 , b 9 -A, and b 9 -2A of A8YA, and b 13 of A20YVFL are observed indicating that even these large b-type ions form macrocyclic structures.

Tenascin-X promotes epithelial-to-mesenchymal transition by activating latent TGF-β

PubMed Central

Alcaraz, Lindsay B.; Exposito, Jean-Yves; Chuvin, Nicolas; Pommier, Roxane M.; Cluzel, Caroline; Martel, Sylvie; Sentis, Stéphanie; Bartholin, Laurent; Lethias, Claire

2014-01-01

Transforming growth factor β (TGF-β) isoforms are secreted as inactive complexes formed through noncovalent interactions between the bioactive TGF-β entity and its N-terminal latency-associated peptide prodomain. Extracellular activation of the latent TGF-β complex is a crucial step in the regulation of TGF-β function for tissue homeostasis. We show that the fibrinogen-like (FBG) domain of the matrix glycoprotein tenascin-X (TNX) interacts physically with the small latent TGF-β complex in vitro and in vivo, thus regulating the bioavailability of mature TGF-β to cells by activating the latent cytokine into an active molecule. Activation by the FBG domain most likely occurs through a conformational change in the latent complex and involves a novel cell adhesion–dependent mechanism. We identify α11β1 integrin as a cell surface receptor for TNX and show that this integrin is crucial to elicit FBG-mediated activation of latent TGF-β and subsequent epithelial-to-mesenchymal transition in mammary epithelial cells. PMID:24821840

The C-terminal heavy-chain domain of botulinum neurotoxin a is not the only site that binds neurons, as the N-terminal heavy-chain domain also plays a very active role in toxin-cell binding and interactions.

PubMed

Ayyar, B Vijayalakshmi; Aoki, K Roger; Atassi, M Zouhair

2015-04-01

Botulinum neurotoxins (BoNTs) possess unique specificity for nerve terminals. They bind to the presynaptic membrane and then translocate intracellularly, where the light-chain endopeptidase cleaves the SNARE complex proteins, subverting the synaptic exocytosis responsible for acetylcholine release to the synaptic cleft. This inhibits acetylcholine binding to its receptor, causing paralysis. Binding, an obligate event for cell intoxication, is believed to occur through the heavy-chain C-terminal (HC) domain. It is followed by toxin translocation and entry into the cell cytoplasm, which is thought to be mediated by the heavy-chain N-terminal (HN) domain. Submolecular mapping analysis by using synthetic peptides spanning BoNT serotype A (BoNT/A) and mouse brain synaptosomes (SNPs) and protective antibodies against toxin from mice and cervical dystonia patients undergoing BoNT/A treatment revealed that not only regions of the HC domain but also regions of the HN domain are involved in the toxin binding process. Based on these findings, we expressed a peptide corresponding to the BoNT/A region comprising HN domain residues 729 to 845 (HN729-845). HN729-845 bound directly to mouse brain SNPs and substantially inhibited BoNT/A binding to SNPs. The binding involved gangliosides GT1b and GD1a and a few membrane lipids. The peptide bound to human or mouse neuroblastoma cells within 1 min. Peptide HN729-845 protected mice completely against a lethal BoNT/A dose (1.05 times the 100% lethal dose). This protective activity was obtained at a dose comparable to that of the peptide from positions 967 to 1296 in the HC domain. These findings strongly indicate that HN729-845 and, by extension, the HN domain are fully programmed and equipped to bind to neuronal cells and in the free state can even inhibit the binding of the toxin. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

An N-terminal glycine-rich sequence contributes to retrovirus trimer of hairpins stability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilson, Kirilee A.; Maerz, Anne L.; Baer, Severine

2007-08-10

Retroviral transmembrane proteins (TMs) contain a glycine-rich segment linking the N-terminal fusion peptide and coiled coil core. Previously, we reported that the glycine-rich segment (Met-326-Ser-337) of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, is a determinant of membrane fusion function [K.A. Wilson, S. Baer, A.L. Maerz, M. Alizon, P. Poumbourios, The conserved glycine-rich segment linking the N-terminal fusion peptide to the coiled coil of human T-cell leukemia virus type 1 transmembrane glycoprotein gp21 is a determinant of membrane fusion function, J. Virol. 79 (2005) 4533-4539]. Here we show that the reduced fusion activity of an I334A mutantmore » correlated with a decrease in stability of the gp21 trimer of hairpins conformation, in the context of a maltose-binding protein-gp21 chimera. The stabilizing influence of Ile-334 required the C-terminal membrane-proximal sequence Trp-431-Ser-436. Proline substitution of four of five Gly residues altered gp21 trimer of hairpins stability. Our data indicate that flexibility within and hydrophobic interactions mediated by this region are determinants of gp21 stability and membrane fusion function.« less

Structural basis for substrate recognition by the human N-terminal methyltransferase 1

DOE PAGES

Dong, Cheng; Mao, Yunfei; Tempel, Wolfram; ...

2015-11-05

α-N-terminal methylation represents a highly conserved and prevalent post-translational modification, yet its biological function has remained largely speculative. The recent discovery of α-N-terminal methyltransferase 1 (NTMT1) and its physiological substrates propels the elucidation of a general role of α-N-terminal methylation in mediating DNA-binding ability of the modified proteins. The phenotypes, observed from both NTMT1 knockdown in breast cancer cell lines and knockout mouse models, suggest the potential involvement of α-N-terminal methylation in DNA damage response and cancer development. In this study, we report the first crystal structures of human NTMT1 in complex with cofactor S-adenosyl-L-homocysteine (SAH) and six substrate peptides,more » respectively, and reveal that NTMT1 contains two characteristic structural elements (a β hairpin and an N-terminal extension) that contribute to its substrate specificity. Our complex structures, coupled with mutagenesis, binding, and enzymatic studies, also present the key elements involved in locking the consensus substrate motif XPK (X indicates any residue type other than D/E) into the catalytic pocket for α-N-terminal methylation and explain why NTMT1 prefers an XPK sequence motif. We propose a catalytic mechanism for α-N-terminal methylation. Overall, this study gives us the first glimpse of the molecular mechanism of α-N-terminal methylation and potentially contributes to the advent of therapeutic agents for human diseases associated with deregulated α-N-terminal methylation.« less

ACTH-like peptides increase pain sensitivity and antagonize opiate analgesia

NASA Technical Reports Server (NTRS)

Heybach, J. P.; Vernikos, J.

1981-01-01

The role of the pituitary and of ACTH in pain sensitivity was investigated in the rat. Pain sensitivity was assessed by measuring paw-lick and jump latencies in response to being placed on a grid at 55 C. Hypophysectomy reduced pain sensitivity, and this effect was reversed by the intracerebroventricular (ICV) injection of the opiate antagonist naloxone. Similarly, the analgesia produced by a dose of morphine was antagonized by the administration of ACTH or alpha-MSH. The peripheral injection of ACTH or alpha-MSH in normal rats did not increase pain sensitivity. However, ACTH administered ICV increased pain sensivity within 10 min. The results indicate that the pituitary is the source of an endogenous opiate antagonist and hyperalgesic factor and that this factor is ACTH or an ACTH-like peptide. This activity resides in the N-terminal portion of the ACTH molecule since ACTH sub 4-10 is not active in this respect, nor does this activity require a free N-terminal serine since alpha-MSH appears to be almost as potent as the ACTH sub 1-24 peptide. It is concluded that ACTH-like peptides of pituitary origin act as endogenous hyperalgesic and opiate antagonistic factors.

Targeting Cancer Protein Profiles with Split-Enzyme Reporter Fragments to Achieve Chemical Resolution for Molecular Imaging

DTIC Science & Technology

2014-11-01

near-infrared fluorophore, Cy5.5, linked with up to three units of amino-ethoxy-ethoxy- acid (AEEA) at the N-terminal amine of the peptide. Table 1...RPMI or Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco), respectively, and supplemented with 10% FBS and 1% penicillin–streptomycin. The cells were...peptide, compound 6, using the amino acid residues of the parent peptide (compound 5) in random order. Compound 2 targeted the tumor efficiently

Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GPI Ectodomain Shedding

DTIC Science & Technology

2008-12-23

glycoprotein precursor (GPC) signal peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain...consistent with viral TM fusion proteins [9,10]. GPC con- tains a 58 residue hydrophobic N-terminal signal peptide (SP), which directs the precursor to the...including GPC, GP1, and GP2. Various signal peptides , purification tags, and modifications to internal domains were employed for the generation and

N-terminal ProBNP distribution and correlations with biological characteristics in apparently healthy Greek population: ATTICA study.

PubMed

Fragopoulou, Elizabeth; Panagiotakos, Demosthenes B; Pitsavos, Christos; Chrysohoou, Christina; Nomikos, Tzortzis; Evangelopoulos, Angelos; Katsagoni, Christina; Skoumas, John; Antonopoulou, Smaragdi; Stefanadis, Christodoulos

2010-05-01

Brain natriuretic peptides are widely used as biomarkers of cardiovascular diseases and mainly heart failure. However, these markers are often found to be high even in apparently healthy participants, and little is known about which factors contribute to physiological change in plasma brain natriuretic peptide (BNP) and amino-terminal pro-B-type natriuretic peptide (NTproBNP) concentration in general populations. In this study, a random subsample of the ATTICA study was used (486 individuals) and serum NT-proBNP was measured. Approximately 20% of the participants had no detectable NT-proBNP values. Women had higher values of NT-proBNP than men (median [25th-75th percentiles]: 30.2 [15.8-54.3] vs 14.9 [4.0-28.1] pg/mL, P < .001]. Amino-terminal pro-B-type natriuretic peptide values were positively correlated with age (rho = .140, P = .006) and inversely with body mass index (BMI; rho = -.142, P = .005), creatinine (Cr) clearance (rho = -.349, P < .001), and hemoglobin (rho = -.249, P < .001) values. Linear regression analysis revealed that gender is the main contributor of NT-proBNP levels, followed by age, BMI, and Cr values.

A Blocking Group Scan Using a Spherical Organometallic Complex Identifies an Unprecedented Binding Mode with Potent Activity In Vitro and In Vivo for the Opioid Peptide Dermorphin.

PubMed

Strack, Martin; Bedini, Andrea; Yip, King T; Lombardi, Sara; Siegmund, Daniel; Stoll, Raphael; Spampinato, Santi M; Metzler-Nolte, Nils

2016-10-04

Herein, the selective enforcement of one particular receptor-ligand interaction between specific domains of the μ-selective opioid peptide dermorphin and the μ opioid receptor is presented. For this, a blocking group scan is described which exploits the steric demand of a bis(quinolinylmethyl)amine rhenium(I) tricarbonyl complex conjugated to a number of different, strategically chosen positions of dermorphin. The prepared peptide conjugates lead to the discovery of two different binding modes: An expected N-terminal binding mode corresponds to the established view of opioid peptide binding, whereas an unexpected C-terminal binding mode is newly discovered. Surprisingly, both binding modes provide high affinity and agonistic activity at the μ opioid receptor in vitro. Furthermore, the unprecedented C-terminal binding mode shows potent dose-dependent antinociception in vivo. Finally, in silico docking studies support receptor activation by both dermorphin binding modes and suggest a biological relevance for dermorphin itself. Relevant ligand-protein interactions are similar for both binding modes, which is in line with previous protein mutation studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Signatures of Mechanically Interlocked Topology of Lasso Peptides by Ion Mobility-Mass Spectrometry: Lessons from a Collection of Representatives

NASA Astrophysics Data System (ADS)

Fouque, Kevin Jeanne Dit; Lavanant, Hélène; Zirah, Séverine; Hegemann, Julian D.; Zimmermann, Marcel; Marahiel, Mohamed A.; Rebuffat, Sylvie; Afonso, Carlos

2017-02-01

Lasso peptides are characterized by a mechanically interlocked structure, where the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. Their compact and stable structures have a significant impact on their biological and physical properties and make them highly interesting for drug development. Ion mobility - mass spectrometry (IM-MS) has shown to be effective to discriminate the lasso topology from their corresponding branched-cyclic topoisomers in which the C-terminal tail is unthreaded. In fact, previous comparison of the IM-MS data of the two topologies has yielded three trends that allow differentiation of the lasso fold from the branched-cyclic structure: (1) the low abundance of highly charged ions, (2) the low change in collision cross sections (CCS) with increasing charge state and (3) a narrow ion mobility peak width. In this study, a three-dimensional plot was generated using three indicators based on these three trends: (1) mean charge divided by mass (ζ), (2) relative range of CCS covered by all protonated molecules (ΔΩ/Ω) and (3) mean ion mobility peak width (δΩ). The data were first collected on a set of twenty one lasso peptides and eight branched-cyclic peptides. The indicators were obtained also for eight variants of the well-known lasso peptide MccJ25 obtained by site-directed mutagenesis and further extended to five linear peptides, two macrocyclic peptides and one disulfide constrained peptide. In all cases, a clear clustering was observed between constrained and unconstrained structures, thus providing a new strategy to discriminate mechanically interlocked topologies.

Metal-Assisted Channel Stabilization: Disposition of a Single Histidine on the N-terminus of Alamethicin Yields Channels with Extraordinarily Long Lifetimes

PubMed Central

Noshiro, Daisuke; Asami, Koji; Futaki, Shiroh

2010-01-01

Abstract Alamethicin, a member of the peptaibol family of antibiotics, is a typical channel-forming peptide with a helical structure. The self-assembly of the peptide in the membranes yields voltage-dependent channels. In this study, three alamethicin analogs possessing a charged residue (His, Lys, or Glu) on their N-termini were designed with the expectation of stabilizing the transmembrane structure. A slight elongation of channel lifetime was observed for the Lys and Glu analogs. On the other hand, extensive stabilization of certain channel open states was observed for the His analog. This stabilization was predominantly observed in the presence of metal ions such as Zn2+, suggesting that metal coordination with His facilitates the formation of a supramolecular assembly in the membranes. Channel stability was greatly diminished by acetylation of the N-terminal amino group, indicating that the N-terminal amino group also plays an important role in metal coordination. PMID:20441743

Selective neurotensin-derived internally quenched fluorogenic substrates for neurolysin (EC 3.4.24.16): comparison with thimet oligopeptidase (EC 3.4.24.15) and neprilysin (EC 3.4.24.11).

PubMed

Oliveira, V; Campos, M; Hemerly, J P; Ferro, E S; Camargo, A C; Juliano, M A; Juliano, L

2001-05-15

Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for neurolysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15 or TOP), and neprilysin (EC 3.4.24.11 or NEP). Abz-LYENKPRRPYILQ-EDDnp (where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine and Abz is ortho-aminobenzoic acid) was derived from neurotensin by the introduction of Q-EDDnp at the C-terminal end of peptide and by the substitution of the pyroglutamic (pE) residue at N-terminus for Abz and a series of shorter peptides was obtained by deletion of amino acids residues from C-terminal, N-terminal, or both sides. Neurolysin and TOP hydrolyzed the substrates at P--Y or Y--I or R--R bonds depending on the sequence and size of the peptides, while NEP cleaved P-Y or Y-I bonds according to its S'(1) specificity. One of these substrates, Abz-NKPRRPQ-EDDnp was a specific and sensitive substrate for neurolysin (k(cat) = 7.0 s(-1), K(m) = 1.19 microM and k(cat)/K(m) = 5882 mM(-1). s(-1)), while it was completely resistant to NEP and poorly hydrolyzed by TOP and also by prolyl oligopeptidase (EC 3.4.21.26). Neurolysin concentrations as low as 1 pM were detected using this substrate under our conditions and its analogue Abz-NKPRAPQ-EDDnp was hydrolyzed by neurolysin with k(cat) = 14.03 s(-1), K(m) = 0.82 microM, and k(cat)/K(m) = 17,110 mM(-1). s(-1), being the best substrate so far described for this peptidase. Copyright 2001 Academic Press.

Structural basis of protein translocation by the Vps4-Vta1 AAA ATPase

PubMed Central

Monroe, Nicole; Han, Han; Shen, Peter S; Sundquist, Wesley I; Hill, Christopher P

2017-01-01

Many important cellular membrane fission reactions are driven by ESCRT pathways, which culminate in disassembly of ESCRT-III polymers by the AAA ATPase Vps4. We report a 4.3 Å resolution cryo-EM structure of the active Vps4 hexamer with its cofactor Vta1, ADP·BeFx, and an ESCRT-III substrate peptide. Four Vps4 subunits form a helix whose interfaces are consistent with ATP binding, is stabilized by Vta1, and binds the substrate peptide. The fifth subunit approximately continues this helix but appears to be dissociating. The final Vps4 subunit completes a notched-washer configuration as if transitioning between the ends of the helix. We propose that ATP binding propagates growth at one end of the helix while hydrolysis promotes disassembly at the other end, so that Vps4 ‘walks’ along ESCRT-III until it encounters the ordered N-terminal domain to destabilize the ESCRT-III lattice. This model may be generally applicable to other protein-translocating AAA ATPases. DOI: http://dx.doi.org/10.7554/eLife.24487.001 PMID:28379137

The Human Leukocyte Antigen (HLA)-B27 Peptidome in Vivo, in Spondyloarthritis-susceptible HLA-B27 Transgenic Rats and the Effect of Erap1 Deletion.

PubMed

Barnea, Eilon; Melamed Kadosh, Dganit; Haimovich, Yael; Satumtira, Nimman; Dorris, Martha L; Nguyen, Mylinh T; Hammer, Robert E; Tran, Tri M; Colbert, Robert A; Taurog, Joel D; Admon, Arie

2017-04-01

HLA-B27 is a class I major histocompatibility (MHC-I) allele that confers susceptibility to the rheumatic disease ankylosing spondylitis (AS) by an unknown mechanism. ERAP1 is an aminopeptidase that trims peptides in the endoplasmic reticulum for binding to MHC-I molecules. ERAP1 shows genetic epistasis with HLA-B27 in conferring susceptibility to AS. Male HLA-B27 transgenic rats develop arthritis and serve as an animal model of AS, whereas female B27 transgenic rats remain healthy. We used large scale quantitative mass spectrometry to identify over 15,000 unique HLA-B27 peptide ligands, isolated after immunoaffinity purification of the B27 molecules from the spleens of HLA-B27 transgenic rats. Heterozygous deletion of Erap1, which reduced the Erap1 level to less than half, had no qualitative or quantitative effects on the B27 peptidome. Homozygous deletion of Erap1 affected approximately one-third of the B27 peptidome but left most of the B27 peptidome unchanged, suggesting the possibility that some of the HLA-B27 immunopeptidome is not processed in the presence of Erap1. Deletion of Erap1 was permissive for the AS-like phenotype, increased mean peptide length and increased the frequency of C-terminal hydrophobic residues and of N-terminal Ala, Ser, or Lys. The presence of Erap1 increased the frequency of C-terminal Lys and Arg, of Glu and Asp at intermediate residues, and of N-terminal Gly. Several peptides of potential interest in AS pathogenesis, previously identified in human cell lines, were isolated. However, rats susceptible to arthritis had B27 peptidomes similar to those of non-susceptible rats, and no peptides were found to be uniquely associated with arthritis. Whether specific B27-bound peptides are required for AS pathogenesis remains to be determined. Data are available via ProteomeXchange with identifier PXD005502. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Engineering RNA phage MS2 virus-like particles for peptide display

NASA Astrophysics Data System (ADS)

Jordan, Sheldon Keith

Phage display is a powerful and versatile technology that enables the selection of novel binding functions from large populations of randomly generated peptide sequences. Random sequences are genetically fused to a viral structural protein to produce complex peptide libraries. From a sufficiently complex library, phage bearing peptides with practically any desired binding activity can be physically isolated by affinity selection, and, since each particle carries in its genome the genetic information for its own replication, the selectants can be amplified by infection of bacteria. For certain applications however, existing phage display platforms have limitations. One such area is in the field of vaccine development, where the goal is to identify relevant epitopes by affinity-selection against an antibody target, and then to utilize them as immunogens to elicit a desired antibody response. Today, affinity selection is usually conducted using display on filamentous phages like M13. This technology provides an efficient means for epitope identification, but, because filamentous phages do not display peptides in the high-density, multivalent arrays the immune system prefers to recognize, they generally make poor immunogens and are typically useless as vaccines. This makes it necessary to confer immunogenicity by conjugating synthetic versions of the peptides to more immunogenic carriers. Unfortunately, when introduced into these new structural environments, the epitopes often fail to elicit relevant antibody responses. Thus, it would be advantageous to combine the epitope selection and immunogen functions into a single platform where the structural constraints present during affinity selection can be preserved during immunization. This dissertation describes efforts to develop a peptide display system based on the virus-like particles (VLPs) of bacteriophage MS2. Phage display technologies rely on (1) the identification of a site in a viral structural protein that is present on the surface of the virus particle and can accept foreign sequence insertions without disruption of protein folding and viral particle assembly, and (2) on the encapsidation of nucleic acid sequences encoding both the VLP and the peptide it displays. The experiments described here are aimed at satisfying the first of these two requirements by engineering efficient peptide display at two different sites in MS2 coat protein. First, we evaluated the suitability of the N-terminus of MS2 coat for peptide insertions. It was observed that random N-terminal 10-mer fusions generally disrupted protein folding and VLP assembly, but by bracketing the foreign sequences with certain specific dipeptides, these defects could be suppressed. Next, the suitability of a coat protein surface loop for foreign sequence insertion was tested. Specifically, random sequence peptides were inserted into the N-terminal-most AB-loop of a coat protein single-chain dimer. Again we found that efficient display required the presence of appropriate dipeptides bracketing the peptide insertion. Finally, it was shown that an N-terminal fusion that tended to interfere specifically with capsid assembly could be efficiently incorporated into mosaic particles when co-expressed with wild-type coat protein.

UVnovo: A De Novo Sequencing Algorithm Using Single Series of Fragment Ions via Chromophore Tagging and 351 nm Ultraviolet Photodissociation Mass Spectrometry

PubMed Central

Robotham, Scott A.; Horton, Andrew P.; Cannon, Joe R.; Cotham, Victoria C.; Marcotte, Edward M.; Brodbelt, Jennifer S.

2016-01-01

De novo peptide sequencing by mass spectrometry represents an important strategy for characterizing novel peptides and proteins, in which a peptide’s amino acid sequence is inferred directly from the precursor peptide mass and tandem mass spectrum (MS/MS or MS3) fragment ions, without comparison to a reference proteome. This method is ideal for organisms or samples lacking a complete or well-annotated reference sequence set. One of the major barriers to de novo spectral interpretation arises from confusion of N- and C-terminal ion series due to the symmetry between b and y ion pairs created by collisional activation methods (or c, z ions for electron-based activation methods). This is known as the ‘antisymmetric path problem’ and leads to inverted amino acid subsequences within a de novo reconstruction. Here, we combine several key strategies for de novo peptide sequencing into a single high-throughput pipeline: high efficiency carbamylation blocks lysine side chains, and subsequent tryptic digestion and N-terminal peptide derivatization with the ultraviolet chromophore AMCA yields peptides susceptible to 351 nm ultraviolet photodissociation (UVPD). UVPD-MS/MS of the AMCA-modified peptides then predominantly produces y ions in the MS/MS spectra, specifically addressing the antisymmetric path problem. Finally, the program UVnovo applies a random forest algorithm to automatically learn from and then interpret UVPD mass spectra, passing results to a hidden Markov model for de novo sequence prediction and scoring. We show this combined strategy provides high performance de novo peptide sequencing, enabling the de novo sequencing of thousands of peptides from an E. coli lysate at high confidence. PMID:26938041

NMR investigations of the dual targeting peptide of Thr-tRNA synthetase and its interaction with the mitochondrial Tom20 receptor in Arabidopsis thaliana.

PubMed

Ye, Weihua; Spånning, Erika; Unnerståle, Sofia; Gotthold, David; Glaser, Elzbieta; Mäler, Lena

2012-10-01

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins containing an N-terminal targeting peptide and are imported into mitochondria through the import machineries, the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mitochondrial membrane (TIM). The N-terminal targeting peptide of precursor proteins destined for the mitochondrial matrix is recognized by the Tom20 receptor and plays an important role in the import process. Protein import is usually organelle specific, but several plant proteins are dually targeted into mitochondria and chloroplasts using an ambiguous dual targeting peptide. We present NMR studies of the dual targeting peptide of Thr-tRNA synthetase and its interaction with Tom20 in Arabidopsis thaliana. Our findings show that the targeting peptide is mostly unstructured in buffer, with a propensity to form α-helical structure in one region, S6-F27, and a very weak β-strand propensity for Q34-Q38. The α-helical structured region has an amphiphilic character and a φχχφφ motif, both of which have previously been shown to be important for mitochondrial import. Using NMR we have mapped out two regions in the peptide that are important for Tom20 recognition: one of them, F9-V28, overlaps with the amphiphilic region, and the other comprises residues L30-Q39. Our results show that the targeting peptide may interact with Tom20 in several ways. Furthermore, our results indicate a weak, dynamic interaction. The results provide for the first time molecular details on the interaction of the Tom20 receptor with a dual targeting peptide. © 2012 The Authors Journal compilation © 2012 FEBS.

Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein

PubMed Central

Patnaik, Bharat Bhusan; Kim, Dong Hyun; Oh, Seung Han; Song, Yong-Su; Chanh, Nguyen Dang Minh; Kim, Jong Sun; Jung, Woo-jin; Saha, Atul Kumar; Bindroo, Bharat Bhushan; Han, Yeon Soo

2012-01-01

Background Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens. Methodology/Principal Findings Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense. Conclusions/Significance The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba. PMID:23284650

Natriuretic peptides: Diagnostic and therapeutic use

PubMed Central

Pandit, Kaushik; Mukhopadhyay, Pradip; Ghosh, Sujoy; Chowdhury, Subhankar

2011-01-01

Natriuretic peptides (NPs) are hormones which are mainly secreted from heart and have important natriuretic and kaliuretic properties. There are four different groups NPs identified till date [atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and dendroaspis natriuretic peptide, a D-type natriuretic peptide (DNP)], each with its own characteristic functions. The N-terminal part of the prohormone of BNP, NT-proBNP, is secreted alongside BNP and has been documented to have important diagnostic value in heart failure. NPs or their fragments have been subjected to scientific observation for their diagnostic value and this has yielded important epidemiological data for interpretation. However, little progress has been made in harnessing the therapeutic potential of these cardiac hormones. PMID:22145138

Hydrophobic benzyl amines as supports for liquid-phase C-terminal amidated peptide synthesis: application to the preparation of ABT-510.

PubMed

Matsumoto, Emiko; Fujita, Yuko; Okada, Yohei; Kauppinen, Esko I; Kamiya, Hidehiro; Chiba, Kazuhiro

2015-09-01

C-terminal amidation is one of the most common modification of peptides and frequently found in bioactive peptides. However, the C-terminal modification must be creative, because current chemical synthetic techniques of peptides are dominated by the use of C-terminal protecting supports. Therefore, it must be carried out after the removal of such supports, complicating reaction work-up and product isolation. In this context, hydrophobic benzyl amines were successfully added to the growing toolbox of soluble tag-assisted liquid-phase peptide synthesis as supports, leading to the total synthesis of ABT-510 (2). Although an ethyl amide-forming type was used in the present work, different types of hydrophobic benzyl amines could also be simply designed and prepared through versatile reductive aminations in one step. The standard acidic treatment used in the final deprotection step for peptide synthesis gave the desired C-terminal secondary amidated peptide with no epimerization. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

A simulation investigation on interaction mechanism between Ebola nucleoprotein and VP35 peptide.

PubMed

Ding, Jing-Na; Zhang, Yan-Jun; Zhong, Hui; Ao, Cheng-Cheng; Han, Ju-Guang

2018-03-01

Ebola viruses (EBOV) will induce acute hemorrhagic fever, which is fatal to humans and nonhuman primates. The combination of EBOV VP35 peptide with nucleoprotein N-terminal (NPNTD) is proposed based on static crystal structures in recent studies, but VP35 binding mechanism and conformational dynamics are still unclear. This investigation, using Molecular Dynamic (MD) simulation and Molecular Mechanics Generalized Born Surface Area (MM-GB/SA) energy calculation, more convincingly proves the greater roles of the protein binding mechanisms than do hints from the static crystal structure observations. Conformational analysis of the systems demonstrate that combination with VP35 may lead to the conformational transition of NPNTD from "open" to "closed" state. According to the analyses of binding free energies and their decomposition, VP35 residue R37 plays a crucial role in wild type as well as mutant systems. Mutations of I29 and L33 to aspartate as well as M34 to proline affect binding affinity mainly through influencing electrostatic interaction, which is closely related to H-bonds formation. In addition, mutations mainly affect β-hairpin and loop regions, among which, M34P may have the greatest influence to the binding. This study may provide specific binding mechanisms between VP35 peptide and NPNTD, especially some important residues concerning binding.

Synthesis of N-peptide-6-amino-D-luciferin Conjugates.

PubMed

Kovács, Anita K; Hegyes, Péter; Szebeni, Gábor J; Nagy, Lajos I; Puskás, László G; Tóth, Gábor K

2018-01-01

A general strategy for the synthesis of N -peptide-6-amino-D-luciferin conjugates has been developed. The applicability of the strategy was demonstrated with the preparation of a known substrate, N -Z-Asp-Glu-Val-Asp-6-amino-D-luciferin ( N -Z-DEVD-aLuc). N -Z-DEVD-aLuc was obtained via a hybrid liquid/solid phase synthesis method, in which the appropriately protected C-terminal amino acid was coupled to 6-amino-2-cyanobenzothiazole and the resulting conjugate was reacted with D-cysteine in order to get the protected amino acid-6-amino-D-luciferin conjugate, which was then attached to resin. The resulting loaded resin was used for the solid-phase synthesis of the desired N -peptide-6-amino-D-luciferin conjugate without difficulties, which was then attested with NMR spectroscopy and LC-MS, and successfully tested in a bioluminescent system.

Synthesis of N-peptide-6-amino-D-luciferin Conjugates

PubMed Central

Kovács, Anita K.; Hegyes, Péter; Szebeni, Gábor J.; Nagy, Lajos I.; Puskás, László G.; Tóth, Gábor K.

2018-01-01

A general strategy for the synthesis of N-peptide-6-amino-D-luciferin conjugates has been developed. The applicability of the strategy was demonstrated with the preparation of a known substrate, N-Z-Asp-Glu-Val-Asp-6-amino-D-luciferin (N-Z-DEVD-aLuc). N-Z-DEVD-aLuc was obtained via a hybrid liquid/solid phase synthesis method, in which the appropriately protected C-terminal amino acid was coupled to 6-amino-2-cyanobenzothiazole and the resulting conjugate was reacted with D-cysteine in order to get the protected amino acid-6-amino-D-luciferin conjugate, which was then attached to resin. The resulting loaded resin was used for the solid-phase synthesis of the desired N-peptide-6-amino-D-luciferin conjugate without difficulties, which was then attested with NMR spectroscopy and LC-MS, and successfully tested in a bioluminescent system. PMID:29725588

Intravitreal injection or topical eye-drop application of a μ-calpain C2L domain peptide protects against photoreceptor cell death in Royal College of Surgeons' rats, a model of retinitis pigmentosa.

PubMed

Ozaki, Taku; Nakazawa, Mitsuru; Yamashita, Tetsuro; Sorimachi, Hiroyuki; Hata, Shoji; Tomita, Hiroshi; Isago, Hitomi; Baba, Ayaka; Ishiguro, Sei-Ichi

2012-11-01

Mitochondrial μ-calpain initiates apoptosis-inducing factor (AIF)-dependent apoptosis in retinal photoreceptor degeneration. Mitochondrial μ-calpain inhibitors may represent therapeutic targets for the disease. Therefore, we sought to identify inhibitors of mitochondrial calpains and determine their effects in Royal College of Surgeons' (RCS) rats, an animal model of retinitis pigmentosa (RP). We synthesized 20-mer peptides of the C2-like (C2L) domain of μ-calpain. Two μ-calpain peptides N2 and N9 inhibited mitochondrial μ-calpain activity (IC(50); 892 and 498nM, respectively), but not other proteases. Western blotting showed that 50μM of both μ-calpain peptides caused specific degradation of mitochondrial μ-calpain. Three-dimensional structure of calpains suggested that the peptides N2 and N9 corresponded to the regions forming salt bridges between the protease core domain 2 and the C2L domain. We determined the inhibitory regions of μ-calpain peptides N2 and N9 using 10-mers, and one peptide, N2-10-2, inhibited the activity of mitochondrial μ-calpain (IC(50); 112nM). We next conjugated the peptide N2-10-2 to the C-terminal of HIV-1 tat (HIV), a cell-penetrating peptide. Using isolated rat liver mitochondria, 50μM HIV-conjugated μ-calpain N2-10-2 peptide (HIV-Nμ, IC(50); 285nM) significantly inhibited AIF truncation. The intravitreal injection of 20mM HIV-Nμ also prevented retinal photoreceptor apoptosis determined by TUNEL staining, and preserved retinal function assessed by electroretinography in RCS rats. Topical application of 40mM HIV-Nμ also prevented apoptosis of retinal photoreceptors in RCS rats. Our results demonstrate that HIV-Nμ, a peptide inhibitor of mitochondrial μ-calpain, offers a new modality for treating RP. Copyright © 2012 Elsevier B.V. All rights reserved.

AntiAngioPred: A Server for Prediction of Anti-Angiogenic Peptides.

PubMed

Ettayapuram Ramaprasad, Azhagiya Singam; Singh, Sandeep; Gajendra P S, Raghava; Venkatesan, Subramanian

2015-01-01

The process of angiogenesis is a vital step towards the formation of malignant tumors. Anti-angiogenic peptides are therefore promising candidates in the treatment of cancer. In this study, we have collected anti-angiogenic peptides from the literature and analyzed the residue preference in these peptides. Residues like Cys, Pro, Ser, Arg, Trp, Thr and Gly are preferred while Ala, Asp, Ile, Leu, Val and Phe are not preferred in these peptides. There is a positional preference of Ser, Pro, Trp and Cys in the N terminal region and Cys, Gly and Arg in the C terminal region of anti-angiogenic peptides. Motif analysis suggests the motifs "CG-G", "TC", "SC", "SP-S", etc., which are highly prominent in anti-angiogenic peptides. Based on the primary analysis, we developed prediction models using different machine learning based methods. The maximum accuracy and MCC for amino acid composition based model is 80.9% and 0.62 respectively. The performance of the models on independent dataset is also reasonable. Based on the above study, we have developed a user-friendly web server named "AntiAngioPred" for the prediction of anti-angiogenic peptides. AntiAngioPred web server is freely accessible at http://clri.res.in/subramanian/tools/antiangiopred/index.html (mirror site: http://crdd.osdd.net/raghava/antiangiopred/).

Hydrocarbon-stapled lipopeptides exhibit selective antimicrobial activity.

PubMed

Jenner, Zachary B; Crittenden, Christopher M; Gonzalez, Martín; Brodbelt, Jennifer S; Bruns, Kerry A

2017-05-01

Antimicrobial peptides (AMPs) occur widely in nature and have been studied for their therapeutic potential. AMPs are of interest due to the large number of possible chemical structural combinations using natural and unnatural amino acids, with varying effects on their biological activities. Using physicochemical properties from known naturally occurring amphipathic cationic AMPs, several hydrocarbon-stapled lipopeptides (HSLPs) were designed, synthesized, and tested for antimicrobial properties. Peptides were chemically modified by N-terminal acylation, C-terminal amidation, and some were hydrocarbon stapled by intramolecular olefin metathesis. The effects of peptide length, amphipathic character, and stapling on antimicrobial activity were tested against Escherichia coli, three species of Gram-positive bacteria (Staphylococcus aureus, Bacillus megaterium, and Enterococcus faecalis), and two strains of Candida albicans. Peptides were shown to disrupt liposomes of different phospholipid composition, as measured by leakage of a fluorescent compound from vesicles. Peptides with (S)-2-(4'-pentenyl)-alanine substituted for l-alanine in a reference peptide showed a marked increase in antimicrobial activity, hemolysis, and membrane disruption. Stapled peptides exhibited slightly higher antimicrobial potency; those with greatest hydrophobic character showed the greatest hemolysis and liposome leakage, but lower antimicrobial activity. The results support a model of HSLPs as membrane-disruptive AMPs with potent antimicrobial activity and relatively low hemolytic potential at biologically active peptide concentrations. © 2017 Wiley Periodicals, Inc.

Solid-Phase Synthesis of Diverse Peptide Tertiary Amides By Reductive Amination

PubMed Central

Pels, Kevin; Kodadek, Thomas

2015-01-01

The synthesis of libraries of conformationally-constrained peptide-like oligomers is an important goal in combinatorial chemistry. In this regard an attractive building block is the N-alkylated peptide, also known as peptide tertiary amide (PTA). PTAs are strongly biased conformationally due to allylic 1,3 strain interactions. We report here an improved synthesis of these species on solid supports through the use of reductive amination chemistry using amino acid-terminated, bead-displayed oligomers and diverse aldehydes. The utility of this chemistry is demonstrated by the synthesis of a library of 10,000 mixed peptoid-PTA oligomers. PMID:25695359

Solid-phase synthesis of diverse peptide tertiary amides by reductive amination.

PubMed

Pels, Kevin; Kodadek, Thomas

2015-03-09

The synthesis of libraries of conformationally constrained peptide-like oligomers is an important goal in combinatorial chemistry. In this regard an attractive building block is the N-alkylated peptide, also known as a peptide tertiary amide (PTA). PTAs are conformationally constrained because of allylic 1,3 strain interactions. We report here an improved synthesis of these species on solid supports through the use of reductive amination chemistry using amino acid-terminated, bead-displayed oligomers and diverse aldehydes. The utility of this chemistry is demonstrated by the synthesis of a library of 10,000 mixed peptoid-PTA oligomers.

Interaction of a putative BH3 domain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Dong-Hwa; Ha, Ji-Hyang; Kim, Yul

Highlights: {yields} Identification of a conserved BH3 motif in C-terminal coiled coil region of nCLU. {yields} The nCLU BH3 domain binds to BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. {yields} A conserved binding mechanism of nCLU BH3 and the other pro-apoptotic BH3 peptides with Bcl-X{sub L}. {yields} The absolutely conserved Leu323 and Asp328 of nCLU BH3 domain are critical for binding to Bcl-X{sub L.} {yields} Molecular understanding of the pro-apoptotic function of nCLU as a novel BH3-only protein. -- Abstract: Clusterin (CLU) is a multifunctional glycoprotein that is overexpressed in prostate and breast cancers. Although CLU is knownmore » to be involved in the regulation of apoptosis and cell survival, the precise molecular mechanism underlying the pro-apoptotic function of nuclear CLU (nCLU) remains unclear. In this study, we identified a conserved BH3 motif in C-terminal coiled coil (CC2) region of nCLU by sequence analysis and characterized the molecular interaction of the putative nCLU BH3 domain with anti-apoptotic Bcl-2 family proteins by nuclear magnetic resonance (NMR) spectroscopy. The chemical shift perturbation data demonstrated that the nCLU BH3 domain binds to pro-apoptotic BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. A structural model of the Bcl-X{sub L}/nCLU BH3 peptide complex reveals that the binding mode is remarkably similar to those of other Bcl-X{sub L}/BH3 peptide complexes. In addition, mutational analysis confirmed that Leu323 and Asp328 of nCLU BH3 domain, absolutely conserved in the BH3 motifs of BH3-only protein family, are critical for binding to Bcl-X{sub L}. Taken altogether, our results suggest a molecular basis for the pro-apoptotic function of nCLU by elucidating the residue specific interactions of the BH3 motif in nCLU with anti-apoptotic Bcl-2 family proteins.« less

Studies on Aculeines: Synthetic Strategy to the Fully Protected Protoaculeine B, the N-Terminal Amino Acid of Aculeine B.

PubMed

Shiozaki, Hiroki; Miyahara, Masayoshi; Otsuka, Kazunori; Miyako, Kei; Honda, Akito; Takasaki, Yuichi; Takamizawa, Satoshi; Tukada, Hideyuki; Ishikawa, Yuichi; Sakai, Ryuichi; Oikawa, Masato

2018-05-23

A synthetic strategy for accessing protoaculeine B (1), the N-terminal amino acid of the highly modified peptide toxin aculeine, was developed via the synthesis of the fully protected natural homologue of 1 with a 12-mer poly(propanediamine). The synthesis of mono(propanediamine) analog 2, as well as core amino acid 3, was demonstrated by this strategy. New amino acid 3 induced convulsions in mice; however, compound 2 showed no such activity.

Pyrylium-based dye and charge tagging in proteomics.

PubMed

Bayer, Malte; König, Simone

2016-11-01

The pyrylium group is a selective reagent for ε-amino groups in proteins. In particular, for fluorescence labeling, a number of advantages over traditional N-hydroxysuccinimidyl ester chemistry were recognized such as the rapid prestaining procedure. Here, we have investigated the labeling reaction for the fluorogenic pyrylium dye Py-1 using liquid chromatography coupled to MS with the aim of determining its specificity and possible side products. Peptides containing no, one, and two lysine residue and a choice of no or one cysteine residue were labeled with Py-1 at yields > 30%. Gas phase fragmentation proved both labeling of lysine residues as well as that of the N-terminus also in peptides that contained a lysine residue. Evidence for cysteine labeling was not found, but several other products were detected such as the results of rearrangements with adjacent acidic amino acids. Apart from the use as a fluorogenic label, Py-1 recommends itself for N-terminal charge tagging as alternative to the commonly used quaternary ammonium salts. Predominantly a- and b-type ion series were observed for N-terminally labeled peptides. Further applications include chromophore tagging since the labeled product is not only fluorescent but also colored red. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization and mapping of cDNA encoding aspartate aminotransferase in rice, Oryza sativa L.

PubMed

Song, J; Yamamoto, K; Shomura, A; Yano, M; Minobe, Y; Sasaki, T

1996-10-31

Fifteen cDNA clones, putatively identified as encoding aspartate aminotransferase (AST, EC 2.6.1.1.), were isolated and partially sequenced. Together with six previously isolated clones putatively identified to encode ASTs (Sasaki, et al. 1994, Plant Journal 6, 615-624), their sequences were characterized and classified into 4 cDNA species. Two of the isolated clones, C60213 and C2079, were full-length cDNAs, and their complete nucleotide sequences were determined. C60213 was 1612 bp long and its deduced amino acid sequence showed 88% homology with that of Panicum miliaceum L. mitochondrial AST. The C60213-encoded protein had an N-terminal amino acid sequence that was characteristic of a mitochondrial transit peptide. On the other hand, C2079 was 1546 bp long and had 91% amino acid sequence homology with P. miliaceum L. cytosolic AST but lacked in the transit peptide sequence. The homologies of nucleotide sequences and deduced amino acid sequences of C2079 and C60213 were 54% and 52%, respectively. C2079 and C60213 were mapped on chromosomes 1 and 6, respectively, by restriction fragment length polymorphism linkage analysis. Northern blot analysis using C2079 as a probe revealed much higher transcript levels in callus and root than in green and etiolated shoots, suggesting tissue-specific variations of AST gene expression.

Optimization of Acidothermus Celluloyticus Endoglucanase (E1) Production in Transgenic Tobacco Plants by Transcriptional, Post-transcription and Post-Translational Modification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Ziyu; Hooker, Brian S.; Quesenberry, Ryan D.

2005-10-01

Biochemical characteristics of Acidothermus cellulolyticus endoglucanase (E1) and its physiological effects in transgenic tobacco (Nicotiana tabacum) has been studied previously. In an attempt to obtain a high level of production of intact E1 in transgenic plants, the E1 gene was expressed under the control of strong Mac promoter (a hybrid promoter of manopine synthase promoter and cauliflower mosaic virus 35S promoter enhancer region) or tomato Rubisco small subunit (RbcS-3C) promoter with different 5’ untranslated leader (UTL) sequence and targeted to different subcellular comartmentations with various transit peptides. The expression of E1 protein in transgenic tobacco plants was determined via E1more » activity, protein immunobloting, and RNA gel-blotting analyses. Effects of different transit peptides on E1 protein production and its stability were examined in transgenic tobacco plants carrying one of six transgene expression vectors with the same (Mac) promoter and transcription terminator (Tmas). Transgenic tobacco plants with apoplast transit peptide (Mm-apo) had the highest average E1 activity and protein accumulation , while E1 protein was more stable in transgenic plants with no transit peptide (Mm) than others. The E1 expression under tomato RbcS-3C promoter was higher than that under Mac promoter based on the average E1 activity, E1 protein accumulation, and RNA gel-blotting. The E1 expression was increased more than two fold when the 5’-UTL of alfalfa mosaic virus RNA4 gene replaced the UTL of RbcS-3C promoter, while the UTL of alfalfa mosaic virus RNA4 gene was less effective than the UTL of Mac promoter. The optimal combination of promoter, 5’-UTL, and subcellular compartmentation (transit peptide) for E1 protein production in transgenic tobacco plants are discussed.« less

Amino-acid sequence and predicted three-dimensional structure of pea seed (Pisum sativum) ferritin.

PubMed Central

Lobreaux, S; Yewdall, S J; Briat, J F; Harrison, P M

1992-01-01

The iron storage protein, ferritin, is widely distributed in the living kingdom. Here the complete cDNA and derived amino-acid sequence of pea seed ferritin are described, together with its predicted secondary structure, namely a four-helix-bundle fold similar to those of mammalian ferritins, with a fifth short helix at the C-terminus. An N-terminal extension of 71 residues contains a transit peptide (first 47 residues) responsible for plastid targetting as in other plant ferritins, and this is cleaved before assembly. The second part of the extension (24 residues) belongs to the mature subunit; it is cleaved during germination. The amino-acid sequence of pea seed ferritin is aligned with those of other ferritins (49% amino-acid identity with H-chains and 40% with L-chains of human liver ferritin in the aligned region). A three-dimensional model has been constructed by fitting the aligned sequence to the coordinates of human H-chains, with appropriate modifications. A folded conformation with an 11-residue helix is predicted for the N-terminal extension. As in mammalian ferritins, 24 subunits assemble into a hollow shell. In pea seed ferritin, its N-terminal extension is exposed on the outside surface of the shell. Within each pea subunit is a ferroxidase centre resembling those of human ferritin H-chains except for a replacement of Glu-62 by His. The channel at the 4-fold-symmetry axes defined by E-helices, is predicted to be hydrophilic in plant ferritins, whereas it is hydrophobic in mammalian ferritins. Images Fig. 3. Fig. 5. Fig. 6. PMID:1472006

Propeptide big-endothelin, N-terminal-pro brain natriuretic peptide and mortality. The Ludwigshafen risk and cardiovascular health (LURIC) study.

PubMed

Gergei, Ingrid; Krämer, Bernhard K; Scharnagl, Hubert; Stojakovic, Tatjana; März, Winfried; Mondorf, Ulrich

The endothelin system (Big-ET-1) is a key regulator in cardiovascular (CV) disease and congestive heart failure (CHF). We have examined the incremental value of Big-ET-1 in predicting total and CV mortality next to the well-established CV risk marker N-Terminal Pro-B-Type Natriuretic Peptide (NT-proBNP). Big-ET-1 and NT-proBNP were determined in 2829 participants referred for coronary angiography (follow-up 9.9 years). Big-ET-1 is an independent predictor of total, CV mortality and death due to CHF. The conjunct use of Big-ET-1 and NT-proBNP improves the risk stratification of patients with intermediate to high risk of CV death and CHF. Big-ET-1improves risk stratification in patients referred for coronary angiography.

The use of N-terminal pro-brain natriuretic Peptide to evaluate vascular disease in elderly patients with mental illness.

PubMed

Nilsson, Karin; Gustafson, Lars; Hultberg, Björn

2012-01-01

Serum N-terminal pro-brain natriuretic peptide (NT-proBNP) is regarded as a sensitive marker of cardiovascular disease. Vascular disease plays an important role in cognitive impairment. In 447 elderly patients with mental illness, serum NT-proBNP level and the presence or absence of vascular disease according to the medical record were used to categorize patients in different subgroups of vascular disease. Patients with vascular disease and elevated serum NT-proBNP level had a lower cognition level, shorter survival time, lower renal function and a higher percentage of pathological brain imaging than patients with vascular disease and normal NT-proBNP level. Thus, elevated serum NT-proBNP level might be helpful to detect patients who have a more severe cardiovascular disease.

Combined Measurement of Soluble ST2 and Amino-Terminal Pro-B-Type Natriuretic Peptide Provides Early Assessment of Severity in Cardiogenic Shock Complicating Acute Coronary Syndrome.

PubMed

Tolppanen, Heli; Rivas-Lasarte, Mercedes; Lassus, Johan; Sadoune, Malha; Gayat, Etienne; Pulkki, Kari; Arrigo, Mattia; Krastinova, Evguenia; Sionis, Alessandro; Parissis, John; Spinar, Jindrich; Januzzi, James; Harjola, Veli-Pekka; Mebazaa, Alexandre

2017-07-01

Mortality in cardiogenic shock complicating acute coronary syndrome is high, and objective risk stratification is needed for rational use of advanced therapies such as mechanical circulatory support. Traditionally, clinical variables have been used to judge risk in cardiogenic shock. The aim of this study was to assess the added value of serial measurement of soluble ST2 and amino-terminal pro-B-type natriuretic peptide to clinical parameters for risk stratification in cardiogenic shock. CardShock (www.clinicaltrials.gov NCT01374867) is a prospective European multinational study of cardiogenic shock. The main study introduced CardShock risk score, which is calculated from seven clinical variables at baseline, and was associated with short-term mortality. Nine tertiary care university hospitals. Patients with cardiogenic shock caused by acute coronary syndrome (n=145). In this substudy, plasma samples from the study patients were analyzed at eight time points during the ICU or cardiac care unit stay. Additional prognostic value of the biomarkers was assessed with incremental discrimination improvement. The combination of soluble ST2 and amino-terminal pro-B-type natriuretic peptide showed excellent discrimination for 30-day mortality (area under the curve, 0.77 at 12 hr up to 0.93 at 5-10 d after cardiogenic shock onset). At 12 hours, patients with both biomarkers elevated (soluble ST2, ≥ 500 ng/mL and amino-terminal pro-B-type natriuretic peptide, ≥ 4,500 ng/L) had higher 30-day mortality (79%) compared to those with one or neither biomarkers elevated (31% or 10%, respectively; p < 0.001). Combined measurement of soluble ST2 and amino-terminal pro-B-type natriuretic peptide at 12 hours added value to CardShock risk score, correctly reclassifying 11% of patients. The combination of results for soluble ST2 and amino-terminal pro-B-type natriuretic peptide provides early risk assessment beyond clinical variables in patients with acute coronary syndrome-related cardiogenic shock and may help therapeutic decision making in these patients.

Expansion of ribosomally produced natural products: a nitrile hydratase- and Nif11-related precursor family

PubMed Central

2010-01-01

Background A new family of natural products has been described in which cysteine, serine and threonine from ribosomally-produced peptides are converted to thiazoles, oxazoles and methyloxazoles, respectively. These metabolites and their biosynthetic gene clusters are now referred to as thiazole/oxazole-modified microcins (TOMM). As exemplified by microcin B17 and streptolysin S, TOMM precursors contain an N-terminal leader sequence and C-terminal core peptide. The leader sequence contains binding sites for the posttranslational modifying enzymes which subsequently act upon the core peptide. TOMM peptides are small and highly variable, frequently missed by gene-finders and occasionally situated far from the thiazole/oxazole forming genes. Thus, locating a substrate for a particular TOMM pathway can be a challenging endeavor. Results Examination of candidate TOMM precursors has revealed a subclass with an uncharacteristically long leader sequence closely related to the enzyme nitrile hydratase. Members of this nitrile hydratase leader peptide (NHLP) family lack the metal-binding residues required for catalysis. Instead, NHLP sequences display the classic Gly-Gly cleavage motif and have C-terminal regions rich in heterocyclizable residues. The NHLP family exhibits a correlated species distribution and local clustering with an ABC transport system. This study also provides evidence that a separate family, annotated as Nif11 nitrogen-fixing proteins, can serve as natural product precursors (N11P), but not always of the TOMM variety. Indeed, a number of cyanobacterial genomes show extensive N11P paralogous expansion, such as Nostoc, Prochlorococcus and Cyanothece, which replace the TOMM cluster with lanthionine biosynthetic machinery. Conclusions This study has united numerous TOMM gene clusters with their cognate substrates. These results suggest that two large protein families, the nitrile hydratases and Nif11, have been retailored for secondary metabolism. Precursors for TOMMs and lanthionine-containing peptides derived from larger proteins to which other functions are attributed, may be widespread. The functions of these natural products have yet to be elucidated, but it is probable that some will display valuable industrial or medical activities. PMID:20500830

Release of free amino acids upon oxidation of peptides and proteins by hydroxyl radicals.

PubMed

Liu, Fobang; Lai, Senchao; Tong, Haijie; Lakey, Pascale S J; Shiraiwa, Manabu; Weller, Michael G; Pöschl, Ulrich; Kampf, Christopher J

2017-03-01

Hydroxyl radical-induced oxidation of proteins and peptides can lead to the cleavage of the peptide, leading to a release of fragments. Here, we used high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and pre-column online ortho-phthalaldehyde (OPA) derivatization-based amino acid analysis by HPLC with diode array detection and fluorescence detection to identify and quantify free amino acids released upon oxidation of proteins and peptides by hydroxyl radicals. Bovine serum albumin (BSA), ovalbumin (OVA) as model proteins, and synthetic tripeptides (comprised of varying compositions of the amino acids Gly, Ala, Ser, and Met) were used for reactions with hydroxyl radicals, which were generated by the Fenton reaction of iron ions and hydrogen peroxide. The molar yields of free glycine, aspartic acid, asparagine, and alanine per peptide or protein varied between 4 and 55%. For protein oxidation reactions, the molar yields of Gly (∼32-55% for BSA, ∼10-21% for OVA) were substantially higher than those for the other identified amino acids (∼5-12% for BSA, ∼4-6% for OVA). Upon oxidation of tripeptides with Gly in C-terminal, mid-chain, or N-terminal positions, Gly was preferentially released when it was located at the C-terminal site. Overall, we observe evidence for a site-selective formation of free amino acids in the OH radical-induced oxidation of peptides and proteins, which may be due to a reaction pathway involving nitrogen-centered radicals.

Small peptides patterned after the N-terminus domain of SNAP25 inhibit SNARE complex assembly and regulated exocytosis.

PubMed

Blanes-Mira, Clara; Merino, Jaime M; Valera, Elvira; Fernández-Ballester, Gregorio; Gutiérrez, Luis M; Viniegra, Salvador; Pérez-Payá, Enrique; Ferrer-Montiel, Antonio

2004-01-01

Synthetic peptides patterned after the C-terminus of synaptosomal associated protein of 25 kDa (SNAP25) efficiently abrogate regulated exocytosis. In contrast, the use of SNAP25 N-terminal-derived peptides to modulate SNAP receptors (SNARE) complex assembly and neurosecretion has not been explored. Here, we show that the N-terminus of SNAP25, specially the segment that encompasses 22Ala-44Ile, is essential for the formation of the SNARE complex. Peptides patterned after this protein domain are potent inhibitors of SNARE complex formation. The inhibitory activity correlated with their propensity to adopt an alpha-helical secondary structure. These peptides abrogated SNARE complex formation only when added previous to the onset of aggregate assembly. Analysis of the mechanism of action revealed that these peptides disrupted the binary complex formed by SNAP25 and syntaxin. The identified peptides inhibited Ca2+-dependent exocytosis from detergent-permeabilized excitable cells. Noteworthy, these amino acid sequences markedly protected intact hippocampal neurones against hypoglycaemia-induced, glutamate-mediated excitotoxicity with a potency that rivalled that displayed by botulinum neurotoxins. Our findings indicate that peptides patterned after the N-terminus of SNAP25 are potent inhibitors of SNARE complex formation and neuronal exocytosis. Because of their activity in intact neurones, these cell permeable peptides may be hits for antispasmodic and analgesic drug development.

Galactinol synthase from kidney bean cotyledon and zucchini leaf. Purification and N-terminal sequences.

PubMed Central

Liu, J J; Odegard, W; de Lumen, B O

1995-01-01

Galactinol synthase (GS) was purified 1591-fold with a 3.9% recovery from the cotyledon of kidney bean (Phaseolus vulgaris) by a novel scheme consisting of ammonium sulfate fractionation followed by diethylaminoethyl, Affi-Gel Blue, and UDP-hexanolamine affinity chromatography. The purified enzyme had a specific activity of 8.75 mumol mg-1 min-1, a pH optimum of 7.0, and requirements for manganese ion and DTT. The enzyme exhibited a Km = 0.4 mM for UDP-galactose and a Km = 4.5 mM for myo-inositol. It was identified as a 38-kD peptide that co-purified with a 41- and a 43-kD peptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purification to homogeneity was achieved by isolating the 38-kD peptide from the SDS-PAGE gel. To clarify conflicting reports in the literature about the relative molecular mass of purified GS from zucchini leaf (Cucurbita pepo), a similar scheme with modified eluting conditions was used to purify GS from this source. Zucchini leaf GS was purified to homogeneity and identified as a 36-kD peptide on SDS-PAGE. Partial N-terminal sequences of the 38-kD peptide from kidney bean cotyledon and the 36-kD peptide from zucchini leaf were obtained. To facilitate identification of GS during the purification, an assay utilizing thin-layer chromatography and an isotopic analytic imaging scanner was developed. PMID:7480343

Detection of site specific glycosylation in proteins using flow cytometry†

PubMed Central

Jayakumar, Deepak; Marathe, Dhananjay D.; Neelamegham, Sriram

2009-01-01

We tested the possibility that it is possible to express unique peptide probes on cell surfaces and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can be released from these cells by protease, and the resulting peptide can be readily captured and detected using cytometry-bead assays. Using this strategy, the peptide was immunoprecipitated onto beads bearing mAbs against either the poly-histidine sequence or the human PSGL-1. The carbohydrate epitope associated with the released peptide was detected using HECA-452 and CSLEX-1, monoclonal antibodies that recognize the sialyl Lewis-X epitope. Finally, the peptide released from cells could be separated and enriched using nickel chelate beads. Overall, such an approach that combines recombinant protein expression with flow cytometry, may be useful to quantify changes in site-specific glycosylation for basic science and clinical applications. PMID:19735085

Influence of proline upon the folding and geometry of the WALP19 transmembrane peptide.

PubMed

Thomas, Rachel; Vostrikov, Vitaly V; Greathouse, Denise V; Koeppe, Roger E

2009-12-22

The orientations, geometries, and lipid interactions of designed transmembrane (TM) peptides have attracted significant experimental and theoretical interest. Because the amino acid proline will introduce a known discontinuity into an alpha helix, we have sought to measure the extent of helix kinking caused by a single proline within the isolated TM helical domain of WALP19. For this purpose, we synthesized acetyl-GWWLALALAP(10)ALALALWWA-ethanolamide and included pairs of deuterated alanines by using 60-100% Fmoc-l-Ala-d(4) at selected sequence positions. Solid-state deuterium ((2)H) magnetic resonance spectra from oriented, hydrated samples (1/40, peptide/lipid; using several lipids) reveal signals from many of the alanine backbone C(alpha) deuterons as well as the alanine side-chain C(beta) methyl groups, whereas signals from C(alpha) deuterons generally have not been observed for similar peptides without proline. It is conceivable that altered peptide dynamics may be responsible for the apparent "unmasking" of the backbone resonances in the presence of the proline. Data analysis using the geometric analysis of labeled alanines (GALA) method reveals that the peptide helix is distorted due to the presence of the proline. To provide additional data points for evaluating the segmental tilt angles of the two halves of the peptide, we substituted selected leucines with l-Ala-d(4). Using this approach, we were able to deduce that the apparent average tilt of the C-terminal increases from approximately 4 degrees to approximately 12 degrees when Pro(10) is introduced. The segment N-terminal to proline is more complex and possibly is more dynamically flexible; Leu to Ala mutations within the N-terminal segment alter the average orientations of alanines in both segments. Nevertheless, in DOPC, we could estimate an apparent kink angle of approximately 19 degrees . Together, the results suggest that the central proline influences not only the geometry but also the dynamics of the membrane-spanning peptide. The results make up an important basis for understanding the functional role of proline in several families of membrane proteins.

Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for delta opioid binding sites.

PubMed

Erspamer, V; Melchiorri, P; Falconieri-Erspamer, G; Negri, L; Corsi, R; Severini, C; Barra, D; Simmaco, M; Kreil, G

1989-07-01

Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus Phyllomedusa, that have a higher affinity and selectivity for delta opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of Phyllomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, [D-Ala2]deltorphins I and II, show an even higher affinity for delta receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phyllomedusa skin, dermorphin, which is highly selective for mu-opioid receptors. These peptides all have the N-terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both mu and delta opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin.

Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for delta opioid binding sites.

PubMed Central

Erspamer, V; Melchiorri, P; Falconieri-Erspamer, G; Negri, L; Corsi, R; Severini, C; Barra, D; Simmaco, M; Kreil, G

1989-01-01

Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus Phyllomedusa, that have a higher affinity and selectivity for delta opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of Phyllomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, [D-Ala2]deltorphins I and II, show an even higher affinity for delta receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phyllomedusa skin, dermorphin, which is highly selective for mu-opioid receptors. These peptides all have the N-terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both mu and delta opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin. PMID:2544892

Kinetic and structural characterization of amyloid-β peptide hydrolysis by human angiotensin-1-converting enzyme.

PubMed

Larmuth, Kate M; Masuyer, Geoffrey; Douglas, Ross G; Schwager, Sylva L; Acharya, K Ravi; Sturrock, Edward D

2016-03-01

Angiotensin-1-converting enzyme (ACE), a zinc metallopeptidase, consists of two homologous catalytic domains (N and C) with different substrate specificities. Here we report kinetic parameters of five different forms of human ACE with various amyloid beta (Aβ) substrates together with high resolution crystal structures of the N-domain in complex with Aβ fragments. For the physiological Aβ(1-16) peptide, a novel ACE cleavage site was found at His14-Gln15. Furthermore, Aβ(1-16) was preferentially cleaved by the individual N-domain; however, the presence of an inactive C-domain in full-length somatic ACE (sACE) greatly reduced enzyme activity and affected apparent selectivity. Two fluorogenic substrates, Aβ(4-10)Q and Aβ(4-10)Y, underwent endoproteolytic cleavage at the Asp7-Ser8 bond with all ACE constructs showing greater catalytic efficiency for Aβ(4-10)Y. Surprisingly, in contrast to Aβ(1-16) and Aβ(4-10)Q, sACE showed positive domain cooperativity and the double C-domain (CC-sACE) construct no cooperativity towards Aβ(4-10)Y. The structures of the Aβ peptide-ACE complexes revealed a common mode of peptide binding for both domains which principally targets the C-terminal P2' position to the S2' pocket and recognizes the main chain of the P1' peptide. It is likely that N-domain selectivity for the amyloid peptide is conferred through the N-domain specific S2' residue Thr358. Additionally, the N-domain can accommodate larger substrates through movement of the N-terminal helices, as suggested by the disorder of the hinge region in the crystal structures. Our findings are important for the design of domain selective inhibitors as the differences in domain selectivity are more pronounced with the truncated domains compared to the more physiological full-length forms. The atomic coordinates and structure factors for N-domain ACE with Aβ peptides 4-10 (5AM8), 10-16 (5AM9), 1-16 (5AMA), 35-42 (5AMB) and (4-10)Y (5AMC) complexes have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ, USA (http://www.rcsb.org/). © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Design, Synthesis, and Enzymatic Evaluation of Novel ZnO Quantum Dot-Based Assay for Detection of Proteinase 3 Activity.

PubMed

Popow-Stellmaszyk, Jadwiga; Bajorowicz, Beata; Malankowska, Anna; Wysocka, Magdalena; Klimczuk, Tomasz; Zaleska-Medynska, Adriana; Lesner, Adam

2018-05-16

Herein, the synthesis and application of functionalized quantum dot-based protease probes is described. Such probes are composed of nontoxic ZnO nanocrystals decorated by amino groups followed by linker and labeled peptide attachment. Spherical NH 2 -terminated ZnO quantum dots (QDs) with the average size ranging from 4 to 8 nm and strong emission centered at 530 nm were prepared using the sol-gel method. The fluorescence of ZnO QDs was quenched by the BHQ1 moiety present on the N-terminal amino group of the peptide. The enzymatic cleavage of the peptide mediated by the proteinase 3 (PR3) bond resulted in an increase in the QD probe fluorescence. This observation was verified using both model and biological systems; and the picomolar detection limit was found to be more than 30 times lower than that of the previously reported internally quenched peptide (a decrease in detection limit from 43 to 1.3 pmol was observed).

Phage display and selection of lanthipeptides on the carboxy-terminus of the gene-3 minor coat protein.

PubMed

Urban, Johannes H; Moosmeier, Markus A; Aumüller, Tobias; Thein, Marcus; Bosma, Tjibbe; Rink, Rick; Groth, Katharina; Zulley, Moritz; Siegers, Katja; Tissot, Kathrin; Moll, Gert N; Prassler, Josef

2017-11-15

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products with drug-like properties. To fully exploit the potential of RiPPs as peptide drug candidates, tools for their systematic engineering are required. Here we report the engineering of lanthipeptides, a subclass of RiPPs characterized by multiple thioether cycles that are enzymatically introduced in a regio- and stereospecific manner, by phage display. This was achieved by heterologous co-expression of linear lanthipeptide precursors fused to the widely neglected C-terminus of the bacteriophage M13 minor coat protein pIII, rather than the conventionally used N-terminus, along with the modifying enzymes from distantly related bacteria. We observe that C-terminal precursor peptide fusions to pIII are enzymatically modified in the cytoplasm of the producing cell and subsequently displayed as mature cyclic peptides on the phage surface. Biopanning of large C-terminal display libraries readily identifies artificial lanthipeptide ligands specific to urokinase plasminogen activator (uPA) and streptavidin.

Direct peptide bioconjugation/PEGylation at tyrosine with linear and branched polymeric diazonium salts.

PubMed

Jones, Mathew W; Mantovani, Giuseppe; Blindauer, Claudia A; Ryan, Sinead M; Wang, Xuexuan; Brayden, David J; Haddleton, David M

2012-05-02

Direct polymer conjugation at peptide tyrosine residues is described. In this study Tyr residues of both leucine enkephalin and salmon calcitonin (sCT) were targeted using appropriate diazonium salt-terminated linear monomethoxy poly(ethylene glycol)s (mPEGs) and poly(mPEG) methacrylate prepared by atom transfer radical polymerization. Judicious choice of the reaction conditions-pH, stoichiometry, and chemical structure of diazonium salt-led to a high degree of site-specificity in the conjugation reaction, even in the presence of competitive peptide amino acid targets such as histidine, lysines, and N-terminal amine. In vitro studies showed that conjugation of mPEG(2000) to sCT did not affect the peptide's ability to increase intracellular cAMP induced in T47D human breast cancer cells bearing sCT receptors. Preliminary in vivo investigation showed preserved ability to reduce [Ca(2+)] plasma levels by mPEG(2000)-sCT conjugate in rat animal models. © 2012 American Chemical Society

Antihypertensive properties of lactoferricin B-derived peptides.

PubMed

Ruiz-Giménez, Pedro; Ibáñez, Aida; Salom, Juan B; Marcos, Jose F; López-Díez, Jose Javier; Vallés, Salvador; Torregrosa, Germán; Alborch, Enrique; Manzanares, Paloma

2010-06-09

A set of eight lactoferricin B (LfcinB)-derived peptides was examined for inhibitory effects on angiotensin I-converting enzyme (ACE) activity and ACE-dependent vasoconstriction, and their hypotensive effect in spontaneously hypertensive rats (SHR). Peptides were derived from different elongations both at the C-terminal and N-terminal ends of the representative peptide LfcinB(20-25), which is known as the LfcinB antimicrobial core. All of the eight LfcinB-derived peptides showed in vitro inhibitory effects on ACE activity with different IC(50) values. Moreover, seven of them showed ex vivo inhibitory effects on ACE-dependent vasoconstriction. No clear correlation between in vitro and ex vivo inhibitory effects was found. Only LfcinB(20-25) and one of its fragments, F1, generated after a simulated gastrointestinal digestion, showed significant antihypertensive effects in SHR after oral administration. Remarkably, F1 did not show any effect on ACE-dependent vasoconstriction in contrast to the inhibitory effect showed by LfcinB(20-25). In conclusion, two LfcinB-derived peptides lower blood pressure and exhibit potential as orally effective antihypertensive compounds, yet a complete elucidation of the mechanism(s) involved deserves further ongoing research.

NT-ProBNP and Troponin T and Risk of Rapid Kidney Function Decline and Incident CKD in Elderly Adults

PubMed Central

Katz, Ronit; Dalrymple, Lorien; de Boer, Ian; DeFilippi, Christopher; Kestenbaum, Bryan; Park, Meyeon; Sarnak, Mark; Seliger, Stephen; Shlipak, Michael

2015-01-01

Background and objectives Elevations in N-terminal pro–B-type natriuretic peptide and high-sensitivity troponin T are associated with poor cardiovascular outcomes. Whether elevations in these cardiac biomarkers are associated with decline in kidney function was evaluated. Design, setting, participants, & measurements N-terminal pro–B-type natriuretic peptide and troponin T were measured at baseline in 3752 participants free of heart failure in the Cardiovascular Health Study. eGFR was determined from the Chronic Kidney Disease Epidemiology Collaboration equation using serum cystatin C. Rapid decline in kidney function was defined as decline in serum cystatin C eGFR≥30%, and incident CKD was defined as the onset of serum cystatin C eGFR<60 among those without CKD at baseline (n=2786). Cox regression models were used to examine the associations of each biomarker with kidney function decline adjusting for demographics, baseline serum cystatin C eGFR, diabetes, and other CKD risk factors. Results In total, 503 participants had rapid decline in serum cystatin C eGFR over a mean follow-up time of 6.41 (1.81) years, and 685 participants developed incident CKD over a mean follow-up time of 6.41 (1.74) years. Participants in the highest quartile of N-terminal pro–B-type natriuretic peptide (>237 pg/ml) had an 67% higher risk of rapid decline and 38% higher adjusted risk of incident CKD compared with participants in the lowest quartile (adjusted hazard ratio for serum cystatin C eGFR rapid decline, 1.67; 95% confidence interval, 1.25 to 2.23; hazard ratio for incident CKD, 1.38; 95% confidence interval, 1.08 to 1.76). Participants in the highest category of troponin T (>10.58 pg/ml) had 80% greater risk of rapid decline compared with participants in the lowest category (adjusted hazard ratio, 1.80; 95% confidence interval, 1.35 to 2.40). The association of troponin T with incident CKD was not statistically significant (hazard ratio, 1.17; 95% confidence interval, 0.92 to 1.50). Conclusions Elevated N-terminal pro–B-type natriuretic peptide and troponin T are associated with rapid decline of kidney function and incident CKD. Additional studies are needed to evaluate the mechanisms that may explain this association. PMID:25605700

Biomarkers and mortality after transient ischemic attack and minor ischemic stroke: population-based study.

PubMed

Greisenegger, Stefan; Segal, Helen C; Burgess, Annette I; Poole, Debbie L; Mehta, Ziyah; Rothwell, Peter M

2015-03-01

Premature death after transient ischemic attack or stroke is more often because of heart disease or cancer than stroke. Previous studies found blood biomarkers not usefully predictive of nonfatal stroke but possibly of all-cause death. This association might be explained by potentially treatable occult cardiac disease or cancer. We therefore aimed to validate the association of a panel of biomarkers with all-cause death, particularly cardiac death and cancer death, despite the absence of associations with risk of nonfatal vascular events. Fifteen biomarkers were measured in 929 consecutive patients in a population-based study (Oxford Vascular Study), recruited from 2002 and followed up to 2013. Associations were determined by Cox regression. Model discrimination was assessed by c-statistic and the integrated discrimination improvement. During 5560 patient-years of follow-up, none of the biomarkers predicted risk of nonfatal vascular events. However, soluble tumor necrosis factor α receptor-1, von Willebrand factor, heart-type fatty-acid-binding protein, and N-terminal pro-B-type natriuretic peptide were independently predictive of all-cause death (n=361; adjusted hazard ratio per SD, 95% confidence interval: heart-type fatty-acid-binding protein: 1.31, 1.12-1.56, P=0.002; N-terminal pro-B-type natriuretic peptide: 1.34, 1.11-1.62, P=0.002; soluble tumor necrosis factor α receptor-1: 1.45, 1.26-1.66, P=0.02; von Willebrand factor: 1.19, 1.04-1.36, P=0.01). The independent contribution of the four biomarkers taken together added prognostic information and improved model discrimination (integrated discrimination improvement=0.028, P=0.0001). N-terminal pro-B-type natriuretic peptide was most predictive of vascular death (adjusted hazard ratio=1.80, 95% confidence interval, 1.34-2.41, P<0.0001), whereas heart-type fatty-acid-binding protein predicted cancer deaths (1.64, 1.26-2.12, P=0.0002). Associations were strongest in patients without known prior cardiac disease or cancer. Several biomarkers predicted death of any cause after transient ischemic attack and minor stroke. N-terminal pro-B-type natriuretic peptide and heart-type fatty-acid-binding protein might improve patient selection for additional screening for occult cardiac disease or cancer, respectively. However, our results require validation in future studies. © 2015 American Heart Association, Inc.

Proline kink angle distributions for GWALP23 in lipid bilayers of different thicknesses.

PubMed

Rankenberg, Johanna M; Vostrikov, Vitaly V; DuVall, Christopher D; Greathouse, Denise V; Koeppe, Roger E; Grant, Christopher V; Opella, Stanley J

2012-05-01

By using selected (2)H and (15)N labels, we have examined the influence of a central proline residue on the properties of a defined peptide that spans lipid bilayer membranes by solid-state nuclear magnetic resonance (NMR) spectroscopy. For this purpose, GWALP23 (acetyl-GGALW(5)LALALALALALALW(19)LAGA-ethanolamide) is a suitable model peptide that employs, for the purpose of interfacial anchoring, only one tryptophan residue on either end of a central α-helical core sequence. Because of its systematic behavior in lipid bilayer membranes of differing thicknesses [Vostrikov, V. V., et al. (2010) J. Biol. Chem. 285, 31723-31730], we utilize GWALP23 as a well-characterized framework for introducing guest residues within a transmembrane sequence; for example, a central proline yields acetyl-GGALW(5)LALALAP(12)ALALALW(19)LAGA-ethanolamide. We synthesized GWALP23-P12 with specifically placed (2)H and (15)N labels for solid-state NMR spectroscopy and examined the peptide orientation and segmental tilt in oriented DMPC lipid bilayer membranes using combined (2)H GALA and (15)N-(1)H high-resolution separated local field methods. In DMPC bilayer membranes, the peptide segments N-terminal and C-terminal to the proline are both tilted substantially with respect to the bilayer normal, by ~34 ± 5° and 29 ± 5°, respectively. While the tilt increases for both segments when proline is present, the range and extent of the individual segment motions are comparable to or smaller than those of the entire GWALP23 peptide in bilayer membranes. In DMPC, the proline induces a kink of ~30 ± 5°, with an apparent helix unwinding or "swivel" angle of ~70°. In DLPC and DOPC, on the basis of (2)H NMR data only, the kink angle and swivel angle probability distributions overlap those of DMPC, yet the most probable kink angle appears to be somewhat smaller than in DMPC. As has been described for GWALP23 itself, the C-terminal helix ends before Ala(21) in the phospholipids DMPC and DLPC yet remains intact through Ala(21) in DOPC. The dynamics of bilayer-incorporated, membrane-spanning GWALP23 and GWALP23-P12 are less extensive than those observed for WALP family peptides that have more than two interfacial Trp residues.

Proline Kink Angle Distributions for GWALP23 in Lipid Bilayers of Different Thickness†

PubMed Central

Rankenberg, Johanna M.; Vostrikov, Vitaly V.; DuVall, Christopher D.; Greathouse, Denise V.; Koeppe, Roger E.; Grant, Christopher V.; Opella, Stanley J.

2013-01-01

By using selected 2H and 15N labels, we have examined the influence of a central proline residue upon the properties of a defined peptide that spans lipid bilayer membranes by solid-state NMR spectroscopy. For this purpose, GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-ethanolamide) is a suitable model peptide that employs—for the purpose of interfacial anchoring—only one tryptophan residue on either end of a central alpha-helical core sequence. Because of its systematic behavior in lipid bilayer membranes of differing thickness (see J. Biol. Chem. 285, 31723), we utilize GWALP23 as a well-characterized framework for introducing guest residues within a transmembrane sequence; for example, a central proline yields acetyl-GGALW5LALALAP12ALALALW19LAGA-ethanolamide. We synthesized the GWALP23-P12 with specifically placed 2H and 15N labels for solid-state NMR spectroscopy, and examined the peptide orientation and segmental tilt in oriented DMPC lipid bilayer membranes using combined (2H)-GALA and (15N-1H) high resolution separated local field methods. In DMPC bilayer membranes, the peptide segments N-terminal and C-terminal to the proline are both tilted substantially with respect to the bilayer normal, by about 34° and 29° (± 5°), respectively. While the tilt increases for both segments when proline is present, the range and extent of the individual segment motions are comparable or less than those of the entire GWALP23 peptide in bilayer membranes. In DMPC, the proline induces a kink of about 30° (± 5°), with an apparent helix unwinding or “swivel” angle of about 70°. In DLPC and DOPC, based on 2H NMR data only, the kink angle and swivel angle probability distributions overlap those of DMPC, yet the most probable kink angle appears somewhat smaller than in DMPC. As has been described for GWALP23 itself, the C-terminal helix ends before Ala-21 in the phospholipids DMPC and DLPC, yet remains intact through Ala-21 in DOPC. The dynamics of bilayer-incorporated, membrane-spanning GWALP23 and GWALP23-P12 are less extensive than observed for WALP-family peptides that have more than two interfacial Trp residues. PMID:22489564

Structural Basis for Hormone Recognition by the Human CRFR2[alpha] G Protein-coupled Receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pal, Kuntal; Swaminathan, Kunchithapadam; Xu, H. Eric

2012-05-09

The mammalian corticotropin releasing factor (CRF)/urocortin (Ucn) peptide hormones include four structurally similar peptides, CRF, Ucn1, Ucn2, and Ucn3, that regulate stress responses, metabolism, and cardiovascular function by activating either of two related class B G protein-coupled receptors, CRFR1 and CRFR2. CRF and Ucn1 activate both receptors, whereas Ucn2 and Ucn3 are CRFR2-selective. The molecular basis for selectivity is unclear. Here, we show that the purified N-terminal extracellular domains (ECDs) of human CRFR1 and the CRFR2{alpha} isoform are sufficient to discriminate the peptides, and we present three crystal structures of the CRFR2{alpha} ECD bound to each of the Ucn peptides.more » The CRFR2{alpha} ECD forms the same fold observed for the CRFR1 and mouse CRFR2{beta} ECDs but contains a unique N-terminal {alpha}-helix formed by its pseudo signal peptide. The CRFR2{alpha} ECD peptide-binding site architecture is similar to that of CRFR1, and binding of the {alpha}-helical Ucn peptides closely resembles CRF binding to CRFR1. Comparing the electrostatic surface potentials of the ECDs suggests a charge compatibility mechanism for ligand discrimination involving a single amino acid difference in the receptors (CRFR1 Glu104/CRFR2{alpha} Pro-100) at a site proximate to peptide residue 35 (Arg in CRF/Ucn1, Ala in Ucn2/3). CRFR1 Glu-104 acts as a selectivity filter preventing Ucn2/3 binding because the nonpolar Ala-35 is incompatible with the negatively charged Glu-104. The structures explain the mechanisms of ligand recognition and discrimination and provide a molecular template for the rational design of therapeutic agents selectively targeting these receptors.« less

Serine Protease PrtA from Streptococcus pneumoniae Plays a Role in the Killing of S. pneumoniae by Apolactoferrin ▿

PubMed Central

Mirza, Shaper; Wilson, Landon; Benjamin, William H.; Novak, Jan; Barnes, Stephen; Hollingshead, Susan K.; Briles, David E.

2011-01-01

It is known that apolactoferrin, the iron-free form of human lactoferrin, can kill many species of bacteria, including Streptococcus pneumoniae. Lactoferricin, an N-terminal peptide of apolactoferrin, and fragments of it are even more bactericidal than apolactoferrin. In this study we found that apolactoferrin must be cleaved by a serine protease in order for it to kill pneumococci. The serine protease inhibitors were able to block killing by apolactoferrin but did not block killing by a lactoferrin-derived peptide. Thus, the killing of pneumococci by apolactoferrin appears to require a protease to release a lactoferricin-like peptide(s). Incubation of apolactoferrin with growing pneumococci resulted in a 12-kDa reduction in its molecular mass, of which about 7 to 8 kDa of the reduction was protease dependent. Capsular type 2 and 19F strains with mutations in the gene encoding the major cell wall-associated serine protease, prtA, lost much of their ability to degrade apolactoferrin and were relatively resistant to killing by apolactoferrin (P < 0.001). Recombinant PrtA was also able to cleave apolactoferrin, reducing its mass by about 8 kDa, and greatly enhance the killing activity of the solution containing the apolactoferrin and its cleavage products. Mass spectroscopy revealed that PrtA makes a major cut between amino acids 78 and 79 of human lactoferrin, removing the N-terminal end of the molecule (about 8.6 kDa). The simplest interpretation of these data is that the mechanism by which apolactoferrin kills Streptococcus pneumoniae requires the release of a lactoferricin-like peptide(s) and that it is this peptide(s), and not the intact apolactoferrin, which kills pneumococci. PMID:21422179

Structural specificity of mucosal-cell transport and metabolism of peptide drugs: implication for oral peptide drug delivery

NASA Technical Reports Server (NTRS)

Bai, J. P.; Amidon, G. L.

1992-01-01

The brush border membrane of intestinal mucosal cells contains a peptide carrier system with rather broad substrate specificity and various endo- and exopeptidase activities. Small peptide (di-/tripeptide)-type drugs with or without an N-terminal alpha-amino group, including beta-lactam antibiotics and angiotensin-converting enzyme (ACE) inhibitors, are transported by the peptide transporter. Polypeptide drugs are hydrolyzed by brush border membrane proteolytic enzymes to di-/tripeptides and amino acids. Therefore, while the intestinal brush border membrane has a carrier system facilitating the absorption of di-/tripeptide drugs, it is a major barrier limiting oral availability of polypeptide drugs. In this paper, the specificity of peptide transport and metabolism in the intestinal brush border membrane is reviewed.

Simultaneous Detection of Human C-Terminal p53 Isoforms by Single Template Molecularly Imprinted Polymers (MIPs) Coupled with Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)-Based Targeted Proteomics.

PubMed

Jiang, Wenting; Liu, Liang; Chen, Yun

2018-03-06

Abnormal expression of C-terminal p53 isoforms α, β, and γ can cause the development of cancers including breast cancer. To date, much evidence has demonstrated that these isoforms can differentially regulate target genes and modulate their expression. Thus, quantification of individual isoforms may help to link clinical outcome to p53 status and to improve cancer patient treatment. However, there are few studies on accurate determination of p53 isoforms, probably due to sequence homology of these isoforms and also their low abundance. In this study, a targeted proteomics assay combining molecularly imprinted polymers (MIPs) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for simultaneous quantification of C-terminal p53 isoforms. Isoform-specific surrogate peptides (i.e., KPLDGEYFTLQIR (peptide-α) for isoform α, KPLDGEYFTLQDQTSFQK (peptide-β) for isoform β, and KPLDGEYFTLQMLLDLR (peptide-γ) for isoform γ) were first selected and used in both MIPs enrichment and mass spectrometric detection. The common sequence KPLDGEYFTLQ of these three surrogate peptides was used as single template in MIPs. In addition to optimization of imprinting conditions and characterization of the prepared MIPs, binding affinity and cross-reactivity of the MIPs for each surrogate peptide were also evaluated. As a result, a LOQ of 5 nM was achieved, which was >15-fold more sensitive than that without MIPs. Finally, the assay was validated and applied to simultaneous quantitative analysis of C-terminal p53 isoforms α, β, and γ in several human breast cell lines (i.e., MCF-10A normal cells, MCF-7 and MDA-MB-231 cancer cells, and drug-resistant MCF-7/ADR cancer cells). This study is among the first to employ single template MIPs and cross-reactivity phenomenon to select isoform-specific surrogate peptides and enable simultaneous quantification of protein isoforms in LC-MS/MS-based targeted proteomics.

NMR solution structure of the N-terminal domain of hERG and its interaction with the S4-S5 linker

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Qingxin; Gayen, Shovanlal; Chen, Angela Shuyi

Research highlights: {yields} The N-terminal domain (NTD, eag domain) containing 135 residues of hERG was expressed and purified from E. coli cells. {yields} Solution structure of NTD was determined with NMR spectroscopy. {yields} The alpha-helical region (residues 13-23) was demonstrated to possess the characteristics of an amphipathic helix. {yields} NMR titration confirmed the interaction between NTD and the peptide from the S4-S5 linker. -- Abstract: The human Ether-a-go-go Related Gene (hERG) potassium channel mediates the rapid delayed rectifier current (IKr) in the cardiac action potential. Mutations in the 135 amino acid residue N-terminal domain (NTD) cause channel dysfunction or mis-translocation.more » To study the structure of NTD, it was overexpressed and purified from Escherichia coli cells using affinity purification and gel filtration chromatography. The purified protein behaved as a monomer under purification conditions. Far- and near-UV, circular dichroism (CD) and solution nuclear magnetic resonance (NMR) studies showed that the purified protein was well-folded. The solution structure of NTD was obtained and the N-terminal residues 13-23 forming an amphipathic helix which may be important for the protein-protein or protein-membrane interactions. NMR titration experiment also demonstrated that residues from 88 to 94 in NTD are important for the molecular interaction with the peptide derived from the S4-S5 linker.« less

Unexpected Hydrolytic Instability of N-Acylated Amino Acid Amides and Peptides

PubMed Central

2015-01-01

Remote amide bonds in simple N-acyl amino acid amide or peptide derivatives 1 can be surprisingly unstable hydrolytically, affording, in solution, variable amounts of 3 under mild acidic conditions, such as trifluoroacetic acid/water mixtures at room temperature. This observation has important implications for the synthesis of this class of compounds, which includes N-terminal-acylated peptides. We describe the factors contributing to this instability and how to predict and control it. The instability is a function of the remote acyl group, R2CO, four bonds away from the site of hydrolysis. Electron-rich acyl R2 groups accelerate this reaction. In the case of acyl groups derived from substituted aromatic carboxylic acids, the acceleration is predictable from the substituent’s Hammett σ value. N-Acyl dipeptides are also hydrolyzed under typical cleavage conditions. This suggests that unwanted peptide truncation may occur during synthesis or prolonged standing in solution when dipeptides or longer peptides are acylated on the N-terminus with electron-rich aromatic groups. When amide hydrolysis is an undesired secondary reaction, as can be the case in the trifluoroacetic acid-catalyzed cleavage of amino acid amide or peptide derivatives 1 from solid-phase resins, conditions are provided to minimize that hydrolysis. PMID:24617596

The C- and N-Terminal Residues of Synthetic Heptapeptide Ion Channels Influence Transport Efficacy Through Phospholipid Bilayers

PubMed Central

Djedovič, Natasha; Ferdani, Riccardo; Harder, Egan; Pajewska, Jolanta; Pajewski, Robert; Weber, Michelle E.; Schlesinger, Paul H.; Gokel, George W.

2008-01-01

The synthetic peptide, R2N-COCH2OCH2CO-Gly-Gly-Gly-Pro-Gly-Gly-Gly-OR’, was shown to be selective for Cl- over K+ when R is n-octadecyl and R’ is benzyl. Nineteen heptapeptides have now been prepared in which the N-terminal and C-terminal residues have been varied. All of the N-terminal residues are dialkyl but the C-terminal chains are esters, 2° amides, or 3° amides. The compounds having varied N-terminal anchors and C-terminal benzyl groups are as follows: 1, R = n-propyl; 2, R = n-hexyl; 3, R = n-octyl; 4, R = n-decyl; 5, R = n-dodecyl; 6, R = n-tetradecyl; 7, R = n-hexadecyl; 8, R = n-octadecyl. Compounds 9-19 have R = n-octadecyl and C-terminal residues as follows: 9, OR’ = OCH2CH3; 10, OR’ = OCH(CH3)2; 11, OR’ = O(CH2)6CH3; 12, OR’ = OCH2-c-C6H11; 13, OR’ = O(CH2)9CH3; 14, OR’ = O (CH2)17CH3; 15, NR’2 = N[(CH2)6CH3]2; 16, NHR’ = NH(CH2)9CH3; 17, NR’2 = N[(CH2)9CH3]2; 18, NHR’ = NH(CH2)17CH3; 19, NR’2 = N[(CH2)17CH3]2. The highest anion transport activities were observed as follows. For the benzyl esters whose N-terminal residues were varied, i.e. 1-8, compound 3 was most active. For the C18 anchored esters 10-14, n-heptyl ester 11 was most active. For the C18 anchored, C-terminal amides 15-19, di-n-decylamide 17 was most active. It was concluded that both the C- and N-terminal anchors were important for channel function in the bilayer but that activity was lost unless only one of the two anchoring groups was dominant. PMID:19633728

Production of cecropin A antimicrobial peptide in rice seed endosperm

PubMed Central

2014-01-01

Background Cecropin A is a natural antimicrobial peptide that exhibits rapid, potent and long-lasting lytic activity against a broad spectrum of pathogens, thus having great biotechnological potential. Here, we report a system for producing bioactive cecropin A in rice seeds. Results Transgenic rice plants expressing a codon-optimized synthetic cecropin A gene drived by an endosperm-specific promoter, either the glutelin B1 or glutelin B4 promoter, were generated. The signal peptide sequence from either the glutelin B1 or the glutelin B4 were N-terminally fused to the coding sequence of the cecropin A. We also studied whether the presence of the KDEL endoplasmic reticulum retention signal at the C-terminal has an effect on cecropin A subcellular localization and accumulation. The transgenic rice plants showed stable transgene integration and inheritance. We show that cecropin A accumulates in protein storage bodies in the rice endosperm, particularly in type II protein bodies, supporting that the glutelin N-terminal signal peptides play a crucial role in directing the cecropin A to this organelle, independently of being tagged with the KDEL endoplasmic reticulum retention signal. The production of cecropin A in transgenic rice seeds did not affect seed viability or seedling growth. Furthermore, transgenic cecropin A seeds exhibited resistance to infection by fungal and bacterial pathogens (Fusarium verticillioides and Dickeya dadantii, respectively) indicating that the in planta-produced cecropin A is biologically active. Conclusions Rice seeds can sustain bioactive cecropin A production and accumulation in protein bodies. The system might benefit the production of this antimicrobial agent for subsequent applications in crop protection and food preservation. PMID:24755305

Discrepancies between conformational distributions of a polyalanine peptide in solution obtained from molecular dynamics force fields and amide I' band profiles.

PubMed

Verbaro, Daniel; Ghosh, Indrajit; Nau, Werner M; Schweitzer-Stenner, Reinhard

2010-12-30

Structural preferences in the unfolded state of peptides determined by molecular dynamics still contradict experimental data. A remedy in this regard has been suggested by MD simulations with an optimized Amber force field ff03* ( Best, R. Hummer, G. J. Phys. Chem. B 2009 , 113 , 9004 - 9015 ). The simulations yielded a statistical coil distribution for alanine which is at variance with recent experimental results. To check the validity of this distribution, we investigated the peptide H-A(5)W-OH, which with the exception of the additional terminal tryptophan is analogous to the peptide used to optimize the force fields ff03*. Electronic circular dichroism, vibrational circular dichroism, and infrared spectroscopy as well as J-coupling constants obtained from NMR experiments were used to derive the peptide's conformational ensemble. Additionally, Förster resonance energy transfer between the terminal chromophores of the fluorescently labeled peptide analogue H-Dbo-A(5)W-OH was used to determine its average length, from which the end-to-end distance of the unlabeled peptide was estimated. Qualitatively, the experimental (3)J(H(N),C(α)), VCD, and ECD indicated a preference of alanine for polyproline II-like conformations. The experimental (3)J(H(N),C(α)) for A(5)W closely resembles the constants obtained for A(5). In order to quantitatively relate the conformational distribution of A(5) obtained with the optimized AMBER ff03* force field to experimental data, the former was used to derive a distribution function which expressed the conformational ensemble as a mixture of polyproline II, β-strand, helical, and turn conformations. This model was found to satisfactorily reproduce all experimental J-coupling constants. We employed the model to calculate the amide I' profiles of the IR and vibrational circular dichroism spectrum of A(5)W, as well as the distance between the two terminal peptide carbonyls. This led to an underestimated negative VCD couplet and an overestimated distance between terminal carbonyl groups. In order to more accurately account for the experimental data, we changed the distribution parameters based on results recently obtained for the alanine-based tripeptides. The final model, which satisfactorily reproduced amide I' profiles, J-coupling constant, and the end-to-end distance of A(5)W, reinforces alanine's high structural preference for polyproline II. Our results suggest that distributions obtained from MD simulations suggesting a statistical coil-like distribution for alanine are still based on insufficiently accurate force fields.

Microinjection of recombinant O-GlcNAc transferase potentiates Xenopus oocytes M-phase entry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dehennaut, Vanessa; EA 4020, Laboratoire de Regulation des Signaux de Division, USTL, IFR147, Villeneuve d'Ascq; Hanoulle, Xavier

2008-05-02

In order to understand the importance of the cytosolic and nuclear-specific O-linked N-acetylglucosaminylation (O-GlcNAc) on cell cycle regulation, we recently reported that inhibition of O-GlcNAc transferase (OGT) delayed or blocked Xenopus laevis oocyte germinal vesicle breakdown (GVBD). Here, we show that increased levels of the long OGT isoform (ncOGT) accelerate X. laevis oocyte GVBD. A N-terminally truncated isoform (sOGT) with a similar in vitro catalytic activity towards a synthetic CKII-derived peptide had no effect, illustrating the important role played by the N-terminal tetratrico-peptide repeats. ncOGT microinjection in the oocytes increases both the speed and extent of O-GlcNAc addition, leads tomore » a quicker activation of the MPF and MAPK pathways and finally results in a faster GVBD. Microinjection of anti-OGT antibodies leads to a delay of the GVBD kinetics. Our results hence demonstrate that OGT is a key molecule for the timely progression of the cell cycle.« less

Influence of shifting positions of Ser, Thr, and Cys residues in prenisin on the efficiency of modification reactions and on the antimicrobial activities of the modified prepeptides.

PubMed

Lubelski, Jacek; Overkamp, Wout; Kluskens, Leon D; Moll, Gert N; Kuipers, Oscar P

2008-08-01

Since the recent discovery that the nisin modification and transport machinery can be used to produce and modify peptides unrelated to nisin, specific questions arose concerning the specificity of the modification enzymes involved and the limits of their promiscuity with respect to the dehydration and cyclization processes. The nisin leader peptide has been postulated to fulfill a recognition and binding function required for these modifications. Here, we investigated whether the relative positions of the modifiable residues in the nisin prepeptide, with respect to the leader peptide, could influence the efficiency of their modification. We conducted a systematic study on the insertion of one to four alanines in front of either ring A or ring D to change the "reading frame" of modifiable residues, resulting in altered distance and topology of the modifiable residues relative to the leader. The insertion of N-terminal and hinge-located Ala residues had only a modest influence on the modification efficiency, demonstrating that the "phasing" of these residues relative to the leader peptide is not a critical factor in determining modification. However, in all cases, but especially with the N-terminal insertions, the antimicrobial activities of the fully modified nisin species were decreased.

The leader peptide of mutacin 1140 has distinct structural components compared to related class I lantibiotics

PubMed Central

Escano, Jerome; Stauffer, Byron; Brennan, Jacob; Bullock, Monica; Smith, Leif

2014-01-01

Lantibiotics are ribosomally synthesized peptide antibiotics composed of an N-terminal leader peptide that promotes the core peptide's interaction with the post translational modification (PTM) enzymes. Following PTMs, mutacin 1140 is transported out of the cell and the leader peptide is cleaved to yield the antibacterial peptide. Mutacin 1140 leader peptide is structurally unique compared to other class I lantibiotic leader peptides. Herein, we further our understanding of the structural differences of mutacin 1140 leader peptide with regard to other class I leader peptides. We have determined that the length of the leader peptide is important for the biosynthesis of mutacin 1140. We have also determined that mutacin 1140 leader peptide contains a novel four amino acid motif compared to related lantibiotics. PTM enzyme recognition of the leader peptide appears to be evolutionarily distinct from related class I lantibiotics. Our study on mutacin 1140 leader peptide provides a basis for future studies aimed at understanding its interaction with the PTM enzymes. PMID:25400246

Benzoisothiazolone Organo/Copper-Cocatalyzed Redox Dehydrative Construction of Amides and Peptides from Carboxylic Acids using (EtO)3P as the Reductant and O2 in Air as the Terminal Oxidant.

PubMed

Liebeskind, Lanny S; Gangireddy, Pavankumar; Lindale, Matthew G

2016-06-01

Carboxylic acids and amine/amino acid reactants can be converted to amides and peptides at neutral pH within 5-36 h at 50 °C using catalytic quantities of a redox-active benzoisothiazolone and a copper complex. These catalytic "oxidation-reduction condensation" reactions are carried out open to dry air using O2 as the terminal oxidant and a slight excess of triethyl phosphite as the reductant. Triethyl phosphate is the easily removed byproduct. These simple-to-run catalytic reactions provide practical and economical procedures for the acylative construction of C-N bonds.

Plastoglobule-Targeting Competence of a Putative Transit Peptide Sequence from Rice Phytoene Synthase 2 in Plastids.

PubMed

You, Min Kyoung; Kim, Jin Hwa; Lee, Yeo Jin; Jeong, Ye Sol; Ha, Sun-Hwa

2016-12-22

Plastoglobules (PGs) are thylakoid membrane microdomains within plastids that are known as specialized locations of carotenogenesis. Three rice phytoene synthase proteins (OsPSYs) involved in carotenoid biosynthesis have been identified. Here, the N-terminal 80-amino-acid portion of OsPSY2 (PTp) was demonstrated to be a chloroplast-targeting peptide by displaying cytosolic localization of OsPSY2(ΔPTp):mCherry in rice protoplast, in contrast to chloroplast localization of OsPSY2:mCherry in a punctate pattern. The peptide sequence of a PTp was predicted to harbor two transmembrane domains eligible for a putative PG-targeting signal. To assess and enhance the PG-targeting ability of PTp, the original PTp DNA sequence ( PTp ) was modified to a synthetic DNA sequence ( stPTp ), which had 84.4% similarity to the original sequence. The motivation of this modification was to reduce the GC ratio from 75% to 65% and to disentangle the hairpin loop structures of PTp . These two DNA sequences were fused to the sequence of the synthetic green fluorescent protein (sGFP) and drove GFP expression with different efficiencies. In particular, the RNA and protein levels of stPTp-sGFP were slightly improved to 1.4-fold and 1.3-fold more than those of sGFP, respectively. The green fluorescent signals of their mature proteins were all observed as speckle-like patterns with slightly blurred stromal signals in chloroplasts. These discrete green speckles of PTp - sGFP and stPTp - sGFP corresponded exactly to the red fluorescent signal displayed by OsPSY2:mCherry in both etiolated and greening protoplasts and it is presumed to correspond to distinct PGs. In conclusion, we identified PTp as a transit peptide sequence facilitating preferential translocation of foreign proteins to PGs, and developed an improved PTp sequence, a s tPTp , which is expected to be very useful for applications in plant biotechnologies requiring precise micro-compartmental localization in plastids.

Structure and Mode-of-Action of the Two-Peptide (Class-IIb) Bacteriocins.

PubMed

Nissen-Meyer, Jon; Oppegård, Camilla; Rogne, Per; Haugen, Helen Sophie; Kristiansen, Per Eugen

2010-03-01

This review focuses on the structure and mode-of-action of the two-peptide (class-IIb) bacteriocins that consist of two different peptides whose genes are next to each other in the same operon. Optimal antibacterial activity requires the presence of both peptides in about equal amounts. The two peptides are synthesized as preforms that contain a 15-30 residue double-glycine-type N-terminal leader sequence that is cleaved off at the C-terminal side of two glycine residues by a dedicated ABC-transporter that concomitantly transfers the bacteriocin peptides across cell membranes. Two-peptide bacteriocins render the membrane of sensitive bacteria permeable to a selected group of ions, indicating that the bacteriocins form or induce the formation of pores that display specificity with respect to the transport of molecules. Based on structure-function studies, it has been proposed that the two peptides of two-peptide bacteriocins form a membrane-penetrating helix-helix structure involving helix-helix-interacting GxxxG-motifs that are present in all characterized two-peptide bacteriocins. It has also been suggested that the membrane-penetrating helix-helix structure interacts with an integrated membrane protein, thereby triggering a conformational alteration in the protein, which in turn causes membrane-leakage. This proposed mode-of-action is similar to the mode-of-action of the pediocin-like (class-IIa) bacteriocins and lactococcin A (a class-IId bacteriocin), which bind to a membrane-embedded part of the mannose phosphotransferase permease in a manner that causes membrane-leakage and cell death.

Molecular cloning of crustins from the hemocytes of Brazilian penaeid shrimps.

PubMed

Rosa, Rafael Diego; Bandeira, Paula Terra; Barracco, Margherita Anna

2007-09-01

Crustins are antimicrobial peptides initially identified in the hemocytes of the crab Carcinus maenas (11.5-kDa peptide or carcinin) and recently also recognized in penaeid shrimps and other crustacean species. The aim of this study was to identify sequences encoding for crustins from the hemocytes of four Brazilian penaeid species: Farfantepenaeus paulensis, Farfantepenaeus subtilis, Farfantepenaeus brasiliensis and Litopenaeus schmitti. Using primers based on consensus nucleotide alignment of crustins from different crustaceans, cDNA sequences coding for crustins in all indigenous penaeid species were amplified. The obtained four crustin sequences encoded for peptides containing a hydrophobic N-terminal region rich in glycine repeats and a C-terminal part with 12 cysteine residues and a conserved whey acidic protein domain. All obtained crustin sequences showed high amino acidic similarity among each other and with crustins from litopenaeid shrimps (76-98%). This is the first report of crustins in native Brazilian penaeid shrimps.

Radioimmunoassay of neuropeptide Y.

PubMed

Allen, J M; Yeats, J C; Adrian, T E; Bloom, S R

1984-01-01

The development of a radioimmunoassay to the newly isolated peptide, neuropeptide Y is described. Four separate antisera have been developed using different immunisation schedules. Two of these antisera (YNI and YNIO) are directed to the C-terminal region of the peptide and cross-react with the related peptide PYY, whereas YN7 is specific being directed to the N-terminal region of NPY, YN6 is similarly specific for NPY, but is unable to bind the available fragments. These four antisera provide similar results for determination of NPY immunoreactivity within porcine brain extracts, however YN6 consistently undervalues all extracts from the other species examined (human, rat, guinea pig, cat and mouse). Chromatographic analysis by means of reverse phase high pressure liquid chromatography (HPLC) shows that NPY immunoreactivity of human extracts elutes in an earlier position than the porcine standard. It seems likely therefore that human and porcine NPY differ in their amino acid sequences.

Neuropeptide Y mRNA and peptide in the night-migratory redheaded bunting brain.

PubMed

Devraj, Singh; Kumari, Yatinesh; Rastogi, Ashutosh; Rani, Sangeeta; Kumar, Vinod

2013-11-01

This study investigated the distribution of neuropeptide Y (NPY) in the brain of the night-migratory redheaded bunting (Emberiza bruniceps). We first cloned the 275-bp NPY gene in buntings, with ≥95% homology with known sequences from other birds. The deduced peptide sequence contained all conserved 36 amino acids chain of the mature NPY peptide, but lacked 6 amino acids that form the NPY signal peptide. Using digosigenin-labeled riboprobe prepared from the cloned sequence, the brain cells that synthesize NPY were identified by in-situ hybridization. The NPY peptide containing cell bodies and terminals (fibers) were localized by immunocytochemistry. NPY mRNA and peptide were widespread throughout the bunting brain. This included predominant pallial and sub-pallial areas (cortex piriformis, cortex prepiriformis, hyperpallium apicale, hippocampus, globus pallidus) and thalamic and hypothalamic nuclei (organum vasculosum laminae terminalis, nucleus (n.) dorsolateralis anterior thalami, n. rotundus, n. infundibularis) including the median eminence and hind brain (n. pretectalis, n. opticus basalis, n. reticularis pontis caudalis pars gigantocellularis). The important structures with only NPY-immunoreactive fibers included the olfactory bulb, medial and lateral septal areas, medial preoptic nucleus, medial suprachiasmatic nucleus, paraventricular nucleus, ventromedial hypothalamic nucleus, optic tectum, and ventro-lateral geniculate nucleus. These results demonstrate that NPY is possibly involved in the regulation of several physiological functions (e.g. daily timing feeding, and reproduction) in the migratory bunting.

Gene structure, transcripts and calciotropic effects of the PTH family of peptides in Xenopus and chicken.

PubMed

Pinheiro, Pedro L C; Cardoso, João C R; Gomes, Ana S; Fuentes, Juan; Power, Deborah M; Canário, Adelino V M

2010-12-01

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken. The PTH-L gene is present throughout the vertebrates with the exception of placental mammals. Gene structure of PTH and PTH-L seems to be conserved in vertebrates while PTHrP gene structure is divergent and has acquired new exons and alternative promoters. Splice variants of PTHrP and PTH-L are common in Xenopus and chicken and transcripts of the former have a widespread tissue distribution, although PTH-L is more restricted. PTH is widely expressed in fish tissue but from Xenopus to mammals becomes largely restricted to the parathyroid gland. The N-terminal (1-34) region of PTH, PTHrP and PTH-L in Xenopus and chicken share high sequence conservation and the capacity to modify calcium fluxes across epithelia suggesting a conserved role in calcium metabolism possibly via similar receptors. The parathyroid hormone family contains 3 principal members, PTH, PTHrP and the recently identified PTH-L. In teleosts there are 5 genes which encode PTHrP (2), PTH (2) and PTH-L and in tetrapods there are 3 genes (PTHrP, PTH and PTH-L), the exception is placental mammals which have 2 genes and lack PTH-L. It is hypothesized that genes of the PTH family appeared at approximately the same time during the vertebrate radiation and evolved via gene duplication/deletion events. PTH-L was lost from the genome of eutherian mammals and PTH, which has a paracrine distribution in lower vertebrates, became the product of a specific endocrine tissue in Amphibia, the parathyroid gland. The PTHrP gene organisation diverged and became more complex in vertebrates and retained its widespread tissue distribution which is congruent with its paracrine nature.

A classical molecular dynamics investigation of the free energy and structure of short polyproline conformers

NASA Astrophysics Data System (ADS)

Moradi, Mahmoud; Babin, Volodymyr; Roland, Christopher; Sagui, Celeste

2010-09-01

Folded polyproline peptides can exist as either left-(PPII) or right-handed (PPI) helices, depending on their environment. In this work, we have characterized the conformations and the free energy landscapes of Ace-(Pro)n-Nme, n =2,3,…,9, and 13 peptides both in vacuo and in an implicit solvent environment. In order to enhance the sampling provided by regular molecular dynamics simulations, we have used the recently developed adaptively biased molecular dynamics method—which provides an accurate description of the free energy landscapes in terms of a set of relevant collective variables—combined with Hamiltonian and temperature replica exchange molecular dynamics methods. The collective variables, which are chosen so as to reflect the stable structures and the "slow modes" of the polyproline system, were based primarily on properties of length and of the cis/trans isomerization associated with the prolyl bonds. Results indicate that the space of peptide structures is characterized not just by pure PPII and PPI structures, but rather by a broad distribution of stable minima with similar free energies. These results are in agreement with recent experimental work. In addition, we have used steered molecular dynamics methods in order to quantitatively estimate the free energy difference of PPI and PPII for peptides of the length n =2,…,5 in vacuo and implicit water and qualitatively investigate transition pathways and mechanisms for the PPII to PPI transitions. A zipper-like mechanism, starting from either the center of the peptide or the amidated end, appear to be the most likely mechanisms for the PPII→PPI transition for the longer peptides.

Identification and Functional Characterization of N-Terminally Acetylated Proteins in Drosophila melanogaster

PubMed Central

Gerrits, Bertran; Roschitzki, Bernd; Mohanty, Sonali; Niederer, Eva M.; Laczko, Endre; Timmerman, Evy; Lange, Vinzenz; Hafen, Ernst; Aebersold, Ruedi; Vandekerckhove, Joël; Basler, Konrad; Ahrens, Christian H.; Gevaert, Kris; Brunner, Erich

2009-01-01

Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species. PMID:19885390

Conformational study of the hydroxyproline-O-glycosidic linkage: sugar-peptide orientation and prolyl amide isomerization in (α/β)-galactosylated 4(R/S)-hydroxyproline.

PubMed

Naziga, Emmanuel B; Schweizer, Frank; Wetmore, Stacey D

2012-01-19

Glycosylation is a frequent post-translational modification of proteins that has been shown to influence protein structure and function. Glycosylation of hydroxyproline occurs widely in plants, but is absent in humans and animals. Previous experimental studies on model amides have indicated that α/β-galactosylation of 4R-hydroxyproline (Hyp) has no measurable effect on prolyl amide isomerization, while a 7% increase in the trans isomer population, as well as a 25-50% increase in the isomerization rate, was observed for the 4S stereoisomer (hyp). In this work, molecular dynamics simulations in explicit water and implicit solvent DFT optimizations are used to examine the structure of the hydroxyproline-O-galactosyl linkage and the effect of glycosylation on the structure and cis/trans isomerization of the peptide backbone. The calculations show two major minima with respect to the glycosidic linkage in all compounds. The C(γ)-exo puckering observed in 4R compounds projects the sugar away from the peptide backbone, while a twisted C(γ)-endo/C(β)-exo pucker in the 4S compounds brings the peptide and sugar rings together and leads to an intramolecular hydrogen-bonding interaction that is sometimes bridged by a water molecule. This hydrogen bond changes the conformation of the peptide backbone, inducing a favorable n → π* interaction between the oxygen lone pair from the prolyl N-terminal amide and the C═O, which explains the observed increase in trans isomer population in α/β-galactosylated 4S-hydroxyproline. Our results provide the first molecular level information about this important glycosidic linkage, as well as provide an explanation for the previously observed increase in trans isomer population in 4S-hyp compounds. Moreover, this study provides evidence that sugar-mediated long-range hydrogen bonding between hydroxyl groups and the carbonyl peptide backbone can modify the properties of N-terminal prolyl cis/trans isomerization in peptides.

Molecular characterization of aspartylglucosaminidase, a lysosomal hydrolase upregulated during strobilation in the moon jellyfish, Aurelia aurita.

PubMed

Tsujita, Natsumi; Kuwahara, Hiroyuki; Koyama, Hiroki; Yanaka, Noriyuki; Arakawa, Kenji; Kuniyoshi, Hisato

2017-05-01

The life cycle of the moon jellyfish, Aurelia aurita, alternates between a benthic asexual polyp stage and a planktonic sexual medusa (jellyfish) stage. Transition from polyp to medusa is called strobilation. To investigate the molecular mechanisms of strobilation, we screened for genes that are upregulated during strobilation using the differential display method and we identified aspartylglucosaminidase (AGA), which encodes a lysosomal hydrolase. Similar to AGAs from other species, Aurelia AGA possessed an N-terminal signal peptide and potential N-glycosylation sites. The genomic region of Aurelia AGA was approximately 9.8 kb in length and contained 12 exons and 11 introns. Quantitative RT-PCR analysis revealed that AGA expression increased during strobilation, and was then decreased in medusae. To inhibit AGA function, we administered the lysosomal acidification inhibitors, chloroquine or bafilomycin A1, to animals during strobilation. Both inhibitors disturbed medusa morphogenesis at the oral end, suggesting involvement of lysosomal hydrolases in strobilation.

A prohormone convertase cleavage site within a predicted alpha-helix mediates sorting of the neuronal and endocrine polypeptide VGF into the regulated secretory pathway.

PubMed

Garcia, Angelo L; Han, Shan-Kuo; Janssen, William G; Khaing, Zin Z; Ito, Timothy; Glucksman, Marc J; Benson, Deanna L; Salton, Stephen R J

2005-12-16

Distinct intracellular pathways are involved in regulated and constitutive protein secretion from neuronal and endocrine cells, yet the peptide signals and molecular mechanisms responsible for targeting and retention of soluble proteins in secretory granules are incompletely understood. By using confocal microscopy and subcellular fractionation, we examined trafficking of the neuronal and endocrine peptide precursor VGF that is stored in large dense core vesicles and undergoes regulated secretion. VGF cofractionated with secretory vesicle membranes but was not detected in detergent-resistant lipid rafts. Deletional analysis using epitope-tagged VGF suggested that the C-terminal 73-amino acid fragment of VGF, containing two predicted alpha-helical loops and four potential prohormone convertase (PC) cleavage sites, was necessary and sufficient with an N-terminal signal peptide-containing domain, for large dense core vesicle sorting and regulated secretion from PC12 and INS-1 cells. Further transfection analysis identified the sorting sequence as a compact C-terminal alpha-helix and embedded 564RRR566 PC cleavage site; mutation of the 564RRR566 PC site in VGF-(1-65): GFP:VGF-(545-617) blocked regulated secretion, whereas disruption of the alpha-helix had no effect. Mutation of the adjacent 567HFHH570 motif, a charged region that might enhance PC cleavage in acidic environments, also blocked regulated release. Finally, inhibition of PC cleavage in PC12 cells using the membrane-permeable synthetic peptide chloromethyl ketone (decanoyl-RVKR-CMK) blocked regulated secretion of VGF. Our studies define a critical RRR-containing C-terminal domain that targets VGF into the regulated pathway in neuronal PC12 and endocrine INS-1 cells, providing additional support for the proposed role that PCs and their cleavage sites play in regulated peptide secretion.

Elucidation of the Binding Mode of the Carboxyterminal Region of Peptide YY to the Human Y2 Receptor.

PubMed

Xu, Bo; Vasile, Silvana; Østergaard, Søren; Paulsson, Johan F; Pruner, Jasna; Åqvist, Johan; Wulff, Birgitte S; Gutiérrez-de-Terán, Hugo; Larhammar, Dan

2018-04-01

Understanding the agonist-receptor interactions in the neuropeptide Y (NPY)/peptide YY (PYY) signaling system is fundamental for the design of novel modulators of appetite regulation. We report here the results of a multidisciplinary approach to elucidate the binding mode of the native peptide agonist PYY to the human Y 2 receptor, based on computational modeling, peptide chemistry and in vitro pharmacological analyses. The preserved binding orientation proposed for full-length PYY and five analogs, truncated at the amino terminus, explains our pharmacological results where truncations of the N-terminal proline helix showed little effect on peptide affinity. This was followed by receptor mutagenesis to investigate the roles of several receptor positions suggested by the modeling. As a complement, PYY-(3-36) analogs were synthesized with modifications at different positions in the common PYY/NPY C-terminal fragment ( 32 TRQRY 36 -amide). The results were assessed and interpreted by molecular dynamics and Free Energy Perturbation (FEP) simulations of selected mutants, providing a detailed map of the interactions of the PYY/NPY C-terminal fragment with the transmembrane cavity of the Y 2 receptor. The amidated C-terminus would be stabilized by polar interactions with Gln288 6.55 and Tyr219 5.39 , while Gln130 3.32 contributes to interactions with Q 34 in the peptide and T 32 is close to the tip of TM7 in the receptor. This leaves the core, α -helix of the peptide exposed to make potential interactions with the extracellular loops. This model agrees with most experimental data available for the Y 2 system and can be used as a basis for optimization of Y 2 receptor agonists. Copyright © 2018 by The Author(s).

The quantum chemical causality of pMHC-TCR biological avidity: Peptide atomic coordination data and the electronic state of agonist N termini.

PubMed

Antipas, Georgios S E; Germenis, Anastasios E

2015-06-01

The quantum state of functional avidity of the synapse formed between a peptide-Major Histocompatibility Complex (pMHC) and a T cell receptor (TCR) is a subject not previously touched upon. Here we present atomic pair correlation meta-data based on crystalized tertiary structures of the Tax (HTLV-1) peptide along with three artificially altered variants, all of which were presented by the (Class I) HLA-A201 protein in complexation with the human (CD8(+)) A6TCR. The meta-data reveal the existence of a direct relationship between pMHC-TCR functional avidity (agonist/antagonist) and peptide pair distribution function (PDF). In this context, antagonist peptides are consistently under-coordinated in respect to Tax. Moreover, Density Functional Theory (DFT) datasets in the BLYP/TZ2P level of theory resulting from relaxation of the H species on peptide tertiary structures reveal that the coordination requirement of agonist peptides is also expressed as a physical observable of the protonation state of their N termini: agonistic peptides are always found to retain a stable ammonium (NH3 (+)) terminal group while antagonist peptides are not.

The structure of lactoferrin-binding protein B from Neisseria meningitidis suggests roles in iron acquisition and neutralization of host defences

PubMed Central

Brooks, Cory L.; Arutyunova, Elena; Lemieux, M. Joanne

2014-01-01

Pathogens have evolved a range of mechanisms to acquire iron from the host during infection. Several Gram-negative pathogens including members of the genera Neisseria and Moraxella have evolved two-component systems that can extract iron from the host glycoproteins lactoferrin and transferrin. The homologous iron-transport systems consist of a membrane-bound transporter and an accessory lipoprotein. While the mechanism behind iron acquisition from transferrin is well understood, relatively little is known regarding how iron is extracted from lactoferrin. Here, the crystal structure of the N-terminal domain (N-lobe) of the accessory lipoprotein lactoferrin-binding protein B (LbpB) from the pathogen Neisseria meningitidis is reported. The structure is highly homologous to the previously determined structures of the accessory lipoprotein transferrin-binding protein B (TbpB) and LbpB from the bovine pathogen Moraxella bovis. Docking the LbpB structure with lactoferrin reveals extensive binding interactions with the N1 subdomain of lactoferrin. The nature of the interaction precludes apolactoferrin from binding LbpB, ensuring the specificity of iron-loaded lactoferrin. The specificity of LbpB safeguards proper delivery of iron-bound lactoferrin to the transporter lactoferrin-binding protein A (LbpA). The structure also reveals a possible secondary role for LbpB in protecting the bacteria from host defences. Following proteolytic digestion of lactoferrin, a cationic peptide derived from the N-terminus is released. This peptide, called lactoferricin, exhibits potent antimicrobial effects. The docked model of LbpB with lactoferrin reveals that LbpB interacts extensively with the N-terminal lactoferricin region. This may provide a venue for preventing the production of the peptide by proteolysis, or directly sequestering the peptide, protecting the bacteria from the toxic effects of lactoferricin. PMID:25286931

Follow-up of congenital heart disease patients with subclinical hypothyroidism.

PubMed

Martínez-Quintana, Efrén; Rodríguez-González, Fayna

2015-08-01

Subclinical hypothyroidism or mild thyroid failure is a common problem in patients without known thyroid disease. Demographic and analytical data were collected in 309, of which 181 were male and 128 were female, congenital heart disease (CHD) patients. CHD patients with thyroid-stimulating hormone above 5.5 mIU/L were also followed up from an analytical point of view to determine changes in serum glucose, cholesterol, N-terminal pro b-type natriuretic peptide, and C-reactive protein concentrations. Of the CHD patients, 35 (11.3%) showed thyroid-stimulating hormone concentration above 5.5 mIU/L. Of them, 27 were followed up during 2.4±1.2 years - 10 were under thyroid hormone replacement treatment, and 17 were not. Of the 27 patients (25.9%), 7 with subclinical hypothyroidism had positive anti-thyroid peroxidase, and 3 of them (42.8%) with positive anti-thyroid peroxidase had Down syndrome. Down syndrome and hypoxaemic CHD patients showed higher thyroid-stimulating hormone concentrations than the rest of the congenital patients (p<0.001). No significant differences were observed in serum thyroxine, creatinine, uric acid, lipids, C-reactive protein, or N-terminal pro b-type natriuretic peptide concentrations before and after the follow-up in those CHD patients with thyroid-stimulating hormone above 5.5 mIU/L whether or not they received levothyroxine therapy. CHD patients with subclinical hypothyroidism showed no significant changes in serum thyroxine, cholesterol, C-reactive protein, or N-terminal pro b-type natriuretic peptide concentrations whether or not they were treated with thyroid hormone replacement therapy.

A novel antimicrobial peptide derived from modified N-terminal domain of bovine lactoferrin: design, synthesis, activity against multidrug-resistant bacteria and Candida.

PubMed

Mishra, Biswajit; Leishangthem, Geeta Devi; Gill, Kamaldeep; Singh, Abhay K; Das, Swagata; Singh, Kusum; Xess, Immaculata; Dinda, Amit; Kapil, Arti; Patro, Ishan K; Dey, Sharmistha

2013-02-01

Lactoferrin (LF) is believed to contribute to the host's defense against microbial infections. This work focuses on the antibacterial and antifungal activities of a designed peptide, L10 (WFRKQLKW) by modifying the first eight N-terminal residues of bovine LF by selective homologous substitution of amino acids on the basis of hydrophobicity, L10 has shown potent antibacterial and antifungal properties against clinically isolated extended spectrum beta lactamases (ESBL), producing gram-negative bacteria as well as Candida strains with minimal inhibitory concentrations (MIC) ranging from 1 to 8 μg/mL and 6.5 μg/mL, respectively. The peptide was found to be least hemolytic at a concentration of 800 μg/mL. Interaction with lipopolysaccharide (LPS) and lipid A (LA) suggests that the peptide targets the membrane of gram-negative bacteria. The membrane interactive nature of the peptide, both antibacterial and antifungal, was further confirmed by visual observations employing electron microscopy. Further analyses, by means of propidium iodide based flow cytometry, also supported the membrane permeabilization of Candida cells. The peptide was also found to possess anti-inflammatory properties, by virtue of its ability to inhibit cyclooxygenase-2 (COX-2). L10 therefore emerges as a potential therapeutic remedial solution for infections caused by ESBL positive, gram-negative bacteria and multidrug-resistant (MDR) fungal strains, on account of its multifunctional activities. This study may open up new approach to develop and design novel antimicrobials. Copyright © 2012 Elsevier B.V. All rights reserved.

Peptide structure: Its effect on penetration into human hair.

PubMed

Silva, Carla J S M; Vasconcelos, Andreia; Cavaco-Paulo, Artur

2007-01-01

The influence of the peptide structure on its penetration inside hair was studied, together with the effect of hair bleaching (oxidation). For that reason, the outcome of positioning a charged sequence (KAKAK) either at the N or C terminal on hair penetration has been studied for peptides with 17 residues each. It was observed that the penetration of these peptides into hair was driven by electrostatic interactions, where the position of the charged group at the peptide structure was of major importance. The penetration was only achieved for damaged hair due to its higher negative charge at the membrane surface. It was also observed that the peptides were able to restore the original tensile strength of bleached hair. Consequently, the knowledge of hair surface properties is of extreme importance when designing peptides directed for hair treatment.

Anomalous Insulator-Metal Transition in Boron Nitride-Graphene Hybrid Atomic Layers

DTIC Science & Technology

2012-08-13

REPORT Anomalous insulator-metal transition in boron nitride-graphene hybrid atomic layers 14 . ABSTRACT 16. SECURITY CLASSIFICATION OF: The study of...from the DFT calculation. The calculated transmission through a N terminated zigzag edged h-BN nanodomain embedded in graphene is shown in Fig. 14 , with...Energy ε − ε F (eV) 0 0.5 1 1.5 2 Tr an sm is si on FIG. 14 . (Color online) Transmission through a N terminated zigzag edged h-BN nanodomain embedded in

Effects of alkali or acid treatment on the isomerization of amino acids.

PubMed

Ohmori, Taketo; Mutaguchi, Yuta; Doi, Katsumi; Ohshima, Toshihisa

2012-10-01

The effect of alkali treatment on the isomerization of amino acids was investigated. The 100×D/(D+L) values of amino acids from peptide increased with increase in the number of constituent amino acid residues. Furthermore, the N-terminal amino acid of a dipeptide was isomerized to a greater extent than the C-terminal residue. Copyright © 2012. Published by Elsevier B.V.

The proteolytic processing site of the precursor of lysyl oxidase.

PubMed Central

Cronshaw, A D; Fothergill-Gilmore, L A; Hulmes, D J

1995-01-01

The precise cleavage site of the N-terminal propeptide region of the precursor of lysyl oxidase has not yet been established, due to N-terminal blocking of the mature protein. Using a combination of peptide fragmentation, amino acid sequencing, time-of-flight m.s. and partial chemical unblocking procedures, it is shown that the mature form of lysyl oxidase begins at residue Asp-169 of the precursor protein (numbered according to the human sequence). The cleavage site is 28 residues to the C-terminal side of the site previously suggested on the basis of apparant molecular mass by SDS/PAGE, with the consequence that the two putative, N-linked glycosylation sites and the position of the Arg/Gln sequence polymorphism are now all in the precursor region. PMID:7864821

Incorporation of N-amidino-pyroglutamic acid into peptides using intramolecular cyclization of alpha-guanidinoglutaric acid.

PubMed

Burov, Sergey; Moskalenko, Yulia; Dorosh, Marina; Shkarubskaya, Zoya; Panarin, Evgeny

2009-11-01

N-terminal modification of peptides by unnatural amino acids significantly affects their enzymatic stability, conformational properties and biological activity. Application of N-amidino-amino acids, positively charged under physiological conditions, can change peptide conformation and its affinity to the corresponding receptor. In this article, we describe synthesis of short peptides, containing a new building block-N-amidino-pyroglutamic acid. Although direct guanidinylation of pyroglutamic acid and oxidation of N-amidino-proline using RuO(4) did not produce positive results, N-amidino-Glp-Phe-OH was synthesized on Wang polymer by cyclization of alpha-guanidinoglutaric acid residue. In the course of synthesis, it was found that literature procedure of selective Boc deprotection using TMSOTf/TEA reagent is accompanied by concomitant side reaction of triethylamine alkylation by polymer linker fragment. It should be mentioned that independently from cyclization time and coupling agent (DIC or HCTU), the lactam formation was incomplete. Separation of the cyclic product from the linear precursor was achieved by HPLC in ammonium formate buffer at pH 6. HPLC analysis showed N-amidino-Glp-Phe-OH stability at acidic and physiological pH and fast ring opening in water solution at pH 9. The suggested method of N-amidino-Glp residue formation can be applied in the case of short peptide chains, whereas synthesis of longer ones will require fragment condensation approach.

Crystal structure of an EfPDF complex with Met-Ala-Ser based on crystallographic packing.

PubMed

Nam, Ki Hyun; Kim, Kook-Han; Kim, Eunice Eun Kyeong; Hwang, Kwang Yeon

2009-04-17

PDF (peptide deformylase) plays a critical role in the production of mature proteins by removing the N-formyl polypeptide of nascent proteins in the prokaryote cell system. This protein is essential for bacterial growth, making it an attractive target for the design of new antibiotics. Accordingly, PDF has been evaluated as a drug target; however, architectural mechanism studies of PDF have not yet fully elucidated its molecular function. We recently reported the crystal structure of PDF produced by Enterococcus faecium [K.H. Nam, J.I. Ham, A. Priyadarshi, E.E. Kim, N. Chung, K.Y. Hwang, "Insight into the antibacterial drug design and architectural mechanism of peptide recognition from the E. faecium peptide deformylase structure", Proteins 74 (2009) 261-265]. Here, we present the crystal structure of the EfPDF complex with MAS (Met-Ser-Ala), thereby not only delineating the architectural mechanism for the recognition of mimic-peptides by N-terminal cleaved expression peptide, but also suggesting possible targets for rational design of antibacterial drugs. In addition to their implications for drug design, these structural studies will facilitate elucidation of the architectural mechanism responsible for the peptide recognition of PDF.

Critical Structural and Functional Roles for the N-Terminal Insertion Sequence in Surfactant Protein B Analogs

PubMed Central

Walther, Frans J.; Waring, Alan J.; Hernandez-Juviel, Jose M.; Gordon, Larry M.; Wang, Zhengdong; Jung, Chun-Ling; Ruchala, Piotr; Clark, Andrew P.; Smith, Wesley M.; Sharma, Shantanu; Notter, Robert H.

2010-01-01

Background Surfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., ∼residues 8–25 and 63–78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1–7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity. Methodology/Results FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary α-helix and secondary β-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a “saposin-like” fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B. Conclusion Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B. PMID:20084172

Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation

DOE PAGES

Colbert, Karen N.; Hattendorf, Douglas A.; Weiss, Thomas M.; ...

2013-07-15

In neurons, soluble N-ethylmaleimide–sensitive factor attachment receptor (SNARE) proteins drive the fusion of synaptic vesicles to the plasma membrane through the formation of a four-helix SNARE complex. Members of the Sec1/Munc18 protein family regulate membrane fusion through interactions with the syntaxin family of SNARE proteins. The neuronal protein Munc18a interacts with a closed conformation of the SNARE protein syntaxin1a (Syx1a) and with an assembled SNARE complex containing Syx1a in an open conformation. The N-peptide of Syx1a (amino acids 1–24) has been implicated in the transition of Munc18a-bound Syx1a to Munc18a-bound SNARE complex, but the underlying mechanism is not understood. Inmore » addition, we report the X-ray crystal structures of Munc18a bound to Syx1a with and without its native N-peptide (Syx1aΔN), along with small-angle X-ray scattering (SAXS) data for Munc18a bound to Syx1a, Syx1aΔN, and Syx1a L165A/E166A (LE), a mutation thought to render Syx1a in a constitutively open conformation. We show that all three complexes adopt the same global structure, in which Munc18a binds a closed conformation of Syx1a. We also identify a possible structural connection between the Syx1a N-peptide and SNARE domain that might be important for the transition of closed-to-open Syx1a in SNARE complex assembly. Although the role of the N-peptide in Munc18a-mediated SNARE complex assembly remains unclear, our results demonstrate that the N-peptide and LE mutation have no effect on the global conformation of the Munc18a–Syx1a complex.« less

Effect of chain length on the conformation and T cell recognition of synthetic hemagglutinin fragments

NASA Astrophysics Data System (ADS)

Tóth, Gábor K.; Holly, Sándor; Majer, Zsuzsa; Hollósi, Miklós; Rajnavölgyi, Éva; Laczkó, Ilona

2000-01-01

Circular dichroism and Fourier-transform infrared spectroscopies were used to compare the conformational mobility of 13-mer peptides covering the 317-329 region of the envelope protein hemagglutinin of human influenza A virus subtypes H1, H2 and H3 with that of their truncated deca- and nonapeptide analogs. These peptides were demonstrated to bind to the murine I-E d major histocompatibility complex encoded class II and human HLA-B*2705 class I molecules. Despite the amino acid substitutions in the three 13-mer subtype sequences, no significant differences in the conformational properties could be shown. Deletion of the N-terminal three residues resulted in a shift to an increased α-helical conformer population in the 317-329 H1 peptide and the breakage of the 3 10 or weakly H-bonded (nascent) α-helix in the H2 and H3 peptides. The conformational change observed upon deletion did not influence the efficiency of I-E d-peptide interaction, however, the C-terminal Arg had a beneficial effect both on MHC class II and class I binding without causing any remarkable change in solution conformation.

Characterization of the macrocyclase involved in the biosynthesis of RiPP cyclic peptides in plants.

PubMed

Chekan, Jonathan R; Estrada, Paola; Covello, Patrick S; Nair, Satish K

2017-06-20

Enzymes that can catalyze the macrocyclization of linear peptide substrates have long been sought for the production of libraries of structurally diverse scaffolds via combinatorial gene assembly as well as to afford rapid in vivo screening methods. Orbitides are plant ribosomally synthesized and posttranslationally modified peptides (RiPPs) of various sizes and topologies, several of which are shown to be biologically active. The diversity in size and sequence of orbitides suggests that the corresponding macrocyclases may be ideal catalysts for production of cyclic peptides. Here we present the biochemical characterization and crystal structures of the plant enzyme PCY1 involved in orbitide macrocyclization. These studies demonstrate how the PCY1 S9A protease fold has been adapted for transamidation, rather than hydrolysis, of acyl-enzyme intermediates to yield cyclic products. Notably, PCY1 uses an unusual strategy in which the cleaved C-terminal follower peptide from the substrate stabilizes the enzyme in a productive conformation to facilitate macrocyclization of the N-terminal fragment. The broad substrate tolerance of PCY1 can be exploited as a biotechnological tool to generate structurally diverse arrays of macrocycles, including those with nonproteinogenic elements.

Myticalins: A Novel Multigenic Family of Linear, Cationic Antimicrobial Peptides from Marine Mussels (Mytilus spp.).

PubMed

Leoni, Gabriele; De Poli, Andrea; Mardirossian, Mario; Gambato, Stefano; Florian, Fiorella; Venier, Paola; Wilson, Daniel N; Tossi, Alessandro; Pallavicini, Alberto; Gerdol, Marco

2017-08-22

The application of high-throughput sequencing technologies to non-model organisms has brought new opportunities for the identification of bioactive peptides from genomes and transcriptomes. From this point of view, marine invertebrates represent a potentially rich, yet largely unexplored resource for de novo discovery due to their adaptation to diverse challenging habitats. Bioinformatics analyses of available genomic and transcriptomic data allowed us to identify myticalins, a novel family of antimicrobial peptides (AMPs) from the mussel Mytilus galloprovincialis , and a similar family of AMPs from Modiolus spp., named modiocalins. Their coding sequence encompasses two conserved N-terminal (signal peptide) and C-terminal (propeptide) regions and a hypervariable central cationic region corresponding to the mature peptide. Myticalins are taxonomically restricted to Mytiloida and they can be classified into four subfamilies. These AMPs are subject to considerable interindividual sequence variability and possibly to presence/absence variation. Functional assays performed on selected members of this family indicate a remarkable tissue-specific expression (in gills) and broad spectrum of activity against both Gram-positive and Gram-negative bacteria. Overall, we present the first linear AMPs ever described in marine mussels and confirm the great potential of bioinformatics tools for the de novo discovery of bioactive peptides in non-model organisms.

Characterization of melanocortin NDP-MSH agonist peptide fragments at the mouse central and peripheral melanocortin receptors.

PubMed

Haskell-Luevano, C; Holder, J R; Monck, E K; Bauzo, R M

2001-06-21

The central melanocortin receptors, melanocortin-4 (MC4R) and melanocortin-3 (MC3R), are involved in the regulation of satiety and energy homeostasis. The MC4R in particular has become a pharmaceutical industry drug target due to its direct involvement in the regulation of food intake and its potential therapeutic application for the treatment of obesity-related diseases. The melanocortin receptors are stimulated by the native ligand, alpha-melanocyte stimulating hormone (alpha-MSH). The potent and enzymatically stable analogue NDP-MSH (Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH(2)) is a lead peptide for the identification of melanocortin amino acids important for receptor molecular recognition and stimulation. We have synthesized nine peptide fragments of NDP-MSH, deleting N- and C-terminal amino acids to determine the "minimally active" sequence of NDP-MSH. Additionally, five peptides were synthesized to study stereochemical inversion at the Phe 7 and Trp 9 positions in attempts to increase tetra- and tripeptide potencies. These peptide analogues were pharmacologically characterized at the mouse melanocortin MC1, MC3, MC4, and MC5 receptors. This study has identified the Ac-His-DPhe-Arg-Trp-NH(2) tetrapeptide as possessing 10 nM agonist activity at the brain MC4R. The tripeptide Ac-DPhe-Arg-Trp-NH(2) possessed micromolar agonist activities at the MC1R, MC4R, and MC5R but only slight stimulatory activity was observed at the MC3R (at up to 100 microM concentration). This study has also examined to importance of both N- and C-terminal NDP-MSH amino acids at the different melanocortin receptors, providing information for drug design and identification of putative ligand-receptor interactions.

Serum Stabilities of Short Tryptophan- and Arginine-Rich Antimicrobial Peptide Analogs

PubMed Central

Nguyen, Leonard T.; Chau, Johnny K.; Perry, Nicole A.; de Boer, Leonie; Zaat, Sebastian A. J.; Vogel, Hans J.

2010-01-01

Background Several short antimicrobial peptides that are rich in tryptophan and arginine residues were designed with a series of simple modifications such as end capping and cyclization. The two sets of hexapeptides are based on the Trp- and Arg-rich primary sequences from the “antimicrobial centre” of bovine lactoferricin as well as an antimicrobial sequence obtained through the screening of a hexapeptide combinatorial library. Methodology/Principal Findings HPLC, mass spectrometry and antimicrobial assays were carried out to explore the consequences of the modifications on the serum stability and microbicidal activity of the peptides. The results show that C-terminal amidation increases the antimicrobial activity but that it makes little difference to its proteolytic degradation in human serum. On the other hand, N-terminal acetylation decreases the peptide activities but significantly increases their protease resistance. Peptide cyclization of the hexameric peptides was found to be highly effective for both serum stability and antimicrobial activity. However the two cyclization strategies employed have different effects, with disulfide cyclization resulting in more active peptides while backbone cyclization results in more proteolytically stable peptides. However, the benefit of backbone cyclization did not extend to longer 11-mer peptides derived from the same region of lactoferricin. Mass spectrometry data support the serum stability assay results and allowed us to determine preferred proteolysis sites in the peptides. Furthermore, isothermal titration calorimetry experiments showed that the peptides all had weak interactions with albumin, the most abundant protein in human serum. Conclusions/Significance Taken together, the results provide insight into the behavior of the peptides in human serum and will therefore aid in advancing antimicrobial peptide design towards systemic applications. PMID:20844765

Left-handed helical preference in an achiral peptide chain is induced by an L-amino acid in an N-terminal type II β-turn.

PubMed

De Poli, Matteo; De Zotti, Marta; Raftery, James; Aguilar, Juan A; Morris, Gareth A; Clayden, Jonathan

2013-03-15

Oligomers of the achiral amino acid Aib adopt helical conformations in which the screw-sense may be controlled by a single N-terminal residue. Using crystallographic and NMR techniques, we show that the left- or right-handed sense of helical induction arises from the nature of the β-turn at the N terminus: the tertiary amino acid L-Val induces a left-handed type II β-turn in both the solid state and in solution, while the corresponding quaternary amino acid L-α-methylvaline induces a right-handed type III β-turn.

Structure-activity relationships of sialogogic heptapeptides analogous to physalaemin.

PubMed

Gao, C; Abe, K

2000-05-01

The rationale behind this study was to determine in detail which amino acids in physalaemin are crucial to its sialogogue activity, with a view of synthesizing new sialogogues which might be of use in the treatment of dry mouth. With the progressive elimination of amino acids, one by one, from the C- and N-terminal regions, 126 heptapeptides were newly synthesized by the multipin peptide method, for comparison with II naturally occurring tachykinins. The C-terminal amide in position II was essential for salivation, but not the pyrolidine group or the N-terminal amino acid residues in positions I to 4. In 18 heptapeptides in which M in position II (MII) was replaced by another amino acid, one by one, none caused salivation. In 18 heptapeptides, in which L10 or G9 was replaced, three peptides caused salivation but none had significantly increased secretory activities. In 18 heptapeptides in which Y8 was replaced, four caused salivation but only one (I) had significantly increased secretory activity. In 18 heptapeptides in which F7 was replaced, only Y caused salivation but with significantly reduced secretory activity. In contrast, in 18 heptapeptides in which K6 and N5 were replaced, most caused salivation and some of them had significantly increased secretory activities. It is concluded that the sequence FYGLM-NH2 conserved in the C-terminal region of physalaemin is optimal, that amides in position II and F7 are very important for salivation, but that K6 and N5 can be replaced by some other amino acids, resulting in increased secretory activities.

Crystallization and X-ray analysis of the T = 4 particle of hepatitis B capsid protein with an N-terminal extension

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Wen Siang; McNae, Iain W.; Ho, Kok Lian

2007-08-01

Hepatitis B virus capsids have significant potential as carriers for immunogenic peptides. The crystal structure of the T = 4 particle of hepatitis B core protein containing an N-terminal extension reveals that the fusion peptide is exposed on the exterior of the particle. Hepatitis B core (HBc) particles have been extensively exploited as carriers for foreign immunological epitopes in the development of multicomponent vaccines and diagnostic reagents. Crystals of the T = 4 HBc particle were grown in PEG 20 000, ammonium sulfate and various types of alcohols. A temperature jump from 277 or 283 to 290 K was foundmore » to enhance crystal growth. A crystal grown using MPD as a cryoprotectant diffracted X-rays to 7.7 Å resolution and data were collected to 99.6% completeness at 8.9 Å. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 352.3, b = 465.5, c = 645.0 Å. The electron-density map reveals a protrusion that is consistent with the N-terminus extending out from the surface of the capsid. The structure presented here supports the idea that N-terminal insertions can be exploited in the development of diagnostic reagents, multicomponent vaccines and delivery vehicles into mammalian cells.« less

Age-dependent loss of the C-terminal amino acid from alpha crystallin

NASA Technical Reports Server (NTRS)

Emmons, T.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

1992-01-01

Antiserum made against the C-terminal region of alpha-A crystallin was used to monitor the purification of a tryptic peptide containing the C-terminus of the molecule from fetal versus adult bovine lenses. Mass spectral analysis of the peptide preparations obtained from these lenses demonstrated the presence of a peptide (T20) containing an intact C-terminus from fetal lenses and the presence of an additional peptide (T20') from older lenses that contained a cleaved C-terminal serine. These results demonstrate an age-dependent processing of alpha-A crystallin in the bovine lens, resulting in removal of the C-terminal amino acid residue.

Non-peptidic antagonists of the CGRP receptor, BIBN4096BS and MK-0974, interact with the calcitonin receptor-like receptor via methionine-42 and RAMP1 via tryptophan-74.

PubMed

Miller, Philip S; Barwell, James; Poyner, David R; Wigglesworth, Mark J; Garland, Stephen L; Donnelly, Dan

2010-01-01

The receptor for calcitonin gene-related peptide (CGRP) has been the target for the development of novel small molecule antagonists for the treatment of migraine. Two such antagonists, BIBN4096BS and MK-0974, have shown great promise in clinical trials and hence a deeper understanding of the mechanism of their interaction with the receptor is now required. The structure of the CGRP receptor is unusual since it is comprised of a hetero-oligomeric complex between the calcitonin receptor-like receptor (CRL) and an accessory protein (RAMP1). Both the CLR and RAMP1 components have extracellular domains which interact with each other and together form part of the peptide-binding site. It seems likely that the antagonist binding site will also be located on the extracellular domains and indeed Trp-74 of RAMP1 has been shown to form part of the binding site for BIBN4096BS. However, despite a chimeric study demonstrating the role of the N-terminal domain of CLR in antagonist binding, no specific residues have been identified. Here we carry out a mutagenic screen of the extreme N-terminal domain of CLR (residues 23-63) and identify a mutant, Met-42-Ala, which displays 48-fold lower affinity for BIBN4096BS and almost 900-fold lower affinity for MK-0974. In addition, we confirm that the Trp-74-Lys mutation at human RAMP1 reduces BIBN4096BS affinity by over 300-fold and show for the first time a similar effect for MK-0974 affinity. The data suggest that the non-peptide antagonists occupy a binding site close to the interface of the N-terminal domains of CLR and RAMP1. Copyright 2009 Elsevier Inc. All rights reserved.

Prognostic prediction of troponins in cardiac myxoma: case study with literature review

PubMed Central

Yuan, Shi-Min

2015-01-01

Objective It was supposed that troponins in cardiac myxoma patients might be in a same fashion as in the conditions without myocardial injury. In order to verify this hypothesis, troponins in cardiac myxoma patients were discussed by presenting a comprehensive retrieval of the literature with incorporating the information of a recent patient. Methods Postoperative detections of troponin I, creatine kinase isoenzyme MB (CK-MB) and N-terminal pro-B-type natriuretic peptide revealed elevated troponin I and CK-MB and normal N-terminal pro-B-type natriuretic peptide. Postoperative troponin I and CK-MB shared a same trend, reaching a peak value at postoperative hour 2, gradually decreased on postoperative day 1, and reached a plateau on postoperative days 7 and 13. A significant correlation could be noted between the postoperative values of the two indicators (Y=0.0714X + 0.6425, r2=0.9111, r=0.9545, P=0.0116). No significant linear correlation between troponin I and N-terminal pro-B-type natriuretic peptide were found. Literature review of troponins in cardiac myxoma patients revealed the uncomplicated patients had a normal or only slightly elevated troponin before open heart surgery. However, the complicated patients (with cerebral or cardiac events) showed a normal preoperative troponin in 3 (23.1%) and an elevated troponin in 10 (76.9%) patients (χ2=7.54, P=0.0169, Fisher's exact test). The overall quantitative result of troponin I was 2.45±2.53 µg/L, and that of troponin T was 3.10±4.29 mg/L, respectively. Conclusion Troponins are not necessarily elevated in patients with a cardiac myxoma without coronary syndrome. By contrast, patients with a cardiac myxoma with an elevated troponin may herald the presence of an associated coronary event. An old cerebral infarct does not necessarily cause an elevation of troponin or B-type natriuretic peptide, or new neurological events, but might lead to a delayed awakening. PMID:26107461

Radiolabeled Escherichia coli heat-stable enterotoxin analogs for in vivo imaging of colorectal cancer

NASA Astrophysics Data System (ADS)

Giblin, M. F.; Sieckman, G. L.; Owen, N. K.; Hoffman, T. J.; Forte, L. R.; Volkert, W. A.

2005-12-01

The human Escherichia coli heat-stable enterotoxin (STh, amino acid sequence N1SSNYCCELCCNPACTGCY19) binds specifically to the guanylate cyclase C (GC-C) receptor, which is present in high density on the apical surface of normal intestinal epithelial cells as well as on the surface of human colon cancer cells. In the current study, two STh analogs were synthesized and evaluated in vitro and in vivo. Both analogs shared identical 6-19 core sequences, and had N-terminal pendant DOTA moieties. The analogs differed in the identity of a 6 amino acid peptide sequence intervening between DOTA and the 6-19 core. In one analog, the peptide was an RGD-containing sequence found in human fibronectin (GRGDSP), while in the other this peptide sequence was randomly scrambled (GRDSGP). The results indicated that the presence of the human fibronectin sequence in the hybrid peptide did not affect tumor localization in vivo.

Structures of E. coli peptide deformylase bound to formate: insight into the preference for Fe2+ over Zn2+ as the active site metal.

PubMed

Jain, Rinku; Hao, Bing; Liu, Ren-Peng; Chan, Michael K

2005-04-06

E. coli peptide deformylase (PDF) catalyzes the deformylation of nascent polypeptides generated during protein synthesis. While PDF was originally thought to be a zinc enzyme, subsequent studies revealed that the active site metal is iron. In an attempt to understand this unusual metal preference, high-resolution structures of Fe-, Co-, and Zn-PDF were determined in complex with its deformylation product, formate. In all three structures, the formate ion binds the metal and forms hydrogen-bonding interactions with the backbone nitrogen of Leu91, the amide side chain of Gln50, and the carboxylate side chain of Glu133. One key difference, however, is how the formate binds the metal. In Fe-PDF and Co-PDF, formate binds in a bidentate fashion, while in Zn-PDF, it binds in a monodentate fashion. Importantly, these structural results provide the first clues into the origins of PDF's metal-dependent activity differences. On the basis of these structures, we propose that the basis for the higher activity of Fe-PDF stems from the better ability of iron to bind and activate the tetrahedral transition state required for cleavage of the N-terminal formyl group.

The early mature part of bacterial twin-arginine translocation (Tat) precursor proteins contributes to TatBC receptor binding.

PubMed

Ulfig, Agnes; Freudl, Roland

2018-05-11

The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in the binding of the proteins to the membrane-associated TatBC receptor complex. In addition, the hydrophobic region in the Tat signal peptides also contributes to TatBC binding, but whether regions beyond the signal-peptide cleavage site are involved in this process is unknown. Here, we analyzed the contribution of the early mature protein part of the Escherichia coli trimethylamine N -oxide reductase (TorA) to productive TatBC receptor binding. We identified substitutions in the 30 amino acids immediately following the TorA signal peptide (30aa-region) that restored export of a transport-defective TorA[KQ]-30aa-MalE precursor, in which the RR residues had been replaced by a lysine-glutamine pair. Some of these substitutions increased the hydrophobicity of the N-terminal part of the 30aa-region and thereby likely enhanced hydrophobic substrate-receptor interactions within the hydrophobic TatBC substrate-binding cavity. Another class of substitutions increased the positive net charge of the region's C-terminal part, presumably leading to strengthened electrostatic interactions between the mature substrate part and the cytoplasmic TatBC regions. Furthermore, we identified substitutions in the C-terminal domains of TatB following the transmembrane segment that restored transport of various transport-defective TorA-MalE derivatives. Some of these substitutions most likely affected the orientation or conformation of the flexible, carboxy-proximal helices of TatB. Therefore, we propose that a tight accommodation of the folded mature region by TatB contributes to productive binding of Tat substrates to TatBC. © 2018 Ulfig and Freudl.

Nano-scale characterization of the dynamics of the chloroplast Toc translocon.

PubMed

Reddick, L Evan; Chotewutmontri, Prakitchai; Crenshaw, Will; Dave, Ashita; Vaughn, Michael; Bruce, Barry D

2008-01-01

Translocons are macromolecular nano-scale machines that facilitate the selective translocation of proteins across membranes. Although common in function, different translocons have evolved diverse molecular mechanisms for protein translocation. Subcellular organelles of endosymbiotic origin such as the chloroplast and mitochondria had to evolve/acquire translocons capable of importing proteins whose genes were transferred to the host genome. These gene products are expressed on cytosolic ribosomes as precursor proteins and targeted back to the organelle by an N-terminal extension called the transit peptide or presequence. In chloroplasts the transit peptide is specifically recognized by the Translocon of the Outer Chloroplast membrane (Toc) which is composed of receptor GTPases that potentially function as gate-like switches, where GTP binding and hydrolysis somehow facilitate preprotein binding and translocation. Compared to other translocons, the dynamics of the Toc translocon are probably more complex and certainly less understood. We have developed biochemical/biophysical, imaging, and computational techniques to probe the dynamics of the Toc translocon at the nanoscale. In this chapter we provide detailed protocols for kinetic and binding analysis of precursor interactions in organeller, measurement of the activity and nucleotide binding of the Toc GTPases, native electrophoretic analysis of the assembly/organization of the Toc complex, visualization of the distribution and mobility of Toc apparatus on the surface of chloroplasts, and conclude with the identification and molecular modeling Toc75 POTRA domains. With these new methodologies we discuss future directions of the field.

The First Step of Gibberellin Biosynthesis in Pumpkin Is Catalyzed by at Least Two Copalyl Diphosphate Synthases Encoded by Differentially Regulated Genes

PubMed Central

Smith, Maria W.; Yamaguchi, Shinjiro; Ait-Ali, Tahar; Kamiya, Yuji

1998-01-01

The first step in gibberellin biosynthesis is catalyzed by copalyl diphosphate synthase (CPS) and ent-kaurene synthase. We have cloned from pumpkin (Cucurbita maxima L.) two cDNAs, CmCPS1 and CmCPS2, that each encode a CPS. Both recombinant fusion CmCPS proteins were active in vitro. CPS are translocated into plastids and processed by cleavage of transit peptides. For CmCPS1 and CmCPS2, the putative transit peptides cannot exceed the first 99 and 107 amino acids, respectively, because longer N-terminal deletions abolished activity. Levels of both CmCPS transcripts were strictly regulated in an organ-specific and developmental manner. Both transcripts were almost undetectable in leaves and were abundant in petioles. CmCPS1 transcript levels were high in young cotyledons and low in roots. In contrast, CmCPS2 transcripts were undetectable in cotyledons but present at significant levels in roots. In hypocotyls, apices, and petioles, CmCPS1 transcript levels decreased with age much more rapidly than those of CmCPS2. We speculate that CmCPS1 expression is correlated with the early stages of organ development, whereas CmCPS2 expression is correlated with subsequent growth. In contrast, C. maxima ent-kaurene synthase transcripts were detected in every organ at almost constant levels. Thus, ent-kaurene biosynthesis may be regulated through control of CPS expression. PMID:9847116

Mapping Protein–Protein Interactions of the Resistance-Related Bacterial Zeta Toxin–Epsilon Antitoxin Complex (ε2ζ2) with High Affinity Peptide Ligands Using Fluorescence Polarization

PubMed Central

Fernández-Bachiller, María Isabel; Brzozowska, Iwona; Odolczyk, Norbert; Zielenkiewicz, Urszula; Zielenkiewicz, Piotr; Rademann, Jörg

2016-01-01

Toxin–antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta–Epsilon toxin–antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε2ζ2 complex. Three α helices of Zeta forming the protein–protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε2ζ2 complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay. PMID:27438853

Mapping Protein-Protein Interactions of the Resistance-Related Bacterial Zeta Toxin-Epsilon Antitoxin Complex (ε₂ζ₂) with High Affinity Peptide Ligands Using Fluorescence Polarization.

PubMed

Fernández-Bachiller, María Isabel; Brzozowska, Iwona; Odolczyk, Norbert; Zielenkiewicz, Urszula; Zielenkiewicz, Piotr; Rademann, Jörg

2016-07-16

Toxin-antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta-Epsilon toxin-antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε₂ζ₂ complex. Three α helices of Zeta forming the protein-protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε₂ζ₂ complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay.

Enhancement of the Enterocin CRL35 Activity by a Synthetic Peptide Derived from the NH2-Terminal Sequence

PubMed Central

Saavedra, Lucila; Minahk, Carlos; de Ruiz Holgado, Aída P.; Sesma, Fernando

2004-01-01

The enterocin CRL35 biosynthetic gene cluster was cloned and sequenced. The sequence was revealed to be highly identical to that of the mundticin KS gene cluster (S. Kawamoto, J. Shima, R. Sato, T. Eguchi, S. Ohmomo, J. Shibato, N. Horikoshi, K. Takeshita, and T. Sameshima, Appl. Environ. Microbiol. 68:3830-3840, 2002). Short synthetic peptides were designed based on the bacteriocin sequence and were evaluated in antimicrobial competitive assays. The peptide KYYGNGVSCNKKGCS produced an enhancement of enterocin CRL35 antimicrobial activity in a buffer system. PMID:15215149

Pharmacologic study of C-terminal fragments of frog skin calcitonin gene-related peptide.

PubMed

Ladram, Ali; Besné, Isabelle; Breton, Lionel; de Lacharrière, Olivier; Nicolas, Pierre; Amiche, Mohamed

2008-07-01

The calcitonin gene-related peptide from the skin of the frog Phyllomedusa bicolor (pbCGRP) is a 37-residue neuropeptide that differs from human alpha CGRP (halphaCGRP) at 16 positions. The affinities of the C-terminal fragments of pbCGRP and halphaCGRP were evaluated in SK-N-MC cells: pbCGRP(8-37) (K(i)=0.2nM) and pbCGRP(27-37) (K(i)=95nM) were, respectively, 3 times and 20 times more potent than the human fragments halphaCGRP(8-37) and halphaCGRP(27-37). Their antagonistic potencies were measured in SK-N-MC and Col 29 cells, and the rat vas deferens. pbCGRP(8-37) inhibited the halphaCGRP-stimulated production of cAMP by SK-N-MC and Col 29 cells 3 to 4 times more strongly than halphaCGRP(8-37). Thus pbCGRP(8-37) is the most potent CGRP-1 competitive antagonist of all the natural sequences reported to date. pbCGRP(27-37) was also as potent as [D(31), A(34), F(35)] halphaCGRP(27-37), a prototypic antagonist analog derived from structure-activity relationship studies of halphaCGRP(8-37).

Cloning and sequencing of a gene encoding the 69-kDa extracellular chitinase of Janthinobacterium lividum.

PubMed

Gleave, A P; Taylor, R K; Morris, B A; Greenwood, D R

1995-09-15

Janthinobacterium lividum secretes a major 56-kDa chitinase and a minor 69-kDa chitinase. A chitinase gene was defined on a 3-kb fragment of clone pRKT10, by virtue of fluorescent colonies in the presence of 4-methylumbelliferyl-beta-D-N,N',N"-chitotrioside. Nucleotide sequencing revealed an 1998-bp open reading frame with the potential to encode a 69,716-Da protein with amino acid sequences similar to those in other chitinases, suggesting it encodes the minor chitinase (Chi69). Chitinase activity of Escherichia coli (pRKT10) lysates was detected mainly in the periplasmic fraction and immunoblotting detected a 70-kDa protein in this fraction. Chi69 has an N-terminal secretory leader peptide preceding two probable chitin-binding domains and a catalytic domain. These functional domains are separated by linker regions of proline-threonine repeats. Amino acid sequencing of cyanogen bromide cleavage-derived peptides from the major 56-kDa chitinase suggested that Chi69 may be a precursor of Chi56. In addition, an N-terminally truncated version of Chi69 retained chitinase activity as expected if in vivo processing of Chi69 generates Chi56.

Cellular origin of the Bufo arenarum sperm receptor gp75, a ZP2 family member: its proteolysis after fertilization.

PubMed

Scarpeci, Sonia L; Sanchez, Mercedes L; Cabada, Marcelo O

2008-04-01

The egg envelope is an extracellular matrix that surrounds oocytes. In frogs and mammals, a prominent feature of envelope modification following fertilization is the N-terminal proteolysis of the envelope glycoproteins, ZPA [ZP (zona pellucida) A]. It was proposed that ZPA N-terminal proteolysis leads to a conformational change in egg envelope glycoproteins, resulting in the prevention of polyspermy. Bufo arenarum VE (vitelline envelope) is made up of at least four glycoproteins: gp120 (glycoprotein 120), gp75, gp41 and gp38. The aim of the present study was to identify and characterize the baZPA (B. arenarum ZPA homologue). Also, our aim was to evaluate its integrity and functional significance during fertilization. VE components were labelled with FITC in order to study their sperm-binding capacity. The assay showed that gp75, gp41 and gp38 possess sperm-binding activity. We obtained a full-length cDNA of 2062 bp containing one ORF (open reading frame) with a sequence for 687 amino acids. The predicted amino acid sequence had close similarity to that of mammalian ZPA. This result indicates that gp75 is the baZPA. Antibodies raised against an N-terminal sequence recognized baZPA and inhibited sperm-baZPA extracted from VE binding. This protein does not induce the acrosome reaction in homologue sperm. Northern-blot studies indicated that the transcript is exclusively expressed in the ovary. In situ hybridization studies confirmed this and pointed to previtellogenic oocytes and follicle cells surrounding the oocyte as the source of the transcript. baZPA was cleaved during fertilization and the N-terminal peptide fragment remained disulfide bonded to the glycoprotein moiety following proteolysis. From the sequence analysis, it was possible to consider that gp75 is the baZPA. It is expressed by previtellogenic oocytes and follicle cells. Also, it can be considered as a sperm receptor that undergoes N-terminal proteolysis during fertilization. The N-terminal peptide could be necessary for sperm binding.

Crystal Structure and Function of PqqF Protein in the Pyrroloquinoline Quinone Biosynthetic Pathway.

PubMed

Wei, Qiaoe; Ran, Tingting; Ma, Chencui; He, Jianhua; Xu, Dongqing; Wang, Weiwu

2016-07-22

Pyrroloquinoline quinone (PQQ) has received considerable attention due to its numerous important physiological functions. PqqA is a precursor peptide of PQQ with two conserved residues: glutamate and tyrosine. After linkage of the Cγ of glutamate and Cϵ of tyrosine by PqqE, these two residues are hypothesized to be cleaved from PqqA by PqqF. The linked glutamate and tyrosine residues are then used to synthesize PQQ. Here, we demonstrated that the pqqF gene is essential for PQQ biosynthesis as deletion of it eliminated the inhibition of prodigiosin production by glucose. We further determined the crystal structure of PqqF, which has a closed clamshell-like shape. The PqqF consists of two halves composed of an N- and a C-terminal lobe. The PqqF-N and PqqF-C lobes form a chamber with the volume of the cavity of ∼9400 Å(3) The PqqF structure conforms to the general structure of inverzincins. Compared with the most thoroughly characterized inverzincin insulin-degrading enzyme, the size of PqqF chamber is markedly smaller, which may define the specificity for its substrate PqqA. Furthermore, the 14-amino acid-residue-long tag formed by the N-terminal tag from expression vector precisely protrudes into the counterpart active site; this N-terminal tag occupies the active site and stabilizes the closed, inactive conformation. His-48, His-52, Glu-129 and His-14 from the N-terminal tag coordinate with the zinc ion. Glu-51 acts as a base catalyst. The observed histidine residue-mediated inhibition may be applicable for the design of a peptide for the inhibition of M16 metalloproteases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Crystal Structure and Function of PqqF Protein in the Pyrroloquinoline Quinone Biosynthetic Pathway*

PubMed Central

Wei, Qiaoe; Ran, Tingting; Ma, Chencui; He, Jianhua; Xu, Dongqing; Wang, Weiwu

2016-01-01

Pyrroloquinoline quinone (PQQ) has received considerable attention due to its numerous important physiological functions. PqqA is a precursor peptide of PQQ with two conserved residues: glutamate and tyrosine. After linkage of the Cγ of glutamate and Cϵ of tyrosine by PqqE, these two residues are hypothesized to be cleaved from PqqA by PqqF. The linked glutamate and tyrosine residues are then used to synthesize PQQ. Here, we demonstrated that the pqqF gene is essential for PQQ biosynthesis as deletion of it eliminated the inhibition of prodigiosin production by glucose. We further determined the crystal structure of PqqF, which has a closed clamshell-like shape. The PqqF consists of two halves composed of an N- and a C-terminal lobe. The PqqF-N and PqqF-C lobes form a chamber with the volume of the cavity of ∼9400 Å3. The PqqF structure conforms to the general structure of inverzincins. Compared with the most thoroughly characterized inverzincin insulin-degrading enzyme, the size of PqqF chamber is markedly smaller, which may define the specificity for its substrate PqqA. Furthermore, the 14-amino acid-residue-long tag formed by the N-terminal tag from expression vector precisely protrudes into the counterpart active site; this N-terminal tag occupies the active site and stabilizes the closed, inactive conformation. His-48, His-52, Glu-129 and His-14 from the N-terminal tag coordinate with the zinc ion. Glu-51 acts as a base catalyst. The observed histidine residue-mediated inhibition may be applicable for the design of a peptide for the inhibition of M16 metalloproteases. PMID:27231346

A peptide sequence on carcinoembryonic antigen binds to a 80kD protein on Kupffer cells.

PubMed

Thomas, P; Petrick, A T; Toth, C A; Fox, E S; Elting, J J; Steele, G

1992-10-30

Clearance of carcinoembryonic antigen (CEA) from the circulation is by binding to Kupffer cells in the liver. We have shown that CEA binding to Kupffer cells occurs via a peptide sequence YPELPK representing amino acids 107-112 of the CEA sequence. This peptide sequence is located in the region between the N-terminal and the first immunoglobulin like loop domain. Using native CEA and peptides containing this sequence complexed with a heterobifunctional crosslinking agent and ligand blotting with biotinylated CEA and NCA we have shown binding to an 80kD protein on the Kupffer cell surface. This binding protein may be important in the development of hepatic metastases.

Comparison of expression and enzymatic properties of Aspergillus oryzae lysine aminopeptidases ApsA and ApsB.

PubMed

Marui, Junichiro; Matsushita-Morita, Mayumi; Tada, Sawaki; Hattori, Ryota; Suzuki, Satoshi; Amano, Hitoshi; Ishida, Hiroki; Yamagata, Youhei; Takeuchi, Michio; Kusumoto, Ken-Ichi

2012-08-01

The apsA and apsB genes encoding family M1 aminopeptidases were identified in the industrial fungus Aspergillus oryzae. The apsB was transcriptionally up-regulated up to 2.5-fold in response to the deprivation of nitrogen or carbon sources in growth media, while up-regulation of apsA was less significant. The encoded proteins were bacterially expressed and purified to characterize their enzymatic properties. ApsA and ApsB were optimally active at pH 7.0 and 35 °C and stable at pH ranges of 6-10 and 4-10, respectively, up to 40 °C. The enzymes were inhibited by bestatin and EDTA, as has been reported for family M1 aminopeptidases that characteristically contain a zinc-binding catalytic motif. Both enzymes preferentially liberated N-terminal lysine, which is an essential amino acid and an important additive to animal feed. Enzymes that efficiently release N-terminal lysine from peptides could be useful for food and forage industries. Examination of the reactivity toward peptide substrate of varying length revealed that ApsB exhibited broader substrate specificity than ApsA although the reactivity of ApsB decreased as the length of peptide substrate decreased.

Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris.

PubMed

Huang, Yang-Bin; Li, Jiang; Gao, Xin; Sun, Jiu-Ru; Lu, Yi; Feng, Tao; Fei, Jian; Cui, Da-Fu; Xia, Qi-Chang; Ren, Jun; Zhang, You-Shang

2006-08-01

In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Heme impairs the ball-and-chain inactivation of potassium channels.

PubMed

Sahoo, Nirakar; Goradia, Nishit; Ohlenschläger, Oliver; Schönherr, Roland; Friedrich, Manfred; Plass, Winfried; Kappl, Reinhard; Hoshi, Toshinori; Heinemann, Stefan H

2013-10-15

Fine-tuned regulation of K(+) channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K(+) channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K(+) channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K(+) current with an apparent EC50 value of ∼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore.

The C-terminal region of Trypanosoma cruzi MASPs is antigenic and secreted via exovesicles

PubMed Central

De Pablos, Luis Miguel; Díaz Lozano, Isabel María; Jercic, Maria Isabel; Quinzada, Markela; Giménez, Maria José; Calabuig, Eva; Espino, Ana Margarita; Schijman, Alejandro Gabriel; Zulantay, Inés; Apt, Werner; Osuna, Antonio

2016-01-01

Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected and emerging tropical disease, endemic to South America and present in non-endemic regions due to human migration. The MASP multigene family is specific to T. cruzi, accounting for 6% of the parasite’s genome and plays a key role in immune evasion. A common feature of MASPs is the presence of two conserved regions: an N-terminal region codifying for signal peptide and a C-terminal (C-term) region, which potentially acts as GPI-addition signal peptide. Our aim was the analysis of the presence of an immune response against the MASP C-term region. We found that this region is highly conserved, released via exovesicles (EVs) and has an associated immune response as revealed by epitope affinity mapping, IFA and inhibition of the complement lysis assays. We also demonstrate the presence of a fast IgM response in Balb/c mice infected with T. cruzi. Our results reveal the presence of non-canonical secreted peptides in EVs, which can subsequently be exposed to the immune system with a potential role in evading immune system targets in the parasite. PMID:27270330

The leader peptide of mutacin 1140 has distinct structural components compared to related class I lantibiotics.

PubMed

Escano, Jerome; Stauffer, Byron; Brennan, Jacob; Bullock, Monica; Smith, Leif

2014-12-01

Lantibiotics are ribosomally synthesized peptide antibiotics composed of an N-terminal leader peptide that promotes the core peptide's interaction with the post translational modification (PTM) enzymes. Following PTMs, mutacin 1140 is transported out of the cell and the leader peptide is cleaved to yield the antibacterial peptide. Mutacin 1140 leader peptide is structurally unique compared to other class I lantibiotic leader peptides. Herein, we further our understanding of the structural differences of mutacin 1140 leader peptide with regard to other class I leader peptides. We have determined that the length of the leader peptide is important for the biosynthesis of mutacin 1140. We have also determined that mutacin 1140 leader peptide contains a novel four amino acid motif compared to related lantibiotics. PTM enzyme recognition of the leader peptide appears to be evolutionarily distinct from related class I lantibiotics. Our study on mutacin 1140 leader peptide provides a basis for future studies aimed at understanding its interaction with the PTM enzymes. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Alarin but not its alternative-splicing form, GALP (Galanin-like peptide) has antimicrobial activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wada, Akihiro, E-mail: a-wada@nagasaki-u.ac.jp; Wong, Pooi-Fong; Hojo, Hironobu

Highlights: • Alarin inhibits the growth of E. coli but not S. aureus. • Alarin’s potency is comparable to LL-37 in inhibiting the growth of E. coli. • Alarin can cause bacterial membrane blebbing. • Alalin does not induce hemolysis on erythrocytes. -- Abstract: Alarin is an alternative-splicing form of GALP (galanin-like peptide). It shares only 5 conserved amino acids at the N-terminal region with GALP which is involved in a diverse range of normal brain functions. This study seeks to investigate whether alarin has additional functions due to its differences from GALP. Here, we have shown using a radialmore » diffusion assay that alarin but not GALP inhibited the growth of Escherichia coli (strain ML-35). The conserved N-terminal region, however, remained essential for the antimicrobial activity of alarin as truncated peptides showed reduced killing effect. Moreover, alarin inhibited the growth of E. coli in a similar potency as human cathelicidin LL-37, a well-studied antimicrobial peptide. Electron microscopy further showed that alarin induced bacterial membrane blebbing but unlike LL-37, it did not cause hemolysis of erythrocytes. In addition, alarin is only active against the gram-negative bacteria, E. coli but not the gram-positive bacteria, Staphylococcus aureus. Thus, these data suggest that alarin has potentials as an antimicrobial and should be considered for the development in human therapeutics.« less

Big defensins, a diverse family of antimicrobial peptides that follows different patterns of expression in hemocytes of the oyster Crassostrea gigas.

PubMed

Rosa, Rafael D; Santini, Adrien; Fievet, Julie; Bulet, Philippe; Destoumieux-Garzón, Delphine; Bachère, Evelyne

2011-01-01

Big defensin is an antimicrobial peptide composed of a highly hydrophobic N-terminal region and a cationic C-terminal region containing six cysteine residues involved in three internal disulfide bridges. While big defensin sequences have been reported in various mollusk species, few studies have been devoted to their sequence diversity, gene organization and their expression in response to microbial infections. Using the high-throughput Digital Gene Expression approach, we have identified in Crassostrea gigas oysters several sequences coding for big defensins induced in response to a Vibrio infection. We showed that the oyster big defensin family is composed of three members (named Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3) that are encoded by distinct genomic sequences. All Cg-BigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain that resembles vertebrate β-defensins. Both domains are encoded by separate exons. We found that big defensins form a group predominantly present in mollusks and closer to vertebrate defensins than to invertebrate and fungi CSαβ-containing defensins. Moreover, we showed that Cg-BigDefs are expressed in oyster hemocytes only and follow different patterns of gene expression. While Cg-BigDef3 is non-regulated, both Cg-BigDef1 and Cg-BigDef2 transcripts are strongly induced in response to bacterial challenge. Induction was dependent on pathogen associated molecular patterns but not damage-dependent. The inducibility of Cg-BigDef1 was confirmed by HPLC and mass spectrometry, since ions with a molecular mass compatible with mature Cg-BigDef1 (10.7 kDa) were present in immune-challenged oysters only. From our biochemical data, native Cg-BigDef1 would result from the elimination of a prepropeptide sequence and the cyclization of the resulting N-terminal glutamine residue into a pyroglutamic acid. We provide here the first report showing that big defensins form a family of antimicrobial peptides diverse not only in terms of sequences but also in terms of genomic organization and regulation of gene expression.

Big Defensins, a Diverse Family of Antimicrobial Peptides That Follows Different Patterns of Expression in Hemocytes of the Oyster Crassostrea gigas

PubMed Central

Rosa, Rafael D.; Santini, Adrien; Fievet, Julie; Bulet, Philippe; Destoumieux-Garzón, Delphine; Bachère, Evelyne

2011-01-01

Background Big defensin is an antimicrobial peptide composed of a highly hydrophobic N-terminal region and a cationic C-terminal region containing six cysteine residues involved in three internal disulfide bridges. While big defensin sequences have been reported in various mollusk species, few studies have been devoted to their sequence diversity, gene organization and their expression in response to microbial infections. Findings Using the high-throughput Digital Gene Expression approach, we have identified in Crassostrea gigas oysters several sequences coding for big defensins induced in response to a Vibrio infection. We showed that the oyster big defensin family is composed of three members (named Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3) that are encoded by distinct genomic sequences. All Cg-BigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain that resembles vertebrate β-defensins. Both domains are encoded by separate exons. We found that big defensins form a group predominantly present in mollusks and closer to vertebrate defensins than to invertebrate and fungi CSαβ-containing defensins. Moreover, we showed that Cg-BigDefs are expressed in oyster hemocytes only and follow different patterns of gene expression. While Cg-BigDef3 is non-regulated, both Cg-BigDef1 and Cg-BigDef2 transcripts are strongly induced in response to bacterial challenge. Induction was dependent on pathogen associated molecular patterns but not damage-dependent. The inducibility of Cg-BigDef1 was confirmed by HPLC and mass spectrometry, since ions with a molecular mass compatible with mature Cg-BigDef1 (10.7 kDa) were present in immune-challenged oysters only. From our biochemical data, native Cg-BigDef1 would result from the elimination of a prepropeptide sequence and the cyclization of the resulting N-terminal glutamine residue into a pyroglutamic acid. Conclusions We provide here the first report showing that big defensins form a family of antimicrobial peptides diverse not only in terms of sequences but also in terms of genomic organization and regulation of gene expression. PMID:21980497

Binding of transducin and transducin-derived peptides to rhodopsin studies by attenuated total reflection-Fourier transform infrared difference spectroscopy.

PubMed Central

Fahmy, K

1998-01-01

Fourier transform infrared difference spectroscopy combined with the attenuated total reflection technique allows the monitoring of the association of transducin with bovine photoreceptor membranes in the dark. Illumination causes infrared absorption changes linked to formation of the light-activated rhodopsin-transducin complex. In addition to the spectral changes normally associated with meta II formation, prominent absorption increases occur at 1735 cm-1, 1640 cm-1, 1550 cm-1, and 1517 cm-1. The D2O sensitivity of the broad carbonyl stretching band around 1735 cm-1 indicates that a carboxylic acid group becomes protonated upon formation of the activated complex. Reconstitution of rhodopsin into phosphatidylcholine vesicles has little influence on the spectral properties of the rhodopsin-transducin complex, whereas pH affects the intensity of the carbonyl stretching band. AC-terminal peptide comprising amino acids 340-350 of the transducin alpha-subunit reproduces the frequencies and isotope sensitivities of several of the transducin-induced bands between 1500 and 1800 cm-1, whereas an N-terminal peptide (aa 8-23) does not. Therefore, the transducin-induced absorption changes can be ascribed mainly to an interaction between the transducin-alpha C-terminus and rhodopsin. The 1735 cm-1 vibration is also seen in the complex with C-terminal peptides devoid of free carboxylic acid groups, indicating that the corresponding carbonyl group is located on rhodopsin. PMID:9726932

Analysis of the two-peptide bacteriocins lactococcin G and enterocin 1071 by site-directed mutagenesis.

PubMed

Oppegård, Camilla; Fimland, Gunnar; Thorbaek, Lisbeth; Nissen-Meyer, Jon

2007-05-01

The two peptides (Lcn-alpha and Lcn-beta) of the two-peptide bacteriocin lactococcin G (Lcn) were changed by stepwise site-directed mutagenesis into the corresponding peptides (Ent-alpha and Ent-beta) of the two-peptide bacteriocin enterocin 1071 (Ent), and the potencies and specificities of the various hybrid constructs were determined. Both Lcn and, to a lesser extent, Ent were active against all the tested lactococcal strains, but only Ent was active against the tested enterococcal strains. The two bacteriocins thus differed in their relative potencies to various target cells, despite their sequence similarities. The hybrid combination Lcn-alpha+Ent-beta had low potency against all strains tested, indicating that these two peptides do not interact optimally. The reciprocal hybrid combination (i.e., Ent-alpha+Lcn-beta), in contrast, was highly potent, indicating that these two peptides may form a functional antimicrobial unit. In fact, this hybrid combination (Ent-alpha+Lcn-beta) was more potent against lactococcal strains than wild-type Ent was (i.e., Ent-alpha+Ent-beta), but it was inactive against enterococcal strains (in contrast to Ent but similar to Lcn). The observation that Ent-alpha is more active against lactococci in combination with Lcn-beta and more active against enterococci in combination with Ent-beta suggests that the beta peptide is an important determinant of target cell specificity. Especially the N-terminal residues of the beta peptide seem to be important for specificity, since Ent-alpha combined with an Ent-beta variant with Ent-to-Lcn mutations at positions 1 to 4, 7, 9, and 10 was >150-fold less active against enterococcal strains but one to four times more active against lactococcal strains than Ent-alpha+Ent-beta. Moreover, Ent-to-Lcn single-residue mutations in the region spanning residues 1 to 7 in Ent-beta had a more detrimental effect on the activity against enterococci than on that against lactococcal strains. Of the single-residue mutations made in the N-terminal region of the alpha peptide, the Ent-to-Lcn mutations N8Q and P12R in Ent-alpha influenced specificity, as follows: the N8Q mutation had no effect on activity against tested enterococcal strains but increased the activity 2- to 4-fold against the tested lactococcal strains, and the P12R mutation reduced the activity >150-fold and only approximately 2-fold against enterococcal and lactococcal strains, respectively. Changing residues in the C-terminal half/part of the Lcn peptides (residues 20 to 39 and 25 to 35 in Lcn-alpha and Lcn-beta, respectively) to those found in the corresponding Ent peptides did not have a marked effect on the activity, but there was an approximately 10-fold or greater reduction in the activity upon also introducing Lcn-to-Ent mutations in the mid-region (residues 8 to 19 and 9 to 24 in Lcn-alpha and Lcn-beta, respectively). Interestingly, the Lcn-to-Ent F19L+G20A mutation in an Lcn-Ent-beta hybrid peptide was more detrimental when the altered peptide was combined with Lcn-alpha (>10-fold reduction) than when it was combined with Ent-alpha ( approximately 2-fold reduction), suggesting that residues 19 and 20 (which are part of a GXXXG motif) in the beta peptide may be involved in a specific interaction with the cognate alpha peptide. It is also noteworthy that the K2P and A7P mutations in Lcn-beta reduced the activity only approximately 2-fold, suggesting that the first seven residues in the beta peptides do not form an alpha-helix.

Use of N-terminal prohormone brain natriuretic peptide assay for etiologic diagnosis of acute dyspnea in elderly patients.

PubMed

Berdagué, Philippe; Caffin, Pierre-Yves; Barazer, Isabelle; Vergnes, Christine; Sedighian, Shahin; Letrillard, Sébastien; Pilossof, Romain; Goutorbe, Frédéric; Piot, Christophe; Reny, Jean-Luc

2006-03-01

B-type peptide assay (brain natriuretic peptide [BNP] and N-terminal prohormone brain natriuretic peptide [NT-proBNP]) is useful for the diagnosis of heart failure (HF), but few data are available on the use of these markers in elderly subjects. The aim of this study was to evaluate NT-proBNP assay for the diagnosis of acute left HF in patients older than 70 years hospitalized for acute dyspnea. We prospectively enrolled 256 elderly patients with acute dyspnea. They were categorized by 2 cardiologists unaware of NT-proBNP values into a cardiac dyspnea subgroup (left HF) and a noncardiac dyspnea subgroup (all other causes). Mean age was 81 +/- 7 years, and 52% of the patients were women. The diagnoses made in the emergency setting were incorrect or uncertain in 45% of cases. The median NT-proBNP value was higher (P < .0001) in patients with cardiac dyspnea (n = 142; 7906 pg/mL) than in patients with noncardiac dyspnea (n = 112; 1066 pg/mL). The area under the receiver operating characteristic curve was 0.86 (95% CI 0.81-0.91). At a cutoff of 2000 pg/mL, NT-proBNP had a sensitivity of 86%, a specificity of 71%, and an overall accuracy of 80% for cardiac dyspnea. The use of 2 cutoffs (< 1200 and > 4500 pg/mL) resulted in an 8% error rate and a gray area englobing 32% of values. NT-proBNP appears to be a sensitive and specific means of distinguishing pulmonary from cardiac causes of dyspnea in elderly patients. An optimal diagnostic strategy requires the use of 2 cutoffs and further investigations of patients with values in the gray area.

Hydrolysis by somatic angiotensin-I converting enzyme of basic dipeptides from a cholecystokinin/gastrin and a LH-RH peptide extended at the C-terminus with gly-Arg/Lys-arg, but not from diarginyl insulin.

PubMed

Isaac, R E; Michaud, A; Keen, J N; Williams, T A; Coates, D; Wetsel, W C; Corvol, P

1999-06-01

Endoproteolytic cleavage of protein prohormones often generates intermediates extended at the C-terminus by Arg-Arg or Lys-Arg, the removal of which by a carboxypeptidase (CPE) is normally an important step in the maturation of many peptide hormones. Recent studies in mice that lack CP activity indicate the existence of alternative tissue or plasma enzymes capable of removing C-terminal basic residues from prohormone intermediates. Using inhibitors of angiotensin I-converting enzyme (ACE) and CP, we show that both these enzymes in mouse serum can remove the basic amino acids from the C-terminus of CCK5-GRR and LH-RH-GKR, but only CP is responsible for converting diarginyl insulin to insulin. ACE activity removes C-terminal dipeptides to generate the Gly-extended peptides, whereas CP hydrolysis gives rise to CCK5-GR and LH-RH-GK, both of which are susceptible to the dipeptidyl carboxypeptidase activity of ACE. Somatic ACE has two similar protein domains (the N-domain and the C-domain), each with an active site that can display different substrate specificities. CCK5-GRR is a high-affinity substrate for both the N-domain and C-domain active sites of human sACE (Km of 9.4 microm and 9.0 microm, respectively) with the N-domain showing greater efficiency (kcat : Km ratio of 2.6 in favour of the N-domain). We conclude that somatic forms of ACE should be considered as alternatives to CPs for the removal of basic residues from some Arg/Lys-extended peptides.

Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage.

PubMed Central

Kwong, M. Y.; Harris, R. J.

1994-01-01

Under favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 degrees C will specifically cleave on the C-terminal side of succinimides without cleavage at Asn-Gly bonds; yields are typically approximately 50%. N-terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site. PMID:8142891

Isolation and Properties of Floral Defensins from Ornamental Tobacco and Petunia1

PubMed Central

Lay, Fung T.; Brugliera, Filippa; Anderson, Marilyn A.

2003-01-01

The flowers of the solanaceous plants ornamental tobacco (Nicotiana alata) and petunia (Petunia hybrida) produce high levels of defensins during the early stages of development. In contrast to the well-described seed defensins, these floral defensins are produced as precursors with C-terminal prodomains of 27 to 33 amino acids in addition to a typical secretion signal peptide and central defensin domain of 47 or 49 amino acids. Defensins isolated from N. alata and petunia flowers lack the C-terminal domain, suggesting that it is removed during or after transit through the secretory pathway. Immunogold electron microscopy has been used to demonstrate that the N. alata defensin is deposited in the vacuole. In addition to the eight canonical cysteine residues that define the plant defensin family, the two petunia defensins have an extra pair of cysteines that form a fifth disulfide bond and hence define a new subclass of this family of proteins. Expression of the N. alata defensin NaD1 is predominantly flower specific and is most active during the early stages of flower development. NaD1 transcripts accumulate in the outermost cell layers of petals, sepals, anthers, and styles, consistent with a role in protection of the reproductive organs against potential pathogens. The floral defensins inhibit the growth of Botrytis cinerea and Fusarium oxysporum in vitro, providing further support for a role in protection of floral tissues against pathogen invasion. PMID:12644678

The expanded amelogenin polyproline region preferentially binds to apatite versus carbonate and promotes apatite crystal elongation

PubMed Central

Gopinathan, Gokul; Jin, Tianquan; Liu, Min; Li, Steve; Atsawasuwan, Phimon; Galang, Maria-Therese; Allen, Michael; Luan, Xianghong; Diekwisch, Thomas G. H.

2014-01-01

The transition from invertebrate calcium carbonate-based calcite and aragonite exo- and endoskeletons to the calcium phosphate-based vertebrate backbones and jaws composed of microscopic hydroxyapatite crystals is one of the great revolutions in the evolution of terrestrial organisms. To identify potential factors that might have played a role in such a transition, three key domains of the vertebrate tooth enamel protein amelogenin were probed for calcium mineral/protein interactions and their ability to promote calcium phosphate and calcium carbonate crystal growth. Under calcium phosphate crystal growth conditions, only the carboxy-terminus augmented polyproline repeat peptide, but not the N-terminal peptide nor the polyproline repeat peptide alone, promoted the formation of thin and parallel crystallites resembling those of bone and initial enamel. In contrast, under calcium carbonate crystal growth conditions, all three amelogenin-derived polypeptides caused calcium carbonate to form fused crystalline conglomerates. When examined for long-term crystal growth, polyproline repeat peptides of increasing length promoted the growth of shorter calcium carbonate crystals with broader basis, contrary to the positive correlation between polyproline repeat element length and apatite mineralization published earlier. To determine whether the positive correlation between polyproline repeat element length and apatite crystal growth versus the inverse correlation between polyproline repeat length and calcium carbonate crystal growth were related to the binding affinity of the polyproline domain to either apatite or carbonate, a parallel series of calcium carbonate and calcium phosphate/apatite protein binding studies was conducted. These studies demonstrated a remarkable binding affinity between the augmented amelogenin polyproline repeat region and calcium phosphates, and almost no binding to calcium carbonates. In contrast, the amelogenin N-terminus bound to both carbonate and apatite, but preferentially to calcium carbonate. Together, these studies highlight the specific binding affinity of the augmented amelogenin polyproline repeat region to calcium phosphates versus calcium carbonate, and its unique role in the growth of thin apatite crystals as they occur in vertebrate biominerals. Our data suggest that the rise of apatite-based biominerals in vertebrates might have been facilitated by a rapid evolution of specialized polyproline repeat proteins flanked by a charged domain, resulting in apatite crystals with reduced width, increased length, and tailored biomechanical properties. PMID:25426079

Biochemistry of terminal deoxynucleotidyltransferase. Identification and unity of ribo- and deoxyribonucleoside triphosphate binding site in terminal deoxynucleotidyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pandey, V.N.; Modak, M.J.

Terminal deoxynucleotidyltransferase is the only DNA polymerase that is strongly inhibited in the presence of ATP. We have labeled calf terminal deoxynucleotidyltransferase with (/sup 32/P)ATP in order to identify its binding site in terminal deoxynucleotidyltransferase. The specificity of ATP cross-linking to terminal deoxynucleotidyltransferase is shown by the competitive inhibition of the overall cross-linking reaction by deoxynucleoside triphosphates, as well as the ATP analogs Ap4A and Ap5A. Tryptic peptide mapping of (/sup 32/P)ATP-labeled enzyme revealed a peptide fraction that contained the majority of cross-linked ATP. The properties, chromatographic characteristics, amino acid composition, and sequence analysis of this peptide fraction were identicalmore » with those found associated with dTTP cross-linked terminal deoxynucleotidyl-transferase peptide. The involvement of the same 2 cysteine residues in the crosslinking of both nucleotides further confirmed the unity of the ATP and dTTP binding domain that contains residues 224-237 in the primary amino acid sequence of calf terminal deoxynucleotidyltransferase.« less

Inhibition of Vascular c-Jun N-Terminal Kinase 2 Improves Obesity-Induced Endothelial Dysfunction After Roux-en-Y Gastric Bypass.

PubMed

Doytcheva, Petia; Bächler, Thomas; Tarasco, Erika; Marzolla, Vincenzo; Engeli, Michael; Pellegrini, Giovanni; Stivala, Simona; Rohrer, Lucia; Tona, Francesco; Camici, Giovanni G; Vanhoutte, Paul M; Matter, Christian M; Lutz, Thomas A; Lüscher, Thomas F; Osto, Elena

2017-11-14

Roux-en-Y gastric bypass (RYGB) reduces obesity-associated comorbidities and cardiovascular mortality. RYGB improves endothelial dysfunction, reducing c-Jun N-terminal kinase (JNK) vascular phosphorylation. JNK activation links obesity with insulin resistance and endothelial dysfunction. Herein, we examined whether JNK1 or JNK2 mediates obesity-induced endothelial dysfunction and if pharmacological JNK inhibition can mimic RYGB vascular benefits. After 7 weeks of a high-fat high-cholesterol diet, obese rats underwent RYGB or sham surgery; sham-operated ad libitum-fed rats received, for 8 days, either the control peptide D-TAT or the JNK peptide inhibitor D-JNKi-1 (20 mg/kg per day subcutaneous). JNK peptide inhibitor D-JNKi-1 treatment improved endothelial vasorelaxation in response to insulin and glucagon-like peptide-1, as observed after RYGB. Obesity increased aortic phosphorylation of JNK2, but not of JNK1. RYGB and JNK peptide inhibitor D-JNKi-1 treatment blunted aortic JNK2 phosphorylation via activation of glucagon-like peptide-1-mediated signaling. The inhibitory phosphorylation of insulin receptor substrate-1 was reduced, whereas the protein kinase B/endothelial NO synthase pathway was increased and oxidative stress was decreased, resulting in improved vascular NO bioavailability. Decreased aortic JNK2 phosphorylation after RYGB rapidly improves obesity-induced endothelial dysfunction. Pharmacological JNK inhibition mimics the endothelial protective effects of RYGB. These findings highlight the therapeutic potential of novel strategies targeting vascular JNK2 against the severe cardiovascular disease associated with obesity. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

High peripheral levels of h-FABP are associated with poor prognosis in end-stage heart failure patients with mechanical circulatory support.

PubMed

Cabiati, Manuela; Caselli, Chiara; Caruso, Raffaele; Prescimone, Tommaso; Verde, Alessandro; Botta, Luca; Parodi, Oberdan; Del Ry, Silvia; Giannessi, Daniela

2013-06-01

To associate the time-course of h-FABP and N-terminal pro B-type natriuretic peptide (NT-proBNP)after left ventricular assist device (LVAD) implantation to outcome in end-stage heart failure patients. Patients (n = 14, NYHA class III/IV; left ventricular ejection fraction <25% were enrolled; ten survived up to 1 month after LVAD (survivors) and four died of multiorgan failure within 2 weeks (nonsurvivors). Blood samples were obtained at admission; at 4, 24 and 72 h; and at 1 and 4 weeks after LVAD. h-FABP significantly increases after surgery, decreasing since 72 h in all patients. At 72 h all survivor patients present h-FABP lower than the median value. N-terminal pro B-type natriuretic peptide is not associated with patient outcome at any time. High h-FABP levels, indicating the presence of more severe myocardial damage, are associated with a poor prognosis in patients with LVAD, suggesting that an early cardiac injury marker could improve the prediction of clinical outcome.

Morintides: cargo-free chitin-binding peptides from Moringa oleifera.

PubMed

Kini, Shruthi G; Wong, Ka H; Tan, Wei Liang; Xiao, Tianshu; Tam, James P

2017-03-31

Hevein-like peptides are a family of cysteine-rich and chitin-binding peptides consisting of 29-45 amino acids. Their chitin-binding property is essential for plant defense against fungi. Based on the number of cysteine residues in their sequences, they are divided into three sub-families: 6C-, 8C- and 10C-hevein-like peptides. All three subfamilies contain a three-domain precursor comprising a signal peptide, a mature hevein-like peptide and a C-terminal domain comprising a hinge region with protein cargo in 8C- and 10C-hevein-like peptides. Here we report the isolation and characterization of two novel 8C-hevein-like peptides, designated morintides (mO1 and mO2), from the drumstick tree Moringa oleifera, a drought-resistant tree belonging to the Moringaceae family. Proteomic analysis revealed that morintides comprise 44 amino acid residues and are rich in cysteine, glycine and hydrophilic amino acid residues such as asparagine and glutamine. Morintides are resistant to thermal and enzymatic degradation, able to bind to chitin and inhibit the growth of phyto-pathogenic fungi. Transcriptomic analysis showed that they contain a three-domain precursor comprising an endoplasmic reticulum (ER) signal sequence, a mature peptide domain and a C-terminal domain. A striking feature distinguishing morintides from other 8C-hevein-like peptides is a short and protein-cargo-free C-terminal domain. Previously, a similar protein-cargo-free C-terminal domain has been observed only in ginkgotides, the 8C-hevein-like peptides from a gymnosperm Ginkgo biloba. Thus, morintides, with a cargo-free C-terminal domain, are a stand-alone class of 8C-hevein-like peptides from angiosperms. Our results expand the existing library of hevein-like peptides and shed light on molecular diversity within the hevein-like peptide family. Our work also sheds light on the anti-fungal activity and stability of 8C-hevein-like peptides.

Carrier protein influences immunodominance of a known epitope: implication in peptide vaccine design.

PubMed

Ghosh, Moumita; Solanki, Ashish K; Roy, Koushik; Dhoke, Reema R; Ashish; Roy, Syamal

2013-09-23

We investigated how the processing of a given antigen by antigen presenting cells (APC) is dictated by the conformation of the antigen and how this governs the immunodominance hierarchy. To address the question, a known immunodominant sequence of bacteriophage lambda repressor N-terminal sequence 12-26 [λR(12-26)] was engineered at the N and C termini of a heterologous leishmanial protein, Kinetoplastid membrane protein-11 (KMP-11); the resulting proteins were defined as N-KMP-11 and C-KMP-11 respectively. The presence of λR(12-26) in N-KMP-11 and C-KMP-11 was established by western blot analysis with antibody to λR(12-26) peptide. N-KMP-11 but not C-KMP-11 could stimulate the anti λR(12-26) T-cell clonal population very efficiently in the presence of APCs. Priming of BALB/c mice with N-KMP-11 or C-KMP-11 generated similar levels of anti-KMP-11 IgG, but anti-λR(12-26) specific IgG was observed only upon priming with N-KMP-11. Interestingly, uptake of both N-KMP-11 and C-KMP-11 by APCs was similar but catabolism of N-KMP-11 but not C-KMP-11 was biphasic and fast at the initial time point. Kratky plots of small angle X-ray scattering showed that while N-KMP-11 adopts flexible Gaussian type of topology, C-KMP-11 prefers Globular nature. To show that KMP-11 is not unique as a carrier protein, an epitope (SPITBTNLBTMBK) of Plasmodium yoelii (PY) apical membrane protein 1[AMA-1 (136-148)], is placed at the C and N terminals of a dominant T-cell epitope of ovalbumin protein OVA(323-339) and the resulting peptides are defined as PY-OVA and OVA-PY respectively. Interestingly, only OVA-PY could stimulate anti-OVA T-cells and produce IgG response upon priming of BALB/c mice with it. Thus for rational design of peptide vaccine it is important to place the dominant epitope appropriately in the context of the carrier protein. Copyright © 2013 Elsevier Ltd. All rights reserved.

Autoantibodies in Serum of Systemic Scleroderma Patients: Peptide-Based Epitope Mapping Indicates Increased Binding to Cytoplasmic Domains of CXCR3.

PubMed

Recke, Andreas; Regensburger, Ann-Katrin; Weigold, Florian; Müller, Antje; Heidecke, Harald; Marschner, Gabriele; Hammers, Christoph M; Ludwig, Ralf J; Riemekasten, Gabriela

2018-01-01

Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera ( N  = 32, with positive reactivity with CXCR3) to healthy controls ( N  = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients ( N  = 31) from normal healthy controls ( N  = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc sera. Based upon our results, we could devise a novel ELISA concept that may be helpful for monitoring of SSc patients.

Preparation and properties of pure, full-length IclR protein of Escherichia coli. Use of time-of-flight mass spectrometry to investigate the problems encountered.

PubMed Central

Donald, L. J.; Chernushevich, I. V.; Zhou, J.; Verentchikov, A.; Poppe-Schriemer, N.; Hosfield, D. J.; Westmore, J. B.; Ens, W.; Duckworth, H. W.; Standing, K. G.

1996-01-01

IclR protein, the repressor of the aceBAK operon of Escherichia coli, has been examined by time-of-flight mass spectrometry, with ionization by matrix assisted laser desorption or by electrospray. The purified protein was found to have a smaller mass than that predicted from the base sequence of the cloned iclR gene. Additional measurements were made on mixtures of peptides derived from IclR by treatment with trypsin and cyanogen bromide. They showed that the amino acid sequence is that predicted from the gene sequence, except that the protein has suffered truncation by removal of the N-terminal eight or, in some cases, nine amino acid residues. The peptide bond whose hydrolysis would remove eight residues is a typical target for the E. coli protease OmpT. We find that, by taking precautions to minimize Omp T proteolysis, or by eliminating it through mutation of the host strain, we can isolate full-length IclR protein (lacking only the N-terminal methionine residue). Full-length IclR is a much better DNA-binding protein than the truncated versions: it binds the aceBAK operator sequence 44-fold more tightly, presumably because of additional contacts that the N-terminal residues make with the DNA. Our experience thus demonstrates the advantages of using mass spectrometry to characterize newly purified proteins produced from cloned genes, especially where proteolysis or other covalent modification is a concern. This technique gives mass spectra from complex peptide mixtures that can be analyzed completely, without any fractionation of the mixtures, by reference to the amino acid sequence inferred from the base sequence of the cloned gene. PMID:8844850

Characterization of the tachykinin neurokinin-2 receptor in the human urinary bladder by means of selective receptor antagonists and peptidase inhibitors.

PubMed

Giuliani, S; Patacchini, R; Barbanti, G; Turini, D; Rovero, P; Quartara, L; Giachetti, A; Maggi, C A

1993-11-01

The tachykinin (NK2) receptor-mediating contraction of the human isolated bladder to NKA was investigated by studying the affinities of eight structurally different receptor-selective antagonists (linear peptides, cyclic peptides and pseudopeptides, nonpeptide NK2 receptor antagonists). The affinities of the antagonists were compared to those measured for the same ligands at the NK2 receptors previously characterized in the rabbit pulmonary artery and hamster trachea. In the presence of a cocktail of peptidase inhibitors (bestatin captopril and thiorphan, 1 microM each) no significant correlation was found between pA2 values measured in the human bladder vs. those measured in the other two NK2 receptor-bearing preparation. In the presence of the aminopeptidase inhibitor amastatin, however, pA2 values of linear antagonists bearing an N-terminal Asp residue MEN 10,207 and MEN 10,376 were significantly enhanced and these pA2 values were used for analysis; a significant correlation was found between pA2 values measured in the human urinary bladder and rabbit pulmonary artery. The pseudopeptide analog of NKA (4-10), MDL 28,564 which also bears a N-terminal Asp residue behaved as an agonist and its action was enhanced by amastatin. We conclude that the NK2 receptor-mediating contraction of the human urinary bladder smooth muscle is similar to that previously characterized in the rabbit pulmonary artery (NK2A receptor category); in the human bladder smooth muscle an amastatin-sensitive peptidase (possibly aminopeptidase A) limits biological activity of linear peptide derivatives of NKA bearing a N-terminal Asp residue.

Interaction of Munc18c and syntaxin4 facilitates invadopodium formation and extracellular matrix invasion of tumor cells.

PubMed

Brasher, Megan I; Martynowicz, David M; Grafinger, Olivia R; Hucik, Andrea; Shanks-Skinner, Emma; Uniacke, James; Coppolino, Marc G

2017-09-29

Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell-extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification and characterization of two arasin-like peptides in red swamp crayfish Procambarus clarkii.

PubMed

Chai, Lian-Qin; Li, Wan-Wan; Wang, Xian-Wei

2017-11-01

Antimicrobial peptides (AMPs) are small effectors in host defense by directly targeting microorganisms or by indirectly modulating immune responses. In the present study, two arasin like AMPs, named as Pc-arasin1 and Pc-arasin2, were identified in red swamp crayfish Procambarus clarkii with sequence similarity to the arasins found in Hyas araneus. Both Pc-arasins consisted of signal peptide, N-terminal proline-rich region and C-terminal region containing four conserved cysteine residues. The similarity of two Pc-arasins was 44.44%, and Pc-arasin2 contained several additional residues in the N-terminus. Multiple alignment of arasin family suggested the conservation of the C-terminus and the variation of the N-terminus of Pc-arasins. Both AMPs were found hemocytes-specific, and the expression could be induced the challenge of bacteria, espeacially by the pathogenic bacterium Aeromonas hydrophila. Knockdown of each Pc-arasin expression by double strand RNA would suppress the host immunity against A. hydrophila, and the commercially synthetic Pc-arasins could rescue the knockdown consequence. Both synthetic peptide showed broad antimicrobial activity towards 3 Gram-positive bacterium and 3 Gram-negative bacterium, and the minimal inhibitory concentrations varied from 6.25 μM to 50 μM. These results presented new data about the sequence, expression and function of arasin family, and emphasized the role of this family in host immune response against bacterial pathogens. The characterization of Pc-arasins also provided potential of therapeutic agent development for disease control in aquaculture based on these two newly identified AMPs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Role of electrostatic interactions in binding of peptides and intrinsically disordered proteins to their folded targets. 1. NMR and MD characterization of the complex between the c-Crk N-SH3 domain and the peptide Sos.

PubMed

Xue, Yi; Yuwen, Tairan; Zhu, Fangqiang; Skrynnikov, Nikolai R

2014-10-21

Intrinsically disordered proteins (IDPs) often rely on electrostatic interactions to bind their structured targets. To obtain insight into the mechanism of formation of the electrostatic encounter complex, we investigated the binding of the peptide Sos (PPPVPPRRRR), which serves as a minimal model for an IDP, to the c-Crk N-terminal SH3 domain. Initially, we measured ¹⁵N relaxation rates at two magnetic field strengths and determined the binding shifts for the complex of Sos with wild-type SH3. We have also recorded a 3 μs molecular dynamics (MD) trajectory of this complex using the Amber ff99SB*-ILDN force field. The comparison of the experimental and simulated data shows that MD simulation consistently overestimates the strength of salt bridge interactions at the binding interface. The series of simulations using other advanced force fields also failed to produce any satisfactory results. To address this issue, we have devised an empirical correction to the Amber ff99SB*-ILDN force field whereby the Lennard-Jones equilibrium distance for the nitrogen-oxygen pair across the Arg-to-Asp and Arg-to-Glu salt bridges has been increased by 3%. Implementing this correction resulted in a good agreement between the simulations and the experiment. Adjusting the strength of salt bridge interactions removed a certain amount of strain contained in the original MD model, thus improving the binding of the hydrophobic N-terminal portion of the peptide. The arginine-rich C-terminal portion of the peptide, freed from the effect of the overstabilized salt bridges, was found to interconvert more rapidly between its multiple conformational states. The modified MD protocol has also been successfully used to simulate the entire binding process. In doing so, the peptide was initially placed high above the protein surface. It then arrived at the correct bound pose within ∼2 Å of the crystallographic coordinates. This simulation allowed us to analyze the details of the dynamic binding intermediate, i.e., the electrostatic encounter complex. However, an experimental characterization of this transient, weakly populated state remains out of reach. To overcome this problem, we designed the double mutant of c-Crk N-SH3 in which mutations Y186L and W169F abrogate tight Sos binding and shift the equilibrium toward the intermediate state resembling the electrostatic encounter complex. The results of the combined NMR and MD study of this engineered system will be reported in the next part of this paper.

Simultaneous prediction of binding free energy and specificity for PDZ domain-peptide interactions

NASA Astrophysics Data System (ADS)

Crivelli, Joseph J.; Lemmon, Gordon; Kaufmann, Kristian W.; Meiler, Jens

2013-12-01

Interactions between protein domains and linear peptides underlie many biological processes. Among these interactions, the recognition of C-terminal peptides by PDZ domains is one of the most ubiquitous. In this work, we present a mathematical model for PDZ domain-peptide interactions capable of predicting both affinity and specificity of binding based on X-ray crystal structures and comparative modeling with R osetta. We developed our mathematical model using a large phage display dataset describing binding specificity for a wild type PDZ domain and 91 single mutants, as well as binding affinity data for a wild type PDZ domain binding to 28 different peptides. Structural refinement was carried out through several R osetta protocols, the most accurate of which included flexible peptide docking and several iterations of side chain repacking and backbone minimization. Our findings emphasize the importance of backbone flexibility and the energetic contributions of side chain-side chain hydrogen bonds in accurately predicting interactions. We also determined that predicting PDZ domain-peptide interactions became increasingly challenging as the length of the peptide increased in the N-terminal direction. In the training dataset, predicted binding energies correlated with those derived through calorimetry and specificity switches introduced through single mutations at interface positions were recapitulated. In independent tests, our best performing protocol was capable of predicting dissociation constants well within one order of magnitude of the experimental values and specificity profiles at the level of accuracy of previous studies. To our knowledge, this approach represents the first integrated protocol for predicting both affinity and specificity for PDZ domain-peptide interactions.

CLE-like (CLEL) peptides control the pattern of root growth and lateral root development in Arabidopsis.

PubMed

Meng, Ling; Buchanan, Bob B; Feldman, Lewis J; Luan, Sheng

2012-01-31

CLE peptides, named for the CLV3/ESR-related peptide family, participate in intercellular-signaling pathways. Here we investigated members of the CLE-like (CLEL) gene family that encode peptide precursors recently designated as root growth factors [Matsuzaki Y et al. (2010) Science 329:1065-1067]. CLEL precursors share a similar domain structure with CLE precursors (i.e., they contain a putative N-terminal signal peptide and a C-terminal conserved 13-amino-acid CLEL motif with a variable middle portion). Our evidence shows that, unlike root growth factor, CLEL peptides are (i) unmodified and (ii) function in the regulation of the direction of root growth and lateral root development. Overexpression of several CLEL genes in Arabidopsis resulted in either long roots or long and wavy roots that also showed altered lateral root patterning. Exogenous application of unmodified synthetic 13-amino-acid peptides derived from two CLEL motifs resulted in similar phenotypic changes in roots of wild-type plants. In CLEL peptide-induced long roots, the root apical meristem (RAM) was enlarged and consisted of an increased number of cells, compared with wild-type root apical meristems. The wavy-root phenotype appeared to be independent of other responses of the roots to the environment (e.g., gravitropism, phototropism, and thigmotropism). Results also showed that the inhibition of lateral initiation by CLEL overexpression was not overcome by the application of auxin. These findings establish CLEL as a peptide family with previously unrecognized regulatory functions controlling the pattern of root growth and lateral root development in plants.

Inclusion of Zinc Oxide Nanoparticles into Virus-Like Peptide Nanocapsules Self-Assembled from Viral β-Annulus Peptide

PubMed Central

Fujita, Seiya; Matsuura, Kazunori

2014-01-01

A viral β-annulus peptide connected with a zinc oxide (ZnO)-binding sequence (HCVAHR) at its N-terminal was synthesized, and the inclusion behavior of quantum-sized ZnO nanoparticles into the peptide nanocapsules formed by self-assembly of the peptide in water was investigated. Dynamic light scattering (DLS) measurements showed that ZnO nanoparticles (approximately 10 nm) in the presence of the peptide (0.1 mM) formed assemblies with an average size of 48 ± 24 nm, whereas ZnO nanoparticles in the absence of the peptide formed large aggregates. Transmission electron microscopy (TEM) observations of the ZnO nanoparticles in the presence of the peptide revealed that ZnO nanoparticles were encapsulated into the peptide nanocapsules with a size of approximately 50 nm. Fluorescence spectra of a mixture of the peptide and ZnO nanoparticles suggested that the ZnO surface and the peptide interact. Template synthesis of ZnO nanoparticles with the peptide nanocapsules afforded larger nanoparticles (approximately 40 nm), which are not quantum-sized ZnO. PMID:28344248

Factors associated with the development of atrial fibrillation in patients with rheumatic mitral stenosis.

PubMed

Ozaydin, Mehmet; Turker, Yasin; Varol, Ercan; Alaca, Sule; Erdogan, Dogan; Yilmaz, Nigar; Dogan, Abdullah

2010-06-01

The aim of this study was to evaluate the factors associated with the development of atrial fibrillation (AF) in patients with rheumatic mitral stenosis (MS). A total of 146 consecutive patients with rheumatic MS were screened. They were accepted to be in AF group and sinus rhythm group according to their rhythm in the baseline ECG. After screening, 38 patients were excluded due to hyperthyroidism (n = 13), chronic obstructive pulmonary disease (n = 22), malignancy (n = 2) and rheumatoid arthritis (n = 1). Therefore, remaining 108 patients, 74 of whom in sinus rhythm (MS-SR) and 34 of whom in AF (MS-AF) constituted study population. Fourty age- and gender-matched patients constituted control group. Factors associated with development of AF in multivariable analysis included High sensitivity C reactive protein (P = 0.005; odds ratio, 3.44; 95% confidence interval, 1.44-8.22), N-terminal of brain natriuretic peptide precursor (P < 0.0001; odds ratio, 1.03; 95% confidence interval, 1.02-1.06) and left atrial diameter (P < 0.0001; odds ratio, 1.68; 95% confidence interval, 1.32-2.14). Present study suggests that High sensitivity C reactive protein, N-terminal of brain natriuretic peptide precursor and left atrial diameter are associated with development AF in patients with MS.

The Neurotrophin-Inducible Gene Vgf Regulates Hippocampal Function and Behavior Through a BDNF-Dependent Mechanism

PubMed Central

Bozdagi, Ozlem; Rich, Erin; Tronel, Sophie; Sadahiro, Masato; Patterson, Kamara; Shapiro, Matthew L.; Alberini, Cristina M.; Huntley, George W.; Salton, Stephen R. J.

2009-01-01

VGF is a neurotrophin-inducible, activity-regulated gene product that is expressed in CNS and PNS neurons, where it is processed into peptides and secreted. VGF synthesis is stimulated by BDNF, a critical regulator of hippocampal development and function, and two VGF C-terminal peptides increase synaptic activity in cultured hippocampal neurons. To assess VGF function in the hippocampus, we tested heterozygous and homozygous VGF knockout mice in two different learning tasks, assessed long-term potentiation (LTP) and depression (LTD) in hippocampal slices from VGF mutant mice, and investigated how VGF C-terminal peptides modulate synaptic plasticity. Treatment of rat hippocampal slices with the VGF-derived peptide TLQP62 resulted in transient potentiation through a mechanism that was selectively blocked by the BDNF scavenger TrkB-Fc, the Trk tyrosine kinase inhibitor K252a (100 nM), and by tPASTOP, an inhibitor of tissue plasminogen activator (tPA), an enzyme involved in pro-BDNF cleavage to BDNF, but was not blocked by the NMDA receptor antagonist APV, anti-p75NTR function-blocking antiserum, nor by prior tetanic stimulation. Although LTP was normal in slices from VGF knockout mice, LTD could not be induced, and VGF mutant mice were impaired in hippocampal-dependent spatial learning and contextual fear conditioning tasks. Our studies indicate that the VGF C-terminal peptide TLQP62 modulates hippocampal synaptic transmission through a BDNF-dependent mechanism, and that VGF deficiency in mice impacts synaptic plasticity and memory in addition to depressive behavior. PMID:18815270

Evolution of Rubisco activase gene in plants.

PubMed

Nagarajan, Ragupathi; Gill, Kulvinder S

2018-01-01

Rubisco activase of plants evolved in a stepwise manner without losing its function to adapt to the major evolutionary events including endosymbiosis and land colonization. Rubisco activase is an essential enzyme for photosynthesis, which removes inhibitory sugar phosphates from the active sites of Rubisco, a process necessary for Rubisco activation and carbon fixation. The gene probably evolved in cyanobacteria as different species differ for its presence. However, the gene is present in all other plant species. At least a single gene copy was maintained throughout plant evolution; but various genome and gene duplication events, which occurred during plant evolution, increased its copy number in some species. The exons and exon-intron junctions of present day higher plant's Rca, which is conserved in most species seem to have evolved in charophytes. A unique tandem duplication of Rca gene occurred in a common grass ancestor, and the two genes evolved differently for gene structure, sequence, and expression pattern. At the protein level, starting with a primitive form in cyanobacteria, RCA of chlorophytes evolved by integrating chloroplast transit peptide (cTP), and N-terminal domains to the ATPase, Rubisco recognition and C-terminal domains. The redox regulated C-terminal extension (CTE) and the associated alternate splicing mechanism, which splices the RCA-α and RCA-β isoforms were probably gained from another gene in charophytes, conserved in most species except the members of Solanaceae family.

Traceless β-mercaptan-assisted activation of valinyl benzimidazolinones in peptide ligations† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc04148a

PubMed Central

Wang, Yinglu; Han, Lin; Yuan, Ning; Wang, Hanxuan; Li, Hongxing; Liu, Jinrong; Chen, Huan

2018-01-01

Peptidyl thioesters or their surrogates with C-terminal β-branched hydrophobic amino acid residues usually exhibit poor reactivities in ligation reactions. Thus, activation using exogenous additives is required to ensure an acceptable reaction efficiency. Herein, we report a traceless ligation at Val-Xaa sites under mild thiol additive-free reaction conditions, whereby the introduction of β-mercaptan on the C-terminal valine residue effectively activates the otherwise unreactive N-acyl-benzimidazolinone (Nbz), and enables the use of a one-pot ligation–desulfurization strategy to generate the desired peptide products. The orthogonality between β-thiovaline-Nbz and a conventional alkyl thioester, as well as the convenient access to the former from readily available penicillamine, also allowed expedited assembly of the peptidic hormone β-LPH and hPTH analogues, based on a kinetically controlled one-pot three-segment ligation and desulfurization strategy. PMID:29675240

A Review on Bradykinin-Related Peptides Isolated from Amphibian Skin Secretion

PubMed Central

Xi, Xinping; Li, Bin; Chen, Tianbao; Kwok, Hang Fai

2015-01-01

Amphibian skin secretion has great potential for drug discovery and contributes hundreds of bioactive peptides including bradykinin-related peptides (BRPs). More than 50 BRPs have been reported in the last two decades arising from the skin secretion of amphibian species. They belong to the families Ascaphidae (1 species), Bombinatoridae (3 species), Hylidae (9 speices) and Ranidae (25 species). This paper presents the diversity of structural characteristics of BRPs with N-terminal, C-terminal extension and amino acid substitution. The further comparison of cDNA-encoded prepropeptides between the different species and families demonstrated that there are various forms of kininogen precursors to release BRPs and they constitute important evidence in amphibian evolution. The pharmacological activities of isolated BRPs exhibited unclear structure–function relationships, and therefore the scope for drug discovery and development is limited. However, their diversity shows new insights into biotechnological applications and, as a result, comprehensive and systematic studies of the physiological and pharmacological activities of BRPs from amphibian skin secretion are needed in the future. PMID:25793726

Structural Basis for the Effective Myostatin Inhibition of the Mouse Myostatin Prodomain-Derived Minimum Peptide.

PubMed

Asari, Tomo; Takayama, Kentaro; Nakamura, Akari; Shimada, Takahiro; Taguchi, Akihiro; Hayashi, Yoshio

2017-01-12

Myostatin inhibition is one of the promising strategies for treating muscle atrophic disorders, including muscular dystrophy. It is well-known that the myostatin prodomain derived from the myostatin precursor acts as an inhibitor of mature myostatin. In our previous study, myostatin inhibitory minimum peptide 1 (WRQNTRYSRIEAIKIQILSKLRL-amide) was discovered from the mouse myostatin prodomain. In the present study, alanine scanning of 1 demonstrated that the key amino acid residues for the effective inhibitory activity are rodent-specific Tyr and C-terminal aliphatic residues, in addition to N-terminal Trp residue. Subsequently, we designed five Pro-substituted peptides and examined the relationship between secondary structure and inhibitory activity. As a result, we found that Pro-substitutions of Ala or Gln residues around the center of 1 significantly decreased both α-helicity and inhibitory activity. These results suggested that an α-helical structure possessing hydrophobic faces formed around the C-terminus is important for inhibitory activity.

Plasmids encoding therapeutic agents

DOEpatents

Keener, William K [Idaho Falls, ID

2007-08-07

Plasmids encoding anti-HIV and anti-anthrax therapeutic agents are disclosed. Plasmid pWKK-500 encodes a fusion protein containing DP178 as a targeting moiety, the ricin A chain, an HIV protease cleavable linker, and a truncated ricin B chain. N-terminal extensions of the fusion protein include the maltose binding protein and a Factor Xa protease site. C-terminal extensions include a hydrophobic linker, an L domain motif peptide, a KDEL ER retention signal, another Factor Xa protease site, an out-of-frame buforin II coding sequence, the lacZ.alpha. peptide, and a polyhistidine tag. More than twenty derivatives of plasmid pWKK-500 are described. Plasmids pWKK-700 and pWKK-800 are similar to pWKK-500 wherein the DP178-encoding sequence is substituted by RANTES- and SDF-1-encoding sequences, respectively. Plasmid pWKK-900 is similar to pWKK-500 wherein the HIV protease cleavable linker is substituted by a lethal factor (LF) peptide-cleavable linker.

The Cu(II) affinity of the N-terminus of human copper transporter CTR1: Comparison of human and mouse sequences.

PubMed

Bossak, Karolina; Drew, Simon C; Stefaniak, Ewelina; Płonka, Dawid; Bonna, Arkadiusz; Bal, Wojciech

2018-05-01

Copper Transporter 1 (CTR1) is a homotrimeric membrane protein providing the main route of copper transport into eukaryotic cells from the extracellular milieu. Its N-terminal extracellular domain, rich in His and Met residues, is considered responsible for directing copper into the transmembrane channel. Most of vertebrate CTR1 proteins contain the His residue in position three from N-terminus, creating a well-known Amino Terminal Cu(II)- and Ni(II)-Binding (ATCUN) site. CTR1 from humans, primates and many other species contains the Met-Asp-His (MDH) sequence, while some rodents including mouse have the Met-Asn-His (MNH) N-terminal sequence. CTR1 is thought to collect Cu(II) ions from blood copper transport proteins, including albumin, but previous reports indicated that the affinity of N-terminal peptide/domain of CTR1 is significantly lower than that of albumin, casting serious doubt on this aspect of CTR1 function. Using potentiometry and spectroscopic techniques we demonstrated that MDH-amide, a tripeptide model of human CTR1 N-terminus, binds Cu(II) with K of 1.3 × 10 13  M -1 at pH 7.4, ~13 times stronger than Human Serum Albumin (HSA), and MNH-amide is even stronger, K of 3.2 × 10 14  M -1 at pH 7.4. These results indicate that the N-terminus of CTR1 may serve as intermediate binding site during Cu(II) transfer from blood copper carriers to the transporter. MDH-amide, but not MNH-amide also forms a low abundance complex with non-ATCUN coordination involving the Met amine, His imidazole and Asp carboxylate. This species might assist Cu(II) relay down the peptide chain or its reduction to Cu(I), both steps necessary for the CTR1 function. Copyright © 2018 Elsevier Inc. All rights reserved.

The evolution of energy-transducing systems: Studies with archaebacteria

NASA Technical Reports Server (NTRS)

Stan-Lotter, Helga

1993-01-01

N-ethylmaleimide (NEM) inhibits the ATPase of H. saccharovorum in a nucleotide protectable manner. The bulk of 14C-NEM is incorporated into subunit 1. Inhibition kinetics indicated a single binding site. To determine the sequence around this site, cyanogen bromide peptides of NEM-labeled ATPase enzyme were prepared and separated on Tris-Tricine gels. Autoradiography indicated that the NEM binding site is probably located in a fragment of Mr 10-12 K. This result will be confirmed by N-terminal sequencing of the peptide. Since the cysteinyl residue, to which NEM is bound, may be located at the C-terminal end, purification and proteolytic treatment of the 10 K peptide will be required. One inhibitor of V-type ATPases, fluoresceinisothiocyanate (FITC) inhibited also the ATPase of H. saccharovorum. Preliminary results indicated protection against inhibition by nucleotides. Localization of the binding sited to the major subunits is in progress. An extraction procedure for the membrane sector of the ATPase complex of H. saccharovorum yielded a preparation which was enriched in a peptide of Mr 5 500. Experiments to test the immunological crossreaction with subunit c from the Escherichia coli F-type ATPase and the labeling with 14C-DCCD are currently carried out. Polyclonal antiserum to the smaller of the major subunits of the ATPase from H. saccharovorum (subunit ll) reacts in Western blots strongly with the alpha and beta subunits of the F1 ATPase of E. coli, suggesting highly conserved regions on both types of ATPases. To elucidate further the regions of homology, cyanogen bromide peptides of the beta subunits were prepared for sequence analysis.

Concerted In Vitro Trimming of Viral HLA-B27-Restricted Ligands by Human ERAP1 and ERAP2 Aminopeptidases

PubMed Central

Lorente, Elena; Barriga, Alejandro; Johnstone, Carolina; Mir, Carmen; Jiménez, Mercedes; López, Daniel

2013-01-01

In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. In this study, the possible cooperative effect of generating five naturally processed HLA-B27 ligands by both proteases was analyzed. We identified differences in the products obtained with increased detection of natural HLA-B27 ligands by comparing double versus single enzyme digestions by mass spectrometry analysis. These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2. PMID:24223975

Concerted in vitro trimming of viral HLA-B27-restricted ligands by human ERAP1 and ERAP2 aminopeptidases.

PubMed

Lorente, Elena; Barriga, Alejandro; Johnstone, Carolina; Mir, Carmen; Jiménez, Mercedes; López, Daniel

2013-01-01

In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. In this study, the possible cooperative effect of generating five naturally processed HLA-B27 ligands by both proteases was analyzed. We identified differences in the products obtained with increased detection of natural HLA-B27 ligands by comparing double versus single enzyme digestions by mass spectrometry analysis. These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2.

N-terminal pro-B-type Natriuretic Peptide in three different mechanisms of dysnatremia onset after a child's craniopharyngioma surgery.

PubMed

Spatenkova, Vera; Hradil, Jan; Suchomel, Petr

2017-10-01

Craniopharyngioma, due to its sellar location, can be perioperatively complicated by different types of dysnatremia. We present a rare postoperative onset of a combination of three different mechanisms of dysnatremia with N-terminal pro-B-type Natriuretic Peptide (NT-proBNP) and renal function parameters in a boy with a good outcome after craniopharyngioma surgery: 1/ Central diabetes insipidus (CDI) onset immediately after the operation, hypernatremia with peak serum sodium (SNa) 158 mmol/l) caused by free water polyuria (electrolyte-free water clearance, EWC 0.104 ml/s), NT-proBNP 350 pg/ml; 2/ cerebral salt wasting (CSW) onset on day 7, hyponatremia (SNa 128 mmol/l) with hypoosmolality (measured serum osmolality, SOsm 265 mmol/kg) caused by natriuresis (sodium - daily output 605 mmol/day, fractional excretion 0.035), NT-proBNP 191 pg/ml; 3/ Polydypsia onset on day 11 caused hyponatremia (SNa 132 mmol/l), EWC 0.015, NT-proBNP 68 pg/ml.

Pulmonary transit time measurement by contrast-enhanced ultrasound in left ventricular dyssynchrony.

PubMed

Herold, Ingeborg H F; Saporito, Salvatore; Mischi, Massimo; van Assen, Hans C; Bouwman, R Arthur; de Lepper, Anouk G W; van den Bosch, Harrie C M; Korsten, Hendrikus H M; Houthuizen, Patrick

2016-06-01

Pulmonary transit time (PTT) is an indirect measure of preload and left ventricular function, which can be estimated using the indicator dilution theory by contrast-enhanced ultrasound (CEUS). In this study, we first assessed the accuracy of PTT-CEUS by comparing it with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Secondly, we tested the hypothesis that PTT-CEUS correlates with the severity of heart failure, assessed by MRI and N-terminal pro-B-type natriuretic peptide (NT-proBNP). Twenty patients referred to our hospital for cardiac resynchronization therapy (CRT) were enrolled. DCE-MRI, CEUS, and NT-proBNP measurements were performed within an hour. Mean transit time (MTT) was obtained by estimating the time evolution of indicator concentration within regions of interest drawn in the right and left ventricles in video loops of DCE-MRI and CEUS. PTT was estimated as the difference of the left and right ventricular MTT. Normalized PTT (nPTT) was obtained by multiplication of PTT with the heart rate. Mean PTT-CEUS was 10.5±2.4s and PTT-DCE-MRI was 10.4±2.0s (P=0.88). The correlations of PTT and nPTT by CEUS and DCE-MRI were strong; r=0.75 (P=0.0001) and r=0.76 (P=0.0001), respectively. Bland-Altman analysis revealed a bias of 0.1s for PTT. nPTT-CEUS correlated moderately with left ventricle volumes. The correlations for PTT-CEUS and nPTT-CEUS were moderate to strong with NT-proBNP; r=0.54 (P=0.022) and r=0.68 (P=0.002), respectively. (n)PTT-CEUS showed strong agreement with that by DCE-MRI. Given the good correlation with NT-proBNP level, (n)PTT-CEUS may provide a novel, clinically feasible measure to quantify the severity of heart failure. NCT01735838. © 2016 The authors.

Entropic Anomaly Observed in Lipid Polymorphisms Induced by Surfactant Peptide SP-B(1-25).

PubMed

Tran, Nhi; Kurian, Justin; Bhatt, Avni; McKenna, Robert; Long, Joanna R

2017-10-05

The N-terminal 25 amino-acid residues of pulmonary surfactant protein B (SP-B 1-25 ) induces unusual lipid polymorphisms in a model lipid system, 4:1 DPPC/POPG, mirroring the lipid composition of native pulmonary surfactant. It is widely suggested that SP-B 1-25 -induced lipid polymorphisms within the alveolar aqueous subphase provide a structural platform for rapid lipid adsorption to the air-water interface. Here, we characterize in detail the phase behavior of DPPC and POPG in hydrated lipid assemblies containing therapeutic levels of SP-B 1-25 using 2 H and 31 P solid state NMR spectroscopy. The appearance of a previously observed isotropic lipid phase is found to be highly dependent on the thermal cycling of the samples. Slow heating of frozen samples leads to phase separation of DPPC into a lamellar phase whereas POPG lipids interact with the peptide to form an isotropic phase at physiologic temperature. Rapid heating of frozen samples to room temperature leads to strongly isotropic phase behavior for both DPPC and POPG lipids, with DPPC in exchange between isotropic and interdigitated phases. 31 P T 2 relaxation times confirm the isotropic phase to be consistent with a lipid cubic phase. The observed phases exhibit thermal stability up to physiologic temperature (37 °C) and are consistent with the formation of a ripple phase containing a large number of peptide-induced membrane structural defects enabling rapid transit of lipids between lipid lamellae. The coexistance of a lipid cubic phase with interdigitated lipids suggests a specific role for the highly conserved N-terminus of SP-B in stabilizing this unusual lipid polymorphism.

Critical Nucleus Structure and Aggregation Mechanism of the C-terminal Fragment of Copper-Zinc Superoxide Dismutase Protein.

PubMed

Zou, Yu; Sun, Yunxiang; Zhu, Yuzhen; Ma, Buyong; Nussinov, Ruth; Zhang, Qingwen

2016-03-16

The aggregation of the copper-zinc superoxide dismutase (SOD1) protein is linked to familial amyotrophic lateral sclerosis, a progressive neurodegenerative disease. A recent experimental study has shown that the (147)GVIGIAQ(153) SOD1 C-terminal segment not only forms amyloid fibrils in isolation but also accelerates the aggregation of full-length SOD1, while substitution of isoleucine at site 149 by proline blocks its fibril formation. Amyloid formation is a nucleation-polymerization process. In this study, we investigated the oligomerization and the nucleus structure of this heptapeptide. By performing extensive replica-exchange molecular dynamics (REMD) simulations and conventional MD simulations, we found that the GVIGIAQ hexamers can adopt highly ordered bilayer β-sheets and β-barrels. In contrast, substitution of I149 by proline significantly reduces the β-sheet probability and results in the disappearance of bilayer β-sheet structures and the increase of disordered hexamers. We identified mixed parallel-antiparallel bilayer β-sheets in both REMD and conventional MD simulations and provided the conformational transition from the experimentally observed parallel bilayer sheets to the mixed parallel-antiparallel bilayer β-sheets. Our simulations suggest that the critical nucleus consists of six peptide chains and two additional peptide chains strongly stabilize this critical nucleus. The stabilized octamer is able to recruit additional random peptides into the β-sheet. Therefore, our simulations provide insights into the critical nucleus formation and the smallest stable nucleus of the (147)GVIGIAQ(153) peptide.

Targeted Type 1 phototherapeutic agents using azido-peptide bioconjugates

NASA Astrophysics Data System (ADS)

Rajagopalan, Raghavan; Achilefu, Samuel I.; Jimenez, Hermo N.; Webb, Elizabeth G.; Schmidt, Michelle A.; Bugaj, Joseph E.; Dorshow, Richard B.

2001-07-01

Five peptides binding to somatostatin and bombesin receptors were conjugated to 4-azido-2,3,4,6-tetrafluorophenylbenzoic acid, a Type 1 photosensitizer, at the N-terminal position. The receptor affinities were determined by competition binding assay using two different pancreatic tumor cell lines, CA20948 and AR42-J, that expresses somatostatin-2 (SST-2) and bombesin receptors receptively. All compounds exhibited high receptor specificity, i.e., the IC50 values ranged between 1.0 to 64.0 nM. These conjugates may be useful for targeted Type 1 phototherapy via the generation of nitrenes at the cell surfaces expressing these receptors.

Localization of the major antigenic determinant of EDP208 pili at the N-terminus of the pilus protein.

PubMed Central

Worobec, E A; Taneja, A K; Hodges, R S; Paranchych, W

1983-01-01

Trypsin digestion of pilin monomers from EDP208 conjugative pili causes cleavage of Lys12 to yield an N-terminal dodecapeptide, ET1 (Mr approximately equal to 1,500), and the remaining C-terminal fragment, ER (Mr approximately equal to 10,000). Using the amino acid sequence for ET1 provided by Frost et al. (J. Bacteriol. 153:950-954), we synthesized the N-terminal dodecapeptide chemically, conjugated it to bovine serum albumin, and subjected it to immunological studies. Antisera prepared against intact EDP208 pili as well as against the synthetic ET1-BSA conjugate were used in experiments involving an enzyme-linked immunosorbant assay and electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Both experimental approaches showed strong reactivity between the synthetic dodecapeptide and antiserum raised against whole pili. It was also found that antiserum raised against the synthetic peptide was reactive against intact pilus protein, indicating that the N-terminal dodecapeptide is an important antigenic determinant of the EDP208 pilus protein. Additional studies showed that the C-terminal fragment, ER, may contain one or two additional antigenic sites. Images PMID:6185467

Localization of the major antigenic determinant of EDP208 pili at the N-terminus of the pilus protein.

PubMed

Worobec, E A; Taneja, A K; Hodges, R S; Paranchych, W

1983-02-01

Trypsin digestion of pilin monomers from EDP208 conjugative pili causes cleavage of Lys12 to yield an N-terminal dodecapeptide, ET1 (Mr approximately equal to 1,500), and the remaining C-terminal fragment, ER (Mr approximately equal to 10,000). Using the amino acid sequence for ET1 provided by Frost et al. (J. Bacteriol. 153:950-954), we synthesized the N-terminal dodecapeptide chemically, conjugated it to bovine serum albumin, and subjected it to immunological studies. Antisera prepared against intact EDP208 pili as well as against the synthetic ET1-BSA conjugate were used in experiments involving an enzyme-linked immunosorbant assay and electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Both experimental approaches showed strong reactivity between the synthetic dodecapeptide and antiserum raised against whole pili. It was also found that antiserum raised against the synthetic peptide was reactive against intact pilus protein, indicating that the N-terminal dodecapeptide is an important antigenic determinant of the EDP208 pilus protein. Additional studies showed that the C-terminal fragment, ER, may contain one or two additional antigenic sites.

Conformational and Functional Effects Induced by D- and L-Amino Acid Epimerization on a Single Gene Encoded Peptide from the Skin Secretion of Hypsiboas punctatus

PubMed Central

de Magalhães, Mariana T. Q.; Barbosa, Eder A.; Prates, Maura V.; Verly, Rodrigo M.; Munhoz, Victor Hugo O.; de Araújo, Ivan E.; Bloch, Carlos

2013-01-01

Skin secretion of Hypsiboas punctatus is the source of a complex mixture of bioactive compounds where peptides and small proteins prevail, similarly to many other amphibians. Among dozens of molecules isolated from H. punctatus in a proteomic based approach, we report here the structural and functional studies of a novel peptide named Phenylseptin (FFFDTLKNLAGKVIGALT-NH2) that was purified as two naturally occurring D- and L-Phes configurations. The amino acid epimerization and C-terminal amidation for both molecules were confirmed by a combination of techniques including reverse-phase UFLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty lowest-energy 1H NMR structures of each peptide revealed a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of their respective side chains. These structural divergences were considered to be the primary cause of the in vitro quantitative differences in antimicrobial activities between the two molecules. Finally, both molecules elicited equally aversive reactions in mice when delivered orally, an effect that depended entirely on peripheral gustatory pathways. PMID:23565145

Characterization of a novel alpha-amidated decapeptide derived from proopiomelanocortin-A in the trout pituitary.

PubMed

Tollemer, H; Leprince, J; Bailhache, T; Chauveau, I; Vandesande, F; Tonon, M C; Jego, P; Vaudry, H

1997-01-01

Two complementary DNAs encoding distinct forms of POMC have been characterized in the trout pituitary. One of the POMC variants (POMC-A) possesses a C-terminal extension of 25 amino acids, which has no equivalent in other POMCs described to date. This C-terminal peptide contains three pairs of basic amino acids, suggesting that it may be the precursor of multiple processed peptides. In addition, the presence of a C-terminal glycine residue suggests that some of the processing products may be alpha-amidated. To characterize the molecular forms of the peptides generated from the C-terminal domain of trout POMC-A, we have developed specific antibodies against the C-terminal pentapeptide YHFQG and its alpha-amidated derivative YHFQ-NH2. Immunocytochemical labeling of pituitary sections with antibodies against YHFQ-NH2 revealed the presence of numerous immunoreactive cells in the pars intermedia and the rostral pars distalis. In contrast, the antibodies against YHFQG produced only weak immunostaining. HPLC analysis combined with RIA detection revealed that extracts of the pars intermedia and pars distalis contain several peptides derived from the C-terminal extension of trout POMC-A, with the predominant molecular form exhibiting the same retention time as ALGERKYHFQ-NH2. Tryptic digestion of this major form produced a peptide that coeluted with YHFQ-NH2. These data indicate that the processing of the C-terminal extension of trout POMC-A generates several novel peptides including the decapeptide amide ALGERKYHFQ-NH2.

Identification of critical residues of subunit H in its interaction with subunit E of the A-ATP synthase from Methanocaldococcus jannaschii.

PubMed

Gayen, Shovanlal; Balakrishna, Asha M; Biuković, Goran; Yulei, Wu; Hunke, Cornelia; Grüber, Gerhard

2008-04-01

The boomerang-like H subunit of A(1)A(0) ATP synthase forms one of the peripheral stalks connecting the A(1) and A(0) sections. Structural analyses of the N-terminal part (H1-47) of subunit H of the A(1)A(0) ATP synthase from Methanocaldococcus jannaschii have been performed by NMR spectroscopy. Our initial NMR structural calculations for H1-47 indicate that amino acid residues 7-44 fold into a single alpha-helical structure. Using the purified N- (E1-100) and C-terminal domains (E101-206) of subunit E, NMR titration experiments revealed that the N-terminal residues Met1-6, Lys10, Glu11, Ala15, Val20 and Glu24 of H1-47 interact specifically with the N-terminal domain E1-100 of subunit E. A more detailed picture regarding the residues of E1-100 involved in this association was obtained by titration studies using the N-terminal peptides E1-20, E21-40 and E41-60. These data indicate that the N-terminal tail E41-60 interacts with the N-terminal amino acids of H1-47, and this has been confirmed by fluorescence correlation spectroscopy results. Analysis of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of the central stalk subunit F in the presence and absence of E101-206 show no obvious interaction between the C-terminal domain of E and subunit F. The data presented provide, for the first time, structural insights into the interaction of subunits E and H, and their arrangement within A(1)A(0) ATP synthase.

NMR assignments of the N-terminal domain of Nephila clavipes spidroin 1

PubMed Central

Parnham, Stuart; Gaines, William A.; Duggan, Brendan M.; Marcotte, William R.

2011-01-01

The building blocks of spider dragline silk are two fibrous proteins secreted from the major ampullate gland named spidroins 1 and 2 (MaSp1, MaSp2). These proteins consist of a large central domain composed of approximately 100 tandem copies of a 35–40 amino acid repeat sequence. Non-repetitive N and C-terminal domains, of which the C-terminal domain has been implicated to transition from soluble and insoluble states during spinning, flank the repetitive core. The N-terminal domain until recently has been largely unknown due to difficulties in cloning and expression. Here, we report nearly complete assignment for all 1H, 13C, and 15N resonances in the 14 kDa N-terminal domain of major ampullate spidroin 1 (MaSp1-N) of the golden orb-web spider Nephila clavipes. PMID:21152998

Artificial 64-Residue HIV-1 Enhancer-Binding Peptide Is a Potent Inhibitor of Viral Replication in HIV-1-Infected Cells.

PubMed

Oufir, Mouhssin; Bisset, Leslie R; Hoffmann, Stefan R K; Xue, Gongda; Klauser, Stephan; Bergamaschi, Bianca; Gervaix, Alain; Böni, Jürg; Schüpbach, Jörg; Gutte, Bernd

2011-01-01

An artificial HIV-1 enhancer-binding peptide was extended by nine consecutive arginine residues at the C-terminus and by the nuclear localization signal of SV40 large T antigen at the N-terminus. The resulting synthetic 64-residue peptide was found to bind to the two enhancers of the HIV-1 long terminal repeat, cross the plasma membrane and the nuclear envelope of human cells, and suppress the HIV-1 enhancer-controlled expression of a green fluorescent protein reporter gene. Moreover, HIV-1 replication is inhibited by this peptide in HIV-1-infected CEM-GFP cells as revealed by HIV-1 p24 ELISA and real-time RT-PCR of HIV-1 RNA. Rapid uptake of this intracellular stable and inhibitory peptide into the cells implies that this peptide may have the potential to attenuate HIV-1 replication in vivo.

Artificial 64-Residue HIV-1 Enhancer-Binding Peptide Is a Potent Inhibitor of Viral Replication in HIV-1-Infected Cells

PubMed Central

Oufir, Mouhssin; Bisset, Leslie R.; Hoffmann, Stefan R. K.; Xue, Gongda; Klauser, Stephan; Bergamaschi, Bianca; Gervaix, Alain; Böni, Jürg; Schüpbach, Jörg; Gutte, Bernd

2011-01-01

An artificial HIV-1 enhancer-binding peptide was extended by nine consecutive arginine residues at the C-terminus and by the nuclear localization signal of SV40 large T antigen at the N-terminus. The resulting synthetic 64-residue peptide was found to bind to the two enhancers of the HIV-1 long terminal repeat, cross the plasma membrane and the nuclear envelope of human cells, and suppress the HIV-1 enhancer-controlled expression of a green fluorescent protein reporter gene. Moreover, HIV-1 replication is inhibited by this peptide in HIV-1-infected CEM-GFP cells as revealed by HIV-1 p24 ELISA and real-time RT-PCR of HIV-1 RNA. Rapid uptake of this intracellular stable and inhibitory peptide into the cells implies that this peptide may have the potential to attenuate HIV-1 replication in vivo. PMID:22312334

Structure and Membrane Interactions of the Antibiotic Peptide Dermadistinctin K by Multidimensional Solution and Oriented 15N and 31P Solid-State NMR Spectroscopy

PubMed Central

Verly, Rodrigo M.; Moraes, Cléria Mendonça de; Resende, Jarbas M.; Aisenbrey, Christopher; Bemquerer, Marcelo Porto; Piló-Veloso, Dorila; Valente, Ana Paula; Almeida, Fábio C.L.; Bechinger, Burkhard

2009-01-01

DD K, a peptide first isolated from the skin secretion of the Phyllomedusa distincta frog, has been prepared by solid-phase chemical peptide synthesis and its conformation was studied in trifluoroethanol/water as well as in the presence of sodium dodecyl sulfate and dodecylphosphocholine micelles or small unilamellar vesicles. Multidimensional solution NMR spectroscopy indicates an α-helical conformation in membrane environments starting at residue 7 and extending to the C-terminal carboxyamide. Furthermore, DD K has been labeled with 15N at a single alanine position that is located within the helical core region of the sequence. When reconstituted into oriented phosphatidylcholine membranes the resulting 15N solid-state NMR spectrum shows a well-defined helix alignment parallel to the membrane surface in excellent agreement with the amphipathic character of DD K. Proton-decoupled 31P solid-state NMR spectroscopy indicates that the peptide creates a high level of disorder at the level of the phospholipid headgroup suggesting that DD K partitions into the bilayer where it severely disrupts membrane packing. PMID:19289046

Discovery of Novel Bacterial Cell-Penetrating Phylloseptins in Defensive Skin Secretions of the South American Hylid Frogs, Phyllomedusa duellmani and Phyllomedusa coelestis

PubMed Central

Yang, Nan; Li, Lei; Wu, Di; Gao, Yitian; Xi, Xinping; Zhou, Mei; Wang, Lei; Chen, Tianbao; Shaw, Chris

2016-01-01

Phylloseptin (PS) peptides, derived from South American hylid frogs (subfamily Phyllomedusinae), have been found to have broad-spectrum antimicrobial activities and relatively low haemolytic activities. Although PS peptides have been identified from several well-known and widely-distributed species of the Phyllomedusinae, there remains merit in their study in additional, more obscure and specialised members of this taxon. Here, we report the discovery of two novel PS peptides, named PS-Du and PS-Co, which were respectively identified for the first time and isolated from the skin secretions of Phyllomedusa duellmani and Phyllomedusa coelestis. Their encoding cDNAs were cloned, from which it was possible to deduce the entire primary structures of their biosynthetic precursors. Reversed-phase high-performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS) analyses were employed to isolate and structurally-characterise respective encoded PS peptides from skin secretions. The peptides had molecular masses of 2049.7 Da (PS-Du) and 1972.8 Da (PS-Co). They shared typical N-terminal sequences and C-terminal amidation with other known phylloseptins. The two peptides exhibited growth inhibitory activity against E. coli (NCTC 10418), as a standard Gram-negative bacterium, S. aureus (NCTC 10788), as a standard Gram-positive bacterium and C. albicans (NCPF 1467), as a standard pathogenic yeast, all as planktonic cultures. Moreover, both peptides demonstrated the capability of eliminating S. aureus biofilm. PMID:27589802

Lantibiotic engineering: molecular characterization and exploitation of lantibiotic-synthesizing enzymes for peptide engineering.

PubMed

Nagao, Jun-ichi; Aso, Yuji; Shioya, Kouki; Nakayama, Jiro; Sonomoto, Kenji

2007-01-01

Lanthionine-containing peptide antibiotics called lantibiotics are produced by a large number of Gram-positive bacteria. Nukacin ISK-1 produced by Staphylococcus warneri ISK-1 is type-A(II) lantibiotic. Ribosomally synthesized nukacin ISK-1 prepeptide (NukA) consists of an N-terminal leader peptide followed by a C-terminal propeptide moiety that undergoes several post-translational modification events including unusual amino acid formation by the modification enzyme NukM, cleavage of leader peptide and export by the dual functional ABC transporter NukT, finally yielding a biologically active peptide. Unusual amino acids in lantibiotics contribute to biological activity and also structural stability against proteases. Thus, lantibiotic-synthesizing enzymes have a high potentiality for peptide engineering by introduction of unusual amino acids into desired peptides with altering biological and physicochemical properties, e.g., activity and stability, termed lantibiotic engineering. We report the establishment of a heterologous expression of nukacin ISK-1 biosynthetic gene cluster by the nisin-controlled expression system and discuss our recent progress in understanding of the biosynthetic enzymes for nukacin ISK-1 such as localization, molecular interaction in biophysical and biochemical aspects. Substrate specificity of the lantibiotic-synthesizing enzymes was evaluated by complementation of the biosynthetic enzymes (LctM and LctT) of closely related lantibiotic lacticin 481 for nukacin ISK-1 biosynthesis. We further explored a rapid and powerful tool for introduction of unusual amino acids by co-expression of hexa-histidine-tagged NukA and NukM in Escherichia coli.

Engineering the N-terminal end of CelA results in improved performance and growth of Caldicellulosiruptor bescii on crystalline cellulose

DOE PAGES

Kim, Sun -Ki; Chung, Daehwan; Himmel, Michael E.; ...

2016-12-26

Here, CelA is the most abundant enzyme secreted by Caldicellulosiruptor bescii and has been shown to outperform mixtures of commercially available exo- and endoglucanases in vitro. CelA contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. Here, repeated aspartate residues were introduced into the N-terminal ends of CelA GH9 and GH48 domains to improve secretion efficiency and/or catalytic efficiency of CelA. Among several constructs, the highest activity on carboxymethylcellulose (CMC), 0.81 ± 0.03 mg/mL was observed for the C.more » bescii strain containing CelA with 5-aspartate tag at the N-terminal end of GH9 domain – an 82% increase over wild type CelA. In addition, Expression of CelA with N-terminal repeated aspartate residues in C. bescii results in a dramatic increase in its ability to grow on Avicel.« less

The role of polyglutamine expansion and protein context in disease-related huntingtin/lipid interactions

NASA Astrophysics Data System (ADS)

Burke, Kathleen Anne

Huntington's Disease (HD) is a neurodegenerative disorder that is defined by the accumulation of nanoscale aggregates comprised of the huntingtin (htt) protein. Aggregation is directly caused by an expanded polyglutamine (polyQ) domain in htt, leading to a diverse population of aggregate species, such as oligomers, fibrils, and annular aggregates. Furthermore, the length of this polyQ domain is directly related to onset and severity of disease. The first 17 amino acids on the N-terminus (N17) and the polyproline domain on the C-terminal side of the polyQ domain have been shown to further modulate the aggregation process. Additionally, N17 appears to have lipid binding properties as htt interacts with a variety of membrane-containing structures present in cells, such as organelles, and interactions with these membrane surfaces may further modulate htt aggregation. To investigate the interaction between htt exon1 and lipid bilayers, in situ atomic force microscopy (AFM) was used to directly monitor the aggregation of htt exon1 constructs with varying Q-length (35Q, 46Q, 51Q, and myc- 53Q) or synthetic peptides with different polyQ domain flanking sequences (KK-Q35-KK, KK-Q 35-P10-KK, N17-Q35-KK, and N 17-Q35-P10-KK) on supported lipid membranes comprised of total brain lipid extract. The exon1 fragments accumulated on the lipid membranes, causing disruption of the membrane, in a polyQ dependent manner. By adding N-terminal tags to the htt exon1 fragments, the interaction with the lipid bilayer was impeded. The KK-Q35-KK and KK-Q 35-P10-KK peptides had no appreciable interaction with lipid bilayers. Interestingly, polyQ peptides with the N17 flanking sequence interacted with the bilayer. N17-Q35-KK formed discrete aggregates on the bilayer, but there was minimal membrane disruption. The N17-Q35-P10-KK peptide interacted more aggressively with the lipid bilayer in a manner reminiscent of the htt exon1 proteins.

24. Photocopy of construction drawing (microfilm in NJ TRANSIT archive, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

24. Photocopy of construction drawing (microfilm in NJ TRANSIT archive, Newark, N.J., uncatalogued), Elevations & Sections, 1930. - Delaware, Lackawanna & Western Railroad Freight & Rail Yard, Multiple Unit Light Inspection Shed, New Jersey Transit Hoboken Terminal Rail Yard, Hoboken, Hudson County, NJ

25. Photocopy of construction drawing (microfilm in NJ TRANSIT archive, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

25. Photocopy of construction drawing (microfilm in NJ TRANSIT archive, Newark, N.J., uncatalogued), Floor Plan, 1930. - Delaware, Lackawanna & Western Railroad Freight & Rail Yard, Multiple Unit Light Inspection Shed, New Jersey Transit Hoboken Terminal Rail Yard, Hoboken, Hudson County, NJ

The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast).

PubMed

Yamaguchi, K; von Knoblauch, K; Subramanian, A R

2000-09-15

Identification of all the protein components of a plastid (chloroplast) ribosomal 30 S subunit has been achieved, using two-dimensional gel electropholesis, high performance liquid chromatography purification, N-terminal sequencing, polymerase chain reaction-based screening of cDNA library, nucleotide sequencing, and mass spectrometry (electrospray ionization, matrix-assisted laser desorption/ionization time-of-flight, and reversed-phase HPLC coupled with electrospray ionization mass spectrometry). 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1-S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases. 12 of the 25 plastid 30 S ribosomal proteins (PRPs) are encoded in the plastid genome, whereas the remaining 13 are encoded by the nuclear genome. Post-translational transit peptide cleavage sites for the maturation of the 13 cytosolically synthesized PRPs, and post-translational N-terminal processing in the maturation of the 12 plastid synthesized PRPs are described. Post-translational modifications in several PRPs were observed: alpha-N-acetylation of S9, N-terminal processings leading to five mature forms of S6 and two mature forms of S10, C-terminal and/or internal modifications in S1, S14, S18, and S19, leading to two distinct forms differing in mass and/or charge (the corresponding modifications are not observed in E. coli). The four PSRPs in spinach plastid 30 S ribosomal subunit (PSRP-1, 26.8 kDa, pI 6.2; PSRP-2, 21.7 kDa, pI 5.0; PSRP-3, 13.8 kDa, pI 4.9; PSRP-4, 5.2 kDa, pI 11.8) comprise 16% (67.6 kDa) of the total protein mass of the 30 S subunit (429.3 kDa). PSRP-1 and PSRP-3 show sequence similarities with hypothetical photosynthetic bacterial proteins, indicating their possible origins in photosynthetic bacteria. We propose the hypothesis that PSRPs form a "plastid translational regulatory module" on the 30 S ribosomal subunit structure for the possible mediation of nuclear factors on plastid translation.

Cu(II) Binding to the Peptide Ala-His-His, a Chimera of the Canonical Cu(II)-Binding Motifs Xxx-His and Xxx-Zzz-His.

PubMed

Gonzalez, Paulina; Vileno, Bertrand; Bossak, Karolina; El Khoury, Youssef; Hellwig, Petra; Bal, Wojciech; Hureau, Christelle; Faller, Peter

2017-12-18

Peptides and proteins with the N-terminal motifs NH 2 -Xxx-His and NH 2 -Xxx-Zzz-His form well-established Cu(II) complexes. The canonical peptides are Gly-His-Lys and Asp-Ala-His-Lys (from the wound healing factor and human serum albumin, respectively). Cu(II) is bound to NH 2 -Xxx-His via three nitrogens from the peptide and an external ligand in the equatorial plane (called 3N form here). In contrast, Cu(II) is bound to NH 2 -Xxx-Zzz-His via four nitrogens from the peptide in the equatorial plane (called 4N form here). These two motifs are not mutually exclusive, as the peptides with the sequence NH 2 -Xxx-His-His contain both of them. However, this chimera has never been fully explored. In this work, we use a multispectroscopic approach to analyze the Cu(II) binding to the chimeric peptide Ala-His-His (AHH). AHH is capable of forming the 3N- and 4N-type complexes in a pH dependent manner. The 3N form predominates at pH ∼ 4-6.5 and the 4N form at ∼ pH 6.5-10. NMR experiments showed that at pH 8.5, where Cu(II) is almost exclusively bound in the 4N form, the Cu(II)-exchange between AHH or the amidated AHH-NH 2 is fast, in comparison to the nonchimeric 4N form (AAH). Together, the results show that the chimeric AHH can access both Cu(II) coordination types, that minor changes in the second (or further) coordination sphere can impact considerably the equilibrium between the forms, and that Cu kinetic exchange is fast even when Cu-AHH is mainly in the 4N form.

Isolation of dermatoxin from frog skin, an antibacterial peptide encoded by a novel member of the dermaseptin genes family.

PubMed

Amiche, M; Seon, A A; Wroblewski, H; Nicolas, P

2000-07-01

A 32-residue peptide, named dermatoxin, has been extracted from the skin of a single specimen of the tree frog Phyllomedusa bicolor, and purified to homogeneity using a four-step protocol. Mass spectral analysis and sequencing of the purified peptide, as well as chemical synthesis and cDNA analysis were consistent with the structure: SLGSFLKGVGTTLASVGKVVSDQF GKLLQAGQ. This peptide proved to be bactericidal towards mollicutes (wall-less eubacteria) and Gram-positive eubacteria, and also, though to a lesser extent, towards Gram-negative eubacteria. Measurement of the bacterial membrane potential revealed that the plasma membrane is the primary target of dermatoxin. Observation of bacterial cells using reflected light fluorescence microscopy after DNA-staining was consistent with a mechanism of cell killing based upon the alteration of membrane permeability rather than membrane solubilization, very likely by forming ion-conducting channels through the plasma membrane. CD spectroscopy and secondary structure predictions indicated that dermatoxin assumes an amphipathic alpha-helical conformation in low polarity media which mimic the lipophilicity of the membrane of target microorganisms. PCR analysis coupled with cDNA cloning and sequencing revealed that dermatoxin is expressed in the skin, the intestine and the brain. Preprodermatoxin from the brain and the intestine have the same sequence as the skin preproform except for two amino-acid substitutions in the preproregion of the brain precursor. The dermatoxin precursor displayed the characteristic features of preprodermaseptins, a family of peptide precursors found in the skin of Phyllomedusa ssp. Precursors of this family have a common N-terminal preproregion followed by markedly different C-terminal domains that give rise to 19-34-residue peptide antibiotics named dermaseptins B and phylloxin, and to the D-amino-acid-containing opioid heptapeptides dermorphins and deltorphins. Because the structures and cidal mechanisms of dermatoxin, dermaseptins B and phylloxin are very different, dermatoxin extends the repertoire of structurally and functionally diverse peptides derived from the rapidly evolving C-terminal domains of precursors of the dermaseptins family.

C-terminal amino acid residue loss for deprotonated peptide ions containing glutamic acid, aspartic acid, or serine residues at the C-terminus.

PubMed

Li, Zhong; Yalcin, Talat; Cassady, Carolyn J

2006-07-01

Deprotonated peptides containing C-terminal glutamic acid, aspartic acid, or serine residues were studied by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer with ion production by electrospray ionization (ESI). Additional studies were performed by post source decay (PSD) in a matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometer. This work included both model peptides synthesized in our laboratory and bioactive peptides with more complex sequences. During SORI-CID and PSD, [M - H]- and [M - 2H]2- underwent an unusual cleavage corresponding to the elimination of the C-terminal residue. Two mechanisms are proposed to occur. They involve nucleophilic attack on the carbonyl carbon of the adjacent residue by either the carboxylate group of the C-terminus or the side chain carboxylate group of C-terminal glutamic acid and aspartic acid residues. To confirm the proposed mechanisms, AAAAAD was labelled by 18O specifically on the side chain of the aspartic acid residue. For peptides that contain multiple C-terminal glutamic acid residues, each of these residues can be sequentially eliminated from the deprotonated ions; a driving force may be the formation of a very stable pyroglutamatic acid neutral. For peptides with multiple aspartic acid residues at the C-terminus, aspartic acid residue loss is not sequential. For peptides with multiple serine residues at the C-terminus, C-terminal residue loss is sequential; however, abundant loss of other neutral molecules also occurs. In addition, the presence of basic residues (arginine or lysine) in the sequence has no effect on C-terminal residue elimination in the negative ion mode.

Role of VGF-derived carboxy-terminal peptides in energy balance and reproduction: analysis of "humanized" knockin mice expressing full-length or truncated VGF.

PubMed

Sadahiro, Masato; Erickson, Connor; Lin, Wei-Jye; Shin, Andrew C; Razzoli, Maria; Jiang, Cheng; Fargali, Samira; Gurney, Allison; Kelley, Kevin A; Buettner, Christoph; Bartolomucci, Alessandro; Salton, Stephen R

2015-05-01

Targeted deletion of VGF, a secreted neuronal and endocrine peptide precursor, produces lean, hypermetabolic, and infertile mice that are resistant to diet-, lesion-, and genetically-induced obesity and diabetes. Previous studies suggest that VGF controls energy expenditure (EE), fat storage, and lipolysis, whereas VGF C-terminal peptides also regulate reproductive behavior and glucose homeostasis. To assess the functional equivalence of human VGF(1-615) (hVGF) and mouse VGF(1-617) (mVGF), and to elucidate the function of the VGF C-terminal region in the regulation of energy balance and susceptibility to obesity, we generated humanized VGF knockin mouse models expressing full-length hVGF or a C-terminally deleted human VGF(1-524) (hSNP), encoded by a single nucleotide polymorphism (rs35400704). We show that homozygous male and female hVGF and hSNP mice are fertile. hVGF female mice had significantly increased body weight compared with wild-type mice, whereas hSNP mice have reduced adiposity, increased activity- and nonactivity-related EE, and improved glucose tolerance, indicating that VGF C-terminal peptides are not required for reproductive function, but 1 or more specific VGF C-terminal peptides are likely to be critical regulators of EE. Taken together, our results suggest that human and mouse VGF proteins are largely functionally conserved but that species-specific differences in VGF peptide function, perhaps a result of known differences in receptor binding affinity, likely alter the metabolic phenotype of hVGF compared with mVGF mice, and in hSNP mice in which several C-terminal VGF peptides are ablated, result in significantly increased activity- and nonactivity-related EE.

Role of VGF-Derived Carboxy-Terminal Peptides in Energy Balance and Reproduction: Analysis of “Humanized” Knockin Mice Expressing Full-Length or Truncated VGF

PubMed Central

Sadahiro, Masato; Erickson, Connor; Lin, Wei-Jye; Shin, Andrew C.; Razzoli, Maria; Jiang, Cheng; Fargali, Samira; Gurney, Allison; Kelley, Kevin A.; Buettner, Christoph

2015-01-01

Targeted deletion of VGF, a secreted neuronal and endocrine peptide precursor, produces lean, hypermetabolic, and infertile mice that are resistant to diet-, lesion-, and genetically-induced obesity and diabetes. Previous studies suggest that VGF controls energy expenditure (EE), fat storage, and lipolysis, whereas VGF C-terminal peptides also regulate reproductive behavior and glucose homeostasis. To assess the functional equivalence of human VGF1–615 (hVGF) and mouse VGF1–617 (mVGF), and to elucidate the function of the VGF C-terminal region in the regulation of energy balance and susceptibility to obesity, we generated humanized VGF knockin mouse models expressing full-length hVGF or a C-terminally deleted human VGF1–524 (hSNP), encoded by a single nucleotide polymorphism (rs35400704). We show that homozygous male and female hVGF and hSNP mice are fertile. hVGF female mice had significantly increased body weight compared with wild-type mice, whereas hSNP mice have reduced adiposity, increased activity- and nonactivity-related EE, and improved glucose tolerance, indicating that VGF C-terminal peptides are not required for reproductive function, but 1 or more specific VGF C-terminal peptides are likely to be critical regulators of EE. Taken together, our results suggest that human and mouse VGF proteins are largely functionally conserved but that species-specific differences in VGF peptide function, perhaps a result of known differences in receptor binding affinity, likely alter the metabolic phenotype of hVGF compared with mVGF mice, and in hSNP mice in which several C-terminal VGF peptides are ablated, result in significantly increased activity- and nonactivity-related EE. PMID:25675362

The enzymatic processing of α-dystroglycan by MMP-2 is controlled by two anchoring sites distinct from the active site

PubMed Central

Fasciglione, Giovanni Francesco; Sbardella, Diego; Sciandra, Francesca; Casella, MariaLuisa; Camerini, Serena; Crescenzi, Marco; Gori, Alessandro; Tarantino, Umberto; Cozza, Paola; Brancaccio, Andrea; Coletta, Massimo; Bozzi, Manuela

2018-01-01

Dystroglycan (DG) is a membrane receptor, belonging to the dystrophin-glycoprotein complex (DGC) and formed by two subunits, α-dystroglycan (α-DG) and β-dystroglycan (β -DG). The C-terminal domain of α-DG and the N-terminal extracellular domain of β -DG are connected, providing a link between the extracellular matrix and the cytosol. Under pathological conditions, such as cancer and muscular dystrophies, DG may be the target of metalloproteinases MMP-2 and MMP-9, contributing to disease progression. Previously, we reported that the C-terminal domain α-DG (483–628) domain is particularly susceptible to the catalytic activity of MMP-2; here we show that the α-DG 621–628 region is required to carry out its complete digestion, suggesting that this portion may represent a MMP-2 anchoring site. Following this observation, we synthesized an α-DG based-peptide, spanning the (613–651) C-terminal region. The analysis of the kinetic and thermodynamic parameters of the whole and the isolated catalytic domain of MMP-2 (cdMMP-2) has shown its inhibitory properties, indicating the presence of (at least) two binding sites for the peptide, both located within the catalytic domain, only one of the two being topologically distinct from the catalytic active groove. However, the different behavior between whole MMP-2 and cdMMP-2 envisages the occurrence of an additional binding site for the peptide on the hemopexin-like domain of MMP-2. Interestingly, mass spectrometry analysis has shown that α-DG (613–651) peptide is cleavable even though it is a very poor substrate of MMP-2, a feature that renders this molecule a promising template for developing a selective MMP-2 inhibitor. PMID:29447293

N-terminal fatty acylated His-dPhe-Arg-Trp-NH(2) tetrapeptides: influence of fatty acid chain length on potency and selectivity at the mouse melanocortin receptors and human melanocytes.

PubMed

Todorovic, Aleksandar; Holder, Jerry Ryan; Bauzo, Rayna M; Scott, Joseph Walker; Kavanagh, Renny; Abdel-Malek, Zalfa; Haskell-Luevano, Carrie

2005-05-05

The melanocortin system is involved in the regulation of a diverse number of physiologically important pathways including pigmentation, feeding behavior, weight and energy homeostasis, inflammation, and sexual function. All the endogenous melanocortin agonist ligands possess the conserved His-Phe-Arg-Trp tetrapeptide sequence that is postulated to be important for melanocortin receptor molecular recognition and stimulation. Previous studies by our laboratory resulted in the discovery that increasing alkyl chain length at the N-terminal "capping" region of the His-dPhe-Arg-Trp-NH(2) tetrapeptide resulted in a 100-fold increased melanocortin receptor agonist potency. This study was undertaken to systematically evaluate the pharmacological effects of increasing N-capping alkyl chain length of the CH(3)(CH(2))(n)CO-His-dPhe-Arg-Trp-NH(2) (n = 6-16) tetrapeptide template. Twelve analogues were synthesized and pharmacologically characterized at the mouse melanocortin receptors MC1R and MC3R-MC5R and human melanocytes known to express the MC1R. These peptides demonstrated melanocortin receptor selectivity profiles different from those of previously published tetrapeptides. The most notable results of enhanced ligand potency (20- to 200-fold) and receptor selectivity were observed at the MC1R. Tetrapeptides that possessed greater than nine alkyl groups were superior to alpha-MSH in terms of the stimulation of human melanocyte tyrosinase activity. Additionally, the n-pentadecanoyl derivative had a residual effect on tyrosinase activity that existed for at least 4 days after the peptide was removed from the human melanocyte culture medium. These data demonstrate the utility, potency, and residual effect of melanocortin tetrapeptides by adding N-terminal fatty acid moieties.

Identification and characterization of a calmodulin binding domain in the plasma membrane Ca2+-ATPase from Trypanosoma equiperdum.

PubMed

Ramírez-Iglesias, José Rubén; Pérez-Gordones, María Carolina; Del Castillo, Jesús Rafael; Mijares, Alfredo; Benaim, Gustavo; Mendoza, Marta

2018-05-09

The plasma membrane Ca 2+ -ATPase (PMCA) from trypanosomatids lacks a classical calmodulin (CaM) binding domain, although CaM stimulated activities have been detected by biochemical assays. Recently we proposed that the Trypanosoma equiperdum CaM-sensitive PMCA (TePMCA) contains a potential 1-18 CaM-binding motif at the C-terminal region of the pump. In the present study, we evaluated the potential CaM-binding motifs using CaM from Trypanosoma cruzi and either the recombinant full length TePMCA C-terminal sequence (P14) or synthetic peptides comprising different regions of the C-terminal domain. We demonstrated that P14 and a synthetic peptide corresponding to residues 1037-1062 (which contains the predicted 1-18 binding motif) competed efficiently for binding to TcCaM, exhibiting similar IC 50 s of 200 nM. A stable complex of this peptide and TcCaM was formed in the presence of Ca 2+ , as determined by native-polyacrylamide gel electrophoresis. A predicted structure obtained by molecular docking showed an interaction of the 1-18 binding motif with the Ca 2+ /CaM complex. Moreover, when the peptide was incubated with CaM and Ca 2+ , a blue shift in the tryptophan fluorescence spectrum (from 350 to 329 nm) was observed. Substitutions at W 1039 and F 1056 , strongly decreased both CaM-peptide interaction and the complex assembly. Our results demonstrated the presence of a functional 1-18 motif at the TePMCA C-terminal domain. Furthermore, on the basis of spectrofluorometric assays and the resulting structure modeled by docking we propose that the L 1042 and W 1060 residues might also participate as anchors to form a 1-4-18-22 motif. Copyright © 2018 Elsevier B.V. All rights reserved.

Two non-redundant fragments in the N-terminal peptide of human cytosolic methionyl-tRNA synthetase were indispensable for the multi-synthetase complex incorporation and enzyme activity.

PubMed

He, Ran; Zu, Li-Dong; Yao, Peng; Chen, Xin; Wang, En-Duo

2009-02-01

In human cytoplasm, nine aminoacyl-tRNA synthetases (aaRSs) and three protein factors form a multi-synthetase complex (MSC). Human cytosolic methionyl-tRNA synthetase (hcMetRS) is a component of the MSC. Sequence alignment revealed that hcMetRS has an N-terminal extension of 267 amino acid residues. This extension can be divided into three sub-domains: GST-like, GN, and GC sub-domains. The effect of each sub-domain in the N-terminal extension of hcMetRS on enzymatic activity and incorporation into the MSC was studied. The results of cellular assay showed that the GST-like sub-domain was responsible for the incorporation of hcMetRS into the MSC. The entire N-terminal extension of hcMetRS is indispensable for the enzymatic activity. Deletion mutagenesis revealed that a seven-amino acid motif within the sub-domain GC was important for the activity of amino acid activation. A conserved proline residue within the seven-amino acid motif was crucial, while the other six residues were moderately important for the amino acid activation activity. Thus, the last 15 residues of previously defined N-terminal extension of hcMetRS was a part of the catalytic domain; whereas the first 252 residues of hcMetRS constitute the N-terminal extended domain of hcMetRS. The formerly defined N-terminal extension of hcMetRS possesses two functions of two different domains.

Quantitative modeling of peptide binding to TAP using support vector machine.

PubMed

Diez-Rivero, Carmen M; Chenlo, Bernardo; Zuluaga, Pilar; Reche, Pedro A

2010-01-01

The transport of peptides to the endoplasmic reticulum by the transporter associated with antigen processing (TAP) is a necessary step towards determining CD8 T cell epitopes. In this work, we have studied the predictive performance of support vector machine models trained on single residue positions and residue combinations drawn from a large dataset consisting of 613 nonamer peptides of known affinity to TAP. Predictive performance of these TAP affinity models was evaluated under 10-fold cross-validation experiments and measured using Pearson's correlation coefficients (R(p)). Our results show that every peptide position (P1-P9) contributes to TAP binding (minimum R(p) of 0.26 +/- 0.11 was achieved by a model trained on the P6 residue), although the largest contributions to binding correspond to the C-terminal end (R(p) = 0.68 +/- 0.06) and the P1 (R(p) = 0.51 +/- 0.09) and P2 (0.57 +/- 0.08) residues of the peptide. Training the models on additional peptide residues generally improved their predictive performance and a maximum correlation (R(p) = 0.89 +/- 0.03) was achieved by a model trained on the full-length sequences or a residue selection consisting of the first 5 N- and last 3 C-terminal residues of the peptides included in the training set. A system for predicting the binding affinity of peptides to TAP using the methods described here is readily available for free public use at http://imed.med.ucm.es/Tools/tapreg/. (c) 2009 Wiley-Liss, Inc.

A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

PubMed

Skottrup, Peter Durand; Sørensen, Grete; Ksiazek, Miroslaw; Potempa, Jan; Riise, Erik

2012-01-01

Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18) by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15), shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP) was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG) could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48). Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.