Further screening of the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa

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

Vaithinathan, R.; Berson, E.L.; Dryja, T.P.

Here the authors report 8 novel mutations and 8 previously reported mutations found from further analysis of the rhodopsin gene in a large set of additional patients with autosomal dominant retinitis pigmentosa. Leukocyte DNA was purified from 122 unrelated patients with autosomal dominant retinitis pigmentosa who were not included in previous analyses. The coding region and splice donor and acceptor sites of the rhodopsin gene were screened for mutations using single-strand conformation polymorphism analysis and direct genomic sequencing. They found 29 patients with varient bands that were due to mutations. Sequence analysis showed that 20 cases each had 1 ofmore » 9 previously published mutations: Pro23His, Thr58Arg, Gly89Asp, Pro171Leu, Glu181Lys, Pro347Leu, Phe45Leu, Arg135Trp, and Lys296Glu. In 9 other cases, they found 8 novel mutations. One was a 3-bp deletion (Cys264-del), and the rest were point mutations resulting in an altered amino acid: Gly51Arg (GGC [yields] CGC), Cys110Tyr (TCG [yields] TAC), Gly114Asp (GGC [yields] GAC), Ala164Glu (GCG [yields] GAG), Pro171Ser (CCA [yields] TCA), Val345Leu (GTG [yields] CTG), and Pro347Gln (CCG [yields] CAG). Each of these novel mutations was found in only one family except for Gly51Arg, which was found in two. In every family tested, the mutation cosegregated with the disease. However, in pedigree D865 only one affected member was available for analysis. About two-thirds of the mutations affect amino acids in transmembrane domains, yet only one-half of opsin's residues are in these regions. One-third of the mutations alter residues in the extracellular/intradiscal space, which includes only 25% of the protein.« less

Inhibition of polyphenol oxidases activity by various dipeptides.

PubMed

Girelli, Anna M; Mattei, Enrico; Messina, Antonella; Tarola, Anna M

2004-05-19

In an effort to develop natural and nontoxic inhibitors on the activity of mushroom polyphenol oxidase (PPO) the effect of various glycyl-dipeptides (GlyAsp, GlyGly, GlyHis, GlyLeu, GlyLys, GlyPhe, GlyPro, GlyTyr) was investigated. The inhibition study with dihydroxyphenylalanine (DOPA) as substrate is based on separation of the enzymatic reaction components by reversed phase HPLC and the UV detection of the dopachrome formed. The results have evidenced that several of tested dipeptides inhibited PPO activity in the range of 20-40% while GlyPro and GlyLeu had no effect. The study has also permitted the characterization of the following kinetic pattern: a linear-mixed-type mechanism for GlyAsp, GlyGly, GlyLys, and GlyPhe and a hyperbolic-mixed-type for GlyTyr. It was not possible to identify the inhibition mechanism for GlyHis, although it affects PPO activity. In addition the effects of GlyAsp, GlyLys and GlyHis were evaluated for lessening the browning of fresh Golden Delicious apple and Irish White Skinned potato. The effectiveness of such inhibitors was determined by the difference between the colors observed in the dipeptide-treated sample and the controls using the color space CIE-Lab system. The % browning inhibition on potato (20-50%) was greater than of apple (20-30%) by the all tested dipeptides. Only GlyLys presented the significant value of 50%.

Purification and characterization of galanin and scyliorhinin I from the hybrid sturgeon, Scaphirhynchus platorynchus x Scaphirhynchus albus (Acipenseriformes).

PubMed

Wang, Y; Barton, B A; Thim, L; Nielsen, P F; Conlon, J M

1999-01-01

The sturgeons (order Acipenseriformes) are extant representatives of a group of ancient Actinopterygian (ray-finned) fish. Galanin and scyliorhinin I (a tachykinin with limited structural similarity to mammalian substance P) have been isolated from an extract of the gastrointestinal tract of a sturgeon (an F1 hybrid between the shovelnose sturgeon, Scaphirhynchus platorynchus, and the pallid sturgeon, Scaphirhynchus albus). The primary structure of sturgeon galanin (Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu10-Leu-Gly-Pro-His-Ala-Val -As p-Gly-His-Arg20-Ser-Leu-Ser-Asp-Lys-His-Gly-Leu-Pro.NH2) contains only two amino acid substitutions (Ser23 --> Asn and Pro29 --> Ala) compared with galanin from the bowfin, Amia calva (Amiiformes), but five amino acid substitutions compared with galanin from the trout (Teleostei). Similarly, the sturgeon tachykinin (Ser-Lys-Tyr-His-Gln-Phe-Tyr-Gly-Leu-Met.NH2) contains only one amino acid substitution (Tyr3 --> Ser) compared with scyliorhinin I previously isolated from bowfin stomach but five amino acid substitutions compared with trout substance P. The data support the hypothesis that the Acipenseriformes and the basal Neopterygians (gars and bowfin) share a close phylogenetic relationship. Copyright 1999 Academic Press.

Gallium-67-labeled lactam bridge-cyclized alpha-MSH peptides with enhanced melanoma uptake and reduced renal uptake.

PubMed

Guo, Haixun; Gallazzi, Fabio; Miao, Yubin

2012-06-20

The purpose of this study was to examine the melanoma targeting and pharmacokinetic properties of (67)Ga-DOTA-GGNle-CycMSHhex {(67)Ga-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and (67)Ga-NOTA-GGNle-CycMSHhex {(67)Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and compare with (67)Ga-DOTA-GlyGlu-CycMSH {(67)Ga-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]} we previously reported. DOTA-GGNle-CycMSHhex and NOTA-GGNle-CycMSHhex were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSHhex was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSHhex. The melanoma targeting and pharmacokinetic properties of (67)Ga-NOTA-GGNle-CycMSHhex and (67)Ga-DOTA-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex displayed comparable MC1 receptor binding affinities (1.6 vs 2.1 nM) in B16/F1 melanoma cells. Both (67)Ga-NOTA-GGNle-CycMSHhex and (67)Ga-DOTA-GGNle-CycMSHhex exhibited dramatically enhanced melanoma uptake and reduced renal uptake than (67)Ga-DOTA-GlyGlu-CycMSH in B16/F1 melanoma-bearing C57 mice. Furthermore, (67)Ga-NOTA-GGNle-CycMSHhex exhibited more favorable radiolabeling conditions (>85% radiolabeling yields started at 37 °C), as well as higher tumor/kidney uptake ratios than (67)Ga-DOTA-GGNle-CycMSHhex at 0.5, 2, and 24 h postinjection. High melanoma uptake coupled with low renal uptake highlighted the potential of (67)Ga-NOTA-GGNle-CycMSHhex for melanoma imaging and therapy.

Gallium-67-Labeled Lactam Bridge-Cyclized Alpha-MSH Peptides with Enhanced Melanoma Uptake and Reduced Renal Uptake

PubMed Central

Guo, Haixun; Gallazzi, Fabio; Miao, Yubin

2012-01-01

The purpose of this study was to examine the melanoma targeting and pharmacokinetic properties of 67Ga-DOTA-GGNle-CycMSHhex {67Ga-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-Gly-Gly-Nle-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2} and 67Ga-NOTA-GGNle-CycMSHhex {67Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2} and compare with 67Ga-DOTA-GlyGlu-CycMSH {67Ga-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-dPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]} we previously reported. DOTA-GGNle-CycMSHhex and NOTA-GGNle-CycMSHhex were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSHhex was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSHhex. The melanoma targeting and pharmacokinetic properties of 67Ga-NOTA-GGNle-CycMSHhex and 67Ga-DOTA-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex displayed comparable MC1 receptor binding affinities (1.6 vs. 2.1 nM) in B16/F1 melanoma cells. Both 67Ga-NOTA-GGNle-CycMSHhex and 67Ga-DOTA-GGNle-CycMSHhex exhibited dramatically enhanced melanoma uptake and reduced renal uptake than 67Ga-DOTA-GlyGlu-CycMSH in B16/F1 melanoma-bearing C57 mice. Furthermore, 67Ga-NOTA-GGNle-CycMSHhexexhibited more favorable radiolabeling conditions (> 85% radiolabeling yields started at 37°C), as well as higher tumor/kidney uptake ratios than 67Ga-DOTA-GGNle-CycMSHhex at 0.5, 2 and 24 h post-injection. High melanoma uptake coupled with low renal uptake highlighted the potential of 67Ga-NOTA-GGNle-CycMSHhexfor melanoma imaging and therapy. PMID:22621181

111In-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone peptide analogues for melanoma imaging.

PubMed

Miao, Yubin; Gallazzi, Fabio; Guo, Haixun; Quinn, Thomas P

2008-02-01

The purpose of this study was to examine the influence of the lactam bridge cyclization on melanoma targeting and biodistribution properties of the radiolabeled conjugates. Two novel lactam bridge-cyclized alpha-MSH peptide analogues, DOTA-CycMSH (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]) and DOTA-GlyGlu-CycMSH (DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]), were synthesized and radiolabeled with (111)In. The internalization and efflux of (111)In-labeled CycMSH peptides were examined in B16/F1 melanoma cells. The melanoma targeting properties, pharmacokinetics, and SPECT/CT imaging of (111)In-labeled CycMSH peptides were determined in B16/F1 melanoma-bearing C57 mice. Both (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH exhibited fast internalization and extended retention in B16/F1 cells. The tumor uptake values of (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH were 9.53+/-1.41% injected dose/gram (% ID/g) and 10.40+/-1.40% ID/g at 2 h postinjection, respectively. Flank melanoma tumors were clearly visualized with (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH by SPECT/CT images at 2 h postinjection. Whole-body clearance of the peptides was fast, with greater than 90% of the radioactivities cleared through urinary system by 2 h postinjection. There was low radioactivity (<0.8% ID/g) accumulated in blood and normal organs except kidneys at all time points investigated. Introduction of a negatively charged linker (-Gly-Glu-) into the peptide sequence decreased the renal uptake by 44% without affecting the tumor uptake at 4 h postinjection. High receptor-mediated melanoma uptakes coupled with fast whole-body clearance in B16/F1 melanoma-bearing C57 mice demonstrated the feasibility of using (111)In-labeled lactam bridge-cyclized alpha-MSH peptide analogues as a novel class of imaging probes for receptor-targeting melanoma imaging.

Comparative biochemical and computational study of the role of naturally occurring mutations at Ambler positions 104 and 170 in GES β-lactamases.

PubMed

Kotsakis, Stathis D; Miriagou, Vivi; Tzelepi, Eva; Tzouvelekis, Leonidas S

2010-11-01

In GES-type β-lactamases, positions 104 and 170 are occupied by Glu or Lys and by Gly, Asn, or Ser, respectively. Previous studies have indicated an important role of these amino acids in the interaction with β-lactams, although their precise role, especially that of residue 104, remains uncertain. In this study, we constructed GES-1 (Glu104, Gly170), GES-2 (Glu104, Asn170), GES-5 (Glu104, Ser170), GES-6 (Lys104, Ser170), GES-7 (Lys104, Gly170), and GES-13 (Lys104, Asn170) by site-specific mutagenesis and compared their hydrolytic properties. Isogenic comparisons of β-lactam resistance levels conferred by these GES variants were also performed. Data indicated the following patterns: (i) Lys104-containing enzymes exhibited enhanced hydrolysis of oxyimino-cephalosporins and reduced efficiency against imipenem in relation to enzymes possessing Glu104, (ii) Asn170-containing enzymes showed reduced hydrolysis rates of penicillins and older cephalosporins, (iii) Ser170 enabled GES to hydrolyze cefoxitin efficiently, and (iv) Asn170 and Ser170 increased the carbapenemase character of GES enzymes but reduced their activity against ceftazidime. Molecular dynamic simulations of GES apoenzyme models, as well as construction of GES structures complexed with cefoxitin and an achiral ceftazidime-like boronic acid, provided insights into the catalytic behavior of the studied mutants. There were indications that an increased stability of the hydrogen bonding network of Glu166-Lys73-Ser70 and an altered positioning of Trp105 correlated with the substrate spectra, especially with acylation of GES by imipenem. Furthermore, likely effects of Ser170 on GES interactions with cefoxitin and of Lys104 on interactions with oxyimino-cephalosporins were revealed. Overall, the data unveiled the importance of residues 104 and 170 in the function of GES enzymes.

Primary structure of a guanyl-specific ribonuclease from the fungus Penicillium brevicompactum

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

Kulikov, V.A.; Shlyapnikov, S.V.; Yakovlev, G.I.

1986-01-01

By the automatic Edman degradation of the intact S-carboxymethylated protein and a mixture of the products of its proteolytic cleavage at Arg, Lys, and Glu residues, together with results on the kinetics of the proteolysis of the protein under the action of carboxypeptidase Y, the primary structure of the extracellular guanyl-specific RNase of the fungus Penicillium brevicompactum has been determined. The RNase contains 102 amino acid residues: 7 Asp, 7 Asn, 9 Thr, 11 Ser, 4 Glu, 1 Gln, 4 Pro, 10 Gly, 11 Ala, 4 Cys, 7 Val, 4 Ile, 3 Leu, 9 Tyr, 5 Phe, 2 Lys, 3more » His, 1 Arg (M/sub r/ 10,801). It has been established that four hemicystine residues of the P. compactum RNase form, in pairs, two disulfide bonds« less

Probing the binding of Cu(2+) ions to a fragment of the Aβ(1-42) polypeptide using fluorescence spectroscopy, isothermal titration calorimetry and molecular dynamics simulations.

PubMed

Makowska, Joanna; Żamojć, Krzysztof; Wyrzykowski, Dariusz; Żmudzińska, Wioletta; Uber, Dorota; Wierzbicka, Małgorzata; Wiczk, Wiesław; Chmurzyński, Lech

2016-09-01

Steady-state and time-resolved fluorescence quenching measurements supported by isothermal titration calorimetry (ITC) and molecular dynamics simulations (MD), with the NMR-derived restraints, were used to investigate the interactions of Cu(2+) ions with a fragment of the Aβ(1-42) polypeptide, Aβ(5-16) with the following sequence: Ac-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-NH2, denoted as HZ1. The studies presented in this paper, when compared with our previous results (Makowska et al., Spectrochim. Acta A 153: 451-456), show that the affinity of the peptide to metal ions is conformation-dependent. All the measurements were carried out in 20mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution, pH6.0. The Stern-Volmer equations, along with spectroscopic observations, were used to determine the quenching and binding parameters. The obtained results unequivocally suggest that Cu(2+) ions quench the fluorescence of HZ1 only through a static quenching mechanism, in contrast to the fragment from the N-terminal part of the FPB28 protein, with sequence Ac-Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr- NH2 (D9) and its derivative with a single point mutation: Ac-Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr- NH2 (D9_M), where dynamic quenching occurred. The thermodynamic parameters (ΔITCH, ΔITCS) for the interactions between Cu(2+) ions and the HZ1 peptide were determined from the calorimetric data. The conditional thermodynamic parameters suggest that, under the experimental conditions, the formation of the Cu(2+)-HZ1 complex is both an enthalpy and entropy driven process. Copyright © 2016 Elsevier B.V. All rights reserved.

Enzymes processing somatostatin precursors: an Arg-Lys esteropeptidase from the rat brain cortex converting somatostatin-28 into somatostatin-14.

PubMed Central

Gluschankof, P; Morel, A; Gomez, S; Nicolas, P; Fahy, C; Cohen, P

1984-01-01

The post-translational proteolytic conversion of somatostatin-14 precursors was studied to characterize the enzyme system responsible for the production of the tetradecapeptide either from its 15-kDa precursor protein or from its COOH-terminal fragment, somatostatin-28. A synthetic undecapeptide Pro-Arg-Glu-Arg-Lys-Ala-Gly-Ala-Lys-Asn-Tyr(NH2), homologous to the amino acid sequence of the octacosapeptide at the putative Arg-Lys cleavage locus, was used as substrate, after 125I labeling on the COOH-terminal tyrosine residue. A 90-kDa proteolytic activity was detected in rat brain cortex extracts after molecular sieve fractionation followed by ion exchange chromatography. The protease released the peptide 125I-Ala-Gly-Ala-Lys-Asn-Tyr(NH2) from the synthetic undecapeptide substrate and converted somatostatin-28 into somatostatin-14 under similar conditions (pH 7.0). Under these experimental conditions, the product tetradecapeptide was not further degraded by the enzyme. In contrast, the purified 15-kDa hypothalamic precursor remained unaffected when exposed to the proteolytic enzyme under identical conditions. It is concluded that this Arg-Lys esteropeptidase from the brain cortex may be involved in the in vivo processing of the somatostatin-28 fragment of prosomatostatin into somatostatin-14, the former species being an obligatory intermediate in a two-step proteolytic mechanism leading to somatostatin-14. PMID:6149550

Prosomatostatin-I is processed to somatostatin-26 and somatostatin-14 in the pancreas of the bowfin, Amia calva.

PubMed

Wang, Y; Youson, J H; Conlon, J M

1993-08-13

With the exception of the Agnatha (lampreys and hagfishes), somatostatin-14 is the predominant molecular form of somatostatin in the pancreas of species from all classes of vertebrates yet studied. The pancreas of the holostean fish, Amia calva (bowfin; order Amiiformes) contained somatostatin-like immunoreactivity that was resolved by reversed phase HPLC in two components. The primary structure of the more abundant peptide (somatostatin-26) was established as: Ser-Ala-Asn-Pro-Ala5-Leu-Ala-Pro-Arg-Glu10-Arg-Lys-Ala-Gly-+ ++Cys15-Lys-Asn-Phe- Phe-Trp20-Lys-Thr-Phe-Thr-Ser25-Cys. This amino acid sequence shows one substitution (Leu for Met at position 6) and two deletions compared with mammalian somatostatin-28. The minor component was identical to somatostatin-14. The data show that the pathway of post-translational processing of prosomatostatin-I in the bowfin pancreas is appreciably different from the corresponding pathway in teleost fish and higher vertebrates.

Effect of DOTA position on melanoma targeting and pharmacokinetic properties of 111In-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone peptide.

PubMed

Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Prossnitz, Eric R; Sklar, Larry A; Miao, Yubin

2009-11-01

The purpose of this study was to examine the effect of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) position on melanoma targeting and pharmacokinetics of radiolabeled lactam bridge-cyclized alpha-melanocyte stimulating hormone (alpha-MSH) peptide. A novel lactam bridge-cyclized alpha-MSH peptide, Ac-GluGlu-CycMSH[DOTA] {Ac-Glu-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Lys(DOTA)]}, was synthesized using standard 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry. DOTA was directly attached to the alpha-amino group of Lys in the cyclic ring, while the N-terminus of the peptide was acetylated to generate Ac-GluGlu-CycMSH[DOTA]. The MC1 receptor binding affinity of Ac-GluGlu-CycMSH[DOTA] was determined in B16/F1 melanoma cells. Melanoma targeting and pharmacokinetic properties of Ac-GluGlu-CycMSH[DOTA]-111In were determined in B16/F1 melanoma-bearing C57 mice and compared to that of 111In-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp] (111In-DOTA-GlyGlu-CycMSH; DOTA was coupled to the N-terminus of the peptide). Ac-GluGlu-CycMSH[DOTA] displayed 0.6 nM MC1 receptor binding affinity in B16/F1 cells. Ac-GluGlu-CycMSH[DOTA]-111In was readily prepared with greater than 95% radiolabeling yield. Ac-GluGlu-CycMSH[DOTA]-111In exhibited high tumor uptake (11.42 +/- 2.20% ID/g 2 h postinjection) and prolonged tumor retention (9.42 +/- 2.41% ID/g 4 h postinjection) in B16/F1 melanoma-bearing C57 mice. The uptake values for nontarget organs were generally low (<1.3% ID/g) except for the kidneys 2, 4, and 24 h postinjection. DOTA position exhibited profound effect on melanoma targeting and pharmacokinetic properties of Ac-GluGlu-CycMSH[DOTA]-111In, providing a new insight into the design of lactam bridge-cyclized peptide for melanoma imaging and therapy.

In vitro effect of short peptides on expression of interleukin-2 gene in splenocytes.

PubMed

Kazakova, T B; Barabanova, S V; Khavinson, V Kh; Glushikhina, M S; Parkhomenko, E P; Malinin, V V; Korneva, E A

2002-06-01

Synthetic peptides Vilon (Lys-Glu), Epithalon (Ala-Glu-Asp-Gly), and Cortagen (Ala-Glu-Asp-Pro) in vitro activated interleukin-2 mRNA synthesis in splenocytes from CBA mice in the absence of specific inductors. The intensity of interleukin-2 mRNA synthesis in splenocytes depended on the type, concentration, and duration of treatment with the peptides. Vilon and Epithalon were most potent, while Cortagen produced a less pronounced effect on interleukin-2 mRNA synthesis.

Isolation and structural elucidation of antioxidant peptides from oyster (Saccostrea cucullata) protein hydrolysate.

PubMed

Umayaparvathi, S; Meenakshi, S; Vimalraj, V; Arumugam, M; Balasubramanian, T

2014-01-01

Protein derived from the oyster (Saccostrea cucullata) was hydrolyzed using protease from Bacillus cereus SU12 for isolation of antioxidant peptides. The oyster hydrolysate exhibited a strong antioxidant potential in DPPH (85.7±0.37%) followed by Hydrogen peroxide radical scavenging activity (81.6±0.3%), Hydroxyl radical-scavenging activity (79.32±0.6%), Reducing power assay (2.63±0.2 OD at 700nm). Due to the high antioxidant potential, hydrolysate was fractionated in Sephadex G-25 gel filtration chromatography. The active peptide fraction was further purified by UPLC-MS. Totally 7 antioxidant peptides were collected. Among 7 peptides (SCAP 1-7), 3 peptides (SCAP 1, 3 and 7) had highest scavenging ability on DPPH radicals. The amino acid sequence and molecular mass of purified antioxidant peptides (SCAP1, SCAP3 and SCAP7) were determined by Q-TOF ESI mass spectroscopy and structures of the peptides were Leu-Ala-Asn-Ala-Lys (MW=515.29Da), Pro-Ser-Leu-Val-Gly-Arg-Pro-Pro-Val-Gly-Lys-Leu-Thr-Leu (MW=1432.89Da) and Val-Lys-Val-Leu-Leu-Glu-His-Pro-Val-Leu (MW=1145.75Da), respectively. The unique amino acid composition and sequence in the peptides might play an important role in expression of their antioxidant activity. The results of this study suggest that oyster protein hydrolysate is good source of natural antioxidants.

Purification and in vitro antioxidative effects of giant squid muscle peptides on free radical-mediated oxidative systems.

PubMed

Rajapakse, Niranjan; Mendis, Eresha; Byun, Hee-Guk; Kim, Se-Kwon

2005-09-01

Low molecular weight peptides obtained from ultrafiltration (UF) of giant squid (Dosidicus gigas) muscle protein were studied for their antioxidative effects in different in vitro oxidative systems. The most potent two peptides, Asn-Ala-Asp-Phe-Gly-Leu-Asn-Gly-Leu-Glu-Gly-Leu-Ala (1307 Da) and Asn-Gly-Leu-Glu-Gly-Leu-Lys (747 Da), exhibited their antioxidant potential to act as chain-breaking antioxidants by inhibiting radical-mediated peroxidation of linoleic acid, and their activities were closer to highly active synthetic antioxidant, butylated hydroxytoluene. Addition of these peptides could enhance the viability of cytotoxic embryonic lung fibroblasts significantly (P<.05) at a low concentration of 50 microg/ml, and it was presumed due to the suppression of radical-induced oxidation of membrane lipids. Electron spin trapping studies revealed that the peptides were potent scavengers of free radicals in the order of carbon-centered (IC(50) 396.04 and 304.67 microM), hydroxyl (IC(50) 497.32 and 428.54 microM) and superoxide radicals (IC(50) 669.34 and 573.83 microM). Even though the exact molecular mechanism for scavenging of free radicals was unclear, unusually high hydrophobic amino acid composition (more than 75%) of giant squid muscle peptides was presumed to be involved in the observed activities.

Effects of amino acids on melanoma targeting and clearance properties of Tc-99m-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone peptides.

PubMed

Flook, Adam M; Yang, Jianquan; Miao, Yubin

2013-11-14

The purpose of this study was to examine the effects of amino acids on melanoma targeting and clearance properties of new (99m)Tc-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-Lys-(Arg(11))CCMSH {c[Arg-Ser-Asp-DTyr-Asp]-Lys-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RNleD-Lys-(Arg(11))CCMSH, RPheD-Lys-(Arg(11))CCMSH, and RdPheD-Lys-(Arg(11))CCMSH peptides were synthesized and evaluated for their melanocortin-1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of (99m)Tc-RSD-Lys-(Arg(11))CCMSH, (99m)Tc-RFD-Lys-(Arg(11))CCMSH, and (99m)Tc-RfD-Lys-(Arg(11))CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The substitution of Gly with Ser, Phe, and dPhe increased the MC1 receptor binding affinities of the peptides, whereas the substitution of Gly with Nle decreased the MC1 receptor binding affinity of the peptide. (99m)Tc-RSD-Lys-(Arg(11))CCMSH exhibited the highest melanoma uptake (18.01 ± 4.22% ID/g) and the lowest kidney and liver uptake among these (99m)Tc-peptides. The B16/F1 melanoma lesions could be clearly visualized by SPECT/CT using (99m)Tc-RSD-Lys-(Arg(11))CCMSH as an imaging probe. It is desirable to reduce the renal uptake of (99m)Tc-RSD-Lys-(Arg(11))CCMSH to facilitate its potential therapeutic application.

Effects of Amino Acids on Melanoma Targeting and Clearance Properties of Tc-99m-Labeled Arg-X-Asp-Conjugated α-Melanocyte Stimulating Hormone Peptides

PubMed Central

Flook, Adam M.; Yang, Jianquan; Miao, Yubin

2013-01-01

The purpose of this study was to examine the effects of amino acids on melanoma targeting and clearance properties of new 99mTc-labeled Arg-X-Asp-conjugated alpha-melanocyte stimulating hormone (α-MSH) peptides. RSD-Lys-(Arg11)CCMSH {c[Arg-Ser-Asp-dTyr-Asp]-Lys-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RNleD-Lys-(Arg11)CCMSH, RPheD-Lys-(Arg11)CCMSH and RdPheD-Lys-(Arg11)CCMSH peptides were synthesized and evaluated for their melanocortin-1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of 99mTc-RSD-Lys-(Arg11)CCMSH, 99mTc-RFD-Lys-(Arg11)CCMSH and 99mTc-RfD-Lys-(Arg11)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The substitution of Gly with Ser, Phe and dPhe increased the MC1 receptor binding affinities of the peptides, whereas the substitution of Gly with Nle decreased the MC1 receptor binding affinity of the peptide. 99mTc-RSD-Lys-(Arg11)CCMSH exhibited the highest melanoma uptake (18.01 ± 4.22% ID/g) and the lowest kidney and liver uptake among these 99mTc-peptides. The B16/F1 melanoma lesions could be clearly visualized by SPECT/CT using 99mTc-RSD-Lys-(Arg11)CCMSH as an imaging probe. It is desirable to reduce the renal uptake of 99mTc-RSD-Lys-(Arg11)CCMSH to facilitate its potential therapeutic application. PMID:24131154

Effects of short peptides on thymocyte blast transformation and signal transduction along the sphingomyelin pathway.

PubMed

Khavinson, V Kh; Rybakina, E G; Malinin, V V; Pivanovich, I Yu; Shanin, S N; Korneva, E A

2002-05-01

Immunomodulating effects of synthetic peptides Vilon (Lys-Glu), Epithalon (Ala-Glu-Asp-Gly), and Cortagen (Ala-Glu-Asp-Pro) and possible involvement of the sphingomyelin signal transduction pathway in their effects in mouse thymocytes were studied. Vilon produced the most potent comitogenic effect on thymocyte proliferation and modulated comitogenic activity of interleukin-1b. Epithalon was less potent, while Cortagen produced no such effects. Vilon produced a more pronounced stimulatory effect on sphingomyelinase activity in mouse thymocyte membranes compared to Epithalon and Cortagen.

Structural studies of α-melanocyte-stimulating hormone and a novel β-melanocyte-stimulating hormone from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias

PubMed Central

Bennett, Hugh P. J.; Lowry, Philip J.; McMartin, Colin; Scott, Alexander P.

1974-01-01

A melanocyte-stimulating hormone (MSH) has been isolated from extracts of the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias by gel-filtration and ion-exchange chromatography. It had approximately 1% of the potency of mammalian α-MSH on bioassays in vitro on frog skin and dogfish skin. Sequence analysis revealed it to be a hexadecapeptide with the following primary structure: Asp-Gly-Asp-Asp-Tyr-Lys-Phe-Gly-His-Phe-Arg-Trp-Ser-Val-Pro-Leu. It appears to be related to the β-MSH species of mammalian species but has only the sequence -His-Phe-Arg-Trp- in common with the heptapeptide core -Met-Glu-His-Phe-Arg-Trp-Gly- which is characteristic not only of the MSH peptides but also of the adrenocorticotrophins and lipotrophins studied so far. An α-MSH was also isolated, 50% of which was amidated at the C-terminus group. Sequence data from this study taken in conjunction with those from a previous study (Lowry & Chadwick, 1970b) revealed it to be a tridecapeptide which is identical with the N-terminal sequence of dogfish adrenocorticotrophin. PMID:4375978

An antimicrobial peptide from the skin secretions of the mountain chicken frog Leptodactylus fallax (Anura:Leptodactylidae).

PubMed

Rollins-Smith, Louise A; King, Jay D; Nielsen, Per F; Sonnevend, Agnes; Conlon, J Michael

2005-01-15

A 25 amino-acid-residue, C-terminally alpha-amidated peptide with antimicrobial activity, which has been termed fallaxin, was isolated in high yield from the norepinephrine-stimulated skin secretions of the mountain chicken frog Leptodactylus fallax (Anura:Leptodactylidae). The amino acid sequence of the peptide (Gly-Val-Val-Asp-Ile-Leu-Lys-Gly-Ala-Ala-Lys-Asp-Ile-Ala-Gly-His-Leu-Ala-Ser-Lys-Val-Met-Asn-Lys-Leu.NH2) shows structural similarity with members of the ranatuerin-2 family previously isolated from the skins of frogs of the genus Rana that are only distantly related to the Leptodactylidae. This observation is consistent with the hypothesis that many frog skin antimicrobial peptides are related evolutionarily, having arisen from multiple duplications of an ancestral gene that existed before the radiation of the different families. Fallaxin inhibited the growth of reference strains of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae) but with relatively low potency (MIC> or =20 microM) and was inactive against the Gram-positive bacterium (Staphylococcus aureus) and the yeast Candida albicans. The hemolytic activity of fallaxin was very low (HC50>200 microM). A second peptide, comprising residues (1-22) of fallaxin, was also isolated from the skin secretions but this component was inactive against the microorganisms tested.

DNA-fiber EPR investigation of the influence of amino-terminal residue stereochemistry on the DNA binding orientation of Cu(II)•Gly-Gly-His-derived metallopeptides

PubMed Central

Hamada, Hirokazu; Abe, Yuko; Nagane, Ryoichi; Fang, Ya-Yin; Lewis, Mark A.; Long, Eric C.; Chikira, Makoto

2007-01-01

DNA fiber EPR was used to investigate the DNA binding stabilities and orientations of Cu(II)•Gly-Gly-His-derived metallopeptides containing d- vs. l-amino acid substitutions in the first peptide position. This examination included studies of Cu(II)•d-Arg-Gly-His and Cu(II)•d-Lys-Gly-His for comparison to metallopeptides containing l-Arg/Lys substitutions, and also the diastereoisomeric pairs Cu(II)•d/l-Pro-Gly-His and Cu(II)•d/l-Pro-Lys-His. Results indicated that l-Arg/Lys to d-Arg/Lys substitutions considerably randomized the orientation of the metallopeptides on DNA whereas the replacement of l-Pro by d-Pro in Cu(II)•l-Pro-Gly-His caused a decrease in randomness. The difference in the extent of randomness of d- vs. l-Pro-Gly-His complexes was diminished through the substitution of Gly for Lys in the middle peptide position, supporting the notion that the ε-amino group of Lys triggered further randomization, likely through hydrogen bonding or electrostatic interactions that disrupt binding of the metallopeptide equatorial plane and the DNA. The relationship between the stereochemistry of amino acid residues and the binding and reaction of M(II)•Xaa-Xaa’-His metallopeptides with DNA are also discussed. PMID:17706784

Isolation and structural analysis of antihypertensive peptides that exist naturally in Gouda cheese.

PubMed

Saito, T; Nakamura, T; Kitazawa, H; Kawai, Y; Itoh, T

2000-07-01

Seven kinds of ripened cheeses (8-mo-aged and 24-mo-aged Gouda, Emmental, Blue, Camembert, Edam, and Havarti) were homogenized with distilled water, and water-soluble peptides were prepared by C-18 hydrophobic chromatography. The inhibitory activity to angiotensin I-converting enzyme and decrease in the systolic blood pressure in spontaneously hypertensive rats were measured before and after oral administration of each peptide sample. The strongest depressive effect in the systolic blood pressure (-24.7 mm Hg) and intensive inhibitory activity to angiotensin I-converting enzyme (75.7%) were detected in the peptides from 8-mo-aged Gouda cheese. Four peptides were isolated by HPLC with reverse-phase and gel filtration modes. Their chemical structures and origins, clarified by combination analyses of protein sequencing, amino acid composition, and mass spectrometry, were as follows: peptide A, Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln [alpha(s1)-casein (CN), B-8P; f 1-9]; peptide B, Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln (alpha(s1)-CN, B-8P; f 1-13); peptide F, Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn (beta-CN, A2-5P; f 60-68); and peptide G, Met-Pro-Phe-Pro-Lys-Tyr-Pro-Val-Gln-Pro-Phe (beta-CN, A2-5P; f 109-119). Peptides A and F, which were chemically synthesized, showed potent angiotensin I-converting enzyme inhibitory activity with little antihypertensive effects.

Distribution of stable free radicals among amino acids of isolated soy proteins.

PubMed

Lei, Qingxin; Liebold, Christopher M; Boatright, William L; Shah Jahan, M

2010-09-01

Application of deuterium sulfide to powdered isolated soy proteins (ISP) was used to quench stable free radicals and produce a single deuterium label on amino acids where free radicals reside. The deuterium labels rendered increases of isotope ratio for the specific ions of radical-bearing amino acids. Isotope ratio measurements were achieved by gas chromatography/mass spectrometry (GC/MS) analyses after the amino acids were released by acidic hydrolysis and converted to volatile derivatives with propyl chloroformate. The isotope enrichment data showed the stable free radicals were located on Ala, Gly, Leu, Ile, Asx (Asp+Asn), Glx (Glu+Gln), and Trp but not on Val, Pro, Met, Phe, Lys, and His. Due to the low abundance of Ser, Thr, and Cys derivatives and the impossibility to accurately measure their isotope ratios, the radical bearing status for these amino acids remained undetermined even though their derivatives were positively identified from ISP hydrolysates. The relative isotope enrichment for radical-bearing amino acids Ala, Gly, Leu, Ile, Asx (Asp+Asn), Glx (Glu+Gln), and Trp were 8.67%, 2.96%, 2.90%, 3.94%, 6.03%, 3.91%, and 21.48%, respectively. Isotope ratio increase for Tyr was also observed but further investigation revealed such increase was mainly from nonspecific deuterium-hydrogen exchange not free radical quenching. The results obtained from the present study provide important information for a better understanding of the mechanisms of free radical formation and stabilization in "dry" ISP.

Interaction model of steviol glycosides from Stevia rebaudiana (Bertoni) with sweet taste receptors: A computational approach.

PubMed

Mayank; Jaitak, Vikas

2015-08-01

Docking studies were performed on natural sweeteners from Stevia rebaudiana by constructing homology models of T1R2 and T1R3 subunits of human sweet taste receptors. Ramachandran plot, PROCHECK results and ERRAT overall quality factor were used to validate the quality of models. Furthermore, docking results of steviol glycosides (SG's) were correlated significantly with data available in the literature which enabled to predict the exact sweetness rank order of SG's. The binding pattern indicated that Asn 44, Ans 52, Ala 345, Pro 343, Ile 352, Gly 346, Gly 47, Ala 354, Ser 336, Thr 326 and Ser 329 are the main interacting amino acid residues in case of T1R2 and Arg 56, Glu 105, Asp 215, Asp 216, Glu 148, Asp 258, Lys 255, Ser 104, Glu 217, Leu 51, Arg 52 for T1R3, respectively. Amino acids interact with SG's mainly by forming hydrogen bonds with the hydroxyl group of glucose moieties. Significant variation in docked poses of all the SG's were found. In this study, we have proposed the mechanism of the sweetness of the SG's in the form of multiple point stimulation model by considering the diverse binding patterns of various SG's, as well as their structural features. It will give further insight in understanding the differences in the quality of taste and will be used to improve the taste of SG's using semi-synthetic approaches. Copyright © 2015 Elsevier Ltd. All rights reserved.

Functional variants in intercellular adhesion molecule-1 and toll-like receptor-4 genes are more frequent in children with febrile urinary tract infection with renal parenchymal involvement.

PubMed

Hussein, Almontaser; Saad, Khaled; Askar, Eman; Zahran, Asmaa M; Farghaly, Hekma; Metwalley, Kotb; Elderwy, Ahmad A

2018-02-01

We studied the functional polymorphisms of intercellular adhesion molecule-1 (ICAM-1) and toll-like receptor-4 (TLR-4) genes and risk of acute pyelonephritis (APN) in children attending Assiut University Children's Hospitals, Egypt, from 2011 to 2015. Urinary tract infections (UTIs) were diagnosed in 380 children: 98 had APN and 282 had lower UTIs. Four single-nucleotide polymorphisms in ICAM-1 and TLR-4 genes were genotyped in all subjects: ICAM-1 rs1799969 Gly241Arg, ICAM-1 rs5498 Glu469Lys, TLR-4 rs4896791 Thr399Ile and TLR-4 rs4896790 Asp299Gly. Patients with APN were significantly more likely to have AA genotype of the ICAM-1 rs5498 (1462 A/G) polymorphism (p = 0.04) than children with lower UTIs and the TLR-4 Asp299Gly GG genotype (p = 0.002) and G allele (p = 0.006) than healthy controls. The association with the ICAM-1 Glu469Lys (1462A/G) was less evident. The GG genotype was associated with a modest relative risk of 1.4 (p = 0.1) of developing APN, but was not an independent odds ratio, at 1.2 (p = 0.48). Functional variants in ICAM-1 and TLR-4 genes were increasingly common in children with febrile UTIs with renal parenchymal involvement, but the ICAM-1 Glu469Lys (1462A/G) association was less evident. TLR4 Asp299Gly might independently increase renal parenchymal infection rather than renal scarring. ©2017 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

From topical antidote against skin irritants to a novel counter-irritating and anti-inflammatory peptide

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

Brodsky, Berta; Erlanger-Rosengarten, Avigail; Proscura, Elena

2008-06-15

The primary purpose of the present study was to investigate the mechanism of the counter-irritating activity of topical iodine against skin lesions induced by chemical and thermal stimuli. The hypothesis that iodine exerts its activity by inducing an endogenous anti-inflammatory factor was confirmed by exposing guinea pig skin to heat stimulus followed by topical iodine treatment and skin extraction. Injection of the extract into naive guinea pigs reduced heat-induced irritation by 69%. The protective factor, identified as a new nonapeptide (histone H2A 36-44, H-Lys-Gly-Asn-Tyr-Ala-Glu-Arg-Ileu-Ala-OH), caused reduction of 40% in irritation score in heat-exposed guinea pigs. The murine analog (H-Lys-Gly-His-Tyr-Ala-Glu-Arg-Val-Gly-OH, termedmore » IIIM1) reduced sulfur mustard (SM)-induced ear swelling at a dose-dependent bell-shape manner reaching peak activity of 1 mg/kg. Cultured keratinocytes transfected with the peptide were more resistant towards SM than the control cells. The peptide suppressed oxidative burst in activated neutrophils in a concentration-dependent manner. In addition, the peptide reduced glucose oxidase-induced skin edema in mice at a dose-dependent bell-shape manner. Apart from thermal and chemical-induced skin irritation this novel peptide might be of potential use in chronic dermal disorders such as psoriasis and pemphigus as well as non-dermal inflammatory diseases like multiple sclerosis, arthritis and colitis.« less

Characterization of two peptides isolated from the venom of social wasp Chartergellus communis (Hymenoptera: Vespidae): Influence of multiple alanine residues and C-terminal amidation on biological effects.

PubMed

Lopes, Kamila Soares; Campos, Gabriel Avohay Alves; Camargo, Luana Cristina; de Souza, Adolfo Carlos Barros; Ibituruna, Beatriz Vasconcelos; Magalhães, Ana Carolina Martins; da Rocha, Lucas Ferreira; Garcia, Alessa Bembom; Rodrigues, Mosar Correa; Ribeiro, Dagon Manoel; Costa, Michelle Cruz; López, Manuel Humberto Mera; Nolli, Luciana Marangni; Zamudio-Zuniga, Fernando; Possani, Lourival Domingos; Schwartz, Elisabeth Ferroni; Mortari, Márcia Renata

2017-09-01

Chatergellus communis is a wasp species endemic to the neotropical region and its venom constituents have never been described. In this study, two peptides from C. communis venom, denominated Communis and Communis-AAAA, were chemically and biologically characterized. In respect to the chemical characterization, the following amino acid sequences and molecular masses were identified: Communis: Ile-Asn-Trp-Lys-Ala-Ile-Leu-Gly-Lys-Ile-Gly-Lys-COOH (1340.9Da) Communis-AAAA: Ile-Asn-Trp-Lys-Ala-Ile-Leu-Gly-Lys-Ile-Gly-Lys-Ala-Ala-Ala-Ala-Val-Xle-NH 2 (1836.3Da). Furthermore, their biological effects were compared, accounting for the differences in structural characteristics between the two peptides. To this end, three biological assays were performed in order to evaluate the hyperalgesic, edematogenic and hemolytic effects of these molecules. Communis-AAAA, unlike Communis, showed a potent hemolytic activity with EC 50 =142.6μM. Moreover, the highest dose of Communis-AAAA (2nmol/animal) induced hyperalgesia in mice. On the other hand, Communis (10nmol/animal) was able to induce edema but did not present hemolytic or hyperalgesic activity. Although both peptides have similarities in linear structures, we demonstrated the distinct biological effects of Communis and Communis-AAAA. This is the first study with Chartegellus communis venom, and both Communis and Communis-AAAA are unpublished peptides. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2

NASA Technical Reports Server (NTRS)

Chang, D.; Haynes, J. I. 2nd; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

A nuclear localization signal (NLS) has been identified in the C-terminal (Glu307-Glu-Asp-Gly-Pro-Gln-Lys-Lys-Lys-Arg-Arg-Leu318) amino acid sequence of the polyomavirus minor capsid protein VP2. The importance of this amino acid sequence for nuclear transport of newly synthesized VP2 was demonstrated by a genetic "subtractive" study using the constructs pSG5VP2 (expressing full-length VP2) and pSG5 delta 3VP2 (expressing truncated VP2, lacking amino acids Glu307-Leu318). These constructs were transfected into COS-7 cells, and the intracellular localization of the VP2 protein was determined by indirect immunofluorescence. These studies revealed that the full-length VP2 was localized in the nucleus, while the truncated VP2 protein was localized in the cytoplasm and not transported to the nucleus. A biochemical "additive" approach was also used to determine whether this sequence could target nonnuclear proteins to the nucleus. A synthetic peptide identical to VP2 amino acids Glu307-Leu318 was cross-linked to the nonnuclear proteins bovine serum albumin (BSA) or immunoglobulin G (IgG). The conjugates were then labeled with fluorescein isothiocyanate and microinjected into the cytoplasm of NIH 3T6 cells. Both conjugates localized in the nucleus of the microinjected cells, whereas unconjugated BSA and IgG remained in the cytoplasm. Taken together, these genetic subtractive and biochemical additive approaches have identified the C-terminal sequence of polyoma-virus VP2 (containing amino acids Glu307-Leu318) as the NLS of this protein.

The pharmacological properties of the novel peptide BPC 157 (PL-10).

PubMed

Sikiric, P

1999-01-01

The reported beneficial effects of the gastric mucosal derived pentadecapeptide BPC 157 (Gly Glu Pro Pro Pro Gly Lys Pro Ala Asp Asp Ala Gly Leu Val, M.W. 1419) on different organ lesions are reviewed. Apart from the effects on various gastrointestinal lesions, the potentially beneficial effect on pancreas, liver injuries, endothelium and heart damage, i.e. dysrhythmias following reoxygenation, and blood pressure, along with effect on experimental acute/chronic inflammation, wound and fracture (pseudoarthrosis) healing are described. It appears that these beneficial effects all together provide a particular network reflecting activity of a special peptidergic defence system. In support of this concept, it appears that there are interactions of this pentadecapeptide with many important systems (namely, dopamine-, NO-, prostaglandin-, somatosensory neurone-systems), that could provide a basis for the observed protective effects. Moreover, since disturbance of these systems' functions (i.e. dopamine-, NO-, somatosensory neuronal-system) which manifest either over-activity or as inhibition, may contribute to the multiple lesions in different organs. The reported evidence that this pentadecapeptide is able to counteract both their over-action, and their inhibition, may suggest this pentadecapeptide as a new, but most probably essential physiological defence system and that should be further investigated.

Changes of CFTR functional measurements and clinical improvements in cystic fibrosis patients with non p.Gly551Asp gating mutations treated with ivacaftor.

PubMed

Mesbahi, Myriam; Shteinberg, Michal; Wilschanski, Michael; Hatton, Aurelie; Nguyen-Khoa, Thao; Friedman, Hannah; Cohen, Michael; Escabasse, Virginie; Le Bourgeois, Muriel; Lucidi, Vicenzina; Sermet-Gaudelus, Isabelle; Bassinet, Laurence; Livnat, Galit

2017-01-01

Ivacaftor, a CFTR potentiator, has been found to improve CFTR function and clinical outcomes in patients with cystic fibrosis (CF) gating mutations. We investigated the effects of ivacaftor on CFTR functional measurement in CF patients carrying gating mutations other than p.Gly551Asp. Two siblings aged 13 and 12 carrying the p.Ser549Asn mutation, two sisters (45 and 43years old) compound heterozygotes for p.Asp1152His and p.Gly1244Glu, a 37year old man homozygous for the p.Gly1244Glu mutation, and a 7year old girl with p.Arg352Gln and p.Gly1244Glu mutations commenced treatment with ivacaftor. NPD was performed in all the patients and approached normal for four patients who had also clinical improvement (p.Ser549Asn compound heterozygotes, and p.Asp1152His/p.Gly1244Glu siblings). Beta-adrenergic sweat chloride secretion performed in thep.Asp1152His/p.Gly1244Glu patients improved significantly. The p.Gly1244Glu mutation homozygous patient, who had undergone an ileal resection with ileostomy and enterocutaneous fistula, did not respond clinically to ivacaftor and did not modify his sweat test. These results highlight the importance of different CFTR activity measurements to explore CFTR modulator efficacy. Copyright © 2016. Published by Elsevier B.V.

Binding of Cu(II) ions to peptides studied by fluorescence spectroscopy and isothermal titration calorimetry.

PubMed

Makowska, Joanna; Żamojć, Krzysztof; Wyrzykowski, Dariusz; Uber, Dorota; Wierzbicka, Małgorzata; Wiczk, Wiesław; Chmurzyński, Lech

2016-01-15

Steady-state and time-resolved fluorescence quenching measurements supported by Isothermal Titration Calorimetry (ITC) were used to study the interactions of Cu(2+) with four peptides. Two of them were taken from the N-terminal part of the FBP28 protein (formin binding protein) WW domain: Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH2 (D9) and its mutant Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr-NH2 (D9_M) as well as two mutated peptides from the B3 domain of the immunoglobulin binding protein G derived from Streptococcus: Asp-Val-Ala-Thr-Tyr-Thr-NH2 (J1) and Glu-Val-Ala-Thr-Tyr-Thr-NH2 (J2). The measurements were carried out at 298.15K in 20mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution with a pH of 6. The fluorescence of all peptides was quenched by Cu(2+) ions. The stoichiometry, conditional stability constants and thermodynamic parameters for the interactions of the Cu(2+) ions with D9 and D9_M were determined from the calorimetric data. The values of the conditional stability constants were additionally determined from fluorescence quenching measurements and compared with those obtained from calorimetric studies. There was a good correlation between data obtained from the two techniques. On the other hand, the studies revealed that J1 and J2 do not exhibit an affinity towards metal ions. The obtained results prove that fluorescence quenching experiments may be successfully used in order to determine stability constants of complexes with fluorescent ligands. Finally, based on the obtained results, the coordinating properties of the peptides towards the Cu(2+) ions are discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Binding of Cu(II) ions to peptides studied by fluorescence spectroscopy and isothermal titration calorimetry

NASA Astrophysics Data System (ADS)

Makowska, Joanna; Żamojć, Krzysztof; Wyrzykowski, Dariusz; Uber, Dorota; Wierzbicka, Małgorzata; Wiczk, Wiesław; Chmurzyński, Lech

2016-01-01

Steady-state and time-resolved fluorescence quenching measurements supported by Isothermal Titration Calorimetry (ITC) were used to study the interactions of Cu2 + with four peptides. Two of them were taken from the N-terminal part of the FBP28 protein (formin binding protein) WW domain: Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH2 (D9) and its mutant Tyr-Lys-Thr-Ala-Asn-Gly-Lys-Thr-Tyr-NH2 (D9_M) as well as two mutated peptides from the B3 domain of the immunoglobulin binding protein G derived from Streptococcus: Asp-Val-Ala-Thr-Tyr-Thr-NH2 (J1) and Glu-Val-Ala-Thr-Tyr-Thr-NH2 (J2). The measurements were carried out at 298.15 K in 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution with a pH of 6. The fluorescence of all peptides was quenched by Cu2 + ions. The stoichiometry, conditional stability constants and thermodynamic parameters for the interactions of the Cu2 + ions with D9 and D9_M were determined from the calorimetric data. The values of the conditional stability constants were additionally determined from fluorescence quenching measurements and compared with those obtained from calorimetric studies. There was a good correlation between data obtained from the two techniques. On the other hand, the studies revealed that J1 and J2 do not exhibit an affinity towards metal ions. The obtained results prove that fluorescence quenching experiments may be successfully used in order to determine stability constants of complexes with fluorescent ligands. Finally, based on the obtained results, the coordinating properties of the peptides towards the Cu2 + ions are discussed.

Severity of experimental traumatic brain injury modulates changes in concentrations of cerebral free amino acids.

PubMed

Amorini, Angela Maria; Lazzarino, Giacomo; Di Pietro, Valentina; Signoretti, Stefano; Lazzarino, Giuseppe; Belli, Antonio; Tavazzi, Barbara

2017-03-01

In this study, concentrations of free amino acids (FAA) and amino group containing compounds (AGCC) following graded diffuse traumatic brain injury (mild TBI, mTBI; severe TBI, sTBI) were evaluated. After 6, 12, 24, 48 and 120 hr aspartate (Asp), glutamate (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), histidine (His), glycine (Gly), threonine (Thr), citrulline (Cit), arginine (Arg), alanine (Ala), taurine (Tau), γ-aminobutyrate (GABA), tyrosine (Tyr), S-adenosylhomocysteine (SAH), l-cystathionine (l-Cystat), valine (Val), methionine (Met), tryptophane (Trp), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), ornithine (Orn), lysine (Lys), plus N-acetylaspartate (NAA) were determined in whole brain extracts (n = 6 rats at each time for both TBI levels). Sham-operated animals (n = 6) were used as controls. Results demonstrated that mTBI caused modest, transient changes in NAA, Asp, GABA, Gly, Arg. Following sTBI, animals showed profound, long-lasting modifications of Glu, Gln, NAA, Asp, GABA, Ser, Gly, Ala, Arg, Citr, Tau, Met, SAH, l-Cystat, Tyr and Phe. Increase in Glu and Gln, depletion of NAA and Asp increase, suggested a link between NAA hydrolysis and excitotoxicity after sTBI. Additionally, sTBI rats showed net imbalances of the Glu-Gln/GABA cycle between neurons and astrocytes, and of the methyl-cycle (demonstrated by decrease in Met, and increase in SAH and l-Cystat), throughout the post-injury period. Besides evidencing new potential targets for novel pharmacological treatments, these results suggest that the force acting on the brain tissue at the time of the impact is the main determinant of the reactions ignited and involving amino acid metabolism. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Familial hypofibrinogenaemia associated with heterozygous substitution of a conserved arginine residue; Bbeta255 Arg-->His (Fibrinogen Merivale).

PubMed

Maghzal, Ghassan J; Brennan, Stephen O; Fellowes, Andrew P; Spearing, Ruth; George, Peter M

2003-02-21

Sequencing of all three fibrinogen genes from an individual with hypofibrinogenaemia led to the identification of two new point mutations in the Bbeta gene. Family studies showed the mutations Bbeta255 Arg-->His (Fibrinogen Merivale) and Bbeta148 Lys-->Asn (Fibrinogen Merivale II) were on different alleles and that only the Bbeta255 Arg-->His mutation segregated with hypofibrinogenaemia. Three simple heterozygotes for this mutation had mean fibrinogen concentrations of 1.4 mg/ml, while heterozygotes for the Bbeta148 Lys-->Asn mutation had normal fibrinogen concentrations. ESI MS analysis of endoproteinase Asp-N digests of Bbeta chains showed that the Bbeta255 Arg-->His substitution was not expressed in plasma, confirming it as the cause of the hypofibrinogenaemia. The Bbeta148 Lys-->Asn chains, on the other hand, were equally expressed with wild-type Bbeta chains in simple heterozygotes. Genotype analysis failed to detect either substitution in 182 healthy controls. Arg(255) is located in the first strand of the five-stranded sheet that forms the main feature of the betaD domain and appears to form an essential H bond with Gly(414). Both the Arg and Gly are absolutely conserved, not only in all known Bbeta chains, but also in all homologous alphaE and gamma chains and in all fibrinogen-related proteins. Protein instability from loss of this contact could easily explain the association of this mutation with hypofibrinogenaemia.

Ceftazidime-Resistant Enterobacteriaceae Isolates from Three Polish Hospitals: Identification of Three Novel TEM- and SHV-5-Type Extended-Spectrum β-Lactamases

PubMed Central

Gniadkowski, Marek; Schneider, Ines; Jungwirth, Renate; Hryniewicz, Waleria; Bauernfeind, Adolf

1998-01-01

Twelve ceftazidime-resistant isolates of the family Enterobacteriaceae (11 Klebsiella pneumoniae isolates and 1 Escherichia coli isolate) were collected in 1995 from three Polish hospitals located in different cities. All were identified as producers of extended-spectrum β-lactamases (ESBLs). Detailed analysis of their β-lactamase contents revealed that six of them expressed SHV-5-like ESBLs. The remaining six were found to produce three different TEM enzymes, each characterized by a pI value of 6.0 and specified by new combinations of amino acid substitutions. The amino acid substitutions compared to the TEM-1 β-lactamase sequence were Gly238Ser, Glu240Lys, and Thr265Met for TEM-47; Leu21Phe, Gly238Ser, Glu240Lys, and Thr265Met for TEM-48; and Leu21Phe, Gly238Ser, Glu240Lys, Thr265Met, and Ser268Gly for TEM-49. The new TEM β-lactamases, TEM-47, TEM-48, and TEM-49, belong to a subfamily of TEM-2-related enzymes. Genes coding for TEM-47 and TEM-49 could have originated from the TEM-48-encoding sequence by various single genetic events. The new TEM derivatives probably document the already advanced microevolution of ESBLs ongoing in Polish hospitals, in a majority of which no monitoring of ESBL producers was performed before 1996. PMID:9517925

Structural analysis of peptides that fill sites near the active center of the two different enzyme molecules by artificial intelligence and computer simulations

NASA Astrophysics Data System (ADS)

Nishiyama, Katsuhiko

2018-05-01

Using artificial intelligence, the binding styles of 167 tetrapeptides were predicted in the active site of papain and cathepsin K. Five tetrapeptides (Asn-Leu-Lys-Trp, Asp-Gln-Trp-Gly, Cys-Gln-Leu-Arg, Gln-Leu-Trp-Thr and Arg-Ser-Glu-Arg) were found to bind sites near the active center of both papain and cathepsin K. These five tetrapeptides have the potential to also bind sites of other cysteine proteases, and structural characteristics of these tetrapeptides should aid the design of a common inhibitor of cysteine proteases. Smart application of artificial intelligence should accelerate data mining of important complex systems.

Effects of peptides on generation of reactive oxygen species in subcellular fractions of Drosophila melanogaster.

PubMed

Khavinson, V K; Myl'nikov, S V; Oparina, T I; Arutyunyan, A V

2001-07-01

We studied the effects of Epithalon (Ala-Glu-Asp-Gly) and Vilon (Lys-Glu) on free radical processes in highly inbred HA(+)line of Drosophila melanogaster. Vilon inhibited generation of reactive oxygen species in mitochondria, but stimulated this process in the cytosol. We found sex- and age-related differences in the generation of reactive oxygen species and cytosol antioxidant activity.

Investigation of jumbo squid (Dosidicus gigas) skin gelatin peptides for their in vitro antioxidant effects.

PubMed

Mendis, Eresha; Rajapakse, Niranjan; Byun, Hee-Guk; Kim, Se-Kwon

2005-09-09

Peptides derived from tryptic hydrolysate of jumbo squid (Dosidicus gigas) skin gelatin were assessed for their antioxidant properties in different in vitro assay systems. The hydrolysate itself exhibited a strong lipid peroxidation inhibition and it was much higher than that of natural antioxidant, alpha-tocopherol. In addition, it could scavenge highly active free radicals in oxidative systems, in the order of hydroxyl and carbon-centered radicals. Two representative peptides with comparatively higher antioxidant potency were purified and characterized as Phe-Asp-Ser-Gly-Pro-Ala-Gly-Val-Leu (880.18 Da) and Asn-Gly-Pro-Leu-Gln-Ala-Gly-Gln-Pro-Gly-Glu-Arg (1241.59 Da). Furthermore, viability of radical-mediated oxidation-induced human lung fibroblasts was enhanced following the treatment of two peptides. However it did not exhibit substantial ion chelation, and we presumed that the observed radical scavenging potency of these peptides play a vital role for their strong antioxidant activity. Based on our results we suggest that hydrophobic amino acids present in peptide sequences contributed greatly for observed antioxidant activities.

Fluorimetric assay of the neurotensin-degrading metalloendopeptidase, endopeptidase 24.16.

PubMed

Dauch, P; Barelli, H; Vincent, J P; Checler, F

1991-12-01

Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp (Mcc = 3-carboxy-7-methoxycoumarin; Dnp = dinitrophenyl), a quenched fluorimetric substrate originally designed as a probe to measure Pz-peptidase (also called endopeptidase 24.15), was examined as a putative substrate of the neurotensin-degrading neutral metalloendopeptidase, endopeptidase 24.16. During the purification of endopeptidase 24.16 the neurotensin(1-10) and neurotensin(11-13) formation due to this enzyme was coeluted with Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp-hydrolysing activity. By both fluorimetric and h.p.l.c. analyses, we observed that the latter activity was dose-dependently and completely abolished by neurotensin with an IC50 value (2.6 microM) that closely corresponds to the affinity of purified endopeptidase 24.16 for neurotensin (Km = 2.5 microM). Furthermore, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp hydrolysis was inhibited by a series of dipeptides with a rank of order of potencies that parallels that observed in competition experiments of tritiated neurotensin hydrolysis by brain and intestinal endopeptidase 24.16. Altogether, these data clearly demonstrate that, in addition to Pz-peptidase, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp also behaves as a substrate of endopeptidase 24.16, with a Km of about 26 microM. In addition, we show that, even in crude membrane preparations, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp behaves as a useful tool to monitor and accurately quantify endopeptidase 24.16.

Fluorimetric assay of the neurotensin-degrading metalloendopeptidase, endopeptidase 24.16.

PubMed Central

Dauch, P; Barelli, H; Vincent, J P; Checler, F

1991-01-01

Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp (Mcc = 3-carboxy-7-methoxycoumarin; Dnp = dinitrophenyl), a quenched fluorimetric substrate originally designed as a probe to measure Pz-peptidase (also called endopeptidase 24.15), was examined as a putative substrate of the neurotensin-degrading neutral metalloendopeptidase, endopeptidase 24.16. During the purification of endopeptidase 24.16 the neurotensin(1-10) and neurotensin(11-13) formation due to this enzyme was coeluted with Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp-hydrolysing activity. By both fluorimetric and h.p.l.c. analyses, we observed that the latter activity was dose-dependently and completely abolished by neurotensin with an IC50 value (2.6 microM) that closely corresponds to the affinity of purified endopeptidase 24.16 for neurotensin (Km = 2.5 microM). Furthermore, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp hydrolysis was inhibited by a series of dipeptides with a rank of order of potencies that parallels that observed in competition experiments of tritiated neurotensin hydrolysis by brain and intestinal endopeptidase 24.16. Altogether, these data clearly demonstrate that, in addition to Pz-peptidase, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp also behaves as a substrate of endopeptidase 24.16, with a Km of about 26 microM. In addition, we show that, even in crude membrane preparations, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp behaves as a useful tool to monitor and accurately quantify endopeptidase 24.16. PMID:1747117

Gallium-67-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone peptide for primary and metastatic melanoma imaging.

PubMed

Guo, Haixun; Yang, Jianquan; Shenoy, Nalini; Miao, Yubin

2009-12-01

The purpose of this study was to examine the melanoma imaging properties of a novel 67Ga-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone (alpha-MSH) peptide. A lactam bridge-cyclized alpha-MSH peptide, DOTA-GlyGlu-CycMSH {DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]}, was synthesized and radiolabeled with 67Ga. The melanoma targeting and pharmacokinetic properties of 67Ga-DOTA-GlyGlu-CycMSH were determined in B16/F1 flank primary melanoma-bearing and B16/F10 pulmonary metastatic melanoma-bearing C57 mice. Flank primary melanoma and pulmonary metastatic melanoma imaging were performed by small animal single photon emission computed tomography (SPECT)/CT using 67Ga-DOTA-GlyGlu-CycMSH as an imaging probe. 67Ga-DOTA-GlyGlu-CycMSH was readily prepared with greater than 95% radiolabeling yield. 67Ga-DOTA-GlyGlu-CycMSH exhibited substantial tumor uptake (12.93 +/- 1.63%ID/g at 2 h postinjection) and prolonged tumor retention (5.02 +/- 1.35%ID/g at 24 h postinjection) in B16/F1 melanoma-bearing C57 mice. The uptake values for nontarget organs were generally low (<0.30%ID/g) except for the kidneys at 2, 4, and 24 h postinjection. 67Ga-DOTA-GlyGlu-CycMSH exhibited significantly (p < 0.05) higher uptakes (1.44 +/- 0.75%ID/g at 2 h postinjection and 1.49 +/- 0.69%ID/g at 4 h postinjection) in metastatic melanoma-bearing lung than those in normal lung (0.15 +/- 0.10%ID/g and 0.17 +/- 0.11%ID/g at 2 and 4 h postinjection, respectively). Both flank primary B16/F1 melanoma and B16/F10 pulmonary melanoma metastases were clearly visualized by SPECT/CT using 67Ga-DOTA-GlyGlu-CycMSH as an imaging probe 2 h postinjection. 67Ga-DOTA-GlyGlu-CycMSH exhibited favorable melanoma targeting and imaging properties, highlighting its potential as an effective imaging probe for early detection of primary and metastatic melanoma.

Development and characterisation of highly antibiotic resistant Bartonella bacilliformis mutants

PubMed Central

Gomes, Cláudia; Martínez-Puchol, Sandra; Ruiz-Roldán, Lidia; Pons, Maria J.; del Valle Mendoza, Juana; Ruiz, Joaquim

2016-01-01

The objective was to develop and characterise in vitro Bartonella bacilliformis antibiotic resistant mutants. Three B. bacilliformis strains were plated 35 or 40 times with azithromycin, chloramphenicol, ciprofloxacin or rifampicin discs. Resistance-stability was assessed performing 5 serial passages without antibiotic pressure. MICs were determined with/without Phe-Arg-β-Napthylamide and artesunate. Target alterations were screened in the 23S rRNA, rplD, rplV, gyrA, gyrB, parC, parE and rpoB genes. Chloramphenicol and ciprofloxacin resistance were the most difficult and easiest (>37.3 and 10.6 passages) to be selected, respectively. All mutants but one selected with chloramphenicol achieved high resistance levels. All rifampicin, one azithromycin and one ciprofloxacin mutants did not totally revert when cultured without antibiotic pressure. Azithromycin resistance was related to L4 substitutions Gln-66 → Lys or Gly-70 → Arg; L4 deletion Δ62–65 (Lys-Met-Tyr-Lys) or L22 insertion 83::Val-Ser-Glu-Ala-His-Val-Gly-Lys-Ser; in two chloramphenicol-resistant mutants the 23S rRNA mutation G2372A was detected. GyrA Ala-91 → Val and Asp-95 → Gly and GyrB Glu474 → Lys were detected in ciprofloxacin-resistant mutants. RpoB substitutions Gln-527 → Arg, His-540 → Tyr and Ser-545 → Phe plus Ser-588 → Tyr were detected in rifampicin-resistant mutants. In 5 mutants the effect of efflux pumps on resistance was observed. Antibiotic resistance was mainly related to target mutations and overexpression of efflux pumps, which might underlie microbiological failures during treatments. PMID:27667026

A molecular dynamics simulation study decodes the Zika virus NS5 methyltransferase bound to SAH and RNA analogue.

PubMed

Chuang, Chih-Hung; Chiou, Shean-Jaw; Cheng, Tian-Lu; Wang, Yeng-Tseng

2018-04-20

Since 2015, widespread Zika virus outbreaks in Central and South America have caused increases in microcephaly cases, and this acute problem requires urgent attention. We employed molecular dynamics and Gaussian accelerated molecular dynamics techniques to investigate the structure of Zika NS5 protein with S-adenosyl-L-homocysteine (SAH) and an RNA analogue, namely 7-methylguanosine 5'-triphosphate (m7GTP). For the binding motif of Zika virus NS5 protein and SAH, we suggest that the four Zika NS5 substructures (residue orders: 101-112, 54-86, 127-136 and 146-161) and the residues (Ser56, Gly81, Arg84, Trp87, Thr104, Gly106, Gly107, His110, Asp146, Ile147, and Gly148) might be responsible for the selectivity of the new Zika virus drugs. For the binding motif of Zika NS5 protein and m7GTP, we suggest that the three Zika NS5 substructures (residue orders: 11-31, 146-161 and 207-218) and the residues (Asn17, Phe24, Lys28, Lys29, Ser150, Arg213, and Ser215) might be responsible for the selectivity of the new Zika virus drugs.

A novel prosthetic group for site-selective labeling of peptides for positron emission tomography.

PubMed

Olberg, Dag Erlend; Hjelstuen, Ole Kristian; Solbakken, Magne; Arukwe, Joseph; Karlsen, Hege; Cuthbertson, Alan

2008-06-01

Efficient methodologies for the radiolabeling of peptides with [(18)F]fluoride are a prerequisite to enabling commercialization of peptide-containing radiotracers for positron emission tomography (PET) imaging. It was the purpose of this study to investigate a novel chemoselective ligation reaction comprising conjugation of an [(18)F]-N-methylaminooxy-containing prosthetic group to a functionalized peptide. Twelve derivatives of general formula R1-CO-NH-Lys-Gly-Phe-Gly-Lys-OH were synthesized where R1 was selected from a short list of moieties anticipated to be reactive toward the N-methylaminooxy group. Conjugation reactions were initially carried out with nonradioactive precursors to assess, in a qualitative manner, their general suitability for PET chemistry with only the most promising pairings progressing to full radiochemical assessment. Best results were obtained for the ligation of O-[2-(2-[(18)F]fluoroethoxy)ethyl]-N-methyl-N-hydroxylamine 18 to the maleimidopropionyl-Lys-Gly-Phe-Gly-Lys-OH precursor 10 in acetate buffer (pH 5) after 1 h at 70 degrees C. The non-decay-corrected isolated yield was calculated to be 8.5%. The most encouraging result was observed with the combination 18 and 4-(2-nitrovinyl)benzoyl-Lys-Gly-Phe-Gly-Lys-OH, 9, where the conjugation reaction proceeded rapidly to completion at 30 degrees C after only 5 min. The corresponding non-decay-corrected radiochemical yield for the isolated (18)F-labeled product 27 was 12%. The preliminary results from this study demonstrate the considerable potential of this novel strategy for the radiolabeling of peptides.

Diastereoselective DNA Cleavage Recognition by Ni(II)•Gly-Gly-His Derived Metallopeptides

PubMed Central

Fang, Ya-Yin; Claussen, Craig A.; Lipkowitz, Kenny B.; Long, Eric C.

2008-01-01

Site-selective DNA cleavage by diastereoisomers of Ni(II)•Gly-Gly-His-derived metallopeptides was investigated through high-resolution gel analyses and molecular dynamics simulations. Ni(II)•L-Arg-Gly-His and Ni(II)•D-Arg-Gly-His (and their respective Lys analogues) targeted A/T-rich regions; however, the L-isomers consistently modified a sub-set of available nucleotides within a given minor groove site while the D-isomers differed in both their sites of preference and ability to target individual nucleotides within some sites. In comparison, Ni(II)•L-Pro-Gly-His and Ni(II)•D-Pro-Gly-His were unable to exhibit a similar diastereoselectivity. Simulations of the above systems, along with Ni(II)•Gly-Gly-His, indicated that the stereochemistry of the amino-terminal amino acid produces either an isohelical metallopeptide that associates stably at individual DNA sites (L-Arg or L-Lys) or, with D-Arg and D-Lys, a non-complementary metallopeptide structure that cannot fully employ its side chain nor amino-terminal amine as a positional stabilizing moiety. In contrast, amino-terminal Pro-containing metallopeptides of either stereochemistry, lacking an extended side chain directed toward the minor groove, did not exhibit a similar diastereoselectivity. While the identity and stereochemistry of amino acids located in the amino-terminal peptide position influenced DNA cleavage, metallopeptide diastereoisomers containing L- and D-Arg (or Lys) within the second peptide position did not exhibit diastereoselective DNA cleavage patterns; simulations indicated that a positively-charged amino acid in this location alters the interaction of the metallopeptide equatorial plane and the minor groove leading to an interaction similar to Ni(II)•Gly-Gly-His. PMID:16522100

Penultimate proline in neuropeptides.

PubMed

Glover, Matthew S; Bellinger, Earl P; Radivojac, Predrag; Clemmer, David E

2015-08-18

A recent ion mobility spectrometry-mass spectrometry (IMS-MS) study revealed that tryptic peptide ions containing a proline residue at the second position from the N-terminus (i.e., penultimate proline) frequently adopt multiple conformations, owing to the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds [J. Am. Soc. Mass Spectrom. 2015, 26, 444]. Here, we present a statistical analysis of a neuropeptide database that illustrates penultimate proline residues are frequently found in neuropeptides. In order to probe the effect of penultimate proline on neuropeptide conformations, IMS-MS experiments were performed on two model peptides in which penultimate proline residues were known to be important for biological activity: the N-terminal region of human neuropeptide Y (NPY1-9, Tyr(1)-Pro(2)-Ser(3)-Lys(4)-Pro(5)-Asp(6)-Asn(7)-Pro(8)-Gly(9)-NH2) and a tachykinin-related peptide (CabTRP Ia, Ala(1)-Pro(2)-Ser(3)-Gly(4)-Phe(5)-Leu(6)-Gly(7)-Met(8)-Arg(9)-NH2). From these studies, it appears that penultimate prolines allow neuropeptides to populate multiple conformations arising from the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds. Although it is commonly proposed that the role of penultimate proline residues is to protect peptides from enzymatic degradation, the present results indicate that penultimate proline residues also are an important means of increasing the conformational heterogeneity of neuropeptides.

Evaluation of a novel Arg-Gly-Asp-conjugated α-melanocyte stimulating hormone hybrid peptide for potential melanoma therapy.

PubMed

Yang, Jianquan; Guo, Haixun; Gallazzi, Fabio; Berwick, Marianne; Padilla, R Steven; Miao, Yubin

2009-08-19

The purpose of this study was to determine whether Arg-Gly-Asp (RGD)-conjugated α-melanocyte stimulating hormone (α-MSH) hybrid peptide could be employed to target melanocortin-1 (MC1) receptor for potential melanoma therapy. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys(3,4,10), DPhe(7), Arg(11)]α-MSH(3-13) {(Arg(11))CCMSH} to generate RGD-Lys-(Arg(11))CCMSH hybrid peptide. The MC1 receptor binding affinity of RGD-Lys-(Arg(11))CCMSH was determined in B16/F1 melanoma cells. The internalization and efflux, melanoma targeting and pharmacokinetic properties and single photon emission computed tomography/CT (SPECT/CT) imaging of (99m)Tc-RGD-Lys-(Arg(11))CCMSH were determined in B16/F1 melanoma cells and melanoma-bearing C57 mice. Clonogenic cytotoxic effect of RGD-Lys-(Arg(11))CCMSH was examined in B16/F1 melanoma cells. RGD-Lys-(Arg(11))CCMSH displayed 2.1 nM MC1 receptor binding affinity. (99m)Tc-RGD-Lys-(Arg(11))CCMSH showed rapid internalization and extended retention in B16/F1 cells. The cellular uptake of (99m)Tc-RGD-Lys-(Arg(11))CCMSH was MC1 receptor-mediated. (99m)Tc-RGD-Lys-(Arg(11))CCMSH exhibited high tumor uptake (14.83 ± 2.94% ID/g 2 h postinjection) and prolonged tumor retention (7.59 ± 2.04% ID/g 24 h postinjection) in B16/F1 melanoma-bearing mice. Nontarget organ uptakes were generally low except for the kidneys. Whole-body clearance of (99m)Tc-RGD-Lys-(Arg(11))CCMSH was rapid, with approximately 62% of the injected radioactivity cleared through the urinary system by 2 h postinjection. Flank melanoma tumors were clearly imaged by small animal SPECT/CT using (99m)Tc-RGD-Lys-(Arg(11))CCMSH as an imaging probe 2 h postinjection. Single treatment (3 h incubation) with 100 nM of RGD-Lys-(Arg(11))CCMSH significantly (p < 0.05) decreased the clonogenic survival of B16/F1 cells by 65% compared to the untreated control cells. Favorable melanoma targeting property of (99m)Tc-RGD-Lys-(Arg(11))CCMSH and remarkable cytotoxic effect of RGD-Lys-(Arg(11))CCMSH in B16/F1 cells warranted the further evaluation of (188)Re-labeled α-MSH hybrid peptides as novel therapeutic peptides for melanoma treatment once the strategies of amino acid coinjection or structural modification of peptide sequence substantially reduce the renal uptake.

Sequence composition and environment effects on residue fluctuations in protein structures

NASA Astrophysics Data System (ADS)

Ruvinsky, Anatoly M.; Vakser, Ilya A.

2010-10-01

Structure fluctuations in proteins affect a broad range of cell phenomena, including stability of proteins and their fragments, allosteric transitions, and energy transfer. This study presents a statistical-thermodynamic analysis of relationship between the sequence composition and the distribution of residue fluctuations in protein-protein complexes. A one-node-per-residue elastic network model accounting for the nonhomogeneous protein mass distribution and the interatomic interactions through the renormalized inter-residue potential is developed. Two factors, a protein mass distribution and a residue environment, were found to determine the scale of residue fluctuations. Surface residues undergo larger fluctuations than core residues in agreement with experimental observations. Ranking residues over the normalized scale of fluctuations yields a distinct classification of amino acids into three groups: (i) highly fluctuating-Gly, Ala, Ser, Pro, and Asp, (ii) moderately fluctuating-Thr, Asn, Gln, Lys, Glu, Arg, Val, and Cys, and (iii) weakly fluctuating-Ile, Leu, Met, Phe, Tyr, Trp, and His. The structural instability in proteins possibly relates to the high content of the highly fluctuating residues and a deficiency of the weakly fluctuating residues in irregular secondary structure elements (loops), chameleon sequences, and disordered proteins. Strong correlation between residue fluctuations and the sequence composition of protein loops supports this hypothesis. Comparing fluctuations of binding site residues (interface residues) with other surface residues shows that, on average, the interface is more rigid than the rest of the protein surface and Gly, Ala, Ser, Cys, Leu, and Trp have a propensity to form more stable docking patches on the interface. The findings have broad implications for understanding mechanisms of protein association and stability of protein structures.

Proteolytic processing of the pro beta chain of beta-hexosaminidase occurs at basic residues contained within an exposed disulfide loop structure.

PubMed

Sagherian, C; Poroszlay, S; Vavougios, G; Mahuran, D

1993-01-01

Lysosomal beta-hexosaminidase (EC 3.2.1.52) occurs as two major isozymes, Hex A (alpha beta) and Hex B (beta beta). The alpha and beta subunits are encoded by the HEXA and HEXB genes, respectively. Extensive homology in both the gene structures and deduced primary sequences demonstrate their common evolutionary origin. While undergoing similar proteolytic modifications in the lysosome, the pro beta polypeptide is additionally cleaved internally to produce the mature 24-30 kilodalton beta b and beta a chains. Previous data have suggested that this processing event occurs somewhere between residues Ser311 and Lys315. In this report we demonstrate that this area is located in a hydrophilic disulfide-loop structure (between Cys309 and Cys360). The cleavage event is prevented by the deletion through in vitro mutagenesis of the Arg312-Gln-Asn-Lys tetrapeptide or by its substitution with the aligned alpha residues (Gly-Ser-Glu-Pro). Reintroduction of either Arg312 or Lys315 reinstates the processing. Furthermore, we show that this area is not involved in lysosomal targeting of pro-Hex B, or in the increased stability or the variation in substrate specificity of the beta as compared with the alpha subunit. Our data suggest the presence of a novel lysosomal endoprotease. Like other endoproteases it is specific for basic amino acids; however, it cleaves on the amino-terminal side rather than the conventional carboxy-terminal side of such residues and then only if they are fully exposed to the lysosomal environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Iron depletion strategy for targeted cancer therapy: utilizing the dual roles of neutrophil gelatinase-associated lipocalin protein.

PubMed

Tang, Hsin-Chieh; Chang, Pei-Chun; Chen, Yu-Chian

2016-01-01

Decreasing iron uptake and increasing iron efflux may result in cell death by oxidative inactivation of vital enzymes. Applying the dual function of neutrophil gelatinase-associated lipocalin (NGAL) could achieve the goal of iron depletion in the cancer cells. Tyr106, Lys125 or Lys134 was the key binding site for NGAL protein to sequester iron-chelating siderophores. In this study, we employed all bioactive peptides in peptide databank to dock with the siderophore-binding sites of NGAL protein by virtual screening. In addition, we performed molecular dynamics (MD) simulation to observe the molecular character and structural variation of ligand-protein interaction. Glu-Glu-Lys-Glu (EEKE), Glu-Glu-Asp-Cys-Lys (EEDCK), and Gly-Glu-Glu-Cys-Asp (GEECD) were selected preliminarily by rigorous scoring functions for further investigation. GEECD was excluded due to higher binding total energy than the others. Moreover, we also excluded EEKE due to larger influence to the stability of binding residues by the information of root mean square fluctuation (RMSF) and principal component analysis (PCA). Thus, we suggested that EEDCK was the potential bioactive peptide which had been proved to inhibit malignant cells for targeted cancer therapy. Graphical Abstract Perspective drug design of occupying the siderophore-binding sites of NGAL outside the cell temporarily by a potential short peptide until NGAL enters into the cell, and releasing the siderophore-binding sites inside the cell.

Penetration of short fluorescence-labeled peptides into the nucleus in HeLa cells and in vitro specific interaction of the peptides with deoxyribooligonucleotides and DNA.

PubMed

Fedoreyeva, L I; Kireev, I I; Khavinson, V Kh; Vanyushin, B F

2011-11-01

Marked fluorescence in cytoplasm, nucleus, and nucleolus was observed in HeLa cells after incubation with each of several fluorescein isothiocyanate-labeled peptides (epithalon, Ala-Glu-Asp-Gly; pinealon, Glu-Asp-Arg; testagen, Lys-Glu-Asp-Gly). This means that short biologically active peptides are able to penetrate into an animal cell and its nucleus and, in principle they may interact with various components of cytoplasm and nucleus including DNA and RNA. It was established that various initial (intact) peptides differently affect the fluorescence of the 5,6-carboxyfluorescein-labeled deoxyribooligonucleotides and DNA-ethidium bromide complexes. The Stern-Volmer constants characterizing the degree of fluorescence quenching of various single- and double-stranded fluorescence-labeled deoxyribooligonucleotides with short peptides used were different depending on the peptide primary structures. This indicates the specific interaction between short biologically active peptides and nucleic acid structures. On binding to them, the peptides discriminate between different nucleotide sequences and recognize even their cytosine methylation status. Judging from corresponding constants of the fluorescence quenching, the epithalon, pinealon, and bronchogen (Ala-Glu-Asp-Leu) bind preferentially with deoxyribooligonucleotides containing CNG sequence (CNG sites are targets for cytosine DNA methylation in eukaryotes). Epithalon, testagen, and pinealon seem to preferentially bind with CAG- but bronchogen with CTG-containing sequences. The site-specific interactions of peptides with DNA can control epigenetically the cell genetic functions, and they seem to play an important role in regulation of gene activity even at the earliest stages of life origin and in evolution.

Penicillium sp. mitigates Fusarium-induced biotic stress in sesame plants.

PubMed

Radhakrishnan, Ramalingam; Pae, Suk-Bok; Shim, Kang-Bo; Baek, In-Youl

2013-07-01

Fusarium-infected sesame plants have significantly higher contents of amino acids (Asp, Thr, Ser, Asn, Glu, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Try, Arg, and Pro), compared with their respective levels in the healthy control. These higher levels of amino acids induced by Fusarium infection were decreased when Penicillium was co-inoculated with Fusarium. Compared with the control, Fusarium-infected plants showed higher contents of palmitic (8%), stearic (8%), oleic (7%), and linolenic acids (4%), and lower contents of oil (4%) and linoleic acid (11%). Co-inoculation with Penicillium mitigated the Fusarium-induced changes in fatty acids. The total chlorophyll content was lower in Fusarium- and Penicillium-infected plants than in the healthy control. The accumulation of carotenoids and γ-amino butyric acid in Fusarium-infected plants was slightly decreased by co-inoculation with Penicillium. Sesamin and sesamolin contents were higher in Penicillium- and Fusarium- infected plants than in the control. To clarify the mechanism of the biocontrol effect of Penicillium against Fusarium by evaluating changes in primary and secondary metabolite contents in sesame plants.

Deficiencies in pro-thyrotropin-releasing hormone processing and abnormalities in thermoregulation in Cpefat/fat mice.

PubMed

Nillni, Eduardo A; Xie, Weihua; Mulcahy, Lawrence; Sanchez, Vanesa C; Wetsel, William C

2002-12-13

Cpe(fat/fat) mice are obese, diabetic, and infertile. They have a mutation in carboxypeptidase E (CPE), an enzyme that converts prohormone intermediates to bioactive peptides. The Cpe(fat) mutation leads to rapid degradation of the enzyme. To test whether pro-thyrotropin-releasing hormone (TRH) conversion to TRH involves CPE, processing was examined in the Cpe(fat/fat) mouse. Hypothalamic TRH is depressed by at least 75% compared with wild-type controls. Concentrations of pro-TRH forms are increased in homozygotes. TRH-[Gly(4)-Lys(5)-Arg(6)] and TRH-[Gly(4)-Lys(5)] represent approximately 45% of the total TRH-like immunoreactivity in Cpe(fat/fat) mice; they constitute approximately 1% in controls. Levels of TRH-[Gly(4)] were depressed in homozygotes. Because the hypothalamus contains some TRH, another carboxypeptidase must be responsible for processing. Immunocytochemical studies indicate that TRH neurons contain CPE- and carboxypeptidase D-like immunoreactivity. Recombinant CPE or carboxypeptidase D can convert synthetic TRH-[Gly(4)-Lys(5)] and TRH-[Gly(4)-Lys(5)-Arg(6)] to TRH-[Gly(4)]. When Cpe(fat/fat) mice are exposed to cold, they cannot maintain their body temperatures, and this loss is associated with hypothalamic TRH depletion and reduction in thyroid hormone. These findings demonstrate that the Cpe(fat) mutation can affect not only carboxypeptidase activity but also endoproteolysis. Because Cpe(fat/fat) mice cannot sustain a cold challenge, and because alterations in the hypothalamic-pituitary-thyroid axis can affect metabolism, deficits in pro-TRH processing may contribute to the obese and diabetic phenotype in these mice.

Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity.

PubMed

Joseph, Christine G; Wilczynski, Andrzej; Holder, Jerry R; Xiang, Zhimin; Bauzo, Rayna M; Scott, Joseph W; Haskell-Luevano, Carrie

2003-12-01

Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111-113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7-9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle4-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH2 peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.

Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr

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

Deutscher, J.; Pevec, B.; Beyreuther, K.

1986-10-21

The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolyptic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system,more » HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction. The site of ATP-dependent phosphorylation in HPr of S faecalis has now been determined. (/sup 32/P)P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, they obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, they isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-. Thus, the site of ATP-dependent phosphorylation was determined to be Ser-46 within the primary structure of HPr.« less

Xeroderma pigmentosum group D polymorphisms and esophageal cancer susceptibility: a meta-analysis based on case-control studies.

PubMed

Yang, Rong; Zhang, Chong; Malik, Armah; Shen, Zhi-Da; Hu, Jian; Wu, Yi-He

2014-11-28

To clarify the effects of the xeroderma pigmentosum group D (XPD) Asp312Asn and Lys751Gln gene polymorphisms on the risk of esophageal cancer (EC). A computerised literature search was conducted to identify the relevant studies from the PUBMED and EMBASE databases, reviews, and reference lists of relevant articles. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the associations between the XPD Asp312Asn and/or Lys751Gln polymorphisms and EC susceptibility. Statistical analyses were performed using the software Stata 12.0. A fixed or random effects model was selected based on a heterogeneity test. Publication bias was estimated using funnel plots and Egger's linear regression method. Subgroup analyses were performed based on histological type and ethnicity. Thirteen case-control studies with a total of 10 comparisons for the Asp312Asn polymorphism, including 2373 cases and 3175 controls, and 15 comparisons for the Lys751Gln polymorphism, including 3226 cases and 5237 controls, were recruited for the meta-analysis. In terms of the XPD Asp312Asn polymorphism, significantly increased EC risks were identified in the Asp/Asn vs Asp/Asp comparison (OR = 1.17, 95%CI: 1.02-1.33, P = 0.03) and in the dominant-model comparison (Asn/Asn+Asp/Asn vs Asp/Asp: OR = 1.18, 95%CI: 1.04-1.34, P = 0.01). However, no significant associations were found in the Asn/Asn vs Asp/Asp comparison (OR = 1.30, 95%CI: 1.00-1.70, P = 0.05) or in the recessive-model comparison (Asn/Asn vs Asp/Asn + Asp/Asp: OR = 1.17, 95%CI: 0.91-1.50, P = 0.22). In terms of the XPD Lys751Gln polymorphism, a significant association with EC susceptibility was found under the recessive model (Gln/Gln vs Lys/Gln+Lys/Lys: OR = 1.21, 95%CI: 1.02-1.43, P = 0.03). However, no associations were identified in the other comparisons (co-dominant model: Lys/Gln vs Lys/Lys: OR = 1.11, 95%CI: 0.94-1.31, P = 0.20; Gln/Gln vs Lys/Lys: OR = 1.31, 95%CI: 0.98-1.75, P = 0.07; dominant model: OR = 1.14, 95%CI: 0.96-1.35, P = 0.14). The results of this meta-analysis suggest that the XPD Asp312Asn and Lys751Gln gene polymorphisms are associated with a significantly increased risk for EC.

An antibody reactive to the Gly63–Lys68 epitope of NT-proBNP exhibits O-glycosylation-independent binding

PubMed Central

Lee, Yujean; Kim, Hyori; Chung, Junho

2014-01-01

The N-terminal fragment of prohormone brain natriuretic peptide (NT-proBNP) is a commonly used biomarker for the diagnosis of congestive heart failure, although its biological function is not well known. NT-proBNP exhibits heavy O-linked glycosylation, and it is quite difficult to develop an antibody that exhibits glycosylation-independent binding. We developed an antibody that binds to the recombinant NT-proBNP protein and its deglycosylated form with similar affinities in an enzyme immunoassay. The epitope was defined as Gly63–Lys68 based on mimetic peptide screening, site-directed mutagenesis and a competition assay with a peptide mimotope. The nearest O-glycosylation residues are Thr58 and Thr71; therefore, four amino acid residues intervene between the epitope and those residues in both directions. In conclusion, we report that an antibody reactive to Gly63–Lys68 of NT-proBNP exhibits O-glycosylation-independent binding. PMID:25236766

Short cell-penetrating peptides: a model of interactions with gene promoter sites.

PubMed

Khavinson, V Kh; Tarnovskaya, S I; Linkova, N S; Pronyaeva, V E; Shataeva, L K; Yakutseni, P P

2013-01-01

Analysis of the main parameters of molecular mechanics (number of hydrogen bonds, hydrophobic and electrostatic interactions, DNA-peptide complex minimization energy) provided the data to validate the previously proposed qualitative models of peptide-DNA interactions and to evaluate their quantitative characteristics. Based on these estimations, a three-dimensional model of Lys-Glu and Ala-Glu-Asp-Gly peptide interactions with DNA sites (GCAG and ATTTC) located in the promoter zones of genes encoding CD5, IL-2, MMP2, and Tram1 signal molecules.

A fluorescently labeled undecapeptide derived from a protein in royal jelly of the honeybee-royalisin-for specific detection of oxidized low-density lipoprotein.

PubMed

Sato, Akira; Unuma, Hiroto; Yamazaki, Yoji; Ebina, Keiichi

2018-06-01

The probes for detection of oxidized low-density lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques are expected to facilitate the diagnosis, prevention, and treatment of atherosclerosis. Recently, we have reported that a heptapeptide (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6) coupled through the ε-amino group of N-terminal Lys to fluorescein isothiocyanate (FITC), (FITC)KP6, can be useful as a fluorescent probe for specific detection of ox-LDL. In the present study, to develop a novel fluorescent peptide for specific detection of ox-LDL, we investigated the interaction (with ox-LDL) of an undecapeptide corresponding to positions 41 to 51 of a potent antimicrobial protein (royalisin, which consists of 51 residues; from royal jelly of honeybees), conjugated at the N-terminus to FITC in the presence of 6-amino-n-caproic acid (AC) linker, (FITC-AC)-royalisin P11, which contains both sequences, Phe-Lys-Asp and Asp-Lys-Tyr, similar to Tyr-Lys-Asp in (FITC)KP6. The (FITC-AC)-royalisin P11 bound with high specificity to ox-LDL in a dose-dependent manner, through the binding to major lipid components in ox-LDL (lysophosphatidylcholine and oxidized phosphatidylcholine). In contrast, a (FITC-AC)-shuffled royalisin P11 peptide, in which sequences Phe-Lys-Asp and Asp-Lys-Tyr were modified to Lys-Phe-Asp and Asp-Tyr-Lys, respectively, hardly bound to LDL and ox-LDL. These findings strongly suggest that (FITC-AC)-royalisin P11 may be an effective fluorescent probe for specific detection of ox-LDL and that royalisin from the royal jelly of honeybees may play a role in the treatment of atherosclerosis through the specific binding of the region at positions 41 to 51 to ox-LDL. Copyright © 2018 European Peptide Society and John Wiley & Sons, Ltd.

Amino acid metabolism during exercise in trained rats: the potential role of carnitine in the metabolic fate of branched-chain amino acids.

PubMed

Ji, L L; Miller, R H; Nagle, F J; Lardy, H A; Stratman, F W

1987-08-01

The influence of endurance training and an acute bout of exercise on plasma concentrations of free amino acids and the intermediates of branched-chain amino acid (BCAA) metabolism were investigated in the rat. Training did not affect the plasma amino acid levels in the resting state. Plasma concentrations of alanine (Ala), aspartic acid (Asp), asparagine (Asn), arginine (Arg), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val) were significantly lower, whereas glutamate (Glu), glycine (Gly), ornithine (Orn), tryptophan (Trp), tyrosine (Tyr), creatinine, urea, and ammonia levels were unchanged, after one hour of treadmill running in the trained rats. Plasma concentration of glutamine (Glu), the branched-chain keto acids (BCKA) and short-chain acyl carnitines were elevated with exercise. Ratios of plasma BCAA/BCKA were dramatically lowered by exercise in the trained rats. A decrease in plasma-free carnitine levels was also observed. These data suggest that amino acid metabolism is enhanced by exercise even in the trained state. BCAA may only be partially metabolized within muscle and some of their carbon skeletons are released into the circulation in forms of BCKA and short-chain acyl carnitines.

A molecular dynamics study of the three-dimensional model of human synovial fluid phospholipase A2--transition state mimic complexes.

PubMed

Hariprasad, V; Kulkarni, V M

1996-01-01

Different modes of binding of transition state mimics: amide, phosphonate and difluoro ketone, to human synovial fluid phospholipase A2 (HSF PLA2) are studies by molecular dynamics simulations computed in solvent. The results are analysed in the light of primary binding sites. Hydrogen bonding interaction plays an important role for amino acids such as Gly32, Val30, and Glu55, apart from the well known active site residues viz Asp48, Gly25, Gly29, Gly31, His27, His47, Lys62, Phe23, Asn114 and Tyr112. In addition, the hydrogen bonding interaction between Sn-1 tetrahedral phosphonate group of amide and difluoro ketone inhibitors and crystallographic water molecules (H2O 523, H2O 524 and H2O 401) seems to have a significant role. Many of the active site charged residues display considerable movement upon ligand binding. The structural effects of ligand binding were analyzed from RMS deviations of C alpha in the resulting energy-minimized average structures of the receptor-ligand complexes. The values of the RMS deviations differ among the HSF PLA2s, in a pattern that is not the same for the three complexes. This suggests that ligands with different pharmacological efficacies induce different types of conformational changes of the receptor. Our active-orientation model is, at least qualitatively, consistent with experimental data and should be useful for the rational design of more potent inhibitors.

Variability of the protein sequences of lcrV between epidemic and atypical rhamnose-positive strains of Yersinia pestis.

PubMed

Anisimov, Andrey P; Panfertsev, Evgeniy A; Svetoch, Tat'yana E; Dentovskaya, Svetlana V

2007-01-01

Sequencing of lcrV genes and comparison of the deduced amino acid sequences from ten Y. pestis strains belonging mostly to the group of atypical rhamnose-positive isolates (non-pestis subspecies or pestoides group) showed that the LcrV proteins analyzed could be classified into five sequence types. This classification was based on major amino acid polymorphisms among LcrV proteins in the four "hot points" of the protein sequences. Some additional minor polymorphisms were found throughout these sequence types. The "hot points" corresponded to amino acids 18 (Lys --> Asn), 72 (Lys --> Arg), 273 (Cys --> Ser), and 324-326 (Ser-Gly-Lys --> Arg) in the LcrV sequence of the reference Y. pestis strain CO92. One possible explanation for polymorphism in amino acid sequences of LcrV among different strains is that strain-specific variation resulted from adaptation of the plague pathogen to different rodent and lagomorph hosts.

DehydroalanylGly, a new post translational modification resulting from the breakdown of glutathione.

PubMed

Friedrich, Michael G; Wang, Zhen; Schey, Kevin L; Truscott, Roger J W

2018-04-01

The human body contains numerous long-lived proteins which deteriorate with age, typically by racemisation, deamidation, crosslinking and truncation. Previously we elucidated one reaction responsible for age-related crosslinking, the spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine and cysteine. This resulted in non-disulphide covalent crosslinks. The current paper outlines a novel posttranslational modification (PTM) in human proteins, which involves the addition of dehydroalanylglycine (DHAGly) to Lys residues. Human lens digests were examined by mass spectrometry for the presence of (DHA)Gly (+144.0535 Da) adducts to Lys residues. Peptide model studies were undertaken to elucidate the mechanism of formation. In the lens, this PTM was detected at 18 lysine sites in 7 proteins. Using model peptides, a pathway for its formation was found to involve initial formation of the glutathione degradation product, γ-Glu(DHA)Gly from oxidised glutathione (GSSG). Once the Lys adduct formed, the Glu residue was lost in a hydrolytic mechanism apparently catalysed by the ε-amino group of the Lys. This discovery suggests that within cells, the functional groups of amino acids in proteins may be susceptible to modification by reactive metabolites derived from GSSG. Our finding demonstrates a novel +144.0535 Da PTM arising from the breakdown of oxidised glutathione. Copyright © 2018. Published by Elsevier B.V.

Haloperidol-stomach lesions attenuation by pentadecapeptide BPC 157, omeprazole, bromocriptine, but not atropine, lansoprazole, pantoprazole, ranitidine, cimetidine and misoprostol in mice.

PubMed

Bilic, I; Zoricic, I; Anic, T; Separovic, J; Stancic-Rokotov, D; Mikus, D; Buljat, G; Ivankovic, D; Aralica, G; Prkacin, I; Perovic, D; Mise, S; Rotkvic, I; Petek, M; Rucman, R; Seiwerth, S; Sikiric, P

2001-03-09

The focus was on haloperidol (central dopamine antagonist)-stomach lesion, a longly described suitable counterpart of dopamine blocker cysteamine-duodenal lesion. In this, the contribution of blockade of central/peripheral dopamine receptors and prostaglandins synthesis, along with influence of antiulcer agents was evaluated in mice. Male NMRI Hannnover mice were sacrificed 24 h after haloperidol (25 mg/kg b.w. i.p., given alone or with saline (haloperidol+saline) (i) or in combination (ii,iii)). Supporting central dopamine predominance for haloperidol stomach lesion induction, co-administration of peripheral dopamine receptor antagonist domperidone (5 mg/kg i.p.) (haloperidol+ domperidone) (ii), or prostaglandin synthesis inhibitor indomethacin (10 mg/kg s.c.) (haloperidol+ indomethacin) (iii) did not aggravate this lesion. (i) In haloperidol+saline challenged mice the lesions were inhibited by co-administration (/kg i.p.) of a gastric pentadecapeptide BPC 157, GlyGluProProProGlyLysProAlaAspAspAlaGlyLeuVal, M.W. 1419 (10 microg, 10 ng, 10 pg, but not 1 pg, 100 fg, 10 fg), bromocriptine (10 mg), omeprazole (10 mg, 100 mg, but not 1 mg). Atropine (10, 100, 200 mg), pirenzepine (10, 100, 200 mg), misoprostol (10, 100, 200 microg), pantoprazole (1, 10, 100 mg), lansoprazole (0.1, 1, 10 mg), cimetidine (10, 100, 200 mg) and ranitidine (10, 100, 200 mg) were not effective. (ii) Dopamine peripheral blockade influence: in haloperidol+domperidone mice, previously effective bromocriptine, pentadecapeptide BPC 157 (10 microg) or omeprazole (10 mg) did not attenuate stomach lesions. (iii) Prostaglandins synthesis blockade effect: in haloperidol+indomethacin mice, previously effective agents, bromocriptine or omeprazole were not active, while BPC 157 effect was only lessened.

Development of a physiologically based pharmacokinetic model to predict the effects of flavin-containing monooxygenase 3 (FMO3) polymorphisms on itopride exposure.

PubMed

Zhou, Wangda; Humphries, Helen; Neuhoff, Sibylle; Gardner, Iain; Masson, Eric; Al-Huniti, Nidal; Zhou, Diansong

2017-09-01

Itopride, a substrate of FMO3, has been used for the symptomatic treatment of various gastrointestinal disorders. Physiologically based pharmacokinetic (PBPK) modeling was applied to evaluate the impact of FMO3 polymorphism on itopride pharmacokinetics (PK). The Asian populations within the Simcyp simulator were updated to incorporate information on the frequency, activity and abundance of FMO3 enzyme with different phenotypes. A meta-analysis of relative enzyme activities suggested that FMO3 activity in subjects with homozygous Glu158Lys and Glu308Gly mutations (Lys158 and Gly308) in both alleles is ~47% lower than those carrying two wild-type FMO3 alleles. Individuals with homozygous Lys158 and Gly308 mutations account for about 5% of the total population in Asian populations. A CL int of 9 μl/min/pmol was optimised for itopride via a retrograde approach as human liver microsomal results would under-predict its clearance by ~7.9-fold. The developed itopride PBPK model was first verified with three additional clinical studies in Korean and Japanese subjects resulting in a predicted clearance of 52 to 69 l/h, which was comparable to those observed (55 to 88 l/h). The model was then applied to predict plasma concentration-time profiles of itopride in Chinese subjects with wild type or homozygous Lys158 and Gly308 FMO3 genotypes. The ratios of predicted to observed AUC of itopride in subjects with each genotype were 1.23 and 0.94, respectively. In addition, the results also suggested that for FMO3 metabolised drugs with a safety margin of 2 or more, proactive genotyping FMO3 to exclude subjects with homozygous Lys158/Gly308 alleles may not be necessary. Copyright © 2017 John Wiley & Sons, Ltd.

Potent and selective agonists of alpha-melanotropin (alphaMSH) action at human melanocortin receptor 5; linear analogs of alpha-melanotropin.

PubMed

Bednarek, Maria A; MacNeil, Tanya; Tang, Rui; Fong, Tung M; Cabello, M Angeles; Maroto, Marta; Teran, Ana

2007-05-01

Alpha-melanotropin, Ac-Ser(1)-Tyr-Ser-Met-Glu-His(6)-Phe(7)-Arg(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2)(1), is a non-selective endogenous agonist for the melanocortin receptor 5; the receptor present in various peripheral tissues and in the brain, cortex and cerebellum. Most of the synthetic analogs of alphaMSH, including a broadly used and more potent the NDP-alphaMSH peptide, Ac-Ser(1)-Tyr-Ser-Nle(4)-Glu-His(6)-D-Phe(7)-Arg(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2), are also not particularly selective for MC5R. To elucidate physiological functions of the melanocortin receptor 5 in rodents and humans, the receptor subtype selective research tools are needed. We report herein syntheses and pharmacological evaluation in vitro of several analogs of NDP-alphaMSH which are highly potent and specific agonists for the human MC5R. The new linear peptides, of structures and solubility properties similar to those of the endogenous ligand alphaMSH, are exemplified by compound 7, Ac-Ser(1)-Tyr-Ser-Met-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2) (Oic: octahydroindole-2-COOH, 4,4'-Bip: 4,4'-biphenylalanine, Pip: pipecolic acid), shortly NODBP-alphaMSH, which has an IC(50)=0.74 nM (binding assay) and EC(50)=0.41 (cAMP production assay) at hMC5R nM and greater than 3500-fold selectivity with respect to the melanocortin receptors 1b, 3 and 4. A shorter peptide derived from NODBP-alphaMSH: Ac-Nle-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8)-Trp(9) -NH(2) (17) was measured to be an agonist only 10-fold less potent at hMC5R than the full length parent peptide. In the structure of this smaller analog, the Nle-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8) segment was found to be critical for high agonist potency, while the C-terminal Trp(9) residue was shown to be required for high hMC5R selectivity versus hMC1b,3,4R.

Molecular characterization, transcriptional profiling, and antibacterial potential of G-type lysozyme from seahorse (Hippocampus abdominalis).

PubMed

Ko, Jiyeon; Wan, Qiang; Bathige, S D N K; Lee, Jehee

2016-11-01

Lysozymes are a family of enzymes that catalyze the hydrolysis of bacterial cell wall, acting as antimicrobial effectors of the innate immune system. In the present study, an ortholog of goose-type lysozyme (ShLysG) from the big-belly seahorse (Hippocampus abdominalis) was identified and characterized structurally and functionally. The full-length cDNA sequence (1213 bp) of ShLysG is comprised of an open reading frame made up of 552 bp, encoding a polypeptide of 184 amino acid (aa) with a predicted molecular mass of 20 kDa. In silico analysis of ShLysG revealed the absence of signal peptide and the presence of a characteristic bacterial soluble lytic transglycosylase (SLT) domain bearing three catalytic residues (Glu 71 , Asp 84 , and Asp 95 ) and seven N-acetyl-d-glucosamine binding sites (Glu 71 , Asp 95 , Tyr 98 , His 99 , Ile 117 , Tyr 145 , and Asn 146 ). Homology analysis demonstrated that the aa sequence of ShLysG shared 60.7-67.4% identity and 72.6-79.3% similarity with the orthologs of other teleosts. Phylogenetic analysis of ShLysG indicated a closest relationship with the ortholog from Gadus morhua. In healthy seahorse, ShLysG mRNA showed a constitutive expression in all the tissues examined, with the highest expression in kidney and the least expression in liver. The ShLysG mRNA levels were also shown significant elevation upon the bacterial and pathogen-associated molecular pattern (PAMPs) challenges. Furthermore, lytic activities of ShLysG recombinant protein were detected against several Gram-negative and Gram-positive bacterial species. Taken together, these results suggest that ShLysG might possess a potential immune defensive role against invading microbial pathogens in seahorse. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effect of Vilon and Epithalon on glucose and glycine absorption in various regions of small intestine in aged rats.

PubMed

Khavinson, V Kh; Egorova, V V; Timofeeva, N M; Malinin, V V; Gordova, L A; Gromova, L V

2002-05-01

Vilon (Lys-Glu) and Epithalon (Ala-Glu-Asp-Gly) administered orally for 1 month improved transport characteristics of the small intestine in aged rats. Vilon enhanced passive glucose accumulation in the serous fluid in inverted sac made from the distal region of the small intestine, while Epithalon enhanced this process in the medial region. Vilon stimulated active glucose accumulation in the serous sac of the medial small intestine, Epithalon - in the proximal and distal small intestinal segments. Glycine absorption increased only in the proximal intestinal segment under the effect of Epithalon.

Transient trimethylaminuria related to menstruation

PubMed Central

Shimizu, Makiko; Cashman, John R; Yamazaki, Hiroshi

2007-01-01

Background Trimethylaminuria, or fish odor syndrome, includes a transient or mild malodor caused by an excessive amount of malodorous trimethylamine as a result of body secretions. Herein, we describe data to support the proposal that menses can be an additional factor causing transient trimethylaminuria in self-reported subjects suffering from malodor and even in healthy women harboring functionally active flavin-containing monooxygenase 3 (FMO3). Methods FMO3 metabolic capacity (conversion of trimethylamine to trimethylamine N-oxide) was defined as the urinary ratio of trimethylamine N-oxide to total trimethylamine. Results Self-reported Case (A) that was homozygous for inactive Arg500stop FMO3, showed decreased metabolic capacity of FMO3 (i.e., ~10% the unaffected metabolic capacity) during 120 days of observation. For Case (B) that was homozygous for common [Glu158Lys; Glu308Gly] FMO3 polymorphisms, metabolic capacity of FMO3 was almost ~90%, except for a few days surrounding menstruation showing < 40% metabolic capacity. In comparison, three healthy control subjects that harbored heterozygous polymorphisms for [Glu158Lys; Glu308Gly] FMO3 or homozygous for wild FMO3 showed normal (> 90%) metabolic capacity, however, on days around menstruation the FMO3 metabolic capacity was decreased to ~60–70%. Conclusion Together, these results indicate that abnormal FMO3 capacity is caused by menstruation particularly in the presence, in homozygous form, of mild genetic variants such as [Glu158Lys; Glu308Gly] that cause a reduced FMO3 function. PMID:17257434

Purification and characterization of angiotensin I converting enzyme inhibition peptides from sandworm Sipunculus nudus

NASA Astrophysics Data System (ADS)

Sun, Xueping; Wang, Man; Liu, Buming; Sun, Zhenliang

2017-10-01

Three angiotensin I converting enzyme (ACE) inhibition peptides were isolated from sandworm Sipunculus nudus protein hydrolysate prepared using protamex. Consecutive purification methods, including size exclusion chromatography and reverse-phase high performance liquid chromatography (RP-HPLC), were used to isolate the ACE inhibition peptides. The amino acid sequences of the peptides were identified as Ile-Asn-Asp, Val-Glu-Pro-Gly and Leu-Ala-Asp-Glu-Phe. The IC50 values of the purified peptides for ACE inhibition activity were 34.72 μmol L-1, 20.55 μmol L-1 and 22.77 μmol L-1, respectively. These results suggested that S. nudus proteins contain specific peptides that can be released by enzymatic hydrolysis. This study may provide an experimental basis for further systematic research, rational development and clinical utilization of sandworm resources.

Differentiating founder and chronic HIV envelope sequences

PubMed Central

Maher, Stephen; Mota, Talia; Suzuki, Kazuo; Kelleher, Anthony D.

2017-01-01

Significant progress has been made in characterizing broadly neutralizing antibodies against the HIV envelope glycoprotein Env, but an effective vaccine has proven elusive. Vaccine development would be facilitated if common features of early founder virus required for transmission could be identified. Here we employ a combination of bioinformatic and operations research methods to determine the most prevalent features that distinguish 78 subtype B and 55 subtype C founder Env sequences from an equal number of chronic sequences. There were a number of equivalent optimal networks (based on the fewest covarying amino acid (AA) pairs or a measure of maximal covariance) that separated founders from chronics: 13 pairs for subtype B and 75 for subtype C. Every subtype B optimal solution contained the founder pairs 178–346 Asn-Val, 232–236 Thr-Ser, 240–340 Lys-Lys, 279–315 Asp-Lys, 291–792 Ala-Ile, 322–347 Asp-Thr, 535–620 Leu-Asp, 742–837 Arg-Phe, and 750–836 Asp-Ile; the most common optimal pairs for subtype C were 644–781 Lys-Ala (74 of 75 networks), 133–287 Ala-Gln (73/75) and 307–337 Ile-Gln (73/75). No pair was present in all optimal subtype C solutions highlighting the difficulty in targeting transmission with a single vaccine strain. Relative to the size of its domain (0.35% of Env), the α4β7 binding site occurred most frequently among optimal pairs, especially for subtype C: 4.2% of optimal pairs (1.2% for subtype B). Early sequences from 5 subtype B pre-seroconverters each exhibited at least one clone containing an optimal feature 553–624 (Ser-Asn), 724–747 (Arg-Arg), or 46–293 (Arg-Glu). PMID:28187204

Dansylated octapeptide Dns-Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn inhibits the proliferation rate of HL-60 cells.

PubMed

Marsili, V; Nardicchi, V; Lupidi, G; Brozzetti, A; Gianfranceschi, G L

1996-12-01

Small acidic phosphorylated chromatin peptides show regulatory activity on gene expression. The peptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn, synthesized on the basis of structural and biochemical studies, shows functional properties in vitro (phosphorylation by casein kinase II, control of DNA transcription by RNA polymerase II, inhibition of proliferation and promotion of differentiation in some cell lines) very similar to those of native chromatin peptides. In this report we show that the dansylated octapeptide Dns-Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn remarkably inhibits cell growth of the HL-60 cell line. The biological effect of the peptide seems to be considerably higher than that shown by the nondansylated peptide, and it cannot be attributed to a toxic effect of the Dns group. The measurement of uptake of 3H-labelled Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn demonstrates that it is unable to pass through the HL-60 cell membrane. It is our considered opinion that the addition of hydrophobic groups to the peptide N-terminus should increase the biological activity by improving its transport through the cellular membrane.

Engineering acidic Streptomyces rubiginosus D-xylose isomerase by rational enzyme design.

PubMed

Waltman, Mary Jo; Yang, Zamin Koo; Langan, Paul; Graham, David E; Kovalevsky, Andrey

2014-02-01

To maximize bioethanol production from lignocellulosic biomass, all sugars must be utilized. Yeast fermentation can be improved by introducing the d-xylose isomerase enzyme to convert the pentose sugar d-xylose, which cannot be fermented by Saccharomyces cerevisiae, into the fermentable ketose d-xylulose. The low activity of d-xylose isomerase, especially at the low pH required for optimal fermentation, limits its use. A rational enzyme engineering approach was undertaken, and seven amino acid positions were replaced to improve the activity of Streptomyces rubiginosus d-xylose isomerase towards its physiological substrate at pH values below 6. The active-site design was guided by mechanistic insights and the knowledge of amino acid protonation states at low pH obtained from previous joint X-ray/neutron crystallographic experiments. Tagging the enzyme with 6 or 12 histidine residues at the N-terminus resulted in a significant increase in the active-site affinity towards substrate at pH 5.8. Substituting an asparagine at position 215, which hydrogen bonded to the metal-bound Glu181 and Asp245, with an aspartate gave a variant with almost an order of magnitude lower KM than measured for the native enzyme, with a 4-fold increase in activity. Other studied variants showed similar (Asp57Asn, Glu186Gln/Asn215Asp), lower (Asp57His, Asn247Asp, Lys289His, Lys289Glu) or no (Gln256Asp, Asp287Asn, ΔAsp287) activity in acidic conditions relative to the native enzyme.

Primary structure of pancreatic polypeptide from four species of Perissodactyla (Przewalski's horse, zebra, rhino, tapir).

PubMed

Henry, J S; Lance, V A; Conlon, J M

1991-12-01

Pancreatic polypeptide (PP) has been purified from extracts of the pancreas of four species of odd-toed ungulates (Perissodactyla): Przewalski's horse, mountain zebra, white rhinoceros, and mountain tapir. The amino acid sequence of Przewalski's horse pancreatic polypeptide was established as Ala-Pro-Met-Glu-Pro-Val-Tyr-Pro-Gly-Asp10-Asn- Ala-Thr-Pro-Glu-Gln-Met-Ala-Gln-Tyr20-Ala-Ala-Glu-Leu-Arg-Arg-Tyr- Ile-Asn-Met30 - Leu-Thr-Arg-Pro-Arg-Tyr.NH2. Zebra PP was identical to Przewalski's horse PP, rhinoceros PP contained three substitutions relative to the horse (Ser for Ala1, Leu for Met3, and Glu for Gln16), and tapir PP contained one substitution relative to the horse (Leu for Met3). On the basis of morphological characteristics and the fossil record, the rhinocerotids are classified with the tapirids in the suborder Ceratomorpha, whereas the horse and zebra belong to a separate suborder, Hippomorpha. On the basis of structural similarity of the PP molecules, however, it would appear that the tapir is more closely related to the horse than to the rhinoceros. These observations provide a further example of the need for extreme caution when inferring taxonomic or phylogenetic relationships between species from the structures of homologous peptides.

Synthesis and antibacterial activity of new peptides from Alfalfa RuBisCO protein hydrolysates and mode of action via a membrane damage mechanism against Listeria innocua.

PubMed

Kobbi, Sabrine; Nedjar, Naima; Chihib, Nourdine; Balti, Rafik; Chevalier, Mickael; Silvain, Amandine; Chaabouni, Semia; Dhulster, Pascal; Bougatef, Ali

2018-02-01

In this work we evaluated the mode of action of six new synthesized peptides (Met-Asp-Asn; Glu-leu-Ala-Ala-Ala-Cys; Leu-Arg-Asp-Asp-Phe; Gly-Asn-Ala-Pro-Gly-Ala-Val-Ala; Ala-Leu-Arg-Met-Ser-Gly and Arg-Asp-Arg-Phe-Leu), previously identified, from the most active peptide fractions of RuBisCO peptic hydrolysate against Listeria innocua via a membrane damage mechanism. Antibacterial effect and the minimum inhibitory concentrations (MIC) of these peptides were evaluated against six strains and their hemolytic activities towards bovine erythrocytes were determined. Prediction of the secondary structure of peptides indicated that these new antibacterial peptides are characterized by a short peptide chains (3-8 amino acid) and a random coli structure. Moreover, it was observed that one key characteristic of antibacterial peptides is the presence of specific amino acids such as cysteine, glycine, arginine and aspartic acid. In addition the determination of the extracellular potassium concentration revealed that treatment with pure RuBisCO peptides could cause morphological changes of L. innocua and destruction of the cell integrity via irreversible membrane damage. The results could provide information for investigating the antibacterial model of antibacterial peptides derived from RuBisCO protein hydrolysates. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Peptides and CCL11 and HMGB1 as molecular markers of aging: literature review and own data].

PubMed

Khavinson, V Kh; Kuznik, B I; Tarnovskaia, S I; Lin'kova, N S

2014-01-01

Cytokines CCL11 (eotaxin) and HMGB1 (alarmin1) are molecular markers of ageing and neurological, cardiovascular and immune diseases. Created in St. Petersburg Institute of Bioregulation and Gerontology short peptides are known to regulate gene expression and protein synthesis. They promote the mortality decrease and slowdown the development of pathology in the elderly. The article presents the proposed role of dipeptide vilon (Lys-Glu) and tetrapeptide epitalon (Ala-Glu-Asp-Gly) in CCL11 and HMGB1 genes regulation as activators of their expression. Geroprotective action of vilon and epitalon probably realizes in suppression of these genes.

[Peptide Ala-Glu-Asp-Gly and interferon gamma: their role in immune response during aging].

PubMed

Lin'kova, N S; Kuznik, B I; Khavinson, V Kh

2012-01-01

The decrease of lymphocyte interferon gamma expression during aging is one of the main mechanisms leading to the immunodeficiency state in the elderly. Cell penetrating geroprotective peptide Ala-Glu-Asp-Gly has the capability to activate the proliferation of lymphocytes in thymus during its aging. The nucleotide sequence which is complementary contacted with peptide Ala-Glu-Asp-Gly was found in promoter region of interferon gamma gene. Thus, the immune protection of this peptide can be explained by its activation of the interferon gamma production in T-cells.

Hydrogen Exchange During Cell-Free Incorporation of Deuterated Amino Acids and an Approach to its Inhibition

PubMed Central

Tonelli, M.; Singarapu, K. K.; Markley, J. L.; Makino, S.; Sahu, S .C.; Matsubara, Y.; Endo, Y.; Kainosho, M.

2012-01-01

Perdeuteration, selective deuteration, and stereo array isotope labeling (SAIL) are valuable strategies for NMR studies of larger proteins and membrane proteins. To minimize scrambling of the label, it is best to use cell-free methods to prepare selectively labeled proteins. However, when proteins are prepared from deuterated amino acids by cell-free translation in H2O, exchange reactions can lead to contamination of 2H sites by 1H from the solvent. Examination of a sample of SAIL-chlorella ubiquitin prepared by Escherichia coli cell-free synthesis revealed that exchange had occurred at several residues (mainly at Gly, Ala, Asp, Asn, Glu, and Gln). We present results from a study aimed at identifying the exchanging sites and level of exchange and at testing a strategy for minimizing 1H contamination during wheat germ cell-free translation of proteins produced from deuterated amino acids by adding known inhibitors of transaminases (1 mM aminooxyacetic acid) and glutamate synthetase (0.1 mM L-methionine sulfoximine). By using a wheat germ cell-free expression system, we produced [U-2H,15N]-chlorella ubiquitin without and with added inhibitors, and [U-15N]-chlorella ubiquitin as a reference to determine the extent of deuterium incorporation. We also prepared a sample of [U-13C,15N]-chlorella ubiquitin, for use in assigning the sites of exchange. The added inhibitors did not reduce the protein yield and were successful in blocking hydrogen exchange at Cα sites with the exception of Gly. We discovered, in addition, that partial exchange occurred with or without the inhibitors at certain side-chain methyl and methylene groups: Ala-Hβ, Asn-Hβ, Asp-Hβ, Gln-Hγ, Glu-Hγ, and Lys-Hε. The side-chain labeling pattern, in particular the mixed chiral labeling resulting from partial exchange at certain sites, should be of interest in studies of large proteins, protein complexes, and membrane proteins. PMID:21984356

Bone Matrix Proteins: Isolation and Characterization of a Novel Cell-binding Keratan Sulfate Proteoglycan (Osteoadherin) from Bovine Bone

PubMed Central

Wendel, Mikael; Sommarin, Yngve; Heinegård, Dick

1998-01-01

A small cell-binding proteoglycan for which we propose the name osteoadherin was extracted from bovine bone with guanidine hydrochloride–containing EDTA. It was purified to homogeneity using a combination of ion-exchange chromatography, hydroxyapatite chromatography, and gel filtration. The Mr of the proteoglycan was 85,000 as determined by SDS-PAGE. The protein is rich in aspartic acid, glutamic acid, and leucine. Two internal octapeptides from the proteoglycan contained the sequences Glu-Ile-Asn-Leu-Ser-His-Asn-Lys and Arg-Asp-Leu-Tyr-Phe-Asn-Lys-Ile. These sequences are not previously described, and support the notion that osteoadherin belongs to the family of leucine-rich repeat proteins. A monospecific antiserum was raised in rabbits. An enzyme-linked immunosorbent assay was developed, and showed the osteoadherin content of bone extracts to be 0.4 mg/g of tissue wet weight, whereas none was found in extracts of various other bovine tissues. Metabolic labeling of primary bovine osteoblasts followed by immunoprecipitation showed the cells to synthesize and secrete the proteoglycan. Digesting the immunoprecipitated osteoadherin with N-glycosidase reduced its apparent size to 47 kD, thus showing the presence of several N-linked oligosaccharides. Digestion with keratanase indicated some of the oligosaccharides to be extended to keratan sulfate chains. In immunohistochemical studies of the bovine fetal rib growth plate, osteoadherin was exclusively identified in the primary bone spongiosa. Osteoadherin binds to hydroxyapatite. A potential function of this proteoglycan is to bind cells, since we showed it to be as efficient as fibronectin in promoting osteoblast attachment in vitro. The binding appears to be mediated by the integrin αvβ3, since this was the only integrin isolated by osteoadherin affinity chromatography of surface-iodinated osteoblast extracts. PMID:9566981

[Rhythm of protein synthesis in cultures of hepatocytes from rats of different ages. Norm and effect of the peptide livagen].

PubMed

Brodskiĭ, V Ia; Khavinson, V Kh; Zolotarev, Iu A; Nechaeva, N V; Malinin, V V; Novikova, T E; Gvazava, I G; Fateeva, V I

2001-01-01

The circumhoralian rhythm of protein synthesis was determined in a monolayer culture of hepatocytes from rats at the age of 1 to 24 months and weighing from 45 to 480 g, respectively. The peptide lyvagen (Lys-Glu-Asp-Ala) obtained by directed chemical synthesis on the basis of amino acid analysis of the liver polypeptide preparations increased the level of protein synthesis in the hepatocytes from rats of different ages; the highest effect was observed in the cells of old animals. In old rats, lyvagen increased the amplitude of protein synthesis fluctuations. The peptide epitalon (Ala-Glu-Asp-Gly) constructed on the basis of analysis of the epiphysis peptides did not change the intensity of protein synthesis in the cultured hepatocytes.

Dipeptidyl-peptidase IV inhibitory activity of peptides derived from tuna cooking juice hydrolysates.

PubMed

Huang, Shih-Li; Jao, Chia-Ling; Ho, Kit-Pan; Hsu, Kuo-Chiang

2012-05-01

The in vitro DPP-IV inhibitory activity of isolated peptides from of tuna cooking juice hydrolyzed by Protease XXIII (PR) and orientase (OR) was determined. The results showed that the peptide fractions with the molecular weight over 1,422 Da possessed the greatest DPP-IV inhibitory activity. The amino acid sequences of the three peptides isolated from PR and OR hydrolysates were identified by MALDI-TOF/TOF MS/MS, and they were Pro-Gly-Val-Gly-Gly-Pro-Leu-Gly-Pro-Ile-Gly-Pro-Cys-Tyr-Glu (1412.7 Da), Cys-Ala-Tyr-Gln-Trp-Gln-Arg-Pro-Val-Asp-Arg-Ile-Arg (1690.8 Da) and Pro-Ala-Cys-Gly-Gly-Phe-Try-Ile-Ser-Gly-Arg-Pro-Gly (1304.6 Da), while they showed the dose-dependent inhibition effect of DPP-IV with IC(50) values of 116.1, 78.0 and 96.4 μM, respectively. In vitro simulated gastrointestinal digestion retained or even improved the DPP-IV inhibitory activities of the three peptides. The results suggest that tuna cooking juice would be a good precursor of DPP-IV inhibitor, and the DPP-IV inhibitory peptides can successfully passed through the digestive tract. Copyright © 2012 Elsevier Inc. All rights reserved.

Engineering of isoamylase: improvement of protein stability and catalytic efficiency through semi-rational design.

PubMed

Li, Youran; Zhang, Liang; Ding, Zhongyang; Gu, Zhenghua; Shi, Guiyang

2016-01-01

Isoamylase catalyzes the hydrolysis of α-1,6-glycosidic linkages in glycogen, amylopectin and α/β-limit dextrins. A semi-rational design strategy was performed to improve catalytic properties of isoamylase from Bacillus lentus. Three residues in vicinity of the essential residues, Arg505, Asn513, and Gly608, were chosen as the mutation sites and were substituted by Ala, Pro, Glu, and Lys, respectively. Thermal stability of the mutant R505P and acidic stability of the mutant R505E were enhanced. The k cat /K m values of the mutant G608V have been promoted by 49%, and the specific activity increased by 33%. This work provides an effective strategy for improving the catalytic activity and stability of isoamylase, and the results obtained here may be useful for the improvement of catalytic properties of other α/β barrel enzymes.

Autosomal Dominant Retinal Dystrophies Caused by a Founder Splice Site Mutation, c.828+3A>T, in PRPH2 and Protein Haplotypes in trans as Modifiers

PubMed Central

Shankar, Suma P.; Hughbanks-Wheaton, Dianna K.; Birch, David G.; Sullivan, Lori S.; Conneely, Karen N.; Bowne, Sara J.; Stone, Edwin M.; Daiger, Stephen P.

2016-01-01

Purpose We determined the phenotypic variation, disease progression, and potential modifiers of autosomal dominant retinal dystrophies caused by a splice site founder mutation, c.828+3A>T, in the PRPH2 gene. Methods A total of 62 individuals (19 families) harboring the PRPH2 c.828+3A>T mutation, had phenotype analysis by fundus appearance, electrophysiology, and visual fields. The PRPH2 haplotypes in trans were sequenced for potential modifying variants and generalized estimating equations (GEE) used for statistical analysis. Results Several distinct phenotypes caused by the PRPH2 c.828+3A>T mutation were observed and fell into two clinical categories: Group I (N = 44) with mild pattern dystrophies (PD) and Group II (N = 18) with more severe cone-rod dystrophy (CRD), retinitis pigmentosa (RP), and central areolar chorioretinal dystrophy (CACD). The PRPH2 Gln304-Lys310-Asp338 protein haplotype in trans was found in Group I only (29.6% vs. 0%), whereas the Glu304-Lys310-Gly338 haplotype was predominant in Group II (94.4% vs. 70.4%). Generalized estimating equations analysis for PD versus the CRD/CACD/RP phenotypes in individuals over 43 years alone with the PRPH2 haplotypes in trans and age as predictors, adjusted for correlation within families, confirmed a significant effect of haplotype on severity (P = 0.03) with an estimated odds ratio of 7.16 (95% confidence interval [CI] = [2.8, 18.4]). Conclusions The PRPH2 c.828+3A>T mutation results in multiple distinct phenotypes likely modified by protein haplotypes in trans; the odds of having the CACD/RP-like phenotype (versus the PD phenotype) are 7.16 times greater with a Glu304-Lys310-Gly338 haplotype in trans. Further functional studies of the modifying haplotypes in trans and PRPH2 splice variants may offer therapeutic targets. PMID:26842753

The effect of perinatal anoxia on amino acid metabolism in the developing brain. Part II: The effect of perinatal anoxia on the free amino acid patterns in CSF of infants and children.

PubMed

Kaneko, K

1985-01-01

To clarify the effects of perinatal anoxia on the subsequent amino acid metabolism in the brain of children, free amino acid levels in the cerebrospinal fluid (CSF) were determined in 15 children diagnosed as having cerebral palsy and/or mental retardation with perinatal anoxia, and 58 control children without anoxia, aged from 4 days to 12 yrs. There was no significant difference in total amino acid levels between anoxic children and the controls. In the controls, the Gln level in CSF was high, Arg, Asp and Glu levels in CSF were almost the same during infancy and childhood, and the levels of Orn, Lys, His, Tau, Thr, Ser, Asn, Gly, Ala, Val, Met, Ile, Leu, Tyr and Phe in CSF decreased with age until pre-school age. In the newborns and infants among the anoxic children, the levels of most free amino acids in CSF were relatively high compared with those of the controls and, except Glu and Gln, decreased with age during infancy. The Orn, His, Gly, Tyr and Phe levels in CSF of anoxic children were lower than those of the controls in older infants. These results suggest that perinatal anoxia affected free amino acid patterns in CSF of newborns and infants and that the subsequent disturbance of amino acid metabolism in their brains remained after birth.

Red meat and poultry intake, polymorphisms in the nucleotide excision repair and mismatch repair pathways and colorectal cancer risk

PubMed Central

Joshi, Amit D.; Corral, Román; Siegmund, Kimberly D.; Haile, Robert W.; Le Marchand, Loïc; Martínez, Maria Elena; Ahnen, Dennis J.; Sandler, Robert S.; Lance, Peter; Stern, Mariana C.

2009-01-01

Diets high in red meat have been consistently associated with colorectal cancer (CRC) risk and may result in exposure to carcinogens that cause DNA damage [i.e polycyclic aromatic hydrocarbons, heterocyclic amines (HCAs) and N-nitroso compounds]. Using a family-based study, we investigated whether polymorphisms in the nucleotide excision repair (NER) (ERCC1 3′ untranslated region (UTR) G/T, XPD Asp312Asn and Lys751Gln, XPC intron 11 C/A, XPA 5′ UTR C/T, XPF Arg415Gln and XPG Asp1104His) and mismatch repair (MLH1 Ile219Val and MSH2 Gly322Asp) pathways modified the association with red meat and poultry intake. We tested for gene–environment interactions using case-only analyses (n = 577) and compared the results using case-unaffected sibling comparisons (n = 307 sibships). Increased risk of CRC was observed for intake of more than or equal to three servings per week of red meat [odds ratio (OR) = 1.8, 95% confidence interval (CI) = 1.3–2.5)] or high-temperature cooked red meat (OR = 1.6, 95% CI = 1.1–2.2). Intake of red meat heavily brown on the outside or inside increased CRC risk only among subjects who carried the XPD codon 751 Lys/Lys genotype (case-only interaction P = 0.006 and P = 0.001, respectively, for doneness outside or inside) or the XPD codon 312 Asp/Asp genotype (case-only interaction P = 0.090 and P < 0.001, respectively). These interactions were stronger for rectal cancer cases (heterogeneity test P = 0.002 for XPD Asp312Asn and P = 0.03 for XPD Lys751Gln) and remained statistically significant after accounting for multiple testing. Case-unaffected sibling analyses were generally supportive of the case-only results. These findings highlight the possible contribution of diets high in red meat to the formation of lesions that elicit the NER pathway, such as carcinogen-induced bulky adducts. PMID:19029193

A fibronectin receptor on Candida albicans mediates adherence of the fungus to extracellular matrix

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

Klotz, S.A.; Smith, R.L.

1991-03-01

Binding of fibronectin, an extracellular matrix (ECM) protein, to Candida albicans was measured, and adherence of the fungus to immobilized ECM proteins, fibronectin, laminin, types I and IV collagen, and subendothelial ECM was studied. 125I-labeled fibronectin was inhibited from binding to the fungus by unlabeled human plasma fibronectin and by Arg-Gly-Asp (RGD), Gly-Arg-Gly-Glu-Ser-Pro (GRGESP), and Gly-Arg-Gly-Asp-Thr-Pro (GRGDTP), but binding was not inhibited by Gly-Arg-Gly-Asp-Ser-Pro. Soluble fibronectin, RGD, GRGESP, and GRGDTP also inhibited fungal adherence to the individual immobilized ECM proteins in a complex pattern, but only soluble fibronectin (10(-7) M) inhibited fungal adherence to subendothelial ECM. Thus, C. albicans possessesmore » at least one type of cell surface receptor for binding soluble fibronectin that can be inhibited with peptides. This receptor apparently is used to bind the fungus to immobilized ECM proteins and to subendothelial ECM and may play a role in the initiation of disseminated disease by bloodborne fungi by providing for adherence of the microorganisms to ECM proteins.« less

Antagonists of substance P. Further modifications of substance P antagonists obtained by replacing either positions 7, 9 or 7, 8 and 11 of SP with D-amino acid residues.

PubMed

Dutta, A S; Gormley, J J; Graham, A S; Briggs, I; Growcott, J W; Jamieson, A

1986-07-01

Antagonists of SP and the C-terminal (6-11)-hexapeptide have been obtained by multiple D-amino acid substitutions in various positions of SP and by protecting the N alpha-Arg1 and N epsilon Lys3 amino groups with benzyloxycarbonyl groups. On the guinea pig ileum a number of these antagonized both SP and the hexapeptide. Except [N alpha-Z-Arg1,D-Pro2,N epsilon-Z-Lys3,Asn5,Arg6,D-Phe7,D-Trp9]-SP-OMe (4) and the corresponding amide 7, which were more potent antagonists of SP than the hexapeptide, all the others, e.g., [N alpha-Z-Arg1,D-Pro2,4,N epsilon-Z-Lys3,D-Phe7,8,Sar9,D-Met11]-SP-OMe (9), [N alpha-Z-Arg1,D-Pro2,4,N epsilon-Z-Lys3,D-Phe7,8,Sar9,MeLeu10,D-Met11]-SP -OMe (11), were more potent antagonists of the hexapeptide. On the rat spinal cord preparation, most of the antagonists were only active against the hexapeptide. A few antagonized SP, but these also reduced carbachol or both carbachol and glutamate responses. Two of the antagonists, [D-Pro2,Asn5,Lys6,D-Phe7,D-Trp9]-SP-OMe (2) and [Boc-D-Pro4,D-Phe7,8,Sar9,D-Met11]-SP(4-11)-OMe (10), were inactive on the ileum but still antagonized the hexapeptide on the spinal cord. The smallest peptides to antagonize SP and the hexapeptide were two heptapeptides, 6 and 21, [Z-Asn5,Arg6,D-Phe7,8,Gly9 psi (CH2S)D-Leu10,D-Met11]-SP(5-11)-OMe (21) being more potent than 6. None of the antagonists showed significant analgesic activity without side effects. Some of the antagonists were shown to release histamine from isolated rat peritoneal cells.

Novel angiotensin I-converting enzyme inhibitory peptides isolated from Alcalase hydrolysate of mung bean protein.

PubMed

Li, Guan-Hong; Wan, Ju-Zhen; Le, Guo-Wei; Shi, Yong-Hui

2006-08-01

Mung bean protein isolates were hydrolyzed for 2 h by Alcalase. The generated hydrolysate showed angiotensin I-converting enzyme (ACE) inhibitory activity with the IC(50) value of 0.64 mg protein/ml. Three kinds of novel ACE inhibitory peptides were isolated from the hydrolysate by Sephadex G-15 and reverse-phase high performance liquid chromatography (RP-HPLC). These peptides were identified by amino acid composition analysis and matrix assisted-laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), as Lys-Asp-Tyr-Arg-Leu, Val-Thr-Pro-Ala-Leu-Arg and Lys-Leu-Pro-Ala-Gly-Thr-Leu-Phe with the IC(50) values of 26.5 microM, 82.4 microM and 13.4 microM, respectively. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

Peptide (Lys-Leu) and amino acids (Lys and Leu) supplementations improve physiological activity and fermentation performance of brewer's yeast during very high-gravity (VHG) wort fermentation.

PubMed

Yang, Huirong; Zong, Xuyan; Cui, Chun; Mu, Lixia; Zhao, Haifeng

2017-12-22

Lys and Leu were generally considered as the key amino acids for brewer's yeast during beer brewing. In the present study, peptide Lys-Leu and a free amino acid (FAA) mixture of Lys and Leu (Lys + Leu) were supplemented in 24 °P wort to examine their effects on physiological activity and fermentation performance of brewer's yeast during very high-gravity (VHG) wort fermentation. Results showed that although both peptide Lys-Leu and their FAA mixture supplementations could increase the growth and viability, intracellular trehalose and glycerol content, wort fermentability, and ethanol content for brewer's yeast during VHG wort fermentation, and peptide was better than their FAA mixture at promoting growth and fermentation for brewer's yeast when the same dose was kept. Moreover, peptide Lys-Leu supplementation significantly increased the assimilation of Asp, but decreased the assimilation of Gly, Ala, Val, (Cys)2, Ile, Leu, Tyr, Phe, Lys, Arg, and Pro. However, the FAA mixture supplementation only promoted the assimilation of Lys and Leu, while reduced the absorption of total amino acids to a greater extent. Thus, the peptide Lys-Leu was more effective than their FAA mixture on the improvement of physiological activity, fermentation performance, and nitrogen metabolism of brewer's yeast during VHG wort fermentation. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Polymorphisms in XPD (Asp312Asn and Lys751Gln) genes, sunburn and arsenic-related skin lesions.

PubMed

McCarty, Kathleen M; Smith, Thomas J; Zhou, Wei; Gonzalez, Ernesto; Quamruzzaman, Quazzi; Rahman, Mahmuder; Mahiuddin, Golam; Ryan, Louise; Su, Li; Christiani, David C

2007-08-01

Single-nucleotide polymorphisms in genes related to DNA repair capacity and ultraviolet exposure have not been well investigated in relation to skin lesions associated with arsenic exposure. This population based case-control study, of 600 cases and 600 controls, frequency matched on age and gender in Pabna, Bangladesh, in 2001-2002, investigated the association and potential effect modification between polymorphisms in Xeroderma Pigmentosum complementation group D (XPD) (Lys751Gln and Asp312Asn) genes, tendency to sunburn and arsenic-related skin lesions. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). No significant association was observed between skin lesions and the XPD 312 Asp/Asn (adjusted OR = 0.87, 95% CI = 0.65-1.15) Asn/Asn (adjusted OR = 0.76, 95% CI = 0.50-1.15) (referent Asp/Asp); XPD 751 Lys/Gln (adjusted OR = 0.92, 95% CI = 0.69-1.23) Gln/Gln (adjusted OR = 0.98, 95% CI = 0.66-1.45) (referent Lys/Lys). While we did not observe any evidence of effect modification of these polymorphisms on the association between well arsenic concentration and skin lesions, we did observe effect modification between these polymorphisms and sunburn tendency and arsenic-related skin lesions. Individuals with the heterozygote or homozygote variant forms (Asp/Asn or Asn/Asn) had half the risk of skin lesions (OR = 0.45, 95% CI = 0.29-0.68) compared with those with the wild-type XPDAsp312Asn genotype (Asp/Asp) and individuals with heterozygote or homozygote variant forms (Lys/Gln or Gln/Gln) had half the risk of skin lesions (OR = 0.47, 95% CI = 0.31-0.72) compared with those with the wild-type XPDLys751Gln genotype (Lys/Lys), within the least sensitive strata of sunburn severity. We observed effect modification on the multiplicative scale for XPD 751 and XPD 312. XPD polymorphisms modified the relationship between tendency to sunburn and skin lesions in an arsenic exposed population. Further study is necessary to explore the effect of XPD polymorphisms and sun exposure on risk of arsenic-related skin lesions.

EDN1 Lys198Asn is associated with diabetic retinopathy in type 2 diabetes

PubMed Central

Li, Haitao; Louey, Janice W.C.; Choy, Kwong Wai; Liu, David T.L.; Chan, Wai Man; Chan, Yiu Man; Fung, Nicholas S.K.; Fan, Bao Jian; Baum, Larry; Chan, Juliana C.N.; Lam, Dennis S.C.

2008-01-01

Purpose We tested the hypothesis that genetic variants in vasoactive and angiogenic factors regulating the retina vasculature contribute to the development of diabetic retinopathy (DR). Methods A case-control study was performed to study the genetic association between DR and polymorphic variants of EDN1 (Lys198Asn), LTA (IVS1–80C>A, IVS1–206G>C, IVS1–252A>G), eNOS (Glu298Asp), and ITGA2 (BgI II) in a Chinese population with type 2 diabetes mellitus. A well defined population with type 2 diabetes, consisting of 127 controls and 216 DR patients, was recruited. Results A higher frequency of the Asn/Asn genotype of EDN1 was found in individuals with at least 10 years of diabetes and no retinopathy (controls) compared with DR patients with any duration of diabetes (DR: 2.3%; control: 11.0%; p=0.0002). The Asn allele was also more frequent in controls than DR patients (DR: 16.4%; control: 29.5%; p=0.007). Multiple logistic regression analysis showed that the Asn/Asn genotype was the factor most significantly associated with reduced risk of DR (odds ratio=0.19; 95% CI: 0.07-0.53; p=0.002) and with late onset of diabetes (Asn/Asn: 59 years; Lys/Lys + Lys/Asn: 53 years; p=0.02). Moreover, the Lys/Lys genotype was more common among patients with nonproliferative (75.7%) than proliferative DR (56.9%; p=0.008). The distributions of Lys198Asn alleles in hypertension did not differ from normotensive subjects. No associations between DR and polymorphisms of LTA, eNOS, or ITGA2 were detected, and there were no detectable gene-gene or gene-environmental interactions among the polymorphisms. Conclusions The Asn/Asn genotype of EDN1 was associated with a reduced risk of DR and with delayed onset of type 2 diabetes. PMID:18806884

Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang.

PubMed Central

Kim, W; Choi, K; Kim, Y; Park, H; Choi, J; Lee, Y; Oh, H; Kwon, I; Lee, S

1996-01-01

Bacillus sp. strain CK 11-4, which produces a strongly fibrinolytic enzyme, was screened from Chungkook-Jang, a traditional Korean fermented-soybean sauce. The fibrinolytic enzyme (CK) was purified from supernatant of Bacillus sp. strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity. The optimum temperature and pH were 70 degrees C and 10.5, respectively, and the molecular weight was 28,200 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 14 amino acids of the N-terminal sequence of CK are Ala-Gin-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp. This sequence is identical to that of subtilisin Carlsberg and different from that of nattokinase, but CK showed a level of fibrinolytic activity that was about eight times higher than that of subtilisin Carlsberg. The amidolytic activity of CK increased about twofold at the initial state of the reaction when CK enzyme was added to a mixture of plasminogen and substrate (H-D-Val-Leu-Lys-pNA). A similar result was also obtained from fibrin plate analysis. PMID:8779587

Combining UV photodissociation action spectroscopy with electron transfer dissociation for structure analysis of gas-phase peptide cation-radicals.

PubMed

Shaffer, Christopher J; Pepin, Robert; Tureček, František

2015-12-01

We report the first example of using ultraviolet (UV) photodissociation action spectroscopy for the investigation of gas-phase peptide cation-radicals produced by electron transfer dissociation. z-Type fragment ions (●) Gly-Gly-Lys(+), coordinated to 18-crown-6-ether (CE), are generated, selected by mass and photodissociated in the 200-400 nm region. The UVPD action spectra indicate the presence of valence-bond isomers differing in the position of the Cα radical defect, (α-Gly)-Gly-Lys(+) (CE), Gly-(α-Gly)-Lys(+) (CE) and Gly-Gly-(α-Lys(+))(CE). The isomers are readily distinguishable by UV absorption spectra obtained by time-dependent density functional theory (TD-DFT) calculations. In contrast, conformational isomers of these radical types are calculated to have similar UV spectra. UV photodissociation action spectroscopy represents a new tool for the investigation of transient intermediates of ion-electron reactions. Specifically, z-type cation radicals are shown to undergo spontaneous hydrogen atom migrations upon electron transfer dissociation. Copyright © 2015 John Wiley & Sons, Ltd.

Role of different β-turns in β-hairpin conformation and stability studied by optical spectroscopy.

PubMed

Wu, Ling; McElheny, Dan; Setnicka, Vladimír; Hilario, Jovencio; Keiderling, Timothy A

2012-01-01

Model β-hairpin peptides based on variations in the turn sequence of Cochran's tryptophan zipper peptide, SWTWENGKWTWK, were studied using electronic circular dichroism (ECD), fluorescence, and infrared (IR) spectroscopies. The trpzip2 Asn-Gly turn sequence was substituted with Thr-Gly, Aib-Gly, (D)Pro-Gly, and Gly-Asn (trpzip1) to study the impact of turn stability on β-hairpin formation. Stability and conformational changes of these hairpins were monitored by thermodynamic analyses of the temperature variation of both FTIR (amide I') and ECD spectral intensities. These changes were fit to a two-state model which yielded different T(m) values, representing the folding/unfolding process, for hairpins with different β-turns. Different β-turns show systematic contributions to hairpin structure formation, and their inclusion in hairpin design can modify the folding pathways. Aib-Gly or (D)Pro-Gly sequences stabilize the turn resulting in residual Trp-Trp interaction at high temperatures, but at the same time the β-structure (cross strand H-bonds) can become less stable due to constraints of the turn, as seen for (D)Pro-Gly. The structure of the Aib-Gly turn containing hairpin was determined by NMR and was shown to be like trpzip2 (Asn-Gly turn) as regards turn and strand geometries, but to differ from trpzip1 (Gly-Asn turn). The Munoz and Eaton statistical mechanically derived multistate model, tested as an alternate point of view, represented contributions from H-bonds and hydrophobic interactions as well as conformational change as interdependent. Use of different spectral methods that vary in dependence on these physical interactions along with the structural variations provided insight to the complex folding pathways of these small, well-folded peptides. Copyright © 2011 Wiley Periodicals, Inc.

Mullinamides A and B, New Cyclopeptides Produced by the Ruth Mullins Coal Mine Fire Isolate Streptomyces sp. RM-27-46

PubMed Central

Wang, Xiachang; Shaaban, Khaled A.; Elshahawi, Sherif I.; Ponomareva, Larissa V.; Sunkara, Manjula; Copley, Gregory C.; Hower, James C.; Morris, Andrew J.; Kharel, Madan K.; Thorson, Jon S.

2014-01-01

Two new cyclopeptides, mullinamides A [cyclo-(-l-Gly-l-Glu-l-Val-l-Ile-l-Pro-)] and B [cyclo-(-l-Glu-l-Met-l-Pro-)] were isolated from the crude extract of terrestrial Streptomyces sp. RM-27-46 along with the three known cyclopeptides surugamide A [cyclo-(-l-Ile-d-Ile-l-Lys-l-Ile-d-Phe-d-Leu-l-Ile-d-Ala-)], cyclo-(-l-Pro-l-Phe-) and cyclo-(-l-Pro-l-Leu-). The structures of the new compounds were elucidated by the cumulative analyses of NMR spectroscopy and high resolution mass spectrometry. While mullinamides A and B displayed no appreciable antimicrobial/fungal activity or cytotoxicity, this study highlights the first reported antibacterial activity of surugamide A. PMID:24713874

Molecularly imprinted polymers for RGD selective recognition and separation.

PubMed

Papaioannou, Emmanuel; Koutsas, Christos; Liakopoulou-Kyriakides, Maria

2009-03-01

Molecularly imprinted polymers that could recognize the tripeptide Arg-Gly-Asp have been produced with the use of two functional monomers and three different cross-linkers, respectively. Methacrylic acid and acrylamide were used as functional monomers and the role of the ethylene glycol dimethacrylate, trimethylpropane trimethacrylate and N,N'-methylene-bisacrylamide as crosslinking monomers, was investigated on their recognition capability. The % net rebinding and the imprinting factor values were obtained, giving for the methacrylic acid-trimethylpropane trimethacrylate polymer the highest values 12.3% and 2.44, respectively. In addition, this polymer presented lower dissociation constant (K(D)) value and the higher B (max)% of theoretical total binding sites than all the other polymers. Rebinding experiments with Lys-Gly-Asp, an analogue of Arg-Gly-Asp, and other different peptides, such as cholecystokinin C-terminal tri- and pentapeptide and gramicidin, further indicated the selectivity of methacrylic acid-trimethylpropane trimethacrylate copolymer for Arg-Gly-Asp giving specific selectivity factor values 1.27, 1.98, 1.31 and 1.67, respectively.

Differential effects of C- and N-terminal substance P metabolites on the release of amino acid neurotransmitters from the spinal cord: potential role in nociception.

PubMed

Skilling, S R; Smullin, D H; Larson, A A

1990-04-01

Extensive evidence implicates Substance P [SP(1-11)] as a primary afferent neurotransmitter or modulator of nociceptive information, and there is increasing evidence that the excitatory amino acids aspartate (Asp) and glutamate (Glu) may also act as nociceptive neurotransmitters. We have previously demonstrated that nociceptive stimulation (metatarsal injection of formalin) caused a tetrodotoxin (TTX)-sensitive release of Asp and a TTX-insensitive release of Glu from the dorsal spinal cord. We have also shown release of Asp and Glu following the direct infusion of SP(1-11), suggesting that formalin-induced Asp or Glu changes could be secondary to an initial release of SP(1-11). In contrast to nociception, pretreatment with TTX, reported here, had no effect on the SP(1-11)-induced release of Asp, suggesting a presynaptic mechanism. Behavioral experiments, in both our laboratory, and others, now suggest that the N-terminal products of SP metabolism play a distinct role in the modulation of SP(1-11) nociception, possibly through an interaction with an opiate receptor. To test the hypothesis that N- and C-terminal fragments of SP produce opposite effects on biochemical events potentially involved in nociception, we compared the effects of infusion of the N-terminal metabolite SP(1-7) and the C-terminal metabolite SP(5-11) on changes in the ECF concentration of amino acids in the spinal cord as a measure of their apparent release, using microdialysis. Intradiaylsate infusion of SP(5-11) increased the release of Asp, Glu, asparagine (Asn), glycine (Gly), and taurine (Tau). The changes in Asp, Glu, and Tau were similar in direction and magnitude to changes produced by SP(1-11) or formalin injection, further supporting the hypothesis that the C-terminal is responsible for the nociceptive effects of SP(1-11). In contrast, infusion of SP(1-7) significantly decreased the release of Asn, Tau, Glu, and Gly. This inhibition of amino acid release is consistent with the hypothesis that N-terminal metabolites produce opposite effects to those of C-terminal metabolites of SP(1-11). The decreases in Glu, Asn, Gly, and Tau following SP(1-7) infusion were significantly reduced by i.p. or intradialysate naloxone. Systemic naloxone had no significant effects on the SP(5-11)-induced amino acid changes; however, it did inhibit the SP(1-11)-induced increase in Asp and Glu. Intradialysate naloxone had no effect on the SP(1-11)-induced increases.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetic polymorphisms of xeroderma pigmentosum group D gene Asp312Asn and Lys751Gln and susceptibility to prostate cancer: a systematic review and meta-analysis.

PubMed

Ma, Qingtong; Qi, Can; Tie, Chong; Guo, Zhanjun

2013-11-10

Many studies have reported the role of xeroderma pigmentosum group D (XPD) with prostate cancer risk, but the results remained controversial. To derive a more precise estimation of the relationship, a meta-analysis was performed. Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated to assess the association between XPD Asp312Asn and Lys751Gln polymorphisms and prostate cancer risk. A total of 8 studies including 2620 cases and 3225 controls described Asp312Asn genotypes, among which 10 articles involving 3230 cases and 3582 controls described Lys751Gln genotypes and were also involved in this meta-analysis. When all the eligible studies were pooled into this meta-analysis, a significant association between prostate cancer risk and XPD Asp312Asn polymorphism was found. For Asp312Asn polymorphism, in the stratified analysis by ethnicity and source of controls, prostate cancer risk was observed in co-dominant, dominant and recessive models, while no evidence of any associations of XPD Lys751Gln polymorphism with prostate cancer was found in the overall or subgroup analyses. Our meta-analysis supports that the XPD Asp312Asn polymorphism contributed to the risk of prostate cancer from currently available evidence. However, a study with a larger sample size is needed to further evaluate gene-environment interaction on XPD Asp312Asn and Lys751Gln polymorphisms and prostate cancer risk. © 2013.

Design and characterization of hirulogs: A novel class of bivalent peptide inhibitors of thrombin

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

Maraganore, J.M.; Bourdon, P.; Jablonski, J.

1990-07-31

A novel class of synthetic peptides has been designed that inhibit the thrombin catalytic site and exhibit specificity for the anion-binding exosite (ABE) of {alpha}-thrombin. These peptides, called hirulogs, consist of (i) an active-site specificity sequence with a restricted Arg-Pro scissile bond, (ii) a polymeric linker of glycyl residues from 6 to 18 {angstrom} in length, and (iii) an ABE recognition sequence such as that in the hirudin C-terminus. Hirulog-1 ((D-Phe)-Pro-Arg-Pro-(Gly){sub 4}-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Tyr-Leu) inhibits the thrombin-catalyzed hydrolysis of a tripeptide p-nitroanilide substrate with K{sub i} = 2.3 nM. In contrast, the synthetic C-terminal hirudin peptide S-Hir{sub 53-64}, which binds to themore » thrombin ABE, blocked the fibrinogen clotting activity of the enzyme with K{sub i} = 144 nM but failed to inhibit the hydrolysis of p-nitroanilide substrates at concentrations as high as 1 mM. Hirulog-1, but not S-Hir{sub 53-64}, was found to inhibit the incorporation of ({sup 14}C)diisopropyl fluorophosphate in thrombin. Hirulog-1 appears specific for thrombin as it lacks inhibitory activities toward human factor Xa, human plasmin, and bovine trypsin at inhibitor:enzyme concentrations 3 orders of magnitude higher than those required to inhibit thrombin. The optimal inhibitory activity of hirulog-1 depends upon all three components of its structure. Comparison of anticoagulant activities of hirulog-1, hirudin, and S-Hir{sub 53-64} showed that the synthetic hirulog-1 is 2-fold more potent than hirudin and 100-fold more active than S-Hir{sub 53-64} in increasing the activated partial thromboplastin time of normal human plasma.« less

Polymorphisms in XPD (Asp312Asn and Lys751Gln) genes, sunburn and arsenic-related skin lesions

PubMed Central

McCarty, Kathleen M.; Smith, Thomas J.; Zhou, Wei; Gonzalez, Ernesto; Quamruzzaman, Quazzi; Rahman, Mahmuder; Mahiuddin, Golam; Ryan, Louise; Su, Li; Christiani, David C.

2013-01-01

Background Single-nucleotide polymorphisms in genes related to DNA repair capacity and ultraviolet exposure have not been well investigated in relation to skin lesions associated with arsenic exposure. This population based case–control study, of 600 cases and 600 controls, frequency matched on age and gender in Pabna, Bangladesh, in 2001–2002, investigated the association and potential effect modification between polymorphisms in Xeroderma Pigmentosum complementation group D (XPD) (Lys751Gln and Asp312Asn) genes, tendency to sunburn and arsenic-related skin lesions. Methods Unconditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Result No significant association was observed between skin lesions and the XPD 312 Asp/Asn (adjusted OR = 0.87, 95% CI = 0.65–1.15) Asn/Asn (adjusted OR = 0.76, 95% CI = 0.50–1.15) (referent Asp/Asp); XPD 751 Lys/Gln (adjusted OR = 0.92, 95% CI = 0.69–1.23) Gln/Gln (adjusted OR = 0.98, 95% CI = 0.66–1.45) (referent Lys/Lys). While we did not observe any evidence of effect modification of these polymorphisms on the association between well arsenic concentration and skin lesions, we did observe effect modification between these polymorphisms and sunburn tendency and arsenic-related skin lesions. Individuals with the heterozygote or homozygote variant forms (Asp/Asn or Asn/Asn) had half the risk of skin lesions (OR = 0.45, 95% CI = 0.29–0.68) compared with those with the wild-type XPDAsp312Asn genotype (Asp/Asp) and individuals with heterozygote or homozygote variant forms (Lys/Gln or Gln/Gln) had half the risk of skin lesions (OR = 0.47, 95% CI = 0.31–0.72) compared with those with the wild-type XPDLys751Gln genotype (Lys/Lys), within the least sensitive strata of sunburn severity. We observed effect modification on the multiplicative scale for XPD 751 and XPD 312. Conclusion XPD polymorphisms modified the relationship between tendency to sunburn and skin lesions in an arsenic exposed population. Further study is necessary to explore the effect of XPD polymorphisms and sun exposure on risk of arsenic-related skin lesions. PMID:17470448

Covalent structure of chicken pepsinogen.

PubMed

Baudys, M; Kostka, V

1983-10-17

Chicken pepsinogen is a glycoprotein consisting of a single polypeptide chain and containing the following 367 amino acid residues: Asp23, Asn16, Thr26, Ser41, Glu14, Gln11, Pro18, Gly31, Ala17, Cys7, Val25, Met9, Ile23, Leu28, Tyr22, Phe20, His8, Lys17, Arg7, Trp4. The Mr-value of the protein is 42 074. This value includes the carbohydrate moiety of the protein, i.e. Man3, (GlcNAc)7, (-SO3H)5. The primary fragmentation of the molecule was effected by limited trypsinolysis at arginine residues after preceding modification of the lysines with citraconic anhydride. All eight peptides expected in theory were obtained and their size, amino acid composition, and N-terminal amino acid sequence were characterized. To elucidate the amino acid sequence of these large fragments the latter were subjected to secondary cleavage by CNBr, trypsin (after removal of the protecting groups from the lysines), the proteinase from Staphylococcus aureus V8 strain, alpha-chymotrypsin, hydroxylamine, or dilute acid; the resulting peptides were isolated by gel permeation and ion-exchange chromatography and by the fingerprint techniques. Overlaps at sites of the arginine residues were obtained in an earlier study [Baudys, M. & Kostka, V. (1982) Collect. Czech. Chem. Commun. 47, 2814-2832]. Chicken pepsinogen shows the highest degree of homology with the primary structures of pepsinogens A. The internal homologies are apparent in the neighborhood of the two active aspartic acid residues. We have assigned tentatively chicken pepsinogen to the group of pepsinogens A (EC 3.4.23.1); this assignment is a result both of our sequence studies and of an investigation of the kinetic characteristics of the enzyme.

Production of antioxidant and ACE-inhibitory peptides from Kluyveromyces marxianus protein hydrolysates: Purification and molecular docking.

PubMed

Mirzaei, Mahta; Mirdamadi, Saeed; Ehsani, Mohamad Reza; Aminlari, Mahmoud

2018-04-01

Kluyveromyces marxianus protein hydrolysates were prepared by two different sonicated-enzymatic (trypsin and chymotrypsin) hydrolysis treatments to obtain antioxidant and ACE-inhibitory peptides. Trypsin and chymotrypsin hydrolysates obtained by 5 h, exhibited the highest antioxidant and ACE-inhibitory activities. After fractionation using ultrafiltration and reverse phase high performance liquid chromatography (RP-HPLC) techniques, two new peptides were identified. One fragment (LL-9, MW = 1180 Da) with the amino acid sequence of Leu-Pro-Glu-Ser-Val-His-Leu-Asp-Lys showed significant ACE inhibitory activity (IC 50  = 22.88 μM) while another peptide fragment (VL-9, MW = 1118 Da) with the amino acid sequence of Val-Leu-Ser-Thr-Ser-Phe-Pro-Pro-Lys showed the highest antioxidant and ACE inhibitory properties (IC 50  = 15.20 μM, 5568 μM TE/mg protein). The molecular docking studies revealed that the ACE inhibitory activities of VL-9 is due to interaction with the S2 (His513, His353, Glu281) and S'1 (Glu162) pockets of ACE and LL-9 can fit perfectly into the S1 (Thr345) and S2 (Tyr520, Lys511, Gln281) pockets of ACE. Copyright © 2017. Published by Elsevier B.V.

Effect of vilon and epithalon on activity of enzymes in epithelial and subepithelial layers in small intestine of old rats.

PubMed

Khavinson, V Kh; Timofeeva, N M; Malinin, V V; Gordova, L A; Nikitina, A A

2002-12-01

Per os administration of Vilon (Lys-Glu) or Epithalon (Ala-Glu-Asp-Gly) to aged Wistar rats for 1 month significantly increased activity of membrane enzymes maltase and alkaline phosphatase in epithelial layer of the small intestine. In addition, Vilon significantly increased activity of cytosolic glycyl-L-leucine dipeptidase in the stromal and seromuscular layers of the small intestine in comparison with the control rats not treated with this agent. These findings suggest improvement of trophic and barrier functions of the small intestine and corroborate the hypothesis on the existence of not only epithelial, but also subepithelial enzymatic barrier supporting the enzyme system in the small intestine, especially in aged animals.

Products of cholecystokinin (CCK)-octapeptide proteolysis interact with central CCK receptors.

PubMed

Steardo, L; Knight, M; Tamminga, C A; Chase, T N

1985-03-15

Peptidases present in central nervous system (CNS) synaptic membranes, hydrolyze the neuroactive peptide cholecystokinin-octapeptide (CCK-8; Asp-Tyr-SO3H-Met-Gly-Trp-Met-Asp-Phe-NH2). In order to determine the pathway of degradation, synthetic CCK-8 was incubated at 37 degrees C with purified synaptic membranes; at various intervals reaction samples were removed from the reaction mixture and analysed by high-performance liquid chromatography to identify and quantify the peptide fragments. The results indicate an initial endopeptidase cleavage at the Met-Gly bond producing CCK-5 (Gly-Trp-Met-Asp-Phe-NH2). The carboxyl-terminal pentapeptide is further proteolysed to CCK-4 (Trp-Met-Asp-Phe-NH2) by a puromycin-sensitive aminopeptidase and to CCK-3 (Met-Asp-Phe-NH2) and Gly-Trp by an endopeptidase action. CCK-3 and CCK-2 appear to be relatively stable end-products. Moreover, these proteolytic fragments are shown to bind to the CCK receptor in brain with varying potencies.

De novo mutations of the ATP6V1A gene cause developmental encephalopathy with epilepsy.

PubMed

Fassio, Anna; Esposito, Alessandro; Kato, Mitsuhiro; Saitsu, Hirotomo; Mei, Davide; Marini, Carla; Conti, Valerio; Nakashima, Mitsuko; Okamoto, Nobuhiko; Olmez Turker, Akgun; Albuz, Burcu; Semerci Gündüz, C Nur; Yanagihara, Keiko; Belmonte, Elisa; Maragliano, Luca; Ramsey, Keri; Balak, Chris; Siniard, Ashley; Narayanan, Vinodh; Ohba, Chihiro; Shiina, Masaaki; Ogata, Kazuhiro; Matsumoto, Naomichi; Benfenati, Fabio; Guerrini, Renzo

2018-06-01

V-type proton (H+) ATPase (v-ATPase) is a multi-subunit proton pump that regulates pH homeostasis in all eukaryotic cells; in neurons, v-ATPase plays additional and unique roles in synapse function. Through whole exome sequencing, we identified de novo heterozygous mutations (p.Pro27Arg, p.Asp100Tyr, p.Asp349Asn, p.Asp371Gly) in ATP6V1A, encoding the A subunit of v-ATPase, in four patients with developmental encephalopathy with epilepsy. Early manifestations, observed in all patients, were developmental delay and febrile seizures, evolving to encephalopathy with profound delay, hypotonic/dyskinetic quadriparesis and intractable multiple seizure types in two patients (p.Pro27Arg, p.Asp100Tyr), and to moderate delay with milder epilepsy in the other two (p.Asp349Asn, p.Asp371Gly). Modelling performed on the available prokaryotic and eukaryotic structures of v-ATPase predicted p.Pro27Arg to perturb subunit interaction, p.Asp100Tyr to cause steric hindrance and destabilize protein folding, p.Asp349Asn to affect the catalytic function and p.Asp371Gly to impair the rotation process, necessary for proton transport. We addressed the impact of p.Asp349Asn and p.Asp100Tyr mutations on ATP6V1A expression and function by analysing ATP6V1A-overexpressing HEK293T cells and patients' lymphoblasts. The p.Asp100Tyr mutant was characterized by reduced expression due to increased degradation. Conversely, no decrease in expression and clearance was observed for p.Asp349Asn. In HEK293T cells overexpressing either pathogenic or control variants, p.Asp349Asn significantly increased LysoTracker® fluorescence with no effects on EEA1 and LAMP1 expression. Conversely, p.Asp100Tyr decreased both LysoTracker® fluorescence and LAMP1 levels, leaving EEA1 expression unaffected. Both mutations decreased v-ATPase recruitment to autophagosomes, with no major impact on autophagy. Experiments performed on patients' lymphoblasts using the LysoSensor™ probe revealed lower pH of endocytic organelles for p.Asp349Asn and a reduced expression of LAMP1 with no effect on the pH for p.Asp100Tyr. These data demonstrate gain of function for p.Asp349Asn characterized by an increased proton pumping in intracellular organelles, and loss of function for p.Asp100Tyr with decreased expression of ATP6V1A and reduced levels of lysosomal markers. We expressed p.Asp349Asn and p.Asp100Tyr in rat hippocampal neurons and confirmed significant and opposite effects in lysosomal labelling. However, both mutations caused a similar defect in neurite elongation accompanied by loss of excitatory inputs, revealing that altered lysosomal homeostasis markedly affects neurite development and synaptic connectivity. This study provides evidence that de novo heterozygous ATP6V1A mutations cause a developmental encephalopathy with a pathomechanism that involves perturbations of lysosomal homeostasis and neuronal connectivity, uncovering a novel role for v-ATPase in neuronal development.

Purification, characterization, and biological activity of a substance P-related peptide from the gut of the Australian lungfish, Neoceratodus forsteri.

PubMed

Liu, Lu; Conlon, J Michael; Joss, Jean M P; Burcher, Elizabeth

2002-01-01

A peptide with mammalian substance P (SP)-like immunoreactivity was isolated from an extract of the spiral intestine of the Australian lungfish, Neoceratodus forsteri. The primary structure of this peptide was established as Lys-Pro-Arg-Pro-Asp-Glu-Phe-Tyr-Gly-Leu-Met . NH2, showing 64% identity with mammalian SP. In isolated preparations of lungfish foregut circular muscle, lungfish SP produced a slow, long-lasting tonic contraction, with a pD2 value of 8.19. Lungfish midgut circular muscle preparations responded to lungfish SP rapidly and in a more complex manner. There was an increase in the frequency of spontaneous activity (pD2 = 8.76), associated with diminished amplitude of the spontaneous contractions (pD2 = 9.24), also coupled in some preparations with a tonic contraction (pD2 = 8.43). The response patterns of foregut and midgut circular muscle to acetylcholine (ACh) were very similar to those seen to lungfish SP. Lungfish SP and ACh, however, had very weak effects on both foregut and midgut longitudinal muscle. These data demonstrate that lungfish SP may be a physiologically important regulator of gastrointestinal motility in Neoceratodus. This study further confirmed that the structures of SP-related peptides have been strongly conserved under the pressure of vertebrate evolution, particularly in preserving the functionally important sequence, Phe-Xaa-Gly-Leu-Met . amide, at the C-terminus. The sequence of lungfish SP is identical to that of bufokinin, a SP-related peptide previously isolated from the intestine of the cane toad, Bufo marinus, supporting the hypothesis that lungfishes and amphibians share a common ancestor.

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme III/sup mtl/ of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme II/sup mtl/ of Escherichia coli

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

Reiche, B.; Frank, R.; Deutscher, J.

1988-08-23

Enzyme III/sup mtl/ is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, the authors report the isolation of III/sup mtl/ from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of III/sup mtl/ with (/sup 32/P)PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase GLu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp-Asp. The correspondingmore » peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which they assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the III/sup mtl/ proteins was found to be 15,000. They have also determined the N-terminal sequence of both proteins. Comparison of the III/sup mtl/ peptide sequences and the C-terminal part of the enzyme II/sup mtl/ of Escherichia coli reveals considerable sequence homology, which supports the suggestion that II/sup mtl/ of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II.« less

Aspartic acid 405 contributes to the substrate specificity of aminopeptidase B.

PubMed

Fukasawa, Kayoko M; Hirose, Junzo; Hata, Toshiyuki; Ono, Yukio

2006-09-26

Aminopeptidase B (EC 3.4.11.6, ApB) specifically cleaves in vitro the N-terminal Arg or Lys residue from peptides and synthetic derivatives. Ap B was shown to have a consensus sequence found in the metallopeptidase family. We determined the putative zinc binding residues (His324, His328, and Glu347) and the essential Glu325 residue for the enzyme using site-directed mutagenesis (Fukasawa, K. M., et al. (1999) Biochem. J. 339, 497-502). To identify the residues binding to the amino-terminal basic amino acid of the substrate, rat cDNA encoding ApB was cloned into pGEX-4T-3 so that recombinant protein was expressed as a GST fusion protein. Twelve acidic amino acid residues (Glu or Asp) in ApB were replaced with a Gln or Asn using site-directed mutagenesis. These mutants were isolated to characterize the kinetic parameters of enzyme activity toward Arg-NA and compare them to those of the wild-type ApB. The catalytic efficiency (kcat/Km) of the mutant D405N was 1.7 x 10(4) M(-1) s(-1), markedly decreased compared with that of the wild-type ApB (6.2 x 10(5) M(-1) s(-1)). The replacement of Asp405 with an Asn residue resulted in the change of substrate specificity such that the specific activity of the mutant D405N toward Lys-NA was twice that toward Arg-NA (in the case of wild-type ApB; 0.4). Moreover, when Asp405 was replaced with an Ala residue, the kcat/Km ratio was 1000-fold lower than that of the wild-type ApB for hydrolysis of Arg-NA; in contrast, in the hydrolysis of Tyr-NA, the kcat/Km ratios of the wild-type (1.1 x 10(4) M(-1) s(-1)) and the mutated (8.2 x 10(3) M(-1) s(-1)) enzymes were similar. Furthermore, the replacement of Asp-405 with a Glu residue led to the reduction of the kcat/Km ratio for the hydrolysis of Arg-NA by a factor of 6 and an increase of that for the hydrolysis of Lys-NA. Then the kcat/Km ratio of the D405E mutant for the hydrolysis of Lys-NA was higher than that for the hydrolysis of Arg-NA as opposed to that of wild-type ApB. These data strongly suggest that the Asp 405 residue is involved in substrate binding via an interaction with the P1 amino group of the substrate's side chain.

Purification and structural characterization of vasoactive intestinal polypeptide from the trout and bowfin.

PubMed

Wang, Y; Conlon, J M

1995-04-01

Vasoactive intestinal polypeptide (VIP) was purified from extracts of the stomachs of the rainbow trout, Oncorhynchus mykiss, and the bowfin, Amia calva. The primary structure of VIP from both species was the same: His-Ser-Asp-Ala-Ile-Phe-Thr-Asp-Asn-Tyr10- Ser-Arg-Phe-Arg-Lys-Gln-Met-Ala-Val-Lys20-Lys-Tyr-Leu-Asn-Ser-Val- Leu-Thr. This amino acid sequence shows only one amino acid substitution (Val5-->Ile) compared with the common sequence of VIP from the chicken, alligator, and European green frog. The structural identity of VIP from the trout and bowfin is consistent with the close phylogenetic relationship between the Salmoniformes and the Amiiformes and the data indicate that pressure to conserve the complete primary structure of VIP during vertebrate evolution has been very strong.

Impact of microencapsulated peptidase (Aspergillus oryzae) on cheddar cheese proteolysis and its biologically active peptide profile.

PubMed

Seneweera, Saman; Kailasapathy, Kaila

2011-07-01

We investigated the delivery of calcium-alginate encapsulated peptidase (Flavourzyme(®), Aspergillus oryzae) on proteolysis of Cheddar cheese. Physical and chemical characteristics such as moisture, pH and fat content were measured, and no differences were found between control and experimental cheese at day 0. SDS-PAGE analysis clearly showed that proteolysis of α and k casein was significantly accelerated after three months of maturity in the experimental cheese. A large number of low molecular weight peptides were found in the water soluble fraction of the experimental cheeses and some of these peptides were new. N-terminal amino acid sequence analysis identified these as P(1), Leu-Thu-Glu; P(3), Asp-Val-Pro-Ser-Glu) and relatively abundant stable peptides P(2), P(4), Arg-Pro-Lys-His-Pro-Ile; P(5), Arg-Pro-Lys-His-Pro-Ile-Lys and P(6). These peptides were mainly originated from αs1-CN and β-CN. Three of the identified peptides (P(1), P(2), P(3) and P(4)) are known to biologically active and P(1) and P(3) were only present in experimental cheese suggesting that experimental cheese has improved health benefits.

Ranalexin. A novel antimicrobial peptide from bullfrog (Rana catesbeiana) skin, structurally related to the bacterial antibiotic, polymyxin.

PubMed

Clark, D P; Durell, S; Maloy, W L; Zasloff, M

1994-04-08

Antimicrobial peptides comprise a diverse class of molecules used in host defense by plants, insects, and animals. In this study we have isolated a novel antimicrobial peptide from the skin of the bullfrog, Rana catesbeiana. This 20 amino acid peptide, which we have termed Ranalexin, has the amino acid sequence: NH2-Phe-Leu-Gly-Gly-Leu-Ile-Lys-Ile-Val-Pro-Ala-Met-Ile-Cys-Ala-Val-Thr- Lys-Lys - Cys-COOH, and it contains a single intramolecular disulfide bond which forms a heptapeptide ring within the molecule. Structurally, Ranalexin resembles the bacterial antibiotic, polymyxin, which contains a similar heptapeptide ring. We have also cloned the cDNA for Ranalexin from a metamorphic R. catesbeiana tadpole cDNA library. Based on the cDNA sequence, it appears that Ranalexin is initially synthesized as a propeptide with a putative signal sequence and an acidic amino acid-rich region at its amino-terminal end. Interestingly, the putative signal sequence of the Ranalexin cDNA is strikingly similar to the signal sequence of opioid peptide precursors isolated from the skin of the South American frogs Phyllomedusa sauvagei and Phyllomedusa bicolor. Northern blot analysis and in situ hybridization experiments demonstrated that Ranalexin mRNA is first expressed in R. catesbeiana skin at metamorphosis and continues to be expressed into adulthood.

A novel hemoglobin variant found on the α1 chain: Hb KSVGH (HBA1: p.Lys57_Gly58insSerHisGlySerAlaGlnValLys).

PubMed

Wang, Mei-Chun; Tsai, Kuo-Wang; Chu, Chih-Hsun; Yu, Ming-Sun; Lam, Hing-Chung

2015-01-01

Glycosylated hemoglobin (Hb A1C) is a crucial indicator for the long-term control and the diagnosis of diabetes. However, the presence of hemoglobin (Hb) variants may affect the measured value of Hb A1C and result in an abnormal graph trend and inconsistency between the clinical blood sugar test and Hb A1C values. In this study, laboratory data of 41,267 patients with diabetes were collected. The Hb A1C levels and the graph results were examined. We identified 74 cases containing abnormal Hb A1C graph trends. The conducted blood cell counts and capillary Hb electrophoresis were used to analyze Hb variants. We also determined gene variation for the Hb variants by a sequence approach. Fifteen different types of Hb variants were identified in this study. Among these, we found a novel variant in which the α1 subunit of Hb showed an insertion of 24 nucleotides (nts) between the 56th and 57th residues. We named this novel variant Hb Kaohsiung Veterans General Hospital (Hb KSVGH) (HBA1: p.Lys57_Gly58insSerHisGlySerAlaGlnValLys).

[Effects of epithalon and cortagene on immunity and hemostasis in neonatally hypophysectomized chicken and old birds].

PubMed

Kuznik, B I; Pateiuk, A V; Baranchugova, L M; Rusaeva, N S

2008-01-01

It has been found that chicken hypophysectomized early in the neonatal period develop anemia, cellular and humoral immune deficiency, hypercoagulation and inhibited fibrinolysis by their 45th postnatal day. An analogous operation performed on old birds produces less significant changes in erythrocytes, immunity and hemostasis. Injections of epithalon tetrapeptide (Ala-Glu-Asp-Gly) administered to either hypophysectomized chicken or old birds during a period of 40 days completely eliminate the shifts registered in erythrocytes, immunity and hemostasis, while injections of cortagene (Ala-Glu-Asp-Pro) which is distinguished from epithalon by a different terminal aminoacid (with Gly being replaced by Pro) do not affect the parameters studied.

Sodium caprate enables the blood pressure-lowering effect of Ile-Pro-Pro and Leu-Lys-Pro in spontaneously hypertensive rats by indirectly overcoming PepT1 inhibition.

PubMed

Gleeson, John P; Frías, Jesús M; Ryan, Sinéad M; Brayden, David J

2018-04-20

The tripeptides, Ile-Pro-Pro (IPP) and Leu-Lys-Pro (LKP), inhibit angiotensin-converting enzyme (ACE) resulting in lowered blood pressure. Our hypothesis was that the medium chain fatty acid permeation enhancer, sodium caprate (C 10 ), may prevent the decrease in permeability of the tripeptides when PepT1 is inhibited by glycyl-sarcosine (Gly-Sar), a situation that may occur in the presence of food hydrolysates. Using Caco-2 monolayers and isolated rat jejunal tissue, the apparent permeability coefficients (P app ) of [ 3 H]-IPP and [ 3 H]-LKP were assessed in the presence of Gly-Sar with and without C 10 . Gly-Sar decreased the P app of both tripeptides across monolayers and isolated jejunal tissue, but C 10 restored it. C 10 likely increased the paracellular permeability of the tripeptides, as indicated by immunofluorescence changes in tight junction proteins in Caco-2 monolayers accompanied by a concentration-dependent decrease in transepithelial electrical resistance (TEER). [ 3 H]-IPP and [ 3 H]-LKP were orally-gavaged to normal rats with Gly-Sar, C 10 , or with a mixture. Plasma levels of both peptides were reduced by Gly-Sar to less than half that of the levels detected in its absence, but were restored when C 10 was co-administered. In spontaneously hypertensive rats (SHRs), unlabelled IPP and LKP lowered blood pressure when delivered either by i.v. or oral routes. Oral gavage of Gly-Sar reduced the hypotensive action of peptides in SHRs, but the effect was restored in the presence of C 10 . In conclusion, there was a reduction in the hypotensive effects of IPP and LKP in SHRs when intestinal PepT1 was inhibited by Gly-Sar, but C 10 may circumvent this by enhancing paracellular permeability. Copyright © 2018 Elsevier B.V. All rights reserved.

Salmonella typhimurium gyrA mutations associated with fluoroquinolone resistance.

PubMed Central

Reyna, F; Huesca, M; González, V; Fuchs, L Y

1995-01-01

Spontaneous quinolone-resistant mutants obtained from Salmonella typhimurium Su694 were screened for mutations by direct DNA sequencing of an amplified PCR gyrA fragment. Substitutions Ser-83-->Phe (Ser83Phe), Ser83Tyr, Asp87Tyr, and Asp87Asn and double mutation Ala67Pro-Gly81Ser, which resulted in decreased sensitivities to ciprofloxacin, enoxacin, pefloxacin, norfloxacin, ofloxacin, and nalidixic acid, were found. The levels of resistance to quinolones for each mutant were determined. PMID:7492118

A novel type of peptidoglycan-binding domain highly specific for amidated D-Asp cross-bridge, identified in Lactobacillus casei bacteriophage endolysins.

PubMed

Regulski, Krzysztof; Courtin, Pascal; Kulakauskas, Saulius; Chapot-Chartier, Marie-Pierre

2013-07-12

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-L-alanine amidase, whereas Lc-Lys-2 is a γ-D-glutamyl-L-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with D-Ala(4)→D-Asx-L-Lys(3) in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting D-Ala(4)→L-Ala-(L-Ala/L-Ser)-L-Lys(3); moreover, they do not lyse the L. lactis mutant containing only the nonamidated D-Asp cross-bridge, i.e. D-Ala(4)→D-Asp-L-Lys(3). In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 L-Lys(3)-D-Asn-L-Lys(3) bridges replacing the wild-type 4→3 D-Ala(4)-D-Asn-L-Lys(3) bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly D-Asn but not PG with only the nonamidated D-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the D-Asn interpeptide bridge of PG.

Biochemical characterisation of the chlamydial MurF ligase, and possible sequence of the chlamydial peptidoglycan pentapeptide stem.

PubMed

Patin, Delphine; Bostock, Julieanne; Chopra, Ian; Mengin-Lecreulx, Dominique; Blanot, Didier

2012-06-01

Chlamydiaceae are obligate intracellular bacteria that do not synthesise detectable peptidoglycan although they possess an almost complete arsenal of genes encoding peptidoglycan biosynthetic activities. In this paper, the murF gene from Chlamydia trachomatis was shown to be capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-tripeptide:D-Ala-D-Ala ligase activity. Recombinant MurF from C. trachomatis was overproduced and purified from E. coli. It exhibited ATP-dependent UDP-MurNAc-X-γ-D-Glu-meso-A(2)pm:D-Ala-D-Ala ligase activity in vitro. No significant difference of kinetic parameters was seen when X was L-Ala, L-Ser or Gly. The L-Lys-containing UDP-MurNAc-tripeptide was a poorer substrate as compared to the meso-A(2)pm-containing one. Based on the respective substrate specificities of the chlamydial MurC, MurE, MurF and Ddl enzymes, a sequence L-Ala/L-Ser/Gly-γ-D-Glu-meso-A(2)pm-D-Ala-D-Ala is expected for the chlamydial pentapeptide stem, with Gly at position 1 being less likely.

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

Functional map of arrestin-1 at single amino acid resolution

PubMed Central

Ostermaier, Martin K.; Peterhans, Christian; Jaussi, Rolf; Deupi, Xavier; Standfuss, Jörg

2014-01-01

Arrestins function as adapter proteins that mediate G protein-coupled receptor (GPCR) desensitization, internalization, and additional rounds of signaling. Here we have compared binding of the GPCR rhodopsin to 403 mutants of arrestin-1 covering its complete sequence. This comprehensive and unbiased mutagenesis approach provides a functional dimension to the crystal structures of inactive, preactivated p44 and phosphopeptide-bound arrestins and will guide our understanding of arrestin–GPCR complexes. The presented functional map quantitatively connects critical interactions in the polar core and along the C tail of arrestin. A series of amino acids (Phe375, Phe377, Phe380, and Arg382) anchor the C tail in a position that blocks binding of the receptor. Interaction of phosphates in the rhodopsin C terminus with Arg29 controls a C-tail exchange mechanism in which the C tail of arrestin is released and exposes several charged amino acids (Lys14, Lys15, Arg18, Lys20, Lys110, and Lys300) for binding of the phosphorylated receptor C terminus. In addition to this arrestin phosphosensor, our data reveal several patches of amino acids in the finger (Gln69 and Asp73–Met75) and the lariat loops (L249–S252 and Y254) that can act as direct binding interfaces. A stretch of amino acids at the edge of the C domain (Trp194–Ser199, Gly337–Gly340, Thr343, and Thr345) could act as membrane anchor, binding interface for a second rhodopsin, or rearrange closer to the central loops upon complex formation. We discuss these interfaces in the context of experimentally guided docking between the crystal structures of arrestin and light-activated rhodopsin. PMID:24449856

Functional map of arrestin-1 at single amino acid resolution.

PubMed

Ostermaier, Martin K; Peterhans, Christian; Jaussi, Rolf; Deupi, Xavier; Standfuss, Jörg

2014-02-04

Arrestins function as adapter proteins that mediate G protein-coupled receptor (GPCR) desensitization, internalization, and additional rounds of signaling. Here we have compared binding of the GPCR rhodopsin to 403 mutants of arrestin-1 covering its complete sequence. This comprehensive and unbiased mutagenesis approach provides a functional dimension to the crystal structures of inactive, preactivated p44 and phosphopeptide-bound arrestins and will guide our understanding of arrestin-GPCR complexes. The presented functional map quantitatively connects critical interactions in the polar core and along the C tail of arrestin. A series of amino acids (Phe375, Phe377, Phe380, and Arg382) anchor the C tail in a position that blocks binding of the receptor. Interaction of phosphates in the rhodopsin C terminus with Arg29 controls a C-tail exchange mechanism in which the C tail of arrestin is released and exposes several charged amino acids (Lys14, Lys15, Arg18, Lys20, Lys110, and Lys300) for binding of the phosphorylated receptor C terminus. In addition to this arrestin phosphosensor, our data reveal several patches of amino acids in the finger (Gln69 and Asp73-Met75) and the lariat loops (L249-S252 and Y254) that can act as direct binding interfaces. A stretch of amino acids at the edge of the C domain (Trp194-Ser199, Gly337-Gly340, Thr343, and Thr345) could act as membrane anchor, binding interface for a second rhodopsin, or rearrange closer to the central loops upon complex formation. We discuss these interfaces in the context of experimentally guided docking between the crystal structures of arrestin and light-activated rhodopsin.

Analysis of Isoaccepting Transfer Ribonucleic Acid Species of Bacillus subtilis: Chromatographic Differences Between Transfer Ribonucleic Acids from Spores and Cells in Exponential Growth

PubMed Central

Vold, Barbara S.

1973-01-01

Differences between the transfer ribonucleic acid (tRNA) of spores and exponentially growing cells of Bacillus subtilis 168 were compared by co-chromatography on reversed-phase column RPC-5. This system gave excellent resolution of isoaccepting species in 1 to 2 hr using a 200-ml gradient. Two methods were used to extract spore tRNAs, a procedure using a Braun homogenizer and a pretreatment with dithiothreitol followed by lysis with lysozyme. Where changes were observed, column elution profiles of spore tRNAs were independent of the extraction method used. Three kinds of changes between the profiles of vegetative cell tRNA and spore tRNA were observed: (i) no change; phe-, val-, ala-, asp-, ileu-, pro-, met-, fmet-, and his-tRNAs, (ii) a change in the ratio of existing peaks; gly-, tyr-, leu-, ser-, thr-, aspn-, and arg-tRNAs, and (iii) the appearance or disappearance of unique peaks; lys-, glu-, and trp-tRNAs. PMID:4632322

Isolation and structure elucidation of neuropeptides of the AKH/RPCH family in long-horned grasshoppers (Ensifera).

PubMed

Gäde, G

1992-11-01

An identical neuropeptide was isolated by reversed-phase high-performance liquid chromatography from the corpora cardiaca of the king cricket, Libanasidus vittatus, and the two armoured ground crickets, Heterodes namaqua and Acanthoproctus cervinus. The crude gland extracts had adipokinetic activity in migratory locusts, hypertrehalosaemic activity in American cockroaches and a slight hypertrehalosaemic, but no adipokinetic, effect in armoured ground crickets. The primary structure of this neuropeptide was determined by pulsed-liquid phase sequencing employing Edman chemistry after enzymically deblocking the N-terminal 5-oxopyrrolidine-2-carboxylic acid residue. The C-terminus was also blocked, as indicated by the lack of digestion by carboxypeptidase A. The peptide was assigned the structure [symbol: see text]Glu-Leu-Asn-Phe-Ser-Thr-Gly-TrpNH2, previously designated Scg-AKH-II. The corpora cardiaca of the cricket Gryllodes sigillatus contained a neuropeptide which differed in retention time from the one isolated from the king and armoured ground crickets. The structure was assigned as [symbol: see text]Glu-Val-Asn-Phe-Ser-Thr-Gly-TrpNH2, previously designated Grb-AKH. This octapeptide caused hyperlipaemia in its donor species. The presence of the same peptide, Scg-AKH-II, in the two primitive infraorders of Ensifera, and the different peptide, Grb-AKH, in the most advanced infraorder of Ensifera, supports the evolutionary trends assigned formerly from morphological and physiological evidence.

Cloning and characterization of the novel D-aspartyl endopeptidase, paenidase, from Paenibacillus sp. B38.

PubMed

Nirasawa, Satoru; Nakahara, Kazuhiko; Takahashi, Saori

2018-02-27

Paenidase is the first microorganism-derived D-aspartyl endopeptidase that specifically recognizes an internal D-Asp residue to cleave [D-Asp]-X peptide bonds. Using peptide sequences obtained from the protein, we performed PCR with degenerate primers to amplify the paenidase I-encoding gene. Nucleotide sequencing revealed that mature paenidase I consists of 322 amino acid residues and that the protein is encoded as a pro-protein with a 197-amino-acid N-terminal extension compared to the mature protein. Paenidase I exhibits amino acid sequence similarity to several penicillin-binding proteins. In addition, paenidase I was classified into peptidase family S12 based on a MEROPS database search. Family S12 contains serine-type D-Ala-D-Ala carboxypeptidases that have three active site residues (Ser, Lys, and Tyr) in the conserved motifs Ser-Xaa-Thr-Lys and Tyr-Xaa-Asn. These motifs were conserved in the primary structure of paenidase I, and the role of these residues was confirmed by site-directed mutagenesis.

Molecular recognition of avirulence protein (avrxa5) by eukaryotic transcription factor xa5 of rice (Oryza sativa L.): insights from molecular dynamics simulations.

PubMed

Dehury, Budheswar; Maharana, Jitendra; Sahoo, Bikash Ranjan; Sahu, Jagajjit; Sen, Priyabrata; Modi, Mahendra Kumar; Barooah, Madhumita

2015-04-01

The avirulence gene avrxa5 of bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) recognized by the resistant rice lines having corresponding resistance (xa5) gene in a gene-for-gene manner. We used a combinatorial approach involving protein-protein docking, molecular dynamics (MD) simulations and binding free energy calculations to gain novel insights into the gene-for-gene mechanism that governs the direct interaction of R-Avr protein. From the best three binding poses predicted by molecular docking, MD simulations were performed to explore the dynamic binding mechanism of xa5 and avrxa5. Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) techniques were employed to calculate the binding free energy and to uncover the thriving force behind the molecular recognition of avrxa5 by eukaryotic transcription factor xa5. Binding free energy analysis revealed van der Waals term as the most constructive component that favors the xa5 and avrxa5 interaction. In addition, hydrogen bonds (H-bonds) and essential electrostatic interactions analysis highlighted amino acid residues Lys54/Asp870, Lys56/Ala868, Lys56/Ala866, Lys56/Glu871, Ile59/His862, Gly61/Phe858, His62/Arg841, His62/Leu856, Ser101/Ala872 and Ser105/Asp870 plays pivotal role for the energetically stability of the R-Avr complex. Insights gained from the present study are expected to unveil the molecular mechanisms that define the transcriptional activator mediated transcriptome modification in host plants. Copyright © 2015 Elsevier Inc. All rights reserved.

[Effect of new peptide bioregulators livagen and epitalon on enkephalin-degrading enzymes in human serum].

PubMed

Kost, N V; Sokolov, O Iu; Gabaeva, M V; Zolotarev, Iu A; Malinin, V V; Khavinson, V Kh

2003-01-01

The effect of new peptide bioregulators--Livagen (Lys-Glu-Asp-Ala) and Epitalon (Ala-Glu-Asp-Gly)--on endogenous opioid system was studied, particularly, their ability to change the activity of enkephalin-degrading enzymes from serum and interact with opioid receptors of the brain membrane fraction. Enkephalinase activity was assayed in vitro by the rate of 3H-Leu-enkephalin hydrolysis in the presence of the tested peptides. Livagen and Epitalon inhibited enkephalin-degrading enzymes from human serum. Livagen proved to be more efficient also as compared to well-known peptidase inhibitors such as puromycin, leupeptin, and D-PAM. The dose-inhibitory effect curves for Livagen and Epitalon were plotted; their IC50 equaled 20 and 500 microM, respectively. The interaction between the peptides and opioid receptors was estimated using a radioreceptor method with [3H][D-Ala2, D-Leu5]-enkephalin. No interaction was observed between the tested peptides and mu- or delta-opioid receptors of the membrane fraction from the rat brain.

Solution 1H NMR determination of secondary structure for the three-iron form of ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus.

PubMed

Teng, Q; Zhou, Z H; Smith, E T; Busse, S C; Howard, J B; Adams, M W; La Mar, G N

1994-05-24

Two-dimensional 1H NMR data have been used to make sequence-specific assignments and define the secondary structure of the three-iron form of the oxidized ferredoxin, Fd, from the hyperthermophilic archaeon Pyrococcus furiosus, Pf. Signals for at least some protons were located for 65 of the 66 amino acids in the sequence, in spite of the paramagnetic (S = 1/2) ground state, but not all could be assigned. Unassigned and missing signals could be qualitatively correlated with the expected proximity of the protons to the paramagnetic cluster. The secondary structure was deduced from qualitative analysis of the 2D nuclear Overhauser effect, which identified two antiparallel beta-sheets, one triple-stranded including Ala1-Ser5, Val39-Glu41, and Thr62-Ala66, and one double-stranded consisting of Glu26-Asn28 and Lys32-Glu34, as well as an alpha-helix involving Glu43-Glu54. Three tight type I turns are located at residues Asp7-Thr10, Pro22-Phe25, and Asp29-Gly31. Comparison with the crystal structure of Desulfovibrio gigas, Dg, Fd (Kissinger et al., 1991) reveals a very similar folding topology, although several secondary structural elements are extended in Pf relative to Dg Fd. Thus the beta-sheet involving the two termini is expanded to include the two terminal residues and incorporates a third strand from the internal loop that is lengthened by several insertions in Pf relative to Dg Fd. The double-stranded beta-sheet in the interior of Pf Fd is lengthened slightly due to a much tighter type I turn between the two strands. The helix near the C-terminus is three residues longer in Pf than in Dg Fd, as well as being shifted toward the N-terminus. The disulfide link between the two nonligating Cys residues (Cys21 and Cys48) is conserved in Pf Fd, but the link near the C-terminus is in the middle of the long alpha-helix in Pf Fd, instead of at the N-terminus of the helix as in Dg Fd. The extensions of the beta-sheets and alpha-helix increase the number of main-chain hydrogen bonds in Pf Fd by approximately 8 relative to those in Dg Fd and likely contribute to its remarkable thermostability (it is unaffected by anaerobic incubation at 95 degrees C for 24 h).(ABSTRACT TRUNCATED AT 400 WORDS)

Radiolabeling of a cyclic RGD (cyclo Arg-Gly-Asp-d-Tyr-Lys) peptide using sodium hypochlorite as an oxidizing agent.

PubMed

Doll, Stephanie; Woolum, Karen; Kumar, Krishan

2016-09-01

A simple and rapid nonradioactive iodide labeling/radiolabeling method for peptides, using an inexpensive oxidizing agent such as sodium hypochlorite and a cyclic peptide, cRGDyK (cyclo Arg-Gly-Asp-d-Tyr-Lys), was developed in this work. Labeling reaction was optimized by conducting experiments under variable ratios of the reagents, the reaction times, and the pH. The study demonstrated that radiolabeling of the cyclic peptide was fast and pH independent. Monoiodinated and di-iodinated cRGDyK were formed under all conditions and varied with the ratio of the reagents and the reaction time. Total percent of the iodinated cRGDyK (monoiodinated and di-iodinated cRGDyK) varied between 44 and 100 depending on the reaction conditions. Excess cyclic peptide over equal molar ratio of sodium iodide and sodium hypochlorite yielded in predominant amounts of monoiodinated cRGDyK, ie, >60% under 2:1:1 ratio and ~88% under 5:1:1 ratio of cRGDyK:sodium iodide:sodium hypochlorite. Copyright © 2016 John Wiley & Sons, Ltd.

Sungsanpin, a lasso peptide from a deep-sea streptomycete.

PubMed

Um, Soohyun; Kim, Young-Joo; Kwon, Hyuknam; Wen, He; Kim, Seong-Hwan; Kwon, Hak Cheol; Park, Sunghyouk; Shin, Jongheon; Oh, Dong-Chan

2013-05-24

Sungsanpin (1), a new 15-amino-acid peptide, was discovered from a Streptomyces species isolated from deep-sea sediment collected off Jeju Island, Korea. The planar structure of 1 was determined by 1D and 2D NMR spectroscopy, mass spectrometry, and UV spectroscopy. The absolute configurations of the stereocenters in this compound were assigned by derivatizations of the hydrolysate of 1 with Marfey's reagents and 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate, followed by LC-MS analysis. Careful analysis of the ROESY NMR spectrum and three-dimensional structure calculations revealed that sungsanpin possesses the features of a lasso peptide: eight amino acids (-Gly(1)-Phe-Gly-Ser-Lys-Pro-Ile-Asp(8)-) that form a cyclic peptide and seven amino acids (-Ser(9)-Phe-Gly-Leu-Ser-Trp-Leu(15)) that form a tail that loops through the ring. Sungsanpin is thus the first example of a lasso peptide isolated from a marine-derived microorganism. Sungsanpin displayed inhibitory activity in a cell invasion assay with the human lung cancer cell line A549.

A Sucrose-derived Scaffold for Multimerization of Bioactive Peptides

PubMed Central

Rao, Venkataramanarao; Alleti, Ramesh; Xu, Liping; Tafreshi, Narges K.; Morse, David L.; Gillies, Robert J.; Mash, Eugene A.

2011-01-01

A spherical molecular scaffold bearing eight terminal alkyne groups was synthesized in one step from sucrose. One or more copies of a tetrapeptide azide, either N3(CH2)5(C=O)-His-dPhe-Arg-Trp-NH2 (MSH4) or N3(CH2)5(C=O)-Trp-Met-Asp-Phe-NH2 (CCK4), were attached to the scaffold via the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Competitive binding assays using Eu-labeled probes based on the superpotent ligands Ser-Tyr-Ser-Nle-Glu-His-dPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 (NDP-α-MSH) and Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2 (CCK8) were used to study the interactions of monovalent and multivalent MSH4 and CCK4 constructs with Hek293 cells engineered to overexpress MC4R and CCK2R. All of the monovalent and multivalent MSH4 constructs exhibited binding comparable to that of the parental ligand, suggesting that either the ligand spacing was inappropriate for multivalent binding, or MSH4 is too weak a binder for a second “anchoring” binding event to occur before the monovalently-bound construct is released from the cell surface. In contrast with this behavior, monovalent CCK4 constructs were significantly less potent than the parental ligand, while multivalent CCK4 constructs were as or more potent than the parental ligand. These results are suggestive of multivalent binding, which may be due to increased residence times for monovalently bound CCK4 constructs on the cell surface relative to MSH4 constructs, the greater residence time being necessary for the establishment of multivalent binding. PMID:21940174

A sucrose-derived scaffold for multimerization of bioactive peptides.

PubMed

Rao, Venkataramanarao; Alleti, Ramesh; Xu, Liping; Tafreshi, Narges K; Morse, David L; Gillies, Robert J; Mash, Eugene A

2011-11-01

A spherical molecular scaffold bearing eight terminal alkyne groups was synthesized in one step from sucrose. One or more copies of a tetrapeptide azide, either N(3)(CH(2))(5)(CO)-His-DPhe-Arg-Trp-NH(2) (MSH4) or N(3)(CH(2))(5)(CO)-Trp-Met-Asp-Phe-NH(2) (CCK4), were attached to the scaffold via the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Competitive binding assays using Eu-labeled probes based on the superpotent ligands Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH(2) (NDP-α-MSH) and Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH(2) (CCK8) were used to study the interactions of monovalent and multivalent MSH4 and CCK4 constructs with Hek293 cells engineered to overexpress MC4R and CCK2R. All of the monovalent and multivalent MSH4 constructs exhibited binding comparable to that of the parental ligand, suggesting that either the ligand spacing was inappropriate for multivalent binding, or MSH4 is too weak a binder for a second 'anchoring' binding event to occur before the monovalently-bound construct is released from the cell surface. In contrast with this behavior, monovalent CCK4 constructs were significantly less potent than the parental ligand, while multivalent CCK4 constructs were as or more potent than the parental ligand. These results are suggestive of multivalent binding, which may be due to increased residence times for monovalently bound CCK4 constructs on the cell surface relative to MSH4 constructs, the greater residence time being necessary for the establishment of multivalent binding. Copyright © 2011 Elsevier Ltd. All rights reserved.

Effects of the Amino Acid Linkers on the Melanoma-Targeting and Pharmacokinetic Properties of Indium-111-labeled Lactam Bridge-Cyclized α-MSH Peptides

PubMed Central

Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin

2011-01-01

The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma targeting and pharmacokinetic properties of novel 111In-labeled lactam bridge-cyclized DOTA-[X]-CycMSHhex {1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2, X=GlyGlyNle, GlyGluNle or NleGlyGlu} peptides. Methods Three novel DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex and DOTA-NleGE-CycMSHhex peptides were designed and synthesized. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma targeting and pharmacokinetic properties of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. Results DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex displayed 2.1 and 11.5 nM MC1 receptor binding affinities, whereas DOTA-NleGE-CycMSHhex showed 873.4 nM MC1 receptor binding affinity. The introduction of the -GlyGly- linker maintained high melanoma uptake while decreased the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex. The tumor uptake values of 111In-DOTA-GGNle-CycMSHhex were 19.05 ± 5.04 and 18.6 ± 3.56 % injected dose/gram (%ID/g) at 2 and 4 h post-injection. 111In-DOTA-GGNle-CycMSHhex exhibited 28, 32 and 42% less renal uptake values than 111In-DOTA-Nle-CycMSHhex we reported previously, and 61, 65 and 68% less liver uptake values than 111In-DOTA-Nle-CycMSHhex at 2, 4 and 24 h post-injection, respectively. Conclusion The amino acid linkers exhibited the profound effects on the melanoma targeting and pharmacokinetic properties of the 111In-labeled lactam bridge-cyclized α-MSH peptides. Introduction of the -GlyGly- linker maintained high melanoma uptake while reducing the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex, highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide for melanoma treatment when labeled with a therapeutic radionuclide. PMID:21421725

Probing electrostatic interactions and ligand binding in aspartyl-tRNA synthetase through site-directed mutagenesis and computer simulations.

PubMed

Thompson, Damien; Lazennec, Christine; Plateau, Pierre; Simonson, Thomas

2008-05-15

Faithful genetic code translation requires that each aminoacyl-tRNA synthetase recognise its cognate amino acid ligand specifically. Aspartyl-tRNA synthetase (AspRS) distinguishes between its negatively-charged Asp substrate and two competitors, neutral Asn and di-negative succinate, using a complex network of electrostatic interactions. Here, we used molecular dynamics simulations and site-directed mutagenesis experiments to probe these interactions further. We attempt to decrease the Asp/Asn binding free energy difference via single, double and triple mutations that reduce the net positive charge in the active site of Escherichia coli AspRS. Earlier, Glutamine 199 was changed to a negatively-charged glutamate, giving a computed reduction in Asp affinity in good agreement with experiment. Here, Lysine 198 was changed to a neutral leucine; then, Lys198 and Gln199 were mutated simultaneously. Both mutants are predicted to have reduced Asp binding and improved Asn binding, but the changes are insufficient to overcome the initial, high specificity of the native enzyme, which retains a preference for Asp. Probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments, we found no detectable activity for the mutant enzymes, indicating weaker Asp binding and/or poorer transition state stabilization. The simulations show that the mutations' effect is partly offset by proton uptake by a nearby histidine. Therefore, we performed additional simulations where the nearby Histidines 448 and 449 were mutated to neutral or negative residues: (Lys198Leu, His448Gln, His449Gln), and (Lys198Leu, His448Glu, His449Gln). This led to unexpected conformational changes and loss of active site preorganization, suggesting that the AspRS active site has a limited structural tolerance for electrostatic modifications. The data give insights into the complex electrostatic network in the AspRS active site and illustrate the difficulty in engineering charged-to-neutral changes of the preferred ligand. 2007 Wiley-Liss, Inc.

Preparation of umami octopeptide with recombined Escherichia coli: Feasibility and challenges.

PubMed

Zhao, Liming; Zhang, Yin; Venkitasamy, Chandrasekar; Pan, Zhongli; Zhang, Longyi; Guo, Siya; Xiong, Wei; Xia, Hu; Wenlong, Liu; Xinhua, Gou

2018-01-01

The taste of umami peptide H-Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala-OH (LGAGGSLA) is controversial. One possible reason for this controversy is the use of chemically synthesized LGAGGSLA to confirm its taste. To explore other ways to further confirm the flavor of LGAGGSLA, we developed a new strategy to prepare a bio-source peptide by adopting a gene engineering method to express LGAGGSLA in recombinant Escherichia coli. In our previous work, we structured the LGAGGSLA recombinant expression system and optimized the culturing conditions for preparing a fusion protein. However, the fusion protein was not cleaved by enterokinase to obtain LGAGGSLA. Because the cleavage conditions of commercial enterokinase were not specific and recombinant engineered bacteria had the potential to be used in industrial processes, in this addendum, we calculated the mass and volume yields of key processing steps in the preparation of LGAGGSLA, and established a model of cleavage conditions with the cleavage ratio of LGAGGSLA. When the LGAGGSLA was confirmed to show umami taste, it is considered as a new umami or umami enhancer. The gene information of LGAGGSLA should have a great potential in the development of new flavor product and food product containing high umami flavor.

Optimization of hydrolysis conditions, isolation, and identification of neuroprotective peptides derived from seahorse Hippocampus trimaculatus.

PubMed

Pangestuti, Ratih; Ryu, Bomi; Himaya, Swa; Kim, Se-Kwon

2013-08-01

Hippocampus trimaculatus is one of the most heavily traded seahorse species for traditional medicine purposes in many countries. In the present study, we showed neuroprotective effects of peptide derived from H. trimaculatus against amyloid-β42 (Aβ42) toxicity which are central to the pathogenesis of Alzheimer's diseases (AD). Firstly, H. trimaculatus was separately hydrolyzed by four different enzymes and tested for their protective effect on Aβ42-induced neurotoxicity in differentiated PC12 cells. Pronase E hydrolysate exerted highest protection with cell viability value of 88.33 ± 3.33 %. Furthermore, we used response surface methodology to optimize pronase E hydrolysis conditions and found that temperature at 36.69 °C with the hydrolysis time 20.01 h, enzyme to substrate (E/S) ratio of 2.02 % and pH 7.34 were the most optimum conditions. Following several purification steps, H. trimaculatus-derived neuroprotective peptides (HTP-1) sequence was identified as Gly-Thr-Glu-Asp-Glu-Leu-Asp-Lys (906.4 Da). HTP-1 protected PC12 cells from Aβ42-induced neuronal death with the cell viability value of 85.52 ± 2.22 % and up-regulated pro-survival gene (Bcl-2) expressions. These results suggest that HTP-1 has the potential to be used in treatment of neurodegenerative diseases, particularly AD. Identification, characterization, and synthesis of bioactive components derived from H. trimaculatus have the potential to replace or at least complement the use of seahorse as traditional medicine, which further may become an approach to minimize seahorse exploitation in traditional medicine.

Purification and sequence of rat oxyntomodulin.

PubMed Central

Collie, N L; Walsh, J H; Wong, H C; Shively, J E; Davis, M T; Lee, T D; Reeve, J R

1994-01-01

Structural information about rat enteroglucagon, intestinal peptides containing the pancreatic glucagon sequence, has been based previously on cDNA, immunologic, and chromatographic data. Our interests in testing the physiological actions of synthetic enteroglucagon peptides in rats required that we identify precisely the forms present in vivo. From knowledge of the proglucagon gene sequence, we synthesized an enteroglucagon C-terminal octapeptide common to both proposed enteroglucagon forms, glicentin and oxyntomodulin, but sharing no sequence overlap with glucagon. We then developed a radioimmunoassay using antibodies raised against the octapeptide that was specific for enteroglucagon peptides without cross-reacting with glucagon. Rat intestine was extracted, and one presumptive enteroglucagon form was purified by following the enteroglucagon C-terminal octapeptide-like immunoreactivity through several HPLC purification steps. Structural characterization of the material by amino acid composition, microsequence, and mass spectral analyses identified the peptide as rat oxyntomodulin. The 37-residue peptide consists of pancreatic glucagon plus the C-terminal extension, Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala. This now permits synthesis of an unambiguous duplicate of endogenous rat oxyntomodulin for physiological studies. Images PMID:7937770

Human beta-endorphin: synthesis and biological activity of analogs with substitutions in positions 2, 9, 18, 27 and 31.

PubMed

Yeung, H W; Yamashiro, D; Tseng, L F; Chang, W C; Li, C H

1981-02-01

Four analogs of the opioid peptide human beta-endorphin (Bh-EP) have been synthesized: [D-Lys9, Phe27, Gly31]-beta h-EP, [D-PHe18,Phe27,Gly31]-beta h-EP, [D-Thr2,D-Lys9,Phe27,Gly31]-beta h-EP, and [D-Thr2,D-Phe18,Phe27,Gly31]-beta h-EP. All are practically indistinguishable from beta h-EP in the guinea pig ileum assay. All show diminished analgesic potency in the mouse tail-flick assay.

Chemical properties and reactive oxygen and nitrogen species quenching activities of dry sugar-amino acid maillard reaction mixtures exposed to baking temperatures.

PubMed

Chen, Xiu-Min; Liang, Ningjian; Kitts, David D

2015-10-01

Maillard reaction products (MRPs) derived from 10 different, dry sugar-amino acid reaction model systems were examined for changes in color index (E), sugar loss, and formation of α-dicarbonyl compounds; the changes were correlated with relative activities to quench both reactive oxygen (ROS) and reactive nitrogen (RNS) species. Reducing sugars, xylose, ribose, fructose, glucose, and non-reducing sucrose were reacted with glycine (Xyl-Gly, Rib-Gly, Fru-Gly, Glc-Gly, and Suc-Gly), or lysine (Xyl-Lys, Rib-Lys, Fru-Lys, Glc-Lys, and Suc-Lys), respectively, at temperatures of 150°C and 180°C for time periods ranging from 5 to 60min. ROS quenching capacity was negatively correlated with color index (E) (r=-0.604, P<0.001), and positively correlated with sugar loss (r=0.567, P<0.001). MRPs also exhibited activity to quench RNS as assessed by nitric oxide (NO) inhibition in differentiated Caco-2 cells that were induced with interferon-γ (IFN-γ) and phorbol ester (PMA) cocktail. We also showed a correlation between RNS and color index, sugar loss, and ROS quenching activities for MR mixtures that were heated for a short time (e.g. 10min) at 150°C. MRP quenching of ROS was largely influenced by sugar type, whereas, RNS quenching was dependent more so on the interaction between reactants and reaction conditions used to generate MRPs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Isolation: analysis and properties of three bradykinin-potentiating peptides (BPP-II, BPP-III, and BPP-V) from Bothrops neuwiedi venom.

PubMed

Ferreira, L A; Galle, A; Raida, M; Schrader, M; Lebrun, I; Habermehl, G

1998-04-01

In the course of systematic investigations on low-molecular-weight compounds from the venom of Crotalidae and Viperidae, we have isolated and characterized at least three bradykinin-potentiating peptides (BPP-II, BPP-III, and BPP-V) from Bothrops neuwiedi venom by gel filtration on Sephadex G-25 M, Sephadex G-10 followed by HPLC. The peptides showed bradykinin-potentiating action on isolated guinea-pig ileum, for which the BPP-V was more active than of BPP-II, and BPP-III, rat arterial blood pressure, and a relevant angiotensin-converting enzyme (ACE) competitive inhibiting activity. The kinetic studies showed a Ki of the order of 9.7 x 10(-3) microM to BPP-II, 7 x 10(-3) microM to BPP-III, and 3.3 x 10(-3) microM to BPP-V. The amino acid sequence of the BPP-III has been determined to be pGlu-Gly-Gly-Trp-Pro-Arg-Pro-Gly-Pro-Glu-Ile-Pro-Pro, and the amino acid compositions of the BPP-II and BPP-V by amino acid analysis were 2Glu-2Gly-1Arg-4Pro-1Ile and 2Glu-2Gly-1Ser-3Pro-2Val-1Ile, with molecular weight of 1372, 1046, and 1078, respectively.

Gastric mucosal lesions induced by complete dopamine system failure in rats. The effects of dopamine agents, ranitidine, atropine, omeprazole and pentadecapeptide BPC 157.

PubMed

Sikiric, P; Separovic, J; Buljat, G; Anic, T; Stancic-Rokotov, D; Mikus, D; Duplancic, B; Marovic, A; Zoricic, I; Prkacin, I; Lovric-Bencic, M; Aralica, G; Ziger, T; Perovic, D; Jelovac, N; Dodig, G; Rotkvic, I; Mise, S; Seiwerth, S; Turkovic, B; Grabarevic, Z; Petek, M; Rucman, R

2000-01-01

Up to now, for gastric lesions potentiation or induction, as well as determination of endogenous dopamine significance, dopamine antagonist or dopamine vesicle depletor were given separately. Therefore, without combination studies, the evidence for dopamine significance remains split on either blockade of dopamine post-synaptic receptor or inhibition of dopamine storage, essentially contrasting with endogenous circumstances, where both functions could be simultaneously disturbed. For this purpose, a co-administration of reserpine and haloperidol, a dopamine granule depletor combined with a dopamine antagonist with pronounced ulcerogenic effect, was tested, and the rats were sacrificed 24 h after injurious agent(s) administration. Haloperidol (5 mg x kg(-1) b.w. i.p.), given alone, produced the lesions in all rats. Reserpine (5 mg x kg(-1) b.w. i.p.), given separately, also produced lesions. When these agents were given together, the lesions were apparently larger than in the groups injured with separate administration of either haloperidol or reserpine alone. Along with our previous results, when beneficial agents were co-administered, all dopaminomimetics (bromocriptine 10 mg, apomophine 1 mg, amphetamine 20 mg x kg(-1) i.p.) apparently attenuated the otherwise consistent haloperidol-gastric lesions. Likewise, an apparent inhibition of the reserpine-lesions was noted as well. However, if they were given in rats injured with combination of haloperidol and reserpine, their otherwise prominent beneficial effects were absent. Ranitidine (10 mg), omeprazole (10 mg), atropine (10 mg), pentadecapeptide BPC 157 (Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val) (10 microg or 10 ng x kg(-1) i.p.) evidently prevented both haloperidol-gastric lesions and reserpine-gastric lesions. Confronted with potentiated lesions following a combination of haloperidol and reserpine, these agents maintained their beneficial effects, noted in the rats treated with either haloperidol or reserpine alone. The failure of dopaminomimetics could be most likely due to more extensive inhibition of endogenous dopamine system activity, and need for remained endogenous dopamine for their salutary effect, whereas the beneficial activities of ranitidine, omeprazole, atropine, pentadecapeptide BPC 157 following dopamine system inhibition by haloperidol+reserpine suggest their corresponding systems parallel those of dopamine system, and they may function despite extensive inhibition of endogenous dopamine system activity.

Salt-bridging effects on short amphiphilic helical structure and introducing sequence-based short beta-turn motifs.

PubMed

Guarracino, Danielle A; Gentile, Kayla; Grossman, Alec; Li, Evan; Refai, Nader; Mohnot, Joy; King, Daniel

2018-02-01

Determining the minimal sequence necessary to induce protein folding is beneficial in understanding the role of protein-protein interactions in biological systems, as their three-dimensional structures often dictate their activity. Proteins are generally comprised of discrete secondary structures, from α-helices to β-turns and larger β-sheets, each of which is influenced by its primary structure. Manipulating the sequence of short, moderately helical peptides can help elucidate the influences on folding. We created two new scaffolds based on a modestly helical eight-residue peptide, PT3, we previously published. Using circular dichroism (CD) spectroscopy and changing the possible salt-bridging residues to new combinations of Lys, Arg, Glu, and Asp, we found that our most helical improvements came from the Arg-Glu combination, whereas the Lys-Asp was not significantly different from the Lys-Glu of the parent scaffold, PT3. The marked 3 10 -helical contributions in PT3 were lessened in the Arg-Glu-containing peptide with the beginning of cooperative unfolding seen through a thermal denaturation. However, a unique and unexpected signature was seen for the denaturation of the Lys-Asp peptide which could help elucidate the stages of folding between the 3 10 and α-helix. In addition, we developed a short six-residue peptide with β-turn/sheet CD signature, again to help study minimal sequences needed for folding. Overall, the results indicate that improvements made to short peptide scaffolds by fine-tuning the salt-bridging residues can enhance scaffold structure. Likewise, with the results from the new, short β-turn motif, these can help impact future peptidomimetic designs in creating biologically useful, short, structured β-sheet-forming peptides.

Role of the conserved amino acids of the 'SDN' loop (Ser130, Asp131 and Asn132) in a class A beta-lactamase studied by site-directed mutagenesis.

PubMed

Jacob, F; Joris, B; Lepage, S; Dusart, J; Frère, J M

1990-10-15

Ser130, Asp131 and Asn132 ('SDN') are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.

Myelin basic protein stimulates plasminogen activation via tissue plasminogen activator following binding to independent l-lysine-containing domains.

PubMed

Gonzalez-Gronow, Mario; Fiedler, Jenny L; Farias Gomez, Cristian; Wang, Fang; Ray, Rupa; Ferrell, Paul D; Pizzo, Salvatore V

2017-08-26

Myelin basic protein (MBP) is a key component of myelin, the specialized lipid membrane that encases the axons of all neurons. Both plasminogen (Pg) and tissue-type plasminogen activator (t-PA) bind to MBP with high affinity. We investigated the kinetics and mechanisms involved in this process using immobilized MBP and found that Pg activation by t-PA is significantly stimulated by MBP. This mechanism involves the binding of t-PA via a lysine-dependent mechanism to the Lys 91 residue of the MBP NH 2 -terminal region Asp 82 -Pro 99 , and the binding of Pg via a lysine-dependent mechanism to the Lys 122 residue of the MBP COOH-terminal region Leu 109 -Gly 126 . In this context, MBP mimics fibrin and because MBP is a plasmin substrate, our results suggest direct participation of the Pg activation system on MBP physiology. Copyright © 2017 Elsevier Inc. All rights reserved.

Alkylation of an active-site cysteinyl residue during substrate-dependent inactivation of Escherichia coli S-adenosylmethionine decarboxylase.

PubMed

Diaz, E; Anton, D L

1991-04-23

S-Adenosylmethionine decarboxylase from Escherichia coli is a member of a small class of enzymes that uses a pyruvoyl prosthetic group. The pyruvoyl group is proposed to form a Schiff base with the substrate and then act as an electron sink facilitating decarboxylation. We have previously shown that once every 6000-7000 turnovers the enzyme undergoes an inactivation that results in a transaminated pyruvoyl group and the formation of an acrolein-like species from the methionine moiety. The acrolein then covalently alkylates the enzyme [Anton, D. L., & Kutny, R. (1987) Biochemistry 26, 6444]. After reduction of the alkylated enzyme with NaBH4, a tryptic peptide with the sequence Ala-Asp-Ile-Glu-Val-Ser-Thr-[S-(3-hydroxypropyl)Cys]-Gly-Val-Ile-Ser-Pro - Leu-Lys was isolated. This corresponds to acrolein alkylation of a cysteine residue in the second tryptic peptide from the NH2 terminal of the alpha-subunit [Anton, D. L., & Kutny, R. (1987) J. Biol. Chem. 262, 2817-2822]. The modified residue derived is from Cys-140 of the proenzyme [Tabor, C. W., & Tabor, H. (1987) J. Biol. Chem. 262, 16037-16040] and lies in the only sequence conserved between rat liver and E. coli S-adenosylmethionine decarboxylase [Pajunen et al. (1988) J. Biol. Chem. 263, 17040-17049]. We suggest that the alkylated Cys residue could have a role in the catalytic mechanism.

Access of Hydrogen-Radicals to the Peptide-Backbone as a Measure for Estimating the Flexibility of Proteins Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

PubMed Central

Takayama, Mitsuo; Nagoshi, Keishiro; Iimuro, Ryunosuke; Inatomi, Kazuma

2014-01-01

A factor for estimating the flexibility of proteins is described that uses a cleavage method of “in-source decay (ISD)” coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The MALDI-ISD spectra of bovine serum albumin (BSA), myoglobin and thioredoxin show discontinuous intense ion peaks originating from one-side preferential cleavage at the N-Cα bond of Xxx-Asp, Xxx-Asn, Xxx-Cys and Gly-Xxx residues. Consistent with these observations, Asp, Asn and Gly residues are also identified by other flexibility measures such as B-factor, turn preference, protection and fluorescence decay factors, while Asp, Asn, Cys and Gly residues are identified by turn preference factor based on X-ray crystallography. The results suggest that protein molecules embedded in/on MALDI matrix crystals partly maintain α-helix and that the reason some of the residues are more susceptible to ISD (Asp, Asn, Cys and Gly) and others less so (Ile and Val) is because of accessibility of the peptide backbone to hydrogen-radicals from matrix molecules. The hydrogen-radical accessibility in MALDI-ISD could therefore be adopted as a factor for measuring protein flexibility. PMID:24828203

Flexible xxx-asp/asn and gly-xxx residues of equine cytochrome C in matrix-assisted laser desorption/ionization in-source decay mass spectrometry.

PubMed

Takayama, Mitsuo

2012-01-01

The backbone flexibility of a protein has been studied from the standpoint of the susceptibility of amino acid residues to in-source decay (ISD) in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Residues more susceptible to MALDI-ISD, namely Xxx-Asp/Asn and Gly-Xxx, were identified from the discontinuous intense peak of c'-ions originating from specific cleavage at N-Cα bonds of the backbone of equine cytochrome c. The identity of the residues susceptible to ISD was consistent with the known flexible backbone amides as estimated by hydrogen/deuterium exchange (HDX) experiments. The identity of these flexible amino acid residues (Asp, Asn, and Gly) is consistent with the fact that these residues are preferred in flexible secondary structure free from intramolecular hydrogen-bonded structures such as α-helix and β-sheet. The MALDI-ISD spectrum of equine cytochrome c gave not only intense N-terminal side c'-ions originating from N-Cα bond cleavage at Xxx-Asp/Asn and Gly-Xxx residues, but also C-terminal side complement z'-ions originating from the same cleavage sites. The present study implies that MALDI-ISD can give information about backbone flexibility of proteins, comparable with the protection factors estimated by HDX.

The decapod red pigment-concentrating hormone (Panbo-RPCH) is the first identified neuropeptide of the order Plecoptera and is interpreted as homoplastic character state.

PubMed

Gäde, Gerd; Marco, Heather G

2015-09-15

This paper presents the first neuropeptide structure, identified by mass spectrometry, in two species of Plectoptera (stoneflies) and in one species of the coleopteran family Lycidae. In all three species, the octapeptide Panbo-RPCH (first identified in Pandalus borealis as a red pigment-concentrating hormone: pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp amide) is present. A review of the literature available on invertebrate neuropeptides that are identified or predicted from expressed sequence tags, transcriptome shotgun assemblies, and from fully sequenced genomes, show that Panbo-RPCH is found in Malacostraca (Crustacea) and certain hemipteran Heteroptera (Insecta). To date, Panbo-RPCH has not been shown present in non-Malacostracan crustaceans, nor in basal taxa of the Insecta (Archaeognatha, Zygentoma, Ephemeroptera, Odonata). The present data adds to knowledge on the distribution of Panbo-RPCH, and when taking into account the most accepted, current phylogenetics of the Crustacea-Hexapoda relationship, this distribution of Panbo-RPCH in Malacostraca, Plecoptera, some hemipteran Heteroptera and in Coleoptera (Lycidae) can best be explained by homoplasy, implying parallel evolution. Copyright © 2015 Elsevier Inc. All rights reserved.

Increasing the availability of threonine, isoleucine, valine, and leucine relative to lysine while maintaining an ideal ratio of lysine:methionine alters mammary cellular metabolites, mammalian target of rapamycin signaling, and gene transcription.

PubMed

Dong, X; Zhou, Z; Wang, L; Saremi, B; Helmbrecht, A; Wang, Z; Loor, J J

2018-06-01

Amino acids not only serve as precursors for protein synthesis but also function as signaling molecules that can regulate the mammalian target of rapamycin (mTOR) pathway. Methionine and Lys are the most-limiting AA for milk production and a ratio of ∼3:1 Lys:Met in the metabolizable protein has been determined to be ideal. Besides Met and Lys, recent studies have evaluated Ile, Leu, Val, and Thr as potentially limiting for milk protein synthesis. The objective of this experiment was to determine if varying the ratio of Lys:Thr, Lys:Ile, Lys:Val, and Lys:Leu while maintaining an ideal ratio of Lys:Met and fixed ratio of other essential AA (IPAA) elicits changes in intracellular metabolites, gene transcription related to protein synthesis, and phosphorylation status of mTOR pathway proteins. Immortalized bovine mammary epithelial cell line (MAC-T) cells were incubated for 12 h (n = 5 replicates/treatment) with IPAA (2.9:1 Lys:Met; 1.8:1 Lys:Thr; 2.38:1 Lys:His; 1.23:1 Lys:Val; 1.45:1 Lys:Ile; 0.85:1 Lys:Leu; 2.08:1 Lys:Arg) or IPAA supplemented with Thr, Ile, Val, and Leu to achieve a Lys:Thr 1.3:1 (LT1.3), Lys:Ile 1.29:1 (LI1.29), Lys:Val 1.12:1 (LV1.12), or Lys:Leu 0.78:1 (LL0.78). Compared with IPAA, metabolomics via gas chromatography-mass spectrometry revealed that increases in availability of Thr, Ile, Val, and Leu led to greater concentrations of essential AA (Leu, Ile, Thr), nonessential AA (Gly, Glu, Gln, Ser, Pro, Asp), and various metabolites including uric acid, phosphoric acid, N-acetylglutamic acid, and intermediates of glycolysis and the tricarboxylic acid cycle. Compared with other treatments, LV1.12 led to greater phosphorylation status of serine/threonine kinase B (Akt), mTORC1, and ribosomal protein S6 and lower phosphorylation of α subunit of eukaryotic translation initiation factor 2. In addition, LV1.12 upregulated abundance of CSN2 and both the abundance and promoter methylation of CSN1S1. Although LI1.29 led to the second highest response in mTORC1 phosphorylation status, it resulted in the lowest phosphorylation of Akt and eEF2 and mRNA abundance of CSN2 and various AA transporters (SLC7A5, SLC36A1, SLC38A2, SLC38A9, SLC43A1). Overall, data indicate that an increase in Val at an ideal ratio of Lys:Met could further enhance milk protein synthesis by altering intracellular concentrations of essential AA and metabolites that could play a regulatory role, increasing phosphorylation status of mTORC1 and key signaling proteins, and upregulation of AA transporters. The Authors. Published by FASS Inc. and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Genetic polymorphisms in glutathione S-transferase (GST) superfamily and arsenic metabolism in residents of the Red River Delta, Vietnam

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

Agusa, Tetsuro; Center for Marine Environmental Studies; Iwata, Hisato, E-mail: iwatah@agr.ehime-u.ac.j

To elucidate the role of genetic factors in arsenic metabolism, we investigated associations of genetic polymorphisms in the members of glutathione S-transferase (GST) superfamily with the arsenic concentrations in hair and urine, and urinary arsenic profile in residents in the Red River Delta, Vietnam. Genotyping was conducted for GST omega1 (GSTO1) Ala140Asp, Glu155del, Glu208Lys, Thr217Asn, and Ala236Val, GST omega2 (GSTO2) Asn142Asp, GST pi1 (GSTP1) Ile105Val, GST mu1 (GSTM1) wild/null, and GST theta1 (GSTT1) wild/null. There were no mutation alleles for GSTO1 Glu208Lys, Thr217Asn, and Ala236Val in this population. GSTO1 Glu155del hetero type showed higher urinary concentration of As{sup V} thanmore » the wild homo type. Higher percentage of DMA{sup V} in urine of GSTM1 wild type was observed compared with that of the null type. Strong correlations between GSTP1 Ile105Val and arsenic exposure level and profile were observed in this study. Especially, heterozygote of GSTP1 Ile105Val had a higher metabolic capacity from inorganic arsenic to monomethyl arsenic, while the opposite trend was observed for ability of metabolism from As{sup V} to As{sup III}. Furthermore, other factors including sex, age, body mass index, arsenic level in drinking water, and genotypes of As (+ 3 oxidation state) methyltransferase (AS3MT) were also significantly co-associated with arsenic level and profile in the Vietnamese. To our knowledge, this is the first study indicating the associations of genetic factors of GST superfamily with arsenic metabolism in a Vietnamese population.« less

Genetic polymorphisms in glutathione S-transferase (GST) superfamily and arsenic metabolism in residents of the Red River Delta, Vietnam.

PubMed

Agusa, Tetsuro; Iwata, Hisato; Fujihara, Junko; Kunito, Takashi; Takeshita, Haruo; Minh, Tu Binh; Trang, Pham Thi Kim; Viet, Pham Hung; Tanabe, Shinsuke

2010-02-01

To elucidate the role of genetic factors in arsenic metabolism, we investigated associations of genetic polymorphisms in the members of glutathione S-transferase (GST) superfamily with the arsenic concentrations in hair and urine, and urinary arsenic profile in residents in the Red River Delta, Vietnam. Genotyping was conducted for GST omega1 (GSTO1) Ala140Asp, Glu155del, Glu208Lys, Thr217Asn, and Ala236Val, GST omega2 (GSTO2) Asn142Asp, GST pi1 (GSTP1) Ile105Val, GST mu1 (GSTM1) wild/null, and GST theta1 (GSTT1) wild/null. There were no mutation alleles for GSTO1 Glu208Lys, Thr217Asn, and Ala236Val in this population. GSTO1 Glu155del hetero type showed higher urinary concentration of As(V) than the wild homo type. Higher percentage of DMA(V) in urine of GSTM1 wild type was observed compared with that of the null type. Strong correlations between GSTP1 Ile105Val and arsenic exposure level and profile were observed in this study. Especially, heterozygote of GSTP1 Ile105Val had a higher metabolic capacity from inorganic arsenic to monomethyl arsenic, while the opposite trend was observed for ability of metabolism from As(V) to As(III). Furthermore, other factors including sex, age, body mass index, arsenic level in drinking water, and genotypes of As (+3 oxidation state) methyltransferase (AS3MT) were also significantly co-associated with arsenic level and profile in the Vietnamese. To our knowledge, this is the first study indicating the associations of genetic factors of GST superfamily with arsenic metabolism in a Vietnamese population. Copyright 2009 Elsevier Inc. All rights reserved.

KLF1 mutations are relatively more common in a thalassemia endemic region and ameliorate the severity of β-thalassemia

PubMed Central

Liu, Dun; Zhang, Xinhua; Yu, Lihua; Cai, Ren; Ma, Xiaoxia; Zheng, Chengguang; Zhou, Yuqiu; Liu, Qiji; Wei, Xiaofeng; Lin, Li; Yan, Tizhen; Huang, Jiwei; Mohandas, Narla; An, Xiuli

2014-01-01

Mutations in human Krüppel-like factor 1 (KLF1) have recently been reported to be responsible for increased fetal hemoglobin (HbF) and hemoglobin A2 (HbA2). Because increased HbF and HbA2 levels are important features of β-thalassemia, we examined whether there is any relationship between KLF1 mutation and β-thalassemia in China. To do this, we first studied the incidence of KLF1 mutations in 2 Chinese populations: 3839 individuals from a thalassemia endemic region in south China and 1190 individuals from a nonthalassemia endemic region in north China. Interestingly, we found that the prevalence of KLF1 mutations is significantly higher in the thalassemia endemic region than that in nonthalassemia endemic region (1.25% vs 0.08%). Furthermore, we identified 7 functional variants including 4 previously reported (p.Gly176AlafsX179, p.Ala298Pro, p.Thr334Arg, and c.913+1G>A) and 3 novel variants (p.His299Asp, p.Cys341Tyr, and p.Glu5Lys) in southern China. The 2 most common mutations, p.Gly176AlafsX179 and p.His299Asp, accounted for 90.6% of the total. We found that zinc-finger mutations in KLF1 were selectively represented in 12 β-thalassemia intermedia patients and resulted in significantly different transfusion-free survival curves. Our findings suggest that KLF1 mutations occur selectively in the presence of β-thalassemia to increase the production of HbF, which in turn ameliorates the clinical severity of β-thalassemia. PMID:24829204

Peptidomic analysis of skin secretions supports separate species status for the tailed frogs, Ascaphus truei and Ascaphus montanus

USGS Publications Warehouse

Conlon, J.M.; Bevier, C.R.; Coquet, L.; Leprince, J.; Jouenne, T.; Vaudry, H.; Hossack, B.R.

2007-01-01

The tailed frog Ascaphus truei Stejneger, 1899 is the most primitive extant anuran and the sister taxon to the clade of all other living frogs. The species occupies two disjunct ranges in the Northwest region of North America: the Cascade Mountains and coastal area from British Columbia to Northern California, and an inland range in the northern Rocky Mountains and the Blue and Wallowa mountains. A previous study led to the isolation of eight peptides with antimicrobial activity (termed the ascaphins) from skin secretions of A. truei from the coastal range. The present study has used peptidomic analysis to identify the products of orthologous ascaphin genes in electrically-stimulated skin secretions from inland range specimens. Structural characterization of the peptides demonstrated that ascaphins from the inland range contained the following amino acid substitutions compared with orthologs from the coastal range frogs: ascaphin-1 (Ala12 → Glu), ascaphin-3 (Asp4 → Glu), ascaphin-4 (Ala19 → Ser), ascaphin-5 (Lys12 → Thr), and ascaphin-7 (Gly8 → Ser and Ser20 → Asn). Orthologs of ascaphins-2, -6, and -8 were not identified but a paralog of ascaphin-5, identical to ascaphin-5 from coastal range frogs, was found. The data support the claims, derived from analysis of the nucleotide sequences of mitochondrial genes, that the inland populations of the tailed frog should be recognized as a distinct species, the Rocky Mountain tailed frog Ascaphus montanus and that the divergence of the species from A. truei probably occurred in the late Miocene (approximately 10 Mya).

Potent μ-Opioid Receptor Agonists from Cyclic Peptides Tyr-c[D-Lys-Xxx-Tyr-Gly]: Synthesis, Biological, and Structural Evaluation.

PubMed

Li, Yangmei; Cazares, Margret; Wu, Jinhua; Houghten, Richard A; Toll, Laurence; Dooley, Colette

2016-02-11

To optimize the structure of a μ-opioid receptor ligand, analogs H-Tyr-c[D-Lys-Xxx-Tyr-Gly] were synthesized and their biological activity was tested. The analog containing a Phe(3) was identified as not only exhibiting binding affinity 14-fold higher than the original hit but also producing agonist activity 3-fold more potent than morphine. NMR study suggested that a trans conformation at D-Lys(2)-Xxx(3) is crucial for these cyclic peptides to maintain high affinity, selectivity, and functional activity toward the μ-opioid receptor.

XPD Asp312Asn and Lys751Gln polymorphisms and breast cancer susceptibility: a meta-analysis.

PubMed

Yan, Yulan; Liang, Hongjie; Light, Morning; Li, Taijie; Deng, Yan; Li, Meng; Li, Shan; Qin, Xue

2014-03-01

The association between xeroderma pigmentosum complementation group D (XPD) Asp312Asn and Lys751Gln gene polymorphisms and breast cancer risk has been widely reported, but the results were inconsistent. In order to derive a more precise estimation of the relationship, a meta-analysis was performed. A comprehensive search strategy was conducted towards the electronic databases including Medline, PubMed, Web of Science, Embase, and Chinese Biomedical Literature Database (Chinese). The association between the XPD polymorphism and breast cancer risk was conducted by odds ratios (ORs) and 95% confidence intervals (95% CIs). A total of 22 studies with 18,136 cases and 18,351 controls were included in our meta-analysis. Among these, 12 studies with 7,667 cases and 7,480 controls for Asp312Asn polymorphism and 20 studies with 10,469 cases and 10,871 controls for Lys751Gln polymorphism. With regard to Asp312Asn polymorphism, no significantly associated was found with breast cancer risk. However, significant association was found between Lys751Gln polymorphism and breast cancer risk under all genetic models in overall populations (C vs. A-OR = 1.10, 95% CI = 1.04-1.17, P = 0.002; CC vs. AA-OR = 1.17, 95% CI = 1.06-1.30, P = 0.003; AC vs. AA-OR = 1.06, 95% CI = 1.01-1.12, P = 0.032; CC vs. AC/AA-OR = 1.17, 95% CI = 1.04-1.32, P = 0.009; CC/AC vs. AA-OR = 1.07, 95% CI = 1.02-1.12, P = 0.005). In subgroup analysis base on ethnicity, significance was found in Caucasians and mix. The results suggest that XPD Asp312Asn polymorphism was not associated with breast cancer. The XPD Lys751Gln polymorphism significantly increased breast cancer risk, especially for Caucasian and mix.

Identification of Genetic Defects Underlying FXII Deficiency in Four Unrelated Chinese Patients.

PubMed

Yang, Lihong; Wang, Yingyu; Zhou, Jianpin; Cheng, Xiaoli; Hao, Xiuping; Xie, Haixiao; Jin, Yanhui; Wang, Mingshan

2016-01-01

Congenital factor XII (FXII) dexFB01;ciency is a rare autosomal recessive disorder, characterized by a great variability in its clinical manifestations. In this study, we screened for mutations in the F12 gene of 4 unrelated patients with FXII coagulant activity <10% of that of normal human plasma. To investigate the molecular defects in these FXII-deficient patients, we performed FXII mutation screening. By sequencing all coding exons as well as xFB02;anking intronic regions of the F12 gene, 6 different mutations, including 3 missense mutations (Gly341Arg, Glu502Lys and Gly542 Ser), 1 insertion (7142insertC) and 2 deletions (5741-5742 delCA and 6753-6755delACA), were identixFB01;ed on the F12 gene. Three of them (Gly341Arg, 5741-5742delCA and 6753-6755delACA) are reported here for the first time. Computer-based algorithms predicted these missense mutations to be deleterious. This study has increased our knowledge of the mutational spectrum underlying FXII deficiency. © 2016 S. Karger AG, Basel.

Reaction Mechanism of Isopentenyl Phosphate Kinase: A QM/MM Study.

PubMed

McClory, James; Timson, David J; Singh, Warispreet; Zhang, Jian; Huang, Meilan

2017-12-14

Isopentenyl phosphate kinase (IPK) catalyzes the Mg 2+ -ATP dependent phosphorylation reactions to produce isopentenyl diphosphate, an important precursor in the synthesis of isopentenols. However, the position of the divalent metal ion in the crystal structures of IPK in complex with ATP and its native substrate IP has not been definitively resolved, and as a result ambiguity surrounds the catalytic mechanism of IP, limiting its exploitation as a biofuel and in drug design. Here we report the catalytically competent structure in complex with the metal ion Mg 2+ and elucidate the phosphorylation reaction mechanism using molecular dynamic simulations and density functional theory-based quantum mechanics/molecular mechanics calculations (B97d/AMBER99). Comparing the substrate-bound and substrate-free IPK complexes, we observed that substrate binding results in significant conformational change of three residues Lys204, Glu207, and Lys211 located on the αG helix to form a strong salt bridge network with Asp145, which in turn tethers the invariant Ser142 via H-bond interaction. The conformational change shuts the subtrate entrance channel formed between the αG and αE helices. Further, we demonstrate the phosphorylation reaction occurs with a reaction barrier of 17.58 kcal/mol, which is in agreement with the previous experimental kinetic data. We found that a highly conserved Gly8 on a glycine-rich loop, together with Lys14, stabilizes the transition state.

Reaction between the Pt(II)-complexes and the amino acids of the β-amyloid peptide

NASA Astrophysics Data System (ADS)

Novato, Willian T. G.; Stroppa, Pedro Henrique F.; Da Silva, Adilson D.; Botezine, Naiara P.; Machado, Flávia C.; Costa, Luiz Antônio S.; Dos Santos, Hélio F.

2017-01-01

Reaction between [Pt(ophen)Cl2] and HIS was monitored and the solvolysis (k1) and Cl/HIS ligand exchange (k2) rate constants obtained. The k1 and k2 were (6.2 ± 0.4) × 10-5 s-1 and 52.8 × 10-2 M-1 s-1, respectively. The corresponding calculated values were 47.5 × 10-5 s-1 and 52.2 × 10-2 M-1 s-1, in agreement with the experiment. Calculations were used to establish the reactivity order for a set of amino acids: MET ∼ LYS ∼ HIS(ε) > GLU ∼ ASP >> ASN ∼ GLN. In spite of the similar reactivity among MET, LYS and HIS, the thermodynamics suggests the reactions with LYS and HIS more favorable than with MET. Therefore, N-containing amino acids should be potential targets of Pt(II)-complexes in β-amyloid.

Screening of LDLR and APOB gene mutations in Mexican patients with homozygous familial hypercholesterolemia.

PubMed

Hernández Flores, Teresita De Jesús; González García, Juan Ramón; Colima Fausto, Ana Gabriela; Vázquez Cárdenas, Norma Alejandra; Sánchez López, Yoaly; Zarate Morales, César Augusto; Magaña Torres, María Teresa

Familial hypercholesterolemia (FH) is an autosomal dominant disorder that causes accumulation of serum low-density lipoprotein cholesterol and premature cardiovascular disease. It is mainly related to mutations in the LDLR gene. Homozygous FH (HoFH) patients have the most severe form of the disease accounting for a worldwide prevalence of 1:1,000,000. In Mexico, at least 5 cases of HoFH have been reported. The aim of this study was to describe the clinical, biochemical, and molecular data observed in patients with HoFH phenotype. We included 13 patients, belonging to 11 families, with clinical and biochemical diagnoses suggestive of HoFH. Molecular analyses of the LDLR and APOB genes were performed by means of polymerase chain reaction followed by Sanger sequencing. The causal mutation of HoFH was found in 8 of 11 unrelated patients. Excepting 1, all were true homozygotes. Six different variants in LDLR were identified: c.-139delCTCCCCCTGC, p.Glu140Lys, p.Asp360His, p.Asn405Lys, p.Ala755Glyfs*7, and p.Leu759Serfs*6. Of these, p.Asp360His and p.Asn405Lys were detected for the first time in Mexico; p.Leu759Serfs*6 showed to be the most frequent (43.7% of the alleles 7/16), and c.-139delCTCCCCCTGC is a new variant located in the promoter region. This work increases knowledge of biochemical and genetic features in Mexican patients with HoFH. A novel mutation in the LDLR gene promoter was detected: c.-139delCTCCCCCTGC, which possibly inhibits its expression. Copyright © 2018 National Lipid Association. Published by Elsevier Inc. All rights reserved.

A computational analysis of SARS cysteine proteinase-octapeptide substrate interaction: implication for structure and active site binding mechanism

PubMed Central

Phakthanakanok, Krongsakda; Ratanakhanokchai, Khanok; Kyu, Khin Lay; Sompornpisut, Pornthep; Watts, Aaron; Pinitglang, Surapong

2009-01-01

Background SARS coronavirus main proteinase (SARS CoVMpro) is an important enzyme for the replication of Severe Acute Respiratory Syndrome virus. The active site region of SARS CoVMpro is divided into 8 subsites. Understanding the binding mode of SARS CoVMpro with a specific substrate is useful and contributes to structural-based drug design. The purpose of this research is to investigate the binding mode between the SARS CoVMpro and two octapeptides, especially in the region of the S3 subsite, through a molecular docking and molecular dynamics (MD) simulation approach. Results The one turn α-helix chain (residues 47–54) of the SARS CoVMpro was directly involved in the induced-fit model of the enzyme-substrate complex. The S3 subsite of the enzyme had a negatively charged region due to the presence of Glu47. During MD simulations, Glu47 of the enzyme was shown to play a key role in electrostatic bonding with the P3Lys of the octapeptide. Conclusion MD simulations were carried out on the SARS CoVMpro-octapeptide complex. The hypothesis proposed that Glu47 of SARS CoVMpro is an important residue in the S3 subsite and is involved in binding with P3Lys of the octapeptide. PMID:19208150

Correlation of Specific Mutations in Line Probe Assay (LPA) and Drug Susceptibility Test (DST) with respect to Fluoroquinolone Resistance in Drug Resistant Mycobacterium tuberculosis

PubMed Central

Agrawal, Umang; Savaj, Pratik; Davda, Kanishka; Rodrigues, Camilla; Soman, Rajeev

2017-01-01

Abstract Background This study was done to investigate the utility of specific fluoroquinolone mutations in LPA in predicting the susceptibility in DST at WHO recommended Critical Concentrations of 0.5 and 2 µg/dL of moxifloxacin within a short time frame as provided by LPA. Methods In a retrospective study performed at a tertiary care hospital of Mumbai, India from October 2015 to February 2017, consecutive samples demonstrating fluoroquinolone resistance by LPA were selected. The LPA kit used was Hain Lifescience Genotype MTBDRsl (Version 1). It detects the following mutations in gyrA gene: MUT1: Ala90Val, MUT2: Ser91Pro, MUT3A: Asp94Ala, MUT3B: Asp94Asn/Tyr, MUT3C: Asp94Gly, MUT3D: Asp94His. The causal mutation was noted. For 89 of these samples, DST had been requested and results with Critical Concentration of 0.5µg/dL and 2µg/dL for moxifloxacin were available Results The 89 samples studied were as follows: Sputum (n = 60), paravertebral soft tissue (n = 2), bronchoalveolar fluid (n = 2), cerebrospinal fluid (n = 1), endotracheal tube secretion (n = 1), pleural fluid (n = 1) and site not recorded (22). 3 of these samples had double mutations. Results are as follows. Mutation in gyrA gene Number of samples (n) Susceptible at 0.5 µg/dL [n (%)] Susceptible at 2 µg/dL [n (%)] MUT1 (Ala90Val) 18 6(33.33) 16(88.89) MUT2 (Ser91Pro) 2 0(0) 1(50) MUT3A (Asp94Asn) 13 3(23.07) 11(84.61) MUT3B (Asp94Asn/Tyr) 6 1(16.67) 4(66.67) MUT3C (Asp94Gly) 51 4(8) 43(84.31) MUT3D (Asp94His) 2 0(0) 2(100) Conclusion This study showed a higher proportion of M. tuberculosis susceptibility at 2 µg/dL rather than at 0.5 µg/dL, to moxifloxacin for gyrA mutations Ala90Val (MUT1), Asp94Ala (MUT3A), Asp94Gly (MUT3C), Asp94His (MUT3D) but not for Ser91Pro (MUT2) and Asp94Asn/Tyr (MUT3B). However, the number of samples with Ser91Pro (MUT2) and Asp94Asn/Tyr (MUT3B) mutations was too small for meaningful conclusion. This susceptibility at a higher critical concentration of moxifloxacin may have clinical implications for use of high dose moxifloxacin. Since this information is available within a short time frame as provided by LPA, a more effective regimen could be devised 4 to 8 weeks earlier than after results of DST. This may result in faster sputum conversion and prevent amplification of resistance. Disclosures All authors: No reported disclosures.

Sequestosome-1 (SQSTM1) sequence variants in ALS cases in the UK: prevalence and coexistence of SQSTM1 mutations in ALS kindred with PDB.

PubMed

Kwok, Chun T; Morris, Alex; de Belleroche, Jacqueline S

2014-04-01

Mutations in the SQSTM1 gene have been reported to be associated with amyotrophic lateral sclerosis (ALS). We sought to determine the frequency of these mutations in a UK familial ALS (FALS) cohort. Sequences of all eight exons of the SQSTM1 gene were analysed in index cases from 61 different FALS kindred lacking known FALS mutations. Six exonic variants c.463G>A, p.(Glu155Lys), c.822G>C, p.(Glu274Asp), c.888G>T, p.(=), c.954C>T, p.(=), c.1038G>A, p.(=) and c.1175C>T, p.(Pro392Leu) were identified in five FALS index cases, three of which were non-synonymous and three were synonymous. One index case harboured three variants (c.822G>C, c.888G>T and c.954C>T), and a second index case harboured two variants (c.822G>C and c.954C>T). Only the p.(Pro392Leu) and p.(Glu155Lys) mutations were predicted to be pathogenic. In one p.(Pro392Leu) kindred, the carrier developed both ALS and Paget's disease of bone (PDB), and, in the p.(Glu155Lys) kindred, the father of the proband developed PDB. All p.(Pro392Leu) carriers were heterozygous for a previously reported founder haplotype for PDB, where this mutation has an established causal effect. The frequency of the p.(Pro392Leu) mutation in this UK FALS cohort was 2.3% and 0.97% overall including three previously screened FALS cohorts. Our results confirm the presence of the p.(Pro392Leu) SQSTM1 mutation in FALS. This mutation is the most common SQSTM1 mutation found in ALS to date, and a likely pathogenicity is supported by having an established causal role in PDB. The occurrence of the same mutation in ALS and PDB is indicative of a common pathogenic pathway that converges on protein homeostasis.

[Parallel analysis of c-Fos protein and interleukin-2 expression in hypothalamic cells under different influence].

PubMed

Barabanova, S V; Artiukhina, Z E; Ovchinnikova, K T; Abramova, T V; Kazakova, T B; Khavinson, V Kh; Malinin, V V; Korneva, E A

2007-02-01

The objective of this work was to perform a parallel analysis of activation of the rat anterior hypothalamus cells as judged by c-Fos protein expression, and of the expression of interleukin-2 (IL-2) under different influences, i. e., mild stress (handling) and adaptation to it, and intranasal administration of saline and the peptides Vilon (Lys-Glu) and Epithalon (Ala-Glu-Asp-Gly). Changes in the counts of cells positive for c-Fos- and IL-2 proteins were studied in structures of the lateral (LHA) area, anterior (AHN), supraoptic (SO) and paraventricular (PVH) nuclei of Wistar rat hypothalamus. Quantity of the interleukin-2-positive and c-Fos-positive cells was calculated. The findings were: a negative correlation between the activation of cells and the amount of IL-2 in the cells in the hypothalamic structures under study, and the specific patterns of changes in the counts of cells positive for c-Fos and IL-2 under stress and adaptation to stress.

Missense SLC25A38 variations play an important role in autosomal recessive inherited sideroblastic anemia

PubMed Central

Kannengiesser, Caroline; Sanchez, Mayka; Sweeney, Marion; Hetet, Gilles; Kerr, Briedgeen; Moran, Erica; Fuster Soler, Jose L.; Maloum, Karim; Matthes, Thomas; Oudot, Caroline; Lascaux, Axelle; Pondarré, Corinne; Sevilla Navarro, Julian; Vidyatilake, Sudharma; Beaumont, Carole; Grandchamp, Bernard; May, Alison

2011-01-01

Background Congenital sideroblastic anemias are rare disorders with several genetic causes; they are characterized by erythroblast mitochondrial iron overload, differ greatly in severity and some occur within a syndrome. The most common cause of non-syndromic, microcytic sideroblastic anemia is a defect in the X-linked 5-aminolevulinate synthase 2 gene but this is not always present. Recently, variations in the gene for the mitochondrial carrier SLC25A38 were reported to cause a non-syndromic, severe type of autosomal-recessive sideroblastic anemia. Further evaluation of the importance of this gene was required to estimate the proportion of patients affected and to gain further insight into the range and types of variations involved. Design and Methods In three European diagnostic laboratories sequence analysis of SLC25A38 was performed on DNA from patients affected by congenital sideroblastic anemia of a non-syndromic nature not caused by variations in the 5-aminolevulinate synthase 2 gene. Results Eleven patients whose ancestral origins spread across several continents were homozygous or compound heterozygous for ten different SLC25A38 variations causing premature termination of translation (p.Arg117X, p.Tyr109LeufsX43), predicted splicing alteration (c.625G>C; p.Asp209His) or missense substitution (p.Gln56Lys, p.Arg134Cys, p.Ile147Asn, p.Arg187Gln, p.Pro190Arg, p.Gly228Val, p.Arg278Gly). Only three of these variations have been described previously (p.Arg117X, p.Tyr109LeufsX43 and p.Asp209His). All new variants reported here are missense and affect conserved amino acids. Structure modeling suggests that these variants may influence different aspects of transport as described for mutations in other mitochondrial carrier disorders. Conclusions Mutations in the SLC25A38 gene cause severe, non-syndromic, microcytic/hypochromic sideroblastic anemia in many populations. Missense mutations are shown to be of importance as are mutations that affect protein production. Further investigation of these mutations should shed light on structure-function relationships in this protein. PMID:21393332

Enhancement of the thrombolytic efficacy of prourokinase by lys-plasminogen in a dog model of arterial thrombosis.

PubMed

Badylak, S F; Voytik, S L; Henkin, J; Burke, S E; Sasahara, A A; Simmons, A

1991-05-01

Current findings suggest that the efficacy of thrombolytic therapy may be limited by the availability of active forms of plasminogen at the thrombus site. The purpose of this study was to determine if the systemic administration of 0.5 mg kg-1 glu-plasminogen (glu-plg) or 0.5 mg kg-1 lys-plasminogen (lys-plg) could safely increase the efficacy of a single intravenous bolus injection of 50,000 U kg-1 prourokinase (proUK) in a dog model of arterial thrombosis. Thrombolysis was measured by monitoring the continuous decrement of 125I-gamma emissions from a radiolabeled thrombus. Reflow was evaluated by direct visual examination. Forty dogs (mean wt 10.3 +/- 2 kg) were randomly sorted into 4 groups of 10 each. The dogs in each group were given either saline plus saline, saline plus proUK, glu-plg plus proUK, or lys-plg plus proUK 60 minutes after formation of an occlusive arterial thrombus. Ninety minutes after drug administration the dogs receiving saline plus proUK, glu-plg plus proUK, and the lys-plg plus proUK showed greater thrombolysis (41%, 43%, and 66%, respectively) than the control (saline plus saline) group (15%, P less than 0.01). The lys-plg plus proUK treatment caused greater lysis than the saline plus proUK or the glu-plg plus proUK treatment (P less than 0.05). All of the dogs (10/10) receiving lys-plg plus proUK had patent vessels at the end of the 90 minute monitoring period, whereas only 4/10 and 5/10 vessels were patent in the saline plus proUK and glu-plg plus proUK groups, respectively. None of the dogs in the saline plus saline group had patent vessels. No significant changes were observed in the various coagulation parameters tested for any of the 4 treatment groups. The results show that lys-plg can safely increase the thrombolytic efficacy of proUK.

Identification of Dipeptidyl-Peptidase (DPP)5 and DPP7 in Porphyromonas endodontalis, Distinct from Those in Porphyromonas gingivalis.

PubMed

Nishimata, Haruka; Ohara-Nemoto, Yuko; Baba, Tomomi T; Hoshino, Tomonori; Fujiwara, Taku; Shimoyama, Yu; Kimura, Shigenobu; Nemoto, Takayuki K

2014-01-01

Dipeptidyl peptidases (DPPs) that liberate dipeptides from the N-terminal end of oligopeptides are crucial for the growth of Porphyromonas species, anaerobic asaccharolytic gram negative rods that utilize amino acids as energy sources. Porphyromonas endodontalis is a causative agent of periapical lesions with acute symptoms and Asp/Glu-specific DPP11 has been solely characterized in this organism. In this study, we identified and characterized two P. endodontalis DPPs, DPP5 and DPP7. Cell-associated DPP activity toward Lys-Ala-4-methylcoumaryl-7-amide (MCA) was prominent in P. endodontalis ATCC 35406 as compared with the Porphyromonas gingivalis strains ATCC 33277, 16-1, HW24D1, ATCC 49417, W83, W50, and HNA99. The level of hydrolysis of Leu-Asp-MCA by DPP11, Gly-Pro-MCA by DPP4, and Met-Leu-MCA was also higher than in the P. gingivalis strains. MER236725 and MER278904 are P. endodontalis proteins belong to the S9- and S46-family peptidases, respectively. Recombinant MER236725 exhibited enzymatic properties including substrate specificity, and salt- and pH-dependence similar to P. gingivalis DPP5 belonging to the S9 family. However, the kcat/Km figure (194 µM-1·sec-1) for the most potent substrate (Lys-Ala-MCA) was 18.4-fold higher as compared to the P. gingivalis entity (10.5 µM-1·sec-1). In addition, P. endodontalis DPP5 mRNA and protein contents were increased several fold as compared with those in P. gingivalis. Recombinant MER278904 preferentially hydrolyzed Met-Leu-MCA and exhibited a substrate specificity similar to P. gingivalis DPP7 belonging to the S46 family. In accord with the deduced molecular mass of 818 amino acids, a 105-kDa band was immunologically detected, indicating that P. endodontalis DPP7 is an exceptionally large molecule in the DPP7/DPP11/S46 peptidase family. The enhancement of four DPP activities was conclusively demonstrated in P. endodontalis, and remarkable Lys-Ala-MCA-hydrolysis was achieved by qualitative and quantitative potentiation of the DPP5 molecule.

Identification of Dipeptidyl-Peptidase (DPP)5 and DPP7 in Porphyromonas endodontalis, Distinct from Those in Porphyromonas gingivalis

PubMed Central

Nishimata, Haruka; Ohara-Nemoto, Yuko; Baba, Tomomi T.; Hoshino, Tomonori; Fujiwara, Taku; Shimoyama, Yu; Kimura, Shigenobu; Nemoto, Takayuki K.

2014-01-01

Dipeptidyl peptidases (DPPs) that liberate dipeptides from the N-terminal end of oligopeptides are crucial for the growth of Porphyromonas species, anaerobic asaccharolytic gram negative rods that utilize amino acids as energy sources. Porphyromonas endodontalis is a causative agent of periapical lesions with acute symptoms and Asp/Glu-specific DPP11 has been solely characterized in this organism. In this study, we identified and characterized two P. endodontalis DPPs, DPP5 and DPP7. Cell-associated DPP activity toward Lys-Ala-4-methylcoumaryl-7-amide (MCA) was prominent in P. endodontalis ATCC 35406 as compared with the Porphyromonas gingivalis strains ATCC 33277, 16-1, HW24D1, ATCC 49417, W83, W50, and HNA99. The level of hydrolysis of Leu-Asp-MCA by DPP11, Gly-Pro-MCA by DPP4, and Met-Leu-MCA was also higher than in the P. gingivalis strains. MER236725 and MER278904 are P. endodontalis proteins belong to the S9- and S46-family peptidases, respectively. Recombinant MER236725 exhibited enzymatic properties including substrate specificity, and salt- and pH-dependence similar to P. gingivalis DPP5 belonging to the S9 family. However, the k cat/K m figure (194 µM−1·sec−1) for the most potent substrate (Lys-Ala-MCA) was 18.4-fold higher as compared to the P. gingivalis entity (10.5 µM−1·sec−1). In addition, P. endodontalis DPP5 mRNA and protein contents were increased several fold as compared with those in P. gingivalis. Recombinant MER278904 preferentially hydrolyzed Met-Leu-MCA and exhibited a substrate specificity similar to P. gingivalis DPP7 belonging to the S46 family. In accord with the deduced molecular mass of 818 amino acids, a 105-kDa band was immunologically detected, indicating that P. endodontalis DPP7 is an exceptionally large molecule in the DPP7/DPP11/S46 peptidase family. The enhancement of four DPP activities was conclusively demonstrated in P. endodontalis, and remarkable Lys-Ala-MCA-hydrolysis was achieved by qualitative and quantitative potentiation of the DPP5 molecule. PMID:25494328

Preparation of antioxidative corn protein hydrolysates, purification and evaluation of three novel corn antioxidant peptides.

PubMed

Jin, Du-Xin; Liu, Xiao-Lan; Zheng, Xi-Qun; Wang, Xiao-Jie; He, Jun-Fang

2016-08-01

Corn gluten meal is a major co-product of corn wet milling. Corn gluten meal was hydrolyzed with Alcalase, Flavourzyme, Alcalase+Flavourzyme and Flavourzyme+Alcalase. At the substrate concentration of 10%, corn protein hydrolysate catalyzed by Alcalase had a degree of hydrolysis of 17.83%, which was higher than that by Flavourzyme (3.65%). The hydrolysate catalyzed by Alcalase+Flavourzyme exhibited better antioxidant activities and was further purified. Three novel antioxidant peptides were purified by a series of chromatographic techniques. Sequences of the three peptides were identified as Cys-Ser-Gln-Ala-Pro-Leu-Ala, Tyr-Pro-Lys-Leu-Ala-Pro-Asn-Glu and Tyr-Pro-Gln-Leu-Leu-Pro-Asn-Glu, respectively. Among the three peptides, Cys-Ser-Gln-Ala-Pro-Leu-Ala exhibited good reducing power and excellent scavenging capacities for DPPH radical and superoxide anion radical, with IC50 values of 0.116 and 0.39mg/ml, respectively. The results from our study indicate antioxidant potency of corn protein hydrolysates and peptides separated from corn gluten meal and can provide basic understanding for the application of corn protein hydrolysates as natural antioxidants. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation of New Tc-99m-Labeled Arg-X-Asp-Conjugated Alpha-Melanocyte Stimulating Hormone Peptides for Melanoma Imaging

PubMed Central

Flook, Adam M.; Yang, Jianquan; Miao, Yubin

2013-01-01

The purpose of this study was to examine the melanoma targeting and imaging properties of two new 99mTc-labeled Arg-X-Asp-conjugated alpha-melanocyte stimulating hormone (α-MSH) peptides. RTD-Lys-(Arg11)CCMSH {c[Asp-Arg-Thr-Asp-DTyr]-Lys-Cys-Cys-Glu-His-DPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2} and RVD-Lys-(Arg11)CCMSH peptides were synthesized and their melanocortin-1 (MC1) receptor binding affinities were determined in B16/F1 melanoma cells. The biodistribution and melanoma imaging properties of 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The IC50 values of RTD-Lys-(Arg11)CCMSH and RVD-Lys-(Arg11)CCMSH were 0.7 ± 0.07 and 1.0 ± 0.3 nM in B16/F1 melanoma cells. Both 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH displayed high melanoma uptake. 99mTc-RTD-Lys-(Arg11)CCMSH exhibited the peak tumor uptake of 18.77 ± 5.13% ID/g at 2 h post-injection, whereas 99mTc-RVD-Lys-(Arg11)CCMSH reached the peak tumor uptake of 19.63 ± 4.68% ID/g at 4 h post-injection. Both 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH showed low accumulation in normal organs (<1.7% ID/g) except for the kidneys at 2 h post-injection. The renal uptake of 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH was 135.14 ± 23.62 and 94.01 ± 18.31% ID/g at 2 h post-injection, respectively. The melanoma lesions were clearly visualized by SPECT/CT using either 99mTc-RTD-Lys-(Arg11)CCMSH or 99mTc-RVD-Lys-(Arg11)CCMSH as an imaging probe at 2 h post-injection. Overall, the introduction of Thr or Val residue retained high melanoma uptake of 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH. However, high renal uptake of 99mTc-RTD-Lys-(Arg11)CCMSH and 99mTc-RVD-Lys-(Arg11)CCMSH need to be reduced to facilitate their future applications. PMID:23885640

Evaluation of new Tc-99m-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone peptides for melanoma imaging.

PubMed

Flook, Adam M; Yang, Jianquan; Miao, Yubin

2013-09-03

The purpose of this study was to examine the melanoma targeting and imaging properties of two new (99m)Tc-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RTD-Lys-(Arg(11))CCMSH {c[Asp-Arg-Thr-Asp-DTyr]-Lys-Cys-Cys-Glu-His-DPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2} and RVD-Lys-(Arg(11))CCMSH peptides were synthesized, and their melanocortin-1 (MC1) receptor binding affinities were determined in B16/F1 melanoma cells. The biodistribution and melanoma imaging properties of (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The IC50 values of RTD-Lys-(Arg(11))CCMSH and RVD-Lys-(Arg(11))CCMSH were 0.7 ± 0.07 and 1.0 ± 0.3 nM in B16/F1 melanoma cells. Both (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH displayed high melanoma uptake. (99m)Tc-RTD-Lys-(Arg(11))CCMSH exhibited the highest tumor uptake of 18.77 ± 5.13% ID/g at 2 h postinjection, whereas (99m)Tc-RVD-Lys-(Arg(11))CCMSH reached the highest tumor uptake of 19.63 ± 4.68% ID/g at 4 h postinjection. Both (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH showed low accumulation in normal organs (<1.7% ID/g) except for the kidneys at 2 h postinjection. The renal uptake of (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH was 135.14 ± 23.62 and 94.01 ± 18.31% ID/g at 2 h postinjection, respectively. The melanoma lesions were clearly visualized by single-photon emission computed tomography (SPECT)/CT using either (99m)Tc-RTD-Lys-(Arg(11))CCMSH or (99m)Tc-RVD-Lys-(Arg(11))CCMSH as an imaging probe at 2 h postinjection. Overall, the introduction of Thr or Val residue retained high melanoma uptake of (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH. However, high renal uptake of (99m)Tc-RTD-Lys-(Arg(11))CCMSH and (99m)Tc-RVD-Lys-(Arg(11))CCMSH need to be reduced to facilitate their future applications.

Short Exogenous Peptides Regulate Expression of CLE, KNOX1, and GRF Family Genes in Nicotiana tabacum.

PubMed

Fedoreyeva, L I; Dilovarova, T A; Ashapkin, V V; Martirosyan, Yu Ts; Khavinson, V Kh; Kharchenko, P N; Vanyushin, B F

2017-04-01

Exogenous short biologically active peptides epitalon (Ala-Glu-Asp-Gly), bronchogen (Ala-Glu-Asp-Leu), and vilon (Lys-Glu) at concentrations 10 -7 -10 -9  M significantly influence growth, development, and differentiation of tobacco (Nicotiana tabacum) callus cultures. Epitalon and bronchogen, in particular, both increase growth of calluses and stimulate formation and growth of leaves in plant regenerants. Because the regulatory activity of the short peptides appears at low peptide concentrations, their action to some extent is like that of the activity of phytohormones, and it seems to have signaling character and epigenetic nature. The investigated peptides modulate in tobacco cells the expression of genes including genes responsible for tissue formation and cell differentiation. These peptides differently modulate expression of CLE family genes coding for known endogenous regulatory peptides, the KNOX1 genes (transcription factor genes) and GRF (growth regulatory factor) genes coding for respective DNA-binding proteins such as topoisomerases, nucleases, and others. Thus, at the level of transcription, plants have a system of short peptide regulation of formation of long-known peptide regulators of growth and development. The peptides studied here may be related to a new generation of plant growth regulators. They can be used in the experimental botany, plant molecular biology, biotechnology, and practical agronomy.

Quantum Mechanics and Molecular Mechanics Study of the Catalytic Mechanism of Human AMSH-LP Domain Deubiquitinating Enzymes.

PubMed

Zhu, Wenyou; Liu, Yongjun; Ling, Baoping

2015-08-25

Deubiquitinating enzymes (DUBs) catalyze the cleavage of the isopeptide bond in polyubiquitin chains to control and regulate the deubiquitination process in all known eukaryotic cells. The human AMSH-LP DUB domain specifically cleaves the isopeptide bonds in the Lys63-linked polyubiquitin chains. In this article, the catalytic mechanism of AMSH-LP has been studied using a combined quantum mechanics and molecular mechanics method. Two possible hydrolysis processes (Path 1 and Path 2) have been considered. Our calculation results reveal that the activation of Zn(2+)-coordinated water molecule is the essential step for the hydrolysis of isopeptide bond. In Path 1, the generated hydroxyl first attacks the carbonyl group of Gly76, and then the amino group of Lys63 is protonated, which is calculated to be the rate limiting step with an energy barrier of 13.1 kcal/mol. The energy barrier of the rate limiting step and the structures of intermediate and product are in agreement with the experimental results. In Path 2, the protonation of amino group of Lys63 is prior to the nucleophilic attack of activated hydroxyl. The two proton transfer processes in Path 2 correspond to comparable overall barriers (33.4 and 36.1 kcal/mol), which are very high for an enzymatic reaction. Thus, Path 2 can be ruled out. During the reaction, Glu292 acts as a proton transfer mediator, and Ser357 mainly plays a role in stabilizing the negative charge of Gly76. Besides acting as a Lewis acid, Zn(2+) also influences the reaction by coordinating to the reaction substrates (W1 and Gly76).

Evolution of the insulin molecule: insights into structure-activity and phylogenetic relationships.

PubMed

Conlon, J M

2001-07-01

The conformation of insulin in the crystalline state has been known for more than 30 years but there remains uncertainty regarding the biologically active conformation and the structural features that constitute the receptor-binding domain. The primary structure of insulin has been determined for at least 100 vertebrate species. In addition to the invariant cysteines, only ten amino acids (GlyA1, IleA2, ValA3, TyrA19, LeuB6, GlyB8, LeuB11, ValB12, GlyB23 and PheB24) have been fully conserved during vertebrate evolution. This observation supports the hypothesis derived from alanine-scanning mutagenesis studies that five of these invariant residues (IleA2, ValA3, TyrA19, GlyB23, and Phe24) interact directly with the receptor and five additional conserved residues (LeuB6, GlyB8, LeuB11, GluB13 and PheB25) are important in maintaining the receptor-binding conformation. With the exception of the hagfish, only conservative substitutions are found at B13 (Glu --> Asp) and B25(Phe --> Tyr). In contrast, amino acid residues that were also considered to be important in receptor binding based upon the crystal structure of insulin (GluA4, GlnA5, AsnA21, TyrB16, TyrB26) have been much less well conserved and are probably not components of the receptor-binding domain. The hypothesis that LeuA13 and LeuB17 form part of a second receptor-binding site in the insulin molecule finds some support in terms of their conservation during vertebrate evolution, although the site is probably absent in some hystricomorph insulins. In general, the amino acid sequences of insulins are not useful in cladistic analyses especially when evolutionary distant taxa are compared but, among related species in a particular order or family, the presence of unusual structural features in the insulin molecule may permit a meaningful phylogenetic inference. For example, analysis of insulin sequences supports monophyletic status for Dipnoi, Elasmobranchii, Holocephali and Petromyzontiformes.

Formation of crystalline nanoparticles by iron binding to pentapeptide (Asp-His-Thr-Lys-Glu) from egg white hydrolysates.

PubMed

Sun, Na; Cui, Pengbo; Li, Dongmei; Jin, Ziqi; Zhang, Shuyu; Lin, Songyi

2017-09-20

A novel peptide from egg white, Asp-His-Thr-Lys-Glu (DHTKE), contains specific amino acids associated with iron binding. The present study aims to better understand the molecular basis of interactions between the DHTKE peptide and iron ions. The ultraviolet-visible and fluorescence spectra indicate an interaction between the DHTKE peptide and iron ions, which leads to the formation of a DHTKE-iron complex. Notably, Asp, Glu, His, and Lys in the DHTKE peptide play crucial roles in the formation of the DHTKE-iron complex, and the iron-binding site of the DHTKE peptide corresponds primarily to the amide and carboxyl groups. The DHTKE peptide can bind iron ions in a 1 : 2 ratio with a binding constant of 1.312 × 10 5 M -1 . Moreover, the DHTKE-iron complex belongs to thermodynamically stable nanoparticles that are present in the crystalline structure, which might be attributed to peptide folding induced by iron binding. Meanwhile, the DHTKE-iron complex exhibits a relatively high iron-releasing percentage and exerts excellent solubility in the human gastrointestinal tract in vitro. This suggests a potential application of peptides containing Asp, Glu, His, or Lys residues as potential iron supplements.

Structure and biological activities of eumenine mastoparan-AF (EMP-AF), a new mast cell degranulating peptide in the venom of the solitary wasp (Anterhynchium flavomarginatum micado).

PubMed

Konno, K; Hisada, M; Naoki, H; Itagaki, Y; Kawai, N; Miwa, A; Yasuhara, T; Morimoto, Y; Nakata, Y

2000-11-01

A new mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF), was isolated from the venom of the solitary wasp Anterhynchium flavomarginatum micado, the most common eumenine wasp found in Japan. The structure was analyzed by FAB-MS/MS together with Edman degradation, which was corroborated by solid-phase synthesis. The sequence of EMP-AF, Ile-Asn-Leu-Leu-Lys-Ile-Ala-Lys-Gly-Ile-Ile-Lys-Ser-Leu-NH(2), was similar to that of mastoparan, a mast cell degranulating peptide from a hornet venom; tetradecapeptide with C-terminus amidated and rich in hydrophobic and basic amino acids. In fact, EMP-AF exhibited similar activity to mastoparan in stimulating degranulation from rat peritoneal mast cells and RBL-2H3 cells. It also showed significant hemolytic activity in human erythrocytes. Therefore, this is the first example that a mast cell degranulating peptide is found in the solitary wasp venom. Besides the degranulation and hemolytic activity, EMP-AF also affects on neuromuscular transmission in the lobster walking leg preparation. Three analogs EMP-AF-1 approximately 3 were snythesized and biologically tested together with EMP-AF, resulting in the importance of the C-terminal amide structure for biological activities.

Effect of lysine at C-terminus of the Dmt-Tic opioid pharmacophore.

PubMed

Balboni, Gianfranco; Onnis, Valentina; Congiu, Cenzo; Zotti, Margherita; Sasaki, Yusuke; Ambo, Akihiro; Bryant, Sharon D; Jinsmaa, Yunden; Lazarus, Lawrence H; Trapella, Claudio; Salvadori, Severo

2006-09-07

Substitution of Gly with side-chain-protected or unprotected Lys in lead compounds containing the opioid pharmacophore Dmt-Tic [H-Dmt-Tic-Gly-NH-CH(2)-Ph, mu agonist/delta antagonist; H-Dmt-Tic-Gly-NH-Ph, mu agonist/delta agonist; and H-Dmt-Tic-NH-CH(2)-Bid, delta agonist (Bid = 1H-benzimidazole-2-yl)] yielded a new series of compounds endowed with distinct pharmacological activities. Compounds (1-10) included high delta- (Ki(delta) = 0.068-0.64 nM) and mu-opioid affinities (Ki(mu) = 0.13-5.50 nM), with a bioactivity that ranged from mu-opioid agonism {10, H-Dmt-Tic-NH-CH[(CH2)4-NH2]-Bid (IC50 GPI = 39.7 nM)} to a selective mu-opioid antagonist [3, H-Dmt-Tic-Lys-NH-CH2-Ph (pA2(mu) = 7.96)] and a selective delta-opioid antagonist [5, H-Dmt-Tic-Lys(Ac)-NH-Ph (pA2(delta) = 12.0)]. The presence of a Lys linker provides new lead compounds in the formation of opioid peptidomimetics containing the Dmt-Tic pharmacophore with distinct agonist and/or antagonist properties.

Influence of Glu/Arg, Asp/Arg, and Glu/Lys Salt Bridges on α-Helical Stability and Folding Kinetics.

PubMed

Meuzelaar, Heleen; Vreede, Jocelyne; Woutersen, Sander

2016-06-07

Using a combination of ultraviolet circular dichroism, temperature-jump transient-infrared spectroscopy, and molecular dynamics simulations, we investigate the effect of salt bridges between different types of charged amino-acid residue pairs on α-helix folding. We determine the stability and the folding and unfolding rates of 12 alanine-based α-helical peptides, each of which has a nearly identical composition containing three pairs of positively and negatively charged residues (either Glu(-)/Arg(+), Asp(-)/Arg(+), or Glu(-)/Lys(+)). Within each set of peptides, the distance and order of the oppositely charged residues in the peptide sequence differ, such that they have different capabilities of forming salt bridges. Our results indicate that stabilizing salt bridges (in which the interacting residues are spaced and ordered such that they favor helix formation) speed up α-helix formation by up to 50% and slow down the unfolding of the α-helix, whereas salt bridges with an unfavorable geometry have the opposite effect. Comparing the peptides with different types of charge pairs, we observe that salt bridges between side chains of Glu(-) and Arg(+) are most favorable for the speed of folding, probably because of the larger conformational space of the salt-bridging Glu(-)/Arg(+) rotamer pairs compared to Asp(-)/Arg(+) and Glu(-)/Lys(+). We speculate that the observed impact of salt bridges on the folding kinetics might explain why some proteins contain salt bridges that do not stabilize the final, folded conformation. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Selection of a platinum-binding sequence in a loop of a four-helix bundle protein.

PubMed

Yagi, Sota; Akanuma, Satoshi; Kaji, Asumi; Niiro, Hiroya; Akiyama, Hayato; Uchida, Tatsuya; Yamagishi, Akihiko

2018-02-01

Protein-metal hybrids are functional materials with various industrial applications. For example, a redox enzyme immobilized on a platinum electrode is a key component of some biofuel cells and biosensors. To create these hybrid materials, protein molecules are bound to metal surfaces. Here, we report the selection of a novel platinum-binding sequence in a loop of a four-helix bundle protein, the Lac repressor four-helix protein (LARFH), an artificial protein in which four identical α-helices are connected via three identical loops. We created a genetic library in which the Ser-Gly-Gln-Gly-Gly-Ser sequence within the first inter-helical loop of LARFH was semi-randomly mutated. The library was then subjected to selection for platinum-binding affinity by using the T7 phage display method. The majority of the selected variants contained the Tyr-Lys-Arg-Gly-Tyr-Lys (YKRGYK) sequence in their randomized segment. We characterized the platinum-binding properties of mutant LARFH by using quartz crystal microbalance analysis. Mutant LARFH seemed to interact with platinum through its loop containing the YKRGYK sequence, as judged by the estimated exclusive area occupied by a single molecule. Furthermore, a 10-residue peptide containing the YKRGYK sequence bound to platinum with reasonably high affinity and basic side chains in the peptide were crucial in mediating this interaction. In conclusion, we have identified an amino acid sequence, YKRGYK, in the loop of a helix-loop-helix motif that shows high platinum-binding affinity. This sequence could be grafted into loops of other polypeptides as an approach to immobilize proteins on platinum electrodes for use as biosensors among other applications. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Dietary l-Lysine Prevents Arterial Calcification in Adenine-Induced Uremic Rats

PubMed Central

Shimomura, Akihiro; Matsui, Isao; Hamano, Takayuki; Ishimoto, Takuya; Katou, Yumiko; Takehana, Kenji; Inoue, Kazunori; Kusunoki, Yasuo; Mori, Daisuke; Nakano, Chikako; Obi, Yoshitsugu; Fujii, Naohiko; Takabatake, Yoshitsugu; Nakano, Takayoshi; Tsubakihara, Yoshiharu; Rakugi, Hiromi

2014-01-01

Vascular calcification (VC) is a life-threatening complication of CKD. Severe protein restriction causes a shortage of essential amino acids, and exacerbates VC in rats. Therefore, we investigated the effects of dietary l-lysine, the first-limiting amino acid of cereal grains, on VC. Male Sprague-Dawley rats at age 13 weeks were divided randomly into four groups: low-protein (LP) diet (group LP), LP diet+adenine (group Ade), LP diet+adenine+glycine (group Gly) as a control amino acid group, and LP diet+adenine+l-lysine·HCl (group Lys). At age 18 weeks, group LP had no VC, whereas groups Ade and Gly had comparable levels of severe VC. l-Lysine supplementation almost completely ameliorated VC. Physical parameters and serum creatinine, urea nitrogen, and phosphate did not differ among groups Ade, Gly, and Lys. Notably, serum calcium in group Lys was slightly but significantly higher than in groups Ade and Gly. Dietary l-lysine strongly suppressed plasma intact parathyroid hormone in adenine rats and supported a proper bone-vascular axis. The conserved orientation of the femoral apatite in group Lys also evidenced the bone-protective effects of l-lysine. Dietary l-lysine elevated plasma alanine, proline, arginine, and homoarginine but not lysine. Analyses in vitro demonstrated that alanine and proline inhibit apoptosis of cultured vascular smooth muscle cells, and that arginine and homoarginine attenuate mineral precipitations in a supersaturated calcium/phosphate solution. In conclusion, dietary supplementation of l-lysine ameliorated VC by modifying key pathways that exacerbate VC. PMID:24652795

Identification of Transglutaminase Reactive Residues in Human Osteopontin and Their Role in Polymerization

PubMed Central

Christensen, Brian; Zachariae, Elias D.; Scavenius, Carsten; Thybo, Morten; Callesen, Morten M.; Kløverpris, Søren; Oxvig, Claus; Enghild, Jan J.; Sørensen, Esben S.

2014-01-01

Osteopontin (OPN) is a highly posttranslationally modified protein present in several tissues where it is implicated in numerous physiological processes. OPN primarily exerts its functions through interaction with integrins via the Arg-Gly-Asp and Ser-Val-Val-Tyr-Gly-Leu-Arg sequences located in the N-terminal part of the protein. OPN can be polymerized by the cross-linking enzyme transglutaminase 2 (TG2), and polymerization has been shown to enhance the biological activity of OPN. However, little is known about the reactivity and location of the glutamine and lysine residues involved in the TG2-mediated modification of OPN. Here we show that TG2 catalyses the incorporation of 5-(Biotinamido)pentylamine at glutamines in both the N- and C-terminal parts of OPN, whereas TG2 primarily incorporated the glutamine-donor peptide biotinyl-TVQQEL-OH into the C-terminal part of OPN. By mass spectrometric analyses we identified Gln34, Gln42, Gln193 and Gln248 as the major TG2 reactive glutamines in OPN. The distribution of reactive Gln and Lys residues in OPN proved to be important, as the full-length protein but not the physiologically highly active integrin-binding N-terminal part of OPN were able to polymerize in a TG2-mediated reaction. Collectively, these data provide important new molecular knowledge about the mechanism of OPN polymerization. PMID:25419572

Helical peptides with three pairs of Asp-Arg and Glu-Arg residues in different orientations and spacings.

PubMed Central

Huyghues-Despointes, B. M.; Scholtz, J. M.; Baldwin, R. L.

1993-01-01

The helix-stabilizing effects of repeating pairs of Asp-Arg and Glu-Arg residues have been characterized using a peptide system of the same design used earlier to study Glu-Lys (Marqusee, S. & Baldwin, R.L., 1987, Proc. Natl. Acad. Sci. USA 84, 8898-8902) and Asp-Lys ion pairs (Marqusee, S. & Baldwin, R.L., 1990, In Protein Folding [Gierasch, L.M. & King, J., Eds.], pp. 85-94, AAAS, Washington, D.C.). The consequences of breaking ion pair and charge-helix dipole interactions by titration to pH 2 have been compared with the results of screening these interactions with NaCl at pH 7.0 and pH 2.5. The four peptides in each set contain three pairs of acidic (A) and basic (B) residues spaced either i, i + 4 or i, i + 3 apart. In one peptide of each kind the pairwise order of residues is AB, with the charges oriented favorably to the helix macrodipole, and in the other peptide the order is BA. The results are as follows: (1) Remarkably, both Asp-Arg and Glu-Arg peptides show the same pattern of helix stabilization at pH 7.0 found earlier for Glu-Lys and Asp-Lys peptides: i + 4 AB > i + 4 BA approximately i + 3 AB > i + 3 BA. (2) The ion pairs and charge-helix dipole interactions cannot be cleanly separated, but the results suggest that both interactions make important contributions to helix stability.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8443591

A novel KRT5 mutation associated with generalized severe epidermolysis bullosa simplex in a 2-year-old Chinese boy

PubMed Central

Zhang, Jia; Yan, Ming; Liang, Jianying; Li, Ming; Yao, Zhirong

2016-01-01

Mutations in keratin 5 (KRT5) or KRT14 genes are responsible for the most severe form of epidermolysis bullosa simplex (EBS), which is EBS generalized severe (EBS-gen sev). To date, only four pathogenic mutations (p.Arg165Ser and p.Lys199Asn in KRT5; p.Arg125Cys and p.Arg125His in KRT14) have been reported to be responsible for EBS-gen sev in the Chinese population. In the present study, a 2-year-old Chinese boy was clinically suspected to suffer from EBS, and thus Sanger sequencing was performed in the extracted genomic DNA samples from the patient, his parents and 100 healthy controls. A novel de novo heterozygous missense mutation c.503A>G (p.Glu168Gly) located at the N-terminal end segment of the 1A domain in KRT5 was identified by molecular analysis. In silico analysis tools were used to predict the pathogenicity of the novel missense mutation. A diagnosis of EBS-gen sev was thus confirmed according to the clinical presentations and molecular results. PMID:27882080

Effects of feeding reduced crude protein diets on growth performance, nitrogen excretion, and plasma uric acid concentration of broiler chicks during the starter period.

PubMed

Kriseldi, R; Tillman, P B; Jiang, Z; Dozier, W A

2018-05-01

An experiment (2 trials) was conducted to determine the effects of feeding reduced crude protein (CP) diets to Ross × Ross 708 male broilers while maintaining adequate essential amino acid (AA) concentrations on growth performance, nitrogen excretion, and plasma uric acid (UA) concentration during the starter period. In trial 1, 11 dietary treatments were fed from 1 to 18 d of age containing 1.20% digestible Lys. Diet 1 (23.2% CP) was formulated with DL-Met, L-Lys, and L-Thr to contain 1.70 total Gly + Ser to digestible Lys ratio whereas diets 2 (23.4% CP) to 11 were formulated with additional Gly to contain 1.90 total Gly + Ser to digestible Lys ratio. Free AA were added sequentially in the order of limitation (L-Val, L-Ile, L-Arg, L-Trp, L-His, L-Phe, and L-Leu) from diets 3 to 10 to decrease CP content from 22.6 to 18.8%, respectively. In diet 11, L-Gln was added to increase the CP content to 23.4%. Feed conversion of broilers fed diet 2 was lower (P < 0.05) than those consuming diets 6 to 11 from 1 to 17 d of age. Nitrogen excretion (mg/b/d) decreased (P < 0.001) by 14.1% when broilers were fed diet 4 compared with birds fed diet 2 from 15 to 16 d of age. Broilers fed diet 4 had lower (P = 0.011) plasma UA concentration than birds fed diet 2 at 18 d of age. In trial 2, 8 dietary treatments containing 1.25% digestible Lys and 1.70 total Gly + Ser to digestible Lys ratio were fed from 1 to 21 d of age. Diet 1 (24.0% CP) was supplemented with DL-Met, L-Lys, and L-Thr. Free AA (L-Val, Gly, L-Ile, L-Arg, L-Trp, L-His, and L-Phe) were sequentially supplemented in the order of limitation to decrease CP content in diets 2 to 8 from 23.8 to 20.3%. Broilers fed diet 1 had higher (P < 0.05) body weight gain and lower (P < 0.05) feed conversion when compared with diet 7 or 8. Plasma UA concentration of broiler provided diets 4 to 8 was lower (P < 0.05) compared with diet 1 at 21 d of age. Placing a minimum on dietary CP percentage may not be necessary when proper AA ratios are implemented in diet formulation.

Modular Small Diameter Vascular Grafts with Bioactive Functionalities

PubMed Central

Neufurth, Meik; Wang, Xiaohong; Tolba, Emad; Dorweiler, Bernhard; Schröder, Heinz C.; Link, Thorben; Diehl-Seifert, Bärbel; Müller, Werner E. G.

2015-01-01

We report the fabrication of a novel type of artificial small diameter blood vessels, termed biomimetic tissue-engineered blood vessels (bTEBV), with a modular composition. They are composed of a hydrogel scaffold consisting of two negatively charged natural polymers, alginate and a modified chitosan, N,O-carboxymethyl chitosan (N,O-CMC). Into this biologically inert scaffold two biofunctionally active biopolymers are embedded, inorganic polyphosphate (polyP) and silica, as well as gelatin which exposes the cell recognition signal, Arg-Gly-Asp (RGD). These materials can be hardened by exposure to Ca2+ through formation of Ca2+ bridges between the polyanions, alginate, N,O-CMC, and polyP (alginate-Ca2+-N,O-CMC-polyP). The bTEBV are formed by pressing the hydrogel through an extruder into a hardening solution, containing Ca2+. In this universal scaffold of the bTEBV biomaterial, polycations such as poly(l-Lys), poly(d-Lys) or a His/Gly-tagged RGD peptide (three RGD units) were incorporated, which promote the adhesion of endothelial cells to the vessel surface. The mechanical properties of the biopolymer material (alginate-Ca2+-N,O-CMC-polyP-silica) revealed a hardness (elastic modulus) of 475 kPa even after a short incubation period in CaCl2 solution. The material of the artificial vascular grafts (bTEBVs with an outer size 6 mm and 1.8 mm, and an inner diameter 4 mm and 0.8 mm, respectively) turned out to be durable in 4-week pulsatile flow experiments at an alternating pressure between 25 and 100 mbar (18.7 and 75.0 mm Hg). The burst pressure of the larger (smaller) vessels was 850 mbar (145 mbar). Incorporation of polycationic poly(l-Lys), poly(d-Lys), and especially the His/Gly-tagged RGD peptide, markedly increased the adhesion of human, umbilical vein/vascular endothelial cells, EA.HY926 cells, to the surface of the hydrogel. No significant effect of the polyP samples on the clotting of human plasma is measured. We propose that the metabolically degradable polymeric scaffold bTEBV is a promising biomaterial for future prosthetic vascular grafts. PMID:26204529

Fragment-based screen against HIV protease.

PubMed

Perryman, Alexander L; Zhang, Qing; Soutter, Holly H; Rosenfeld, Robin; McRee, Duncan E; Olson, Arthur J; Elder, John E; Stout, C David

2010-03-01

We have employed a fragment-based screen against wild-type (NL4-3) HIV protease (PR) using the Active Sight fragment library and X-ray crystallography. The experiments reveal two new binding sites for small molecules. PR was co-crystallized with fragments, or crystals were soaked in fragment solutions, using five crystal forms, and 378 data sets were collected to 2.3-1.3 A resolution. Fragment binding induces a distinct conformation and specific crystal form of TL-3 inhibited PR during co-crystallization. One fragment, 2-methylcyclohexanol, binds in the 'exo site' adjacent to the Gly(16)Gly(17)Gln(18)loop where the amide of Gly(17)is a specific hydrogen bond donor, and hydrophobic contacts occur with the side chains of Lys(14)and Leu(63). Another fragment, indole-6-carboxylic acid, binds on the 'outside/top of the flap' via hydrophobic contacts with Trp(42), Pro(44), Met(46), and Lys(55), a hydrogen bond with Val(56), and a salt-bridge with Arg(57). 2-acetyl-benzothiophene also binds at this site. This study is the first fragment-based crystallographic screen against HIV PR, and the first time that fragments were screened against an inhibitor-bound drug target to search for compounds that both bind to novel sites and stabilize the inhibited conformation of the target.

Fragment-Based Screen against HIV Protease

PubMed Central

Perryman, A. L.; Zhang, Q.; Soutter, H. H.; Rosenfeld, R.; McRee, D. E.; Olson, A. J.; Elder, J. E.; Stout, C. D.

2009-01-01

We have employed a fragment-based screen against wild-type (NL4-3) HIV protease (PR) using the Active Sight fragment library and X-ray crystallography. The experiments reveal two new binding sites for small molecules. PR was co-crystallized with fragments, or crystals were soaked in fragment solutions, using five crystal forms, and 378 data sets were collected to 2.3-1.3 Å resolution. Fragment binding induces a distinct conformation and specific crystal form of TL-3 inhibited PR during co-crystallization. One fragment, 2-methylcyclohexanol, binds in the ‘exo site’ adjacent to the Gly16Gly17Gln18 loop where the amide of Gly17 is a specific hydrogen bond donor, and hydrophobic contacts occur with the side chains of Lys14 and Leu63. Another fragment, indole-6-carboxylic acid, binds on the ‘outside/top of the flap’ via hydrophobic contacts with Trp42, Pro44, Met46, and Lys55, a hydrogen bond with Val56, and a salt-bridge with Arg57. 2-acetyl-benzothiophene also binds at this site. This study is the first fragment-based crystallographic screen against HIV PR, and the first time that fragments were screened against an inhibitor-bound drug target to search for compounds that both bind to novel sites and stabilize the inhibited conformation of the target. PMID:20659109

A conserved modified wobble nucleoside (mcm5s2U) in lysyl-tRNA is required for viability in yeast

PubMed Central

Björk, Glenn R.; Huang, Bo; Persson, Olof P.; Byström, Anders S.

2007-01-01

Transfer RNAs specific for Gln, Lys, and Glu from all organisms (except Mycoplasma) and organelles have a 2-thiouridine derivative (xm5s2U) as wobble nucleoside. These tRNAs read the A- and G-ending codons in the split codon boxes His/Gln, Asn/Lys, and Asp/Glu. In eukaryotic cytoplasmic tRNAs the conserved constituent (xm5-) in position 5 of uridine is 5-methoxycarbonylmethyl (mcm5). A protein (Tuc1p) from yeast resembling the bacterial protein TtcA, which is required for the synthesis of 2-thiocytidine in position 32 of the tRNA, was shown instead to be required for the synthesis of 2-thiouridine in the wobble position (position 34). Apparently, an ancient member of the TtcA family has evolved to thiolate U34 in tRNAs of organisms from the domains Eukarya and Archaea. Deletion of the TUC1 gene together with a deletion of the ELP3 gene, which results in the lack of the mcm5 side chain, removes all modifications from the wobble uridine derivatives of the cytoplasmic tRNAs specific for Gln, Lys, and Glu, and is lethal to the cell. Since excess of the unmodified form of these three tRNAs rescued the double mutant elp3 tuc1, the primary function of mcm5s2U34 seems to be to improve the efficiency to read the cognate codons rather than to prevent mis-sense errors. Surprisingly, overexpression of the mcm5s2U-lacking tRNALys alone was sufficient to restore viability of the double mutant. PMID:17592039

The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1

NASA Technical Reports Server (NTRS)

Chang, D.; Haynes, J. I. 2nd; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

A nuclear localization signal (NLS) has been identified in the N-terminal (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) amino acid sequence of the polyomavirus major capsid protein VP1. The importance of this amino acid sequence for nuclear transport of VP1 protein was demonstrated by a genetic "subtractive" study using the constructs pSG5VP1 (full-length VP1) and pSG5 delta 5'VP1 (truncated VP1, lacking amino acids Ala1-Cys11). These constructs were used to transfect COS-7 cells, and expression and intracellular localization of the VP1 protein was visualized by indirect immunofluorescence. These studies revealed that the full-length VP1 was expressed and localized in the nucleus, while the truncated VP1 protein was localized in the cytoplasm and not transported to the nucleus. These findings were substantiated by an "additive" approach using FITC-labeled conjugates of synthetic peptides homologous to the NLS of VP1 cross-linked to bovine serum albumin or immunoglobulin G. Both conjugates localized in the nucleus after microinjection into the cytoplasm of 3T6 cells. The importance of individual amino acids found in the basic sequence (Lys3-Arg-Lys5) of the NLS was also investigated. This was accomplished by synthesizing three additional peptides in which lysine-3 was substituted with threonine, arginine-4 was substituted with threonine, or lysine-5 was substituted with threonine. It was found that lysine-3 was crucial for nuclear transport, since substitution of this amino acid with threonine prevented nuclear localization of the microinjected, FITC-labeled conjugate.

Dietary L-lysine prevents arterial calcification in adenine-induced uremic rats.

PubMed

Shimomura, Akihiro; Matsui, Isao; Hamano, Takayuki; Ishimoto, Takuya; Katou, Yumiko; Takehana, Kenji; Inoue, Kazunori; Kusunoki, Yasuo; Mori, Daisuke; Nakano, Chikako; Obi, Yoshitsugu; Fujii, Naohiko; Takabatake, Yoshitsugu; Nakano, Takayoshi; Tsubakihara, Yoshiharu; Isaka, Yoshitaka; Rakugi, Hiromi

2014-09-01

Vascular calcification (VC) is a life-threatening complication of CKD. Severe protein restriction causes a shortage of essential amino acids, and exacerbates VC in rats. Therefore, we investigated the effects of dietary l-lysine, the first-limiting amino acid of cereal grains, on VC. Male Sprague-Dawley rats at age 13 weeks were divided randomly into four groups: low-protein (LP) diet (group LP), LP diet+adenine (group Ade), LP diet+adenine+glycine (group Gly) as a control amino acid group, and LP diet+adenine+l-lysine·HCl (group Lys). At age 18 weeks, group LP had no VC, whereas groups Ade and Gly had comparable levels of severe VC. l-Lysine supplementation almost completely ameliorated VC. Physical parameters and serum creatinine, urea nitrogen, and phosphate did not differ among groups Ade, Gly, and Lys. Notably, serum calcium in group Lys was slightly but significantly higher than in groups Ade and Gly. Dietary l-lysine strongly suppressed plasma intact parathyroid hormone in adenine rats and supported a proper bone-vascular axis. The conserved orientation of the femoral apatite in group Lys also evidenced the bone-protective effects of l-lysine. Dietary l-lysine elevated plasma alanine, proline, arginine, and homoarginine but not lysine. Analyses in vitro demonstrated that alanine and proline inhibit apoptosis of cultured vascular smooth muscle cells, and that arginine and homoarginine attenuate mineral precipitations in a supersaturated calcium/phosphate solution. In conclusion, dietary supplementation of l-lysine ameliorated VC by modifying key pathways that exacerbate VC. Copyright © 2014 by the American Society of Nephrology.

Analysis of the major epitope of the alpha2 chain of bovine type I collagen in children with bovine gelatin allergy.

PubMed

Hori, Hisae; Hattori, Shunji; Inouye, Sakae; Kimura, Akinori; Irie, Shinkichi; Miyazawa, Hiroshi; Sakaguchi, Masahiro

2002-10-01

Anaphylaxis to measles, mumps, and rubella vaccines has been reported. It has been found that most of these reactions to live vaccines are caused by type I allergy with the bovine gelatin present in the vaccines as an allergen. Gelatin mainly includes denatured type I collagen, which consists of alpha1 and alpha2 chains. We previously reported that allergic reactions to gelatin are caused by the type I collagen alpha2 (alpha2[I]) chain. To aid in the development of gelatin that has little or no allergenicity in human subjects, we investigated epitopes of bovine alpha2(I) chain with use of IgE in gelatin-sensitive children. Serum samples were collected from 15 patients who had systemic allergic reactions to vaccines and high levels of specific IgE to bovine gelatin. Eleven overlapping recombinant proteins that cover bovine alpha2(I) were prepared with a bacterial expression vector. We examined IgE reactivity to these recombinant proteins by means of ELISA. Fifteen peptides covering a major reactive recombinant protein were synthesized. The IgE-reacting epitope was identified by means of IgE-ELISA inhibition with these synthetic peptides and pooled serum from the patients. We found that of the 15 patients, 13 showed IgE reactivity to a recombinant protein (no. 3) spanning the central region of the collagenous domain ((418)Gly-(662)Pro). Furthermore, all 13 patients showed IgE reactivity to the 4-kd recombinant protein (no. 3a) spanning the region from (461)Pro to (500)Glu. In IgE-ELISA inhibition we found that a minimum IgE epitope of gelatin allergen was composed of the 10-amino-acid sequence (485)Ile-Pro-Gly-Glu-Phe-Gly-Leu-Pro-Gly-Pro(494). This sequence is not observed in the human type I collagen alpha1 and alpha2 chains, nor is it found in the bovine type I collagen alpha1 chain. We found that Ile-Pro-Gly-Glu-Phe-Gly-Leu-Pro-Gly-Pro is a major IgE epitope of the alpha2 chain of bovine type I collagen in patients with gelatin allergy. The degree of anaphylaxis to gelatin in vaccines might be reduced by digestion of this IgE-binding site in gelatin.

Structural characterization of core-bradavidin in complex with biotin

PubMed Central

Agrawal, Nitin; Määttä, Juha A. E.; Kulomaa, Markku S.; Hytönen, Vesa P.; Johnson, Mark S.; Airenne, Tomi T.

2017-01-01

Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 (“Brad-tag”) act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E. coli, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin–Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin. PMID:28426764

Diversity of the causal genes in hearing impaired Algerian individuals identified by whole exome sequencing

PubMed Central

Ammar-Khodja, Fatima; Bonnet, Crystel; Dahmani, Malika; Ouhab, Sofiane; Lefèvre, Gaelle M; Ibrahim, Hassina; Hardelin, Jean-Pierre; Weil, Dominique; Louha, Malek; Petit, Christine

2015-01-01

The genetic heterogeneity of congenital hearing disorders makes molecular diagnosis expensive and time-consuming using conventional techniques such as Sanger sequencing of DNA. In order to design an appropriate strategy of molecular diagnosis in the Algerian population, we explored the diversity of the involved mutations by studying 65 families affected by autosomal recessive forms of nonsyndromic hearing impairment (DFNB forms), which are the most prevalent early onset forms. We first carried out a systematic screening for mutations in GJB2 and the recurrent p.(Arg34*) mutation in TMC1, which were found in 31 (47.7%) families and 1 (1.5%) family, respectively. We then performed whole exome sequencing in nine of the remaining families, and identified the causative mutations in all the patients analyzed, either in the homozygous state (eight families) or in the compound heterozygous state (one family): (c.709C>T: p.(Arg237*)) and (c.2122C>T: p.(Arg708*)) in OTOF, (c.1334T>G: p.(Leu445Trp)) in SLC26A4, (c.764T>A: p.(Met255Lys)) in GIPC3, (c.518T>A: p.(Cys173Ser)) in LHFPL5, (c.5336T>C: p.(Leu1779Pro)) in MYO15A, (c.1807G>T: p.(Val603Phe)) in OTOA, (c.6080dup: p.(Asn2027Lys*9)) in PTPRQ, and (c.6017del: p.(Gly2006Alafs*13); c.7188_7189ins14: p.(Val2397Leufs*2)) in GPR98. Notably, 7 of these 10 mutations affecting 8 different genes had not been reported previously. These results highlight for the first time the genetic heterogeneity of the early onset forms of nonsyndromic deafness in Algerian families. PMID:26029705

Glycyl-alanyl-histidine protects PC12 cells against hydrogen peroxide toxicity.

PubMed

Shimura, Hideki; Tanaka, Ryota; Shimada, Yoshiaki; Yamashiro, Kazuo; Hattori, Nobutaka; Urabe, Takao

2017-11-22

Peptides with cytoprotective functions, including antioxidants and anti-infectives, could be useful therapeutics. Carnosine, β-alanine-histidine, is a dipeptide with anti-oxidant properties. Tripeptides of Ala-His-Lys, Pro-His-His, or Tyr-His-Tyr are also of interest in this respect. We synthesized several histidine-containing peptides including glycine or alanine, and tested their cytoprotective effects on hydrogen peroxide toxicity for PC12 cells. Of all these peptides (Gly-His-His, Ala-His-His, Ala-His-Ala, Ala-Ala-His, Ala-Gly-His, Gly-Ala-His (GAH), Ala-His-Gly, His-Ala-Gly, His-His-His, Gly-His-Ala, and Gly-Gly-His), GAH was found to have the strongest cytoprotective activity. GAH decreased lactate dehydrogenase (LDH) leakage, apoptosis, morphological changes, and nuclear membrane permeability changes against hydrogen peroxide toxicity in PC12 cells. The cytoprotective activity of GAH was superior to that of carnosine against hydrogen peroxide toxicity in PC12 cells. GAH also protected PC12 cells against damage caused by actinomycin D and staurosporine. Additionally, it was found that GAH also protected SH-SY5Y and Jurkat cells from damage caused by hydrogen peroxide, as assessed by LDH leakage. Thus, a novel tripeptide, GAH, has been identified as having broad cytoprotective effects against hydrogen peroxide-induced cell damage.

Changes in Amino Acid Profile in Roots of Glyphosate Resistant and Susceptible Soybean (Glycine max) Induced by Foliar Glyphosate Application.

PubMed

Moldes, Carlos Alberto; Cantarelli, Miguel Angel; Camiña, José Manuel; Tsai, Siu Mui; Azevedo, Ricardo Antunes

2017-10-11

Amino acid profiles are useful to analyze the responses to glyphosate in susceptible and resistant soybean lines. Comparisons of profiles for 10 amino acids (Asp, Asn, Glu, Gln, Ser, His, Gly, Thr, Tyr, Leu) by HPLC in soybean roots were performed in two near isogenic pairs (four varieties). Foliar application of glyphosate was made to soybean plants after 5 weeks of seeding. Roots of four varieties were collected at 0 and 72 h after glyphosate application (AGA) for amino acid analysis by HPLC. Univariate analysis showed a significant increase of several amino acids in susceptible as well as resistant soybean lines; however, amino acids from the major pathways of carbon (C) and nitrogen (N) metabolism, such as Asp, Asn, Glu and Gln, and Ser, increased significantly in susceptible varieties at 72 h AGA. Multivariate analysis using principal component analysis (2D PCA and 3D PCA) allowed different groups to be identified and discriminated based on the soybean genetic origin, showing the amino acid responses on susceptible and resistant varieties. Based on the results, it is possible to infer that the increase of Asn, Asp, Glu, Gln, and Ser in susceptible varieties would be related to the deregulation of C and N metabolism, as well as changes in the growth mechanisms regulated by Ser.

New peptidomimetics of insulin.

PubMed

Maslov, D L; Lokhov, P G; Abakumova, O Yu; Tsvetkova, T A; Prozorovskiy, V N

2002-08-01

New peptidomimetics that have been obtained in the course of our experimental work show distinct insulin-like activity both in vitro and in vivo. The first peptidomimetic (PM 1) is essentially a decapeptide in which sites of A (20-21) and B (19-26) chains of insulin are linked by the peptides bond (Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Cys-Asn). The second peptidomimetic (PM 2) has similar set of amino acid residues, except that two aromatic amino acids corresponding to the residues of B chain of insulin (B24 and B26) have been replaced with their D optical isomers (Cys-Gly-Glu-Arg-Gly-DPhe-Phe-DTyr-Cys-Asn). The third peptidomimetic (PM 3) has been obtained through acylation of N-terminal of PM 1 by the use of palmitic acid. The peptidomimetic incorporating D aromatic amino acids (PM 2) was demonstrated to exhibit more pronounced hypoglycemic impact, while the acylation of decapeptide tends to prolong the effective time of peptidomimetic influence in vivo.

(±)-Japonones A and B, two pairs of new enantiomers with anti-KSHV activities from Hypericum japonicum

NASA Astrophysics Data System (ADS)

Hu, Linzhen; Zhu, Hucheng; Li, Lei; Huang, Jinfeng; Sun, Weiguang; Liu, Junjun; Li, Hua; Luo, Zengwei; Wang, Jianping; Xue, Yongbo; Zhang, Yu; Zhang, Yonghui

2016-06-01

Two pairs of new enantiomers with unusual 5,5-spiroketal cores, termed (±)-japonones A and B [(±)-1 and (±)-2], were obtained from Hypericum japonicum Thunb. The absolute configurations of (±)-1 and (±)-2 were characterized by extensive analyses of spectroscopic data and calculated electronic circular dichroism (ECD) spectra, the application of modified Mosher’s methods, and the assistance of quantum chemical predictions (QCP) of 13C NMR chemical shifts. Among these metabolites, (+)-1 exhibited some inhibitory activity on Kaposi’s sarcoma associated herpesvirus (KSHV). Virtual screening of (±)-1 and (±)-2 were conducted using the Surflex-Dock module in the Sybyl software, and (+)-1 exhibited ability to bind with ERK to form key interactions with residues Lys52, Pro56, Ile101, Asp165, Gly167 and Val99.

Fibronectin tetrapeptide is target for syphilis spirochete cytadherence

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

Thomas, D.D.; Baseman, J.B.; Alderete, J.F.

1985-11-01

The syphilis bacterium, Treponema pallidum, parasitizes host cells through recognition of fibronectin (Fn) on cell surfaces. The active site of the Fn molecule has been identified as a four-amino acid sequence, arg-gly-asp-ser (RGDS), located on each monomer of the cell-binding domain. The synthetic heptapeptide gly-arg-gly-asp-ser-pro-cys (GRGDSPC), with the active site sequence RGDS, specifically competed with SVI-labeled cell-binding domain acquisition by T. pallidum. Additionally, the same heptapeptide with the RGDS sequence diminished treponemal attachment to HEp-2 and HT1080 cell monolayers. Related heptapeptides altered in one key amino acid within the RGDS sequence failed to inhibit Fn cell-binding domain acquisition or parasitismmore » of host cells by T. pallidum. The data support the view that T. pallidum cytadherence of host cells is through recognition of the RGDS sequence also important for eukaryotic cell-Fn binding.« less

Cathepsin D immobilized capillary reactors for on-flow screening assays.

PubMed

Cornelio, Vivian Estevam; de Moraes, Marcela Cristina; Domingues, Vanessa de Cassia; Fernandes, João Batista; da Silva, Maria Fátima das Gracas Fernandes; Cass, Quezia Bezerra; Vieira, Paulo Cezar

2018-03-20

The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (K M  = 81.9 ± 7.49 μmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts. Copyright © 2018 Elsevier B.V. All rights reserved.

Albumin Redhill (-1 Arg, 320 Ala----Thr): a glycoprotein variant of human serum albumin whose precursor has an aberrant signal peptidase cleavage site.

PubMed

Brennan, S O; Myles, T; Peach, R J; Donaldson, D; George, P M

1990-01-01

Albumin Redhill is an electrophoretically slow genetic variant of human serum albumin that does not bind 63Ni2+ and has a molecular mass 2.5 kDa higher than normal albumin. Its inability to bind Ni2+ was explained by the finding of an additional residue of Arg at position -1. This did not explain the molecular basis of the genetic variation (since proalbumin contains adjacent Arg residues at -1 and -2) or the increase in apparent molecular mass. Fractionation of tryptic digests on concanavalin A-Sepharose followed by peptide mapping of the bound and unbound fractions and sequence analysis of the glycopeptides identified a mutation of 320 Ala----Thr. This introduces an Asn-Tyr-Thr oligosaccharide attachment sequence centered on Asn-318 and explains the increase in molecular mass. This, however, did not satisfactorily explain the presence of the additional Arg residue at position -1. DNA sequencing of polymerase chain reaction-amplified genomic DNA encoding the prepro sequence of albumin indicated an additional mutation of -2 Arg----Cys. This introduces a prepro sequence, Met-Lys-Trp-Val-Thr-Phe-Ile-Ser-Leu-Leu-Phe-Leu-Phe-Ser-Ser-Ala-Tyr- Ser-Arg-Gly-Val-Phe-Cys-Arg (cf.-Tyr-Ser-Arg-Gly-Val-Phe-Arg-Arg- in normal human pre-proalbumin). We propose that the new Phe-Cys-Arg sequence in the propeptide is an aberrant signal peptidase cleavage site and that the signal peptidase cleaves the propeptide of albumin Redhill in the lumen of the endoplasmic reticulum before it reaches the Golgi vesicles, the site of the diarginyl-specific proalbumin convertase.

Molecular analysis of Cypriot patients with Glutaric aciduria type I: identification of two novel mutations.

PubMed

Georgiou, Theodoros; Nicolaidou, Paola; Hadjichristou, Anastasia; Ioannou, Rodothea; Dionysiou, Maria; Siama, Elli; Chappa, Georgia; Anastasiadou, Violetta; Drousiotou, Anthi

2014-09-01

The purpose of this study was to identify the mutations in the glutaryl-CoA dehydrogenase gene (GCDH) in ten Cypriot patients with Glutaric aciduria type I (GAI). Molecular analysis of the GCDH gene was performed by direct sequencing of the patients' genomic DNA. In silico tools were applied to predict the effect of the novel variants on the structure and function of the protein. All disease alleles were characterized (mutation detection rate 100%). Five missense mutations were identified: c.192G>T (p.Glu64Asp) and c.803G>T (p.Gly268Val), which are novel, and three previously described mutations, c.1123T>C (p.Cys375Arg), c.1204C>T (p.Arg402Trp) and c.1286C>T (p.Thr429Met). Two novel mutations, p.Glu64Asp and p.Gly268Val, account for the majority of disease alleles (76.5%) in Cypriot patients with Glutaric aciduria type I. A founder effect for the p.Glu64Asp and the p.Gly268Val can be suggested based on the place of origin of the carriers of these mutations. Identification of the causative mutations of GAI in Cypriot patients will facilitate carrier detection as well as post- and pre-natal diagnosis. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

[Mechanisms of beta-lactam and quinolone resistance in Haemophilus influenzae].

PubMed

Ubukata, Kimiko

2012-02-01

Haemophilus influenzae is one of the important pathogens causing respiratory tract infections, pneumonia, and meningitis. Genotypic(g) beta-lactamase-nonproducing ampicillin resistance (gBLNAR) H. influenzae has rapidly increased since 2000 years in Japan. The resistant percentage exceeded 60% in Hib isolates from meningitis in 2009. The affinity of beta-lactam antibiotics for penicillin-binding proteins-3 (PBP3) that involved in septal peptidoglycan synthesis deceased in the resistant strains. Three amino acid substitutions, Ser385Thr, Asn526Lys and Arg517His in PBP3 encoded by ftsI gene are especially responsible for beta-lactam resistance in the gBLNAR. Susceptibilities of cephalosporin agents including cefotaxime for gBLNAR were apparently decreased than the ampicillin and carbapenem antibiotics. Though fluoroquinolone resistant isolates are rare (< 1%) in H. influenzae, strains of levofloxacin and ciprofloxacin MIC with > or = 8 microg/mL were isolated from elderly patients with CAP. These strains possessed amino acid substitutions of Ser84Phe and Asp88Asn in GyrA and Glu88Lys in ParC. It is important to practice rapidly identification of these resistant strains at routine work.

Antioxidant activities of red tilapia (Oreochromis niloticus) protein hydrolysates as influenced by thermolysin and alcalase

NASA Astrophysics Data System (ADS)

Daud, Nur'Aliah; Babji, Abdul Salam; Yusop, Salma Mohamad

2013-11-01

The hydrolysis process was performed on fish meat from Red Tilapia (Oreochromis niloticus) by enzymes thermolysin and alcalase under optimum conditions. The hydrolysis was performed from 0 - 4 hours at 37°C. Hydrolysates after 2 hours incubation with thermolysin and alcalase had degree of hydrolysis of 76.29 % and 63.49 %, respectively. The freeze dried protein hydrolysate was tested for peptide content and characterized with respect to amino acid composition. The result of increased peptide content in Red Tilapia (O. Niloticus) hydrolysates obtained was directly proportional to the increase activities of different proteolytic enzymes. The result of amino acid composition showed that the sample used contained abundant Gly, Ala, Asp, Glu, Lys and Leu in residues or peptide sequences. Both enzymatic hydrolysates were tested for anti-oxidant activity with DPPH and ABTS assay. Alcalase yielded higher anti-oxidative activity than Thermolysin hydrolysates after 1 hour incubation, but both enzymes hydrolysates showed a significant decrease of anti-oxidant activity after 2 hours of incubation. Hydrolysates from Red Tilapia may contribute as a health promoting ingredient in functional foods to reduce oxidation stress caused by accumulated free radicals.

Stoichiometry and pH dependence of the rabbit proton-dependent oligopeptide transporter PepT1.

PubMed

Steel, A; Nussberger, S; Romero, M F; Boron, W F; Boyd, C A; Hediger, M A

1997-02-01

1. The intestinal H(+)-coupled peptide transporter PepT1, displays a broad substrate specificity and accepts most charged and neutral di- and tripeptides. To study the proton-to-peptide stoichiometry and the dependence of the kinetic parameters on extracellular pH (pHo), rabbit PepT1 was expressed in Xenopus laevis oocytes and used for uptake studies of radiolabelled neutral and charged dipeptides, voltage-clamp analysis and intracellular pH measurements. 2. PepT1 did not display the substrate-gated anion conductances that have been found to be characteristic of members of the Na(+)- and H(+)-coupled high-affinity glutamate transporter family. In conjunction with previous data on the ion dependence of PepT1, it can therefore be concluded that peptide-evoked charge fluxes of PepT1 are entirely due to H+ movement. 3. Neutral, acidic and basic dipeptides induced intracellular acidification. The rate of acidification, the initial rates of the uptake of radiolabelled peptides and the associated charge fluxes gave proton-substrate coupling ratios of 1:1, 2:1 and 1:1 for neutral, acidic and basic dipeptides, respectively. 4. Maximal transport of the neutral and charged dipeptides Gly-Leu, Gly-Glu, Gly-Lys and Ala-Lys occurred at pHo 5.5, 5.2, 6.2 and 5.8, respectively. The Imax values were relatively pHo independent but the apparent affinity (Km(app) values for these peptides were shown to be highly pHo dependent. 5. Our data show that at physiological pH (pHo 5.5-6.0) PepT1 prefers neutral and acidic peptides. The shift in transport maximum for the acidic peptide Gly-Glu to a lower pH value suggests that acidic dipeptides are transported in the protonated form. The shift in the transport maxima of the basic dipeptides to higher pH values may involve titration of a side-chain on the transporter molecule (e.g. protonation of a histidine group). These considerations have led us to propose a model for coupled transport of neutral, acidic and basic dipeptides.

Potent inhibition of endopeptidase 24.16 and endopeptidase 24.15 by the phosphonamide peptide N-(phenylethylphosphonyl)-Gly-L-Pro-L-aminohexanoic acid.

PubMed Central

Barelli, H; Dive, V; Yiotakis, A; Vincent, J P; Checler, F

1992-01-01

A phosphonamide peptide, N-(phenylethylphosphonyl)-Gly-L-Pro-L-aminohexanoic acid, previously shown to block Clostridium histolyticum collagenases, was examined as a putative inhibitor of endopeptidase 24.16 and endopeptidase 24.15. Hydrolysis of two endopeptidase 24.16 substrates, i.e. 3-carboxy-7-methoxycoumarin (Mcc)-Pro-Leu-Gly-Pro-D-Lys-dinitrophenyl (Dnp) and neurotensin, were completely and dose-dependently inhibited by the phosphonamide inhibitor with KI values of 0.3 and 0.9 nM respectively. In addition, the phosphonamide peptide inhibited the hydrolysis of benzoyl (Bz)-Gly-Ala-Ala-Phe-(pAB) p-aminobenzoate and neurotensin by endopeptidase 24.15 with about a 10-fold lower potency (KI values of 5 and 7.5 nM respectively). The selectivity of this inhibitor towards several exo- and endo-peptidases belonging to the zinc-containing metallopeptidase family established that a 1 microM concentration of this inhibitor was unable to affect leucine aminopeptidase, carboxypeptidase A, angiotensin-converting enzyme and endopeptidase 24.11. The present paper therefore reports on the first hydrophilic highly potent endopeptidase 24.16 inhibitor and describes the most potent inhibitory agent directed towards endopeptidase 24.15 developed to date. These tools should allow one to assess the contribution of endopeptidase 24.16 and endopeptidase 24.15 to the physiological inactivation of neurotensin as well as other neuropeptides. PMID:1332678

Two duplicated chicken-type lysozyme genes in disc abalone Haliotis discus discus: molecular aspects in relevance to structure, genomic organization, mRNA expression and bacteriolytic function.

PubMed

Umasuthan, Navaneethaiyer; Bathige, S D N K; Kasthuri, Saranya Revathy; Wan, Qiang; Whang, Ilson; Lee, Jehee

2013-08-01

Lysozymes are crucial antibacterial proteins that are associated with catalytic cleavage of peptidoglycan and subsequent bacteriolysis. The present study describes the identification of two lysozyme genes from disc abalone Haliotis discus discus and their characterization at sequence-, genomic-, transcriptional- and functional-levels. Two cDNAs and BAC clones bearing lysozyme genes were isolated from abalone transcriptome and BAC genomic libraries, respectively and sequences were determined. Corresponding deduced amino acid sequences harbored a chicken-type lysozyme (LysC) family profile and exhibited conserved characteristics of LysC family members including active residues (Glu and Asp) and GS(S/T)DYGIFQINS motif suggested that they are LysC counterparts in disc abalone and designated as abLysC1 and abLysC2. While abLysC1 represented the homolog recently reported in Ezo abalone [1], abLysC2 shared significant identity with LysC homologs. Unlike other vertebrate LysCs, coding sequence of abLysCs were distributed within five exons interrupted by four introns. Both abLysCs revealed a broader mRNA distribution with highest levels in mantle (abLysC1) and hepatopancreas (abLysC2) suggesting their likely main role in defense and digestion, respectively. Investigation of temporal transcriptional profiles post-LPS and -pathogen challenges revealed induced-responses of abLysCs in gills and hemocytes. The in vitro muramidase activity of purified recombinant (r) abLysCs proteins was evaluated, and findings indicated that they are active in acidic pH range (3.5-6.5) and over a broad temperature range (20-60 °C) and influenced by ionic strength. When the antibacterial spectra of (r)abLysCs were examined, they displayed differential activities against both Gram positive and Gram negative strains providing evidence for their involvement in bacteriolytic function in abalone physiology. Copyright © 2013 Elsevier Ltd. All rights reserved.

Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.

PubMed

Reith, A D; Ellis, C; Lyman, S D; Anderson, D M; Williams, D E; Bernstein, A; Pawson, T

1991-09-01

Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c-kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c-kit gene products with cytoplasmic signalling proteins, using transient c-kit expression assays in COS cells. In addition to the previously identified c-kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in-frame insertion, Gly-Asn-Asn-Lys, within the extracellular domain, was detected in murine mast cell cultures and mid-gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3' kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP-associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association with PI-3' kinase and PLC gamma 1. Introduction of the point substitutions associated with W37 (Glu582----Lys) or W41 (Val831----Met) mutant alleles into c-kit expression constructs abolished (W37) or reduced (W41) the Steel factor-induced association of the Kit receptor with signalling proteins in a manner proportional to the overall severity of the corresponding W mutant phenotype. These data suggest a diversity of normal Kit signalling pathways and indicate that W mutant phenotypes result from primary defects in the Kit receptor that affect its interaction with cytoplasmic signalling proteins.

Functional and pharmacological evaluation of novel GLA variants in Fabry disease identifies six (two de novo) causative mutations and two amenable variants to the chaperone DGJ.

PubMed

Ferri, Lorenzo; Malesci, Duccio; Fioravanti, Antonella; Bagordo, Gaia; Filippini, Armando; Ficcadenti, Anna; Manna, Raffaele; Antuzzi, Daniela; Verrecchia, Elena; Donati, Ilaria; Mignani, Renzo; Cavicchi, Catia; Guerrini, Renzo; Morrone, Amelia

2018-06-01

Allelic heterogeneity is an important feature of the GLA gene for which almost 900 known genetic variants have been discovered so far. Pathogenetic GLA variants cause alpha-galactosidase A (α-Gal A) enzyme deficiency leading to the X-linked lysosomal storage disorder Fabry disease (FD). Benign GLA intronic and exonic variants (e.g. pseudodeficient p.Asp313Tyr) have also been described. Some GLA missense variants, previously deemed to be pathogenetic (e.g. p.Glu66Gln and p.Arg118Cys), they have been reclassified as benign after re-evaluation by functional and population studies. Hence, the functional role of novel GLA variants should be investigated to assess their clinical relevance. We identified six GLA variants in 4 males and 2 females who exhibited symptoms of FD: c.159C>G p.(Asn53Lys), c.400T>C p.(Tyr134His), c.680G>C (p.Arg227Pro), c.815A>T p.(Asn272Ile), c.907A>T p.(Ile303Phe) and c.1163_1165delTCC (p.Leu388del). We evaluated their impact on the α-Gal A protein by bioinformatic analysis and homology modelling, by analysis of the GLA mRNA, and by site-directed mutagenesis and in vitro expression studies. We also measured their responsiveness to the pharmacological chaperone DGJ. The six detected GLA variants cause deficient α-Gal A activity and impairment or loss of the protein wild-type structure. We found p.Asn53Lys and p.Ile303Phe variants to be susceptible to DGJ. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Charge stabilization and entropy reduction of central lysine residues in fructose-bisphosphate aldolase.

PubMed

St-Jean, Miguel; Blonski, Casimir; Sygusch, Jurgen

2009-06-02

Fructose-1,6-bisphosphate muscle aldolase is an essential glycolytic enzyme that catalyzes reversible carbon-carbon bond formation by cleaving fructose 1,6-bisphosphate to yield dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde phosphate. To elucidate the mechanistic role of conserved amino acid Asp-33, Asn-33 and Ser-33 mutants were examined by kinetic and structural analyses. The mutations significantly compromised enzymatic activity and carbanion oxidation in presence of DHAP. Detailed structural analysis demonstrated that, like native crystals, Asp-33 mutant crystals, soaked in DHAP solutions, trapped Schiff base-derived intermediates covalently attached to Lys-229. The mutant structures, however, exhibited an abridged conformational change with the helical region (34-65) flanking the active site as well as pK(a) reductions and increased side chain disorder by central lysine residues, Lys-107 and Lys-146. These changes directly affect their interaction with the C-terminal Tyr-363, consistent with the absence of active site binding by the C-terminal region in the presence of phosphate. Lys-146 pK(a) reduction and side chain disorder would further compromise charge stabilization during C-C bond cleavage and proton transfer during enamine formation. These mechanistic impediments explain diminished catalytic activity and a reduced level of carbanion oxidation and are consistent with rate-determining proton transfer observed in the Asn-33 mutant. Asp-33 reduces the entropic cost and augments the enthalpic gain during catalysis by rigidifying Lys-107 and Lys-146, stabilizing their protonated forms, and promoting a conformational change triggered by substrate or obligate product binding, which lower kinetic barriers in C-C bond cleavage and Schiff base-enamine interconversion.

Pentadecapeptide BPC 157 attenuates disturbances induced by neuroleptics: the effect on catalepsy and gastric ulcers in mice and rats.

PubMed

Jelovac, N; Sikiric, P; Rucman, R; Petek, M; Marovic, A; Perovic, D; Seiwerth, S; Mise, S; Turkovic, B; Dodig, G; Miklic, P; Buljat, G; Prkacin, I

1999-08-20

A gastric pentadecapeptide, BPC 157, with the amino acid sequence, Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val, MW 1419, known to have a variety of protective effects in gastrointestinal tract and other organs, was recently shown to particularly affect dopamine systems. For instance, it blocks the stereotypy produced acutely by amphetamine in rats, and the development of haloperidol-induced supersensitivity to amphetamine in mice. Consequently, whether pentadecapeptide BPC 157, that by itself has no cataleptogenic effect in normal animals, may attenuate the immediate effects of neuroleptics application, particularly catalepsy, was the focus of the present report. Prominent catalepsy, otherwise consistently seen in the mice treated with haloperidol (0.625, 1.25, 2.5, 5.0 and 10.0 mg/kg b.w., i.p.) and fluphenazine (0.3125, 0.625, 1.25, 2.5 and 5.0 mg/kg b.w., i.p.) after 1.5, 3, 4.5, 6 and 7.5 h following administration, was markedly attenuated when pentadecapeptide BPC 157 (10 microg or 10 ng/kg b.w., i.p.) was coadministered with the neuroleptic. The number of cataleptic mice was markedly lower throughout most of the experimental period. Moreover, on challenge with lower doses of neuroleptics, catalepsy appearance was postponed and the mice, otherwise cataleptic since the earliest period, became cataleptic later, not before 3 or 4.5 h after neuroleptic administration, especially if protected with higher pentadecapeptide dose. Besides catalepsy, coadministration of the pentadecapeptide BPC 157, given in the above mentioned doses, reduced not only catalepsy but somatosensory disorientation (for 7.5 h after administration of a neuroleptic, assessed at intervals of 1.5 h, by a simple scoring system [0-5]) in haloperidol- or fluphenazine-challenged mice as it did in mice treated with sulpiride (20, 40, 80 and 160 mg/kg b.w., i.p.) or with clozapine (25, 50 and 100 mg/kg b.w., i.p.), in which case catalepsy was absent. In other experiments, considering the gastric origin of this pentadecapeptide, the focus was shifted to the evidence that a dose of haloperidol, cataleptogenic due to dopamine receptors blockade, induces gastric ulcers in rats. Coadministration of pentadecapeptide BPC 157 (10 microg, 10 ng, 1.0 ng, 100 pg/kg b.w., i.p.) to rats completely inhibited the lesions otherwise regularly evident 24 h after haloperidol (5.0 mg/kg b.w., i.p.) in control rats (18 of 20 rats had gastric lesions). This activity accompanied the antagonism of the haloperidol catalepsy in rats (assessed at 60-min intervals from I to 5 h after haloperidol), when 10-microg- or 10-ng regimens were given (lower doses could not influence catalepsy). Together, these findings indicate that pentadecapeptide BPC 157 fully interacts with the dopamine system, both centrally and peripherally, or at least, that BPC 157 interferes with some steps involved in catalepsy and/or ulcer formation.

Impact of phenylalanine and urea applications to Tempranillo and Monastrell vineyards on grape amino acid content during two consecutive vintages.

PubMed

Garde-Cerdán, Teresa; Gutiérrez-Gamboa, Gastón; Portu, Javier; Fernández-Fernández, José Ignacio; Gil-Muñoz, Rocío

2017-12-01

Nitrogen plays a key role in the fermentation and secondary metabolites formation. The aim was to study the influence of vine nitrogen applications on grape amino acid composition. Nitrogen sources applied to Tempranillo and Monastrell grapevines were phenylalanine and urea, during two seasons. Results showed that the application of these compounds had little effect on grape amino acid composition, regardless of variety and vintage. This could be due to the fact that vineyards did not present nitrogenous requirements. Thus, variety was the determining factor in Asp, Glu, Gln, Cit, Met, Gly, Gaba, Val, Ile, and Leu while season was the factor that most affected Thr, Arg, Ala, and Lys due its implication on berry ripening. The concentration of the remaining amino acids was influenced by two or three of the factors studied. Therefore, when the vineyard has adequate nitrogen nutritional status, grape amino acid content was determined by variety and vintage. Copyright © 2017 Elsevier Ltd. All rights reserved.

Conformational relaxation and water penetration coupled to ionization of internal groups in proteins.

PubMed

Damjanović, Ana; Brooks, Bernard R; García-Moreno, Bertrand

2011-04-28

Molecular dynamics simulations were used to examine the effects of ionization of internal groups on the structures of eighteen variants of staphylococcal nuclease (SNase) with internal Lys, Asp, or Glu. In most cases the RMSD values of internal ionizable side chains were larger when the ionizable moieties were charged than when they were neutral. Calculations of solvent-accessible surface area showed that the internal ionizable side chains were buried in the protein interior when they were neutral and moved toward crevices and toward the protein-water interface when they were charged. The only exceptions are Lys-36, Lys-62, and Lys-103, which remained buried even after charging. With the exception of Lys-38, the number of internal water molecules surrounding the ionizable group increased upon charging: the average number of water oxygen atoms within the first hydration shell increased by 1.7 for Lys residues, by 5.2 for Asp residues, and by 3.2 for Glu residues. The polarity of the microenvironment of the ionizable group also increased when the groups were charged: the average number of polar atoms of any kind within the first hydration shell increased by 2.7 for Lys residues, by 4.8 for Asp residues, and by 4.0 for Glu residues. An unexpected correlation was observed between the absolute value of the shifts in pK(a) values measured experimentally, and several parameters of structural relaxation: the net difference in the polarity of the microenvironment of the charged and neutral forms of the ionizable groups, the net difference in hydration of the charged and neutral forms of the ionizable groups, and the difference in RMSD values of the charged and neutral forms of the ionizable groups. The effects of ionization of internal groups on the conformation of the backbone were noticeable but mostly small and localized to the area immediately next to the internal ionizable moiety. Some variants did exhibit local unfolding.

Crosslinking Constraints and Computational Models as Complementary Tools in Modeling the Extracellular Domain of the Glycine Receptor

PubMed Central

Liu, Zhenyu; Szarecka, Agnieszka; Yonkunas, Michael; Speranskiy, Kirill; Kurnikova, Maria; Cascio, Michael

2014-01-01

The glycine receptor (GlyR), a member of the pentameric ligand-gated ion channel superfamily, is the major inhibitory neurotransmitter-gated receptor in the spinal cord and brainstem. In these receptors, the extracellular domain binds agonists, antagonists and various other modulatory ligands that act allosterically to modulate receptor function. The structures of homologous receptors and binding proteins provide templates for modeling of the ligand-binding domain of GlyR, but limitations in sequence homology and structure resolution impact on modeling studies. The determination of distance constraints via chemical crosslinking studies coupled with mass spectrometry can provide additional structural information to aid in model refinement, however it is critical to be able to distinguish between intra- and inter-subunit constraints. In this report we model the structure of GlyBP, a structural and functional homolog of the extracellular domain of human homomeric α1 GlyR. We then show that intra- and intersubunit Lys-Lys crosslinks in trypsinized samples of purified monomeric and oligomeric protein bands from SDS-polyacrylamide gels may be identified and differentiated by MALDI-TOF MS studies of limited resolution. Thus, broadly available MS platforms are capable of providing distance constraints that may be utilized in characterizing large complexes that may be less amenable to NMR and crystallographic studies. Systematic studies of state-dependent chemical crosslinking and mass spectrometric identification of crosslinked sites has the potential to complement computational modeling efforts by providing constraints that can validate and refine allosteric models. PMID:25025226

Computational and experimental investigations into the conformations of cyclic tetra-α/β-peptides.

PubMed

Oakley, Mark T; Oheix, Emmanuel; Peacock, Anna F A; Johnston, Roy L

2013-07-11

We present a combined computational and experimental study of the energy landscapes of cyclic tetra-α/β-peptides. We have performed discrete path sampling calculations on a series of cyclic tetra-α/β-peptides to obtain the relative free energies and barriers to interconversion of their conformers. The most stable conformers of cyclo-[(β-Ala-Gly)2] contain all-trans peptide groups. The relative energies of the cis isomers and the cis-trans barriers are lower than in acyclic peptides but not as low as in the highly strained cyclic α-peptides. For cyclic tetra-α/β-peptides containing a single proline residue, of the type cyclo-[β-Ala-Xaa-β-Ala-Pro], the energy landscapes show that the most stable isomers containing cis and trans β-Ala-Pro have similar free energies and are separated by barriers of approximately 15 kcal mol(-1). We show that the underlying energy landscapes of cyclo-[β-Ala-Lys-β-Ala-Pro] and cyclo-[β-Ala-Ala-β-Ala-Pro] are similar, allowing the substitution of the flexible side chain of Lys with Ala to reduce the computational demand of our calculations. However, the steric bulk of the Val side chain in cyclo-[β-Ala-Val-β-Ala-Pro] affects the conformations of the ring, leading to significant differences between its energy landscape and that of cyclo-[β-Ala-Ala-β-Ala-Pro]. We have synthesized the cyclic peptide cyclo-[β-Ala-Lys-β-Ala-Pro], and NMR spectroscopy shows the presence of conformers that interconvert slowly on the NMR time scale at temperatures up to 80 °C. Calculated circular dichroism (CD) spectra for the proposed major isomer of cyclo-[β-Ala-Ala-β-Ala-Pro] are in good agreement with the experimental spectra of cyclo-[β-Ala-Lys-β-Ala-Pro], suggesting that the Ala cyclic tetrapeptide is a viable model for the Lys analogue.

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

James Graham, Robert Leslie; Graham, Ciaren; McClean, Stephen

A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin actionmore » at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.« less

A theoretical elucidation of glucose interaction with HSA's domains.

PubMed

Nasiri, Rasoul; Bahrami, Homayoon; Zahedi, Mansour; Moosavi-Movahedi, Ali Akbar; Sattarahmady, Naghmeh

2010-10-01

The interaction of different domains belonging to Human Serum Albumin (HSA) with open form of glucose have been investigated using molecular dynamics simulation methods. Applying docking, primary structures involving interaction of some residues with glucose have been obtained. Subsequently, equilibrium geometries at 300 K and minimum geometries have been determined for each of aforementioned structures by employing MD simulation and simulated annealing. The stability of species has been evaluated using a SAWSA v2.0 model. Ultimately, NBO analysis has been carried out to specify possible hydrogen bonding regarding the HSA interaction with glucose. Results obtained show that glucose can interact with Lys195, Lys199, and Glu153. In these interactions, each lysine forms an H-bonding with glucose. The H-bonding is obtained by stretching of N-H bond belonging to NH(3)(+) group of lysine along an oxygen atom of glucose. In addition, the above mentioned lysines are protonated, and there is an electrostatic interaction between glucose with Lys195 or Lys199. In addition, an H-bonding is formed between O atom of -COO group belonging to Glu153 and H atom of OH group belonging to glucose. Because, the N-H group of Lys195 interacts with the O atom of latter OH group, reaction of Lys195 is more desirable than that of Lys199. In fact, glucose is placed in the vicinity of Lys195 along with electrostatic interaction and H-bonding to Lys195 and Lys199 as well as H-bonding with Glu153, which subsequently reacts with Lys195. Thus, Lys195 is the primary site in reaction of glucose with HSA.

GC Glu416Asp and Thr420Lys polymorphisms contribute to gastrointestinal cancer susceptibility in a Chinese population

PubMed Central

Zhou, Liqing; Zhang, Xiaojiao; Chen, Xuechao; Liu, Li; Lu, Chao; Tang, Xiaohu; Shi, Juan; Li, Meng; Zhou, Mo; Zhang, Zhouwei; Xiao, Lingchen; Yang, Ming

2012-01-01

Vitamin D has potent anticancer properties, especially against gastrointestinal cancers. Group-specific component (GC), a key member of vitamin D pathway proteins, could bind to and transport vitamin D to target organs. As a polymorphic protein, two common coding single nucleotide polymorphisms (SNP) [Glu416Asp (rs7041) and Thr420Lys (rs4588)] were identified in its gene. These SNPs have been associated to circulating vitamin D levels and several cancer risks in different populations. However, there is no report on their role in gastrointestinal cancer development among Chinese to date. Therefore, we examined the association between these variants and risk of gastrointestinal cancers in a case-control cohort including 964 patients with four gastrointestinal cancers (hepatocellular carcinoma, esophageal cancer, gastric cancer and colorectal cancer) and 1187 controls. Odds ratios and 95% confidence intervals were estimated by logistic regression. We found that GC Thr420Lys polymorphism has significant impact on the risk of developing gastrointestinal cancers, especially colorectal cancer. Additionally, subjects who carrying GC Asp416-Lys420 haplotype, which contains the at-risk 420Lys allele, also showed significantly increased risk to develop gastrointestinal cancers. In conclusion, our study demonstrated that common genetic variants and haplo-types in GC may influence individual susceptibility to gastrointestinal cancers in Chinese population. PMID:22328951

Mannose-binding lectin (MBL) mutants are susceptible to matrix metalloproteinase proteolysis: potential role in human MBL deficiency.

PubMed

Butler, Georgina S; Sim, Derek; Tam, Eric; Devine, Dana; Overall, Christopher M

2002-05-17

Mannose-binding lectin (MBL) plays a critical role in innate immunity. Point mutations in the collagen-like domain (R32C, G34D, or G37E) of MBL cause a serum deficiency, predisposing patients to infections and diseases such as rheumatoid arthritis. We examined whether MBL mutants show enhanced susceptibility to proteolysis by matrix metalloproteinases (MMPs), which are important mediators in inflammatory tissue destruction. Human and rat MBL were resistant to proteolysis in the native state but were cleaved selectively within the collagen-like domain by multiple MMPs after heat denaturation. In contrast, rat MBL with mutations homologous to those of the human variants (R23C, G25D, or G28E) was cleaved efficiently without denaturation in the collagen-like domain by MMP-2 and MMP-9 (gelatinases A and B) and MMP-14 (membrane type-1 MMP), as well as by MMP-1 (collagenase-1), MMP-8 (neutrophil collagenase), MMP-3 (stromelysin-1), neutrophil elastase, and bacterial collagenase. Sites and order of cleavage of the rat MBL mutants for MMP-2 and MMP-9 were: Gly(45)-Lys(46) --> Gly(51)-Ser(52) --> Gly(63)-Gln(64) --> Asn(80)-Met(81) which differed from that of MMP-14, Gly(39)-Leu(40) --> Asn(80)-Met(81), revealing that the MMPs were not functionally interchangeable. These sites were homologous to those cleaved in denatured human MBL. Hence, perturbation of the collagen-like structure of MBL by natural mutations or by denaturation renders MBL susceptible to MMP cleavage. MMPs are likely to contribute to MBL deficiency in individuals with variant alleles and may also be involved in clearance of MBL and modulation of the host response in normal individuals.

Angiotensin-converting enzyme: I. New strategies for assay

PubMed Central

Ryan, James W.; Chung, Alfred; Ryan, Una S.

1980-01-01

The disposition of converting enzyme (kininase II) on the luminal surface of pulmonary endothelial cells is well established. Further, it is known that there is a net conversion of angiotensin I into angiotensin II as blood passes through the lungs. However, little is known about modulations of converting enzyme activity that may arise through, e.g., changes in the quality of inhalants, blood flow, or blood oxygenation. There are few data on the effects of lung disease. A major barrier to studies to examine for pathophysiologic modulations of converting enzyme is that of assay. The enzyme can be measured in terms of the rate of formation of angiotensin II from a known quantity of angiotensin I. However, both peptides are biologically active, and lungs contain other enzymes capable of degrading them. We have developed a series of radiolabeled, acylated tripeptides to improve our ability to examine for changes in the net converting enzyme of intact lungs. The enzyme, a dipeptidyl carboxypeptidase, is capable of removing C-terminal dipeptides from a variety of oligopeptides. We have prepared benzoyl-Gly-Gly-Gly (I), benzoyl-Pro-Phe-Arg (II), benzoyl-Gly-His-Leu (III), benzoyl-Phe-Ala-Pro (IV), and benzoyl-Phe-His-Leu (V), each containing a 3H-atom in the para position of the benzoyl moiety. Substrates I and III have been used previously in photometric assays of low sensitivity. II is the acylated C-terminal tripeptide of bradykinin, IV is an acylated tripeptide analog of BPP5a (

Characterization of endopeptidase activity of tripeptidyl peptidase-I/CLN2 protein which is deficient in classical late infantile neuronal ceroid lipofuscinosis.

PubMed

Ezaki, J; Takeda-Ezaki, M; Oda, K; Kominami, E

2000-02-24

Endopeptidase activities of the CLN2 gene product (Cln2p)/tripeptidyl peptidase I (TPP-I), purified from rat spleen, were studied using the synthetic fluorogenic substrates. We designed and constructed decapeptides, based on the known sequence cleavage specificities of bacterial pepstatin-insensitive carboxyl proteases (BPICP). MOCAc-Gly-Lys-Pro-Ile-Pro-Phe-Phe-Arg-Leu-Lys(Dnp)r-NH(2) is readily hydrolyzed by Cln2p/TPP-I (K(cat)/K(m) = 7.8 s(-1) mM(-1)). The enzyme had a maximal activity at pH 3.0 for an endopeptidase substrate, but at pH 4.5 with respect to tripeptidyl peptidase activity. Both endopeptidase and tripeptidyl peptidase activities were strongly inhibited by Ala-Ala-Phe-CH(2)Cl, but not inhibited by tyrostatin, an inhibitor of bacterial pepstatin-insensitive carboxyl proteases, pepstatin, or inhibitors of serine proteases. Fibroblasts from classical late infantile neuronal ceroid lipofuscinosis patients have less than 5% of the normal tripeptidyl peptidase activity and pepstatin-insensitive endopeptidase activity. Cln2p/TPP-I is a unique enzyme with both tripeptidyl peptidase and endopeptidase activities for certain substrate specificity. Copyright 2000 Academic Press.

New cytotoxic cyclic peptides and dianthramide from Dianthus superbus.

PubMed

Hsieh, Pei-Wen; Chang, Fang-Rong; Wu, Ching-Chung; Wu, Kuen-Yuh; Li, Chien-Ming; Chen, Su-Li; Wu, Yang-Chang

2004-09-01

Four new cyclic peptides, dianthins C-F (1-4), and a new dianthramide, 4-methoxydianthramide B (5), were isolated from the MeOH extract of the traditional Chinese medicinal plant Dianthus superbus. The sequences of cyclic peptides 1-4 were elucidated as cyclo(Gly(1)-Pro(2)-Phe(3)-Tyr(4)-Val(5)-Ile(6)-), cyclo(Gly(1)-Ser(2)-Leu(3)-Pro(4)-Pro(5)-Ile(6)-Phe(7)-), cyclo(Gly(1)-Pro(2)-Ile(3)-Ser(4)-Phe(5)-Val(6)-), and cyclo(Gly(1)-Pro(2)-Phe(3)-Val(4)-Phe(5)-) on the basis of ESI tandem mass fragmentation analysis, chemical evidence, and extensive 2D NMR methods. The conformation of compound 1 was established as an alpha-helix by CD analysis. Furthermore, compounds 3 and 5 showed cytotoxicities toward the Hep G2 cancer cell line with IC(50) values of 2.37 and 4.08, respectively.

Targeted 'next-generation' sequencing in anophthalmia and microphthalmia patients confirms SOX2, OTX2 and FOXE3 mutations.

PubMed

Jimenez, Nelson Lopez; Flannick, Jason; Yahyavi, Mani; Li, Jiang; Bardakjian, Tanya; Tonkin, Leath; Schneider, Adele; Sherr, Elliott H; Slavotinek, Anne M

2011-12-28

Anophthalmia/microphthalmia (A/M) is caused by mutations in several different transcription factors, but mutations in each causative gene are relatively rare, emphasizing the need for a testing approach that screens multiple genes simultaneously. We used next-generation sequencing to screen 15 A/M patients for mutations in 9 pathogenic genes to evaluate this technology for screening in A/M. We used a pooled sequencing design, together with custom single nucleotide polymorphism (SNP) calling software. We verified predicted sequence alterations using Sanger sequencing. We verified three mutations - c.542delC in SOX2, resulting in p.Pro181Argfs*22, p.Glu105X in OTX2 and p.Cys240X in FOXE3. We found several novel sequence alterations and SNPs that were likely to be non-pathogenic - p.Glu42Lys in CRYBA4, p.Val201Met in FOXE3 and p.Asp291Asn in VSX2. Our analysis methodology gave one false positive result comprising a mutation in PAX6 (c.1268A > T, predicting p.X423LeuextX*15) that was not verified by Sanger sequencing. We also failed to detect one 20 base pair (bp) deletion and one 3 bp duplication in SOX2. Our results demonstrated the power of next-generation sequencing with pooled sample groups for the rapid screening of candidate genes for A/M as we were correctly able to identify disease-causing mutations. However, next-generation sequencing was less useful for small, intragenic deletions and duplications. We did not find mutations in 10/15 patients and conclude that there is a need for further gene discovery in A/M.

Targeted 'Next-Generation' sequencing in anophthalmia and microphthalmia patients confirms SOX2, OTX2 and FOXE3 mutations

PubMed Central

2011-01-01

Background Anophthalmia/microphthalmia (A/M) is caused by mutations in several different transcription factors, but mutations in each causative gene are relatively rare, emphasizing the need for a testing approach that screens multiple genes simultaneously. We used next-generation sequencing to screen 15 A/M patients for mutations in 9 pathogenic genes to evaluate this technology for screening in A/M. Methods We used a pooled sequencing design, together with custom single nucleotide polymorphism (SNP) calling software. We verified predicted sequence alterations using Sanger sequencing. Results We verified three mutations - c.542delC in SOX2, resulting in p.Pro181Argfs*22, p.Glu105X in OTX2 and p.Cys240X in FOXE3. We found several novel sequence alterations and SNPs that were likely to be non-pathogenic - p.Glu42Lys in CRYBA4, p.Val201Met in FOXE3 and p.Asp291Asn in VSX2. Our analysis methodology gave one false positive result comprising a mutation in PAX6 (c.1268A > T, predicting p.X423LeuextX*15) that was not verified by Sanger sequencing. We also failed to detect one 20 base pair (bp) deletion and one 3 bp duplication in SOX2. Conclusions Our results demonstrated the power of next-generation sequencing with pooled sample groups for the rapid screening of candidate genes for A/M as we were correctly able to identify disease-causing mutations. However, next-generation sequencing was less useful for small, intragenic deletions and duplications. We did not find mutations in 10/15 patients and conclude that there is a need for further gene discovery in A/M. PMID:22204637

[Rapid detection of hot spot mutations of FGFR3 gene with PCR-high resolution melting assay].

PubMed

Li, Shan; Wang, Han; Su, Hua; Gao, Jinsong; Zhao, Xiuli

2017-08-10

To identify the causative mutations in five individuals affected with dyschondroplasia and develop an efficient procedure for detecting hot spot mutations of the FGFR3 gene. Genomic DNA was extracted from peripheral blood samples with a standard phenol/chloroform method. PCR-Sanger sequencing was used to analyze the causative mutations in the five probands. PCR-high resolution melting (HRM) was developed to detect the identified mutations. A c.1138G>A mutation in exon 8 was found in 4 probands, while a c.1620C>G mutation was found in exon 11 of proband 5 whom had a mild phenotype. All patients were successfully distinguished from healthy controls with the PCR-HRM method. The results of HRM analysis were highly consistent with that of Sanger sequencing. The Gly380Arg and Asn540Lys are hot spot mutations of the FGFR3 gene among patients with ACH/HCH. PCR-HRM analysis is more efficient for detecting hot spot mutations of the FGFR3 gene.

Substitution of the Lys Linker with the β-Ala Linker Dramatically Decreased the Renal Uptake of 99mTc-Labeled Arg-X-Asp-Conjugated and X-Ala-Asp-Conjugated α-Melanocyte Stimulating Hormone Peptides

PubMed Central

2015-01-01

The purpose of this study was to examine whether the substitution of the Lys linker with the β-Ala could reduce the renal uptake of 99mTc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-β-Ala-(Arg11)CCMSH (1) {c[Arg-Ser-Asp-dTyr-Asp]-β-Ala-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RTD-β-Ala-(Arg11)CCMSH (2), RVD-β-Ala-(Arg11)CCMSH (3), RAD-β-Ala-(Arg11)CCMSH (4), NAD-β-Ala-(Arg11)CCMSH (5), and EAD-β-Ala-(Arg11)CCMSH (6) peptides were synthesized and evaluated for their melanocortin 1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of their 99mTc-conjugates were determined in B16/F1 melanoma-bearing C57 mice. The substitution of the Lys linker with β-Ala linker dramatically reduced the renal uptake of all six 99mTc-peptides. 99mTc-4 exhibited the highest melanoma uptake (15.66 ± 6.19% ID/g) and the lowest kidney uptake (20.18 ± 3.86% ID/g) among these 99mTc-peptides at 2 h postinjection. The B16/F1 melanoma lesions could be clearly visualized by single photon emission computed tomography (SPECT)/CT using 99mTc-4 as an imaging probe. PMID:25290883

Substitution of the Lys linker with the β-Ala linker dramatically decreased the renal uptake of 99mTc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone peptides.

PubMed

Flook, Adam M; Yang, Jianquan; Miao, Yubin

2014-11-13

The purpose of this study was to examine whether the substitution of the Lys linker with the β-Ala could reduce the renal uptake of (99m)Tc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-β-Ala-(Arg(11))CCMSH (1) {c[Arg-Ser-Asp-dTyr-Asp]-β-Ala-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RTD-β-Ala-(Arg(11))CCMSH (2), RVD-β-Ala-(Arg(11))CCMSH (3), RAD-β-Ala-(Arg(11))CCMSH (4), NAD-β-Ala-(Arg(11))CCMSH (5), and EAD-β-Ala-(Arg(11))CCMSH (6) peptides were synthesized and evaluated for their melanocortin 1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of their (99m)Tc-conjugates were determined in B16/F1 melanoma-bearing C57 mice. The substitution of the Lys linker with β-Ala linker dramatically reduced the renal uptake of all six (99m)Tc-peptides. (99m)Tc-4 exhibited the highest melanoma uptake (15.66 ± 6.19% ID/g) and the lowest kidney uptake (20.18 ± 3.86% ID/g) among these (99m)Tc-peptides at 2 h postinjection. The B16/F1 melanoma lesions could be clearly visualized by single photon emission computed tomography (SPECT)/CT using (99m)Tc-4 as an imaging probe.

Antitumor and Antimicrobial Activity of Some Cyclic Tetrapeptides and Tripeptides Derived from Marine Bacteria

PubMed Central

Chakraborty, Subrata; Tai, Dar-Fu; Lin, Yi-Chun; Chiou, Tzyy-Wen

2015-01-01

Marine derived cyclo(Gly-l-Ser-l-Pro-l-Glu) was selected as a lead to evaluate antitumor-antibiotic activity. Histidine was chosen to replace the serine residue to form cyclo(Gly-l-His-l-Pro-l-Glu). Cyclic tetrapeptides (CtetPs) were then synthesized using a solution phase method, and subjected to antitumor and antibiotic assays. The benzyl group protected CtetPs derivatives, showed better activity against antibiotic-resistant Staphylococcus aureus in the range of 60–120 μM. Benzyl group protected CtetPs 3 and 4, exhibited antitumor activity against several cell lines at a concentration of 80–108 μM. However, shortening the size of the ring to the cyclic tripeptide (CtriP) scaffold, cyclo(Gly-l-Ser-l-Pro), cyclo(Ser-l-Pro-l-Glu) and their analogues showed no antibiotic or antitumor activity. This phenomenon can be explained from their backbone structures. PMID:25988520

Antitumor and antimicrobial activity of some cyclic tetrapeptides and tripeptides derived from marine bacteria.

PubMed

Chakraborty, Subrata; Tai, Dar-Fu; Lin, Yi-Chun; Chiou, Tzyy-Wen

2015-05-15

Marine derived cyclo(Gly-l-Ser-l-Pro-l-Glu) was selected as a lead to evaluate antitumor-antibiotic activity. Histidine was chosen to replace the serine residue to form cyclo(Gly-l-His-l-Pro-l-Glu). Cyclic tetrapeptides (CtetPs) were then synthesized using a solution phase method, and subjected to antitumor and antibiotic assays. The benzyl group protected CtetPs derivatives, showed better activity against antibiotic-resistant Staphylococcus aureus in the range of 60-120 μM. Benzyl group protected CtetPs 3 and 4, exhibited antitumor activity against several cell lines at a concentration of 80-108 μM. However, shortening the size of the ring to the cyclic tripeptide (CtriP) scaffold, cyclo(Gly-l-Ser-l-Pro), cyclo(Ser-l-Pro-l-Glu) and their analogues showed no antibiotic or antitumor activity. This phenomenon can be explained from their backbone structures.

Toxicity of leucine-containing peptides in Escherichia coli caused by circumvention of leucine transport regulation.

PubMed Central

Tavori, H; Kimmel, Y; Barak, Z

1981-01-01

A variety of leucine-containing peptides (LCP), Phe-Leu, Gly-Leu, Pro-Leu, Ala-Leu, Ala-Leu-Lys, Leu-Phe-Ala, Leu-Leu-Leu, and Leu-Gly-Gly, inhibited the growth of a prototrophic strain of Escherichia coli K-12 at concentrations between 0.05 and 0.28 mM. Toxicity requires normal uptake of peptides. When peptide transport was impaired by mutations, strains became resistant to the respective LCP. Inhibition of growth occurred immediately after the addition of LCP, and was relieved when 0.4 mM isoleucine was added. The presence of Gly-Leu in the medium correlated with the inhibition of growth, and the bacteria began to grow at the normal rate 70 min after Gly-Leu became undetectable. Disappearance of the peptide corresponded with the appearance of free leucine and glycine in the medium. The concentration of leucine inside the LCP-treated bacteria was higher than that in the leucine-treated and the control cultures. We suggest that entry of LCP into the cells via peptide transport systems circumvents the regulation of leucine transport, thereby causing abnormality high concentrations of leucine inside the cells. This accumulation of leucine interferes with the biosynthesis of isoleucine and inhibits the growth of the bacteria. Images PMID:7012134

Structure characterization of lipocyclopeptide antibiotics, aspartocins A, B & C, by ESI-MSMS and ESI-nozzle-skimmer-MSMS.

PubMed

Siegel, Marshall M; Kong, Fangming; Feng, Xidong; Carter, Guy T

2009-12-01

Three lipocyclopeptide antibiotics, aspartocins A (1), B (2), and C (3), were obtained from the aspartocin complex by HPLC separation methodology. Their structures were elucidated using previously published chemical degradation results coupled with spectroscopic studies including ESI-MS, ESI-Nozzle Skimmer-MSMS and NMR. All three aspartocin compounds share the same cyclic decapeptide core of cyclo [Dab2 (Asp1-FA)-Pip3-MeAsp4-Asp5-Gly6-Asp7-Gly8-Dab9-Val10-Pro11]. They differ only in the fatty acid side chain moiety (FA) corresponding to (Z)-13-methyltetradec-3-ene-carbonyl, (+,Z)-12-methyltetradec-3-ene-carbonyl and (Z)-12-methyltridec-3-ene-carbonyl for aspartocins A (1), B (2), and C (3), respectively. All of the sequence ions were observed by ESI-MSMS of the doubly charged parent ions. However, a number of the sequence ions observed were of low abundance. To fully sequence the lipocyclopeptide antibiotic structures, these low abundance sequence ions together with complementary sequence ions were confirmed by ESI-Nozzle-Skimmer-MSMS of the singly charged linear peptide parent fragment ions H-Asp5-Gly6-Asp7-Gly8-Dab9-Val10-Pro11-Dab2(1+)-Asp1-FA. Cyclization of the aspartocins was demonstrated to occur via the beta-amino group of Dab2 from ions of moderate intensity in the ESI-MSMS spectra. As the fatty acid moieties do not undergo internal fragmentations under the experimental ESI mass spectral conditions used, the 14 Da mass difference between the fatty acid moieties of aspartocins A (1) and B (2) versus aspartocin C (3) was used as an internal mass tag to differentiate fragment ions containing fatty acid moieties and those not containing the fatty acid moieties. The most numerous and abundant fragment ions observed in the tandem mass spectra are due to the cleavage of the tertiary nitrogen amide of the pipecolic acid residue-3 (16 fragment ions) and the proline residue-11 (7 fragment ions). In addition, the neutral loss of ethanimine from alpha,beta-diaminobutyric acid residue 9 was observed for the parent molecular ion and for 7 fragment ions. Copyright 2009 John Wiley & Sons, Ltd.

Propensities of peptides containing the Asn-Gly segment to form β-turn and β-hairpin structures.

PubMed

Kang, Young Kee; Yoo, In Kee

2016-09-01

The propensities of peptides that contain the Asn-Gly segment to form β-turn and β-hairpin structures were explored using the density functional methods and the implicit solvation model in CH2 Cl2 and water. The populations of preferred β-turn structures varied depending on the sequence and solvent polarity. In solution, β-hairpin structures with βI' turn motifs were most preferred for the heptapeptides containing the Asn-Gly segment regardless of the sequence of the strands. These preferences in solution are consistent with the corresponding X-ray structures. The sequence, H-bond strengths, solvent polarity, and conformational flexibility appeared to interact to determine the preferred β-hairpin structure of each heptapeptide, although the β-turn segments played a role in promoting the formation of β-hairpin structures and the β-hairpin propensity varied. In the heptapeptides containing the Asn-Gly segment, the β-hairpin formation was enthalpically favored and entropically disfavored at 25°C in water. The calculated results for β-turns and β-hairpins containing the Asn-Gly segment imply that these structural preferences may be useful for the design of bioactive macrocyclic peptides containing β-hairpin mimics and the design of binding epitopes for protein-protein and protein-nucleic acid recognitions. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 653-664, 2016. © 2016 Wiley Periodicals, Inc.

A glycine-leucine-rich peptide structurally related to the plasticins from skin secretions of the frog Leptodactylus laticeps (Leptodactylidae).

PubMed

Conlon, J Michael; Abdel-Wahab, Yasser H A; Flatt, Peter R; Leprince, Jérôme; Vaudry, Hubert; Jouenne, Thierry; Condamine, Eric

2009-05-01

A glycine-leucine-rich peptide was isolated from norepinephrine-stimulated skin secretions of the Sante Fe frog Leptodactylus laticeps (Leptodactylidae) whose primary structure (Gly-Leu-Val-Asn-Gly-Leu-Leu-Ser-Ser-Val-Leu-Gly-Gly-Gly-Gln-Gly-Gly-Gly-Gly-Leu-Leu-Gly-Gly-Ile-Leu) contains the (GXXXG)(3) motif found in the plasticins, previously identified only in phyllomedusid frogs (Hylidae). Circular dichroism studies showed that the secondary structure of the peptide, termed plasticin-L1, was markedly solvent-dependent displaying a random coil conformation in water, a beta-sheet structure in methanol, and an alpha-helical conformation in 50% trifluoroethanol-water. A synthetic replicate of the peptide did not inhibit the growth of Escherichia coli or Staphylococcus aureus or lyse human erythrocytes at concentrations up to 500 microM. At relatively high concentrations (>or=1 microM), the peptide produced a significant (P<0.05), although modest (139% of basal rate at 3 microM), increase in the rate of glucose-induced release of insulin from rat clonal BRIN-BD11 beta cells without increasing the rate of release of lactate dehydrogenase. A peptide, termed ocellatin-L2 was also identified in the skin secretion that was identical to the previously described ocellatin-L1 except for the substitution Asn(23)-->Asp. Ocellatin-L2 was devoid of antimicrobial and hemolytic activity but also showed significant activity in stimulating insulin release from BRIN-BD11 cells (181% of basal rate at 3 microM).

Medium-Chain Acyl-CoA Deficiency: Outlines from Newborn Screening, In Silico Predictions, and Molecular Studies

PubMed Central

Catarzi, Serena; Caciotti, Anna; Thusberg, Janita; Tonin, Rodolfo; Malvagia, Sabrina; la Marca, Giancarlo; Pasquini, Elisabetta; Cavicchi, Catia; Ferri, Lorenzo; Donati, Maria A.; Baronio, Federico; Guerrini, Renzo; Mooney, Sean D.; Morrone, Amelia

2013-01-01

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is a disorder of fatty acid oxidation characterized by hypoglycemic crisis under fasting or during stress conditions, leading to lethargy, seizures, brain damage, or even death. Biochemical acylcarnitines data obtained through newborn screening by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were confirmed by molecular analysis of the medium-chain acyl-CoA dehydrogenase (ACADM) gene. Out of 324.000 newborns screened, we identified 14 MCADD patients, in whom, by molecular analysis, we found a new nonsense c.823G>T (p.Gly275∗) and two new missense mutations: c.253G>C (p.Gly85Arg) and c.356T>A (p.Val119Asp). Bioinformatics predictions based on both phylogenetic conservation and functional/structural software were used to characterize the new identified variants. Our findings confirm the rising incidence of MCADD whose existence is increasingly recognized due to the efficacy of an expanded newborn screening panel by LC-MS/MS making possible early specific therapies that can prevent possible crises in at-risk infants. We noticed that the “common” p.Lys329Glu mutation only accounted for 32% of the defective alleles, while, in clinically diagnosed patients, this mutation accounted for 90% of defective alleles. Unclassified variants (UVs or VUSs) are especially critical when considering screening programs. The functional and pathogenic characterization of genetic variants presented here is required to predict their medical consequences in newborns. PMID:24294134

Toll like receptor-4 gene polymorphisms in patients with solitary cysticercus granuloma.

PubMed

Singh, Akhilesh; Garg, Ravindra Kumar; Jain, Amita; Malhotra, Hardeep Singh; Prakash, Shantanu; Verma, Rajesh; Sharma, Praveen Kumar

2015-08-15

Solitary cysticercus granuloma (SCG) of the brain is the most common type of neurocysticercosis in India. In this study, we evaluated TLR4 polymorphisms in patients with SCG. One-hundred-forty-three patients with SCG and 134 controls were enrolled. Assessment for TLR4 Asp299Gly and Thr399Ile polymorphism was done. TLR4 genotype was determined by PCR-sequencing chain termination method. The patients were followed for 6 months. Asp/Gly (P=0.024) and Thr/Ile (P=0.004) genotypes were significantly associated with the SCG. The Gly (Asp/Gly plus Gly/Gly) genotype (P=0.025) and Ile (Thr/Ile plus Ile/Ile) genotype (P=0.008) were significantly associated with the SCG. Gly/Gly and Ile/Ile genotypes were not significantly associated with SCG (P=0.767 for Gly/Gly, P=0.936 for Ile/Ile). At 6 months, TLR4 299Asp/Gly (P=0.02) and 399Ile/Thr (P=0.023) polymorphisms were significantly associated with the calcification or persistence of SCG. TLR4 polymorphisms are associated with the susceptibility to infection with SCG. Copyright © 2015 Elsevier B.V. All rights reserved.

Role of Protein Dimeric Interface in Allosteric Inhibition of N-Acetyl-Aspartate Hydrolysis by Human Aspartoacylase.

PubMed

Kots, Ekaterina D; Lushchekina, Sofya V; Varfolomeev, Sergey D; Nemukhin, Alexander V

2017-08-28

The results of molecular modeling suggest a mechanism of allosteric inhibition upon hydrolysis of N-acetyl-aspartate (NAA), one of the most abundant amino acid derivatives in brain, by human aspartoacylase (hAsp). Details of this reaction are important to suggest the practical ways to control the enzyme activity. Search for allosteric sites using the Allosite web server and SiteMap analysis allowed us to identify substrate binding pockets located at the interface between the subunits of the hAsp dimer molecule. Molecular docking of NAA to the pointed areas at the dimer interface predicted a specific site, in which the substrate molecule interacts with the Gly237, Arg233, Glu290, and Lys292 residues. Analysis of multiple long-scaled molecular dynamics trajectories (the total simulation time exceeded 1.5 μs) showed that binding of NAA to the identified allosteric site induced significant rigidity to the protein loops with the amino acid side chains forming gates to the enzyme active site. Application of the protein dynamical network algorithms showed that substantial reorganization of the signal propagation pathways of intersubunit communication in the dimer occurred upon allosteric NAA binding to the remote site. The modeling approaches provide an explanation to the observed decrease of the reaction rate of NAA hydrolysis by hAsp at high substrate concentrations.

Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli NAD+-dependent DNA ligase (LigA).

PubMed

Zhu, Hui; Shuman, Stewart

2005-04-01

NAD+-dependent DNA ligase (LigA) is essential for bacterial growth and a potential target for antimicrobial drug discovery. Here we queried the role of 14 conserved amino acids of Escherichia coli LigA by alanine scanning and thereby identified five new residues within the nucleotidyltransferase domain as being essential for LigA function in vitro and in vivo. Structure activity relationships were determined by conservative mutagenesis for the Glu-173, Arg-200, Arg-208, and Arg-277 side chains, as well as four other essential side chains that had been identified previously (Lys-115, Asp-117, Asp-285, and Lys-314). In addition, we identified Lys-290 as important for LigA activity. Reference to the structure of Enterococcus faecalis LigA allowed us to discriminate three classes of essential/important side chains that: (i) contact NAD+ directly (Lys-115, Glu-173, Lys-290, and Lys-314); (ii) comprise the interface between the NMN-binding domain (domain Ia) and the nucleotidyltransferase domain or comprise part of a nick-binding site on the surface of the nucleotidyltransferase domain (Arg-200 and Arg-208); or (iii) stabilize the active site fold of the nucleotidyltransferase domain (Arg-277). Analysis of mutational effects on the isolated ligase adenylylation and phosphodiester formation reactions revealed different functions for essential side chains at different steps of the DNA ligase pathway, consistent with the proposal that the active site is serially remodeled as the reaction proceeds.

Inhibitors of tripeptidyl peptidase II. 2. Generation of the first novel lead inhibitor of cholecystokinin-8-inactivating peptidase: a strategy for the design of peptidase inhibitors.

PubMed

Ganellin, C R; Bishop, P B; Bambal, R B; Chan, S M; Law, J K; Marabout, B; Luthra, P M; Moore, A N; Peschard, O; Bourgeat, P; Rose, C; Vargas, F; Schwartz, J C

2000-02-24

The cholecystokinin-8 (CCK-8)-inactivating peptidase is a serine peptidase which has been shown to be a membrane-bound isoform of tripeptidyl peptidase II (EC 3.4.14.10). It cleaves the neurotransmitter CCK-8 sulfate at the Met-Gly bond to give Asp-Tyr(SO(3)H)-Met-OH + Gly-Trp-Met-Asp-Phe-NH(2). In seeking a reversible inhibitor of this peptidase, the enzymatic binding subsites were characterized using a fluorimetric assay based on the hydrolysis of the artificial substrate Ala-Ala-Phe-amidomethylcoumarin. A series of di- and tripeptides having various alkyl or aryl side chains was studied to determine the accessible volume for binding and to probe the potential for hydrophobic interactions. From this initial study the tripeptides Ile-Pro-Ile-OH (K(i) = 1 microM) and Ala-Pro-Ala-OH (K(i) = 3 microM) and dipeptide amide Val-Nvl-NHBu (K(i) = 3 microM) emerged as leads. Comparison of these structures led to the synthesis of Val-Pro-NHBu (K(i) = 0.57 microM) which served for later optimization in the design of butabindide, a potent reversible competitive and selective inhibitor of the CCK-8-inactivating peptidase. The strategy for this work is explicitly described since it illustrates a possible general approach for peptidase inhibitor design.

The hypertrehalosemic neuropeptides of cicadas are structural isomers-evidence by ion mobility mass spectrometry.

PubMed

König, Simone; Marco, Heather; Gäde, Gerd

2017-11-01

It has been known for more than 20 years that the neurosecretory glands of the cicadas, the corpora cardiaca, synthesize two isobaric peptides with hypertrehalosemic activity. Both decapeptides have exactly the same amino acid sequence (pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-Gly-Asn-NH 2 ) and mass but differ in their retention time in reversed-phase liquid chromatography. A synthetic peptide with the same sequence elutes together with the second more hydrophobic peptide peak of the natural cicada extract. It is not clear what modification is causing the described observations. Therefore, in the current study, ion mobility separation in conjunction with high-resolution mass spectrometry was used to investigate this phenomenon as it was sensitive to changes in conformation. It detected different drift times in buffer gas for both the intact peptides and some of their fragment ions. Based on the ion mobility and fragment ion intensity of the corresponding ions, it is concluded that the region Pro 6 -Ser 7 -Trp 8 contains a structural feature differing from the L-amino acids present in the known peptide. Whether the conformer is the result of racemization or other biochemical processes needs to be further investigated.

The importance of four histidine residues in isocitrate lyase from Escherichia coli.

PubMed Central

Diehl, P; McFadden, B A

1994-01-01

By site-directed mutagenesis, substitutions were made for His-184 (H-184), H-197, H-266, and H-306 in Escherichia coli isocitrate lyase. Of these changes, only mutations of H-184 and H-197 appreciably reduced enzyme activity. Mutation of H-184 to Lys, Arg, or Leu resulted in an inactive isocitrate lyase, and mutation of H-184 to Gln resulted in an enzyme with 0.28% activity. Nondenaturing polyacrylamide gel electrophoresis demonstrated that isocitrate lyase containing the Lys, Arg, Gln, and Leu substitutions at H-184 was assembled poorly into the tetrameric subunit complex. Mutation of H-197 to Lys, Arg, Leu, and Gln resulted in an assembled enzyme with less than 0.25% wild-type activity. Five substitutions for H-266 (Asp, Glu, Val, Ser, and Lys), four substitutions for H-306 (Asp, Glu, Val, and Ser), and a variant in which both H-266 and H-306 were substituted for showed little or no effect on enzyme activity. All the H-197, H-266, and H-306 mutants supported the growth of isocitrate lyase-deficient E. coli JE10 on acetate as the sole carbon source; however, the H-184 mutants did not. Images PMID:8300547

The efficacy of a new 6-phytase obtained from Buttiauxella spp. expressed in Trichoderma reesei on digestibility of amino acids, energy, and nutrients in pigs fed a diet based on corn, soybean meal, wheat middlings, and corn distillers' dried grains with solubles.

PubMed

Adedokun, S A; Owusu-Asiedu, A; Ragland, D; Plumstead, P; Adeola, O

2015-01-01

Sixteen cannulated pigs were used to evaluate the effect of a new 6-phytase derived from Buttiauxella spp. and expressed in Trichoderma reesei on apparent ileal digestibility (AID) of AA and apparent total tract digestibility (ATTD) of DM, N, Ca, P, Na, Mg, K, Cl, and energy. Pigs were fed 4 diets for 2 periods in a crossover design. Within each period, there were 4 blocks of 4 pigs per block with each diet represented within each block. The average initial BW in periods 1 and 2 were 22 and 30 kg, respectively. Each period lasted 9 d with fecal collection on d 5 and 6 and a 12-h ileal digesta collection on d 7, 8, and 9. Pigs received a daily feed allowance of approximately 4.5% of their BW. The experimental diets were based on corn, soybean meal, wheat middlings, and corn distillers dried grain with solubles. Phytase was added at 0; 500; 1,000; or 2,000 phytase units/kg of diet to a basal diet that contained 205, 15, 5.4, and 10 g of CP, Lys, total P (1.6 g of nonphytate P), and Ca/kg diet, respectively. The addition of phytase improved (P < 0.05) AID of DM, N, Ca, and P. Increasing phytase supplementation linearly and quadratically increased (P < 0.05) AID of P and Ca, respectively, with AID of Ca showing a tendency for a linear increase (P = 0.053). Phytase supplementation of the basal diet improved (P < 0.05) AID of P from 46 to 62%. Phytase supplementation increased (P < 0.05) ATTD of DM, N, Ca, P, Mg, K, and energy. Contrasts showed that phytase supplementation of the basal diet increased (P < 0.05) AID for 8 indispensable AA (Arg, His, Ile, Leu, Lys, Phe, Thr, and Val), 6 dispensable AA (Ala, Asp, Cys, Glu, Ser, and Tyr), as well as for total AA. Furthermore, phytase supplementation to the basal diet showed a tendency (P < 0.10) to increase ileal digestibility of Gly. Ileal digestibility of Met, Trp, and Pro were not affected by phytase supplementation. Increasing the level of phytase supplementation resulted in linear increases (P < 0.05) in AID of 6 indispensable AA (Arg, Ile, Leu, Lys, Phe, and Val) and 1 dispensable AA (Asp) with 4 AA (His, Cys, Glu, and Tyr) showing a tendency for linear increase (P < 0.10) in AID of AA. The results from this study showed that in addition to increasing P and Ca utilization, the new Buttiauxella 6-phytase expressed in Trichoderma reesei enhanced ileal digestibility of N and several AA in growing pigs in a dose-dependent manner.

Identification and Expression Profile Analysis of Antimicrobial Peptide/Protein in Asian Corn Borer, Ostrinia furnacalis (Guenée)

PubMed Central

Zhang, Mingming; Zhou, Fan; Chu, Yuan; Zhao, Zhangwu; An, Chunju

2013-01-01

Antimicrobial peptides/proteins (AMPs) are a group of immune proteins that exhibit strong antibiotic properties against numerous infectious bacterial strains. They are evolutionarily conserved and present in every kingdom and phylum, ranging from prokaryotes to humans. We analyzed the transciptome from the larvae of Asian corn borer, Ostrinia furnacalis (Guenée), and identified several putative AMP transcripts, OfgLys5, OfgLys6, OfgLys10, OfgAtt, and OfgIID. OfgLys5, OfgLys6, and OfgLys10 are all highly homologous with c-type lysozymes, and OfgAtt shows significant identities with Lepidoptera attacin. The amino acid sequence of OfgLys5 and OfgLys6 possessed all conserved features critical for fundamental structure and function of c-type lysozyme, including the two catalytic sites, Glu32 and Asp50. OfgAtt is a typical glycine-rich protein. The antimicrobial activity of O. furnacalis hemolymph increased significantly after injection with Escherichia coli, Micrococcus luteus, or Beauveria bassiana. OfgAtt, IDD, and Lys6 are expressed at low level prior to the challenge, but strongly induced against Gram-positive and negative bacteria, and fungi. Under the same inducement conditions, the transcripts of these three genes elevated most when fifth instar larvae were injected. Therefore, O. furnacalis larvae are induced to produce antimicrobial materials in the hemolymph after the infection, and increase of lysozyme and attacin may contribute to the antimicrobial activity. PMID:24155672

Evolution of TEM-type enzymes: biochemical and genetic characterization of two new complex mutant TEM enzymes, TEM-151 and TEM-152, from a single patient.

PubMed

Robin, Frédéric; Delmas, Julien; Schweitzer, Cédric; Tournilhac, Olivier; Lesens, Olivier; Chanal, Catherine; Bonnet, Richard

2007-04-01

Two clinical isolates of Escherichia coli, CF1179 and CF1295, were isolated from a patient hospitalized in the hematology unit of the University Hospital of Clermont-Ferrand, Clermont-Ferrand, France. They were resistant to penicillin-clavulanate combinations and to ceftazidime. The double-disk synergy test was positive only for isolate CF1179. Molecular comparison of the isolates showed that they were clonally related. E. coli recombinant strains exhibiting the resistance phenotype of the clinical strains were obtained by cloning. The clones corresponding to strains CF1179 and CF1295 produced TEM-type beta-lactamases with pI values of 5.7 and 5.3, respectively. Sequencing analysis revealed two novel blaTEM genes encoding closely related complex mutant TEM enzymes, designated TEM-151 (pI 5.3) and TEM-152 (pI 5.7). These two genes also harbored a new promoter region which presented a 9-bp deletion. The two novel beta-lactamases differed from the parental enzyme, TEM-1, by the substitution Arg164His, previously observed in extended-spectrum beta-lactamases (ESBLs), and by the substitutions Met69Val and Asn276Asp, previously observed in the inhibitor-resistant penicillinase TEM-36/IRT-7. They differed by two amino acid substitutions: TEM-152 harbored a Glu240Lys ESBL-type substitution and TEM-151 had an Ala284Gly substitution. Functional analysis of TEM-151 and TEM-152 showed that both enzymes had hydrolytic activity against ceftazidime (kcat, 5 and 16 s-1, respectively). TEM-152 was more resistant than TEM-151 to the inhibitor clavulanic acid (50% inhibitory concentrations, 1 versus 0.17 microM). These results confirm the evolution of TEM-type enzymes toward complex enzymes harboring the two kinds of substitutions which confer an extended spectrum of action against beta-lactam antibiotics and resistance to inhibitors.

Size-exclusion chromatography of tea tannins and intercepting potentials of peptides for the inhibition of trypsin-caseinolytic activity by tea tannins.

PubMed

Kasai, Naoya; Nakatsubo, Genki

2006-07-12

Molecular-weight distribution and characterization of tea tannin were investigated by high-performance liquid chromatography and the equivalent preparative exclusion gel chromatography using Sephadex G-25. The characteristics of the fractions were studied regarding the amounts of terminal catechin, sugar, and gallic acid, the color reaction of the Folin-Chiocalteu reagent, the UV absorbance, and the inhibition activity for the trypsin-caseinolytic activity per weight. Furthermore, we investigated the intercepting activities of the inhibition by the amino acids, peptides, their analogues, poly(ethylene glycol)s (PEGs), and histatin 5 using the inhibition of trypsin-caseinolytic activity by tea. Arg, Lys, and their peptides had strong intercepting activities for the inhibition, but only a weak activity was detected in the Pro peptides or gelatin-like peptides of (Pro-Pro-Gly)(n) (n = 5 or 10). The guanidyl group of Arg and the amino methylene group of Lys were important for the intercepting activity, but the activity was weakly dependent upon the peptide bond formation. The intercepting activity of the peptides or PEG exponentially increased with the number of polymerizations. Histatin 5 did not have a remarkably strong intercepting activity considering the peptide length. The activity of the synthetic histatin 5 in which all of the Lys and Arg were substituted by Ala was at the same level as histatin 5.

Localization and characterization of the calsequestrin-binding domain of triadin 1. Evidence for a charged beta-strand in mediating the protein-protein interaction.

PubMed

Kobayashi, Y M; Alseikhan, B A; Jones, L R

2000-06-09

Triadin is an integral membrane protein of the junctional sarcoplasmic reticulum that binds to the high capacity Ca(2+)-binding protein calsequestrin and anchors it to the ryanodine receptor. The lumenal domain of triadin contains multiple repeats of alternating lysine and glutamic acid residues, which have been defined as KEKE motifs and have been proposed to promote protein associations. Here we identified the specific residues of triadin responsible for binding to calsequestrin by mutational analysis of triadin 1, the major cardiac isoform. A series of deletional fusion proteins of triadin 1 was generated, and by using metabolically labeled calsequestrin in filter-overlay assays, the calsequestrin-binding domain of triadin 1 was localized to a single KEKE motif comprised of 25 amino acids. Alanine mutagenesis within this motif demonstrated that the critical amino acids of triadin binding to calsequestrin are the even-numbered residues Lys(210), Lys(212), Glu(214), Lys(216), Gly(218), Gln(220), Lys(222), and Lys(224). Replacement of the odd-numbered residues within this motif by alanine had no effect on calsequestrin binding to triadin. The results suggest a model in which residues 210-224 of triadin form a beta-strand, with the even-numbered residues in the strand interacting with charged residues of calsequestrin, stabilizing a "polar zipper" that links the two proteins together. This small, highly charged beta-strand of triadin may tether calsequestrin to the junctional face membrane, allowing calsequestrin to sequester Ca(2+) in the vicinity of the ryanodine receptor during Ca(2+) uptake and Ca(2+) release.

Characterization of molecular interactions between Zika virus protease and peptides derived from the C-terminus of NS2B.

PubMed

Li, Yan; Loh, Ying Ru; Hung, Alvin W; Kang, CongBao

2018-06-21

Zika virus (ZIKV) protease is a two-component complex in which NS3 contains the catalytic triad and NS2B cofactor region is important for protease folding and activity. A protease construct-eZiPro without the transmembrane domains of NS2B was designed. Structural study on eZiPro reveals that the Thr-Gly-Lys-Arg (TGKR) sequence at the C-terminus of NS2B binds to the active site after cleavage. The bZiPro construct only contains NS2B cofactor region and the N-terminus of NS3 without any artificial linker or protease cleavage site, giving rise to an empty pocket accessible to substrate and inhibitor binding. Herein, we demonstrate that the TGKR sequence of NS2B in eZiPro is dynamic. Peptides from NS2B with various lengths exhibit different binding affinities to bZiPro. TGKR binding to the active site in eZiPro does not affect protease binding to small-molecule compounds. Our results suggest that eZiPro will also be useful for evaluating small-molecule protease inhibitors. Copyright © 2018 Elsevier Inc. All rights reserved.

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.

An intrinsically fluorescent dendrimer as a nanoprobe of cell transport.

PubMed

Al-Jamal, Khuloud T; Ruenraroengsak, Pakatip; Hartell, Nicholas; Florence, Alexander T

2006-07-01

Dendrimers, spherical or quasi-spherical synthetic polymers in the nano-size range, have found useful applications as prospective carriers in drug and gene delivery. The investigation of dendrimer uptake by cells has been previously achieved by the incorporation of a fluorescent dye to the dendrimer either by chemical conjugation or by physical interaction. Here we describe the synthesis of two intrinsically fluorescent lysine based cationic dendrimers which lack a fluorophore, but which has sufficient fluorescence intensity to be detected at low concentrations. The nomenclature used to describe our compounds results in, for example the 6th generation dendrimer being notated as Gly-Lys(63) (NH2)(64); Gly denotes that the compound has a glycine in the core coupled to 63 lysine branching units (Lys(63)) and that the surface has 64 free amino groups (NH2)(64). The use of these dendrimers in probing transport avoids the need for fluorescent tagging with its attendant problems. The uptake of Gly-Lys(63) (NH2)(64) into Caco-2 cells was followed using confocal microscopy. Being cationic, it first adsorbs to the cell surface, enters the cytoplasm and reaches the nucleus within 35-45 min. Estimates of the diffusion coefficient of the dendrimer within the cell cytoplasm leads to a value of 6.27 ( +/- 0.49) x 10(-11) cm(2) s(-1), which is up to 1000 times lower than the diffusion coefficient of the dendrimer in water. Intrinsically fluorescent dendrimers of different size and charge are useful probes of transport in cells.

Amyloid-β peptide structure in aqueous solution varies with fragment size

NASA Astrophysics Data System (ADS)

Wise-Scira, Olivia; Xu, Liang; Kitahara, Taizo; Perry, George; Coskuner, Orkid

2011-11-01

Various fragment sizes of the amyloid-β (Aβ) peptide have been utilized to mimic the properties of the full-length Aβ peptide in solution. Among these smaller fragments, Aβ16 and Aβ28 have been investigated extensively. In this work, we report the structural and thermodynamic properties of the Aβ16, Aβ28, and Aβ42 peptides in an aqueous solution environment. We performed replica exchange molecular dynamics simulations along with thermodynamic calculations for investigating the conformational free energies, secondary and tertiary structures of the Aβ16, Aβ28, and Aβ42 peptides. The results show that the thermodynamic properties vary from each other for these peptides. Furthermore, the secondary structures in the Asp1-Lys16 and Asp1-Lys28 regions of Aβ42 cannot be completely captured by the Aβ16 and Aβ28 fragments. For example, the β-sheet structures in the N-terminal region of Aβ16 and Aβ28 are either not present or the abundance is significantly decreased in Aβ42. The α-helix and β-sheet abundances in Aβ28 and Aβ42 show trends - to some extent - with the potential of mean forces but no such trend could be obtained for Aβ16. Interestingly, Arg5 forms salt bridges with large abundances in all three peptides. The formation of a salt bridge between Asp23-Lys28 is more preferred over the Glu22-Lys28 salt bridge in Aβ28 but this trend is vice versa for Aβ42. This study shows that the Asp1-Lys16 and Asp1-Lys28 regions of the full length Aβ42 peptide cannot be completely mimicked by studying the Aβ16 and Aβ28 peptides.

Molecular cloning of the pheromone biosynthesis-activating neuropeptide in Helicoverpa zea.

PubMed Central

Davis, M T; Vakharia, V N; Henry, J; Kempe, T G; Raina, A K

1992-01-01

Pheromone biosynthesis-activating neuropeptide (PBAN) regulates sex pheromone biosynthesis in female Helicoverpa (Heliothis) zea. Two oligonucleotide probes representing two overlapping amino acid regions of PBAN were used to screen 2.5 x 10(5) recombinant plaques, and a positive recombinant clone was isolated. Sequence analysis of the isolated clone showed that the PBAN gene is interrupted after the codon encoding amino acid 14 by a 0.63-kilobase (kb) intron. Preceding the PBAN amino acid sequence is a 10-amino acid sequence containing a pentapeptide Phe-Thr-Pro-Arg-Leu, which is followed by a Gly-Arg-Arg processing site. Immediately after the PBAN amino acid sequence is a Gly-Arg processing site and a short stretch of 10 amino acids. This 10-amino acid sequence contains a repeat of the PBAN C-terminal pentapeptide Phe-Ser-Pro-Arg-Leu and is terminated by another Gly-Arg processing site. It is suggested that the PBAN gene in H. zea might carry, besides PBAN, a 7- and an 8-residue amidated peptide, which share with PBAN the core C-terminal pentapeptide Phe-(Ser or Thr)-Pro-Arg-Leu-NH2. The C-terminal pentapeptide sequence of PBAN represents the minimum sequence required for pheromonotropic activity in H. zea and also bears a high degree of homology to the pyrokinin family of insect peptides with myotropic activity. It is possible that the putative heptapeptide and octapeptide might be new members of the pyrokinin family, with pheromonotropic and/or myotropic activities. Thus, the PBAN gene products, besides affecting sexual behavior, might have broad influence on many biological processes in H. zea. Images PMID:1729680

Molecular Analysis of Glucose-6-Phosphate Dehydrogenase Gene Mutations in Bangladeshi Individuals.

PubMed

Sarker, Suprovath Kumar; Islam, Md Tarikul; Eckhoff, Grace; Hossain, Mohammad Amir; Qadri, Syeda Kashfi; Muraduzzaman, A K M; Bhuyan, Golam Sarower; Shahidullah, Mohammod; Mannan, Mohammad Abdul; Tahura, Sarabon; Hussain, Manzoor; Akhter, Shahida; Nahar, Nazmun; Shirin, Tahmina; Qadri, Firdausi; Mannoor, Kaiissar

2016-01-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked human enzyme defect of red blood cells (RBCs). Individuals with this gene defect appear normal until exposed to oxidative stress which induces hemolysis. Consumption of certain foods such as fava beans, legumes; infection with bacteria or virus; and use of certain drugs such as primaquine, sulfa drugs etc. may result in lysis of RBCs in G6PD deficient individuals. The genetic defect that causes G6PD deficiency has been identified mostly as single base missense mutations. One hundred and sixty G6PD gene mutations, which lead to amino acid substitutions, have been described worldwide. The purpose of this study was to detect G6PD gene mutations in hospital-based settings in the local population of Dhaka city, Bangladesh. Qualitative fluorescent spot test and quantitative enzyme activity measurement using RANDOX G6PDH kit were performed for analysis of blood specimens and detection of G6PD-deficient participants. For G6PD-deficient samples, PCR was done with six sets of primers specific for G6PD gene. Automated Sanger sequencing of the PCR products was performed to identify the mutations in the gene. Based on fluorescence spot test and quantitative enzyme assay followed by G6PD gene sequencing, 12 specimens (11 males and one female) among 121 clinically suspected patient-specimens were found to be deficient, suggesting a frequency of 9.9% G6PD deficiency. Sequencing of the G6PD-deficient samples revealed c.C131G substitution (exon-3: Ala44Gly) in six samples, c.G487A substitution (exon-6:Gly163Ser) in five samples and c.G949A substitution (exon-9: Glu317Lys) of coding sequence in one sample. These mutations either affect NADP binding or disrupt protein structure. From the study it appears that Ala44Gly and Gly163Ser are the most common G6PD mutations in Dhaka, Bangladesh. This is the first study of G6PD mutations in Bangladesh.

Molecular Analysis of Glucose-6-Phosphate Dehydrogenase Gene Mutations in Bangladeshi Individuals

PubMed Central

Sarker, Suprovath Kumar; Hossain, Mohammad Amir; Qadri, Syeda Kashfi; Muraduzzaman, A. K. M.; Bhuyan, Golam Sarower; Shahidullah, Mohammod; Mannan, Mohammad Abdul; Tahura, Sarabon; Hussain, Manzoor; Akhter, Shahida; Nahar, Nazmun; Shirin, Tahmina; Qadri, Firdausi; Mannoor, Kaiissar

2016-01-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked human enzyme defect of red blood cells (RBCs). Individuals with this gene defect appear normal until exposed to oxidative stress which induces hemolysis. Consumption of certain foods such as fava beans, legumes; infection with bacteria or virus; and use of certain drugs such as primaquine, sulfa drugs etc. may result in lysis of RBCs in G6PD deficient individuals. The genetic defect that causes G6PD deficiency has been identified mostly as single base missense mutations. One hundred and sixty G6PD gene mutations, which lead to amino acid substitutions, have been described worldwide. The purpose of this study was to detect G6PD gene mutations in hospital-based settings in the local population of Dhaka city, Bangladesh. Qualitative fluorescent spot test and quantitative enzyme activity measurement using RANDOX G6PDH kit were performed for analysis of blood specimens and detection of G6PD-deficient participants. For G6PD-deficient samples, PCR was done with six sets of primers specific for G6PD gene. Automated Sanger sequencing of the PCR products was performed to identify the mutations in the gene. Based on fluorescence spot test and quantitative enzyme assay followed by G6PD gene sequencing, 12 specimens (11 males and one female) among 121 clinically suspected patient-specimens were found to be deficient, suggesting a frequency of 9.9% G6PD deficiency. Sequencing of the G6PD-deficient samples revealed c.C131G substitution (exon-3: Ala44Gly) in six samples, c.G487A substitution (exon-6:Gly163Ser) in five samples and c.G949A substitution (exon-9: Glu317Lys) of coding sequence in one sample. These mutations either affect NADP binding or disrupt protein structure. From the study it appears that Ala44Gly and Gly163Ser are the most common G6PD mutations in Dhaka, Bangladesh. This is the first study of G6PD mutations in Bangladesh. PMID:27880809

Pathogenetic role of Arg-Gly-Asp-recognizing integrins in acute renal failure. off.

PubMed Central

Goligorsky, M S; DiBona, G F

1993-01-01

Reorientation of the alpha 3 subunit of integrins from predominantly basal to the apical cell surface of cultured renal tubular epithelial cells subjected to oxidant stress has previously been demonstrated. The present study was designed to assess functional competence of ectopically expressed apical integrins. Cell-cell adhesion assay revealed enhanced cytoatractant properties of stressed cells. Stressed epithelial cells exhibited specific recognition and binding of laminin-coated latex beads. These processes were inhibited with the peptide Gly-Arg-Gly-Asp-Asn-Pro (GRGDNP) suggesting a role of RGD-recognizing integrins in augmented adhesion to stressed cells. Given that such enhanced adhesion in in vivo acute renal failure may govern tubular obstruction by desquamated epithelium, a physiological marker of patency of tubular lumen, proximal tubular pressure, was monitored in rats subjected to 60 min of renal ischemia followed by reperfusion. Proximal tubular pressure increased 2-fold after 2 hr of reperfusion in animals that had undergone 60 min of ischemia. Infusion of GRGDNP into the renal artery during reperfusion period virtually abolished an increase in proximal tubular pressure observed in ischemic acute renal failure. These in vitro and in vivo findings are consistent with the hypothesis that RGD-recognizing integrins play an important role in the pathogenesis of tubular obstruction in ischemic acute renal failure. Images Fig. 2 Fig. 3 PMID:8516318

Characterization of Novel Missense Variants of SERPINA1 Gene Causing Alpha-1 Antitrypsin Deficiency.

PubMed

Matamala, Nerea; Lara, Beatriz; Gomez-Mariano, Gema; Martínez, Selene; Retana, Diana; Fernandez, Taiomara; Silvestre, Ramona Angeles; Belmonte, Irene; Rodriguez-Frias, Francisco; Vilar, Marçal; Sáez, Raquel; Iturbe, Igor; Castillo, Silvia; Molina-Molina, María; Texido, Anna; Tirado-Conde, Gema; Lopez-Campos, Jose Luis; Posada, Manuel; Blanco, Ignacio; Janciauskiene, Sabina; Martinez-Delgado, Beatriz

2018-06-01

The SERPINA1 gene is highly polymorphic, with more than 100 variants described in databases. SERPINA1 encodes the alpha-1 antitrypsin (AAT) protein, and severe deficiency of AAT is a major contributor to pulmonary emphysema and liver diseases. In Spanish patients with AAT deficiency, we identified seven new variants of the SERPINA1 gene involving amino acid substitutions in different exons: PiSDonosti (S+Ser14Phe), PiTijarafe (Ile50Asn), PiSevilla (Ala58Asp), PiCadiz (Glu151Lys), PiTarragona (Phe227Cys), PiPuerto Real (Thr249Ala), and PiValencia (Lys328Glu). We examined the characteristics of these variants and the putative association with the disease. Mutant proteins were overexpressed in HEK293T cells, and AAT expression, polymerization, degradation, and secretion, as well as antielastase activity, were analyzed by periodic acid-Schiff staining, Western blotting, pulse-chase, and elastase inhibition assays. When overexpressed, S+S14F, I50N, A58D, F227C, and T249A variants formed intracellular polymers and did not secrete AAT protein. Both the E151K and K328E variants secreted AAT protein and did not form polymers, although K328E showed intracellular retention and reduced antielastase activity. We conclude that deficient variants may be more frequent than previously thought and that their discovery is possible only by the complete sequencing of the gene and subsequent functional characterization. Better knowledge of SERPINA1 variants would improve diagnosis and management of individuals with AAT deficiency.

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

Prediction of beta-turns at over 80% accuracy based on an ensemble of predicted secondary structures and multiple alignments.

PubMed

Zheng, Ce; Kurgan, Lukasz

2008-10-10

beta-turn is a secondary protein structure type that plays significant role in protein folding, stability, and molecular recognition. To date, several methods for prediction of beta-turns from protein sequences were developed, but they are characterized by relatively poor prediction quality. The novelty of the proposed sequence-based beta-turn predictor stems from the usage of a window based information extracted from four predicted three-state secondary structures, which together with a selected set of position specific scoring matrix (PSSM) values serve as an input to the support vector machine (SVM) predictor. We show that (1) all four predicted secondary structures are useful; (2) the most useful information extracted from the predicted secondary structure includes the structure of the predicted residue, secondary structure content in a window around the predicted residue, and features that indicate whether the predicted residue is inside a secondary structure segment; (3) the PSSM values of Asn, Asp, Gly, Ile, Leu, Met, Pro, and Val were among the top ranked features, which corroborates with recent studies. The Asn, Asp, Gly, and Pro indicate potential beta-turns, while the remaining four amino acids are useful to predict non-beta-turns. Empirical evaluation using three nonredundant datasets shows favorable Q total, Q predicted and MCC values when compared with over a dozen of modern competing methods. Our method is the first to break the 80% Q total barrier and achieves Q total = 80.9%, MCC = 0.47, and Q predicted higher by over 6% when compared with the second best method. We use feature selection to reduce the dimensionality of the feature vector used as the input for the proposed prediction method. The applied feature set is smaller by 86, 62 and 37% when compared with the second and two third-best (with respect to MCC) competing methods, respectively. Experiments show that the proposed method constitutes an improvement over the competing prediction methods. The proposed prediction model can better discriminate between beta-turns and non-beta-turns due to obtaining lower numbers of false positive predictions. The prediction model and datasets are freely available at http://biomine.ece.ualberta.ca/BTNpred/BTNpred.html.

Prediction of beta-turns at over 80% accuracy based on an ensemble of predicted secondary structures and multiple alignments

PubMed Central

Zheng, Ce; Kurgan, Lukasz

2008-01-01

Background β-turn is a secondary protein structure type that plays significant role in protein folding, stability, and molecular recognition. To date, several methods for prediction of β-turns from protein sequences were developed, but they are characterized by relatively poor prediction quality. The novelty of the proposed sequence-based β-turn predictor stems from the usage of a window based information extracted from four predicted three-state secondary structures, which together with a selected set of position specific scoring matrix (PSSM) values serve as an input to the support vector machine (SVM) predictor. Results We show that (1) all four predicted secondary structures are useful; (2) the most useful information extracted from the predicted secondary structure includes the structure of the predicted residue, secondary structure content in a window around the predicted residue, and features that indicate whether the predicted residue is inside a secondary structure segment; (3) the PSSM values of Asn, Asp, Gly, Ile, Leu, Met, Pro, and Val were among the top ranked features, which corroborates with recent studies. The Asn, Asp, Gly, and Pro indicate potential β-turns, while the remaining four amino acids are useful to predict non-β-turns. Empirical evaluation using three nonredundant datasets shows favorable Qtotal, Qpredicted and MCC values when compared with over a dozen of modern competing methods. Our method is the first to break the 80% Qtotal barrier and achieves Qtotal = 80.9%, MCC = 0.47, and Qpredicted higher by over 6% when compared with the second best method. We use feature selection to reduce the dimensionality of the feature vector used as the input for the proposed prediction method. The applied feature set is smaller by 86, 62 and 37% when compared with the second and two third-best (with respect to MCC) competing methods, respectively. Conclusion Experiments show that the proposed method constitutes an improvement over the competing prediction methods. The proposed prediction model can better discriminate between β-turns and non-β-turns due to obtaining lower numbers of false positive predictions. The prediction model and datasets are freely available at . PMID:18847492

May TLR4 Asp299Gly and IL17 His161Arg polymorphism be associated with progression of primary measles infection to subacute sclerosing panencephalitis?

PubMed

Karakas-Celik, Sevim; Piskin, Ibrahim Etem; Keni, Mehmet Fatih; Calık, Mustafa; Iscan, Akın; Dursun, Ahmet

2014-09-01

SSPE is a progressive neurological disorder of children. Only some of the children who are infected with measles virus develop SSPE, which supports individual variation. TLR-2 and TLR-4 play an important role in innate immunity by recognizing envelope proteins of MV. Another important cytokine that plays an important role in orchestrating innate immune function is IL-17. The purpose of our study is to elucidate whether the TLR2, TLR4, IL17F and IL17A gene polymorphisms are susceptibility genes for the development of SSPE. Using the PCR-RFLP methods, the single nucleotide polymorphisms of TLR2 (Arg753Gln, Arg677Trp, -194 to -174 del), TLR4 (Asp299Gly and Thr399Ile) IL17F (His161Arg, Glu126Gly) and IL17A were studied in 54 patients with SSPE and 81 healthy controls. For Asp299Gly polymorphism of the TLR4 gene we found that there were no control individuals who were homozygous carriers of the Gly/Gly genotype, and the risk for SSPE increased at approximately 4.7 fold for the heterozygous carriers of the Asp/Gly genotype (OR 4.727, 95%-CI 1.192-18.742; P=0.01), when compared to healthy controls. Also our findings demonstrate that homozygosity for the Arg161 variant of the IL17F His161Arg polymorphism is inversely associated with development of SSPE (OR 0.114 95%-CI 0.026-0.494; P<0.001). In conclusion, it is suggested that variation in susceptibility to SSPE disease may be in part due to variations in TLR4 and IL17 function resulting from polymorphisms of TLR4 Asp299Gly and IL17F His161Arg. Copyright © 2014 Elsevier B.V. All rights reserved.

Structural and Functional Characterization of a New Double Variant Haemoglobin (HbG-Philadelphia/Duarte α(2)β(2)).

PubMed

Fais, Antonella; Casu, Mariano; Ruggerone, Paolo; Ceccarelli, Matteo; Porcu, Simona; Era, Benedetta; Anedda, Roberto; Sollaino, Maria Carla; Galanello, Renzo; Corda, Marcella

2011-01-01

WE REPORT THE FIRST CASE OF COSEGREGATION OF TWO HAEMOGLOBINS (HBS): HbG-Philadelphia [α68(E17)Asn → Lys] and HbDuarte [β62(E6)Ala → Pro]. The proband is a young patient heterozygous also for β°-thalassaemia. We detected exclusively two haemoglobin variants: HbDuarte and HbG-Philadelphia/Duarte. Functional study of the new double variant HbG-Philadelphia/Duarte exhibited an increase in oxygen affinity, with a slight decrease of cooperativity and Bohr effect. This functional behaviour is attributed to β62Ala → Pro instead of α68Asn → Lys substitution. Indeed, HbG-Philadelphia isolated in our laboratory from blood cells donor carrier for this variant is not affected by any functional modification, whereas purified Hb Duarte showed functional properties very similar to the double variant. NMR and MD simulation studies confirmed that the presence of Pro instead of Ala at the β62 position produces displacement of the E helix and modifications of the tertiary structure. The substitution α68(E17)Asn → Lys does not cause significant structural and dynamical modifications of the protein. A possible structure-based rational of substitution effects is suggested.

Processing of mammalian preprogastrin-releasing peptide.

PubMed

Reeve, J R; Cuttitta, F; Vigna, S R; Shively, J E; Walsh, J H

1988-01-01

The processing of preprogastrin-releasing peptide in mammalian tissues and in cultured cells takes place at discrete sites (Figure 6). Signal peptidase cleaves away the signal peptide from the amino terminus of gastrin-releasing peptide. An exopeptidase activity may remove dipeptides from the amino terminus. The amidation site (not shown in Fig. 6; see Fig. 2) has the same general sequence (Gly-Lys-Lys) seen for other amidated peptides. Cleavage after single basic residues yields gene-related products from Form I or II preproGRP. A unique non-basic cleavage yields a gene-related product from Form III preproGRP. The processing that occurs to form GRP, GRP, and GRP gene-related peptides is shown in Figure 7. ProGRP is cleaved by a series of enzymes to form GRP with an amidated carboxyl-terminal methionine (indicated by an asterisk in Fig. 7). GRP is cleaved to form the decapeptide GRP. The carboxyl-terminal flanking peptides of all three mRNA translation products are cleaved to form several gastrin-releasing peptide gene-related products. Knowledge of the processing of gastrin-releasing peptide and its gene-related products will allow synthesis of duplicates of the stored forms of these peptides, which can then be used for biological testing.

Control of rectification and permeation by two distinct sites after the second transmembrane region in Kir2.1 K+ channel

PubMed Central

Kubo, Yoshihiro; Murata, Yoshimichi

2001-01-01

The rectification property of the inward rectifier K+ channel is chiefly due to the block of outward current by cytoplasmic Mg2+ and polyamines. In the cloned inward rectifier K+ channel Kir2.1 (IRK1), Asp172 in the second transmembrane region (M2) and Glu224 in the putative cytoplasmic region after M2 are reported to be critical for the sensitivity to these blockers. However, the difference in the inward rectification properties between Kir2.1 and a very weak inward rectifier sWIRK could not be explained by differences at these two sites. Following sequence comparison of Kir2.1 and sWIRK, we focused this study on Glu299 located in the centre of the putative cytoplasmic region after M2. Single-point mutants of Kir2.1 (Glu224Gly and Glu299Ser) and a double-point mutant (Glu224Gly-Glu299Ser) were made and expressed in Xenopus oocytes or in HEK293T cells. Their electrophysiological properties were compared with those of wild-type (WT) Kir2.1 and the following observations were made. (a) Glu299Ser showed a weaker inward rectification, a slower activation upon hyperpolarization, a slower decay of the outward current upon depolarization, a lower sensitivity to block by cytoplasmic spermine and a smaller single-channel conductance than WT. (b) The features of Glu224Gly were similar to those of Glu299Ser. (c) In the double mutant (Glu224Gly-Glu299Ser), the differences from WT described above were more prominent. These results demonstrate that Glu299 as well as Glu224 control rectification and permeation, and suggest the possibility that the two sites contribute to the inner vestibule of the channel pore. The slowing down of the on- and off-blocking processes by mutation of these sites implies that Glu224 and Glu299 function to facilitate the entry (and exit) of spermine to (and from) the blocking site. PMID:11251047

E9-Im9 Colicin DNase−Immunity Protein Biomolecular Association in Water: A Multiple-Copy and Accelerated Molecular Dynamics Simulation Study

PubMed Central

2008-01-01

Protein−protein transient and dynamic interactions underlie all biological processes. The molecular dynamics (MD) of the E9 colicin DNase protein, its Im9 inhibitor protein, and their E9-Im9 recognition complex are investigated by combining multiple-copy (MC) MD and accelerated MD (aMD) explicit-solvent simulation approaches, after validation with crystalline-phase and solution experiments. Im9 shows higher flexibility than its E9 counterpart. Im9 displays a significant reduction of backbone flexibility and a remarkable increase in motional correlation upon E9 association. Im9 loops 23−31 and 54−64 open with respect to the E9-Im9 X-ray structure and show high conformational diversity. Upon association a large fraction (∼20 nm2) of E9 and Im9 protein surfaces become inaccessible to water. Numerous salt bridges transiently occurring throughout our six 50 ns long MC-MD simulations are not present in the X-ray model. Among these Im9 Glu31−E9 Arg96 and Im9 Glu41−Lys89 involve interface interactions. Through the use of 10 ns of Im9 aMD simulation, we reconcile the largest thermodynamic impact measured for Asp51Ala mutation with Im9 structure and dynamics. Lys57 acts as an essential molecular switch to shift Im9 surface loop towards an ideal configuration for E9 inhibition. This is achieved by switching Asp60−Lys57 and Asp62−Lys57 hydrogen bonds to Asp51−Lys57 salt bridge. E9-Im9 recognition involves shifts of conformational distributions, reorganization of intramolecular hydrogen bond patterns, and formation of new inter- and intramolecular interactions. The description of key transient biological interactions can be significantly enriched by the dynamic and atomic-level information provided by computer simulations. PMID:19053689

Conformational analysis of the N-terminal sequence Met1 Val60 of the tyrosine hydroxylase

NASA Astrophysics Data System (ADS)

Alieva, Irada N.; Mustafayeva, Narmina N.; Gojayev, Niftali M.

2006-03-01

Molecular mechanics method and molecular dynamics (MD) simulation techniques are used to study the behavior and the effect of the amino acids substitution on structure and molecular dynamics of the specific portion of Met1-Val60 amino acid residues from N-terminal regulatory domain of the tyrosine hydroxylase (TH) and its mutants in which the positively charged arginine residues at positions 37 and 38 were replaced by electrically neutral Gly and negatively charged Glu, and serine residue at position 40 was replaced by Ala or Asp residue. Our study allowed us to make the following conclusions: (i) the higher conformational flexibility of the Met1-Arg16 sequence is revealed in comparision to other part of the N-terminus; (ii) the stretch of amino acid residues Met30-Ser40 within the N-terminus forms β-turn so that two α-helices (residues 16-29 and residues 41-60) are paralel one another; (ii) the significant differences that are observed for the Arg37→Gly37, Arg37-Arg38→Glu37-Glu38 mutant segments indicates that the positive charge of the Arg37 and Arg38 residues is one of the main factor that maintains the characteristic of the turn; (ii) no major conformational changes are observed between Ser40→Ala40, and Ser40→Asp40 mutant segments.

Reactivity of cosmetic UV filters towards skin proteins: model studies with Boc-lysine, Boc-Gly-Phe-Gly-Lys-OH, BSA and gelatin.

PubMed

Stiefel, C; Schwack, W

2014-12-01

Organic UV filters are used as active ingredients in most sunscreens and also in a variety of daily care products. Their good (photo) stability is of special interest to guarantee protective function and to prevent interactions with the human skin. Due to the mostly electrophilic character of the UV filters, reactions with nucleophilic protein moieties like lysine side chains are conceivable. Prior studies showed that the UV filters octocrylene (OCR), butyl methoxydibenzoylmethane (BM-DBM), ethylhexyl salicylate (EHS), ethylhexyl methoxycinnamate (EHMC), benzophenone-3 (BP-3), ethylhexyl triazone (EHT) and dibenzoylmethane (DBM) were able to covalently bind to an HPTLC amino phase and the amino acid models ethanolamine and butylamine after slightly heating and/or radiation. Boc-protected lysine, the tetrapeptide Boc-Gly-Phe-Gly-Lys-OH, bovine serum albumin (BSA) and porcine gelatin were used as more complex models to determine the reactivity of the mentioned UV filters towards skin proteins under thermal or UV irradiation conditions. After gentle heating at 37°C, benzophenone imines were identified as reaction products of BP-3 and OCR with Boc-lysine and the tetrapeptide, whereas DBM and BM-DBM yielded enamines. For EHMC, a Michael-type reaction occurred, which resulted in addition of Boc-lysine or the tetrapeptide to the conjugated double bond. Ester aminolysis of EHS and EHT mainly afforded the corresponding amides. Reactions of the UV filters with BSA changed the UV spectrum of BSA, generally associated with an increase of the absorption strength in the UVA or UVB range. For all protein models, the UV filters showed an increasing reactivity in the order EHT < EHMC < EHS < BP-3 < OCR < DBM < BM-DBM. Especially the UV absorbers BM-DBM, OCR and BP-3, which are seen as common allergens or photoallergens, showed a high reactivity towards the different skin protein models. As the formation of protein adducts is recognized as important key element in the induction of skin sensitization, the results of this study can contribute to a better understanding of the underlying chemical mechanisms of such reactions. © 2014 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Rare and Common Variants in CARD14, Encoding an Epidermal Regulator of NF-kappaB, in Psoriasis

PubMed Central

Jordan, Catherine T.; Cao, Li; Roberson, Elisha D.O.; Duan, Shenghui; Helms, Cynthia A.; Nair, Rajan P.; Duffin, Kristina Callis; Stuart, Philip E.; Goldgar, David; Hayashi, Genki; Olfson, Emily H.; Feng, Bing-Jian; Pullinger, Clive R.; Kane, John P.; Wise, Carol A.; Goldbach-Mansky, Raphaela; Lowes, Michelle A.; Peddle, Lynette; Chandran, Vinod; Liao, Wilson; Rahman, Proton; Krueger, Gerald G.; Gladman, Dafna; Elder, James T.; Menter, Alan; Bowcock, Anne M.

2012-01-01

Psoriasis is a common inflammatory disorder of the skin and other organs. We have determined that mutations in CARD14, encoding a nuclear factor of kappa light chain enhancer in B cells (NF-kB) activator within skin epidermis, account for PSORS2. Here, we describe fifteen additional rare missense variants in CARD14, their distribution in seven psoriasis cohorts (>6,000 cases and >4,000 controls), and their effects on NF-kB activation and the transcriptome of keratinocytes. There were more CARD14 rare variants in cases than in controls (burden test p value = 0.0015). Some variants were only seen in a single case, and these included putative pathogenic mutations (c.424G>A [p.Glu142Lys] and c.425A>G [p.Glu142Gly]) and the generalized-pustular-psoriasis mutation, c.413A>C (p.Glu138Ala); these three mutations lie within the coiled-coil domain of CARD14. The c.349G>A (p.Gly117Ser) familial-psoriasis mutation was present at a frequency of 0.0005 in cases of European ancestry. CARD14 variants led to a range of NF-kB activities; in particular, putative pathogenic variants led to levels >2.5× higher than did wild-type CARD14. Two variants (c.511C>A [p.His171Asn] and c.536G>A [p.Arg179His]) required stimulation with tumor necrosis factor alpha (TNF-α) to achieve significant increases in NF-kB levels. Transcriptome profiling of wild-type and variant CARD14 transfectants in keratinocytes differentiated probably pathogenic mutations from neutral variants such as polymorphisms. Over 20 CARD14 polymorphisms were also genotyped, and meta-analysis revealed an association between psoriasis and rs11652075 (c.2458C>T [p.Arg820Trp]; p value = 2.1 × 10−6). In the two largest psoriasis cohorts, evidence for association increased when rs11652075 was conditioned on HLA-Cw∗0602 (PSORS1). These studies contribute to our understanding of the genetic basis of psoriasis and illustrate the challenges faced in identifying pathogenic variants in common disease. PMID:22521419

Comparison of non-volatile umami components in chicken soup and chicken enzymatic hydrolysate.

PubMed

Kong, Yan; Yang, Xiao; Ding, Qi; Zhang, Yu-Yu; Sun, Bao-Guo; Chen, Hai-Tao; Sun, Ying

2017-12-01

Umami taste is an important part to the taste of chicken. To isolate and identify non-volatile umami compounds, fractions from chicken soup and hydrolysate were prepared and analyzed. Amino acids were analyzed by amino acid analyzer. Organic acids and nucleotides were determined by ultra-performance liquid chromatography. Separation procedures utilizing ultrafiltration, Sephadex G-15 and reversed-phase high-performance liquid chromatography were used to isolate umami taste peptides. Combined with sensory evaluation and LC-Q-TOF-MS, the amino acid sequences of 12 oligopeptides were determined. The amount of taste compounds was higher in chicken enzymatic hydrolysate than that of chicken soup. Eight oligopeptides from chicken enzymatic hydrolysate were identified, including Ala-Asp, Ala-Met, His-Ser, Val-Glu, Ala-Glu, Asp-Ala-Gly, Glu-Asp and Ala-Glu-Ala. Four oligopeptides from chicken soup were identified, including Val-Thr, Ala-His, Ala-Phe and Thr-Glu. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Logical OR Redundancy within the Asx-Pro-Asx-Gly Type 1 {Beta}-Turn Motif

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

Lee, Jihun; Dubey, Vikash Kumar; Longo, Lian M.

2008-04-19

Turn secondary structure is essential to the formation of globular protein architecture. Turn structures are, however, much more complex than either {alpha}-helix or {beta}-sheet, and the thermodynamics and folding kinetics are poorly understood. Type I {beta}-turns are the most common type of reverse turn, and they exhibit a statistical consensus sequence of Asx-Pro-Asx-Gly (where Asx is Asp or Asn). A comprehensive series of individual and combined Asx mutations has been constructed within three separate type I 3:5 G1 bulge {beta}-turns in human fibroblast growth factor-1, and their effects on structure, stability, and folding have been determined. The results show amore » fundamental logical OR relationship between the Asx residues in the motif, involving H-bond interactions with main-chain amides within the turn. These interactions can be modulated by additional interactions with residues adjacent to the turn at positions i + 4 and i + 6. The results show that the Asx residues in the turn motif make a substantial contribution to the overall stability of the protein, and the Asx logical OR relationship defines a redundant system that can compensate for deleterious point mutations. The results also show that the stability of the turn is unlikely to be the prime determinant of formation of turn structure in the folding transition state.« less

Oxidation-induced Structural Changes of Ceruloplasmin Foster NGR Motif Deamidation That Promotes Integrin Binding and Signaling

PubMed Central

Barbariga, Marco; Curnis, Flavio; Spitaleri, Andrea; Andolfo, Annapaola; Zucchelli, Chiara; Lazzaro, Massimo; Magnani, Giuseppe; Musco, Giovanna; Corti, Angelo; Alessio, Massimo

2014-01-01

Asparagine deamidation occurs spontaneously in proteins during aging; deamidation of Asn-Gly-Arg (NGR) sites can lead to the formation of isoAsp-Gly-Arg (isoDGR), a motif that can recognize the RGD-binding site of integrins. Ceruloplasmin (Cp), a ferroxidase present in the cerebrospinal fluid (CSF), contains two NGR sites in its sequence: one exposed on the protein surface (568NGR) and the other buried in the tertiary structure (962NGR). Considering that Cp can undergo oxidative modifications in the CSF of neurodegenerative diseases, we investigated the effect of oxidation on the deamidation of both NGR motifs and, consequently, on the acquisition of integrin binding properties. We observed that the exposed 568NGR site can deamidate under conditions mimicking accelerated Asn aging. In contrast, the hidden 962NGR site can deamidate exclusively when aging occurs under oxidative conditions, suggesting that oxidation-induced structural changes foster deamidation at this site. NGR deamidation in Cp was associated with gain of integrin-binding function, intracellular signaling, and cell pro-adhesive activity. Finally, Cp aging in the CSF from Alzheimer disease patients, but not in control CSF, causes Cp deamidation with gain of integrin-binding function, suggesting that this transition might also occur in pathological conditions. In conclusion, both Cp NGR sites can deamidate during aging under oxidative conditions, likely as a consequence of oxidative-induced structural changes, thereby promoting a gain of function in integrin binding, signaling, and cell adhesion. PMID:24366863

Recombinant Brucella abortus gene expressing immunogenic protein

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

Mayfield, J.E.; Tabatabai, L.B.

This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

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

Qasim, Mohammad A., E-mail: qasimm@ipfw.edu; Song, Jikui; Markley, John L.

Research highlights: {yields} Large pK shifts in ionizable groups when buried in the protein interior. {yields} Substrate dependent shifts in pH optimum for serine proteases. {yields} Lys side chain is a stronger acid in serine protease S{sub 1} pocket than Asp side chain. -- Abstract: Enzymatic hydrolysis of the synthetic substrate succinyl-Ala-Ala-Pro-Xxx-pNA (where Xxx = Leu, Asp or Lys) catalyzed by bovine chymotrypsin (CHYM) or Streptomyces griseus protease B (SGPB) has been studied at different pH values in the pH range 3-11. The pH optima for substrates having Leu, Asp, and Lys have been found to be 7.5-8.0, 5.5-6.0, andmore » {approx}10, respectively. At the normally reported pH optimum (pH 7-8) of CHYM and SGPB, the substrate with Leu at the reactive site is more than 25,000-fold more reactive than that with Asp. However, when fully protonated, Asp is nearly as good a substrate as Leu. The pK values of the side chains of Asp and Lys in the hydrophobic S{sub 1} pocket of CHYM and SGPB have been calculated from pH-dependent hydrolysis data and have been found to be about 9 for Asp and 7.4 and 9.7 for Lys for CHYM and SGPB, respectively. The results presented in this communication suggest a possible application of CHYM like enzymes in cleaving peptide bonds contributed by acidic amino acids between pH 5 and 6.« less

Antihypertensive activity of transgenic rice seed containing an 18-repeat novokinin peptide localized in the nucleolus of endosperm cells.

PubMed

Wakasa, Yuhya; Zhao, Hui; Hirose, Sakiko; Yamauchi, Daiki; Yamada, Yuko; Yang, Lijun; Ohinata, Kousaku; Yoshikawa, Masaaki; Takaiwa, Fumio

2011-09-01

Novokinin (Arg-Pro-Leu-Lys-Pro-Trp, RPLKPW) is a new potent antihypertensive peptide based on the sequence of ovokinin (2-7) derived from ovalbumin. We previously generated transgenic rice seeds in which eight novokinin were fused to storage protein glutelins (GluA2 and GluC) for expression. Oral administration of these seeds to spontaneously hypertensive rats (SHRs) reduced systolic blood pressures at a dose of 1 g seed/kg of SHR. Here, 10- or 18-tandem repeats of novokinin with an endoplasmic reticulum (ER) retention signal (Lys-Asp-Glu-Leu, KDEL) at the C terminus were directly expressed in rice under the control of the glutelin promoter containing its signal peptide. Only small amounts of the 18-repeat novokinin accumulated, and it was unexpectedly deposited in the nucleolus. This abnormal intracellular localization was explained by an endogenous signal for nuclear localization. The GFP reporter protein fused to this sequence targeted to nuclei by a transient assay using onion epidermal cells. Transgenic seed expressing the 18-repeat novokinin exhibited significantly higher antihypertensive activity after a single oral dose to SHR even at one-quarter the amount (0.25 g/kg) of the transgenic rice seed expressing the fusion construct; though, its novokinin content was much lower (1/5). Furthermore, in a long-term administration for 5 weeks, even a smaller dose (0.0625 g/kg) of transgenic seeds could confer antihypertensive activity. This high antihypertensive activity may be attributed to differences in digestibility of expressed products by gastrointestinal enzymes and the unique intracellular localization. These results indicate that accumulation of novokinin as a tandemly repeated structure in transgenic rice is more effective than as a fusion-type structure. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology and Blackwell Publishing Ltd.

Hexapeptide fragment of carcinoembryonic antigen which acts as an agonist of heterogeneous ribonucleoprotein M.

PubMed

Palermo, Nicholas Y; Thomas, Peter; Murphy, Richard F; Lovas, Sándor

2012-04-01

Colorectal cancers with metastatic potential secrete the glycoprotein carcinoembryonic antigen (CEA). CEA has been implicated in colorectal cancer metastasis by inducing Kupffer cells to produce inflammatory cytokines which, in turn, make the hepatic micro-environment ideal for tumor cell implantation. CEA binds to the heterogeneous ribonucleoprotein M (hnRNP M) which acts as a cell surface receptor in Kupffer cells. The amino acid sequence in CEA, which binds the hnRNP M receptor, is Tyr-Pro-Glu-Leu-Pro-Lys. In this study, the structure of Ac-Tyr-Pro-Glu-Leu-Pro-Lys-NH₂ (YPELPK) was investigated using electronic circular dichroism, vibrational circular dichroism, and molecular dynamics simulations. The binding of the peptide to hnRNP M was also investigated using molecular docking calculations. The biological activity of YPELPK was studied using differentiated human THP-1 cells, which express hnRNP M on their surface and secrete IL-6 when stimulated by CEA. YPELPK forms a stable polyproline-II helix and stimulates IL-6 production of THP-1 cells at micromolar concentrations. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

Association of Amino Acid Substitutions in Penicillin-Binding Protein 3 with β-Lactam Resistance in β-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae

PubMed Central

Ubukata, Kimiko; Shibasaki, Yumi; Yamamoto, Kentarou; Chiba, Naoko; Hasegawa, Keiko; Takeuchi, Yasuo; Sunakawa, Keisuke; Inoue, Matsuhisa; Konno, Masatoshi

2001-01-01

The affinity of [3H]benzylpenicillin for penicillin-binding protein (PBP) 3A was reduced in 25 clinical isolates of β-lactamase-negative ampicillin (AMP)-resistant (BLNAR) Haemophilus influenzae for which the AMP MIC was ≥1.0 μg/ml. The affinities of PBP 3B and PBP 4 were also reduced in some strains. The sequences of the ftsI gene encoding the transpeptidase domain of PBP 3A and/or PBP 3B and of the dacB gene encoding PBP 4 were determined for these strains and compared to those of AMP-susceptible Rd strains. The BLNAR strains were classified into three groups on the basis of deduced amino acid substitutions in the ftsI gene, which is thought to be involved in septal peptidoglycan synthesis. His-517, near the conserved Lys-Thr-Gly (KTG) motif, was substituted for Arg-517 in group I strains (n = 9), and Lys-526 was substituted for Asn-526 in group II strains (n = 12). In group III strains (n = 4), three residues (Met-377, Ser-385, and Leu-389), positioned near the conserved Ser-Ser-Asn (SSN) motif, were replaced with Ile, Thr, and Phe, respectively, in addition to the replacement with Lys-526. The MICs of cephem antibiotics with relatively high affinities for PBP 3A and PBP 3B were higher than those of AMP and meropenem for group III strains. The MICs of β-lactams for H. influenzae transformants into which the ftsI gene from BLNAR strains was introduced were as high as those for the donors, and PBP 3A and PBP 3B showed decreased affinities for β-lactams. There was no clear relationship between 7-bp deletions in the dacB gene and AMP susceptibility. Even though mutations in another gene(s) may be involved in β-lactam resistance, these data indicate that mutations in the ftsI gene are the most important for development of resistance to β-lactams in BLNAR strains. PMID:11353613

Geroprotective effect of ala-glu-asp-gly peptide in male rats exposed to different illumination regimens.

PubMed

Vinogradova, I A; Bukalev, A V; Zabezhinski, M A; Semenchenko, A V; Khavinson, V Kh; Anisimov, V N

2008-04-01

Exposure of male rats to permanent or natural illumination of North-Western Russia accelerated their death in comparison with animals exposed to standard (12 h) light. Permanent illumination promoted the development of spontaneous tumors in comparison with the standard photoregimen. Injection of epithalone (synthetic Ala-Glu-Asp-Gly peptide; subcutaneously 0.1 microg/rat 5 times a week from the age of 4 months until natural death) virtually did not change the mean lifespan of male rats, but was associated with a significant (p<0.05) normalization of population aging rate and hence, time of mortality rate doubling in groups exposed to natural or constant illumination. Epithalone injected to rats exposed to any photoregimen significantly inhibited the development of spontaneous tumors, primarily testicular leydigomas and leukemias.

Active diuretic peptidomimetic insect kinin analogs that contain Beta-turn mimetic motif 4-aminopyroglutamate and lack native peptide bonds

USDA-ARS?s Scientific Manuscript database

The multifunctional arthropod 'insect kinins' share the evolutionarily conserved C-terminal pentapeptide core sequence Phe-X1-X2-Trp-Gly-NH2, where X1 = His, Asn, Ser, or Tyr and X2 = Ser, Pro, or Ala. Insect kinins regulate diuresis in many species of insects, including the cricket. Insect kinins...

Helixconstraints and amino acid substitution in GLP-1 increase cAMP and insulin secretion but not beta-arrestin 2 signaling.

PubMed

Plisson, Fabien; Hill, Timothy A; Mitchell, Justin M; Hoang, Huy N; de Araujo, Aline D; Xu, Weijun; Cotterell, Adam; Edmonds, David J; Stanton, Robert V; Derksen, David R; Loria, Paula M; Griffith, David A; Price, David A; Liras, Spiros; Fairlie, David P

2017-02-15

Glucagon-like peptide (GLP-1) is an endogenous hormone that induces insulin secretion from pancreatic islets and modified forms are used to treat diabetes mellitus type 2. Understanding how GLP-1 interacts with its receptor (GLP-1R) can potentially lead to more effective drugs. Modeling and NMR studies of the N-terminus of GLP-1 suggest a β-turn between residues Glu9-Phe12 and a kinked alpha helix between Val16-Gly37. N-terminal turn constraints attenuated binding affinity and activity (compounds 1-8). Lys-Asp (i, i+4) crosslinks in the middle and at the C-terminus increased alpha helicity and cAMP stimulation without much effect on binding affinity or beta-arrestin 2 recruitment (compounds 9-18). Strategic positioning of helix-inducing constraints and amino acid substitutions (Tyr16, Ala22) increased peptide helicity and produced ten-fold higher cAMP potency (compounds 19-28) over GLP-1(7-37)-NH 2 . The most potent cAMP activator (compound 23) was also the most potent inducer of insulin secretion. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Acylated apelin-13 amide analogues exhibit enzyme resistance and prolonged insulin releasing, glucose lowering and anorexic properties.

PubMed

O'Harte, Finbarr P M; Parthsarathy, Vadivel; Hogg, Christopher; Flatt, Peter R

2017-12-15

The adipokine, apelin has many biological functions but its activity is curtailed by rapid plasma degradation. Fatty acid derived apelin analogues represent a new and exciting avenue for the treatment of obesity-diabetes. This study explores four novel fatty acid modified apelin-13 analogues, namely, (Lys 8 GluPAL)apelin-13 amide, pGlu(Lys 8 GluPAL)apelin-13 amide, Lys 8 GluPAL(Tyr 13 )apelin-13 and Lys 8 GluPAL(Val 13 )apelin-13. Fatty acid modification extended the half-life of native apelin-13 to >24 h in vitro. pGlu(Lys 8 GluPAL)apelin-13 amide was the most potent insulinotropic analogue in BRIN-BD11 cells and isolated islets with maximal stimulatory effects of up to 2.7-fold (p < .001). (Lys 8 GluPAL)apelin-13 amide (1.9-fold) and Lys 8 GluPAL(Tyr 13 )apelin-13 (1.7-fold) were less effective, whereas Lys 8 GluPAL(Val 13 )apelin-13 had an inhibitory effect on insulin secretion. Similarly, pGlu(Lys 8 GluPAL)apelin-13 amide was most potent in increasing beta-cell intracellular Ca 2+ concentrations (1.8-fold, p < .001) and increasing glucose uptake in 3T3-L1 adipocytes (2.3-fold, p < .01). Persistent biological action was observed with both pGlu(Lys 8 GluPAL)apelin-13 amide and (Lys 8 GluPAL)apelin-13 amide significantly reducing blood glucose (39-43%, p < .01) and enhancing insulin secretion (43-56%, p < .001) during glucose tolerance tests in diet-induced obese mice. pGlu(Lys 8 GluPAL)apelin-13 amide and (Lys 8 GluPAL)apelin-13 amide also inhibited feeding (28-40%, p < .001), whereas Lys 8 GluPAL(Val 13 )apelin-13 increased food intake (8%, p < .05) in mice. These data indicate that novel enzymatically stable analogues of apelin-13 may be suitable for future development as therapeutic agents for obesity-diabetes. Copyright © 2017 Elsevier Inc. All rights reserved.

Selective coupling of methotrexate to peptide hormone carriers through a gamma-carboxamide linkage of its glutamic acid moiety: benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate activation in salt coupling.

PubMed Central

Nagy, A; Szoke, B; Schally, A V

1993-01-01

A convenient synthetic method is described for the preparation of peptide-methotrexate (MTX) conjugates in which MTX is coupled selectively through the gamma-carboxyl group of its glutamic acid moiety to a free amino group in peptide analogs. The syntheses of a somatostatin analog-MTX conjugate (MTX-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2) (AN-51) and two conjugates of analogs of luteinizing hormone-releasing hormone (LH-RH) with MTX [Glp-His-Trp-Ser-Tyr-D-Lys(MTX)-Leu-Arg-Pro-Gly-NH2] (AJ-04) and [Ac-Ser-Tyr-D-Lys(MTX)-Leu-Arg-Pro-NH-Et] AJ-51 are presented as examples. Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) was used in the synthesis for activation of 4-amino-4-deoxy-N10-methylpteroic acid, which reacted with the potassium salt of glutamic acid alpha-tert-butyl ester in dimethyl sulfoxide to form the suitably protected MTX derivative. This synthesis provides an example of the high suitability of BOP reagent for the salt-coupling method. The selectively protected MTX derivative was then coupled to the different peptide carriers and deprotected under relatively mild conditions by trifluoroacetic acid. The conjugates of MTX with hormonal analogs are suitable for targeting to various tumors that possess receptors for the peptide moieties. PMID:8101004

Purification and characterization of novel antioxidant peptides from enzymatic hydrolysates of tilapia (Oreochromis niloticus) skin gelatin.

PubMed

Zhang, Yufeng; Duan, Xiu; Zhuang, Yongliang

2012-11-01

To obtain hydrolysates with high degree of hydrolysis (DH) and scavenging radical activity, tilapia skin gelatin (TSG) was hydrolyzed by properase E and multifect neutral. The optimum hydrolysis condition of each enzyme was determined using the orthogonal experiment, and double-enzyme hydrolysis was further applied. The results showed the tilapia skin gelatin hydrolysate (TSGH) obtained by progressive hydrolysis using multifect neutral and properase E had the highest DH and hydroxyl radical scavenging activity. The IC(50) values of TSGH on scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide anion radical (·O(2)) and hydroxyl radical (·OH) activities were also determined. TSGH was further purified using gel filtration chromatography, ion exchange chromatography, and RP-HPLC. The peptides were identified using nano-LC-ESI mass spectrometry. Finally, two antioxidant peptides were identified and the amino acid sequences were Glu-Gly-Leu (317.33 Da) and Tyr-Gly-Asp-Glu-Tyr (645.21 Da), respectively. The IC(50) values of two peptides on hydroxyl radical scavenging activities were 4.61 μg mL(-1)and 6.45 μg mL(-1), respectively. Therefore, the results demonstrated that the hydrolysates of TSG prepared by multifect neutral and properase E could serve as a source of peptides with high antioxidant activity. It provided a scientific basis for the preparation of antioxidant peptides. Copyright © 2012 Elsevier Inc. All rights reserved.

Functional roles and efficiencies of the thioredoxin boxes of calcium-binding proteins 1 and 2 in protein folding.

PubMed Central

Kramer, B; Ferrari, D M; Klappa, P; Pöhlmann, N; Söling, H D

2001-01-01

The rat luminal endoplasmic-recticulum calcium-binding proteins 1 and 2 (CaBP1 and CaBP2 respectively) are members of the protein disulphide-isomerase (PDI) family. They contain two and three thioredoxin boxes (Cys-Gly-His-Cys) respectively and, like PDI, may be involved in the folding of nascent proteins. We demonstrate here that CaBP1, similar to PDI and CaBP2, can complement the lethal phenotype of the disrupted Saccharomyces cerevisiae PDI gene, provided that the natural C-terminal Lys-Asp-Glu-Leu sequence is replaced by His-Asp-Glu-Leu. Both the in vitro RNase AIII-re-activation assays and in vivo pro-(carboxypeptidase Y) processing assays using CaBP1 and CaBP2 thioredoxin (trx)-box mutants revealed that, whereas the three trx boxes in CaBP2 seem to be functionally equivalent, the first trx box of CaBP1 is significantly more active than the second trx box. Furthermore, only about 65% re-activation of denatured reduced RNase AIII could be obtained with CaBP1 or CaBP2 compared with PDI, and the yield of PDI-catalysed reactions was significantly reduced in the presence of either CaBP1 or CaBP2. In contrast with PDI, neither CaBP1 nor CaBP2 could catalyse the renaturation of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a redox-independent process, and neither protein had any effect on the PDI-catalysed refolding of GAPDH. Furthermore, although PDI can bind peptides via its b' domain, a property it shares with PDIp, the pancreas-specific PDI homologue, and although PDI can bind malfolded proteins such as 'scrambled' ribonuclease, no such interactions could be detected for CaBP2. We conclude that: (1) both CaBP2 and CaBP1 lack peptide-binding activity for GAPDH attributed to the C-terminal region of the a' domain of PDI; (2) CaBP2 lacks the general peptide-binding activity attributed to the b' domain of PDI; (3) interaction of CaBP2 with substrate (RNase AIII) is different from that of PDI and substrate; and (4) both CaBP2 and CaBP1 may promote oxidative folding by different kinetic pathways. PMID:11415439

AMPA receptor flip/flop mutants affecting deactivation, desensitization, and modulation by cyclothiazide, aniracetam, and thiocyanate.

PubMed

Partin, K M; Fleck, M W; Mayer, M L

1996-11-01

AMPA receptor GluRA subunits with mutations at position 750, a residue shown previously to control allosteric regulation by cyclothiazide, were analyzed for modulation of deactivation and desensitization by cyclothiazide, aniracetam, and thiocyanate. Point mutations from Ser to Asn, Ala, Asp, Gly, Gln, Met, Cys, Thr, Leu, Val, and Tyr were constructed in GluRAflip. The last four of these mutants were not functional; S750D was active only in the presence of cyclothiazide, and the remaining mutants exhibited altered rates of deactivation and desensitization for control responses to glutamate, and showed differential modulation by cyclothiazide and aniracetam. Results from kinetic analysis are consistent with aniracetam and cyclothiazide acting via distinct mechanisms. Our experiments demonstrate for the first time the functional importance of residue 750 in regulating intrinsic channel-gating kinetics and emphasize the biological significance of alternative splicing in the M3-M4 extracellular loop.

The adipokinetic hormone of Mantodea in comparison to other Dictyoptera.

PubMed

Gäde, Gerd; Marco, Heather G

2017-03-01

Six species of the order Mantodea (praying mantises) are investigated for the presence and sequence of putative adipokinetic hormones (AKHs). The selected species span a wide evolutionary range of various families and subfamilies of the clade Mantodea. The corpora cardiaca of the different species are dissected, methanolic extracts prepared, peptides separated by liquid chromatography, and AKHs detected and sequenced by ion trap mass spectrometry. All six species investigated contain an octapeptide with the primary structure pGlu-Val-Asn-Phe-Thr-Pro-Asn-Trp amide, which is code-named Emppe-AKH and had been found earlier in three other species of Mantodea. Conspecific bioassays with the species Creoboter sp. (family Hymenopodidae) reveal an adipokinetic but not a hypertrehalosemic function of Emppe-AKH. Comparison with other members of the Dictyoptera (cockroaches, termites) show that Emppe-AKH is only found in certain termites, which have been recently placed into the Blattaria (cockroaches) as sister group to the family Cryptocercidae. Termites and cockroaches both show biodiversity in the sequence of AKHs, and some cockroach species even contain two AKHs. In contrast, all praying mantises-irrespective of their phylogenetic position-synthesize uniformly only one and the same octapeptide Emppe-AKH. © 2017 Wiley Periodicals, Inc.

A docking study of enhanced intracellular survival protein from Mycobacterium tuberculosis with human DUSP16/MKP-7.

PubMed

Yoon, Hye Jin; Kim, Kyoung Hoon; Yang, Jin Kuk; Suh, Se Won; Kim, Hyunsik; Jang, Soonmin

2013-11-01

The intracellular pathogen Mycobacterium tuberculosis (Mtb) causes tuberculosis, and one of its secreted effector proteins, called enhanced intracellular survival (Eis) protein, enhances its survival in macrophages. Mtb Eis activates JNK-specific dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase-7 (MKP-7) through the acetylation on Lys55, thus inactivating JNK by dephosphorylation. Based on the recently reported crystal structure of Mtb Eis, a docking model for the binding of Mtb Eis to DUSP16/MKP-7 was generated. In the docking model, the substrate helix containing Lys55 of DUSP16/MKP-7 fits nicely into the active-site cleft of Mtb Eis; the twisted β-sheet of Eis domain II embraces the substrate helix from one side. Most importantly, the side-chain of Lys55 is inserted toward acetyl-CoA and the resulting distance is 4.6 Å between the NZ atom of Lys55 and the carbonyl carbon of the acetyl group in acetyl-CoA. The binding of Mtb Eis and DUSP16/MKP-7 is maintained by strong electrostatic interactions. The active-site cleft of Mtb Eis has a negatively charged surface formed by Asp25, Glu138, Asp286, Glu395 and the terminal carboxylic group of Phe396. In contrast, DUSP16/MKP-7 contains five basic residues, Lys52, Lys55, Arg56, Arg57 and Lys62, which point toward the negatively charged surface of the active-site pocket of Mtb Eis. Thus, the current docking model suggests that the binding of DUSP16/MKP-7 to Mtb Eis should be established by charge complementarity in addition to a very favorable geometric arrangement. The suggested mode of binding requires the dissociation of the hexameric Mtb Eis into dimers or monomers. This study may be useful for future studies aiming to develop inhibitors of Mtb Eis as a new anti-tuberculosis drug candidate.

Excision repair cross-complementing group 2/Xeroderma pigmentousm complementation group D (ERCC2/XPD) genetic variations and susceptibility to diffuse large B cell lymphoma in Egypt.

PubMed

El-Din, Mennat Allah Kamal; Khorshied, Mervat Mamdooh; El-Saadany, Zainab Ali; El-Banna, Marwa Ahmed; Reda Khorshid, Ola M

2013-12-01

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous neoplasm. Although several genetic and environmental factors have been postulated, no obvious risk factors have been emerged for DLBCL in the general population. DNA repair systems are responsible for maintaining the integrity of the genome and protecting it against genetic alterations that can lead to malignant transformation. The current study aimed at investigating the possible role of ERCC2/XPD Arg156Arg, Asp312Asn and Lys751Gln genetic polymorphisms as risk factors for DLBCL in Egypt. The study included 81 DLBCL patients and 100 healthy controls. Genotyping of the studied genetic polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism technique. Our results revealed that there was no statistical difference encountered in the distribution of -Asp312Asn and -Lys751Gln polymorphic genotypes between DLBCL cases and controls, thus it could not considered as molecular risk factors for DLBCL in Egyptians. However, Arg156Arg polymorphism at exon-6 conferred twofold increased risk of DLBCL (OR 2.034, 95 %CI 1.015-4.35, p = 0.43), and the risk increased when co-inherited with Lys751Gln at exon-23 (OR 3.304, 95 %CI 1.113-9.812, p = 0.038). In conclusion, ERCC2/XPD Arg156Arg polymorphism might be considered as a genetic risk factor for DLBCL in Egyptians, whether alone or conjoined with Lys751Gln.

Mechanism of protein splicing of the Pyrococcus abyssi lon protease intein

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

O'Brien, Kevin M.; Schufreider, Ann K.; McGill, Melissa A.

2010-12-17

Research highlights: {yields} The Pyrococcus abyssi lon protease intein promotes efficient protein splicing. {yields} Inteins with mutations that interfere with individual steps of splicing do not promote unproductive side reactions. {yields} The intein splices with Lys in place of the highly conserved penultimate His. {yields} The intein is flanked by a Gly-rich region at its C terminus that may increase the efficiency of the third step of splicing, Asn cyclization coupled to peptide bond cleavage. -- Abstract: Protein splicing is a post-translational process by which an intervening polypeptide, the intein, excises itself from the flanking polypeptides, the exteins, coupled tomore » ligation of the exteins. The lon protease of Pyrococcus abyssi (Pab) is interrupted by an intein. When over-expressed as a fusion protein in Escherichia coli, the Pab lon protease intein can promote efficient protein splicing. Mutations that block individual steps of splicing generally do not lead to unproductive side reactions, suggesting that the intein tightly coordinates the splicing process. The intein can splice, although it has Lys in place of the highly conserved penultimate His, and mutants of the intein in the C-terminal region lead to the accumulation of stable branched-ester intermediate.« less

Mutant botrocetin-2 inhibits von Willebrand factor-induced platelet agglutination.

PubMed

Matsui, T; Hori, A; Hamako, J; Matsushita, F; Ozeki, Y; Sakurai, Y; Hayakawa, M; Matsumoto, M; Fujimura, Y

2017-03-01

Essentials Botrocetin-2 (Bot2) binds to von Willebrand factor (VWF) and induces platelet agglutination. We identified Bot2 residues that are required for binding to VWF and glycoprotein (GP) Ib. We produced a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Mutant Bot2 could be used as a potential anti-thrombotic reagent to block VWF-GPIb interaction. Background Botrocetin-2 (Bot2) is a botrocetin-like protein composed of α and β subunits that have been cloned from the snake Bothrops jararaca. Bot2 binds specifically to von Willebrand factor (VWF), and the complex induces glycoprotein (GP) Ib-dependent platelet agglutination. Objectives To exploit Bot2's VWF-binding capacity in order to attempt to create a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Methods and Results Several point mutations were introduced into Bot2 cDNA, and the recombinant protein (recombinant Bot2 [rBot2]) was purified on an anti-botrocetin column. The mutant rBot2 with either Ala at Asp70 in the β subunit (Aspβ70Ala), or Argβ115Ala and Lysβ117Ala, showed reduced platelet agglutination-inducing activity. rBot2 with Aspβ70Ala showed little binding activity towards immobilized VWF on an ELISA plate, whereas rBot2 with Argβ115Ala/Lysβ117Ala showed reduced binding activity towards GPIb (glycocalicin) after forming a complex with VWF. rBot2 point-mutated to oppositely charged Glu at both Argβ115 and Lysβ117 showed normal binding activity towards VWF but no platelet-agglutinating activity. Furthermore, this doubly mutated protein inhibited ristocetin-induced or high shear stress-induced platelet aggregation, and restrained thrombus formation under flow conditions. Conclusions Asp70 in the β subunit of botrocetin is important for VWF binding, and Arg115 and Lys117 in the β subunit are essential for interaction with GPIb. Doubly mutated rBot2, with Argβ115Glu and Lysβ117Glu, repels GPIb and might have potential as an antithrombotic reagent that specifically blocks VWF function. This is the first report on an artificial botrocetin that can inhibit the VWF-GPIb interaction. © 2017 International Society on Thrombosis and Haemostasis.

Requirement of catalytic-triad and related amino acids for the acyltransferase activity of Tanacetum cinerariifolium GDSL lipase/esterase TcGLIP for ester-bond formation in pyrethrin biosynthesis.

PubMed

Kikuta, Yukio; Yamada, Gen; Mitsumori, Tomonori; Takeuchi, Takayuki; Nakayama, Koji; Katsuda, Yoshio; Hatanaka, Akikazu; Matsuda, Kazuhiko

2013-01-01

We have recently discovered that a GDSL lipase/esterase (TcGLIP) in Tanacetum cinerariifolium catalyzed acyltransferase activity to form an ester bond in the natural insecticide, pyrethrin. TcGLIP contained Ser40 in Block I, Gly64 in Block II, Asn168 in Block III and Asp318 and His321 in Block V, suggesting underlying hydrolase activity, although little is known about their role in acyltransferase activity. We expressed TcGLIP here in Esherichia coli as a fusion with maltose-binding protein (MBP), part of the fusion being cleaved with a protease to obtain MBP-free TcGLIP. A kinetic analysis revealed that the MBP moiety scarcely influenced the kinetic parameters. The effects on acyltransferase activity of mutations of Gly64, Asn168, Asp318 and His321 were investigated by using MBP-fused TcGLIP. Mutations of these amino acids markedly reduced the acyltransferase activity, suggesting their critical role in the production of pyrethrins.

Differential Ubiquitin Binding by the Acidic Loops of Ube2g1 and Ube2r1 Enzymes Distinguishes Their Lys-48-ubiquitylation Activities*

PubMed Central

Choi, Yun-Seok; Lee, Yun-Ju; Lee, Seo-Yeon; Shi, Lei; Ha, Jung-Hye; Cheong, Hae-Kap; Cheong, Chaejoon; Cohen, Robert E.; Ryu, Kyoung-Seok

2015-01-01

The ubiquitin E2 enzymes, Ube2g1 and Ube2r1, are able to synthesize Lys-48-linked polyubiquitins without an E3 ligase but how that is accomplished has been unclear. Although both E2s contain essential acidic loops, only Ube2r1 requires an additional C-terminal extension (184–196) for efficient Lys-48-ubiquitylation activity. The presence of Tyr-102 and Tyr-104 in the Ube2g1 acidic loop enhanced both ubiquitin binding and Lys-48-ubiquitylation and distinguished Ube2g1 from the otherwise similar truncated Ube2r11–183 (Ube2r1C). Replacement of Gln-105–Ser-106–Gly-107 in the acidic loop of Ube2r1C (Ube2r1CYGY) by the corresponding residues from Ube2g1 (Tyr-102–Gly-103–Tyr-104) increased Lys-48-ubiquitylation activity and ubiquitin binding. Two E2∼UB thioester mimics (oxyester and disulfide) were prepared to characterize the ubiquitin binding activity of the acidic loop. The oxyester but not the disulfide derivative was found to be a functional equivalent of the E2∼UB thioester. The ubiquitin moiety of the Ube2r1CC93S-[15N]UBK48R oxyester displayed two-state conformational exchange, whereas the Ube2r1CC93S/YGY-[15N]UBK48R oxyester showed predominantly one state. Together with NMR studies that compared UBK48R oxyesters of the wild-type and the acidic loop mutant (Y102G/Y104G) forms of Ube2g1, in vitro ubiquitylation assays with various mutation forms of the E2s revealed how the intramolecular interaction between the acidic loop and the attached donor ubiquitin regulates Lys-48-ubiquitylation activity. PMID:25471371

SIFamide peptides in clawed lobsters and freshwater crayfish (Crustacea, Decapoda, Astacidea): a combined molecular, mass spectrometric and electrophysiological investigation.

PubMed

Dickinson, Patsy S; Stemmler, Elizabeth A; Cashman, Christopher R; Brennan, Henry R; Dennison, Bobbi; Huber, Kristen E; Peguero, Braulio; Rabacal, Whitney; Goiney, Christopher C; Smith, Christine M; Towle, David W; Christie, Andrew E

2008-04-01

Recently, we identified the peptide VYRKPPFNGSIFamide (Val(1)-SIFamide) in the stomatogastric nervous system (STNS) of the American lobster Homarus americanus using matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry (MALDI-FTMS). Given that H. americanus is the only species thus far shown to possess this peptide, and that a second SIFamide isoform, Gly(1)-SIFamide, is broadly conserved in other decapods, including another astacidean, the crayfish Procambarus clarkii, we became interested both in confirming our identification of Val(1)-SIFamide via molecular methods and in determining the extent to which this isoform is conserved within other members of the infraorder Astacidea. Here, we present the identification and characterization of an H. americanus prepro-SIFamide cDNA that encodes the Val(1) isoform. Moreover, we demonstrate via MALDI-FTMS the presence of Val(1)-SIFamide in a second Homarus species, Homarus gammarus. In contrast, only the Gly(1) isoform was detected in the other astacideans investigated, including the lobster Nephrops norvegicus, a member of the same family as Homarus, and the crayfish Cherax quadricarinatus, P. clarkii and Pacifastacus leniusculus, which represent members of each of the extant families of freshwater astacideans. These results suggest that Val(1)-SIFamide may be a genus (Homarus)-specific isoform. Interestingly, both Val(1)- and Gly(1)-SIFamide possess an internal dibasic site, Arg(3)-Lys(4), raising the possibility of the ubiquitously conserved isoform PPFNGSIFamide. However, this octapeptide was not detected via MALDI-FTMS in any of the investigated species, and when applied to the isolated STNS of H. americanus possessed little bioactivity relative to the full-length Val(1) isoform. Thus, it appears that the dodeca-variants Val(1)- and Gly(1)-SIFamide are the sole bioactive isoforms of this peptide family in clawed lobsters and freshwater crayfish.

The C-terminal domain of glyceraldehyde 3-phosphate dehydrogenase plays an important role in suppression of tRNALys3 packaging into human immunodeficiency virus type-1 particles.

PubMed

Kishimoto, Naoki; Onitsuka-Kishimoto, Ayano; Iga, Nozomi; Takamune, Nobutoki; Shoji, Shozo; Misumi, Shogo

2016-12-01

Human immunodeficiency virus type-1 (HIV-1) requires the packaging of human tRNA Lys3 as a primer for effective viral reverse transcription. Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) suppresses the packaging efficiency of tRNA Lys3 . Although the binding of GAPDH to Pr55 gag is important for the suppression mechanism, it remains unclear which domain of GAPDH is responsible for the interaction with Pr55 gag . In this study, we show that Asp 256 , Lys 260 , Lys 263 and Glu 267 of GAPDH are important for the suppression of tRNA Lys3 packaging. Yeast two-hybrid analysis demonstrated that the C -terminal domain of GAPDH (151-335) interacts with both the matrix region (MA; 1-132) and capsid N -terminal domain (CA-NTD; 133-282). The D256R, K263E or E267R mutation of GAPDH led to the loss of the ability to bind to wild-type (WT) MA, and the D256R/K260E double mutation of GAPDH resulted in the loss of detectable binding activity to WT CA-NTD. In contrast, R58E, Q59A or Q63A of MA, and E76R or R82E of CA-NTD abrogated the interaction with the C -terminal domain of GAPDH. Multiple-substituted GAPDH mutant (D256R/K260E/K263E/E267R) retained the oligomeric formation with WT GAPDH in HIV-1 producing cells, but the incorporation level of the hetero-oligomer was decreased in viral particles. Furthermore, the viruses produced from cells expressing the D256R/K260E/K263E/E267R mutant restored tRNA Lys3 packaging efficiency because the mutant exerted a dominant negative effect by preventing WT GAPDH from binding to MA and CA-NTD and improved the reverse transcription. These findings indicate that the amino acids Asp 256 , Lys 260 , Lys 263 and Glu 267 of GAPDH is essential for the mechanism of tRNA Lys3 -packaging suppression and the D256R/K260E/K263E/E267R mutant of GAPDH acts in a dominant negative manner to suppress tRNA Lys3 packaging.

Very Short Peptides with Stable Folds

PubMed Central

Eidenschink, Lisa; Kier, Brandon L.; Huggins, Kelly N. L.; Andersen, Niels H.

2008-01-01

By combining a favorable turn sequence with a turn flanking Trp/Trp interaction and a C-terminal H-bonding interaction between a backbone amide and an i - 2 Trp ring, a particularly stable (ΔGU > 7 kJ/mol) truncated hairpin, Ac-WI-(D-Pro-D-Asn)-KWTG-NH2, results. In this construct and others with a W-(4-residue turn)-W motif in severely truncated hairpins, the C-terminal Trp is the edge residue in a well-defined face-to-edge (FtE) aryl/aryl interaction. Longer hairpins and those with six-residue turns retain the reversed “edge-to-face” Trp/Trp geometry first observed for the trpzip peptides. Mutational studies suggest that the W-(4-residue turn)-W interaction provides at least 3 kJ/mol of stabilization in excess of that due to the greater β-propensity of Trp. The β-propensity of Trp is context dependent; but, for the systems studied, always greater than that of Thr (by 0.4 - 4.7 kJ/mol). At non-H-bonded positions remote from the turn, two alternative edge-to-face geometries are observed and there is no evidence of additional stabilization due to the Trp/Trp interaction. The NMR structuring shift diagnostics of edge-to-face Trp/Trp, Trp/Lys π-cation, and Trp/Gly-HN interactions have been defined. The latter can give rise to > 3 ppm upfield shifts for the Gly-HN in -WXnG- units both in turns (n = 2) and at the C-termini (n = 1) of hairpins. Terminal YTG units result in somewhat smaller shifts (extrapolated to 2 ppm for 100% folding). In peptides with both the EtF and FtE W/W interaction geometries, Trp to Tyr mutations indicate that Trp is the preferred “face” residue in aryl/aryl pairings, presumably due to its greater π basicity. PMID:18831035

In silico approaches and proportional odds model towards identifying selective ADAM17 inhibitors from anti-inflammatory natural molecules.

PubMed

Borah, Pallab Kumar; Chakraborty, Sourav; Jha, Anupam N; Rajkhowa, Sanchaita; Duary, Raj Kumar

2016-11-01

ADAM metallopeptidase domain 17 (ADAM17) is an attractive target for the development of new anti-inflammatory drugs. We aimed to identify selective inhibitors of ADAM17 against matrix metalloproteinase enzymes (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, and MMP-16) which have substantial structural similarity. Target proteins were docked with 29 anti-inflammatory natural molecule ligands and a known selective inhibitor IK682. The ligands were screened based on Lipinski rules, interaction with the ADAM17 active site cavity, and then ranked using the proportional odds model multinomial logistic regression. Silymarin was the most selective inhibitor of ADAM17 exhibiting H-bonding with Glu 406, Gly 349, Glu 398, Asn 447, Tyr 433, and Lys 432. Molecular dynamics simulations were carried out for 10ns. The root mean square deviation (RMSD), root mean squared fluctuations (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), and H-bonding indicated the induced metastability. A comparison of the principal component analysis revealed that the silymarin complex also explored lesser region compared to IK682 complex. A control study on ADAM17 protein (2OI0) is included. These observations present silymarin (widely present in plants such as milk thistle (Silybum maianum), wild artichokes (Cynara cardunculus), turmeric (Curcuma longa) roots, coriander (Coriandrum sativum) seeds, etc.) as a promising natural template for development of ADAM17 selective drugs. Copyright © 2016 Elsevier Inc. All rights reserved.

Ampicillin-Resistant Non-β-Lactamase-Producing Haemophilus influenzae in Spain: Recent Emergence of Clonal Isolates with Increased Resistance to Cefotaxime and Cefixime▿

PubMed Central

García-Cobos, Silvia; Campos, José; Lázaro, Edurne; Román, Federico; Cercenado, Emilia; García-Rey, César; Pérez-Vázquez, María; Oteo, Jesús; de Abajo, Francisco

2007-01-01

The sequence of the ftsI gene encoding the transpeptidase domain of penicillin-binding protein 3 (PBP 3) was determined for 354 nonconsecutive Haemophilus influenzae isolates from Spain; 17.8% of them were ampicillin susceptible, 56% were β-lactamase nonproducing ampicillin resistant (BLNAR), 15.8% were β-lactamase producers and ampicillin resistant, and 10.4% displayed both resistance mechanisms. The ftsI gene sequences had 28 different mutation patterns and amino acid substitutions at 23 positions. Some 93.2% of the BLNAR strains had amino acid substitutions at the Lys-Thr-Gly (KTG) motif, the two most common being Asn526 to Lys (83.9%) and Arg517 to His (9.3%). Amino acid substitutions at positions 377, 385, and 389, which conferred cefotaxime and cefixime MICs 10 to 60 times higher than those of susceptible strains, were found for the first time in Europe. In 72 isolates for which the repressor acrR gene of the AcrAB efflux pump was sequenced, numerous amino acid substitutions were found. Eight isolates with ampicillin MICs of 0.25 to 2 μg/ml showed changes that predicted the early termination of the acrR reading frame. Pulsed-field gel electrophoresis analysis demonstrated that most BLNAR strains were genetically diverse, although clonal dissemination was detected in a group of isolates presenting with increased resistance to cefotaxime and cefixime. Background antibiotic use at the community level revealed a marked trend toward increased amoxicillin-clavulanic acid consumption. BLNAR H. influenzae strains have arisen by vertical and horizontal spread and have evolved to adapt rapidly to the increased selective pressures posed by the use of oral penicillins and cephalosporins. PMID:17470649

Total enzymatic synthesis of cholecystokinin CCK-5.

PubMed

Xiang, H; Xiang, G Y; Lu, Z M; Guo, L; Eckstein, H

2004-08-01

This paper describes the enzymatic synthesis of the C-terminal fragment H-Gly-Trp-Met-Asp-Phe-NH2 of cholecystokinin. Immobilized enzymes were used for the formation of all peptide bonds except thermolysin. Beginning the synthesis with phenylacetyl (PhAc) glycine carboxamidomethyl ester (OCam) and H-Trp-OMe by using immobilized papain as biocatalyst in buffered ethyl acetate, the dipeptide methyl ester was then coupled directly with Met-OEt.HCl by alpha-chymotrypsin/Celite 545 in a solvent free system. For the 3+2 coupling PhAc-Gly-Trp-Met-OEt had to be converted into its OCam ester. The other fragment H-Asp(OMe)-Phe-NH2 resulted from the coupling of Cbo-Asp(OMe)-OH with H-Phe-NH2.HCl and thermolysin as catalyst, followed by catalytic hydrogenation. Finally PhAc-Gly-Trp-Met-Asp-Phe-NH2 was obtained in a smooth reaction from PhAc-Gly-Trp-Met-OCam and H-Asp(OMe)-Phe-NH2 with alpha-chymotrypsin/Celite 545 in acetonitrile, followed by basic hydrolysis of the beta-methyl ester. The PhAc-group is removed with penicillin G amidase and CCK-5 is obtained in an overall isolated yield of 19.6%.

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.

Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from Lactobacillus sakei

PubMed Central

Sanz, Yolanda; Toldrá, Fidel

2001-01-01

An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu2+, Hg2+, and Zn2+). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. Km s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 μM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 μM, respectively. Among peptides, β-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed. PMID:11282638

Key role of hydrazine to the interaction between oxaloacetic against phosphoenolpyruvic carboxykinase (PEPCK): ONIOM calculations.

PubMed

Prajongtat, Pongthep; Phromyothin, Darinee Sae-Tang; Hannongbua, Supa

2013-08-01

The interactions between oxaloacetic (OAA) and phosphoenolpyruvic carboxykinase (PEPCK) binding pocket in the presence and absence of hydrazine were carried out using quantum chemical calculations, based on the two-layered ONIOM (ONIOM2) approach. The complexes were partially optimized by ONIOM2 (B3LYP/6-31G(d):PM6) method while the interaction energies between OAA and individual residues surrounding the pocket were performed at the MP2/6-31G(d,p) level of theory. The calculated interaction energies (INT) indicated that Arg87, Gly237, Ser286, and Arg405 are key residues for binding to OAA with the INT values of -1.93, -2.06, -2.47, and -3.16 kcal mol(-1), respectively. The interactions are mainly due to the formation of hydrogen bonding interactions with OAA. Moreover, using ONIOM2 (B3LYP/6-31G(d):PM6) applied on the PEPCKHS complex, two proton transfers were observed; first, the proton was transferred from the carboxylic group of OAA to hydrazine while the second one was from Asp311 to Lys244. Such reactions cause the generation of binding strength of OAA to the pocket via electrostatic interaction. The orientations of Lys243, Lys244, His264, Asp311, Phe333, and Arg405 were greatly deviated after hydrazine incorporation. These indicate that hydrazine plays an important role in terms of not only changing the conformation of the binding pocket, but is also tightly bound to OAA resulting in its conformation change in the pocket. The understanding of such interaction can be useful for the design of hydrazine-based inhibitor for antichachexia agents.

Mapping of the gene encoding the melanocortin-1 ([alpha]-melanocyte stimulating hormone) receptor (MC1R) to human chromosome 16q24. 3 by fluorescence in situ hybridization

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

Gantz, I.; Yamada, Tadataka; Tashiro, Takao

1994-01-15

[alpha]-Melanocyte stimulating hormone ([alpha]-MSH), a hormone originally named for its ability to regulate pigmentation of melanocytes, is a 13-amino-acid post-translational product of the pro-opiomelanocortin (POMC) gene. [alpha]-MSH and the other products of POMC processing, which share the core heptapeptide amino acid sequence Met-Glu (Gly)-His-Phe-Arg-Trp-Gly (Asp), the adrenocorticotropic hormone (ACTH), [beta]-MSH, and [gamma]-MSH, are collectively referred to as melanocortins. While best known for their effects on the melanocyte (pigmentation) and adrenal cortical cells (steroidogenesis), melanocortins have been postulated to function in diverse activities, including enhancement of learning and memory, control of the cardiovascular system, analgesia, thermoregulation, immunomodulation, parturition, and neurotrophism. Tomore » identify the chromosomal band encoding the human melanocortin-1 receptor gene, 1 [mu]g of an EMBL clone coding region of the human MC1R and approximately 15 kb of surrounding DNA was labeled with biotin and hybridized to human metaphase chromosomes as previously described. The results indicate that the human MC1R gene is localized to 16q24.3. 15 refs., 1 fig.« less

Chemical-exchange-saturation-transfer magnetic resonance imaging to map gamma-aminobutyric acid, glutamate, myoinositol, glycine, and asparagine: Phantom experiments

NASA Astrophysics Data System (ADS)

Oh, Jang-Hoon; Kim, Hyug-Gi; Woo, Dong-Cheol; Jeong, Ha-Kyu; Lee, Soo Yeol; Jahng, Geon-Ho

2017-03-01

The physical and technical development of chemical-exchange-saturation-transfer (CEST) magnetic resonance imaging (MRI) using clinical 3 T MRI was explored with the goal of mapping asparagine (Asn), gamma-aminobutyric acid (GABA), glutamate (Glu), glycine (Gly), and myoinositol (MI), which exist in the brain. Phantoms with nine different conditions at concentrations of 10, 30, and 50 mM and pH values of 5.6, 6.2, and 7.4 were prepared for the five target molecules to evaluate the dependence of the CEST effect in the concentration, the pH, and the amplitude of the applied radiofrequency field B1. CEST images in the offset frequency range of ±6 parts per million (ppm) were acquired using a pulsed radio-frequency saturation scheme with a clinical 3 T MRI system. A voxel-based main magnetic field B0 inhomogeneity correction, where B0 is the center frequency offset at zero ppm, was performed by using the spline interpolation method to fit the full Z-spectrum to estimate the center frequency. A voxel-based CEST asymmetry map was calculated to evaluate amide (-NH), amine (-NH2), and hydroxyl (-OH) groups for the five target molecules. The CEST effect for Glu, GABA, and Gly clearly increased with increasing concentrations. The CEST effect for MI was minimal, with no noticeable differences at different concentrations. The CEST effect for Glu and Gly increased with increasing acidity. The highest CEST asymmetry for GABA was observed at pH 6.2. The CEST effect for Glu, GABA, and Gly increased with increasing B1 amplitude. For all target molecules, the CEST effect for the human 3 T MRI system increased with increasing concentration and B1 amplitude, but varied with pH, depending on the characteristics of the molecules. The CEST effect for MI may be not suitable with clinical MRI systems. These results show that CEST imaging in the brain with the amine protons by using 3 T MRI is possible for several neuronal diseases.

Divergence of Structure and Function in the Haloacid Dehalogenase Enzyme Superfamily: Bacteroides thetaiotaomicron BT2127 is an Inorganic Pyrophosphatase+

PubMed Central

Huang, Hua; Yury, Patskovsky; Toro, Rafael; Farelli, Jeremiah D.; Pandya, Chetanya; Almo, Steven C.; Allen, Karen N.; Dunaway-Mariano, Debra

2012-01-01

The explosion of protein sequence information requires that current strategies for function assignment must evolve to complement experimental approaches with computationally-based function prediction. This necessitates the development of strategies based on the identification of sequence markers in the form of specificity determinants and a more informed definition of orthologues. Herein, we have undertaken the function assignment of the unknown Haloalkanoate Dehalogenase superfamily member BT2127 (Uniprot accession # Q8A5V9) from Bacteroides thetaiotaomicron using an integrated bioinformatics/structure/mechanism approach. The substrate specificity profile and steady-state rate constants of BT2127 (with kcat/Km value for pyrophosphate of ∼1 × 105 M−1 s−1), together with the gene context, supports the assigned in vivo function as an inorganic pyrophosphatase. The X-ray structural analysis of the wild-type BT2127 and several variants generated by site-directed mutagenesis shows that substrate discrimination is based, in part, on active site space restrictions imposed by the cap domain (specifically by residues Tyr76 and Glu47). Structure guided site directed mutagenesis coupled with kinetic analysis of the mutant enzymes identified the residues required for catalysis, substrate binding, and domain-domain association. Based on this structure-function analysis, the catalytic residues Asp11, Asp13, Thr113, and Lys147 as well the metal binding residues Asp171, Asn172 and Glu47 were used as markers to confirm BT2127 orthologues identified via sequence searches. This bioinformatic analysis demonstrated that the biological range of BT2127 orthologue is restricted to the phylum Bacteroidetes/Chlorobi. The key structural determinants in the divergence of BT2127 and its closest homologue β-phosphoglucomutase control the leaving group size (phosphate vs. glucose-phosphate) and the position of the Asp acid/base in the open vs. closed conformations. HADSF pyrophosphatases represent a third mechanistic and fold type for bacterial pyrophosphatases. PMID:21894910

A novel mutation in KIF5A in a Malian family with spastic paraplegia and sensory loss.

PubMed

Guinto, Cheick O; Diarra, Salimata; Diallo, Salimata; Cissé, Lassana; Coulibaly, Thomas; Diallo, Seybou H; Taméga, Abdoulaye; Chen, Ke-Lian; Schindler, Alice B; Bagayoko, Koumba; Simaga, Assiatou; Blackstone, Craig; Fischbeck, Kenneth H; Landouré, Guida

2017-04-01

Hereditary spastic paraplegias (HSPs) are well-characterized disorders but rarely reported in Africa. We evaluated a Malian family in which three individuals had HSP and distal muscle atrophy and sensory loss. HSP panel testing identified a novel heterozygous missense mutation in KIF5A (c.1086G>C, p.Lys362Asn) that segregated with the disease (SPG10). Lys362 is highly conserved across species and Lys362Asn is predicted to be damaging. This study shows that HSPs are present in sub-Saharan Africa, although likely underdiagnosed. Increasing efficiency and decreasing costs of DNA sequencing will make it more feasible to diagnose HSPs in developing countries.

[Risk of type 2 diabetes mellitus in the Kyrgyz population in the presence of ADIPOQ (G276T), KCNJ11 (Glu23Lys), TCF7L2 (IVS3C>T) gene polymorphisms].

PubMed

Isakova, Zh T; Talaibekova, E T; Asambaeva, D A; Kerimkulova, A S; Lunegova, O S; Aldasheva, N M; Aldashev, A A

To analyze the association of genotype combinations of the polymorphic markers G276T in the ADIPOQ gene, Glu23Lys in the KCNJ11 gene, and IVS3C>T in the TCF7L2 gene with the development of type 2 diabetes mellitus (T2DM) in the Kyrgyz population. The investigation enrolled 23 Kyrgyz people, of whom there were 114 patients with T2DM and 109 without T2DM (a control group). T2DM was diagnosed in accordance with the WHO criteria (1999). The genotypes of ADIPOQ (G276T), KCNJ11 (Glu23Lys), and TCF7L2 (IVS3C>T) gene polymorphisms were identified using the restriction fragment length polymorphism analysis. When typing at the polymorphic loci G276T in the ADIPOQ gene, Glu23Lys in the KCNJ11 gene, and IVS3C>T in the TCF7L2 gene, the development of T2DM in the Kyrgyz population was associated with the T allele (odds ratio (OR), 1.68; p=0.025), the heterozygous G276T genotype (OR 1,8; p=0.036) in the ADIPOQ gene; the 23Lys allele (OR, 1.62; p=0.019) in the KCNJ11 gene; a two-locus genotype combination in the genes ADIPOQ/KCNJ11: G276T/Glu23Lys (OR, 4.88; p=0.0013), G276G/Lys23Lys (OR, 4.65; p=0.019), G276T/Glu23Glu (OR, 3.10; p=0.022), a two-locus genotype combination in the genes ADIPOQ/TCF7L2: G276T/СС (OR, 1.97; p=0.04); two-locus genotype combinations in the genes KCNJ11/TCF7L2: Lys23Lys/CC (ОR, 2.65; p=0.042), Glu23Lys/CT (OR, 3.88; p=0.027); and a three-locus genotype combination in the genes ADIPOQ/KCNJ11/TCF7L2: G276T/Glu23Lys/CT (OR, 14.48; p=0.02). The development of T2DM in the Kyrgyz population is genetically determined by ADIPOQ (G276T) gene, KCNJ11 (Glu23Lys), and TCF7L (IVS3C>T) gene polymorphisms with the predisposing value of the T allele of the heterozygous G276T genotype in the ADIPOQ gene; the 23Lys allele in the KCNJ1 gene; as well as by genotype combinations in the genes ADIPOQ/KCNJ11 (G276T/Glu23Lys, G276G/Lys23Lys, G276T/Glu23Glu); ADIPOQ/TCF7L2 (G276T/SS); KCNJ11/TCF7L2 (Lys23Lys/CC, Glu23Lys/CT); ADIPOQ/KCNJ11/TCF7L2 (G276T/Glu23Lys /CT). The IVS3C>T locus in the TCF7L2 gene is not independently statistically significantly associated with the development of T2DM; however, its predisposing effect has been identified in its combination with the variant genotypes of the polymorphic loci G276T in the ADIPOQ gene and Glu23Lys in the KCNJ11 gene.

A new anti-MRSA antibiotic complex, WAP-8294A II. Structure characterization of minor components by ESI LCMS and MS/MS.

PubMed

Kato, Azusa; Hirata, Haruhisa; Ohashi, Yoshitami; Fujii, Kiyonaga; Mori, Kenji; Harada, Ken-ichi

2011-05-01

The anti-MRSA antibiotic, WAP-8294A, was isolated from the fermentation broth of Lysobacter sp. The major component, WAP-8294A2, is composed of 1 mol of Gly, L-Leu, L-Glu, D-Asn, D-Trp, D-threo-β-hydroxyasparagine, N-Me-D-Phe and N-Me-L-Val, and 2 mol of L-Ser, D-Orn and D-3-hydroxy-7-Me-octanoic acid. The structure of the WAP-8294A2 was mainly determined as a cyclic depsipeptide by 2D NMR experiments. However, it was difficult to use the NMR experiment to determine the minor components, A1, A4 and Ax13, isolated in small amounts. In the present study, ESI MS/MS was applied to the structure elucidation of these minor components. The structures of these minor components were determined on the basis of the fragmentation pattern of the product ions of WAP-8294A2 in the ESI MS/MS. As a result, it was confirmed that A1 and A4 had the same amino acid sequence as A2, while A1 and A4 had the 3-OH-octanoic acid and 3-OH-8-Me-nonanoic acid, respectively, in the place of the 3-OH-7-Me-octanoic acid in A2. In the structure of Ax13, it was found that Gly of A2 was changed to β-Ala of Ax13. © 2011 Japan Antibiotics Research Association All rights reserved

Peptide/protein-polymer conjugates: synthetic strategies and design concepts.

PubMed

Gauthier, Marc A; Klok, Harm-Anton

2008-06-21

This feature article provides a compilation of tools available for preparing well-defined peptide/protein-polymer conjugates, which are defined as hybrid constructs combining (i) a defined number of peptide/protein segments with uniform chain lengths and defined monomer sequences (primary structure) with (ii) a defined number of synthetic polymer chains. The first section describes methods for post-translational, or direct, introduction of chemoselective handles onto natural or synthetic peptides/proteins. Addressed topics include the residue- and/or site-specific modification of peptides/proteins at Arg, Asp, Cys, Gln, Glu, Gly, His, Lys, Met, Phe, Ser, Thr, Trp, Tyr and Val residues and methods for producing peptides/proteins containing non-canonical amino acids by peptide synthesis and protein engineering. In the second section, methods for introducing chemoselective groups onto the side-chain or chain-end of synthetic polymers produced by radical, anionic, cationic, metathesis and ring-opening polymerization are described. The final section discusses convergent and divergent strategies for covalently assembling polymers and peptides/proteins. An overview of the use of chemoselective reactions such as Heck, Sonogashira and Suzuki coupling, Diels-Alder cycloaddition, Click chemistry, Staudinger ligation, Michael's addition, reductive alkylation and oxime/hydrazone chemistry for the convergent synthesis of peptide/protein-polymer conjugates is given. Divergent approaches for preparing peptide/protein-polymer conjugates which are discussed include peptide synthesis from synthetic polymer supports, polymerization from peptide/protein macroinitiators or chain transfer agents and the polymerization of peptide side-chain monomers.

Alterations in Topoisomerase IV and DNA Gyrase in Quinolone-Resistant Mutants of Mycoplasma hominis Obtained In Vitro

PubMed Central

Bébéar, Cécile M.; Renaudin, Hélène; Charron, Alain; Bové, Joseph M.; Bébéar, Christiane; Renaudin, Joel

1998-01-01

Mycoplasma hominis mutants were selected stepwise for resistance to ofloxacin and sparfloxacin, and their gyrA, gyrB, parC, and parE quinolone resistance-determining regions were characterized. For ofloxacin, four rounds of selection yielded six first-, six second-, five third-, and two fourth-step mutants. The first-step mutants harbored a single Asp426→Asn substitution in ParE. GyrA changes (Ser83→Leu or Trp) were found only from the third round of selection. With sparfloxacin, three rounds of selection generated 4 first-, 7 second-, and 10 third-step mutants. In contrast to ofloxacin resistance, GyrA mutations (Ser83→Leu or Ser84→Trp) were detected in the first-step mutants prior to ParC changes (Glu84→Lys), which appeared only after the second round of selection. Further analysis of eight multistep-selected mutants of M. hominis that were previously described (2) revealed that they carried mutations in ParE (Asp426→Asn), GyrA (Ser83→Leu) and ParE (Asp426→Asn), GyrA (Ser83→Leu) and ParC (Ser80→Ile), or ParC (Ser80→Ile) alone, depending on the fluoroquinolone used for selection, i.e., ciprofloxacin, norfloxacin, ofloxacin, or pefloxacin, respectively. These data indicate that in M. hominis DNA gyrase is the primary target of sparfloxacin whereas topoisomerase IV is the primary target of pefloxacin, ofloxacin, and ciprofloxacin. PMID:9736554

Efficient Absorption of X-Hydroxyproline (Hyp)-Gly after Oral Administration of a Novel Gelatin Hydrolysate Prepared Using Ginger Protease.

PubMed

Taga, Yuki; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

2016-04-13

Recent studies have reported that oral intake of gelatin hydrolysate has various beneficial effects, such as reduction of joint pain and lowering of blood sugar levels. In this study, we produced a novel gelatin hydrolysate using a cysteine-type ginger protease having unique substrate specificity with preferential peptide cleavage with Pro at the P2 position. Substantial amounts of X-hydroxyproline (Hyp)-Gly-type tripeptides were generated up to 2.5% (w/w) concomitantly with Gly-Pro-Y-type tripeptides (5%; w/w) using ginger powder. The in vivo absorption of the ginger-degraded gelatin hydrolysate was estimated using mice. The plasma levels of collagen-derived oligopeptides, especially X-Hyp-Gly, were significantly high (e.g., 2.3-fold for Glu-Hyp-Gly, p < 0.05) compared with those of the control gelatin hydrolysate, which was prepared using gastrointestinal proteases and did not contain detectable X-Hyp-Gly. This study demonstrated that orally administered X-Hyp-Gly was effectively absorbed into the blood, probably due to the high protease resistance of this type of tripeptide.

Isolation of an inhibitory insulin-like growth factor (IGF) binding protein from bone cell-conditioned medium: A potential local regulator of IGF action

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

Mohan, S.; Bautista, C.M.; Wergedal, J.

1989-11-01

Inhibitory insulin-like growth factor binding protein (In-IGF-BP) has been purified to homogeneity from medium conditioned by TE89 human osteosarcoma cells by two different methods using Sephadex G-100 gel filtration, FPLC Mono Q ion-exchange, HPLC C{sub 4} reverse-phase, HPLC CN reverse-phase and affinity chromatographies. In-IGF-BP thus purified appeared to be homogeneous and unique by the following criteria. (i) N-terminal sequence analysis yielded a unique sequence (Asp-Glu-Ala-Ile-His-Cys-Pro-Pro-Glu-Ser-Glu-Ala-Lys-Leu-Ala). (ii) Amino acid composition of In-IGF-BP revealed marked differences with the amino acid compositions of other known PBs. (iii) In-IGF-BP exhibited a single band with molecular mass of 25 kDa under reducing conditions on sodiummore » dodecyl sulfate/polyacrylamide gels. IGF-I and IGF-II but not insulin displaced the binding of {sup 125}I-labeled IGF-I or {sup 125}I-labeled IGF-II binding to In-IGF-BP. In-IGF-BP inhibited basal, IGF-stimulated bone cell proliferation and serum-stimulated bone cell proliferation. Forskolin increases synthesis of In-IGF-BP in TE85 human osteosarcoma cells in a dose-dependent manner. Based on these findings, the authors conclude that In-IGF-BP is a protein that has a unique sequence and significant biological actions on bone cells.« less

Induced Fit and the Catalytic Mechanism of Isocitrate Dehydrogenase†

PubMed Central

Gonçalves, Susana; Miller, Stephen P.; Carrondo, Maria A.; Dean, Anthony M.; Matias, Pedro M.

2012-01-01

NADP+ dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42) belongs to a large family of α-hydroxyacid oxidative β-decarboxylases that catalyze similar three-step reactions, with dehydrogenation to an oxaloacid intermediate preceding β-decarboxylation to an enol intermediate followed by tautomerization to the final α-ketone product. A comprehensive view of the induced fit needed for catalysis is revealed on comparing the first “fully closed” crystal structures of a pseudo-Michaelis complex of wild-type Escherichia coli IDH (EcoIDH) and the “fully closed” reaction product complex of the K100M mutant with previously obtained “quasi-closed” and “open” conformations. Conserved catalytic residues, binding the nicotinamide ring of NADP+ and the metal-bound substrate, move as rigid bodies during domain closure by a hinge motion that spans the central β-sheet in each monomer. Interactions established between Thr105 and Ser113, which flank the “phosphorylation loop”, and the nicotinamide mononucleotide moiety of NADP+ establish productive coenzyme binding. Electrostatic interactions of a Lys100-Leu103-Asn115-Glu336 tetrad play a pivotal role in assembling a catalytically competent active site. As predicted, Lys230* is positioned to deprotonate/reprotonate the α-hydroxyl in both reaction steps and Tyr160 moves into position to protonate C3 following β-decarboxylation. A proton relay from the catalytic triad Tyr160-Asp307-Lys230* connects the α-hydroxyl of isocitrate to the bulk solvent to complete the picture of the catalytic mechanism. PMID:22891681

[GENOMIC VARIABILITY IN PATIENTS WITH DUCTAL FORM OF BREAST CANCER AND THE POSSIBILITY OF CORRECTION THE PEPTIDE BIOREGULATOR AND METAL IONS].

PubMed

Jokhadze, T; Monaselidze, J; Nemsadze, G; Buadze, T; Gaiozishvili, M; Lezhava, T

2017-01-01

Level of genome stability (structural aberrations, aneuploidy and fragile sites) was studied in cells of the lymphocyte culture of ductal breast cancer patients (DBC). Was studied the correctional influence of separate and combinative action of peptide bioregulator (Ala-Glu-Asp-Gly) and heavy metal - nickel. It is shown that DBC patients are characterized by high level of genome instability, which is the result of the chromatin changing state. The used tests makes it possible to conclude that in the case of this form of cancer subordinates to specific epigenetic variation as a hetero- also euchromatic regions of genome. The agents - peptide bioregulator (Ala-Glu-Asp-Gly) and nickel ions, used in cell culture of ductal breast cancer patients, revealed the protective effect what indicates the prospects to further study for their involving purpose in combined therapy of this form of cancer.

The potential role of toll-like receptor 4 Asp299Gly polymorphism and its association with recurrent cystic echinococcosis in postoperative patients.

PubMed

Noori, Jafar; Spotin, Adel; Ahmadpour, Ehsan; Mahami-Oskouei, Mahmoud; Sadeghi-Bazargani, Homayoun; Kazemi, Tohid; Sakhinia, Ebrahim; Aghebati-Maleki, Leili; Shahrivar, Firooz

2018-06-01

The study of pathogenesis mechanisms of larval stages in the Taeniidae has recently focused on host genetic factors, particularly toll-like receptor (TLR) variations. However, the potential role of TLR4 polymorphism in hydatidosis has not yet been sufficiently elucidated in postoperative patients. In this case-control investigation, 80 patients from Iran, including 40 with acute hydatidosis (AH) and 40 with recurrent hydatidosis (RH), and 80 ethnically matched controls were evaluated from February 2015 to February 2017. Hydatidosis patients were confirmed using radiological, immunological, and histopathological examinations. Genotyping of Asp299Gly and Thr399Ile of TLR4 single-nucleotide polymorphisms was determined by restriction fragment length polymorphism, sequencing, and phylogenetic strategies. The heterozygous mutant-type TLR4 Asp299Gly genotype indicated a tendency to be associated with the occurrence of RH (P = 0.060) and conferred a 3-fold risk for susceptibility. There was no difference in genotype frequency of Asp299Gly between patients with AH and healthy controls (P = 0.42; OR, 1.82; 95% CI, 0.11-30.1%). Interestingly, a frequency of the G allele (12%: Gly) was observed to be a risk factor for susceptibility to RH patients (P = 0.050; OR, 7.08; 95% CI, 0.97-51.5%). A relative genetic variability of TLR4 Asp299Gly was found in RH patients (haplotype diversity: 0.700) compared to AH patients and healthy controls (Hd: 0.000). The Asp299Gly genotype was dominantly identified in patients with hepatic hydatid cysts. The TLR4 Thr399Ile codon was not detected except in a patient with a pulmonary hydatid cyst. The current findings enhance our knowledge regarding the TLR4 Asp299Gly polymorphism potentially leading to the development of RH, by skewing the immune system towards a Th2 response. Identification of the Asp299Gly codon may be a diagnostic hallmark in RH patients who have undergone unsuccessful postoperative intervention. However, further studies with a higher case number are needed on ethnic population from various geographic regions, in order to confirm this hypothesis.

Indacaterol/glycopyrronium is cost-effective compared to salmeterol/fluticasone in COPD: FLAME-based modelling in a Swedish population.

PubMed

Bjermer, Leif; van Boven, Job F M; Costa-Scharplatz, Madlaina; Keininger, Dorothy L; Gutzwiller, Florian S; Lisspers, Karin; Mahon, Ronan; Olsson, Petter; Roche, Nicolas

2017-12-11

This study assessed the cost-effectiveness of indacaterol/glycopyrronium (IND/GLY) versus salmeterol/fluticasone (SFC) in chronic obstructive pulmonary disease (COPD) patients with moderate to very severe airflow limitation and ≥1 exacerbation in the preceding year. A previously published and validated patient-level simulation model was adapted using clinical data from the FLAME trial and real-world cost data from the ARCTIC study. Costs (total monetary costs comprising drug, maintenance, exacerbation, and pneumonia costs) and health outcomes (life-years (LYs), quality-adjusted life-years (QALYs)) were projected over various time horizons (1, 5, 10 years, and lifetime) from the Swedish payer's perspective and were discounted at 3% annually. Uncertainty in model input values was studied through one-way and probabilistic sensitivity analyses. Subgroup analyses were also performed. IND/GLY was associated with lower costs and better outcomes compared with SFC over all the analysed time horizons. Use of IND/GLY resulted in additional 0.192 LYs and 0.134 QALYs with cost savings of €1211 compared with SFC over lifetime. The net monetary benefit (NMB) was estimated to be €8560 based on a willingness-to-pay threshold of €55,000/QALY. The NMB was higher in the following subgroups: severe (GOLD 3), high risk and more symptoms (GOLD D), females, and current smokers. IND/GLY is a cost-effective treatment compared with SFC in COPD patients with mMRC dyspnea grade ≥ 2, moderate to very severe airflow limitation, and ≥1 exacerbation in the preceding year.

Combined effects of ankylosing spondylitis-associated ERAP1 polymorphisms outside the catalytic and peptide-binding sites on the processing of natural HLA-B27 ligands.

PubMed

Martín-Esteban, Adrian; Gómez-Molina, Patricia; Sanz-Bravo, Alejandro; López de Castro, José A

2014-02-14

ERAP1 polymorphism involving residues 528 and 575/725 is associated with ankylosing spondylitis among HLA-B27-positive individuals. We used four recombinant variants to address the combined effects of the K528R and D575N polymorphism on the processing of HLA-B27 ligands. The hydrolysis of a fluorogenic substrate, Arg-528/Asp-575 < Lys-528/Asp-575 < Arg-528/Asn-575 < Lys-528/Asn-575, indicated that the relative activity of variants carrying Arg-528 or Lys-528 depends on residue 575. Asp-575 conferred lower activity than Asn-575, but the difference depended on residue 528. The same hierarchy was observed with synthetic precursors of HLA-B27 ligands, but the effects were peptide-dependent. Sometimes the epitope yields were variant-specific at all times. For other peptides, concomitant generation and destruction led to similar epitope amounts with all the variants at long, but not at short, digestion times. The generation/destruction balance of two related HLA-B27 ligands was analyzed in vitro and in live cells. Their relative yields at long digestion times were comparable with those from HLA-B27-positive cells, suggesting that ERAP1 was a major determinant of the abundance of these peptides in vivo. The hydrolysis of fluorogenic and peptide substrates by an HLA-B27 ligand or a shorter peptide, respectively, was increasingly inhibited as a function of ERAP1 activity, indicating that residues 528 and 575 affect substrate inhibition of ERAP1 trimming. The significant and complex effects of co-occurring ERAP1 polymorphisms on multiple HLA-B27 ligands, and their potential to alter the immunological and pathogenetic features of HLA-B27 as a function of the ERAP1 context, explain the epistatic association of both molecules in ankylosing spondylitis.

Combined Effects of Ankylosing Spondylitis-associated ERAP1 Polymorphisms Outside the Catalytic and Peptide-binding Sites on the Processing of Natural HLA-B27 Ligands*

PubMed Central

Martín-Esteban, Adrian; Gómez-Molina, Patricia; Sanz-Bravo, Alejandro; López de Castro, José A.

2014-01-01

ERAP1 polymorphism involving residues 528 and 575/725 is associated with ankylosing spondylitis among HLA-B27-positive individuals. We used four recombinant variants to address the combined effects of the K528R and D575N polymorphism on the processing of HLA-B27 ligands. The hydrolysis of a fluorogenic substrate, Arg-528/Asp-575 < Lys-528/Asp-575 < Arg-528/Asn-575 < Lys-528/Asn-575, indicated that the relative activity of variants carrying Arg-528 or Lys-528 depends on residue 575. Asp-575 conferred lower activity than Asn-575, but the difference depended on residue 528. The same hierarchy was observed with synthetic precursors of HLA-B27 ligands, but the effects were peptide-dependent. Sometimes the epitope yields were variant-specific at all times. For other peptides, concomitant generation and destruction led to similar epitope amounts with all the variants at long, but not at short, digestion times. The generation/destruction balance of two related HLA-B27 ligands was analyzed in vitro and in live cells. Their relative yields at long digestion times were comparable with those from HLA-B27-positive cells, suggesting that ERAP1 was a major determinant of the abundance of these peptides in vivo. The hydrolysis of fluorogenic and peptide substrates by an HLA-B27 ligand or a shorter peptide, respectively, was increasingly inhibited as a function of ERAP1 activity, indicating that residues 528 and 575 affect substrate inhibition of ERAP1 trimming. The significant and complex effects of co-occurring ERAP1 polymorphisms on multiple HLA-B27 ligands, and their potential to alter the immunological and pathogenetic features of HLA-B27 as a function of the ERAP1 context, explain the epistatic association of both molecules in ankylosing spondylitis. PMID:24352655

Towards a prevention program for β-thalassemia. The molecular spectrum in East Java, Indonesia.

PubMed

Hernanda, Pratika Yuhyi; Tursilowati, Luluk; Arkesteijn, Sandra G J; Ugrasena, I Dewa Gede; Larasati, Marian C Shanty; Soeatmadji, Sentot Mustajab; Giordano, Piero C; Harteveld, Cornelis L

2012-01-01

Defining the spectrum of specific thalassemia mutations is an important issue when planning prevention programs in large multi ethnic countries as is Indonesia. In a first attempt to define the prevalence of the common mutations in East Java we selected a cohort of 17 transfusion-dependent patients attending the Dr. Soetomo Hospital, Surabaya, Indonesia. After basic diagnostics we performed direct DNA sequencing for all β-globin genes. The results obtained on 34 independent chromosomes revealed the following prevalence rates: c.79 G>A p. Glu27Lys (Hb E) 47.0%; c.92+5G>C (IVS-I-5 G>C) 20.6%; c.109_110 delC p.Pro37Leu fs X7 [codon 35 (-C)] 17.6%; c.46del T p.Trp16Gly fsX4 [codon 15 (-T)] 5.9%; c.126_129delCTTT p. Phe42Leu fs X19 (codons 41/42) 2.9%; c.316-197 C>T [IVS-II-654 (C>T)] 2.9%; c*112 A>G (PolyA) 2.9%. Our preliminary results show that the distribution of the prevalent mutations in our cohort is quite homogeneous but with different forms than previously reported. This indicates that more studies on a larger scale and in different geographical areas are needed to refine our provisional results and to characterize the molecular background of the disease in the whole country.

Entropy and enthalpy of interaction between amino acid side chains in nanopores

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

Vaitheeswaran, S., E-mail: vaithee05@gmail.com; Thirumalai, D.; Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742

2014-12-14

Understanding the stabilities of proteins in nanopores requires a quantitative description of confinement induced interactions between amino acid side chains. We use molecular dynamics simulations to study the nature of interactions between the side chain pairs ALA-PHE, SER-ASN, and LYS-GLU in bulk water and in water-filled nanopores. The temperature dependence of the bulk solvent potentials of mean force and the interaction free energies in cylindrical and spherical nanopores is used to identify the corresponding entropic and enthalpic components. The entropically stabilized hydrophobic interaction between ALA and PHE in bulk water is enthalpically dominated upon confinement depending on the relative orientationsmore » between the side chains. In the case of SER-ASN, hydrogen bonded configurations that are similar in bulk water are thermodynamically distinct in a cylindrical pore, thus making rotamer distributions different from those in the bulk. Remarkably, salt bridge formation between LYS-GLU is stabilized by entropy in contrast to the bulk. Implications of our findings for confinement-induced alterations in protein stability are briefly outlined.« less

Evidence that a functional fertilin-like ADAM plays a role in human sperm-oolemmal interactions.

PubMed

Bronson, R A; Fusi, F M; Calzi, F; Doldi, N; Ferrari, A

1999-05-01

Fertilin is a protein initially identified in guinea pig spermatozoa; it is the prototype of a larger family of conserved, proteins designated as a disintegrin and a metalloproteinase (ADAM). These heterodimers which consist of alpha and beta subunits, containing metalloproteinase-like and disintegrin-like domains, appear to play a role in mammalian fertilization. Peptides derived from the disintegrin domains of two ADAMs, fertilin and cyritestin, interfere with gamete adhesion and sperm-egg membrane fusion in non-human species. It has been suggested that fertilin-beta binds to an oolemmal integrin, and it is proposed that the tripeptide FEE (Phe-Glu-Glu) is the integrin recognition sequence in human fertilin-beta. We evaluated whether fertilin beta plays a role in human fertilization by studying the effects of a linear octapeptide containing the FEE sequence, SFEECDLP, and a scrambled octapeptide with the same amino acids, SFPCEDEL, on the incorporation of human spermatozoa by human zona-free eggs. The effects of G4120, a potent RGD-containing (Arg-Gly-Asp) thioether-bridged cyclic peptide which blocks both fibronectin and vitronectin receptors, and the relationship between FEE- and RGD-receptor interactions on sperm-egg interactions were also studied. The FEE-containing peptide, but not the scrampled peptide, inhibited sperm adhesion to oocytes and their penetration, over the range 1-5 microM. The inhibition induced by SFEECDLP was reversible and occurred only in the presence of peptide itself. The G4120 peptide exhibited 10-fold less inhibitory effects on sperm adhesion and penetration than did SFEECDLP. When combined, SFEECDLP and G4120 exhibited strong inhibition of both adhesion and penetration at concentrations that individually had been ineffective, suggesting co-operation between the two receptor-ligand interactions during fertilization. We propose that a fertilin-like molecule is functionally active on human spermatozoa and that its interaction with an oolemmal integrin receptor plays a role in fertilization in humans.

An endo-acting proline-specific oligopeptidase from Treponema denticola ATCC 35405: evidence of hydrolysis of human bioactive peptides.

PubMed Central

Mäkinen, P L; Mäkinen, K K; Syed, S A

1994-01-01

An endo-acting proline-specific oligopeptidase (prolyl oligopeptidase [POPase], EC 3.4.21.26) was purified to homogeneity from the Triton X-100 extracts of cells of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised five successive fast protein liquid chromatography steps. The POPase is a cell-associated 75- to 77-kDa protein with an isoelectric point of ca. 6.5. The enzyme hydrolyzed (optimum pH 6.5) the Pro-pNA bond in carbobenzoxy-Gly-Pro-p-nitroanilide (Z-Gly-Pro-pNA) and bonds at the carboxyl side of proline in several human bioactive peptides, such as bradykinin, substance P, neurotensin, angiotensins, oxytocin, vasopressin, and human endothelin fragment 22-38. The minimum hydrolyzable peptide size was tetrapeptide P3P2P1P'1, while the maximum substrate size was ca. 3 kDa. An imino acid residue in position P1 was absolutely necessary. The hydrolysis of Z-Gly-Pro-pNA was potently inhibited by the following, with the Ki(app) (in micromolar) in parentheses: insulin B-chain (0.7), human endothelin-1 (0.5), neuropeptide Y (1.7), substance P (32.0), T-kinin (4.0), neurotensin (5.0), and bradykinin (16.0). Chemical modification and inhibition studies suggest that the POPase is a serine endopeptidase whose activity depends on the catalytic triad of COOH ... Ser ... His but not on a metal. The amino acid sequence around the putative active-site serine is Gly-Gly-Ser-Asn-Pro-Gly. The enzyme is suggested to contain a reactive cysteinyl residue near the active site. Amino acid residues 4 to 24 of the first 24 N-terminal residues showed a homology of 71% with the POPase precursor from Flavobacterium meningosepticum and considerable homology with the Aeromonas hydrophila POPase. The ready hydrolysis of human bioactive peptides at bonds involving an imino acid residue suggests that enzymes like POPase may contribute to the chronicity of periodontal infections by participating in the peptidolytic processing of those peptides. Images PMID:7523301

Processing, stability, and kinetic parameters of C5a peptidase from Streptococcus pyogenes.

PubMed

Anderson, Elizabeth T; Wetherell, Michael G; Winter, Laurie A; Olmsted, Stephen B; Cleary, Patrick P; Matsuka, Yury V

2002-10-01

A recombinant streptococcal C5a peptidase was expressed in Escherichia coli and its catalytic properties and thermal stability were subjected to examination. It was shown that the NH2-terminal region of C5a peptidase (Asn32-Asp79/Lys90) forms the pro-sequence segment. Upon maturation the propeptide is hydrolyzed either via an autocatalytic intramolecular cleavage or by exogenous protease streptopain. At pH 7.4 the enzyme exhibited maximum activity in the narrow range of temperatures between 40 and 43 degrees C. The process of heat denaturation of C5a peptidase investigated by fluorescence and circular dichroism spectroscopy revealed that the protein undergoes biphasic unfolding transition with Tm of 50 and 70 degrees C suggesting melting of different parts of the molecule with different stability. Unfolding of the less stable structures was accompanied by the loss of proteolytic activity. Using synthetic peptides corresponding to the COOH-terminus of human complement C5a we demonstrated that in vitro peptidase catalyzes hydrolysis of two His67-Lys68 and Ala58-Ser59 peptide bonds. The high catalytic efficiency obtained for the SQLRANISHKDMQLGR extended peptide compared to the poor hydrolysis of its derivative Ac-SQLRANISH-pNA that lacks residues at P2'-P7' positions, suggest the importance of C5a peptidase interactions with the P' side of the substrate.

Comparative analysis of the expression of c-Fos and interleukin-2 proteins in hypothalamus cells during various treatments.

PubMed

Barabanova, S V; Artyukhina, Z E; Ovchinnikova, K T; Abramova, T V; Kazakova, T B; Khavinson, V Kh; Malinin, V V; Korneva, E A

2008-03-01

The aim of the present work was to perform a combined analysis of the degree of activation of the anterior hypothalamus of the rat and expression of the interleukin-2 gene during treatments of different types: mild stress ("handling") and adaption to it, as well as intranasal administration of physiological saline and the peptides Vilon (Lys-Glu) and Epitalon (Ala-Glu-Asp-Gly). Changes in the numbers of c-Fos-and IL-2-positive cells in structures of the lateral area (LHA) and anterior (AHN), supraoptic (SON), and paraventricular (PVN) nuclei of the hypothalamus in Wistar rats. Ratios of the quantities of c-Fos-and IL-2-positive cells were determined in intact animals and after activation of brain cells initiated by different treatments; the influences of adaptation to handling on the nature of changes in the expression of these proteins was also studied. Combined analysis of the intensity of expression of these two proteins - c-Fos, a marker of neuron activation and a trans-factor for the IL-2 cytokine gene and other inducible genes, and IL-2 - in intact animals and after various treatments showed that the process of cell activation in most of the hypothalamic structures studied correlated with decreases in the quantity of IL-2-positive cells in these structures; different patterns of changes in the numbers of c-Fos-and IL-2-positive cells were seen in response to different treatments in conditions of stress and adaptation to it.

Structural insight of dopamine β-hydroxylase, a drug target for complex traits, and functional significance of exonic single nucleotide polymorphisms.

PubMed

Kapoor, Abhijeet; Shandilya, Manish; Kundu, Suman

2011-01-01

Human dopamine β-hydroxylase (DBH) is an important therapeutic target for complex traits. Several single nucleotide polymorphisms (SNPs) have also been identified in DBH with potential adverse physiological effect. However, difficulty in obtaining diffractable crystals and lack of a suitable template for modeling the protein has ensured that neither crystallographic three-dimensional structure nor computational model for the enzyme is available to aid rational drug design, prediction of functional significance of SNPs or analytical protein engineering. Adequate biochemical information regarding human DBH, structural coordinates for peptidylglycine alpha-hydroxylating monooxygenase and computational data from a partial model of rat DBH were used along with logical manual intervention in a novel way to build an in silico model of human DBH. The model provides structural insight into the active site, metal coordination, subunit interface, substrate recognition and inhibitor binding. It reveals that DOMON domain potentially promotes tetramerization, while substrate dopamine and a potential therapeutic inhibitor nepicastat are stabilized in the active site through multiple hydrogen bonding. Functional significance of several exonic SNPs could be described from a structural analysis of the model. The model confirms that SNP resulting in Ala318Ser or Leu317Pro mutation may not influence enzyme activity, while Gly482Arg might actually do so being in the proximity of the active site. Arg549Cys may cause abnormal oligomerization through non-native disulfide bond formation. Other SNPs like Glu181, Glu250, Lys239 and Asp290 could potentially inhibit tetramerization thus affecting function. The first three-dimensional model of full-length human DBH protein was obtained in a novel manner with a set of experimental data as guideline for consistency of in silico prediction. Preliminary physicochemical tests validated the model. The model confirms, rationalizes and provides structural basis for several biochemical data and claims testable hypotheses regarding function. It provides a reasonable template for drug design as well.

Prevalence of Factor V Genetic Variants Associated With Indian APCR Contributing to Thrombotic Risk.

PubMed

Sharma, Amit; Bhakuni, Teena; Biswas, Arijit; Ranjan, Ravi; Kumar, Ravi; Kishore, Kamal; Mahapatra, Manoranjan; Jairajpuri, Mohamad Aman; Saxena, Renu

2017-09-01

Phenotypic resistance to activated protein C (APC) is a complex mechanism associated with increased thrombosis risk. Activated protein C resistance (APCR) is mainly influenced by FV Leiden mutation, and various other single nucleotide polymorphisms (SNPs) in FV gene are known to be associated with APCR. The aim of present study was to investigate the incidence and assess possible mechanisms of APCR in Indian patients with deep vein thrombosis (DVT). Three hundred and ten Doppler-proven patients with DVT were screened for APCR, and 50 APCR positive patients and 50 controls were typed for FV Leiden , Hong Kong, Cambridge, HR2 haplotype, Glu666Asp, Ala485Lys, and Liverpool using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or allele specific PCR. FV Leiden was commonest cause of APCR (50%) in Indian patients with DVT being statistically significant ( P = .001) compared to controls. FV Liverpool, FV Glu666Asp and FV Ala485Lys were studied for the first time in Indian population. FV Liverpool, FV Glu666Asp, Hong Kong, and Cambridge were found to be absent. High frequency of Ala485Lys in patients shows that it might be a risk factor contributing to APCR in Indian patients with DVT. HR2 haplotype was not associated with APCR; however, presence of homozygous HR2 haplotype in patients only indicates the role it might play in Indian APCR population. In conclusion, contribution of FV Leiden causing APCR in Indian population is not as strong as previously reported in Western countries. The presence of other SNPs observed in the present study requires such studies on larger sample size to understand the molecular basis of defect.

Structure of the carboxypeptidase B complex with N-sulfamoyl-L-phenylalanine - a transition state analog of non-specific substrate.

PubMed

Akparov, Valery; Timofeev, Vladimir; Khaliullin, Ilyas; Švedas, Vytas; Kuranova, Inna

2018-03-01

Carboxypeptidase B (EC 3.4.17.2) (CPB) is commonly used in the industrial insulin production and as a template for drug design. However, its ability to discriminate substrates with hydrophobic, hydrophilic, and charged side chains is not well understood. We report structure of CPB complex with a transition state analog N-sulfamoyl-L-phenylalanine solved at 1.74Å. The study provided an insight into structural basis of CPB substrate specificity. Ligand binding is affected by structure-depended conformational changes of Asp255 in S1'-subsite, interactions with Asn144 and Arg145 in C-terminal binding subsite, and Glu270 in the catalytic center. Side chain of the non-specific substrate analog SPhe in comparison with that of specific substrate analog SArg (reported earlier) not only loses favorable electrostatic interactions and two hydrogen bonds with Asp255 and three fixed water molecules, but is forced to be in the unfavorable hydrophilic environment. Thus, Ser207, Gly253, Tyr248, and Asp255 residues play major role in the substrate recognition by S1'-subsite.

Structure-guided modification of Rhizomucor miehei lipase for production of structured lipids.

PubMed

Zhang, Jun-Hui; Jiang, Yu-Yan; Lin, Ying; Sun, Yu-Fei; Zheng, Sui-Ping; Han, Shuang-Yan

2013-01-01

To improve the performance of yeast surface-displayed Rhizomucor miehei lipase (RML) in the production of human milk fat substitute (HMFS), we mutated amino acids in the lipase substrate-binding pocket based on protein hydrophobicity, to improve esterification activity. Five mutants: Asn87Ile, Asn87Ile/Asp91Val, His108Leu/Lys109Ile, Asp256Ile/His257Leu, and His108Leu/Lys109Ile/Asp256Ile/His257Leu were obtained and their hydrolytic and esterification activities were assayed. Using Discovery Studio 3.1 to build models and calculate the binding energy between lipase and substrates, compared to wild-type, the mutant Asp256Ile/His257Leu was found to have significantly lower energy when oleic acid (3.97 KJ/mol decrease) and tripalmitin (7.55 KJ/mol decrease) were substrates. This result was in accordance with the esterification activity of Asp256Ile/His257Leu (2.37-fold of wild-type). The four mutants were also evaluated for the production of HMFS in organic solvent and in a solvent-free system. Asp256Ile/His257Leu had an oleic acid incorporation of 28.27% for catalyzing tripalmitin and oleic acid, and 53.18% for the reaction of palm oil with oleic acid. The efficiency of Asp256Ile/His257Leu was 1.82-fold and 1.65-fold that of the wild-type enzyme for the two reactions. The oleic acid incorporation of Asp256Ile/His257Leu was similar to commercial Lipozyme RM IM for palm oil acidolysis with oleic acid. Yeast surface-displayed RML mutant Asp256Ile/His257Leu is a potential, economically feasible catalyst for the production of structured lipids.

Water Dynamics in Egg White Peptide, Asp-His-Thr-Lys-Glu, Powder Monitored by Dynamic Vapor Sorption and LF-NMR.

PubMed

Yang, Shuailing; Liu, Xuye; Jin, Yan; Li, Xingfang; Chen, Feng; Zhang, Mingdi; Lin, Songyi

2016-03-16

Water absorbed into the bulk amorphous structure of peptides can have profound effects on their properties. Here, we elucidated water dynamics in Asp-His-Thr-Lys-Glu (DHTKE), an antioxidant peptide derived from egg white ovalbumin, using water dynamic vapor sorption (DVS) and low-field nuclear magnetic resonance (LF-NMR). The DVS results indicated that parallel exponential kinetics model fitted well to the data of sorption kinetics behavior of DHTKE. Four different proton fractions with different mobilities were identified based on the degree of interaction between peptide and water. The water could significantly change the proton distribution and structure of the sample. The different phases of moisture absorption were reflected in the T2 parameters. In addition, the combined water content was dominant in the hygroscopicity of DHTKE. This study provides an effective real-time monitoring method for water mobility and distribution in synthetic peptides, and this method may have applications in promoting peptide quality assurance.

Leptin receptor Lys656Asn polymorphism is associated with decreased leptin response and weight loss secondary to a lifestyle modification in obese patients.

PubMed

de Luis Roman, Daniel; de la Fuente, Rocio Aller; Sagrado, Manuel Gonzalez; Izaola, Olatz; Vicente, Rosa Conde

2006-10-01

Human obesity is characterized by high levels of leptin, and it has been suggested that obese patients may be leptin resistant. The aim of our study was to investigate the influence of Lys656Asn polymorphism in the leptin receptor gene on leptin response and weight loss secondary to a lifestyle modification (Mediterranean hypocaloric diet and exercise) in obese patients. A population of 67 obese (body mass index >30) nondiabetic outpatients was analyzed in a prospective way. Before and after 3 months of lifestyle modification program, bipolar electrical bioimpedance, blood pressure, and a serial assessment of nutritional intake with 3 days written food records and biochemical analysis were performed. The lifestyle modification program consisted of a hypocaloric diet (1520 kcal, 52% carbohydrates, 25% lipids and 23% proteins). The exercise program consisted of aerobic exercise for at least three times per week (60 min each). Statistical analysis was performed for the combined Lys656/Asn656 and Asn656/Asn656 as mutant group and type Lys656/Lys 656 as wild-type second group. Sixty seven patients gave informed consent and were enrolled in the study. The mean age was 45.7 +/- 16.6 years and the mean BMI 34.1 +/- 5.1, with 18 males (26.9%) and 49 females (73.1%). Thirty six patients (10 males/26 females) (46.8%) had the genotype Lys656/Lys 656 (wild-type group) and 31 patients (8 males/23 females) (46.3%) Lys656/Asn656 (n = 28, 41.8%) or Asn656/Asn656 (n = 3, 4.5%) (mutant group). The percentage of responders (weight loss) was similar in both groups (91.7 vs. 87.1%). In wild-type group (responders and nonresponders), BMI, weight, fat mass, systolic blood pressure and waist circumference decreased. In mutant group, BMI, weight and waist circumference decreased. No differences were detected between basal values in both groups. Only leptin levels decreased significantly in wild-type group (11.5%; p <0.05) (57.3 +/- 31.5 ng/mL vs. 45.8 +/- 29.3 ng/mL; p <0.05). In mutant group, leptin increased without statistical differences (0.44%; ns). Patients with Asn656 allele of LEPR gene have a different response than wild-type patients, and Lys656Lys patients have a significant decrease in weight, BMI, fat mass, waist circumference, systolic blood pressure and leptin levels.

Alteration of high and low spin equilibrium by a single mutation of amino acid 209 in mouse cytochromes P450.

PubMed

Iwasaki, M; Juvonen, R; Lindberg, R; Negishi, M

1991-02-25

The identities of the amino acid at position 209 are most critical in determining specific coumarin 7- and steroid 15 alpha-hydroxylase activity in P450coh and P450(15)alpha, respectively. This system, therefore, provides us with an excellent model to study the structural basis for P450 specificity as a monooxygenase. We expressed in Saccharomyces cerevisiae a series of the mutated P450s in which residue 209 was substituted with the various amino acids and characterized the spectral property and hydroxylase activity of these mutated P450s. The positioning of a hydrophobic residue including Phe, Leu, and Val at position 209 resulted in shifting the P450 to the high-spin state, while a charged amino acid such as Lys or Asp produced the low-spin form. Moreover, a P450 with Asn or Gly in this position exhibited spectra indicating a mixture of the high- and low-spin forms. This spin alteration, depending upon the hydrophobicity and size of residue at position 209, indicates that this position is likely to reside close to the sixth axial ligand on the distal surface of the heme in these P450s. This proximity of residue 209 to the ligand may explain the critical role of this residue in determining the hydroxylase specificity and activity of these P450s.

Characterization of an extracellular lipase from the biocontrol fungus, Nomuraea rileyi MJ, and its toxicity toward Spodoptera litura.

PubMed

Supakdamrongkul, Piyaporn; Bhumiratana, Amaret; Wiwat, Chanpen

2010-11-01

An extracellular lipase from Nomuraea rileyi MJ was purified 23.9-fold with 1.69% yield by ammonium sulfate precipitation followed by Sephacryl S-100 HR column chromatography. By mass spectrometry and SDS-polyacrylamide gel electrophoresis, the molecular weight of the homogenous lipase was 81kDa. The N-terminal sequence was determined as LeuSerValGluGlnThrLysLeuSerLysLeuAlaTyrAsnAsp and it showed no homology to sequences of known lipases. The optimum pH and temperature for activity were 8.0 and 35°C, respectively. The enzyme was stable in the pH range 7.0-9.0 and at 15-35°C for 1h. Higher activity was observed in the presence of surfactants, Na(+), NH(4)(+) ions, NaN(3) and ethylenediaminetetraacetic acid (EDTA), while Co(2+) and Cu(2+) ions, cysteine and dithiothreitol (DTT) strongly inhibited activity. The purified lipase hydrolyzed both synthetic and natural triglycerides with maximum activity for trilaurin and coconut oil, respectively. It also hydrolyzed esters of p-nitrophenol (pNP) with highest activity for p-nitrophenyl caprate (pNPCA). The purified lipase was found to promote N. rileyi spore germination in vitro in that germination reached 98% in conidial suspensions containing purified lipase at 2.75 U. Moreover, it enhanced toxicity of N. rileyi toward Spodoptera litura larvae with mortality via topical application reaching 63.3% at 4-10days post-treatment which calculated to be 2.7 times higher than the mortality obtained using conidial suspensions alone. Copyright © 2010 Elsevier Inc. All rights reserved.

Novel Peptide Ligands of RGS4 from a Focused One-Bead, One-Compound Library

PubMed Central

Roof, Rebecca A.; Sobczyk-Kojiro, Katarzyna; Turbiak, Anjanette J.; Roman, David L.; Pogozheva, Irina D.; Blazer, Levi L.; Neubig, Richard R.; Mosberg, Henry I.

2010-01-01

Regulators of G Protein Signaling (RGS) accelerate GTP hydrolysis by Gα subunits and profoundly inhibit signaling by G protein-coupled receptors. The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. We previously reported the design, mechanistic evaluation and structure-activity relationships (SAR) of a disulfide-containing cyclic peptide inhibitor of RGS4, YJ34 (Ac-Val-Lys-c[Cys-Thr-Gly-Ile-Cys]-Glu-NH2, S-S) (Roof, et al. Chem Biol Drug Des 2006; 67:266-274). Using a focused one-bead, one-compound (OBOC) peptide library that contains features known to be necessary for the activity of YJ34, we now identify peptides that bind to RGS4. Six peptides showed confirmed binding to RGS4 by flow cytometry. Two analogs of peptide 2, (Gly-Thr-c[Cys-Phe-Gly-Thr-Cys]-Trp-NH2, S-S with a free or acetylated N-terminus) inhibited RGS4-stimulated Gαo GTPase activity at 25–50 μM. They selectively inhibit RGS4 but not RGS7, RGS16 and RGS19. Their inhibition of RGS4 does not depend on cysteine-modification of RGS4, as they do not lose activity when all cysteines are removed from RGS4. Peptide 2 has been modeled to fit in the same binding pocket predicted for YJ34 but in the reverse orientation. PMID:18637987

Self and viral peptides can initiate lysis by autologous natural killer cells.

PubMed

Mandelboim, O; Wilson, S B; Valés-Gómez, M; Reyburn, H T; Strominger, J L

1997-04-29

Natural killer (NK) cells are inhibited by specific allotypes of class I major histocompatibility complex ligands recognized by polymorphic inhibitory receptors (e.g., NKIR1 and NKIR2). NK1- and NK2-specific clones recognize two groups of HLA-C allotypes that are distinguished by a dimorphism at residue 80 in the alpha1 helix (alphaLys-80 and alphaAsn-80, respectively). "Empty" HLA-Cw7 expressed in peptide transporter-deficient cells and HLA-Cw7 loaded with several peptides each functioned as inhibitory ligands for NK2 lines and clones. However, loading of HLA-Cw7 with two other peptides derived from glutamic acid decarboxylase or coxsackie virus (each of which has been associated with autoimmune diabetes mellitus) abrogated this inhibitory recognition. Both peptides contained Lys at P8 of the epitope. Substitution of P8 with Ala or two other basic amino acids, His and Arg, resulted in peptides that were inhibitory, as were peptides with P8 Val, Glu, or Asn. The manner in which a Lys at P8 might affect recognition is discussed, together with a hypothesis for a novel mechanism by which an autoimmune disease might be initiated.

Identification and Characterization of a Gene stp17 Located on the Linear Plasmid pBSSB1 as an Enhanced Gene of Growth and Motility in Salmonella enterica Serovar Typhi

PubMed Central

Zhang, Haifang; Zhu, Yunxia; Xie, Xiaofang; Wang, Min; Du, Hong; Xu, Shungao; Zhang, Ying; Gong, Mingyu; Ni, Bin; Xu, Huaxi; Huang, Xinxiang

2016-01-01

The linear plasmid pBSSB1 mediates the flagellar phase variation in H:z66 positive Salmonella enterica serovar Typhi (S. Typhi). The gene named stp17 (S. Typhi plasmid number 17 gene) is located on pBSSB1 and encodes the protein STP17. The expression pattern at the protein-level and function of STP17 remains unknown. In this study, the recombinant protein STP17His6 was expressed, purified and used to prepare the polyclonal anti-STP17 antibody. We detected protein-level expression of stp17 in S. Typhi and further investigated the protein expression characteristics of stp17 in different growth phases by western blot analysis. The effects of STP17 on bacterial growth and motility were analyzed. In addition, the structure of STP17 was predicted and the active site of STP17 was identified by site-directed mutagenesis. The results showed that STP17 was expressed stably in the wild type strain of S. Typhi. STP17 expression at the protein level peaks when cultures reach an OD600 value of 1.2. The growth rate and motility of the Δstp17 strain were significantly decreased compared with the wild type strain (P < 0.05) and this phenotype was restored in the stp17 complementary strain. Moreover, the growth rate and motility of the stp17 over-expression strain was greater than the wild type strain. STP17 contains nine Helix segments, six Stand segments and some Coil segments in the secondary structural level. The top-ranked 3-D structure of STP17 predicted by I-TASSER contains a putative ATPase domain and the amino acid residues of GLY16, GLY19, LYS20, ASN133, LYS157, and LYS158 may be the active site residues of STP17. Finally, STP17 was able to catalyze the ATP to ADP reaction, suggesting that STP17 may be an ATPase. To our knowledge, this is the first report describing the protein expression characteristics of STP17 in S. Typhi, showing that STP17 promotes bacterial growth and motility, which may be associated with its potential ATPase activity. PMID:27761429

Novel RS1 mutations associated with X-linked juvenile retinoschisis

PubMed Central

YI, JUNHUI; LI, SHIQIANG; JIA, XIAOYUN; XIAO, XUESHAN; WANG, PANFENG; GUO, XIANGMING; ZHANG, QINGJIONG

2012-01-01

To identify mutations in the retinoschisin (RS1) gene in families with X-linked retinoschisis (XLRS). Twenty families with XLRS were enrolled in this study. All six coding exons and adjacent intronic regions of RS1 were amplified by polymerase chain reaction (PCR). The nucleotide sequences of the amplicons were determined by Sanger sequencing. Ten hemizygous mutations in RS1 were detected in patients from 14 of the 20 families. Four of the ten mutations were novel, including c:176G>A (p:Cys59Tyr) in exon 3, c:531T>G (p:Tyr177X), c:607C>G (p:Pro203Ala) and c:668G>A (p:Cys223Tyr) in exon 6. These four novel mutations were not present in 176 normal individuals. The remaining six were recurrent mutations, including c:214G>A (p:Glu72Lys), c:304C>T (p:Arg102Trp), c:436G>A (p:Glu146Lys), c:544C>T (p:Arg182Cys), c:599G>A (p:Arg200His) and c:644A>T (p:Glu215Val). Our study expanded the mutation spectrum of RS1 and enriches our understanding of the molecular basis of XLRS. PMID:22245991

Novel RS1 mutations associated with X-linked juvenile retinoschisis.

PubMed

Yi, Junhui; Li, Shiqiang; Jia, Xiaoyun; Xiao, Xueshan; Wang, Panfeng; Guo, Xiangming; Zhang, Qingjiong

2012-04-01

To identify mutations in the retinoschisin (RS1) gene in families with X-linked retinoschisis (XLRS). Twenty families with XLRS were enrolled in this study. All six coding exons and adjacent intronic regions of RS1 were amplified by polymerase chain reaction (PCR). The nucleotide sequences of the amplicons were determined by Sanger sequencing. Ten hemizygous mutations in RS1 were detected in patients from 14 of the 20 families. Four of the ten mutations were novel, including c:176G>A (p:Cys59Tyr) in exon 3, c:531T>G (p:Tyr177X), c:607C>G (p:Pro203Ala) and c:668G>A (p:Cys223Tyr) in exon 6. These four novel mutations were not present in 176 normal individuals. The remaining six were recurrent mutations, including c:214G>A (p:Glu72Lys), c:304C>T (p:Arg102Trp), c:436G>A (p:Glu146Lys), c:544C>T (p:Arg182Cys), c:599G>A (p:Arg200His) and c:644A>T (p:Glu215Val). Our study expanded the mutation spectrum of RS1 and enriches our understanding of the molecular basis of XLRS.

The action of neutrophil serine proteases on elastin and its precursor.

PubMed

Heinz, Andrea; Jung, Michael C; Jahreis, Günther; Rusciani, Anthony; Duca, Laurent; Debelle, Laurent; Weiss, Anthony S; Neubert, Reinhard H H; Schmelzer, Christian E H

2012-01-01

This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P(1), which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr. CG shows a strong preference for the charged amino acid Lys at P(1) in tropoelastin, whereas Lys was not identified at P(1) in CG digests of elastin due to extensive cross-linking at Lys residues in mature elastin. All three serine proteases showed a clear preference for Pro at P(2) and P(4)'. With respect to the liberation of potentially bioactive peptides from elastin, the study revealed that all three serine proteases have a similar ability to release bioactive sequences, with CG producing the highest number of these peptides. In bioactivity studies, potentially bioactive peptides that have not been investigated on their bioactivity to date, were tested. Three new bioactive GxxPG motifs were identified; GVYPG, GFGPG and GVLPG. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Codon usage analysis of photolyase encoding genes of cyanobacteria inhabiting diverse habitats.

PubMed

Rajneesh; Pathak, Jainendra; Kannaujiya, Vinod K; Singh, Shailendra P; Sinha, Rajeshwar P

2017-07-01

Nucleotide and amino acid compositions were studied to determine the genomic and structural relationship of photolyase gene in freshwater, marine and hot spring cyanobacteria. Among three habitats, photolyase encoding genes from hot spring cyanobacteria were found to have highest GC content. The genomic GC content was found to influence the codon usage and amino acid variability in photolyases. The third position of codon was found to have more effect on amino acid variability in photolyases than the first and second positions of codon. The variation of amino acids Ala, Asp, Glu, Gly, His, Leu, Pro, Gln, Arg and Val in photolyases of three different habitats was found to be controlled by first position of codon (G1C1). However, second position (G2C2) of codon regulates variation of Ala, Cys, Gly, Pro, Arg, Ser, Thr and Tyr contents in photolyases. Third position (G3C3) of codon controls incorporation of amino acids such as Ala, Phe, Gly, Leu, Gln, Pro, Arg, Ser, Thr and Tyr in photolyases from three habitats. Photolyase encoding genes of hot spring cyanobacteria have 85% codons with G or C at third position, whereas marine and freshwater cyanobacteria showed 82 and 60% codons, respectively, with G or C at third position. Principal component analysis (PCA) showed that GC content has a profound effect in separating the genes along the first major axis according to their RSCU (relative synonymous codon usage) values, and neutrality analysis indicated that mutational pressure has resulted in codon bias in photolyase genes of cyanobacteria.

Purification and partial characterization of canine S100A12.

PubMed

Heilmann, Romy M; Suchodolski, Jan S; Steiner, Jörg M

2010-12-01

Canine S100A12 (cS100A12) is a calcium-binding protein of the S100 superfamily of EF-hand proteins, and its expression is restricted to neutrophils and monocytes. Interaction of S100A12 with the receptor for advanced glycation end products (RAGE) has been suggested to play a central role in inflammation. Moreover, S100A12 has been shown to represent a sensitive and specific marker for gastrointestinal inflammation in humans. Only human, porcine, bovine, and rabbit S100A12 have been purified to date, and an immunoassay for the quantification of S100A12 is available only for humans. Therefore, the aim of this study was to develop a protocol for the purification of S100A12 and to partially characterize this protein in the dog (Canis lupus familiaris) as a prelude to the development of an immunologic method for its detection and quantification in canine serum and fecal specimens. Leukocytes were isolated from canine whole blood by dextran sedimentation, and canine S100A12 was extracted from the cytosol fraction of these cells. Further purification of cS100A12 comprised of ammonium sulfate precipitation, hydrophobic interaction chromatography, and strong cation- and anion-exchange column chromatography. Canine S100A12 was successfully purified from canine whole blood. The relative molecular mass of the protein was estimated at 10,379.5 and isoelectric focusing revealed an isoelectric point of 6.0. The approximate specific absorbance of cS100A12 at 280 nm was determined to be 1.78 for a 1 mg/ml solution. The N-terminal AA sequence of the first 15 residues of cS100A12 was Thr-Lys-Leu-Glu-Asp-His-X-Glu-Gly-Ile-Val-Asp-Val-Phe-His, and revealed 100% identity with the predicted protein sequence available through the canine genome project. Sequence homology for the 14 N-terminal residues identified for cS100A12 with those of feline, bovine, porcine, and human S100A12 was 78.6%. We conclude that canine S100A12 can be successfully purified from canine whole blood using the described methods. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

The Gly-54-->Asp allelic form of human mannose-binding protein (MBP) fails to bind MBP-associated serine protease.

PubMed Central

Matsushita, M; Ezekowitz, R A; Fujita, T

1995-01-01

The human mannose-binding protein (MBP) is a pattern recognition molecule that appears to play a role in initial host defence. MBP activates the complement cascade and it may act as an opsonin both in the absence and in the presence of complement. A number of distinct MBP allelic forms exist in different population groups. An allele that occurs in 5-7% of Caucasians was identified by an inability to activate the complement system. A homozygous mutation at base pair 230 of the MBP gene results in a Gly-to-Asp substitution at the fifth collagen repeat. It appears that the resultant protein, MBPD, is able to form high-order multimers that bind bacteria but do not support complement activation. Recently a novel serine protease, the MBP-associated serine protease (MASP), has been described. MBP-MASP complexes circulate in serum and result in the direct activation of a novel complement pathway (lectin pathway) in the absence of the first complement components. In this study we demonstrate that MASP and its proenzyme proMASP are unable to bind to recombinant (r)MBPD. This lack of a MASP-rMBPD association corresponds to a failure of the Gly-54-->Asp form of MBP to activate complement. Our results provide a biochemical basis for the functional deficit in the Gly-54-->Asp allelic form of MBP and suggest that the proMASP/MASP binding site maps to the fifth collagen repeat of MBP. Images Figure 1 PMID:7487919

First-second shell interactions in metal binding sites in proteins: a PDB survey and DFT/CDM calculations.

PubMed

Dudev, Todor; Lin, Yen-lin; Dudev, Minko; Lim, Carmay

2003-03-12

The role of the second shell in the process of metal binding and selectivity in metalloproteins has been elucidated by combining Protein Data Bank (PDB) surveys of Mg, Mn, Ca, and Zn binding sites with density functional theory/continuum dielectric methods (DFT/CDM). Peptide backbone groups were found to be the most common second-shell ligand in Mg, Mn, Ca, and Zn binding sites, followed (in decreasing order) by Asp/Glu, Lys/Arg, Asn/Gln, and Ser/Thr side chains. Aromatic oxygen- or nitrogen-containing side chains (Tyr, His, and Trp) and sulfur-containing side chains (Cys and Met) are seldom found in the second coordination layer. The backbone and Asn/Gln side chain are ubiquitous in the metal second coordination layer as their carbonyl oxygen and amide hydrogen can act as a hydrogen-bond acceptor and donor, respectively, and can therefore partner practically every first-shell ligand. The second most common outer-shell ligand, Asp/Glu, predominantly hydrogen bonds to a metal-bound water or Zn-bound histidine and polarizes the H-O or H-N bond. In certain cases, a second-shell Asp/Glu could affect the protonation state of the metal ligand. It could also energetically stabilize a positively charged metal complex more than a neutral ligand such as the backbone and Asn/Gln side chain. As for the first shell, the second shell is predicted to contribute to the metal selectivity of the binding site by discriminating between metal cations of different ionic radii and coordination geometries. The first-shell-second-shell interaction energies decay rapidly with increasing solvent exposure of the metal binding site. They are less favorable but are of the same order of magnitude as compared to the respective metal-first-shell interaction energies. Altogether, the results indicate that the structure and properties of the second shell are dictated by those of the first layer. The outer shell is apparently designed to stabilize/protect the inner-shell and complement/enhance its properties.

Truncation studies of alpha-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity.

PubMed

Haskell-Luevano, C; Sawyer, T K; Hendrata, S; North, C; Panahinia, L; Stum, M; Staples, D J; Castrucci, A M; Hadley, M F; Hruby, V J

1996-01-01

Truncation studies of alpha-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity: PEPTIDES 17(6) 995-1002, 1996.-Systematic analysis of fragment derivatives of the superpotent alpha-MSH analogue. Ac-Ser.Tyr-Ser-Nle4-Glu- His-DPhe7-Arg-Trp-Gly-Lys-Pro-Val-NH2(NDP-MSH), led to the discovery of tripeptide agonists possessing prolonged bioactivity in the frog skin assay. Of particular significance to this discovery was Ac-DPhe-Arg-DTrp-NH2, which was the most potent tripeptide in this series exhibiting sustained melanotropic activity. Different pharmacophore models appear to exist that are dependent on the substructure and stereochemistry of the MSH(6-9) "active site." The tripeptides Ac-DPhe-Arg-Trp-NH2, Ac-DPhe-Arg-DTrp-NH2, and Ac-DPhe-DArg-Trp-NH2 stereo-chemical combinations require only Phe7-Xaa8-Trp9, whereas Ac-DPhe-DArg-DTrp-NH2, Ac-Phe-Arg-DTrp-NH2, and Ac-Phe-Arg-Trp-NH2 additionally require His4 for minimal biological activity. Ac-DPhe-Arg-DTrp-NH2 represents a novel prototype lead for the development of MSH-based peptidomimetic agonists.

[Glycemic variability and short-term outcome in critically ill].

PubMed

Zhang, L P; Guo, Y B; Zhou, L H

2016-06-07

To analyze the association of blood glucose variability and short-term outcome in critically ill. The retrospective study including 552 patients admitted to the Affiliated Hospital of Inner Mongolia Medical University from January 2013 to March 2015. The initial blood glucose (GluAdm), the first 24 hours average blood glucose(GluMV1d) and glycemic lability index(GLI1d), 72-hour average blood glucose (GluMV3d) and glycemic lability index(GLI3d) were recorded. The receiver operating characteristic curve (ROC curve) was applied to evaluate the association between GluAdm, GLI1d, GLI3d and APACHE Ⅱ score and prognosis. The levels of APACHE Ⅱ, GluAdm, GLI 1d, GLI 3d of nonsurvivors were higher than those of survivors[(23.2±5.2) vs (16.7±4.4), (12.3±5.2)mmol/L vs(9.2±2.2)mmol/L, (23.3±12.2)vs(12.3±11.1), (21.6±19.3)vs(13.2±9.9), P<0.05]; there was no statistically significant difference between GluMV1d and GluMV3d; when ROC was applied, and the area under the curve (AUC) of APACHEⅡ, GLI1d and GLI3d were 0.826±0.035, 0.726±0.052 and 0.786±0.046, which were significantly higher than the GluMV1d and GluMV3d (0.412±0.031, 0.425±0.026, P<0.05) .It is correlated between GluAdm, GLI1d, GLI3d and the 28-day mortality, ICU days and total time of hospitalization. Blood glucose variability is closely related with the mortality in critical ill patients, GLI1d, GLI3d and APACHEⅡ score of critically ill patients have a similar predictive value in the short-term prognosis.

Hb Hope [beta136(H14)Gly-->Asp (GGT-->GAT)]: interactions with Hb S [beta6(A3)Glu-->Val (GAG-->GTG)], other variant hemoglobins and thalassemia.

PubMed

Ingle, John; Adewoye, Adeboye; Dewan, Robert; Okoli, Michael; Rollins, Lamarr; Eung, Shawn H; Luo, Hong-Yuan; Chui, David H K; Steinberg, Martin H

2004-01-01

Hb Hope [beta136(H14)Gly-->Asp (GGT-->GAT)] was first described in an African-American family in 1965. Since then, it has been found in combination with several different globin gene mutations in many other families of divergent ethnic backgrounds. The basis for its relatively frequent occurrences remains unexplained. This variant hemoglobin (Hb) is mildly unstable and has reduced oxygen affinity, but is generally innocuous clinically. This variant Hb can present as a confounding factor in arriving at a correct diagnosis by either electrophoresis or high performance liquid chromatography (HPLC), particularly during the neonatal period. DNA-based diagnostics can help solve this potential problem.

Inhibitors of tripeptidyl peptidase II. 3. Derivation of butabindide by successive structure optimizations leading to a potential general approach to designing exopeptidase inhibitors.

PubMed

Ganellin, C Robin; Bishop, Paul B; Bambal, Ramesh B; Chan, Suzanne M T; Leblond, Bertrand; Moore, Andrew N J; Zhao, Lihua; Bourgeat, Pierre; Rose, Christiane; Vargas, Froylan; Schwartz, Jean-Charles

2005-11-17

The cholecystokinin-8 (CCK-8)-inactivating peptidase is a serine peptidase that has been shown to be a membrane-bound isoform of tripeptidyl peptidase II (EC 3.4.14.10). It cleaves the neurotransmitter CCK-8 sulfate at the Met-Gly bond to give Asp-Tyr(SO3H)-Met-OH + Gly-Trp-Met-Asp-Phe-NH2. Starting from Val-Pro-NHBu, a dipeptide of submicromolar affinity that had previously been generated to serve as a lead, successive optimization at P3, P1, and then P2 gave Abu-Pro-NHBu (18, Ki = 80 nM). Further transformation (by making a benzologue) gave the indoline analogue, butabindide (33) as a reversible inhibitor having nanomolar affinity (Ki = 7 nM). Retrospective analysis suggested the possibility of a general approach to designing exopeptidase inhibitors starting from the structure of the first hydrolysis product. Application of this approach to CCK-8 led to Abu-Phe-NHBu (37), but this only had Ki = 9.4 microM. Molecular modeling, to determine the minimum energy conformations and explain the 1000-fold better affinity of butabindide, indicated that 37 cannot access the likely active conformation of butabindide.

Roles of the conserved cytoplasmic region and non-conserved carboxy-terminal region of SecE in Escherichia coli protein translocase.

PubMed

Kontinen, V P; Yamanaka, M; Nishiyama, K; Tokuda, H

1996-06-01

SecE, an essential membrane component of the Escherichia coli protein translocase, consists of 127 amino acid residues. Only a part of the second putative cytoplasmic region comprising some 13 residues is essential for the SecE function as long as the proper topological arrangement is retained. The Trp84 and Pro85 residues of this region are conserved in all eubacterial SecE homologues. The conservation of positively charged residues corresponding to Arg80 and Lys81 is also substantial. We deleted or replaced these residues to assess their roles in the SecE function. Deletion of the Arg80-Lys81 dipeptide did not abolish the SecE function whereas that of Trp84 or Pro85 caused a loss of the function. Strikingly, however, replacement of Pro85 with either Gly, Ser, or Ala, and that of Trp84 with Lys did not abolish the SecE function. These results indicate that the strong conservation of these residues does not reflect their obligatory requirement for the SecE function. A chimeric SecE possessing the cytoplasmic region of the E. coli SecE and the following region of the Bacillus subtilis SecE was able to form the translocation machinery together with SecA, SecY, and SecG. Although a Leu to Arg mutation at position 108 has been thought to cause a loss of signal recognition fidelity and thereby suppress a signal sequence defect, the same mutation at position 111 caused a complete loss of the function. The levels of SecY and SecG in the secEcsE501 mutant, which expresses SecE at a decreased level and is sensitive to low temperature, increased upon the expression of functional SecE derivatives, irrespective of the site of mutation, suggesting that the levels of SecY and SecG are co-operatively determined by the level of functional, but not non-functional, SecE. Based on these results, the SecE function in the translocase is discussed.

ESR1 ligand binding domain mutations in hormone-resistant breast cancer

PubMed Central

Toy, Weiyi; Shen, Yang; Won, Helen; Green, Bradley; Sakr, Rita A.; Will, Marie; Li, Zhiqiang; Gala, Kinisha; Fanning, Sean; King, Tari A.; Hudis, Clifford; Chen, David; Taran, Tetiana; Hortobagyi, Gabriel; Greene, Geoffrey; Berger, Michael; Baselga, Jose; Chandarlapaty, Sarat

2013-01-01

Seventy percent of breast cancers express estrogen receptor (ER) and most of these are sensitive to ER inhibition. However, many such tumors become refractory to inhibition of estrogen action in the metastatic setting for unknown reasons. We conducted a comprehensive genetic analysis of two independent cohorts of metastatic ER+ breast tumors and identified mutations in the ligand binding domain (LBD) of ESR1 in 14/80 cases. These included highly recurrent mutations p.Tyr537Ser/Asn and p.Asp538Gly. Molecular dynamics simulations suggest the Tyr537Ser and Asp538Gly structures lead to hydrogen bonding of the mutant amino acid with Asp351, thus favoring the receptor’s agonist conformation. Consistent with this model, mutant receptors drive ER-dependent transcription and proliferation in the absence of hormone and reduce the efficacy of ER antagonists. These data implicate LBD mutant forms of ER in mediating clinical resistance to hormonal therapy and suggest that more potent ER antagonists may have significant therapeutic benefit. PMID:24185512

Preferential amino acid sequences in alumina-catalyzed peptide bond formation.

PubMed

Bujdák, J; Rode, B M

2002-05-21

The catalytic effect of activated alumina on amino acid condensation was investigated. The readiness of amino acids to form peptide sequences was estimated on the basis of the yield of dipeptides and was found to decrease in the order glycine (Gly), alanine (Ala), leucine (Leu), valine (Val), proline (Pro). For example, approximately 15% Gly was converted to the dipeptide (Gly(2)), 5% to cyclic anhydride (cyc(Gly(2))) and small amounts of tri- (Gly(3)) and tetrapeptide (Gly(4)) were formed after 28 days. On the other hand, only trace amounts of Pro(2) were formed from proline under the same conditions. Preferential formation of certain sequences was observed in the mixed reaction systems containing two amino acids. For example, almost ten times more Gly-Val than Val-Gly was formed in the Gly+Val reaction system. The preferred sequences can be explained on the basis of an inductive effect that side groups have on the nucleophilicity and electrophilicity, respectively, of the amino and carboxyl groups. A comparison with published data of amino acid reactions in other reaction systems revealed that the main trends of preferential sequence formation were the same as those described for the salt-induced peptide formation (SIPF) reaction. The results of this work and other previously published papers show that alumina and related mineral surfaces might have played a crucial role in the prebiotic formation of the first peptides on the primitive earth.

Tetrapod-type [Asp1] angiotensin is present in a holostean fish, Amia calva.

PubMed

Takei, Y; Itahara, Y; Butler, D G; Watanabe, T X; Oudit, G Y

1998-05-01

The renin-angiotensin system has been identified in various vertebrates, from elasmobranchs to mammals. Tetrapod (amphibians to mammals) angiotensin (ANG) has Asp at the N-terminus, but Asp is replaced by Asn in elasmobranch and teleost fish. ANG I has been isolated from incubates of plasma and kidney extracts of the bowfin Amia calva, a holostean fish, using the eel vasopressor activity as an assay system; its sequence was found to be H-Asp-Arg-Val-Tyr-Val-His-Pro-Phe-Asn-Leu-OH after sequence analysis, mass spectrometry, and comparison with the synthetic peptide. This sequence is identical to bullfrog ANG I. [Asn1] ANG I was not detected. Thus the bowfin is the first fish species which contains only [Asp1] ANG I. The bowfin ANG I and II were no more vasopressor than eel peptides in the bowfin, indicating that bowfin ANG II receptors do not distinguish between [Asp1] and [Asn1] peptides. In the rat, bowfin ANG I and rat [Ile5, His9] ANG I have equipressor activities when examined in different animals, but the vasopressor activity of bowfin ANG I decreased following rat ANG I in the same animals, although the activity of rat ANG I was unaffected after bowfin ANG I. The present study directly demonstrates the presence of the renin-angiotensin system in a holostean fish and showed that its ANG II receptors have not yet fully coevolved with the homologous [Asp1] peptide.

Retardation of ice crystallization by short peptides

NASA Astrophysics Data System (ADS)

Kim, Jun Soo; Yethiraj, Arun

2009-03-01

The effect of short peptides on the growth of ice crystals is studied using molecular dynamics simulations. The simulations focus on two sequences (Gly-Pro-Ala-Gly and Gly-Gly-Ala-Gly) that are found in collagen hydrolysate, a substance that is known to retard crystal growth. In the absence of peptides, the growth of ice crystal in the solution with the ice/water interface is observed in at a rate comparable to the experimental data. When peptides are present in the liquid phase, the crystal growth is retarded to a significant extent compared to the pure water. It is found that Gly-Pro-Ala-Gly is more effective (crystallization is up to 5 times slower than in its absence) than Gly-Gly-Ala-Gly (up to 3 times slower) implying that the role of the proline residue is important. The mechanism can be understood in the nature of binding of the peptides to the growing crystal.

Viscosity-Lowering Effect of Amino Acids and Salts on Highly Concentrated Solutions of Two IgG1 Monoclonal Antibodies.

PubMed

Wang, Shujing; Zhang, Ning; Hu, Tao; Dai, Weiguo; Feng, Xiuying; Zhang, Xinyi; Qian, Feng

2015-12-07

Monoclonal antibodies display complicated solution properties in highly concentrated (>100 mg/mL) formulations, such as high viscosity, high aggregation propensity, and low stability, among others, originating from protein-protein interactions within the colloidal protein solution. These properties severely hinder the successful development of high-concentration mAb solution for subcutaneous injection. We hereby investigated the effects of several small-molecule excipients with diverse biophysical-chemical properties on the viscosity, aggregation propensity, and stability on two model IgG1 (JM1 and JM2) mAb formulations. These excipients include nine amino acids or their salt forms (Ala, Pro, Val, Gly, Ser, HisHCl, LysHCl, ArgHCl, and NaGlu), four representative salts (NaCl, NaAc, Na2SO4, and NH4Cl), and two chaotropic reagents (urea and GdnHCl). With only salts or amino acids in their salt-forms, significant decrease in viscosity was observed for JM1 (by up to 30-40%) and JM2 (by up to 50-80%) formulations, suggesting charge-charge interaction between the mAbs dictates the high viscosity of these mAbs formulations. Most of these viscosity-lowering excipients did not induce substantial protein aggregation or changes in the secondary structure of the mAbs, as evidenced by HPLC-SEC, DSC, and FT-IR analysis, even in the absence of common protein stabilizers such as sugars and surfactants. Therefore, amino acids in their salt-forms and several common salts, such as ArgHCl, HisHCl, LysHCl, NaCl, Na2SO4, and NaAc, could potentially serve as viscosity-lowering excipients during high-concentration mAb formulation development.

Mechanical insights of oxythiamine compound as potent inhibitor for human transketolase-like protein 1 (TKTL1 protein).

PubMed

Mariadasse, Richard; Biswal, Jayashree; Jayaprakash, Prajisha; Rao, Guru Raj; Choubey, Sanjay Kumar; Rajendran, Santhosh; Jeyakanthan, Jeyaraman

2016-01-01

Transketolase is a connecting link between glycolytic and pentose phosphate pathway, which is considered as the rate-limiting step due to synthesis of large number of ATP molecule and it can be proposed as a plausible target facilitating the growth of cancerous cells suggesting its potential role in cancer. Oxythiamine, an antimetabolite has been proved to be an efficient anticancerous compound in vitro, but its structural elucidation of the inhibitory mechanism has not yet been done against the human transketolase-like 1 protein (TKTL1). The three-dimensional (3D) structure of TKTL1 protein was modeled and subjected for refinement, stability and validation. Based on the reported homologs of transketolase (TKT), the active site residues His46, Ser49, Ser52, Ser53, Ile56, Leu82, Lys84, Leu123, Ser125, Glu128, Asp154, His160, Thr216 and Lys218 were identified and considered for molecular-modeling studies. Docking studies reveal the H-bond interactions with residues Ser49 and Lys218 that could play a major role in the activity of TKTL1. Molecular dynamics (MD) simulation study was performed to reveal the comparative stability of both native and complex forms of TKTL1. MD trajectory at 30 ns, confirm the role of active site residues Ser49, Lys84, Glu128, His160 and Lys218 in suppressing the activity of TKTL1. Glu128 is observed to be the most important residue for deprotonation state of the aminopyrimidine moiety and preferred to be the site of inhibitory action. Thus, the proposed mechanism of inhibition through in silico studies would pave the way for structure-oriented drug designing against cancer.

Comparative Functional Alanine Positional Scanning of the α-Melanocyte Stimulating Hormone and NDP-Melanocyte Stimulating Hormone Demonstrates Differential Structure-Activity Relationships at the Mouse Melanocortin Receptors.

PubMed

Todorovic, Aleksandar; Ericson, Mark D; Palusak, Ryan D; Sorensen, Nicholas B; Wood, Michael S; Xiang, Zhimin; Haskell-Luevano, Carrie

2016-07-20

The melanocortin system has been implicated in the regulation of various physiological functions including melanogenesis, steroidogenesis, energy homeostasis, and feeding behavior. Five melanocortin receptors have been identified to date and belong to the family of G protein-coupled receptors (GPCR). Post-translational modification of the proopiomelanocortin (POMC) prohormone leads to the biosynthesis of the endogenous melanocortin agonists, including α-melanocyte stimulating hormone (α-MSH), β-MSH, γ-MSH, and adrenocorticotropic hormone (ACTH). All the melanocortin agonists derived from the POMC prohormone contain a His-Phe-Arg-Trp tetrapeptide sequence that has been implicated in eliciting the pharmacological responses at the melanocortin receptors. Herein, an alanine (Ala) positional scan is reported for the endogenous α-MSH ligand and the synthetic, more potent, NDP-MSH peptide (Ac-Ser(1)-Tyr(2)-Ser(3)-Nle(4)-Glu(5)-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH2) at the cloned mouse melanocortin receptors to test the assumption that the structure-activity relationships of one ligand would apply to the other. Several residues outside of the postulated pharmacophore altered potency at the melanocortin receptors, most notably the 1560-, 37-, and 15-fold potency loss when the Glu(5) position of α-MSH was substituted with Ala at the mMC1R, mMC3R, and mMC4R, respectively. Importantly, the altered potencies due to Ala substitutions in α-MSH did not necessarily correlate with equivalent Ala substitutions in NDP-MSH, indicating that structural modifications and corresponding biological activities in one of these melanocortin ligands may not be predictive for the other agonist.

A common structural motif in immunopotentiating peptides with sequences present in human autoantigens. Elicitation of a response mediated by monocytes and Th1 cells.

PubMed

López-Moratalla, N; Ruíz, E; López-Zabalza, M J; Santiago, E

1996-12-16

We have found a common structural motif in human autoantigens, heat shock proteins and viral proteins. Peptides modelled after sequences present in those molecules were synthesized and immunomodulating properties tested. They share a core of 15 amino acid residues and a common pattern ('2-6-11' motif) characterized by requirements at fixed positions with respect to a Pro (position 6); an apolar residue or a Lys at position 2; and a Glu, Asp or Lys at position 11. Any of these peptides, when added to cultures of lymphomononuclear cells, caused the activation of monocytes manifested by a release of IL-1 alpha, IL-1 beta and TNF alpha. A release of INF gamma and IL-2 took also place; this release was abolished by anti-DR antibodies. Neither IL-4 nor IL-5 could be detected. This suggests a presentation by APCs and the appearance of cells with a Th1 phenotype. Monocytes and Th1 cells freshly obtained from 12 patients of Graves' disease, 8 of Hashimoto's disease and 8 of primary biliary cirrhosis exhibited activation features similar to those found in cells from healthy subjects incubated in the presence of peptides with a "2-6-11' motif and representing fragments of autoantigens. Their immunopotentiating properties suggest their involvement in the initiation or progression of the autoimmune response mediated by activated monocytes and Th1 cells.

NMR structure of the Arctic mutation of the Alzheimer's Aβ(1-40) peptide docked to SDS micelles

NASA Astrophysics Data System (ADS)

Usachev, K. S.; Filippov, A. V.; Khairutdinov, B. I.; Antzutkin, O. N.; Klochkov, V. V.

2014-11-01

The “Arctic” point mutation of the Alzheimer's amyloid β-peptide is a rare mutation leading to an early onset of Alzheimer's disease. The peptide may interact with neuronal membranes, where it can provide its toxic effects. We used 2D NMR spectroscopy to investigate the conformation of the “Arctic” mutant of Aβ1-40 Alzheimer's amyloid peptide in sodium dodecyl sulfate micelle solutions, which are the type of amphiphilic structures mimicking some properties of biomembranes. The study showed that the Arctic mutant of Aβ1-40 interacts with the surface of SDS micelles mainly through the Leu17-Asn27 310-helical region, while the Ile31-Val40 region is buried in the hydrophobic interior of the micelle. In contrast, wild-type Aβ1-40 interacts with SDS micelles through the Lys16-Asp23 α-helical region and Gly29-Met35. Both the Arctic mutant and the wild-type Aβ1-40 peptides interactions with SDS micelles are hydrophobic in nature. Aβ peptides are thought to be capable of forming pores in biomembranes that can cause changes in neuronal and endothelial cell membrane permeability. It has also been shown that Aβ peptides containing the “Arctic” mutation are more neurotoxic and aggregate more readily than the wild-type Aβ peptides at physiological conditions. Here, we propose that the extension of the helical structure of Leu17-Asn27 and a high aliphaticity (neutrality) of the C-terminal region in the Arctic Aβ peptides are consistent with the idea that formation of ion-permeable pores by Aβ oligomers may be one of prevailing mechanisms of a larger neuronal toxicity of the Arctic Aβ compared to the wild-type Aβ peptides, independent of oxidative damage and lipid peroxidation.

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.

DNA stabilization at the Bacillus subtilis PolX core—a binding model to coordinate polymerase, AP-endonuclease and 3′-5′ exonuclease activities

PubMed Central

Baños, Benito; Villar, Laurentino; Salas, Margarita; de Vega, Miguel

2012-01-01

Family X DNA polymerases (PolXs) are involved in DNA repair. Their binding to gapped DNAs relies on two conserved helix-hairpin-helix motifs, one located at the 8-kDa domain and the other at the fingers subdomain. Bacterial/archaeal PolXs have a specifically conserved third helix-hairpin-helix motif (GFGxK) at the fingers subdomain whose putative role in DNA binding had not been established. Here, mutagenesis at the corresponding residues of Bacillus subtilis PolX (PolXBs), Gly130, Gly132 and Lys134 produced enzymes with altered DNA binding properties affecting the three enzymatic activities of the protein: polymerization, located at the PolX core, 3′-5′ exonucleolysis and apurinic/apyrimidinic (AP)-endonucleolysis, placed at the so-called polymerase and histidinol phosphatase domain. Furthermore, we have changed Lys192 of PolXBs, a residue moderately conserved in the palm subdomain of bacterial PolXs and immediately preceding two catalytic aspartates of the polymerization reaction. The results point to a function of residue Lys192 in guaranteeing the right orientation of the DNA substrates at the polymerization and histidinol phosphatase active sites. The results presented here and the recently solved structures of other bacterial PolX ternary complexes lead us to propose a structural model to account for the appropriate coordination of the different catalytic activities of bacterial PolXs. PMID:22844091

Function and solution structure of huwentoxin-IV, a potent neuronal tetrodotoxin (TTX)-sensitive sodium channel antagonist from Chinese bird spider Selenocosmia huwena.

PubMed

Peng, Kuan; Shu, Qin; Liu, Zhonghua; Liang, Songping

2002-12-06

We have isolated a highly potent neurotoxin from the venom of the Chinese bird spider, Selenocosmia huwena. This 4.1-kDa toxin, which has been named huwentoxin-IV, contains 35 residues with three disulfide bridges: Cys-2-Cys-17, Cys-9-Cys-24, and Cys-16-Cys-31, assigned by a chemical strategy including partial reduction of the toxin and sequence analysis of the modified intermediates. It specifically inhibits the neuronal tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channel with the IC(50) value of 30 nm in adult rat dorsal root ganglion neurons, while having no significant effect on the tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel. This toxin seems to be a site I toxin affecting the sodium channel through a mechanism quite similar to that of TTX: it suppresses the peak sodium current without altering the activation or inactivation kinetics. The three-dimensional structure of huwentoxin-IV has been determined by two-dimensional (1)H NMR combined with distant geometry and simulated annealing calculation by using 527 nuclear Overhauser effect constraints and 14 dihedral constraints. The resulting structure is composed of a double-stranded antiparallel beta-sheet (Leu-22-Ser-25 and Trp-30-Tyr-33) and four turns (Glu-4-Lys-7, Pro-11-Asp-14, Lys-18-Lys-21 and Arg-26-Arg-29) and belongs to the inhibitor cystine knot structural family. After comparison with other toxins purified from the same species, we are convinced that the positively charged residues of loop IV (residues 25-29), especially residue Arg-26, must be crucial to its binding to the neuronal tetrodotoxin-sensitive voltage-gated sodium channel.

[Peptide fragments of chemokine domain of fractalkine: effect on human monocyte migration].

PubMed

Kukhtina, N B; Aref'eva, T I; Ruleva, N Iu; Sidorova, M V; Az'muko, A A; Bespalova, Zh D; Krasnikova, T L

2012-01-01

Leukocyte chemotaxis to the area of tissue damage is mediated by chemokines. According to the primary structure, chemokines are divided into four families, fractalkine (CX3CL1) is the only one member of CX3C family and the only membrane-bound chemokine. Fractalkine molecule includes the extracellular N-terminal chemokine domain, mucin-like rod, the transmembrane and the intracellular domains. In membrane-bound state fractalkine has the properties of an adhesion molecule. Chemokine domain of fractalkine (CDF) is released from cell membrane by proteolysis, and this soluble form acts as a chemoattractant for leukocytes expressing fractalkine receptor CX3CR1. Fractalkine is involved in development of a number of pathological processes caused by inflammation, and therefore a search for fractalkine inhibitors is very important. For this purpose we identified several antigenic determinants--the fragments of CDF, and the following peptides were synthesized--P41-52 H-Leu-Glu-Thr-Arg-Gln-His-Arg-Leu-Phe-Cys-Ala-Asp-NH2, P53-60 H-Pro-Lys-Glu-Gln-Trp-Val-Lys-Asp-NH2 and P60-71 H-Asp-Ala-Met-Gln-His-Leu-Asp-Arg-Gln-Ala-Ala-Ala-NH2. The peptide effects on adhesion and migration of human peripheral blood monocytes expressing fractalkine receptors were investigated. In the presence of CDF and P41-52 we observed the increased adhesion and migration of monocytes compared with spontaneous values. Peptides P53-60 and P60-71 significantly inhibited monocyte adhesion and migration stimulated by CDF. Since the chemotactic activity of chemokines was shown to be dependent on their binding to glycosaminoglycans of the cell surface and extracellular matrix, the effect ofpeptides on the interaction of CDF with heparin was analyzed by ELISA. Peptide P41-52 competed with CDF for heparin binding, while peptides P53-60 and P60-71 had no significant activity.

Behavioural and biochemical responses following activation of midbrain dopamine pathways by receptor selective neurokinin agonists.

PubMed

Elliott, P J; Mason, G S; Stephens-Smith, M; Hagan, R M

1991-06-01

Preferential activation of mesolimbic and nigro-striatal dopamine (DA) pathways by receptor-selective and peptidase-resistant neurokinin (NK) agonists is reported. The DA cell body region of the mesolimbic pathway appears to be activated by NK agonists selective for NK-1 and NK-3 receptors whereas the DA cell bodies in the substantia nigra are under an excitatory NK-2 receptor-mediated influence. Stimulation of the mesolimbic DA pathway by NK-1 (Ava[L-Pro9,N-Me-Leu10]SP (7-11) [GR73632]) or NK-3 (Senktide) agonists increase locomotor activity. Additional studies showed that this elevated motor response observed after intra-VTA infusion of GR73632 was accompanied by a corresponding increase in DA turnover in the terminal fields of this pathway. Similarly, unilateral activation of the nigro-striatal DA pathway by NK-2 selective agonists (Ava (D-Pro9) SP (7-11) [GR51667] or [Lys3,Gly8,R-Lac-Leu9]NKA (3-10) [GR64349]) elicit contralateral rotational activity and an increase in DA turnover in the ipsilateral striatum. The rotational response was attenuated by prior administration of an NK-2 antagonist (cyclo (Gln, Trp, Phe, Gly, Leu, Met)] L-659877]) into the nigra. Peripheral injection of haloperidol, a DA antagonist, also blocked the NK-2 agonist induced rotations.

Structurally conserved water molecules in ribonuclease T1.

PubMed

Malin, R; Zielenkiewicz, P; Saenger, W

1991-03-15

In the high resolution (1.7-1.9 A) crystal structures of ribonuclease T1 (RNase T1) in complex with guanosine, guanosine 2'-phosphate, guanylyl 2',5'-guanosine, and vanadate, there are 30 water sites in nearly identical (+/- 1 A) positions that are considered conserved. One water is tightly bound to Asp76(O delta), Thr93(O gamma), Cys6(O), and Asn9(N); another bridges two loops by hydrogen-bonding to Tyr68(O eta) and to Ser35(N), Asn36(N); a loop structure is stabilized by two waters coordinated to Gly31(O) and His27(N delta), and by water bound to cis-Pro39(O). Most notable is a hydrogen-bonded chain of 10 water molecules. Waters 1-5 of this chain are inaccessible to solvent, are anchored at Trp59(N), and stitch together the loop formed by segments 60-68; waters 5-8 coordinate to Ca2+, and waters 9 and 10 hydrogen-bond to N-terminal side chains of the alpha-helix. The water chain and two conserved water molecules are bound to amino acids adjacent to the active site residues His40, Glu58, Arg77, and His92; they are probably involved in maintaining their spatial orientation required for catalysis. Water sites must be considered in genetic engineering; the mutation Trp59Tyr, which probably influences the 10-water chain, doubles the catalytic activity of RNase T1.

An Amino Acid Code for Irregular and Mixed Protein Packing

PubMed Central

Joo, Hyun; Chavan, Archana; Fraga, Keith; Tsai, Jerry

2015-01-01

To advance our understanding of protein tertiary structure, the development of the knob-socket model is completed in an analysis of the packing in irregular coil and turn secondary structure packing as well as between mixed secondary structure. The knob-socket model simplifies packing based on repeated patterns of 2 motifs: a 3 residue socket for packing within 2° structure and a 4 residue knob-socket for 3° packing. For coil and turn secondary structure, knob-sockets allow identification of a correlation between amino acid composition and tertiary arrangements in space. Coil contributes almost as much as α-helices to tertiary packing. Irregular secondary structure involves 3 residue cliques of consecutive contacting residues or XYZ sockets. In irregular sockets, Gly, Pro, Asp and Ser are favored, while Cys, His, Met and Trp are not. For irregular knobs, the preference order is Arg, Asp, Pro, Asn, Thr, Leu, and Gly, while Cys, His, Met and Trp are not. In mixed packing, the knob amino acid preferences are a function of the socket that they are packing into, whereas the amino acid composition of the sockets does not depend on the secondary structure of the knob. A unique motif of a coil knob with an XYZ β-sheet socket may potentially function to inhibit β-sheet extension. In addition, analysis of the preferred crossing angles for strands within a β-sheet and mixed α-helices/β-sheets identifies canonical packing patterns useful in protein design. Lastly, the knob-socket model abstracts the complexity of protein tertiary structure into an intuitive packing surface topology map. PMID:26370334

Analysis of the interaction between Bacillus coagulans and Bacillus thuringiensis S-layers and calcium ions by XRD, light microscopy, and FTIR.

PubMed

Babolmorad, Ghazal; Emtiazi, Giti; Emamzadeh, Rahman

2014-05-01

S-layer is a self-assemble regularly crystalline surface that covers major cell wall component of many bacteria and archaea and exhibits a high metal-binding capacity. We have studied the effect of the calcium ions and type of solid support (glass or mica) on the structure of the S-layers from Bacillus coagulans HN-68 and Bacillus thuringiensis MH14 upon simple methods based on light microscopy and AFM. Furthermore, the Fourier transform infrared spectroscopy (FTIR) study is indicated that the calcium-S-layer interaction occurred mainly through the carboxylate groups of the side chains of aspartic acid (Asp) and glutamic acid (Glu) and nitrogen atoms of Lys, Asn, and histidine (His) amino acids and N-H groups of the peptide backbone. Studied FTIR revealed that inner faces of S-layer are mainly negative, and outer faces of S-layer are mainly positive. Probably, calcium ions with positive charges bound to the carboxyl groups of Glu and Asp. Accordingly, calcium ions are anchored in the space between the inner faces of S-layer with negative charge and the surface of mica with negative charge. This leads to regular arrangement of the S-layer subunits.

Influence of secondary structure on in-source decay of protein in matrix-assisted laser desorption/ionization mass spectrometry.

PubMed

Takayama, Mitsuo; Osaka, Issey; Sakakura, Motoshi

2012-01-01

The susceptibility of the N-Cα bond of the peptide backbone to specific cleavage by in-source decay (ISD) in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) was studied from the standpoint of the secondary structure of three proteins. A naphthalene derivative, 5-amino-1-naphtol (5,1-ANL), was used as the matrix. The resulting c'-ions, which originate from the cleavage at N-Cα bonds in flexible secondary structures such as turn and bend, and are free from intra-molecular hydrogen-bonded α-helix structure, gave relatively intense peaks. Furthermore, ISD spectra of the proteins showed that the N-Cα bonds of specific amino acid residues, namely Gly-Xxx, Xxx-Asp, and Xxx-Asn, were more susceptible to MALDI-ISD than other amino acid residues. This is in agreement with the observation that Gly, Asp and Asn residues usually located in turns, rather than α-helix. The results obtained indicate that protein molecules embedded into the matrix crystal in the MALDI experiments maintain their secondary structures as determined by X-ray crystallography, and that MALDI-ISD has the capability for providing information concerning the secondary structure of protein.

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.

Meta-analysis of ADH1B and ALDH2 polymorphisms and esophageal cancer risk in China.

PubMed

Zhang, Guo-Hong; Mai, Rui-Qin; Huang, Bo

2010-12-21

To evaluate whether alcohol dehydrogenase-1B (ADH1B) His47Arg and aldehyde dehydrogenase-2 (ALDH2) Glu487Lys polymorphism is involved in the esophageal squamous cell carcinoma (ESCC) risk in Chinese Han population. Seven studies of ADH1B and ALDH2 genotypes in Chinese Han population in 1450 cases and 2459 controls were included for meta-analysis. Stratified analyses were carried out to determine the gene-alcohol and gene-gene interaction with ESCC risk. Potential sources of heterogeneity between studies were explored, and publication bias was also evaluated. Individuals with ADH1B arginine (Arg)/Arg genotype showed 3.95-fold increased ESCC risk in the recessive genetic model [Arg/Arg vs Arg/histidine (His) + His/His: odds ratio (OR) = 3.95, 95% confidence interval (CI): 2.76-5.67]. Significant association was found in the dominant model for ALDH2 lysine (Lys) allele [glutamate (Glu)/Lys + Lys/Lys vs Glu/Glu: OR = 2.00, 95% CI: 1.54-2.61]. Compared with the non-alcoholics, Arg/Arg (OR = 25.20, 95% CI: 10.87-53.44) and Glu/Lys + Lys/Lys (OR = 21.47, 95% CI: 6.44-71.59) were found to interact with alcohol drinking to increase the ESCC risk. ADH1B Arg+ and ALDH2 Lys+ had a higher risk for ESCC (OR = 7.09, 95% CI: 2.16-23.33). The genetic variations of ADH1B His47Arg and ALDH2 Glu487Lys are susceptible loci for ESCC in Chinese Han population and interact substantially with alcohol consumption. The individuals carrying both risky genotypes have a higher baseline risk of ESCC.

Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae.

PubMed

Moon, Jaewoong; Liu, Z Lewis

2015-04-01

The aldehyde reductase gene ARI1 is a recently characterized member of an intermediate subfamily within the short-chain dehydrogenase/reductase (SDR) superfamily that clarified mechanisms of in situ detoxification of 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde by Saccharomyces cerevisiae. Uncharacterized open reading frames (ORFs) are common among tolerant candidate genes identified for lignocellulose-to-advanced biofuels conversion. This study presents partially purified proteins of two ORFs, YDR541C and YGL039W, and direct enzyme assay evidence against aldehyde-inhibitory compounds commonly encountered during lignocellulosic biomass fermentation processes. Each of the partially purified proteins encoded by these ORFs showed a molecular mass of approximately 38 kDa, similar to Ari1p, a protein encoded by aldehyde reductase gene. Both proteins demonstrated strong aldehyde reduction activities toward 14 aldehyde substrates, with high levels of reduction activity for Ydr541cp toward both aromatic and aliphatic aldehydes. While Ydr541cp was observed to have a significantly higher specific enzyme activity at 20 U/mg using co-factor NADPH, Ygl039wp displayed a NADH preference at 25 U/mg in reduction of butylaldehyde. Amino acid sequence analysis identified a characteristic catalytic triad, Ser, Tyr and Lys; a conserved catalytic motif of Tyr-X-X-X-Lys; and a cofactor-binding sequence motif, Gly-X-X-Gly-X-X-Ala, near the N-terminus that are shared by Ydr541cp, Ygl039wp, Yol151wp/GRE2 and Ari1p. Findings of aldehyde reductase genes contribute to the yeast gene annotation and aids development of the next-generation biocatalyst for advanced biofuels production. Copyright © 2015 John Wiley & Sons, Ltd.

Spontaneous chemical reversion of an active site mutation: deamidation of an asparagine residue replacing the catalytic aspartic acid of glutamate dehydrogenase.

PubMed

Paradisi, Francesca; Dean, Jonathan L E; Geoghegan, Kieran F; Engel, Paul C

2005-03-08

A mutant (D165N) of clostridial glutamate dehydrogenase (GDH) in which the catalytic Asp is replaced by Asn surprisingly showed a residual 2% of wild-type activity when purified after expression in Escherichia coli at 37 degrees C. This low-level activity also displayed Michaelis constants for substrates that were remarkably similar to those of the wild-type enzyme. Expression at 8 degrees C gave a mutant enzyme preparation 1000 times less active than the first preparation, but progressively, over 2 weeks' incubation at 37 degrees C in sealed vials, this enzyme regained 90% of the specific activity of wild type. This suggested that the mutant might undergo spontaneous deamidation. Mass spectrometric analysis of tryptic peptides derived from D165N samples treated in various ways showed (i) that the Asn is in place in D165N GDH freshly prepared at 8 degrees C; (ii) that there is a time-dependent reversion of this Asn to Asp over the 2-week incubation period; (iii) that detectable deamidation of other Asn residues, in Asn-Gly sequences, mainly occurred in sample workup rather than during the 2-week incubation; (iv) that there is no significant deamidation of other randomly chosen Asn residues in this mutant over the same period; and (v) that when the protein is denatured before incubation, no deamidation at Asn-165 is detectable. It appears that this deamidation depends on the residual catalytic machinery of the mutated GDH active site. A literature search indicates that this finding is not unique and that Asn may not be a suitable mutational replacement in the assessment of putative catalytic Asp residues by site-directed mutagenesis.

Binding constants of phenylalanine for the four mononucleotides

NASA Technical Reports Server (NTRS)

Khaled, M. A.; Mullins, D. W., Jr.; Lacey, J. C., Jr.

1984-01-01

Earlier work has shown that several properties of amino acids correlate directly with properties of their anticodonic nucleotides. Furthermore, in precipitation studies with thermal proteinoids and homopolyribonucleotides, an anticodonic preference was displayed between Lys-rich, Pro-rich and Gly-rich thermal proteinoids and their anticodonic polyribonucleotides. However, Phe-rich thermal proteinoid displayed a preference for its codonic nucleotide, poly U. This inconsistency seemed to be explained by a folding in of the hydrophobic residues of Phe causing the proteinoid to appear more hydrophilic. The present work used nuclear magnetic resonance techniques to resolve a limited question: to which of the four nucleotides does Phe bind most strongly? The results show quite clearly that Phe binds most strongly to its anticodonic nucleotide, AMP.

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

Tests of linkage and/or association of the LEPR gene polymorphisms with obesity phenotypes in Caucasian nuclear families.

PubMed

Liu, Yong-Jun; Rocha-Sanchez, Sonia M S; Liu, Peng-Yuan; Long, Ji-Rong; Lu, Yan; Elze, Leo; Recker, Robert R; Deng, Hong-Wen

2004-04-13

Genetic variations in the leptin receptor (LEPR) gene have been conceived to affect body weight in general populations. In this study, using the tests implemented in the statistical package QTDT, we evaluated association and/or linkage of the LEPR gene with obesity phenotypes in a large sample comprising 1,873 subjects from 405 Caucasian nuclear families. Obesity phenotypes tested include body mass index (BMI), fat mass, percentage fat mass (PFM), and lean mass, with the latter three measured by dual-energy X-ray absorptiometry (DXA). Three single nucleotide polymorphisms (SNPs), namely Lys109Arg (A/G), Lys656Asn (G/C), Pro1019Pro (G/A), in the LEPR gene were analyzed. Significant linkage disequilibrium (0.394 < or = |D'| < or = 0.688, P < 0.001) was observed between pairs of the three SNPs. No significant population stratification was found for any SNP/phenotype. In single-locus analyses, evidence of association was observed for Lys656Asn with lean mass (P = 0.002) and fat mass (P = 0.015). The contribution of this polymorphism to the phenotypic variation of lean mass and fat mass was 2.63% and 1.15%, respectively. Subjects carrying allele G at the Lys656Asn site had, on average, 3.16% higher lean mass and 2.71% higher fat mass than those without it. In the analyses for haplotypes defined by the three SNPs, significant associations were detected between haplotype GCA (P = 0.005) and lean mass. In addition, marginally significant evidence of association was observed for this haplotype with fat mass (P = 0.012). No statistically significant linkage was found, largely due to the limited power of the linkage approach to detect small genetic effects in our data sets. Our results suggest that the LEPR gene polymorphisms contribute to variation in obesity phenotypes.

Mutations in the histamine N-methyltransferase gene, HNMT, are associated with nonsyndromic autosomal recessive intellectual disability

PubMed Central

Heidari, Abolfazl; Tongsook, Chanakan; Najafipour, Reza; Musante, Luciana; Vasli, Nasim; Garshasbi, Masoud; Hu, Hao; Mittal, Kirti; McNaughton, Amy J. M.; Sritharan, Kumudesh; Hudson, Melissa; Stehr, Henning; Talebi, Saeid; Moradi, Mohammad; Darvish, Hossein; Arshad Rafiq, Muhammad; Mozhdehipanah, Hossein; Rashidinejad, Ali; Samiei, Shahram; Ghadami, Mohsen; Windpassinger, Christian; Gillessen-Kaesbach, Gabriele; Tzschach, Andreas; Ahmed, Iltaf; Mikhailov, Anna; Stavropoulos, D. James; Carter, Melissa T.; Keshavarz, Soraya; Ayub, Muhammad; Najmabadi, Hossein; Liu, Xudong; Ropers, Hans Hilger; Macheroux, Peter; Vincent, John B.

2015-01-01

Histamine (HA) acts as a neurotransmitter in the brain, which participates in the regulation of many biological processes including inflammation, gastric acid secretion and neuromodulation. The enzyme histamine N-methyltransferase (HNMT) inactivates HA by transferring a methyl group from S-adenosyl-l-methionine to HA, and is the only well-known pathway for termination of neurotransmission actions of HA in mammalian central nervous system. We performed autozygosity mapping followed by targeted exome sequencing and identified two homozygous HNMT alterations, p.Gly60Asp and p.Leu208Pro, in patients affected with nonsyndromic autosomal recessive intellectual disability from two unrelated consanguineous families of Turkish and Kurdish ancestry, respectively. We verified the complete absence of a functional HNMT in patients using in vitro toxicology assay. Using mutant and wild-type DNA constructs as well as in silico protein modeling, we confirmed that p.Gly60Asp disrupts the enzymatic activity of the protein, and that p.Leu208Pro results in reduced protein stability, resulting in decreased HA inactivation. Our results highlight the importance of inclusion of HNMT for genetic testing of individuals presenting with intellectual disability. PMID:26206890

Hemoglobin analyses in the Netherlands reveal more than 80 different variants including six novel ones.

PubMed

van Zwieten, Rob; Veldthuis, Martijn; Delzenne, Barend; Berghuis, Jeffrey; Groen, Joke; Ait Ichou, Fatima; Clifford, Els; Harteveld, Cornelis L; Stroobants, An K

2014-01-01

More than 20,000 blood samples of individuals living in The Netherlands and suspected of hemolytic anemia or diabetes were analyzed by high resolution cation exchange high performance liquid chromatography (HPLC). Besides common disease-related hemoglobins (Hbs), rare variants were also detected. The variant Hbs were retrospectively analyzed by capillary zone electrophoresis (CZE) and by isoelectric focusing (IEF). For unambiguous identification, the globin genes were sequenced. Most of the 80 Hb variants detected by initial screening on HPLC were also separated by capillary electrophoresis (CE), but a few variants were only detectable with one of these methods. Some variants were unstable, had thalassemic properties or increased oxygen affinity, and some interfered with Hb A2 measurement, detection of sickle cell Hb or Hb A1c quantification. Two of the six novel variants, Hb Enschede (HBA2: c.308G  > A, p.Ser103Asn) and Hb Weesp (HBA1: c.301C > T, p.Leu101Phe), had no clinical consequences. In contrast, two others appeared clinically significant: Hb Ede (HBB: c.53A > T, p.Lys18Met) caused thalassemia and Hb Waterland (HBB: c.428C > T, pAla143Val) was related to mild polycytemia. Hb A2-Venlo (HBD: c.193G > A, p.Gly65Ser) and Hb A2-Rotterdam (HBD: c.38A > C, p.Asn13Thr) interfered with Hb A2 quantification. This survey shows that HPLC analysis followed by globin gene sequencing of rare variants is an effective method to reveal Hb variants.

Molecular analysis of markers associated with chloroquine and sulfadoxine/pyrimethamine resistance in Plasmodium falciparum malaria parasites from southeastern Côte-d'Ivoire by the time of Artemisinin-based Combination Therapy adoption in 2005.

PubMed

Ako, Berenger Aristide; Offianan, André Toure; Johansson, Marnie; Penali, Louis Koné; Nguetta, Simon-Pierre Assanvo; Sibley, Carol Hopkin

2012-01-01

Artemisin-based combination therapies became the recommended therapy in Côte-d'Ivoire in 2005, but both chloroquine (CQ) and sulfadoxine/pyrimethamine (SP) have been heavily used for many decades. Despite this long history, little is known about the geographical distribution of drug resistance-conferring genotypes outside the capital city of Abidjan. In this work, we compared the prevalence of drug-resistant genotypes in Bonoua, an urban area, and Samo, a rural agricultural area, in southeastern Côte-d'Ivoire, about 59 km from Abidjan. Samples were collected from symptomatic patients in both sites during the rainy season in 2005. Genomic DNA was isolated and codons associated with resistance to CQ and SP were analyzed: pfcrt codons Cys-72-Ser, Val-73-Val, Met-74-Ile, Arg-75-Glu, Lys-76-Thr; pfdhfr codons Ala-16-Val, Arg-51-Ile, Cys-59-Arg, Ser-108-Arg/Thr, and Ile-164-Leu; pfdhps codons Ser-436-Ala, Ala-437-Gly, Lys-540-Glu, Ala-581-Gly, and Ala-613-Thr/Ser. A limited number of genotypes were found in Bonoua compared with Samo. In both sites, the triple-mutant allele CVIET of pfcrt predominated: 100% in Bonoua and 86.2% in Samo. The wild-type allele, NCSI of pfdhfr, was common - 50% in Bonoua and 38.7% in Samo - but the triple-mutant IRNI and double-mutant NRNI were also frequent (IRNI, 32.6% in Bonoua and 19.4% in Samo; NRNI, 15.2% in Bonoua and 9.7% in Samo). In Samo, a wide range of different genotypes of Pfdhps was observed, with alleles carrying the Gly-437 codon fixed in Bonoua and comprising 73% of the isolates in Samo. Although these two sites are only 8 km apart, they belonged to very different ecological environments. The overall prevalence of alleles of single-nucleotide polymorphisms associated with resistance to CQ and SP in both locations was among the highest of the region by 2005, although the more rural site showed a more diverse set of alleles and mixed infections. Continued surveillance of these markers will be a useful tool for drug policy, as both CQ and SP are still frequently used years after withdrawal, and SP is recommended by the World Health Organization for intermittent preventive therapy for pregnant women and infants. Data analyzed herein are among the first to be generated during the year of artemisin-based combination-therapy introduction in Côte-d'Ivoire and could be of some interest for malaria policy-makers.

Molecular Dynamics of Mouse Acetylcholinesterase Complexed with Huperzine A

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

Tara, Sylvia; Helms, Volkhard H.; Straatsma, TP

1999-03-16

Two molecular dynamics simulations were performed for a modeled complex of mouse acetylcholinesterase liganded with huperzine A (HupA). Analysis of these simulations shows that HupA shifts in the active site toward Tyr 337 and Phe 338, and that several residues in the active site area reach out to make hydrogen bonds with the inhibitor. Rapid fluctuations of the gorge width are observed, ranging from widths that allow substrate access to the active site, to pinched structures that do not allow access of molecules as small as water. Additional openings or channels to the active site are found. One opening ismore » formed in the side wall of the active site gorge by residues Val 73, Asp 74, Thr 83, Glu 84, and Asn 87. Another opening is formed at the base of the gorge by residues Trp 86, Val 132, Glu 202, Gly 448, and Ile 451. Both of these openings have been observed separately in the Torpedo californica form of the enzyme. These channels could allow transport of waters and ions to and from the bulk solution.« less

Effects of peptides on proliferative activity of retinal and pigmented epithelial cells.

PubMed

Khavinson, V Kh; Zemchikhina, V N; Trofimova, S V; Malinin, V V

2003-06-01

We studied the effects of Retinalamin (polypeptide preparation isolated from the retina) and a synthetic peptide Epithalon (Ala-Glu-Asp-Gly) on proliferative activity of retinal and pigmented epithelial cells. Experiments showed that Retinalamin and Epithalon (in certain concentrations) tissue-specifically stimulated proliferation of retinal and pigmented epithelial cell in culture.

Effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of 111In-labeled lactam bridge-cyclized alpha-MSH peptides.

PubMed

Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin

2011-04-01

The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of (111)In-labeled lactam bridge-cyclized DOTA-[X]-CycMSH(hex) {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH(2); X = GGNle, GENle, or NleGE; GG = -Gly-Gly- and GE = -Gly-Glu-} peptides. Three novel peptides (DOTA-GGNle-CycMSH(hex), DOTA-GENle-CycMSH(hex), and DOTA-NleGE-CycMSH(hex)) were designed and synthesized. The melanocortin-1 (MC1) receptor-binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma-targeting and pharmacokinetic properties of (111)In-DOTA-GGNle-CycMSH(hex) and (111)In-DOTA-GENle-CycMSH(hex) were determined in B16/F1 melanoma-bearing C57 mice. DOTA-GGNle-CycMSH(hex) and DOTA-GENle-CycMSH(hex) displayed 2.1 and 11.5 nM MC1 receptor-binding affinities, whereas DOTA-NleGE-CycMSH(hex) showed 873.4 nM MC1 receptor-binding affinity. The introduction of the -GG- linker maintained high melanoma uptake while decreasing kidney and liver uptake of (111)In-DOTA-GGNle-CycMSH(hex). The tumor uptake of (111)In-DOTA-GGNle-CycMSH(hex) was 19.05 ± 5.04 and 18.6 ± 3.56 percentage injected dose per gram at 2 and 4 h after injection, respectively. (111)In-DOTA-GGNle-CycMSH(hex) exhibited 28%, 32%, and 42% less kidney uptake than (111)In-DOTA-Nle-CycMSH(hex) we reported previously, and 61%, 65%, and 68% less liver uptake than (111)In-DOTA-Nle-CycMSH(hex) at 2, 4, and 24 h after injection, respectively. The amino acid linkers exhibited profound effects on the melanoma-targeting and pharmacokinetic properties of the (111)In-labeled lactam bridge-cyclized α-melanocyte-stimulating hormone peptides. Introduction of the -GG- linker maintained high melanoma uptake while reducing kidney and liver uptake of (111)In-DOTA-GGNle-CycMSH(hex), highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide for melanoma treatment when labeled with a therapeutic radionuclide.

Association between Toll-like receptor 4 Asp299Gly polymorphism and coronary heart disease susceptibility.

PubMed

Wu, B W; Zhu, J; Shi, H M; Jin, B; Wen, Z C

2017-08-07

Published data on the association between Toll-like receptor 4 (TLR4) Asp299Gly polymorphism and coronary heart disease (CHD) susceptibility are inconclusive. To derive a more precise estimation of the relationship, a meta-analysis was performed. English-language studies were identified by searching PubMed and Embase databases (up to November 2016). All epidemiological studies were regarding Caucasians because no TLR4 Asp/Gly and Gly/Gly genotypes have been detected in Asians. A total of 20 case-control studies involving 14,416 cases and 10,764 controls were included in the meta-analysis. Overall, no significant associations were found between TLR4 Asp299Gly polymorphism and CHD susceptibility in the dominant model (OR=0.89; 95%CI=0.74 to 1.06; P=0.20) pooled in the meta-analysis. In the subgroup analysis by CHD, non-significant associations were found in cases compared to controls. When stratified by control source, no significantly decreased risk was found in the additive model or dominant model. The present meta-analysis suggests that the TLR4 Asp299Gly polymorphism was not associated with decreased CHD risk in Caucasians.

EEC- and ADULT-associated TP63 mutations exhibit functional heterogeneity toward P63 responsive sequences.

PubMed

Monti, Paola; Russo, Debora; Bocciardi, Renata; Foggetti, Giorgia; Menichini, Paola; Divizia, Maria T; Lerone, Margherita; Graziano, Claudio; Wischmeijer, Anita; Viadiu, Hector; Ravazzolo, Roberto; Inga, Alberto; Fronza, Gilberto

2013-06-01

TP63 germ-line mutations are responsible for a group of human ectodermal dysplasia syndromes, underlining the key role of P63 in the development of ectoderm-derived tissues. Here, we report the identification of two TP63 alleles, G134V (p.Gly173Val) and insR155 (p.Thr193_Tyr194insArg), associated to ADULT and EEC syndromes, respectively. These alleles, along with previously identified G134D (p.Gly173Asp) and R204W (p.Arg243Trp), were functionally characterized in yeast, studied in a mammalian cell line and modeled based on the crystal structure of the P63 DNA-binding domain. Although the p.Arg243Trp mutant showed both complete loss of transactivation function and ability to interfere over wild-type P63, the impact of p.Gly173Asp, p.Gly173Val, and p.Thr193_Tyr194insArg varied depending on the response element (RE) tested. Interestingly, p.Gly173Asp and p.Gly173Val mutants were characterized by a severe defect in transactivation along with interfering ability on two DN-P63α-specific REs derived from genes closely related to the clinical manifestations of the TP63-associated syndromes, namely PERP and COL18A1. The modeling of the mutations supported the distinct functional effect of each mutant. The present results highlight the importance of integrating different functional endpoints that take in account the features of P63 proteins' target sequences to examine the impact of TP63 mutations and the associated clinical variability. © 2013 Wiley Periodicals, Inc.

Impact of the green tea ingredient epigallocatechin gallate and a short pentapeptide (Ile-Ile-ala-Glu-Lys) on the structural organization of mixed micelles and the related uptake of cholesterol.

PubMed

Giangreco, Francesco; Höfinger, Siegfried; Bakalis, Evangelos; Zerbetto, Francesco

2018-06-07

High levels of blood cholesterol are conventionally linked to an increased risk of developing cardiovascular disease (Grundy, 1986). Here we examine the molecular mode of action of natural products with known cholesterol-lowering activity, such as for example the green tea ingredient epigallocatechin gallate and a short pentapeptide, Ile-Ile-Ala-Glu-Lys. Molecular Dynamics simulations are used to gain insight into the formation process of mixed micelles and, correspondingly, how active agents epigallocatechin gallate and Ile-Ile-Ala-Glu-Lys could possibly interfere with it. Self-assembly of physiological micelles occurs on the order of 35-50 ns; most of the structural properties of mixed micelles are unaffected by epigallocatechin gallate or Ile-Ile-Ala-Glu-Lys which integrate into the micellar surface; the diffusive motion of constituting lipids palmitoyl-oleoyl-phosphatidylcholine and cholesterol is significantly down-regulated by both epigallocatechin gallate and Ile-Ile-Ala-Glu-Lys; CONCLUSIONS: The molecular mode of action of natural compounds epigallocatechin gallate and Ile-Ile-Ala-Glu-Lys is a significant down-regulation of the diffusive motion of micellar lipids. Natural compounds like the green tea ingredient epigallocatechin gallate and a short pentapeptide, Ile-Ile-Ala-Glu-Lys, lead to a significant down-regulation of the diffusive motion of micellar lipids thereby modulating cholesterol absorption into physiological micelles. Copyright © 2018. Published by Elsevier B.V.

A Novel Missense Mutation p.Gly162Glu of the Gene MYL2 Involved in Hypertrophic Cardiomyopathy: A Pedigree Analysis of a Proband.

PubMed

Renaudin, Pauline; Janin, Alexandre; Millat, Gilles; Chevalier, Philippe

2018-04-01

Hypertrophic cardiomyopathy (HCM), a common and clinically heterogeneous disease characterized by unexplained ventricular myocardial hypertrophy, is mostly caused by mutations in sarcomeric genes. Identifying the genetic cause is important for management, therapy, and genetic counseling. A molecular diagnosis was performed on a 51-year-old woman diagnosed with HCM using a next-generation sequencing workflow based on a panel designed for sequencing the most prevalent cardiomyopathy-causing genes. Segregation analysis was performed on the woman's family. A novel myosin regulatory light chain (MYL2) missense variant, NM_000432.3:c485G>A, p.Gly162Glu, was identified and firstly considered as a putative pathogenic mutation. Among the 27 family members tested, 16 were carriers for the MYL2-p.Gly162Glu mutation, of whom 12 with the phenotype were positive. None of the 11 family members without mutation had cardiomyopathy. Genetic analysis combined with a segregation study allowed us to classify this novel MYL2 variation, p.Gly162Glu, as a novel pathogenic mutation leading to a familial form of HCM. Due to absence of fast in vitro approaches to evaluate the functional impact of missense variants on HCM-causing genes, segregation studies remain, when possible, the easiest approach to evaluate the putative pathogenicity of novel gene variants, more particularly missense ones.

Inhibition of angiogenesis in vitro by Arg-Gly-Asp-containing synthetic peptide.

PubMed Central

Nicosia, R. F.; Bonanno, E.

1991-01-01

This study was designed to evaluate the effect of the synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) on angiogenesis in serum-free collagen gel culture of rat aorta. The GRGDS peptide contains the amino acid sequence Arg-Gly-Asp (RGD), which has been implicated as a recognition site in interactions between extracellular matrix (ECM) molecules and cell membrane receptors. RGD-containing synthetic peptides are known to inhibit attachment of endothelial cells to substrates, but their effect on angiogenesis has not been fully characterized. Aortic explants embedded in collagen gel in the absence of GRGDS generated branching microvessels through a process of endothelial migration and proliferation. Addition of GRGDS to the culture medium caused a marked inhibition of angiogenesis. In contrast, GRGES, a control peptide lacking the RGD sequence, failed to inhibit angiogenesis. The inhibitory effect of GRGDS was nontoxic and reversible. The angiogenic activity of aortic explants previously inhibited with GRGDS could be restored by incubating the cultures in GRGDS-free medium. These findings suggest that angiogenesis is an anchorage-dependent process that can be inhibited by interfering with the attachment of endothelial cells to the ECM. It also indicates that synthetic peptides can be used as probes to study the mechanisms by which the ECM regulates angiogenesis. Images Figure 1 PMID:1707235

A periplasmic D-alanyl-D-alanine dipeptidase in the gram-negative bacterium Salmonella enterica.

PubMed

Hilbert, F; García-del Portillo, F; Groisman, E A

1999-04-01

The VanX protein is a D-alanyl-D-alanine (D-Ala-D-Ala) dipeptidase essential for resistance to the glycopeptide antibiotic vancomycin. While this enzymatic activity has been typically associated with vancomycin- and teicoplainin-resistant enterococci, we now report the identification of a D-Ala-D-Ala dipeptidase in the gram-negative species Salmonella enterica. The Salmonella enzyme is only 36% identical to VanX but exhibits a similar substrate specificity: it hydrolyzes D-Ala-D-Ala, DL-Ala-DL-Phe, and D-Ala-Gly but not the tripeptides D-Ala-D-Ala-D-Ala and DL-Ala-DL-Lys-Gly or the dipeptides L-Ala-L-Ala, N-acetyl-D-Ala-D-Ala, and L-Leu-Pro. The Salmonella dipeptidase gene, designated pcgL, appears to have been acquired by horizontal gene transfer because pcgL-hybridizing sequences were not detected in related bacterial species and the G+C content of the pcgL-containing region (41%) is much lower than the overall G+C content of the Salmonella chromosome (52%). In contrast to wild-type Salmonella, a pcgL mutant was unable to use D-Ala-D-Ala as a sole carbon source. The pcgL gene conferred D-Ala-D-Ala dipeptidase activity upon Escherichia coli K-12 but did not allow growth on D-Ala-D-Ala. The PcgL protein localizes to the periplasmic space of Salmonella, suggesting that this dipeptidase participates in peptidoglycan metabolism.

XPD polymorphisms: effects on DNA repair proficiency.

PubMed

Lunn, R M; Helzlsouer, K J; Parshad, R; Umbach, D M; Harris, E L; Sanford, K K; Bell, D A

2000-04-01

XPD codes for a DNA helicase involved in transcription and nucleotide excision repair. Rare XPD mutations diminish nucleotide excision repair resulting in hypersensitivity to UV light and increased risk of skin cancer. Several polymorphisms in this gene have been identified but their impact on DNA repair is not known. We compared XPD genotypes at codons 312 and 751 with DNA repair proficiency in 31 women. XPD genotypes were measured by PCR-RFLP. DNA repair proficiency was assessed using a cytogenetic assay that detects X-ray induced chromatid aberrations (breaks and gaps). Chromatid aberrations were scored per 100 metaphase cells following incubation at 37 degrees C (1.5 h after irradiation) to allow for repair of DNA damage. Individuals with the Lys/Lys codon 751 XPD genotype had a higher number of chromatid aberrations (132/100 metaphase cells) than those having a 751Gln allele (34/100 metaphase cells). Individuals having greater than 60 chromatid breaks plus gaps were categorized as having sub-optimal repair. Possessing a Lys/Lys751 genotype increased the risk of sub-optimal DNA repair (odds ratio = 7.2, 95% confidence interval = 1.01-87.7). The Asp312Asn XPD polymorphism did not appear to affect DNA repair proficiency. These results suggest that the Lys751 (common) allele may alter the XPD protein product resulting in sub-optimal repair of X-ray-induced DNA damage.

Molecular recognition and regulation of human angiotensin-I converting enzyme (ACE) activity by natural inhibitory peptides

PubMed Central

Masuyer, Geoffrey; Schwager, Sylva L. U.; Sturrock, Edward D.; Isaac, R. Elwyn; Acharya, K. Ravi

2012-01-01

Angiotensin-I converting enzyme (ACE), a two-domain dipeptidylcarboxypeptidase, is a key regulator of blood pressure as a result of its critical role in the renin-angiotensin-aldosterone and kallikrein-kinin systems. Hence it is an important drug target in the treatment of cardiovascular diseases. ACE is primarily known for its ability to cleave angiotensin I (Ang I) to the vasoactive octapeptide angiotensin II (Ang II), but is also able to cleave a number of other substrates including the vasodilator bradykinin and N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), a physiological modulator of hematopoiesis. For the first time we provide a detailed biochemical and structural basis for the domain selectivity of the natural peptide inhibitors of ACE, bradykinin potentiating peptide b and Ang II. Moreover, Ang II showed selective competitive inhibition of the carboxy-terminal domain of human somatic ACE providing evidence for a regulatory role in the human renin-angiotensin system (RAS). PMID:23056909

NMNAT1 variants cause cone and cone-rod dystrophy.

PubMed

Nash, Benjamin M; Symes, Richard; Goel, Himanshu; Dinger, Marcel E; Bennetts, Bruce; Grigg, John R; Jamieson, Robyn V

2018-03-01

Cone and cone-rod dystrophies (CD and CRD, respectively) are degenerative retinal diseases that predominantly affect the cone photoreceptors. The underlying disease gene is not known in approximately 75% of autosomal recessive cases. Variants in NMNAT1 cause a severe, early-onset retinal dystrophy called Leber congenital amaurosis (LCA). We report two patients where clinical phenotyping indicated diagnoses of CD and CRD, respectively. NMNAT1 variants were identified, with Case 1 showing an extremely rare homozygous variant c.[271G > A] p.(Glu91Lys) and Case 2 compound heterozygous variants c.[53 A > G];[769G > A] p.(Asn18Ser);(Glu257Lys). The detailed variant analysis, in combination with the observation of an associated macular atrophy phenotype, indicated that these variants were disease-causing. This report demonstrates that the variants in NMNAT1 may cause CD or CRD associated with macular atrophy. Genetic investigations of the patients with CD or CRD should include NMNAT1 in the genes examined.

Optical backbone-sidechain charge transfer transitions in proteins sensitive to secondary structure and modifications.

PubMed

Mandal, I; Paul, S; Venkatramani, R

2018-04-17

The absorption of light by proteins can induce charge transfer (CT) transitions in the UV-visible range of the electromagnetic spectrum. Metal-ligand complexes or active site prosthetic groups which absorb in the visible region exhibit prominent CT transitions. Furthermore, the protein backbone also exhibits CT transitions in the far UV range. In this manuscript, we present a detailed computational study of new near UV-visible CT transitions that involve amino acids with charged side chains. Specifically, using time dependent density functional theory calculations, we examine the absorption spectra of naturally charged amino acids (Lys, Glu, Arg, Asp and His), extracted from solution phase protein structures generated by classical molecular dynamics simulations, and phosphorylated amino acids (Tyr, Thr and Ser) from experimentally determined protein structures. We show that amino acids with charged sidechains present a directed electronic donor-bridge-acceptor paradigm, with the lowest energy optical excitations demonstrating peptide backbone-sidechain charge separations. The UV-visible spectral range of the backbone-sidechain CT transitions is determined by the chemical nature of the donor, bridge and acceptor groups within each amino acid, amino acid conformation and the protein secondary structure where the amino acids are located. Photoinduced CT occurs in opposite directions for the anionic and cationic amino acids along the ground state dipole moment vector for the chromophores. We find that photoinduced charge separation is more facile for the anionic amino acids (Asp, Glu, pSer, pThr and pTyr) relative to that for the cationic amino acids (Lys, Arg and Hsp). Our results provide a foundation for the development of spectroscopic markers based on the recently proposed Protein Charge Transfer Spectra (ProCharTS) which are relevant for the study of DNA-binding or intrinsically disordered proteins that are rich in charged amino acids.

Design of novel neurokinin 1 receptor antagonists based on conformationally constrained aromatic amino acids and discovery of a potent chimeric opioid agonist-neurokinin 1 receptor antagonist.

PubMed

Ballet, Steven; Feytens, Debby; Buysse, Koen; Chung, Nga N; Lemieux, Carole; Tumati, Suneeta; Keresztes, Attila; Van Duppen, Joost; Lai, Josephine; Varga, Eva; Porreca, Frank; Schiller, Peter W; Vanden Broeck, Jozef; Tourwé, Dirk

2011-04-14

A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3',5'-(CF(3))(2)-Bn], 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], and 23 [Ac-Tic-NMe-3',5'-(CF(3))(2)-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], which combines the N terminus of the established Dmt(1)-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH(2)) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, that is, Dmt-D-Arg-Aba-Gly-NH(2) (36), also proved to be an extremely potent and balanced μ and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity.

A Novel High-Molecular-Mass Bacteriocin Produced by Enterococcus faecium: Biochemical Features and Mode of Action.

PubMed

Vasilchenko, A S; Vasilchenko, A V; Valyshev, A V; Rogozhin, E A

2018-02-08

Discovery of a novel bacteriocin is always an event in sciences, since cultivation of most bacterial species is a general problem in microbiology. This statement is reflected by the fact that number of bacteriocins is smaller for tenfold comparing to known antimicrobial peptides. We cultivated Enterococcus faecium on simplified medium to reduce amount of purification steps. This approach allows to purify the novel heavy weight bacteriocin produced by E. faecium ICIS 7. The novelty of this bacteriocin, named enterocin-7, was confirmed by N-terminal sequencing and by comparing the structural-functional properties with available data. Purified enterocin-7 is characterized by a sequence of amino acid residues having no homology in UniProt/SwissProt/TrEMBL databases: NH2 - Asp - Ala - His - Leu - Ser - Glu - Val - Ala - Glu - Arg - Phe - Glu - Asp - Leu - Gly. Isolated thermostable protein has a molecular mass of 65 kDa, which allows it to be classified into class III in bacteriocin classification schemes. Enterocin-7 displayed a broad spectrum of activity against some Gram-positive and Gram-negative microorganisms. Fluorescent microscopy and spectroscopy showed the permeabilizing mechanism of the action of enterocin-7, which is realized within a few minutes.

Naloxone-blocked depriming effect of anxiolytic selank on apomorphine-induced behavioral manifestations of hyperfunction of dopamine system.

PubMed

Meshavkin, V K; Kost, N V; Sokolov, O Yu; Zolotarev, Yu A; Myasoedov, N F; Zozulya, A A

2006-11-01

Peptide anxiolytic selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro) applied intraperitoneally in doses of 0.01, 0.1, 1.0, and 10.0 mg/kg to mice reduces behavioral manifestations of dopaminergic system induced by apomorphine in the verticalization test. This effect was comparable to that of atypical antipsychotic olanzapine in near-therapeutic doses (0.1 and 1.0 mg/kg, intraperitoneally) and was blocked with nonselective opioid receptor antagonist naloxone (10 mg/kg, intraperitoneally). Radioreceptor assay showed that selank did not displace nonselective D2-dopamine receptor antagonist (3)H-spiperone (EC50>100 microM) and delta- and micro-opioid receptor ligand 3H-DADLE (EC50>40 microM) from specific binding sites on rat brain membranes. It is hypothesized that the revealed behavioral effect of selank is mediated by its modulating effect on the endogenous opioid system and specifically, by its effect on activity of enkephalin-degrading enzymes.

Detection of new SHV-12, SHV-5 and SHV-2a variants of extended spectrum Beta-lactamase in Klebsiella pneumoniae in Egypt

PubMed Central

2013-01-01

Background Klebsiella pneumoniae outbreaks possessing extended-spectrum β-lactamase- (ESBL) mediated resistance to third-generation cephalosporins have increased significantly in hospital and community settings worldwide. The study objective was to characterize prevalent genetic determinants of TEM, SHV and CTX-M types ESBL activity in K. pneumoniae isolates from Egypt. Methods Sixty five ESBL-producing K. pneumoniae strains, isolated from nosocomial and community-acquired infections from 10 Egyptian University hospitals (2000–2003), were confirmed with double disc-synergy method and E-test. blaTEM, blaSHV and blaCTX-m genes were identified by PCR and DNA sequencing. Pulsed-field gel electrophoresis (PFGE) was conducted for genotyping. Results All isolates displayed ceftazidime and cefotaxime resistance. blaTEM and blaSHV genes were detected in 98% of the isolates’ genomes, while 11% carried blaCTX-m. DNA sequencing revealed plasmid-borne SHV-12,-5,-2a (17%), CTX-m-15 (11%), and TEM-1 (10%) prevalence. Among SHV-12 (n=8), one isolate displayed 100% blaSHV-12 amino acid identity, while others had various point mutations: T17G (Leu to Arg, position 6 of the enzyme: n=2); A8T and A10G (Tyr and Ile to Phe and Val, positions 3 and 4, respectively: n=4), and; A703G (Lys to Glu 235: n=1). SHV-5 and SHV-2a variants were identified in three isolates: T17G (n=1); A703G and G705A (Ser and Lys to Gly and Glu: n=1); multiple mutations at A8T, A10G, T17G, A703G and G705A (n=1). Remarkably, 57% of community-acquired isolates carried CTX-m-15. PFGE demonstrated four distinct genetic clusters, grouping strains of different genetic backgrounds. Conclusions This is the first study demonstrating the occurrence of SHV-12, SHV-5 and SHV-2a variants in Egypt, indicating the spread of class A ESBL in K. pneumoniae through different mechanisms. PMID:23866018

Requirement of standardized ileal digestible valine to lysine ratio for 8- to 14-kg pigs.

PubMed

Soumeh, E A; van Milgen, J; Sloth, N M; Corrent, E; Poulsen, H D; Nørgaard, J V

2015-08-01

The objective was to define the Val requirement for weaned piglets in the context of reducing the dietary protein content. A dose-response experiment was conducted to estimate the standardized ileal digestible (SID) Val to Lys ratio required to support the optimum growth of post-weaned piglets. In this study, 96 pigs weighing 8 kg were allotted to one of six dietary treatments (16 pigs for each dietary treatment) and were housed individually. Diets were formulated to provide 0.58, 0.62, 0.66, 0.70, 0.74 and 0.78 SID Val : Lys by adding graded levels of crystalline l-Val to the 0.58 SID Val : Lys diet. Lysine was sub-limiting and supplied 90% of the recommendation (10.95 g SID Lys/kg equal to 11.8 g/kg total Lys). Average daily feed intake (ADFI), average daily gain (ADG) and gain to feed ratio (G : F) were determined during a 14-day period of ad libitum feeding. Blood and urine samples were taken at the end of each week (day 7 and 14 of the experiment) 3 h after feeding the experimental diets. The maximum ADFI and ADG were obtained in pigs fed the 0.78 SID Val : Lys diet; it was not different from the results of pigs fed 0.70 SID Val : Lys diet. The highest G : F was obtained in pigs fed 0.70 SID Val : Lys. The plasma concentration of Val increased linearly (P<0.001) as the dietary SID Val : Lys increased. The increasing dietary Val : Lys also resulted in a linear increase in Cys (P<0.001) and a quadratic increase in Arg (P=0.003), Lys (P=0.05) and Phe (P=0.009). The plasma Gly showed a quadratic decrease (P=0.05) as the dietary Val : Lys increased. Neither plasma nor urinary urea to creatinine ratio was affected by treatment. The minimum SID Val : Lys required to maximize ADFI, ADG and G : F was estimated at 0.67 SID Val : Lys by a broken-line model, and at 0.71 SID Val : Lys by a curvilinear plateau model. The Val deficiency caused a reduction in ADFI, and Val supplementation above the requirement did not impair animal performance. In conclusion, 0.70 SID Val : Lys is suggested as the Val requirement for 8 to 14 kg individually housed pigs.

Quinolone resistance-associated amino acid substitutions affect enzymatic activity of Mycobacterium leprae DNA gyrase.

PubMed

Yamaguchi, Tomoyuki; Yokoyama, Kazumasa; Nakajima, Chie; Suzuki, Yasuhiko

2017-07-01

Quinolones are important antimicrobials for treatment of leprosy, a chronic infectious disease caused by Mycobacterium leprae. Although it is well known that mutations in DNA gyrase are responsible for quinolone resistance, the effect of those mutations on the enzymatic activity is yet to be studied in depth. Hence, we conducted in vitro assays to observe supercoiling reactions of wild type and mutated M. leprae DNA gyrases. DNA gyrase with amino acid substitution Ala91Val possessed the highest activity among the mutants. DNA gyrase with Gly89Cys showed the lowest level of activity despite being found in clinical strains, but it supercoiled DNA like the wild type does if applied at a sufficient concentration. In addition, patterns of time-dependent conversion from relaxed circular DNA into supercoiled DNA by DNA gyrases with clinically unreported Asp95Gly and Asp95Asn were observed to be distinct from those by the other DNA gyrases.

Hydrolysis of substance P in the presence of the osteosarcoma cell line SaOS-2: release of free amino acids.

PubMed

Cavazza, Antonella; Marini, Mario; Roda, L Giorgio; Tarantino, Umberto; Valenti, Angela

2011-12-01

The possible hydrolysis of substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met) in presence of the osteoblastic cell line SaOS-2 was measured by capillary electrophoresis coupled to mass detection. The results obtained indicate that a very rapid disappearance of the intact undecapeptide was associated to a slower appearance of seven of its eight component amino acids. These results can be interpreted as indicating that an extremely fast hydrolysis of substance P by endopeptidases, which released peptidic by-products, was followed by a noticeably slower secondary degradation which released free amino acids. In decreasing quantitative importance, these phenomena appear to originate by the hydrolysis of the Pro(4)-Gln(5) bond, followed by C-terminal sequential degradation of the Arg(1)-Pro(4) tetrapeptide; by the hydrolysis of or Phe(7)-Phe(8) bond (or, possibly, of Gln(6)-Phe(7)) leading to release of free Phe and Gln; by hydrolysis of the Gly(9)-Leu(10) bond with subsequent release of Met and Leu. Results obtained appear to be compatible with the expression by SaOS-2 cells of enzymes already known to catalyze substance P hydrolysis, together with an apparent low efficiency of aminopeptidases. Because of the activity of C-terminal fragments on NK1 receptors, the delay between primary hydrolysis of substance P and secondary hydrolysis of its peptidic fragments indicated by the data shown implies a possible persistence of substance P physiological effects even after degradation of the intact peptide.

Recognizable cerebellar dysplasia associated with mutations in multiple tubulin genes

PubMed Central

Oegema, Renske; Cushion, Thomas D.; Phelps, Ian G.; Chung, Seo-Kyung; Dempsey, Jennifer C.; Collins, Sarah; Mullins, Jonathan G.L.; Dudding, Tracy; Gill, Harinder; Green, Andrew J.; Dobyns, William B.; Ishak, Gisele E.; Rees, Mark I.; Doherty, Dan

2015-01-01

Mutations in alpha- and beta-tubulins are increasingly recognized as a major cause of malformations of cortical development (MCD), typically lissencephaly, pachygyria and polymicrogyria; however, sequencing tubulin genes in large cohorts of MCD patients has detected tubulin mutations in only 1–13%. We identified patients with a highly characteristic cerebellar dysplasia but without lissencephaly, pachygyria and polymicrogyria typically associated with tubulin mutations. Remarkably, in seven of nine patients (78%), targeted sequencing revealed mutations in three different tubulin genes (TUBA1A, TUBB2B and TUBB3), occurring de novo or inherited from a mosaic parent. Careful re-review of the cortical phenotype on brain imaging revealed only an irregular pattern of gyri and sulci, for which we propose the term tubulinopathy-related dysgyria. Basal ganglia (100%) and brainstem dysplasia (80%) were common features. On the basis of in silico structural predictions, the mutations affect amino acids in diverse regions of the alpha-/beta-tubulin heterodimer, including the nucleotide binding pocket. Cell-based assays of tubulin dynamics reveal various effects of the mutations on incorporation into microtubules: TUBB3 p.Glu288Lys and p.Pro357Leu do not incorporate into microtubules at all, whereas TUBB2B p.Gly13Ala shows reduced incorporation and TUBA1A p.Arg214His incorporates fully, but at a slower rate than wild-type. The broad range of effects on microtubule incorporation is at odds with the highly stereotypical clinical phenotype, supporting differential roles for the three tubulin genes involved. Identifying this highly characteristic phenotype is important due to the low recurrence risk compared with the other (recessive) cerebellar dysplasias and the apparent lack of non-neurological medical issues. PMID:26130693

When silence is noise: infantile-onset Barth syndrome caused by a synonymous substitution affecting TAZ gene transcription.

PubMed

Ferri, L; Dionisi-Vici, C; Taurisano, R; Vaz, F M; Guerrini, R; Morrone, A

2016-11-01

Barth syndrome (BTHS) is an X-linked inborn error of metabolism which affects males. The main manifestations are cardiomyopathy, myopathy, hypotonia, growth delay, intermittent neutropenia and 3-methylglutaconic aciduria. Diagnosis is confirmed by mutational analysis of the TAZ gene and biochemical dosage of the monolysocardiolipin/tetralinoleoyl cardiolipin (MLCL:L4-CL) ratio. We report a 6-year-old boy who presented with severe hypoglycemia, lactic acidosis and severe dilated cardiomyopathy soon after birth. The MLCL:L4-CL ratio confirmed BTHS (3.90 on patient's fibroblast, normal: 0-0.3). Subsequent sequencing of the TAZ gene revealed only the new synonymous variant NM_000116.3 (TAZ):c.348C>T p.(Gly116Gly), which did not appear to affect the protein sequence. In silico prediction analysis suggested the new c.348C>T nucleotide change could alter the TAZ mRNA splicing processing. We analyzed TAZ mRNAs in the patient's fibroblasts and found an abnormal skipping of 24 bases (NM_000116.3:c.346_371), with the consequent ablation of 8 amino acid residues in the tafazzin protein (NP_000107.1:p.Lys117_Gly124del). Molecular analysis of at risk female family members identified the patient's sister and mother as heterozygous carriers. Apparently harmless synonymous variants in the TAZ gene can damage gene expression. Such findings widen our knowledge of molecular heterogeneity in BTHS. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Folding and Function of a T4 Lysozyme Containing 10 Consecutive Alanines Illustrate the Redundancy of Information in an Amino Acid Sequence

NASA Astrophysics Data System (ADS)

Heinz, Dirk W.; Baase, Walt A.; Matthews, Brian W.

1992-05-01

Single and multiple Xaa -> Ala substitutions were constructed in the α-helix comprising residues 39-50 in bacteriophage T4 lysozyme. The variant with alanines at 10 consecutive positions (A40-49) folds normally and has activity essentially the same as wild type, although it is less stable. The crystal structure of this polyalanine mutant displays no significant change in the main-chain atoms of the helix when compared with the wild-type structure. The individual substitutions of the solvent-exposed residues Asn-40, Ser-44, and Glu-45 with alanine tend to increase the thermostability of the protein, whereas replacements of the buried or partially buried residues Lys-43 and Leu-46 are destabilizing. The melting temperature of the lysozyme in which Lys-43 and Leu-46 are retained and positions 40, 44, 45, 47, and 48 are substituted with alanine (i.e., A40-42/44-45/47-49) is increased by 3.1^circC relative to wild type at pH 3.0, but reduced by 1.6^circC at pH 6.7. In the case of the charged amino acids Glu-45 and Lys-48, the changes in melting temperature indicate that the putative salt bridge between these two residues contributes essentially nothing to the stability of the protein. The results clearly demonstrate that there is considerable redundancy in the sequence information in the polypeptide chain; not every amino acid is essential for folding. Also, further evidence is provided that the replacement of fully solvent-exposed residues within α-helices with alanines may be a general way to increase protein stability. The general approach may permit a simplification of the protein folding problem by retaining only amino acids proven to be essential for folding and replacing the remainder with alanine.

Proline 235 plays a key role in the regulation of the oligomeric states of Thermotoga maritima Arginine Binding Protein.

PubMed

Smaldone, Giovanni; Vigorita, Marilisa; Ruggiero, Alessia; Balasco, Nicole; Dattelbaum, Jonathan D; D'Auria, Sabato; Del Vecchio, Pompea; Graziano, Giuseppe; Vitagliano, Luigi

2016-07-01

The Arginine Binding Protein isolated from Thermotoga maritima (TmArgBP) is a protein endowed with several peculiar properties. We have previously shown that TmArgBP dimerization is a consequence of the swapping of the C-terminal helix. Here we explored the structural determinants of TmArgBP domain swapping and oligomerization. In particular, we report a mutational analysis of the residue Pro235, which is located in the hinge region of the swapping dimer. This residue was either replaced with a Gly-Lys dipeptide (TmArgBP(P235GK)) or a Gly residue (TmArgBP(P235G)). Different forms of these mutants were generated and extensively characterized using biophysical techniques. For both TmArgBP(P235GK) and TmArgBP(P235G) mutants, the occurrence of multiple oligomerization states (monomers, dimers and trimers) was detected. The formation of well-folded monomeric forms for these mutants indicates that the dimerization through C-terminal domain swapping observed in wild-type TmArgBP is driven by conformational restraints imposed by the presence of Pro235 in the hinge region. Molecular dynamics studies corroborate this observation by showing that Gly235 assumes conformational states forbidden for Pro residues in the TmArgBP(P235G) monomer. Unexpectedly, the trimeric forms present: (a) peculiar circular dichroism spectra, (b) a great susceptibility to heating, and (c) the ability to bind the Thioflavin T dye. The present findings clearly demonstrate that single-point mutations have an important impact on the TmArgBP oligomerization process. In a wider context, they also indicate that proteins endowed with an intrinsic propensity to swap have an easy access to states with altered structural and, possibly, functional properties. Copyright © 2016. Published by Elsevier B.V.

A Cyclic Tetrapeptide (“Cyclodal”) and Its Mirror-Image Isomer Are Both High-Affinity μ Opioid Receptor Antagonists

PubMed Central

Weltrowska, Grazyna; Nguyen, Thi M.-D.; Chung, Nga N.; Wood, JodiAnne; Ma, Xiaoyu; Guo, Jason; Wilkes, Brian C.; Ge, Yang; Laferrière, André; Coderre, Terence J.; Schiller, Peter W.

2016-01-01

Head-to-tail cyclization of the μ opioid receptor (MOR) agonist [Dmt1]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2 (9; Dmt = 2′,6′-dimethyltyrosine) resulted in a highly active, selective MOR antagonist, c[-d-Arg-Phe-Lys-Dmt-] (1) (“cyclodal”), with subnanomolar binding affinity. A docking study of cyclodal using the crystal structure of MOR in the inactive form showed a unique binding mode with the two basic residues of the ligand forming salt bridges with the Asp127 and Glu229 receptor residues. Cyclodal showed high plasma stability and was able to cross the blood–brain barrier to reverse morphine-induced, centrally mediated analgesia when given intravenously. Surprisingly, the mirror-image isomer (optical antipode) of cyclodal, c[-Arg-d-Phe-d-Lys-d-Dmt-] (2), also turned out to be a selective MOR antagonist with 1 nM binding affinity, and thus, these two compounds represent the first example of mirror image opioid receptor ligands with both optical antipodes having high binding affinity. Reduction of the Lys-Dmt peptide bond in cyclodal resulted in an analogue, c[-d-Arg-Phe-LysΨ[CH2NH]Dmt-] (8), with MOR agonist activity. PMID:27676089

Evolution subverting essentiality: Dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease virus

PubMed Central

Martínez, Miguel A.; Verdaguer, Nuria; Mateu, Mauricio G.; Domingo, Esteban

1997-01-01

Aphthoviruses use a conserved Arg-Gly-Asp triplet for attachment to host cells and this motif is believed to be essential for virus viability. Here we report that this triplet—which is also a widespread motif involved in cell-to-cell adhesion—can become dispensable upon short-term evolution of the virus harboring it. Foot-and-mouth disease virus (FMDV), which was multiply passaged in cell culture, showed an altered repertoire of antigenic variants resistant to a neutralizing monoclonal antibody. The altered repertoire includes variants with substitutions at the Arg-Gly-Asp motif. Mutants lacking this sequence replicated normally in cell culture and were indistinguishable from the parental virus. Studies with individual FMDV clones indicate that amino acid replacements on the capsid surface located around the loop harboring the Arg-Gly-Asp triplet may mediate in the dispensability of this motif. The results show that FMDV quasispecies evolving in a constant biological environment have the capability of rendering totally dispensable a receptor recognition motif previously invariant, and to ensure an alternative pathway for normal viral replication. Thus, variability of highly conserved motifs, even those that viruses have adapted from functional cellular motifs, can contribute to phenotypic flexibility of RNA viruses in nature. PMID:9192645

Itch/β-arrestin2-dependent non-proteolytic ubiquitylation of SuFu controls Hedgehog signalling and medulloblastoma tumorigenesis.

PubMed

Infante, Paola; Faedda, Roberta; Bernardi, Flavia; Bufalieri, Francesca; Lospinoso Severini, Ludovica; Alfonsi, Romina; Mazzà, Daniela; Siler, Mariangela; Coni, Sonia; Po, Agnese; Petroni, Marialaura; Ferretti, Elisabetta; Mori, Mattia; De Smaele, Enrico; Canettieri, Gianluca; Capalbo, Carlo; Maroder, Marella; Screpanti, Isabella; Kool, Marcel; Pfister, Stefan M; Guardavaccaro, Daniele; Gulino, Alberto; Di Marcotullio, Lucia

2018-03-07

Suppressor of Fused (SuFu), a tumour suppressor mutated in medulloblastoma, is a central player of Hh signalling, a pathway crucial for development and deregulated in cancer. Although the control of Gli transcription factors by SuFu is critical in Hh signalling, our understanding of the mechanism regulating this key event remains limited. Here, we show that the Itch/β-arrestin2 complex binds SuFu and induces its Lys63-linked polyubiquitylation without affecting its stability. This process increases the association of SuFu with Gli3, promoting the conversion of Gli3 into a repressor, which keeps Hh signalling off. Activation of Hh signalling antagonises the Itch-dependent polyubiquitylation of SuFu. Notably, different SuFu mutations occurring in medulloblastoma patients are insensitive to Itch activity, thus leading to deregulated Hh signalling and enhancing medulloblastoma cell growth. Our findings uncover mechanisms controlling the tumour suppressive functions of SuFu and reveal that their alterations are implicated in medulloblastoma tumorigenesis.

Evolution of Enzymatic Activities in the Enolase Superfamily: D-Tartrate Dehydratase from Bradyrhizobium japonicum

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

Yew,W.; Fedorov, A.; Fedorov, E.

2006-01-01

We focus on the assignment of function to and elucidation of structure-function relationships for a member of the mechanistically diverse enolase superfamily encoded by the Bradyrhizobium japonicum genome (bll6730; GI:27381841). As suggested by sequence alignments, the active site contains the same functional groups found in the active site of mandelate racemase (MR) that catalyzes a 1,1-proton transfer reaction: two acid/base catalysts, Lys 184 at the end of the second {beta}-strand, and a His 322-Asp 292 dyad at the ends of the seventh and sixth -strands, respectively, as well as ligands for an essential Mg{sup 2+}, Asp 213, Glu 239, andmore » Glu 265 at the ends of the third, fourth, and fifth {beta}-strands, respectively. We screened a library of 46 acid sugars and discovered that only D-tartrate is dehydrated, yielding oxaloacetate as product. The kinetic constants (k{sub cat} = 7.3 s{sup -1}; k{sub cat}/K{sub M} = 8.5 x 10{sup 4} M{sup -1} s{sup -1}) are consistent with assignment of the D-tartrate dehydratase (TarD) function. The kinetic phenotypes of mutants as well as the structures of liganded complexes are consistent with a mechanism in which Lys 184 initiates the reaction by abstraction of the {alpha}-proton to generate a Mg{sup 2+}-stabilized enediolate intermediate, and the vinylogous -elimination of the 3-OH group is general acid-catalyzed by the His 322, accomplishing the anti-elimination of water. The replacement of the leaving group by solvent-derived hydrogen is stereorandom, suggesting that the enol tautomer of oxaloacetate is the product; this expectation was confirmed by its observation by {sup 1}H NMR spectroscopy. Thus, the TarD-catalyzed reaction is a 'simple' extension of the two-step reaction catalyzed by MR: base-catalyzed proton abstraction to generate a Mg{sup 2+}-stabilized enediolate intermediate followed by acid-catalyzed decomposition of that intermediate to yield the product.« less

Severe hypertriglyceridemia in a patient heterozygous for a lipoprotein lipase gene allele with two novel missense variants.

PubMed

Kassner, Ursula; Salewsky, Bastian; Wühle-Demuth, Marion; Szijarto, Istvan Andras; Grenkowitz, Thomas; Binner, Priska; März, Winfried; Steinhagen-Thiessen, Elisabeth; Demuth, Ilja

2015-09-01

Rare monogenic hyperchylomicronemia is caused by loss-of-function mutations in genes involved in the catabolism of triglyceride-rich lipoproteins, including the lipoprotein lipase gene, LPL. Clinical hallmarks of this condition are eruptive xanthomas, recurrent pancreatitis and abdominal pain. Patients with LPL deficiency and severe or recurrent pancreatitis are eligible for the first gene therapy treatment approved by the European Union. Therefore the precise molecular diagnosis of familial hyperchylomicronemia may affect treatment decisions. We present a 57-year-old male patient with excessive hypertriglyceridemia despite intensive lipid-lowering therapy. Abdominal sonography showed signs of chronic pancreatitis. Direct DNA sequencing and cloning revealed two novel missense variants, c.1302A>T and c.1306G>A, in exon 8 of the LPL gene coexisting on the same allele. The variants result in the amino-acid exchanges p.(Lys434Asn) and p.(Gly436Arg). They are located in the carboxy-terminal domain of lipoprotein lipase that interacts with the glycosylphosphatidylinositol-anchored HDL-binding protein (GPIHBP1) and are likely of functional relevance. No further relevant mutations were found by direct sequencing of the genes for APOA5, APOC2, LMF1 and GPIHBP1. We conclude that heterozygosity for damaging mutations of LPL may be sufficient to produce severe hypertriglyceridemia and that chylomicronemia may be transmitted in a dominant manner, at least in some families.

Severe hypertriglyceridemia in a patient heterozygous for a lipoprotein lipase gene allele with two novel missense variants

PubMed Central

Kassner, Ursula; Salewsky, Bastian; Wühle-Demuth, Marion; Szijarto, Istvan Andras; Grenkowitz, Thomas; Binner, Priska; März, Winfried; Steinhagen-Thiessen, Elisabeth; Demuth, Ilja

2015-01-01

Rare monogenic hyperchylomicronemia is caused by loss-of-function mutations in genes involved in the catabolism of triglyceride-rich lipoproteins, including the lipoprotein lipase gene, LPL. Clinical hallmarks of this condition are eruptive xanthomas, recurrent pancreatitis and abdominal pain. Patients with LPL deficiency and severe or recurrent pancreatitis are eligible for the first gene therapy treatment approved by the European Union. Therefore the precise molecular diagnosis of familial hyperchylomicronemia may affect treatment decisions. We present a 57-year-old male patient with excessive hypertriglyceridemia despite intensive lipid-lowering therapy. Abdominal sonography showed signs of chronic pancreatitis. Direct DNA sequencing and cloning revealed two novel missense variants, c.1302A>T and c.1306G>A, in exon 8 of the LPL gene coexisting on the same allele. The variants result in the amino-acid exchanges p.(Lys434Asn) and p.(Gly436Arg). They are located in the carboxy-terminal domain of lipoprotein lipase that interacts with the glycosylphosphatidylinositol-anchored HDL-binding protein (GPIHBP1) and are likely of functional relevance. No further relevant mutations were found by direct sequencing of the genes for APOA5, APOC2, LMF1 and GPIHBP1. We conclude that heterozygosity for damaging mutations of LPL may be sufficient to produce severe hypertriglyceridemia and that chylomicronemia may be transmitted in a dominant manner, at least in some families. PMID:25585702

Inhibitory effect of the peptide epitalon on the development of spontaneous mammary tumors in HER-2/neu transgenic mice.

PubMed

Anisimov, Vladimir N; Khavinson, Vladimir K H; Provinciali, Mauro; Alimova, Irina N; Baturin, Dmitri A; Popovich, Irina G; Zabezhinski, Mark A; Imyanitov, Eugeni N; Mancini, Romina; Franceschi, Claudio

2002-09-01

Female FVB/N HER-2/neu transgenic mice from the age of 2 months were subcutaneously injected with saline, the peptide Epitalon(R) (Ala-Glu-Asp-Gly) or with the peptide Vilon(R) (Lys-Glu) in a single dose of 1 microg/mouse for 5 consecutive days every month. Epitalon treatment reduced the cumulative number and the maximum size of tumors (p < 0.05). Furthermore, the number of mice bearing 1 mammary tumor was increased, whereas the number of mice bearing 2 or more mammary tumors was reduced in Epitalon-treated in comparison to saline-treated animals (p < 0.05). The size but not the number of lung metastases was reduced in Epitalon-treated compared to saline-treated mice (p < 0.05). The treatment with Vilon produced significant negative effects when compared to the control group, with an increased incidence of mammary cancer development (p < 0.05), a shorter mean latent period of tumors (p < 0.05) and an increased cumulative number of tumors (p < 0.05). A 3.7-fold reduction in the expression of HER-2/neu mRNA was found in mammary tumors from HER-2/neu transgenic mice treated with Epitalon compared to control animals. The expression of mRNA for HER-2/neu was also partially reduced in Vilon-treated mice, but it remained significantly higher in Vilon- than in Epitalon-treated animals (1.9-fold increase). The data demonstrate the inhibitory effect of Epitalon in the development of spontaneous mammary tumors in HER-2/neu mice, suggesting that a downregulation of HER-2/neu gene expression in mammary adenocarcinoma may be responsible, at least in part, for the antitumor effect of the peptide. Copyright 2002 Wiley-Liss, Inc.

Active Site Characterization of Proteases Sequences from Different Species of Aspergillus.

PubMed

Morya, V K; Yadav, Virendra K; Yadav, Sangeeta; Yadav, Dinesh

2016-09-01

A total of 129 proteases sequences comprising 43 serine proteases, 36 aspartic proteases, 24 cysteine protease, 21 metalloproteases, and 05 neutral proteases from different Aspergillus species were analyzed for the catalytically active site residues using MEROPS database and various bioinformatics tools. Different proteases have predominance of variable active site residues. In case of 24 cysteine proteases of Aspergilli, the predominant active site residues observed were Gln193, Cys199, His364, Asn384 while for 43 serine proteases, the active site residues namely Asp164, His193, Asn284, Ser349 and Asp325, His357, Asn454, Ser519 were frequently observed. The analysis of 21 metalloproteases of Aspergilli revealed Glu298 and Glu388, Tyr476 as predominant active site residues. In general, Aspergilli species-specific active site residues were observed for different types of protease sequences analyzed. The phylogenetic analysis of these 129 proteases sequences revealed 14 different clans representing different types of proteases with diverse active site residues.

Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site

PubMed Central

Kumar, Hemant; Finer-Moore, Janet S.; Kaback, H. Ronald; Stroud, Robert M.

2015-01-01

The X-ray crystal structure of a conformationally constrained mutant of the Escherichia coli lactose permease (the LacY double-Trp mutant Gly-46→Trp/Gly-262→Trp) with bound p-nitrophenyl-α-d-galactopyranoside (α-NPG), a high-affinity lactose analog, is described. With the exception of Glu-126 (helix IV), side chains Trp-151 (helix V), Glu-269 (helix VIII), Arg-144 (helix V), His-322 (helix X), and Asn-272 (helix VIII) interact directly with the galactopyranosyl ring of α-NPG to provide specificity, as indicated by biochemical studies and shown directly by X-ray crystallography. In contrast, Phe-20, Met-23, and Phe-27 (helix I) are within van der Waals distance of the benzyl moiety of the analog and thereby increase binding affinity nonspecifically. Thus, the specificity of LacY for sugar is determined solely by side-chain interactions with the galactopyranosyl ring, whereas affinity is increased by nonspecific hydrophobic interactions with the anomeric substituent. PMID:26157133

Protease-activated receptor 2 agonist increases cell proliferation and invasion of human pancreatic cancer cells

PubMed Central

XIE, LIQUN; DUAN, ZEXING; LIU, CAIJU; ZHENG, YANMIN; ZHOU, JING

2015-01-01

The aim of this study was to determine the expression of protease-activated receptor 2 (PAR-2) in the human pancreatic cancer cell line SW1990, and to evaluate its effect on cell proliferation and invasion. The expression of PAR-2 protein and mRNA in SW1990 cells was determined by immunocytochemistry and reverse transcription polymerase chain reaction (PCR), respectively. MTT and cell invasion and migration assays, as well as semi-quantitative PCR and zymography analysis, were additionally performed. PAR-2 mRNA was significantly upregulated in the cells treated with trypsin or the PAR-2 activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) (P<0.01), but not in the Val-Lys-Gly-Ile-Leu-Ser group (P>0.05). Trypsin and SLIGKV significantly promoted SW1990 cell proliferation in a dose- and time-dependent manner (P<0.05). Compared with the control group, trypsin and SLIGKV significantly increased the mRNA expression (P<0.01) and gelatinolytic activity (P<0.01) of matrix metalloproteinase (MMP)-2. In conclusion, PAR-2 is expressed in SW1990 cells. PAR-2 activation may promote the invasion and migration of human pancreatic cancer cells by increasing MMP-2 expression. PMID:25452809

The study on the relationship between IRS-1 Gly972Arg and IRS-2 Gly1057Asp polymorphisms and type 2 diabetes in the Kurdish ethnic group in West Iran.

PubMed

Haghani, Karimeh; Bakhtiyari, Salar

2012-11-01

An association between the IRS-1 Gly972Arg and IRS-2 Gly1057Asp polymorphisms and type 2 diabetes mellitus (T2DM) in different ethnic groups is controversial. We aimed to identify the association of these polymorphisms with T2DM in the Kurdish ethnic group of Iran. Study groups included 336 T2DM and 341 normoglycemic subjects. Genotyping was determined by polymerase chain reaction-restriction fragment length polymorphism. Genotypic and allelic frequencies were then evaluated. GR and RR genotypes of IRS-1 Gly972Arg variant gave a higher risk for T2DM (odds ratios [OR]=1.76 and OR=3.86, respectively). IRS-1 Gly972Arg polymorphism was found to be significantly associated with T2DM (OR=1.63) for the dominant model (GG vs. GR+RR). GD genotypes of the IRS-2 Gly1057Asp variant gave a higher risk for T2DM (OR=1.63). The dominant model analysis of the IRS-2 Gly1057Asp genotypes (GG vs. GD+DD) also showed an enhanced association with T2DM (OR=1.69). Among several combinations, GR/GD gave the highest risk for T2DM (OR=3.1). Other combinations were also significantly associated with T2DM, including, GR/GG (OR=1.86), RR/GG (OR=1.76), GG/GD (OR=1.83), and GG/DD (OR=2.35). HbA1c, serum triglyceride, and systolic blood pressure were higher in the control subjects with GR+RR genotypes compared with the GG genotype. Among the T2DM subjects, fasting plasma glucose was significantly lower in subjects with the GG genotype in relation to those with the GR+RR genotypes. Normoglycemic subjects carrying GD+DD genotypes of IRS-2 Gly1057Asp variation had a significantly higher fasting plasma glucose and total cholesterol, as compared with those with the GG genotype. Our findings revealed that IRS-1 Gly972Arg and IRS-2 Gly1057Asp polymorphisms are associated with T2DM in the Kurdish ethnic group.

Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis

PubMed Central

Zaprasis, Adrienne; Bleisteiner, Monika; Kerres, Anne; Hoffmann, Tamara

2014-01-01

The data presented here reveal a new facet of the physiological adjustment processes through which Bacillus subtilis can derive osmostress protection. We found that the import of proteogenic (Glu, Gln, Asp, Asn, and Arg) and of nonproteogenic (Orn and Cit) amino acids and their metabolic conversion into proline enhances growth under otherwise osmotically unfavorable conditions. Osmoprotection by amino acids depends on the functioning of the ProJ-ProA-ProH enzymes, but different entry points into this biosynthetic route are used by different amino acids to finally yield the compatible solute proline. Glu, Gln, Asp, and Asn are used to replenish the cellular pool of glutamate, the precursor for proline production, whereas Arg, Orn, and Cit are converted into γ-glutamic semialdehyde/Δ1-pyrroline-5-carboxylate, an intermediate in proline biosynthesis. The import of Glu, Gln, Asp, Asn, Arg, Orn, and Cit did not lead to a further increase in the size of the proline pool that is already present in osmotically stressed cells. Hence, our data suggest that osmoprotection of B. subtilis by this group of amino acids rests on the savings in biosynthetic building blocks and energy that would otherwise have to be devoted either to the synthesis of the proline precursor glutamate or of proline itself. Since glutamate is the direct biosynthetic precursor for proline, we studied its uptake and found that GltT, an Na+-coupled symporter, is the main uptake system for both glutamate and aspartate in B. subtilis. Collectively, our data show how effectively B. subtilis can exploit environmental resources to derive osmotic-stress protection through physiological means. PMID:25344233

Mutations in the histamine N-methyltransferase gene, HNMT, are associated with nonsyndromic autosomal recessive intellectual disability.

PubMed

Heidari, Abolfazl; Tongsook, Chanakan; Najafipour, Reza; Musante, Luciana; Vasli, Nasim; Garshasbi, Masoud; Hu, Hao; Mittal, Kirti; McNaughton, Amy J M; Sritharan, Kumudesh; Hudson, Melissa; Stehr, Henning; Talebi, Saeid; Moradi, Mohammad; Darvish, Hossein; Arshad Rafiq, Muhammad; Mozhdehipanah, Hossein; Rashidinejad, Ali; Samiei, Shahram; Ghadami, Mohsen; Windpassinger, Christian; Gillessen-Kaesbach, Gabriele; Tzschach, Andreas; Ahmed, Iltaf; Mikhailov, Anna; Stavropoulos, D James; Carter, Melissa T; Keshavarz, Soraya; Ayub, Muhammad; Najmabadi, Hossein; Liu, Xudong; Ropers, Hans Hilger; Macheroux, Peter; Vincent, John B

2015-10-15

Histamine (HA) acts as a neurotransmitter in the brain, which participates in the regulation of many biological processes including inflammation, gastric acid secretion and neuromodulation. The enzyme histamine N-methyltransferase (HNMT) inactivates HA by transferring a methyl group from S-adenosyl-l-methionine to HA, and is the only well-known pathway for termination of neurotransmission actions of HA in mammalian central nervous system. We performed autozygosity mapping followed by targeted exome sequencing and identified two homozygous HNMT alterations, p.Gly60Asp and p.Leu208Pro, in patients affected with nonsyndromic autosomal recessive intellectual disability from two unrelated consanguineous families of Turkish and Kurdish ancestry, respectively. We verified the complete absence of a functional HNMT in patients using in vitro toxicology assay. Using mutant and wild-type DNA constructs as well as in silico protein modeling, we confirmed that p.Gly60Asp disrupts the enzymatic activity of the protein, and that p.Leu208Pro results in reduced protein stability, resulting in decreased HA inactivation. Our results highlight the importance of inclusion of HNMT for genetic testing of individuals presenting with intellectual disability. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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.

Interaction between lysine 102 and aspartate 338 in the insect amino acid cotransporter KAAT1.

PubMed

Castagna, M; Soragna, A; Mari, S A; Santacroce, M; Betté, S; Mandela, P G; Rudnick, G; Peres, A; Sacchi, V F

2007-10-01

KAAT1 is a lepidopteran neutral amino acid transporter belonging to the NSS super family (SLC6), which has an unusual cation selectivity, being activated by K(+) and Li(+) in addition to Na(+). We have previously demonstrated that Asp338 is essential for KAAT1 activation by K(+) and for the coupling of amino acid and driver ion fluxes. By comparing sequences of NSS family members, site-directed mutagenesis, and expression in Xenopus laevis oocytes, we identified Lys102 as a residue likely to interact with Asp338. Compared with wild type, the single mutants K102V and D338E each showed altered leucine uptake and transport-associated currents in the presence of both Na(+) and K(+). However, in K102V/D338E double mutant, the K102V mutation reversed both the inhibition of Na(+)-dependent transport and the block in K(+)-dependent transport that characterize the D338E mutant. K(+)-dependent leucine currents were not observed in any mutants with D338E. In the presence of the oxidant Cu(II) (1,10-phenanthroline)(3), we observed specific and reversible inhibition of K102C/D338C mutant, but not of the corresponding single cysteine mutants, suggesting that these residues are sufficiently close to form a disulfide bond. Thus both structural and functional evidence suggests that these two residues interact. Similar results have been obtained mutating the bacterial transporter homolog TnaT. Asp338 corresponds to Asn286, a residue located in the Na(+) binding site in the recently solved crystal structure of the NSS transporter LeuT(Aa) (41). Our results suggest that Lys102, interacting with Asp338, could contribute to the spatial organization of KAAT1 cation binding site and permeation pathway.

The adipokinetic hormone of the coleopteran suborder Adephaga: Structure, function, and comparison of distribution in other insects.

PubMed

Gäde, Gerd; Marco, Heather G

2017-07-01

The aim of the current study is to identify the adipokinetic hormone(s) (AKHs) of a basal suborder of the species-rich Coleoptera, the Adephaga, and possibly learn more about the ancestral AKH of beetles. Moreover, we wanted to compare the ancestral AKH with AKHs of more advanced beetles, of which a number are pest insects. This would allow us to assess whether AKH mimetics would be suitable as insecticides, that is, be harmful to the pest species but not to the beneficial species. Nine species of the Adephaga were investigated and all synthesize only one octapeptide in the corpus cardiacum, as revealed by Edman degradation sequencing techniques or by mass spectrometry. The amino acid sequence pGlu-Leu-Asn-Phe-Ser-Thr-Gly-Trp corresponds to Schgr-AKH-II that was first identified in the desert locust. It is assumed that Schgr-AKH-II-the peptide of a basal beetle clade-is the ancestral AKH for beetles. Some other beetle families, as well as some Hymenoptera (including honey bees) also contain this peptide, whereas most of the pest beetle species have different AKHs. This argues that those peptides and their receptors should be explored for developing mimetics with insecticidal properties. A scenario where Schgr-AKH-II (the only AKH of Adephaga) is used as basic molecular structure to derive almost all other known beetle AKHs via single step mutations is very likely, and supports the interpretation that Schgr-AKH-II is the ancestral AKH of Coleoptera. © 2017 Wiley Periodicals, Inc.

A thermostable Gloeophyllum trabeum xylanase with potential for the brewing industry.

PubMed

Wang, Xiaoyu; Luo, Huiying; Yu, Wangning; Ma, Rui; You, Shuai; Liu, Weina; Hou, Lingyu; Zheng, Fei; Xie, Xiangming; Yao, Bin

2016-05-15

A xylanase gene of glycoside hydrolase family 10, GtXyn10, was cloned from Gloeophyllum trabeum CBS 900.73 and expressed in Pichia pastoris GS115. Purified recombinant GtXyn10 exhibited significant activities to xylan (100.0%), lichenan (11.2%), glucan (15.2%) and p-nitrophenol-β-cellobiose (18.6%), demonstrated the maximum xylanase and glucanase activities at pH 4.5-5.0 and 75°C, retained stability over the pH range of 2.0-7.5 and at 70°C, and was resistant to pepsin and trypsin, most metal ions and SDS. Multiple sequence alignment and modeled-structure analysis identified a unique Gly48 in GtXyn10, and site-directed mutagenesis of Gly48 to Lys improved the temperature optimum up to 80°C. Under simulated mashing conditions, GtXyn10 (80U) reduced the mash viscosity by 12.8% and improved the filtration rate by 31.3%. All these properties above make GtXyn10 attractive for potential applications in the feed and brewing industries. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification of dipeptidyl peptidase-IV inhibitory peptides from mare whey protein hydrolysates.

PubMed

Song, J J; Wang, Q; Du, M; Ji, X M; Mao, X Y

2017-09-01

Inhibition of dipeptidyl peptidase-IV (DPP-IV) activity is a promising strategy for treatment of type 2 diabetes. In the current study, DPP-IV inhibitory peptides were identified from mare whey protein hydrolysates obtained by papain. The results showed that all the mare whey protein hydrolysates obtained at various hydrolysis durations possessed more potent DPP-IV inhibitory activity compared with intact whey protein. The 4-h hydrolysates showed the greatest DPP-IV inhibitory activity with half-maximal inhibitory concentration of 0.18 mg/mL. The 2 novel peptides from 4-h hydrolysate fractions separated by successive chromatographic steps were characterized by liquid chromatography-electrospray ionization tandem mass spectrometry. The novel peptides Asn-Leu-Glu-Ile-Ile-Leu-Arg and Thr-Gln-Met-Val-Asp-Glu-Glu-Ile-Met-Glu-Lys-Phe-Arg, which corresponded to β-lactoglobulin 1 f(71-77) and β-lactoglobulin 1 f(143-155), demonstrated DPP-IV inhibitory activity with half-maximal inhibitory concentrations of 86.34 and 69.84 μM, respectively. The DPP-IV inhibitory activity of the 2 peptides was retained or even improved after simulated gastrointestinal digestion in vitro. Our findings indicate that mare whey protein-derived peptides may possess potential as functional food ingredients in the management of type 2 diabetes. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Flexible Xxx–Asp/Asn and Gly–Xxx Residues of Equine Cytochrome c in Matrix-Assisted Laser Desorption/Ionization In-Source Decay Mass Spectrometry

PubMed Central

Takayama, Mitsuo

2012-01-01

The backbone flexibility of a protein has been studied from the standpoint of the susceptibility of amino acid residues to in-source decay (ISD) in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Residues more susceptible to MALDI-ISD, namely Xxx–Asp/Asn and Gly–Xxx, were identified from the discontinuous intense peak of c′-ions originating from specific cleavage at N–Cα bonds of the backbone of equine cytochrome c. The identity of the residues susceptible to ISD was consistent with the known flexible backbone amides as estimated by hydrogen/deuterium exchange (HDX) experiments. The identity of these flexible amino acid residues (Asp, Asn, and Gly) is consistent with the fact that these residues are preferred in flexible secondary structure free from intramolecular hydrogen-bonded structures such as α-helix and β-sheet. The MALDI-ISD spectrum of equine cytochrome c gave not only intense N-terminal side c′-ions originating from N–Cα bond cleavage at Xxx–Asp/Asn and Gly–Xxx residues, but also C-terminal side complement z′-ions originating from the same cleavage sites. The present study implies that MALDI-ISD can give information about backbone flexibility of proteins, comparable with the protection factors estimated by HDX. PMID:24349908

Binding Energies of the Proton-Bound Amino Acid Dimers Gly·Gly, Ala·Ala, Gly·Ala, and Lys·Lys Measured by Blackbody Infrared Radiative Dissociation

PubMed Central

Price, William D.; Schnier, Paul D.

2005-01-01

Arrhenius activation energies in the zero-pressure limit for dissociation of gas-phase proton-bound homodimers of N,N-dimethylacetamide (N,N-DMA), glycine, alanine, and lysine and the heterodimer alanine·glycine were measured using blackbody infrared radiative dissociation (BIRD). In combination with master equation modeling of the kinetic data, binding energies of these dimers were determined. A value of 1.25 ± 0.05 eV is obtained for N,N-DMA and is in excellent agreement with that reported in the literature. The value obtained from the truncated Boltzmann model is significantly higher, indicating that the assumptions of this model do not apply to these ions. This is due to the competitive rates of photon emission and dissociation for these relatively large ions. The binding energies of the amino acid dimers are ~1.15 ± 0.05 eV and are indistinguishable despite the difference in their gas-phase basicity and structure. The threshold dissociation energies can be accurately modeled using a range of dissociation parameters and absorption/emission rates. However, the absolute values of the dissociation rates depend more strongly on the absorption/emission rates. For N,N-DMA and glycine, an accurate fit was obtained using frequencies and transition dipole moments calculated at the ab initio RHF/2-31G* and MP2/2-31G* level, respectively. In order to obtain a similar accuracy using values obtained from AM1 semiempirical calculations, it was necessary to multiply the transition dipole moments by a factor of 3. These results demonstrate that in combination with master equation modeling, BIRD can be used to obtain accurate threshold dissociation energies of relatively small ions of biological interest. PMID:17235378

Characteristics of mutants designed to incorporate a new ion pair into the structure of a cold adapted subtilisin-like serine proteinase.

PubMed

Sigurdardóttir, Anna Gudný; Arnórsdóttir, Jóhanna; Thorbjarnardóttir, Sigrídur H; Eggertsson, Gudmundur; Suhre, Karsten; Kristjánsson, Magnús M

2009-03-01

Structural comparisons of VPR, a subtilisin-like serine proteinase from a psychrotrophic Vibrio species and a thermophilic homologue, aqualysin I, have led us to hypothesize about the roles of different residues in the temperature adaptation of the enzymes. Some of these hypotheses are now being examined by analysis of mutants of the enzymes. The selected substitutions are believed to increase the stability of the cold adapted enzyme based on structural analysis of the thermostable structure. We report here on mutants, which were designed to incorporate an ion pair into the structure of VPR. The residues Asp17 and Arg259 are assumed to form an ion pair in aqualysin I. The cold adapted VPR contains Asn (Asn15) and Lys (Lys257) at corresponding sites in its structure. In VPR, Asn 15 is located on a surface loop with its side group pointing towards the side chain of Lys257. By substituting Asn15 by Asp (N15D) it was considered feasible that a salt bridge would form between the oppositely charged groups. To mimic further the putative salt bridge from the thermophile enzyme the corresponding double mutant (N15D/K257R) was also produced. The N15D mutation increased the thermal stability of VPR by approximately 3 degrees C, both in T(50%) and T(m). Addition of the K257R mutation did not however, increase the stability of the double mutant any further. Despite this stabilization of the VPR mutants the catalytic activity (k(cat)) against the substrate Suc-AAPF-NH-Np was increased in the mutants. Molecular dynamics simulations on wild type and the two mutant proteins suggested that indeed a salt bridge was formed in both cases. Furthermore, a truncated form of the N15D mutant (N15DDeltaC) was produced, lacking a 15 residue long C-terminal extended sequence not present in the thermophilic enzyme. In wild type VPR this supposedly moveable, negatively charged arm on the protein molecule might interfere with the new salt bridge introduced as a result of the N15D mutation. Removal of the C-terminal arm improved the thermal stability (T(m) approximately +1.5 degrees C) of the truncated enzyme (VPRDeltaC) as compared to the wild type VPR. Introduction of the N15D substitution into VPRDeltaC improved the thermal stability further by about 3 degrees C, or to about the same extent as in the wild type. However, contrary to what was observed for the wild type, the introduction of the putative salt bridge did not affect the catalytic properties (k(cat)) of the C-terminal truncated enzyme.

Simulations of the myosin II motor reveal a nucleotide-state sensing element that controls the recovery stroke.

PubMed

Koppole, Sampath; Smith, Jeremy C; Fischer, Stefan

2006-08-18

During the recovery stroke, the myosin motor is primed for the next power stroke by a 60 degree rotation of its lever arm. This reversible motion is coupled to the activation of the ATPase function of myosin through conformational changes along the relay helix, which runs from the Switch-2 loop near the ATP to the converter domain carrying the lever arm. Via a hydrogen bond between the side-chain of Asn475 on the relay helix and the Gly457/Ser456 peptide group on the Switch-2, the rotation of the converter domain is coupled to the formation of a hydrogen bond between Gly457 and gamma-phosphate that is essential for ATP hydrolysis. Here, molecular dynamics simulations of Dictyostelium discoideum myosin II in the two end conformations of the recovery stroke with different nucleotide states (ATP, ADP x Pi, ADP) reveal that the side-chain of Asn475 breaks away from Switch-2 upon ATP hydrolysis to make a hydrogen bond with Tyr573. This sensing of the nucleotide state is achieved by a small displacement of the cleaved gamma-phosphate towards Gly457 which in turn pushes Asn475 away. The sensing plays a dual role by (i) preventing the wasteful reversal of the recovery stroke while the nucleotide is in the ADP x Pi state, and (ii) decoupling the relay helix from Switch-2, thus allowing the power stroke to start upon initial binding to actin while Gly457 of Switch-2 keeps interacting with the Pi (known to be released only later after tight actin binding). A catalytically important salt bridge between Arg238 (on Switch-1) and Glu459 (on Switch-2), which covers the hydrolysis site, is seen to form rapidly when ATP is added to the pre-recovery stroke conformer and remains stable after the recovery stroke, indicating that it has a role in shaping the ATP binding site by induced fit.

l-lysine production by Bacillus methanolicus: Genome-based mutational analysis and l-lysine secretion engineering.

PubMed

Nærdal, Ingemar; Netzer, Roman; Irla, Marta; Krog, Anne; Heggeset, Tonje Marita Bjerkan; Wendisch, Volker F; Brautaset, Trygve

2017-02-20

Bacillus methanolicus is a methylotrophic bacterium with an increasing interest in academic research and for biotechnological applications. This bacterium was previously applied for methanol-based production of l-glutamate, l-lysine and the five-carbon diamine cadaverine by wild type, classical mutant and recombinant strains. The genomes of two different l-lysine secreting B. methanolicus classical mutant strains, NOA2#13A52-8A66 and M168-20, were sequenced. We focused on mutational mapping in genes present in l-lysine and other relevant amino acid biosynthetic pathways, as well as in the primary cell metabolism important for precursor supply. In addition to mutations in the aspartate pathway genes dapG, lysA and hom-1, new mutational target genes like alr, proA, proB1, leuC, odhA and pdhD were identified. Surprisingly, no mutations were found in the putative l-lysine transporter gene lysE MGA3 . Inspection of the wild type B. methanolicus strain PB1 genome sequence identified two homologous putative l-lysine transporter genes, lysE PB1 and lysE2 PB1 . The biological role of these putative l-lysine transporter genes, together with the heterologous l-lysine exporter gene lysE Cg from Corynebacterium glutamicum, were therefore investigated. Our results demonstrated that the titer of secreted l-lysine in B. methanolicus was significantly increased by overexpression of lysE Cg while overexpression of lysE MGA3 , lysE PB1 and lysE2 PB1 had no measurable effect. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure-activity relationship of cyclic peptide penta-c[Asp-His(6)-DPhe(7)-Arg(8)-Trp(9)-Lys]-NH(2) at the human melanocortin-1 and -4 receptors: His(6) substitution.

PubMed

Cheung, Adrian Wai-Hing; Danho, Waleed; Swistok, Joseph; Qi, Lida; Kurylko, Grazyna; Rowan, Karen; Yeon, Mitch; Franco, Lucia; Chu, Xin-Jie; Chen, Li; Yagaloff, Keith

2003-04-07

A series of MT-II related cyclic peptides, based on potent but non-selective hMC4R agonist (Penta-c[Asp-His(6)-DPhe(7)-Arg(8)-Trp(9)-Lys]-NH(2)) was prepared in which His(6) residue was systematically substituted. Two of the most interesting peptides identified in this study are Penta-c[Asp-5-ClAtc-DPhe-Arg-Trp-Lys]-NH(2) and Penta-c[Asp-5-ClAtc-DPhe-Cit-Trp-Lys]-NH(2) which are potent hMC4R agonists and are either inactive or weak partial agonists (not tested for their antagonist activities) in hMC1R, hMC3R and hMC5R agonist assays.

Functional analysis of four naturally occurring variants of human constitutive androstane receptor.

PubMed

Ikeda, Shinobu; Kurose, Kouichi; Jinno, Hideto; Sai, Kimie; Ozawa, Shogo; Hasegawa, Ryuichi; Komamura, Kazuo; Kotake, Takeshi; Morishita, Hideki; Kamakura, Shiro; Kitakaze, Masafumi; Tomoike, Hitonobu; Tamura, Tomohide; Yamamoto, Noboru; Kunitoh, Hideo; Yamada, Yasuhide; Ohe, Yuichiro; Shimada, Yasuhiro; Shirao, Kuniaki; Kubota, Kaoru; Minami, Hironobu; Ohtsu, Atsushi; Yoshida, Teruhiko; Saijo, Nagahiro; Saito, Yoshiro; Sawada, Jun-ichi

2005-01-01

The human constitutive androstane receptor (CAR, NR1I3) is a member of the orphan nuclear receptor superfamily that plays an important role in the control of drug metabolism and disposition. In this study, we sequenced all the coding exons of the NR1I3 gene for 334 Japanese subjects. We identified three novel single nucleotide polymorphisms (SNPs) that induce non-synonymous alterations of amino acids (His246Arg, Leu308Pro, and Asn323Ser) residing in the ligand-binding domain of CAR, in addition to the Val133Gly variant, which was another CAR variant identified in our previous study. We performed functional analysis of these four naturally occurring CAR variants in COS-7 cells using a CYP3A4 promoter/enhancer reporter gene that includes the CAR responsive elements. The His246Arg variant caused marked reductions in both transactivation of the reporter gene and in the response to 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO), which is a human CAR-specific agonist. The transactivation ability of the Leu308Pro variant was also significantly decreased, but its responsiveness to CITCO was not abrogated. The transactivation ability and CITCO response of the Val133Gly and Asn323Ser variants did not change as compared to the wild-type CAR. These data suggest that the His246Arg and Leu308Pro variants, especially His246Arg, may influence the expression of drug-metabolizing enzymes and transporters that are transactivated by CAR.

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

Otto, H.; Marti, T.; Holz, M.

Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3-((3-cholamidopropyl)dimethylammonio)-1- propanesulfonate micelles at pH 7.3. In the Asp-85----Ala and Asp-85----Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting amore » role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85----Ala and Asp-85----Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but clearly precedes the rise of M. The amplitude of the early reversed photovoltage component in the Asp-85----Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212----Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 microsecond, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base.« less

Effect of nicotinamide on amino acids content in bone collagen depending on biological availability of vitamins in diabetic rats.

PubMed

Guzyk, M M; Sergiichuk, Iu T; Dyakun, K O; Yanitska, L V; Kuchmerovska, T M

2014-01-01

Connective tissue is highly susceptible to imbalances induced by diabetes. Diabetes-related osteopenia, decreased bone strength etc. may be associated with altered metabolism of various collagens: Although it is assumed that alterations in collagen amino acids (AA) may strongly affect protein properties andphysiological functions, however, very limited evidences are present at the moment regarding AA composition of bone type I collagen and its relevance to abnormal availability of vitamins which are necessary for collagen synthesis in diabetes. We have tested whether nicotinamide (NAm) can influence type Icollagen formation and AA composition as well as vitamins availability in diabetes. After 4 weeks of STZ-induced diabetes (60 mg/ kg) male Wistar rats were injected for 2 weeks with/without NAm (200 mg/kg b. w). Acid extraction of type I collagen from the bones was performed with following stepwise salting out. The content of type I collagen after its acid extraction from the bones was estimated by the amounts of hydroxyproline. Amino acids were assayed by cation exchange chromatography Diabetes-associated changes in AA composition of type I collagen mainly affect those amino acids which are known to be involved in helix formation and cross-linking of the molecules. Diabetes was found to significantly reduce bone collagen contents of o-Pro, Gly, Ala, o-Lys and Pro, whereas Lys, His, Arg, Glu, Thr, Leu, Phe contents were elevated (P < 0.05). NAm treatment was able to partially normalise AA contents. In diabetes, blood serum and hepatic vitamin C and B3 contents were shown to be significantly lowered, whereas a-tocopherol was slightly increased compared with control (P < 0.05). Restoration of circulatory and liver vitamin C and B3 was observed. The data demonstrate the close relationship between the diabetes-associated decrease in type I collagen deposition, altered amino acids metabolism and impaired availability of vitamins, which are necessary for collagen synthesis. Thus, NAm might be a useful agent for treatment of bone failures related to diabetes.

Azadirachtin(A) distinctively modulates subdomain 2 of actin - novel mechanism to induce depolymerization revealed by molecular dynamics study.

PubMed

Pravin Kumar, R; Roopa, L; Sudheer Mohammed, M M; Kulkarni, Naveen

2016-12-01

Azadirachtin(A) (AZA), a potential insecticide from neem, binds to actin and induces depolymerization in Drosophila. AZA binds to the pocket same as that of Latrunculin A (LAT), but LAT inhibits actin polymerization by stiffening the actin structure and affects the ADP-ATP exchange. The mechanism by which AZA induces actin depolymerization is not clearly understood. Therefore, different computational experiments were conducted to delineate the precise mechanism of AZA-induced actin depolymerization. Molecular dynamics studies showed that AZA strongly interacted with subdomain 2 and destabilized the interactions between subdomain 2 of one actin and subdomains 1 and 4 of the adjacent actin, causing the separation of actin subunits. The separation was observed between subdomain 3 of subunit n and subdomain 4 of subunit n + 2. However, the specific triggering point for the separation of the subunits was the destabilization of direct interactions between subdomain 2 of subunit n (Arg39, Val45, Gly46 and Arg62) and subdomain 4 of subunit n + 2 (Asp286, Ile287, Asp288, Ile289, Asp244 and Lys291). These results reveal a unique mechanism of an actin filament modulator that induces depolymerization. This mechanism of AZA can be used to design similar molecules against mammalian actins for cancer therapy.

Design of novel neurokinin 1 receptor antagonists based on conformationally constrained aromatic amino acids and discovery of a potent chimeric opioid agonist-neurokinin 1 receptor antagonist

PubMed Central

Ballet, Steven; Feytens, Debby; Buysse, Koen; Chung, Nga N.; Lemieux, Carole; Tumati, Suneeta; Keresztes, Attila; Van Duppen, Joost; Lai, Josephine; Varga, Eva; Porreca, Frank; Schiller, Peter W.; Broeck, Jozef Vanden; Tourwé, Dirk

2011-01-01

A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3′,5′-(CF3)2-Bn], 20 [Ac-Aba-Gly-NMe-3′,5′-(CF3)2-Bn] and 23 [Ac-Tic-NMe-3′,5′-(CF3)2-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3′,5′-(CF3)2-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3′,5′-(CF3)2-Bn], which combines the N-terminus of the established Dmt1-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH2) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, i.e. Dmt-D-Arg-Aba-Gly-NH2 36, also proved to be an extremely potent and balanced μ- and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity. PMID:21413804

Effect of Ala-Glu-Asp-Gly peptide on life span and development of spontaneous tumors in female rats exposed to different illumination regimes.

PubMed

Vinogradova, I A; Bukalev, A V; Zabezhinski, M A; Semenchenko, A V; Khavinson, V Kh; Anisimov, V N

2007-12-01

The effects of Ala-Glu-Asp-Gly peptide (Epithalon) on the life span and development of spontaneous tumors were studied in female rats exposed to standard, natural for North-Western Russia, and constant illumination. The mean life span of animals exposed to constant or natural illumination decreased by 13.5 and 25.5%, the maximum by 9 and 7 months, respectively, and spontaneous tumors developed much more rapidly than in animals living under conditions of the standard light regimen. Epithalon (0.1 microg daily 5 times a week from the age of 4 months) did not change the life span of rats living under conditions of standard day/night regimen, while in rats exposed to the natural and constant light it promoted prolongation of the maximum life span by 95 and 24 days, respectively. Epithalon prolonged the mean life span of the last 10% of rats exposed to natural and constant illumination, treated with Epithalon, by 137 and 43 days, respectively. This peptide exhibited virtually no effect on the development of spontaneous tumors in rats exposed to standard and constant illumination, but significantly inhibited their development in rats exposed to natural light.

Clinical and functional characterization of TNNT2 mutations identified in patients with dilated cardiomyopathy

PubMed Central

Hershberger, Ray E.; Pinto, Jose Renato; Parks, Sharie B.; Kushner, Jessica D.; Li, Duanxiang; Ludwigsen, Susan; Cowan, Jason; Morales, Ana; Parvatiyar, Michelle S.; Potter, James D.

2009-01-01

Background A key issue for cardiovascular genetic medicine is ascertaining if a putative mutation indeed causes dilated cardiomyopathy (DCM). This is critically important as genetic DCM, usually presenting with advanced, life-threatening disease, may be preventable with early intervention in relatives known to carry the mutation. Methods and Results We recently undertook bidirectional resequencing of TNNT2, the cardiac troponin T gene, in 313 probands with DCM. We identified six TNNT2 protein-altering variants in nine probands, all who had early onset, aggressive disease. Additional family members of mutation carriers were then studied when available. Four of the nine probands had DCM without a family history, and five had familial DCM. Only one mutation (Lys210del) could be attributed as definitively causative from prior reports. Four of the five missense mutations were novel (Arg134Gly, Arg151Cys, Arg159Gln, Arg205Trp), and one was previously reported with hypertrophic cardiomyopathy (Glu244Asp). Based on the clinical, pedigree and molecular genetic data these five mutations were considered possibly or likely disease causing. To further clarify their potential pathophysiologic impact, we undertook functional studies of these mutations in cardiac myocytes reconstituted with mutant troponin T proteins. We observed decreased Ca2+ sensitivity of force development, a hallmark of DCM, in support of the conclusion that these mutations are disease-causing. Conclusions We conclude that the combination of clinical, pedigree, molecular genetic and functional data strengthen the interpretation of TNNT2 mutations in DCM. PMID:20031601

Design of a new peptidomimetic agonist for the melanocortin receptors based on the solution structure of the peptide ligand, Ac-Nle-cyclo[Asp-Pro-DPhe-Arg-Trp-Lys]-NH(2).

PubMed

Fotsch, Christopher; Smith, Duncan M; Adams, Jeffrey A; Cheetham, Janet; Croghan, Michael; Doherty, Elizabeth M; Hale, Clarence; Jarosinski, Mark A; Kelly, Michael G; Norman, Mark H; Tamayo, Nuria A; Xi, Ning; Baumgartner, James W

2003-07-21

The solution structure of a potent melanocortin receptor agonist, Ac-Nle-cyclo[Asp-Pro-DPhe-Arg-Trp-Lys]-NH(2) (1) was calculated using distance restraints determined from 1H NMR spectroscopy. Eight of the lowest energy conformations from this study were used to identify non-peptide cores that mimic the spatial arrangement of the critical tripeptide region, DPhe-Arg-Trp, found in 1. From these studies, compound 2a, containing the cis-cyclohexyl core, was identified as a functional agonist of the melanocortin-4 receptor (MC4R) with an IC(50) and EC(50) below 10 nM. Compound 2a also showed 36- and 7-fold selectivity over MC3R and MC1R, respectively, in the binding assays. Subtle changes in cyclohexane stereochemistry and removal of functional groups led to analogues with lower affinity for the MC receptors.

Synthetic Peptide Drugs for Targeting Skin Cancer: Malignant Melanoma and Melanotic Lesions.

PubMed

Eberle, Alex N; Rout, Bhimsen; Qi, Mei Bigliardi; Bigliardi, Paul L

2017-01-01

Peptides play decisive roles in the skin, ranging from host defense responses to various forms of neuroendocrine regulation of cell and organelle function. Synthetic peptides conjugated to radionuclides or photosensitizers may serve to identify and treat skin tumors and their metastatic forms in other organs of the body. In the introductory part of this review, the role and interplay of the different peptides in the skin are briefly summarized, including their potential application for the management of frequently occurring skin cancers. Special emphasis is given to different targeting options for the treatment of melanoma and melanotic lesions. Radionuclide Targeting: α-Melanocyte-stimulating hormone (α-MSH) is the most prominent peptide for targeting of melanoma tumors via the G protein-coupled melanocortin-1 receptor that is (over-)expressed by melanoma cells and melanocytes. More than 100 different linear and cyclic analogs of α-MSH containing chelators for 111In, 67/68Ga, 64Cu, 90Y, 212Pb, 99mTc, 188Re were synthesized and examined with experimental animals and in a few clinical studies. Linear Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys-NH2 (NAP-amide) and Re-cyclized Cys- Cys-Glu-His-D-Phe-Arg-Trp-Cys-Arg-Pro-Val-NH2 (Re[Arg11]CCMSH) containing different chelators at the N- or C-terminus served as lead compounds for peptide drugs with further optimized characteristics. Alternatively, melanoma may be targeted with radiopeptides that bind to melanin granules occurring extracellularly in these tumors. Photosensitizer targeting: A more recent approach is the application of photosensitizers attached to the MSH molecule for targeted photodynamic therapy using LED or coherent laser light that specifically activates the photosensitizer. Experimental studies have demonstrated the feasibility of this approach as a more gentle and convenient alternative compared to radionuclides. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Dipeptide frequency/bias analysis identifies conserved sites of nonrandomness shared by cysteine-rich motifs.

PubMed

Campion, S R; Ameen, A S; Lai, L; King, J M; Munzenmaier, T N

2001-08-15

This report describes the application of a simple computational tool, AAPAIR.TAB, for the systematic analysis of the cysteine-rich EGF, Sushi, and Laminin motif/sequence families at the two-amino acid level. Automated dipeptide frequency/bias analysis detects preferences in the distribution of amino acids in established protein families, by determining which "ordered dipeptides" occur most frequently in comprehensive motif-specific sequence data sets. Graphic display of the dipeptide frequency/bias data revealed family-specific preferences for certain dipeptides, but more importantly detected a shared preference for employment of the ordered dipeptides Gly-Tyr (GY) and Gly-Phe (GF) in all three protein families. The dipeptide Asn-Gly (NG) also exhibited high-frequency and bias in the EGF and Sushi motif families, whereas Asn-Thr (NT) was distinguished in the Laminin family. Evaluation of the distribution of dipeptides identified by frequency/bias analysis subsequently revealed the highly restricted localization of the G(F/Y) and N(G/T) sequence elements at two separate sites of extreme conservation in the consensus sequence of all three sequence families. The similar employment of the high-frequency/bias dipeptides in three distinct protein sequence families was further correlated with the concurrence of these shared molecular determinants at similar positions within the distinctive scaffolds of three structurally divergent, but similarly employed, motif modules.

Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base.

PubMed

Otto, H; Marti, T; Holz, M; Mogi, T; Stern, L J; Engel, F; Khorana, H G; Heyn, M P

1990-02-01

Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate micelles at pH 7.3. In the Asp-85----Ala and Asp-85----Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting a role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85----Ala and Asp-85----Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but clearly precedes the rise of M. The amplitude of the early (less than 0.2 microseconds) reversed photovoltage component in the Asp-85----Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212----Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 microsecond, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base. In the Arg-82----Gln mutant, no net transient proton release was observed, whereas, in the Arg-82----Ala mutant, uptake and release were reversed. The pK shift of the purple-to-blue transition in the Asp-85----Glu, Arg-82----Ala, and Arg-82----Gln mutants and the similarity in the photocycle and photoelectrical signals of the Asp-85----Ala, Asp-85----Asn, and Asp-212----Asn mutants suggest the interaction between Asp-85, Arg-82, Asp-212, and the Schiff base as essential for proton release.

Aliicoccus persicus gen. nov., sp. nov., a halophilic member of the Firmicutes isolated from a hypersaline lake.

PubMed

Amoozegar, Mohammad Ali; Bagheri, Maryam; Makhdoumi-Kakhki, Ali; Didari, Maryam; Schumann, Peter; Nikou, Mahdi Moshtaghi; Sánchez-Porro, Cristina; Ventosa, Antonio

2014-06-01

A novel Gram-staining-positive, moderately halophilic bacterium, designated strain A76(T), was isolated from a brine sample of the hypersaline lake Aran-Bidgol in Iran. Cells were strictly aerobic, coccus-shaped, non-motile, non-sporulating, and catalase- and oxidase-positive. Strain A76(T) grew between pH 7.0 and 10.0 (optimal growth at pH 8.0), between 20 and 45 °C (optimal growth at 35 °C) and at salinities of 0.5 to 12.5% (w/v) NaCl (optimal growth at 7.5%, w/v, NaCl). On the basis of 16S rRNA gene sequence analysis, strain A76(T) was shown to belong to the phylum Firmicutes with sequence similarities of 94.1, 93.1 and 91.1%, to the type species of the genera Jeotgalicoccus, Salinicoccus and Nosocomiicoccus, respectively. The DNA G+C content of this new isolate was 38.8 mol%. The major cellular fatty acids of strain A76(T) were anteiso-C(15 : 0) and iso-C(15 : 0), and its polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, a glycolipid, an unknown lipid and two unknown phospholipids. The isoprenoid quinones were MK-6 (94%), MK-5 (3%) and MK-7 (3%). The amino acid constituents of the cell wall were Lys, Asp, Gly, Glu and Ala. The physiological, biochemical and phylogenetic differences between strain A76(T) and type strains of taxa with validly published names suggest that this strain represents a novel species in a novel genus within the family Staphylococcaceae, for which the name Aliicoccus persicus gen. nov., sp. nov. is proposed. The type strain of Aliicoccus persicus is strain A76(T) ( = CECT 8508(T) = DSM 28306(T) = IBRC-M 10081(T)). © 2014 IUMS.

Receptor-Mediated Melanoma Targeting with Radiolabeled α-Melanocyte-Stimulating Hormone: Relevance of the Net Charge of the Ligand.

PubMed

Bapst, Jean-Philippe; Eberle, Alex N

2017-01-01

A majority of melanotic and amelanotic melanomas overexpress melanocortin type 1 receptors (MC1Rs) for α-melanocyte-stimulating hormone. Radiolabeled linear or cyclic analogs of α-MSH have a great potential as diagnostic or therapeutic tools for the management of malignant melanoma. Compounds such as [ 111 In]DOTA-NAP-amide exhibit high affinity for the MC1R in vitro , good tumor uptake in vivo , but they may suffer from relatively high kidney uptake and retention in vivo . We have shown previously that the introduction of negative charges into radiolabeled DOTA-NAP-amide peptide analogs may enhance their excretion and reduce kidney retention. To address the question of where to place negative charges within the ligand, we have extended these studies by designing two novel peptides, Ac-Nle-Asp-His-d-Phe-Arg-Trp-Gly-Lys(DOTA)-d-Asp-d-Asp-OH (DOTA-NAP-d-Asp-d-Asp) with three negative charges at the C -terminal end (overall net charge of the molecule -2) and DOTA-Gly-Tyr(P)-Nle-Asp-His-d-Phe-Arg-Trp-NH 2 (DOTA-Phospho-MSH 2-9 ) with two negative charges in the N -terminal region (net charge -1). The former peptide showed markedly reduced receptor affinity and biological activity by >10-fold compared to DOTA-NAP-amide as reference compound, and the latter peptide displayed similar bioactivity and receptor affinity as the reference compound. The uptake by melanoma tumor tissue of [ 111 In]DOTA-Phospho-MSH 2-9 was 7.33 ± 0.47 %ID/g 4 h after injection, i.e., almost equally high as with [ 111 In]DOTA-NAP-amide. The kidney retention was 2.68 ± 0.18 %ID/g 4 h after injection and hence 44% lower than that of [ 111 In]DOTA-NAP-amide. Over an observation period from 4 to 48 h, the tumor-to-kidney ratio of [ 111 In]DOTA-Phospho-MSH 2-9 was 35% more favorable than that of the reference compound. In a comparison of DOTA-NAP-d-Asp-d-Asp, DOTA-Phospho-MSH 2-9 and DOTA-NAP-amide with five previously published analogs of DOTA-NAP-amide that altogether cover a range of peptides with an overall net charge between +2 and -2, we now demonstrate that a net charge of -1, with the extra negative charges preferably placed in the N -terminal region, has led to the lowest kidney uptake and retention. Charges of +2 or -2 markedly increased kidney uptake and retention. In conclusion, the novel DOTA-Phospho-MSH 2-9 may represent a new lead compound for negatively charged linear MC1R ligands that can be further developed into a clinically relevant melanoma targeting radiopeptide.

Receptor-Mediated Melanoma Targeting with Radiolabeled α-Melanocyte-Stimulating Hormone: Relevance of the Net Charge of the Ligand

PubMed Central

Bapst, Jean-Philippe; Eberle, Alex N.

2017-01-01

A majority of melanotic and amelanotic melanomas overexpress melanocortin type 1 receptors (MC1Rs) for α-melanocyte-stimulating hormone. Radiolabeled linear or cyclic analogs of α-MSH have a great potential as diagnostic or therapeutic tools for the management of malignant melanoma. Compounds such as [111In]DOTA-NAP-amide exhibit high affinity for the MC1R in vitro, good tumor uptake in vivo, but they may suffer from relatively high kidney uptake and retention in vivo. We have shown previously that the introduction of negative charges into radiolabeled DOTA-NAP-amide peptide analogs may enhance their excretion and reduce kidney retention. To address the question of where to place negative charges within the ligand, we have extended these studies by designing two novel peptides, Ac-Nle-Asp-His-d-Phe-Arg-Trp-Gly-Lys(DOTA)-d-Asp-d-Asp-OH (DOTA-NAP-d-Asp-d-Asp) with three negative charges at the C-terminal end (overall net charge of the molecule −2) and DOTA-Gly-Tyr(P)-Nle-Asp-His-d-Phe-Arg-Trp-NH2 (DOTA-Phospho-MSH2-9) with two negative charges in the N-terminal region (net charge −1). The former peptide showed markedly reduced receptor affinity and biological activity by >10-fold compared to DOTA-NAP-amide as reference compound, and the latter peptide displayed similar bioactivity and receptor affinity as the reference compound. The uptake by melanoma tumor tissue of [111In]DOTA-Phospho-MSH2-9 was 7.33 ± 0.47 %ID/g 4 h after injection, i.e., almost equally high as with [111In]DOTA-NAP-amide. The kidney retention was 2.68 ± 0.18 %ID/g 4 h after injection and hence 44% lower than that of [111In]DOTA-NAP-amide. Over an observation period from 4 to 48 h, the tumor-to-kidney ratio of [111In]DOTA-Phospho-MSH2-9 was 35% more favorable than that of the reference compound. In a comparison of DOTA-NAP-d-Asp-d-Asp, DOTA-Phospho-MSH2-9 and DOTA-NAP-amide with five previously published analogs of DOTA-NAP-amide that altogether cover a range of peptides with an overall net charge between +2 and −2, we now demonstrate that a net charge of −1, with the extra negative charges preferably placed in the N-terminal region, has led to the lowest kidney uptake and retention. Charges of +2 or −2 markedly increased kidney uptake and retention. In conclusion, the novel DOTA-Phospho-MSH2-9 may represent a new lead compound for negatively charged linear MC1R ligands that can be further developed into a clinically relevant melanoma targeting radiopeptide. PMID:28491052

Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

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

Wartchow, C.A.; Wang, Peng; Bednarski, M.D.

1995-12-31

The synthesis of oligopeptides using stable carbohydrate protease conjugates (CPCs) was examined in acetonitrile solvent systems. CPC[{alpha}-chymotrypsin] was used for the preparation of peptides containing histidine, phenylalanine, tryptophan in the P{sub 1} position in 60-93% yield. The CPC[{alpha}-chymotrypsin]-catalyzed synthesis of octamer Z-Gly-Gly-Phe-Gly-Gly-Phe-Gly-Gly-OEt from Z-Gly-Gly-Phe-Gly-Gly-Phe-OMe was achieved in 71% yield demonstrating that synthesis peptides containing both hydrophylic and hydrophobic amino acids. The P{sub 2} specificity of papain for aromatic residues was utilized for the 2 + 3 coupling of Z-Tyr-Gly-OMe to H{sub 2}N-Gly-Phe-Leu-OH to generate the leucine enkephalin derivative in 79% yield. Although papain is nonspecific for the hydrolysis of N-benzyloxycarbonylmore » amino acid methyl esters in aqueous solution, the rates of synthesis for these derivitives with nucleophile leucine tert-butyl ester differed by nearly 2 orders of magnitude. CPC[thermolysin] was used to prepare the aspartame precursor Z-Asp-Phe-OMe in 90% yield. The increased stability of CPCs prepared from periodate-modified poly(2-methacryl- amido-2-deoxy-D-glucose), poly(2-methacrylamido-2-deoxy-D-galactose), and poly(5-methacryl-amido-5-deoxy-D-ribose), carbohydrate materials designed to increase the aldehyde concentration in aqueous solution, suggests that the stability of CPCs is directly related to the aldehyde concentration of the carbohydrate material. Periodate oxidation of poly(2-methacrylamido-2-deoxy-D-glucose) followed by covalent attachment to {alpha}-chymotrypsin gave a CPC with catalytic activity in potassium phosphate buffer at 90{degrees}C for 2 h. 1 fig., 1 tab., 40 refs.« less

Peptide dendrimer/lipid hybrid systems are efficient DNA transfection reagents: structure--activity relationships highlight the role of charge distribution across dendrimer generations.

PubMed

Kwok, Albert; Eggimann, Gabriela A; Reymond, Jean-Louis; Darbre, Tamis; Hollfelder, Florian

2013-05-28

Efficient DNA delivery into cells is the prerequisite of the genetic manipulation of organisms in molecular and cellular biology as well as, ultimately, in nonviral gene therapy. Current reagents, however, are relatively inefficient, and structure-activity relationships to guide their improvement are hard to come by. We now explore peptide dendrimers as a new type of transfection reagent and provide a quantitative framework for their evaluation. A collection of dendrimers with cationic and hydrophobic amino acid motifs (such as KK, KA, KH, KL, and LL) distributed across three dendrimer generations was synthesized by a solid-phase protocol that provides ready access to dendrimers in milligram quantities. In conjunction with a lipid component (DOTMA/DOPE), the best reagent, G1,2,3-KL ((LysLeu)8(LysLysLeu)4(LysLysLeu)2LysGlySerCys-NH2), improves transfection by 6-10-fold over commercial reagents under their respective optimal conditions. Emerging structure-activity relationships show that dendrimers with cationic and hydrophobic residues distributed in each generation are transfecting most efficiently. The trigenerational dendritic structure has an advantage over a linear analogue worth up to an order of magnitude. The success of placing the decisive cationic charge patterns in inner shells rather than previously on the surface of macromolecules suggests that this class of dendrimers significantly differs from existing transfection reagents. In the future, this platform may be tuned further and coupled to cell-targeting moieties to enhance transfection and cell specificity.

Peptide Dendrimer/Lipid Hybrid Systems Are Efficient DNA Transfection Reagents: Structure–Activity Relationships Highlight the Role of Charge Distribution Across Dendrimer Generations

PubMed Central

2013-01-01

Efficient DNA delivery into cells is the prerequisite of the genetic manipulation of organisms in molecular and cellular biology as well as, ultimately, in nonviral gene therapy. Current reagents, however, are relatively inefficient, and structure–activity relationships to guide their improvement are hard to come by. We now explore peptide dendrimers as a new type of transfection reagent and provide a quantitative framework for their evaluation. A collection of dendrimers with cationic and hydrophobic amino acid motifs (such as KK, KA, KH, KL, and LL) distributed across three dendrimer generations was synthesized by a solid-phase protocol that provides ready access to dendrimers in milligram quantities. In conjunction with a lipid component (DOTMA/DOPE), the best reagent, G1,2,3-KL ((LysLeu)8(LysLysLeu)4(LysLysLeu)2LysGlySerCys-NH2), improves transfection by 6–10-fold over commercial reagents under their respective optimal conditions. Emerging structure–activity relationships show that dendrimers with cationic and hydrophobic residues distributed in each generation are transfecting most efficiently. The trigenerational dendritic structure has an advantage over a linear analogue worth up to an order of magnitude. The success of placing the decisive cationic charge patterns in inner shells rather than previously on the surface of macromolecules suggests that this class of dendrimers significantly differs from existing transfection reagents. In the future, this platform may be tuned further and coupled to cell-targeting moieties to enhance transfection and cell specificity. PMID:23682947

Minimization and Optimization of Designed β-Hairpin Folds

PubMed Central

Andersen, Niels H.; Olsen, Katherine A.; Fesinmeyer, R. Matthew; Tan, Xu; Hudson, F. Michael; Eidenschink, Lisa A.; Farazi, Shabnam R.

2011-01-01

Mimimized β hairpins have provided additional data on the geometric preferences of Trp interactions in TW-loop-WT motifs. This motif imparts significant fold stability to peptides as short as 8 residues. High-resolution NMR structures of a 16- (KKWTWNPATGKWTWQE, ΔGU298 ≥ +7 kJ/mol) and 12-residue (KTWNPATGKWTE, ΔGU298 = +5.05 kJ/mol) hairpin reveal a common turn geometry and edge-to-face (EtF) packing motif and a cation-π interaction between Lys1 and the Trp residue nearest the C-terminus. The magnitude of a CD exciton couplet (due to the two Trp residues) and the chemical shifts of a Trp Hε3 site (shifted upfield by 2.4 ppm due to the EtF stacking geometry) provided near-identical measures of folding. CD melts of representative peptides with the –TW-loop-WT- motif provided the thermodynamic parameters for folding, which reflect enthalpically driven folding at laboratory temperatures with a small ΔCp for unfolding (+420 JK−1/mol). In the case of Asx-Pro-Xaa-Thr-Gly-Xaa loops, mutations established that the two most important residues in this class of direction-reversing loops are Asx and Gly: mutation to alanine is destabilizing by about 6 and 2 kJ/mol, respectively. All indicators of structuring are retained in a minimized 8-residue construct (Ac-WNPATGKW-NH2) with the fold stability reduced to ΔGU278 = −0.7 kJ/mol. NMR and CD comparisons indicate that -TWXNGKWT- (X = S, I) sequences also forms the same hairpin-stabilizing W/W interaction. PMID:16669679

Experimental verification of force fields for molecular dynamics simulations using Gly-Pro-Gly-Gly.

PubMed

Aliev, Abil E; Courtier-Murias, Denis

2010-09-30

Experimental NMR verification of MD simulations using 12 different force fields (AMBER, CHARMM, GROMOS, and OPLS-AA) and 5 different water models has been undertaken to identify reliable MD protocols for structure and dynamics elucidations of small open chain peptides containing Gly and Pro. A conformationally flexible tetrapeptide Gly-Pro-Gly-Gly was selected for NMR (3)J-coupling, chemical shift, and internuclear distance measurements, followed by their calculations using 2 μs long MD simulations in water. In addition, Ramachandran population maps for Pro-2 and Gly-3 residues of GPGG obtained from MD simulations were used for detailed comparisons with similar maps from the protein data bank (PDB) for large number of Gly and Pro residues in proteins. The MD simulations revealed strong dependence of the populations and geometries of preferred backbone and side chain conformations, as well as the time scales of the peptide torsional transitions on the force field used. On the basis of the analysis of the measured and calculated data, AMBER99SB is identified as the most reliable force field for reproducing NMR measured parameters, which are dependent on the peptide backbone and the Pro side chain geometries and dynamics. Ramachandran maps showing the dependence of conformational populations as a function of backbone ϕ/ψ angles for Pro-2 and Gly-3 residues of GPGG from MD simulations using AMBER99SB, AMBER03, and CHARMM were found to resemble similar maps for Gly and Pro residues from the PDB survey. Three force fields (AMBER99, AMBER99ϕ, and AMBER94) showed the least satisfactory agreement with both the solution NMR and the PDB survey data. The poor performance of these force fields is attributed to their propensity to overstabilize helical peptide backbone conformations at the Pro-2 and Gly-3 residues. On the basis of the similarity of the MD and PDB Ramachandran plots, the following sequence of transitions is suggested for the Gly backbone conformation: α(L) ⇆ β(PR) ⇆ β(S) ⇆ β(P) ⇆ α, where backbone secondary structures α(L) and α are associated with helices and turns, β(P) and β(PR) correspond to the left- and right-handed polyproline II structures and β(S) denotes the fully stretched backbone conformation. Compared to the force field dependence, less significant, but noteworthy, variations in the populations of the peptide backbone conformations were observed. For different solvent models considered, a correlation was noted between the number of torsional transitions in GPGG and the water self-diffusion coefficient on using TIP3P, TIP4P, and TIP5P models. In addition to MD results, we also report DFT derived Karplus relationships for Gly and Pro residues using B972 and B3LYP functionals.

Changes in composition and content of food-derived peptide in human blood after daily ingestion of collagen hydrolysate for 4 weeks.

PubMed

Shigemura, Yasutaka; Suzuki, Asahi; Kurokawa, Mihoko; Sato, Yoshio; Sato, Kenji

2018-03-01

Daily ingestion of collagen hydrolysate for a long period improves skin and joint conditions. It has been speculated that the beneficial effects are exerted by food-derived hydroxyproline (Hyp) peptides, which are detected in human blood after single ingestions. In the present study, to investigate the effect of long-term ingestion of collagen hydrolysate on Hyp peptides profile in blood, the concentrations of Hyp-peptides in human blood before and after daily ingestion for a long period were examined. Hyp-peptides increased to a maximum level at 1 h after ingestion and reverted to their initial levels within 24 h during experimental period. Pro-Gly and Hyp-peptides such as Pro-Hyp-Gly, Pro-Hyp, Ile-Hyp, Leu-Hyp, Hyp-Gly, Glu-Hyp and Ala-Hyp were identified in the blood after ingestion of collagen hydrolysate at 4.5 g day -1 for 4 weeks. For the whole period, Pro-Hyp was the leading compound. The compositional rate of Hyp-Gly showed a tendency to increase, while that of Pro-Hyp tended to decrease after daily ingestion. The present results indicate that daily ingestion of collagen hydrolysate for a long period can change compositional rate of Hyp peptides in human blood. This fact suggests that long-term ingestion of collagen hydrolysate might change exo- or endo-type protease activity in the digestive tract, which may consequently promote beneficial effects. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

PAX3 mutations and clinical characteristics in Chinese patients with Waardenburg syndrome type 1.

PubMed

Wang, Juan; Li, Shiqiang; Xiao, Xueshan; Wang, Panfeng; Guo, Xiangming; Zhang, Qingjiong

2010-06-22

To detect paired box gene 3 (PAX3) mutations and associated phenotypes in Chinese patients with Waardenburg syndrome type 1 (WS1). Five unrelated families with suspected WS1 were selected from our Genomic DNA Repository for Hereditary Eye Diseases. The coding and adjacent intronic regions of PAX3 were amplified by polymerase chain reaction and the amplicons were then analyzed by cycle sequencing. Variations detected were further evaluated in available family members as well as one hundred controls with heteroduplex-single strand conformational polymorphism (heteroduplex-SSCP) analysis and/or clone sequencing. Three novel and two known mutations in PAX3 were detected in five patients, respectively: c.567_586+17del (p.Asp189_Gln505delinsGluGlyGlyAlaLeuAlaGly), c.456_459dupTTCC (p.Ile154PhefsX162), c.795_800delCTGGTT (p.Trp266_Phe267del), c.799T>A (p.Phe267Ile), and c.667C>T (p.Arg223X). Two novel mutations proved to be de novo as their parents did not carry the mutations. All five patients with PAX3 mutations had dystopia canthorum and different iris color and fundi between their two eyes. However, none had white forelock, skin hypopigmentation, and deafness. Our findings expand the frequency and spectrum of PAX3 mutations and ethnic-related phenotypes in Chinese patients with WS1. De novo mutations in PAX3 have not been reported before.

PAX3 mutations and clinical characteristics in Chinese patients with Waardenburg syndrome type 1

PubMed Central

Wang, Juan; Li, Shiqiang; Xiao, Xueshan; Wang, Panfeng; Guo, Xiangming

2010-01-01

Purpose To detect paired box gene 3 (PAX3) mutations and associated phenotypes in Chinese patients with Waardenburg syndrome type 1 (WS1). Methods Five unrelated families with suspected WS1 were selected from our Genomic DNA Repository for Hereditary Eye Diseases. The coding and adjacent intronic regions of PAX3 were amplified by polymerase chain reaction and the amplicons were then analyzed by cycle sequencing. Variations detected were further evaluated in available family members as well as one hundred controls with heteroduplex-single strand conformational polymorphism (heteroduplex-SSCP) analysis and/or clone sequencing. Results Three novel and two known mutations in PAX3 were detected in five patients, respectively: c.567_586+17del (p.Asp189_Gln505delinsGluGlyGlyAlaLeuAlaGly), c.456_459dupTTCC (p.Ile154PhefsX162), c.795_800delCTGGTT (p.Trp266_Phe267del), c.799T>A (p.Phe267Ile), and c.667C>T (p.Arg223X). Two novel mutations proved to be de novo as their parents did not carry the mutations. All five patients with PAX3 mutations had dystopia canthorum and different iris color and fundi between their two eyes. However, none had white forelock, skin hypopigmentation, and deafness. Conclusions Our findings expand the frequency and spectrum of PAX3 mutations and ethnic-related phenotypes in Chinese patients with WS1. De novo mutations in PAX3 have not been reported before. PMID:20664692

Comprehensive Maturity Onset Diabetes of the Young (MODY) Gene Screening in Pregnant Women with Diabetes in India.

PubMed

Doddabelavangala Mruthyunjaya, Mahesh; Chapla, Aaron; Hesarghatta Shyamasunder, Asha; Varghese, Deny; Varshney, Manika; Paul, Johan; Inbakumari, Mercy; Christina, Flory; Varghese, Ron Thomas; Kuruvilla, Kurien Anil; V Paul, Thomas; Jose, Ruby; Regi, Annie; Lionel, Jessie; Jeyaseelan, L; Mathew, Jiji; Thomas, Nihal

2017-01-01

Pregnant women with diabetes may have underlying beta cell dysfunction due to mutations/rare variants in genes associated with Maturity Onset Diabetes of the Young (MODY). MODY gene screening would reveal those women genetically predisposed and previously unrecognized with a monogenic form of diabetes for further clinical management, family screening and genetic counselling. However, there are minimal data available on MODY gene variants in pregnant women with diabetes from India. In this study, utilizing the Next generation sequencing (NGS) based protocol fifty subjects were screened for variants in a panel of thirteen MODY genes. Of these subjects 18% (9/50) were positive for definite or likely pathogenic or uncertain MODY variants. The majority of these variants was identified in subjects with autosomal dominant family history, of whom five were in women with pre-GDM and four with overt-GDM. The identified variants included one patient with HNF1A Ser3Cys, two PDX1 Glu224Lys, His94Gln, two NEUROD1 Glu59Gln, Phe318Ser, one INS Gly44Arg, one GCK, one ABCC8 Arg620Cys and one BLK Val418Met variants. In addition, three of the seven offspring screened were positive for the identified variant. These identified variants were further confirmed by Sanger sequencing. In conclusion, these findings in pregnant women with diabetes, imply that a proportion of GDM patients with autosomal dominant family history may have MODY. Further NGS based comprehensive studies with larger samples are required to confirm these finding.

Comprehensive Maturity Onset Diabetes of the Young (MODY) Gene Screening in Pregnant Women with Diabetes in India

PubMed Central

Hesarghatta Shyamasunder, Asha; Varghese, Deny; Varshney, Manika; Paul, Johan; Inbakumari, Mercy; Christina, Flory; Varghese, Ron Thomas; Kuruvilla, Kurien Anil; V. Paul, Thomas; Jose, Ruby; Regi, Annie; Lionel, Jessie; Jeyaseelan, L.; Mathew, Jiji; Thomas, Nihal

2017-01-01

Pregnant women with diabetes may have underlying beta cell dysfunction due to mutations/rare variants in genes associated with Maturity Onset Diabetes of the Young (MODY). MODY gene screening would reveal those women genetically predisposed and previously unrecognized with a monogenic form of diabetes for further clinical management, family screening and genetic counselling. However, there are minimal data available on MODY gene variants in pregnant women with diabetes from India. In this study, utilizing the Next generation sequencing (NGS) based protocol fifty subjects were screened for variants in a panel of thirteen MODY genes. Of these subjects 18% (9/50) were positive for definite or likely pathogenic or uncertain MODY variants. The majority of these variants was identified in subjects with autosomal dominant family history, of whom five were in women with pre-GDM and four with overt-GDM. The identified variants included one patient with HNF1A Ser3Cys, two PDX1 Glu224Lys, His94Gln, two NEUROD1 Glu59Gln, Phe318Ser, one INS Gly44Arg, one GCK, one ABCC8 Arg620Cys and one BLK Val418Met variants. In addition, three of the seven offspring screened were positive for the identified variant. These identified variants were further confirmed by Sanger sequencing. In conclusion, these findings in pregnant women with diabetes, imply that a proportion of GDM patients with autosomal dominant family history may have MODY. Further NGS based comprehensive studies with larger samples are required to confirm these finding PMID:28095440

Solid-phase synthesis of a nucleopeptide from the linking site of adenovirus-2 nucleoprotein, -Ser(p5'CATCAT)-Gly-Asp-. Convergent versus stepwise strategy.

PubMed Central

Robles, J; Pedroso, E; Grandas, A

1995-01-01

The synthesis of a nucleopeptide with the sequence -Ser(p5'CATCAT)-Gly-Asp- has been undertaken by either convergent or stepwise solid-phase strategies, both of which use base-labile permanent protecting groups. The coupling of phosphitylated protected peptides onto oligonucleotide-resins did not afford the desired nucleopeptide, which was nevertheless obtained after oligonucleotide elongation at the hydroxyl group of the resin-bound peptide and deprotection under mild basic conditions. A preliminary study on the stability of different nucleopeptides to bases is also reported. PMID:7479079

Evolution of amino acid metabolism inferred through cladistic analysis.

PubMed

Cunchillos, Chomin; Lecointre, Guillaume

2003-11-28

Because free amino acids were most probably available in primitive abiotic environments, their metabolism is likely to have provided some of the very first metabolic pathways of life. What were the first enzymatic reactions to emerge? A cladistic analysis of metabolic pathways of the 16 aliphatic amino acids and 2 portions of the Krebs cycle was performed using four criteria of homology. The analysis is not based on sequence comparisons but, rather, on coding similarities in enzyme properties. The properties used are shared specific enzymatic activity, shared enzymatic function without substrate specificity, shared coenzymes, and shared functional family. The tree shows that the earliest pathways to emerge are not portions of the Krebs cycle but metabolisms of aspartate, asparagine, glutamate, and glutamine. The views of Horowitz (Horowitz, N. H. (1945) Proc. Natl. Acad. Sci. U. S. A. 31, 153-157) and Cordón (Cordón, F. (1990) Tratado Evolucionista de Biologia, Aguilar, Madrid, Spain), according to which the upstream reactions in the catabolic pathways and the downstream reactions in the anabolic pathways are the earliest in evolution, are globally corroborated; however, with some exceptions. These are due to later opportunistic connections of pathways (actually already suggested by these authors). Earliest enzymatic functions are mostly catabolic; they were deaminations, transaminations, and decarboxylations. From the consensus tree we extracted four time spans for amino acid metabolism development. For some amino acids catabolism and biosynthesis occurred at the same time (Asp, Glu, Lys, Leu, Ala, Val, Ile, Pro, Arg). For others ultimate reactions that use amino acids as a substrate or as a product are distinct in time, with catabolism preceding anabolism for Asn, Gln, and Cys and anabolism preceding catabolism for Ser, Met, and Thr. Cladistic analysis of the structure of biochemical pathways makes hypotheses in biochemical evolution explicit and parsimonious.

Partial purification and functional properties of an endoprotease from bovine neurosecretory granules cleaving proocytocin/neurophysin peptides at the basic amino acid doublet.

PubMed

Clamagirand, C; Creminon, C; Fahy, C; Boussetta, H; Cohen, P

1987-09-22

An enriched preparation of neurosecretory granules from bovine pituitary neural lobes was used as a source of processing enzymes possibly involved in the cleavage of the proocytocin/neurophysin precursor. A synthetic eicosapeptide reproducing the entire (1-20) sequence of the NH2-terminal domain of the bovine ocytocin/neurophysin precursor was used as a substrate to monitor an endoprotease activity cleaving at the Lys11-Arg12 doublet. The 58-kDa endoprotease detected in the lysate of neurohypophyseal granules produced a single cleavage, after the doublet, at the Arg12-Ala13 peptide bond. This endoprotease with pHi 6.9 and 7.2 exhibits maximal activity at pH around neutrality (7.0) and was strongly inhibited by divalent cation chelating agents [ethylenediaminetetraacetic acid and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',-N'-tetraacetic acid] and to some extent by p-(chloromercuri)benzoate and p-(chloromercuri)benzenesulfonic acid, while phenylmethanesulfonyl fluoride and pepstatin were not active. This endoprotease action was sensitive to any modification of the substrate at either basic amino acid of the doublet since replacement of either L-Lys11 or L-Arg12 by D-Lys or D-Arg and by L-Nle abolished the cleavage reaction. In contrast, reversal of the polarity of the doublet in [Arg11,Lys12]proocytocin/neurophysin(1-20) had no effect on the mode of endoproteolytic cleavage as well as modifications of Gly10 (replaced by Ala10). It is concluded that the selectivity of this endoprotease, which may be involved in the primary event occurring in proocytocin/neurophysin processing, is strictly dependent upon the integrity of the basic doublet but that other parameters determined by the amino acid sequence around this doublet may play an important role.

Differentiation of highly virulent strains of Streptococcus suis serotype 2 according to glutamate dehydrogenase electrophoretic and sequence type.

PubMed

Kutz, Russell; Okwumabua, Ogi

2008-10-01

The glutamate dehydrogenase (GDH) enzymes of 19 Streptococcus suis serotype 2 strains, consisting of 18 swine isolates and 1 human clinical isolate from a geographically varied collection, were analyzed by activity staining on a nondenaturing gel. All seven (100%) of the highly virulent strains tested produced an electrophoretic type (ET) distinct from those of moderately virulent and nonvirulent strains. By PCR and nucleotide sequence determination, the gdh genes of the 19 strains and of 2 highly virulent strains involved in recent Chinese outbreaks yielded a 1,820-bp fragment containing an open reading frame of 1,344 nucleotides, which encodes a protein of 448 amino acid residues with a calculated molecular mass of approximately 49 kDa. The nucleotide sequences contained base pair differences, but most were silent. Cluster analysis of the deduced amino acid sequences separated the isolates into three groups. Group I (ETI) consisted of the seven highly virulent isolates and the two Chinese outbreak strains, containing Ala(299)-to-Ser, Glu(305)-to-Lys, and Glu(330)-to-Lys amino acid substitutions compared with groups II and III (ETII). Groups II and III consisted of moderately virulent and nonvirulent strains, which are separated from each other by Tyr(72)-to-Asp and Thr(296)-to-Ala substitutions. Gene exchange studies resulted in the change of ETI to ETII and vice versa. A spectrophotometric activity assay for GDH did not show significant differences between the groups. These results suggest that the GDH ETs and sequence types may serve as useful markers in predicting the pathogenic behavior of strains of this serotype and that the molecular basis for the observed differences in the ETs was amino acid substitutions and not deletion, insertion, or processing uniqueness.

Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding*

PubMed Central

Xia, Chuanwu; Hamdane, Djemel; Shen, Anna L.; Choi, Vivian; Kasper, Charles B.; Pearl, Naw May; Zhang, Haoming; Im, Sang-Choul; Waskell, Lucy; Kim, Jung-Ja P.

2011-01-01

The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp147 and Arg514 in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP+ revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP+ shows movement of the Gly631–Asn635 loop. In the NADP+-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP+ shows movement of the Gly631–Asn635 loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly631–Asn635 loop movement controls NADPH binding and NADP+ release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners. PMID:21345800

Mapping of epitopes for autoantibodies to the type 1 diabetes autoantigen IA-2 by peptide phage display and molecular modeling: overlap of antibody and T cell determinants.

PubMed

Dromey, James A; Weenink, Sarah M; Peters, Günther H; Endl, Josef; Tighe, Patrick J; Todd, Ian; Christie, Michael R

2004-04-01

IA-2 is a major target of autoimmunity in type 1 diabetes. IA-2 responsive T cells recognize determinants within regions represented by amino acids 787-817 and 841-869 of the molecule. Epitopes for IA-2 autoantibodies are largely conformational and not well defined. In this study, we used peptide phage display and homology modeling to characterize the epitope of a monoclonal IA-2 Ab (96/3) from a human type 1 diabetic patient. This Ab competes for IA-2 binding with Abs from the majority of patients with type 1 diabetes and therefore binds a region close to common autoantibody epitopes. Alignment of peptides obtained after screening phage-displayed peptide libraries with purified 96/3 identified a consensus binding sequence of Asn-x-Glu-x-x-(aromatic)-x-x-Gly. The predicted surface on a three-dimensional homology model of the tyrosine phosphatase domain of IA-2 was analyzed for clusters of Asn, Glu, and aromatic residues and amino acids contributing to the epitope investigated using site-directed mutagenesis. Mutation of each of amino acids Asn(858), Glu(836), and Trp(799) reduced 96/3 Ab binding by >45%. Mutations of these residues also inhibited binding of serum autoantibodies from IA-2 Ab-positive type 1 diabetic patients. This study identifies a region commonly recognized by autoantibodies in type 1 diabetes that overlaps with dominant T cell determinants.

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

Steiner, B.; Cousot, D.; Trzeciak, A.

The platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a member of the integrin receptor family that recognizes adhesive proteins containing the Arg-Gly-Asp (RGD) sequence. In the present study the binding characteristics of the synthetic hexapeptide Tyr-Asn-Arg-Gly-Asp-Ser (YNRGDS, a sequence present in the fibrinogen alpha-chain at position 570-575) to purified GP IIb-IIIa were determined by equilibrium dialysis. The binding of 125I-YNRGDS to GP IIb-IIIa was specific, saturable, and reversible. The apparent dissociation constant was 1.0 +/- 0.2 microM, and the maximal binding capacity was 0.92 +/- 0.02 mol of 125I-YNRGDS/mol of GP IIb-IIIa, indicating that GP IIb-IIIa contains a single bindingmore » site for RGD peptides. The binding of 125I-YNRGDS to purified GP IIb-IIIa showed many of the characteristics of fibrinogen binding to activated platelets: the binding was inhibited by fibrinogen, by the monoclonal antibody A2A9, and by the dodecapeptide from the C terminus of the fibrinogen gamma-chain. In addition, the binding of 125I-YNRGDS to GP IIb-IIIa was divalent cation-dependent. Our data suggest that two divalent cation binding sites must be occupied for YNRGDS to bind: one site is specific for calcium and is saturated at 1 microM free Ca2+, whereas the other site is less specific and reaches saturation at millimolar concentrations of either Ca2+ or Mg2+. The results of the present study support the hypothesis that the RGD domains within the adhesive proteins are responsible for their binding to GP IIb-IIIa.« less

Evaluation of the membrane lipid selectivity of the pea defensin Psd1.

PubMed

Gonçalves, Sónia; Teixeira, Alexandre; Abade, João; de Medeiros, Luciano Neves; Kurtenbach, Eleonora; Santos, Nuno C

2012-05-01

Psd1, a 46 amino acid residues defensin isolated from the pea Pisum sativum seeds, exhibits anti-fungal activity by a poorly understood mechanism of action. In this work, the interaction of Psd1 with biomembrane model systems of different lipid compositions was assessed by fluorescence spectroscopy. Partition studies showed a marked lipid selectivity of this antimicrobial peptide (AMP) toward lipid membranes containing ergosterol (the main sterol in fungal membranes) or specific glycosphingolipid components, with partition coefficients (K(p)) reaching uncommonly high values of 10(6). By the opposite, Psd1 does not partition to cholesterol-enriched lipid bilayers, such as mammalian cell membranes. The Psd1 mutants His36Lys and Gly12Glu present a membrane affinity loss relative to the wild type. Fluorescence quenching data obtained using acrylamide and membrane probes further clarify the mechanism of action of this peptide at the molecular level, pointing out the potential therapeutic use of Psd1 as a natural antimycotic agent. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

Synergistic algicidal effect and mechanism of two diketopiperazines produced by Chryseobacterium sp. strain GLY-1106 on the harmful bloom-forming Microcystis aeruginosa

NASA Astrophysics Data System (ADS)

Guo, Xingliang; Liu, Xianglong; Pan, Jianliang; Yang, Hong

2015-10-01

A potent algicidal bacterium isolated from Lake Taihu, Chryseobacterium sp. strain GLY-1106, produces two algicidal compounds: 1106-A (cyclo(4-OH-Pro-Leu)) and 1106-B (cyclo(Pro-Leu)). Both diketopiperazines showed strong algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. Interestingly, these two algicidal compounds functioned synergistically. Compared with individual treatment, combined treatment with cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) significantly enhanced algicidal activity, accelerated the increase in intracellular reactive oxygen species (ROS) levels in M. aeruginosa, and further decreased the activities of antioxidases, effective quantum yield and maximal electron transport rate of M. aeruginosa. The results also showed that the algicidal characteristics of cyclo(4-OH-Pro-Leu) are distinct from those of cyclo(Pro-Leu). Cyclo(4-OH-Pro-Leu) mainly interrupted the flux of electron transport in the cyanobacterial photosynthetic system, whereas cyclo(Pro-Leu) mainly inhibited the activity of cyanobacterial intracellular antioxidases. A possible algicidal mechanism for the synergism between cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) is proposed, which is in accordance with their distinct algicidal characteristics in individual and combined treatment. These findings suggest that synergism between algicidal compounds might be used as an effective strategy for the future control of Microcystis blooms.

Synergistic algicidal effect and mechanism of two diketopiperazines produced by Chryseobacterium sp. strain GLY-1106 on the harmful bloom-forming Microcystis aeruginosa

PubMed Central

Guo, Xingliang; Liu, Xianglong; Pan, Jianliang; Yang, Hong

2015-01-01

A potent algicidal bacterium isolated from Lake Taihu, Chryseobacterium sp. strain GLY-1106, produces two algicidal compounds: 1106-A (cyclo(4-OH-Pro-Leu)) and 1106-B (cyclo(Pro-Leu)). Both diketopiperazines showed strong algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. Interestingly, these two algicidal compounds functioned synergistically. Compared with individual treatment, combined treatment with cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) significantly enhanced algicidal activity, accelerated the increase in intracellular reactive oxygen species (ROS) levels in M. aeruginosa, and further decreased the activities of antioxidases, effective quantum yield and maximal electron transport rate of M. aeruginosa. The results also showed that the algicidal characteristics of cyclo(4-OH-Pro-Leu) are distinct from those of cyclo(Pro-Leu). Cyclo(4-OH-Pro-Leu) mainly interrupted the flux of electron transport in the cyanobacterial photosynthetic system, whereas cyclo(Pro-Leu) mainly inhibited the activity of cyanobacterial intracellular antioxidases. A possible algicidal mechanism for the synergism between cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) is proposed, which is in accordance with their distinct algicidal characteristics in individual and combined treatment. These findings suggest that synergism between algicidal compounds might be used as an effective strategy for the future control of Microcystis blooms. PMID:26423356

Synergistic algicidal effect and mechanism of two diketopiperazines produced by Chryseobacterium sp. strain GLY-1106 on the harmful bloom-forming Microcystis aeruginosa.

PubMed

Guo, Xingliang; Liu, Xianglong; Pan, Jianliang; Yang, Hong

2015-10-01

A potent algicidal bacterium isolated from Lake Taihu, Chryseobacterium sp. strain GLY-1106, produces two algicidal compounds: 1106-A (cyclo(4-OH-Pro-Leu)) and 1106-B (cyclo(Pro-Leu)). Both diketopiperazines showed strong algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. Interestingly, these two algicidal compounds functioned synergistically. Compared with individual treatment, combined treatment with cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) significantly enhanced algicidal activity, accelerated the increase in intracellular reactive oxygen species (ROS) levels in M. aeruginosa, and further decreased the activities of antioxidases, effective quantum yield and maximal electron transport rate of M. aeruginosa. The results also showed that the algicidal characteristics of cyclo(4-OH-Pro-Leu) are distinct from those of cyclo(Pro-Leu). Cyclo(4-OH-Pro-Leu) mainly interrupted the flux of electron transport in the cyanobacterial photosynthetic system, whereas cyclo(Pro-Leu) mainly inhibited the activity of cyanobacterial intracellular antioxidases. A possible algicidal mechanism for the synergism between cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) is proposed, which is in accordance with their distinct algicidal characteristics in individual and combined treatment. These findings suggest that synergism between algicidal compounds might be used as an effective strategy for the future control of Microcystis blooms.

Analysis of Arg-Gly-Asp mimetics and soluble receptor of tumour necrosis factor as therapeutic modalities for concanavalin A induced hepatitis in mice.

PubMed Central

Bruck, R; Shirin, H; Hershkoviz, R; Lider, O; Kenet, G; Aeed, H; Matas, Z; Zaidel, L; Halpern, Z

1997-01-01

BACKGROUND/AIMS: It has been shown that synthetic non-peptidic analogues of Arg-Gly-Asp, a major cell adhesive ligand of extracellular matrix, prevented an increase in serum aminotransferase activity, as a manifestation of concanavalin A induced liver damage in mice. This study examined the effects of an Arg-Gly-Asp mimetic on liver histology and cytokine release in response to concanavalin A administration, and the efficacy of soluble receptor of tumour necrosis factor (TNF) alpha in preventing hepatitis in this model of liver injury. METHODS: Mice were pretreated with either the Arg-Gly-Asp mimetic SF-6,5 or recombinant soluble receptor of TNF alpha before their inoculation with 10 mg/kg concanavalin A. Liver enzymes, histology, and the serum values of TNF alpha and interleukin (IL)6 were examined. RESULTS: The histopathological damage in the liver, and the concanavalin A induced release of TNF alpha and IL6 were significantly inhibited by the synthetic Arg-Gly-Asp mimetic (p < 0.001). Liver injury, manifested by the increase in serum aminotransferase and cytokines, as well as by histological manifestations of hepatic damage, was effectively prevented by pretreatment of the mice with the soluble TNF receptor (p < 0.001). CONCLUSIONS: This study confirms the efficacy of a synthetic Arg-Gly-Asp mimetic and soluble TNF receptor in the prevention of immune mediated liver damage in mice. Images PMID:9155591

Variation of clinical expression in patients with Stargardt dystrophy and sequence variations in the ABCR gene.

PubMed

Fishman, G A; Stone, E M; Grover, S; Derlacki, D J; Haines, H L; Hockey, R R

1999-04-01

To report the spectrum of ophthalmic findings in patients with Stargardt dystrophy or fundus flavimaculatus who have a specific sequence variation in the ABCR gene. Twenty-nine patients with Stargardt dystrophy or fundus flavimaculatus from different pedigrees were identified with possible disease-causing sequence variations in the ABCR gene from a group of 66 patients who were screened for sequence variations in this gene. Patients underwent a routine ocular examination, including slitlamp biomicroscopy and a dilated fundus examination. Fluorescein angiography was performed on 22 patients, and electroretinographic measurements were obtained on 24 of 29 patients. Kinetic visual fields were measured with a Goldmann perimeter in 26 patients. Single-strand conformation polymorphism analysis and DNA sequencing were used to identify variations in coding sequences of the ABCR gene. Three clinical phenotypes were observed among these 29 patients. In phenotype I, 9 of 12 patients had a sequence change in exon 42 of the ABCR gene in which the amino acid glutamic acid was substituted for glycine (Gly1961Glu). In only 4 of these 9 patients was a second possible disease-causing mutation found on the other ABCR allele. In addition to an atrophic-appearing macular lesion, phenotype I was characterized by localized perifoveal yellowish white flecks, the absence of a dark choroid, and normal electroretinographic amplitudes. Phenotype II consisted of 10 patients who showed a dark choroid and more diffuse yellowish white flecks in the fundus. None exhibited the Gly1961Glu change. Phenotype III consisted of 7 patients who showed extensive atrophic-appearing changes of the retinal pigment epithelium. Electroretinographic cone and rod amplitudes were reduced. One patient showed the Gly1961Glu change. A wide variation in clinical phenotype can occur in patients with sequence changes in the ABCR gene. In individual patients, a certain phenotype seems to be associated with the presence of a Gly1961Glu change in exon 42 of the ABCR gene. The identification of correlations between specific mutations in the ABCR gene and clinical phenotypes will better facilitate the counseling of patients on their visual prognosis. This information will also likely be important for future therapeutic trials in patients with Stargardt dystrophy.

NTS2-selective neurotensin mimetics with tetrahydrofuran amino acids.

PubMed

Simeth, Nadja A; Bause, Manuel; Dobmeier, Michael; Kling, Ralf C; Lachmann, Daniel; Hübner, Harald; Einsiedel, Jürgen; Gmeiner, Peter; König, Burkhard

2017-01-01

Stimulation of the NTS2 neurotensin receptor causes antipsychotic effects and leads to a promotion of the μ-opioid-independent antinociception, which is important in the modulation of tonic pain sensitivity. We report the synthesis and properties of a small library of peptidic agonists based on the active neurotensin fragment NT(8-13). Two tetrahydrofuran amino acid derivatives were synthesized to replace Tyr 11 in NT(8-13). Additionally, Arg 8 , Arg 9 , and Ile 12 of the lead peptide were exchanged by Lys, Lys, and Gly, respectively. The new compounds showed substantial NTS2 binding affinity and up to 1000-fold selectivity over NTS1. The highest selectivity (K i (NTS2): 29nM, K i (NTS1): 35,000nM) was observed for the peptide analog 17R trans . Copyright © 2016 Elsevier Ltd. All rights reserved.

Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site.

PubMed Central

Vandenberg, R J; French, C R; Barry, P H; Shine, J; Schofield, P R

1992-01-01

The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily. PMID:1311851

Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry

PubMed Central

Jahouh, Farid; Hou, Shu-jie; Kováč, Pavol; Banoub, Joseph H.

2012-01-01

We present herein an efficient mass spectrometric method for the localization of the glycation sites of a model neoglycoconjugate vaccine formed by a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases, and the digests were then analyzed by MALDI-TOF/TOF-CID-MS/MS and nano-LC-ESI-QqTOF-CID-MS/MS. The sequences of the unknown peptides analyzed by MALDI-TOF/TOF-CID-MS/MS, following digestion with the GluC V8 endoproteinase, allowed us to recognize three glycopeptides whose glycation occupancies were, respectively, on Lys 235, Lys 420, and Lys 498. Similarly, the same analysis was performed on the tryptic digests, which permitted us to recognize two glycation sites on Lys 100 and Lys 374. In addition, we have also used LC-ESI-QqTOF-CID-MS/MS analysis for the identification of the tryptic digests. However, this analysis identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate–protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during the synthetic carbohydrate–spacer–carrier protein conjugation. Likewise, the LC-ESI-QqTOF-MS/MS analysis of the GluC V8 digest also identified 17 different glycation sites on the synthetic glycoconjugate. PMID:22012665

Elucidation of interactions of Alzheimer amyloid beta peptides (Abeta40 and Abeta42) with insulin degrading enzyme: a molecular dynamics study.

PubMed

Bora, Ram Prasad; Prabhakar, Rajeev

2010-05-11

In this study, interactions of the two full-length Alzheimer amyloid beta peptides (Abeta40 and Abeta42) with the fully active form of insulin degrading enzyme (IDE) through unrestrained, all-atom MD simulations have been investigated. This enzyme is a Zn-containing metallopeptidase that catalyzes the degradation of the monomeric forms of these peptides, and this process is critical for preventing the progression of Alzheimer's disease (AD). The available X-ray structures of the free and small fragment-bound (Asp1-Glu3 and Lys16-Asp23 of Abeta40 and Asp1-Glu3 and Lys16-Glu22 of Abeta42) mutated forms of IDE and NMR structures of the full-length Abeta40 and Abeta42 have been used to build the starting structures for these simulations. The most representative structures derived from the Abeta40-IDE and Abeta42-IDE simulations accurately reproduced the locations of the active site Zn(2+) metal and small fragments of the substrates and their interactions with the enzyme from the X-ray structures. The remaining fragments of both the substrates were found to interact with IDE through several hydrogen bonding, pi-pi, CH-pi, and NH-pi interactions. In comparison to Abeta40, Abeta42 is more flexible and interacts through a smaller number (17-22) of hydrogen bonds in the catalytic chamber of IDE. Both the substrates adopted more beta-sheet character in the IDE environment, an observation that is in line with experiments. Their structural characteristics inside IDE are significantly different than the ones observed in aqueous solution. The atomistic level details provided by these simulations can help in the elucidation of binding and degrading mechanisms of the Abeta peptides by IDE.

Evaluating the role of acidic, basic, and polar amino acids and dipeptides on a molecular electrocatalyst for H 2 oxidation

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

Boralugodage, Nilusha Priyadarshani; Arachchige, Rajith Jayasingha; Dutta, Arnab

Amino acids and peptides have been shown to have a significant influence on the H2 production and oxidation reactivity of Ni(P R 2N R’ 2) 2, where P R 2N R’ 2 = 1,5-diaza-3,7-diphosphacyclooctane, R is either phenyl (Ph) or cyclohexyl (Cy), and R’ is either an amino acid or peptide. Most recently, the Ni(P Cy 2Naminoacid 2) 2 complexes (CyAA) have shown enhanced H 2 oxidation rates, water solubility, and in the case of arginine (CyArg) and phenylalanine (CyPhe), electrocatalytic reversibility. Both the backbone –COOH and side chain interactions were shown to be critical to catalytic performance. Here wemore » further investigate the roles of the outer coordination sphere by evaluating amino acids with acidic, basic, and hydrophilic side chains, as well as dipeptides which combine multiple successful features from previous complexes. Six new complexes were prepared, three containing single amino acids: aspartic acid (CyAsp), lysine (CyLys), and serine (CySer) and three containing dipeptides: glycine-phenylalanine (Cy(GlyPhe)), phenylalanine-glycine (Cy(PheGly)), and aspartic acid-phenylananine (Cy(AspPhe)). The resulting catalytic performance demonstrates that complexes need both interactions between side chain and –COOH groups for fast, efficient catalysis. The fastest of all of the catalysts, Cy(AspPhe), had both of these features, while the other dipeptide complexes with an amide replacing the -COOH were both slower; however, the amide group was demonstrated to participate in the proton pathway when side chain interactions are present to position it. Both the hydrophilic and basic side chains, notably lacking in side chain interactions, significantly increased the overpotential, with only modest increases in TOF. Of all of the complexes, only CyAsp was reversible at room temperature, and only in water, the first of these complexes to demonstrate room temperature reversibility in water. These results continue to provide and solidify design rules for controlling reactivity and efficiency of Ni(P 2N 2) 2 complexes with the outer coordination sphere.« less

N-Terminal Cu-Binding Motifs (Xxx-Zzz-His, Xxx-His) and Their Derivatives: Chemistry, Biology and Medicinal Applications.

PubMed

Gonzalez, Paulina; Bossak, Karolina; Stefaniak, Ewelina; Hureau, Christelle; Raibaut, Laurent; Bal, Wojciech; Faller, Peter

2018-06-07

Peptides and proteins with N-terminal amino acid sequences NH 2 -Xxx-His (XH) and NH 2 -Xxx-Zzz-His (XZH) form well-established high-affinity Cu II -complexes. Key examples are Asp-Ala-His (in serum albumin) and Gly-His-Lys, the wound healing factor. This opens a straightforward way to add a high-affinity Cu II -binding site to almost any peptide or protein, by chemical or recombinant approaches. Thus, these motifs, NH 2 -Xxx-Zzz-His in particular, have been used to equip peptides and proteins with a multitude of functions based on the redox activity of Cu, including nuclease, protease, glycosidase, or oxygen activation properties, useful in anticancer or antimicrobial drugs. More recent research suggests novel biological functions, mainly based on the redox inertness of Cu II in XZH, like PET imaging (with 64 Cu), chelation therapies (for instance in Alzheimer's disease and other types of neurodegeneration), antioxidant units, Cu transporters and activation of biological functions by strong Cu II binding. This Review gives an overview of the chemical properties of Cu-XH and -XZH motifs and discusses the pros and cons of the vastly different biological applications, and how they could be improved depending on the application. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lysine and the Na+/K+ Selectivity in Mammalian Voltage-Gated Sodium Channels.

PubMed

Li, Yang; Liu, Huihui; Xia, Mengdie; Gong, Haipeng

2016-01-01

Voltage-gated sodium (Nav) channels are critical in the generation and transmission of neuronal signals in mammals. The crystal structures of several prokaryotic Nav channels determined in recent years inspire the mechanistic studies on their selection upon the permeable cations (especially between Na+ and K+ ions), a property that is proposed to be mainly determined by residues in the selectivity filter. However, the mechanism of cation selection in mammalian Nav channels lacks direct explanation at atomic level due to the difference in amino acid sequences between mammalian and prokaryotic Nav homologues, especially at the constriction site where the DEKA motif has been identified to determine the Na+/K+ selectivity in mammalian Nav channels but is completely absent in the prokaryotic counterparts. Among the DEKA residues, Lys is of the most importance since its mutation to Arg abolishes the Na+/K+ selectivity. In this work, we modeled the pore domain of mammalian Nav channels by mutating the four residues at the constriction site of a prokaryotic Nav channel (NavRh) to DEKA, and then mechanistically investigated the contribution of Lys in cation selection using molecular dynamics simulations. The DERA mutant was generated as a comparison to understand the loss of ion selectivity caused by the K-to-R mutation. Simulations and free energy calculations on the mutants indicate that Lys facilitates Na+/K+ selection by electrostatically repelling the cation to a highly Na+-selective location sandwiched by the carboxylate groups of Asp and Glu at the constriction site. In contrast, the electrostatic repulsion is substantially weakened when Lys is mutated to Arg, because of two intrinsic properties of the Arg side chain: the planar geometric design and the sparse charge distribution of the guanidine group.

Minimum requirements for inhibition of smooth-muscle myosin light-chain kinase by synthetic peptides.

PubMed Central

Hunt, J T; Floyd, D M; Lee, V G; Little, D K; Moreland, S

1989-01-01

Although the amino acid residues that are important for peptide substrates of myosin light-chain kinase have been reported, those that are important for peptide inhibitors of this enzyme have not previously been investigated. Synthetic peptides based on the sequence Lys11-Lys12-Arg13-Ala-Ala-Arg16-Ala-Thr-Ser19 -Asn-Val21-Phe22-Ala of the chicken gizzard myosin light chain were tested as inhibitors of pig carotid-artery myosin light-chain kinase. The basic amino acid residues of the known myosin light-chain kinase inhibitor Lys-Lys-Arg-Ala-Ala-Arg-Ala-Thr-Ser-NH2 (IC50 = 14 microM) [Pearson, Misconi & Kemp (1986) J. Biol. Chem. 261, 25-27] were shown to be the important residues that contribute to inhibitor potency, as evidence by the finding that the hexapeptide Lys-Lys-Arg-Ala-Ala-Arg-NH2 had an IC50 value of 22 microM. This indicates that binding of the phosphorylatable serine residue to myosin light-chain kinase, which is of obvious importance for a substrate, does not enhance the potency of an inhibitor. With the aim of preparing more potent inhibitors, peptides Lys-Lys-Arg-Ala-Ala-Arg-Ala-Ala-Xaa-NH2 were prepared with a variety of amino acids substituted for the phosphorylatable serine residue. None of these peptides was a more potent inhibitor than the serine peptide. PMID:2920029

Molecular basis of a novel adaptation to hypoxic-hypercapnia in a strictly fossorial mole.

PubMed

Campbell, Kevin L; Storz, Jay F; Signore, Anthony V; Moriyama, Hideaki; Catania, Kenneth C; Payson, Alexander P; Bonaventura, Joseph; Stetefeld, Jörg; Weber, Roy E

2010-07-16

Elevated blood O(2) affinity enhances survival at low O(2) pressures, and is perhaps the best known and most broadly accepted evolutionary adjustment of terrestrial vertebrates to environmental hypoxia. This phenotype arises by increasing the intrinsic O(2) affinity of the hemoglobin (Hb) molecule, by decreasing the intracellular concentration of allosteric effectors (e.g., 2,3-diphosphoglycerate; DPG), or by suppressing the sensitivity of Hb to these physiological cofactors. Here we report that strictly fossorial eastern moles (Scalopus aquaticus) have evolved a low O(2) affinity, DPG-insensitive Hb - contrary to expectations for a mammalian species that is adapted to the chronic hypoxia and hypercapnia of subterranean burrow systems. Molecular modelling indicates that this functional shift is principally attributable to a single charge altering amino acid substitution in the beta-type delta-globin chain (delta136Gly-->Glu) of this species that perturbs electrostatic interactions between the dimer subunits via formation of an intra-chain salt-bridge with delta82Lys. However, this replacement also abolishes key binding sites for the red blood cell effectors Cl-, lactate and DPG (the latter of which is virtually absent from the red cells of this species) at delta82Lys, thereby markedly reducing competition for carbamate formation (CO(2) binding) at the delta-chain N-termini. We propose this Hb phenotype illustrates a novel mechanism for adaptively elevating the CO(2) carrying capacity of eastern mole blood during burst tunnelling activities associated with subterranean habitation.

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

Xiong, J.-P.; Stehle, T.; Zhang, R.

The structural basis for the divalent cation-dependent binding of heterodimeric alpha beta integrins to their ligands, which contain the prototypical Arg-Gly-Asp sequence, is unknown. Interaction with ligands triggers tertiary and quaternary structural rearrangements in integrins that are needed for cell signaling. Here we report the crystal structure of the extracellular segment of integrin alpha Vbeta 3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence. The ligand binds at the major interface between the alpha V and beta 3 subunits and makes extensive contacts with both. Both tertiary and quaternary changes are observed in the presence of ligand. Themore » tertiary rearrangements take place in beta A, the ligand-binding domain of beta 3; in the complex, beta A acquires two cations, one of which contacts the ligand Asp directly and the other stabilizes the ligand-binding surface. Ligand binding induces small changes in the orientation of alpha V relative to beta 3.« less

Transport and metabolism of delta sleep-inducing peptide in cultured human intestinal epithelial cell monolayers.

PubMed

Augustijns, P F; Borchardt, R T

1995-12-01

A cultured human intestinal epithelial (Caco-2) cell monolayer was used to study the transport and metabolism of delta sleep-inducing peptide [DSIP (Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu)]. DSIP is of interest because it has been reported to be capable of permeating biological barriers (e.g. blood-brain barrier), and this property has been related to its solution conformation. When applied to the apical (AP) side of Caco-2 cell monolayers, DSIP was rapidly metabolized (8.2 +/- 1.1% remaining after a 2-hr incubation), affording Trp as the major metabolite and Trp-Ala as a minor metabolite. When DSIP was added to the basolateral (BL) side of the monolayer, the same metabolites were detected, but the peptide was more stable (70.6 +/- 3.0% remaining after a 2-hr incubation). Inclusion of bestatin, an inhibitor of aminopeptidases, at concentrations up to 0.29 mM with DSIP on the AP side of the Caco-2 cell monolayer increased the stability of the peptide only slightly but dramatically altered the distribution of the metabolites (Trp-Ala became the major metabolite, and Trp became the minor metabolite). Inclusion of other aminopeptidase inhibitors (e.g. amastatin, puromycin) alone, dipeptidylpeptidase IV inhibitors (e.g. diprotin A, Gly-Pro) alone, inhibitors of proteases that require heavy metals for proper activity (e.g. EDTA, 1,10-phenanthroline) alone, or cysteine protease inhibitors (e.g. leupeptin) alone did not lead to significant stabilization of the peptide. However, inclusion of a combination of 0.29 mM bestatin and 1 mM diprotin A with DSIP on the AP side of the monolayers resulted in a substantial increase in the stability of the peptide (83.2 +/- 3.7% remaining after a 2-hr incubation). However, under these conditions, a new metabolite (Trp-Ala-Gly-Gly-Asp-Ala-Ser) was observed with a formation that could be inhibited by inclusion of 1 mM captopril, an inhibitor of peptidyl dipeptidase A. Therefore, the stability of DSIP could be further increased (95.1 +/- 1.6% remaining after a 2-hr incubation) by incubating the peptide with 0.29 mM bestatin, 1 mM diprotin A, and 1 mM captopril. However, even when the major metabolic pathways were inhibited on the AP side of the cell monolayer, no DSIP was detected on the BL side of a Caco-2 cell monolayer. These results suggest that a yet unidentified metabolic pathway is preventing the AP-to-BL flux of DSIP or that DSIP has lower "intrinsic" ability to permeate across cultured intestinal epithelial cells than across cultured brain endothelial cells, a cell culture model of the blood-brain barrier.

Identification of residues that control Li+ versus Na+ dependent Ca2+ exchange at the transport site of the mitochondrial NCLX.

PubMed

Roy, Soumitra; Dey, Kuntal; Hershfinkel, Michal; Ohana, Ehud; Sekler, Israel

2017-06-01

The Na + /Ca 2+ /Li + exchanger (NCLX) is a member of the Na + /Ca 2+ exchanger family. NCLX is unique in its capacity to transport both Na + and Li + , unlike other members, which are Na + selective. The major aim of this study was twofold, i.e., to identify NCLX residues that confer Li + or Na + selective Ca 2+ transport and map their putative location on NCLX cation transport site. We combined molecular modeling to map transport site of NCLX with euryarchaeal H + /Ca 2+ exchanger, CAX_Af, and fluorescence analysis to monitor Li + versus Na + dependent mitochondrial Ca 2+ efflux of transport site mutants of NCLX in permeabilized cells. Mutation of Asn149, Pro152, Asp153, Gly176, Asn467, Ser468, Gly494 and Asn498 partially or strongly abolished mitochondrial Ca 2+ exchange activity in intact cells. In permeabilized cells, N149A, P152A, D153A, N467Q, S468T and G494S demonstrated normal Li + /Ca 2+ exchange activity but a reduced Na + /Ca 2+ exchange activity. On the other hand, D471A showed dramatically reduced Li + /Ca 2+ exchange, but Na + /Ca 2+ exchange activity was unaffected. Finally, simultaneous mutation of four putative Ca 2+ binding residues was required to completely abolish both Na + /Ca 2+ and Li + /Ca 2+ exchange activities. We identified distinct Na + and Li + selective residues in the NCLX transport site. We propose that functional segregation in Li + and Na + sites reflects the functional properties of NCLX required for Ca 2+ exchange under the unique membrane potential and ion gradient across the inner mitochondrial membrane. The results of this study provide functional insights into the unique Li + and Na + selectivity of the mitochondrial exchanger. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech. Copyright © 2017 Elsevier B.V. All rights reserved.

Tobacco chloroplast tRNA(UUU) gene contains a 2.5-kilobase-pair intron: An open reading frame and a conserved boundary sequence in the intron.

PubMed

Sugita, M; Shinozaki, K; Sugiura, M

1985-06-01

The nucleotide sequence of a tRNA(Lys)(UUU) gene on tobacco (Nicotiana tabacum) chloroplast DNA has been determined. This gene is located 215 base pairs upstream from the gene for the 32,000-dalton thylakoid membrane protein on the same DNA strand and has a 2526-base-pair intron in the anticodon loop. The intron boundary sequence does not follow the G-U/A-G rule but is similar to those of tobacco chloroplast split genes for tRNA(Gly)(UCC) and ribosomal proteins L2 and S12. The intron contains one major open reading frame of 509 codons. The codon usage in the open reading frame resembles those observed in the genes for tobacco chloroplast proteins so far analyzed. The primary transcript of this tRNA gene is 2.7 kilobases long.

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.

Engineering an Affinity-Enhanced Peptide through Optimization of Cyclization Chemistry.

PubMed

Ngambenjawong, Chayanon; Pineda, Julio Marco B; Pun, Suzie H

2016-12-21

Peptide cyclization is a strategy used to improve stability and activity of peptides. The most commonly used cyclization method is disulfide bridge formation of cysteine-containing peptides, as is typically found in nature. Over the years, an increasing number of alternative chemistries for peptide cyclization with improved efficiency, kinetics, orthogonality, and stability have been reported. However, there has been less appreciation for the opportunity to fine-tune peptide activity via the diverse chemical entities introduced at the site of linkage by different cyclization strategies. Here, we demonstrate how cyclization optimization of an M2 "anti-inflammatory" macrophage-binding peptide (M2pep) resulted in a significant increase in binding affinity of the optimized analog to M2 macrophages while maintaining binding selectivity compared to M1 "pro-inflammatory" macrophages. In this study, we report synthesis and evaluation of four cyclic M2pep(RY) analogs with diverse cyclization strategies: (1) Asp-[amide]-Lys, (2) azido-Lys-[triazole(copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC))]-propargyl-Gly, (3) Cys-[decafluorobiphenyl (DFBP)]-Cys, and (4) Cys-[decafluorobiphenyl sulfone (DFS)]-Cys, whereby the chemical entity or linker at the linkage site is shown in the square bracket and is between the residues involved in cyclization. These peptides are compared to a disulfide-cyclized M2pep(RY) that we previously reported as a serum-stable, affinity-enhanced analog to the original linear M2pep. DFBP-cyclized M2pep(RY) exhibits the highest binding activity to M2 macrophages with apparent dissociation constant (K D ) about 2.03 μM compared to 36.3 μM for the original disulfide-cyclized M2pep(RY) and 220 μM for the original linear peptide. DFS-cyclized M2pep(RY) also binds more strongly than the original cyclized analog, whereas amide- and triazole-cyclized M2pep(RY) analogs bind less strongly. We verified that DFBP alone has negligible binding to M2 macrophages and the incorporation of diphenylalanine to the original sequence improves binding activity at the expense of solubility and increased toxicity. In conclusion, we report development of cyclic M2pep(RY) analogs with diverse cyclization strategies leading to the discovery of DFBP-cyclized M2pep(RY) with enhanced M2 macrophage-binding activity.

Near UV-Visible electronic absorption originating from charged amino acids in a monomeric protein† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc00880e

PubMed Central

Prasad, Saumya; Mandal, Imon; Singh, Shubham; Paul, Ashim; Mandal, Bhubaneswar

2017-01-01

Electronic absorption spectra of proteins are primarily characterized over the ultraviolet region (185–320 nm) of the electromagnetic spectrum. While recent studies on peptide aggregates have revealed absorption beyond 350 nm, monomeric proteins lacking aromatic amino acids, disulphide bonds, and active site prosthetic groups are expected to remain optically silent beyond 250 nm. Here, in a joint theoretical and experimental investigation, we report the distinctive UV-Vis absorption spectrum between 250 nm [ε = 7338 M–1 cm–1] and 800 nm [ε = 501 M–1 cm–1] in a synthetic 67 residue protein (α3C), in monomeric form, devoid of aromatic amino acids. Systematic control studies with high concentration non-aromatic amino acid solutions revealed significant absorption beyond 250 nm for charged amino acids which constitute over 50% of the sequence composition in α3C. Classical atomistic molecular dynamics (MD) simulations of α3C reveal dynamic interactions between multiple charged sidechains of Lys and Glu residues present in α3C. Time-dependent density functional theory calculations on charged amino acid residues sampled from the MD trajectories of α3C reveal that the distinctive absorption features of α3C may arise from two different types of charge transfer (CT) transitions involving spatially proximal Lys/Glu amino acids. Specifically, we show that the charged amino (NH3+)/carboxylate (COO–) groups of Lys/Glu sidechains act as electronic charge acceptors/donors for photoinduced electron transfer either from/to the polypeptide backbone or to each other. Further, the sensitivity of the CT spectra to close/far/intermediate range of encounters between sidechains of Lys/Glu owing to the three dimensional protein fold can create the long tail in the α3C absorption profile between 300 and 800 nm. Finally, we experimentally demonstrate the sensitivity of α3C absorption spectrum to temperature and pH-induced changes in protein structure. Taken together, our investigation significantly expands the pool of spectroscopically active biomolecular chromophores and adds an optical 250–800 nm spectral window, which we term ProCharTS (Protein Charge Transfer Spectra), for label free probes of biomolecular structure and dynamics. PMID:28970921

Eco-Friendly Insecticide Discovery via Peptidomimetics: Design, Synthesis, and Aphicidal Activity of Novel Insect Kinin Analogues.

PubMed

Zhang, Chuanliang; Qu, Yanyan; Wu, Xiaoqing; Song, Dunlun; Ling, Yun; Yang, Xinling

2015-05-13

Insect kinin neuropeptides are pleiotropic peptides that are involved in the regulation of hindgut contraction, diuresis, and digestive enzyme release. They share a common C-terminal pentapeptide sequence of Phe(1)-Xaa(2)-Yaa(3)-Trp(4)-Gly(5)-NH2 (where Xaa(2) = His, Asn, Phe, Ser, or Tyr; Yaa(3) = Pro, Ser, or Ala). Recently, the aphicidal activity of insect kinin analogues has attracted the attention of researchers. Our previous work demonstrated that the sequence-simplified insect kinin pentapeptide analogue Phe-Phe-[Aib]-Trp-Gly-NH2 could retain good aphicidal activity and be the lead compound for the further discovery of eco-friendly insecticides which encompassed a broad array of biochemicals derived from micro-organisms and other natural sources. Using the peptidomimetics strategy, we chose Phe-Phe-[Aib]-Trp-Gly-NH2 as the lead compound, and we designed and synthesized three series, including 31 novel insect kinin analogues. The aphicidal activity of the new analogues against soybean aphid was determined. The results showed that all of the analogues exhibited aphicidal activity. Of particular interest was the analogue II-1, which exhibited improved aphicidal activity with an LC50 of 0.019 mmol/L compared with the lead compound (LC50 = 0.045 mmol/L) or the commercial insecticide pymetrozine (LC50 = 0.034 mmol/L). This suggests that the analogue II-1 could be used as a new lead for the discovery of potential eco-friendly insecticides.

Cell-mediated remodeling of biomimetic encapsulating hydrogels triggered by adipogenic differentiation of adipose stem cells.

PubMed

Clevenger, Tracy N; Luna, Gabriel; Boctor, Daniel; Fisher, Steven K; Clegg, Dennis O

2016-01-01

One of the most common regenerative therapies is autologous fat grafting, which frequently suffers from unexpected volume loss. One approach is to deliver adipose stem cells encapsulated in the engineered hydrogels supportive of cell survival, differentiation, and integration after transplant. We describe an encapsulating, biomimetic poly(ethylene)-glycol hydrogel, with embedded peptides for attachment and biodegradation. Poly(ethylene)-glycol hydrogels containing an Arg-Gly-Asp attachment sequence and a matrix metalloprotease 3/10 cleavage site supported adipose stem cell survival and showed remodeling initiated by adipogenic differentiation. Arg-Gly-Asp-matrix metalloprotease 3/10 cleavage site hydrogels showed an increased number and area of lacunae or holes after adipose stem cell differentiation. Image analysis of adipose stem cells in Arg-Gly-Asp-matrix metalloprotease 3/10 cleavage site hydrogels showed larger Voronoi domains, while cell density remained unchanged. The differentiated adipocytes residing within these newly remodeled spaces express proteins and messenger RNAs indicative of adipocytic differentiation. These engineered scaffolds may provide niches for stem cell differentiation and could prove useful in soft tissue regeneration.

On the Split Personality of Penultimate Proline

PubMed Central

Glover, Matthew S.; Shi, Liuqing; Fuller, Daniel R.; Arnold, Randy J.; Radivojac, Predrag; Clemmer, David E.

2014-01-01

The influence of the position of the amino acid proline in polypeptide sequences is examined by a combination of ion mobility spectrometry-mass spectrometry (IMS-MS), amino acid substitutions, and molecular modeling. The results suggest that when proline exists as the second residue from the N-terminus (i.e., penultimate proline), two families of conformers are formed. We demonstrate the existence of these families by a study of a series of truncated and mutated peptides derived from the 11-residue peptide Ser1-Pro2-Glu3-Leu4-Pro5-Ser6-Pro7-Gln8-Ala9-Glu10-Lys11. We find that every peptide from this sequence with a penultimate proline residue has multiple conformations. Substitution of Ala for Pro residues indicates that multiple conformers arise from the cis- trans isomerization of Xaa1–Pro2 peptide bonds as Xaa–Ala peptide bonds are unlikely to adopt the cis isomer, and examination of spectra from a library of 58 peptides indicates that ~80% of sequences show this effect. A simple mechanism suggesting that the barrier between the cis-and trans-proline forms is lowered because of low steric impedance is proposed. This observation may have interesting biological implications as well, and we note that a number of biologically active peptides have penultimate proline residues. PMID:25503299

Combination of ALDH2 and ADH1B polymorphisms is associated with smoking initiation: A large-scale cross-sectional study in a Japanese population.

PubMed

Masaoka, Hiroyuki; Ito, Hidemi; Gallus, Silvano; Watanabe, Miki; Yokomizo, Akira; Eto, Masatoshi; Matsuo, Keitaro

2017-04-01

Aldehyde dehydrogenase 2 (ALDH2; rs671, Glu504Lys) and alcohol dehydrogenase 1B (ADH1B; rs1229984, His47Arg) polymorphisms are known to strongly influence alcohol　drinking behavior. Given evidence of an association between smoking and drinking behaviors, we hypothesized that ALDH2/ADH1B polymorphisms might also be associated with smoking initiation, and conducted a cross-sectional study to examine this hypothesis. Study subjects were first-visit outpatients diagnosed not to have cancer at Aichi Cancer Center Hospital between 2001 and 2005, including 4141 never smokers and 2912 ever smokers. Unconditional logistic regression models were applied to estimate odds ratios (OR) and 95% confidence intervals (CI) for smoking initiation by comparing ever smokers with never smokers. Excessive alcohol drinking was associated with a higher likelihood of ever smoking. After adjustment for drinking behaviors, compared to individuals with ALDH2 Glu/Glu, the ORs of ever smoking were 1.71 (95% CI, 1.49-1.95) and 2.28 (1.81-2.87) among those with ALDH2 Glu/Lys and Lys/Lys, respectively. Combination of ALDH2 Lys/Lys and ADH1B His/His (i.e., the most alcohol-intolerant subpopulation) showed the highest OR [2.44 (1.84-3.23)], whereas combination of ALDH2 Glu/Glu and ADH1B Arg/Arg (i.e., the most alcohol-tolerant subpopulation) showed the lowest OR [0.83 (0.57-1.21)] compared with ALDH2 Glu/Glu and ADH1B His/His. Besides the amount and frequency of alcohol drinking, the combination of ALDH2 and ADH1B polymorphisms predicts smoking initiation. This study suggests that alcohol tolerance regulated by ALDH2 and ADH1B polymorphisms is associated with smoking initiation, and facilitates the development of targeted interventions to reduce smoking prevalence. Copyright © 2017 Elsevier B.V. All rights reserved.

Association of a common vitamin D-binding protein polymorphism with inflammatory bowel disease.

PubMed

Eloranta, Jyrki J; Wenger, Christa; Mwinyi, Jessica; Hiller, Christian; Gubler, Christoph; Vavricka, Stephan R; Fried, Michael; Kullak-Ublick, Gerd A

2011-09-01

Inflammatory bowel diseases (IBDs), Crohn's disease, and ulcerative colitis (UC), are multifactorial disorders, characterized by chronic inflammation of the intestine. A number of genetic components have been proposed to contribute to IBD pathogenesis. In this case-control study, we investigated the association between two common vitamin D-binding protein (DBP) genetic variants and IBD susceptibility. These two single nucleotide polymorphisms (SNPs) in exon 11 of the DBP gene, at codons 416 (GAT>GAG; Asp>Glu) and 420 (ACG>AAG; Thr>Lys), have been previously suggested to play roles in the etiology of other autoimmune diseases. Using TaqMan SNP technology, we have genotyped 884 individuals (636 IBD cases and 248 non-IBD controls) for the two DBP variants. On statistical analysis, we observed that the DBP 420 variant Lys is less frequent in IBD cases than in non-IBD controls (allele frequencies, P=0.034; homozygous carrier genotype frequencies, P=0.006). This inverse association between the DBP 420 Lys and the disease remained significant, when non-IBD participants were compared with UC (homozygous carrier genotype frequencies, P=0.022) or Crohn's disease (homozygous carrier genotype frequencies, P=0.016) patients separately. Although the DBP position 416 alone was not found to be significantly associated with IBD, the haplotype DBP_2, consisting of 416 Asp and 420 Lys, was more frequent in the non-IBD population, particularly notably when compared with the UC group (Odds ratio, 4.390). Our study adds DBP to the list of potential genes that contribute to the complex genetic etiology of IBD, and further emphasizes the association between vitamin D homeostasis and intestinal inflammation.

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli

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

Nicholas, R.A.; Suzuki, H.; Hirota, Y.

This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with (/sup 14/C)penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed thatmore » the serine residue in the middle of the sequence was covalently bonded to the (/sup 14/C)penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented.« less

The purification and characterisation of novel dipeptidyl peptidase IV-like activity from bovine serum.

PubMed

Buckley, Seamus J; Collins, Patrick J; O'Connor, Brendan F

2004-07-01

The discovery of a potentially novel proline-specific peptidase from bovine serum is presented which is capable of cleaving the dipeptidyl peptidase IV (DPIV) substrate Gly-Pro-MCA. The enzyme was isolated and purified with the use of Phenyl Sepharose Hydrophobic Interaction, Sephacryl S-300 Gel Filtration, and Q-Sephacryl Anion Exchange, producing an overall purification factor of 257. SDS PAGE resulted in a monomeric molecular mass of 158kDa while size exclusion chromatography generated a native molecular mass of 328kDa. The enzyme remained active over a broad pH range with a distinct preference for a neutral pH range of 7-8.5. Chromatofocusing and isoelectric focusing (IEF) revealed the enzyme's isoelectric point to be 4.74. DPIV-like activity was not inhibited by serine protease inhibitors but was by the metallo-protease inhibitors, the phenanthrolines. The enzyme was also partially inhibited by bestatin. Substrate specificity studies proved that the enzyme is capable of sequential cleavage of bovine beta-Casomorphin and Substance P. The peptidase cleaved the standard DPIV substrate, Gly-Pro-MCA with a K(M) of 38.4 microM, while Lys-Pro-MCA was hydrolysed with a K(M) of 103 microM. The DPIV-like activity was specifically inhibited by both Diprotin A and B, non-competitively, generating a K(i) of 1.4 x 10(-4) M for both inhibitors. Ile-Thiazolidide and Ile-Pyrrolidide both inhibited competitively with an inhibition constant of 3.7 x 10(-7) and 7.5 x 10(-7) M, respectively. It is concluded that bovine serum DPIV-like activity share many biochemical properties with DPIV and DPIV-like enzymes but not exclusively, suggesting that the purified peptidase may play an important novel role in bioactive oligopeptide degradation.

Cost-effectiveness of umeclidinium compared with tiotropium and glycopyrronium as monotherapy for chronic obstructive pulmonary disease: a UK perspective.

PubMed

Shah, Dhvani; Driessen, Maurice; Risebrough, Nancy; Baker, Timothy; Naya, Ian; Briggs, Andrew; Ismaila, Afisi S

2018-01-01

Cost-effectiveness of once-daily umeclidinium bromide (UMEC) was compared with once-daily tiotropium (TIO) and once-daily glycopyrronium (GLY) in patients with chronic obstructive pulmonary disease (COPD) from a UK National Health Service (NHS) perspective. A linked-equation model was implemented to estimate COPD progression, associated healthcare costs, exacerbations rates, life years (LY) and quality-adjusted LY (QALYs). Statistical risk equations for endpoints and resource use were derived from the ECLIPSE and TORCH studies, respectively. Treatment effects [mean (standard error)] at 12 weeks on forced expiratory volume in 1 s and St George's Respiratory Questionnaire score were obtained from the intention-to-treat populations of two head-to-head studies [GSK study identifiers 201316 (NCT02207829) and 201315 (NCT02236611)] which compared UMEC 62.5 mcg with TIO 18 mcg and UMEC 62.5 mcg with GLY 50 mcg, respectively. Treatment costs reflect UK list prices (2016) and NHS unit costs; UMEC and GLY prices being equal and less than TIO. A lifetime horizon, discounted costs and effects at 3.5% were used. Sensitivity analyses were performed to evaluate the robustness of variations in input parameters and assumptions in the model. Over a lifetime horizon, UMEC was predicted to increase LYs (+ 0.195; 95% confidence interval [CI]: 0.069, 0.356) and QALYs (+ 0.118; 95% CI: 0.055, 0.191) and reduce the number of annual exacerbations (- 0.053; 95% CI: - 0.171, 0.028) compared with TIO, with incremental cost savings of £460/patient (95% CI: - £645, - £240). Compared with GLY, UMEC increased LYs (+ 0.124; 95% CI: 0.015, 0.281) and QALYs (+ 0.101; 95% CI: 0.043, 0.179) and reduced annual exacerbation (- 0.033; 95% CI: - 0.135, 0.017) at an additional cost of £132/patient (95% CI: £12, £330), resulting in an incremental cost-effectiveness ratio of £1310/QALY (95% CI: £284, £2060). Similar results were observed in alternative time horizons and additional sensitivity analyses. For treatment of patients with COPD in the UK over a lifetime horizon, treatment with UMEC dominates treatment with TIO, providing both improved health outcomes and cost savings. In comparison with GLY, treatment with UMEC achieved improved health outcomes but was associated with a higher cost. Trial registration 201316, NCT02207829; 201315, NCT02236611.

Immunologically active peptides capable of inducing immunization against malaria and genes encoding therefor

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

Dame, J.B.; Williams, J.L.; McCutchan, T.F.

An antimalarial immunogenic stimulant is described comprising an immunogenic carrier and a peptide sequence of between 2 and 1000 consecutive repeats of a sequence Asn-X-Y-Pro, wherein X is Ala or Val and Y is Asn or Asp.

Characterization of the Pivotal Carbon Metabolism of Streptococcus suis Serotype 2 under ex Vivo and Chemically Defined in Vitro Conditions by Isotopologue Profiling*

PubMed Central

Willenborg, Jörg; Huber, Claudia; Koczula, Anna; Lange, Birgit; Eisenreich, Wolfgang; Valentin-Weigand, Peter; Goethe, Ralph

2015-01-01

Streptococcus suis is a neglected zoonotic pathogen that has to adapt to the nutritional requirements in the different host niches encountered during infection and establishment of invasive diseases. To dissect the central metabolic activity of S. suis under different conditions of nutrient availability, we performed labeling experiments starting from [13C]glucose specimens and analyzed the resulting isotopologue patterns in amino acids of S. suis grown under in vitro and ex vivo conditions. In combination with classical growth experiments, we found that S. suis is auxotrophic for Arg, Gln/Glu, His, Leu, and Trp in chemically defined medium. De novo biosynthesis was shown for Ala, Asp, Ser, and Thr at high rates and for Gly, Lys, Phe, Tyr, and Val at moderate or low rates, respectively. Glucose degradation occurred mainly by glycolysis and to a minor extent by the pentose phosphate pathway. Furthermore, the exclusive formation of oxaloacetate by phosphoenolpyruvate (PEP) carboxylation became evident from the patterns in de novo synthesized amino acids. Labeling experiments with S. suis grown ex vivo in blood or cerebrospinal fluid reflected the metabolic adaptation to these host niches with different nutrient availability; however, similar key metabolic activities were identified under these conditions. This points at the robustness of the core metabolic pathways in S. suis during the infection process. The crucial role of PEP carboxylation for growth of S. suis in the host was supported by experiments with a PEP carboxylase-deficient mutant strain in blood and cerebrospinal fluid. PMID:25575595

Purification and biochemical characterization of three myotoxins from Bothrops mattogrossensis snake venom with toxicity against Leishmania and tumor cells.

PubMed

de Moura, Andréa A; Kayano, Anderson M; Oliveira, George A; Setúbal, Sulamita S; Ribeiro, João G; Barros, Neuza B; Nicolete, Roberto; Moura, Laura A; Fuly, Andre L; Nomizo, Auro; da Silva, Saulo L; Fernandes, Carla F C; Zuliani, Juliana P; Stábeli, Rodrigo G; Soares, Andreimar M; Calderon, Leonardo A

2014-01-01

Bothrops mattogrossensis snake is widely distributed throughout eastern South America and is responsible for snakebites in this region. This paper reports the purification and biochemical characterization of three new phospholipases A2 (PLA2s), one of which is presumably an enzymatically active Asp49 and two are very likely enzymatically inactive Lys49 PLA2 homologues. The purification was obtained after two chromatographic steps on ion exchange and reverse phase column. The 2D SDS-PAGE analysis revealed that the proteins have pI values around 10, are each made of a single chain, and have molecular masses near 13 kDa, which was confirmed by MALDI-TOF mass spectrometry. The N-terminal similarity analysis of the sequences showed that the proteins are highly homologous with other Lys49 and Asp49 PLA2s from Bothrops species. The PLA2s isolated were named BmatTX-I (Lys49 PLA2-like), BmatTX-II (Lys49 PLA2-like), and BmatTX-III (Asp49 PLA2). The PLA2s induced cytokine release from mouse neutrophils and showed cytotoxicity towards JURKAT (leukemia T) and SK-BR-3 (breast adenocarcinoma) cell lines and promastigote forms of Leishmania amazonensis. The structural and functional elucidation of snake venoms components may contribute to a better understanding of the mechanism of action of these proteins during envenomation and their potential pharmacological and therapeutic applications.

Seed and Foliar Application of Amino Acids Improve Variables of Nitrogen Metabolism and Productivity in Soybean Crop.

PubMed

Teixeira, Walquíria F; Fagan, Evandro B; Soares, Luis H; Soares, Jérssica N; Reichardt, Klaus; Neto, Durval D

2018-01-01

The application of amino acids in crops has been a common practice in recent years, although most of the time they are associated with products based on algae extracts or on fermented animal or vegetable wastes. However, little is known about the isolated effect of amino acids on the development of crops. Therefore, the objective of this research was to evaluate the effect of the application of isolated amino acids on the in some steps of the soybean nitrogen metabolism and on productivity. Experiments were carried out in a greenhouse and in the field with the application of the amino acids glutamate (Glu), phenylalanine (Phe), cysteine (Cys) and glycine (Gly) and as a set (Glu+Phe+Cys+Gly), as seed treatment (ST), as foliar application (FA) and both (ST+FA), at the V 4 growth stage. Evaluations consisted of nitrate reductase and urease activities, nitrate, ureide, total amino acids and total nitrogen content in leaves, and productivity. The application of Glu to leaves, Cys as ST and a mixture of Glu+Cys+Phe+Gly as ST+FA in the greenhouse experiment increased the total amino acids content. In the field experiment all treatments increased the amino acid content in leaves. At the V 6 stage in the field experiment, all modes of Gly application, Glu as ST and FA, Cys and Phe as ST+FA and Glu+Cys+Phe+Gly as FA increased the nitrate content in leaves. In the greenhouse, application of Cys and Phe as ST increased the production of soybean plants by at least 21%. The isolated application of Cys, Phe, Gly, Glu and the set of these amino acids as ST increased the productivity of soybean plants in the field experiment by at least 22%.