RESUMO
The purpose of this study was to evaluate the effects of anginex on tumour angiogenesis assessed by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) on a clinical 1.5 Tesla MR system and with the clinically available contrast agent gadopentetate dimeglumine. C57BL/6 mice carrying B16F10 melanomas were treated with anginex, TNP-470 or saline. Tumour growth curves and microvessel density (MVD) were recorded to establish the effects of treatment. DCE-MRI was performed on day 16 after tumour inoculation, and the endothelial transfer coefficients of the microvessel permeability surface-area product (K(PS)) were calculated using a two-compartment model. Both anginex and TNP-470 resulted in smaller tumour volumes (P<0.0001) and lower MVD (P <0.05) compared to saline. Treatment with anginex resulted in a 64% reduction (P<0.01) of tumour K(PS) and TNP-470 resulted in a 44% reduction (P=0.17), compared to saline. DCE-MRI with a clinically available, small-molecular contrast agent can therefore be used to evaluate the angiostatic effects of anginex and TNP-470 on tumour angiogenesis.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Melanoma/irrigação sanguínea , Neovascularização Patológica/prevenção & controle , Animais , Meios de Contraste , Gadolínio DTPA , Imageamento por Ressonância Magnética/métodos , Melanoma/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/diagnóstico , Peptídeos , ProteínasRESUMO
A simple method is presented to accurately determine (15)N-[(1)H] NOEs in biomolecules in the presence of H(N)-water proton chemical exchange. Three measurements are required: one with nonselective proton saturation and two with different water saturation conditions to determine the equilibrium value of the (15)N signal. This approach is exemplified with data on two peptides, one helix-forming 17-mer and one compactly folded 56-mer. Results indicate that (15)N-[(1)H] NOEs determined using the standard approach with short recycle times (3 to 4 s) can be significantly in error when H(N)-water proton chemical exchange is relatively rapid, water proton relaxation is relatively slow, and (15)N-[(1)H] NOEs are away from the value of -1. This new method avoids such inaccuracies resulting from the use of short recycle times.
Assuntos
Proteínas/química , Água/química , Algoritmos , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética , PrótonsRESUMO
Methane monooxygenase (MMO) is a non-heme-iron-containing enzyme which consists of 3 protein components: a hydroxylase (MMOH), an NAD(P)H-linked reductase (MMOR), and a 138-residue regulatory protein, component B (MMOB). Here, NMR spectroscopy has been used to derive interactions between MMOB and reduced and oxidized states of MMOH (245 kDa). Differential broadening of MMOB resonances in 1H-15N HSQC spectra acquired at different molar ratios of MMOH indicates interaction of both proteins, with MMOB binding more tightly to oxidized MMOH as observed previously. The most broadened backbone NH resonances suggest which residues in MMOB are part of the MMOH-binding interface, particularly when those residues are spatially close or clustered in the structure of MMOB. Although a number of different residues in MMOB appear to be involved in interacting with oxidized- and reduced-MMOH, some are identical. The two most common segments, proximal in the structure of MMOB, are beta-strand 1 with turn 1 (residues 36-46) and alpha-helix 3 going into loop 2 (residues 101-112). In addition, the N-terminus of MMOB is observed to be involved in binding to MMOH in either redox state. This is most strongly evidenced by use of a synthetic N-terminal peptide from MMOB (residues 1-29) in differential broadening 1H TOCSY studies with MMOH. Binding specificity is demonstrated by displacement of the peptide from MMOH by parent MMOB, indicating that the peptide binds in or near the normal site of N-terminal binding. The N-terminus is also observed to be functionally important. Steady-state kinetic studies show that neither a delta2-29 MMOB deletion mutant (which in fact does bind to MMOH), the N-terminal peptide, nor a combination of the two elicit the effector functions of MMOB. Furthermore, transient kinetic studies indicate that none of the intermediates of the MMOH catalytic cycle are observed if either the delta2-29 MMOB mutant or the N-terminal peptide is used in place of MMOB, suggesting that deletion of the N-terminus prevents reaction of reduced MMOH with O2 that initiates catalysis.
Assuntos
Metaloproteínas/química , Methylosinus trichosporium/enzimologia , Oxigenases/química , Sequência de Aminoácidos , Metaloproteínas/genética , Metaloproteínas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Mutação , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Oxigenases/genética , Oxigenases/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
A new approach to visualizing spectral densities and analyzing NMR relaxation data has been developed. By plotting the spectral density function, J(omega), as F(omega)=2 omega J(omega) on the log-log scale, the distribution of motional correlation times can be easily visualized. F(omega) is calculated from experimental data using a multi-Lorentzian expansion that is insensitive to the number of Lorentzians used and allows contributions from overall tumbling and internal motions to be separated without explicitly determining values for correlation times and their weighting coefficients. To demonstrate the approach, (15)N and (13)C NMR relaxation data have been analyzed for backbone NH and C(alpha)H groups in an alpha-helix-forming peptide 17mer and in a well-folded 138-residue protein, and the functions F(omega) have been calculated and deconvoluted for contributions from overall tumbling and internal motions. Overall tumbling correlation time distribution maxima yield essentially the same overall correlation times obtained using the Lipari-Szabo model and other standard NMR relaxation data analyses. Internal motional correlational times for NH and C(alpha)H bond motions fall in the range from 100 ps to about 1 ns. Slower overall molecular tumbling leads to better separation of internal motional correlation time distributions from those of overall tumbling. The usefulness of the approach rests in its ability to visualize spectral densities and to define and separate frequency distributions for molecular motions.
Assuntos
Espectroscopia de Ressonância Magnética , Peptídeos/química , Dobramento de Proteína , Matemática , Movimento (Física) , Fatores de TempoRESUMO
A first-in-class non-peptide antagonist of the motilin receptor was identified through electronic screening of our corporate database against a 3D pharmacophore. The pharmacophore was developed from the motilin 22 residue endogenous peptide using NMR structural data, principles of peptide folding, and peptide structure activity relationships. The NMR data supported helical content within the peptide, and both the hydrophobic staple and N-capping box motifs were identified in the motilin sequence. The conformational features of these motifs were imposed on the peptide structure, providing a constrained conformer as a starting point for database searching. A trisubstituted cyclopentene lead was identified directly from the electronic search. Compounds in this series inhibit the binding of 125I-motilin to human antral smooth muscle membrane and antagonize motilin-induced intracellular calcium mobilization in cells expressing the human motilin receptor. A potent compound developed through optimization, RWJ 68023, is active in binding and cell-based functional assays and is also effective in inhibiting motilin-induced contractility in segments of rabbit duodenum. This orally active compound is currently undergoing clinical evaluation for the treatment of gastrointestinal disorders associated with altered motility.
Assuntos
Receptores dos Hormônios Gastrointestinais/antagonistas & inibidores , Receptores de Neuropeptídeos/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Duodeno/efeitos dos fármacos , Duodeno/fisiologia , Motilidade Gastrointestinal/efeitos dos fármacos , Humanos , Conformação Molecular , Dados de Sequência Molecular , Motilina/metabolismo , CoelhosRESUMO
For the designed peptide 33mer, beta pep-4, formation of beta-sheet structure [Ilyina, Roongta and Mayo (1997) Biochemistry 36, 5245--5250] is thermodynamically linked to self-association. Dimers and tetramers are stabilized by interactions between hydrophobic residues lying on the hydrophobic faces of the amphipathic monomer subunits. The present study investigates the effects on folding and self-association of the substitution of two key hydrophobic residues (Ile(20) and Val(22)) at the beta-sheet sandwich interface of beta pep-4. Single-site (I20L, I20V, I20A, V22L, V22I and V22A; where I20L corresponds to the substitution of Ile(20) with leucine etc.) and double-site (I20L/V22L and I20V/V22I) variants have been investigated. Like parent beta pep-4, all variants can form dimers and tetramers. NOESY data indicate that the overall beta-sheet fold and intersubunit beta-strand alignments are the same in all variant tetramers. CD data for all variants indicate mostly beta-sheet character in dimers and random coil character in monomers. Only for the V22I variant is the beta-sheet fold stabilized in the monomer state. Pulse-field gradient NMR-derived diffusion coefficients, measured as a function of peptide concentration, provide a means for deriving the distribution of monomer, dimer and tetramer states and, therefore, equilibrium association constants. Relative thermodynamic stabilities, which vary no more than approx. 0.5 kcal/mol (where 1 kcal identical with 4.184 kJ) from peptide to peptide, are I20V/V22I>I20V>I20L/V22L=beta pep-4 (Delta G(D) of 7.5 kcal/mol)=I20L=I20A>V22I>V22L>V22A for dimer formation and I20V>I20L/V22L>I20L>beta pep-4 (Delta G(T) of 6 kcal/mol)>V22I>I20V/V22I>V22L>I20A>V22A for tetramer formation. For the most part, dimer and/or tetramer stabilities are enhanced by the presence of valine and leucine and are attenuated by the presence of isoleucine and alanine.
Assuntos
Dobramento de Proteína , Proteínas/química , Proteínas Recombinantes , Sequência de Aminoácidos , Substituição de Aminoácidos , Dicroísmo Circular , Dimerização , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Polímeros/química , Estrutura Secundária de Proteína , Proteínas/síntese química , Temperatura , TermodinâmicaRESUMO
13C-NMR relaxation experiments (T(1), T(2), T(1)(rho), and NOE) were performed on selectively enriched residues in two peptides, one hydrophobic staple alpha-helix-forming peptide GFSKAELAKARAAKRGGY and one beta-hairpin-forming peptide RGITVNGKTYGR, in water and in water/trifluoroethanol (TFE). Exchange contributions, R(ex), to spin-spin relaxation rates for (13)C(alpha) and (13)C(beta) groups were derived and were ascribed to be mainly due to peptide folding-unfolding. To evaluate the exchange time, tau(ex), from R(ex), the chemical shift difference between folded and unfolded states, Deltadelta, and the populations of these states, p(i), were determined from the temperature dependence of (13)C chemical shifts. For both peptides, values for tau(ex) fell in the 1 micros to 10 micros range. Under conditions where the peptides are most folded (water/TFE, 5 degrees C), tau(ex) values for all residues in each respective peptide were essentially the same, supporting the presence of a global folding-unfolding exchange process. Rounded-up average tau(ex) values were 4 micros for the helix peptide and 9 micros for the hairpin peptide. This 2-3-fold difference in exchange times between helix and hairpin peptides is consistent with that observed for folding-unfolding of other small peptides.
Assuntos
Peptídeos/química , Sequência de Aminoácidos , Isótopos de Carbono , Dicroísmo Circular , Computação Matemática , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Peptídeos/síntese química , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Temperatura , TermodinâmicaRESUMO
Novel beta-sheet-forming peptide 33-mers, betapep peptides, have been designed by using a combination approach employing basic folding principles and incorporating short sequences from the beta-sheet domains of anti-angiogenic proteins. One of these designed peptides (betapep-25), named anginex, was observed to be potently anti-angiogenic. Anginex specifically inhibits vascular endothelial cell proliferation and induces apoptosis in these cells, as shown by flow-cytometric detection of sub-diploid cells, TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-nick-end labelling) analysis and cell morphology. Anginex also inhibits endothelial cell adhesion to and migration on different extracellular matrix components. Inhibition of angiogenesis in vitro is demonstrated in the sprout-formation assay and in vivo in the chick embryo chorio-allantoic membrane angiogenesis assay. Comparison of active and inactive betapep sequences allows structure-function relationships to be deduced. Five hydrophobic residues and two lysines appear to be crucial to activity. This is the first report of a designed peptide having a well-defined biological function as a novel cytokine, which may be an effective anti-angiogenic agent for therapeutic use against various pathological disorders, such as neoplasia, rheumatoid arthritis, diabetic retinopathy and restenosis.
Assuntos
Neovascularização Fisiológica/efeitos dos fármacos , Proteínas/síntese química , Sequência de Aminoácidos , Angiostatinas , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/farmacologia , Cicloexanos , Endostatinas , Endotélio Vascular/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , O-(Cloroacetilcarbamoil)fumagilol , Fragmentos de Peptídeos/farmacologia , Peptídeos , Plasminogênio/farmacologia , Proteínas/farmacologia , Sesquiterpenos/farmacologia , Trombospondina 1/farmacologiaRESUMO
Although the pseudohalide thiocyanate (SCN(-)) is the preferred substrate for eosinophil peroxidase (EPO) in fluids of physiologic halide composition, the product(s) of this reaction have not been directly identified, and mechanisms underlying their cytotoxic potential are poorly characterized. We used nuclear magnetic resonance spectroscopy (NMR), electrospray ionization mass spectrometry, and quantitative chemical analysis to identify the principal reaction products of both the EPO/SCN(-)/H(2)O(2) system and activated eosinophils as roughly equimolar amounts of OSCN(-) (hypothiocyanite) and OCN(-) (cyanate). Red blood cells exposed to increasing concentrations of OSCN(-)/OCN(-) are first depleted of glutathione, after which glutathione S-transferase and glyceraldehyde-3-phosphate dehydrogenase then ATPases undergo sulfhydryl (SH) reductant-reversible inactivation before lysing. OSCN(-)/OCN(-) inactivates red blood cell membrane ATPases 10-1000 times more potently than do HOCl, HOBr, and H(2)O(2). Exposure of glutathione S-transferase to [(14)C]OSCN(-)/OCN(-) causes SH reductant-reversible disulfide bonding and covalent isotope labeling. We propose that EPO/SCN(-)/H(2)O(2) reaction products comprise a potential SH-targeted cytotoxic system that functions in striking contrast to HOCl, the highly but relatively indiscriminantly reactive product of the neutrophil myeloperoxidase system.
Assuntos
Eosinófilos/enzimologia , Eritrócitos/efeitos dos fármacos , Oxidantes/metabolismo , Oxidantes/toxicidade , Peroxidases/metabolismo , Tiocianatos/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Cianatos/metabolismo , Peroxidase de Eosinófilo , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Eritrócitos/enzimologia , Eritrócitos/metabolismo , Glutationa/metabolismo , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Oxidantes/química , Peroxidase/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Temperatura , Acetato de Tetradecanoilforbol/farmacologia , Tiocianatos/química , Tiocianatos/toxicidadeRESUMO
Novel beta-sheet-forming peptide 33 mers, beta pep peptides, have been designed by using a combination approach employing basic folding principles and incorporating short sequences or proposed key residues from the beta-sheet domains of interleukin-8 (IL-8), platelet factor-4 (PF4) and bactericidal/permeability increasing protein (B/PI). Since PF4 and B/PI are anti-angiogenic and IL-8 is angiogenic, the library of 30 beta pep peptides was assayed for the ability to affect the growth of endothelial cells. Results indicate that five beta pep peptides (beta pep-2, 7, 8, 21 and 25) demonstrate greater than 50% anti-proliferative activity at 30 micrograms/ml, and one of those (beta pep-25) is similarly active at 10 micrograms/ml. Insight into the mechanism of action was probed in an apoptosis assay. Anti-proliferative activity was found to be correlated with the induction of apoptosis. For example, at 100 micrograms/ml beta pep-25 induces 85% of endothelial cells to undergo apoptosis within 2 days. These effects from beta pep peptides appear to be selective for endothelial cell (EC) because normal cells (fibroblasts and leukocytes) and various tumor cells are not significantly affected at peptide concentrations used in this study. Comparison of active and inactive beta pep sequences allows structure-function relationships to be deduced. Five hydrophobic residues and two lysines appear to be crucial to activity. This research contributes to the development of novel anti-angiogenic peptides.
Assuntos
Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Proteínas de Membrana , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos , Apoptose/efeitos dos fármacos , Proteínas Sanguíneas/química , Bovinos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Desenho de Fármacos , Humanos , Interleucina-8/química , Dados de Sequência Molecular , Peptídeos/síntese química , Fator Plaquetário 4/química , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Angiogenesis, or the formation of new vasculature out of preexisting capillaries, is a sequence of events that is essential in the normal physiological processes of tissue growth and in a broad spectrum of pathologies. The diseases in which angiogenesis plays a key role are divided into diseases that are characterized by hypoxia/ ischemia and diseases that are dependent on neovascularization. The formerpathologies may benefit from therapeutic angiogenesis stimulation. This review concentrates on the different strategies to inhibit angiogenesis in diseases that are characterized by excessive angiogenesis, for example, cancer, arthritis, diabetic retinopathy, and inflammatory diseases. These diseases are dependent on the development of newvasculature, and hence, a large variety of different strategies to inhibit angiogenesis are underwayin laboratories throughout the world. At present, over250 angiogenesis inhibitors are described, and approximately half of them display activity in in vivo models. A large percentage of these molecules are natural, nonnatural, or synthetic so-called small molecules. Others are of protein origin, either endogenous or exogenous by nature. The authors highlight the current knowledge on the development of angiostatic proteins and peptides and their potential in the treatment of disease.
Assuntos
Inibidores da Angiogênese/fisiologia , Inibidores da Angiogênese/farmacologia , Angiostatinas , Animais , Antineoplásicos/farmacologia , Autoantígenos/metabolismo , Adesão Celular/fisiologia , Inibição de Migração Celular , Colágeno/farmacologia , Colágeno Tipo IV/metabolismo , Endostatinas , Fatores de Crescimento Endotelial/antagonistas & inibidores , Endotélio Vascular/crescimento & desenvolvimento , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Humanos , Integrinas/antagonistas & inibidores , Integrinas/metabolismo , Linfocinas/antagonistas & inibidores , Inibidores de Metaloproteinases de Matriz , Oligopeptídeos/farmacologia , Endopeptidase Neutra Reguladora de Fosfato PHEX , Fragmentos de Peptídeos/farmacologia , Plasminogênio/farmacologia , Proteínas/farmacologia , Trombospondina 1/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
NMR relaxation-derived spectral densities provide information on molecular and internal motions occurring on the picosecond to nanosecond time scales. Using (13)C and (15)N NMR relaxation parameters [T(1), T(2), and NOE] acquired at four Larmor frequencies (for (13)C: 62.5, 125, 150, and 200 MHz), spectral densities J(0), J(omega(C)), J(omega(H)), J(omega(H) + omega(C)), J(omega(H) - omega(C)), J(omega(N)), J(omega(H) + omega(N)), and J(omega(H) - omega(N)) were derived as a function of frequency for (15)NH, (13)C(alpha)H, and (13)C(beta)H(3) groups of an alanine residue in an alpha-helix-forming peptide. This extensive relaxation data set has allowed derivation of highly defined (13)C and (15)N spectral density maps. Using Monte Carlo minimization, these maps were fit to a spectral density function of three Lorentzian terms having six motional parameters: tau(0), tau(1), tau(2), c(0), c(1), and c(2), where tau(0), tau(1) and tau(2) are correlation times for overall tumbling and for slower and faster internal motions, and c(0), c(1), and c(2) are their weighting coefficients. Analysis of the high-frequency portion of these maps was particularly informative, especially when deriving motional parameters of the side-chain methyl group for which the order parameter is very small and overall tumbling motions do not dominate the spectral density function. Overall correlation times, tau(0), are found to be in nanosecond range, consistent with values determined using the Lipari-Szabo model-free approach. Internal motional correlation times range from picoseconds for methyl group rotation to nanoseconds for backbone N-H, C(alpha)-H, and C(alpha)-C(beta) bond motions. General application of this approach will allow greater insight into the internal motions in peptides and proteins.
Assuntos
Peptídeos/química , Algoritmos , Sequência de Aminoácidos , Isótopos de Carbono , Difusão , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Método de Monte Carlo , Isótopos de Nitrogênio , Estrutura Secundária de ProteínaRESUMO
A series of designed peptide 33-mers (betapep peptides) areknown to be bactericidal [Mayo, Haseman, Ilyina and Gray (1998)Biochim. Biophys. Acta 1425, 81-92]. Here dodecapeptides (SC-1-SC-8), which 'walk through' the sequence ofbetapep-25, were investigated for their ability to kill Gram-negativeand -positive bacteria and to neutralize endotoxin. SC-4 (KLFKRHLKWKI I-NH(2); the -NH(2) at the right of each sequenceindicates amidation of the C-terminal carboxylate group) is the mosteffective, more so than betapep-25, at killing Gram-negative bacteriawith nanomolar LD(50) values. Against Gram-positive bacteria,SC-4 also shows good activity with submicromolar LD(50)values. Leakage studies indicate rapid bacterial membrane permeability,with t(1/2) valuesof 10-15 min. SC-4 in the micromolar range also effectivelyneutralizes endotoxin and is not haemolytic below 10(-4)M. For all SC peptides, CD and NMR data indicate the presence of both 3(10)- and alpha-helix. For SC-4, nuclear-Overhauser-effect-based computational modelling yields an amphipathic helix with K1, K4,R5, and K8 arrayed on the same face (K is lysine, R is arginine). Activity differences among SC peptides and single-site variants of SC-4allow some structure-function relationships to be deduced. Relative to other known bactericidal peptides in the linear peptide,helix-forming category, SC-4 is the most potent broad-spectrumantibacterial identified to date. The present study contributes to thedevelopment of agents involved in combating the ever-recurring problemof drug-resistant micro-organisms.
Assuntos
Antibacterianos/farmacologia , Endotoxinas/antagonistas & inibidores , Peptídeos/química , Pseudomonas aeruginosa/efeitos dos fármacos , Sequência de Aminoácidos , Dicroísmo Circular , Hemólise/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
Bactericidal/permeability-increasing protein (BPI) has been known for some time to function in killing bacteria and in neutralizing the effects of bacterial endotoxin lipopolysaccharide. In the present study, BPI is found to be a novel endogenous inhibitor of angiogenesis. Within the sub-muM range, BPI shows a concentration-dependent inhibition of endothelial cell (EC) proliferation that is mediated by cell detachment and subsequent induction of apoptosis. As measured by flow cytometric analysis of the percentage of subdiploid cells, apoptosis induction was half-maximal at about 250 nmol/L BPI. Apoptosis was confirmed by quantification of cells with nuclear fragmentation. Apoptosis was found to be EC specific. In an in vitro collagen gel-based angiogenesis assay, BPI at 1.8 micromol/L inhibited tube formation by 81% after only 24 hours. Evidence for in vivo inhibition of angiogenesis was obtained, using the chorioallantoic membrane assay in which BPI was seen to be significantly effective at concentrations as low as 180 nmol/L. This newly discovered function of BPI might provide a possible therapeutic modality for the treatment of various pathologic disorders that depend on angiogenesis.
Assuntos
Apoptose/fisiologia , Atividade Bactericida do Sangue , Proteínas Sanguíneas/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Proteínas de Membrana , Neovascularização Fisiológica/fisiologia , Alantoide/irrigação sanguínea , Animais , Peptídeos Catiônicos Antimicrobianos , Apoptose/efeitos dos fármacos , Proteínas Sanguíneas/antagonistas & inibidores , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Córion/irrigação sanguínea , Fragmentação do DNA , Eritrócitos/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Heparina/farmacologia , Humanos , Leucócitos/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Veias UmbilicaisRESUMO
Although ethanol has been reported to affect cholesterol homeostasis in biological membranes, the molecular mechanism of action is unknown. Here, nuclear magnetic resonance (NMR) spectroscopic techniques have been used to investigate possible direct interactions between ethanol and cholesterol in various low dielectric solvents (acetone, methanol, isopropanol, DMF, DMSO, chloroform, and CCl(4)). Measurement of (13)C chemical shifts, spin-lattice and multiplet relaxation times, as well as self-diffusion coefficients, indicates that ethanol interacts weakly, yet specifically, with the HC-OH moiety and the two flanking methylenes in the cyclohexanol ring of cholesterol. This interaction is most strong in the least polar-solvent carbon tetrachloride where the ethanol-cholesterol equilibrium dissociation constant is estimated to be 2 x 10(-3) M. (13)C-NMR spin-lattice relaxation studies allow insight into the geometry of this complex, which is best modeled with the methyl group of ethanol sandwiched between the two methylenes in the cyclohexanol ring and the hydroxyl group of ethanol hydrogen bonded to the hydroxyl group of cholesterol.
Assuntos
Colesterol/química , Etanol/química , Solventes/química , 2-Propanol/química , Acetona/química , Tetracloreto de Carbono/química , Clorofórmio/química , Difusão , Dimetil Sulfóxido/química , Dimetilformamida/química , Hidroxibutiratos/química , Espectroscopia de Ressonância Magnética , Metanol/química , Modelos Químicos , Modelos Moleculares , Pentanonas/químicaRESUMO
Advances in the methods used for the chemical synthesis of peptides, and in the accumulation and analysis of numerous X-ray crystallographic and nuclear magnetic resonance protein and peptide structures, have enabled the design and construction of peptides for investigating basic folding principles, making novel structural motifs, modifying functional activities, developing pharmaceutical drugs and creating new biomaterials. The focus of this review is on recent trends in the design and construction of synthetic peptides for basic science and for various biotechnological applications.
Assuntos
Bioquímica/métodos , Materiais Biocompatíveis , Biotecnologia/métodos , Peptídeos/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Biotecnologia/tendências , Enzimas/metabolismo , Mimetismo Molecular , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/metabolismo , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Dobramento de ProteínaRESUMO
Peptide GFSKAELAKARAAKRGGY folds in an alpha-helical conformation that is stabilized by formation of a hydrophobic staple motif and an N-terminal capping box (Munoz V. Blanco FJ, Serrano L, 1995, Struct Biol 2:380-385). To investigate backbone and side-chain internal motions within the helix and hydrophobic staple, residues F2, A5, L7, A8, and A10 were selectively 13C- and 15N-enriched and NMR relaxation experiments were performed in water and in water/trifluoroethanol (TFE) solution at four Larmor frequencies (62.5, 125, 150, and 200 MHz for 13C). Relaxation data were analyzed using the model free approach and an anisotropic diffusion model. In water, angular variances of motional vectors range from 10 to 20 degrees and backbone phi,psi bond rotations for helix residues A5, L7, A8, and A10 are correlated indicating the presence of Calpha-H, Calpha-Cbeta, and N-H rocking-type motions along the helix dipole axis. L7 side-chain CbetaH2 and CgammaH motions are also correlated and as motionally restricted as backbone CalphaH, suggesting considerable steric hindrance with neighboring groups. In TFE which stabilizes the fold, internal motional amplitudes are attenuated and rotational correlations are increased. For the side chain of hydrophobic staple residue F2, wobbling-in-a-cone type motions dominate in water, whereas in TFE, the Cbeta-Cgamma bond and phenyl ring fluctuate more simply about the Calpha-Cbeta bond. These data support the Daragan-Mayo model of correlated bond rotations (Daragan VA, Mayo KH, 1996, J Phys Chem 100:8378-8388) and contribute to a general understanding of internal motions in peptides and proteins.
Assuntos
Carbono/química , Nitrogênio/química , Peptídeos/química , Sequência de Aminoácidos , Anisotropia , Espectroscopia de Ressonância Magnética , Modelos Químicos , Modelos Estatísticos , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Prótons , Temperatura , Termodinâmica , Trifluoretanol/química , Água/químicaRESUMO
alpha-Chemokines are known heparin-binding proteins. Here, a heparin dodecasaccharide (H12) was purified and used in NMR studies to investigate binding to growth-related protein-alpha (Gro-alpha) and to platelet factor-4-M2 (PF4-M2), an N-terminal chimera of PF4. Pulsed field gradient NMR was used to derive diffusion coefficients as the protein (monomer):H12 ratio was varied. In the absence of H12, both PF4-M2 and Gro-alpha give diffusion coefficients consistent with the presence of mostly dimers. As the PF4-M2:H12 ratio is increased from 1:6 to 2:1, the diffusion coefficient increases, indicating dissociation to the monomer state. On addition of H12 to either protein, (15)N/(1)H heteronuclear single quantum coherence NMR data demonstrate loss of (1)H resonance dispersion and intensity, particularly at protein:H12 ratios of 2:1 to 4:1, indicating significant perturbation to native structures. For Gro-alpha in particular, (1)H resonance dispersion appears random coil-like. At these same ratios, circular dichroism (CD) data show general retention of secondary structure elements with a slight shift to additional helix formation. Random coil NMR resonance dispersion suggests a shift to a less compact, partially folded, and/or more flexible state. Further addition of H12 causes resonance intensity and dispersion to return making NMR spectra appear native-like. At low PF4-M2:H12 ratios, loss of resonance intensity for residues proximal to Arg-20 and Arg-22 in three-dimensional NMR HCCH-TOCSY spectra suggests that the Arg-20-Arg-22 loop either interacts most strongly with H12 and/or that binding at this site is heterogeneous. This domain was previously shown to be crucial to heparin binding. Of particular interest to the biology of PF4-heparin complex formation, heparin-induced thrombocytopenia antibody binding occurs at about the same PF4-M2:H12 ratio as does this transition to a partially folded PF4-M2 state, strongly suggesting that heparin-induced thrombocytopenia antibody recognizes a less folded, lower aggregate state of the protein.
Assuntos
Fatores Quimiotáticos/metabolismo , Substâncias de Crescimento/metabolismo , Heparina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Fator Plaquetário 4/metabolismo , Trombocitopenia/sangue , Quimiocina CXCL1 , Quimiocinas CXC/química , Quimiocinas CXC/metabolismo , Fatores Quimiotáticos/química , Dissacarídeos/química , Dissacarídeos/metabolismo , Substâncias de Crescimento/química , Heparina/química , Humanos , Fator Plaquetário 4/química , Dobramento de ProteínaRESUMO
Methane monooxygenase (MMO) is a nonheme iron-containing enzyme which consists of three protein components: a hydroxylase (MMOH), an NADH-linked reductase (MMOR), and a small "B" component (MMOB) which plays a regulatory role. Here, 1H, 13C, 15N heteronuclear 2D and 3D NMR spectroscopy has been used to derive the solution structure of the 138 amino acid MMOB protein in the monomer state. Pulse field gradient NMR self-diffusion measurements indicate predominant formation of dimers at 1 mM MMOB and monomers at or below 0.2 mM. MMOB is active as a monomer. Aggregate exchange broadening and limited solubility dictated that multidimensional heteronuclear NMR experiments had to be performed at a protein concentration of 0.2 mM. Using 1340 experimental constraints (1182 NOEs, 98 dihedrals, and 60 hydrogen bonding) within the well-folded part of the protein (residues 36-126), MMOB structural modeling produced a well-defined, compact alpha/beta fold which consists of three alpha-helices and six antiparallel beta-strands arranged in two domains: a betaalphabetabeta and a betaalphaalphabetabeta. Excluding the ill-defined N- and C-terminal segments (residues 1-35 and 127-138), RMS deviations are 1.1 A for backbone atoms and 1.6 A for all non-hydrogen atoms. Compared to the lower resolution NMR structure for the homologous protein P2 from the Pseudomonas sp. CF600 phenol hydroxylase system (RMSD = 2.48 A for backbone atoms) (Qian, H., Edlund, U., Powlowski, J., Shingler, V., and Sethson, I. (1997) Biochemistry, 36, 495-504), that of MMOB reveals a considerably more compact protein. In particular, MMOB lacks the large "doughnut" shaped cavity reported for the P2 protein. This difference may result from the limited number of long-range NOEs that were available for use in the modeling of the P2 structure. This NMR-derived structure of MMOB, therefore, presents the first high-resolution structure of a small protein effector of a nonheme oxygenase system.
Assuntos
Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oxigenases/química , Sequência de Aminoácidos , Simulação por Computador , Cristalografia por Raios X , Methanococcaceae/enzimologia , Dados de Sequência Molecular , Oxigenases/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Soluções , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Neutrophil-activating peptide 2 (NAP-2), which demonstrates a range of proinflammatory activities, is a 72-residue protein belonging to the alpha-chemokine family. Although NAP-2, like other alpha-chemokines, is known to self-associate into dimers and tetramers, it has been shown that the monomeric form is physiologically active. Here we investigate the solution structure of monomeric NAP-2 by multi-dimensional 1H-NMR and 15N-NMR spectroscopy and computational modelling. The NAP-2 monomer consists of an amphipathic, triple-stranded, anti-parallel beta-sheet on which is folded a C-terminal alpha-helix and an aperiodic N-terminal segment. The backbone fold is essentially the same as that found in other alpha-chemokines. 15N T1, T2 and nuclear Overhauser effects (NOEs) have been measured for backbone NH groups and used in a model free approach to calculate order parameters and conformational exchange terms that map out motions of the backbone. N-terminal residues 1 to 17 and the C-terminus are relatively highly flexible, whereas the beta-sheet domain forms the most motionally restricted part of the fold. Conformational exchange occurring on the millisecond time scale is noted at the top of the C-terminal helix and at proximal residues from beta-strands 1 and 2 and the connecting loop. Dissociation to the monomeric state is apparently responsible for increased internal mobility in NAP-2 compared with dimeric and tetrameric states in other alpha-chemokines.