RESUMO
Langerhans cell histiocytosis (LCH) has a broad spectrum of clinical behaviors; some cases are self-limited, whereas others involve multiple organs and cause significant mortality. Although Langerhans cells in LCH are clonal, their benign morphology and their lack (to date) of reported recurrent genomic abnormalities have suggested that LCH may not be a neoplasm. Here, using 2 orthogonal technologies for detecting cancer-associated mutations in formalin-fixed, paraffin-embedded material, we identified the oncogenic BRAF V600E mutation in 35 of 61 archived specimens (57%). TP53 and MET mutations were also observed in one sample each. BRAF V600E tended to appear in younger patients but was not associated with disease site or stage. Langerhans cells stained for phospho-mitogen-activated protein kinase kinase (phospho-MEK) and phospho-extracellular signal-regulated kinase (phospho-ERK) regardless of mutation status. High prevalence, recurrent BRAF mutations in LCH indicate that it is a neoplastic disease that may respond to RAF pathway inhibitors.
Assuntos
Predisposição Genética para Doença , Histiocitose de Células de Langerhans/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Adolescente , Adulto , Substituição de Aminoácidos , Antígenos CD1/metabolismo , Criança , Pré-Escolar , Análise Mutacional de DNA , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Imunofluorescência , Genótipo , Histiocitose de Células de Langerhans/metabolismo , Histiocitose de Células de Langerhans/patologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Adulto JovemRESUMO
Binding of chemokines to glycosaminoglycans (GAGs) represents a crucial step in leukocyte attraction and activation. Since chemokine oligomerisation was shown to be important for GAG binding, the apparent oligomerisation constant of RANTES was determined to be 225 nM using fluorescence anisotropy. In the presence of heparan sulfate, chemokine oligomerisation was found to be promoted by the glycosaminoglycan as expressed in the increase in cooperativity and a shift towards higher melting temperatures in thermal unfolding experiments. In surface plasmon resonance investigations of RANTES-GAG binding kinetics using a heparan sulfate-coated chip, GAG-induced oligomerisation led to a bell-shaped (bi-phasic) Scatchard plot referring to cooperativity in the chemokine-GAG interaction. This was absent in the oligomerisation deficient RANTES mutants N46R and Q48K. We have further investigated the dependence of RANTES-GAG dissociation constants on oligosaccharide chain length by performing isothermal fluorescence titrations with size-defined heparin and heparan sulfate oligosaccharides as chemokine ligands. Heparin dp18 and heparan sulfate dp14 yielded the highest affinities with Kd values of 31.7 nM and 42.9 nM, respectively. Far-UV CD spectroscopy revealed a significant conformational change of RANTES upon heparan sulfate binding which is suggested to be a pre-requisite for oligomerisation and thus for optimal GPCR activation in vivo. This was shown by the impaired chemotactic activity of the RANTES N46R and Q48K mutants.
Assuntos
Quimiocina CCL5/metabolismo , Heparitina Sulfato/metabolismo , Linhagem Celular Tumoral , Quimiocina CCL5/química , Quimiocina CCL5/genética , Quimiotaxia , Dicroísmo Circular , Escherichia coli/genética , Polarização de Fluorescência , Heparitina Sulfato/química , Humanos , Cinética , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Cloreto de Sódio/química , Ressonância de Plasmônio de SuperfícieRESUMO
2-Substituted derivatives of diphenylpyraline and their 1-phenyl and 1-phenethyl analogues have been prepared in several steps from dihydropyridine-2(1H)-thiones. The structures of all new compounds have been confirmed by NMR spectroscopy. Their activity against Mycobacterium tuberculosis H(37)Rv as well as their cytotoxicity against human cells (HEK-293) have been determined via in vitro assays. The antimycobacterial potency was in general increased by substitution in ring position 2. The most promising modifications were a 2-isopropyl derivative and a 1,2-diphenyl analogue.
Assuntos
Antituberculosos/farmacologia , Proliferação de Células/efeitos dos fármacos , Piperidinas/química , Antituberculosos/síntese química , Antituberculosos/química , Células Cultivadas , Rim/citologia , Rim/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Binding of interleukin-8 (IL-8) to glycosaminoglycans (GAGs) on the surface of endothelial cells is crucial for the recruitment of neutrophils to an inflammatory site. Deriving structural knowledge about this interaction from in silico docking experiments has proved difficult because of the high flexibility and the size of GAGs. Therefore, we developed a docking method that takes into account ligand and protein flexibility by running approximately 15,000 molecular dynamics simulations of the docking event with different initial orientations of the binding partners. The method was shown to successfully reproduce the residues of basic fibroblast growth factor involved in GAG binding. Docking of a heparin hexasaccharide to IL-8 gave an interaction interface involving the basic residues His18, Lys20, Arg60, Lys64, Lys67, and Arg68. By subjecting IL-8 single-site mutants, in which these amino acids were replaced by alanine, to isothermal fluorescence titrations, the affinities for heparin were determined to be wtIL-8 > IL-8(H18A) >> IL-8(R68A) > IL-8(K67A) >> IL-8(K20A) > IL-8(R60A) >> IL-8(K64A). A comparison with the binding energies calculated from the model revealed high values for wtIL-8 and the H18A mutant and significantly lower but similar energies for the remaining mutants. Connecting the two fully sulfated hexasaccharides bound to each of the two IL-8 monomers in the dimeric chemokine by an N-acetylated dodecasaccharide gave a complex structure in which the GAG molecule aligned in a parallel fashion to the N-terminal alpha-helices of IL-8 like a horseshoe. A 5-ns molecular dynamics simulation of this complex confirmed its structural stability and revealed a reorientation in both binding sites where a disaccharide became the central binding unit. Isothermal fluorescence titration experiments using differently sulfated heparin disaccharides confirmed that a single disaccharide can indeed bind IL-8 with high affinity.
Assuntos
Heparina/química , Heparina/metabolismo , Interleucina-8/química , Interleucina-8/metabolismo , Modelos Moleculares , Substituição de Aminoácidos , Sítios de Ligação , Simulação por Computador , Dissacarídeos/química , Dissacarídeos/metabolismo , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fluorescência , Interleucina-8/genética , Mutagênese Sítio-Dirigida , Mutação/genética , Termodinâmica , TitulometriaRESUMO
Binding to glycosaminoglycans (GAGs) is a necessary prerequisite for the biological activity of the proinflammatory chemokine RANTES in vivo. We have applied protein engineering methods to modulate equilibrium-binding affinity as well as binding kinetics of RANTES towards its GAG ligand which also altered the chemokine's oligomerization behavior. Out of 10 mutants, A22K and H23K were chosen for further in vitro and in vivo characterization because their stability was comparable with wild-type (wt) RANTES. In chemical cross-linking experiments, A22K gave higher and H23K lower molecular weight aggregates compared with wtRANTES as shown on SDS-PAGE. All mutants contained an N-terminal methionine residue, a well-described G-protein-coupled receptor (GPCR) antagonistic modification, which resulted in the mutants' inability to induce monocyte chemotaxis. In surface plasmon resonance experiments using immobilized heparan sulfate (HS) and physiological buffer conditions, Met-RANTES exhibited a significantly longer residual time on the GAG chip compared with the other RANTES variants. In Scatchard plot analysis, RANTES gave a bi-phasic, bell-shaped curve suggesting 'creation' of ligand-binding sites on the protein during HS interaction. This was not observed in the mutants' Scatchard plots which gave K(d) values of 317.5 and 44.5 nM for the A22K and H23K mutants, respectively. The mutants were subsequently tested for their inhibitory effect in a rat model of autoimmune uveitis where only H23K exhibited a transient improvement of the clinical disease score. H23K is therefore proposed to be a GPCR-inactive GAG antagonist which displaces the wt chemokine from its natural HS-proteoglycan co-receptor. The protein engineering approach presented here opens new ways for the treatment of RANTES-related diseases.
Assuntos
Quimiocina CCL5/metabolismo , Glicosaminoglicanos/antagonistas & inibidores , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Animais , Arrestina/administração & dosagem , Doenças Autoimunes/metabolismo , Quimiocina CCL5/química , Quimiocina CCL5/genética , Modelos Animais de Doenças , Escherichia coli/genética , Glicosaminoglicanos/metabolismo , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Dobramento de Proteína , Estabilidade Proteica , Ratos , Ratos Endogâmicos Lew , Proteínas Recombinantes/genética , Uveíte/induzido quimicamente , Uveíte/metabolismoRESUMO
VEGF165 binding to endothelial cells is potentiated by glycosaminoglycans (GAGs). Here, we have investigated the impact of VEGF165 N-glycosylation on GAG binding. Although glycosylated VEGF165 bound to heparin with only slightly higher affinity than non-glycosylated VEGF165, the natural ligand heparan sulfate induced a conformational change only in the glycosylated protein. Unfolding studies of the VEGF proteins indicated a stabilising effect of heparin on the growth factor structure.