RESUMEN
Leukocyte recruitment from the blood into injured tissues during inflammatory diseases is the result of sequential events involving chemokines binding to their GPC receptors as well as to their glycosaminoglycan (GAG) co-receptors. The induction and the crucial role of MCP-1/CCL2 in the course of diseases that feature monocyte-rich infiltrates have been validated in many animal models, and several MCP-1/CCL2 as well as CCR2 antagonists have since been generated. However, despite some of them being shown to be efficacious in a number of animal models, many failed in clinical trials, and therapeutically interfering with the activity of this chemokine is not yet possible. We have therefore generated novel MCP-1/CCL2 mutants with increased GAG binding affinity and knocked out CCR2 activity, which were designed to interrupt the MCP-1/CCL2-related signaling cascade. We provide evidence that our lead mutant MCP-1(Y13A/S21K/Q23R) exhibits a 4-fold higher affinity toward the natural MCP-1 GAG ligand heparan sulfate and that it shows a complete deficiency in activating CCR2 on THP-1 cells. Furthermore, a significantly longer residual time on GAG ligands was observed by surface plasmon resonance. Finally, we were able to show that MCP-1(Y13A/S21K/Q23R) had a mild ameliorating effect on experimental autoimmune uveitis and that a marginal effect on oral tolerance in the group co-fed with Met-MCP-1(Y13A/S21K/Q23R) plus immunogenic peptide PDSAg was observed. These results suggest that disrupting wild type chemokine-GAG interactions by a chemokine-based antagonist can result in anti-inflammatory activity that could have potential therapeutic implications.
Asunto(s)
Antiinflamatorios/farmacología , Quimiocina CCL2/química , Quimiocina CCL2/metabolismo , Quimiocinas/metabolismo , Quimiotaxis , Humanos , Cinética , Leucocitos/metabolismo , Ligandos , Monocitos/metabolismo , Mutación , Péptidos/química , Unión Proteica , Transducción de Señal , Espectrometría de Fluorescencia/métodos , Resonancia por Plasmón de SuperficieRESUMEN
Alpha-synuclein is an intrinsically unstructured protein that binds to membranes, forms fibrils, and is involved in neurodegeneration. We used a reconstituted in vitro system to show that the molecular chaperone Hsp90 influenced alpha-synuclein vesicle binding and amyloid fibril formation, two processes that are tightly coupled to alpha-synuclein folding. Binding of Hsp90 to monomeric alpha-synuclein occurred in the low micromolar range, involving regions of alpha-synuclein that are critical for vesicle binding and amyloidogenesis. As a consequence, both processes were affected. In the absence of ATP, the accumulation of non-amyloid alpha-synuclein oligomers prevailed over fibril formation, whereas ATP favored fibril growth. This suggests that Hsp90 modulates the assembly of alpha-synuclein in an ATP-dependent manner. We propose that Hsp90 affects these folding processes by restricting conformational fluctuations of alpha-synuclein.
Asunto(s)
Amiloide/química , Proteínas HSP90 de Choque Térmico/química , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Amiloide/genética , Amiloide/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Cinética , Modelos Biológicos , Enfermedad de Parkinson/genética , Unión Proteica , Pliegue de Proteína , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
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.
Asunto(s)
Quimiocina CCL5/metabolismo , Heparitina Sulfato/metabolismo , Línea Celular Tumoral , Quimiocina CCL5/química , Quimiocina CCL5/genética , Quimiotaxis , Dicroismo Circular , Escherichia coli/genética , Polarización de Fluorescencia , Heparitina Sulfato/química , Humanos , Cinética , Unión Proteica , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Cloruro de Sodio/química , Resonancia por Plasmón de SuperficieRESUMEN
Streptococcus pyogenes is one of the most common human pathogens and possesses diverse mechanisms to evade the human immune defence. One example of its immune evasion is the degradation of the chemokine IL (interleukin)-8 by ScpC, a serine proteinase that prevents the recruitment of neutrophils to an infection site. By applying the ANTIGENome technology and using human serum antibodies, we identified Spy0416, annotated as ScpC, as a prominent antigen that induces protective immune responses in animals. We demonstrate here for the first time that the recombinant form of Spy0416 is capable of IL-8 degradation in vitro in a concentration- and time-dependent manner. Mutations in the conserved amino acid residues of the catalytic triad of Spy0416 completely abolished in vitro activity. However, the isolated predicted proteinase domain does not exhibit IL-8-degrading activity, but is dependent on the presence of the C-terminal region of Spy0416. Binding to IL-8 is mainly mediated by the catalytic domain. However, the C-terminal region modulates substrate binding, indicating that the proteolytic activity is amenable to regulation via the non-catalytic regions. The specificity for human substrates is not restricted to IL-8, since we also detected in vitro protease activity for another CXC chemokine GRO-alpha (growth-related oncogene alpha), but not for NAP-2 (neutrophil-activating protein 2), SDF (stromal-cell-derived factor)-1alpha, PF-4 (platelet factor 4), I-TAC (interferon-gamma-inducible T-cell alpha-chemoattractant), IP-10 (interferon-gamma-inducible protein 10) and MCP-1 (monocyte chemoattractant protein 1). The degradation of two human CXC chemokines in vitro, the high sequence conservation, the immunogenicity of the protein in humans and the shown protection in animal studies suggest that Spy0416 is a promising vaccine candidate for the prevention of infections by S. pyogenes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Quimiocinas/metabolismo , Serina Endopeptidasas/metabolismo , Streptococcus pyogenes/enzimología , Proteínas Bacterianas/genética , Quimiocinas CXC/metabolismo , Immunoblotting , Interleucina-8/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/genética , Streptococcus pyogenes/genética , Especificidad por Sustrato , Resonancia por Plasmón de SuperficieRESUMEN
3-O-sulfation of heparan sulfate (HS) is the rarest modification within heparan sulfate biosynthesis resulting in unique biological activities. Heparan sulfate d-glucosaminyl 3-O-sulfotransferase-3A (3-OST-3A) (EC 2.8.2.23) generates a binding site for the envelope glycoprotein D (gD) of herpes simplex virus 1. We have expressed the sulfotransferase domain of the human heparan sulfate 3-OST-3A isoform in Escherichia coli and subsequently purified the active enzyme which was found to be present as an oligomer under nonreducing conditions. The activity of the enzyme was tested by a novel gD-dependent gel mobility assay. A biophysical characterisation of 3-OST-3A was performed to study ligand binding and ligand-induced structural changes. Interestingly, the natural substrate HS did not cause a secondary structural change in the enzyme, whereas heparin and chondroitin sulfate did, both of which also exhibited similar high affinity binding to 3-OST-3A compared to HS as detected by isothermal fluorescence titrations. In cross-link assays, only HS was found to induce high molecular aggregates of 3-OST-3A whereas other GAG ligands did not or even inhibited enzyme oligomerisation like the K5 polysaccharide, which was nevertheless found to bind to the enzyme. We therefore conclude that since 3-OST-3A is able to bind also non-substrate GAG ligands with high affinity, discrimination among ligands is triggered by protein oligomerisation.
Asunto(s)
Glicosaminoglicanos/metabolismo , Sulfotransferasas/metabolismo , Dicroismo Circular , Escherichia coli/genética , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Ligandos , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfotransferasas/química , Sulfotransferasas/genéticaRESUMEN
K5 lyase of coliphage K5A degrades the K5 polysaccharide of encapsulated E. coli strains expressing the K5 antigen thereby contributing to virus binding and infection. We have investigated the affinities of the recombinant enzyme for different GAG ligands by isothermal fluorescence titrations and correlated them with substrate processing and protein structural changes. Chondroitin sulfate (CS) and heparan sulfate (HS) bound to K5 lyase with a Kd of 0.5 microM whereas heparin exhibited a Kd=1.1 microM. The natural substrate K5 polysaccharide displayed a similar apparent affinity as CS and HS but was the only ligand of the enzyme which induced a large structural rearrangement of the protein as detected by far-UV CD spectroscopy. Since significant enzymatic degradation was only found for the K5 polysaccharide peaking at 44 degrees C, but binding was also detected for heparin, we propose that the K5 lyase is able to discriminate between specific (acetylated/non-sulfated) and unspecific (acetylated/sulfated) ligands by its heparin binding motif in the C-terminus. This is proposed to be the origin for the enzyme's residual HS degrading activity.
Asunto(s)
Proteínas de Escherichia coli/química , Polisacárido Liasas/química , Sulfatos de Condroitina/metabolismo , Dicroismo Circular , Estabilidad de Enzimas , Proteínas de Escherichia coli/metabolismo , Heparitina Sulfato/metabolismo , Calor , Cinética , Polisacárido Liasas/metabolismo , Desnaturalización Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rayos UltravioletaRESUMEN
Application of reverse transcription-PCR to total RNA prepared from TNF-alpha (tumour necrosis factor-alpha)-stimulated HUVECs (human umbilical vein endothelial cells) revealed that the syndecan-2 mRNA was up-regulated by this inflammatory stimulus. By immunoprecipitation using an anti-syndecan-2 antibody on TNF-alpha-stimulated HUVEC lysates, inflammation-induced interleukin-8 was found to be an interaction partner of this HS (heparan sulphate) proteoglycan, but not of any other syndecan on these cells. The glycosylated [Syn2(ect)(+HS)] and non-glycosylated [Syn2(ect)(-HS)] forms of Syn2(ect) (the syndecan-2 ectodomain) were purified from a stably transfected human cell line and from a bacterial expression system respectively. By CD spectroscopy, Syn2(ect) was found to adopt an all-beta secondary structure. The dissociation constant of Syn2(ect)(+HS) with respect to interleukin-8 binding was determined by isothermal fluorescence titrations to be 23 nM. Despite its lack of HS chains, Syn2(ect)(-HS) exhibited significant binding to the chemokine, with a K (d) of >1 microM. Thus, in addition to glycosaminoglycan binding, protein-protein contacts might also contribute to the chemokine-proteoglycan interaction.
Asunto(s)
Endotelio Vascular/metabolismo , Interleucina-8/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoglicanos/metabolismo , Sitios de Unión , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Glicosilación , Humanos , Glicoproteínas de Membrana/química , Estructura Secundaria de Proteína , Proteoglicanos/química , Sindecano-2 , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Acute renal allograft damage is caused by early leukocyte infiltration which is mediated in part by chemokines presented by glycosaminoglycan (GAG) structures on endothelial surfaces. CXCL8 can recruit neutrophils and induce the firm arrest of monocytes on activated endothelial cells. A human CXCL8-based antagonist (dnCXCL8) designed to generate a dominant-negative mutant protein with enhanced binding to GAG structures and reduced CXCR1/2 receptor binding ability was tested in models of early allograft injury. The agent displayed enhanced binding to GAG structures in vitro and could antagonize CXCL8-induced firm adhesion of monocytes as well as neutrophils to activated microvascular endothelium in physiologic flow assays. In a rat model of acute renal damage, dnCXCL8 treatment limited proximal tubular damage and reduced granulocyte infiltration. In a Fischer 344 (RT1(lvl)) to Lewis (RT1(l)) rat acute renal allograft model, dnCXCL8 was found to reduce monocyte and CD8+ T-cell infiltration into glomeruli and to limit tubular interstitial inflammation and tubulitis in vivo. Early treatment of allografts with agents like dnCXCL8 may help reduce acute allograft damage and preserve renal morphology and thereby help limit chronic dysfunction.
Asunto(s)
Glicosaminoglicanos/inmunología , Rechazo de Injerto/tratamiento farmacológico , Interleucina-8/antagonistas & inhibidores , Interleucina-8/farmacología , Trasplante de Riñón/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Glicosaminoglicanos/genética , Rechazo de Injerto/inmunología , Humanos , Interleucina-8/genética , Interleucina-8/inmunología , Monocitos/inmunología , Neutrófilos/inmunología , Ratas , Ratas Endogámicas F344 , Ratas Endogámicas Lew , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/inmunología , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/inmunología , Trasplante HomólogoRESUMEN
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.
Asunto(s)
Quimiocina CCL5/metabolismo , Glicosaminoglicanos/antagonistas & inhibidores , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Animales , Arrestina/administración & dosificación , Enfermedades Autoinmunes/metabolismo , Quimiocina CCL5/química , Quimiocina CCL5/genética , Modelos Animales de Enfermedad , Escherichia coli/genética , Glicosaminoglicanos/metabolismo , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Pliegue de Proteína , Estabilidad Proteica , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes/genética , Uveítis/inducido químicamente , Uveítis/metabolismoRESUMEN
Chemokine function in vivo depends on the presentation by structures of the extracellular matrix or on endothelial surfaces. CCL5 contains two clusters of basic amino acid residues ((44)RKNR(47) and (55)KKWVR(59)) implicated in presentation of the protein. While (44)RKNR(47) has been shown to moderate CCL5 binding to glycosaminoglycans (GAGs), no direct role for the basic residues in the so called 50s loop ((55)KKWVR(59)) as a presentation structure has been published to date. In ex vivo studies both regions were found to be necessary for direct tissue binding suggesting a role for (55)KKWVR(59). In vitroT lymphocyte and monocyte induced firm adhesion under flow, as well as leukocyte recruitment to the peritoneal cavity in vivo was reduced in the 50s mutant. The binding of the 50s mutant to endothelial cells was significantly reduced as compared to the wild type protein demonstrated by ELISA. The 50s mutant had little impact on GAG binding in vitro. These data suggest that functional CCL5 presentation is mediated through both the 40s as well as the 50s loop with differential functions of the two loops of clusters of basic residues.
Asunto(s)
Secuencias de Aminoácidos , Quimiocina CCL5/metabolismo , Secuencia de Aminoácidos , Animales , Adhesión Celular , Línea Celular , Movimiento Celular , Quimiocina CCL5/química , Quimiocina CCL5/genética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Femenino , Citometría de Flujo , Glicosaminoglicanos/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos/citología , Leucocitos/metabolismo , Ratones , Ratones Endogámicos BALB C , Mutación , Oligosacáridos/metabolismo , Unión ProteicaRESUMEN
Since many Hsp90 client proteins are key players in tumour pathways, the ubiquitylation and subsequent degradation of Hsp90-substrates as a consequence of pharmacologically inhibiting Hsp90 represents an innovative approach for cancer therapy. We therefore identified Hsp90-binding proteins which accumulated as ubiquityl-tagged aggregates in the detergent insoluble fraction of HeLa cells as a consequence of simultaneously inhibiting Hsp90 and the proteasome. 2-DE followed by nanoLC-MS/MS of trypsinised protein spots provided the Hsp90-dependent ubiquitylated proteome which was finally annotated and functionally classified. The overall picture thus obtained emphasised the well-established role of Hsp90 in stabilising proteins involved in gene transcription and signal transduction. It also provided a novel Hsp90-related link to metabolic pathways as the inhibition of Hsp90 caused the ubiquitylation of a significant amount of metabolic enzymes. These findings serve to support cumulating indications which attribute Hsp90 to diverse stabilising functions beyond signal transduction and gene transcription.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Proteoma/metabolismo , Ubiquitina/metabolismo , Electroforesis en Gel Bidimensional , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Células HeLa , Humanos , Leupeptinas/farmacología , Macrólidos/farmacología , Unión Proteica , Proteómica , SolubilidadRESUMEN
Glycosylation is the most frequent PTM and contributes significantly to the function of proteins depending on the type of glycosylation. Especially glycan structures like the glycosaminoglycans are considered to constitute themselves the major function of the glycoconjugate which is therefore termed proteoglycan. Here we review recent views on and novel tools for analysing the proteoglycanome, which are directly related to the type of glycanation under investigation. We define the major function of the proteoglycanome to be its interaction with various proteins in many different (patho-)physiological conditions. This is exemplified by the differential glycosaminoglycan-interactome of healthy versus arthritic patient sera.
Asunto(s)
Glicosaminoglicanos/fisiología , Polisacáridos/química , Proteómica , Glicoproteínas/química , Glicosaminoglicanos/químicaRESUMEN
The binding of interleukin-8 (IL-8) to heparan sulfate (HS) proteoglycans on the surface of endothelial cells is crucial for the recruitment of neutrophils to an inflammatory site. Fluorescence anisotropy measurements yielded an IL-8 dimerization constant of 120 nM. The binding affinities, obtained by isothermal fluorescence titration, of size-defined heparin and HS oligosaccharides to the chemokine were found to depend on the oligomerization state of IL-8: high affinity was detected for monomeric and low affinity was detected for dimeric IL-8, referring to a self-regulatory mechanism for its chemoattractant effect. The highest affinity for monomeric IL-8 was detected for the HS octamer with a K(d) < 5 nM whereas the dissociation constants of dimeric IL-8 were found in the medium micromolar range. No indication for increasing affinities for monomeric IL-8 with increasing oligosaccharide chain length was found. Instead, a periodic pattern was obtained for the dissociation constants of the GAG oligosaccharides with respect to chain length, referring to optimum and least optimum chain lengths for IL-8 binding. GAG disaccharides were identified to be the minimum length for chemokine binding. Conformational changes of the dimeric chemokine, determined using CD spectroscopy, were detected only for the IL-8/HS complexes and not for heparin, pointing to an HS-induced activation of the chemokine with respect to receptor binding. Thermal unfolding of IL-8 yielded a single transition at 56 degrees C which was completely prevented by the presence of undigested HS or heparin, indicating structural stabilization, thereby prolonging the biological effect of the chemokine.