RESUMEN
For the treatment of large bone defects, the commonly used technique of autologous bone grafting presents several drawbacks and limitations. With the discovery of the bone-inducing capabilities of bone morphogenetic protein 2 (BMP2), several delivery techniques were developed and translated to clinical applications. Implantation of scaffolds containing adsorbed BMP2 showed promising results. However, off-label use of this protein-scaffold combination caused severe complications due to an uncontrolled release of the growth factor, which has to be applied in supraphysiological doses in order to induce bone formation. Here, we propose an alternative strategy that focuses on the covalent immobilization of an engineered BMP2 variant to biocompatible scaffolds. The new BMP2 variant harbors an artificial amino acid with a specific functional group, allowing a site-directed covalent scaffold functionalization. The introduced artificial amino acid does not alter BMP2's bioactivity in vitro. When applied in vivo, the covalently coupled BMP2 variant induces the formation of bone tissue characterized by a structurally different morphology compared to that induced by the same scaffold containing ab-/adsorbed wild-type BMP2. Our results clearly show that this innovative technique comprises translational potential for the development of novel osteoinductive materials, improving safety for patients and reducing costs.
Asunto(s)
Proteína Morfogenética Ósea 2 , Sustitutos de Huesos , Aminoácidos , Proteína Morfogenética Ósea 2/farmacología , Regeneración Ósea , Colágeno , Humanos , Microesferas , Osteogénesis/genética , Andamios del Tejido/químicaRESUMEN
The self-assembly of block copolymers has captured the interest of scientists for many decades because it can induce ordered structures and help to imitate complex structures found in nature. In contrast to proteins, nature's most functional hierarchical structures, conventional polymers are disperse in their length distribution. Here, we synthesized hydrophilic and hydrophobic polypeptoids via solid-phase synthesis (uniform) and ring-opening polymerization (disperse). Differential scanning calorimetry measurements showed that the uniform hydrophobic peptoids converge to a maximum of the melting temperature at a much lower chain length than their disperse analogs, showing that not only the chain length but also the dispersity has a considerable impact on the thermal properties of those homopolymers. These homopolymers were then coupled to yield amphiphilic block copolypeptoids. SAXS and AFM measurements confirm that the dispersity plays a major role in microphase separation of these macromolecules, and it appears that uniform hydrophobic blocks form more ordered structures.
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Peptoides/química , Rastreo Diferencial de Calorimetría , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía de Fuerza Atómica , Peptoides/síntesis química , Polimerizacion , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Transforming growth factor-ß (TGF-ß) family members, which include TGF-ßs, activins and bone morphogenetic proteins, are pleiotropic cytokines that elicit cell type-specific effects in a highly context-dependent manner in many different tissues. These secreted protein ligands signal via single-transmembrane Type I and Type II serine/threonine kinase receptors and intracellular SMAD transcription factors. Deregulation in signaling has been implicated in a broad array of diseases, and implicate the need for intricate fine tuning in cellular signaling responses. One important emerging mechanism by which TGF-ß family receptor signaling intensity, duration, specificity and diversity are regulated and/or mediated is through cell surface co-receptors. Here, we provide an overview of the co-receptors that have been identified for TGF-ß family members. While some appear to be specific to TGF-ß family members, others are shared with other pathways and provide possible ways for signal integration. This review focuses on novel functions of TGF-ß family co-receptors, which continue to be discovered.
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Activinas/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Activinas/química , Animales , Proteínas Morfogenéticas Óseas/química , Humanos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Receptores de Factores de Crecimiento Transformadores beta/química , Factor de Crecimiento Transformador beta/químicaRESUMEN
Ontogeny of higher organisms as well the regulation of tissue homeostasis in adult individuals requires a fine-balanced interplay of regulating factors that individually trigger the fate of particular cells to either stay undifferentiated or to differentiate towards distinct tissue specific lineages. In some cases, these factors act synergistically to promote certain cellular responses, whereas in other tissues the same factors antagonize each other. However, the molecular basis of this obvious dual signaling activity is still only poorly understood. Bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) are two major signal protein families that have a lot in common: They are both highly preserved between different species, involved in essential cellular functions, and their ligands vastly outnumber their receptors, making extensive signal regulation necessary. In this review we discuss where and how BMP and FGF signaling cross paths. The compiled data reflect that both factors synchronously act in many tissues, and that antagonism and synergism both exist in a context-dependent manner. Therefore, by challenging a generalization of the connection between these two pathways a new chapter in BMP FGF signaling research will be introduced.
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Proteínas Morfogenéticas Óseas/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Animales , Proteínas Morfogenéticas Óseas/genética , Susceptibilidad a Enfermedades , Factores de Crecimiento de Fibroblastos/genética , Humanos , Especificidad de Órganos , Unión ProteicaRESUMEN
Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) in young adults that has serious negative socioeconomic effects. In addition to symptoms caused by CNS pathology, the majority of MS patients frequently exhibit gastrointestinal dysfunction, which was previously either explained by the presence of spinal cord lesions or not directly linked to the autoimmune etiology of the disease. Here, we studied the enteric nervous system (ENS) in a B cell- and antibody-dependent mouse model of MS by immunohistochemistry and electron microscopy at different stages of the disease. ENS degeneration was evident prior to the development of CNS lesions and the onset of neurological deficits in mice. The pathology was antibody mediated and caused a significant decrease in gastrointestinal motility, which was associated with ENS gliosis and neuronal loss. We identified autoantibodies against four potential target antigens derived from enteric glia and/or neurons by immunoprecipitation and mass spectrometry. Antibodies against three of the target antigens were also present in the plasma of MS patients as confirmed by ELISA. The analysis of human colon resectates provided evidence of gliosis and ENS degeneration in MS patients compared to non-MS controls. For the first time, this study establishes a pathomechanistic link between the well-established autoimmune attack on the CNS and ENS pathology in MS, which might provide a paradigm shift in our current understanding of the immunopathogenesis of the disease with broad diagnostic and therapeutic implications.
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Autoanticuerpos/sangre , Enfermedades Gastrointestinales/etiología , Esclerosis Múltiple , Animales , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Sistema Nervioso Entérico/metabolismo , Sistema Nervioso Entérico/patología , Sistema Nervioso Entérico/ultraestructura , Femenino , Adyuvante de Freund/toxicidad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/complicaciones , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Músculo Liso/patología , Músculo Liso/ultraestructura , Proteína Básica de Mielina/inmunología , Proteína Básica de Mielina/metabolismo , Proteína Básica de Mielina/toxicidad , Glicoproteína Mielina-Oligodendrócito/inmunología , Glicoproteína Mielina-Oligodendrócito/toxicidad , Plexo Mientérico/patología , Plexo Mientérico/ultraestructura , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/toxicidad , Tubulina (Proteína)/metabolismoRESUMEN
The regenerative potential of bone is strongly impaired in pathological conditions, such as nonunion fractures. To support bone regeneration various scaffolds have been developed in the past, which have been functionalized with osteogenic growth factors such as bone morphogenetic proteins (BMPs). However, most of them required supra-physiological levels of these proteins leading to burst releases, thereby causing severe side effects. Site-specific, covalent coupling of BMP2 to implant materials might be an optimal strategy in order to overcome these problems. Therefore, we created a BMP-2 variant (BMP2-K3Plk) containing a noncanonical amino acid (propargyl-l-lysine) substitution introduced by genetic code expansion that allows for site-specific and covalent immobilization onto polymeric scaffold materials. To directly compare different coupling strategies, we also produced a BMP2 variant containing an additional cysteine residue (BMP2-A2C) allowing covalent coupling by thioether formation. The BMP2-K3Plk mutant was coupled to functionalized beads by a copper-catalyzed azide-alkyne cycloaddition (CuAAC) either directly or via a short biotin-PEG linker both with high specificity. After exposing the BMP-coated beads to C2C12 cells, ALP expression appeared locally restricted in close proximity to these beads, showing that both coupled BMP2 variants trigger cell differentiation. The advantage of our approach over non-site-directed immobilization techniques is the ability to produce fully defined osteogenic surfaces, allowing for lower BMP2 loads and concomitant higher bioactivities, for example, due to controlled orientation toward BMP2 receptors. Such products might provide superior bone healing capabilities with potential safety advantages as of homogeneous product outcome.
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Proteína Morfogenética Ósea 2/genética , Proteínas Inmovilizadas/química , Andamios del Tejido/química , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Materiales Biocompatibles/química , Proteína Morfogenética Ósea 2/química , Regeneración Ósea/fisiología , Huesos/citología , Huesos/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Clonación Molecular , Humanos , Osteogénesis/fisiología , Polímeros/químicaRESUMEN
Growth and Differentiation Factor 5 (GDF5) is a secreted growth factor that belongs to the Bone Morphogenetic Protein (BMP) family and plays a pivotal role during limb development. GDF5 is a susceptibility gene for osteoarthritis (OA) and mutations in GDF5 are associated with a wide variety of skeletal malformations ranging from complex syndromes such as acromesomelic chondrodysplasias to isolated forms of brachydactylies or multiple synostoses syndrome 2 (SYNS2). Here, we report on a family with an autosomal dominant inherited combination of SYNS2 and additional brachydactyly type A1 (BDA1) caused by a single point mutation in GDF5 (p.W414R). Functional studies, including chondrogenesis assays with primary mesenchymal cells, luciferase reporter gene assays and Surface Plasmon Resonance analysis, of the GDF5(W414R) variant in comparison to other GDF5 mutations associated with isolated BDA1 (p.R399C) or SYNS2 (p.E491K) revealed a dual pathomechanism characterized by a gain- and loss-of-function at the same time. On the one hand insensitivity to the main GDF5 antagonist NOGGIN (NOG) leads to a GDF5 gain of function and subsequent SYNS2 phenotype. Whereas on the other hand, a reduced signaling activity, specifically via the BMP receptor type IA (BMPR1A), is likely responsible for the BDA1 phenotype. These results demonstrate that one mutation in the overlapping interface of antagonist and receptor binding site in GDF5 can lead to a GDF5 variant with pathophysiological relevance for both, BDA1 and SYNS2 development. Consequently, our study assembles another part of the molecular puzzle of how loss and gain of function mutations in GDF5 affect bone development in hands and feet resulting in specific types of brachydactyly and SYNS2. These novel insights into the biology of GDF5 might also provide further clues on the pathophysiology of OA.
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Braquidactilia/genética , Factor 5 de Diferenciación de Crecimiento/genética , Osteoartritis/genética , Sinostosis/genética , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Braquidactilia/fisiopatología , Pollos , Humanos , Ratones , Osteoartritis/fisiopatología , Linaje , Mutación Puntual/genética , Unión Proteica , Transducción de Señal , Sinostosis/fisiopatologíaRESUMEN
BACKGROUND: Bone morphogenetic protein (BMP)-2 and growth and differentiation factor (GDF)-5 are two related transforming growth factor (TGF)-ß family members with important functions in embryonic development and tissue homeostasis. BMP-2 is best known for its osteoinductive properties whereas GDF-5-as evident from its alternative name, cartilage derived morphogenetic protein 1-plays an important role in the formation of cartilage. In spite of these differences both factors signal by binding to the same subset of BMP receptors, raising the question how these different functionalities are generated. The largest difference in receptor binding is observed in the interaction with the type I receptor BMPR-IA. GDF-5, in contrast to BMP-2, shows preferential binding to the isoform BMPR-IB, which is abrogated by a single amino acid (A57R) substitution. The resulting variant, GDF-5 R57A, represents a "BMP-2 mimic" with respect to BMP receptor binding. In this study we thus wanted to analyze whether the two growth factors can induce distinct signals via an identically composed receptor. RESULTS: Unexpectedly and dependent on the cellular context, GDF-5 R57A showed clear differences in its activity compared to BMP-2. In ATDC-5 cells, both ligands induced alkaline phosphatase (ALP) expression with similar potency. But in C2C12 cells, the BMP-2 mimic GDF-5 R57A (and also wild-type GDF-5) clearly antagonized BMP-2-mediated ALP expression, despite signaling in both cell lines occurring solely via BMPR-IA. The BMP-2- antagonizing properties of GDF-5 and GDF-5 R57A could also be observed in vivo when implanting BMP-2 and either one of the two GDF-5 ligands simultaneously at heterotopic sites. CONCLUSIONS: Although comparison of the crystal structures of the GDF-5 R57A:BMPR-IAEC- and BMP-2:BMPR-IAEC complex revealed small ligand-specific differences, these cannot account for the different signaling characteristics because the complexes seem identical in both differently reacting cell lines. We thus predict an additional component, most likely a not yet identified GDF-5-specific co-receptor, which alters the output of the signaling complexes. Hence the presence or absence of this component then switches GDF-5's signaling capabilities to act either similar to BMP-2 or as a BMP-2 antagonist. These findings might shed new light on the role of GDF-5, e.g., in cartilage maintenance and/or limb development in that it might act as an inhibitor of signaling events initiated by other BMPs.
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Proteína Morfogenética Ósea 2/metabolismo , Factor 5 de Diferenciación de Crecimiento/metabolismo , Línea Celular Tumoral , Humanos , Ligandos , Unión Proteica , Conformación Proteica , Transducción de SeñalRESUMEN
Dysregulation of growth and differentiation factor 5 (GDF-5) signalling, a member of the TGF-beta superfamily, is strongly linked to skeletal malformation. GDF-5-mediated signal transduction involves both BMP type I receptors, BMPR-IA and BMPR-IB. However, mutations in either GDF-5 or BMPR-IB lead to similar phenotypes, indicating that in chondrogenesis GDF-5 signalling seems to be exclusively mediated through BMPR-IB. Here, we present structural insights into the GDF-5:BMPR-IB complex revealing how binding specificity for BMPR-IB is generated on a molecular level. In BMPR-IB, a loop within the ligand-binding epitope functions similar to a latch allowing high-affinity binding of GDF-5. In BMPR-IA, this latch is in a closed conformation leading to steric repulsion. The new structural data now provide also a molecular basis of how phenotypically relevant missense mutations in GDF-5 might impair receptor binding and activation.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Factor 5 de Diferenciación de Crecimiento/química , Sitios de Unión , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/química , Línea Celular Tumoral , Cristalografía por Rayos X , Factor 5 de Diferenciación de Crecimiento/metabolismo , Humanos , Modelos Moleculares , Mutación , Conformación Proteica , Sensibilidad y EspecificidadRESUMEN
PURPOSE: Hypertrophic cartilage is an important characteristic of osteoarthritis and can often be found in patients suffering from osteoarthritis. Although the exact pathomechanism remains poorly understood, hypertrophic de-differentiation of chondrocytes also poses a major challenge in the cell-based repair of hyaline cartilage using mesenchymal stromal cells (MSCs). While different members of the transforming growth factor beta (TGF-ß) family have been shown to promote chondrogenesis in MSCs, the transition into a hypertrophic phenotype remains a problem. To further examine this topic we compared the effects of the transcription growth and differentiation factor 5 (GDF-5) and the mutant R57A on in vitro chondrogenesis in MSCs. METHODS: Bone marrow-derived MSCs (BMSCs) were placed in pellet culture and in-cubated in chondrogenic differentiation medium containing R57A, GDF-5 and TGF-ß1 for 21 days. Chondrogenesis was examined histologically, immunohistochemically, through biochemical assays and by RT-qPCR regarding the expression of chondrogenic marker genes. RESULTS: Treatment of BMSCs with R57A led to a dose dependent induction of chondrogenesis in BMSCs. Biochemical assays also showed an elevated glycosaminoglycan (GAG) content and expression of chondrogenic marker genes in corresponding pellets. While treatment with R57A led to superior chondrogenic differentiation compared to treatment with the GDF-5 wild type and similar levels compared to incubation with TGF-ß1, levels of chondrogenic hypertrophy were lower after induction with R57A and the GDF-5 wild type. CONCLUSIONS: R57A is a stronger inducer of chondrogenesis in BMSCs than the GDF-5 wild type while leading to lower levels of chondrogenic hypertrophy in comparison with TGF-ß1.
RESUMEN
Bone Morphogenetic proteins (BMPs) like BMP2 and BMP7 have shown great potential in the treatment of severe bone defects. In recent in vitro studies, BMP9 revealed the highest osteogenic potential compared to other BMPs, possibly due to its unique signaling pathways that differs from other osteogenic BMPs. However, in vivo the bone forming capacity of BMP9-adsorbed scaffolds is not superior to BMP2 or BMP7. In silico analysis of the BMP9 protein sequence revealed that BMP9, in contrast to other osteogenic BMPs such as BMP2, completely lacks so-called heparin binding motifs that enable extracellular matrix (ECM) interactions which in general might be essential for the BMPs' osteogenic function. Therefore, we genetically engineered a new BMP9 variant by adding BMP2-derived heparin binding motifs to the N-terminal segment of BMP9's mature part. The resulting protein (BMP9 HB) showed higher heparin binding affinity than BMP2, similar osteogenic activity in vitro and comparable binding affinities to BMPR-II and ALK1 compared to BMP9. However, remarkable differences were observed when BMP9 HB was adsorbed to collagen scaffolds and implanted subcutaneously in the dorsum of rats, showing a consistent and significant increase in bone volume and density compared to BMP2 and BMP9. Even at 10-fold lower BMP9 HB doses bone tissue formation was observed. This innovative approach of significantly enhancing the osteogenic properties of BMP9 simply by addition of ECM binding motifs, could constitute a valuable replacement to the commonly used BMPs. The possibility to use lower protein doses demonstrates BMP9 HB's high translational potential.
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Despite promising clinical results in osteochondral defect repair, a recently developed bi-layered collagen/collagen-magnesium-hydroxyapatite scaffold has demonstrated less optimal subchondral bone repair. This study aimed to improve the bone repair potential of this scaffold by adsorbing bone morphogenetic protein 2 (BMP-2) and/or platelet-derived growth factor-BB (PDGF-BB) onto said scaffold. The in vitro release kinetics of BMP-2/PDGF-BB demonstrated that PDGF-BB was burst released from the collagen-only layer, whereas BMP-2 was largely retained in both layers. Cell ingrowth was enhanced by BMP-2/PDFG-BB in a bovine osteochondral defect ex vivo model. In an in vivo semi-orthotopic athymic mouse model, adding BMP-2 or PDGF-BB increased tissue repair after four weeks. After eight weeks, most defects were filled with bone tissue. To further investigate the promising effect of BMP-2, a caprine bilateral stifle osteochondral defect model was used where defects were created in weight-bearing femoral condyle and non-weight-bearing trochlear groove locations. After six months, the adsorption of BMP-2 resulted in significantly less bone repair compared with scaffold-only in the femoral condyle defects and a trend to more bone repair in the trochlear groove. Overall, the adsorption of BMP-2 onto a Col/Col-Mg-HAp scaffold reduced bone formation in weight-bearing osteochondral defects, but not in non-weight-bearing osteochondral defects.
RESUMEN
Signaling output of bone morphogenetic proteins (BMPs) is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T) in patients with multiple synostosis syndrome (SYM1) in the BMP-related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP-inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling.
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Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Portadoras/fisiología , Factor 5 de Diferenciación de Crecimiento/genética , Mutación , Animales , Cartílago , Células Cultivadas , Pollos , Retroalimentación Fisiológica , Humanos , Ratones , Sinostosis/genéticaRESUMEN
Screening of various bisquaternary bisnaphthalimides against a variety of human pathogens revealed one compound, designated MT02, with strong inhibitory effects against Gram-positive bacteria. The MICs ranged from 0.31 µg/ml against community-acquired methicillin-resistant Staphylococcus aureus (MRSA) lineage USA300 to 20 µg/ml against Streptococcus pneumoniae. Radioactive whole-cell labeling experiments indicated a strong impact of MT02 on bacterial DNA replication. DNA microarray studies generated a transcriptional signature characterized by stronger expression of genes involved in DNA metabolism, DNA replication, SOS response, and transport of positively charged compounds. Furthermore, surface plasmon resonance and gel retardation experiments demonstrated direct binding of MT02 to DNA in a concentration-dependent, reversible, and non-sequence-specific manner. The data presented suggest that the bisquaternary bisnaphthalimide MT02 exerts anti-Gram-positive activity by binding to DNA and thereby preventing appropriate DNA replication.
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Antibacterianos/farmacología , Proteínas Bacterianas/efectos de los fármacos , Sustancias Intercalantes/farmacología , Antibacterianos/química , Proteínas Bacterianas/genética , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Sustancias Intercalantes/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Staphylococcus aureus/genéticaRESUMEN
Staphylococcus aureus is the most common cause of nosocomial infections. Multiple antibiotic resistance and severe clinical outcomes provide a strong rationale for development of immunoglobulin-based strategies. Traditionally, novel immunological approaches against bacterial pathogens involve antibodies directed against cell surface-exposed virulence-associated epitopes or toxins. In this study, we generated a monoclonal antibody targeting the housekeeping protein IsaA, a suggested soluble lytic transglycosylase of S. aureus, and tested its therapeutic efficacy in two experimental mouse infection models. A murine anti-IsaA antibody of the IgG1 subclass (UK-66P) showed the highest binding affinity in Biacore analysis. This antibody recognized all S. aureus strains tested, including hospital-acquired and community-acquired methicillin-resistant S. aureus strains. Therapeutic efficacy in vivo in mice was analyzed using a central venous catheter-related infection model and a sepsis survival model. In both models, anti-IsaA IgG1 conferred protection against staphylococcal infection. Ex vivo, UK-66P activates professional phagocytes and induces highly microbicidal reactive oxygen metabolites in a dose-dependent manner, resulting in bacterial killing. The study provides proof of concept that monoclonal IgG1 antibodies with high affinity to the ubiquitously expressed, single-epitope-targeting IsaA are effective in the treatment of staphylococcal infection in different mouse models. Anti-IsaA antibodies might be a useful component in an antibody-based therapeutic for prophylaxis or adjunctive treatment of human cases of S. aureus infections.
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Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/uso terapéutico , Antígenos Bacterianos/inmunología , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Animales , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Masculino , Ratones , Sepsis/tratamiento farmacológico , Sepsis/microbiología , Infecciones Estafilocócicas/microbiologíaRESUMEN
PURPOSE: The biologic relevance of human connective tissue growth factor (hCTGF) for primary human tenon fibroblasts (HTFs) was investigated by RNA expression profiling using affymetrix(TM) oligonucleotide array technology to identify genes that are regulated by hCTGF. METHODS: Recombinant hCTGF was expressed in HEK293T cells and purified by affinity and gel chromatography. Specificity and biologic activity of hCTGF was confirmed by biosensor interaction analysis and proliferation assays. For RNA expression profiling HTFs were stimulated with hCTGF for 48h and analyzed using affymetrix(TM) oligonucleotide array technology. Results were validated by real time RT-PCR. RESULTS: hCTGF induces various groups of genes responsible for a wound healing and inflammatory response in HTFs. A new subset of CTGF inducible inflammatory genes was discovered (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1], chemokine [C-X-C motif] ligand 6 [CXCL6], interleukin 6 [IL6], and interleukin 8 [IL8]). We also identified genes that can transmit the known biologic functions initiated by CTGF such as proliferation and extracellular matrix remodelling. Of special interest is a group of genes, e.g., osteoglycin (OGN) and osteomodulin (OMD), which are known to play a key role in osteoblast biology. CONCLUSIONS: This study specifies the important role of hCTGF for primary tenon fibroblast function. The RNA expression profile yields new insights into the relevance of hCTGF in influencing biologic processes like wound healing, inflammation, proliferation, and extracellular matrix remodelling in vitro via transcriptional regulation of specific genes. The results suggest that CTGF potentially acts as a modulating factor in inflammatory and wound healing response in fibroblasts of the human eye.
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Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Cicatrización de Heridas , Quimiocina CXCL1/biosíntesis , Quimiocina CXCL6/biosíntesis , Proteínas de la Matriz Extracelular/biosíntesis , Humanos , Técnicas In Vitro , Inflamación , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Proteoglicanos/biosíntesis , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The binary ligand-receptor complex of human growth and differentiation factor 5 (GDF5) bound to its type I receptor BMP receptor IA (BRIA) was prepared and crystallized. By utilizing the GDF5 variant R57A, which exhibits a high affinity in the subnanomolar range for BRIA, the binary complex of GDF5R57A bound to the extracellular domain of BRIA could be produced and purified. Crystals of this complex belonged to a monoclinic space group: either I2, with unit-cell parameters a = 63.81, b = 62.85, c = 124.99 Å, ß = 95.9°, or C2, with unit-cell parameters a = 132.17, b = 62.78, c = 63.53 Å, ß = 112.8°.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/química , Factor 5 de Diferenciación de Crecimiento/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/aislamiento & purificación , Cristalización , Cristalografía por Rayos X , Factor 5 de Diferenciación de Crecimiento/genética , Factor 5 de Diferenciación de Crecimiento/aislamiento & purificación , Humanos , Ligandos , Mutación , Unión ProteicaRESUMEN
BACKGROUND: Transforming growth factor (TGF)beta superfamily members transduce signals by oligomerizing two classes of serine/threonine kinase receptors, termed type I and type II. In contrast to the large number of ligands only seven type I and five type II receptors have been identified in mammals, implicating a prominent promiscuity in ligand-receptor interaction. Since a given ligand can usually interact with more than one receptor of either subtype, differences in binding affinities and specificities are likely important for the generation of distinct ligand-receptor complexes with different signaling properties. RESULTS: In vitro interaction analyses showed two different prototypes of binding kinetics, 'slow on/slow off' and 'fast on/fast off'. Surprisingly, the binding specificity of ligands to the receptors of one subtype is only moderate. As suggested from the dimeric nature of the ligands, binding to immobilized receptors shows avidity due to cooperative binding caused by bivalent ligand-receptor interactions. To compare these in vitro observations to the situation in vivo, binding studies on whole cells employing homodimeric as well as heterodimeric bone morphogenetic protein 2 (BMP2) mutants were performed. Interestingly, low and high affinity binding sites were identified, as defined by the presence of either one or two BMP receptor (BMPR)-IA receptor chains, respectively. Both sites contribute to different cellular responses in that the high affinity sites allow a rapid transient response at low ligand concentrations whereas the low affinity sites facilitate sustained signaling but higher ligand concentrations are required. CONCLUSION: Binding of a ligand to a single high affinity receptor chain functioning as anchoring molecule and providing sufficient complex stability allows the subsequent formation of signaling competent complexes. Another receptor of the same subtype, and up to two receptors of the other subtype, can then be recruited. Thus, the resulting receptor arrangement can principally consist of four different receptors, which is consistent with our interaction analysis showing low ligand-receptor specificity within one subtype class. For BMP2, further complexity is added by the fact that heterooligomeric signaling complexes containing only one type I receptor chain can also be found. This indicates that despite prominent ligand receptor promiscuity a manifold of diverse signals might be generated in this receptor limited system.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas/química , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Factor 5 de Diferenciación de Crecimiento/metabolismo , Dominios y Motivos de Interacción de Proteínas/fisiología , Receptores de Activinas/química , Receptores de Activinas/genética , Receptores de Activinas/aislamiento & purificación , Receptores de Activinas/metabolismo , Activinas/química , Activinas/genética , Activinas/aislamiento & purificación , Activinas/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Sitios de Unión , Técnicas Biosensibles , Receptores de Proteínas Morfogenéticas Óseas/genética , Receptores de Proteínas Morfogenéticas Óseas/aislamiento & purificación , Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/aislamiento & purificación , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Ácidos Cólicos/química , Detergentes/química , Factor 5 de Diferenciación de Crecimiento/química , Factor 5 de Diferenciación de Crecimiento/genética , Factor 5 de Diferenciación de Crecimiento/aislamiento & purificación , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Cinética , Ligandos , Modelos Biológicos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica , Isoformas de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Bone morphogenetic protein-2 (BMP-2) and other members of the TGF-beta superfamily regulate the development, maintenance and regeneration of tissues and organs. Binding epitopes for these extracellular signaling proteins have been defined, but hot spots specifying binding affinity and specificity have so far not been identified. In this study, mutational and structural analyses show that epitopes of BMP-2 and the BRIA receptor form a new type of protein-protein interface. The main chain atoms of Leu 51 and Asp53 of BMP-2 represent a hot spot of binding to BRIA. The BMP-2 variant L51P was deficient in type I receptor binding only, whereas its overall structure and its binding to type II receptors and modulator proteins, such as noggin, were unchanged. Thus, the L51P substitution converts BMP-2 into a receptor-inactive inhibitor of noggin. These results are relevant for other proteins of the TGF-beta superfamily and provide useful clues for structure-based drug design.
Asunto(s)
Proteínas Morfogenéticas Óseas/química , Proteínas Serina-Treonina Quinasas/química , Receptores de Factores de Crecimiento/química , Factor de Crecimiento Transformador beta , Animales , Proteína Morfogenética Ósea 2 , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1 , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Portadoras , Línea Celular Tumoral , Cristalografía por Rayos X , Enlace de Hidrógeno , Ratones , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/metabolismo , Receptores de Factores de Crecimiento/metabolismoRESUMEN
The ligand-receptor complex of GDF5 bound to its type I and type II receptors BRIB and ActRIIB was produced and crystallized. Crystals of the GDF5-BRIB complex could only be obtained if a ternary complex comprising GDF5, BRIB and the extracellular domain of the type II receptor ActRIIB was used in crystallization; however, the type II receptor ActRIIB was lost during crystallization. Crystals of this complex belonged to the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 76.46, c = 82.78 A. Small changes in the crystallization condition resulted in crystals with a different morphology. These crystals consisted of the full ternary complex GDF5-BRIB-ActRIIB, but only diffracted to low resolution.