ABSTRACT
Interleukin-4 (IL-4) is a hematopoietic cytokine composed by a four-helix bundle stabilized by an antiparallel beta-sheet and three disulfide bonds: Cys3-Cys127, Cys24-Cys65, and Cys46-Cys99. IL-4 is involved in several immune responses associated to infection, allergy, autoimmunity, and cancer. Besides its physiological relevance, IL-4 is often used as a "model" for protein design and engineering. Hence, to understand the role of each disulfide in the structure and dynamics of IL-4, we carried out several spectroscopic analyses (circular dichroism [CD], fluorescence, nuclear magnetic resonance [NMR]), and molecular dynamics (MD) simulations on wild-type IL-4 and four IL-4 disulfide mutants. All disulfide mutants showed loss of structure, altered interhelical angles, and looser core packings, showing that all disulfides are relevant for maintaining the overall fold and stability of the four-helix bundle motif, even at very low pH. In the absence of the disulfide connecting both protein termini Cys3-Cys127, C3T-IL4 showed a less packed protein core, loss of secondary structure (~9%) and fast motions on the sub-nanosecond time scale (lower S2 order parameters and larger τc correlation time), especially at the two protein termini, loops, beginning of helix A and end of helix D. In the absence of Cys24-Cys65, C24T-IL4 presented shorter alpha-helices (14% loss in helical content), altered interhelical angles, less propensity to form the small anti-parallel beta-sheet and increased dynamics. Simultaneously deprived of two disulfides (Cys3-Cys127 and Cys24-Cys65), IL-4 formed a partially folded "molten globule" with high 8-anilino-1-naphtalenesulphonic acid-binding affinity and considerable loss of secondary structure (~50%decrease), as shown by the far UV-CD, NMR, and MD data.
Subject(s)
Disulfides , Interleukin-4 , Protein Conformation , Interleukin-4/chemistry , Disulfides/chemistry , Protein Structure, Secondary , Magnetic Resonance Spectroscopy , Circular DichroismABSTRACT
The antagonist-specific regulation in tissue engineering constitutes important attempts to achieve an improved and rapid bone regeneration by controlling the natural biological response of the natural body growth factors. L51P is molecularly engineered bone morphogentic protein-2 (BMP-2) variant with a substitution of the 51st leucine with a proline residue. L51P is deficient in BMP receptor binding, but maintains its structure and affinity for inhibitory proteins such as noggin, chordin, and gremlin. These modifications convert the BMP-2 variant L51P into a receptor-inactive inhibitor of BMP antagonists. This current approach may prevent the uncontrolled bone overgrowth using high concentration of BMPs and thus regulates the possible growth factor's high-dose side effects. Exploring of L51P biological functions is required to broad our understanding of BMP mutant biological functions and their potential clinical applications. The progress of L51P researches would hopefully lead to the development of multiple applications for using the L51P in bone and fracture healing disorders.
Subject(s)
Bone Morphogenetic Protein 2/metabolism , Carrier Proteins/metabolism , Mutant Proteins/metabolism , Animals , Bone Morphogenetic Protein Receptors, Type I , Humans , Protein Binding , Signal TransductionABSTRACT
Multiple myeloma (MM) represents a haematological cancer characterized by the pathological hyper proliferation of antibody-producing B-lymphocytes. Patients typically suffer from kidney malfunction and skeletal disorders. In the context of MM, the transforming growth factor ß (TGFß) member Activin A was recently identified as a promoter of both accompanying symptoms. Because studies have shown that bone morphogenetic protein (BMP)-2-mediated activities are counteracted by Activin A, we analysed whether BMP2, which also binds to the Activin A receptors ActRII and ActRIIB but activates the alternative SMAD-1/5/8 pathway, can be used to antagonize Activin A activities, such as in the context of MM. Therefore three BMP2 derivatives were generated with modified binding activities for the type II (ActRIIB) and/or type I receptor (BMPRIA) showing either increased or decreased BMP2 activity. In the context of MM these BMP2 muteins show two functionalities since they act as a) an anti-proliferative/apoptotic agent against neoplastic B-cells, b) as a bone-formation promoting growth factor. The molecular basis of both activities was shown in two different cellular models to clearly rely on the properties of the investigated BMP2 muteins to compete for the binding of Activin A to the Activin type II receptors. The experimental outcome suggests new therapeutic strategies using BMP2 variants in the treatment of MM-related pathologies.
Subject(s)
Antineoplastic Agents/therapeutic use , Bone Morphogenetic Protein 2/metabolism , Multiple Myeloma/drug therapy , HumansABSTRACT
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.
Subject(s)
Bone Morphogenetic Protein 2/metabolism , Growth Differentiation Factor 5/metabolism , Cell Line, Tumor , Humans , Ligands , Protein Binding , Protein Conformation , Signal TransductionABSTRACT
BACKGROUND: Alcoholism affects bone repair and this study evaluated the recombinant human BMP-2 (rhBMP-2)/collagen sponge association aiming to improve the bone healing process. The aim of this study was to investigate the action of alcoholism and its effect on the repair of bone defects (BD) performed on rat calvaria after the application of rhBMP-2, either pure or combined with a collagen matrix, using radiographic, histological and immunohistochemical methods. METHODS: We used 80 rats divided into two groups and these into 4 subgroups, each with a waiting period for sacrifice of four and six weeks after the BD (5mm). The groups were divided into: Veh-X) vehicle+BD, Veh-BMP) water+BD+5µg rhBMP-2, Veh-ACS) water+ BD+absorbable collagen sponge, Veh-BMP/ACS) water+BD+5µg rhBMP-2/absorbable collagen sponge, EtOH-X) ethanol+BD, EtOH-BMP) ethanol+BD+5µgrhBMP-2, EtOH-ACS) ethanol+BD+absorbable collagen sponge, and EtOH-BMP/ACS) ethanol+ BD+5µg of rhBMP-2/ absorbable collagen sponge. RESULTS: Radiographically, it was found that after six weeks, for the groups treated with rhBMP-2, independent of the carrier use and ethanol administration, there was more new bone formation (p<0.05). For immunohistochemical analysis, osteocalcin and bone sialoprotein were found to be predominant in groups treated with rhBMP-2. For quantitative stereology, which aims to calculate the volume of new bone, higher values for the groups treated with rhBMP-2 pure or combined with the carrier were found; but for the groups treated with ethanol, a higher bone formation in the groups treated with rhBMP-2 associated with the carrier in the periods of four and six weeks (p<0.001) was found. CONCLUSION: It was concluded that the carrier was effective for rhBMP-2 delivery, even in the presence of ethanol.
Subject(s)
Alcoholism/complications , Bone Diseases/drug therapy , Bone Morphogenetic Protein 2/pharmacology , Collagen/chemistry , Transforming Growth Factor beta/pharmacology , Animals , Bone Diseases/pathology , Disease Models, Animal , Drug Carriers/chemistry , Drug Delivery Systems , Ethanol/administration & dosage , Humans , Integrin-Binding Sialoprotein/metabolism , Male , Osteocalcin/metabolism , Osteogenesis/drug effects , Rats , Rats, Wistar , Recombinant Proteins/pharmacology , Time FactorsABSTRACT
Bone morphogenetic protein 2 (BMP2) is a potent osteoinductive cytokine that plays crucial roles in bone repair. However, large amounts of BMP2 are required to induce sufficient bone formation in humans possibly due to a feedback response of BMP antagonists. The engineered BMP2 variant L51P is deficient in BMP receptor type I activation but maintains affinity for BMP antagonists and can allow for the inactivation of BMP antagonists, and eventually enhance BMP2 action. As hypothesized, simultaneous addition of L51P enhanced the BMP2-induced osteogenesis. To test the ability of L51P to competitively inactivate BMP antagonists, cell binding affinity of BMP2 ligands was investigated in the presence or absence of L51P. Because the BMP antagonists were highly expressed 3 days after exogenous BMP2 stimulation, we collected supernatants from 3-day stimulated cell cultures and used as condition culture media (CM). The results showed a significant decrease in the cell binding of BMP2 ligands when cells were incubated with exogenous BMP2 and CM, whereas L51P addition competitively rescued the suppression of BMP2-to-cell binding induced by CM incubation. In a delayed experimental model, L51P was applied 3 days after exogenous BMP2 stimulation and we could observe a striking enhancement of the BMP2-induced SMAD-1/5/8 phosphorylation and luciferase activity of the Id1 promoter compared to the simultaneous addition of the two factors. These findings provide a deeper insight into the cellular and molecular mechanisms involved in the effect of L51P in suppressing the BMP antagonists and enhancing BMP activity. Additionally, these results demonstrate that L51P is a promising down regulator of BMP-induced negative feedback, which could have a significant impact in future applications of BMP2 in research and clinical settings.
Subject(s)
Bone Morphogenetic Protein 2/antagonists & inhibitors , Osteogenesis/drug effects , Animals , Blotting, Western , Bone Morphogenetic Protein 2/metabolism , Cell Line , Feedback, Physiological/drug effects , Female , Mice , Rats , Rats, Wistar , Real-Time Polymerase Chain Reaction , Recombinant Proteins/pharmacology , X-Ray MicrotomographyABSTRACT
IL-4 signaling into a cell occurs via assembly of a receptor complex that consists of a high-affinity IL-4Rα chain and a low affinity chain, where the low-affinity chain is either γc or IL-13Rα1. It has been previously shown that mutational disruption of the low affinity interface in the IL-4DM (double mutein) yields an antagonist that inhibits IL-4 as well as IL-13-dependent responses. The present study reveals that new types of IL-4 antagonists can be generated by site-specific chemical modification. The chemically modified IL-4 analogues consist of (1) mixed disulfides created by refolding IL-4 cysteine muteins in the presence of different thiol compounds or (2) maleimide conjugates created by modifying cysteine muteins with maleimide derivatives. IL-4 analogues chemically modified at position 121 retain marginal binding affinity to γc or IL-13Rα1 receptor ectodomains during SPR interaction analysis. The biological activity of the analogues is strongly reduced in HEK-Blue IL-4/IL-13 cells as well as in Jurkat cells. Since the IL-4 analogues modified at position 121 have the ability to inhibit γc (IL-4)- and IL13Rα1 (IL-4/IL-13)-dependent responses in Jurkat and HEK-Blue cell lines, they effectively act as IL-4 antagonists. The results of our IL-4 study provide the first example of a cytokine that is transformed into a competitive inhibitor by site-specific chemical modification.
Subject(s)
Interleukin-13/antagonists & inhibitors , Interleukin-4/analogs & derivatives , Interleukin-4/antagonists & inhibitors , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Interleukin-4/chemistry , Jurkat Cells , Models, Molecular , Signal Transduction/drug effects , Structure-Activity RelationshipABSTRACT
Bone Morphogenetic Proteins (BMPs) are secreted protein hormones that act as morphogens and exert essential roles during embryonic development of tissues and organs. Signaling by BMPs occurs via hetero-oligomerization of two types of serine/threonine kinase transmembrane receptors. Due to the small number of available receptors for a large number of BMP ligands ligand-receptor promiscuity presents an evident problem requiring additional regulatory mechanisms for ligand-specific signaling. Such additional regulation is achieved through a plethora of extracellular antagonists, among them members of the Chordin superfamily, that modulate BMP signaling activity by binding. The key-element in Chordin-related antagonists for interacting with BMPs is the von Willebrand type C (VWC) module, which is a small domain of about 50 to 60 residues occurring in many different proteins. Although a structure of the VWC domain of the Chordin-member Crossveinless 2 (CV2) bound to BMP-2 has been determined by X-ray crystallography, the molecular mechanism by which the VWC domain binds BMPs has remained unclear. Here we present the NMR structure of the Danio rerio CV2 VWC1 domain in its unbound state showing that the key features for high affinity binding to BMP-2 is a pre-oriented peptide loop.
Subject(s)
GTPase-Activating Proteins/chemistry , Zebrafish Proteins/chemistry , Zebrafish , Amino Acid Sequence , Amino Acid Substitution , Animals , Bone Morphogenetic Protein 2/chemistry , Conserved Sequence , Cystine/chemistry , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/metabolism , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Folding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
OBJECTIVE: Escherichia coli-derived recombinant human bone morphogenetic protein-2 (E-BMP-2) has been shown to be as effective as mammalian cell-derived BMP-2. However, several in vitro and in vivo experiments are still necessary to validate the effectiveness of E-BMP-2 due to the difference in synthesis process, mainly related to protein nonglycosylation. The objective of this study was to investigate whether biodegradable polylactide-co-glycolide (PLGA) membrane is a suitable carrier for E-BMP-2 delivery for bone regeneration of critical-sized defects in rat calvaria. MATERIALS AND METHODS: First, the osteoinductive effect of E-BMP-2 was confirmed in vitro in mouse bone marrow stromal cells by analysis of osteocalcin mRNA levels, and calcium deposition was detected by alizarin red staining. Before in vivo experiments, the release profile of E-BMP-2 from PLGA membranes was determined by ELISA. E-BMP-2 (0, 1, 5 and 10 µg/µl) was applied for ectopic and orthotopic bone formation and was analyzed by X-ray, micro-CT and histology. RESULTS: Release-profile testing showed that PLGA membrane could retain 94% of the initially applied E-BMP-2. Ectopic bone formation assay revealed that combination of E-BMP-2/PLGA membrane strongly induced bone formation. Stronger osteoinductivity with complete repair of critical-sized defects was observed only with PLGA membranes adsorbed with 5 and 10 µg/µl of E-BMP-2, whereas no bone formation was observed in the groups that received no membrane or 0-µg/µl dose of E-BMP-2. CONCLUSION: PLGA membrane was shown to be a suitable carrier for sustained release of E-BMP-2, and the E-BMP-2/PLGA membrane combination was demonstrated to be efficient in bone regeneration in a model of critical-sized defects.
Subject(s)
Bone Morphogenetic Protein 2/administration & dosage , Bone Morphogenetic Protein 2/chemistry , Bone Regeneration/drug effects , Lactic Acid/chemistry , Polyglycolic Acid/chemistry , Skull/physiology , Transforming Growth Factor beta/administration & dosage , Transforming Growth Factor beta/chemistry , Animals , Escherichia coli/genetics , Escherichia coli/metabolism , Female , Humans , Membranes, Artificial , Mice , Polylactic Acid-Polyglycolic Acid Copolymer , Rats , Rats, Wistar , Recombinant Proteins/administration & dosage , Recombinant Proteins/chemistry , Skull/drug effectsABSTRACT
OBJECTIVE: The aim of this work was to analyse qualitatively and quantitatively the newly formed bone after insertion of rhBMP-2 and protein extracted from Hevea brasiliensis (P-1), associated or not with a carrier in critical bone defects created in Wistar rat calvarial bone, using histological and histomorphometrical analyses. MATERIALS AND METHODS: Eighty-four male Wistar rats were used, divided into two groups, according to the period of time until the sacrifice (2 and 6 weeks). Each one of these groups was subdivided into six groups with seven animals each, according to the treatments: (1) 5 µg of pure rhBMP-2, (2) 5 µg of rhBMP-2/monoolein gel, (3) pure monoolein gel, (4) 5 µg of pure P-1, (5) 5 µg of P-1/monoolein gel and (6) critical bone defect controls. The animals were euthanised and the calvarial bone tissue removed for histological and histomorphometrical analyses. RESULT AND CONCLUSION: The results showed an improvement in the bone healing process using the rhBMP-2 protein, associated or not with a material carrier in relation to the other groups, and this process demonstrated to be time dependent.
Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Hevea , Latex/pharmacology , Osteogenesis/drug effects , Plant Preparations/pharmacology , Skull/drug effects , Analysis of Variance , Animals , Drug Carriers , Glycerides/pharmacology , Latex/isolation & purification , Male , Models, Animal , Plant Preparations/isolation & purification , Rats , Rats, Wistar , Skull/cytologyABSTRACT
Bone morphogenetic proteins (BMP) have been used successfully by orthopedic clinicians to augment bone healing. However, these osteoinductive proteins must be applied at high concentrations to induce bone formation. The limited therapeutic efficacy may be due to the local expression of BMP antagonists such as Noggin that neutralize exogenous and endogenous BMPs. If so, inhibiting BMP antagonists may provide an attractive option to augment BMP induced bone formation. The engineered BMP-2 variant L51P is deficient in BMP receptor type I binding, but maintains its affinity for BMP receptor type II and BMP antagonists including Noggin, Chordin and Gremlin. This modification makes L51P a BMP receptor-inactive inhibitor of BMP antagonists. We implanted ß-tricalcium phosphate ceramics loaded with BMP-2 and/or L51P into a critical size defect model in the rat femur to investigate whether the inhibition of BMP antagonist with L51P enhances the therapeutic efficacy of exogenous BMP-2. Our study reveals that L51P reduces the demand of exogenous BMP-2 to induce bone healing markedly, without promoting bone formation directly when applied alone.
Subject(s)
Bone Morphogenetic Protein 2/antagonists & inhibitors , Bone Morphogenetic Protein 2/pharmacology , Femur/drug effects , Femur/pathology , Mutant Proteins/pharmacology , Protein Engineering , Wound Healing/drug effects , Animals , Calcium Phosphates/pharmacology , Female , Femur/diagnostic imaging , Kinetics , Osteogenesis/drug effects , Radiography , Rats , Rats, Wistar , Staining and LabelingABSTRACT
Bone morphogenetic proteins (BMP) have to be applied at high concentrations to stimulate bone healing. The limited therapeutic efficacy may be due to the local presence of BMP antagonists such as Noggin. Thus, inhibiting BMP antagonists is an attractive therapeutic option. We hypothesized that the engineered BMP2 variant L51P stimulates osteoinduction by antagonizing Noggin-mediated inhibition of BMP2. Primary murine osteoblasts (OB) were treated with L51P, BMP2, and Noggin. OB proliferation and differentiation were quantified with XTT and alkaline phosphatase (ALP) assays. BMP receptor dependent intracellular signaling in OB was evaluated with Smad and p38 MAPK phosphorylation assays. BMP2, Noggin, BMP receptor Ia/Ib/II, osteocalcin, and ALP mRNA expressions were analyzed with real-time PCR. L51P stimulated OB differentiation by blocking Noggin mediated inhibition of BMP2. L51P did not induce OB differentiation directly and did not activate BMP receptor dependent intracellular signaling via the Smad pathway. Treatment of OB cultures with BMP2 but not with L51P resulted in an increased expression of ALP, BMP2, and Noggin mRNA. By inhibiting the BMP antagonist Noggin, L51P enhances BMP2 activity and stimulates osteoinduction without exhibiting direct osteoinductive function. Indirect osteoinduction with L51P seems to be advantageous to osteoinduction with BMP2 as BMP2 stimulates the expression of Noggin thereby self-limiting its own osteoinductive activity. Treatment with L51P is the first protein-based approach available to augment BMP2 induced bone regeneration through inhibition of BMP antagonists. The described strategy may help to decrease the amounts of exogenous BMPs currently required to stimulate bone healing.
Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Carrier Proteins/antagonists & inhibitors , Mutant Proteins/pharmacology , Osseointegration/drug effects , Animals , Blotting, Western , Carrier Proteins/metabolism , Carrier Proteins/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , Gene Expression Regulation/drug effects , Intracellular Space/drug effects , Intracellular Space/metabolism , Mice , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoblasts/metabolism , Phosphorylation/drug effects , Signal Transduction/drug effects , Smad Proteins/metabolismABSTRACT
Interleukin-4 (IL-4) is a prototypical regulator protein of the immune system that is crucial for the pathogenesis and maintenance of asthma and other atopic diseases. It, together with IL-13, uses the IL-4 receptor α chain (IL-4Rα) to signal into immune and other cells. An IL-4 mutein acting as a dual IL-4/IL-13 receptor antagonist is in clinical development. Here, it is described how IL-4 muteins containing a single engineered cysteine with a free thiol can be prepared and used for site-specific chemical modification. The muteins were initially expressed in E. coli, refolded, and purified, but not in a fully reduced nonconjugated form. Attempts to reduce the cysteine chemically failed because the native disulfide bonds of IL-4 were also reduced under similar conditions. Therefore, an enzymatic procedure was developed to reduce glutathionylated IL-4 cysteine muteins employing glutaredoxin and reduced glutathione. Cysteine muteins engineered at four different positions around the IL-4Rα binding site were enzymatically reduced at different rates. All muteins were prepared with free thiol in reasonable yield and were modified by N-ethylmaleimide (NEM) or maleimido-PEG. The effect on IL-4Rα binding of cysteine substitution and of the site-specific modification by glutathione, N-ethylmaleimide (NEM), or a branched 2.36 kDa poly(ethylene glycol) (PEG) will be discussed.
Subject(s)
Cysteine/metabolism , Glutathione Reductase/metabolism , Glutathione/metabolism , Interleukin-4/chemistry , Interleukin-4/metabolism , Protein Engineering , Sulfhydryl Compounds/analysis , Cysteine/chemistry , Humans , Models, Molecular , Molecular Structure , Sulfhydryl Compounds/chemistryABSTRACT
This study analyzed the newly formed bone tissue after application of recombinant human BMP-2 (rhBMP-2) and P-1 (extracted from Hevea brasiliensis) proteins, 2 weeks after the creation of a critical bone defect in male Wistar rats treated or not with a low-intensity laser (GaAlAs 780 nm, 60 mW of power, and energy density dose of 30 J/cm(2)). The animals were divided into two major groups: (1) bone defect plus low-intensity laser treatment and (2) bone defect without laser irradiation. The following subgroups were also analyzed: (a) 5 µg of pure rhBMP-2; (b) 5 µg of pure P-1 fraction; (c) 5 µg of rhBMP-2/monoolein gel; (d) 5 µg of P-1 fraction/monoolein gel; (e) pure monoolein gel. Comparisons of the groups receiving laser treatment with those that did not receive laser irradiation show differences in the areas of new bone tissue. The group treated with 5 µg of rhBMP-2 and laser irradiation was not significantly different (P >0.05) than the nonirradiated group that received the same treatment. The irradiated, rhBMP-2/monoolein gel treatment group showed a lower area of bone formation than the nonirradiated, rhBMP-2/gel monoolein treatment group (P < 0.001). The area of new bone tissue in the other nonirradiated and irradiated groups was not significantly different (P > 0.05). Furthermore, the group that received the 5 µg of rhBMP-2 application showed the greatest bone formation. We conclude that the laser treatment did not interfere with the area of new bone tissue growth and that the greatest stimulus for bone formation involved application of the rhBMP-2 protein.
Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Hevea/chemistry , Low-Level Light Therapy , Osteogenesis/drug effects , Parietal Bone/drug effects , Plant Proteins/pharmacology , Transforming Growth Factor beta/pharmacology , Animals , Bone Morphogenetic Protein 2/administration & dosage , Glycerides/administration & dosage , Humans , Immunohistochemistry , Male , Osteogenesis/radiation effects , Parietal Bone/injuries , Plant Proteins/administration & dosage , Rats , Rats, Wistar , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacology , Skull Fractures/drug therapy , Skull Fractures/radiotherapy , Transforming Growth Factor beta/administration & dosageABSTRACT
BACKGROUND: The aim of this work was to study the new bone tissue formation after bone morphogenetic protein type 2 (rhBMP-2) and P-1 application, using 5 and 10 µg of each, combined to a material carrier, in critical bone defects. METHODS: It was used 70 Wistar rats (male, â¼250 g) that were divided in 10 groups with seven animals on each. Groups are the following: critical bone defect only, pure monoolein gel, 5 µg of pure P-1, 5 µg of pure rhBMP-2, 5 µg of P-1/monoolein gel, 5 µg of rhBMP-2/monoolein gel, 10 µg of pure P-1, 10 µg of pure rhBMP-2, 10 µg of P-1/monoolein gel, 10 µg of rhBMP-2/monoolein gel. Animals were sacrificed after 4 weeks of the surgical procedure and the bone samples were submitted to histological, histomorphometrical, and immunohistochemical evaluations. RESULTS: Animals treated with pure P-1 protein, in both situations with 5 µg and 10 µg, had no significant difference (P > 0.05) for new bone formation; other groups treated with 10 µg were statistically significant (P < 0.05) among themselves and when compared with groups in which it was inserted the monoolein gel or critical bone defect only (P < 0.05). In the group involving the 10 µg rhBMP-2/monoolein gel association, it was observed an extensive bone formation, even when compared with the same treatment without the gel carrier. CONCLUSION: Using this experimental animal model, more new bone tissue was found when it was inserted the rhBMP-2, especially when this protein was combined to the vehicle, and this process seems to be dose dependent.
Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Bone Regeneration/drug effects , Latex/chemistry , Plant Proteins/pharmacology , Transforming Growth Factor beta/pharmacology , Animals , Bone Morphogenetic Protein 2/administration & dosage , Dose-Response Relationship, Drug , Drug Carriers/administration & dosage , Glycerides/administration & dosage , Hevea/chemistry , Humans , Immunohistochemistry , Male , Models, Animal , Plant Proteins/administration & dosage , Rats , Rats, Wistar , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacology , Skull/drug effects , Skull/injuries , Transforming Growth Factor beta/administration & dosageABSTRACT
Growth and differentiation factor 5 (GDF5), a member of the bone morphogenetic protein (BMP) family, is essential for cartilage, bone, and joint formation. Antagonists such as noggin counteract BMP signaling by covering the ligand's BMP type I (BMPRI) and type II (BMPRII, ActRII, ActRIIB) interaction sites. The mutation GDF5-S94N is located within the BMPRII interaction site, the so-called knuckle epitope, and was identified in patients suffering from multiple synostoses syndrome (SYNS). SYNS is characterized by progressive symphalangism, carpal/tarsal fusions, deafness and mild facial dysmorphism. Here we present a novel molecular mechanism of a GDF5 mutation affecting chondrogenesis and osteogenesis. GDF5-S94N exhibits impaired binding to BMPRII causing alleviated Smad and non-Smad signaling and reduced chondrogenic differentiation of ATDC5 cells. Surprisingly, chondrogenesis in mouse micromass cultures was strongly enhanced by GDF5-S94N. By using quantitative techniques (SPR, reporter gene assay, ALP assay, qPCR), we uncovered that this gain of function is caused by strongly reduced affinity of GDF5-S94N to the BMP/GDF antagonist noggin and the consequential lack of noggin inhibition. Thus, since noggin is upregulated during chondrogenic differentiation, GDF5-S94N exceeds the GDF5 action, which results in the phenotypic outcome of SYNS. The detailed molecular characterization of GDF5-S94N as a noggin-resistant growth factor illustrates the potential of GDF5 mutants in applications with defined therapeutical needs.
Subject(s)
Epitopes/genetics , Growth Differentiation Factor 5/chemistry , Growth Differentiation Factor 5/genetics , Mutation/genetics , Synostosis/genetics , Amino Acid Sequence , Animals , Bone Morphogenetic Protein Receptors/metabolism , Carrier Proteins/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , Chondrogenesis/drug effects , Enzyme Activation/drug effects , Humans , Immobilized Proteins/pharmacology , Mice , Molecular Sequence Data , Mutant Proteins/metabolism , Myoblasts/drug effects , Myoblasts/metabolism , Myoblasts/pathology , Osteoblasts/drug effects , Osteoblasts/metabolism , Osteoblasts/pathology , Protein Binding/drug effects , Signal Transduction/drug effects , Smad Proteins/metabolism , Syndrome , Synostosis/enzymology , Synostosis/pathology , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Interleukin-5 (IL-5) is the key mediator for the function of eosinophil granulocytes, whose deregulation is characteristic of hypereosinophilic diseases and presumably contributes to allergic asthma. IL-5 signaling involves two transmembrane receptors, IL-5Rα and the common ß chain, which upon formation of the ternary complex activate the JAK/STAT signaling cascade. To investigate the mechanism underlying ligand-receptor recognition, we determined the structure of IL-5 bound to the extracellular domain of IL-5Rα. IL-5 makes contact with all three fibronectin III-like domains of IL-5Rα, with the receptor architecture resembling a wrench. Mutagenesis data provide evidence that this wrench-like architecture is likely preformed. The structure demonstrates that for steric reasons, homodimeric IL-5 can bind only one receptor molecule, even though two equivalent receptor-binding sites exist. In regard to strong efforts being made to develop IL-5 antagonists for treating asthma and hypereosinophilic diseases, the advances in molecular understanding provided by this structure are of greatest value.
Subject(s)
Interleukin-5 Receptor alpha Subunit/chemistry , Interleukin-5/chemistry , Binding Sites , Humans , Interleukin-5/metabolism , Interleukin-5 Receptor alpha Subunit/metabolism , Ligands , Models, Molecular , Protein ConformationABSTRACT
Site-specific PEGylation offers the possibility to modify a therapeutic protein without interfering with its biological activity. Previously, a preferential N-terminal PEGylation has been reported for several proteins when the reaction was performed at acidic pH. In the present study it was explored if acidic pH favors N-terminal PEGylation of bone morphogenetic protein-2 (BMP-2). PEGylation by poly(ethylene glycol) aldehyde (PEG-AL) or poly(ethylene glycol) carboxymethyl succinimidyl ester (PEG-NHS) was performed at moderate acidic pH of 4. Comparing with PEG-NHS, PEG-AL converted more BMP-2 mainly to mono- or di-PEGylated derivatives at much less molar excess and shorter duration. Analysis of Tryptic fragments of the PEGylated derivatives indicated a partial N-terminal PEGylation specificity. PEG-AL exhibited higher specificity than PEG-NHS. UV spectrometry proved that PEGylation improved the solubility of BMP-2 in PBS. Surface plasmon resonance showed that PEGylation decreased the binding of BMP-2 proteins to a type II receptor. Remarkably, mono-PEGylated BMP-2 with PEG-AL showed higher cellular bioactivity than unmodified protein. Higher N-terminal PEGylation specificity correlates with higher receptor binding affinity and cellular activity. In summary, PEGylation of BMP-2 by PEG-AL and PEG-NHS at acidic pH exhibits a partial N-terminal specificity which however might be sufficient for an efficient site-specific PEGylation process.
Subject(s)
Bone Morphogenetic Protein 2/chemistry , Aldehydes/chemistry , Aldehydes/metabolism , Animals , Binding Sites , Biosensing Techniques , Bone Morphogenetic Protein 2/physiology , Bone Morphogenetic Protein Receptors/metabolism , Cell Line , Cytokines/chemistry , Cytokines/physiology , Drug Compounding , Drug Delivery Systems , Excipients/chemistry , Excipients/metabolism , Humans , Hydrogen-Ion Concentration , Mice , Molecular Conformation , Myoblasts , Polyethylene Glycols/chemistry , Polyethylene Glycols/metabolism , Protein Binding , Proteins/metabolism , Solubility , Trypsin/metabolismABSTRACT
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°.