RESUMO
Muscle wasting and cachexia have long been postulated to be key determinants of cancer-related death, but there has been no direct experimental evidence to substantiate this hypothesis. Here, we show that in several cancer cachexia models, pharmacological blockade of ActRIIB pathway not only prevents further muscle wasting but also completely reverses prior loss of skeletal muscle and cancer-induced cardiac atrophy. This treatment dramatically prolongs survival, even of animals in which tumor growth is not inhibited and fat loss and production of proinflammatory cytokines are not reduced. ActRIIB pathway blockade abolished the activation of the ubiquitin-proteasome system and the induction of atrophy-specific ubiquitin ligases in muscles and also markedly stimulated muscle stem cell growth. These findings establish a crucial link between activation of the ActRIIB pathway and the development of cancer cachexia. Thus ActRIIB antagonism is a promising new approach for treating cancer cachexia, whose inhibition per se prolongs survival.
Assuntos
Receptores de Activinas Tipo II/antagonistas & inibidores , Caquexia/tratamento farmacológico , Atrofia Muscular/tratamento farmacológico , Miocárdio/patologia , Neoplasias/complicações , Receptores de Activinas Tipo II/genética , Ativinas/metabolismo , Animais , Anorexia/tratamento farmacológico , Anorexia/etiologia , Atrofia/tratamento farmacológico , Atrofia/etiologia , Caquexia/etiologia , Feminino , Humanos , Inibinas/genética , Inibinas/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/etiologia , Mioblastos/patologia , Transplante de Neoplasias , Neoplasias/mortalidade , Transdução de Sinais , Transplante Heterólogo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Angiopoietin-2 (Ang2) exhibits broad expression in the remodeling vasculature of human tumors but very limited expression in normal tissues, making it an attractive candidate target for antiangiogenic cancer therapy. To investigate the functional consequences of blocking Ang2 activity, we generated antibodies and peptide-Fc fusion proteins that potently and selectively neutralize the interaction between Ang2 and its receptor, Tie2. Systemic treatment of tumor-bearing mice with these Ang2-blocking agents resulted in tumor stasis, followed by elimination of all measurable tumor in a subset of animals. These effects were accompanied by reduced endothelial cell proliferation, consistent with an antiangiogenic therapeutic mechanism. Anti-Ang2 therapy also prevented VEGF-stimulated neovascularization in a rat corneal model of angiogenesis. These results imply that specific Ang2 inhibition may represent an effective antiangiogenic strategy for treating patients with solid tumors.
Assuntos
Angiopoietina-2/antagonistas & inibidores , Angiopoietina-2/imunologia , Anticorpos/farmacologia , Neoplasias/irrigação sanguínea , Neovascularização Patológica/prevenção & controle , Animais , Proliferação de Células/efeitos dos fármacos , Córnea/irrigação sanguínea , Células Endoteliais/efeitos dos fármacos , Feminino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Testes de Neutralização , Receptores Fc , Proteínas Recombinantes de Fusão/farmacologia , Células Tumorais CultivadasRESUMO
The Fc region has two highly conserved methionine residues, Met 33 (C(H)3 domain) and Met 209 (C(H)3 domain), which are important for the Fc's structure and biological function. To understand the effect of methionine oxidation on the structure and stability of the human IgG1 Fc expressed in Escherichia coli, we have characterized the fully oxidized Fc using biophysical (DSC, CD, and NMR) and bioanalytical (SEC and RP-HPLC-MS) methods. Methionine oxidation resulted in a detectable secondary and tertiary structural alteration measured by circular dichroism. This is further supported by the NMR data. The HSQC spectral changes indicate the structures of both C(H)2 and C(H)3 domains are affected by methionine oxidation. The melting temperature (Tm) of the C(H)2 domain of the human IgG1 Fc was significantly reduced upon methionine oxidation, while the melting temperature of the C(H)3 domain was only affected slightly. The change in the C(H)2 domain T m depended on the extent of oxidation of both Met 33 and Met 209. This was confirmed by DSC analysis of methionine-oxidized samples of two site specific methionine mutants. When incubated at 45 degrees C, the oxidized Fc exhibited an increased aggregation rate. In addition, the oxidized Fc displayed an increased deamidation (at pH 7.4) rate at the Asn 67 and Asn 96 sites, both located on the C(H)2 domain, while the deamidation rates of the other residues were not affected. The methionine oxidation resulted in changes in the structure and stability of the Fc, which are primarily localized to the C(H)2 domain. These changes can impact the Fc's physical and covalent stability and potentially its biological functions; therefore, it is critical to monitor and control methionine oxidation during manufacturing and storage of protein therapeutics.
Assuntos
Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/química , Imunoglobulina G/imunologia , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/genética , Cinética , Metionina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Desnaturação Proteica , Estrutura Terciária de Proteína , TemperaturaRESUMO
Structural properties and folding of interleukin-1 receptor antagonist (IL-1ra), a therapeutically important cytokine with a symmetric beta-trefoil topology, are characterized using optical spectroscopy, high-resolution NMR, and size-exclusion chromatography. Spectral contributions of two tryptophan residues, Trp17 and Trp120, present in the wild-type protein, have been determined from mutational analysis. Trp17 dominates the emission spectrum of IL-1ra, while Trp120 is quenched presumably by the nearby cysteine residues in both folded and unfolded states. The same Trp17 gives rise to two characteristic negative peaks in the aromatic CD. Urea denaturation of the wild-type protein is probed by measuring intrinsic and extrinsic (binding of 1-anilinonaphthalene-8-sulfonic acid) fluorescence, near- and far-UV CD, and 1D and 2D ((1)H-(15)N heteronuclear single quantum coherence (HSQC)) NMR. Overall, the data suggest an essentially two-state equilibrium denaturation mechanism with small, but detectable structural changes within the pretransition region. The majority of the (1)H-(15)N HSQC cross-peaks of the folded state show only a limited chemical shift change as a function of the denaturant concentration. However, the amide cross-peak of Leu31 demonstrates a significant urea dependence that can be fitted to a two-state binding model with a dissociation constant of 0.95+/-0.04 M. This interaction has at least a five times higher affinity than reported values for nonspecific urea binding to denatured proteins and peptides, suggesting that the structural context around Leu31 stabilizes the protein-urea interaction. A possible role of denaturant binding in inducing the pretransition changes in IL-1ra is discussed. Urea unfolding of wild-type IL-1ra is sufficiently slow to enable HPLC separation of folded and unfolded states. Quantitative size-exclusion chromatography has provided a hydrodynamic view of the kinetic denaturation process. Thermodynamic stability and unfolding kinetics of IL-1ra resemble those of structurally and evolutionary close IL-1beta, suggesting similarity of their free energy landscapes.
Assuntos
Proteína Antagonista do Receptor de Interleucina 1/química , Naftalenossulfonato de Anilina , Dicroísmo Circular , Corantes Fluorescentes , Humanos , Indicadores e Reagentes , Proteína Antagonista do Receptor de Interleucina 1/genética , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutação , Desnaturação Proteica , Dobramento de Proteína , Termodinâmica , Triptofano/química , UreiaRESUMO
The interactions of brain-derived neurotrophic factor (BDNF) with the extracellular domain of its receptor (trkB) are investigated by employing isotope-edited Fourier transform IR (FTIR) spectroscopy. The protein secondary structures of individual BDNF and trkB in solutions are compared with those in their complex. The temperature dependence of the secondary structures of BDNF, trkB, and their complex is also investigated. Consistent with the crystal structure, we observe by FTIR spectroscopy that BDNF in solution contains predominantly beta strands (approximately 53%) and relatively low contents of other secondary structures including beta turns (approximately 16%), disordered structures (approximately 12%), and loops (approximately 18%) and is deficient in alpha helix. We also observe that trkB in solution contains mostly beta strands (52%) and little alpha helix. Conformational changes in both BDNF and trkB are observed upon complex formation. Specifically, upon binding of BDNF, the conformational changes in trkB appear to involve mostly beta turns and disordered structures while the majority of the beta-strand conformation remains unchanged. The IR data indicate that some of the disordered structures in the loop regions are likely converted to beta strands upon complex formation. The FTIR spectral data of BDNF, trkB, and their complex indicate that more amide NH groups of trkB undergo H-D exchange within the complex than those of the ligand-free receptor and that the thermal stability of trkB is decreased slightly upon binding of BDNF. The FT-Raman spectra of BDNF, trkB, and their complex show that the six intramolecular disulfide bonds of trkB undergo significant conformational changes upon binding of BDNF as a result of changes in the tertiary structure of trkB. Taken together, the FTIR and Raman data are consistent with the loosening of the tertiary structure of trkB upon binding of BDNF, which leads to more solvent exposure of the amide NH group and decreased thermal stability of trkB. This finding reveals an intriguing structural property of the neurotrophin ligand-receptor complex that is in contrast to other ligand-receptor complexes such as a cytokine-receptor complex that usually shows protection of the amide NH group and increased thermal stability upon complex formation.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/química , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Receptor trkB/química , Receptor trkB/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Células CHO , Isótopos de Carbono , Cricetinae , Meios de Cultivo Condicionados , Deutério/química , Dissulfetos/química , Escherichia coli/genética , Humanos , Isótopos de Nitrogênio , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Temperatura , Transfecção , Água/químicaRESUMO
We have identified a novel chordin-like protein, CHL2, which is structurally most homologous to CHL/neuralin/ventroptin. When injected into Xenopus embryos, CHL2 RNA induced a secondary axis. Recombinant CHL2 protein interacted directly with BMPs in a competitive manner to prevent binding to the type I BMP receptor ectodomain, and inhibited BMP-dependent induction of alkaline phosphatase in C2C12 cells. Thus, CHL2 behaves as a secreted BMP-binding inhibitor. In situ hybridization revealed that CHL2 expression is restricted to chondrocytes of various developing joint cartilage surfaces and connective tissues in reproductive organs. Adult mesenchymal progenitor cells expressed CHL2, and its levels decreased during chondrogenic differentiation. Addition of CHL2 protein to a chondrogenic culture system reduced cartilage matrix deposition. Consistently, CHL2 transcripts were weakly detected in normal adult joint cartilage. However, CHL2 expression was upregulated in middle zone chondrocytes in osteoarthritic joint cartilage (where hypertrophic markers are induced). CHL2 depressed chondrocyte mineralization when added during the hypertrophic differentiation of cultured hyaline cartilage particles. Thus, CHL2 may play negative roles in the (re)generation and maturation of articular chondrocytes in the hyaline cartilage of both developing and degenerated joints.
Assuntos
Proteínas de Transporte/genética , Cartilagem/embriologia , Condrócitos/fisiologia , Osteoartrite/genética , Xenopus/embriologia , Fosfatase Alcalina/biossíntese , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Proteínas Morfogenéticas Ósseas/farmacologia , Proteínas de Transporte/metabolismo , Sequência Conservada , Primers do DNA , Embrião não Mamífero/fisiologia , Proteínas da Matriz Extracelular , Feminino , Humanos , Camundongos , Dados de Sequência Molecular , Placenta/fisiologia , Gravidez , Ratos , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Artemin (ART) signals through the GFR alpha-3/RET receptor complex to support sympathetic neuron development. Here we show that ART also influences autonomic elements in adrenal medulla and enteric and pelvic ganglia. Transgenic mice over-expressing Art throughout development exhibited systemic autonomic neural lesions including fusion of adrenal medullae with adjacent paraganglia, adrenal medullary dysplasia, and marked enlargement of sympathetic (superior cervical and sympathetic chain ganglia) and parasympathetic (enteric, pelvic) ganglia. Changes began by gestational day 12.5 and formed progressively larger masses during adulthood. Art supplementation in wild type adult mice by administering recombinant protein or an Art-bearing retroviral vector resulted in hyperplasia or neuronal metaplasia at the adrenal corticomedullary junction. Expression data revealed that Gfr alpha-3 is expressed during development in the adrenal medulla, sensory and autonomic ganglia and their projections, while Art is found in contiguous mesenchymal domains (especially skeleton) and in certain nerves. Intrathecal Art therapy did not reduce hypalgesia in rats following nerve ligation. These data (1) confirm that ART acts as a differentiation factor for autonomic (chiefly sympathoadrenal but also parasympathetic) neurons, (2) suggest a role for ART overexpression in the genesis of pheochromocytomas and paragangliomas, and (3) indicate that ART is not a suitable therapy for peripheral neuropathy.