RESUMO
Endostatin (ES) is an antiangiogenic protein that exhibits antitumor activity in animal models. However, the activity observed in animals was not observed in human clinical trials. ES-BAX is a fusion protein composed of two functional domains: ES, which presents specificity and is internalized by activated endothelial cells and the proapoptotic BH3 domain of the protein BAX, a peptide inductor of cellular death when internalized. We have previously shown (Chura-Chambi et al., Cell Death Dis, 5, e1371, 2014) that ES-BAX presents improved antitumor activity in relation to wild-type ES. Secondary and tertiary structures of ES-BAX are similar to ES, as indicated by homology-modeling studies and molecular dynamics simulations. Tryptophan intrinsic fluorescence and circular dichroism spectroscopy corroborate these data. 15 N HSQC NMR indicates that ES-BAX is structured, but some ES residues have suffered chemical shift perturbations, suggesting that the BH3 peptide interacts with some parts of the ES protein. ES and ES-BAX present similar stability to thermal denaturation. The production of stable hybrid proteins can be a new approach to the development of therapeutic agents presenting specificity for tumoral endothelium and improved antitumor effect.
Assuntos
Antineoplásicos/química , Endostatinas/química , Proteínas Recombinantes de Fusão/química , Proteína X Associada a bcl-2/química , Endostatinas/genética , Humanos , Espectroscopia de Ressonância Magnética , Domínios Proteicos , Proteínas Recombinantes de Fusão/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Endostatin is a potent antiangiogenic protein derived from the noncollagenous domain 1 (NC1) of collagen XVIII. The mechanism by which endostatin exerts its antiangiogenic effect is still incompletely understood. It has been shown that the 27 amino acid N-terminal fragment of murine endostatin has antitumor, antimigration, and antipermeability activities comparable to the full soluble protein. To understand how this peptide can exert such elaborate function, we performed structural analysis using molecular dynamics to evaluate the behavior of this fragment in aqueous environment. Here, we show that the N-terminal peptide of murine endostatin is able to assume a well-defined structure, folding into a zinc-dependent ß-hairpin conformation. Analyzing the folding mechanism, we were able to understand why the N-terminal peptide of human endostatin with the same length failed to acquire a stable conformation. Conversely, we were able to predict the successful folding of the R4Q mutant and of a shorter form of the human peptide with 25 residues. Finally, we show that the ß-hairpin conformation assumed by the zinc-bound peptide of murine endostatin has a high structural similarity with fragments of another family of angiogenesis inhibitors: the integrin-binding portion of the NC1 domain of collagen IV. Indeed, our docking simulations show that arresten, canstatin, and the endostatin peptide bind to the same spot of αVß3 integrin, suggesting similar interactions via a common binding site on this receptor.
Assuntos
Endostatinas/química , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Inibidores da Angiogênese/química , Inibidores da Angiogênese/metabolismo , Animais , Sítios de Ligação , Colágeno Tipo IV , Colágeno Tipo XVIII/química , Colágeno Tipo XVIII/metabolismo , Endostatinas/metabolismo , Humanos , Integrina alfaVbeta3/química , Integrina alfaVbeta3/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Eletricidade EstáticaRESUMO
High hydrostatic pressure was used for concomitant solubilization and refolding of insoluble endostatin (ES) aggregated as inclusion bodies (IBs). High hydrostatic pressure (200 MPa or 2 kbar) was applied in combination with nondenaturing concentrations of guanidine hydrochloride. High levels of correctly folded ES (90 mg/L culture) were obtained after optimization/standardization of the procedure by applying pressures of 200 MPa for 16 h in 1.5 M guanidine hydrochloride/0.5 mM oxidized glutathione and reduced glutathione. Refolded ES was purified by affinity chromatography on a heparin column and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, size exclusion HPLC, circular dichroism, and intrinsic fluorescence. We demonstrated that high pressure can successfully convert insoluble IBs of ES expressed in Escherichia coli into an ES preparation with native tertiary structure and full biological activity.
Assuntos
Endostatinas/química , Corpos de Inclusão/metabolismo , Dobramento de Proteína , Animais , Western Blotting , Células CHO , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Cricetinae , Cricetulus , Eletroforese em Gel de Poliacrilamida , Endostatinas/genética , Endostatinas/metabolismo , Endostatinas/farmacologia , Humanos , Pressão Hidrostática , Espectrometria de FluorescênciaRESUMO
Extracellular matrix and soluble plasma proteins generate peptides that regulate biological activities such as cell growth, differentiation and migration. Bradykinin, a peptide released from kininogen by kallikreins, stimulates vasodilatation and endothelial cell proliferation. Various classes of substances can potentiate these biological actions of bradykinin. Among them, the best studied are bradykinin potentiating peptides (BPPs) derived from snake venom, which can also strongly inhibit angiotensin I-converting enzyme (ACE) activity. We identified and synthesized sequences resembling BPPs in the vicinity of potential proteolytic cleavage sites in the collagen XVIII molecule, close to endostatin. These peptides were screened as inhibitors of human recombinant wild-type ACE containing two intact functional domains; two full-length ACE mutants containing only a functional C- or N-domain catalytic site; and human testicular ACE, a natural form of the enzyme that only contains the C-domain. The BPP-like peptides inhibited ACE in the micromolar range and interacted preferentially with the C-domain. The proteolytic activity involved in the release of BPP-like peptides was studied in human serum and human umbilical-vein endothelial cells. The presence of enzymes able to release these peptides in blood led us to speculate on a physiological mechanism for the control of ACE activities.
Assuntos
Colágeno Tipo XVIII/metabolismo , Endostatinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A/metabolismo , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Bradicinina/metabolismo , Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Colágeno Tipo XVIII/química , Colágeno Tipo XVIII/genética , Endostatinas/química , Endostatinas/genética , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Peptídeo Hidrolases/metabolismo , Especificidade por SubstratoRESUMO
Endostatin, a carboxy-terminal fragment of collagen XVIII, has been shown to act as an anti-angiogenic agent that specifically inhibits proliferation of endothelial cells and growth of various primary tumors. Here, we describe the expression by Chinese hamster ovary (CHO) cells of murine endostatin and of a tagged-fusion protein, (his)6-met-endostatin. A dicistronic mRNA expression vector was utilized in which endostatin cDNA was inserted upstream of the amplifiable marker gene, dihydrofolate reductase (DHFR). After transfection of the expression vectors, stepwise increments in methotrexate levels in the culture medium were applied, promoting gene amplification and increasing expression levels of the proteins of interest. The expression level of secreted native endostatin was about 78 microg/mL while the one for secreted (his)6-met-endostatin was about 114 microg/mL, for the best expressing clones. Characterization of physico-chemical and immunological activities of the proteins was performed using SDS-PAGE and Western blotting. The biological activities of recombinant endostatins were tested with a cow pulmonary artery endothelial (C-PAE) cell proliferation assay. Both recombinant endostatin and (his)6-met-endostatin inhibited, in a dose-dependent fashion, growth of C-PAE cells stimulated by basic fibroblast growth factor (bFGF).