血管内皮细胞生长因子受体Flt-1结合小肽的融合表达及活性鉴定

血管内皮细胞生长因子 (VEGF)通过结合其酪氨酸激酶受体KDR、fms样酪氨酸激酶 1(Flt 1)调节新生血管形成 ;筛选能封闭VEGF结合Flt 1的小肽 ,可以通过阻断肿瘤血管形成 ,抑制实体瘤生长 .将从噬菌体 12肽库中筛选获得的 2个能与Flt 1结合的阳性噬菌体克隆 (F5 6和F90 )十二肽DNA(36bp)克隆到表达载体pQE4 2中 ,在大肠杆菌M15中稳定表达二氢叶酸还原酶融合蛋白(DHFR F5 6 F90 ) ,经变性、复性后得到纯度达 90 %的可溶性蛋白 .ELISA检测表明 ,DHFR F5 6 F90能结合可溶性受体sFlt 1和血管内皮细胞 ;12 5I VEGF竞争抑制实验显示 ,DHFR F5 6能竞争抑制VEGF同可溶性受体sFlt 1结合 .结果提示 ,F5 6可能是VEGF受体Flt 1的有效拮抗剂 ,具有抗肿瘤新生血管形成的潜在应用前景     

Vascular endothelial growth factor (VEGF) receptor II-derived peptides inhibit VEGF

Vascular endothelial growth factor (VEGF) directly stimulates endothelial cell proliferation and migration via tyrosine kinase receptors of the split kinase domain family. It mediates vascular growth and angiogenesis in the embryo but also in the adult in a variety of physiological and pathological conditions. The potential binding site of VEGF with its receptor was identified using cellulose-bound overlapping peptides of the extracytosolic part of the human vascular endothelial growth factor receptor II (VEGFR II). Thus, a peptide originating from the third globular domain of the VEGFR II comprising residues 247RTELNVGIDFNWEYP261 was revealed as contiguous sequence stretch, which bound 125I-VEGF165. A systematic replacement with L-amino acids within the peptide representing the putative VEGF-binding site on VEGFR II indicates Asp255 as the hydrophilic key residue for binding. The dimerized peptide (RTELNVGIDFNWEYPAS)2K inhibits VEGF165 binding with an IC50 of 0.5 microM on extracellular VEGFR II fragments and 30 microM on human umbilical vein cells. VEGF165-stimulated autophosphorylation of VEGFR II as well as proliferation and migration of microvascular endothelial cells was inhibited by the monomeric peptide RTELNVGIDFNWEYPASK at a half-maximal concentration of 3-10, 0.1, and 0.1 microM, respectively. We conclude that transduction of the VEGF165 signal can be interrupted with a peptide derived from the third Ig-like domain of VEGFR II by blockade of VEGF165 binding to its receptor.     

Tissue Inhibitor of Metalloproteinases-3 Peptides Inhibit Angiogenesis and Choroidal Neovascularization in Mice

Tissue inhibitors of metalloproteinases (TIMPs) while originally characterized as inhibitors of matrix metalloproteinases (MMPs) have recently been shown to have a wide range of functions that are independent of their MMP inhibitory properties. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a potent inhibitor of VEGF-mediated angiogenesis and neovascularization through its ability to block the binding of VEGF to its receptor VEGFR-2. To identify and characterize the anti-angiogenic domain of TIMP-3, structure function analyses and synthetic peptide studies were performed using VEGF-mediated receptor binding, signaling, migration and proliferation. In addition, the ability of TIMP-3 peptides to inhibit CNV in a mouse model was evaluated. We demonstrate that the anti-angiogenic property resides in the COOH-terminal domain of TIMP-3 protein which can block the binding of VEGF specifically to its receptor VEGFR-2, but not to VEGFR-1 similar to the full-length wild-type protein. Synthetic peptides corresponding to putative loop 6 and tail region of TIMP-3 have anti-angiogenic properties as determined by inhibition of VEGF binding to VEGFR-2, VEGF-induced phosphorylation of VEGFR-2 and downstream signaling pathways as well as endothelial cell proliferation and migration in response to VEGF. In addition, we show that intravitreal administration of TIMP-3 peptide could inhibit the size of laser-induced choroidal neovascularization lesions in mice. Thus, we have identified TIMP-3 peptides to be efficient inhibitors of angiogenesis and have a potential to be used therapeutically in diseases with increased neovascularization.     

A novel peptide isolated from a phage display library inhibits tumor growth and metastasis by blocking the binding of vascular endothelial growth factor to its kinase domain receptor

Vascular endothelial growth factor (VEGF), one of the most important angiogenic factors, plays an essential role in both physiological and pathological angiogenesis. The VEGF receptor KDR/Flk-1 (a kinase domain receptor) mediates various biological activities of VEGF related to proliferation, differentiation, and migration of endothelial cells. Here we present a novel peptide designated K237-(HTMYYHHYQHHL), which was isolated from a phage-displayed peptide library, binding to KDR with high affinity and specificity. By interfering with the VEGF-KDR interaction, the peptide K237 inhibited proliferation of cultured primary human umbilical vein endothelial cells induced by recombinant human VEGF(165) in a dose-dependent and cell type-specific manner. The peptide also exerted an anti-angiogenesis activity in vivo as revealed using the chick embryo chorioallantoic membrane angiogenesis assay. Moreover, the peptide K237 significantly inhibited the growth of solid tumors implanted beneath the breasts and their metastases to lungs in severe combined immunodeficient mice. Taken together, these findings suggest that the peptide K237 can functionally disrupt the interaction between VEGF and the KDR receptor and cause potent biological effects that include the inhibition of angiogenesis and tumor growth. As a consequence, this peptide (and its future derivatives) may have use as a potential cancer therapy.     

Vascular endothelial growth factor (VEGF)/VEGF-C mosaic molecules reveal specificity determinants and feature novel receptor binding patterns

Vascular endothelial growth factors (VEGFs) and their receptors play key roles in angiogenesis and lymphangiogenesis. VEGF activates VEGF receptor-1 (VEGFR-1) and VEGFR-2, whereas VEGF-C activates VEGFR-2 and VEGFR-3. We have created a library of VEGF/VEGF-C mosaic molecules that contains factors with novel receptor binding profiles, notably proteins binding to all three VEGF receptors ("super-VEGFs"). The analyzed super-VEGFs show both angiogenic and lymphangiogenic effects in vivo, although weaker than the parental molecules. The composition of the VEGFR-3 binding molecules and scanning mutagenesis revealed determinants of receptor binding and specificity. VEGFR-2 and VEGFR-3 showed striking differences in their requirements for VEGF-C binding; extracellular domain 2 of VEGFR-2 was sufficient, whereas in VEGFR-3, both domains 1 and 2 were necessary.     

A peptide encoded by exon 6 of VEGF (EG3306) inhibits VEGF-induced angiogenesis in vitro and ischaemic retinal neovascularisation in vivo

VEGF is an important mediator of pathological angiogenesis in the eye and is a target for the development of novel anti-angiogenic molecules. In a previous study we identified 12-amino acid peptides derived from exon 6 of VEGF that inhibited VEGF binding to its receptors in HUVECs, endothelial cell functions, and in vitro angiogenesis. Screening of a series of truncated peptides corresponding to the inhibitory region of exon 6 identified a seven amino acid residue peptide, RKRKKSR, as the minimum exon 6-encoded sequence which retains the ability to inhibit VEGF receptor binding and angiogenesis in vitro. The effect of the seven-residue peptide was examined in a mouse model of ischaemic retinal neovascularisation. Administration of the peptide caused a 50% inhibition of retinal neovascularisation and was as effective in inhibiting ischaemic angiogenesis as soluble Flt-1 adenovirus. These results demonstrate that a seven amino acid VEGF exon 6-derived peptide is an effective inhibitor of ocular neovascularisation in vivo, and may have applications in the treatment of pathophysiological ocular neovascularisation in human disease.     

Neuropilin-2 is a receptor for the vascular endothelial growth factor (VEGF) forms VEGF-145 and VEGF-165 [corrected

Neuropilin-1 (np-1) and neuropilin-2 (np-2) are receptors for axon guidance factors belonging to the class 3 semaphorins. np-1 also binds to the 165-amino acid heparin-binding form of VEGF (VEGF(165)) but not to the shorter VEGF(121) form, which lacks a heparin binding ability. We report that human umbilical vein-derived endothelial cells express the a17 and a22 splice forms of the np-2 receptor. Both np-2 forms bind VEGF(165) with high affinity in the presence of heparin (K(D) 1.3 x 10(-10) m) but not VEGF(121). np-2 also binds the heparin-binding form of placenta growth factor. These binding characteristics resemble those of np-1. VEGF(145) is a secreted heparin binding VEGF form that contains the peptide encoded by exon 6 of VEGF but not the peptide encoded by exon 7, which is present in VEGF(165). VEGF(145) binds to np-2 with high affinity (K(D) 7 x 10(-10) m). Surprisingly, VEGF(145) did not bind to np-1. Indeed, VEGF(145) does not bind to MDA-MB-231 breast cancer cells, which predominantly express np-1. By contrast, VEGF(145) binds to human umbilical vein-derived endothelial cells, which express both np-1 and np-2. The binding of VEGF(165) to porcine aortic endothelial cells expressing recombinant np-2 did not affect the proliferation or migration of the cells. Nevertheless, it is possible that VEGF-induced np-2-mediated signaling will take place only in the presence of other VEGF receptors such as VEGF receptor-1 or VEGF receptor-2.     

Homotypic Versican G1 Domain Interactions Enhance Hyaluronan Incorporation into Fibrillin Microfibrils

Versican G1 domain-containing fragments (VG1Fs) have been identified in extracts from the dermis in which hyaluronan (HA)-versican-fibrillin complexes are found. However, the molecular assembly of VG1Fs in the HA-versican-microfibril macrocomplex has not yet been elucidated. Here, we clarify the role of VG1Fs in the extracellular macrocomplex, specifically in mediating the recruitment of HA to microfibrils. Sequential extraction studies suggested that the VG1Fs were not associated with dermal elements through HA binding properties alone. Overlay analyses of dermal tissue sections using the recombinant versican G1 domain, rVN, showed that rVN deposited onto the elastic fiber network. In solid-phase binding assays, rVN bound to isolated nondegraded microfibrils. rVN specifically bound to authentic versican core protein produced by dermal fibroblasts. Furthermore, rVN bound to VG1Fs extracted from the dermis and to nondenatured versican but not to fibrillin-1. Homotypic binding of rVN was also seen. Consistent with these binding properties, macroaggregates containing VG1Fs were detected in high molecular weight fractions of sieved dermal extracts and visualized by electron microscopy, which revealed localization to microfibrils at the microscopic level. Importantly, exogenous rVN enhanced HA recruitment both to isolated microfibrils and to microfibrils in tissue sections in a dose-dependent manner. From these data, we propose that cleaved VG1Fs can be recaptured by microfibrils through VG1F homotypical interactions to enhance HA recruitment to microfibrils.     

A family of leukemia inhibitory factor-binding peptides that can act as antagonists when conjugated to poly(ethylene glycol)

A panel of six na?ve 14-residue random peptide libraries displayed polyvalently on M13 phage was pooled and sorted against human leukemia inhibitory factor (LIF). After four rounds of selection, a single large family of peptides with the consensus sequence XCXXXXG(A/S)(D/E)(W/F)WXCF was found to bind specifically to LIF. Peptides within this family did not bind related members of the interleukin-6 family of cytokines, nor to murine LIF that has 80% sequence identity with human LIF. A representative peptide from this family was synthesized and found to bind to LIF with an affinity of approximately 300 nM. The phage-displayed form of this peptide was able to compete with the LIF receptor alpha chain (LIFR) for binding to LIF; however, the free synthetic peptide was unable to inhibit LIF-LIFR binding or inhibit LIF bioactivity in vitro. Using a panel of human/murine chimeric LIF molecules, the peptide-binding site on LIF was mapped to a groove located between the B and the C helices of the LIF structure, which is distinct from the surfaces involved in binding to receptor. To mimic the effect of the phage particle and convert the free peptide into an antagonist of LIFR binding, a 40 kDa poly(ethylene glycol) (PEG) moiety was conjugated to the synthetic LIF-binding peptide. This PEG-peptide conjugate was found to be both an antagonist of LIF-LIFR binding and of LIF signaling in engineered Ba/F3 cells expressing LIFR and the gp130 coreceptor.     

β-hairpin peptide that targets vascular endothelial growth factor (VEGF) receptors: design, NMR characterization, and biological activity

VEGF receptors have been the target of intense research aimed to develop molecules able to inhibit or stimulate angiogenesis. Based on the x-ray structure of the complex placental growth factor-VEGF receptor 1(D2), we designed a VEGF receptor-binding peptide reproducing the placental growth factor β-hairpin region Gln(87)-Val(100) that is involved in receptor recognition. A conformational analysis showed that the designed peptide adopts the expected fold in pure water. Moreover, a combination of NMR interaction analysis and cell binding studies were used to demonstrate that the peptide targets VEGF receptors. The VEGF receptor 1(D2)-interacting residues were characterized at the molecular level, and they correspond to the residues recognizing the placental growth factor sequence Gln(87)-Val(100). Finally, the peptide biological activity was characterized in vitro and in vivo, and it showed a VEGF-like behavior. Indeed, the peptide activated VEGF-dependent intracellular pathways, induced endothelial cell proliferation and rescue from apoptosis, and promoted angiogenesis in vivo. This compound is one of the few peptides known with proangiogenic activity, which makes it a candidate for the development of a novel peptide-based drug for medical applications in therapeutic angiogenesis.     

Site-directed mutagenesis in the B-neuropilin-2 domain selectively enhances its affinity to VEGF165, but not to semaphorin 3F

Neuropilins (NRPs) are 130-kDa receptors that bind and respond to the class 3 semaphorin family of axon guidance molecules (SEMAs) and to members of the vascular endothelial growth factor (VEGF) family of angiogenic factors. Two NRPs have been reported so far, NRP1 and NRP2. Unlike NRP1, little is known about NRP2 interactions with its ligands, VEGF165 and SEMA3F. Cell binding studies reveal that VEGF165 and SEMA3F bind NRP2 with similar affinities, 5.2 and 3.9 nM, respectively, and are competitive NRP2 ligands. Immunoprecipitation studies show that the B (b1b2) extracellular domain of NRP2 is sufficient for VEGF165 binding, whereas SEMA3F requires both the A (a1a2) and B domains. To identify residues of B-NRP2 involved in VEGF165 binding, point mutations were introduced by site-directed mutagenesis. VEGF165 is a basic protein. Reduction of the electronegative potential of B-NRP2 by exchanging acidic residues for uncharged alanine (B-NRP2 E284A,E291A) in the 280-290 b1-NRP2 loop resulted in a 2-fold reduction in VEGF165 affinity. Conversely, enhancing the electronegative potential (B-NRP2 R287E,N290D and R287E,N290S) significantly increased VEGF165 affinity for B-NRP2 by 8- and 6.6-fold, respectively. The mutagenesis did not affect SEMA3F/B-NRP2 interactions. These results demonstrate that it is possible to alter VEGF165 affinity for NRP2 without affecting SEMA3F affinity. They also identify NRP2 residues involved in VEGF165 binding and suggest that modifications of B-NRP2 could lead to potentially high affinity selective inhibitors of VEGF165/NRP2 interactions.     

Peptide-mediated interference with baculovirus transduction

Baculovirus represents a multifunctional platform with potential for biomedical applications including disease therapies. The importance of F3, a tumor-homing peptide, in baculovirus transduction was previously recognized by the ability of F3 to augment viral binding and gene delivery to human cancer cells following display on the viral envelope. Here, F3 was utilized as a molecular tool to expand understanding of the poorly characterized baculovirus-mammalian cell interactions. Baculovirus-mediated transduction of HepG2 hepatocarcinoma cells was strongly inhibited by coincubating the virus with synthetic F3 or following incorporation of F3 into viral nucleocapsid by genetic engineering, the former suggesting direct interaction of the soluble peptide with the virus particles. Since internalization and nuclear accumulation of the virus were significantly inhibited or delayed, but the kinetics of viral binding, initial uptake, and endosomal release were unaffected, F3 likely interferes with cytoplasmic trafficking and subsequent nuclear transport of the virus. A polyclonal antibody raised against nucleolin, the internalizing receptor of F3, failed to inhibit cellular binding, but considerably reduced viral transduction efficiency, proposing the involvement of nucleolin in baculovirus entry. Together, these results render the F3 peptide a tool for elucidating the mechanism and molecular details conferring to baculovirus-mediated gene transduction in mammalian cells.     

Molecular identification of the human melanocortin-2 receptor responsible for ligand binding and signaling

The melanocortin-2 receptor (MC2R), also known as the adrenocorticotropic hormone (ACTH) receptor, plays an important role in regulating and maintaining adrenocortical function, specifically steroidogenesis. Mutations of the human MC2R (hMC2R) gene have also been identified in humans with familial glucocorticoid deficiency; however, the molecular basis responsible for hMC2R ligand binding and signaling remains unclear. In this study, both truncated ACTH peptides and site-directed mutagenesis studies were used to determine molecular mechanisms of hMC2R binding ACTH and signaling. Our results indicate that ACTH1-16 is the minimal peptide required for hMC2R binding and signaling. Mutations of common melanocortin receptor family amino acid residues E80 in transmembrane domain 2 (TM2), D107 in TM3, F178 in TM4, F235 and H238 in TM6, and F258 in TM7 significantly reduced ACTH-binding affinity and signaling. Furthermore, mutations of unique amino acids D104 and F108 in TM3 and F168 and F178 in TM4 significantly decreased ACTH binding and signaling. In conclusion, our results suggest that the residues in TM2, TM3, and TM6 of hMC2R share similar binding sites with other MCRs but the residues identified in TM4 and TM7 of hMC2R are unique and required for ACTH selectivity. Our study suggests that hMC2R may have a broad binding pocket in which both conserved and unique amino acid residues are required, which may be the reason why alpha-MSH was not able to bind hMC2R.     

Antiangiogenic and antitumor activities of peptide inhibiting the vascular endothelial growth factor binding to neuropilin-1

Neuropilin-1 (NRP-1), a non-tyrosine kinase receptor of vascular endothelial growth factor-165 (VEGF165), was found expressed on endothelial and some tumor cells. Since its overexpression is correlated with tumor angiogenesis and progression, the targeting of NRP-1 could be a potential anti-cancer strategy. To explore this hypothesis, we identified a peptide inhibiting the VEGF165 binding to NRP-1 and we tested whether it was able to inhibit tumor growth and angiogenesis. To prove the target of peptide action, we assessed its effects on binding of radiolabeled VEGF165 to recombinant receptors and to cultured cells expressing only VEGFR-2 (KDR) or NRP-1. Antiangiogenic activity of the peptide was tested in vitro in tubulogenesis assays and in vivo in nude mice xenotransplanted in fat-pad with breast cancer MDA-MB-231 cells. Tumor volumes, vascularity and proliferation indices were determined. The selected peptide, ATWLPPR, inhibited the VEGF165 binding to NRP-1 but not to tyrosine kinase receptors, VEGFR-1 (flt-1) and KDR; nor did it bind to heparin. It diminished the VEGF-induced human umbilical vein endothelial cell proliferation and tubular formation on Matrigel and in co-culture with fibroblasts. Administration of ATWLPPR to nude mice inhibited the growth of MDA-MB-231 xenografts, and reduced blood vessel density and endothelial cell area but did not alter the proliferation indices of the tumor. In conclusion, ATWLPPR, a previously identified KDR-interacting peptide, was shown to inhibit the VEGF165 interactions with NRP-1 but not with KDR and to decrease the tumor angiogenesis and growth, thus validating, in vivo, NRP-1 as a possible target for antiangiogenic and antitumor agents.     

Parallel solid-phase synthesis of a small library of linear and hydrocarbon-bridged analogues of VEGF(81-91): potential biological tools for studying the VEGF/VEGFR-1 interaction

The design, synthesis and binding affinity for VEGFR-1 receptors of a small library of linear and cyclic analogues of the VEGF(81-91) fragment are described. Cyclic 11- and 10-mer peptide derivatives were prepared using parallel solid-phase protocols. The formation of hydrocarbon alkene-bridged cyclic peptides was achieved through optimized ring-closing metathesis reactions from linear derivatives with conveniently located allylGly residues. Alkane-bridged analogues were successfully obtained by ulterior on-resin hydrogenation. Binding assays showed that some of these compounds were able to compete with labeled VEGF for interaction with the VEGFR-1 receptor. Several peptide derivatives, 2, 7 and 8, showed modest but significant binding affinity, indicating that the designed peptide could mimic the VEGF(81-91) fragment and therefore disrupt the VEGF/VEGFR-1 interaction. This fact opens the way for using these peptides as the starting point for biological/pharmacological tools to deeply investigate this protein-protein system.     

Solubilization of active GLP-1 (7–36)amide receptors from RINm5F plasma membranes

Glucagon-like peptide-1 (7–36)amide (GLP-1 (7–36)amide) represents a physiologically important incretin in mammals including man. Receptors for GLP-1 (7–36)amide have been described in RINm5F cells. We have solubilized active GLP-1 (7–36)amide receptors from RINm5F cell membranes utilizing the detergents octyl-β-glucoside and CHAPS; Triton X-100 and Lubrol PX were ineffective. Binding of radiolabeled GLP-1(7–36)amide to the solubilized receptor was inhibited conentration-dependently by addition of unlabeled peptide. Scatchard analysis of binding data revealed a single class of binding sites with K_a= 0.26 ± 0.03 nM and B_max= 65.4 ± 21.24 fmol/mg of protein for the membrane-bound receptor and K_a= 22.54 ± 4.42 μM and B_max= 3.9 ± 0.79 pmol/mg of protein for the solubilized receptor. The binding of the radiolabel to the solubilized receptor was dependent both on the concentrations of mono- and divalent cations and the protein/detergent ratio in the incubation buffer. The membrane bound receptor is sensitive to guanine-nucleotides, however neither GTP-γ-S nor GDP-β-S affected binding or labeled peptide to solubilized receptor. These data indicate that the solubilized receptor may have lost association with its G-protein. In conclusion, the here presented protocol allows solubilization of the GLP-1(7–36)amide receptor in a functional state and opens up the possibility for further molecular characterization of the receptor protein.     

Short PlGF‐derived peptides bind VEGFR‐1 and VEGFR‐2 in vitro and on the surface of endothelial cells

The placental growth factor (PlGF), a member of VEGF family, plays a crucial role in pathological angiogenesis, especially ischemia, inflammation, and cancer. This activity is mediated by its selective binding to VEGF receptor 1 (VEGFR‐1), which occurs predominantly through receptor domains 2 and 3. The PlGF β‐hairpin region spanning residues Q87 to V100 is one of the key binding elements on the protein side. We have undertaken a study on the design, preparation, and functional characterization of the peptide reproducing this region and of a set of analogues where glycine 94, occurring at the corner of the hairpin in the native protein, is replaced by charged as well as hydrophobic residues. Also, some peptides with arginine 96 replaced by other residues have been studied. We find that the parent peptide weakly binds VEGFR‐1, but replacement of G94 with residues bearing H‐bond donating residues significantly improves the affinity. Replacement of R96 instead blocks the interaction between the peptide and the domain. The strongest affinity is observed with the G94H (peptide PlGF‐2) and G94W (peptide PlGF‐10) mutants, while the peptide PlGF‐8, bearing the R96G mutation, is totally inactive. The PlGF‐1 and PlGF‐2 peptides also bind the VEGFR‐2 receptors, though with a reduced affinity, and are able to interfere with the VEGF‐induced receptor signaling on endothelial cells. The peptides also bind VEGFR‐2 on the surface of cells, while PlGF‐8 is inactive. Data suggest that these peptides have potential applications as PlGF/VEGF mimic in various experimental settings.