RESUMO
Aminopeptidase N/CD13, a membrane-bound enzyme upregulated in tumor vasculature and involved in angiogenesis, can be used as a receptor for the targeted delivery of drugs to tumors through ligand-directed targeting approaches. We describe a novel peptide ligand (VGCARRYCS, called "G4") that recognizes CD13 with high affinity and selectivity. Enzymological and computational studies showed that G4 is a competitive inhibitor that binds to the catalytic pocket of CD13 through its N-terminal region. Fusing the peptide C-terminus to tumor necrosis factor-alpha (TNF) or coupling it to a biotin/avidin complex causes loss of binding and inhibitory activity against different forms of CD13, including natural or recombinant ectoenzyme and a membrane form expressed by HL60 promyelocytic leukemia cells (likely due to steric hindrance), but not binding to a membrane form of CD13 expressed by endothelial cells (ECs). Furthermore, G4-TNF systemically administered to tumor-bearing mice exerted anticancer effects through a CD13-targeting mechanism, indicating the presence of a CD13 form in tumor vessels with an accessible binding site. Biochemical studies showed that most CD13 molecules expressed on the surface of ECs are catalytically inactive. Other functional assays showed that these molecules can promote endothelial cell adhesion to plates coated with G4-avidin complexes, suggesting that the endothelial form of CD13 can exert catalytically independent biological functions. In conclusion, ECs express a catalytically inactive form of CD13 characterized by an accessible conformation that can be selectively targeted by G4-protein conjugates. This form of CD13 may represent a specific target receptor for ligand-directed targeted delivery of therapeutics to tumors.
Assuntos
Antígenos CD13 , Células Endoteliais , Leucemia Promielocítica Aguda , Animais , Camundongos , Antígenos CD13/antagonistas & inibidores , LigantesRESUMO
Sotos syndrome is an overgrowth syndrome caused by mutations within the functional domains ofNSD1 gene coding for NSD1, a multidomain protein regulating chromatin structure and gene expression. In particular, PHDVC5HCHNSD1 tandem domain, composed by a classical (PHDV) and an atypical (C5HCH) plant homeo-domain (PHD) finger, is target of several pathological missense-mutations. PHDVC5HCHNSD1 is also crucial for NSD1-dependent transcriptional regulation and interacts with the C2HR domain of transcriptional repressor Nizp1 (C2HRNizp1)in vitro To get molecular insights into the mechanisms dictating the patho-physiological relevance of the PHD finger tandem domain, we solved its solution structure and provided a structural rationale for the effects of seven Sotos syndrome point-mutations. To investigate PHDVC5HCHNSD1 role as structural platform for multiple interactions, we characterized its binding to histone H3 peptides and to C2HRNizp1 by ITC and NMR. We observed only very weak electrostatic interactions with histone H3 N-terminal tails, conversely we proved specific binding to C2HRNizp1 We solved C2HRNizp1 solution structure and generated a 3D model of the complex, corroborated by site-directed mutagenesis. We suggest a mechanistic scenario where NSD1 interactions with cofactors such as Nizp1 are impaired by PHDVC5HCHNSD1 pathological mutations, thus impacting on the repression of growth-promoting genes, leading to overgrowth conditions.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Síndrome de Sotos/genética , Animais , Sítios de Ligação , Proteínas de Transporte/metabolismo , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , Camundongos , Modelos Moleculares , Proteínas Nucleares/metabolismo , Mutação Puntual , Estrutura Terciária de ProteínaRESUMO
NGR (asparagine-glycine-arginine) is a tumor vasculature-homing peptide motif widely used for the functionalization of drugs, nanomaterials and imaging compounds for cancer treatment and diagnosis. Unfortunately, this motif has a strong propensity to undergo rapid deamidation. This reaction, which converts NGR into isoDGR, is associated with receptor switching from CD13 to integrins, with potentially important manufacturing, pharmacological and toxicological implications. It is found that glycine N-methylation of NGR-tagged nanocarriers completely prevents asparagine deamidation without impairing CD13 recognition. Studies in animal models have shown that the methylated NGR motif can be exploited for delivering radiolabeled compounds and nanocarriers, such as tumor necrosis factor-α (TNF)-bearing nanogold and liposomal doxorubicin, to tumors with improved selectivity. These findings suggest that this NGR derivative is a stable and efficient tumor-homing ligand that can be used for delivering functional nanomaterials to tumor vasculature.
RESUMO
BACKGROUND: Graph theory is widely used to dissect structural communication in biomolecular systems. Here, graph theory-based approaches were applied to the headpiece of integrins, adhesion cell-surface receptors that transmit signals across the plasma membranes. METHODS: Protein Structure Network (PSN) analysis incorporating dynamic information either from molecular dynamics simulations or from Elastic Network Models was applied to the ß3 domains from integrins αVß3 and αIIbß3 in their apo and ligand-bound states. RESULTS: Closed and open states of the ß headpiece are characterized by distinct allosteric communication pathways involving highly conserved amino acids at the two different α/ß interfaces in the ßI domain, the closed state being prompted to the closed-to-open transition. In the closed state, pure antagonism is associated with the establishment of communication pathways that start from the ligand, pass through the ß1/α3,α4 interface, and end up in the hybrid domain by involving the Y110-Q82 link, which is weakened in the agonist-bound states. CONCLUSIONS: Allosteric communication in integrins relies on highly conserved and functionally relevant amino acid residues. The αßα-sandwich architecture of integrin ßI domain dictates the structural communication between ligand binding site and hybrid domain. Differently from agonists, pure antagonists are directly involved in allosteric communication pathways and exert long-distance strengthening of the ßI/hybrid interface. Release of the structure network in the ligand binding site is associated with the close-to-open transition accompanying the activation process. GENERAL SIGNIFICANCE: The study strengthens the power of graph-based analyses to decipher allosteric communication intrinsic to protein folds and modified by functionally different ligands.
Assuntos
Integrina beta3/química , Sítios de Ligação , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Dobramento de ProteínaRESUMO
Hemojuvelin (HJV), the coreceptor of the BMP-SMAD pathway that up-regulates hepcidin transcription, is a repulsive guidance molecule (RGMc) which undergoes a complex intracellular processing. Following autoproteolysis, it is exported to the cell surface both as a full-length and a heterodimeric protein. In vitro membrane HJV (m-HJV) is cleaved by the transmembrane serine protease TMPRSS6 to attenuate signalling and to inhibit hepcidin expression. In this study, we investigated the number and position of HJV cleavage sites by mutagenizing arginine residues (R), potential TMPRSS6 targets, to alanine (A). We analysed translation and membrane expression of HJV R mutants and the pattern of fragments they release in the culture media in the presence of TMPRSS6. Abnormal fragments were observed for mutants at arginine 121, 176, 218, 288 and 326. Considering that all variants, except HJV(R121A) , lack autoproteolytic activity and some (HJV(R176A) and HJV(R288A) ) are expressed at reduced levels on cell surface, we identified the fragments originating from either full-length or heterodimeric proteins and defined the residues 121 and 326 as the TMPRSS6 cleavage sites in both isoforms. Using the N-terminal FLAG-tagged HJV, we showed that residue 121 is critical also in the rearrangement of the N-terminal heterodimeric HJV. Exploiting the recently reported RGMb crystallographic structure, we generated a model of HJV that was used as input structure for all-atoms molecular dynamics simulation in explicit solvent. As assessed by in silico studies, we concluded that some arginines in the von Willebrand domain appear TMPRSS6 insensitive, likely because of partial protein structure destabilization.
Assuntos
Arginina/metabolismo , Proteínas Ligadas por GPI/metabolismo , Proteínas de Membrana/metabolismo , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Arginina/química , Arginina/genética , Sítios de Ligação/genética , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Células HeLa , Proteína da Hemocromatose , Humanos , Ligação de Hidrogênio , Proteínas de Membrana/genética , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Proteólise , Serina Endopeptidases/genéticaRESUMO
We combined metadynamics, docking and molecular mechanics/generalised born surface area (MM/GBSA) re-scoring methods to investigate the impact of single and multiple N-methylation on a set of RGD cyclopeptides displaying different affinity for integrin αIIbß3. We rationalised the conformational effects induced by N-methylation and its interplay with receptor affinity, obtaining good agreement with experimental data. This approach can be exploited before entering time-consuming and expensive synthesis and binding experiments.
Assuntos
Peptídeos Cíclicos/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Sítios de Ligação , Ligantes , Espectroscopia de Ressonância Magnética , Metilação , Conformação Molecular , Simulação de Dinâmica Molecular , Peptídeos Cíclicos/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismoRESUMO
The recurrent failures in drug discovery campaigns, the asymmetry between the enormous financial investments and the relatively scarce results have fostered the development of strategies based on complementary methods. In this context in recent years the rigid lock-and-key binding concept had to be revisited in favour of a dynamic model of molecular recognition accounting for conformational changes of both the ligand and the receptor. The high level of complexity required by a dynamic description of the processes underlying molecular recognition requires a multidisciplinary investigation approach. In this perspective, the combination of nuclear magnetic resonance spectroscopy with molecular docking, conformational searches along with molecular dynamics simulations has given new insights into the dynamic mechanisms governing ligand receptor interactions, thus giving an enormous contribution to the identification and design of new and effective drugs. Herein a succinct overview on the applications of both NMR and computational methods to the structural and dynamic characterization of ligand-receptor interactions will be presented.
Assuntos
Antineoplásicos/química , Descoberta de Drogas , Integrina alfaVbeta3/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Integrina alfaVbeta3/antagonistas & inibidores , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Método de Monte Carlo , Ligação Proteica , Conformação Proteica , TermodinâmicaRESUMO
Chemokine heterodimers activate or dampen their cognate receptors during inflammation. The CXCL12 chemokine forms with the fully reduced (fr) alarmin HMGB1 a physiologically relevant heterocomplex (frHMGB1â¢CXCL12) that synergically promotes the inflammatory response elicited by the G-protein coupled receptor CXCR4. The molecular details of complex formation were still elusive. Here we show by an integrated structural approach that frHMGB1â¢CXCL12 is a fuzzy heterocomplex. Unlike previous assumptions, frHMGB1 and CXCL12 form a dynamic equimolar assembly, with structured and unstructured frHMGB1 regions recognizing the CXCL12 dimerization surface. We uncover an unexpected role of the acidic intrinsically disordered region (IDR) of HMGB1 in heterocomplex formation and its binding to CXCR4 on the cell surface. Our work shows that the interaction of frHMGB1 with CXCL12 diverges from the classical rigid heterophilic chemokines dimerization. Simultaneous interference with multiple interactions within frHMGB1â¢CXCL12 might offer pharmacological strategies against inflammatory conditions.
Assuntos
Quimiocina CXCL12 , Proteína HMGB1 , Humanos , Quimiocina CXCL12/metabolismo , Proteína HMGB1/metabolismo , Receptores CXCR4/metabolismo , Inflamação , Transdução de SinaisRESUMO
Ain't got that swing(-out): The cyclopeptide isoDGR is emerging as a new αvß3 integrin binding motif. Agreement between the results of computational and biochemical studies reveals that isoDGR-containing cyclopeptides are true αvß3 integrin antagonists that block αvß3 in its inactive conformation (see scheme). isoDGR-based ligands may give αvß3 antagonists without paradoxical effects.
Assuntos
Integrina alfaVbeta3/antagonistas & inibidores , Simulação de Dinâmica Molecular , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Regulação Alostérica , Integrina alfaVbeta3/metabolismo , Modelos Moleculares , Oligopeptídeos/química , Peptídeos Cíclicos/químicaRESUMO
The SYLF domain is an evolutionary conserved protein domain with phosphatidylinositol binding ability, whose three-dimensional structure is unknown. Here, we present the solution structure and the dynamics characterization of the SYLF domain of the bacterial BPSL1445 protein. BPSL1445 is a seroreactive antigen and a diagnostic marker of Burkholderia pseudomallei, the etiological agent of melioidosis, a severe infectious disease in the tropics. The BPSL1445 SYLF domain (BPSL1445-SYLF) consists of a ß-barrel core, with two flexible loops protruding out of the barrel and three helices packing on its surface. Our structure allows for a more precise definition of the boundaries of the SYLF domain compared to the previously reported one and suggests common ancestry with bacterial EipA domains. We also demonstrate by phosphatidyl-inositol phosphate arrays and nuclear magnetic resonance titrations that BPSL1445-SYLF weakly interacts with phosphoinositides, thus supporting lipid binding abilities of this domain also in prokaryotes.
Assuntos
Proteínas de Bactérias/química , Burkholderia pseudomallei/química , Domínios Proteicos , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Dicroísmo Circular , Ressonância Magnética Nuclear Biomolecular , Fosfatidilinositóis/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Espectrofotometria UltravioletaRESUMO
PHD fingers are small chromatin binding domains, that alone or in tandem work as versatile interaction platforms for diversified activities, ranging from the decoding of the modification status of histone tails to the specific recognition of non-histone proteins. They play a crucial role in their host protein as mutations thereof cause several human malignancies. Thus, PHD fingers are starting to be considered as valuable pharmacological targets. While inhibitors or chemical probes of the histone binding activity of PHD fingers are emerging, their druggability as non-histone interaction platform is still unexplored. In the current study, using a computational and experimental pipeline, we provide proof of concept that the tandem PHD finger of Nuclear receptor-binding SET (Su(var)3-9, Enhancer of zeste, Trithorax) domain protein 1 (PHDVC5HCHNSD1) is ligandable. Combining virtual screening of a small subset of the ZINC database (Zinc Drug Database, ZDD, 2924 molecules) to NMR binding assays and ITC measurements, we have identified Mitoxantrone dihydrochloride, Quinacrine dihydrochloride and Chloroquine diphosphate as the first molecules able to bind to PHDVC5HCHNSD1 and to reduce its documented interaction with the Zinc finger domain (C2HRNizp1) of the transcriptional repressor Nizp1 (NSD1-interacting Zn-finger protein). These results pave the way for the design of small molecules with improved effectiveness in inhibiting this finger-finger interaction.
RESUMO
BACKGROUND: Human Sp140 protein is a leukocyte-specific member of the speckled protein (Sp) family (Sp100, Sp110, Sp140, Sp140L), a class of multi-domain nuclear proteins involved in intrinsic immunity and transcriptional regulation. Sp140 regulates macrophage transcriptional program and is implicated in several haematologic malignancies. Little is known about Sp140 structural domains and its post-translational modifications. METHODS: We used mass spectrometry and biochemical experiments to investigate endogenous Sp140 SUMOylation in Burkitt's Lymphoma cells and Sp140 SUMOylation sites in HEK293T cells, FLAG-Sp140 transfected and His6-SUMO-1T95K infected. NMR spectroscopy and in vitro SUMOylation reactions were applied to investigate the role of Sp140 PHD finger in the SUMOylation of the adjacent BRD. RESULTS: Endogenous Sp140 is a SUMO-1 target, whereby FLAG-Sp140 harbors at least 13 SUMOylation sites distributed along the protein sequence, including the BRD. NMR experiments prove direct binding of the SUMO E2 ligase Ubc9 and SUMO-1 to PHD-BRDSp140. In vitro SUMOylation reactions show that the PHDSp140 behaves as SUMO E3 ligase, assisting intramolecular SUMOylation of the adjacent BRD. CONCLUSIONS: Sp140 is multi-SUMOylated and its PHD finger works as versatile protein-protein interaction platform promoting intramolecular SUMOylation of the adjacent BRD. Thus, combinatorial association of Sp140 chromatin binding domains generates a multifaceted interaction scaffold, whose function goes beyond the canonical histone recognition. GENERAL SIGNIFICANCE: The addition of Sp140 to the increasing lists of multi-SUMOylated proteins opens new perspectives for molecular studies on Sp140 transcriptional activity, where SUMOylation could represent a regulatory route and a docking surface for the recruitment and assembly of leukocyte-specific transcription regulators.
Assuntos
Antígenos Nucleares/química , Antígenos Nucleares/metabolismo , Dedos de Zinco PHD , Domínios Proteicos , Sumoilação , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Células HEK293 , Humanos , Ligação ProteicaRESUMO
Combining 2D STD-NMR, computation, biochemical assays and click-chemistry, we have identified a chromogranin-A derived compound (5) that has high affinity and bi-selectivity for αvß6 and αvß8 integrins and is stable in microsomal preparations. 5 is suitable for nanoparticle functionalization and delivery to cancer cells, holding promise for diagnostic and/or therapeutic applications.
Assuntos
Antígenos de Neoplasias/metabolismo , Cromogranina A/química , Integrinas/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Integrinas/antagonistas & inibidores , Ligantes , Microscopia de Fluorescência , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Ligação ProteicaRESUMO
Influenza A viruses (IAVs) can cause zoonotic infections with pandemic potential when most of the human population is immunologically naive. After a pandemic, IAVs evolve to become seasonal in the human host by acquiring adaptive mutations. We have previously reported that the interferon (IFN)-inducible tripartite motif 22 (TRIM22) protein restricts the replication of seasonal IAVs by direct interaction with the viral nucleoprotein (NP), leading to its polyubiquitination and proteasomal degradation. Here we show that, in contrast to seasonal H1N1 IAVs, the 2009 pandemic H1N1 strain as well as H1N1 strains from the 1930s are resistant to TRIM22 restriction. We demonstrate that arginine-to-lysine substitutions conferring an increased sensitivity to TRIM22-dependent ubiquitination accumulated progressively in the NP of seasonal influenza A (H1N1) viruses between 1918 and 2009. Our findings suggest that during long-term circulation and evolution of IAVs in humans, adaptive mutations are favored at the expense of an increased sensitivity to some components of the innate immune response.IMPORTANCE We have uncovered that long-term circulation of seasonal influenza A viruses (IAV) in the human population resulted in the progressive acquisition of increased sensitivity to a component of the innate immune response: the type I interferon-inducible TRIM22 protein, which acts as a restriction factor by inducing the polyubiquitination of the IAV nucleoprotein (NP). We show that four arginine residues present in the NP of the 1918 H1N1 pandemic strain and early postpandemic strains were progressively substituted for by lysines between 1918 and 2009, rendering NP more susceptible to TRIM22-mediated ubiquitination. Our observations suggest that during long-term evolution of IAVs in humans, variants endowed with increased susceptibility to TRIM22 restriction emerge, highlighting the complexity of selection pressures acting on the NP.
Assuntos
Evolução Molecular , Vírus da Influenza A Subtipo H1N1/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Mutação , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Proteínas do Core Viral/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Arginina/genética , Cães , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Influenza Humana/virologia , Lisina/genética , Células Madin Darby de Rim Canino , Mutagênese Sítio-Dirigida , Proteínas do Nucleocapsídeo , Estrutura Terciária de Proteína , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Replicação ViralRESUMO
Upon engagement of tyrosine kinase receptors, nicotinamide adenine dinucleotide phosphate (NADPH)-oxidases release H2O2 in the extracellular space. We reported previously that aquaporin-8 (AQP8) transports H2O2 across the plasma membrane and is reversibly gated during cell stress, modulating signal strength and duration. We show that AQP8 gating is mediated by persulfidation of cysteine 53 (C53). Treatment with H2S is sufficient to block H2O2 entry in unstressed cells. Silencing cystathionine ß-synthase (CBS) prevents closure, suggesting that this enzyme is the main source of H2S. Molecular modeling indicates that C53 persulfidation displaces a nearby histidine located in the narrowest part of the channel. We propose that H2O2 molecules transported through AQP8 sulfenylate C53, making it susceptible to H2S produced by CBS. This mechanism tunes H2O2 transport and may control signaling and limit oxidative stress.
Assuntos
Aquaporinas/metabolismo , Sulfetos/metabolismo , Sequência de Aminoácidos , Aquaporinas/química , Transporte Biológico , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Peróxido de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/metabolismo , Modelos Biológicos , Conformação Molecular , Oxirredução , Estresse Fisiológico , Sulfetos/químicaRESUMO
The isoDGR sequence is an integrin-binding motif that has been successfully employed as a tumor-vasculature-homing molecule or for the targeted delivery of drugs and diagnostic agents to tumors. In this context, we previously demonstrated that cyclopeptide 2, the product of the conjugation of c(CGisoDGRG) (1) to 4-( N-maleimidomethyl)cyclohexane-1-carboxamide, can be successfully used as a tumor-homing ligand for nanodrug delivery to neoplastic tissues. Here, combining NMR, computational, and biochemical methods, we show that the succinimide ring contained in 2 contributes to stabilizing interactions with αvß3, an integrin overexpressed in the tumor vasculature. Furthermore, we demonstrate that various cyclopeptides containing the isoDGR sequence embedded in different molecular scaffolds do not induce αvß3 allosteric activation and work as pure integrin antagonists. These results could be profitably exploited for the rational design of novel isoDGR-based ligands and tumor-targeting molecules with improved αvß3-binding properties and devoid of adverse integrin-activating effects.
Assuntos
Integrina alfaVbeta3/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Succinimidas/química , Regulação Alostérica , Ligação Competitiva , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células HEK293 , Humanos , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/química , Espectroscopia de Ressonância Magnética , Melanoma/patologia , Simulação de Acoplamento Molecular , Peptídeos Cíclicos/farmacologia , Conformação Proteica , Venenos de Serpentes/farmacologia , Relação Estrutura-Atividade , Tirosina/metabolismoRESUMO
Vascular normalizing strategies, aimed at ameliorating blood vessel perfusion and lessening tissue hypoxia, are treatments that may improve the outcome of cancer patients. Secreted class 3 semaphorins (SEMA3), which are thought to directly bind neuropilin (NRP) co-receptors that, in turn, associate with and elicit plexin (PLXN) receptor signaling, are effective normalizing agents of the cancer vasculature. Yet, SEMA3A was also reported to trigger adverse side effects via NRP1. We rationally designed and generated a safe, parenterally deliverable, and NRP1-independent SEMA3A point mutant isoform that, unlike its wild-type counterpart, binds PLXNA4 with nanomolar affinity and has much greater biochemical and biological activities in cultured endothelial cells. In vivo, when parenterally administered in mouse models of pancreatic cancer, the NRP1-independent SEMA3A point mutant successfully normalized the vasculature, inhibited tumor growth, curbed metastatic dissemination, and effectively improved the supply and anticancer activity of chemotherapy. Mutant SEMA3A also inhibited retinal neovascularization in a mouse model of age-related macular degeneration. In summary, mutant SEMA3A is a vascular normalizing agent that can be exploited to treat cancer and, potentially, other diseases characterized by pathological angiogenesis.