Antonella Bugatti

Identification of an Antiangiogenic FGF2-binding Site in the N Terminus of the Soluble Pattern Recognition Receptor PTX3

Long-pentraxin 3 (PTX3) is a soluble pattern recognition receptor with non-redundant functions in inflammation and innate immunity. PTX3 comprises a pentraxin-like C-terminal domain involved in complement activation via C1q interaction and an N-terminal extension with unknown functions. PTX3 binds fibroblast growth factor-2 (FGF2), inhibiting its pro-angiogenic and pro-restenotic activity. Here, retroviral transduced endothelial cells (ECs) overexpressing the N-terminal fragment PTX3-(1–178) showed reduced mitogenic activity in response to FGF2. Accordingly, purified recombinant PTX3-(1–178) binds FGF2, prevents PTX3/FGF2 interaction, and inhibits FGF2 mitogenic activity in ECs. Also, the monoclonal antibody mAb-MNB4, which recognizes the PTX3-(87–99) epitope, prevents FGF2/PTX3 interaction and abolishes the FGF2 antagonist activity of PTX3. Consistently, the synthetic peptides PTX3-(82–110) and PTX3-(97–110) bind FGF2 and inhibit the interaction of FGF2 with PTX3 immobilized to a BIAcore sensor chip, FGF2-dependent EC proliferation, and angiogenesis in vivo. Thus, the data identify a FGF2-binding domain in the N-terminal extension of PTX3 spanning the PTX3-(97–110) region, pointing to a novel function for the N-terminal extension of PTX3 and underlining the complexity of the PTX3 molecule for modular humoral pattern recognition.

Pradimicin A, a Carbohydrate-Binding Nonpeptidic Lead Compound for Treatment of Infections with Viruses with Highly Glycosylated Envelopes, Such as Human Immunodeficiency Virus

ABSTRACT Pradimicin A (PRM-A), an antifungal nonpeptidic benzonaphtacenequinone antibiotic, is a low-molecular-weight (molecular weight, 838) carbohydrate binding agent (CBA) endowed with a selective inhibitory activity against human immunodeficiency virus (HIV). It invariably inhibits representative virus strains of a variety of HIV-1 clades with X4 and R5 tropisms at nontoxic concentrations. Time-of-addition studies revealed that PRM-A acts as a true virus entry inhibitor. PRM-A specifically interacts with HIV-1 gp120 and efficiently prevents virus transmission in cocultures of HUT-78/HIV-1 and Sup T1 cells. Upon prolonged exposure of HIV-1-infected CEM cell cultures, PRM-A drug pressure selects for mutant HIV-1 strains containing N-glycosylation site deletions in gp120 but not gp41. A relatively long exposure time to PRM-A is required before drug-resistant virus strains emerge. PRM-A has a high genetic barrier, since more than five N-glycosylation site deletions in gp120 are required to afford moderate drug resistance. Such mutated virus strains keep full sensitivity to the other known clinically used anti-HIV drugs. PRM-A represents the first prototype compound of a nonpeptidic CBA lead and, together with peptide-based lectins, belongs to a conceptually novel type of potential therapeutics for which drug pressure results in the selection of glycan deletions in the HIV gp120 envelope.

HIV-1 matrix protein p17 promotes angiogenesis via chemokine receptors CXCR1 and CXCR2

Vascular diseases supported by aberrant angiogenesis have increased incidence in HIV-1–infected patients. Several data suggest that endothelium dysfunction relies on action of HIV-1 proteins rather than on a direct effect of the virus itself. The HIV-1 matrix protein p17 is known to deregulate the biological activity of different immune cells. Recently, p17 was found to mimic IL-8 chemokine activity by binding to the IL-8 receptor CXCR1. Here we show that p17 binds with high affinity to CXCR2, a CXCR1-related receptor, and promotes the formation of capillary-like structures on human endothelial cells (ECs) by interacting with both CXCR1 and CXCR2 expressed on the EC surface. ERK signaling via Akt was defined as the pathway responsible for p17-induced tube formation. Ex vivo and in vivo experimental models confirmed the provasculogenic activity of p17, which was comparable to that induced by VEGF-A. The hypothesis of a major role for p17 in HIV-1–induced aberrant angiogenesis is enforced by the finding that p17 is detected, as a single protein, in blood vessels of HIV-1–patients and in particular in the nucleus of ECs. Localization of p17 in the nucleus of ECs was evidenced also in in vitro experiments, suggesting the internalization of exogenous p17 in ECs by mechanisms of receptor-mediated endocytosis. Recognizing p17 interaction with CXCR1 and CXCR2 as the key event in sustaining EC aberrant angiogenesis could help us to identify new treatment strategies in combating AIDS-related vascular diseases.

Chemically sulfated Escherichia coli K5 polysaccharide derivatives as extracellular HIV-1 Tat protein antagonists

The HIV‐1 transactivating factor (Tat) acts as an extracellular cytokine on target cells, including endothelium. Here, we report about the Tat‐antagonist capacity of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide. O‐sulfated K5 with high sulfation degree (K5‐OS(H)) and N,O‐sulfated K5 with high (K5‐N,OS(H)) or low (K5‐N,OS(L)) sulfation degree, but not unmodified K5, N‐sulfated K5, and O‐sulfated K5 with low sulfation degree, bind to Tat preventing its interaction with cell surface heparan sulfate proteoglycans, cell internalization, and consequent HIV‐LTR‐transactivation. Also, K5‐OS(H) and K5‐N,OS(H) prevent the interaction of Tat to the vascular endothelial growth factor receptor‐2 on endothelial cell (EC) surface. Finally, K5‐OS(H) inhibits αvβ3 integrin/Tat interaction and EC adhesion to immobilized Tat. Consequently, K5‐OS(H) and K5‐N,OS(H) inhibit the angiogenic activity of Tat in vivo. In conclusion, K5 derivatives with distinct sulfation patterns bind extracellular Tat and modulate its interaction with cell surface receptors and affect its biological activities. These findings provide the basis for the design of novel extracellular Tat antagonists with possible implications in anti‐AIDS therapies.

αvβ3-integrin-dependent activation of focal adhesion kinase mediates NF-κB activation and motogenic activity by HIV-1 Tat in endothelial cells

Once in the extracellular environment, the transactivator protein HIV-1 Tat exerts several pleiotropic effects by interacting with different cellular receptors, including integrin αvβ3. Real-time surface plasmon resonance analysis reveals that Tat/αVβ3 interaction occurs with rapid kinetics (association and dissociation rates equal to 1.16×107 M-1 s-1 and 3.78×10-1 s-1, respectively) and high affinity (dissociation constant = 32 nM). Through this interaction, substratum-immobilized Tat promotes adhesion and motogenic activity in endothelial cells. Also, αvβ3/Tat interaction triggers the activation of focal adhesion kinase, RhoA and pp60src. Overexpression of the dominant negative form of focal adhesion kinase, but not of an inactive Leu1034Ser substitution mutant isoform, impairs the activation of focal adhesion kinase and RhoA, but not that of pp60src, without affecting endothelial cell adhesion and spreading. αvβ3/Tat interaction triggers the activation of NF-κB in endothelial cells in a focal adhesion kinase-, RhoA- and pp60src-dependent manner, as shown in dominant negative focal adhesion kinase transfectants or using specific pharmacological inhibitors. Finally, the activation of focal adhesion kinase, RhoA, NF-κB and pp60src are required to mediate the motogenic activity of Tat in endothelial cells. Since Tat accumulates in an immobilized form in the extracellular matrix, these results provide new biochemical and biological insights about αvβ3/Tat interaction exploitable for the design of anti-Tat strategies.

Heparin/Heparan Sulfate Proteoglycans Glycomic Interactome in Angiogenesis: Biological Implications and Therapeutical Use

Angiogenesis, the process of formation of new blood vessel from pre-existing ones, is involved in various intertwined pathological processes including virus infection, inflammation and oncogenesis, making it a promising target for the development of novel strategies for various interventions. To induce angiogenesis, angiogenic growth factors (AGFs) must interact with pro-angiogenic receptors to induce proliferation, protease production and migration of endothelial cells (ECs). The action of AGFs is counteracted by antiangiogenic modulators whose main mechanism of action is to bind (thus sequestering or masking) AGFs or their receptors. Many sugars, either free or associated to proteins, are involved in these interactions, thus exerting a tight regulation of the neovascularization process. Heparin and heparan sulfate proteoglycans undoubtedly play a pivotal role in this context since they bind to almost all the known AGFs, to several pro-angiogenic receptors and even to angiogenic inhibitors, originating an intricate network of interaction, the so called “angiogenesis glycomic interactome”. The decoding of the angiogenesis glycomic interactome, achievable by a systematic study of the interactions occurring among angiogenic modulators and sugars, may help to design novel antiangiogenic therapies with implications in the cure of angiogenesis-dependent diseases.

Heparin-mimicking sulfonic acid polymers as multitarget inhibitors of human immunodeficiency virus type 1 tat and gp120 proteins

ABSTRACT Human immunodeficiency virus (HIV) Tat and gp120 intriguingly share the feature of being basic peptides that, once released by HIV+ cells, bind to polyanionic heparan sulfate proteoglycans (HSPGs) on target uninfected cells, contributing to the onset of AIDS-associated pathologies. To identify multitarget anti-HIV prodrugs, we investigated the gp120 and Tat antagonist potentials of a series of polyanionic synthetic sulfonic acid polymers (SSAPs). Surface plasmon resonance revealed that SSAPs inhibit with a competitive mechanism of action the binding of Tat and gp120 to surface-immobilized heparin, an experimental condition that resembles binding to cellular HSPGs. Accordingly, SSAPs inhibited HSPG-dependent cell internalization and the transactivating activity of Tat. Little is known about the binding of free gp120 to target cells. Here, we identified two classes of gp120 receptors expressed on endothelial cells, one of which was consistent with an HSPG-binding, low-affinity/high-capacity receptor that is inhibited by free heparin. SSAPs inhibited the binding of free gp120 to endothelial cells, as well as its capacity to induce apoptosis in the same cells. In all the assays, poly(4-styrenesulfonic acid) (PSS) proved to be the most potent antagonist of Tat and gp120. Accordingly, PSS bound both proteins with high affinity. In conclusion, SSAPs represent an interesting class of compounds that bind both gp120 and Tat and inhibit their HSPG-dependent cell surface binding and pathological effects. As these activities contribute to both AIDS progression and associated pathologies, SSAPs can be considered prototypic molecules for the development of multitarget drugs for the treatment of HIV infection and AIDS-associated pathologies.

Identification of a Dendrimeric Heparan Sulfate-Binding Peptide That Inhibits Infectivity of Genital Types of Human Papillomaviruses

ABSTRACT Peptide dendrimers consist of a peptidyl branching core and/or covalently attached surface functional units. They show a variety of biological properties, including antiviral activity. In this study, a minilibrary of linear, dimeric, and dendrimeric peptides containing clusters of basic amino acids was evaluated for in vitro activity against human papillomaviruses (HPVs). The peptide dendrimer SB105-A10 was found to be a potent inhibitor of genital HPV types (i.e., types 16, 18, and 6) in pseudovirus-based neutralization assays. The 50% inhibitory concentration was between 2.8 and 4.2 μg/ml (0.59 and 0.88 μM), and no evidence of cytotoxicity was observed. SB105-A10 interacts with immobilized heparin and with heparan sulfates exposed on the cell surface, most likely preventing virus attachment. The findings from this study indicate SB105-A10 to be a leading candidate compound for further development as an active ingredient of a topical microbicide against HPV and other sexually transmitted viral infections.

Membrane association of peroxiredoxin-2 in red cells is mediated by the N-terminal cytoplasmic domain of band 3

Band 3 (B3), the anion transporter, is an integral membrane protein that plays a key structural role by anchoring the plasma membrane to the spectrin-based membrane skeleton in the red cell. In addition, it also plays a critical role in the assembly of glycolytic enzymes to regulate red cell metabolism. However, its ability to recruit proteins that can prevent membrane oxidation has not been previously explored. In this study, using a variety of experimental approaches including cross-linking studies, fluorescence and dichroic measurements, surface plasmon resonance analysis, and proteolytic digestion assays, we document that the antioxidant protein peroxiredoxin-2 (PRDX2), the third most abundant cytoplasmic protein in RBCs, interacts with the cytoplasmic domain of B3. The surface electrostatic potential analysis and stoichiometry measurements revealed that the N-terminal peptide of B3 is involved in the interaction. PRDX2 underwent a conformational change upon its binding to B3 without losing its peroxidase activity. Hemichrome formation induced by phenylhydrazine of RBCs prevented membrane association of PRDX2, implying overlapping binding sites. Documentation of the absence of binding of PRDX2 to B3 Neapolis red cell membranes, in which the initial N-terminal 11 amino acids are deleted, enabled us to conclude that PRDX2 binds to the N-terminal cytoplasmic domain of B3 and that the first 11 amino acids of this domain are crucial for PRDX2 membrane association in intact RBCs. These findings imply yet another important role for B3 in regulating red cell membrane function.

Fibroblast growth factor 2-antagonist activity of a long-pentraxin 3-derived anti-angiogenic pentapeptide

Fibroblast growth factor‐2 (FGF2) plays a major role in angiogenesis. The pattern recognition receptor long‐pentraxin 3 (PTX3) inhibits the angiogenic activity of FGF2. To identify novel FGF2‐antagonistic peptide(s), four acetylated (Ac) synthetic peptides overlapping the FGF2‐binding region PTX3‐(97–110) were assessed for their FGF2‐binding capacity. Among them, the shortest pentapeptide Ac‐ARPCA‐NH2 (PTX3‐[100–104]) inhibits the interaction of FGF2 with PTX3 immobilized to a BIAcore sensorchip and suppresses FGF2‐dependent proliferation in endothelial cells, without affecting the activity of unrelated mitogens. Also, Ac‐ARPCA‐NH2 inhibits angiogenesis triggered by FGF2 or by tumorigenic FGF2‐overexpressing murine endothelial cells in chick and zebrafish embryos, respectively. Accordingly, the peptide hampers the binding of FGF2 to Chinese Hamster ovary cells overexpressing the tyrosine‐kinase FGF receptor‐1 (FGFR1) and to recombinant FGFR1 immobilized to a BIAcore sensorchip without affecting heparin interaction. In all the assays the mutated Ac‐ARPSA‐NH2 peptide was ineffective. In keeping with the observation that hydrophobic interactions dominate the interface between FGF2 and the FGF‐binding domain of the Ig‐like loop D2 of FGFR1, amino acid substitutions in Ac‐ARPCA‐NH2 and saturation transfer difference‐nuclear magnetic resonance analysis of its mode of interaction with FGF2 implicate the hydrophobic methyl groups of the pentapeptide in FGF2 binding. These results will provide the basis for the design of novel PTX3‐derived anti‐angiogenic FGF2 antagonists.