The Basic Domain in HIV-1 Tat Protein as a Target for Polysulfonated Heparin-mimicking Extracellular Tat Antagonists

Rusnati, Marco; Tulipano, Giovanni; Urbinati, Chiara; Tanghetti, Elena; Giuliani, Roberta; Giacca, Mauro; Ciomei, Marina; Corallini, Alfredo; Presta, Marco

doi:10.1074/jbc.273.26.16027

Cited by 106 publications

(147 citation statements)

References 89 publications

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“…Previously, Rusnati et al showed in a competition assay that cyclodextrin sulfate was not able to displace heparin from immobilized HIV TAT protein even at concentrations as high as 300 M [43]. In agreement with its inability to affect the TAT/ heparin interaction [43,44], cyclodextrin sulfate showed no binding to MCP-1. These data suggest that a linear, flexible backbone structure may be a critical determinant for ligand binding to MCP-1.…”

Section: Filtration Trapping Analysis To Identify Mcp-1/ Small Molecumentioning

confidence: 78%

Potential inhibitors of chemokine function: Analysis of noncovalent complexes of cc chemokine and small polyanionic molecules by ESI FT-ICR mass spectrometry

Sweeney

Saad

et al. 2006

J. Am. Soc. Mass Spectrom.

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Chemokines play a critical role in inducing chemotaxis, extravasation, and activation of leukocytes both in routine immunosurveillance and autoimmune diseases. Traditionally, to disrupt chemokine function, strategies have focused on blockage of its interaction with the receptor. Recently, it has been demonstrated that binding to glycosaminoglycans (GAGs) is also required for the in vivo activity of many chemokines. Thus, interference with the GAG-binding of chemokines may offer an alternative, valid, anti-inflammatory strategy. However, the potential of using small polyanions to inhibit the interactions between chemokines and cell surface GAGs has not been fully explored. In this study, a mass spectrometry based filtration trapping assay was utilized to study the interactions between two CCR 2 ligands (MCP-1/CCL2 and MCP-3/CCL7) and a series of low molecular weight, polyanionic molecules. Findings were confirmed by using a hydrophobic trapping assay. The results indicated that Arixtra (fondaparinux sodium), sucrose octasulfate, and suramin were specific binders of the chemokines, while cyclodextrin sulfate, although the most highly sulfated molecule among the ones investigated, showed no binding. The binding stoichiometry of the small molecule ligand was determined from the measured molecular weight of the noncovalent complex. Furthermore, the dissociation constant between MCP-3 and Arixtra was determined by using electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry, which compared favorably with the result of the isothermal titration calorimetry (ITC) assay. The relative binding affinity of these ligands to MCP-3 was also determined using a competitive filtration trapping assay. (J Am Soc Mass Spectrom 2006, 17, 524 -535)

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Section: Filtration Trapping Analysis To Identify Mcp-1/ Small Molecumentioning

confidence: 78%

Potential inhibitors of chemokine function: Analysis of noncovalent complexes of cc chemokine and small polyanionic molecules by ESI FT-ICR mass spectrometry

Sweeney

Saad

et al. 2006

J. Am. Soc. Mass Spectrom.

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“…In these latter cells, PTX-B inhibits the transactivating activity of endogenously produced Tat as well as of its extracellular form. Relevant to this point, a brief exposure (3 h) of the cells to PTX-B is sufficient to abolish Tat responsiveness, and PTX-B retains its inhibitory effect also when added to cells up to 6 h after Tat administration, when a saturating amount of extracellular Tat is already internalized [35] and classical extracellular Tat antagonists are ineffective [35,36]. Thus, PTX-B acts as a "flexible" Tat antagonist without binding Tat nor interfering with the process of Tat uptake by cells.…”

Section: Discussionmentioning

confidence: 92%

Inhibition of intra‐ and extra‐cellular Tat function and HIV expression by pertussis toxin B‐oligomer

et al. 2004

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HIV‐1‐transactivating factor Tat contributes to virus replication and to the onset of AIDS‐associated pathologies by targeting different infected and uninfected cell types. We previously demonstrated that the B‐oligomer of pertussis toxin (PTX‐B) inhibits HIV infection and replication in primary T cells and macrophages and Tat‐dependent HIV‐1 long terminal repeat (LTR) transactivation inT lymphoid Jurkat cells. Here we demonstrate that PTX‐B inhibits Tat‐dependent NF‐κB activation and HIV‐1 LTR‐transactivation in non‐permissive epithelial HL3T1 cells in a phosphatidylinositol 3′‐kinase‐dependent way. PTX‐B exerts its inhibition both when Tat is produced endogenously in transfected cells and in cells incubated with the extracellular Tat protein. In this latter case, PTX‐B does not interfere with extracellular Tat uptake by cells. PTX‐B inhibited also interleukin‐8 secretion and virus expression stimulated in chronically infected U1 promonocytic cells by intra‐ and/or extracellular Tat. The genetically modified holotoxin PT‐9 K/129G retains the capacity to inhibit Tat transactivating activity and HIV replication in both HIV‐permissive and non‐permissive cells. Inconclusion, PTX‐B acts as a "pleiotropic" inhibitor of Tat, and this may significantly contribute to the broad spectrum of anti‐HIV‐1 effects exerted by PTX‐B in different cell types, and suggests PTX‐B and its derivatives as prototypic for the development of anti‐Tat drugs.

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“…The basic domain/NLS of Tat mediates the interaction of extracellular Tat with cell surface HSPGs (Rusnati et al, 1998) that, in turn, is necessary for Tat internalization and transactivating activity (Tyagi et al, 2001). We then investigated if BSA-Tat-NLS exerts its inhibition by binding to cell-surface HSPGs, hampering their interaction with Tat and thus inhibiting Tat internalization.…”

Section: Bsa-tat-nls Binds To Heparin/hspgs and Inhibits Tat Internalmentioning

confidence: 99%

“…86 amino acid HIV-1 Tat was expressed and purified by Escherichia coli as glutathione-S-transferase (GST-Tat) or GST-Tatgreen fluorescent protein (GST-Tat-GFP) fusion proteins (Rusnati et al, 1998(Rusnati et al, , 2000. To avoid Tat oxidation, purified proteins were maintained at −20 • C in Tris 100 mM pH 9.5 containing 250 mM NaCl, 20 mM glutathione and 5 mM dithiothreitol.…”

Section: Reagentsmentioning

confidence: 99%

“…In AIDS patients, HIV reactivation by extracellular Tat may explain the "burst" of replication associated with early phases of HIV infection, where synchronized virion replication takes place (Peterlin et al, 1993). Extracellular Tat uptake by cells occurs via the interaction of cell-surface heparan sulfate proteoglycans (HSPGs) with the basic domain/NLS of Tat (Rusnati et al, 1998;Futaki et al, 2001). Accordingly, this sequence has been exploited as an effective protein transduction domain to mediate cell internalization of a variety of molecules (Fittipaldi and Giacca, 2005;Chauhan et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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BSA conjugates bearing multiple copies of the basic domain of HIV-1 Tat: Prototype for the development of multitarget inhibitors of extracellular Tat

et al. 2010

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a b s t r a c tThe transactivating factor (Tat) of HIV-1 is involved in AIDS progression and associated pathologies. Tat possesses a basic amino acid sequence implicated in heparan sulfate proteoglycan (HSPG)-mediated internalization, nuclear localization and transactivation by Tat and in the interaction of Tat with integrins and with the vascular endothelial growth factor receptor 2 (KDR) (kinase insert domain receptor). A BSA conjugate bearing an average of four copies of a peptide representing the basic domain/nuclear localization signal of Tat (BSA-Tat-NLS) inhibits transactivation by Tat exogenously added to cells but not by Tat endogenously produced after cell transfection with a tat cDNA, indicating that BSA-Tat-NLS does not interfere with Tat at an intracellular level. Surface plasmon resonance (SPR) experiments revealed that BSA-Tat-NLS binds to the HSPG analogue heparin. Accordingly, BSA-Tat-NLS binds to HSPGs of HL3T1 cell surface and inhibits HSPG-dependent Tat internalization. BSA-Tat-NLS retains its inhibitory potential when pre-incubated with HL3T1 cells before Tat administration, possibly by masking cell-surface HSPGs thus preventing Tat binding and internalization. SPR experiments revealed that BSA-Tat-NLS binds also to integrin ␣ v ␤ 3 and KDR. Accordingly, it inhibits pro-angiogenic endothelial cell adhesion to Tat and motogenesis. In conclusion, BSA-Tat-NLS binds/masks three different cell-surface receptors of Tat inhibiting different biological activities. These data point to BSA-Tat-NLS as a prototype for the development of Tat-antagonists endowed with a multitargeted mechanism of action.

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The Basic Domain in HIV-1 Tat Protein as a Target for Polysulfonated Heparin-mimicking Extracellular Tat Antagonists

Cited by 106 publications

References 89 publications

Potential inhibitors of chemokine function: Analysis of noncovalent complexes of cc chemokine and small polyanionic molecules by ESI FT-ICR mass spectrometry

Potential inhibitors of chemokine function: Analysis of noncovalent complexes of cc chemokine and small polyanionic molecules by ESI FT-ICR mass spectrometry

Inhibition of intra‐ and extra‐cellular Tat function and HIV expression by pertussis toxin B‐oligomer

BSA conjugates bearing multiple copies of the basic domain of HIV-1 Tat: Prototype for the development of multitarget inhibitors of extracellular Tat

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