Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

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

1998; Elsevier BV; Volume: 273; Issue: 26 Linguagem: Inglês

10.1074/jbc.273.26.16027

ISSN

1083-351X

Autores

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

Tópico(s)

Viral Infections and Immunology Research

Resumo

Heparin binds extracellular HIV-1 Tat protein and modulates its HIV long terminal repeat (LTR)-transactivating activity (M. Rusnati, D. Coltrini, P. Oreste, G. Zoppetti, A. Albini, D. Noonan, F. d'Adda di Fagagna, M. Giacca, and M. Presta (1997) J. Biol. Chem. 272, 11313–11320). On this basis, the glutathioneS-transferase (GST)-TatR49/52/53/55/56/57Amutant, in which six arginine residues within the basic domain of Tat were mutagenized to alanine residues, was compared with GST-Tat for its capacity to bind immobilized heparin. Dissociation of the GST-TatR49/52/53/55/56/57A·heparin complex occurred at ionic strength significantly lower than that required to dissociate the GST-Tat·heparin complex. Accordingly, heparin binds immobilized GST-Tat and GST-TatR49/52/53/55/56/57A with a dissociation constant equal to 0.3 and 1.0 μm, respectively. Also, the synthetic basic domain Tat-(41–60) competes with GST-Tat for heparin binding. Suramin inhibits [3H]heparin/Tat interaction,125I-GST-Tat internalization, and the LTR-transactivating activity of extracellular Tat in HL3T1 cells and prevents125I-GST-Tat binding and cell proliferation in Tat-overexpressing T53 cells. The suramin derivative14C-PNU 145156E binds immobilized GST-Tat with a dissociation constant 5 times higher than heparin and is unable to bind GST-TatR49/52/53/55/56/57A. Although heparin was an antagonist more potent than suramin, modifications of the backbone structure in selected suramin derivatives originated Tat antagonists whose potency was close to that shown by heparin.In conclusion, suramin derivatives bind the basic domain of Tat, prevent Tat/heparin and Tat/cell surface interactions, and inhibit the biological activity of extracellular Tat. Our data demonstrate that tailored polysulfonated compounds represent potent extracellular Tat inhibitors of possible therapeutic value. Heparin binds extracellular HIV-1 Tat protein and modulates its HIV long terminal repeat (LTR)-transactivating activity (M. Rusnati, D. Coltrini, P. Oreste, G. Zoppetti, A. Albini, D. Noonan, F. d'Adda di Fagagna, M. Giacca, and M. Presta (1997) J. Biol. Chem. 272, 11313–11320). On this basis, the glutathioneS-transferase (GST)-TatR49/52/53/55/56/57Amutant, in which six arginine residues within the basic domain of Tat were mutagenized to alanine residues, was compared with GST-Tat for its capacity to bind immobilized heparin. Dissociation of the GST-TatR49/52/53/55/56/57A·heparin complex occurred at ionic strength significantly lower than that required to dissociate the GST-Tat·heparin complex. Accordingly, heparin binds immobilized GST-Tat and GST-TatR49/52/53/55/56/57A with a dissociation constant equal to 0.3 and 1.0 μm, respectively. Also, the synthetic basic domain Tat-(41–60) competes with GST-Tat for heparin binding. Suramin inhibits [3H]heparin/Tat interaction,125I-GST-Tat internalization, and the LTR-transactivating activity of extracellular Tat in HL3T1 cells and prevents125I-GST-Tat binding and cell proliferation in Tat-overexpressing T53 cells. The suramin derivative14C-PNU 145156E binds immobilized GST-Tat with a dissociation constant 5 times higher than heparin and is unable to bind GST-TatR49/52/53/55/56/57A. Although heparin was an antagonist more potent than suramin, modifications of the backbone structure in selected suramin derivatives originated Tat antagonists whose potency was close to that shown by heparin. In conclusion, suramin derivatives bind the basic domain of Tat, prevent Tat/heparin and Tat/cell surface interactions, and inhibit the biological activity of extracellular Tat. Our data demonstrate that tailored polysulfonated compounds represent potent extracellular Tat inhibitors of possible therapeutic value. tat is a viral regulatory gene of the human immunodeficiency virus type 1 (HIV-1), 1The abbreviations used are: HIV-1, human immunodeficiency virus type 1; CAT, chloramphenicol acetyltransferase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GAG, glycosaminoglycan; GST, glutathione S-transferase; HS, heparan sulfate; FGF, fibroblast growth factor; LTR, long terminal repeat; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin. 1The abbreviations used are: HIV-1, human immunodeficiency virus type 1; CAT, chloramphenicol acetyltransferase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GAG, glycosaminoglycan; GST, glutathione S-transferase; HS, heparan sulfate; FGF, fibroblast growth factor; LTR, long terminal repeat; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin. the etiologic agent of AIDS (1Barrè-Sinoussi F. Chermann J.C. Rey F. Nugeyre M.T. Chamaret S. Gruest J. Dauget C. Axler-Blin C. Vezinet-Brun F. Rouzioux C. Rozenbaum W. Montaigner L. Science. 1983; 220: 868-871Crossref PubMed Scopus (4753) Google Scholar, 2Gallo R.C. Salahuddin S.Z. Popovic M. Shearer G.M. Kaplan M. Haynes B.F. Palker T.J. Redfield R. Oleske J. Safai B. White G. Foster P. Markham P.D. Science. 1984; 224: 500-503Crossref PubMed Scopus (2411) Google Scholar). tat is essential for viral replication, since the Tat protein promotes transcription of the viral genome by interacting with the transactivation responsive element located at the 5′-end of viral mRNAs (3Sodroski J.G. Patarca R. Rosen C. Wong-Staal F. Haseltine W.A. Science. 1985; 229: 74-77Crossref PubMed Scopus (355) Google Scholar, 4Arya S.K Guo C. Josephs S.F. Wong-Staal F. Science. 1985; 229: 69-73Crossref PubMed Scopus (591) Google Scholar, 5Sodroski J.G. Rosen C.A. Wong-Staal F. Salahuddin S.Z. Popovic M. Arya S. Gallo R.C. Haseltine W.A. Science. 1985; 227: 171-173Crossref PubMed Scopus (307) Google Scholar). Tat can be released by HIV-infected cells (6Ensoli B. Buonaguro L. Barillari G. Fiorelli V. Gendelman R. Morgan R.A. Wingfield P. Gallo R.C. J. Virol. 1993; 67: 277-287Crossref PubMed Google Scholar), and significant levels of Tat have been detected in HIV-1-seropositive subjects (7Westendorp M.O. Frank R. Ochsenbauer C. Stricker K. Dhein J. Walczak H. Debatin K.M. Krammer P.H. Nature. 1995; 375: 497-500Crossref PubMed Scopus (913) Google Scholar). As an extracellular molecule, Tat exerts pleiotropic effects on uninfected cells. Extracellular Tat promotes the production of growth factors and cytokines (8Nabell L.M. Raja H. Sayeski P. Paterson A.J. Kudlow J.E. Cell Growth Differ. 1994; 5: 87-93PubMed Google Scholar, 9Zauli G. Davis B.R. Re M.C. Visani C. Furlini G. La Placa M. Blood. 1992; 80: 3035-3043Crossref Google Scholar, 10Lotz M. Clark-Lewis I. Ganu V. J. Cell Biol. 1994; 124: 365-371Crossref PubMed Scopus (77) Google Scholar, 11Opalenik S.R. Shin J.T. Wehby J.N. Mahesh V.K. Thompson J.A. J. Biol. Chem. 1995; 270: 17457-17467Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 12Philippon V. Vellutini C. Gambarelli D. Harkiss G. Arbuthnott G. Metzger D. Roubin R. Filippi P. Virology. 1994; 205: 519-528Crossref PubMed Scopus (133) Google Scholar, 13Purvis S.F. Georges D.L. Williams T.M. Lederman M.M. Cell. Immunol. 1992; 144: 32-42Crossref PubMed Scopus (67) Google Scholar, 14Westendorp M.O. Li-Weber M. Frank R.W. Krammer P.H. J. Virol. 1994; 68: 4177-4185Crossref PubMed Google Scholar, 15Vacca A. Farina M. Maroder M. Alesse E. Screpanti I. Frati L. Gulino A. Biochem. Biophys. Res. Commun. 1994; 205: 467-474Crossref PubMed Scopus (32) Google Scholar, 16Zauli G. Furlini G. Re M.C. Milani D. Capitani S. La Placa M. Microbiologica. 1993; 16: 115-120PubMed Google Scholar, 17Scala G. Ruocco M.R. Ambrosino C. Mallardo M. Giordano V. Baldassarre F. Dragonetti E. Quinto I. Venuta S. J. Exp. Med. 1994; 179: 961-971Crossref PubMed Scopus (232) Google Scholar) and of cytokine receptors (13Purvis S.F. Georges D.L. Williams T.M. Lederman M.M. Cell. Immunol. 1992; 144: 32-42Crossref PubMed Scopus (67) Google Scholar, 18Pocsik E. Higuchi M. Aggarwal B.B. Lymphokine Cytokine Res. 1992; 11: 317-325PubMed Google Scholar, 19Milani D. Zauli G. Neri L.M. Marchisio M. Previati M. Capitani S. J. Gen. Virol. 1993; 74: 2587-2594Crossref PubMed Scopus (40) Google Scholar, 20Puri R.K. Aggarwal B.B. Cancer Res. 1992; 52: 3787-3790PubMed Google Scholar). Tat stimulates proliferation (10Lotz M. Clark-Lewis I. Ganu V. J. Cell Biol. 1994; 124: 365-371Crossref PubMed Scopus (77) Google Scholar,21Albini A. Benelli R. Presta M. Rusnati M. Ziche M. Rubartelli A. Paglialunga G. Bussolino F. Noonan D. Oncogene. 1996; 12: 289-297PubMed Google Scholar, 22Ensoli B. Barillari G. Salahuddin S.Z. Gallo R.C. Wong-Staal F. Nature. 1990; 345: 84-86Crossref PubMed Scopus (798) Google Scholar, 23Barillari G. Bonaguro L. Fiorelli V. Hoffman J. Michaels F. Gallo R.C. Ensoli B. J. Immunol. 1992; 149: 3727-3734PubMed Google Scholar, 24Campioni D. Corallini A. Zauli G. Possati L. Altavilla G. Barbanti-Brodano G. AIDS Res. Hum. Retroviruses. 1995; 11: 1039-1048Crossref PubMed Scopus (66) Google Scholar), migration (21Albini A. Benelli R. Presta M. Rusnati M. Ziche M. Rubartelli A. Paglialunga G. Bussolino F. Noonan D. Oncogene. 1996; 12: 289-297PubMed Google Scholar, 25Lafrenie R.M. Wahl L.M. Epstein J.S. Hewlett I.K. Yamada K.M. Dhawan S. J. Immunol. 1996; 156: 638-1645Google Scholar, 26Lafrenie R.M. Wahl L.M. Epstein J.S. Hewlett I.K. Yamada K.M. Dhawan S. J. Immunol. 1996; 157: 974-977PubMed Google Scholar), and protease production (27Ensoli B. Gendelman R. Markham P. Fiorelli V. Colombini S. Raffeld M. Cafaro A. Chang H.K. Brady J.N. Gallo R.C. Nature. 1994; 271: 674-680Crossref Scopus (537) Google Scholar, 28Corallini A. Campioni D. Rossi C. Albini A. Possati L. Rusnati M. Gazzanelli G. Benelli R. Masiello L. Spacciari V. Presta M. Manello F. Fontanini G. Barbanti-Brodano G. AIDS. 1996; 10: 701-710Crossref PubMed Scopus (42) Google Scholar, 29Rusnati M. Coltrini D. Campioni D. Tanghetti E. Corallini A. Barbanti-Brodano G. Gibellini D. Presta M. AIDS. 1997; 11: 727-736Crossref PubMed Scopus (11) Google Scholar) in different cell types. Extracellular Tat exerts an angiogenic activityin vitro (30Albini A. Barillari G. Benelli R. Gallo R.C. Ensoli B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4838-4842Crossref PubMed Scopus (203) Google Scholar) and in vivo (21Albini A. Benelli R. Presta M. Rusnati M. Ziche M. Rubartelli A. Paglialunga G. Bussolino F. Noonan D. Oncogene. 1996; 12: 289-297PubMed Google Scholar, 28Corallini A. Campioni D. Rossi C. Albini A. Possati L. Rusnati M. Gazzanelli G. Benelli R. Masiello L. Spacciari V. Presta M. Manello F. Fontanini G. Barbanti-Brodano G. AIDS. 1996; 10: 701-710Crossref PubMed Scopus (42) Google Scholar, 31Albini A. Fontanini G. Masiello L. Tacchetti D. Bigini P. Luzzi D.M. Noonan D. Stetler-Stevenson W.G. AIDS. 1994; 8: 1237-1244Crossref PubMed Scopus (138) Google Scholar). In the central nervous system, Tat released by HIV-infected macrophages can induce neurotoxicity by acting directly on neurons (32Strijbos P.J. Zamani M.R. Rothwell N.J. Arbuthnott G. Harkiss G. Neurosci. Lett. 1995; 197: 215-218Crossref PubMed Scopus (25) Google Scholar, 33Sabatier J.M. Vives E. Mabrouk K. Benjouad A. Rochat H. Duval A. Hue B. Bahraoui E. J. Virol. 1991; 65: 961-967Crossref PubMed Google Scholar, 34Magnuson D.S.K. Knudsen B.E. Geiger J.D. Brownstone R.M. Nath A. Ann. Neurol. 1995; 37: 371-380Crossref Scopus (256) Google Scholar, 35Weeks B.S. Lieberman D.M. Johnson B. Roque E. Green M. Loewenstein P. Oldfield E.H. Kleinman H.K. J. Neurosci. Res. 1995; 42: 24-40Crossref Scopus (67) Google Scholar, 36Nath A. Psooy K. Martin C. Knudsen B. Magnuson D.S. Haughey N. Geiger J.D. J. Virol. 1996; 70: 1475-1480Crossref PubMed Google Scholar) or by stimulating different cell types to produce and release neurotoxic molecules (12Philippon V. Vellutini C. Gambarelli D. Harkiss G. Arbuthnott G. Metzger D. Roubin R. Filippi P. Virology. 1994; 205: 519-528Crossref PubMed Scopus (133) Google Scholar, 37Hofman F.M. Dohadwala M.M. Albion D.W. Hinton D.R. Walker S.M. J. Neuroimmunol. 1994; 54: 19-28Abstract Full Text PDF PubMed Scopus (91) Google Scholar, 38Taylor J.P. Cupp C. Diaz A. Chowdhury M. Khalili K. Jimenez S.A. Amini S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9617-9621Crossref PubMed Scopus (90) Google Scholar). Finally, extracellular Tat transforms and immortalizes keratinocyte in culture (39Kim C.M. Vogel J. Jay G.R. Rhim J.S. Oncogene. 1992; 7: 1525-1529PubMed Google Scholar). Thus, extracellular Tat exerts a wide spectrum of biological effects on several cell types, suggesting its implication in different AIDS-associated pathologies including Kaposi's sarcoma (27Ensoli B. Gendelman R. Markham P. Fiorelli V. Colombini S. Raffeld M. Cafaro A. Chang H.K. Brady J.N. Gallo R.C. Nature. 1994; 271: 674-680Crossref Scopus (537) Google Scholar, 41Vogel J. Hinrichs S.H. Reynolds R.K. Luciw P.A. Jay G. Nature. 1988; 335: 606-611Crossref PubMed Scopus (464) Google Scholar), AIDS dementia (42Dewhurst S. Gelbard H.A. Fine S.M. Mol. Med. Today. 1996; 2: 16-23Abstract Full Text PDF PubMed Scopus (39) Google Scholar), and the increased incidence of tumors in AIDS patients (28Corallini A. Campioni D. Rossi C. Albini A. Possati L. Rusnati M. Gazzanelli G. Benelli R. Masiello L. Spacciari V. Presta M. Manello F. Fontanini G. Barbanti-Brodano G. AIDS. 1996; 10: 701-710Crossref PubMed Scopus (42) Google Scholar, 43Corallini A. Altavilla G. Pozzi L. Bignozzi F. Negrini M. Rimessi P. Gualandi F. Barbanti-Brodano G. Cancer Res. 1993; 53: 5569-5575PubMed Google Scholar). The ability of extracellular Tat to induce peripheral blood mononucleated cell apoptosis (7Westendorp M.O. Frank R. Ochsenbauer C. Stricker K. Dhein J. Walczak H. Debatin K.M. Krammer P.H. Nature. 1995; 375: 497-500Crossref PubMed Scopus (913) Google Scholar, 44Li C.J. Friedman D.J. Wang C. Metelev V. Pardee A.B. Science. 1995; 268: 429-431Crossref PubMed Scopus (546) Google Scholar, 45Sastry K.J. Marin M.C. Nehete P.N. McConnell K. el-Naggar A.K. McDonnell T.J. Oncogene. 1996; 13: 487-493PubMed Google Scholar) implicates the protein also in CD4+ cell depletion and in the progression of AIDS. Moreover, extracellular Tat appears to be responsible for the burst of virus replication that takes place at the beginning of HIV infection (46Peterlin B.M. Adams M. Alonso A. Baur A. Ghosh S. Lu X. Luo X. Cullen B.R. Human Retroviruses. Oxford University Press, Oxford1993: 75-100Google Scholar,47Goldstein G. Nat. Med. 1996; 1: 960-964Crossref Scopus (143) Google Scholar). This possibility is supported by the observation that anti-Tat antibody causes a significant delay in HIV-1 replication in infected peripheral blood mononucleated cells (48Re M.C. Furlini G. Vignoli M. Ramazzotti E. Roderigo G. De Rosa V. Zauli G. Lolli S. Capitani S. La Placa M. J. Acquired Immune Defic. Syndr. Hum. Retrov. 1995; 10: 408-416Crossref PubMed Scopus (116) Google Scholar, 49Zauli G. La Placa M. Vignoli M. Re M.C. Gibellini D. Furlini G. Milani D. Marchisio M. Mazzoni M. Capitani S. J. Acquired Immune Defic. Syndr. Hum. Retrov. 1995; 10: 306-316Crossref PubMed Scopus (68) Google Scholar, 50Steinaa L. Sorenson A.M. Nielsen J. Hansen J.-E.S. Arch. Virol. 1994; 139: 263-271Crossref PubMed Scopus (39) Google Scholar) and that an inverse correlation exists between the levels of natural anti-Tat antibody and those of p24 antigen in HIV-seropositive subjects (48Re M.C. Furlini G. Vignoli M. Ramazzotti E. Roderigo G. De Rosa V. Zauli G. Lolli S. Capitani S. La Placa M. J. Acquired Immune Defic. Syndr. Hum. Retrov. 1995; 10: 408-416Crossref PubMed Scopus (116) Google Scholar). Finally, Tat exerts negative effects on the immune system also by repressing the transcription of the major histocompatibility complex class I gene (51Howcroft T.K. Strebel K. Martin M.A. Singer D.S. Science. 1993; 260: 1320-1322Crossref PubMed Scopus (200) Google Scholar), by inhibiting antigen-induced lymphocyte proliferation and generation of functional suppressive CD8+ cells (52Viscidi R.P. Mayur K. Lederman H.M. Frankel A.D. Science. 1989; 246: 1606-1608Crossref PubMed Scopus (319) Google Scholar, 53Lachgar A. Bernard J. Bizzini B. Astgen A. Le Coq H. Fouchard M. Chams V. Feldman M. Burny A. Zagury J.F. Biomed. Pharmacother. 1996; 50: 13-18Crossref PubMed Scopus (6) Google Scholar), by impairing thymocyte development (54Maroder M. Scarpa S. Screpanti I. Stigliano A. Meco D. Vacca A. Stuppia L. Frati L. Modesti A. Gulino A. Cell. Immunol. 1996; 168: 49-58Crossref PubMed Scopus (10) Google Scholar), and by inhibiting interferon-induced iNos activity in macrophages (55Barton C.H. Biggs T.E. Mee T.R. Mann B. J. Gen. Virol. 1996; 77: 1643-1647Crossref PubMed Scopus (25) Google Scholar). Thus, from the bulk of data it emerges that extracellular Tat may be implicated both in the progression of AIDS and in the pathogenesis of several AIDS-associated diseases. On this basis, Tat protein should be considered as a target for novel anti-HIV strategies. Tat protein is a polypeptide of 86–102 amino acids depending on the viral strain, which is encoded by two exons and is translated from multiply spliced 2-kilobase mRNAs (56Schwartz S. Felber B.K. Benko D.M. Fenyo E.M. Pavlakis G.N. J. Virol. 1990; 64: 2519-2529Crossref PubMed Google Scholar). The amino acid sequence 1–72 encoded by the first exon is endowed with full transactivating activity (3Sodroski J.G. Patarca R. Rosen C. Wong-Staal F. Haseltine W.A. Science. 1985; 229: 74-77Crossref PubMed Scopus (355) Google Scholar, 4Arya S.K Guo C. Josephs S.F. Wong-Staal F. Science. 1985; 229: 69-73Crossref PubMed Scopus (591) Google Scholar, 57Cullen B.R. Cell. 1990; 63: 655-657Abstract Full Text PDF PubMed Scopus (139) Google Scholar), while the carboxyl-terminal region encoded by the second exon (amino acids 72–86) is not required either for the transactivating activity of Tat or for the replication of HIV-1 (5Sodroski J.G. Rosen C.A. Wong-Staal F. Salahuddin S.Z. Popovic M. Arya S. Gallo R.C. Haseltine W.A. Science. 1985; 227: 171-173Crossref PubMed Scopus (307) Google Scholar,57Cullen B.R. Cell. 1990; 63: 655-657Abstract Full Text PDF PubMed Scopus (139) Google Scholar). However, this latter region is necessary for different biological activities of Tat (29Rusnati M. Coltrini D. Campioni D. Tanghetti E. Corallini A. Barbanti-Brodano G. Gibellini D. Presta M. AIDS. 1997; 11: 727-736Crossref PubMed Scopus (11) Google Scholar, 58Kim Y.S. Panganiban A.T. J. Virol. 1993; 67: 3739-3747Crossref PubMed Google Scholar, 59Gentz R. Chen C.-H. Rosen C.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 821-824Crossref PubMed Scopus (103) Google Scholar, 60Frankel A.D. Pabo C.O. Cell. 1988; 55: 1189-1193Abstract Full Text PDF PubMed Scopus (2306) Google Scholar) and contains an Arg-Gly-Asp (RGD) motif responsible for Tat binding to integrin receptors (61Vogel B.E. Lee S.-U. Hildebrand A. Craig W. Pierschbacher D. Wong-Staal F. Ruoslahti E. J. Cell Biol. 1993; 121: 461-468Crossref PubMed Scopus (228) Google Scholar, 62Barillari G. Gendelman R. Gallo R.C. Ensoli B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7941-7945Crossref PubMed Scopus (340) Google Scholar). Within the sequence encoded by the first exon of tat is present a so-called basic domain (amino acids 49–57) constituted by a stretch of repeated Arg and Lys residues. This highly immunoreactive region is well conserved among Tat proteins isolated from different strains of HIV-1 (47Goldstein G. Nat. Med. 1996; 1: 960-964Crossref Scopus (143) Google Scholar, 63Gregoire C.J. Loret E.P. J. Biol. Chem. 1996; 271: 22641-22646Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The basic domain is implicated in several aspects of Tat biology. Indeed, it is necessary for Tat stability (64Hauber J. Malim M.H. Cullen B.R. J. Virol. 1989; 63: 1181-1187Crossref PubMed Google Scholar), nuclear and nucleolar delivery (65Endo S. Kubota S. Siomi H. Adachi A. Oroszlan S. Maki M. Hatanaka M. Virus Genes. 1989; 2: 99-110Google Scholar), 2M. Giacca, unpublished observations. 2M. Giacca, unpublished observations. and interaction with nucleic acids (66Puglisi J.D. Tan R. Calnan B.J. Frankel A.D. Williamson J.R. Science. 1992; 257: 76-80Crossref PubMed Scopus (534) Google Scholar). It mediates some of the neurotoxic effects of extracellular Tat (12Philippon V. Vellutini C. Gambarelli D. Harkiss G. Arbuthnott G. Metzger D. Roubin R. Filippi P. Virology. 1994; 205: 519-528Crossref PubMed Scopus (133) Google Scholar, 33Sabatier J.M. Vives E. Mabrouk K. Benjouad A. Rochat H. Duval A. Hue B. Bahraoui E. J. Virol. 1991; 65: 961-967Crossref PubMed Google Scholar, 35Weeks B.S. Lieberman D.M. Johnson B. Roque E. Green M. Loewenstein P. Oldfield E.H. Kleinman H.K. J. Neurosci. Res. 1995; 42: 24-40Crossref Scopus (67) Google Scholar), and it is necessary for the mitogenic, chemotactic, and angiogenic activities of extracellular Tat (21Albini A. Benelli R. Presta M. Rusnati M. Ziche M. Rubartelli A. Paglialunga G. Bussolino F. Noonan D. Oncogene. 1996; 12: 289-297PubMed Google Scholar). The basic domain cooperates with the RGD motif in the interaction of Tat protein with integrins (61Vogel B.E. Lee S.-U. Hildebrand A. Craig W. Pierschbacher D. Wong-Staal F. Ruoslahti E. J. Cell Biol. 1993; 121: 461-468Crossref PubMed Scopus (228) Google Scholar), and it is implicated in Tat binding and activation of the Flk-1/KDR receptor (67Albini A. Soldi R. Giunciuglio D. Giraudo E. Benelli R. Primo L. Noonan D. Salio M. Camussi G. Rockl W. Bussolino F. Nat. Med. 1996; 2: 1371-1375Crossref PubMed Scopus (347) Google Scholar). Tat protein binds to cell surface heparan sulfate (HS) and heparin (68Rusnati M. Coltrini D. Oreste P. Zoppetti G. Albini A. Noonan D. d'Adda di Fagagna F. Giacca M. Presta M. J. Biol. Chem. 1997; 272: 11313-11320Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar,69Mann D.A. Frankel A.D. EMBO J. 1991; 10: 1733-1739Crossref PubMed Scopus (444) Google Scholar). This interaction occurs through the negatively charged sulfate groups of the glycosaminoglycans (GAGs) (68Rusnati M. Coltrini D. Oreste P. Zoppetti G. Albini A. Noonan D. d'Adda di Fagagna F. Giacca M. Presta M. J. Biol. Chem. 1997; 272: 11313-11320Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), suggesting that the positively charged Arg and Lys residues present within the basic domain of Tat may be responsible for the interaction. Soluble polysulfated GAGs can inhibit the transactivating activity of extracellular Tat, and their inhibitory action appears to be directly related to their capacity to bind Tat protein (68Rusnati M. Coltrini D. Oreste P. Zoppetti G. Albini A. Noonan D. d'Adda di Fagagna F. Giacca M. Presta M. J. Biol. Chem. 1997; 272: 11313-11320Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). These observations point to the basic domain of Tat as a preferential molecular target for polysulfated compounds able to interact with extracellular Tat protein and neutralize its biological activity. Suramin is a polysulfonated naphthylurea originally developed for the treatment of trypanosomiasis and onchocerciasis. Suramin has been used recently in the treatment of cancer (70Hawkins M.J. Curr. Opin. Oncol. 1995; 7: 90-93Crossref PubMed Scopus (43) Google Scholar). In vitro, suramin blocks the activity of several growth factors by inhibiting their binding to cognate receptors (71Coffey R.J. Leof E.B. Shipley G. Moses H.L. J. Cell. Physiol. 1987; 132: 143-148Crossref PubMed Scopus (337) Google Scholar, 72Braddock P.S. Hu D.E. Fan T.-P. Stratford I.J. Harris A.L. Bicknell R. Br. J. Cancer. 1994; 69: 890-898Crossref PubMed Scopus (78) Google Scholar, 73Rusnati M. Dell'Era P. Urbinati C. Tanghetti E. Massardi M.L. Nagamine Y. Monti E. Presta M. Mol. Biol. Cell. 1996; 7: 369-381Crossref PubMed Scopus (37) Google Scholar). Suramin inhibits the activity of heparanase (74Nakajima M. DeChavigny A. Johnson C.E. Hamada J. Stein C.A. Nicolson G.L. J. Biol. Chem. 1991; 266: 9661-9666Abstract Full Text PDF PubMed Google Scholar) and of urokinase-type plasminogen activator (29Rusnati M. Coltrini D. Campioni D. Tanghetti E. Corallini A. Barbanti-Brodano G. Gibellini D. Presta M. AIDS. 1997; 11: 727-736Crossref PubMed Scopus (11) Google Scholar, 75Behrendt N. Ronne E. Dano K. J. Biol. Chem. 1993; 268: 5985-5989Abstract Full Text PDF PubMed Google Scholar). Moreover, suramin inhibits cell adhesion and migration (76Zabrenetzky V.S. Kohn E.C. Roberts D.D. Cancer Res. 1990; 50: 5937-5942PubMed Google Scholar). All of these in vitro activities may explain, at least in part, the capacity of suramin to inhibit tumor growth and metastasis in different experimental models (77Sola F. Farao M. Marsiglio A. Mariani M. Grandi M. Invasion Metastasis. 1993; 13: 163-168PubMed Google Scholar, 78Pesenti E. Sola F. Mongelli N. Grandi M. Spreafico F. Br. J. Cancer. 1992; 66: 367-372Crossref PubMed Scopus (122) Google Scholar). As an angiogenesis inhibitor, suramin has been demonstrated to inhibit the activity exerted by fibroblast growth factors (FGFs) and vascular endothelial growth factor on cultured endothelial cells by preventing their interaction with cell surface HS-proteoglycans and tyrosine-kinase receptors and to block their angiogenic activity in different animal models (see Ref. 79Fan T.-P. Jaggar R. Bicknell R. Trends Pharmacol. Sci. 1995; 16: 57-66Abstract Full Text PDF PubMed Scopus (214) Google Scholar and references therein). This is due, at least in part, to the capacity of suramin to bind to the heparin-binding region of the growth factor via one or more of its sulfate groups. Accordingly, suramin is able to mimic heparin/HS for the capacity to protect FGF2 from trypsin digestion. Interestingly, the same capacity was observed for the related polysulfonated compound trypan blue (80Coltrini D. Rusnati M. Zoppetti G. Grazioli G. Naggi A. Presta M. Eur. J. Biochem. 1994; 214: 51-58Crossref Scopus (57) Google Scholar). In this work, we have characterized the interaction of heparin, suramin, and suramin-related compounds with Tat protein, focusing on the role played by the basic domain of Tat in this interaction. The data demonstrate the possibility of synthesizing polysulfonated, heparin-mimicking compounds equipotent to natural heparin in interfering with the biological activity of extracellular Tat. Suramin was from Bayer AG (Leverkusen, Germany). Trypan blue was from Sigma. Heparin (13.6 kDa) was from Laboratori Derivati Organici SpA (Milan, Italy). Chondroitin sulfate C was a gift of M. Del Rosso (University of Florence, Italy). β-Cyclodextrin tetradecasulfate was from Consultants on Glycosaminoglycans (Milan, Italy). The PNU compounds are suramin-related distamycin A derivatives (81Clanton D.J. Buckheit R.W. Terpening S.J. Kiser R. Mongelli N. Borgia A.L. Schultz R. Narayanan V. Bader J.P. Rice W.G. Antivir. Res. 1995; 27: 335-354Crossref PubMed Scopus (50) Google Scholar) (see Fig. 9 for structural details). Anti-Tat polyclonal antibody were from American Biotechnologies/Intracel (London). This antibody recognizes with the same efficiency all of the Tat mutants utilized in this study (data not shown). The synthetic peptides representing the amino acid sequences 1–20, 41–60, and 71–85 of HIV-1 Tat (virus strain HIV-1 LAI) were obtained from the Medical Research Council AIDS Reagent Project (National Institute for Biological Standards and Control, Potters Bar, Herts, UK). The synthetic peptides representing the sequences 103–120 and 103–146 of basic fibroblast growth factor (FGF-2) were kind gifts from A. Baird (Prizm Pharmaceuticals, San Diego, CA). Recombinant wild type HIV-1 Tat and the different Tat mutants were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The respective plasmid constructs are derivatives of plasmid pGST-Tat 2E, which was originally obtained by cloning the coding region of both exons of HIV-1HXB2 Tat in the commercial vector pGEX2T, as already described (82Demarchi F. d'Adda di Fagagna F. Falaschi A. Giacca M. J. Virol. 1996; 70: 4427-4437Crossref PubMed Google Scholar). This construct codes for the wild type 86-amino acid Tat protein. The mutated derivatives include GST-Tat-1e (containing one-exon Tat and comprising the first 72 amino acids of the protein), GST-TatΔ1–21 (containing a deletion of the amino acid sequence 1–21), GST-TatH13E (containing a mutation of histidine 13 to glutamine), and GST-TatK49/52/53/55/56/57A(in which the arginine residues at positions 49, 52, 53, 55, 56, and 57 in the basic domain were mutated to alanine residues). These constructs were obtained by a recombinant polymerase chain reaction procedure using overlapping oligonucleotides corresponding to the mutated sequences; a detailed description of the construction of these mutants as well as of their transcriptional properties will be presented elsewhere. 3M. Giacca, manuscript in preparation. Recombinant fusion proteins were purified to homogeneity from bacterial lysates by glutathione-Sepharose affinity chromatography (Amersham Pharmacia Biotech) according to the manufacturer's instructions, with minor modifications. Briefly, lysates were mixed with 1 ml of a 50% (v/v) slurry of glutathione cross-linked agarose beads (Sigma). The fusion protein was allowed to bind to the beads at 4 °C on a rotating wheel for 1 h. The suspension was then loaded on an empty plastic column, letting the unbound proteins pass through, and the beads were submitted to a high salt wash (0.8 m NaCl) to free the fusion protein from contaminating bacterial nucleic acids. The fusion protein was eluted in 1 ml of 100 mm Tris, pH 8.0, containing 2 mm dithiothreitol and 20 mm free glutathione (Sigma). The purity and integrity of the protein was routinely checked by SDS-polyacrylamide gel electrophoresis and silver staining. Usually, this purification procedure leads to >95% purification of the recombinant proteins. The purified proteins were stored in aliquots at −80 °C until use. The T53 cell

Referência(s)