title: Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat.
abstract: BACKGROUND
  During HIV-1 infection, the Tat protein plays a key role by transactivating the transcription of the HIV-1 proviral DNA. In addition, Tat induces apoptosis of non-infected T lymphocytes, leading to a massive loss of immune competence. This apoptosis is notably mediated by the interaction of Tat with microtubules, which are dynamic components essential for cell structure and division. Tat binds two Zn2+ ions through its conserved cysteine-rich region in vitro, but the role of zinc in the structure and properties of Tat is still controversial.
  
  
  RESULTS
  To investigate the role of zinc, we first characterized Tat apo- and holo-forms by fluorescence correlation spectroscopy and time-resolved fluorescence spectroscopy. Both of the Tat forms are monomeric and poorly folded but differ by local conformational changes in the vicinity of the cysteine-rich region. The interaction of the two Tat forms with tubulin dimers and microtubules was monitored by analytical ultracentrifugation, turbidity measurements and electron microscopy. At 20 degrees C, both of the Tat forms bind tubulin dimers, but only the holo-Tat was found to form discrete complexes. At 37 degrees C, both forms promoted the nucleation and increased the elongation rates of tubulin assembly. However, only the holo-Tat increased the amount of microtubules, decreased the tubulin critical concentration, and stabilized the microtubules. In contrast, apo-Tat induced a large amount of tubulin aggregates.
  
  
  CONCLUSIONS
  Our data suggest that holo-Tat corresponds to the active form, responsible for the Tat-mediated apoptosis.
authors.affiliation: Université Louis Pasteur, Strasbourg 1, Institut Gilbert Laustriat, CNRS, UMR 7175, Département Photophysique des Interactions Biomoléculaires, Illkirch, Cedex, France. caroline.egele@pharma.u-strasbg.fr
authors.forename: Caroline
authors.initials: C
authors.lastname: Egelé
authors.forename: Pascale
authors.initials: P
authors.lastname: Barbier
authors.forename: Pascal
authors.initials: P
authors.lastname: Didier
authors.forename: Etienne
authors.initials: E
authors.lastname: Piémont
authors.forename: Diane
authors.initials: D
authors.lastname: Allegro
authors.forename: Olivier
authors.initials: O
authors.lastname: Chaloin
authors.forename: Sylviane
authors.initials: S
authors.lastname: Muller
authors.forename: Vincent
authors.initials: V
authors.lastname: Peyrot
authors.forename: Yves
authors.initials: Y
authors.lastname: Mély
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019281:Dimerization; D015497:HIV-1 / Q000378:metabolism / Q000472:pathogenicity*; D006801:Humans; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*
chemical_list: D014404:Tubulin; D054322:tat Gene Products, Human Immunodeficiency Virus; D015032:Zinc