Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat.
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.
Egelé
Caroline
C
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
Barbier
Pascale
P
Didier
Pascal
P
Piémont
Etienne
E
Allegro
Diane
D
Chaloin
Olivier
O
Muller
Sylviane
S
Peyrot
Vincent
V
Mély
Yves
Y
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*
D014404:Tubulin; D054322:tat Gene Products, Human Immunodeficiency Virus; D015032:Zinc