Effect of the redox state on HIV-1 tat protein multimerization and cell internalization and trafficking.
The redox state of the cysteine-rich region of the HIV Tat protein is known to play a crucial role in Tat biological activity. In this article, we show that Tat displays two alternative functional states depending on the presence of either one or three reduced sulphydryl groups in the cysteine-rich region, respectively. Using different approaches, a disulfide pattern has been defined for the Tat protein and a specific DTT-dependent breaking order of disulfide bonds highlighted. The Tat redox state deeply influences macrophage protein uptake. Immunoistochemistry analysis shows that the oxidized protein does not enter cells, whereas partially reduced protein reaches the cytosol and, to a limited extent, the nucleus. Finally electrophoretic analysis shows Tat high-molecular weight multi-aggregation, resulting in the loss of biological activity. This is due to strong electrostatic and metal-binding interactions, whereas Tat dimerization involves metal-binding interactions as well as disulfide bond formation.
Pierleoni
Raffaella
R
Department of Molecular Sciences, University of Urbino Carlo Bo, Via Saffi 2, 61029, Urbino, Italy.
Menotta
Michele
M
Antonelli
Antonella
A
Sfara
Carla
C
Serafini
Giordano
G
Dominici
Sabrina
S
Laguardia
Maria Elena
ME
Salis
Annalisa
A
Damonte
Gianluca
G
Banci
Lucia
L
Porcu
Marco
M
Monini
Paolo
P
Ensoli
Barbara
B
Magnani
Mauro
M
D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides; D004705:Endocytosis; D015696:Gene Products, tat / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics*; D006801:Humans; D008264:Macrophages / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D010084:Oxidation-Reduction; D055503:Protein Multimerization; D021381:Protein Transport
D004220:Disulfides; D015696:Gene Products, tat; D003545:Cysteine