Ferrari Aldo, Pellegrini Vittorio, Arcangeli Caterina, Fittipaldi Antonio, Giacca Mauro, Beltram Fabio
NEST-INFM and Scuola Normale Superiore, Piazza dei Cavalieri 7, I-56126, Pisa, Italy.
Mol Ther. 2003 Aug;8(2):284-94. doi: 10.1016/s1525-0016(03)00122-9.
The Tat protein from HIV-1, when fused with heterologous proteins or peptides, can traverse cell membranes. This ability has generated great interest due to potential therapeutic applications. However, the relevant cellular pathway and its dynamics have not been elucidated yet. Here we unravel the intracellular fate of exogenously added Tat fused with green fluorescent protein (GFP) in live HeLa and CHO cells, from the early interaction with the plasma membrane up to the long-term accumulation in the perinuclear region. We demonstrate that the internalization process of full-length Tat and of heterologous proteins fused to the transduction domain of Tat exploits a caveolar-mediated pathway and is inhibited at 4 degrees C. Remarkably, a slow linear movement toward the nucleus of individual GFP-tagged Tat-filled caveolae with an average velocity of 3 micro m/h was observed. No fluorescence was observed in the nucleus, possibly suggesting that Tat fusion protein unfolding is required for nuclear translocation. In addition, early sensitivity to cytochalasin-D treatment indicates the essential role of the actin cytoskeleton in the displacement of Tat vesicles toward the nucleus. Our results imply that HIV-1 Tat mediates the internalization of protein cargos in a slow and temperature-dependent manner by exploiting the caveolar pathway.
来自HIV-1的Tat蛋白与异源蛋白或肽融合时，能够穿越细胞膜。由于其潜在的治疗应用，这种能力引起了极大的关注。然而，相关的细胞途径及其动态过程尚未阐明。在此，我们揭示了在活的HeLa细胞和CHO细胞中，与绿色荧光蛋白（GFP）融合的外源添加Tat的细胞内命运，从其与质膜的早期相互作用直至在核周区域的长期积累。我们证明全长Tat以及与Tat转导结构域融合的异源蛋白的内化过程利用了小窝介导的途径，并且在4℃时受到抑制。值得注意的是，观察到单个充满GFP标记Tat的小窝以平均速度3μm/h缓慢向细胞核线性移动。在细胞核中未观察到荧光，这可能表明Tat融合蛋白的解折叠是核转运所必需的。此外，对细胞松弛素-D处理的早期敏感性表明肌动蛋白细胞骨架在Tat囊泡向细胞核的移位中起重要作用。我们的结果表明，HIV-1 Tat通过利用小窝途径以缓慢且依赖温度的方式介导蛋白质货物的内化。
