Tat-mediated delivery of heterologous proteins into cells.

Abstract

The Tat protein of human immunodeficiency virus 1 (HIV-1) can enter cells efficiently when added exogenously in tissue culture. To assess if Tat can carry other molecules into cells, we chemically cross-linked Tat peptides (residues 1-72 or 37-72) to 3-galactosidase, horseradish peroxidase, RNase A, and domain Ill ofPseudomonas exotoxin A (PE) and monitored uptake colorimetrically or by cytotoxicity. The Tat chimeras were effective on all cell types tested, with staining showing uptake into all cells in each experiment. In mice, treatment with Tat-f-galactosidase chimeras resulted in delivery to several tissues, with high levels in heart, liver, and spleen, low-to-moderate levels in lung and skeletal muscle, and little or no activity in kidney and brain. The primary target within these tissues was the cells surrounding the blood vessels, suggesting endothelial cells, Kupffer cells, and/or splenic macrophages.Tat-mediated uptake may allow the therapeutic delivery of macromolecules previously thought to be impermeable to living cells.The potential for intracellular therapeutic use of proteins, peptides, and oligonucleotides has been limited by the impermeable nature of the cell membrane to these compounds. A wide variety of methods have been proposed for the delivery of proteins and other macromolecules into living cells for either experimental or therapeutic uses, including microinjection, scrape loading, electroporation, liposomes (1-7), bacterial toxins (8)(9)(10), and receptor-mediated endocytosis (11-16). Most of these methods are either inefficient or time-consuming, cause appreciable cell death, or result in uptake into intracellular vesicles without efficient cytoplasmic delivery. Several approaches (15-18) rely on binding of macromolecules to the cell surface, followed by internalization via the endocytic route. However, since proteins that have entered this pathway remain enclosed within lipid vesicles, they do not have access to the cell cytoplasm, most typically the target environment. It seems reasonable to assume that the escape from endocytic vesicles is the ratelimiting step in achieving true cellular delivery, yet many assays fail to measure this.Recently the Tat protein from human immunodeficiency virus 1 (HIV-1) was shown to enter cells when added exogenously (19,20). Tat protein can simply be added to medium at concentrations as low as 1 nM, and biological responses can be detected. Since the assay for this process was the transactivation of a transfected reporter gene, activity reflects those molecules that had been targeted to the nucleus, presumably after cytoplasmic delivery. The mechanism by which Tat traverses a membrane and the precise intracellular location of this event remain unclear. However, Tat binds efficiently to cells, with >107 sites per cell and then is internalized by an adsorptive endocytosis process (20). In characterizing the uptake process using iodinated Tat, Mann and Frankel (20) observed that only 3% of the Tat becameThe publication costs of this article were...

Wide Varieties of Cationic Nanoparticles Induce Defects in Supported Lipid Bilayers

“…TAT, a larger protein in comparison to MSI-78, induces the formation of holes in the bilayer ( Figure 2) at significantly lower concentrations than required for MSI-78. These SLB studies are consistent with cell level studies that have shown both of these biological proteins are internalized, 35,36 and more indirectly, are capable of disrupting cell membranes. [9][10][11][12] A variety of polymers have been used to mimic the ability of natural particles to breach cellular membranes.…”

“…Examples include MSI-78 35 and TAT. 36 The ability to penetrate cell membranes is not only advantageous for the host and virus, respectively, but potentially also a useful tool for scientists seeking to utilize these natural transportation systems for cellular delivery. The interaction between MSI-78 and the SLB was found to be concentration-dependent.…”

Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation

“…[1][2][3][4][5][6][7][8][9][10] These membrane-permeable peptides have been used for the delivery of exogenous molecules such as oligonucleotides and proteins into the cytoplasm and the nucleus of cultured cells. Tat-derived peptides that are chemically crosslinked to b-galactosidase, horseradish peroxidase, and RNAase A were found to be successfully taken up by different cell lines.…”

“…Tat-derived peptides that are chemically crosslinked to b-galactosidase, horseradish peroxidase, and RNAase A were found to be successfully taken up by different cell lines. [1][2][3][4] Furthermore, the intraperitoneal injection of b-galactosidase, fused to the Tat protein, resulted in the delivery of the biologically active fusion protein to all tissues in mice, including the brain. 3 An interesting feature of the membrane-permeable peptides is the mechanism by which they are internalized into cells.…”

Long-term protection against SHIV89.6P replication in HIV-1 Tat vaccinated cynomolgus monkeys

“…Tat is essential for viral replication, transmission and disease progression [1,[4][5][6][7]. In addition, Tat can modulate the expression of cytokines and cellular genes [6][7][8][9][10][11], and chemokine receptors [12][13][14][15], which are responsible for the transmission of macrophageand T-cell-tropic HIV-1 strains, respectively. In addition, the Tat protein also plays key roles in viral pathogenesis and in the pathogenesis of acquired immune deficiency syndrome (AIDS)-associated Kaposi's sarcoma [8,[16][17][18][19][20][21][22].…”