The HIV-1 TAT peptide has been used extensively for directing the intracellular delivery of an assortment of cargo, including DNA, liposomes and macromolecules. For protein delivery, a variety of TAT-fusion proteins have been described which link the TAT coding sequence to the protein coding sequence of interest. Streptavidin represents a potentially useful TAT-fusion protein because it could be used to deliver a wide array of biotinylated cargo. Here we have characterized a TAT-streptavidin (TAT-SA) fusion protein, which retains the ability to bind biotinylated cargo while directing their efficient cellular uptake. Fluorescence activated cell sorting (FACS) analysis and confocal microscopy characterization showed that TAT-SA is internalized by Jurkat T-cells and NIH 3T3 cells alone and when complexed to phycoerythrin, whereas the native streptavidin is not. Additionally, biotinylated alkaline phosphatase is successfully internalized and retains its activity when complexed to TAT-SA and incubated with Jurkat T-cells. Confocal microscopy suggested, however, that internalized TAT-SA and TAT-SA complexes were largely compartmentalized in vesicular compartments, rather than freely diffusing in the cytoplasmic compartment. To effect cytoplasmic delivery, the endosomal releasing polymer, poly(propylacrylic acid) (PPAA), was biotinylated and complexed to TAT-SA. Endosomal release and cytoplasmic delivery of fluorescently labeled TAT-SA complexes with PPAA was shown by the diffuse distribution of fluorescent protein in the cytoplasm. Taken together, these results demonstrate that TAT-SA can be used to direct intracellular delivery of large biotinylated cargo to intracellular compartments and that biotinylated PPAA can direct cytoplasmic delivery where desired.
Background: The cell-penetrating peptide derived from the Human immunodeficiency virus-1 transactivator protein Tat possesses the capacity to promote the effective uptake of various cargo molecules across the plasma membrane in vitro and in vivo. The objective of this study was to characterize the uptake and delivery mechanisms of a novel streptavidin fusion construct, TAT 47-
A key challenge in developing protein therapeutics or imaging agents that work against cytosolic targets is the intracellular delivery barrier. Here, we show that the pH-responsive, membrane-destabilizing polymer, poly (propylacrylic acid) (PPAA), can strongly enhance target cell killing through the intracellular delivery of a functional proapoptotic peptide. The Bak BH3 peptide induces apoptosis via antagonization of suppressor targets such as Bcl-2 and Bcl-xL. A genetically-engineered streptavidin that contains an N-terminal TAT peptide sequence was used to optimize the pinocytotic cell uptake of biotinylated BH3 peptide and end-biotinylated PPAA. Fluorescence microscopic analysis of DAPI-stained HELA cells was used to quantitate apoptosis. Approximately 30% of cells treated with TAT-SA:BH3 complexes revealed morphologically distinct nuclear condensation, a hallmark of apoptosis. The incorporation of biotinylated PPAA had the effect of markedly enhancing the killing effect of BH3 peptides by an additional 55% (p<0.001) to a total cell killing efficiency of 85%. Caspase-3 activity was up-regulated in a TAT-SA:BH3:PPAA dose-dependent manner. The induction of apoptosis with the TAT-SA:BH3:PPAA complex was abrogated with the L78A BH3 peptide, that had been previously shown to knock-out antagonization activity. The caspase and L78A peptide results demonstrate that the delivered BH3 is indeed working through the biologically relevant apoptosis signaling pathway. These studies establish the ability of PPAA to strongly enhance the intracellular delivery of a functional pro-apoptotic peptide. Together with the PPAA, the TAT-SA adaptor complex could prove useful as a carrier of peptide/protein cargo to cultured cells.
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