Nuclear Targeting of Macromolecular Polyanions by an HIV-Tat Derived Peptide

Abstract

New therapies based on gene transfer and protein delivery require a better understanding of the basic mechanisms of macromolecular membrane transport. We have studied cellular uptake of macromolecular polyanions, i.e. DNA and glycosaminoglycans, and a polybasic HIV-Tat derived peptide (GRKKRRQRRRPPQC) using fluorescence assisted cell sorting and confocal fluorescence microscopy. The transactivator of HIV transcription (Tat) peptide stimulated cellular uptake of both DNA and heparan sulfate in a time-, concentration-, and temperature-dependent manner. Peptide-polyanion complexes accumulated in large, acidic, cytoplasmic vesicles formed de novo. This was followed by transfer of polyanion into the nuclear compartment and subsequent disappearance of the endolysosomal vesicles. In the absence of polyanion the Tat peptide displayed rapid accumulation in the nuclear compartment. However, in the presence of polyanion the peptide was almost exclusively retained in cytoplasmic vesicles. Cellsurface proteoglycans played a pivotal role in the uptake of complexes exhibiting a relatively high peptide to polyanion ratio, corresponding to a net positive charge of the complexes. Uptake of polyanions per se or complexes with a relatively low peptide to polyanion ratio was favored by proteoglycan deficiency in the recipient cells, indicating the existence of distinct transport mechanisms. Moreover, expression of full-length HIV-Tat as well as exogenous addition of HIV-Tat peptide resulted in cellular accumulation of endogenous proteoglycans. We conclude that an HIV-Tat derived peptide efficiently targets extraneous DNA and glycosaminoglycans to the nuclear compartment and that proteoglycans serve a regulatory role in these processes, which may have implications for directed gene and drug delivery in vivo.

Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery

“…4C, black bars) suggests a more general role of GRP75 in nonclassical endocytosis. The HIV-TAT-derived transduction peptide, Tat, is a wellknown cell-penetrating peptide (CPP) previously shown to mediate DNA uptake through HSPGs (19). Consistent with a more general role of the GRP75-dependent endocytic pathway in macromolecular delivery, GRP75 inhibition reduced cellular uptake of CPP-DNA complexes, and scFv-αHS M colocalized with CPP-DNA complexes in vesicles (Fig.…”

Glypican-1 Is a Vehicle for Polyamine Uptake in Mammalian Cells

“…These saccharides could then form electroneutral membrane-penetrating complexes with small polybasic compounds such as spermine. Other studies from this laboratory have shown that a polybasic human immunodeficiency virus Tat-derived peptide is taken up by cells as a peptide-HS complex, followed by transfer of HS to the nucleus (42). Cell-surface PG molecules play a pivotal role in this process.…”

Arginine‐rich cell‐penetrating peptides

“…Examples include the TAT peptide from the HIV transactivator protein TAT, Penetratin, a 16 amino acid domain from the Antennapedia protein of Drosophila, a flock house virus (FHV) coat peptide (sequence [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49], and oligoarginines [3,5]. In this review, we focus on TAT peptide, partly because it has attracted the most attention, but also because it is a prototypical example that has many of the essential characteristics of the arginine-rich cell-penetrating peptides.…”