Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool

Abstract

Effective gene therapy depends on the efficient transfer of therapeutic genes to target cells. None of the current technologies, however, satisfy all of the requirements necessary for gene therapy, because the plasma and nuclear membranes of mammalian cells are tight barriers against gene transfer using synthetic delivery systems. The protein transduction domain (PTD) of human immunodeficiency virus type 1 (HIV-1) Tat protein greatly facilitates protein transfer via membrane destabilisation. We synthesised polylysine peptides containing Tat PTD (TAT-pK), or other sequences, and investigated their potential as agents for gene transfer. The synthesised polypeptide TAT-pK retains DNA binding function and mediates delivery of a reporter gene to cultured cells. RGD motif binds with low affinity to alpha integrins which induce cell activation. Two control polypeptides, GGG-pK and RGD-pK, were synthesised and tested, but their gene transfer abilities were weaker than those of TAT-pK. TAT-pK-mediated gene transfer was enhanced in the presence of chloroquine or ammonium chloride, to a greater extent than that of cationic lipid-mediated gene transfer in most cancer cell lines tested. These data suggest that TAT-pK may be a potent candidate delivery vehicle that promotes gene transfer, dependent on the endocytic pathway. We conclude that the TAT-pK/DNA complex is useful as a minimal unit to package therapeutic genes and to transduce them into mammalian cells.

Protein Kinase C Promotes N-Methyl-d-aspartate (NMDA) Receptor Trafficking by Indirectly Triggering Calcium/Calmodulin-dependent Protein Kinase II (CaMKII) Autophosphorylation

“…Tat protein (Tyr-Gly-Arg-Lys-LysArg-Arg-Gln-Arg-Arg-Arg), which was obtained originally from the cell membrane transduction domain of the human immunodeficiency virus type 1 (HIV-1), was fused to the constructed peptides and resulted in fusion peptides Tat-NR2A and Tat-NR2B. This manipulation allowed the constructed peptides to easily cross the membrane and exert their effects intracellularly (39). Tat-NR2A (0.5 M) not only completely blocked PKC-induced potentiation of NR2A expression and NR2A-CaMKII association (NR2A, 1.10 Ϯ 0.12, n ϭ 5, p Ͼ 0.05 compared with control; NR2A-CaMKII, 1.11 Ϯ 0.10, n ϭ 5, p Ͼ 0.05, compared with control, one-way ANOVA LSD test) but also partially blocked enhancement in NR2B or p-CaMKII expression and NR2B-CaMKII association at postsynaptic sites (NR2B, 1.17 Ϯ 0.08, n ϭ 5, p Ͻ 0.05, compared with control; p-CaMKII, 1.19 Ϯ 0.12, n ϭ 5, p Ͻ 0.05; NR2B-CaMKII, 1.24 Ϯ 0.09, n ϭ 5, p Ͻ 0.05, one-way ANOVA LSD test) as indicated by both Western blot and co-IP assay (Fig.…”

Development and characterization of chitosan-PEG-TAT nanoparticles for the intracellular delivery of siRNA

“…7 TAT peptide can be conjugated covalently or non-covalently to cationic polymers like liposomes, poly-l-lysine, poly(ethyleneimine) (PEI). [8][9][10] It may also be used as a carrier of large therapeutic molecules, proteins, small interfering RNA (siRNA), quantum dots etc. 1,[11][12][13] Because of these properties, it is emerging as a new strategy to achieve efficient cellular nuclear targeting.…”

Secretory Transactivating Transcription-apoptin fusion protein induces apoptosis in hepatocellular carcinoma HepG2 cells

“…The Human Immunodeficiency Virus (HIV) Transactivating Transcription (TAT)-derived protein transduction peptide is a small basic peptide that has been successfully shown to deliver a large variety of materials, from small particles to proteins, peptides and nucleic acids, across the cell membrane [10][11][12] . The region that conveys the cell-penetrating properties appears to be confined to a small (11 amino acids) stretch of basic amino acids (aa 47-57, YGRKKRRQRRR) [13] .…”