The taming of the cell penetrating domain of the HIV Tat: myths and realities

Abstract

Protein transduction with cell penetrating peptides over the past several years has been shown to be an effective way of delivering proteins in vitro and now several reports have also shown valuable in vivo applications in correcting disease states. An impressive bioinspired phenomenon of crossing biological barriers came from HIV transactivator Tat protein. Specifically, the protein transduction domain of HIV Tat has been shown to be a potent pleiotropic peptide in protein delivery. Various approaches such as molecular modeling, arginine guanidinium head group structural strategy, multimerization of PTD sequence and phage display system have been applied for taming of the PTD. This has resulted in identification of PTD variants which are efficient in cell membrane penetration and cytoplasmic delivery. In spite of these state of the art technologies, the dilemma of low protein transduction efficiency and target specific delivery of PTD fusion proteins remains unsolved. Moreover, some misconceptions about PTD of Tat in the literature require considerations. We have assembled critical information on secretory, plasma membrane penetration and transcellular properties of Tat and PTD using molecular analysis and available experimental evidences.

Figure 1

Proteolytic map of Tat-PTD with amino acid residues from 48-56 is shown. Full length Tat is 101 amino acids and PTD contains mainly charged amino acids lysines and arginines.

Figure 2

Nuclear expression of Tat in SVGA (astrocytic) cells. SVGA cells were transiently transfected with 2 exon HIV-Tat (full length 101 amino acids) and immunostained 24 hours post transfection by monoclonal Tat-antibody. (A) Nuclear localization of Tat in two daughter cells. (B) DAPI nuclear staining corresponds to cells in panel (A).

Figure 3

Wild type Tat-GFP and mutant (Δ) Tat-GFP expression constructs are shown diagrammatically. A, live cell fluorescence in SVGA cells transfected with Tat-GFP; B, light phase of panel A; C, live cell fluorescence in SVGA cells transfected with mutant Tat (48-56)-GFP showing green fluorescence throughout (diffuse) the cells; D, light phase of panel C.

Figure 4

SVGA cells transiently transfected with Tat-GFP or mutant Tat (dTat)-GFP wherein PTD region (48-56) is deleted and immunostained for nucleoli by using nucleophosmin monoclonal antibody. (A) Tat-GFP (green) expression in the nucleus under green filter; (B) same field as in (A) Tat-GFP (green) and nucleophosmin (red) seen with double filter showing co-localization of Tat and nucleoli; panel (C), DAPI nuclear staining corresponds to panels (A) and (B); panel (D), mutant Tat-GFP shows green fluorescence throughout the cells (diffuse), panel (E) same as (D) showing mutant Tat-GFP (green) and nucleoli (red) under double filter, have lost nucleolar distribution of Tat; panel (F), DAPI nuclear staining corresponds to panel (D) and (E); (G), control cells transfected with vector DNA and seen under green filter and in (H) same as (G) showing nucleolar staining; (I), DAPI nuclear staining for panels (G) and (H).

Figure 5

Transduction of PTD-NS1 fusion protein into neuroblastoma cells and primary neurons. His-PTD-NS1 containing a 3-glycine spacer was produced in bacteria. The affinity-purified protein was added to the culture medium with 50 μM chloroquine. After 24 hours, cells were fixed with 2% paraformaldehyde and immunostained using polyclonal rabbit antibody for NS1 protein (gift from Dr. A. Nieto, Spain). (A) PTD-NS1 protein transduction in N1E cells; (B) PTD-NS1 fusion protein transduction in primary hippocampal neurons.

Figure 6

Schematic diagram showing mechanism of PTD-fusion protein entry into the cell. PTD-fusion protein binds with the heparan sulphate or unknown receptors on the cell surface and enters either via endocytosis or macropinocytosis. Endocytic vesicles fuse with lysosomal vesicles, heparan sulphate from PTD-fusion protein complex is then removed by heparinase present in endosomes, and protein is either released into the cytoplasm or degraded. Chloroquine (lysosomotropic agent) treatment prevents degradation and aids release of fusion protein from lysosomes. The released PTD-fusion protein either stays in the cytoplasm or enters the nucleus, depending on the sequence and nature of the protein under study. Transcellular effect is not possible either for cytoplasmic or nuclear protein, as PTD does not have secretory property.