Transduction of a Functional Domain of the AT ₁ Receptor in Neurons by HIV-Tat PTD

Abstract

Abstract-Despite advances in transgenic and gene transfer technologies, in vivo structure-function studies of the angiotensin II type I receptor (AT 1 R) have revealed limited information on the diverse actions of angiotensin II. Our objective in the present study was to determine if protein transduction technology with the use of the HIV-Tat protein transduction domain could fill this gap. Recombinant HIV-Tat protein transduction domain fused to EGFP and to the third intracellular loop of the AT 1 R was expressed. Incubation of hypothalamus and brainstem neurons with this peptide indicated an efficient transport of the protein to most of the cells. This transduction was accompanied by an increase in neuronal firing rate, an effect similar to that observed with angiotensin II stimulation of the neuronal AT 1 R. The characteristics of the chronotropic effects of recombinant third intracellular loop and its synthetic counterpart were similar and comparable to the effects of angiotensin II on these neurons. In addition, in the presence of the protein kinase C inhibitor calphostin C, the peptide failed to increase firing rate. These observations demonstrated that transduction of neurons with the third intracellular loop of the AT 1 R produces chronotropic effects similar to those induced by angiotensin II.

Tapasin modification on the intracellular epitope HBcAg18–27 enhances HBV-specific CTL immune response and inhibits hepatitis B virus replication in vivo

“…However, the majority of PTD and PTD-conjugated molecules translocates to the nucleus rather than to the cytoplasm after transduction, owing to the functional nuclear localization sequence (NLS). 38,39 A new approach that appears to be the safest is to produce recombinant proteins exogenously and then deliver them systemically or by localized injections into the target cells or tissues. In comparison with PTD, CTP and its fusion partners exhibited a clear preference for cytoplasmic localization, and also markedly enhanced membrane transduction potential.…”

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

“…Subsequently, more detailed analysis of Tat protein [21,47,50,51] identified a PTD of 9-11 aa residues, a basic domain that can transduce itself, as well as the bigger proteins fused with it, into the cells [15,16,46,52]. In vitro PTD-fusion protein is used by direct addition to the cell culture medium, which resulted in [53][54][55][56][57] efficient transduction of PTD-AT1 receptor domain and PTD-BCL-x in neurons [58,59]. In another example, PTD-PDX1 fusion protein (PDX1 is a transcription factor that plays a central role in pancreatic development) has shown remarkable biological activity and induction of insulin production in human embryonic stem cells [60].…”

Development of a soluble PTD-HPV18E7 fusion protein and its functional characterization in eukaryotic cells

“…PTD of 11 amino acid residues, the basic transduction domain, can transduce itself as well as other bigger proteins fused with it into the cells [22][23][24]. Despite the fact that the E7 protein is the focus of many studies in cervical carcinogenesis, the detailed molecular mechanism and biological properties that are linked to the malignant transformation of the HPV-infected cells are still unclear.…”