Transduction of full-length Tat fusion proteins directly into mammalian cells: analysis of T cell receptor activation-induced cell death.

Currently, delivery of expression vectors, proteins, and/or pharmacologically important peptidyl mimetics to target cells is problematic because of the low percentage of cells targeted, overexpression, size constraints, and bioavailability. Concentration-dependent transduction of full-length proteins and domains directly into cells would serve to alleviate these problems. Previous researchers have demonstrated the ability of proteins linked to the human immunodeficiency virus (HIV) Tat transduction domain to transduce into cells; but because of inefficiencies, this methodological potential has not significantly progressed since 1988.

Transforming growth factor beta targeted inactivation of cyclin E:cyclin-dependent kinase 2 (Cdk2) complexes by inhibition of Cdk2 activating kinase activity.

Transforming growth factor beta (TGF-beta)-mediated G(1) arrest previously has been shown to specifically target inactivation of cyclin D:cyclin-dependent kinase (Cdk) 4/6 complexes. We report here that TGF-beta-treated human HepG2 hepatocellular carcinoma cells arrest in G(1), but retain continued cyclin D:Cdk4/6 activity and active, hypophosphorylated retinoblastoma tumor suppressor protein. Consistent with this observation, TGF-beta-treated cells failed to induce p15(INK4b), down-regulate CDC25A, or increase levels of p21(CIP1), p27(KIP1), and p57(KIP2).

In vivo protein transduction: delivery of a biologically active protein into the mouse.

Delivery of therapeutic proteins into tissues and across the blood-brain barrier is severely limited by the size and biochemical properties of the proteins. Here it is shown that intraperitoneal injection of the 120-kilodalton beta-galactosidase protein, fused to the protein transduction domain from the human immunodeficiency virus TAT protein, results in delivery of the biologically active fusion protein to all tissues in mice, including the brain. These results open new possibilities for direct delivery of proteins into patients in the context of protein therapy, as well as for epigenetic experimentation with model organisms.

Killing HIV-infected cells by transduction with an HIV protease-activated caspase-3 protein.

At present, treatment of HIV infection uses small inhibitory molecules that target HIV protease; however, the emergence of resistant HIV strains is increasingly problematic. To circumvent this, we report here a new 'Trojan horse' strategy to kill HIV-infected cells by exploiting HIV protease. We engineered a transducing, modified, apoptosis-promoting caspase-3 protein, TAT-Casp3, that substitutes HIV proteolytic cleavage sites for endogenous ones and efficiently transduces about 100% of cells, but remains inactive in uninfected cells.