Biomedical subjects

Jehangir S Wadia

Publications and source records attributed to Jehangir S Wadia.

6 recordsLinked to original sources

PubMed2005

Cationic TAT peptide transduction domain enters cells by macropinocytosis.

Naturally occurring and synthetic short arginine containing protein transduction domains (PTDs), including HIV1 TAT, poly-Arg and Antp, have been used to deliver a wide variety of macromolecular, biologically active therapeutic cargo into cells, including peptides, proteins, antisense oligonucleotides and liposomes, in vitro and to treat pre-clinical models of cancer and stroke. PTDs enter cells in a rapid, receptor-independent fashion. Recently, large TAT-fusion proteins (in excess of 30,000 Da) were shown to transduce into cells by fluid-phase macropinocytosis, a specialized form of endocytosis that is independent of caveolae, clathrin and dynamin. However, it remains controversial as to whether or not PTD peptides (1000-5000 Da) enter cells via macropinocytosis and/or through an unknown alternative mechanism. Due to strong ionic interactions with the cell surface, previous measurements of PTD peptide internalization were inaccurate. Cationic PTD peptides containing variable numbers of arginine residues and conditions entered cells exclusively through macropinocytosis. In addition, no PTD peptide was found to enter cells at 4 degrees C, a long held assumption of transduction. Taken together, these observations provide a solid scientific basis for the development of novel biologically active transducible anticancer PTD peptide therapeutics.

Animals↗

PubMed2004

Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer.

The direct intracellular delivery of proteins, or active peptide domains, has, until recently, been difficult to achieve due primarily to the bioavailability barrier of the plasma membrane, which effectively prevents the uptake of macromolecules by limiting their passive entry. Traditional approaches to modulate protein function have largely relied on the serendipitous discovery of specific drugs and small molecules which could be delivered easily into the cell. However, the usefulness of these pharmacological agents is limited by their tissue distribution and unlike 'information-rich' macromolecules, they often suffer from poor target specificity, unwanted side-effects, and toxicity. Likewise, the development of molecular techniques, over the past several decades, for gene delivery and expression of proteins has provided for tremendous advances in our understanding of cellular processes but has been of surprisingly little benefit for the management of genetic disorders. Apart from these gains however, the transfer of genetic material into eukaryotic cells either using viral vectors or by non-viral mechanisms such as microinjection, electroporation, or chemical transfection remains problematic. Moreover, in vivo, gene therapy approaches relying on adenoviral vectors are associated with significant difficulties relating to a lack of target specificity and toxicity which have contributed to poor performance in several clinical trials. Remarkably, the recent identification of a particular group of proteins with enhanced ability to cross the plasma membrane in a receptor-independent fashion has led to the discovery of a class of protein domains with cell membrane penetrating properties. The fusion of these protein transduction domain peptide sequences with heterologous proteins is sufficient to cause their rapid transduction into a variety of different cells in a rapid, concentration-dependent manner. Moreover, this novel technique for protein and peptide delivery appears to circumvent many problems associated with DNA and drug based methods. This technique may represent the next paradigm in our ability to modulate cell function and offers a unique avenue for the treatment of disease.

Animals↗

PubMed2004

Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis.

The TAT protein transduction domain (PTD) has been used to deliver a wide variety of biologically active cargo for the treatment of multiple preclinical disease models, including cancer and stroke. However, the mechanism of transduction remains unknown. Because of the TAT PTD's strong cell-surface binding, early assumptions regarding cellular uptake suggested a direct penetration mechanism across the lipid bilayer by a temperature- and energy-independent process. Here we show, using a transducible TAT-Cre recombinase reporter assay on live cells, that after an initial ionic cell-surface interaction, TAT-fusion proteins are rapidly internalized by lipid raft-dependent macropinocytosis. Transduction was independent of interleukin-2 receptor/raft-, caveolar- and clathrin-mediated endocytosis and phagocytosis. Using this information, we developed a transducible, pH-sensitive, fusogenic dTAT-HA2 peptide that markedly enhanced TAT-Cre escape from macropinosomes. Taken together, these observations provide a scientific basis for the development of new, biologically active, transducible therapeutic molecules.

Amiloride↗

PubMed2003

Flow cytometry and GFP: a novel assay for measuring the import and turnover of nuclear-encoded mitochondrial proteins in live PC12 cells.

BACKGROUND: Mitochondrial protein import is typically measured by adding radiolabeled precursor proteins to isolated mitochondria. We have developed a novel, high-throughput method for measuring protein import in live differentiated PC12 cells using a tetracycline (Tet) regulated, nuclear encoded, mitochondrially-targeted GFP fusion protein and flow cytometry. METHODS: We generated a PC12 cell line stably transfected with an inducible GFP fusion protein (GFPmt) targeted to mitochondria. GFPmt PC12 cells were treated with NGF for one week to induce neuronal differentiation in the presence of Tet to silence GFP expression. On day seven GFPmt expression was induced by removal of Tet and these "GFP-on" cells were exposed to sublethal levels of CCCP (2 microM) for 24 h. At 24 h, the cells were harvested in Ca(++)-free PBS and the GFPmt signal in live intact cells was measured using flow cytometry. Since GFPmt is not fluorescent prior to being imported into mitochondria, the GFPmt signal reflected only GFPmt imported to mitochondria. PI was used to gate out contributions from dead cells. Turnover of GFPmt in mitochondria was also assessed; in this case, Tet was added to arrest GFPmt expression in GFP-on cells, and the subsequent decline of the fluorescent signal, in the absence of any new GFP synthesis, was measured by flow cytometry. RESULTS: Exposure to 2 microM CCCP for 24 h caused a 61% +/- 0.4 decline in GFPmt fluorescence compared to controls. This decline corresponded to a 30% +/- 7 decrease in GFPmt protein levels measured by Western blot of mitochondrial fractions, and a 72% +/- 5 decline in the import of newly synthesized GFPmt to mitochondria over a 1 h period 24-h after addition of 2 microM CCCP measured by autoradiography. CCCP partially depolarized mitochondria but was not lethal for up to five days. CONCLUSIONS: This novel GFP-based flow cytometry assay is a rapid and sensitive technique for quantifying protein import to mitochondria in live neuronal cells.

Animals↗

PubMed2003

Modulation of cellular function by TAT mediated transduction of full length proteins.

Due to the barrier imposed by the cell membrane, delivery of macromolecules in excess of 500 Daltons directly into cells remains problematic. However, proteins, which have been evolutionarily selected to perform specific functions, are therefore an attractive therapeutic agent to treat a variety of human diseases. In practice, the direct intracellular delivery of these proteins has, until recently, been difficult to achieve due primarily to the bioavailability barrier of the plasma membrane, which effectively prevents the uptake of the majority of peptides and proteins by limiting their passive entry. However, recent work using small cationic peptides, termed protein transduction domains (PTDs), derived from nucleic acid binding proteins, such as HIV TAT protein or the Dros. m. transcription factor Antp. or synthetic poly-Arginine, have now been shown to deliver a myriad of molecules, including synthetic small molecules, peptides and proteins, into animal models in vivo. Here, we focus on the delivery of biologically active, full length proteins to treat pre-clinical disease models.

Animals↗

PubMed2002

Protein transduction technology.

Intracellular delivery of macromolecules remains problematic because of the bioavailability restriction imposed by the cell membrane. Recent studies on protein transduction domains have circumvented this barrier, however, and have resulted in the delivery of peptides, full-length proteins, iron beads, liposomes, and radioactive isotopes into cells in culture and animal models in vivo.

Animals↗