Jehangir Wadia

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 PTDs 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.

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.

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.

Pathologic Prion Protein Infects Cells by Lipid-Raft Dependent Macropinocytosis

Transmissible spongiform encephalopathies, including variant-Creutzfeldt-Jakob disease (vCJD) in humans and bovine spongiform encephalopathies in cattle, are fatal neurodegenerative disorders characterized by protein misfolding of the host cellular prion protein (PrPC) to the infectious scrapie form (PrPSc). However, the mechanism that exogenous PrPSc infects cells and where pathologic conversion of PrPC to the PrPSc form occurs remains uncertain. Here we report that similar to the mechanism of HIV-1 TAT-mediated peptide transduction, processed mature, full length PrP contains a conserved N-terminal cationic domain that stimulates cellular uptake by lipid raft-dependent, macropinocytosis. Inhibition of macropinocytosis by three independent means prevented cellular uptake of recombinant PrP; however, it did not affect recombinant PrP cell surface association. In addition, fusion of the cationic N-terminal PrP domain to a Cre recombinase reporter protein was sufficient to promote both cellular uptake and escape from the macropinosomes into the cytoplasm. Inhibition of macropinocytosis was sufficient to prevent conversion of PrPC to the pathologic PrPSc form in N2a cells exposed to strain RML PrPSc infected brain homogenates, suggesting that a critical determinant of PrPC conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrPSc infects cells by lipid raft dependent, macropinocytosis.

Chronic exposure to sub‐lethal beta‐amyloid (Aβ) inhibits the import of nuclear‐encoded proteins to mitochondria in differentiated PC12 cells*

Studies on amyloid beta (Aβ|), the peptide thought to play a crucial role in the pathogenesis of Alzheimer’s disease, have implicated mitochondria in Aβ‐mediated neurotoxicity. We used differentiated PC12 cells stably transfected with an inducible green fluorescent protein (GFP) fusion protein containing an N′‐terminal mitochondrial targeting sequence (mtGFP), to examine the effects of sub‐lethal Aβ on the import of nuclear‐encoded proteins to mitochondria. Exposure to sub‐lethal Aβ25–35 (10 μmol/L) for 48 h inhibited mtGFP import to mitochondria; average rates decreased by 20 ± 4%. Concomitant with the decline in mtGFP, cytoplasmic mtGFP increased significantly while mtGFP expression and intramitochondrial mtGFP turnover were unchanged. Sub‐lethal Aβ1–42 inhibited mtGFP import and increased cytoplasmic mtGFP but only after 96 h. The import of two endogenous nuclear‐encoded mitochondrial proteins, mortalin/mtHsp70 and Tom20 also declined. Prior to the decline in import, mitochondrial membrane potential (mmp), and reactive oxygen species levels were unchanged in Aβ‐treated cells versus reverse phase controls. Sustained periods of decreased import were associated with decreased mmp, increased reactive oxygen species, increased vulnerability to oxygen‐glucose deprivation and altered mitochondrial morphology. These findings suggest that an Aβ‐mediated inhibition of mitochondrial protein import, and the consequent mitochondrial impairment, may contribute to Alzheimer’s disease.

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

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.

DNA Delivery into Mammalian Cells Using Bacteriophage λ Displaying the TAT Transduction Domain

The ability to modulate cellular function by the transfer and expression of novel genes or by affecting the levels of endogenous proteins by genetic means has been of tremendous benefit in studying cellular functions and offers great promise in the treatment of a variety of diseases. Consequently, the development of novel and efficient nonviral DNA delivery systems is an important goal. Small cationic peptides, termed peptide/protein transduction domains (PTDs), effectively deliver a wide variety of cargoes, including DNA, into all cells. Expression of the human immunodeficiency virus type 1 (HIV-1) TAT PTD on the surface of bacteriophage λ results in the efficient delivery of plasmid DNA into a variety of cells in a concentration-dependent manner without cytotoxicity. This protocol describes the preparation of recombinant λ particles with a TAT peptide transduction domain sequence on their surface and the use of these particles in delivery of plasmid DNA into a variety of cells.

Structural basis for recognition of the central conserved region of RSV G by neutralizing human antibodies

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly, and yet there remains no effective treatment or vaccine. The surface of the virion is decorated with the fusion glycoprotein (RSV F) and the attachment glycoprotein (RSV G), which binds to CX3CR1 on human airway epithelial cells to mediate viral attachment and subsequent infection. RSV G is a major target of the humoral immune response, and antibodies that target the central conserved region of G have been shown to neutralize both subtypes of RSV and to protect against severe RSV disease in animal models. However, the molecular underpinnings for antibody recognition of this region have remained unknown. Therefore, we isolated two human antibodies directed against the central conserved region of RSV G and demonstrated that they neutralize RSV infection of human bronchial epithelial cell cultures in the absence of complement. Moreover, the antibodies protected cotton rats from severe RSV disease. Both antibodies bound with high affinity to a secreted form of RSV G as well as to a peptide corresponding to the unglycosylated central conserved region. High-resolution crystal structures of each antibody in complex with the G peptide revealed two distinct conformational epitopes that require proper folding of the cystine noose located in the C-terminal part of the central conserved region. Comparison of these structures with the structure of fractalkine (CX3CL1) alone or in complex with a viral homolog of CX3CR1 (US28) suggests that RSV G would bind to CX3CR1 in a mode that is distinct from that of fractalkine. Collectively, these results build on recent studies demonstrating the importance of RSV G in antibody-mediated protection from severe RSV disease, and the structural information presented here should guide the development of new vaccines and antibody-based therapies for RSV.