Steven F. Dowdy

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.

Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration.

Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27 Kip1 induces cell migration

CDK Inhibitors: Cell Cycle Regulators and Beyond

First identified as cell cycle inhibitors mediating the growth inhibitory cues of upstream signaling pathways, the cyclin-CDK inhibitors of the Cip/Kip family p21Cip1, p27Kip1, and p57Kip2 have emerged as multifaceted proteins with functions beyond cell cycle regulation. In addition to regulating the cell cycle, Cip/Kip proteins play important roles in apoptosis, transcriptional regulation, cell fate determination, cell migration and cytoskeletal dynamics. A complex phosphorylation network modulates Cip/Kip protein functions by altering their subcellular localization, protein-protein interactions, and stability. These functions are essential for the maintenance of normal cell and tissue homeostasis, in processes ranging from embryonic development to tumor suppression.

Protein transduction: unrestricted delivery into all cells?

Several proteins can traverse biological membranes through protein transduction. Small sections of these proteins (10-16 residues long) are responsible for this. Linking these domains covalently to compounds, peptides, antisense peptide nucleic acids or 40-nm iron beads, or as in-frame fusions with full-length proteins, lets them enter any cell type in a receptor- and transporter-independent fashion. Moreover, several of these fusions, introduced into mice, were delivered to all tissues, even crossing the blood-brain barrier. These domains thus might let us address new questions and even help in the treatment of human disease.

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.

In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA

The in vivo Tat–β-gal studies in mice show that protein transduction holds a tremendous amount of potential for manipulation of model organisms and protein therapy; however, this needs to be tempered until several important questions are answered. First, Tat–β-gal is 120 kDa and requires assembly into a homotetramer before it is active, although it has a high specific activity and therefore, probably requires a low level of protein to detect in tissue sections. The administration of additional Tat fusion proteins to model organisms that generate phenotypic changes is necessary to get a sense of how much potential in vivo protein transduction really holds.Second, the immunogenicity of transduced proteins is a significant and currently unknown concern. Although future protein therapies will probably comprise proteins that are ∼99% of human origin, the PTD might be antigenic or other aspects of the delivery might initiate an immune response. Several variations of PTDs have been described and therefore an immune response to one PTD might be engineered by alteration of anchor residues or TCR recognition motifs on the PTD to reduce or avoid major histocompatibility complex (MHC) presentation. However, what does the immune system do when all antigen-presenting cells (including macrophages, and T and B cells) are presenting the same foreign antigen? This presents a unique unanswered immunological question. One qualitative piece of evidence does exist; we injected mice once a day for 14 consecutive days with Tat–GFP protein and we failed to see any gross signs of a severe immune response. This observation also suggests that continuous transduction of proteins into all cells of a mouse, including into the brain, are not detrimental to viability.Lastly, transduced proteins appear to be cleared from the body not by conventional mechanisms, but based on the half-life of the protein. Therefore, the half-life of the PTD fusion proteins could be extended or shortened depending on the specific application. The most exciting aspect of this technology is the previously unheard-of ability to address specific mutations and deletions of certain disease-causing genes. We look forward to being able to restore tumour suppressor function or interfere with various oncogenic pathways using this technology in the coming years.

A common E2F-1 and p73 pathway mediates cell death induced by TCR activation.

Strong stimulation of the T-cell receptor (TCR) on cycling peripheral T cells causes their apoptosis by a process called TCR-activation-induced cell death (TCR-AICD). TCR-AICD occurs from a late G1 phase cell-cycle check point independently of the ‘tumour suppressor’ protein p53 (refs 5, 6). Disruption of the gene for the E2F-1 transcription factor, an inducer of apoptosis, causes significant increases in T-cell number and splenomegaly. Here we show that T cells undergoing TCR-AICD induce the p53-related gene p73, another mediator of apoptosis, which is hypermethylated in lymphomas. Introducing a dominant-negative E2F-1 protein or a dominant-negative p73 protein into T cells protects them from TCR-mediated apoptosis, whereas dominant-negative E2F-2, E2F-4 or p53 does not. Furthermore, E2F-1-null or p73-null primary T cells do not undergo TCR-mediated apoptosis either. We conclude that TCR-AICD occurs from a late G1 cell-cycle checkpoint that is dependent on both E2F-1 and p73 activities. These observations indicate that, unlike p53, p73 serves to integrate receptor-mediated apoptotic stimuli.

Efficient siRNA delivery into primary cells by a peptide transduction domain-dsRNA binding domain fusion protein.

RNA interference (RNAi) induced by short interfering RNA (siRNA) allows for discovery research and large-scale screening; however, owing to their size and anionic charge, siRNAs do not readily enter cells. Current approaches do not deliver siRNAs into a high percentage of primary cells without cytotoxicity. Here we report an efficient siRNA delivery approach that uses a peptide transduction domain–double-stranded RNA-binding domain (PTD-DRBD) fusion protein. DRBDs bind to siRNAs with high avidity, masking the siRNAs negative charge and allowing PTD-mediated cellular uptake. PTD-DRBD–delivered siRNA induced rapid RNAi in a large percentage of various primary and transformed cells, including T cells, human umbilical vein endothelial cells and human embryonic stem cells. We observed no cytotoxicity, minimal off-target transcriptional changes and no induction of innate immune responses. Thus, PTD-DRBD–mediated siRNA delivery allows efficient gene silencing in difficult-to-transfect primary cell types.

Cell Penetrating Peptides in Drug Delivery

Protein transduction domains (PTDs) are small cationic peptides that can facilitate the uptake of large, biologically active molecules into mammalian cells. Recent reports have suggested that PTDs may be able to mediate the delivery of cargo to tissues throughout a living organism. Such technology could eliminate the size restrictions on usable drugs, enabling previously unavailable large molecules to modulate in vivo biology and alleviate disease. In this article, we review the evidence that PTDs can be used both to deliver active molecules to pathological tissue in vivo and to treat models of disease such as ischemia, inflammation, and cancer.

Novel p27kip1 C-Terminal Scatter Domain Mediates Rac-Dependent Cell Migration Independent of Cell Cycle Arrest Functions

ABSTRACT Hepatocyte growth factor (HGF) signaling via its receptor, the proto-oncogene Met, alters cell proliferation and motility and has been associated with tumor metastasis. HGF treatment of HepG2 human hepatocellular carcinoma cells induces cell migration concomitant with increased levels of the p27kip1 cyclin-cdk inhibitor. HGF signaling resulted in nuclear export of endogenous p27 to the cytoplasm, via Ser-10 phosphorylation, where it colocalized with F-actin. Introduction of transducible p27 protein (TATp27) was sufficient for actin cytoskeletal rearrangement and migration of HepG2 cells. TATp27 mutational analysis identified a novel p27 C-terminal domain required for cell migration, distinct from the N-terminal cyclin-cyclin-dependent kinase (cdk) binding domain. Loss or disruption of the p27 C-terminal domain abolished both actin rearrangement and cell migration. The cell-scattering activity of p27 occurred independently of its cell cycle arrest functions and required cytoplasmic localization of p27 via Ser-10 phosphorylation. Furthermore, Rac GTPase was necessary for p27-dependent migration but alone was insufficient for HepG2 cell migration. These results predicted a migration defect in p27-deficient cells. Indeed, p27-deficient primary fibroblasts failed to migrate, and reconstitution with TATp27 rescued the motility defect. These observations define a novel role for p27 in cell motility that is independent of its function in cell cycle inhibition.