Intracellular delivery of glutathione S-transferase into mammalian cells

Protein transduction domains (PTDs) derived from human immunodeficiency virus Tat protein and herpes simplex virus VP22 protein are useful for the delivery of non-membrane-permeating polar or large molecules into living cells. In the course of our study aiming at evaluating PTD, we unexpectedly found that the fluorescent-dye-labeled glutathione S-transferase (GST) from Schistosoma japonicum without known PTDs was delivered into COS7 cells. The intracellular transduction of GST was also observed in HeLa, NIH3T3, and PC12 cells, as well as in hippocampal primary neurons, indicating that a wide range of cell types is permissive for GST transduction. Furthermore, we showed that the immunosuppressive peptide VIVIT fused with GST successfully inhibits NFAT activation. These results suggest that GST is a novel PTD which may be useful in the intracellular delivery of biologically active molecules, such as small-molecule drugs, bioactive peptides, or proteins.     

Comparison of protein transduction domains in mediating cell delivery of a secreted CRE protein

Protein transduction domains (PTDs) are versatile peptide sequences that facilitate cell delivery of several cargo molecules including proteins. PTDs usually consist of short stretches of basic amino acids that can cross the plasma membrane and gain entry into cells. Traditionally, to assess PTD mediated protein delivery, PTD-fusion proteins have been used as purified proteins. To overcome the requirement for a protein purification step, we used a secretory signal peptide to allow PTD-CRE fusion proteins to be exported from transfected mammalian cells. PTD induced protein transduction into cells was assessed by a CRE-mediated recombination event that resulted in beta-galactosidase expression. Several PTDs were tested including the prototypic TAT, different TAT variants, Antp, MTS and polyarginine. A negative correlation was observed between the cationic charge on the PTD and the extent of secretion. Poor secretion was found when the PTD charge was greater than +5. One TAT-CRE protein variant had a 14-fold enhancement above CRE alone when added to cells in the presence of chloroquine. This PTD domain also enhanced gene expression after plasmid delivery. These data illustrate that some secreted PTD proteins may be useful reagents to improve protein delivery in mammalian systems and a novel approach to enhancing the response to DNA transfections.     

Revised Role of Glycosaminoglycans in TAT Protein Transduction Domain-mediated Cellular Transduction

Cellular uptake of the human immunodeficiency virus TAT protein transduction domain (PTD), or cell-penetrating peptide, has previously been surmised to occur in a manner dependent on the presence of heparan sulfate proteoglycans that are expressed ubiquitously on the cell surface. These acidic polysaccharides form a large pool of negative charge on the cell surface that TAT PTD binds avidly. Additionally, sulfated glycans have been proposed to aid in the interaction of TAT PTD and other arginine-rich PTDs with the cell membrane, perhaps aiding their translocation across the membrane. Surprisingly, however, TAT PTD-mediated induction of macropinocytosis and cellular transduction occurs in the absence of heparan sulfate and sialic acid. Using labeled TAT PTD peptides and fusion proteins, in addition to TAT PTD-Cre recombination-based phenotypic assays, we show that transduction occurs efficiently in mutant Chinese hamster ovary cell lines deficient in glycosaminoglycans and sialic acids. Similar results were obtained in cells where glycans were enzymatically removed. In contrast, enzymatic removal of proteins from the cell surface completely ablated TAT PTD-mediated transduction. Our findings support the hypothesis that acidic glycans form a pool of charge that TAT PTD binds on the cell surface, but this binding is independent of the PTD-mediated transduction mechanism and the induction of macropinocytotic uptake by TAT PTD.     

Noncovalent protein transduction in plant cells by macropinocytosis

* Protein delivery across cellular membranes or compartments is primarily limited by low biomembrane permeability. * Many protein transduction domains (PTDs) have previously been generated, and covalently cross-linked with cargoes for cellular internalization. * An arginine-rich intracellular delivery (AID) peptide could rapidly deliver fluorescent proteins or beta-galactosidase enzyme into plant and animal cells in a noncovalent fashion. The possible mechanism of this noncovalent protein transduction (NPT) may involve macropinocytosis. * The NPT via a nontoxic AID peptide provides a powerful tool characterized by its simplicity and quickness to have active proteins function in living cells in vivo. This should be of broad utility for functional enzyme assays and protein therapies in both plant biology research as well as biomedical applications.     

The importance of valency in enhancing the import and cell routing potential of protein transduction domain-containing molecules

Protein transduction domains (PTDs) are peptides that afford the internalization of cargo macromolecules (including plasmid DNA, proteins, liposomes, and nanoparticles). In the case of polycationic peptides, the efficiency of PTDs to promote cellular uptake is directly related to their molecular mass or their polyvalent presentation. Similarly, the efficiency of routing to the nucleus increases with the number of nuclear localization signals (NLS) associated with a cargo. The quantitative enhancement, however, depends on the identity of the PTD sequence as well as the targeted cell type. Thus the choice and multivalent presentation of PTD and NLS sequences are important criteria guiding the design of macromolecules intended for specific intracellular localization. This review outlines synthetic and recombinant strategies whereby PTDs and signal sequences can be assembled into multivalent peptide dendrimers and promote the uptake and routing of their cargoes. In particular, the tetramerization domain of the tumour suppressor p53 (p53^tet) is emerging as a useful scaffold to present multiple routing and targeting moieties. Short cationic peptides fused to the 31-residue long p53^tet sequence resulted in tetramers displaying a significant enhancement (up to 1000 fold) in terms of their ability to be imported into cells and delivered to the cell nucleus in relation to their monomeric analogues. The design of future polycationic peptide dendrimers as effective delivering vehicles will need to incorporate selective cell targeting functions and provide solutions to the issue of endosomal entrapment.     

蛋白转导域内在化机制的研究进展

蛋白转导域（protein transduction domain,PTD）可以携带外源生物大分子进入细胞,在分子生物学、细胞生物学的基础研究及生物技术应用中,都展示出良好的前景,应用广泛,但机制不甚明确。已知的PTD均有其关键的特定氨基酸存在和较强的正电荷分布,并具有独特的二级结构及空间构象,这些特殊的结构特征对其内在化机制起决定作用。目前认为巨胞饮作用是PTD入胞的主要机制,PTD在经过细胞表面糖胺聚糖紧密结合快速作用及电荷作用后,由脂筏蛋白介导的巨胞饮作用内在化,然后巨胞饮体脂质双层破裂,使蛋白转导域．大分子释放入胞浆及胞核。     

Influence of protein transduction domains on target-specific chimeric proteins

Direct targeting to the cytoplasm and nucleus using protein transduction domains (PTD) has been described to be efficient but non-cell-type-specific, and only has clinical relevance when the molecule is active exclusively in the diseased cell. The use of PTDs is an attractive mechanism to improve drug delivery. In this work, we designed recombinant proteins that contain epidermal growth factor as ligand to render uptake target cell-specific. We evaluated the potential of several PTDs to induce the cytosolic uptake of the catalytic domain of diphtheria toxin by measuring cytotoxicity. Although PTD-dependent membrane transfer is very low, the proteins exhibited concentration-dependent cytotoxic activity. Higher binding at 4 degrees C compared to 37 degrees C suggests that uptake by the PTDs MTS and TLM occurs via an endocytic pathway. Non-specific binding is predominantly a function of the PTD and greatly increases by substitution of a non-polar glycine with a negatively charged glutamate in the PTD HA2.     

Keratinocytes enriched for stem cells are protected from anoikis via an integrin signaling pathway in a Bcl-2 dependent manner

In zebrafish, the basic helix-loop-helix (bHLH) gene neuroD specifies distinct neurons in the spinal cord. A preliminary experiment indicated that a related bHLH gene, ndr1a, normally expressed only in the olfactory organ in late embryos, also functions as neuroD to induce ectopic formation of spinal cord neurons in early embryos after introduction of its mRNA into early embryos. To define the functional specificity of these bHLH proteins, several mutant forms with selected point mutations in the basic domain were constructed and tested for inducing sensory neurons in the spinal cord. Our data indicate that the functional specificity of NeuroD to define sensory neurons is mainly due to a single residue (asparagine 11) in its basic domain.