RESUMO
The piggyBac (PB) transposable element has recently accumulated enormous attention as a tool for the transgenesis in various eukaryotic organisms. Arginine-rich cell-penetrating peptides (CPPs) are protein transduction domains containing a large amount of basic amino acids that were found to be capable of delivering biologically active macromolecules into living cells. In this study, we demonstrate a strategy, which we called "transposoduction", which is a one-plasmid gene delivery system mediated by the nontoxic CPP-piggyBac transposase (CPP-PBase) fusion protein to accomplish both protein transduction and transposition. CPPs were proven to be able to synchronously deliver covalently linked PBase and noncovalently linked a cis plasmid into human cells. The expression of promoterless reporter genes coding for red (dTomato) and yellow (mOrange) fluorescent proteins (RFP and YFP) with PB elements could be detected in cells treated with the PBase-expressing plasmid after 3 days indicating transposition of coding regions to downstream of endogenous promoter sequences. An enhanced green fluorescent protein (EGFP) plasmid-based excision assay further confirmed the efficiency of the bifunctional CPP-PBase fusion protein. In conclusion, this strategy representing a combinational concept of both protein transduction and mobile transposition may provide tremendous potential for safe and efficient cell line transformation, gene therapy and functional genomics.
Assuntos
Peptídeos Penetradores de Células/administração & dosagem , Técnicas de Transferência de Genes , Transposases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Transfecção , Transposases/genéticaRESUMO
Protein transduction domains comprised of basic amino acid-rich peptides, can efficiently deliver covalently fused macromolecules into cells. Quantum dots (QDs) are luminescent semiconductor nanocrystals that are finding increasing application in biological imaging. Previous studies showed that protein transduction domains mediate the internalization of covalently attached QDs. In this study, we demonstrate that arginine-rich intracellular delivery peptides (cell-penetrating peptides; CPPs), analogs of naturally-occuring protein transduction domains, deliver noncovalently associated QDs into living cells; CPPs dramatically increase the rate and efficiency of cellular uptake of QD probes. The optimal molecular ratio between arginine-rich CPPs and QD cargoes for cellular internalization is approximately 60:1. Upon entry into cells, the QDs are concentrated in the perinuclear region. There is no cytotoxicity following transport of QDs present at concentrations up to 200 nM. The mechanism for arginine-rich CPP/QD complexes to traverse cell membrane appears to involve a combination of internalization pathways. These results provide insight into the mechanism of arginine-rich CPP delivery of noncovalently attached cargoes, and may provide a powerful tool for imaging in vivo.
Assuntos
Peptídeos Penetradores de Células/farmacocinética , Peptídeos/farmacocinética , Pontos Quânticos , Análise de Variância , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Ensaio de Desvio de Mobilidade Eletroforética , Endocitose/efeitos dos fármacos , Humanos , Tamanho da Partícula , Peptídeos/química , Transporte Proteico/efeitos dos fármacos , Espectrometria de FluorescênciaRESUMO
BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor (TNF) family, has shown potent and high selective antitumor activity as a promising therapy for cancer. We have developed an arginine-rich intracellular delivery (AID) peptide-mediated system for nontoxic and efficient gene transfer in cells. MATERIALS AND METHODS: To evaluate antitumor activity and therapeutic potential of TRAIL gene, a bifunctional expression plasmid was constructed encoding the secretory signal peptide of human immunoglobulin kappa (Ig kappa) light chain, the extracellular portion (amino acids 95-281) of human TRAIL and the humanized green fluorescent protein (GFP). RESULTS: We demonstrated that AID peptides were able to effectively deliver TRAIL gene into human lung carcinoma A549 cells. Soluble TRAIL-GFP protein purified from media after gene delivery was further evaluated regarding selective induction of apoptosis in cells. CONCLUSION: AID peptide-mediated DNA transfer provides a potential and convenient tool in nonviral gene therapy.
Assuntos
Apoptose , Terapia Genética , Neoplasias/terapia , Peptídeos/administração & dosagem , Ligante Indutor de Apoptose Relacionado a TNF/genética , Arginina , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Humanos , Marcação In Situ das Extremidades Cortadas , PlasmídeosRESUMO
Protein transduction domains (PTDs) are small peptides with a high content of basic amino acids, and they are responsible for cellular uptake. Many PTDs, including arginine-rich intracellular delivery (AID) peptides, have been shown to transport macromolecules across membranes and into cells. In this study, we demonstrated for the first time that AID peptides could rapidly and efficiently deliver proteins into plant cells in both covalent and noncovalent protein transductions (CNPT) simultaneously. The optimal molecular ratio between an AID peptide carrier and cargo in CNPT was about 3:1. Fluorescence resonance energy transfer (FRET) analysis revealed protein-protein interactions between AID peptide carriers and cargos after CNPT in cells. The possible mechanisms of AID peptides-mediated cellular entry might involve a combination of multiple internalization pathways. Therefore, applications by AID peptide-mediated CNPT may provide a simple and direct transport strategy for delivering two proteins in agricultural systems.