RESUMO
Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Células HeLa , Heparina/metabolismo , Heparina/farmacologia , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Células NIH 3T3 , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Survivina , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The methyl-CpG binding domain (MBD) is a conserved domain in transcriptional factors that binds to methylated CpG dinucleotide DNA sequences in vertebrates. The complex is comprised of an asymmetric MBD monomer and a symmetric DNA duplex. Therefore, in the complex, each strand of the duplex DNA is in contact with the protein at a distinct surface and thus exhibits a different chemical shift in NMR spectra. Two-dimensional chemical exchange spectroscopy revealed the presence of a stochastic exchange of the two strands of the duplex DNA in the complex at a rate of 4 s (-1) at 25 degrees C, which indicates the existence of a motion of the MBD such that the orientation of the MBD becomes reversed with respect to the DNA duplex. Kinetic and thermodynamic analyses using surface plasmon resonance, quartz crystal microbalance, and isothermal titration calorimetry suggest that the reversal of MBD with respect to the DNA duplex takes place without its complete dissociation from DNA, indicating the presence of an intermediate protein-DNA binding state that allows the protein to undergo a flip motion upon DNA.
Assuntos
Ilhas de CpG , DNA/química , Fosfatos de Dinucleosídeos/metabolismo , Termodinâmica , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , DNA/genética , DNA/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fosfatos de Dinucleosídeos/química , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica/genética , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Ressonância de Plasmônio de SuperfícieAssuntos
Corantes Fluorescentes/administração & dosagem , Proteínas de Fluorescência Verde/administração & dosagem , Ressonância Magnética Nuclear Biomolecular , Oócitos/metabolismo , Espectrometria de Fluorescência/métodos , Proteínas de Xenopus/análise , Animais , Técnicas Citológicas , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/químicaRESUMO
Cellular protein delivery is an emerging technique, by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an E. coli expression vector suited for protein cellular delivery experiments. The plasmid is designed to generate a C-terminal fusion with the 12 amino acid HIV-Tat peptide as a protein transduction domain (PTD), whereas the protein N-terminus is fused to an 17-residue peptide lanthanide-binding tag (LBT). LBT is used for both purification by affinity chromatography and fluorescent detection with Tb(3+) as a coordinating metal. We have employed the TA-cloning site between the two tags, LBT and PTD, according to the PRESAT-vector methodology [N. Goda, T. Tenno, H. Takasu, H. Hiroaki, M. Shirakawa, The PRESAT-vector: asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics, Protein Sci. 13 (2004) 652-658], which facilitates unidirectional cloning of any PCR-amplified DNA fragments corresponding to the protein of interest. A simple three-step protocol consisting of affinity purification of LBT/PTD dual-tagged proteins has also been developed, in which the proteins are purified by heparin-, then immobilized Ni(2+)-, and then heparin-affinity chromatography, in this order. The purified protein is ready for protein delivery experiment, and the delivered protein is visible by fluorescent microscopy. Our LBT/PTD dual-tagged PRESAT-vector provides a powerful research tool for exploring cellular functions of proteins in the post-genomic era.
