ABSTRACT
One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.
Subject(s)
Cytosol/metabolism , Drug Carriers/chemistry , Endosomes/metabolism , Spider Venoms/chemistry , Amino Acid Sequence , Amino Acid Substitution , Cell Membrane/metabolism , Drug Liberation , Endocytosis/physiology , HeLa Cells , Humans , Immunoglobulin G/metabolism , Integrases/pharmacokinetics , Pinocytosis/physiology , Protein Engineering , Saporins/pharmacokinetics , Spider Venoms/geneticsABSTRACT
Using native chemical ligation, we constructed a Ca(2+)-gated fusion channel protein consisting of alamethicin and the C-terminal domain of calmodulin. At pH 5.4 and in the absence of Ca(2+), this fusion protein yielded a burst-like channel current with no discrete channel conductance levels. However, Ca(2+) significantly lengthened the specific channel open state and increased the mean channel current, while Mg(2+) produced no significant changes in the channel current. On the basis of 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescent measurement, Ca(2+)-stimulated gating may be related to an increased surface hydrophobicity of the extramembrane segment of the fusion protein.
Subject(s)
Alamethicin/chemistry , Calcium Channels/chemistry , Calcium/metabolism , Calmodulin/chemistry , Alamethicin/chemical synthesis , Alamethicin/metabolism , Amino Acid Sequence , Calcium Channels/chemical synthesis , Calcium Channels/metabolism , Calmodulin/chemical synthesis , Calmodulin/metabolism , Chemistry Techniques, Synthetic , Fluorescence Resonance Energy Transfer , Ion Channel Gating , Molecular Sequence Data , Protein Conformation , Protein Structure, TertiaryABSTRACT
Expressed protein ligation (EPL) is a useful method for the native chemical ligation of proteins with other proteins or peptides. This study assessed the practicability of EPL in the preparation of fusion proteins of enhanced green fluorescent protein (EGFP) with chemically synthesized cell-penetrating peptides (CPPs) for intracellular delivery. Using intein-mediated purification with an affinity chitin-binding tag (IMPACT) system, the thioester of EGFP (EGFP-SR) was prepared. Optimization of the ligation of EGFP-SR with arginine 12-mer (R12) produced the fusion protein in high yield. The EPL procedure also allows the preparation of EGFP-R12 containing a low level of endotoxin (ET), via the satisfactory ET removal of EGFP-SR prior to ligation with the R12 peptide. Fusion proteins of EGFP with R12 and the d-isomer of R12 prepared by EPL showed similar levels of cellular uptake compared to the fusion protein directly expressed in Escherichiacoli.
Subject(s)
Endotoxins , Recombinant Fusion Proteins , Animals , Cell-Penetrating Peptides/biosynthesis , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/genetics , Cell-Penetrating Peptides/isolation & purification , Cell-Penetrating Peptides/pharmacology , Chromatography, Affinity/methods , Escherichia coli , Gene Expression , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/pharmacology , HeLa Cells , Humans , Inteins , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/pharmacologyABSTRACT
pH-Sensitive peptides and polymers have been employed as additives to enhance the cytosolic delivery of drugs and genes by facilitating their endosomal escape. However, little attention has been paid to the intracellular fate of these peptides and polymers. In this study, we explored the possibility of utilizing GALA, a pH-sensitive fusogenic peptide, as a cytosol-targeting vehicle. In combination with cationic liposomes, Lipofectamine 2000 (LF2000), the feasibility of this approach for the cytosolic targeting of proteins and nanoparticles was exemplified through the delivery of avidin (68 kDa) and streptavidin-coated quantum dots (15-20 nm) in serum-containing medium. The use of cationic liposomes is critical to enhance the cell-surface adhesion of the GALA conjugates and eventual endosomal uptake. Circular dichroism studies suggest that the GALA can be liberated from cationic liposomes at a reducing pH to form a helical structure and this may eventually lead to disruption of the endosomal membrane to achieve an efficient leakage of the GALA conjugates into the cytosol.
Subject(s)
Cytosol/metabolism , Lipids/chemistry , Liposomes/chemistry , Liposomes/metabolism , Peptides/chemistry , Peptides/metabolism , Amino Acid Sequence , Animals , Avidin/metabolism , Cell Line , Cricetinae , Diffusion , Endosomes/metabolism , Humans , Hydrogen-Ion Concentration , Lipids/administration & dosage , Liposomes/administration & dosage , Mice , Molecular Sequence Data , Peptides/administration & dosage , Protein Transport , Quantum Dots , Streptavidin/metabolismABSTRACT
Modulation of nitric oxide (NO) production is considered a promising approach to therapy of diseases involving excessive inducible nitric oxide synthase (iNOS) expression, such as certain neuronal diseases. Recombinant arginine deiminase (rADI, EC3.5.3.6) catalyzes the conversion of L-arginine (L-arg), the sole substrate of NOS for NO production, to L-citrulline (L-cit) and ammonia. To understand the effect of the depletion of L-arg by rADI on NO concentration and neuroprotection, a direct coculture of neuron SHSY5Y cells and microglia BV2 cells treated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) was used as a model of iNOS induction. The results showed that rADI preserved cell viability (4-fold higher compared with the cells treated with LPS/IFN-gamma only) by the MTT assay, corresponding with the results of neuronal viability by neuron-specific immunostaining assay. NO production (mean +/- SD) decreased from 67.0 +/- 1.3 to 19.5 +/- 5.5 microM after a 2-day treatment of rADI by the Griess assay; meanwhile, induction of iNOS protein expression by rADI was observed. In addition, rADI substantially preserved the neuronal function of dopamine uptake in the coculture. The replenishment of L-arg in the coculture eliminated the neuroprotective and NO-suppressive effects of rADI in the coculture, indicating that L-arg played a crucial role in the effects of rADI. These results highlight the important role of L-arg in the neuron-microglia coculture in excessive induction of iNOS. Regulation of L-arg by ADI demonstrated that rADI has a potentially therapeutic role in iNOS-related neuronal diseases.
