ABSTRACT
BACKGROUND: In the field of plastic surgery, capsular contracture after silicone breast implant surgery is a major clinical problem. This experimental study confirms that the synthetic tryptophan metabolite N-(3',4'-dimethoxycinnamonyl) anthranilic acid (Tranilast) reduces capsule formation and prevents capsular contracture. METHODS: Eighteen New Zealand white rabbits were divided into 2 groups. In the experimental group, implants were inserted into each rabbit, and oral synthetic tryptophan metabolite was administered daily at a dose of 5 mg/kg in 10 mL of saline. In the control group, rabbits received implants and the same amount of saline without the metabolite. After 2 months, peri-implant tissues were harvested and analyzed. RESULTS: The thickness of the capsules and the inflammatory cell counts were decreased in the experimental group (P < 0.001). The collagen fibers in the experimental group were thinner, less dense, and more organized than in control group. The results of reverse transcription quantitative polymerase chain reaction analysis showed that the genes for transforming growth factor ß1 (P = 0.002), alpha smooth muscle actin (P < 0.001), and collagen types I (P = 0.002) and III (P = 0.004) were underexpressed in the experimental groups. Furthermore, the counts of T-cell immunity-related cytokine presenting cells were decreased in the experimental groups (CD3, 4, 25, 45RA, 45RO, 69, interleukin-2, 4 [P < 0.001], and interferon γ [P = 0.028]). CONCLUSIONS: This study confirms that a synthetic derivative of a tryptophan metabolite decreases capsule formation and prevents capsular contracture by inhibiting the differentiation of fibroblasts to myofibroblasts, selectively inhibiting collagen synthesis, and decreasing specific T-cell immune responses by changing anti-inflammatory cytokine expression.
Subject(s)
Breast Implants/adverse effects , Implant Capsular Contracture/prevention & control , Silicone Gels/adverse effects , ortho-Aminobenzoates/pharmacology , Actins/metabolism , Animals , Collagen/metabolism , Cytokines/metabolism , Disease Models, Animal , Female , Rabbits , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor beta1/metabolismABSTRACT
The modulation of a cell signaling process using a molecular binder followed by an analysis of the cellular response is crucial for understanding its role in the cellular function and developing pharmaceuticals. Herein, we present the modulation of the ERK2-mediated signaling pathway through the cytosolic delivery of a native regulatory protein for ERK2, that is, PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa), and its engineered variants using a bacterial toxin-based delivery system. Based on biochemical and structural analyses, PEA-15 variants with different phosphorylation sites and a high affinity for ERK2 were designed. Semi-rational approach led to about an 830-fold increase in the binding affinity of PEA-15, resulting in more effective modulation of the ERK2-mediated signaling. Our approach enabled an understanding of the cellular function of the ERK2-mediated signaling process and the effect of PEA-15 phosphorylation on its action as an ERK2 blocker. We demonstrated the utility and potential of our approach by showing an efficient cytosolic delivery of these PEA-15 variants and the effective suppression of cell proliferation through the inhibition of the ERK2 function. The present approach can be used broadly for modulating the cell signaling processes and understanding their roles in cellular function, as well as for the development of therapeutics.
Subject(s)
Bacterial Toxins/metabolism , Cytosol/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Phosphoproteins/metabolism , Protein Engineering , Animals , Apoptosis Regulatory Proteins , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , CHO Cells , Cricetulus , Drug Delivery Systems , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , K562 Cells , Mice , Mitogen-Activated Protein Kinase 1/chemistry , Mitogen-Activated Protein Kinase 1/genetics , Molecular Dynamics Simulation , NIH 3T3 Cells , Phosphoproteins/chemistry , Phosphoproteins/geneticsABSTRACT
With the high efficacy of protein-based therapeutics and plenty of intracellular drug targets, cytosolic protein delivery in a cell-specific manner has attracted considerable attention in the field of precision medicine. Herein, we present an intracellular protein delivery system based on a target-specific repebody and the translocation domain of Pseudomonas aeruginosa exotoxin A. The delivery platform was constructed by genetically fusing an EGFR-specific repebody as a targeting moiety to the translocation domain, while a protein cargo was fused to the C-terminal end of the delivery platform. The delivery platform was revealed to efficiently translocate a protein cargo to the cytosol in a target-specific manner. We demonstrate the utility and potential of the delivery platform by showing a remarkable tumor regression with negligible toxicity in a xenograft mice model when gelonin was used as the cytotoxic protein cargo. The present platform can find wide applications to the cell-selective cytosolic delivery of diverse proteins in many areas.
Subject(s)
ADP Ribose Transferases/chemistry , Antineoplastic Agents, Phytogenic/administration & dosage , Bacterial Toxins/chemistry , Drug Carriers/chemistry , Exotoxins/chemistry , Neoplasms/drug therapy , Ribosome Inactivating Proteins, Type 1/administration & dosage , Virulence Factors/chemistry , Animals , Antineoplastic Agents, Phytogenic/therapeutic use , Cell Line, Tumor , Drug Delivery Systems , Humans , Male , Mice , Mice, Inbred BALB C , Models, Molecular , Protein Domains , Ribosome Inactivating Proteins, Type 1/therapeutic use , Pseudomonas aeruginosa Exotoxin AABSTRACT
Age-related macular degeneration (AMD) is the leading cause of vision loss and blindness among people over the age of 60. Vascular endothelial growth factor (VEGF) plays a major role in pathological angiogenesis in AMD. Herein, we present the development of an anti- human VEGF repebody, which is a small-sized protein binder consisting of leucine-rich repeat (LRR) modules. The anti-VEGF repebody selected through a phage-display was shown to have a high affinity and specificity for human VEGF. We demonstrate that this repebody effectively inhibits in vitro angiogenic cellular processes, such as proliferation and migration, by blocking the VEGF-mediated signaling pathway. The repebody was also shown to have a strong suppression effect on choroidal neovascularization (CNV) and vascular leakage in vivo. Our results indicate that the anti-VEGF repebody has a therapeutic potential for treating neovascular AMD as well as other VEGF-involved diseases including diabetic retinopathy and metastatic cancers.
Subject(s)
Antibodies/chemistry , Choroidal Neovascularization/immunology , Leucine/chemistry , Vascular Endothelial Growth Factor A/immunology , Amino Acid Sequence , Angiogenesis Inhibitors/therapeutic use , Animals , Cell Movement , Cell Proliferation , Diabetic Retinopathy/drug therapy , Diabetic Retinopathy/immunology , Extracellular Signal-Regulated MAP Kinases/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Macular Degeneration/immunology , Macular Degeneration/metabolism , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Mutation , Neoplasms/drug therapy , Peptide Library , Signal Transduction , Surface Plasmon ResonanceABSTRACT
The intracellular delivery of proteins with high efficiency in a receptor-specific manner is of great significance in molecular medicine and biotechnology, but remains a challenge. Herein, we present the development of a highly efficient and receptor-specific delivery platform for protein cargos by combining the receptor binding domain of Escherichia coli Shiga-like toxin and the translocation domain of Pseudomonas aeruginosa exotoxin A. We demonstrated the utility and efficiency of the delivery platform by showing a cytosolic delivery of diverse proteins both in vitro and in vivo in a receptor-specific manner. In particular, the delivery system was shown to be effective for targeting an intracellular protein and consequently suppressing the tumor growth in xenograft mice. The present platform can be widely used for intracellular delivery of diverse functional macromolecules with high efficiency in a receptor-specific manner. Biotechnol. Bioeng. 2016;113: 1639-1646. © 2016 Wiley Periodicals, Inc.
Subject(s)
ADP Ribose Transferases/metabolism , Bacterial Toxins/metabolism , Drug Delivery Systems/methods , Exotoxins/metabolism , Intracellular Space/metabolism , Protein Engineering/methods , Recombinant Fusion Proteins/metabolism , Shiga Toxins/metabolism , Virulence Factors/metabolism , ADP Ribose Transferases/chemistry , ADP Ribose Transferases/genetics , Animals , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , Cell Line, Tumor , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Exotoxins/chemistry , Exotoxins/genetics , Female , Mice , Mice, Inbred BALB C , Mice, Nude , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Shiga Toxins/chemistry , Shiga Toxins/genetics , Virulence Factors/chemistry , Virulence Factors/genetics , Pseudomonas aeruginosa Exotoxin AABSTRACT
The cell-specific cytosolic delivery of functional macromolecules with high efficiency is of great significance in molecular medicine and biotechnology. Herein, we present a Shiga-like toxin II-based high-efficiency and receptor-specific intracellular delivery system. We designed and constructed the Shiga-like toxin-based carrier (STC) to comprise the targeting and translocation domains, and used it for delivering a protein cargo. The STC was shown to deliver a protein cargo into the cytosol with high efficiency in a receptor-specific manner, exhibiting much higher efficiency than the most widely used cell-penetrating peptide. The general utility of the STC was demonstrated by modulating the targeting domain. The present delivery platform can be widely used for the intracellular delivery of diverse biomolecules in a receptor-specific and genetically encodable manner.
Subject(s)
Cell Membrane/metabolism , Cytoplasm/metabolism , Green Fluorescent Proteins/administration & dosage , Green Fluorescent Proteins/pharmacokinetics , Nanocapsules/chemistry , Shiga Toxin 2/pharmacokinetics , Animals , Green Fluorescent Proteins/genetics , Humans , Nanocapsules/ultrastructure , Shiga Toxin 2/chemistry , Shiga Toxin 2/geneticsABSTRACT
Glycoprotein Ibα (GpIbα), a family of LRR (leucine-rich repeat) proteins, is a membrane protein on the platelet, and plays an important role in atherothrombotic events. The complex formation of GpIbα with the von Willebrand Factor (vWF) has been revealed to lead to acute coronary syndrome (ACS) or stroke. A considerable attention has been paid to understand the biological functions of GpIbα and its regulation. However, difficulty with the soluble expression of human GpIbα in bacteria has hampered the relevant research. Herein, we present a soluble expression of GpIbα in Escherichiacoli by replacing the N-terminal capping domain of GpIbα with that of Internalin B using a computational approach. The resulting protein was expressed as a soluble form in E. coli, maintaining its structural feature and binding property for vWF. The present approach can be broadly used for the soluble expression of human LRR proteins in E. coli.
