Internalization and intracellular trafficking of a PTD‐conjugated anti‐fibrotic peptide, AZX100, in human dermal keloid fibroblasts
Identifieur interne : 001709 ( Main/Exploration ); précédent : 001708; suivant : 001710Internalization and intracellular trafficking of a PTD‐conjugated anti‐fibrotic peptide, AZX100, in human dermal keloid fibroblasts
Auteurs : Charles R. Flynn [États-Unis] ; Joyce Cheung-Flynn [États-Unis] ; Christopher C. Smoke [États-Unis] ; David Lowry [États-Unis] ; Robert Roberson [États-Unis] ; Michael R. Sheller [États-Unis] ; Colleen M. Brophy [États-Unis]Source :
- Journal of Pharmaceutical Sciences [ 0022-3549 ] ; 2010-07.
English descriptors
- Teeft :
- Aclar disks, Actin, Actin cytoskeleton, Actin cytoskeleton dynamics, Actin deformation, Actin polymerization, Actin rearrangement, Adenylyl cyclase, Adobe photoshop, Adobe systems, Alexa, American peptide, American pharmacists association, Amino, Amino acid, Amino terminus, Arizona state university, Biol, Biol chem, Bodipy, Bodipy lactosylceramide, Bronectin, Brophy, Cacodylate buffer, Capstone therapeutics, Cationic peptides, Caveolae, Caveolin, Caveolin expression, Cell biol, Cell culture, Cell growth, Cell membrane, Cell surface, Cell surface binding, Cell viability, Cells transiently, Cellular, Cellular uptake, Chem, Cholera toxin subunit, Chondroitin sulfate, Clathrinmediated endocytosis, Colocalization, Colocalization results, Colocalize, Complete growth media, Constitutively, Control cells, Cytochalasin, Cytoskeleton, Different cells, Diffuse actin network, Digital camera, Dynamin, Early endosomes, Egfp, Electrophoresis buffer, Encoding, Encoding egfp, Encoding hemagglutinin epitope, Endocytic, Endocytosis, Endocytotic vesicles, Endosomes, Equal amounts, Flynn, Focal adhesion dynamics, Focal adhesions, Fusion protein, Ganglioside, Heparin, Human virus, Hypertrophic scarring, Image series, Immunoblot transfer buffer, Inhibitor, Internalization, Intracellular, Intracellular delivery, Juan bonifacino, July, Keloid, Kinase, Komalavilas, Late endosomes, Leucine mutation, Lipid, Lipid raft, Lipid rafts, Lter sets, Lysine mutation, Lysis buffer, Macropinocytotic regurgitation, Membrane, Membrane integrity, Microtubule, Monoclonal antibodies, Muscle actin, Muscle cells, Mutant, Mutant dynamin, Mutation, Open arrows, Optimized protein transduction domain, Original wound, Osmium tetroxide, Other ptds, Pathway, Peptide, Permeabilized cells, Pharmaceutical, Pharmaceutical sciences, Pharmacological inhibitors, Phosphatase inhibitor cocktail, Phosphorylated, Phosphorylation, Physiol lung cell, Plasma membrane, Plasmid, Plasmid dnas, Plasmid encoding, Plasmids encoding, Potential binding, Propidium iodide, Protein transduction domains, Ptd4, Quiescent cells, Rabbit antibody, Rafts, Scaffolding domain, Skin penetration, Small heat protein, Small heat shock protein, Statistical analysis, Subcellular localization, Supplemental movie, Threonine mutation, Transduction, Transfected transiently, Transfection reagent, Transiently, Transmission electron microscopy, Transport mechanisms, Untreated, Untreated cells, Uorescence, Uorescence micrograph, Uorescence microscopy, Uorescence patterns, Uorescence signal, Uorescent, Uorescent protein, Uorescent proteins, Uptake, Uranyl acetate, Vesicle, Vesicle formation, Vesicles colocalized.
Abstract
A challenge in advanced drug delivery is selectively traversing the plasma membrane, a barrier that prohibits the intracellular delivery of most peptide and nucleic acid‐based therapeutics. A variety of short amino acid sequences termed protein transduction domains (PTDs) first identified in viral proteins have been utilized for over 20 years to deliver proteins nondestructively into cells, however, the mechanisms by which this occurs are varied and cell‐specific. Here we describe the results of live cell imaging experiments with AZX100, a cell‐permeable anti‐fibrotic peptide bearing an “enhanced” PTD (PTD4). We monitored fluorescently labeled AZX100 upon cell surface binding and subsequent intracellular trafficking in the presence of cellular process inhibitors and various well‐defined fluorescently labeled cargos. We conclude that AZX100 enters cells via caveolae rapidly, in a manner that is independent of glycoconjugates, actin/microtubule polymerization, dynamins, multiple GTPases, and clathrin, but is associated with lipid rafts as revealed by methyl‐β‐cylodextrin. AZX100 treatment increases the expression of phospho‐caveolin (Y14), a critical effector of focal adhesion dynamics, suggesting a mechanistic link between caveolin‐1 phosphorylation and actin cytoskeleton dynamics. Our results reveal novel and interesting properties of PTD4 and offer new insight into the cellular mechanisms facilitating an advanced drug delivery tool. © 2010 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:3100–3121, 2010
Url:
DOI: 10.1002/jps.22087
Affiliations:
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<term>Actin polymerization</term>
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<term>American peptide</term>
<term>American pharmacists association</term>
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<term>Bodipy lactosylceramide</term>
<term>Bronectin</term>
<term>Brophy</term>
<term>Cacodylate buffer</term>
<term>Capstone therapeutics</term>
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<term>Caveolin</term>
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<term>Colocalization results</term>
<term>Colocalize</term>
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<term>Cytochalasin</term>
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<term>Encoding hemagglutinin epitope</term>
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<term>Intracellular</term>
<term>Intracellular delivery</term>
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<term>Lysine mutation</term>
<term>Lysis buffer</term>
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<term>Monoclonal antibodies</term>
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<term>Pharmaceutical sciences</term>
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<term>Plasmid encoding</term>
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<term>Propidium iodide</term>
<term>Protein transduction domains</term>
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<term>Quiescent cells</term>
<term>Rabbit antibody</term>
<term>Rafts</term>
<term>Scaffolding domain</term>
<term>Skin penetration</term>
<term>Small heat protein</term>
<term>Small heat shock protein</term>
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<term>Subcellular localization</term>
<term>Supplemental movie</term>
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<term>Transfection reagent</term>
<term>Transiently</term>
<term>Transmission electron microscopy</term>
<term>Transport mechanisms</term>
<term>Untreated</term>
<term>Untreated cells</term>
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<term>Uorescence micrograph</term>
<term>Uorescence microscopy</term>
<term>Uorescence patterns</term>
<term>Uorescence signal</term>
<term>Uorescent</term>
<term>Uorescent protein</term>
<term>Uorescent proteins</term>
<term>Uptake</term>
<term>Uranyl acetate</term>
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<front><div type="abstract" xml:lang="en">A challenge in advanced drug delivery is selectively traversing the plasma membrane, a barrier that prohibits the intracellular delivery of most peptide and nucleic acid‐based therapeutics. A variety of short amino acid sequences termed protein transduction domains (PTDs) first identified in viral proteins have been utilized for over 20 years to deliver proteins nondestructively into cells, however, the mechanisms by which this occurs are varied and cell‐specific. Here we describe the results of live cell imaging experiments with AZX100, a cell‐permeable anti‐fibrotic peptide bearing an “enhanced” PTD (PTD4). We monitored fluorescently labeled AZX100 upon cell surface binding and subsequent intracellular trafficking in the presence of cellular process inhibitors and various well‐defined fluorescently labeled cargos. We conclude that AZX100 enters cells via caveolae rapidly, in a manner that is independent of glycoconjugates, actin/microtubule polymerization, dynamins, multiple GTPases, and clathrin, but is associated with lipid rafts as revealed by methyl‐β‐cylodextrin. AZX100 treatment increases the expression of phospho‐caveolin (Y14), a critical effector of focal adhesion dynamics, suggesting a mechanistic link between caveolin‐1 phosphorylation and actin cytoskeleton dynamics. Our results reveal novel and interesting properties of PTD4 and offer new insight into the cellular mechanisms facilitating an advanced drug delivery tool. © 2010 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:3100–3121, 2010</div>
</front>
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<name sortKey="Brophy, Colleen M" sort="Brophy, Colleen M" uniqKey="Brophy C" first="Colleen M." last="Brophy">Colleen M. Brophy</name>
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<name sortKey="Flynn, Charles R" sort="Flynn, Charles R" uniqKey="Flynn C" first="Charles R." last="Flynn">Charles R. Flynn</name>
<name sortKey="Flynn, Charles R" sort="Flynn, Charles R" uniqKey="Flynn C" first="Charles R." last="Flynn">Charles R. Flynn</name>
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