ABSTRACT
A key regulator of receptor-mediated endocytosis, Rab5, plays a pivotal role in cargo receptor internalization, endosomal maturation, and transduction and degradation of internalized signaling molecules and recycling cargo receptor. Stressful conditions within cells lead to increased Rab5 activation, and increasing evidence correlates Rab5 activity abnormalities with certain diseases. Current antibody-based imaging methods cannot distinguish active Rab5 from total Rab5 population and provide dynamic information on magnitude and duration of Rab5 activation in cellular events and pathogenesis. We report here novel molecular imaging probes that specifically target GTP-bound Rab5 associated with the early endosome membrane in live cells and fixed mouse brain tissues. Our Rab5 activity fluorescent biosensor (RAFB) contains the Rab5 binding domain of the Rab5 effector Rabaptin 5, a fluorophore (a quantum dot or fluorescent dye) and a cell-penetrating peptide for live-cell delivery. The quantum dot conjugated RAFB was able to image the elevated Rab5 activity in both the cortex and hippocampi tissues of a Ts65Dn mouse. A prequenched RAFB based on fluorescence resonance energy transfer (FRET) can image cytosolic active Rab5 in single live cells. This novel method should enable imaging of the biological process in which Rab5 activity is regulated in various cellular systems.
Subject(s)
Biosensing Techniques , Fluorescein-5-isothiocyanate/analogs & derivatives , Peptides/metabolism , rab5 GTP-Binding Proteins/analysis , rab5 GTP-Binding Proteins/metabolism , Animals , Binding Sites , Brain/metabolism , Cell-Penetrating Peptides/metabolism , Cerebral Cortex/metabolism , Cytoplasm/metabolism , Endosomes/metabolism , Fluorescein-5-isothiocyanate/chemistry , Fluorescein-5-isothiocyanate/metabolism , Fluorescence Resonance Energy Transfer , HeLa Cells , Hippocampus/metabolism , Humans , Mice , Peptides/chemistry , Protein Engineering , Quantum Dots , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism , rab5 GTP-Binding Proteins/geneticsABSTRACT
In this paper we report the conjugation of an aromatic moiety (pyrene (P), fluorene (F), or naphthalene (N)) to pentapeptides GAGAS (1), GVPVP (2), VPGVG (3), VTEEI (4), VYGGG (5), and YGFGG (6) to afford peptidic derivatives for exploring pentapeptide-based hydrogels as potential biomaterials. Most of these compounds (1F, 1P, 2F, 2P, 4F, 4P, 4N, 5F, 5N, 6F, 6P, and 6N) behave as molecular hydrogelators and can form hydrogels at minimum concentrations of gelation from 0.5 to 2.8 wt %. The fluorescence spectra of the hydrogels exhibit a significant red shift, indicating the interactions between the aromatic moieties in those hydrogels. The circular dichroism spectra indicate that the self-assembly of the hydrogelators affords helical or beta-sheet-like structures. Transmission and scanning electron microscopic examination reveals the nanofiber networks of these hydrogelators. In addition, rheological measurements show fair to excellent viscoelastic properties of these hydrogels. The balance of intermolecular aromatic-aromatic interactions and hydrogen bonds of these hydrogelators leads to their self-assembly in water and the formation of nanofibers as the matrixes of hydrogels. A total of 6 of these 18 pentapeptide derivatives--1N, 2N, 3F, 3P, 3N, and 5P--fail to form hydrogels under the conditions tested, likely due to unbalanced intermolecular interactions. This work suggests that aromatic-aromatic interactions are useful and favorable forces for creating molecular nanofibers and supramolecular hydrogels.
Subject(s)
Fluorenes/chemistry , Hydrogels/chemistry , Naphthalenes/chemistry , Oligopeptides/chemistry , Pyrenes/chemistry , Amino Acid Sequence , Biocompatible Materials/chemistry , Cell Survival , Circular Dichroism , HeLa Cells , Humans , Nanofibers/chemistry , Nanofibers/ultrastructureABSTRACT
The review describes the use of enzyme catalysis and self-assembly, two essential and ubiquitous processes in biology, to create molecular nanofibers of peptide derivatives at the supramolecular level as potential nanomedicines and biomaterials. In particular, we discuss the use of enzymes to instruct the self-assembly of small peptide derivatives for hydrogelation, which takes place in vitro or in vivo, extra- or intracellularly, as a new strategy to detect the activity of enzymes, screen enzyme inhibitors, type bacteria, develop drug delivery systems, enhance the activity and stability of enzymes, and control the fate of cells.
Subject(s)
Hydrogels , Nanofibers , Peptides/chemistry , Animals , Bacteria/metabolism , Enzyme Stability , Enzymes/chemistry , Enzymes/metabolism , Humans , Hydrogels/chemistry , Molecular Structure , Nanofibers/chemistry , Protein Engineering , Water/chemistryABSTRACT
Composed of a supramolecular hydrogel and a heme model compound, a new type of artificial peroxidase shows high catalytic activity in organic media. The activity of this new type of artificial enzyme is significantly higher than that of the heme model compounds alone. Changes in the distal substituents above the coordinated-metal centers of the model compounds directly modulate catalytic activity. This supramolecular-hydrogel-based artificial enzyme is most active in toluene, reaching about 90% of the nascent activity of horseradish peroxidase. Moreover, this study confirms that the incorporation of the heme models into the nanofibers of gelators accounts for most of the enhancement of catalytic activity.
Subject(s)
Heme/chemistry , Hydrogels/chemistry , Models, Molecular , Peroxidases/metabolism , Catalysis , Microscopy, Electron, Transmission , Spectrophotometry, UltravioletABSTRACT
A simple supramolecular hydrogel based on D-glucosamine, a naturally occurring aminosaccharide, promises new biomaterials for applications such as wound healing.
Subject(s)
Glucosamine/chemical synthesis , Glucosamine/pharmacology , Hydrogels/chemical synthesis , Hydrogels/pharmacology , Wound Healing/drug effects , Animals , Chemical Phenomena , Chemistry, Physical , Circular Dichroism , Fibroblasts/pathology , HeLa Cells , Humans , Indicators and Reagents , Materials Testing , Mice , Mice, Inbred BALB C , Microscopy, Electron, Transmission , Nanoparticles , Paraffin Embedding , Phenylalanine/chemistry , Skin/injuries , Spectrometry, Fluorescence , Wounds and Injuries/drug therapy , Wounds and Injuries/pathologySubject(s)
Amino Acids/chemistry , Enzymes/chemistry , Hydrogels/chemistry , Nanoparticles/chemistry , Nanotechnology/methods , Animals , Biocompatible Materials/chemistry , Body Weight , HeLa Cells , Humans , Hydrogen-Ion Concentration , In Vitro Techniques , Mice , Microscopy, Electron, Transmission , Models, Chemical , RabbitsABSTRACT
This communication describes the use of a molecular hydrogel to immobilize enzymes for catalyzing reactions in an organic solvent to attain superactivity and exceptional stability.
