Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Impact of the Endosomal Escape Activity of Cell-Penetrating Peptides on the Endocytic Pathway.

ABSTRACT: Cell-penetrating peptides (CPPs) are routinely used for the delivery of macromolecules into live human cells. To enter the cytosolic space of cells, CPPs typically permeabilize the membrane of endosomes. In turn, several approaches have been developed to increase the endosomal membrane permeation activity of CPPs so as to improve delivery efficiencies. The endocytic pathway is, however, important in maintaining cellular homeostasis, and understanding how endosomal permeation impacts cells is now critical to define the general utility of CPPs. Herein, we investigate how CPP-based delivery protocols affect the endocytic network. We detect that, in some cases, cell penetration induces the activation of Chmp1b, Galectin-3, and TFEB, which are components of endosomal repair, organelle clearance, and biogenesis pathways, respectively. We also detect that cellular delivery modulates endocytosis and endocytic proteolysis. Remarkably, a multimeric analogue of the prototypical CPP TAT permeabilizes endosomes efficiently without inducing membrane damage responses. These results challenge the notion that reagents that make endosomes leaky are generally toxic. Instead, our data indicates that it is possible to enter cells with minimal deleterious effects.

SUBMITTER: Kondow-McConaghy HM

PROVIDER: S-EPMC7502533 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay.

Project description:Cytosolic transport is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids. Current understanding of cytosolic delivery mechanisms remains limited due to a significant number of conflicting reports, which are compounded by low sensitivity and indirect assays. To resolve this, we develop a highly sensitive Split Luciferase Endosomal Escape Quantification (SLEEQ) assay to probe mechanisms of cytosolic delivery. We apply SLEEQ to evaluate the cytosolic delivery of a range of widely studied cell-penetrating peptides (CPPs) fused to a model protein. We demonstrate that positively charged CPPs enhance cytosolic delivery as a result of increased non-specific cell membrane association, rather than increased endosomal escape efficiency. These findings transform our current understanding of how CPPs increase cytosolic delivery. SLEEQ is a powerful tool that addresses fundamental questions in intracellular drug delivery and will significantly improve the way materials are engineered to increase therapeutic delivery to the cytosol.

| S-EPMC8211857 | biostudies-literature

An l- to d-Amino Acid Conversion in an Endosomolytic Analog of the Cell-penetrating Peptide TAT Influences Proteolytic Stability, Endocytic Uptake, and Endosomal Escape.

Project description:Cell-penetrating peptides (CPPs) are well established as delivery agents for otherwise cell-impermeable cargos. CPPs can also theoretically be used to modulate intracellular processes. However, their susceptibility to proteolytic degradation often limits their utility in these applications. Previous studies have explored the consequences for cellular uptake of converting the residues in CPPs from l- to d-stereochemistry, but conflicting results have been reported and specific steps en route to intracellular activity have not been explored. Here we use dimeric fluorescence TAT as a model CPP to explore the broader consequences of l- to d-stereochemical conversion. We show that inversion of chirality provides protease resistance without altering the overall mode of cellular entry, a process involving endocytic uptake followed by endosomal escape and cytosolic access. However, whereas inversion of chirality reduces endocytic uptake, the d-peptide, once in the endosome, is significantly more prone to escape than its l-counterpart. Moreover, the d-peptide is retained in the cytosol of cells for several days, whereas the l-peptide is degraded within hours. Notably, while the l-peptide is relatively innocuous to cells, the d-peptide exerts a prolonged anti-proliferative activity. Together, our results establish connections between chirality, protease resistance, cellular penetration, and intracellular activity that may be useful for the development of future delivery agents with improved properties.

| S-EPMC5247658 | biostudies-literature

Cytosolic Delivery of Macromolecules in Live Human Cells Using the Combined Endosomal Escape Activities of a Small Molecule and Cell Penetrating Peptides.

Project description:Ineffective cellular delivery is a common problem in numerous biological applications. Developing delivery reagents that work robustly in a variety of experimental settings remains a challenge. Herein, we report how peptides derived from the prototypical cell penetrating peptide TAT can be used in combination with a small molecule, UNC7938, to deliver macromolecules into the cytosol of cells by a simple co-incubation protocol. We establish successful delivery of peptides, DNA plasmids, and a single-chain variable fragment antibody. We also demonstrate that delivery works in hard-to-transfect mammalian cells and under conditions typically inhibitory to cell-penetrating peptides. Mechanistically, UNC7938 destabilizes the membrane of endosomes. This, in turn, enhances the endosome-leakage activity of cell-penetrating peptides and facilitates the endosomal escape of macromolecules initially internalized by mammalian cells via endocytosis. This combined selective membrane-destabilization represents a new chemical space for delivery tools and provides a novel solution to the problem of endosomal entrapment that often limits the effectiveness of reagent-based delivery approaches.

| S-EPMC7008471 | biostudies-literature

Novel cell-penetrating peptide-adaptors effect intracellular delivery and endosomal escape of protein cargos.

Project description:The use of cell-penetrating peptides (CPPs) as biomolecular delivery vehicles holds great promise for therapeutic and other applications, but development has been stymied by poor delivery and lack of endosomal escape. We have developed a CPP-adaptor system capable of efficient intracellular delivery and endosomal escape of user-defined protein cargos. The cell-penetrating sequence of HIV transactivator of transcription was fused to calmodulin, which binds with subnanomolar affinity to proteins containing a calmodulin binding site. Our strategy has tremendous advantage over prior CPP technologies because it utilizes high-affinity non-covalent, but reversible coupling between CPP and cargo. Three different cargo proteins fused to a calmodulin binding sequence were delivered to the cytoplasm of eukaryotic cells and released, demonstrating the feasibility of numerous applications in living cells including alteration of signaling pathways and gene expression.

| S-EPMC4813321 | biostudies-literature

Early endosomal escape of a cyclic cell-penetrating peptide allows effective cytosolic cargo delivery.

Project description:Cyclic heptapeptide cyclo(FΦRRRRQ) (cFΦR4, where Φ is l-2-naphthylalanine) was recently found to be efficiently internalized by mammalian cells. In this study, its mechanism of internalization was investigated by perturbing various endocytic events through the introduction of pharmacologic agents and genetic mutations. The results show that cFΦR4 binds directly to membrane phospholipids, is internalized into human cancer cells through endocytosis, and escapes from early endosomes into the cytoplasm. Its cargo capacity was examined with a wide variety of molecules, including small-molecule dyes, linear and cyclic peptides of various charged states, and proteins. Depending on the nature of the cargos, they may be delivered by endocyclic (insertion of cargo into the cFΦR4 ring), exocyclic (attachment of cargo to the Gln side chain), or bicyclic approaches (fusion of cFΦR4 and cyclic cargo rings). The overall delivery efficiency (i.e., delivery of cargo into the cytoplasm and nucleus) of cFΦR4 was 4-12-fold higher than those of nonaarginine, HIV Tat-derived peptide, or penetratin. The higher delivery efficiency, coupled with superior serum stability, minimal toxicity, and synthetic accessibility, renders cFΦR4 a useful transporter for intracellular cargo delivery and a suitable system for investigating the mechanism of endosomal escape.

| S-EPMC4075989 | biostudies-literature

Cell-Penetrating Peptides Escape the Endosome by Inducing Vesicle Budding and Collapse.

Project description:Cell-penetrating peptides (CPPs) are capable of delivering membrane-impermeable cargoes (including small molecules, peptides, proteins, nucleic acids, and nanoparticles) into the cytosol of mammalian cells and have the potential to revolutionize biomedical research and drug discovery. However, the mechanism of action of CPPs has remained poorly understood, especially how they escape from the endosome into the cytosol following endocytic uptake. We show herein that CPPs exit the endosome by inducing budding and collapse of CPP-enriched vesicles from the endosomal membrane. This mechanism provides a theoretical basis for designing CPPs and other delivery vehicles of improved efficiencies.

| S-EPMC7512842 | biostudies-literature

Efficient cell delivery mediated by lipid-specific endosomal escape of supercharged branched peptides.

Project description:Various densely charged polycationic species, whether of biological or synthetic origin, can penetrate human cells, albeit with variable efficiencies. The molecular underpinnings involved in such transport remain unclear. Herein, we assemble 1, 2 or 3 copies of the HIV peptide TAT on a synthetic scaffold to generate branched cell-permeable prototypes with increasing charge density. We establish that increasing TAT copies dramatically increases the cell penetration efficiency of the peptides while simultaneously enabling the efficient cytosolic delivery of macromolecular cargos. Cellular entry involves the leaky fusion of late endosomal membranes enriched with the anionic lipid BMP. Derivatives with multiple TAT branches induce the leakage of BMP-containing lipid bilayers, liposomal flocculation, fusion and an increase in lamellarity. In contrast, while the monomeric counterpart 1TAT binds to the same extent and causes liposomal flocculation, 1TAT does not cause leakage, induce fusion or a significant increase in lamellarity. Overall, these results indicate that an increase in charge density of these branched structures leads to the emergence of lipid specific membrane-disrupting and cell-penetrating activities.

| S-EPMC5948172 | biostudies-literature

Caspase-activated cell-penetrating peptides reveal temporal coupling between endosomal release and apoptosis in an RGC-5 cell model.

Project description:Caspase-activatable cell-penetrating peptide (CPP) probes, designed for efficient cell uptake and specificity via cleavable intramolecular quenched-fluorophore strategies, show promise for identifying and imaging retinal ganglion cell apoptosis in vivo. However, initial cell uptake and trafficking events cannot be visualized because the probes are designed to be optically quenched in the intact state. To visualize subcellular activation events in real-time during apoptosis, a new series of matched quenched and nonquenched CPP probes were synthesized. In both native and staurosporine-differentiated RGC-5 cells, probe uptake was time- and concentration-dependent through clathrine-, caveolin-, and pinocytosis-mediated endocytic mechanisms. During apoptosis, KcapTR488, a novel dual fluorophore CPP probe, revealed by multispectral imaging a temporal coupling of endosomal release and effector caspase activation in RGC-5 cells. The novel CPPs described herein provide new tools to study spatial and temporal regulation of endosomal permeability during apoptosis.

| S-EPMC3447108 | biostudies-literature

Elucidating the Impact of Payload Conjugation on the Cell-Penetrating Efficiency of the Endosomal Escape Peptide dfTAT: Implications for Future Designs for CPP-Based Delivery Systems.

Project description:Cell-penetrating peptides (CPPs) are promising tools for the intracellular delivery of various biological payloads. However, the impact of payload conjugation on the cell-penetrating activity of CPPs is poorly understood. This study focused on dfTAT, a modified version of the HIV-TAT peptide with enhanced endosomal escape activity, to explore how different payloads affect its cell-penetrating activity. We systematically examined dfTAT conjugated with the SnoopTag/SnoopCatcher pair and found that while smaller payloads such as short peptides do not significantly impair dfTAT's cell delivery activity, larger payloads markedly reduce both its endocytic uptake and endosomal escape efficiency. Our results highlight the role of the payload size and bulk in limiting CPP-mediated delivery. While further research is needed to understand the molecular underpinnings of these effects, our findings pave the way for developing more effective CPP-based delivery systems.

| S-EPMC10644971 | biostudies-literature

Cell-penetrating peptides with intracellular actin-remodeling activity in malignant fibroblasts.

Project description:Cell-penetrating peptides can cross cell membranes and are commonly seen as biologically inert molecules. However, we found that some cell-penetrating peptides could remodel actin cytoskeleton in oncogene-transformed NIH3T3/EWS-Fli cells. These cells have profound actin disorganization related to their tumoral transformation. These arginine- and/or tryptophan-rich peptides could cross cell membrane and induce stress fiber formation in these malignant cells, whereas they had no perceptible effect in non-tumoral fibroblasts. In addition, motility (migration speed, random motility coefficient, wound healing) of the tumor cells could be decreased by the cell-permeant peptides. Although the peptides differently influenced actin polymerization in vitro, they could directly bind monomeric actin as determined by NMR and calorimetry studies. Therefore, cell-penetrating peptides might interact with intracellular protein partners, such as actin. In addition, the fact that they could reverse the tumoral phenotype is of interest for therapeutic purposes.

| S-EPMC2844216 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data