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Cell-penetrating peptides split into two groups based on modulation of intracellular calcium concentration.

ABSTRACT: Cell-penetrating peptides (CPPs) promote the uptake of different cargo molecules, e.g. therapeutic compounds, making the harnessing of CPPs a promising strategy for drug design and delivery. However, the internalization mechanisms of CPPs are still under discussion, and it is not clear how cells compensate the disturbances induced by peptides in the plasma membrane. In this study, we demonstrate that the uptake of various CPPs enhances the intracellular Ca(2+) levels in Jurkat and HeLa cells. The elevated Ca(2+) concentration in turn triggers plasma membrane blebbing, lysosomal exocytosis, and membrane repair response. Our results indicate that CPPs split into two major classes: (i) amphipathic CPPs that modulate the plasma membrane integrity inducing influx of Ca(2+) and activating downstream responses starting from low concentrations; (ii) non-amphipathic CPPs that do not evoke changes at relevant concentrations. Triggering of the membrane repair response may help cells to replace distorted plasma membrane regions and cells can recover from the influx of Ca(2+) if its level is not drastically elevated.

SUBMITTER: Lorents A

PROVIDER: S-EPMC3351292 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Cell-penetrating peptides split into two groups based on modulation of intracellular calcium concentration.

Lorents Annely A Kodavali Praveen Kumar PK Oskolkov Nikita N Langel Ülo Ü Hällbrink Mattias M Pooga Margus M

The Journal of biological chemistry 20120321 20

Cell-penetrating peptides (CPPs) promote the uptake of different cargo molecules, e.g. therapeutic compounds, making the harnessing of CPPs a promising strategy for drug design and delivery. However, the internalization mechanisms of CPPs are still under discussion, and it is not clear how cells compensate the disturbances induced by peptides in the plasma membrane. In this study, we demonstrate that the uptake of various CPPs enhances the intracellular Ca(2+) levels in Jurkat and HeLa cells. Th ...[more]

PMID: 22437827

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Project description:Salmonella infection poses a critical challenge to global public health, and the situation is exacerbated by the increasing prevalence of antibiotic resistance. Bacteriophages (phages) are increasingly being used as antimicrobial agents due to their ability to kill specific bacteria. However, the low cellular uptake of phages has limited their use in treating intracellular bacterial infections. Here, we present a study using engineered phages with cell-penetrating peptides (CPPs) for enhancing the internalization efficiency of phages to inhibit bacterial intracellular infections. Through bioinformatic analysis, we identified a phage-encoded protein harboring an immunoglobulin-like (Ig-like) domain as the potential target for phage display. Using a CRISPR-Cas9-based method, we successfully displayed short peptides on GP94, an Ig-like domain-containing protein, of Salmonella phage selz. We improved phage intracellular uptake in multiple cell types by fusion of various CPPs to GP94. Notably, the phage selzHA-TAT showed promising results in enhancing the intracellular inhibition of Salmonella in different cells. Our research provides a straightforward strategy for displaying CPPs on non-model phages, offering a promising novel and effective therapeutic approach for treating intracellular and drug-resistant bacteria. IMPORTANCE Salmonella infection is a significant threat to global public health, and the increasing prevalence of antibiotic resistance exacerbates the situation. Therefore, finding new and effective ways to combat this pathogen is essential. Phages are natural predators of bacteria and can be used as an alternative to antibiotics to kill specific bacteria, including drug-resistant strains. One significant limitation of using phages as antimicrobial agents is their low cellular uptake, which limits their effectiveness against intracellular bacterial infections. Therefore, finding ways to enhance phage uptake is crucial. Our study provides a straightforward strategy for displaying cell-penetrating peptides on non-model phages, offering a promising novel and effective therapeutic approach for treating intracellular and drug-resistant bacteria. This approach has the potential to address the global challenge of antibiotic resistance and improve public health outcomes.

Project description:Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

Dataset Information

Cell-penetrating peptides split into two groups based on modulation of intracellular calcium concentration.

Publications

Cell-penetrating peptides split into two groups based on modulation of intracellular calcium concentration.

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