Human-protein-derived peptides for intracellular delivery of biomolecules

2012 ◽  
Vol 442 (3) ◽  
pp. 583-593 ◽  
Author(s):  
Alexander K. Haas ◽  
Daniela Maisel ◽  
Juliane Adelmann ◽  
Christoffer von Schwerin ◽  
Ines Kahnt ◽  
...  
Keyword(s):  
Cell Viability ◽  
In Silico ◽  
Cultured Cells ◽  
Cell Penetrating Peptides ◽  
Extracellular Proteins ◽  
Human Protein ◽  
Rna Transfection ◽  
Cell Penetrating ◽  
High Concentrations ◽  
Α Helix

Access of therapeutic biomolecules to cytoplasmic and nuclear targets is hampered by the inability of these molecules to cross biological membranes. Approaches to overcome this hurdle involve CPPs (cell-penetrating peptides) or protein transduction domains. Most of these require rather high concentrations to elicit cell-penetrating functionality, are non-human, pathogen-derived or synthetic entities, and may therefore not be tolerated or even immunogenic. We identified novel human-protein-derived CPPs by a combination of in silico and experimental analyses: polycationic CPP candidates were identified in an in silico library of all 30-mer peptides of the human proteome. Of these peptides, 60 derived from extracellular proteins were evaluated experimentally. Cell viability and siRNA (small interfering RNA) transfection assays revealed that 20 out of the 60 peptides were functional. Three of these showed CPP functionality without interfering with cell viability. A peptide derived from human NRTN (neurturin), which contains an α-helix, performed the best in our screen and was uniformly taken up by cultured cells. Examples for payloads that can be delivered to the cytosol by the NRTN peptide include complexed siRNAs and both N- and C-terminally fused pro-apoptotic peptides.

Stereochemistry as a determining factor for the effect of a cell-penetrating peptide on cellular viability and epithelial integrity

2018 ◽  
Vol 475 (10) ◽  
pp. 1773-1788 ◽  
Author(s):  
Ditlev Birch ◽  
Malene V. Christensen ◽  
Dan Staerk ◽  
Henrik Franzyk ◽  
Hanne Mørck Nielsen
Keyword(s):  
Amino Acids ◽  
Cell Viability ◽  
Mammalian Cells ◽  
Membrane Integrity ◽  
Cell Penetrating Peptides ◽  
Proliferating Cells ◽  
Epithelial Integrity ◽  
Cell Penetrating ◽  
Culture Models ◽  
Well Differentiated

Cell-penetrating peptides (CPPs) comprise efficient peptide-based delivery vectors. Owing to the inherent poor enzymatic stability of peptides, CPPs displaying partial or full replacement of l-amino acids with the corresponding d-amino acids might possess advantages as delivery vectors. Thus, the present study aims to elucidate the membrane- and metabolism-associated effects of l-Penetratin (l-PEN) and its corresponding all-d analog (d-PEN). These effects were investigated when exerted on hepatocellular (HepG2) or intestinal (Caco-2 and IEC-6) cell culture models. The head-to-head comparison of these enantiomeric CPPs included evaluation of their effects on cell viability and morphology, epithelial membrane integrity, and cellular ultrastructure. In all investigated cell models, a rapid decrease in cell viability, pronounced membrane perturbation and an altered ultrastructure were detected upon exposure to d-PEN. At equimolar concentrations, these observations were less pronounced or even absent for cells exposed to l-PEN. Both CPPs remained stable for at least 2 h during exposure to proliferating cells (cultured for 24 h), although d-PEN exhibited a longer half-life when compared with that of l-PEN when exposed to well-differentiated cell monolayers (cultured for 18–20 days). Thus, the stereochemistry of the CPP penetratin significantly influences its effects on cell viability and epithelial integrity when profiled against a panel of mammalian cells.

scholarly journals A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos

2021 ◽  
Author(s):  
Jonathan L. McMurry ◽  
Schuyler B. Gentry ◽  
Scott J. Nowak ◽  
Xuelei Ni ◽  
Stephanie A. Hill ◽  
...  
Keyword(s):  
Real Time ◽  
Cultured Cells ◽  
Flow Chamber ◽  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Calmodulin Binding ◽  
Distance Analysis ◽  
Cell Penetrating ◽  
Broad Array ◽  
Escape Problem

Cell-penetrating peptides (CPPs) are capable of transporting molecules to which they are tethered across cellular membranes. Unsurprisingly, CPPs have attracted attention for their potential drug delivery applications, but several technical hurdles remain to be overcome. Chief among them is the so-called ‘endosomal escape problem,’ i.e. the propensity of CPP-cargo molecules to be endocytosed but remain entrapped in endosomes rather than reaching the cytosol. Previously, a CPP fused to calmodulin that bound calmodulin binding site-containing cargos was shown to efficiently deliver cargos to the cytoplasm, effectively overcoming the endosomal escape problem. The CPP-adaptor, “TAT-CaM,” evinces delivery at nM concentrations and more rapidly than we had previously been able to measure. To better understand the kinetics and mechanism of CPP-adaptor-mediated cargo delivery, a real-time cell penetrating assay was developed in which a flow chamber containing cultured cells was installed on the stage of a confocal microscope to allow for observation ab initio . Also examined in this study was an improved CPP-adaptor that utilizes naked mole rat ( Heterocephalus glaber ) calmodulin in place of human and results in superior internalization, likely due to its lesser net negative charge. Adaptor-cargo complexes were delivered into the flow chamber and fluorescence intensity in the midpoint of baby hamster kidney cells was measured as a function of time. Delivery of 400 nM cargo was observed within seven minutes and fluorescence continued to increase linearly as a function of time. Cargo-only control experiments showed that the minimal uptake which occurred independently of the CPP-adaptor resulted in punctate localization consistent with endosomal entrapment. A distance analysis was performed for cell-penetration experiments in which CPP-adaptor-delivered cargo showing wider dispersions throughout cells as compared to an analogous covalently-bound CPP-cargo. Small molecule endocytosis inhibitors did not have significant effects upon delivery. The real-time assay is an improvement upon static endpoint assays and should be informative in a broad array of applications.

Cell Penetrating Mechanism of Bax Inhibiting Peptides.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4298-4298
Author(s):  
Jose A. Gomez ◽  
Tomoyuki Yoshida ◽  
Minh Lam ◽  
Clark W. Distelhorst ◽  
Shigemi Matsuyama
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Fluorescent Dyes ◽  
Cultured Cells ◽  
Cell Penetrating Peptides ◽  
Dependent Manner ◽  
Cell Penetration ◽  
New Members ◽  
Cell Penetrating ◽  
High Degree

Abstract Plasma membrane is known to have a high degree of selectivity for molecular trafficking, and it does not allow the penetration of peptides larger than 3 amino acids. Previously known exceptions of large peptides that penetrate the plasma membrane are the Arginine rich peptides such as human immunodeficiency virus (HIV)-tat peptides. However, the mechanism of cell penetration of these peptides is largely unknown. Bax Inhibiting Peptides (BIP) are penta-peptides derived from the Bax binding domain of Ku70. At present, three types of BIP have been developed. Those are: VPMLK, VPTLK, and VPALR. All of these three BIPs directly bind Bax and inhibit Bax-mediated cell death in cultured cells as well as in animal study. Surprisingly, BIPs are cell permeable and autonomously enter the cytoplasm of the cells within 1 hr. Therefore BIPs are recognized as new members of cell penetration peptides. In this study, we investigated the mechanism of cell penetration of BIPs. DAMI cells (a human megakaryocyte cell line) and HeLa cells were used to investigate the detailed mechanism of cell penetration of BIPs. To detect the cell entry of BIPs, fluorescent dyes (fluorescein or tetramethylrodhamine) were conjugated to the N-terminus of BIPs and the cytoplasmic localization of BIPs was confirmed by confocal microscopy. Cell Penetration activities of BIPs were detected at 1 uM concentration in the culture medium. The significant accumulation of BIPs in the cytoplasm were detected within 1 hour of incubation both at 4 °C and 37 °C, suggesting that ATP-independent mechanism of cell penetration of BIP exists. However, cellular uptake of BIPs reaches plateau at 100 uM at 4 °C, whereas it increases in a dose dependent manner up to 1 mM at 37 °C without any sign of cytotoxicity. These results suggest that there are at least two mechanisms contributing to the cell penetration of BIPs that are, “ATP-independent (4 °C)” and “ATP-dependent (37 °C)” mechanisms. In addition to BIPs, we generated a series of mutated BIPs that do not bind Bax but retain cell-penetrating activities. We performed competition assay using fluorescence dye-labeled and non-labeled BIP (and the mutant BIPs), and the preliminary results suggest that there is a specific receptor for each peptide for its delivery into the cells. Our data also indicates that BIPs can deliver a cargo molecule (e.g. fluorescent dye) with at least the same molecular weight. Unlike other cell penetrating peptides, BIP has minimum toxicity due to its nature to inhibit Bax-mediated cell death. Along with the new data showing that BIP protects cells from pathological damages in cell culture and animal model, we will discuss the potential application of BIPs as a new type of drug delivery tool.

scholarly journals In silico approaches for designing highly effective cell penetrating peptides

2013 ◽  
Vol 11 (1) ◽  
pp. 74 ◽  
Author(s):  
Ankur Gautam ◽  
Kumardeep Chaudhary ◽  
Rahul Kumar ◽  
Arun Sharma ◽  
Pallavi Kapoor ◽  
...  
Keyword(s):  
In Silico ◽  
Cell Penetrating Peptides ◽  
Cell Penetrating ◽  
Highly Effective

scholarly journals A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0254468
Author(s):  
Schuyler B. Gentry ◽  
Scott J. Nowak ◽  
Xuelei Ni ◽  
Stephanie A. Hill ◽  
Lydia R. Wade ◽  
...  
Keyword(s):  
Real Time ◽  
Cultured Cells ◽  
Flow Chamber ◽  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Calmodulin Binding ◽  
Distance Analysis ◽  
Cell Penetrating ◽  
Broad Array ◽  
Escape Problem

Cell-penetrating peptides (CPPs) are capable of transporting molecules to which they are tethered across cellular membranes. Unsurprisingly, CPPs have attracted attention for their potential drug delivery applications, but several technical hurdles remain to be overcome. Chief among them is the so-called ‘endosomal escape problem,’ i.e. the propensity of CPP-cargo molecules to be endocytosed but remain entrapped in endosomes rather than reaching the cytosol. Previously, a CPP fused to calmodulin that bound calmodulin binding site-containing cargos was shown to efficiently deliver cargos to the cytoplasm, effectively overcoming the endosomal escape problem. The CPP-adaptor, “TAT-CaM,” evinces delivery at nM concentrations and more rapidly than we had previously been able to measure. To better understand the kinetics and mechanism of CPP-adaptor-mediated cargo delivery, a real-time cell penetrating assay was developed in which a flow chamber containing cultured cells was installed on the stage of a confocal microscope to allow for observation ab initio. Also examined in this study was an improved CPP-adaptor that utilizes naked mole rat (Heterocephalus glaber) calmodulin in place of human and results in superior internalization, likely due to its lesser net negative charge. Adaptor-cargo complexes were delivered into the flow chamber and fluorescence intensity in the midpoint of baby hamster kidney cells was measured as a function of time. Delivery of 400 nM cargo was observed within seven minutes and fluorescence continued to increase linearly as a function of time. Cargo-only control experiments showed that the minimal uptake which occurred independently of the CPP-adaptor resulted in punctate localization consistent with endosomal entrapment. A distance analysis was performed for cell-penetration experiments in which CPP-adaptor-delivered cargo showing wider dispersions throughout cells as compared to an analogous covalently-bound CPP-cargo. Small molecule endocytosis inhibitors did not have significant effects upon delivery. The real-time assay is an improvement upon static endpoint assays and should be informative in a broad array of applications.

Human Protein 53-Derived Cell-Penetrating Peptides

2012 ◽  
Vol 18 (4) ◽  
pp. 291-297 ◽  
Author(s):  
Julia Suhorutsenko ◽  
Elo Eriste ◽  
Dana-Maria Copolovici ◽  
Ülo Langel
Keyword(s):  
Cell Penetrating Peptides ◽  
Human Protein ◽  
Cell Penetrating

scholarly journals Glycosylated cell-penetrating peptides and their conjugates to a proapoptotic peptide: preparation by click chemistry and cell viability studies

2009 ◽  
Vol 3 (2) ◽  
pp. 51-65 ◽  
Author(s):  
Laurence Dutot ◽  
Pascaline Lécorché ◽  
Fabienne Burlina ◽  
Rodrigue Marquant ◽  
Vanessa Point ◽  
...  
Keyword(s):  
Cell Viability ◽  
Click Chemistry ◽  
Cell Penetrating Peptides ◽  
Cell Penetrating ◽  
Peptide Preparation

scholarly journals Overlapping Properties of the Short Membrane-Active Peptide BP100 With (i) Polycationic TAT and (ii) α-helical Magainin Family Peptides

2021 ◽  
Vol 11 ◽  
Author(s):  
Christian Mink ◽  
Erik Strandberg ◽  
Parvesh Wadhwani ◽  
Manuel N. Melo ◽  
Johannes Reichert ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Mechanism Of Action ◽  
Helical Structure ◽  
Lipid Vesicles ◽  
Cell Penetrating Peptides ◽  
Cell Uptake ◽  
Active Peptide ◽  
Hiv Tat ◽  
Cell Penetrating ◽  
Α Helix

BP100 is a short, designer-made membrane-active peptide with multiple functionalities: antimicrobial, cell-penetrating, and fusogenic. Consisting of five lysines and 6 hydrophobic residues, BP100 was shown to bind to lipid bilayers as an amphipathic α-helix, but its mechanism of action remains unclear. With these features, BP100 embodies the characteristics of two distinctly different classes of membrane-active peptides, which have been studied in detail and where the mechanism of action is better understood. On the one hand, its amphiphilic helical structure is similar to the pore forming magainin family of antimicrobial peptides, though BP100 is much too short to span the membrane. On the other hand, its length and high charge density are reminiscent of the HIV-TAT family of cell penetrating peptides, for which inverted micelles have been postulated as translocation intermediates, amongst other mechanisms. Assays were performed to test the antimicrobial and hemolytic activity, the induced leakage and fusion of lipid vesicles, and cell uptake. From these results the functional profiles of BP100, HIV-TAT, and the magainin-like peptides magainin 2, PGLa, MSI-103, and MAP were determined and compared. It is observed that the activity of BP100 resembles most closely the much longer amphipathic α-helical magainin-like peptides, with high antimicrobial activity along with considerable fusogenic and hemolytic effects. In contrast, HIV-TAT shows almost no antimicrobial, fusogenic, or hemolytic effects. We conclude that the amphipathic helix of BP100 has a similar membrane-based activity as magainin-like peptides and may have a similar mechanism of action.

Hydrocarbon staple constructing highly efficient α-helix cell-penetrating peptides for intracellular cargo delivery

2020 ◽  
Vol 56 (100) ◽  
pp. 15655-15658
Author(s):  
Shu Li ◽  
Xingjiao Zhang ◽  
Chen Guo ◽  
Yali Peng ◽  
Xiaojing Liu ◽  
...  
Keyword(s):  
Cell Penetrating Peptides ◽  
Highly Efficient ◽  
Cargo Delivery ◽  
Cell Penetrating ◽  
Α Helix

Hydrocarbon staple constructing α-helix cell-penetrating peptides are potent tools for bioactive cargo delivery to the cytosol of cells.

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