Jean-Philippe Pellois

Protein delivery into live cells by incubation with an endosomolytic agent

We report that a tetramethylrhodamine-labeled dimer of the cell-penetrating peptide TAT, dfTAT, penetrates live cells by escaping from endosomes with high efficiency. By mediating endosomal leakage, dfTAT also delivers proteins into cultured cells after a simple co-incubation procedure. We achieved cytosolic delivery in several cell lines and primary cells and observed that only a relatively small amount of material remained trapped inside endosomes. Delivery did not require a binding interaction between dfTAT and a protein, multiple molecules could be delivered simultaneously, and delivery could be repeated. dfTAT-mediated delivery did not noticeably affect cell viability, cell proliferation or gene expression. dfTAT-based intracellular delivery should be useful for cell-based assays, cellular imaging applications and the ex vivo manipulation of cells.

Real-time fluorescence detection of protein transduction into live cells.

We describe a protein probe with multiple fluorescence signals that can unambiguously detect protein translocation into live cells. When combined with fluorescence microscopy, this unique probe design allows for the first time the investigator to visualize and distinguish intact and degraded proteins with high spatial and temporal resolution. Thus, by using this approach, one can now compare the mechanisms and measure the efficiency of different delivery vectors, a prerequisite for the rational design of protein transduction systems with superior properties.

Generation of endosomolytic reagents by branching of cell-penetrating peptides: tools for the delivery of bioactive compounds to live cells in cis or trans.

We describe the synthesis and cellular delivery properties of multivalent and branched delivery systems consisting of cell-penetrating peptides assembled onto a peptide scaffold using native chemical ligation. A trimeric delivery system presenting three copies of the prototypical cell-penetrating peptide TAT shows an endosomolytic activity much higher than its monomeric and dimeric counterparts. This novel reagent promotes the endosomal release of macromolecules internalized into cells by endocytosis, and as a result, it can be used to achieve cytosolic delivery of bioactive but cell-impermeable macromolecules in either cis (covalent conjugation) or trans (simple coincubation).

Conjugation to the Cell-Penetrating Peptide TAT Potentiates the Photodynamic Effect of Carboxytetramethylrhodamine

Background Cell-penetrating peptides (CPPs) can transport macromolecular cargos into live cells. However, the cellular delivery efficiency of these reagents is often suboptimal because CPP-cargo conjugates typically remain trapped inside endosomes. Interestingly, irradiation of fluorescently labeled CPPs with light increases the release of the peptide and its cargos into the cytosol. However, the mechanism of this phenomenon is not clear. Here we investigate the molecular basis of the photo-induced endosomolytic activity of the prototypical CPPs TAT labeled to the fluorophore 5(6)-carboxytetramethylrhodamine (TMR). Methodology/Principal Findings We report that TMR-TAT acts as a photosensitizer that can destroy membranes. TMR-TAT escapes from endosomes after exposure to moderate light doses. However, this is also accompanied by loss of plasma membrane integrity, membrane blebbing, and cell-death. In addition, the peptide causes the destruction of cells when applied extracellularly and also triggers the photohemolysis of red blood cells. These photolytic and photocytotoxic effects were inhibited by hydrophobic singlet oxygen quenchers but not by hydrophilic quenchers. Conclusions/Significance Together, these results suggest that TAT can convert an innocuous fluorophore such as TMR into a potent photolytic agent. This effect involves the targeting of the fluorophore to cellular membranes and the production of singlet oxygen within the hydrophobic environment of the membranes. Our findings may be relevant for the design of reagents with photo-induced endosomolytic activity. The photocytotoxicity exhibited by TMR-TAT also suggests that CPP-chromophore conjugates could aid the development of novel Photodynamic Therapy agents.

MODELING OF THE ENDOSOMOLYTIC ACTIVITY OF HA2-TAT PEPTIDES WITH RED BLOOD CELLS AND GHOSTS

HA2-TAT is a peptide-based delivery agent that combines the pH-sensitive HA2 fusion peptide from influenza and the cell-penetrating peptide TAT from HIV. This chimeric peptide is engineered to induce the cellular uptake of macromolecules into endosomes via the TAT moiety and to respond to the acidifying lumen of endosomes to cause membrane leakage and release of macromolecules into cells via the HA2 moiety. The question of how HA2 and TAT affect the properties of one another remains, however, unanswered, and the behavior of the peptide inside endosomes is mostly uncharacterized. To address these issues, the binding and membrane leakage activity of a glutamic acid-enriched analogue E5-TAT was assessed with red blood cells and giant unilamellar vesicles as membrane models for endosomes. Hemolysis and microscopy assays reveal that E5-TAT binds to membranes in a pH-dependent manner and causes membrane leakage by inducing the formation of pores through which macromolecules can escape. The TAT moiety contributes to this activity by causing a shift in the pH response of E5 and by binding to negatively charged phospholipids. On the other hand, TAT binding to glycosaminoglycans reduces the lytic activity of E5-TAT. Addition of TAT to the C-terminus of E5 can therefore either increase or inhibit the activity of E5 depending on the cellular components present at the membrane. Taken together, these results suggest a model for the endosomolytic activity of the peptide and provide the basis for the molecular design of future delivery agents.

Delivery of macromolecules into live cells by simple co-incubation with a peptide.

The delivery of biosensors and bioactive proteins into live cells can enable the manipulation or probing of intracellular processes in powerful ways. To date, targeting large and/or hydrophilic molecules across the plasma membrane of large numbers of cells rapidly and efficiently remains a challenge. An attractive approach to solving this problem consists of utilizing cell-penetrating peptides (CPPs) such as the prototypical HIV TAT peptide (GRKKRRQRRR) as delivery systems.[1] When conjugated to macromolecules such as peptides, proteins and quantum dots, CPPs have the ability to carry their cargo into cells, both in vitro and in vivo.[2, 3] However, CPPs have been demonstrated to alter the intracellular localization of the macromolecule to which they are conjugated once cytosolic delivery is achieved. For instance, TAT and other arginine and lysine-rich CPPs induce accumulation of their conjugated cargos at the nucleoli of cells (Supplementary Figure S1).[4–6] This results in an undesirable fluorescence signal from improperly targeted biosensors in imaging applications. In addition, it raises the concern that the function of delivered probes or the activity of bioactive agents might be deeply compromised by the delivery peptide. A solution to this problem is to establish a CPP to cargo linkage that can be disrupted once the macromolecule reaches the cytosolic space so as to generate an unmodified species once delivery is achieved. Examples of such strategies include the use of disulfide bonds that can be reduced inside cells or of peptides that bind non-covalently to macromolecules to form carrier peptide/cargo particles.[7–11] This latter strategy has in particular been applied to the delivery of nucleic acid sequences with great success.[12–16] Cationic CPPs can for instance interact electrostatically with negatively charged DNA or RNA sequences to form CPP/nucleic acids complexes that can cross the plasma membrane of cells. Similarly, hydrophobic CPPs that can bind non-covalently to hydrophobic regions of proteins have been used to successfully deliver these macromolecules to live cells.[7, 17] These approaches can however be problematic since the efficiencies of CPP/protein complex formation and delivery are dependent on the identity of the protein cargo. Simple and general delivery methods that would address the aforementioned problems are therefore still required.

Tunable photoactivation of a post-translationally modified signaling protein and its unmodified counterpart in live cells

An ideal technology for direct imaging of post‐translationally modified proteins would be one in which the appearance of a fluorescent signal is linked to a modification dependent protein‐activation event. Herein, we utilize the protein semisynthesis technique, expressed protein ligation (EPL), to prepare caged analogues of the signaling protein Smad2; the function and fluorescence of the analogues were then photocontrolled in a correlated fashion. We show that this strategy permits titration of the cellular levels of active phosphorylated Smad2 in its biologically relevant, full‐length form. We also prepared a nonphosphorylated, caged full‐length Smad2 analogue labeled with an orthogonal fluorophore, and simultaneously imaged the phosphorylated and nonphosphorylated forms of the protein in the same cell. This strategy should enable the dissection of the cellular consequences of post‐translational modifications (PTMs) by direct comparison of the behavior of the modified and unmodified forms of the protein following uncaging.

Photoinduced Membrane Damage of E. coli and S. aureus by the Photosensitizer-Antimicrobial Peptide Conjugate Eosin-(KLAKLAK)2

Background/Objectives Upon irradiation with visible light, the photosensitizer-peptide conjugate eosin-(KLAKLAK)2 kills a broad spectrum of bacteria without damaging human cells. Eosin-(KLAKLAK)2 therefore represents an interesting lead compound for the treatment of local infection by photodynamic bacterial inactivation. The mechanisms of cellular killing by eosin-(KLAKLAK)2, however, remain unclear and this lack of knowledge hampers the development of optimized therapeutic agents. Herein, we investigate the localization of eosin-(KLAKLAK)2 in bacteria prior to light treatment and examine the molecular basis for the photodynamic activity of this conjugate. Methodology/Principal Findings By employing photooxidation of 3,3-diaminobenzidine (DAB), (scanning) transmission electron microscopy ((S)TEM), and energy dispersive X-ray spectroscopy (EDS) methodologies, eosin-(KLAKLAK)2 is visualized at the surface of E. coli and S. aureus prior to photodynamic irradiation. Subsequent irradiation leads to severe membrane damage. Consistent with these observations, eosin-(KLAKLAK)2 binds to liposomes of bacterial lipid composition and causes liposomal leakage upon irradiation. The eosin moiety of the conjugate mediates bacterial killing and lipid bilayer leakage by generating the reactive oxygen species singlet oxygen and superoxide. In contrast, the (KLAKLAK)2 moiety targets the photosensitizer to bacterial lipid bilayers. In addition, while (KLAKLAK)2 does not disrupt intact liposomes, the peptide accelerates the leakage of photo-oxidized liposomes. Conclusions/Significance Together, our results suggest that (KLAKLAK)2 promotes the binding of eosin Y to bacteria cell walls and lipid bilayers. Subsequent light irradiation results in membrane damage from the production of both Type I & II photodynamic products. Membrane damage by oxidation is then further aggravated by the (KLAKLAK)2 moiety and membrane lysis is accelerated by the peptide. These results therefore establish how photosensitizer and peptide act in synergy to achieve bacterial photo-inactivation. Learning how to exploit and optimize this synergy should lead to the development of future bacterial photoinactivation agents that are effective at low concentrations and at low light doses.

A HA2-Fusion Tag Limits the Endosomal Release of its Protein Cargo Despite Causing Endosomal Lysis †

BACKGROUND Protein transduction domains (PTDs) can be fused to a protein to render it cell-permeable. The delivery efficiencies of PTDs are, however, often poor because PTD-protein conjugates cannot escape from endosomes. A potential solution to this problem consists in adding HA2 analogs to the PTD-protein construct as these peptides can cause endosomal lysis upon acidification of the endosomal lumen. To date, however, the utility of HA2-based PTDs has not been clearly established. METHODS We investigate the biophysical and cellular properties of the glutamate-rich HA2 analog E5 fused to the model protein TAT-mCherry. RESULTS E5-TAT-mCherry causes the release of fluorescent dextrans trapped with the protein inside endosomes. Yet, E5-TAT-mCherry itself is not released in the cytosol of cells, indicating that the protein remained trapped inside endosomes even after endosomal lysis takes place. Cytosolic delivery of the protein could be achieved, however, by insertion of a disulfide bond between E5 and its cargo. CONCLUSIONS These results show that E5 causes the retention of its fused protein inside endosomes even after lysis takes place. GENERAL SIGNIFICANCE These data establish that HA2 analogs might not be useful PTDs unless cleavable linkers are engineered between PTD and protein cargo.

Polyarginine Interacts More Strongly and Cooperatively than Polylysine with Phospholipid Bilayers

The interactions of two highly positively charged short peptide sequences with negatively charged lipid bilayers were explored by fluorescence binding assays and all-atom molecular dynamics simulations. The bilayers consisted of mixtures of phosphatidylglycerol (PG) and phosphatidylcholine (PC) lipids as well as a fluorescence probe that was sensitive to the interfacial potential. The first peptide contained nine arginine repeats (Arg9), and the second one had nine lysine repeats (Lys9). The experimentally determined apparent dissociation constants and Hill cooperativity coefficients demonstrated that the Arg9 peptides exhibited weakly anticooperative binding behavior at the bilayer interface at lower PG concentrations, but this anticooperative effect vanished once the bilayers contained at least 20 mol % PG. By contrast, Lys9 peptides showed strongly anticooperative binding behavior at all PG concentrations, and the dissociation constants with Lys9 were approximately 2 orders of magnitude higher than with Arg9. Moreover, only arginine-rich peptides could bind to the phospholipid bilayers containing just PC lipids. These results along with the corresponding molecular dynamics simulations suggested two important distinctions between the behavior of Arg9 and Lys9 that led to these striking differences in binding and cooperativity. First, the interactions of the guanidinium moieties on the Arg side chains with the phospholipid head groups were stronger than for the amino group. This helped facilitate stronger Arg9 binding at all PG concentrations that were tested. However, at PG concentrations of 20 mol % or greater, the Arg9 peptides came into sufficiently close proximity with each other so that favorable like-charge pairing between the guanidinium moieties could just offset the long-range electrostatic repulsions. This led to Arg9 aggregation at the bilayer surface. By contrast, Lys9 molecules experienced electrostatic repulsion from each other at all PG concentrations. These insights may help explain the propensity for cell penetrating peptides containing arginine to more effectively cross cell membranes in comparison with lysine-rich peptides.