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Cargo self-assembly rescues affinity of cell-penetrating peptides to lipid membranes.

ABSTRACT: Although cationic cell-penetrating peptides (CPPs) are able to bind to cell membranes, thus promoting cell internalization by active pathways, attachment of cargo molecules to CPPs invariably reduces their cellular uptake. We show here that CPP binding to lipid bilayers, a simple model of the cell membrane, can be recovered by designing cargo molecules that self-assemble into spherical micelles and increase the local interfacial density of CPP on the surface of the cargo. Experiments performed on model giant unilamellar vesicles under a confocal laser scanning microscope show that a family of thermally responsive elastin-like polypeptides that exhibit temperature-triggered micellization can promote temperature triggered attachment of the micelles to membranes, thus rescuing by self-assembly the cargo-induced loss of the CPP affinity to bio-membranes.

SUBMITTER: Weinberger A

PROVIDER: S-EPMC5338251 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Cargo self-assembly rescues affinity of cell-penetrating peptides to lipid membranes.

Weinberger Andreas A Walter Vivien V MacEwan Sarah R SR Schmatko Tatiana T Muller Pierre P Schroder André P AP Chilkoti Ashutosh A Marques Carlos M CM

Scientific reports 20170306

Although cationic cell-penetrating peptides (CPPs) are able to bind to cell membranes, thus promoting cell internalization by active pathways, attachment of cargo molecules to CPPs invariably reduces their cellular uptake. We show here that CPP binding to lipid bilayers, a simple model of the cell membrane, can be recovered by designing cargo molecules that self-assemble into spherical micelles and increase the local interfacial density of CPP on the surface of the cargo. Experiments performed o ...[more]

PMID: 28262825

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Project description:Cell penetrating peptides (CPPs) are a promising technology for therapeutic delivery of macromolecular cargos. CPPs have generally used covalent linkages to cargo, ensuring a common fate as one molecule. Conversely, our CPP-adaptor, TAT-CaM, noncovalently binds calmodulin binding sequence (CBS)-containing cargos in calcium rich media then dissociates in the calcium-poor endosomal environment following internalization, enhancing endosomal escape relative to standard CPPs. In this study, we report cell entry of positively charged protein cargos that were not increased by TAT-CaM while cargos based on the negatively charged maltose binding protein (MBP) displayed little intrinsic internalization but were internalized by TAT-CaM. In addition, association of positively charged proteins with negatively charged nucleic acids reduced internalization. This evidence points to the dominant role cargo charge plays in apparent CPP effectiveness. There has been little systematic investigation as to how interaction between CPPs and cargos impacts internalization efficiency. Our adaptors provide a tool that allows combinatorial assays to detect emergent properties. Toward this end we added 4 endolytic peptide (EP) sequences between cargo CBS and MBP moieties to create 4 new cargos and between TAT and CaM to create 4 new adaptors. The new cargos were assayed for internalization alone and with a panel of CPP-adaptors to identify combinations that displayed increased internalization efficiency or other properties. Among the most important results, addition of the EP LAH4 improved adaptor performance and provided some CPP capability to cargos. MBP-LAH4-CBS was internalized more effectively by most adaptors, suggesting this sequence has general stimulatory ability. Two other EPs, Aurein 1.2 and HA2, also provided some CPP capability to their MBP cargos but were unexpectedly antagonistic to internalization by most adaptors due to retention of adaptor/cargo complexes on the cell surface. We thus identified LAH4 as stimulator of internalization in both adaptors and cargos and uncovered new functionality for Aurein 1.2 and HA2, which may be related to their identification as EPs. Future experiments will test new endolytic capabilities made possible with combinatorial approaches.

Project description:Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use (2)H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the ?-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. (2)H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct (2)H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ?40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ?80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.

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Cargo self-assembly rescues affinity of cell-penetrating peptides to lipid membranes.

Publications

Cargo self-assembly rescues affinity of cell-penetrating peptides to lipid membranes.

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