Roland Brock

A Comprehensive Model for the Cellular Uptake of Cationic Cell‐penetrating Peptides

The plasma membrane represents an impermeable barrier for most macromolecules. Still some proteins and so‐called cell‐penetrating peptides enter cells efficiently. It has been shown that endocytosis contributes to the import of these molecules. However, conflicting results have been obtained concerning the nature of the endocytic process. In addition, there have been new findings for an endocytosis‐independent cellular entry. In this study, we provide evidence that the Antennapedia‐homeodomain‐derived antennapedia (Antp) peptide, nona‐arginine and the HIV‐1 Tat‐protein‐derived Tat peptide simultaneously use three endocytic pathways: macropinocytosis, clathrin‐mediated endocytosis and caveolae/lipid‐raft‐mediated endocytosis. Antennapedia differs from Tat and R9 by the extent by which the different import mechanisms contribute to uptake. Moreover, at higher concentrations, uptake occurs by a mechanism that originates from spatially restricted sites of the plasma membrane and leads to a rapid cytoplasmic distribution of the peptides. Endocytic vesicles could not be detected, suggesting an endocytosis‐independent mode of uptake. Heparinase treatment of cells negatively affects this import, as does the protein kinase C inhibitor rottlerin, expression of dominant‐negative dynamin and chlorpromazine. This mechanism of uptake was observed for a panel of different cell lines. For Antp, significantly higher peptide concentrations and inhibition of endocytosis were required to induce its uptake. The relevance of these findings for import of biologically active cargos is shown.

Break on through to the Other Side—Biophysics and Cell Biology Shed Light on Cell-Penetrating Peptides

Cell‐penetrating peptides (CPPs) have become widely used vectors for the cellular import of molecules in basic and applied biomedical research. Despite the broad acceptance of these molecules as molecular carriers, the details of the mode of cellular internalization and membrane permeation remain elusive. Within the last two years endocytosis has been demonstrated to be a route of uptake shared by several CPPs. These findings had a significant impact on CPP research. State‐of‐the‐art cell biology is now required to advance the understanding of the intracellular fate of the CPP and cargo molecules. Owing to their presumed ability to cross lipid bilayers, CPPs also represent highly interesting objects of biophysical research. Numerous studies have investigated structure–activity relationships of CPPs with respect to their ability to bind to a lipid bilayer or to cross this barrier. Endocytosis route only relocates the membrane permeation from the cell surface to endocytic compartments. Therefore, biophysical experiments are key to a mechanistic molecular understanding of the cellular uptake of CPPs. However, biophysical investigations have to consider the molecular environment encountered by a peptide inside and outside a cell. In this contribution we will review biophysical and cell‐biology data obtained for several prominent CPPs. Furthermore, we will summarize recent findings on the cell‐penetrating characteristics of antimicrobial peptides and the antimicrobial properties of CPPs. Peptides of both groups have overlapping characteristics. Therefore, both fields may greatly benefit from each other. The review will conclude with a perspective of how biophysics and cell biology may synergize even more efficiently in the future.

TLR1- and TLR6-independent recognition of bacterial lipopeptides.

Bacterial cell walls contain lipoproteins/peptides, which are strong modulators of the innate immune system. Triacylated lipopeptides are assumed to be recognized by TLR2/TLR1-, whereas diacylated lipopeptides use TLR2/TLR6 heteromers for signaling. Following our initial discovery of TLR6-independent diacylated lipopeptides, we could now characterize di- and triacylated lipopeptides (e.g. Pam2C-SK4, Pam3C-GNNDESNISFKEK), which have stimulatory activity in TLR1- and in TLR6-deficient mice. Furthermore, for the first time, we present triacylated lipopeptides with short length ester-bound fatty acids (like PamOct2C-SSNASK4), which induce no response in TLR1-deficient cells. No differences in the phosphorylation of MAP kinases by lipopeptide analogs having different TLR2-coreceptor usage were observed. Blocking experiments indicated that different TLR2 heteromers recognize their specific lipopeptide ligands independently from each other. In summary, a triacylation pattern is necessary but not sufficient to render a lipopeptide TLR1-dependent, and a diacylation pattern is necessary but not sufficient to render a lipopeptide TLR6-dependent. Contrary to the current model, distinct lipopeptides are recognized by TLR2 in a TLR1- and TLR6-independent manner.

Toll-like receptor 6-independent signaling by diacylated lipopeptides.

Bacterial lipopeptides are strong immune modulators that activate early host responses after infection as well as initiating adjuvant effects on the adaptive immune system. These lipopeptides induce signaling in cells of the immune system through Toll‐like receptor 2 (TLR2)–TLR1 or TLR2–TLR6 heteromers. So far it has been thought that triacylated lipopeptides, such as the synthetic N‐palmitoyl‐S‐[2,3‐bis(palmitoyloxy)‐(2RS)‐propyl]‐(R)‐cysteine (Pam3)‐CSK4, signal through TLR2–TLR1 heteromers, whereas diacylated lipopeptides, like the macrophage‐activating lipopeptide from Mycoplasma fermentans (MALP2) or S‐[2,3‐bis(palmitoyloxy)‐(2RS)‐propyl]‐(R)‐cysteine (Pam2)‐CGNNDESNISFKEK, induce signaling through TLR2–TLR6 heteromers. Using new synthetic lipopeptide derivatives we addressed the contribution of the lipid and, in particular, the peptide moieties with respect to TLR2 heteromer usage. In contrast to the current model of receptor usage, not only triacylated lipopeptides, but also diacylated lipopeptides like Pam2CSK4 and the elongated MALP2 analog Pam2CGNNDESNISFKEK‐SK4 (MALP2‐SK4) induced B lymphocyte proliferation and TNF‐α secretion in macrophages in a TLR6‐independent manner as determined with cells from TLR6‐deficient mice. Our results indicate that both the lipid and the N‐terminal peptides of lipoproteins contribute to the specificity of recognition by TLR2 heteromers and are responsible for the ligand–receptor interaction on host cells.

Lipopeptide structure determines TLR2 dependent cell activation level

Bacterial lipoproteins/peptides are composed of di‐O‐acylated‐S‐(2,3‐dihydroxypropyl)‐cysteinyl residues N‐terminally coupled to distinct polypeptides, which can be N‐acylated with a third fatty acid. Using a synthetic lipopeptide library we characterized the contribution of the lipid portion to the TLR2 dependent pattern recognition. We found that the two ester bound fatty acid length threshold is beyond eight C atoms because almost no response was elicited by cellular challenge with analogues carrying shorter acyl chains in HEK293 cells expressing recombinant human TLR2. In contrast, the amide bound fatty acid is of lesser importance. While two ester‐bound palmitic acids mediate a high stimulatory activity of the respective analogue, a lipopeptide carrying one amide‐bound and another ester‐bound palmitic acid molecule was inactive. In addition, species specific LP recognition through murine and human TLR2 depended on the length of the two ester bound fatty acid chains. In conclusion, our results indicate the responsibility of both ester bound acyl chains but not of the amide bound fatty acid molecule for the TLR dependent cellular recognition of canonical triacylated LP, as well as a requirement for a minimal acyl chain length. Thus they might support the explanation of specific immuno‐stimulatory potentials of different microorganisms and provide a basis for rational design of TLR2 specific adjuvants mediating immune activation to distinct levels.

A quantitative validation of fluorophore-labelled cell-permeable peptide conjugates: fluorophore and cargo dependence of import

Cell-permeable peptides were evaluated for a quantitatively controlled import of small molecules. The dependence of the import efficiency on the fluorophore, on the position of the fluorophore as well as on the nature of the cargo were addressed. Cellular uptake was quantitated by flow cytometry and fluorescence correlation microscopy (FCM). Fluorophores with different spectral characteristics, covering the whole visible spectral range, were selected in order to enable the simultaneous detection of several cell-permeable peptide constructs. The transcytosis sequences were based either on the sequence of the Antennapedia homeodomain protein (AntpHD)-derived penetratin peptide or the Kaposi fibroblast growth factor (FGF)-derived membrane translocating sequence (MTS)-peptide. In general, the AntpHD-derived peptides had a three- to fourfold higher import efficiency than the MTS-derived peptides. In spite of the very different physicochemical characteristics of the fluorophores, the import efficiencies for analogues labelled at different positions within the sequence of the import peptides showed a strong positive correlation. However, even for peptide cargos of very similar size, pronounced differences in import efficiency were observed. The use of cell-permeable peptide/cargo constructs for intracellular analyses of structure-function relationships therefore requires the determination of the intracellular concentrations for each construct individually.

One-step analysis of protein complexes in microliters of cell lysate.

We present mix and measure procedures for the analysis of protein complexes in microliters of crude human and mouse cell lysates using fluorescence correlation and crosscorrelation spectroscopy. We labeled interacting endogenous proteins by indirect immunofluorescence with all primary and secondary reagents added in one step. Especially for the screening of compounds interfering with interactions that depend on signaling-induced posttranslational modifications, the approach represents a major advance over existing protocols.

Spatially resolved single bead analysis: homogeneity, diffusion, and adsorption in cross-linked polystyrene.

Spatially resolved single bead analysis in the micrometer range was employed as a tool for evaluating homogeneity, diffusion, and adsorption in solid-phase supported reactions. Fluorescence microscopy (confocal and non-confocal) as well as IR microscopy were used to detect both the distribution of products and the formation of product gradients in representative reactions. For the first time, the optical slices of whole beads obtained by confocal fluorescence microscopy were compared with the fluorescence images of microtome-sliced beads. The experiments revealed that only physical slices of polystyrene beads deliver realistic representations of the distribution of fluorophores, and confirmed-in contrast to a recent report-the homogeneity of functional site distribution in polystyrene beads. Moreover, the pattern of product formation obtained from an acylation reaction as well as from an alkylation reaction were employed as probes to study the impact of bead size, diffusion, and adsorption on the reaction progress. A simulation of the diffusion process was conducted and compared with the experimental results. Diffusional control was found neither in the case of the alkylation nor in the case of the acylation reaction under investigation. As a consequence, the reaction progress was not a function of the bead sizes as proposed in the literature. Interestingly, in the case of rhodamine acylation with substoichiometric amounts an adsorption-controlled reaction was found. This result highlights the significance of adsorptive effects in solid-phase supported chemistry.

Selectivity of competitive multivalent interactions at interfaces.

The development of synthetic, low‐molecular‐weight ligand receptor systems for the selective control of biomolecular interactions remains a major challenge. Binding of oligohistidine peptides to chelators containing Ni2+‐loaded nitrilotriacetic acid (NTA) moieties is one of the most widely used and best‐characterised recognition systems. Recognition units containing multiple NTA moieties (multivalent chelator headgroups, MCHs) recognise oligohistidines with substantially increased binding affinities. Different multivalencies both at the level of the MCH and at that of the oligohistidine ligand provide a powerful means to vary the affinity of the interaction systematically. Here we have explored the selectivity for the binding of different oligohistidines to immobilised MCH. Using microarrays of mono‐, bis‐, tris‐ and tetrakis‐NTA chelators spotted at different surface densities, we explored the ability of these binders to discriminate fluorescently labelled hexa‐ and decahistidine peptides. When hexa‐ and decahistidine were tested alone, the discrimination of ligands showed little dependence either on the nature or on the density of the chelator. In contrast, coincubation of both peptides decreased the affinity of hexahistidine, increased the affinity of decahistidine, and made the binding of decahistidine highly dependent on MCH density. Kinetic binding assays by dual‐colour total internal reflection fluorescence spectroscopy revealed active exchange of His6 by His10 and confirmed the high selectivity towards His10. Our results establish the key role of surface multivalency for the selectivity of multivalent interactions at interfaces.

Detection of antibody peptide interaction using microcantilevers as surface stress sensors

Micromechanical cantilevers were validated for the detection of antibody peptide interactions. For this purpose, gold-coated microcantilevers were chemically functionalized with an analog of the myc-tag decapeptide. Chemoselective covalent coupling of the peptide to the gold surface occurred via the sulfhydryl-group of a C-terminal cystein residue. In order to verify the surface functionalization by epifluorescence microscopy, a carboxyfluorescein reporter group was attached to the N-terminus. Binding of an anti-myc-tag antibody caused a bending of the microcantilever, resulting from a change in surface stress. The bending was recorded optically from the deflection of a laser beam. In order to compensate for bending due to nonspecific interactions, a nonfunctionalized cantilever serving as a reference was measured in parallel. A charge coupled device camera served as a position-sensitive detector, enabling the parallel and simultaneous detection of signals from an array of microcantilevers. The results va...