A peptide-functionalized polymer as a minimal scaffold protein to enhance cluster formation in early T cell signal transduction.

In cellular signal transduction, scaffold proteins provide binding sites to organize signaling proteins into supramolecular complexes and act as nodes in the signaling network. Furthermore, multivalent interactions between the scaffold and other signaling proteins contribute to the formation of protein microclusters. Such microclusters are prominent in early T cell signaling. Here, we explored the minimal structural requirement for a scaffold protein by coupling multiple copies of a proline-rich peptide corresponding to an interaction motif for the SH3 domain of the adaptor protein GADS to an N-(2-hydroxypropyl)methacrylamide polymer backbone. When added to GADS-containing cell lysates, these scaffolds (but not individual peptides) promoted the binding of GADS to peptide microarrays. This can be explained by the cross-linking of GADS into larger complexes. Furthermore, following import into Jurkat T cell leukemia cells, this synthetic scaffold enhanced the formation of microclusters of signaling proteins.

Peptide microarrays to probe for competition for binding sites in a protein interaction network.

Cellular protein interaction networks are a result of the binding preferences of a particular protein and the entirety of interactors that mutually compete for binding sites. Therefore, the reconstruction of interaction networks by the accumulation of interaction networks for individual proteins will greatly overestimate connectivity within the network. Here, we addressed the impact of intracellular complexity on signalling networks using microarrays that carried a collection of peptides binding to the GRB2 SH2 and SH3 domains. Binding patterns and affinities for the recombinant adaptor protein GRB2 were compared with the ones for the protein in cell lysates. Peptide microarrays were titrated with the histidine-tagged recombinant protein, cell lysates or mixtures of both. Indeed, for recombinant GRB2, binding was detected for more peptides than for GRB2 in cell lysates. Moreover, binding was also observed for poor binders. It was impossible to define affinity thresholds for the binding of the recombinant protein to enable a discrimination of physiologically relevant interactions. Titrations of recombinant protein with lysate confirmed competition as the basis for fewer interactions. Importantly, the methods presented here enable the description of physiologically relevant binding patterns for proteins of interest and the identification of those peptide motifs, which are most strongly affected by competition. BIOLOGICAL SIGNIFICANCE: The biological significance of protein-protein interactions can only be addressed in a physiologically meaningful way in the presence of the endogenous proteome which may contain proteins that compete for binding sites. Using peptide microarrays, we here demonstrate for the adaptor protein GRB2 that this competition strongly reduces the number of interactions with other signalling proteins.

Multivalent design of apoptosis-inducing bid-BH3 peptide-oligosaccharides boosts the intracellular activity at identical overall peptide concentrations.

Multivalent peptide-oligosaccharide conjugates were prepared and used to investigate the multivalency effect concerning the activity of Bid-BH3 peptides in live cells. Dextran oligosaccharides were carboxyethylated selectively in the 2-position of the carbohydrate units and activated for the ligation of N-terminally cysteinylated peptides. Ligation through maleimide coupling was found to be superior to the native chemical ligation protocol. Monomeric Bid-BH3 peptides were virtually inactive, whereas pentameric peptide conjugates induced apoptosis up to 20-fold stronger at identical peptide concentrations. Comparison of lowly multivalent and highly multivalent peptide dextrans proved a multivalency effect in life cells which was specific for the BH3 peptide sequence.

The intracellular pharmacokinetics of terminally capped peptides.

With significant progress in delivery technologies, peptides and peptidomimetics are receiving increasing attention as potential therapeutics also for intracellular applications. However, analyses of the intracellular behavior of peptides are a challenge; therefore, knowledge on the intracellular pharmacokinetics of peptides is limited. So far, most research has focused on peptide degradation in the context of antigen processing, rather than on peptide stability. Here, we studied the structure-activity relationship of peptides with respect to intracellular residence time and proteolytic breakdown. The peptides comprised a collection of interaction motifs of SH2 and SH3 domains with different charge but that were of similar size and carried an N-terminal fluorescein moiety. First, we show that electroporation is a highly powerful technique to introduce peptides with different charge and hydrophobicity in uniform yields. Remarkably, the peptides differed strongly in retention of intracellular fluorescence with half-lives ranging from only 1 to more than 10 h. Residence times were greatly increased for retro-inverso peptides, demonstrating that rapid loss of fluorescence is a function of peptide degradation rather than the physicochemical characteristics of the peptide. Differences in proteolytic sensitivity were further confirmed using fluorescence correlation spectroscopy as a separation-free analytical technique to follow degradation in crude cell lysates and also in intact cells. The results provide a straightforward analytical access to a better understanding of the principles of peptide stability inside cells and will therefore greatly assist the development of bioactive peptides.

Coupling to polymeric scaffolds stabilizes biofunctional peptides for intracellular applications.

Here, we demonstrate that coupling to N-hydroxypropyl methacrylamide (HPMA) copolymer greatly enhances the activity of apoptosis-inducing peptides inside cells. Peptides corresponding to the BH3 domain of Bid were coupled to a thioester-activated HPMA (28.5 kDa) via native chemical ligation in a simple one-pot synthesis. Peptides and polymer conjugates were introduced into cells either by electroporation or by conjugation to the cell-penetrating peptide nona-arginine. The molecular basis of the increased activity is elucidated in detail. Loading efficiency and intracellular residence time were assessed by confocal microscopy. Fluorescence correlation spectroscopy was used as a separation-free analytical technique to determine proteolytic degradation in crude cell lysates. HPMA conjugation strongly increased the half-life of the peptides in crude cell lysates and inside cells, revealing proteolytic protection as the basis for higher activity.

Measurements of the intracellular stability of CPPs.

Nowadays, the analysis of the uptake and intracellular distribution of cell-penetrating peptides mostly relies on fluorescence microscopy, using fluorescently labeled CPP analogs. However, fluorescence microscopy does not reveal to which degree fluorescence reflects the intact peptide or only breakdown products. Here, we introduce fluorescence correlation spectroscopy (FCS) as a powerful method to address peptide stability in cells and cell lysates. Measurements in lysates of cells incubated with peptide yield information on degradation of the total cellular peptide content. In combination with protease inhibitors, such measurements enable conclusions on trafficking pathways. Intracellular FCS measurements provide direct information on peptide degradation and association with cellular structures in intact cells.

Simultaneous detection of intracellular target and off-target binding of small molecule cancer drugs at nanomolar concentrations.

BACKGROUND AND PURPOSE: In vitro assays that determine activities of drug candidates with isolated targets have only limited predictive value for activities in cellular assays. Poor membrane permeability and off-target binding are major reasons for such discrepancies. However, it still difficult to directly analyse off-target binding at the same time as target binding, on a subcellular level. Here, we present a combination of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) as a solution to this problem. EXPERIMENTAL APPROACH: The well-established dihydrofolate reductase inhibitor methotrexate and the kinase inhibitors PD173956 and purvalanol B were conjugated via polyethylene glycol linkers with the fluorophore Cy5. The cellular uptake and subcellular distribution of these compounds in single human cancer-derived cells were investigated by confocal laser scanning microscopy. In addition, molecular interactions inside the cell with the respective target proteins and off-target binding were detected simultaneously in the nanomolar range by FCCS and FCS, respectively, using cells expressing green fluorescent protein fusion proteins of dihydrofolate reductase and Abelson kinase 1. KEY RESULTS: Large differences in the interaction patterns were found for these compounds. For methotrexate-Cy5, drug-target interactions could be detected and dissociation constants determined. In contrast, PD173956-Cy5 showed strong interactions with intracellular high-molecular weight structures, other than its target. CONCLUSIONS AND IMPLICATIONS: The combination of FCS and FCCS provides a powerful means to assess subcellular pharmacokinetics and dynamics of drug candidates at nanomolar concentrations.

Cationic cell-penetrating peptides induce ceramide formation via acid sphingomyelinase: implications for uptake.

Cationic cell-penetrating peptides (CPP) are receiving increasing attention as molecular transporters of membrane-impermeable molecules. Import of cationic CPP occurs both via endocytosis and - at higher peptide concentrations - in an endocytosis-independent manner via localized regions of the plasma membrane. At present, this endocytosis-independent import of cationic CPP is not well understood, but has been shown to be sensitive to various pharmacological inhibitors, suggesting a role of an unidentified enzymatic activity. Here, we demonstrate that the direct translocation of cationic CPP depends on a CPP-induced translocation of acid sphingomyelinase (ASMase) to the outer leaflet of the plasma membrane and ceramide formation. The involvement of ASMase in uptake was confirmed by a pharmacological inhibition of ASMase by imipramine and a subsequent rescue of uptake through external addition of sphingomyelinase, and by using ASMase-deficient cells. We also found that the threshold for direct CPP translocation can be lowered through addition of sphingomyelinase and that sphingomyelinase enhances the translocation of R9 coupled to low-molecular weight cargos, but not high-molecular weight cargos. In conclusion, we show that a previously poorly understood mechanism of cationic CPP import depends on the ASMase-dependent formation of ceramide on the outer leaflet of the plasma membrane. To our knowledge, this is the first illustration that a class of delivery vectors operates through the induction of an enzymatic activity that changes the lipid composition of the plasma membrane.

A cell-penetrating peptide derived from human lactoferrin with conformation-dependent uptake efficiency.

The molecular events that contribute to the cellular uptake of cell-penetrating peptides (CPP) are still a matter of intense research. Here, we report on the identification and characterization of a 22-amino acid CPP derived from the human milk protein, lactoferrin. The peptide exhibits a conformation-dependent uptake efficiency that is correlated with efficient binding to heparan sulfate and lipid-induced conformational changes. The peptide contains a disulfide bridge formed by terminal cysteine residues. At concentrations exceeding 10 mum, this peptide undergoes the same rapid entry into the cytoplasm that was described previously for the arginine-rich CPPs nona-arginine and Tat. Cytoplasmic entry strictly depends on the presence of the disulfide bridge. To better understand this conformation dependence, NMR spectroscopy was performed for the free peptide, and CD measurements were performed for free and lipid-bound peptide. In solution, the peptides showed only slight differences in secondary structure, with a predominantly disordered structure both in the presence and absence of the disulfide bridge. In contrast, in complex with large unilamellar vesicles, the conformation of the oxidized and reduced forms of the peptide clearly differed. Moreover, surface plasmon resonance experiments showed that the oxidized form binds to heparan sulfate with a considerably higher affinity than the reduced form. Consistently, membrane binding and cellular uptake of the peptide were reduced when heparan sulfate chains were removed.

The in vitro biological activity of the HLA-DR-binding clinical IgG4 antibody 1D09C3 is a consequence of the disruption of cell aggregates and can be abrogated by Fab arm exchange.

Antibodies of the IgG4 subclass, directed against cell surface antigens have received attention as therapeutic molecules due to their poor induction of the complement system. The MHC class II-directed IgG4 antibody 1D09C3 has been explored for the treatment of lymphomas. The mechanism-of-action is still controversial. Apoptosis induction following HLA-DR engagement has been proposed. However, the validity of these results has been questioned by the observation that antibodies may induce formation of cell aggregates and cell death is induced upon dispersion of these aggregates prior to the quantification of cell death by flow cytometry. Here we address the capacity of 1D09C3 to induce apoptosis in vitro, also taking account of the recently reported Fab arm exchange of IgG4 antibodies. 1D09C3 induces formation of tight cellular aggregates that can only be dispersed at the expense of massive cell damage and death. Using dual color fluorescence cross-correlation spectroscopy (FCCS) we demonstrate that also this antibody undergoes Fab arm exchange in the presence of IgG4. FCCS is a powerful technique to investigate the molecular mechanism of Fab arm exchange using minute amounts of reagents. Following exchange, the functionally monovalent 1D09C3 chimeras loose their ability to induce aggregate formation of HLA-DR-positive cells. Neither functionally monovalent nor bivalent 1D09C3 antibodies induce cell death or apoptosis in myeloma target cells, when microscopy instead of flow cytometry is employed as the analytical technique. Our results indicate that the activity of 1D09C3 in vitro may have been a consequence of assay design rather than an ability to induce HLA-DR-dependent cell death.

HPMA as a scaffold for the modular assembly of functional peptide polymers by native chemical ligation.

Synthetic peptides are valuable tools in fundamental and applied biomedical research. On one hand, these molecules provide highly efficient access to competitive inhibitors of molecular interactions and enzyme substrates by rational design. On the other hand, peptides may serve as powerful vectors to mediate cellular uptake of molecules that otherwise enter cells only poorly. The coupling of both such functionalities provides access to molecules interfering with molecular processes inside the cell. However, the combination of several functionalities on one synthetic peptide may be compromised by problems associated with the synthesis of long peptides. Native chemical ligation enables the chemoselective coupling of fully deprotected functional building blocks. However, peptide thioesters are still not accessible by standard solid-phase peptide synthesis. Here, we demonstrate the cofunctionalization of a thioester-activated N-hydroxypropyl methacrylamide (HPMA) copolymer (28,500 Da) with the cell-penetrating peptide (CPP) nonaarginine and a bioactive peptide as independent building blocks by native chemical ligation. Nonaarginine was employed as a cell-penetrating peptide (CPP), a fluorescein-labeled analogue of a pro-apoptotic peptide as a biofunctional cargo. Incorporation of the fluorescein label enabled the highly sensitive quantification of the coupling stoichiometry by fluorescence correlation spectroscopy (FCS) using 0.4 pmol/12 ng of labeled construct. A construct only bearing the functional cargo peptide required cellular import by electroporation in order to show activity. In contrast, a construct combining all functionalities was active upon incubation of cells, validating the modular nature of the approach.