Roland Brock - Academia.edu (original) (raw)

Papers by Roland Brock

Research paper thumbnail of Alterations in Red Blood Cell Deformability during Storage: A Microfluidic Approach

BioMed Research International, 2014

Red blood cells (RBCs) undergo extensive deformation when travelling through the microcapillaries... more Red blood cells (RBCs) undergo extensive deformation when travelling through the microcapillaries. Deformability, the combined result of properties of the membrane-cytoskeleton complex, the surface area-to-volume ratio, and the hemoglobin content, is a critical determinant of capillary blood flow. During blood bank storage and in many pathophysiological conditions, RBC morphology changes, which has been suggested to be associated with decreased deformability and removal of RBC. While various techniques provide information on the rheological properties of stored RBCs, their clinical significance is controversial. We developed a microfluidic approach for evaluating RBC deformability in a physiologically meaningful and clinically significant manner. Unlike other techniques, our method enables a high-throughput determination of changes in deformation capacity to provide statistically significant data, while providing morphological information at the single-cell level. Our data show that, under conditions that closely mimic capillary dimensions and flow, the capacity to deform and the capacity to relax are not affected during storage in the blood bank. Our data also show that altered cell morphology by itself does not necessarily affect deformability.

Research paper thumbnail of Enhanced Cellular Uptake of Albumin-Based Lyophilisomes when Functionalized with Cell-Penetrating Peptide TAT in HeLa Cells

PLoS ONE, 2014

Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with pote... more Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs) including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers. In the present study, lyophilisomes were modified using CPPs in order to achieve enhanced cellular uptake. Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker. Fluorescent-activated cell sorting (FACS) was utilized to acquire a lyophilisome population with a particle diameter smaller than 1000 nm. Cultured HeLa, OVCAR-3, Caco-2 and SKOV-3 cells were exposed to unmodified lyophilisomes and TAT-conjugated lyophilisomes and examined with FACS. HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy. TAT-conjugation strongly increased binding and cellular uptake of lyophilisomes in a time-dependent manner in vitro, as assessed by FACS. These results were confirmed by confocal microscopy. Transmission electron microscopy indicated rapid cellular uptake of TAT-conjugated lyophilisomes via phagocytosis and/or macropinocytosis. In conclusion, TAT-peptides conjugated to albumin-based lyophilisomes are able to enhance cellular uptake of lyophilisomes in HeLa cells.

Research paper thumbnail of Lyophilisomes as a new generation of drug delivery capsules

International Journal of Pharmaceutics, 2012

Nanoparticulate drug delivery systems are currently explored to overcome critical challenges asso... more Nanoparticulate drug delivery systems are currently explored to overcome critical challenges associated with classical administration forms. In this study, we present a drug delivery system based on a novel class of proteinaceous biodegradable nano/micro capsules, lyophilisomes. Lyophilisomes can be prepared from biomolecules without the need for amphiphilicity. Albumin-based lyophilisomes were prepared by freezing, annealing and lyophilizing, resulting in capsules ranging from 100 to 3000 nm. Lyophilisomes were loaded with the anti-tumor drugs doxorubicin and curcumin using different concentrations and time/temperature regimes. Incubation in 0.1 mg/ml doxorubicin or 1.0 mg/ml curcumin resulted in an entrapment efficiency of 95±1% and 4±1%, respectively. This corresponds to a drug loading of 0.24 mg doxorubicin per milligram albumin and 0.10 mg curcumin per milligram albumin. Drug release profiles from doxorubicin and curcumin-loaded lyophilisomes were studied in culture medium and showed slow release for doxorubicin (2.7% after 72 h), and rapid release for curcumin (55% after 72 h). When applied to cells, non-loaded lyophilisomes did not influence cell viability, even at high concentrations (1 mg/ml). Lyophilisomes were internalized by cells. When loaded with doxorubicin and curcumin, lyophilisomes strongly reduced cell proliferation and viability of SKOV-3 and HeLa cells, respectively, to a level similar or better compared to an equal amount of free drugs. In conclusion, albumin lyophilisomes show potential as (nano)carriers of drugs for tumor cell elimination.

Research paper thumbnail of Structure Analysis and Conformational Transitions of the Cell Penetrating Peptide Transportan 10 in the Membrane-Bound State

PLoS ONE, 2014

Structure analysis of the cell-penetrating peptide transportan 10 (TP10) revealed an exemplary ra... more Structure analysis of the cell-penetrating peptide transportan 10 (TP10) revealed an exemplary range of different conformations in the membrane-bound state. The bipartite peptide (derived N-terminally from galanin and C-terminally from mastoparan) was found to exhibit prominent characteristics of (i) amphiphilic a-helices, (ii) intrinsically disordered peptides, as well as (iii) b-pleated amyloid fibrils, and these conformational states become interconverted as a function of concentration. We used a complementary approach of solid-state 19 F-NMR and circular dichroism in oriented membrane samples to characterize the structural and dynamical behaviour of TP10 in its monomeric and aggregated forms. Nine different positions in the peptide were selectively substituted with either the Lor D-enantiomer of 3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine (CF 3 -Bpg) as a reporter group for 19 F-NMR. Using the L-epimeric analogs, a comprehensive three-dimensional structure analysis was carried out in lipid bilayers at low peptide concentration, where TP10 is monomeric. While the N-terminal region is flexible and intrinsically unstructured within the plane of the lipid bilayer, the Cterminal a-helix is embedded in the membrane with an oblique tilt angle of ,55u and in accordance with its amphiphilic profile. Incorporation of the sterically obstructive D-CF 3 -Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide. At high concentration, these helix-destabilizing C-terminal substitutions promote aggregation into immobile b-sheets, which resemble amyloid fibrils. On the other hand, the obstructive D-CF 3 -Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration. The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the a-helix counteracts the tendency of the unfolded N-terminus to self-assemble into b-pleated fibrils. Citation: Fanghänel S, Wadhwani P, Strandberg E, Verdurmen WPR, Bü rck J, et al. (2014) Structure Analysis and Conformational Transitions of the Cell Penetrating Peptide Transportan 10 in the Membrane-Bound State. PLoS ONE 9(6): e99653.

Research paper thumbnail of Multivalent presentation of the cell-penetrating peptide nona-arginine on a linear scaffold strongly increases its membrane-perturbing capacity

Biochimica et biophysica acta, 2014

Arginine-rich cell-penetrating peptides (CPP) are widely employed as delivery vehicles for a larg... more Arginine-rich cell-penetrating peptides (CPP) are widely employed as delivery vehicles for a large variety of macromolecular cargos. As a mechanism-of-action for induction of uptake cross-linking of heparan sulfates and interaction with lipid head groups have been proposed. Here, we employed a multivalent display of the CPP nona-arginine (R9) on a linear dextran scaffold to assess the impact of heparan sulfate and lipid interactions on uptake and membrane perturbation. Increased avidity through multivalency should potentiate molecular phenomena that may only play a minor role if only individual peptides are used. To this point, the impact of multivalency has only been explored for dendrimers, CPP-decorated proteins and nanoparticles. We reasoned that multivalency on a linear scaffold would more faithfully mimic the arrangement of peptides at the membrane at high local peptide concentrations. On average, five R9 were coupled to a linear dextran backbone. The conjugate displayed a dir...

Research paper thumbnail of A Comprehensive Model for the Cellular Uptake of Cationic Cell-penetrating Peptides

Research paper thumbnail of Quantitative Glucose and ATP Sensing in Mammalian Cells

Pharmaceutical Research, 2011

The functioning and survival of mammalian cells requires an active energy metabolism. Metabolic d... more The functioning and survival of mammalian cells requires an active energy metabolism. Metabolic dysfunction plays an important role in many human diseases, including diabetes, cancer, inherited mitochondrial disorders, and metabolic syndrome. The monosaccharide glucose constitutes a key source of cellular energy. Following its import across the plasma membrane, glucose is converted into pyruvate by the glycolysis pathway. Pyruvate oxidation supplies substrates for the ATP-generating mitochondrial oxidative phosphorylation (OXPHOS) system. To gain cell-biochemical knowledge about the operation and regulation of the cellular energy metabolism in the healthy and diseased state, quantitative knowledge is required about (changes in) metabolite concentrations under (non) steady-state conditions. This information can, for instance, be used to construct more realistic in silico models of cell metabolism, which facilitates understanding the consequences of metabolic dysfunction as well as on- and off-target effects of mitochondrial drugs. Here we review the current state-of-the-art live-cell quantification of two key cellular metabolites, glucose and ATP, using protein-based sensors. The latter apply the principle of FRET (fluorescence resonance energy transfer) and allow measurements in different cell compartments by fluorescence microscopy. We further summarize the properties and applications of the FRET-based sensors, their calibration, pitfalls, and future perspectives.

Research paper thumbnail of Peptide microarrays to probe for competition for binding sites in a protein interaction network

Journal of Proteomics, 2013

Cellular protein interaction networks are a result of the binding preferences of a particular pro... more 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. 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.

Research paper thumbnail of Modulation of neuronal activity by the endogenous pentapeptide QYNAD

European Journal of Neuroscience, 2003

In¯ammation and demyelination both contribute to the neurological de®cits characteristic of multi... more In¯ammation and demyelination both contribute to the neurological de®cits characteristic of multiple sclerosis. Neurological dysfunctions are attributable to in¯ammatory demyelination and, in addition, to soluble factors such as nitric oxide, cytokines and antibodies. QYNAD, an endogenous pentapeptide identi®ed in the cerebrospinal¯uid of patients with demyelinating disorders, has been proposed to promote axonal dysfunction by blocking sodium channels. The present study aimed at characterizing the properties of QYNAD in acutely isolated thalamic neurons in vitro. QYNAD, but not a scrambled peptide (NYDQA), blocked sodium channels in neurons by shifting the steady-state inactivation to more negative potentials. Blocking properties followed a dose±response curve with a maximum effect at 10 mM. A¯uorescently labelled QYNAD analogue with retained biological activity speci®cally stained thalamic neurons, positive for type II sodium channels, thus demonstrating the speci®city of QYNAD binding. Our study con®rms and extends previous observations describing QYNAD as a potent sodium channel-blocking agent. These data as well as our preliminary observations in in vivo experiments in an animal model of in¯ammatory CNS demyelination warrant further in vivo studies in order to clarify the exact pathogenetic role of QYNAD in in¯ammatory neurological diseases.

Research paper thumbnail of Multivalent Design of Apoptosis-Inducing Bid-BH3 Peptide-Oligosaccharides Boosts the Intracellular Activity at Identical Overall Peptide Concentrations

Chemistry - A European Journal, 2012

Multivalent peptide-oligosaccharide conjugates were prepared and used to investigate the multival... more 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.

Research paper thumbnail of Lipoconjugates for the Noncovalent Generation of Microarrays in Biochemical and Cellular Assays

Research paper thumbnail of Break on through to the Other Side-Biophysics and Cell Biology Shed Light on Cell-Penetrating Peptides

ChemBioChem, 2005

Cell-penetrating peptides (CPPs) have become widely used vectors for the cellular import of molec... more 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.

Research paper thumbnail of Geometry sensing by dendritic cells dictates spatial organization and PGE2-induced dissolution of podosomes

Cellular and Molecular Life Sciences, 2012

Assembly and disassembly of adhesion structures such as focal adhesions (FAs) and podosomes regul... more Assembly and disassembly of adhesion structures such as focal adhesions (FAs) and podosomes regulate cell adhesion and differentiation. On antigen-presenting dendritic cells (DCs), acquisition of a migratory and immunostimulatory phenotype depends on podosome dissolution by prostaglandin E 2 (PGE 2 ). Whereas the effects of physico-chemical and topographical cues have been extensively studied on FAs, little is known about how podosomes respond to these signals. Here, we show that, unlike for FAs, podosome formation is not controlled by substrate physico-chemical properties. We demonstrate that cell adhesion is the only prerequisite for podosome formation and that substrate availability dictates podosome density. Interestingly, we show that DCs sense 3-dimensional (3-D) geometry by aligning podosomes along the edges of 3-D micropatterned surfaces. Finally, whereas on a 2-dimensional (2-D) surface PGE 2 causes a rapid increase in activated RhoA levels leading to fast podosome dissolution, 3-D geometric cues prevent PGE 2 -mediated RhoA activation resulting in impaired podosome dissolution even after prolonged stimulation. Our findings indicate that 2-D

Research paper thumbnail of The stoichiometry of peptide-heparan sulfate binding as a determinant of uptake efficiency of cell-penetrating peptides

Cellular and Molecular Life Sciences, 2013

Research paper thumbnail of Molecular Parameters of siRNA–Cell Penetrating Peptide Nanocomplexes for Efficient Cellular Delivery

ACS Nano, 2013

Cell-penetrating peptides (CPPs) are versatile tools for the intracellular delivery of various bi... more Cell-penetrating peptides (CPPs) are versatile tools for the intracellular delivery of various biomolecules, including siRNA.

Research paper thumbnail of Design and self-assembly of simple coat proteins for artificial viruses

Nature Nanotechnology, 2014

Research paper thumbnail of Alterations in Red Blood Cell Deformability during Storage: A Microfluidic Approach

BioMed Research International, 2014

Red blood cells (RBCs) undergo extensive deformation when travelling through the microcapillaries... more Red blood cells (RBCs) undergo extensive deformation when travelling through the microcapillaries. Deformability, the combined result of properties of the membrane-cytoskeleton complex, the surface area-to-volume ratio, and the hemoglobin content, is a critical determinant of capillary blood flow. During blood bank storage and in many pathophysiological conditions, RBC morphology changes, which has been suggested to be associated with decreased deformability and removal of RBC. While various techniques provide information on the rheological properties of stored RBCs, their clinical significance is controversial. We developed a microfluidic approach for evaluating RBC deformability in a physiologically meaningful and clinically significant manner. Unlike other techniques, our method enables a high-throughput determination of changes in deformation capacity to provide statistically significant data, while providing morphological information at the single-cell level. Our data show that, under conditions that closely mimic capillary dimensions and flow, the capacity to deform and the capacity to relax are not affected during storage in the blood bank. Our data also show that altered cell morphology by itself does not necessarily affect deformability.

Research paper thumbnail of Enhanced Cellular Uptake of Albumin-Based Lyophilisomes when Functionalized with Cell-Penetrating Peptide TAT in HeLa Cells

PLoS ONE, 2014

Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with pote... more Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs) including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers. In the present study, lyophilisomes were modified using CPPs in order to achieve enhanced cellular uptake. Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker. Fluorescent-activated cell sorting (FACS) was utilized to acquire a lyophilisome population with a particle diameter smaller than 1000 nm. Cultured HeLa, OVCAR-3, Caco-2 and SKOV-3 cells were exposed to unmodified lyophilisomes and TAT-conjugated lyophilisomes and examined with FACS. HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy. TAT-conjugation strongly increased binding and cellular uptake of lyophilisomes in a time-dependent manner in vitro, as assessed by FACS. These results were confirmed by confocal microscopy. Transmission electron microscopy indicated rapid cellular uptake of TAT-conjugated lyophilisomes via phagocytosis and/or macropinocytosis. In conclusion, TAT-peptides conjugated to albumin-based lyophilisomes are able to enhance cellular uptake of lyophilisomes in HeLa cells.

Research paper thumbnail of Lyophilisomes as a new generation of drug delivery capsules

International Journal of Pharmaceutics, 2012

Nanoparticulate drug delivery systems are currently explored to overcome critical challenges asso... more Nanoparticulate drug delivery systems are currently explored to overcome critical challenges associated with classical administration forms. In this study, we present a drug delivery system based on a novel class of proteinaceous biodegradable nano/micro capsules, lyophilisomes. Lyophilisomes can be prepared from biomolecules without the need for amphiphilicity. Albumin-based lyophilisomes were prepared by freezing, annealing and lyophilizing, resulting in capsules ranging from 100 to 3000 nm. Lyophilisomes were loaded with the anti-tumor drugs doxorubicin and curcumin using different concentrations and time/temperature regimes. Incubation in 0.1 mg/ml doxorubicin or 1.0 mg/ml curcumin resulted in an entrapment efficiency of 95±1% and 4±1%, respectively. This corresponds to a drug loading of 0.24 mg doxorubicin per milligram albumin and 0.10 mg curcumin per milligram albumin. Drug release profiles from doxorubicin and curcumin-loaded lyophilisomes were studied in culture medium and showed slow release for doxorubicin (2.7% after 72 h), and rapid release for curcumin (55% after 72 h). When applied to cells, non-loaded lyophilisomes did not influence cell viability, even at high concentrations (1 mg/ml). Lyophilisomes were internalized by cells. When loaded with doxorubicin and curcumin, lyophilisomes strongly reduced cell proliferation and viability of SKOV-3 and HeLa cells, respectively, to a level similar or better compared to an equal amount of free drugs. In conclusion, albumin lyophilisomes show potential as (nano)carriers of drugs for tumor cell elimination.

Research paper thumbnail of Structure Analysis and Conformational Transitions of the Cell Penetrating Peptide Transportan 10 in the Membrane-Bound State

PLoS ONE, 2014

Structure analysis of the cell-penetrating peptide transportan 10 (TP10) revealed an exemplary ra... more Structure analysis of the cell-penetrating peptide transportan 10 (TP10) revealed an exemplary range of different conformations in the membrane-bound state. The bipartite peptide (derived N-terminally from galanin and C-terminally from mastoparan) was found to exhibit prominent characteristics of (i) amphiphilic a-helices, (ii) intrinsically disordered peptides, as well as (iii) b-pleated amyloid fibrils, and these conformational states become interconverted as a function of concentration. We used a complementary approach of solid-state 19 F-NMR and circular dichroism in oriented membrane samples to characterize the structural and dynamical behaviour of TP10 in its monomeric and aggregated forms. Nine different positions in the peptide were selectively substituted with either the Lor D-enantiomer of 3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine (CF 3 -Bpg) as a reporter group for 19 F-NMR. Using the L-epimeric analogs, a comprehensive three-dimensional structure analysis was carried out in lipid bilayers at low peptide concentration, where TP10 is monomeric. While the N-terminal region is flexible and intrinsically unstructured within the plane of the lipid bilayer, the Cterminal a-helix is embedded in the membrane with an oblique tilt angle of ,55u and in accordance with its amphiphilic profile. Incorporation of the sterically obstructive D-CF 3 -Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide. At high concentration, these helix-destabilizing C-terminal substitutions promote aggregation into immobile b-sheets, which resemble amyloid fibrils. On the other hand, the obstructive D-CF 3 -Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration. The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the a-helix counteracts the tendency of the unfolded N-terminus to self-assemble into b-pleated fibrils. Citation: Fanghänel S, Wadhwani P, Strandberg E, Verdurmen WPR, Bü rck J, et al. (2014) Structure Analysis and Conformational Transitions of the Cell Penetrating Peptide Transportan 10 in the Membrane-Bound State. PLoS ONE 9(6): e99653.

Research paper thumbnail of Multivalent presentation of the cell-penetrating peptide nona-arginine on a linear scaffold strongly increases its membrane-perturbing capacity

Biochimica et biophysica acta, 2014

Arginine-rich cell-penetrating peptides (CPP) are widely employed as delivery vehicles for a larg... more Arginine-rich cell-penetrating peptides (CPP) are widely employed as delivery vehicles for a large variety of macromolecular cargos. As a mechanism-of-action for induction of uptake cross-linking of heparan sulfates and interaction with lipid head groups have been proposed. Here, we employed a multivalent display of the CPP nona-arginine (R9) on a linear dextran scaffold to assess the impact of heparan sulfate and lipid interactions on uptake and membrane perturbation. Increased avidity through multivalency should potentiate molecular phenomena that may only play a minor role if only individual peptides are used. To this point, the impact of multivalency has only been explored for dendrimers, CPP-decorated proteins and nanoparticles. We reasoned that multivalency on a linear scaffold would more faithfully mimic the arrangement of peptides at the membrane at high local peptide concentrations. On average, five R9 were coupled to a linear dextran backbone. The conjugate displayed a dir...

Research paper thumbnail of A Comprehensive Model for the Cellular Uptake of Cationic Cell-penetrating Peptides

Research paper thumbnail of Quantitative Glucose and ATP Sensing in Mammalian Cells

Pharmaceutical Research, 2011

The functioning and survival of mammalian cells requires an active energy metabolism. Metabolic d... more The functioning and survival of mammalian cells requires an active energy metabolism. Metabolic dysfunction plays an important role in many human diseases, including diabetes, cancer, inherited mitochondrial disorders, and metabolic syndrome. The monosaccharide glucose constitutes a key source of cellular energy. Following its import across the plasma membrane, glucose is converted into pyruvate by the glycolysis pathway. Pyruvate oxidation supplies substrates for the ATP-generating mitochondrial oxidative phosphorylation (OXPHOS) system. To gain cell-biochemical knowledge about the operation and regulation of the cellular energy metabolism in the healthy and diseased state, quantitative knowledge is required about (changes in) metabolite concentrations under (non) steady-state conditions. This information can, for instance, be used to construct more realistic in silico models of cell metabolism, which facilitates understanding the consequences of metabolic dysfunction as well as on- and off-target effects of mitochondrial drugs. Here we review the current state-of-the-art live-cell quantification of two key cellular metabolites, glucose and ATP, using protein-based sensors. The latter apply the principle of FRET (fluorescence resonance energy transfer) and allow measurements in different cell compartments by fluorescence microscopy. We further summarize the properties and applications of the FRET-based sensors, their calibration, pitfalls, and future perspectives.

Research paper thumbnail of Peptide microarrays to probe for competition for binding sites in a protein interaction network

Journal of Proteomics, 2013

Cellular protein interaction networks are a result of the binding preferences of a particular pro... more 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. 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.

Research paper thumbnail of Modulation of neuronal activity by the endogenous pentapeptide QYNAD

European Journal of Neuroscience, 2003

In¯ammation and demyelination both contribute to the neurological de®cits characteristic of multi... more In¯ammation and demyelination both contribute to the neurological de®cits characteristic of multiple sclerosis. Neurological dysfunctions are attributable to in¯ammatory demyelination and, in addition, to soluble factors such as nitric oxide, cytokines and antibodies. QYNAD, an endogenous pentapeptide identi®ed in the cerebrospinal¯uid of patients with demyelinating disorders, has been proposed to promote axonal dysfunction by blocking sodium channels. The present study aimed at characterizing the properties of QYNAD in acutely isolated thalamic neurons in vitro. QYNAD, but not a scrambled peptide (NYDQA), blocked sodium channels in neurons by shifting the steady-state inactivation to more negative potentials. Blocking properties followed a dose±response curve with a maximum effect at 10 mM. A¯uorescently labelled QYNAD analogue with retained biological activity speci®cally stained thalamic neurons, positive for type II sodium channels, thus demonstrating the speci®city of QYNAD binding. Our study con®rms and extends previous observations describing QYNAD as a potent sodium channel-blocking agent. These data as well as our preliminary observations in in vivo experiments in an animal model of in¯ammatory CNS demyelination warrant further in vivo studies in order to clarify the exact pathogenetic role of QYNAD in in¯ammatory neurological diseases.

Research paper thumbnail of Multivalent Design of Apoptosis-Inducing Bid-BH3 Peptide-Oligosaccharides Boosts the Intracellular Activity at Identical Overall Peptide Concentrations

Chemistry - A European Journal, 2012

Multivalent peptide-oligosaccharide conjugates were prepared and used to investigate the multival... more 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.

Research paper thumbnail of Lipoconjugates for the Noncovalent Generation of Microarrays in Biochemical and Cellular Assays

Research paper thumbnail of Break on through to the Other Side-Biophysics and Cell Biology Shed Light on Cell-Penetrating Peptides

ChemBioChem, 2005

Cell-penetrating peptides (CPPs) have become widely used vectors for the cellular import of molec... more 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.

Research paper thumbnail of Geometry sensing by dendritic cells dictates spatial organization and PGE2-induced dissolution of podosomes

Cellular and Molecular Life Sciences, 2012

Assembly and disassembly of adhesion structures such as focal adhesions (FAs) and podosomes regul... more Assembly and disassembly of adhesion structures such as focal adhesions (FAs) and podosomes regulate cell adhesion and differentiation. On antigen-presenting dendritic cells (DCs), acquisition of a migratory and immunostimulatory phenotype depends on podosome dissolution by prostaglandin E 2 (PGE 2 ). Whereas the effects of physico-chemical and topographical cues have been extensively studied on FAs, little is known about how podosomes respond to these signals. Here, we show that, unlike for FAs, podosome formation is not controlled by substrate physico-chemical properties. We demonstrate that cell adhesion is the only prerequisite for podosome formation and that substrate availability dictates podosome density. Interestingly, we show that DCs sense 3-dimensional (3-D) geometry by aligning podosomes along the edges of 3-D micropatterned surfaces. Finally, whereas on a 2-dimensional (2-D) surface PGE 2 causes a rapid increase in activated RhoA levels leading to fast podosome dissolution, 3-D geometric cues prevent PGE 2 -mediated RhoA activation resulting in impaired podosome dissolution even after prolonged stimulation. Our findings indicate that 2-D

Research paper thumbnail of The stoichiometry of peptide-heparan sulfate binding as a determinant of uptake efficiency of cell-penetrating peptides

Cellular and Molecular Life Sciences, 2013

Research paper thumbnail of Molecular Parameters of siRNA–Cell Penetrating Peptide Nanocomplexes for Efficient Cellular Delivery

ACS Nano, 2013

Cell-penetrating peptides (CPPs) are versatile tools for the intracellular delivery of various bi... more Cell-penetrating peptides (CPPs) are versatile tools for the intracellular delivery of various biomolecules, including siRNA.

Research paper thumbnail of Design and self-assembly of simple coat proteins for artificial viruses

Nature Nanotechnology, 2014