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Roland Brock

Publications and source records attributed to Roland Brock.

At least 19 recordsLinked to original sources
PubMed2007

A fluorescence-based synthetic LPS sensor.

For the detection of bioanalytes, there is an ongoing search for synthetic sensors to replace enzyme-based assays which are sensitive to contaminants or suboptimal storage conditions. Lipopolysaccharide (LPS), a bacteria-borne endotoxin that may lead to life-threatening conditions such as septic shock, is one such case. Fluorescently labeled analogues of two peptide variants derived from the putative ligand-binding domain of the LPS-binding protein CD14 were developed that detect and discriminate LPS and lipids down to the submicromolar concentration range. Peptides are terminally labeled with carboxyfluorescein and tetramethylrhodamine. For one given peptide, sensitivity and specificity for the detection of LPS and discrimination from other lipids are achieved by spectral signatures that combine changes in the fluorescence resonance energy transfer (FRET) between both dyes and the total emission of tetramethylrhodamine. Alternatively, specificity is obtained by combining the FRET efficiencies of both peptide variants. In comparison to published synthetic LPS sensors, the CD14-derived sensors yield an increase in sensitivity by about 3 orders of magnitude and exhibit specificity for analytes for which the design of synthetic recognition elements is a challenging task. Moreover, one of the sensors enabled the detection of LPS in the presence of up to 50% fetal calf serum, thereby demonstrating the feasibility of this peptide-based approach for clinically relevant samples.

Binding Sites↗
PubMed2006

A network analysis of changes in molecular interactions in cellular signaling.

Multiprotein complexes play an essential role in the propagation and integration of cellular signals. However, systems level analyses of signaling-dependent changes in the pattern of molecular interactions are still missing. Signaling in T-lymphocytes is one prominent example in which multiprotein complexes orchestrate signal transduction. We implemented peptide microarrays comprising a set of interaction motifs of signaling proteins for network-based analyses of signaling-dependent changes in molecular interactions. Lysates of resting or stimulated cells were incubated on these arrays, and the binding of signaling proteins was detected by immunofluorescence. Signaling-dependent complex formation led to changes of signals on the microarrays in two ways. 1) Masking of a binding site of a signaling protein for a peptide on the array resulted in a signal decrease. 2) Interaction of a protein with a second protein, which in turn binds to a peptide on the array, resulted in a signal increase for the first protein. Dissipation of complexes led to the reverse changes. Competition with peptides corresponding to interaction motifs provided detailed information on the architecture of complexes; lack of individual signaling proteins revealed the functional interdependence of interactions in the network. We show that complex formation through phosphorylation of the scaffolding protein LAT (linker for activation of T-cells) acted as a signal amplifier. PLCgamma1 deficiency increased the resting state levels of LAT-dependent complexes and augmented the recruitment of the phosphatase SHPTP2 into complexes. For the analysis of signaling networks, the parallel detection of changes in interactions enabled the identification of functional interdependencies with minimum a priori knowledge.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Lipolanthionine peptides act as inhibitors of TLR2-mediated IL-8 secretion. Synthesis and structure-activity relationships.

Lipoproteins from gram-positive and -negative bacteria, mycoplasma, and shorter synthetic lipopeptide analogues activate cells of the innate immune system via the Toll-like receptor TLR2/TLR1 or TLR2/TLR6 heterodimers. For this reason, these compounds constitute highly active adjuvants for vaccines either admixed or covalently linked. The lanthionine scaffold has structural similarity with the S-(2,3-dihydroxypropyl)cysteine core structure of the lipopeptides. Therefore, lanthionine-based lipopeptide amides were synthesized and probed for activity as potential TLR2 agonists or antagonists. A collection of analytically defined lipolanthionine peptide amides exhibited an inhibitory effect of the TLR2-mediated IL-8 secretion when applied in high molar excess to the agonistic synthetic lipopeptide Pam3Cys-Ser-(Lys)4-OH. Structure-activity relationships revealed the influence of the chirality of the two alpha-carbon atoms, the chain lengths of the attached fatty acids and fatty amines, and the oxidation level of the sulfur atom on the inhibitory activity of the lipolanthionine peptide amides.

Adjuvants, Immunologic↗
PubMed2006

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. Pam(2)C-SK(4), Pam(3)C-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 PamOct(2)C-SSNASK(4)), 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.

Animals↗
PubMed2006

A CD14 domain with lipopolysaccharide-binding and -neutralizing activity.

The interaction of lipopolysaccharide with CD14 plays a key role in signaling that activates an early defense against pathogens but also contributes to the development of sepsis and septic shock. Here we have mapped the entire 356-amino-acid protein with synthetic 20-amino-acid peptides and have identified a new lipopolysaccharide-binding domain with a strong LPS-neutralizing activity. Moreover, analysis of the structure-activity relationship of this peptide, which corresponds to amino acids 81-100 of human CD14, revealed that leucines 87, 91, and 94 are essential for these activities. The functional relevance of these residues was confirmed by cellular expression of mutant CD14 proteins that are no longer able to bind LPS. Furthermore, the peptide provided a basis for the generation of highly soluble analogues with stronger lipopolysaccharide-neutralizing activity.

Binding Sites↗
PubMed2006

A targeted protease substrate for a quantitative determination of protease activities in the endolysosomal pathway.

Inside the cell, proteases act in concert in the degradation of proteins and peptides. In order to understand the significance of an individual proteolytic activity within an ensemble of proteases, protocols and probes are required that enable a quantitative determination of the contribution of a protease to the break-down of a given substrate. Here we present a fluorescence resonance energy transfer-based probe and protocols for a quantitative determination of proteolytic activities inside the endolysosomal compartment. A peptide substrate that is readily cleaved by different cathepsins is flanked by fluorescein and tetramethylrhodamine-labeled lysine residues. Efficient endolysosomal targeting of the substrate is achieved by N-terminal elongation with the cell-penetrating peptide nona-arginine. The proteasome inhibitor lactacystin has a small, but significant effect on the break-down of the substrate, thus demonstrating that only a minor fraction of the peptide reaches the cytoplasm in its intact form. Nona-arginine therefore constitutes a highly efficient low-molecular-weight moiety for targeting the endolysosomal compartment.

Catalysis↗
PubMed2005

Label-free characterization of cell adhesion using reflectometric interference spectroscopy (RIfS).

Reflectometric interference spectroscopy (RIfS) is a label-free, time-resolved technique for detecting interactions of molecules immobilized on a surface with ligands in solution. Here we show that RIfS also permits the detection of the adhesion of tissue culture cells to a functionalized surface in a flow system. Interactions of T cells with other leukocytes or epithelial cells of blood vessels are crucial steps in the regulating immune response and inflammatory reactions. Jurkat T cell leukemia cells rapidly attached to a transducer functionalized with a monoclonal antibody directed against the T cell receptor (TCR)/CD3 complex, followed by activation-dependent cell spreading. RIfS curves were obtained for the Jurkat derivative JCaM 1.6 (which lacks the key signaling protein Lck), cells preincubated with cytochalasin D (an inhibitor of actin polymerization), and for surfaces functionalized with an antibody directed against the coreceptor CD28. These curves differed with respect to the maximum signal and the initial slope of the increase in optical thickness. The testing of chemical inhibitors, cell surface molecules and gene products relevant to a key event in T cell immunity illustrates the potential of label-free techniques for the analysis of activation-dependent cell-surface contacts.

Antibodies↗
PubMed2005

Structural variation of cationic lipids: minimum requirement for improved oligonucleotide delivery into cells.

In vivo transfection efficiency (TE) using cationic liposome/oligonucleotide (ODN) complexes is often hampered by interactions with serum components. Novel cationic lipids with different hydroxyethyl or dihydroxypropyl ammonium backbones, esterified hydrocarbon chains and hydroxy substituents have been synthesized and applied in cationic liposome formulations with and without the helper lipid DOPE (1:1, m/m). Their properties for cellular ODN delivery were determined using fluorescently labeled ODNs (F-ODNs). Cationic lipids with hydrocarbon chains esterified to non-glycerol backbones in non-vicinal configuration were completely ineffective in nuclear ODN-delivery. Instead, an increased cytoplasmic localization of F-ODNs was observed. Cationic lipids equipped with only one hydrocarbon were completely incompetent for cellular ODN delivery. In the absence of serum, all cationic lipids tested with hydrocarbon chains in vicinal configuration esterified to a glycerol backbone (the respective N-(1,2-diacyl-dihydroxypropyl)-N,N,N-trimethyl-ammoniumchlorides or N-(1,2-diacyl-dihydroxypropyl)-N(hydroxyethyl)-N,N-dimethyl-ammoniumchlorides as well as N-(1,2-diacyl-dihydroxypropyl)-N(1,2-dihydroxypropyl)-N,N-dimethyl-ammoniumchlorides with lauroyl, myristoyl, palmitoyl, stearoyl and erucoyl chains) were able to transfect cells when combined with DOPE (20-80% nuclear fluorescence). Remarkably, only the analog esterified with two myristoyl chains was equally effective even in the absence of DOPE. By adding hydroxy groups to the N-alkyl residue, TE under serum conditions was improved yielding transfection rates of 55%, 75% and 90% for 0, 1 or 2 substituted hydroxy groups, respectively. For plasmid DNA, different requirements were identified. Again, the analog with two myristoyl chains was most effective but only in the presence of DOPE. However, the addition of hydroxy groups had no influence on the TE in the presence of serum.

Cations↗
PubMed2005

Cationic cell-penetrating peptides interfere with TNF signalling by induction of TNF receptor internalization.

Cationic cell-penetrating peptides (CPPs) have been used widely as delivery vectors for the import of molecules that otherwise do not cross the plasma membrane of eukaryotic cells. In this work, we demonstrate that the three cationic CPPs, Antennapedia homeodomain-derived peptide (Antp), nona-arginine and Tat-derived peptide, inhibit tumour necrosis factor (TNF)-mediated signal transduction. This inhibition is based on the downregulation of TNF receptors at the cell surface by induction of internalization. In contrast to TNF-dependent receptor internalization, no receptor activation occurs. The receptor downregulation is not restricted to the CPPs. Remarkably, the HIV-1 Tat protein itself also induces the internalization of TNF receptors. The dynamin dependence of the internalization, as well as the fact that epidermal growth factor receptors are also internalized, suggest a general induction of clathrin-dependent endocytosis as the mechanism of action. The significance of these findings for the use of cationic CPPs in the import of bioactive peptides is demonstrated here using a conjugate consisting of Antp and a Smac protein-derived cargo peptide. The cargo alone, when introduced into cells by electroporation, enhanced TNF-induced apoptosis by inhibiting the anti-apoptotic action of IAPs (inhibitor of apoptosis proteins). For the Antp-Smac conjugate at concentrations below 40 muM the inhibitory effect of the Antp peptide compensated for the pro-apoptotic activity of the cargo, and led to the protection of cells against TNF-mediated apoptosis. These data provide important new information for the use of cationic CPPs for the cellular delivery of bioactive molecules.

Apoptosis↗
PubMed2005

High-affinity adaptors for switchable recognition of histidine-tagged proteins.

We aspired to create chemical recognition units, which bind oligohistidine tags with high affinity and stability, as tools for selectively attaching spectroscopic probes and other functional elements to recombinant proteins. Several supramolecular entities containing 2-4 nitrilotriacetic acid (NTA) moieties were synthesized, which additionally contained an amino group, to which fluorescein was coupled as a sensitive reporter probe. These multivalent chelator heads (MCH) (termed bis-, tris-, and tetrakis-NTA) were characterized with respect to their interaction with hexahistidine (H6)- and decahistidine (H10)-tagged targets. Substantially increased binding stability with increasing number of NTA moieties was observed by analytical size exclusion chromatography. The binding enthalpies as determined by isothermal titration calorimetry increased nearly additively with the number of possible coordinative bonds between chelator heads and tags. Yet, a substantial excess of histidines in the oligohistidine tag was required for obtaining fully additive binding enthalpies. Dissociation kinetics of MCH/oligohistidine complexes measured by fluorescence dequenching showed an increase in stability by 4 orders of magnitude compared to that of mono-NTA, and subnanomolar affinity was reached for tris-NTA. The gain in free energy with increasing multivalency was accompanied by an increasing loss of entropy, which was ascribed to the high flexibility of the binding partners. Numerous applications of these MCHs for noncovalent, high affinity, yet reversible tethering of spectroscopic probes and other functional elements to the recombinant proteins can be envisioned.

Calorimetry↗
PubMed2005

Autophagy promotes MHC class II presentation of peptides from intracellular source proteins.

MHC-peptide complexes mediate key functions in adaptive immunity. In a classical view, MHC-I molecules present peptides from intracellular source proteins, whereas MHC-II molecules present antigenic peptides from exogenous and membrane proteins. Nevertheless, substantial crosstalk between these two pathways has been observed. We investigated the influence of autophagy on the MHC-II ligandome and demonstrated that peptide presentation is altered considerably upon induction of autophagy. The presentation of peptides from intracellular and lysosomal source proteins was strongly increased on MHC-II in contrast with peptides from membrane and secreted proteins. In addition, autophagy influenced the MHC-II antigen-processing machinery. Our study illustrates a profound influence of autophagy on the class II peptide repertoire and suggests that this finding has implications for the regulation of CD4(+) T cell-mediated processes.

Amino Acid Sequence↗
PubMed2005

Chemical inhibitors when timing is critical: a pharmacological concept for the maturation of T cell contacts.

Cellular signal transduction proceeds through a complex network of molecular interactions and enzymatic activities. The timing of these molecular events is critical for the propagation of a signal and the generation of a specific cellular response. To define the timing of signalling events, we introduce the combination of high-resolution confocal microscopy with the application of small-molecule inhibitors at various stages of signal transduction in T cells. Inhibitors of Src-family tyrosine kinases and actin dynamics were employed to dissect the role of the lymphocyte-specific tyrosine kinase Lck in the formation and maintenance of T cell receptor/CD3-dependent contacts. Anti-CD3epsilon-coated coverslips served as a highly defined stimulus. The kinetics of the recruitment of the yellow fluorescent protein-tagged signalling protein ZAP-70 were detected by high-resolution confocal microscopy. The analysis revealed that at 5 min after receptor engagement, Lck activity was required for maintenance of contacts. In contrast, after 20 min of receptor engagement, the contacts were Lck-independent. The relevance of the timing of inhibitor application provides a pharmacological concept for the maturation of T cell-substrate contacts.

Animals↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2005

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-alpha 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.

Amino Acid Sequence↗
PubMed2005

Peptide microarrays for the detection of molecular interactions in cellular signal transduction.

The formation of protein complexes is a hallmark of cellular signal transduction. Here, we show that peptide microarrays provide a robust and quantitative means to detect signalling-dependent changes of molecular interactions. Recruitment of a protein into a complex upon stimulation of a cell leads to the masking of an otherwise exposed binding site. In cell lysates this masking can be detected by reduced binding to a microarray carrying a peptide that corresponds to the binding motif of the respective interaction domain. The method is exemplified for the lymphocyte-specific tyrosine kinase 70 kDa zeta-associated protein binding to a bis-phosphotyrosine-motif of the activated T-cell receptor via its tandem SH2 domain. Compared to established techniques, the method provides a significant shortcut to the detection of molecular interactions.

Animals↗
PubMed2005

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.

Animals↗