Multivalent interactions can be applied universally for a targeted strengthening of an interaction between different interfaces or molecules. The binding partners form cooperative, multiple receptor–ligand interactions that are based on individually weak, noncovalent bonds and are thus generally reversible. Hence, multi‐ and polyvalent interactions play a decisive role in biological systems for recognition, adhesion, and signal processes. The scientific and practical realization of this principle will be demonstrated by the development of simple artificial and theoretical models, from natural systems to functional, application‐oriented systems. In a systematic review of scaffold architectures, the underlying effects and control options will be demonstrated, and suggestions will be given for designing effective multivalent binding systems, as well as for polyvalent therapeutics.
Adhesive interactions of leukocytes and endothelial cells initiate leukocyte migration to inflamed tissue and are important for immune surveillance. Acute and chronic inflammatory diseases show a dysregulated immune response and result in a massive efflux of leukocytes that contributes to further tissue damage. Therefore, targeting leukocyte trafficking may provide a potent form of antiinflammatory therapy. Leukocyte migration is initiated by interactions of the cell adhesion molecules E-, L-, and P-selectin and their corresponding carbohydrate ligands. Compounds that efficiently address these interactions are therefore of high therapeutic interest. Based on this rationale we investigated synthetic dendritic polyglycerol sulfates (dPGS) as macromolecular inhibitors that operate via a multivalent binding mechanism mimicking naturally occurring ligands. dPGS inhibited both leukocytic L-selectin and endothelial P-selectin with high efficacy. Size and degree of sulfation of the polymer core determined selectin binding affinity. Administration of dPGS in a contact dermatitis mouse model dampened leukocyte extravasation as effectively as glucocorticoids did and edema formation was significantly reduced. In addition, dPGS interacted with the complement factors C3 and C5 as was shown in vitro and reduced C5a levels in a mouse model of complement activation. Thus, dPGS represent an innovative class of a fully synthetic polymer therapeutics that may be used for the treatment of inflammatory diseases.anti-inflammatory drug | complement inhibition | multiple target binding | multivalent selectin inhibitor | synthetic polymer
We present for the first time the synthesis of sequence-defined monodisperse glycopolymer segments via solid-phase polymer synthesis. Functional building blocks displaying alkyne moieties and hydrophilic ethylenedioxy units were assembled stepwise on solid phase. The resulting polymer segments were conjugated with mannose sugars via 1,3-dipolar cycloaddition. The obtained mono-, di-, and trivalent mannose structures were then subject to Con A lectin binding. Surface plasmon resonance studies showed a nonlinear increase in binding regarding the number and spacing of sugar ligands. The results of Con A lectin binding assays indicate that the chemical composition of the polymeric scaffold strongly contributes to the binding activities as well as the spacing between the ligands and the number of presented mannose units. Our approach now allows for the synthesis of highly defined glycooligomers and glycopolymers with a diversity of properties to investigate systematically multivalent effects of polymeric ligands.
Neurotransmission depends on the exo-endocytosis of synaptic vesicles at active zones. Synaptobrevin 2 [also known as vesicleassociated membrane protein 2 (VAMP2)], the most abundant synaptic vesicle protein and a major soluble NSF attachment protein receptor (SNARE) component, is required for fast calcium-triggered synaptic vesicle fusion. In contrast to the extensive knowledge about the mechanism of SNARE-mediated exocytosis, little is known about the endocytic sorting of synaptobrevin 2. Here we show that synaptobrevin 2 sorting involves determinants within its SNARE motif that are recognized by the ANTH domains of the endocytic adaptors AP180 and clathrin assembly lymphoid myeloid leukemia (CALM). Depletion of CALM or AP180 causes selective surface accumulation of synaptobrevin 2 but not vGLUT1 at the neuronal surface. Endocytic sorting of synaptobrevin 2 is mediated by direct interaction of the ANTH domain of the related endocytic adaptors CALM and AP180 with the N-terminal half of the SNARE motif centered around M46, as evidenced by NMR spectroscopy analysis and site-directed mutagenesis. Our data unravel a unique mechanism of SNARE motif-dependent endocytic sorting and identify the ANTH domain proteins AP180 and CALM as cargo-specific adaptors for synaptobrevin endocytosis. Defective SNARE endocytosis may also underlie the association of CALM and AP180 with neurodevelopmental and cognitive defects or neurodegenerative disorders.clathrin-mediated endocytosis | structure N eurotransmission in the brain depends on the calcium-triggered fusion and recycling of neurotransmitter-filled synaptic vesicles (SVs) with the presynaptic membrane at active zones (1). Following their exocytic insertion into the presynaptic membrane, SV proteins need to be retrieved at a precisely defined stoichiometry by endocytosis, a process involving clathrin, adaptors, and other endocytic proteins (2). Fast calcium-triggered SV fusion critically depends on the SV arginine (R)-soluble NSF attachment protein receptor (SNARE) synaptobrevin [or vesicle-associated membrane protein (VAMP)], which by forming a complex with the plasma membrane glutamine (Q)-SNAREs syntaxin and synaptosomal-associated protein (SNAP)-25 (3) drives neuroexocytosis (4, 5). Synapses lacking synaptobrevin 2 display <1% of wild-type release when stimulated by action potential (AP)-mediated calcium influx (6). Proteomic studies have shown that synaptobrevin 2 is a highly abundant SV protein (7) that is exoendocytically sorted with very high precision (8). Similar observations have been made for other SV proteins, including synaptotagmin and vesicular glutamate transporters (VGLUTs). How such precise sorting of synaptobrevin 2 is achieved has remained enigmatic. Synaptobrevin lacks recognizable linear sorting motifs (9), and unlike other SNARE proteins does not contain a folded N-terminal domain that serves as a targeting determinant in other VAMP family members (10-12).Genetic data have linked synaptobrevin sorting to the function of the AP180 N-terminal homology (...
The vascular endothelial growth factor, VEGF, is an important biomarker for different diseases and clinical disorders. We present a series of optical aptasensor-based sensing platforms for VEGF that include the following: (i) A FRET-based sensor that involves the VEGF-induced separation of aptamer-functionalized quantum dots blocked by a quencher nucleic acid (detection limit 1 nM). (ii) A FRET-based sensor based on the VEGF-induced assembly of the aptamer subunits functionalized with QDs and a dye acceptor (Cy5), respectively (detection limit 12 nM). (iii) A chemiluminescence aptasensor based on VEGF-induced assembly of a hemin/G-quadruplex catalyst (detection limit 18 nM). (iv) A chemiluminescence aptasensor based on the VEGF-stimulated assembly of two aptamer subunits into the hemin/G-quadruplex catalyst (detection limit 2.6 nM). (v) A chemiluminescence resonance energy transfer (CRET) aptasensor based on the VEGF-induced assembly of a semiconductor QDs-hemin/G-quadruplex supramolecular structure (detection limit 875 pM). Furthermore, an amplified optical aptasensor system based on the Exonuclease III (Exo III) recycling of the VEGF analyte was developed. In this system, one aptamer subunit is modified at its 5' and 3' ends with QDs and a black hole quencher, respectively. The VEGF-induced self-assembly of the aptamer subunits result in the digestion of the quencher units and the autonomous recycling of the analyte, while triggering-on the luminescence of the QDs (detection limit 5 pM). The system was implemented to analyze VEGF in human sera samples.
Nucleotide sequence analysis revealed a 1,791-bp open reading frame in the hox gene cluster of the gram-negative chemolithotroph Alcaligenes eutrophus H16. In order to investigate the biological role of this open reading frame, we generated an in-frame deletion allele via a gene replacement strategy. The resulting mutant grew significantly more slowly than the wild type under lithoautotrophic conditions (6.1 versus 4.2 h doubling time). A reduction in the level of the soluble NAD-reducing hydrogenase (60%o of the wild-type activity) was shown to be the cause of the slow lithoautotrophic growth. We used plasmid-borne gene fusions to monitor the expression of the operons encoding the soluble and membrane-bound hydrogenases. The expression of both operons was lower in the mutant than in the wild-type strain. These results suggest that the newly identified gene, designated hoxX, encodes a regulatory component which, in conjunction with the transcriptional activator HoxA, controls hydrogenase synthesis.Alcaligenes eutrophus H16 is a facultative lithoautotroph classified in the ,-purple group of gram-negative bacteria. In the absence of organic substrates, A. eutrophus can grow on a mixture of CO2, H2, and 02 Under these conditions, carbon dioxide is fixed via the reductive pentose phosphate (Calvin) cycle (5). The enzymes which catalyze CO2 fixation are encoded by the cbb (formerly cfx) genes. Two copies of these genes are present: one is located on the bacterial chromosome, and the other is carried on the 450-kb conjugative megaplasmid pHG1 (21,23,26,49). The central energy-yielding process in the lithoautotrophic metabolism of A. eutrophus is the oxidation of molecular hydrogen. This reaction is catalyzed by two enzymes: a heterotetrameric, NAD-reducing hydrogenase (SH) found in the cytoplasm and a heterodimeric hydrogenase (MBH) which is attached to the cytoplasmic membrane (16,33,37,38). Both enzymes are nickel metalloproteins (11). The two hydrogenases are encoded in separate operons located in the hox gene cluster of pHG1 (25,46). The formation of the hydrogen-oxidizing apparatus requires, in addition to the hydrogenase enzymes, a set of accessory proteins instrumental to the maturation of the hydrogenase polypeptides, to nickel uptake, and to the insertion of nickel into the active sites of the enzymes. These accessory proteins are also encoded by genes of the megaplasmid hox cluster (6-8, 10, 24, 25).Both the synthesis of the Calvin cycle enzymes and the formation of the hydrogen-oxidizing apparatus are regulated efficiently (13,27). During growth on preferentially utilized organic substrates, both enzyme systems are tightly repressed; derepression takes place upon transition to lithoautotrophic conditions. However, the two systems are controlled by separate regulatory circuits. The cbb genes are regulated by the product of the cbbR gene, a member of the LysR family of bacterial regulatory proteins (50). Expression of the hox genes is governed by the product of the hoxA locus. Protein HoxA is a member of the ...
A versatile route for the synthesis of highly functionalized, polyanionic macromolecules based on dendritic polyglycerol was applied by means of the Huisgen-Sharpless-Meldal 1,3-dipolar cycloaddition ("click-reaction") of polyglycerolazide precursors and alkyne-functionalized anions such as sulfonates, carboxylates, phosphonates, and bisphosphonates. In addition, the corresponding polyglycerol phosphate has been synthesized via direct hydroxyl interconversion of polyglycerol to the corresponding phosphate with a degree of functionalization >80% by analogy to the synthesis of previously reported polyglycerol sulfates (dPGS). On the basis of the finding that dPGS exhibits high affinity for L- and P-selectin, the potential of these novel polyanionic, multivalent macromolecules of varying anionic nature as L-selectin inhibitors has been evaluated in vitro by means of a competitive concentration dependent binding assay. Affinity of all polyanions toward L-selectin was demonstrated with distinct IC(50) values ranging from the low nanomolar to the high micromolar range. The efficiency of L-selectin inhibition increases in the order carboxylate < phosphate < phosphonate ≈ sulfonate < bisphosphonate < sulfate. Additional DLS and ζ-potential measurements of these polyanions were performed to correlate their binding affinity toward L-selectin with their anionic nature. However, a direct correlation of effective charge and particle size with the determined IC(50) values turned out to require further in-depth studies on the microstructure of the polyanions but clearly indicate an exceptional position of dPGS among the studied dendritic polyelectrolytes.
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