multivalency
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The transcription factor ASCIZ (ATMIN, ZNF822) has an unusually high number of recognition motifs for the product of its main target gene, the hub protein LC8 (DYNLL1). Using a combination of biophysical methods, structural analysis by NMR and electron microscopy, and cellular transcription assays, we developed a model that proposes a concerted role of intrinsic disorder and multiple LC8 binding events in regulating LC8 transcription. We demonstrate that the long intrinsically disordered C-terminal domain of ASCIZ binds LC8 to form a dynamic ensemble of complexes with a gradient of transcriptional activity that is inversely proportional to LC8 occupancy. The preference for low occupancy complexes at saturating LC8 concentrations with both human and Drosophila ASCIZ indicates that negative cooperativity is an important feature of ASCIZ-LC8 interactions. The prevalence of intrinsic disorder and multivalency among transcription factors suggests that formation of heterogeneous, dynamic complexes is a widespread mechanism for tuning transcriptional regulation.
- MeSH
- cytoplazmatické dyneiny chemie genetika metabolismus MeSH
- Drosophila melanogaster růst a vývoj metabolismus fyziologie MeSH
- dyneiny chemie genetika metabolismus MeSH
- lidé MeSH
- proteiny Drosophily chemie genetika metabolismus MeSH
- regulace genové exprese * MeSH
- transkripční faktory chemie genetika metabolismus MeSH
- vnitřně neuspořádané proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Galectins are carbohydrate-binding lectins that modulate the proliferation, apoptosis, adhesion, or migration of cells by cross-linking glycans on cell membranes or extracellular matrix components. Galectin-4 (Gal-4) is a tandem-repeat-type galectin expressed mainly in the epithelial cells of the gastrointestinal tract. It consists of an N- and a C-terminal carbohydrate-binding domain (CRD), each with distinct binding affinities, interconnected with a peptide linker. Compared to other more abundant galectins, the knowledge of the pathophysiology of Gal-4 is sparse. Its altered expression in tumor tissue is associated with, for example, colon, colorectal, and liver cancers, and it increases in tumor progression, and metastasis. There is also very limited information on the preferences of Gal-4 for its carbohydrate ligands, particularly with respect to Gal-4 subunits. Similarly, there is virtually no information on the interaction of Gal-4 with multivalent ligands. This work shows the expression and purification of Gal-4 and its subunits and presents a structure-affinity relationship study with a library of oligosaccharide ligands. Furthermore, the influence of multivalency is demonstrated in the interaction with a model lactosyl-decorated synthetic glycoconjugate. The present data may be used in biomedical research for the design of efficient ligands of Gal-4 with diagnostic or therapeutic potential.
The cytoskeleton consists of polymeric protein filaments with periodic lattices displaying identical binding sites, which establish a multivalent platform for the binding of a plethora of filament-associated ligand proteins. Multivalent ligand proteins can tether themselves to the filaments through one of their binding sites, resulting in an enhanced reaction kinetics for the remaining binding sites. In this Opinion, we discuss a number of cytoskeletal phenomena underpinned by such multivalent interactions, namely (1) generation of entropic forces by filament crosslinkers, (2) processivity of molecular motors, (3) spatial sorting of proteins, and (4) concentration-dependent unbinding of filament-associated proteins. These examples highlight that cytoskeletal filaments constitute the basis for the formation of microenvironments, which cytoskeletal ligand proteins can associate with and, once engaged, can act within at altered reaction kinetics. We thus argue that multivalency is one of the properties crucial for the functionality of the cytoskeleton.
Cellular entry, the first crucial step of viral infection, can be inhibited by molecules adsorbed on the virus surface. However, apart from using stronger affinity, little is known about the properties of such inhibitors that could increase their effectiveness. Our simulations showed that multivalent inhibitors can be designed to be much more efficient than their monovalent counterparts. For example, for our particular simulation model, a single multivalent inhibitor spanning 5 to 6 binding sites is enough to prevent the uptake compared to the required 1/3 of all the receptor binding sites needed to be blocked by monovalent inhibitors. Interestingly, multivalent inhibitors are more efficient in inhibiting the uptake not only due to their increased affinity but mainly due to the co-localization of the inhibited receptor binding sites at the virion's surface. Furthermore, we show that Janus-like inhibitors do not induce virus aggregation. Our findings may be generalized to other uptake processes including bacteria and drug-delivery.
Gangliosides located at the outer leaflet of plasma membrane are molecules that either participate in recognizing of exogenous ligand molecules or exhibit their own receptor activity, which are both essential phenomena for cell communication and signaling as well as for virus and toxin entry. Regulatory mechanisms of lipid-mediated recognition are primarily subjected to the physical status of the membrane in close vicinity of the receptor. Concerning the multivalent receptor activity of the ganglioside GM1, several regulatory strategies dealing with GM1 clustering and cholesterol involvement have been proposed. So far however, merely the isolated issues were addressed and no interplay between them investigated. In this work, several advanced fluorescence techniques such as Z-scan fluorescence correlation spectroscopy, Förster resonance energy transfer combined with Monte Carlo simulations, and a newly developed fluorescence antibunching assay were employed to give a more complex portrait of clustering and cholesterol involvement in multivalent ligand recognition of GM1. Our results indicate that membrane properties have an impact on a fraction of GM1 molecules that is not available for the ligand binding. While at low GM1 densities (~1 %) it is the cholesterol that turns GM1 headgroups invisible, at higher GM1 level (~4 %) it is purely the local density of GM1 molecules that inhibits the recognition. At medium GM1 content, cooperation of the two phenomena occurs. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.
- MeSH
- buněčná membrána metabolismus MeSH
- cholesterol MeSH
- difuze MeSH
- G(M1) gangliosid chemie metabolismus MeSH
- hydraziny metabolismus MeSH
- ligandy MeSH
- metoda Monte Carlo MeSH
- ovce MeSH
- počítačová simulace MeSH
- receptory buněčného povrchu metabolismus MeSH
- rezonanční přenos fluorescenční energie MeSH
- shluková analýza MeSH
- titrace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Clostridium difficile infections cause gastrointestinal disorders and can lead to life-threatening conditions. The symptoms can vary from severe diarrhea to the formation of pseudomembranous colitis and therefore trigger a need for new therapies. The initial step of disease is the binding of the bacterial enterotoxins toxin A and B to the cell surface of epithelial intestinal cells. Scavenging of the toxins is crucial to inhibit their fatal effect in the human body and circumvent the administration of antibiotics. Cell surface glycans are common as ligands for TcdA. Although crucial for carbohydrate-protein interactions, a multivalent presentation of glycans for binding has been hardly considered. Here, we establish a neo-glycoprotein-based glycan library to identify an effective multivalent glycan ligand for TcdA. It comprises 40 different glycan epitopes based on N-acetyllactosamine precursors. Nine structures exhibit strong binding of the receptor domain. Among them, the Lewisy-Lewisx-epitope shows the best performance for binding both the receptor domain and the holotoxin. Therefore, the glycan was synthesized de novo and coupled to BSA as a scaffold for multivalent presentation. The corresponding neo-glycoprotein facilitates the proper scavenging of TcdA in vitro and effectively protects HT29 cells from TcdA-induced cell damage.
Galectin-3 (Gal-3) participates in many cancer-related metabolic processes. The inhibition of overexpressed Gal-3 by, e.g., β-galactoside-derived inhibitors is hence promising for cancer treatment. The multivalent presentation of such inhibitors on a suitable biocompatible carrier can enhance the overall affinity to Gal-3 and favorably modify the interaction with Gal-3-overexpressing cells. We synthesized a library of C-3 aryl-substituted thiodigalactoside inhibitors and their multivalent N-(2-hydroxypropyl)methacrylamide (HPMA)-based counterparts with two different glycomimetic contents. Glycopolymers with a higher content of glycomimetic exhibited a higher affinity to Gal-3 as assessed by ELISA and biolayer interferometry. Among them, four candidates (with 4-acetophenyl, 4-cyanophenyl, 4-fluorophenyl, and thiophen-3-yl substitution) were selected for further evaluation in cancer-related experiments in cell cultures. These glycopolymers inhibited Gal-3-induced processes in cancer cells. The cyanophenyl-substituted glycopolymer exhibited the strongest antiproliferative, antimigratory, antiangiogenic, and immunoprotective properties. The prepared glycopolymers appear to be prospective modulators of the tumor microenvironment applicable in the therapy of Gal-3-associated cancers.
Chemical immunotherapeutic strategies including Antibody Recruiting Molecules (ARMs - bivalent small molecules containing an antibody-binding domain (ABD) and a target-binding domain (TBD)) direct immune-mediated clearance of diseased cells. Anti-cancer ARM function relies on high tumor antigen valency, limiting function against lower antigen expressing tumors. To address this limitation, we report a tunable multivalent immune recruitment (MIR) platform to amplify/stabilize antibody recruitment to cells with lower antigen valencies. An initial set of polymeric ARMs (pARMs) were synthesized and screened to evaluate ABD/TBD copy number, ratio, and steric occlusion on specific immune induction. Most pARMs demonstrated simultaneous high avidity binding to anti-dinitrophenyl antibodies and prostate-specific membrane antigens on prostate cancer. Optimized pARMs mediated enhanced anti-cancer immune function against lower antigen expressing target cells compared to an analogous ARM.
- MeSH
- antigeny * MeSH
- fagocytóza MeSH
- lidé MeSH
- nádory prostaty * MeSH
- protilátky chemie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Galectin-3 (Gal-3), a member of the β-galactoside-binding lectin family, is a tumor biomarker and involved in tumor angiogenesis and metastasis. Gal-3 is therefore considered as a promising target for early cancer diagnosis and anticancer therapy. We here present the synthesis of a library of tailored multivalent neo-glycoproteins and evaluate their Gal-3 binding properties. By the combinatorial use of glycosyltransferases and chemo-enzymatic reactions, we first synthesized a set of N-acetyllactosamine (Galβ1,4GlcNAc; LacNAc type 2)-based oligosaccharides featuring five different terminating glycosylation epitopes, respectively. Neo-glycosylation of bovine serum albumin (BSA) was accomplished by dialkyl squarate coupling to lysine residues resulting in a library of defined multivalent neo-glycoproteins. Solid-phase binding assays with immobilized neo-glycoproteins revealed distinct affinity and specificity of the multivalent glycan epitopes for Gal-3 binding. In particular, neo-glycoproteins decorated with N',N″-diacetyllactosamine (GalNAcβ1,4GlcNAc; LacdiNAc) epitopes showed high selectivity and were demonstrated to capture Gal-3 from human serum with high affinity. Furthermore, neo-glycoproteins with terminal biotinylated LacNAc glycan motif could be utilized as Gal-3 detection agents in a sandwich enzyme-linked immunosorbent assay format. We conclude that, in contrast to antibody-based capture steps, the presented neo-glycoproteins are highly useful to detect functionally intact Gal-3 with high selectivity and avidity. We further gain novel insights into the binding affinity of Gal-3 using tailored multivalent neo-glycoproteins, which have the potential for an application in the context of cancer-related biomedical research.
- MeSH
- aminocukry chemická syntéza chemie metabolismus MeSH
- galektin 3 antagonisté a inhibitory metabolismus MeSH
- glykoproteiny chemická syntéza chemie metabolismus farmakologie MeSH
- glykosylace MeSH
- lidé MeSH
- ligandy MeSH
- oligosacharidy chemická syntéza chemie metabolismus MeSH
- sérový albumin hovězí chemická syntéza chemie metabolismus farmakologie MeSH
- skot MeSH
- techniky kombinatorické chemie MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
