Characterization of the Dynamic Interactions of Transcriptional Activators

Transcription is initiated through a series of coupled binding equilibria between transcriptional activators and their array of protein targets within the transcriptional machinery. However, previous efforts to kinetically characterize these interactions have produced conflicting models for the mechanism of complex formation, which is hampering the discovery of non-natural mimics of their transcriptional activation domains (TADs). Using fluorescence stopped-flow techniques, we determined that the activators Gal4, Gcn4, and VP16 interact with the same coactivator, Med15, via a two-step binding mechanism comprised of a bimolecular association step and a conformational change. We further hypothesized that the life-times of these interactions should be more revealing of differences in activator potency (i.e., transcriptional output); thus, we analyzed the microscopic rate and equilibrium constants defining the individual steps within our mechanism, in order to identify key trends that can differentiate the activators from one another in terms of their ability to recruit the transcriptional machinery to a gene promoter. We determined that it is the favorability of the conformational change step and its partition ratio that correlates with the ability of an activator to stimulate transcription. Future studies will focus on determining how the different structural propensities of the TAD sequences contribute to the stability of the intermediate that they form. Furthermore, another significant challenge in the development of artificial transcription factors (ATFs) is a lack of small molecules that can be used to localize them to a gene promoter in a cellular context. We propose a novel approach to accomplish this task which relies on the interaction of a ligand with an endogenous DNA-bound protein. To this end, we have successfully used an in vitro phage display selection with a random 12 amino acid peptide library to isolate ligands that are capable of interacting with the DNA-binding proteins Gal4 (residues 1-100) and LexA (residues 1-202). Future studies will entail the implementation of a selection with a conformation-constrained peptide library to obtain ligands that may possess increased stability and specificity within the cellular milieu. In particular, protein scaffolds that promote helix stabilization would aid in the future identification of peptidomimetic or small molecule replacements.Ph.D.ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/77780/1/amwands_1.pd

Fucosylated Molecules Competitively Interfere with Cholera Toxin Binding to Host Cells

Cholera toxin (CT) enters host intestinal epithelia cells, and its retrograde transport to the cytosol results in the massive loss of fluids and electrolytes associated with severe dehydration. To initiate this intoxication process, the B subunit of CT (CTB) first binds to a cell surface receptor displayed on the apical surface of the intestinal epithelia. While the monosialoganglioside GM1 is widely accepted to be the sole receptor for CT, intestinal epithelial cell lines also utilize fucosylated glycan epitopes on glycoproteins to facilitate cell surface binding and endocytic uptake of the toxin. Further, l-fucose can competively inhibit CTB binding to intestinal epithelia cells. Here, we use competition binding assays with l-fucose analogs to decipher the molecular determinants for l-fucose inhibition of cholera toxin subunit B (CTB) binding. Additionally, we find that mono- and difucosylated oligosaccharides are more potent inhibitors than l-fucose alone, with the LeY tetrasaccharide emerging as the most potent inhibitor of CTB binding to two colonic epithelial cell lines (T84 and Colo205). Finally, a non-natural fucose-containing polymer inhibits CTB binding two orders of magnitude more potently than the LeY glycan when tested against Colo205 cells. This same polymer also inhibits CTB binding to T84 cells and primary human jejunal epithelial cells in a dose-dependent manner. These findings suggest the possibility that polymeric display of fucose might be exploited as a prophylactic or therapeutic approach to block the action of CT toward the human intestinal epithelium

Fucosylation and protein glycosylation create functional receptors for cholera toxin

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we report that CTB binds cell surface glycoproteins. Relative contributions of gangliosides and glycoproteins to CTB binding depend on cell type, and CTB binds primarily to glycoproteins in colonic epithelial cell lines. Using a metabolically incorporated photocrosslinking sugar, we identified one CTB-binding glycoprotein and demonstrated that the glycan portion of the molecule, not the protein, provides the CTB interaction motif. We further show that fucosylated structures promote CTB entry into a colonic epithelial cell line and subsequent host cell intoxication. CTB-binding fucosylated glycoproteins are present in normal human intestinal epithelia and could play a role in cholera. DOI: http://dx.doi.org/10.7554/eLife.09545.00

Fucosylated Molecules Competitively Interfere with Cholera Toxin Binding to Host Cells

Cholera toxin (CT) enters host intestinal epithelia cells, and its retrograde transport to the cytosol results in the massive loss of fluids and electrolytes associated with severe dehydration. To initiate this intoxication process, the B subunit of CT (CTB) first binds to a cell surface receptor displayed on the apical surface of the intestinal epithelia. While the monosialoganglioside GM1 is widely accepted to be the sole receptor for CT, intestinal epithelial cell lines also utilize fucosylated glycan epitopes on glycoproteins to facilitate cell surface binding and endocytic uptake of the toxin. Further, l-fucose can competively inhibit CTB binding to intestinal epithelia cells. Here, we use competition binding assays with l-fucose analogs to decipher the molecular determinants for l-fucose inhibition of cholera toxin subunit B (CTB) binding. Additionally, we find that mono- and difucosylated oligosaccharides are more potent inhibitors than l-fucose alone, with the LeY tetrasaccharide emerging as the most potent inhibitor of CTB binding to two colonic epithelial cell lines (T84 and Colo205). Finally, a non-natural fucose-containing polymer inhibits CTB binding two orders of magnitude more potently than the LeY glycan when tested against Colo205 cells. This same polymer also inhibits CTB binding to T84 cells and primary human jejunal epithelial cells in a dose-dependent manner. These findings suggest the possibility that polymeric display of fucose might be exploited as a prophylactic or therapeutic approach to block the action of CT toward the human intestinal epithelium

GM1 ganglioside-independent intoxication by Cholera toxin

<div><p>Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (Le^X) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the Le^X glycan <i>in vitro</i> when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to Le^X. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.</p></div

Le^X blocks binding of CTB to human granulocytes but not murine leukocytes.

<p><b>(A-B and D-E)</b> Histograms from flow cytometry analyses of CTB-, G33D- and OVA-binding to granulocytes in human peripheral blood. CTB was pretreated or not with titrated amounts of <b>(A)</b> Le^X-os, <b>(B)</b> GM1-os, <b>(D)</b> os-HSA (not titrated) and <b>(E)</b> Le^X-os and GM1-os. Graphs show the percent of gMFI of CTB binding to the cells where 100% represents CTB staining with no blocking oligosaccharide. <b>(C)</b> Histograms from flow cytometry analyses of CTB-, G33D- and OVA-binding to CD3+ T cells gated from murine splenocytes. CTB was pretreated or not with the indicated os or os-HSA. <b>(A-B)</b> n = 3, <b>(C)</b> One representative out of three independent experiments, <b>(D)</b> n = 8, <b>(E)</b> n = 4–9. Error bars show SD. Each dot represents one donor and significance was calculated using a one-way-ANOVA with Tukey correction compared to CTB without block if not indicated otherwise with bars (**** = p<0,0001, *** = p<0,005, ** = p<0,01).</p

CT induced ion secretion can be inhibited by pretreating the tissue with AAL and PNA.

<p>Human jejunal mucosae were pre-incubated with or without AAL or PNA at the indicated concentrations, mounted in an Ussing chamber and exposed to CT. <b>(A)</b> Dot plot showing percent difference in I_ep to control tissue for jejunal mucosae over time. Each dot represents a mean of 4–7 donors (each treatment for each donor was tested in duplicates) with SEM error bars. Significance was calculated using a two-way-ANOVA with Tukey correction (compared to the CT). * represent CT to CT+AAL comparison and † represent CT to CT+PNA comparison (**** = p<0.0001 and ** = p<0.01). <b>(B)</b> Dot plot showing percent of start I_ep for jejunal mucosae at 180 min. AAL, PNA-treated or untreated tissue were treated with forskolin (or forskolin analog NKH477) bilaterally at 200 min. CT treated tissue were treated with bumetanide at 200 min. Each dot represents a mean of 2–3 donors (each treatment for each donor was tested in duplicates). Error bars show SEM.</p