Numan Oezguen

Measles virus blind to its epithelial cell receptor remains virulent in rhesus monkeys but cannot cross the airway epithelium and is not shed

The current model of measles virus (MV) pathogenesis implies that apical infection of airway epithelial cells precedes systemic spread. An alternative model suggests that primarily infected lymphatic cells carry MV to the basolateral surface of epithelial cells, supporting MV shedding into the airway lumen and contagion. This model predicts that a mutant MV, unable to enter cells through the unidentified epithelial cell receptor (EpR), would remain virulent but not be shed. To test this model, we identified residues of the MV attachment protein sustaining EpR-mediated cell fusion. These nonpolar or uncharged polar residues defined an area located near the binding site of the signaling lymphocytic activation molecule (SLAM), the receptor for MV on lymphatic cells. We then generated an EpR-blind virus maintaining SLAM-dependent cell entry and inoculated rhesus monkeys intranasally. Hosts infected with the selectively EpR-blind MV developed rash and anorexia while averaging slightly lower viremia than hosts infected with wild-type MV but did not shed virus in the airways. The mechanism restricting shedding was characterized using primary well-differentiated human airway epithelial cells. Wild-type MV infected columnar epithelial cells bearing tight junctions only when applied basolaterally, while the EpR-blind virus did not infect these cells. Thus, EpR is probably a basolateral protein, and infection of the airway epithelium is not essential for systemic spread and virulence of MV.

InterProSurf : a web server for predicting interacting sites on protein surfaces

UNLABELLED A new web server, InterProSurf, predicts interacting amino acid residues in proteins that are most likely to interact with other proteins, given the 3D structures of subunits of a protein complex. The prediction method is based on solvent accessible surface area of residues in the isolated subunits, a propensity scale for interface residues and a clustering algorithm to identify surface regions with residues of high interface propensities. Here we illustrate the application of InterProSurf to determine which areas of Bacillus anthracis toxins and measles virus hemagglutinin protein interact with their respective cell surface receptors. The computationally predicted regions overlap with those regions previously identified as interface regions by sequence analysis and mutagenesis experiments. AVAILABILITY The InterProSurf web server is available at http://curie.utmb.edu/

The heads of the measles virus attachment protein move to transmit the fusion-triggering signal

The measles virus entry system, consisting of attachment (hemagglutinin, H) and fusion proteins, operates by means of a variety of natural and targeted receptors; however, the mechanism that triggers fusion of the viral envelope with the plasma membrane is not understood. Here, we tested a model proposing that the two heads of an H dimer, which are covalently linked at their base, after binding two receptor molecules, move relative to each other to transmit the fusion-triggering signal. Indeed, stabilizing the H-dimer interface with additional intermolecular disulfide bonds prevented membrane fusion, an effect that was reversed by a reducing agent. Moreover, a membrane-anchored designated receptor efficiently triggered fusion, provided that it engaged the H dimer at locations proximal to where the natural receptors bind and distal to the H-dimer interface. We discuss how receptors may force H-protein heads to switch partners and transmit the fusion-triggering signal.

Host S-nitrosylation inhibits clostridial small molecule-activated glucosylating toxins

The global prevalence of severe Clostridium difficile infection highlights the profound clinical significance of clostridial glucosylating toxins. Virulence is dependent on the autoactivation of a toxin cysteine protease, which is promoted by the allosteric cofactor inositol hexakisphosphate (InsP6). Host mechanisms that protect against such exotoxins are poorly understood. It is increasingly appreciated that the pleiotropic functions attributed to nitric oxide (NO), including host immunity, are in large part mediated by S-nitrosylation of proteins. Here we show that C. difficile toxins are S-nitrosylated by the infected host and that S-nitrosylation attenuates virulence by inhibiting toxin self-cleavage and cell entry. Notably, InsP6- and inositol pyrophosphate (InsP7)-induced conformational changes in the toxin enabled host S-nitrosothiols to transnitrosylate the toxin catalytic cysteine, which forms part of a structurally conserved nitrosylation motif. Moreover, treatment with exogenous InsP6 enhanced the therapeutic actions of oral S-nitrosothiols in mouse models of C. difficile infection. Allostery in bacterial proteins has thus been successfully exploited in the evolutionary development of nitrosothiol-based innate immunity and may provide an avenue to new therapeutic approaches.

Dynamic Interaction of the Measles Virus Hemagglutinin with Its Receptor Signaling Lymphocytic Activation Molecule (SLAM, CD150)

The interaction of measles virus with its receptor signaling lymphocytic activation molecule (SLAM) controls cell entry and governs tropism. We predicted potential interface areas of the measles virus attachment protein hemagglutinin to begin the investigation. We then assessed the relevance of individual amino acids located in these areas for SLAM-binding and SLAM-dependent membrane fusion, as measured by surface plasmon resonance and receptor-specific fusion assays, respectively. These studies identified one hemagglutinin protein residue, isoleucine 194, which is essential for primary binding. The crystal structure of the hemagglutinin-protein localizes Ile-194 at the interface of propeller blades 5 and 6, and our data indicate that a small aliphatic side chain of residue 194 stabilizes a protein conformation conducive to binding. In contrast, a quartet of residues previously shown to sustain SLAM-dependent fusion is not involved in binding. Instead, our data prove that after binding, this quartet of residues on propeller blade 5 conducts conformational changes that are receptor-specific. Our study sets a structure-based stage for understanding how the SLAM-elicited conformational changes travel through the H-protein ectodomain before triggering fusion protein unfolding and membrane fusion.

Gut microbiota influences low fermentable substrate diet efficacy in children with irritable bowel syndrome

We sought to determine whether a low fermentable substrate diet (LFSD) decreases abdominal pain frequency in children with irritable bowel syndrome (IBS) and to identify potential microbial factors related to diet efficacy. Pain symptoms, stooling characteristics, breath hydrogen and methane, whole intestinal transit time, stool microbiome, and metabolite composition were collected and/or documented in eight children with IBS at baseline and during one week of an LFSD intervention. Pain frequency (P < 0.05), pain severity (P < 0.05), and pain-related interference with activities (P < 0.05) decreased in the subjects while on the LFSD. Responders vs. non-responders: four children (50%) were identified as responders (>50% decrease in abdominal pain frequency while on the LFSD). There were no differences between responders and non-responders with respect to hydrogen production, methane production, stooling characteristics, or gut transit time. Responders were characterized by increased pre-LFSD abundance of bacterial taxa belonging to the genera Sporobacter (P < 0.05) and Subdoligranulum (P < 0.02) and decreased abundance of taxa belonging to Bacteroides (P < 0.05) relative to non-responders. In parallel, stool metabolites differed between responders and non-responders and were associated with differences in microbiome composition. These pilot study results suggest that an LFSD may be effective in decreasing GI symptoms in children with IBS. Microbial factors such as gut microbiome composition and stool metabolites while on the diet may relate to LFSD efficacy.

Nearby Clusters of Hemagglutinin Residues Sustain SLAM-Dependent Canine Distemper Virus Entry in Peripheral Blood Mononuclear Cells

ABSTRACT Signaling lymphocytic activation molecule (SLAM, CD150) is the universal morbillivirus receptor. Based on the identification of measles virus (MV) hemagglutinin (H) amino acids supporting human SLAM-dependent cell entry, we mutated canine distemper virus (CDV) H and identified residues necessary for efficient canine SLAM-dependent membrane fusion. These residues are located in two nearby clusters in a new CDV H structural model. To completely abolish SLAM-dependent fusion, combinations of mutations were necessary. We rescued a SLAM-blind recombinant CDV with six mutations that did not infect ferret peripheral blood mononuclear cells while retaining full infectivity in epithelial cells.

A “moving metal mechanism” for substrate cleavage by the DNA repair endonuclease APE‐1

Apurinic/apyrimidinic endonuclease (APE‐1) is essential for base excision repair (BER) of damaged DNA. Here molecular dynamics (MD) simulations of APE1 complexed with cleaved and uncleaved damaged DNA were used to determine the role and position of the metal ion(s) in the active site before and after DNA cleavage. The simulations started from an energy minimized wild‐type structure of the metal‐free APE1/damaged‐DNA complex (1DE8). A grid search with one Mg2+ ion located two low energy clusters of Mg2+ consistent with the experimentally determined metal ion positions. At the start of the longer MD simulations, Mg2+ ions were placed at different positions as seen in the crystal structures and the movement of the ion was followed over the course of the trajectory. Our analysis suggests a “moving metal mechanism” in which one Mg2+ ion moves from the B‐ (more buried) to the A‐site during substrate cleavage. The anticipated inversion of the phosphate oxygens occurs during the in‐line cleavage reaction. Experimental results, which show competition between Ca2+ and Mg2+ for catalyzing the reaction, and high concentrations of Mg2+ are inihibitory, indicate that both sites cannot be simultaneously occupied for maximal activity. Proteins 2007.

Base of the measles virus fusion trimer head receives the signal that triggers membrane fusion.

Background: A homology model of the trimeric measles virus fusion protein predicts a cavity in the base of the head. Results: Hydrophobic residues required for interactions with the hemagglutinin map to this cavity. Conclusion: The base of the measles virus fusion protein trimer head receives the signal that triggers membrane fusion. Significance: Emerging, re-emerging, and prevalent paramyxoviruses may operate based on similar signal transmission mechanisms. The measles virus (MV) fusion (F) protein trimer executes membrane fusion after receiving a signal elicited by receptor binding to the hemagglutinin (H) tetramer. Where and how this signal is received is understood neither for MV nor for other paramyxoviruses. Because only the prefusion structure of the parainfluenza virus 5 (PIV5) F-trimer is available, to study signal receipt by the MV F-trimer, we generated and energy-refined a homology model. We used two approaches to predict surface residues of the model interacting with other proteins. Both approaches measured interface propensity values for patches of residues. The second approach identified, in addition, individual residues based on the conservation of physical chemical properties among F-proteins. Altogether, about 50 candidate interactive residues were identified. Through iterative cycles of mutagenesis and functional analysis, we characterized six residues that are required specifically for signal transmission; their mutation interferes with fusion, although still allowing efficient F-protein processing and cell surface transport. One residue is located adjacent to the fusion peptide, four line a cavity in the base of the F-trimer head, while the sixth residue is located near this cavity. Hydrophobic interactions in the cavity sustain the fusion process and contacts with H. The cavity is flanked by two different subunits of the F-trimer. Tetrameric H-stalks may be lodged in apposed cavities of two F-trimers. Because these insights are based on a PIV5 homology model, the signal receipt mechanism may be conserved among paramyxoviruses.

Membrane-Protein Interactions Contribute to Efficient 27-Hydroxylation of Cholesterol by Mitochondrial Cytochrome P450 27A1

Mitochondrial cytochrome P450 27A1 (P450 27A1) catalyzes 27-hydroxylation of cholesterol, the first step in the alternative bile acid biosynthetic pathway. Although several crystal structures of P450s are known, no structural information is available for the mammalian, membrane-bound enzymes involved in the removal of cholesterol from the body. We prepared a three-dimensional model of P450 27A1 based on the structure of P450 BM-3. Conservative and non-conservative mutations were introduced at hydrophobic and positively charged residues in the putative F-G loop and the adjacent helix G (positions 219–237). Subcellular distribution of the mutant P450s expressed in Escherichia coli was used as a measure of membrane-protein interactions. Conservative substitutions of residues located on the surface, according to our model, L219V, L219I, Y220F, F223Y, L224I, R229K, V231L, F234Y, K236R, and R237K, weakened the association of the mutant P450s with the membrane and led to the appearance of up to 21% of P450 27A1 in the bacterial cytosol. It is likely that the mutated side chains are involved in binding to membrane phospholipids. Substitutions in the F-G loop did not significantly affect the K m value for cholesterol hydroxylation. However, non-conservative mutants, L219N, Y220A, Y220S, F223A, K226R, and R229A, had significantly impaired catalytic properties, indicating strict requirements for the size and polarity of the side chains at these positions for the catalysis. The results provide insight into the membrane topology of mitochondrial P450s and indicate the importance of membrane-protein interactions in the efficiency of reactions catalyzed by P450 27A1.