Joseph Shiloach

Structural Basis of Toll-Like Receptor 3 Signaling with Double-Stranded RNA

Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA), a molecular signature of most viruses, and triggers inflammatory responses that prevent viral spread. TLR3 ectodomains (ECDs) dimerize on oligonucleotides of at least 40 to 50 base pairs in length, the minimal length required for signal transduction. To establish the molecular basis for ligand binding and signaling, we determined the crystal structure of a complex between two mouse TLR3-ECDs and dsRNA at 3.4 angstrom resolution. Each TLR3-ECD binds dsRNA at two sites located at opposite ends of the TLR3 horseshoe, and an intermolecular contact between the two TLR3-ECD C-terminal domains coordinates and stabilizes the dimer. This juxtaposition could mediate downstream signaling by dimerizing the cytoplasmic Toll interleukin-1 receptor (TIR) domains. The overall shape of the TLR3-ECD does not change upon binding to dsRNA.

The Efficacy of a Salmonella typhi Vi Conjugate Vaccine in Two-to-Five-Year-Old Children

BACKGROUND Typhoid fever is common in developing countries. The licensed typhoid vaccines confer only about 70 percent immunity, do not protect young children, and are not used for routine vaccination. A newly devised conjugate of the capsular polysaccharide of Salmonella typhi, Vi, bound to nontoxic recombinant Pseudomonas aeruginosa exotoxin A (rEPA), has enhanced immunogenicity in adults and in children 5 to 14 years old and has elicited a booster response in children 2 to 4 years old. METHODS In a double-blind, randomized trial, we evaluated the safety, immunogenicity, and efficacy of the Vi-rEPA vaccine in children two to five years old in 16 communes in Dong Thap Province, Vietnam. Each of the 11,091 children received two injections six weeks apart of either Vi-rEPA or a saline placebo. Cases of typhoid, diagnosed by the isolation of S. typhi from blood cultures after 3 or more days of fever (a temperature of 37.5 degrees C or higher), were identified by active surveillance over a period of 27 months. We estimated efficacy by comparing the attack rate of typhoid in the vaccine group with that in the placebo group. RESULTS S. typhi was isolated from 4 of the 5525 children who were fully vaccinated with Vi-rEPA and from 47 of the 5566 children who received both injections of placebo (efficacy, 91.5 percent; 95 percent confidence interval, 77.1 to 96.6; P<0.001). Among the 771 children who received only one injection, there was 1 case of typhoid in the vaccine group and 8 cases in the placebo group. Cases were distributed evenly among all age groups and throughout the study period. No serious adverse reactions were observed. In all 36 children studied four weeks after the second injection of the vaccine, levels of serum IgG Vi antibodies had increased by a factor of 10 or more. CONCLUSIONS The Vi-rEPA conjugate typhoid vaccine is safe and immunogenic and has more than 90 percent efficacy in children two to five years old. The antibody responses and the efficacy suggest that this vaccine should be at least as protective in persons who are more than five years old.

Structure of the agonist-bound neurotensin receptor

Neurotensin (NTS) is a 13-amino-acid peptide that functions as both a neurotransmitter and a hormone through the activation of the neurotensin receptor NTSR1, a G-protein-coupled receptor (GPCR). In the brain, NTS modulates the activity of dopaminergic systems, opioid-independent analgesia, and the inhibition of food intake; in the gut, NTS regulates a range of digestive processes. Here we present the structure at 2.8 Å resolution of Rattus norvegicus NTSR1 in an active-like state, bound to NTS8–13, the carboxy-terminal portion of NTS responsible for agonist-induced activation of the receptor. The peptide agonist binds to NTSR1 in an extended conformation nearly perpendicular to the membrane plane, with the C terminus oriented towards the receptor core. Our findings provide, to our knowledge, the first insight into the binding mode of a peptide agonist to a GPCR and may support the development of non-peptide ligands that could be useful in the treatment of neurological disorders, cancer and obesity.

Poly(γ-d-glutamic acid) protein conjugates induce IgG antibodies in mice to the capsule of Bacillus anthracis: A potential addition to the anthrax vaccine

Both the protective antigen (PA) and the poly(γ-d-glutamic acid) capsule (γdPGA) are essential for the virulence of Bacillus anthracis. A critical level of vaccine-induced IgG anti-PA confers immunity to anthrax, but there is no information about the protective action of IgG anti-γdPGA. Because the number of spores presented by bioterrorists might be greater than encountered in nature, we sought to induce capsular antibodies to expand the immunity conferred by available anthrax vaccines. The nonimmunogenic γdPGA or corresponding synthetic peptides were bound to BSA, recombinant B. anthracis PA (rPA), or recombinant Pseudomonas aeruginosa exotoxin A (rEPA). To identify the optimal construct, conjugates of B. anthracis γdPGA, Bacillus pumilus γdLPGA, and peptides of varying lengths (5-, 10-, or 20-mers), of the d or l configuration with active groups at the N or C termini, were bound at 5–32 mol per protein. The conjugates were characterized by physico-chemical and immunological assays, including GLC-MS and matrix-assisted laser desorption ionization time-of-flight spectrometry, and immunogenicity in 5- to 6-week-old mice. IgG anti-γdPGA and antiprotein were measured by ELISA. The highest levels of IgG anti-γdPGA were elicited by decamers of γdPGA at 10 –20 mol per protein bound to the N- or C-terminal end. High IgG anti-γdPGA levels were elicited by two injections of 2.5 μg of γdPGA per mouse, whereas three injections were needed to achieve high levels of protein antibodies. rPA was the most effective carrier. Anti-γdPGA induced opsonophagocytic killing of B. anthracis tox–, cap+. γdPGA conjugates may enhance the protection conferred by PA alone. γdPGA-rPA conjugates induced both anti-PA and anti-γdPGA.

Dimerization of the class A G protein-coupled neurotensin receptor NTS1 alters G protein interaction

G protein-coupled receptors (GPCRs) have been found as monomers but also as dimers or higher-order oligomers in cells. The relevance of the monomeric or dimeric receptor state for G protein activation is currently under debate for class A rhodopsin-like GPCRs. Clarification of this issue requires the availability of well defined receptor preparations as monomers or dimers and an assessment of their ligand-binding and G protein-coupling properties. We show by pharmacological and hydrodynamic experiments that purified neurotensin receptor NTS1, a class A GPCR, dimerizes in detergent solution in a concentration-dependent manner, with an apparent affinity in the low nanomolar range. At low receptor concentrations, NTS1 binds the agonist neurotensin with a Hill slope of ≈1; at higher receptor concentrations, neurotensin binding displays positive cooperativity with a Hill slope of ≈2. NTS1 monomers activate Gαqβ1γ2, whereas receptor dimers catalyze nucleotide exchange with lower affinity. Our results demonstrate that NTS1 dimerization per se is not a prerequisite for G protein activation.

Effect of glucose supply strategy on acetate accumulation, growth, and recombinant protein production by Escherichia coli BL21 (λDE3) and Escherichia coli JM109

Two Escherichia coli strains, widely used for the production of various recombinant proteins, were compared for their pre‐induction growth and acetate accumulation patterns. The strains studied were E. coli BL21 (λDE3), transformed with a plasmid encoding Pseudomonas exotoxin A, and an E. coli K12 derived strain, JM109, carrying a plasmid encoding maltose‐binding protein fused with HIV protease. Cultures were grown in controlled bench‐top fermentors to the optimal pre‐induction density in both high glucose batch and low glucose fed batch strategies. The results showed the superiority of E. coli BL21 (λDE3) as a host for a recombinant protein expression system. For example, JM109 responds differently to high glucose concentration and to low glucose concentration. Its acetate concentration was as high as 10 g/L in a batch mode and 5 g/L in a fed batch mode. In comparison, strain BL21 (λDE3) reached 2 g/L acetate when grown in batch mode and not more than 1 g/L acetate when grown in a fed batch mode. E. coli BL21 (λDE3), most likely, possesses an acetate self‐control mechanism which makes it possible to grow to the desired pre‐induction density in a high glucose medium using simple batch propagation techniques. Such a technique is cost effective, reproducible, and easy to scale up.

Development of an improved vaccine for anthrax

Bacillus anthracis are aerobic or facultatively anaerobic Gram-positive, nonmotile rods measuring 1.0 μm wide by 3.0–5.0 μm long. Under adverse conditions, B. anthracis form highly resistant endospores (Figure ​(Figure1).1). These are found in soil at sites where infected animals previously died. Interest in the pathogenesis, immunity, and vaccine development for anthrax was heightened by the deliberate contamination of mail with B. anthracis spores soon after the September 11 attacks. At that time, the only US-licensed human vaccine (anthrax vaccine adsorbed, or AVA) was not available because the manufacturer, BioPort Corp., had not received FDA certification of its new manufacturing process. Figure 1 A spore (left) and vegetative cells and a chain of vegetative rod cells of B. anthracis. Electron micrograph courtesy of the Centers for Disease Control and Prevention. Although Pasteur had already demonstrated protection of sheep by injection of heat-attenuated B. anthracis cultures in 1881, our current knowledge of immunity to anthrax in humans remains limited. Widespread vaccination of domesticated animals with attenuated strains such as the Sterne strain began in the 1930s and has virtually abolished anthrax in industrialized countries. In the US, the licensed human vaccine (AVA, newly renamed BioThrax) is an aluminum hydroxide–adsorbed, formalin-treated culture supernatant of a toxigenic, noncapsulated, nonproteolytic B. anthracis strain, V770-NP1-R, derived from the Sterne strain (1). AVA was developed in the early 1950s, when purified components of B. anthracis were not available. Its only demonstrable protective component is the protective antigen (PA) protein (2). A similar culture supernatant–derived human vaccine is produced in the United Kingdom. Data from a 1950s trial of wool-sorters immunized with a vaccine similar to AVA, coupled with long experience with AVA and the United Kingdom vaccine, have shown that a critical level of serum antibodies to the B. anthracis PA confers immunity to anthrax (3, 4). As early as 1959, a British Ministry of Labour report noted that, following the introduction of regular immunization the previous year, the staff of the Government Wool Disinfection Station in Liverpool were free of the disease “despite the high risk to which they are exposed” (5). AVA also protects laboratory animals and cattle from both cutaneous and inhalational challenge with B. anthracis (1, 6, 7). Although safe and efficacious (8), AVA has limitations that justify the widespread interest in developing improved vaccines consisting solely of well-characterized components. First, standardization of AVA is based on the manufacturing process and a potency assay involving protection of guinea pigs challenged intracutaneously with B. anthracis spores (7, 9). PA is not measured in the vaccine, and there is no standardized assay of PA antibodies in animals or humans vaccinated with AVA. These factors probably explain why it has been difficult to maintain consistency of AVA. Second, this vaccine contains other cellular elements that probably contribute to the relatively high rate of local and systemic reactions (8). Finally, the schedule of AVA administration (subcutaneous injections at 0, 2, and 4 weeks and 6, 12, and 18 months with subsequent yearly boosters) is probably not optimal. This schedule, introduced in the 1950s, was designed for rapid induction of immunity (10), but it was recently shown that increasing the interval between the first two injections enhances the level of AVA-induced antibodies to PA (11). Moreover, there is no experimental support for including the injections given at 6, 12, and 18 months.

PROPOSED MECHANISM OF ACETATE ACCUMULATION IN TWO RECOMBINANT ESCHERICHIA COLI STRAINS DURING HIGH DENSITY FERMENTATION

The productivity of Escherichia coli as a producer of recombinant proteins is affected by its metabolic properties, especially by acetate production. Two commercially used E. coli strains, BL21 (lambdaDE3) and JM109, differ significantly in their acetate production during batch fermentation at high initial glucose concentrations. E. coli BL21 grows to an optical density (OD, 600 nm) of 100 and produces no more than 2 g/L acetate, while E. coli JM109 grows to an OD (600 nm) of 80 and produces up to 14 g/L acetate. Even in fed-batch fermentation, when glucose concentration is maintained between 0.5 and 1.0 g/L, JM109 accumulates 4 times more acetate than BL21. To investigate the difference between the two strains, metabolites and enzymes involved in carbon utilization and acetate production were analyzed (isocitrate, ATP, phosphoenolpyruvate, pyruvate, isocitrate lyase, and isocitrate dehydrogenase). The results showed that during batch fermentation isocitrate lyase activity and isocitrate concentration were higher in BL21 than in JM109, while pyruvate concentration was higher in JM109. The activation of the glyoxylate shunt pathway at high glucose concentrations is suggested as a possible explanation for the lower acetate accumulation in E. coli BL21. Metabolic flux analysis of the batch cultures supports the activity of the glyoxylate shunt in E. coli BL21.

THE SOLUTION STRUCTURE OF THE HUMAN ETS1-DNA COMPLEX REVEALS A NOVEL MODE OF BINDING AND TRUE SIDE CHAIN INTERCALATION

Abstract The solution structure of a 24.4 kDa specific complex of the DNA-binding domain (DBD) of the human ETS1 (hETSi) oncoprotein with a 17-mer DNA has been solved by NMR. The interaction of the hETSi DBD with DNA reveals a surprising twist on the general features of helix-turn-helix (HTH)-DNA interactions. Major groove recognition involves the C-terminal two thirds of the HTH recognition helix, while minorgroove recognition occurs via true intercalation of the side chain of Trp-28, which extends from the minor to the major groove. This results in a sharp kink of ∼60° and a widening of the minor groove over one-half turn of the DNA. The orientation of the HTH element of the hETSi DBD with respect to the major groove is significantly rotated relative to other HTH proteins. These observations establish the ETS family of DNA-binding proteins as a distinct family of HTH proteins.

Evidence for an endogenous peptide ligand for the phencyclidine receptor

Porcine brain contained an active factor that competed with [3H]-phencyclidine (PCP) for binding to rat brain membranes. On reverse phase high pressure liquid chromatography, the active material eluted between 38-42% acetonitrile. Gel filtration chromatography of the factor predicted a molecular weight of approximately 3000 daltons. The endogenous substance appeared to be selective for PCP receptors as it did not interact with either benzodiazepine, neurotensin, nor with mu, delta, or kappa opioid receptors. The active material showed a heterogenous distribution in brain, with highest concentrations found in hippocampus and cortex. It is likely to be a small peptide since various proteases eliminated or markedly reduced the potency of the compound in a [3H]-PCP binding assay. The material also possessed PCP-like activity in two bioassays. Like PCP, it induced contralateral rotational behavior after unilateral intranigral injection and depressed spontaneous cell activity after iontophoretic micropressure application in hippocampus and cerebral cortex. Thus, this small peptide is likely to be an endogenous ligand for the PCP receptor.