Aptamer based point of care diagnostic for the detection of food allergens.

Aptamers, due to their small size, strong target affinity, and ease of chemical modification, are ideally suited for molecular detection technologies. Here, we describe successful use of aptamer technology in a consumer device for the detection of peanut antigen in food. The novel aptamer-based protein detection method is robust across a wide variety of food matrices and sensitive to peanut protein at concentrations as low as 12.5 ppm (37.5 µg peanut protein in the sample). Integration of the assay into a sensitive, stable, and consumer friendly portable device will empower users to easily and quickly assess the presence of peanut allergens in foods before eating. With many food reactions occurring outside the home, the type of technology described here has significant potential to improve lives for children and families.

Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase.

Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism.

Desmosterol Increases Lipid Bilayer Fluidity during Hepatitis C Virus Infection.

Hepatitis C virus (HCV) uniquely affects desmosterol homeostasis by increasing its intracellular abundance and affecting its localization. These effects are important for productive viral replication because the inhibition of desmosterol synthesis has an antiviral effect that can be rescued by the addition of exogenous desmosterol. Here, we use subgenomic replicons to show that desmosterol has a major effect on the replication of HCV JFH1 RNA. Fluorescence recovery after photobleaching (FRAP) experiments performed with synthetic supported lipid bilayers demonstrate that the substitution of desmosterol for cholesterol significantly increases the lipid bilayer fluidity, especially in the presence of saturated phospholipids and ceramides. We demonstrate using LC-MS that desmosterol is abundant in the membranes upon which genome replication takes place and that supported lipid bilayers derived from these specialized membranes also exhibit significantly higher fluidity compared to that of negative control membranes isolated from cells lacking HCV. Together, these data suggest a model in which the fluidity-promoting effects of desmosterol on lipid bilayers play a crucial role in the extensive membrane remodeling that takes place in the endoplasmic reticulum during HCV infection. We anticipate that the supported lipid bilayer system described can provide a useful model system in which to interrogate the effects of lipid structure and composition on the biophysical properties of lipid membranes as well as their function in viral processes such as genome replication.

Hepatitis C Virus Selectively Alters the Intracellular Localization of Desmosterol.

Hepatitis C virus (HCV) increases intracellular desmosterol without affecting the steady-state abundance of other sterols, and the antiviral activity of inhibitors of desmosterol synthesis is suppressed by the addition of exogenous desmosterol. These observations suggest a model in which desmosterol has a specific function, direct or indirect, in HCV replication and that HCV alters desmosterol homeostasis to promote viral replication. Here, we use stimulated Raman scattering (SRS) microscopy in combination with isotopically labeled sterols to show that HCV causes desmosterol to accumulate in lipid droplets that are closely associated with the viral NS5A protein and that are visually distinct from the broad distribution of desmosterol in mock-infected cells and the more heterogeneous and disperse lipid droplets to which cholesterol traffics. Localization of desmosterol in NS5A-associated lipid droplets suggests that desmosterol may affect HCV replication via a direct mechanism. We anticipate that SRS microscopy and similar approaches can provide much needed tools to study the localization of specific lipid molecules in cellulo and in vivo.

Targeting host lipid synthesis and metabolism to inhibit dengue and hepatitis C viruses.

Lipids are necessary for every step in the replication cycle of hepatitis C virus (HCV) and dengue virus (DENV), members of the family Flaviviridae. Recent studies have demonstrated that discrete steps in the replication cycles of these viruses can be inhibited by pharmacological agents that target host factors mediating lipid synthesis, metabolism, trafficking, and signal transduction. Despite this, targeting host lipid metabolism and trafficking as an antiviral strategy by blockade of entire pathways may be limited due to host toxicity. Knowledge of the molecular details of lipid structure and function in replication and the mechanisms whereby specific lipids are generated and trafficked to the relevant sites may enable more targeted antiviral strategies without global effects on the host cell. In this review, we discuss lipids demonstrated to be critical to the replication cycles of HCV and DENV and highlight potential areas for anti-viral development. This review article forms part of a symposium on flavivirus drug discovery in Antiviral Research.

The bioactive lipid 4-hydroxyphenyl retinamide inhibits flavivirus replication.

Dengue virus (DENV), a member of the Flaviviridae family, is a mosquito-borne pathogen and the cause of dengue fever. The increasing prevalence of DENV worldwide heightens the need for an effective vaccine and specific antivirals. Due to the dependence of DENV upon the lipid biosynthetic machinery of the host cell, lipid signaling and metabolism present unique opportunities for inhibiting viral replication. We screened a library of bioactive lipids and modulators of lipid metabolism and identified 4-hydroxyphenyl retinamide (4-HPR) (fenretinide) as an inhibitor of DENV in cell culture. 4-HPR inhibits the steady-state accumulation of viral genomic RNA and reduces viremia when orally administered in a murine model of DENV infection. The molecular target responsible for this antiviral activity is distinct from other known inhibitors of DENV but appears to affect other members of the Flaviviridae, including the West Nile, Modoc, and hepatitis C viruses. Although long-chain ceramides have been implicated in DENV replication, we demonstrate that DENV is insensitive to the perturbation of long-chain ceramides in mammalian cell culture and that the effect of 4-HPR on dihydroceramide homeostasis is separable from its antiviral activity. Likewise, the induction of reactive oxygen species by 4-HPR is not required for the inhibition of DENV. The inhibition of DENV in vivo by 4-HPR, combined with its well-established safety and tolerability in humans, suggests that it may be repurposed as a pan-Flaviviridae antiviral agent. This work also illustrates the utility of bioactive lipid screens for identifying critical interactions of DENV and other viral pathogens with host lipid biosynthesis, metabolism, and signal transduction.

Lipid metabolite profiling identifies desmosterol metabolism as a new antiviral target for hepatitis C virus.

Hepatitis C virus (HCV) infection has been clinically associated with serum lipid abnormalities, yet our understanding of the effects of HCV on host lipid metabolism and conversely the function of individual lipids in HCV replication remains incomplete. Using liquid chromatography-mass spectrometry metabolite profiling of the HCV JFH1 cell culture infection model, we identified a significant steady-state accumulation of desmosterol, an immediate precursor to cholesterol. Pharmacological inhibition or RNAi-mediated depletion of DHCR7 significantly reduced steady-state HCV protein expression and viral genomic RNA. Moreover, this effect was reversed when cultures were supplemented with exogenous desmosterol. Together, these observations suggest an intimate connection between HCV replication and desmosterol homeostasis and that the enzymes responsible for synthesis of desmosterol may be novel targets for antiviral design.

Transient weak protein-protein complexes transfer heme across the cell wall of Staphylococcus aureus.

Iron is an essential nutrient for the bacterial pathogen Staphylococcus aureus . Heme in hemoglobin (Hb) is the most abundant source of iron in the human body and during infections is captured by S. aureus using iron-regulated surface determinant (Isd) proteins. A central step in this process is the transfer of heme between the cell wall associated IsdA and IsdC hemoproteins. Biochemical evidence indicates that heme is transferred via an activated IsdA:heme:IsdC heme complex. Transfer is rapid and occurs up to 70,000 times faster than indirect mechanisms in which heme is released into the solvent. To gain insight into the mechanism of transfer, we modeled the structure of the complex using NMR paramagnetic relaxation enhancement (PRE) methods. Our results indicate that IsdA and IsdC transfer heme via an ultraweak affinity "handclasp" complex that juxtaposes their respective 3(10) helices and ß7/ß8 loops. Interestingly, PRE also identified a set of transient complexes that could represent high-energy pre-equilibrium encounter species that form prior to the stereospecific handclasp complex. Targeted amino acid mutagenesis and stopped-flow measurements substantiate the functional relevance of a PRE-derived model, as mutation of interfacial side chains significantly slows the rate of transfer. IsdA and IsdC bind heme using NEAr Transporter (NEAT) domains that are conserved in many species of pathogenic Gram-positive bacteria. Heme transfer in these microbes may also occur through structurally similar transient stereospecific complexes.

Anti-HCV drugs in the pipeline.

Several directly acting and host targeting antivirals that inhibit hepatitis C virus replication have entered clinical trials. Among the most advanced of these are RG7128, an inhibitor of the NS5B polymerase; BMS-790052, an inhibitor of NS5A; and alisporivir, an inhibitor of human cyclophilins. These agents have potent antiviral activity in chronic HCV patients, act additively or synergistically with inhibitors of the HCV NS3/4A protease, and improve the rate of virologic response produced by traditional pegylated interferon plus ribavirin therapy. No cross resistance has been observed; moreover, nucleoside NS5B and cyclophilin inhibitors appear to suppress resistance to non-nucleoside NS5B and NS3/4A inhibitors. Several recent reports of virologic responses produced by combinations of agents that inhibit HCV replication in the absence of interferon provide optimism that eradication of HCV will be possible without interferon in the future.

Anthrax toxin receptor 1/tumor endothelial marker 8: mutation of conserved inserted domain residues overrides cytosolic control of protective antigen binding.

Anthrax toxin receptor 1 (ANTXR1)/tumor endothelial marker 8 (TEM8) is one of two known proteinaceous cell surface anthrax toxin receptors. A metal ion dependent adhesion site (MIDAS) present in the integrin-like inserted (I) domain of ANTXR1 mediates the binding of the anthrax toxin subunit, protective antigen (PA). Here we provide evidence that single point mutations in the I domain can override regulation of ANTXR1 ligand-binding activity mediated by intracellular signals. A previously reported MIDAS mutant of ANTXR1 (T118A) was found to retain normal metal ion binding and secondary structure but failed to bind PA, consistent with a locked inactive state. Conversely, mutation of a conserved I domain phenylalanine residue to a tryptophan (F205W) increased the proportion of cell-surface ANTXR1 that bound PA, consistent with a locked active state. Interestingly, the K(D) and total amount of PA bound by the isolated ANTXR1 I domain were not affected by the F205W mutation, indicating that ANTXR1 is preferentially found in the active state in the absence of inside-out signaling. Circular dichroism (CD) spectroscopy and (1)H-(15)N heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) revealed that structural changes between T118A, F205W, and WT I domains were minor despite a greater than 10(3)-fold difference in their abilities to bind toxin. Regulation of toxin binding has important implications for the design of toxin inhibitors and for the targeting of ANTXR1 for antitumor therapies.

A heteronuclear zero quantum coherence Nz-exchange experiment that resolves resonance overlap and its application to measure the rates of heme binding to the IsdC protein.

Chemical exchange phenomena in NMR spectra can be quantitatively interpreted to measure the rates of ligand binding, as well as conformational and chemical rearrangements. In macromolecules, processes that occur slowly on the chemical shift time scale are frequently studied using 2D heteronuclear ZZ or N(z)-exchange spectroscopy. However, to successfully apply this method, peaks arising from each exchanging species must have unique chemical shifts in both dimensions, a condition that is often not satisfied in protein-ligand binding equilibria for (15)N nuclei. To overcome the problem of (15)N chemical shift degeneracy we developed a heteronuclear zero-quantum (and double-quantum) coherence N(z)-exchange experiment that resolves (15)N chemical shift degeneracy in the indirect dimension. We demonstrate the utility of this new experiment by measuring the heme binding kinetics of the IsdC protein from Staphylococcus aureus. Because of peak overlap, we could not reliably analyze binding kinetics using conventional methods. However, our new experiment resulted in six well-resolved systems that yielded interpretable data. We measured a relatively slow k(off) rate of heme from IsdC (<10 s(-1)), which we interpret as necessary so heme loaded IsdC has time to encounter downstream binding partners to which it passes the heme. The utility of using this new exchange experiment can be easily expanded to (13)C nuclei. We expect our heteronuclear zero-quantum coherence N(z)-exchange experiment will expand the usefulness of exchange spectroscopy to slow chemical exchange events that involve ligand binding.

The structure of the Staphylococcus aureus sortase-substrate complex reveals how the universally conserved LPXTG sorting signal is recognized.

In Gram-positive bacteria, sortase enzymes assemble surface proteins and pili in the cell wall envelope. Sortases catalyze a transpeptidation reaction that joins a highly conserved LPXTG sorting signal within their polypeptide substrate to the cell wall or to other pilin subunits. The molecular basis of transpeptidation and sorting signal recognition are not well understood, because the intermediates of catalysis are short lived. We have overcome this problem by synthesizing an analog of the LPXTG signal whose stable covalent complex with the enzyme mimics a key thioacyl catalytic intermediate. Here we report the solution structure and dynamics of its covalent complex with the Staphylococcus aureus SrtA sortase. In marked contrast to a previously reported crystal structure, we show that SrtA adaptively recognizes the LPXTG sorting signal by closing and immobilizing an active site loop. We have also used chemical shift mapping experiments to localize the binding site for the triglycine portion of lipid II, the second substrate to which surface proteins are attached. We propose a unified model of the transpeptidation reaction that explains the functions of key active site residues. Since the sortase-catalyzed anchoring reaction is required for the virulence of a number of bacterial pathogens, the results presented here may facilitate the development of new anti-infective agents.

Functionally distinct NEAT (NEAr Transporter) domains within the Staphylococcus aureus IsdH/HarA protein extract heme from methemoglobin.

The pathogen Staphylococcus aureus uses iron-regulated surface determinant (Isd) proteins to scavenge the essential nutrient iron from host hemoproteins. The IsdH protein (also known as HarA) is a receptor for hemoglobin (Hb), haptoglobin (Hp), and the Hb-Hp complex. It contains three NEAT (NEAr Transporter) domains: IsdH(N1), IsdH(N2), and IsdH(N3). Here we show that they have different functions; IsdH(N1) binds Hb and Hp, whereas IsdH(N3) captures heme that is released from Hb. The staphylococcal IsdB protein also functions as an Hb receptor. Primary sequence homology to IsdH indicates that it will also employ functionally distinct NEAT domains to bind heme and Hb. We have used site-directed mutagenesis and surface plasmon resonance methods to localize the Hp and Hb binding surface on IsdH(N1). High affinity binding to these structurally unrelated proteins requires residues located within a conserved aromatic motif that is positioned at the end of the beta-barrel structure. Interestingly, this site is quite malleable, as other NEAT domains use it to bind heme. We also demonstrate that the IsdC NEAT domain can capture heme directly from Hb, suggesting that there are multiple pathways for heme transfer across the cell wall.

The IsdC protein from Staphylococcus aureus uses a flexible binding pocket to capture heme.

Staphylococcus aureus scavenges heme-iron from host hemoproteins using iron-regulated surface determinant (Isd) proteins. IsdC is the central conduit through which heme is passed across the cell wall and binds this molecule using a NEAr Transporter (NEAT) domain. NMR spectroscopy was used to determine the structure of IsdC in complex with a heme analog, zinc-substituted protoporphyrin IX (ZnPPIX). The backbone coordinates of the ensemble of conformers representing the structure exhibit a root mean square deviation to the mean structure of 0.53 +/- 0.11 angstroms. IsdC partially buries protoporphyrin within a large hydrophobic pocket that is located at the end of its beta-barrel structure. The central metal ion of the analog adopts a pentacoordinate geometry in which a highly conserved tyrosine residue serves as a proximal ligand. Consistent with the structure and its role in heme transfer across the cell wall, we show that IsdC weakly binds heme (K(D) = 0.34 +/- 0.12 microm) and that ZnPPIX rapidly dissociates from the protein at a rate of 126 +/- 30 s(-1). NMR studies of the apo-form of IsdC reveal that a 3(10) helix within the binding pocket undergoes a flexible to rigid transition as heme is captured. This structural plasticity may increase the efficiency of heme transfer across the cell wall by facilitating protein-protein interactions between apoIsdC and upstream hemoproteins.

Identification and solution structure of a highly conserved C-terminal domain within ORF1p required for retrotransposition of long interspersed nuclear element-1.

Long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons comprise a large fraction of the human and mouse genomes. The mobility of these successful elements requires the protein encoded by open reading frame-1 (ORF1p), which binds single-stranded RNA with high affinity and functions as a nucleic acid chaperone. In this report, we have used limited proteolysis, filter binding, and NMR spectroscopy to characterize the global structure of ORF1p and the three-dimensional structure of a highly conserved RNA binding domain. ORF1p contains three structured regions, a coiled-coil domain, a middle domain of unknown function, and a C-terminal domain (CTD). We show that high affinity RNA binding by ORF1p requires the CTD and residues within an amino acid protease-sensitive segment that joins the CTD to the middle domain. Insights in the mechanism of RNA binding were obtained by determining the solution structure of the CTD, which is shown to adopt a novel fold consisting of a three-stranded beta sheet that is packed against three alpha-helices. An RNA binding surface on the CTD has been localized using chemical shift perturbation experiments and is proximal to residues previously shown to be essential for retrotransposition, RNA binding, and chaperone activity. A similar structure and mechanism of RNA binding is expected for all vertebrate long interspersed nuclear element-1 elements, since residues encoding the middle, protease-sensitive segment, and CTD are highly conserved.

Solution structure of the NEAT (NEAr Transporter) domain from IsdH/HarA: the human hemoglobin receptor in Staphylococcus aureus.

During infections the pathogen Staphylococcus aureus procures the essential nutrient iron from its host using iron-regulated surface determinant (Isd) proteins, which scavenge heme bound iron from host hemoproteins. Four Isd proteins are displayed in the cell wall, where they function as receptors for host proteins and heme. Each of the receptors contains one or more copies of a recently discovered domain called NEAT (NEAr Transporter) that has been shown to mediate protein binding. Here we report the three-dimensional solution structure of the NEAT domain from the IsdH/HarA protein, which is the hemoglobin receptor in the Isd system. This is the first structure of a NEAT domain and reveals that they adopt a beta sandwich fold that consists of two five-stranded antiparallel beta sheets. Although unrelated at the primary sequence level, our results indicate that NEAT domains belong to the immunoglobulin superfamily. Binding studies indicate that two IsdH/HarA NEAT domains bind a single molecule of methemoglobin, while the distantly related NEAT domain from the S. aureus IsdC protein binds only heme. A comparison of their primary sequences in light of the new structure is used to predict the hemoglobin and heme binding surfaces on NEAT domains.

Affinity capillary electrophoresis to examine receptor-ligand interactions.

Afffinity capillary electrophoresis (ACE) is a new analytical technique that has been shown to be an efficient and accurate tool in studying biomolecular noncovalent interactions and determining binding and dissociation constants of formed complexes. ACE uses as its basis the change in migration time of a receptor upon binding to a ligand found in the electrophoresis buffer. Subsequent Scatchard analysis using noninteracting markers realizes a binding constant. Herein, ACE and three modifications in the technique, partial-filling ACE (PFACE), flow through PFACE (FTPFACE), and multiple-step ligand injection ACE (MSLIACE) are used to probe the binding of ristocetin A (Rist A) and vancomycin (Van) from Streptomyces orientalis to D-Ala-D-Ala terminus peptides and carbonic anhydrase B (CAB, E.C.4.2.1.1) to arylsulfonamides.

On-column synthesis coupled to affinity capillary electrophoresis for the determination of binding constants of peptides to glycopeptide antibiotics.

Binding constants of the glycopeptide antibiotics teicoplanin (Teic), ristocetin (Rist), and vancomycin (Van), and their derivatives to D-Ala-D-Ala terminus peptides were determined by on-column ligand and receptor synthesis coupled to affinity capillary electrophoresis (ACE) or partial filling ACE (PFACE). In the first technique, 9-fluorenylmethoxycarbonyl (Fmoc)-amino acid-D-Ala-D-Ala species are first synthesized using on-column techniques. The initial sample plug contains a D-Ala-D-Ala terminus peptide and two non-interacting standards. Plugs two and three contain solutions of Fmoc-amino acid-N-hydroxysuccinimide (NHS) ester and buffer, respectively. Upon electrophoresis, the initial D-Ala-D-Ala peptide reacts with the Fmoc-amino acid NHS ester yielding the Fmoc-amino acid D-Ala-D-Ala peptide. Continued electrophoresis results in the overlap of the glycopeptide in the running buffer and the plug of Fmoc-amino acid-D-Ala-D-Ala peptide and non-interacting markers. Subsequent analysis of the change in the electrophoretic mobility (mu) or relative migration time ratio (RMTR) of the peptide relative to the non-interacting standards, as a function of the concentration of the antibiotic, yields a value for the binding constant. In the second technique, derivatives of the glycopeptides Teic and Rist are first synthesized on-column before analysis by ACE or PFACE. After the column has been partially filled with increasing concentrations of D-Ala-D-Ala terminus peptides, a plug of buffer followed by two separate plugs of reagents are injected. The order of the reagent plugs containing the antibiotic and two non-interacting standards and the anhydride varies with the charge of the glycopeptide. Upon electrophoresis, the antibiotic reacts with the anhydride yielding a derivative of Teic or Rist. Continued electrophoresis results in the overlap of the derivatized antibiotic and the plug of D-Ala-D-Ala peptide. Analysis of the change in RMTR of the new glycopeptide relative to the non-interacting standards, as a function of the concentration of the D-Ala-D-Ala ligand yields a value for the binding constant.

Partial-filling affinity capillary electrophoresis.

Partial-filling affinity capillary electrophoresis (PFACE) is used to examine the binding interactions between two model biological systems: D-Ala-D-Ala terminus peptides to the glycopeptide antibiotic vancomycin (Van) from Streptomyces orientalis, and arylsulfonamides to carbonic anhydrase B (CAB, EC 4.2.1.1, bovine erythrocytes). Using these two systems, modifications in the PFACE technique are demonstrated including flow-through PFACE (FTPFACE), competitive flow-through PFACE (CFTPFACE), on-column ligand synthesis PFACE (OCLSPFACE), and multiple-step ligand injection PFACE (MSLIPFACE). In PFACE small plugs of sample are injected into the capillary column and an equilibrium is established between receptor and ligand during electrophoresis. Binding constants are then obtained by Scatchard analysis using changes in the migration time of the receptor/ligand on changing the concentration of the ligand/receptor. Data demonstrating the quantitative potential of these methods are presented. This review focuses on the unique capabilities of the different PFACE techniques as applied to two model biological systems.