Developmental and reproductive safety evaluation of AV7909 anthrax vaccine candidate in rats.

The AV7909 vaccine, consists of the Anthrax Vaccine Adsorbed (AVA) bulk drug substance and the immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide adjuvant CPG 7909. The purpose of this research was to evaluate the potential maternal, reproductive, and developmental toxicity of AV7909 in rats to support licensure for use in women of childbearing potential. Groups of first generation (F0 ) female Sprague Dawley rats were dosed by intramuscular injection with water for injection, adjuvant or AV7909 at a volume of 0.5 ml/dose. Each rat received three vaccinations: 14 days prior to start of the mating period, on the first day of the mating period and on gestation day (GD) 7. There was no maternal mortality. Body weights, weight gain, and food consumption were comparable between groups. Findings in F0 females were limited to transient injection site edema and nodules consistent with immunostimulatory effects of the vaccine and adjuvant. Administration of AV7909 did not affect mating, fertility, pregnancy, embryo-fetal viability, growth, or morphologic development, parturition, maternal care of offspring or postnatal survival, growth, or development. There was no evidence of systemic inflammation in pregnant rats, based on evaluation of serum concentrations of the acute phase proteins alpha-2-macroglobulin and alpha-1-acid glycoprotein on GD 21. Anthrax lethal toxin-neutralizing antibodies were detected in AV7909-vaccinated F0 females. The antibodies were also detected in the sera of fetuses and F1 pups. Exposure of the fetuses and pups to maternally derived anthrax lethal toxin-neutralizing antibodies was not associated with developmental toxicity.

Repeat Dose Toxicity Study of the AV7909 Anthrax Vaccine Candidate in Juvenile Rats.

AV7909 is a next-generation anthrax vaccine candidate indicated for post-exposure prophylaxis of exposure to Bacillus anthracis. AV7909 consists of the Anthrax Vaccine Adsorbed (AVA) bulk drug substance and the immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide adjuvant, CPG 7909. Safety testing for pediatric population is warranted to support the potential emergency use of AV7909 in children. This study was conducted to investigate the local tolerance and potential systemic toxicity and their reversibility in juvenile rats by repeat intramuscular injections of the AV7909 vaccine candidate. Animals were dosed on postnatal day (PND) 21 (at weaning), PND 28, and PND 35, with the test article (AV7909), the adjuvant alone (Alhydrogel + CPG 7909), or sterile water for injection. Core group animals were necropsied on PND 37 and recovery group on PND 49. Study end points included survival, clinical observations, injection site observations, body weights, clinical pathology (hematology, coagulation, and clinical chemistry), pro-inflammatory biomarker analysis (alpha-2 macroglobulin [A2M] and alpha-1 acid glycoprotein [AGP]), and anatomic pathology. Immune response to vaccination was measured using the high-throughput anthrax lethal toxin neutralization assay (htpTNA). The AV7909 vaccine candidate produced no apparent systemic or local toxicity. The AGP and A2M levels were elevated in both the adjuvant-alone and AV7909 groups at the end of treatment but were comparable to control levels by the end of the recovery period. All animals in the AV7909 group demonstrated a robust neutralizing antibody response. The results indicate that AV7909 has a favorable safety profile in juvenile rats.

Coxiella burnetii serology assays in goat abortion storm.

Many commercial antibody detection enzyme-linked immunosorbent assay (ELISA) kits for Q fever utilize the Nine Mile (Montana tick) strain of Coxiella burnetii as antigen. An ELISA kit manufactured in France employs ovine placenta-sourced antigen and has been used in Europe. Sera from goats experiencing a Q fever abortion storm in the United States were used to compare the sensitivity and specificity of these 2 ELISA formats and the Q fever complement fixation test (CFT). Latent class estimates of sensitivity ranged from 97% to 100% with a specificity of 95-100% for the 2 ELISA kits. Estimates for sensitivity and specificity of the CFT were 89% and 82%, respectively. There was not a significant increase in ELISA sensitivity observed with the ovine-sourced antigen kit in this study. Real-time polymerase chain reactions performed on a portion of the sera found that 15 out of 20 sera were congruent across 4 tests for positive and negative sera.

HNO enhances SERCA2a activity and cardiomyocyte function by promoting redox-dependent phospholamban oligomerization.

AIMS: Nitroxyl (HNO) interacts with thiols to act as a redox-sensitive modulator of protein function. It enhances sarcoplasmic reticular Ca(2+) uptake and myofilament Ca(2+) sensitivity, improving cardiac contractility. This activity has led to clinical testing of HNO donors for heart failure. Here we tested whether HNO alters the inhibitory interaction between phospholamban (PLN) and the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) in a redox-dependent manner, improving Ca(2+) handling in isolated myocytes/hearts. RESULTS: Ventriculocytes, sarcoplasmic reticulum (SR) vesicles, and whole hearts were isolated from control (wildtype [WT]) or PLN knockout (pln(-/-)) mice. Compared to WT, pln(-/-) myocytes displayed enhanced resting sarcomere shortening, peak Ca(2+) transient, and blunted ß-adrenergic responsiveness. HNO stimulated shortening, relaxation, and Ca(2+) transient in WT cardiomyocytes, and evoked positive inotropy/lusitropy in intact hearts. These changes were markedly blunted in pln(-/-) cells/hearts. HNO enhanced SR Ca(2+) uptake in WT but not pln(-/-) SR-vesicles. Spectroscopic studies in insect cell microsomes expressing SERCA2a±PLN showed that HNO increased Ca(2+)-dependent SERCA2a conformational flexibility but only when PLN was present. In cardiomyocytes, HNO achieved this effect by stabilizing PLN in an oligomeric disulfide bond-dependent configuration, decreasing the amount of free inhibitory monomeric PLN available. INNOVATION: HNO-dependent redox changes in myocyte PLN oligomerization relieve PLN inhibition of SERCA2a. CONCLUSIONS: PLN plays a central role in HNO-induced enhancement of SERCA2a activity, leading to increased inotropy/lusitropy in intact myocytes and hearts. PLN remains physically associated with SERCA2a; however, less monomeric PLN is available resulting in decreased inhibition of the enzyme. These findings offer new avenues to improve Ca(2+) handling in failing hearts.

Unique properties of the Mtr4p-poly(A) complex suggest a role in substrate targeting.

Mtr4p is a DEVH-box helicase required for 3'-end processing and degradation of various nuclear RNA substrates. In particular, Mtr4p is essential for the creation of 5.8S rRNA, U4 snRNA, and some snoRNAs and for the degradation of cryptic unstable transcripts (CUTs), aberrant mRNAs, and aberrant tRNAs. Many instances of 3'-end processing require limited polyadenylation to proceed. While polyadenylation can signal degradation in species from bacteria to humans, the mechanism whereby polyadenylated substrates are delivered to the degradation machinery is unknown. Our previous work has shown that Mtr4p preferentially binds poly(A) RNA. We suspect that this preference aids in targeting polyadenylated RNAs to the exosome. In these studies, we have investigated the mechanism underlying the preference of Mtr4p for poly(A) substrates as a means of understanding how Mtr4p might facilitate targeting. Our analysis has revealed that recognition of poly(A) substrates involves sequence-specific changes in the architecture of Mtr4p-RNA complexes. Furthermore, these differences significantly affect downstream activities. In particular, homopolymeric stretches like poly(A) ineffectively stimulate the ATPase activity of Mtr4p and suppress the rate of dissociation of the Mtr4p-RNA complex. These findings indicate that the Mtr4p-poly(A) complex is unique and ideally suited for targeting key substrates to the exosome.

Alternatively expressed domains of AU-rich element RNA-binding protein 1 (AUF1) regulate RNA-binding affinity, RNA-induced protein oligomerization, and the local conformation of bound RNA ligands.

AU-rich element RNA-binding protein 1 (AUF1) binding to AU-rich elements (AREs) in the 3'-untranslated regions of mRNAs encoding many cytokines and other regulatory proteins modulates mRNA stability, thereby influencing protein expression. AUF1-mRNA association is a dynamic paradigm directed by various cellular signals, but many features of its function remain poorly described. There are four isoforms of AUF1 that result from alternative splicing of exons 2 and 7 from a common pre-mRNA. Preliminary evidence suggests that the different isoforms have varied functional characteristics, but no detailed quantitative analysis of the properties of each isoform has been reported despite their differential expression and regulation. Using purified recombinant forms of each AUF1 protein variant, we used chemical cross-linking and gel filtration chromatography to show that each exists as a dimer in solution. We then defined the association mechanisms of each AUF1 isoform for ARE-containing RNA substrates and quantified relevant binding affinities using electrophoretic mobility shift and fluorescence anisotropy assays. Although all AUF1 isoforms generated oligomeric complexes on ARE substrates by sequential dimer association, sequences encoded by exon 2 inhibited RNA-binding affinity. By contrast, the exon 7-encoded domain enhanced RNA-dependent protein oligomerization, even permitting cooperative RNA-binding activity in some contexts. Finally, fluorescence resonance energy transfer-based assays showed that the different AUF1 isoforms remodel bound RNA substrates into divergent structures as a function of protein:RNA stoichiometry. Together, these data describe isoform-specific characteristics among AUF1 ribonucleoprotein complexes, which likely constitute a mechanistic basis for differential functions and regulation among members of this protein family.

Contributions of the histidine side chain and the N-terminal alpha-amino group to the binding thermodynamics of oligopeptides to nucleic acids as a function of pH.

Interactions of histidine with nucleic acid phosphates and histidine pK(a) shifts make important contributions to many protein-nucleic acid binding processes. To characterize these phenomena in simplified systems, we quantified binding of a histidine-containing model peptide HWKK ((+)NH(3)-His-Trp-Lys-Lys-NH(2)) and its lysine analogue KWKK ((+)NH(3)-Lys-Trp-Lys-Lys-NH(2)) to a single-stranded RNA model, polyuridylate (polyU), by changes in tryptophan fluorescence as a function of salt concentration and pH. For both HWKK and KWKK, equilibrium binding constants, K(obs), and magnitudes of log-log salt derivatives, SK(obs) identical with (partial differential logK(obs)/partial differential log[Na(+)]), decreased with increasing pH in the manner expected for a titration curve model in which deprotonation of the histidine and alpha-amino groups weakens binding and reduces its salt-dependence. Fully protonated HWKK and KWKK exhibit the same K(obs) and SK(obs) within uncertainty, and these SK(obs) values are consistent with limiting-law polyelectrolyte theory for +4 cationic oligopeptides binding to single-stranded nucleic acids. The pH-dependence of HWKK binding to polyU provides no evidence for pK(a) shifts nor any requirement for histidine protonation, in stark contrast to the thermodynamics of coupled protonation often seen for these cationic residues in the context of native protein structure where histidine protonation satisfies specific interactions (e.g., salt-bridge formation) within highly complementary binding interfaces. The absence of pK(a) shifts in our studies indicates that additional Coulombic interactions across the nonspecific-binding interface between RNA and protonated histidine or the alpha-amino group are not sufficient to promote proton uptake for these oligopeptides. We present our findings in the context of hydration models for specific vs nonspecific nucleic acid binding.

Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair.

The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIIIbeta and the hLigIIIalpha/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/XRCC4 molecules.

Identification and validation of human DNA ligase inhibitors using computer-aided drug design.

Linking together of DNA strands by DNA ligases is essential for DNA replication and repair. Since many therapies used to treat cancer act by causing DNA damage, there is growing interest in the development of DNA repair inhibitors. Accordingly, virtual database screening and experimental evaluation were applied to identify inhibitors of human DNA ligase I (hLigI). When a DNA binding site within the DNA binding domain (DBD) of hLigI was targeted, more than 1 million compounds were screened from which 192 were chosen for experimental evaluation. In DNA joining assays, 10 compounds specifically inhibited hLigI, 5 of which also inhibited the proliferation of cultured human cell lines. Analysis of the 10 active compounds revealed the utility of including multiple protein conformations and chemical clustering in the virtual screening procedure. The identified ligase inhibitors are structurally diverse and have druglike physical and molecular characteristics making them ideal for further drug development studies.

Local RNA conformational dynamics revealed by 2-aminopurine solvent accessibility.

Acrylamide quenching is widely used to monitor the solvent exposure of fluorescent probes in vitro. Here, we tested the utility of this technique to discriminate local RNA secondary structures using the fluorescent adenine analogue 2-aminopurine (2-AP). Under native conditions, the solvent accessibilities of most 2-AP-labeled RNA substrates were poorly resolved by classical single-population models; rather, a two-state quencher accessibility algorithm was required to model acrylamide-dependent changes in 2-AP fluorescence in structured RNA contexts. Comparing 2-AP quenching parameters between structured and unstructured RNA substrates permitted the effects of local RNA structure on 2-AP solvent exposure to be distinguished from nearest neighbor effects or environmental influences on intrinsic 2-AP photophysics. Using this strategy, the fractional accessibility of 2-AP for acrylamide ( f a) was found to be highly sensitive to local RNA structure. Base-paired 2-AP exhibited relatively poor accessibility, consistent with extensive shielding by adjacent bases. 2-AP in a single-base bulge was uniformly accessible to solvent, whereas the fractional accessibility of 2-AP in a hexanucleotide loop was indistinguishable from that of an unstructured RNA. However, these studies also provided evidence that the f a parameter reflects local conformational dynamics in base-paired RNA. Enhanced base pair dynamics at elevated temperatures were accompanied by increased f a values, while restricting local RNA breathing by adding a C-G base pair clamp or positioning 2-AP within extended RNA duplexes significantly decreased this parameter. Together, these studies show that 2-AP quenching studies can reveal local RNA structural and dynamic features beyond those that can be measured by conventional spectroscopic approaches.

Fluorescence intensity decays of 2-aminopurine solutions: lifetime distribution approach.

The fluorescent adenine analog 2-aminopurine (2AP) has been used extensively to monitor conformational changes and macromolecular binding events involving nucleic acids because its fluorescence properties are highly sensitive to changes in chemical environment. Furthermore, site-specific incorporation of 2AP permits local DNA and RNA conformational events to be discriminated from the global structural changes monitored by UV-Vis spectroscopy and circular dichroism. However, although the steady-state fluorescence properties of 2AP have been well defined in diverse settings, interpretation of 2AP fluorescence lifetime parameters has been hampered by the heterogeneous nature of multiexponential 2AP intensity decays observed across populations of microenvironments. To resolve this problem, we tested the utility of multiexponential versus continuous Lorentzian lifetime distribution models to describe fluorescence intensity decays from 2AP in diverse chemical backgrounds and within the context of RNA. Heterogeneity was introduced into 2AP intensity decays by mixing solvents of differing polarities or by adding quenchers under high viscosity to evaluate the transient effect. Heterogeneity of 2AP fluorescence within the context of a synthetic RNA hairpin was introduced by structural remodeling using a magnesium salt. In each case except folded RNA (which required a bimodal distribution), 2AP lifetime properties were well described by single Lorentzian distribution functions, abrogating the need to introduce additional discrete lifetime subpopulations. Rather, heterogeneity in fluorescence decay processes was accommodated by the breadth of each distribution. This approach also permitted solvent relaxation effects on 2AP emission to be assessed by comparing lifetime distributions at multiple wavelengths. Together, these studies provide a new perspective for the interpretation of 2AP fluorescence lifetime properties that will further the utility of this fluorophore in analyses of the complex and heterogeneous structural microenvironments associated with nucleic acids.

Site-specific variations in RNA folding thermodynamics visualized by 2-aminopurine fluorescence.

The fluorescent base analogue 2-aminopurine (2-AP) is commonly used to study specific conformational and protein binding events involving nucleic acids. Here, combinations of steady-state and time-resolved fluorescence spectroscopy of 2-AP were employed to monitor conformational transitions within a model hairpin RNA from diverse structural perspectives. RNA substrates adopting stable, unambiguous secondary structures were labeled with 2-AP at an unpaired base, within the loop, or inside the base-paired stem. Steady-state fluorescence was monitored as the RNA hairpins made the transitions between folded and unfolded conformations using thermal denaturation, urea titration, and cation-mediated folding. Unstructured control RNA substrates permitted the effects of higher-order RNA structures on 2-AP fluorescence to be distinguished from stimulus-dependent changes in intrinsic 2-AP photophysics and/or interactions with adjacent residues. Thermodynamic parameters describing local conformational changes were thus resolved from multiple perspectives within the model RNA hairpin. These data provided energetic bases for construction of folding mechanisms, which varied among different folding-unfolding stimuli. Time-resolved fluorescence studies further revealed that 2-AP exhibits characteristic signatures of component fluorescence lifetimes and respective fractional contributions in different RNA structural contexts. Together, these studies demonstrate localized conformational events contributing to RNA folding and unfolding that could not be observed by approaches monitoring only global structural transitions.

Specific protein domains mediate cooperative assembly of HuR oligomers on AU-rich mRNA-destabilizing sequences.

The RNA-binding factor HuR is a ubiquitously expressed member of the Hu protein family that binds and stabilizes mRNAs containing AU-rich elements (AREs). Hu proteins share a common domain organization of two tandemly arrayed RNA recognition motifs (RRMs) near the N terminus, followed by a basic hinge domain and a third RRM near the C terminus. In this study, we engineered recombinant wild-type and mutant HuR proteins lacking affinity tags to characterize their ARE-binding properties. Using combinations of electrophoretic mobility shift and fluorescence anisotropy-based binding assays, we show that HuR can bind ARE substrates as small as 13 nucleotides with low nanomolar affinity, but forms cooperative oligomeric protein complexes on ARE substrates of at least 18 nucleotides in length. Analyses of deletion mutant proteins indicated that RRM3 does not contribute to high affinity recognition of ARE substrates, but is required for cooperative assembly of HuR oligomers on RNA. Finally, the hinge domain between RRM2 and RRM3 contributes significant binding energy to HuR.ARE complex formation in an ARE length-dependent manner. The hinge does not enhance RNA-binding activity by increased ion pair formation despite extensive positive charge within this region, and it does not thermodynamically stabilize protein folding. Together, the results define distinct roles for the HuR hinge and RRM3 domains in formation of cooperative HuR.ARE complexes in solution.

Substrate dependence of conformational changes in the RNA-binding domain of tristetraprolin assessed by fluorescence spectroscopy of tryptophan mutants.

Association of tristetraprolin (TTP) with mRNAs containing selected AU-rich mRNA-destabilizing elements (AREs) initiates rapid cytoplasmic degradation of these transcripts. The RNA-binding activity of TTP is mediated by an internal tandem zinc finger domain that preferentially recognizes U-rich RNA ligands containing adjacent UUAU half-sites and is accompanied by conformational changes within the peptide. Here, we have used analogues of the TTP RNA-binding domain containing specific tryptophan substitutions to probe the Zn2+ and RNA substrate dependence of conformational events within individual zinc fingers. Fluorescence methods demonstrate that the N-terminal, but not C-terminal, zinc finger domain adopts a stably folded conformation in the presence of Zn2+. Denaturant titrations suggest that both the N- and C-terminal zinc fingers exhibit limited structural heterogeneity in the absence of RNA substrates, although this is more pronounced for the C-terminal finger. Binding to a cognate ARE substrate induced significant conformational changes within each zinc finger, which also included increased resistance to chemical denaturation. Studies with mutant ARE ligands revealed that a single UUAU half-site was sufficient to induce structural modulation of the N-terminal finger. However, RNA-dependent folding of the C-terminal zinc finger was only observed in the presence of tandem UUAU half-sites, suggesting that the conformation of this domain is linked not only to RNA substrate recognition but also to the ligand occupancy and/or conformational status of the N-terminal finger. Coupled with previous structural and thermodynamic analyses, these data provide a mechanistic framework for discrimination of RNA substrates involving ligand-dependent conformational adaptation of both zinc fingers within the TTP RNA-binding domain.

Interactions of cationic ligands and proteins with small nucleic acids: analytic treatment of the large coulombic end effect on binding free energy as a function of salt concentration.

For nonspecific binding of oligopeptides and other cationic ligands, including proteins, to nucleic acid oligomers, we develop a model capable of quantifying and predicting the salt concentration dependence of the binding free energy (deltaG(o)obs) by way of an analytic treatment of the Coulombic end effect (CEE). Ligands, nucleic acids, and their complexes (species j of valence Zj) are modeled as finite lattices with absolute value(Zj) charged residues; the CEE is quantified by its characteristic length Ne (specified in charged residues) and its consequences for the free energy and ion association of the oligomer. Expressions are developed for the individual site binding constants Ki as a function of position (site number i) of a bound ligand on a nucleic acid and for the observed binding constant Kobs as an ensemble average of Ki. Analysis of deltaG(o)obs = -RT ln Kobs and Sa Kobs identical with (partial differential ln Kobs)/(partial differential ln a(+/-)) for binding of the oligopeptide KWK6 (ZL = +8) to single-stranded (ss) dT(pdT)(absolute value(ZD) oligomers (dT-mers) where ZD = {-6, -10, -11, -14, -15} in the range 0.1-0.25 M Na+ yields Ne = 9.0 +/- 0.8 residues at each end, demonstrating that both KWK6 and the above dT-mers are sufficiently short so that the CEE extends over the entire molecule. The dependences of Kobs and of Sa Kobs on absolute value(ZD) for a given ZL are determined by the difference between 2Ne and the net number of charged residues Q in the complex (Q identical with absolute value(ZD) - ZL). For Q < 2Ne, characteristic of complexes of KWK6 with this set of dT-mers, the distribution of binding free energies deltaG(o)obs = -RT ln Ki for sites along the DNA oligomer is parabolic, and Kobs and Sa Kobs are strongly dependent on absolute value(ZD). For Q > or = 2Ne, the distribution of binding free energies deltaG(o)obs is trapezoidal, and the dependence of Kobs and Sa Kobs on absolute value(ZD) is weaker. Application of the model to nonspecific binding of human DNA polymerase beta to ssDNA demonstrates the significance of the CEE in determining Kobs and Sa Kobs of binding of a cationic site on a protein to a DNA oligomer.