15-deoxy-Delta(12,14)-Prostaglandin J(2) inhibits human soluble epoxide hydrolase by a dual orthosteric and allosteric mechanism

Human soluble epoxide hydrolase (hsEH) is an enzyme responsible for the inactivation of bioactive epoxy fatty acids, and its inhibition is emerging as a promising therapeutical strategy to target hypertension, cardiovascular disease, pain and insulin sensitivity. Here, we uncover the molecular bases of hsEH inhibition mediated by the endogenous 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2). Our data reveal a dual inhibitory mechanism, whereby hsEH can be inhibited by reversible docking of 15d-PGJ2 in the catalytic pocket, as well as by covalent locking of the same compound onto cysteine residues C423 and C522, remote to the active site. Biophysical characterisations allied with in silico investigations indicate that the covalent modification of the reactive cysteines may be part of a hitherto undiscovered allosteric regulatory mechanism of the enzyme. This study provides insights into the molecular modes of inhibition of hsEH epoxy-hydrolytic activity and paves the way for the development of new allosteric inhibitors

Bi-allelic Variants in TKFC Encoding Triokinase/FMN Cyclase Are Associated with Cataracts and Multisystem Disease

We report an inborn error of metabolism caused by TKFC deficiency in two unrelated families. Rapid trio genome sequencing in family 1 and exome sequencing in family 2 excluded known genetic etiologies, and further variant analysis identified rare homozygous variants in TKFC. TKFC encodes a bifunctional enzyme involved in fructose metabolism through its glyceraldehyde kinase activity and in the generation of riboflavin cyclic 4′,5′-phosphate (cyclic FMN) through an FMN lyase domain. The TKFC homozygous variants reported here are located within the FMN lyase domain. Functional assays in yeast support the deleterious effect of these variants on protein function. Shared phenotypes between affected individuals with TKFC deficiency include cataracts and developmental delay, associated with cerebellar hypoplasia in one case. Further complications observed in two affected individuals included liver dysfunction and microcytic anemia, while one had fatal cardiomyopathy with lactic acidosis following a febrile illness. We postulate that deficiency of TKFC causes disruption of endogenous fructose metabolism leading to generation of by-products that can cause cataract. In line with this, an affected individual had mildly elevated urinary galactitol, which has been linked to cataract development in the galactosemias. Further, in light of a previously reported role of TKFC in regulating innate antiviral immunity through suppression of MDA5, we speculate that deficiency of TKFC leads to impaired innate immunity in response to viral illness, which may explain the fatal illness observed in the most severely affected individual

Unscented Kalman filter with parameter identifiability analysis for the estimation of multiple parameters in kinetic models

In systems biology, experimentally measured parameters are not always available, necessitating the use of computationally based parameter estimation. In order to rely on estimated parameters, it is critical to first determine which parameters can be estimated for a given model and measurement set. This is done with parameter identifiability analysis. A kinetic model of the sucrose accumulation in the sugar cane culm tissue developed by Rohwer et al. was taken as a test case model. What differentiates this approach is the integration of an orthogonal-based local identifiability method into the unscented Kalman filter (UKF), rather than using the more common observability-based method which has inherent limitations. It also introduces a variable step size based on the system uncertainty of the UKF during the sensitivity calculation. This method identified 10 out of 12 parameters as identifiable. These ten parameters were estimated using the UKF, which was run 97 times. Throughout the repetitions the UKF proved to be more consistent than the estimation algorithms used for comparison

Transmission of Aerosolized Seasonal H1N1 Influenza A to Ferrets

Influenza virus is a major cause of morbidity and mortality worldwide, yet little quantitative understanding of transmission is available to guide evidence-based public health practice. Recent studies of influenza non-contact transmission between ferrets and guinea pigs have provided insights into the relative transmission efficiencies of pandemic and seasonal strains, but the infecting dose and subsequent contagion has not been quantified for most strains. In order to measure the aerosol infectious dose for 50% (aID50) of seronegative ferrets, seasonal influenza virus was nebulized into an exposure chamber with controlled airflow limiting inhalation to airborne particles less than 5 µm diameter. Airborne virus was collected by liquid impinger and Teflon filters during nebulization of varying doses of aerosolized virus. Since culturable virus was accurately captured on filters only up to 20 minutes, airborne viral RNA collected during 1-hour exposures was quantified by two assays, a high-throughput RT-PCR/mass spectrometry assay detecting 6 genome segments (Ibis T5000™ Biosensor system) and a standard real time RT-qPCR assay. Using the more sensitive T5000 assay, the aID50 for A/New Caledonia/20/99 (H1N1) was approximately 4 infectious virus particles under the exposure conditions used. Although seroconversion and sustained levels of viral RNA in upper airway secretions suggested established mucosal infection, viral cultures were almost always negative. Thus after inhalation, this seasonal H1N1 virus may replicate less efficiently than H3N2 virus after mucosal deposition and exhibit less contagion after aerosol exposure

ARHGEF7 (BETA-PIX) Acts as Guanine Nucleotide Exchange Factor for Leucine-Rich Repeat Kinase 2

Background: Mutations within the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of familial and sporadic Parkinson’s disease. The multidomain protein LRRK2 exhibits overall low GTPase and kinase activity in vitro. Methodology/Principal Findings: Here, we show that the rho guanine nucleotide exchange factor ARHGEF7 and the small GTPase CDC42 are interacting with LRRK2 in vitro and in vivo. GTPase activity of full-length LRRK2 increases in the presence of recombinant ARHGEF7. Interestingly, LRRK2 phosphorylates ARHGEF7 in vitro at previously unknown phosphorylation sites. We provide evidence that ARHGEF7 might act as a guanine nucleotide exchange factor for LRRK2 and that R1441C mutant LRRK2 with reduced GTP hydrolysis activity also shows reduced binding to ARHGEF7. Conclusions/Significance: Downstream effects of phosphorylation of ARHGEF7 through LRRK2 could be (i) a feedback control mechanism for LRRK2 activity as well as (ii) an impact of LRRK2 on actin cytoskeleton regulation. A newly identified familial mutation N1437S, localized within the GTPase domain of LRRK2, further underlines the importance of the GTPas

PRNP Haplotype Associated with Classical BSE Incidence in European Holstein Cattle

Background: Classical bovine spongiform encephalopathy (BSE) is an acquired prion disease of cattle. The bovine prion gene (PRNP) contains regions of both high and low linkage disequilibrium (LD) that appear to be conserved across Bos taurus populations. The region of high LD, which spans the promoter and part of intron 2, contains polymorphic loci that have been associated with classical BSE status. However, the complex genetic architecture of PRNP has not been systematically tested for an association with classical BSE. Methodology/Principal Findings: In this study, haplotype tagging single nucleotide polymorphisms (htSNPs) within PRNP were used to test for association between PRNP haplotypes and BSE disease. A combination of Illumina goldengate assay, sequencing and PCR amplification was used to genotype 18 htSNPs and 2 indels in 95 BSE case and 134 control animals. A haplotype within the region of high LD was found to be associated with BSE unaffected animals (p-value = 0.000114). Conclusion/Significance: A PRNP haplotype association with classical BSE incidence has been identified. This result suggests that a genetic determinant in or near PRNP may influence classical BSE incidence in cattle

Inhibition of Post-Synaptic Kv7/KCNQ/M Channels Facilitates Long-Term Potentiation in the Hippocampus

Activation of muscarinic acetylcholine receptors (mAChR) facilitates the induction of synaptic plasticity and enhances cognitive function. In the hippocampus, M1 mAChR on CA1 pyramidal cells inhibit both small conductance Ca2+-activated KCa2 potassium channels and voltage-activated Kv7 potassium channels. Inhibition of KCa2 channels facilitates long-term potentiation (LTP) by enhancing Ca2+calcium influx through postsynaptic NMDA receptors (NMDAR). Inhibition of Kv7 channels is also reported to facilitate LTP but the mechanism of action is unclear. Here, we show that inhibition of Kv7 channels with XE-991 facilitated LTP induced by theta burst pairing at Schaffer collateral commissural synapses in rat hippocampal slices. Similarly, negating Kv7 channel conductance using dynamic clamp methodologies also facilitated LTP. Negation of Kv7 channels by XE-991 or dynamic clamp did not enhance synaptic NMDAR activation in response to theta burst synaptic stimulation. Instead, Kv7 channel inhibition increased the amplitude and duration of the after-depolarisation following a burst of action potentials. Furthermore, the effects of XE-991 were reversed by re-introducing a Kv7-like conductance with dynamic clamp. These data reveal that Kv7 channel inhibition promotes NMDAR opening during LTP induction by enhancing depolarisation during and after bursts of postsynaptic action potentials. Thus, during the induction of LTP M1 mAChRs enhance NMDAR opening by two distinct mechanisms namely inhibition of KCa2 and Kv7 channels

Propagation of kinetic uncertainties through a canonical topology of the TLR4 signaling network in different regions of biochemical reaction space

<p>Abstract</p> <p>Background</p> <p>Signal transduction networks represent the information processing systems that dictate which dynamical regimes of biochemical activity can be accessible to a cell under certain circumstances. One of the major concerns in molecular systems biology is centered on the elucidation of the robustness properties and information processing capabilities of signal transduction networks. Achieving this goal requires the establishment of causal relations between the design principle of biochemical reaction systems and their emergent dynamical behaviors.</p> <p>Methods</p> <p>In this study, efforts were focused in the construction of a relatively well informed, deterministic, non-linear dynamic model, accounting for reaction mechanisms grounded on standard mass action and Hill saturation kinetics, of the canonical reaction topology underlying Toll-like receptor 4 (TLR4)-mediated signaling events. This signaling mechanism has been shown to be deployed in macrophages during a relatively short time window in response to lypopolysaccharyde (LPS) stimulation, which leads to a rapidly mounted innate immune response. An extensive computational exploration of the biochemical reaction space inhabited by this signal transduction network was performed via local and global perturbation strategies. Importantly, a broad spectrum of biologically plausible dynamical regimes accessible to the network in widely scattered regions of parameter space was reconstructed computationally. Additionally, experimentally reported transcriptional readouts of target pro-inflammatory genes, which are actively modulated by the network in response to LPS stimulation, were also simulated. This was done with the main goal of carrying out an unbiased statistical assessment of the intrinsic robustness properties of this canonical reaction topology.</p> <p>Results</p> <p>Our simulation results provide convincing numerical evidence supporting the idea that a canonical reaction mechanism of the TLR4 signaling network is capable of performing information processing in a robust manner, a functional property that is independent of the signaling task required to be executed. Nevertheless, it was found that the robust performance of the network is not solely determined by its design principle (topology), but this may be heavily dependent on the network's current position in biochemical reaction space. Ultimately, our results enabled us the identification of key rate limiting steps which most effectively control the performance of the system under diverse dynamical regimes.</p> <p>Conclusions</p> <p>Overall, our <it>in silico </it>study suggests that biologically relevant and non-intuitive aspects on the general behavior of a complex biomolecular network can be elucidated only when taking into account a wide spectrum of dynamical regimes attainable by the system. Most importantly, this strategy provides the means for a suitable assessment of the inherent variational constraints imposed by the structure of the system when systematically probing its parameter space.</p

A Yersinia Effector with Enhanced Inhibitory Activity on the NF-κB Pathway Activates the NLRP3/ASC/Caspase-1 Inflammasome in Macrophages

A type III secretion system (T3SS) in pathogenic Yersinia species functions to translocate Yop effectors, which modulate cytokine production and regulate cell death in macrophages. Distinct pathways of T3SS-dependent cell death and caspase-1 activation occur in Yersinia-infected macrophages. One pathway of cell death and caspase-1 activation in macrophages requires the effector YopJ. YopJ is an acetyltransferase that inactivates MAPK kinases and IKKβ to cause TLR4-dependent apoptosis in naïve macrophages. A YopJ isoform in Y. pestis KIM (YopJKIM) has two amino acid substitutions, F177L and K206E, not present in YopJ proteins of Y. pseudotuberculosis and Y. pestis CO92. As compared to other YopJ isoforms, YopJKIM causes increased apoptosis, caspase-1 activation, and secretion of IL-1β in Yersinia-infected macrophages. The molecular basis for increased apoptosis and activation of caspase-1 by YopJKIM in Yersinia-infected macrophages was studied. Site directed mutagenesis showed that the F177L and K206E substitutions in YopJKIM were important for enhanced apoptosis, caspase-1 activation, and IL-1β secretion. As compared to YopJCO92, YopJKIM displayed an enhanced capacity to inhibit phosphorylation of IκB-α in macrophages and to bind IKKβ in vitro. YopJKIM also showed a moderately increased ability to inhibit phosphorylation of MAPKs. Increased caspase-1 cleavage and IL-1β secretion occurred in IKKβ-deficient macrophages infected with Y. pestis expressing YopJCO92, confirming that the NF-κB pathway can negatively regulate inflammasome activation. K+ efflux, NLRP3 and ASC were important for secretion of IL-1β in response to Y. pestis KIM infection as shown using macrophages lacking inflammasome components or by the addition of exogenous KCl. These data show that caspase-1 is activated in naïve macrophages in response to infection with a pathogen that inhibits IKKβ and MAPK kinases and induces TLR4-dependent apoptosis. This pro-inflammatory form of apoptosis may represent an early innate immune response to highly virulent pathogens such as Y. pestis KIM that have evolved an enhanced ability to inhibit host signaling pathways