Upstream Interactions Research Articles

Integrating Proteomics and Transcriptomics Reveals the Potential Pathways of Hippocampal Neuron Apoptosis in Dravet Syndrome Model Mice.

An important component contributing to the onset of epilepsy is the death of hippocampal neurons. Several studies have shown that Dravet syndrome model mice: Scn1a KO mice have a high number of apoptotic neurons following seizures, but the precise mechanism underlying this remains unclear. The aim of this research was to elucidate the potential molecular mechanism of neuronal apoptosis in Scn1a KO mice by integrating proteomics and transcriptomics, with the ultimate goal of offering better neuroprotection. We found that apoptotic processes were enriched in both proteomic and transcriptomic GO analyses, and KEGG results also indicated that differential proteins and genes play a role in neurotransmission, the cell cycle, apoptosis, and neuroinflammation. Then, we examined the upstream and downstream KGML interactions of the pathways to determine the relationship between the two omics, and we found that the HIF-1 signaling pathway plays a significant role in the onset and apoptosis of epilepsy. Meanwhile, the expression of the apoptosis-related protein VHL decreased in this pathway, and the expression of p21 was upregulated. Therefore, this study suggests that VHL/HIF-1α/p21 might be involved in the apoptosis of hippocampal neurons in Scn1a KO mice.

Study of Gas Film Cooling under Large Eddy Simulation: Effect of Forward and Backward Injection on Gas Turbine Cooling Efficiency and Stability

In the design of advanced gas turbines, improving thermal efficiency and power output is crucial achieve energy saving and emission reduction goals. This necessitates raising the inlet temperature, thereby highlighting the importance developing efficient cooling technologies to ensure the longevity of hot components. Gas film cooling, as a widely used traditional cooling method, is subject to the influence of fluid dynamic characteristics, especially in unstable operating environments such as pressure fluctuations in the upstream combustion chamber and interactions between stators and rotors. This study utilized large eddy simulation to analyze the effectiveness of trench film cooling under such unstable conditions, with a specific focus on the time-averaged and transient characteristics of forward and backward injection of cooling air. The study investigated the efficacy of film cooling under pulsating flow conditions, with a blowing ratio of 1.5 under steady-state conditions, utilizing cosine and square waves as pulse boundaries, and a Strouhal number of 0.254. The research findings reveal that: 1) Contrary to the results under steady-state conditions, pulsating reverse injection led to a reduction of over 15% in time-averaged cooling efficiency and a noticeable decrease in the film coverage area compared to forward injection; this phenomenon is determined by the different flow mechanisms under steady-state and pulsating states. 2) Under a certain blowing ratio, the instability of gas film cooling is induced by the temporal fluctuations of near-wall vortices, which could result in rapid temperature changes and component failures. For pulsating forward injection, additional high film cooling unsteadiness outside the slot membrane orifices was observed. In the case of backward injection, pulsations did not lead to a significant increase in instability. These research findings provide a deeper understanding of the impact of gas film cooling on the cooling efficiency and stability of gas turbines, enabling gas film cooling technology to meet the inlet temperature requirements of advanced gas turbines, thereby reducing carbon emissions and contributing to the achievement of China's dual carbon goals.

Asymmetry of oblique shock train and flow control

An oblique shock train generally forms an asymmetric structure in a Mach-2.7 flow field within a duct. To study the flow structure and interaction between oblique shock trains and upstream shocks, a ramp with equal width was installed inside a Mach-2.7 straight duct to generate an incident shock and an oblique shock train interaction. A Schlieren system, transient pressure measurements and particle image velocimetry were used to capture quantitative and qualitative shock structure information. Results show that the asymmetric separation deflection of the oblique shock train occurs randomly in the symmetrical straight duct. The separation deflection of the oblique shock train was steady with upstream shock interactions. Under backpressure conditions, the rate of movement of the oblique shock train increases rapidly when it passes through the separation regions generated by the ramp, and the deflection direction of the asymmetric separation may switch. Based on the characteristics of the oblique shock train and upstream shock interaction, a flow control method was used to generate asymmetric upstream flow conditions, providing active control of the oblique shock train deflection direction.

Does anisotropy hold in mixed traffic conditions?

Traditionally, car-following models have focused on the interaction between leader and follower vehicles. However, some researchers have explored the significance of incorporating upstream vehicle information and considering non-anisotropic traffic flow patterns for homogeneous traffic conditions. This paper presents a comprehensive investigation into the impact of upstream vehicle information on car-following models for mixed traffic conditions. We employ three different approaches, macroscopic, microscopic, and nonparametric modeling, using empirical data to examine the evidence for the existence of non-anisotropy in mixed traffic conditions. We investigate the macroscopic evidence of fundamental diagrams, revealing complex interactions among heterogeneous traffic. The propagation of congestion can exceed the travel speed of the traffic stream itself, emphasizing the intricate nature of traffic dynamics. Our findings demonstrate that incorporating upstream vehicle information significantly enhances the prediction accuracy of car-following models. Particularly in scenarios where heavy vehicles are present upstream, the inclusion of upstream information proves to be crucial. Further, we conduct a microscopic analysis using triplet trajectories involving heavy vehicles to validate our findings, utilizing Edie’s definition. The microscopic validation supports the presence of non-anisotropic fundamental diagrams and reinforces the need to consider upstream interactions in car-following models.

A spatiotemporal analysis of energy use pathways in the construction industry: A study of China

China has experienced rapid urbanization across the country which has required significant energy use and has also posed a series of possible environmental risks. Given the role that the construction industry plays in China's economy and in achieving sustainability, it is paramount to trace and measure the evolutionary pathways of energy induced by China's supply chains. By using a multiregional input–output model, structural path analysis, and exploratory spatial data analysis, this study presents a spatiotemporal analysis of how upstream energy interactions change in the context of rapid urbanization and the Chinese economy. The results show there has been less embodied energy consumption of building materials over time due to significant improvements in energy intensity reduction. The upstream energy distribution exhibited significant spatial heterogeneity within clustered urban agglomerations. Regions with poor resource endowment but highly vibrant economies appear to have wider connections with the national economy, whilst the critical energy flows in resource-exporting regions were only concentrated in energy-intensive sectors. The findings of this study provide improved understanding of energy evolutionary pathways and their complexities from both spatial and temporal perspectives. Such understanding will assist policymakers, especially the local government of each province, to implement effective energy reduction strategies and fairly address the allocation of energy reduction responsibilities.

Reconnection‐Driven Dynamics at Ganymede's Upstream Magnetosphere: 3‐D Global Hall MHD and MHD‐EPIC Simulations

AbstractThe largest moon in the solar system, Ganymede, is the only moon known to possess a strong intrinsic magnetic field and a corresponding magnetosphere. Using the latest version of Space Weather Modeling Framework (SWMF), we study the upstream plasma interactions and dynamics in this sub‐Alfvénic system. Results from the Hall magnetohydrodynamics (MHD) and the coupled MHD with embedded particle‐in‐cell (MHD‐EPIC) models are compared. We find that under steady upstream conditions, magnetopause reconnection occurs in a nonsteady manner, and the energy partition between electrons and ions is different in the two models. Flux ropes of Ganymede's radius in length form on the magnetopause at a rate about 3 min and create spatiotemporal variations in plasma and field properties. Upon reaching proper grid resolutions, the MHD‐EPIC model can resolve both electron and ion kinetics at the magnetopause and show localized nongyrotropic behavior inside the diffusion region. The estimated global reconnection rate from the models is about 80 kV with 60% efficiency, and there is weak evidence of ∼1 min periodicity in the temporal variations due to the dynamic reconnection process.

Serine 19 phosphorylation and 14-3-3 binding regulate phosphorylation and dephosphorylation of tyrosine hydroxylase on serine 31 and serine 40.

Multisite phosphorylation and structural flexibility allow for complex regulation of proteins through cellular signaling. Tyrosine hydroxylase (TH), a key enzyme of catecholamine synthesis, is regulated by multiple neuronal signaling pathways through phosphorylation at serine 19 (Ser19), serine 31 (Ser31), and serine 40 (Ser40) located in the flexible, far N-terminal region of the regulatory domain. Phosphorylated Ser19 (pSer19) provides a binding site for 14-3-3 proteins, a family of multi-target binding adaptor proteins. We hypothesized that pSer19 and 14-3-3 binding can regulate access to the Ser31 and Ser40 sites and modulate the dynamics of their phosphorylation state. To avoid complications from upstream signal interactions and have good control of TH-phosphorylation and 14-3-3 binding stoichiometry, we used purified recombinant human TH and 14-3-3 dimer types. We found that pSer19 strongly stimulated Ser31 phosphorylation (4.6-fold), but inhibited pSer31 dephosphorylation (3.4-fold). Binding of 14-3-3ζ counteracted the stimulatory effect of pSer19 on phosphorylation at Ser31, but amplified the effect on its dephosphorylation. In contrast, phosphorylation at Ser19 had moderate effect on pSer40 dephosphorylation, but 14-3-3ζ binding inhibited dephosphorylation, an effect that was consistent across different homo- and heterodimeric 14-3-3s. Additional phosphorylation of Ser31 or Ser40 had little impact on the binding affinity of pSer19 TH to 14-3-3s. Mathematical modeling was performed to elucidate possible physiological implications of these observations. We propose a role of Ser19 and 14-3-3 proteins as modulators of TH phosphorylation in response to neuronal co-signaling events. These mechanisms add to our understanding of the multifaceted roles of phosphorylation and adaptor proteins in cellular signaling.

TriPOINT: a software tool to prioritize important genes in pathways and their non-coding regulators

SummaryCurrent approaches for pathway analyses focus on representing gene expression levels on graph representations of pathways and conducting pathway enrichment among differentially expressed genes. However, gene expression levels by themselves do not reflect the overall picture as non-coding factors play an important role to regulate gene expression. To incorporate these non-coding factors into pathway analyses and to systematically prioritize genes in a pathway we introduce a new software: Triangulation of Perturbation Origins and Identification of Non-Coding Targets. Triangulation of Perturbation Origins and Identification of Non-Coding Targets is a pathway analysis tool, implemented in Java that identifies the significance of a gene under a condition (e.g. a disease phenotype) by studying graph representations of pathways, analyzing upstream and downstream gene interactions and integrating non-coding regions that may be regulating gene expression levels.Availability and implementationThe TriPOINT open source software is freely available at https://github.uconn.edu/ajt06004/TriPOINT under the GPL v3.0 license.Supplementary information Supplementary data are available at Bioinformatics online.

MYC activation and BCL2L11 silencing by a tumour virus through the large-scale reconfiguration of enhancer-promoter hubs.

Lymphomagenesis in the presence of deregulated MYC requires suppression of MYC-driven apoptosis, often through downregulation of the pro-apoptotic BCL2L11 gene (Bim). Transcription factors (EBNAs) encoded by the lymphoma-associated Epstein-Barr virus (EBV) activate MYC and silence BCL2L11. We show that the EBNA2 transactivator activates multiple MYC enhancers and reconfigures the MYC locus to increase upstream and decrease downstream enhancer-promoter interactions. EBNA2 recruits the BRG1 ATPase of the SWI/SNF remodeller to MYC enhancers and BRG1 is required for enhancer-promoter interactions in EBV-infected cells. At BCL2L11, we identify a haematopoietic enhancer hub that is inactivated by the EBV repressors EBNA3A and EBNA3C through recruitment of the H3K27 methyltransferase EZH2. Reversal of enhancer inactivation using an EZH2 inhibitor upregulates BCL2L11 and induces apoptosis. EBV therefore drives lymphomagenesis by hijacking long-range enhancer hubs and specific cellular co-factors. EBV-driven MYC enhancer activation may contribute to the genesis and localisation of MYC-Immunoglobulin translocation breakpoints in Burkitt's lymphoma.

Allosteric Regulation of Rac1-PAK1 Binding Affinity by Mutant Residues through Molecular Simulations and AFM

Rac1 (Ras-related C3 botulinum toxin substrate 1) is a small member of Rho family of GTPases and PAK1 (p21-activated kinase 1) is a serine/threonine kinase. Switch I region is responsible for the interaction of Rac1 protein with the downstream effectors such as PAK1, whereas Switch II region is important for the upstream activation of Rac1 in its GTP-bound state. Rac1 and PAK1 regulate essential cellular processes such as cytoskeletal dynamics and actin depolymerization. Mutational activation of PAK1 by Rac1 has oncogenic signaling effects in cell proliferation, survival, invasion and metastasis. Here, we aim to explore the Rac1-PAK1 interaction to understand the allosteric regulation of the binding behavior. Q61L, on Switch II, is an oncogenic mutation leading to a constitutively active protein, whereas T17N at GTP/GDP binding site leads to either a nucleotide free or inactive protein. To this, the intrinsic dynamics of the wild type, Q61L and T17N mutant Rac1 proteins is analyzed by the Gaussian Network Model (GNM) and conventional Molecular Dynamics (MD) simulations. Preliminary results reveal that the Q61L and T17N mutations lead to altered dynamics, respectively, as stabilized fluctuations in Switch I region imply a downstream effect on PAK1 and increased fluctuations in Switch I and II regions affect both upstream and downstream interactions. The association of the Q61 and T17 positions with the global hinge sites supports the allosteric coupling between the mutations and Rac1-PAK1 binding. The Y72C oncogenic mutation at a hinge site is to be analyzed in this framework with respect to the binding dynamics of Rac1 and functional implications in oncogenic signaling. The effects of these three mutations on the free energy landscape of Rac1-PAK1 are then to be explored and verified via Atomic Force Microscopy (AFM).

Proteome-wide characterization of signalling interactions in the hippocampal CA4/DG subfield of patients with Alzheimer's disease.

Alzheimer’s disease (AD) is the most common form of dementia; however, mechanisms and biomarkers remain unclear. Here, we examined hippocampal CA4 and dentate gyrus subfields, which are less studied in the context of AD pathology, in post-mortem AD and control tissue to identify possible biomarkers. We performed mass spectrometry-based proteomic analysis combined with label-free quantification for identification of differentially expressed proteins. We identified 4,328 proteins, of which 113 showed more than 2-fold higher or lower expression in AD hippocampi than in control tissues. Five proteins were identified as putative AD biomarkers (MDH2, PCLO, TRRAP, YWHAZ, and MUC19 isoform 5) and were cross-validated by immunoblotting, selected reaction monitoring, and MALDI imaging. We also used a bioinformatics approach to examine upstream signalling interactions of the 113 regulated proteins. Five upstream signalling (IGF1, BDNF, ZAP70, MYC, and cyclosporin A) factors showed novel interactions in AD hippocampi. Taken together, these results demonstrate a novel platform that may provide new strategies for the early detection of AD and thus its diagnosis.

A framework to identify enabling and urgent actions for the 2020 Aichi Targets

In 2010, the parties of the Convention on Biological Diversity (CBD) adopted the Strategic Plan for Biodiversity 2011–2020 with the mission of halting biodiversity loss and enhance the benefits it provides to people. The 20 Aichi Biodiversity Targets (Aichi Targets), which are included in the Strategic Plan, are organized under five Strategic Goals, and provide coherent guidance on how to achieve it. Halfway through the Strategic Plan, it is time to prioritize actions in order to achieve the best possible outcomes for the Aichi Targets in 2020. Actions to achieve one target may influence other targets (downstream interactions); in turn a target may be influenced by actions taken to attain other targets (upstream interactions). We explore the interactions among targets and the time-lags between implemented measures and desired outcomes to develop a framework that can reduce the overall burden associated with the implementation of the Strategic Plan. We identified the targets addressing the underlying drivers of biodiversity loss and the targets aimed at enhancing the implementation of the Strategic Plan as having the highest level of downstream interactions. Targets aimed at improving the status of biodiversity and safeguarding ecosystems followed by targets aimed at reducing the direct pressures on biodiversity and enhancing the benefits to all from biodiversity and ecosystem services, were identified as having the highest levels of upstream interactions. Perhaps one of the most challenging aspects of the Strategic Plan is the need to balance actions for its long-term sustainability with the need for urgent actions to halt biodiversity loss.

Behavior of Multiple-Jet Interactions in a Hypersonic Boundary Layer

The fundamental flow physics of the interaction between an array of fuel jets and a hypersonic boundary layer is investigated. Hydrogen is injected at jet-to-freestream dynamic pressure ratios ranging from 0.350 to 0.875 on a flat plate into a Mach 4.5 crossflow. The injection array consists of four streamwise-aligned flush circular portholes. Both the streamwise spacing and jet-to-freestream dynamic pressure ratio are varied in a parametric study. The injection was performed completely within the boundary layer, with the intention of application to film-cooling drag reduction and boundary-layer combustion. Numerical simulations of four streamwise-aligned transverse sonic injectors in a fully turbulent hypersonic boundary layer revealed a very complex jet interaction flowfield. Variation of the streamwise injector port spacing, along with the jet-to-freestream dynamic pressure ratio, induced a variety of flow structures in the cases investigated. For all downstream interactions, the associated flow behavior was found to be a direct result of both the various upstream effects and interactions between adjacent injectors. Variations in the jet-to-freestream dynamic pressure ratio had a strong effect on the flow behavior. At low injection mass flow rates, coupling of adjacent injectors was small, whereas high mass flow rates increased the effect of jet-to-jet coupling. Variations in the streamwise jet-to-jet spacing were also found to play a critical role in the flow behavior. At very close spacings, intense interactions coupled the behavior of the individual jets; however, at increased spacings, the larger spatial freedom allowed individual jets to develop more naturally, leading to less jet-to-jet interactions. At the maximum spacing investigated, the jet interactions behaved more like discrete jets in crossflow.

Sources of machine-induced background in the ATLAS and CMS detectors at the CERN Large Hadron Collider

One source of experimental background in the CERN Large Hadron Collider (LHC) is particles entering the detectors from the machine. These particles are created in cascades, caused by upstream interactions of beam protons with residual gas molecules or collimators. We estimate the losses on the collimators with SixTrack and simulate the showers with FLUKA and MARS to obtain the flux and distribution of particles entering the ATLAS and CMS detectors. We consider some machine configurations used in the first LHC run, with focus on 3.5TeV operation as in 2011. Results from FLUKA and MARS are compared and a very good agreement is found. An analysis of logged LHC data provides, for different processes, absolute beam loss rates, which are used together with further simulations of vacuum conditions to normalize the results to rates of particles entering the detectors. We assess the relative importance of background from elastic and inelastic beam–gas interactions, and the leakage out of the LHC collimation system, and show that beam–gas interactions are the dominating source of machine-induced background for the studied machine scenarios. Our results serve as a starting point for the experiments to perform further simulations in order to estimate the resulting signals in the detectors.

Chemical fidelity of an RNA polymerase ribozyme

The emergence of catalytically active RNA enzymes (ribozymes) is widely believed to have been an important transition in the origin of life. In the context of a likely heterogeneous chemical environment, substrate specificity and selectivity of these primordial enzymes would have been critical for function. Here we have explored the chemical fidelity, i.e. substrate selectivity and specificity for both single and multiple catalytic steps of the Z RNA polymerase ribozyme – a modern day analogue of the primordial RNA replicase. Using a wide range of nucleotide analogues and ionic conditions, we observe strong energetic but weak geometric discrimination at the incorporation step, indicative of an open active site. In contrast, stringent selectivity is exerted at the extension steps through specific down and upstream interactions with the 3′-terminal nucleoside as well as nascent product and template strands. Our results indicate specificity mechanisms that are found in functionally analogous forms in natural polymerases. They also reveal a level of chemical fidelity over multiple catalytic steps that is remarkable for a comparatively unoptimized enzyme developed de novo from a random sequence pool. The convergent evolution of specificity mechanisms in phylogenetically unrelated proteinaceous polymerases and polymerase ribozymes suggests that chemical as well as informational fidelity are emergent properties of polymerase enzymes.

The Alveolar Epithelial Cell Chemokine Response to Pneumocystis Requires Adaptor Molecule MyD88 and Interleukin-1 Receptor but Not Toll-Like Receptor 2 or 4

Pneumocystis is an opportunistic fungal pathogen that causes pneumonia in a variety of clinical settings. An early step in Pneumocystis infection involves the attachment of organisms to alveolar epithelial cells (AECs). AECs produce chemokines in response to Pneumocystis stimulation, but the upstream host-pathogen interactions that activate AEC signaling cascades are not well-defined. MyD88 is an adaptor molecule required for activation of proinflammatory signaling cascades following Toll-like receptor (TLR)-dependent recognition of conserved molecular patterns on pathogens. To determine whether the TLR/MyD88 pathway is required for the AEC chemokine response to Pneumocystis, wild-type (WT) and MyD88-deficient AECs were incubated with Pneumocystis. As expected, WT AECs produced CCL2 and CXCL2 following Pneumocystis stimulation. In contrast, MyD88-deficient AECs were severely impaired in their ability to respond to Pneumocystis. MyD88-deficient AECs did not display Pneumocystis-induced Jun N-terminal protein kinase activation and produced much less chemokine than Pneumocystis-stimulated WT AECs. Using a panel of TLR agonists, primary murine AECs were found to respond vigorously to TLR2 and TLR4 agonists. However, the AEC chemokine response to Pneumocystis did not require TLR2 or TLR4. Surprisingly, the interleukin-1 receptor (IL-1R) was required for an AEC chemokine response to Pneumocystis. The role of MyD88 in early responses during Pneumocystis infection was supported by in vivo studies demonstrating that MyD88-deficient mice showed impaired Pneumocystis-stimulated chemokine production and impaired inflammatory cell recruitment. These data indicate an important role for MyD88 in the AEC inflammatory response to Pneumocystis.

E. Coli RNA Polymerase: A Molecular DNA Opening Machine

Using “burst” kinetic methods to obtain near-homogeneous populations of transient intermediates for fast footprinting, together with solute probing experiments, we have characterized the series of large-scale conformational changes in E. coli RNA Polymerase (RNAP) and λPR promoter DNA that occur in “isomerization” of the initial recognition complex to form the stable open complex (RPo). We find that RNAP is a molecular isomerization machine that uses wrapping of upstream DNA to place the start-site region of the downstream duplex in the cleft (shown by HO protection and FRET in the closed intermediate I1).1 Then, in the rate determining step, RNAP opens the entire 13 bp initiation bubble (as judged by permanganate reactive thymines) and places the start site base of the template strand in the active site to form a transient open intermediate (I2).2 After this opening/ untwisting step, RNAP stabilizes the open DNA conformation by step-wise assembly of an encircling downstream jaw/ clamp, together with repositioning of the nontemplate strand in the cleft to form the more stable open intermediate I3 and convert it to the stable open complex RPo at λPR.2,3To test these conclusions, we are investigating deletions of the downstream “jaw” of RNAP [B’1149-1190], upstream and/ or downstream DNA, and/ or region 1.1 of σ70 subunit of RNAP [1-98] on the kinetics of formation and lifetime of open complexes. Remarkably, many of these deletions have large effects on the rate-determining, DNA-opening step, suggesting a synergy between upstream, downstream and in-cleft interactions in this sophisticated molecular machine.1. Davis et al. ’07; Drennan, Saecker et al., in preparation2. Gries et al. ‘103. Kontur, Gries et al.’06, ’08, ‘10Supported by NIH GM23467

Differential effects of ethanol on c-jun N-terminal kinase, 14-3-3 proteins, and Bax in postnatal day 4 and postnatal day 7 rat cerebellum

These studies investigated ethanol effects on upstream cellular elements and interactions which contribute to Bax-related apoptosis in neonatal rat cerebellum at ages of peak ethanol sensitivity (postnatal day 4 [P4]), compared to later ages of relative resistance (P7). Analyses were made of basal levels of the pro-apoptotic c-jun N-terminal kinase (JNK), Bax, and the 14-3-3 anchoring proteins, as well as the responsiveness of these substances to ethanol at P4 versus P7. Dimerization of Bax with 14-3-3 was also investigated at the two ages following ethanol treatment, a process which sequesters Bax in the cytosol, thus inhibiting its mitochondrial translocation and disruption of the mitochondrial membrane potential. Cultured cerebellar granule cells were used to examine the protective potential of JNK inhibition on ethanol-mediated cell death. Basal levels of JNK were significantly higher at P4 than P7, but no differences in the other proteins were found. Activated JNK, and cytosolic and mitochondrially-translocated Bax were increased in P4 but not P7 animals following ethanol exposure, while protective 14-3-3 proteins were increased only at P7. Ethanol treatment resulted in decreases in Bax:14-3-3 heterodimers at P4, but not at P7. Inhibition of JNK activity in vitro provided partial protection against ethanol neurotoxicity. Thus, differential temporal vulnerability to ethanol in this CNS region correlates with differences in both levels of apoptosis-related substances (e.g., JNK), and differential cellular responsiveness, favoring apoptosis at the most sensitive age and survival at the resistant age. The upstream elements contributing to this vulnerability can be targets for future therapeutic strategies.

Two Human MYD88 Variants, S34Y and R98C, Interfere with MyD88-IRAK4-Myddosome Assembly

Innate immune receptors detect microbial pathogens and subsequently activate adaptive immune responses to combat pathogen invasion. MyD88 is a key adaptor molecule in both Toll-like receptor (TLR) and IL-1 receptor superfamily signaling pathways. This is illustrated by the fact that human individuals carrying rare, naturally occurring MYD88 point mutations suffer from reoccurring life-threatening infections. Here we analyzed the functional properties of six reported non-synonymous single nucleotide polymorphisms of MYD88 in an in vitro cellular system. Two variants found in the MyD88 death domain, S34Y and R98C, showed severely reduced NF-κB activation due to reduced homo-oligomerization and IRAK4 interaction. Structural modeling highlights Ser-34 and Arg-98 as residues important for the assembly of the Myddosome, a death domain (DD) post-receptor complex involving the DD of MyD88, IRAK4, and IRAK2 or IRAK1. Using S34Y and R98C as functional probes, our data show that MyD88 homo-oligomerization and IRAK4 interaction is modulated by the MyD88 TIR and IRAK4 kinase domain, demonstrating the functional importance of non-DD regions not observed in a recent Myddosome crystal structure. The differential interference of S34Y and R98C with some (IL-1 receptor, TLR2, TLR4, TLR5, and TLR7) but not all (TLR9) MyD88-dependent signaling pathways also suggests that receptor specificities exist at the level of the Myddosome. Given their detrimental effect on signaling, it is not surprising that our epidemiological analysis in several case-control studies confirms that S34Y and R98C are rare variants that may drastically contribute to susceptibility to infection in only few individuals.

Update of human and mouse forkhead box (FOX) gene families

The forkhead box (FOX) proteins are transcription factors that play complex and important roles in processes from development and organogenesis to regulation of metabolism and the immune system. There are 50 FOX genes in the human genome and 44 in the mouse, divided into 19 subfamilies. All human FOX genes have close mouse orthologues, with one exception: the mouse has a single Foxd4, whereas the human gene has undergone a recent duplication to a total of seven (FOXD4 and FOXD4L1 → FOXD4L6). Evolutionarily ancient family members can be found as far back as the fungi and metazoans. The DNA-binding domain, the forkhead domain, is an example of the winged-helix domain, and is very well conserved across the FOX family and across species, with a few notable exceptions in which divergence has created new functionality. Mutations in FOX genes have been implicated in at least four familial human diseases, and differential expression may play a role in a number of other pathologies -- ranging from metabolic disorders to autoimmunity. Furthermore, FOX genes are differentially expressed in a large number of cancers; their role can be either as an oncogene or tumour suppressor, depending on the family member and cell type. Although some drugs that target FOX gene expression or activity, notably proteasome inhibitors, appear to work well, much more basic research is needed to unlock the complex interplay of upstream and downstream interactions with FOX family transcription factors.