The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.     

Soil carbon sequestration and land use change associated with biofuel production: empirical evidence

Soil organic carbon (SOC) change can be a major impact of land use change (LUC) associated with biofuel feedstock production. By collecting and analyzing data from worldwide field observations of major LUCs from cropland, grassland, and forest to lands producing biofuel crops (i.e. corn, switchgrass, Miscanthus, poplar, and willow), we were able to estimate SOC response ratios and sequestration rates and evaluate the effects of soil depth and time scale on SOC change. Both the amount and rate of SOC change were highly dependent on the specific land transition. Irrespective of soil depth or time horizon, cropland conversions resulted in an overall SOC gain of 6–14% relative to initial SOC level, while conversion from grassland or forest to corn (without residue removal) or poplar caused significant carbon loss (9–35%). No significant SOC changes were observed in land converted from grasslands or forests to switchgrass, Miscanthus, or willow. The SOC response ratios were similar in both 0–30 and 0–100 cm soil depths in most cases, suggesting SOC changes in deep soil and that use of top soil only for SOC accounting in biofuel life cycle analysis (LCA) might underestimate total SOC changes. Soil carbon sequestration rates varied greatly among studies and land transition types. Generally, the rates of SOC change tended to be the greatest during the 10 years following land conversion and had declined to approach 0 within about 20 years for most LUCs. Observed trends in SOC change were generally consistent with previous reports. Soil depth and duration of study significantly influence SOC change rates and so should be considered in carbon emission accounting in biofuel LCA. High uncertainty remains for many perennial systems and forest transitions, additional field trials, and modeling efforts are needed to draw conclusions about the site‐ and system‐specific rates and direction of change.     

Synthesis of spirocyclic σ1 receptor ligands as potential PET radiotracers,structure–affinity relationships and in vitro metabolic stability

Several 3H-spiro[[2]benzofuran-1,4′-piperidines] bearing a p-fluorobenzyl residue at the N-atom and various substituents in position 3 of the benzofuran system were synthesized. The crucial reaction steps are the addition of a lithiated benzaldehyde derivative to the p-fluorobenzylpiperidone 5 and the BF₃·OEt₂ catalyzed substitution of the methoxy group of 2a by various nucleophiles. Structure–affinity relationship studies revealed that compounds with two protons (2d), a methoxy group (2a), and a cyano group (2e) in position 3 possess subnanomolar σ₁ affinity (K_i = 0.18 nM, 0.79 nM, 0.86 nM) and high selectivity against the σ₂ subtype. The metabolites of 2a, 2d, and 2e, which were formed upon incubation with rat liver microsomes, were identified. Additionally, the rate of metabolic degradation of 2a, 2d, and 2e was determined and compared with the degradation rate of the non-fluorinated spirocyclic compound 1. For the synthesis of the potential PET tracers [¹⁸F]2a and [¹⁸F]2e two different radiosynthetic approaches were followed.     

Mouse LSECtin as a model for a human Ebola virus receptor

The biochemical properties of mouse LSECtin, a glycan-binding receptor that is a member of the C-type lectin family found on sinusoidal endothelial cells, have been investigated. The C-type carbohydrate-recognition domain of mouse LSECtin, expressed in bacteria, has been used in solid-phase binding assays, and a tetramerized form has been used to probe a glycan array. In spite of sequence differences near the glycan-binding sites, the mouse receptor closely mimics the properties of the human receptor, showing high affinity binding to glycans bearing terminal GlcNAcβ1-2Man motifs. Site-directed mutagenesis has been used to confirm that residues near the binding site that differ between the human and the mouse proteins do not affect this binding specificity. Mouse and human LSECtin have been shown to bind Ebola virus glycoprotein with equivalent affinities, and the GlcNAcβ1-2Man disaccharide has been demonstrated to be an effective inhibitor of this interaction. These studies provide a basis for using mouse LSECtin, and knockout mice lacking this receptor, to model the biological properties of the human receptor.     

Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E‐cadherin to disrupt intercellular adhesion

Mammalian and prokaryotic high‐temperature requirement A (HtrA) proteins are chaperones and serine proteases with important roles in protein quality control. Here, we describe an entirely new function of HtrA and identify it as a new secreted virulence factor from Helicobacter pylori, which cleaves the ectodomain of the cell‐adhesion protein E‐cadherin. E‐cadherin shedding disrupts epithelial barrier functions allowing H. pylori designed to access the intercellular space. We then designed a small‐molecule inhibitor that efficiently blocks HtrA activity, E‐cadherin cleavage and intercellular entry of H. pylori.     

Cryo-Electron Microscopy Structure of an Adenovirus-Integrin Complex Indicates Conformational Changes in both Penton Base and Integrin

A structure of adenovirus type 12 (HAdV12) complexed with a soluble form of integrin αvβ5 was determined by cryo-electron microscopy (cryoEM) image reconstruction. Subnanometer resolution (8 Å) was achieved for the icosahedral capsid with moderate resolution (27 Å) for integrin density above each penton base. Modeling with αvβ3 and α_IIbβ3 crystal structures indicates that a maximum of four integrins fit over the pentameric penton base. The close spacing (∼60 Å) of the RGD protrusions on penton base precludes integrin binding in the same orientation to neighboring RGD sites. Flexible penton-base RGD loops and incoherent averaging of bound integrin molecules explain the moderate resolution observed for the integrin density. A model with four integrins bound to a penton base suggests that integrin might extend one RGD-loop in the direction that could induce a conformational change in the penton base involving clockwise untwisting of the pentamer. A global conformational change in penton base could be one step on the way to the release of Ad vertex proteins during cell entry. Comparison of the cryoEM structure with bent and extended models for the integrin ectodomain reveals that integrin adopts an extended conformation when bound to the Ad penton base, a multivalent viral ligand. These findings shed further light on the structural basis of integrin binding to biologically relevant ligands, as well as on the molecular events leading to HAdV cell entry.A growing number of viruses have been identified as using one of the 24 types of integrin heterodimers as a receptor for cell entry (32). Integrins are cell surface molecules involved in the regulation of adhesion, migration, growth, and differentiation (11). The large multidomained extracellular segments of α and β integrin subunits bind a variety of ligands, including viral ligands, while the smaller intracellular domains interact with cytoskeletal proteins (Fig. ​(Fig.1A).1A). These extracellular and intracellular interactions facilitate bidirectional signaling, with the initiating events occurring either outside of the cell (outside-in signaling) or within the cell (inside-out signaling) (24). Integrin clustering has been established as having an important role in outside-in signaling (9, 19, 20, 44). Clustering results in the formation of focal adhesions, which are organized intracellular complexes, that facilitate downstream signaling cascades within the cell (24).Open in a separate windowFIG. 1.Integrin domains and conformations. (A) Structural domains of integrin αv and β chains, including the extracellular domains, transmembrane-spanning regions, and small cytoplasmic domains, shown in extended schematic forms. The domains are represented as 10Å-resolution density maps based on crystallographic coordinates. The membrane is represented by a gray bar. (Modified from Stewart and Nemerow (32) and reprinted with permission from Elsevier.) (B) Models for soluble αvβ5 integrin with Fos/Jun dimerization domains. Each chain has a six residue glycine-rich linker between the ectodomain and the Fos or Jun dimerization domain. The model of a bent integrin conformation (left) was built as a composite of αvβ3 integrin crystal structures, PDB-IDs 1L5G and 1U8C (42, 43), and the crystal structure of c-Fos/c-Jun bound to DNA, PDB-ID 1FOS (6). The model of an extended integrin conformation (right) is similar to the extended model docked into the HAdV12/αvβ5 cryo structure (Fig. ​(Fig.8B8B).Studies of adenovirus (Ad) interactions with αv integrins provided some of the first evidence of the virus-induced signaling events (13, 14). The Ad penton base capsid protein, which sits at the 12 vertices of the icosahedral capsid, has five prominent Arg-Gly-Asp (RGD) containing loops that are flexible and protrude from the viral surface (31, 48). Receptor-mediated endocytosis of Ad is stimulated by interaction of the RGD-containing penton base with αvβ3 and αvβ5 integrins (34). This interaction leads to receptor clustering, followed by tyrosine phosphorylation/activation of focal adhesion kinase, as well as activation of p130CAS, phosphatidylinositol 3-OH-kinase, and the Rho family of small GTPases, and subsequent actin polymerization and Ad internalization (32). Integrin signaling events also lead to production of proinflammatory cytokines (23) and may result in increased survival of certain host cells through subsequent signaling to protein kinase B (AKT) (25).Multiple studies indicate that after interaction with an RGD-containing ligand a straightening of the integrin extracellular domains occurs, leading to the “extension” or “switchblade” model for integrin activation (16, 45). In the extension model the headpiece domains, which are closest to the RGD interaction site, have a “closed” conformation in the low-affinity, unliganded state. This state is characterized by the close proximity of the α and β subunits at the “knees” or midpoints of the extracellular segments. In contrast, the high-affinity, ligand-bound state in the extension model is distinguished by an “open” headpiece conformation with separation at the knees of the extracellular segments. The location of the RGD binding site between the α-subunit β-propellor and the β-subunit I domain was first visualized in the crystal structure of the αvβ3 extracellular segment with a bound RGD peptide (43). In this structure the RGD site is folded back toward the membrane, and the integrin is in a closed conformation. The closed conformation has also been observed in crystal structures of the αvβ3 ectodomain without an RGD peptide (41) and the α_IIbβ3 ectodomain (47).The open integrin conformation has been characterized as having a large separation of up to ∼70 Å between the knees of α and β subunits (16). Four slightly different open headpiece conformations were observed in crystal structures of the α_IIbβ3 headpiece with bound fibrinogen-mimetic therapeutics (38). These structures show that the change from a closed to an open headpiece conformation is accompanied by a piston-like motion of helix α7 in the β-chain I domain and a large swing of the β-chain hybrid domain of up to 69°, as well as extension and separation of the two integrin chains. Comparison of the available αvβ3 and α_IIbβ3 crystal structures is providing information on the interdomain angle variation and flexibility between domains (47).One aspect of the extension model is that separation of the C-terminal, intracellular portions of the α and β subunits leads to inside-out activation. This concept is supported by nuclear magnetic resonance structures of the cytoplasmic tails of α_IIbβ3 showing that the membrane-proximal helices engage in a weak interaction that can be disrupted by constitutively activating mutations or by talin, a protein found in high concentrations in focal adhesions (33). The concept that the integrin α and β subunits must also separate during outside-in signaling is supported by a study involving a disulfide-bonded mutant of α_IIbβ3 integrin (46). When the α and β subunits are linked in the vicinity of the transmembrane helices the mutant α_IIbβ3 is still able to bind ligand, mediate adhesion, and undergo antibody-induced clustering. However, the disulfide-bonded mutant exhibits defects in focal adhesion formation and focal adhesion kinase activation. Reduction of the disulfide bond or single cysteine mutants rescues signaling.A competing model for integrin activation, called the “deadbolt” model, proposes only small conformational changes in the integrin β-chain I domain upon RGD binding (2). This model is based on crystal structures of the αvβ3 ectodomain with or without an RGD peptide (41, 43). Both of these αvβ3 structures reveal a bent integrin conformation with a closed headpiece conformation. However, the RGD peptide was soaked into a preformed crystal of αvβ3 and crystal contacts may have prevented conformational changes.There are relatively few and only moderate resolution structures of virus-integrin complexes. A moderate resolution cryoEM structure has been determined for the Picornavirus echovirus 1 (EV1) in complex with the I domain of the α2 integrin subunit (39). Docking of crystal structures of EV1 and the α2 I domain into the cryoEM density indicates that the I domain binds within a canyon on the surface of EV1 and that five integrins could potentially bind at one vertex of the icosahedral capsid. Confocal fluorescence microscopy experiments indicated that EV1 causes integrin clustering on human osteosarcoma cells stably transfected with α2 integrin. However, it could not be determined whether the bound integrins were in the inactive (bent) or active (extended) conformation.Moderate resolution (∼21 Å) cryoEM structures of Ad type 2 (HAdV2) and HAdV12 in complex with a soluble form of αvβ5 integrin revealed a ring of integrin density over each penton base capsid protein (5). Better-defined integrin density was observed in the HAdV12/integrin complex, supporting the idea suggested from sequence alignments that the RGD loop of the HAdV12 penton base is shorter and less flexible than that of HAdV2. This study also suggested that the precise spatial arrangement of the five RGD protrusions on the penton base might promote integrin clustering, which may lead to the intracellular signaling events required for virus internalization into a host cell. A similar spacing of RGD-containing integrin-binding sites around the fivefold axis of icosahedral virions has been noted for Ad, foot-and-mouth disease virus, and coxsackievirus A9 (32).We present here a significantly higher-resolution cryoEM structure of HAdV12 complexed with soluble αvβ5 that provides insight into the Ad-integrin interaction. The resolution of the icosahedral capsid portion of the Ad-integrin complex was improved to 8 Å, and the capsid shows clearly resolved α-helices, which allows accurate docking of the penton base crystal structure within the cryoEM density. The resolution of the integrin density is more moderate due to flexibility of the RGD-containing surface loop of penton base and incoherent averaging of integrin heterodimers. Nevertheless, modeling studies with available integrin crystal structures have enabled us to distinguish between a bent or extended conformation (Fig. ​(Fig.1B)1B) when αvβ5 binds to the multivalent ligand presented by the Ad penton base. The cryoEM structural analysis also indicates that integrin induces a conformational change in penton base.     

Relaxation-optimised Hartmann–Hahn transfer using a specifically Tailored MOCCA-XY16 mixing sequence for carbonyl–carbonyl correlation spectroscopy in <Superscript>13</Superscript>C direct detection NMR experiments

Isotropic mixing sequences are one of the key methods to achieve efficient coherence transfer. Among them, the MOCCA-XY16, which keeps the magnetization longitudinal for a significant amount of time, is characterised by favourable relaxation properties. We show here that its adapted version is particularly suited for carbonyl–carbonyl correlations in ¹³C direct detection NMR experiments.     

The essential role of lipids in Alzheimer's disease

In the absence of efficient diagnostic and therapeutic tools, Alzheimer's disease (AD) is a major public health concern due to longer life expectancy in the Western countries. Although the precise cause of AD is still unknown, soluble β-amyloid (Aβ) oligomers are considered the proximate effectors of the synaptic injury and neuronal death occurring in the early stages of AD. Aβ oligomers may directly interact with the synaptic membrane, leading to impairment of synaptic functions and subsequent signalling pathways triggering neurodegeneration. Therefore, membrane structure and lipid status should be considered determinant factors in Aβ-oligomer-induced synaptic and cell injuries, and therefore AD progression. Numerous epidemiological studies have highlighted close relationships between AD incidence and dietary patterns. Among the nutritional factors involved, lipids significantly influence AD pathogenesis. It is likely that maintenance of adequate membrane lipid content could prevent the production of Aβ peptide as well as its deleterious effects upon its interaction with synaptic membrane, thereby protecting neurons from Aβ-induced neurodegeneration. As major constituents of neuronal lipids, n-3 polyunsaturated fatty acids are of particular interest in the prevention of AD valuable diet ingredients whose neuroprotective properties could be essential for designing preventive nutrition-based strategies. In this review, we discuss the functional relevance of neuronal membrane features with respect to susceptibility to Aβ oligomers and AD pathogenesis, as well as the prospective capacities of lipids to prevent or to delay the disease.     

Minimal cut sets in biochemical reaction networks

MOTIVATION: Structural studies of metabolic networks yield deeper insight into topology, functionality and capabilities of the metabolisms of different organisms. Here, we address the analysis of potential failure modes in metabolic networks whose occurrence will render the network structurally incapable of performing certain functions. Such studies will help to identify crucial parts in the network structure and to find suitable targets for repressing undesired metabolic functions. RESULTS: We introduce the concept of minimal cut sets for biochemical networks. A minimal cut set (MCS) is a minimal (irreducible) set of reactions in the network whose inactivation will definitely lead to a failure in certain network functions. We present an algorithm which enables the computation of the MCSs in a given network related to user-defined objective reactions. This algorithm operates on elementary modes. A number of potential applications are outlined, including network verifications, phenotype predictions, assessing structural robustness and fragility, metabolic flux analysis and target identification in drug discovery. Applications are illustrated by the MCSs in the central metabolism of Escherichia coli for growth on different substrates. AVAILABILITY: Computation and analysis of MCSs is an additional feature of the FluxAnalyzer (freely available for academic users upon request, special contracts for industrial companies; see web page below). Supplementary information: http://www.mpi-magdeburg.mpg.de/projects/fluxanalyzer