A conserved structural motif for lipopolysaccharide recognition by procaryotic and eucaryotic proteins

BACKGROUND: Lipopolysaccharide (LPS), a lipoglycan from the outer membrane of Gram-negative bacteria, is an immunomodulatory molecule that stimulates the innate immune response. High levels of LPS cause excessive release of inflammatory mediators and are responsible for the septic shock syndrome. The interaction of LPS with its cognate binding proteins has not, as yet, been structurally elucidated. RESULTS: The X-ray crystallographic structure of LPS in complex with the integral outer membrane protein FhuA from Escherichia coli K-12 is reported. It is in accord with data obtained using mass spectroscopy and nuclear magnetic resonance. Most of the important hydrogen-bonding or electrostatic interactions with LPS are provided by eight positively charged residues of FhuA. Residues in a similar three-dimensional arrangement were searched for in all structurally known proteins using a fast template-matching algorithm, and a subset of four residues was identified that is common to known LPS-binding proteins. CONCLUSIONS: These four residues, three of which form specific interactions with lipid A, appear to provide the structural basis of pattern recognition in the innate immune response. Their arrangement can serve to identify LPS-binding sites on proteins known to interact with LPS, and could serve as a template for molecular modeling of a LPS scavenger designed to reduce the septic shock syndrome.     

The jump shot - a biomechanical analysis focused on lateral ankle ligaments

Handball is one of the top four athletic games with highest injury risks. The jump shot is the most accomplished goal shot technique and the lower extremities are mostly injured. As a basis for ankle sprain simulation, the aim of this study was to extend the ankle region of an existing musculoskeletal full-body model through incorporation of three prominent lateral ankle ligaments: ligamentum fibulotalare anterius (LFTA), ligamentum fibulotalare posterius (LFTP), ligamentum fibulocalcaneare (LFC). The specific objective was to calculate and visualise ligament force scenarios during the jumping and landing phases of controlled jump shots. Recorded kinematic data of performed jump shots and the corresponding ground reaction forces were used to perform inverse dynamics. The calculated peak force of the LFTA (107 N) was found at maximum plantarflexion and of the LFTP (150 N) at maximum dorsiflexion. The peak force of the LFC (190 N) was observed at maximum dorsiflexion combined with maximum eversion. Within the performed jump shots, the LFTA showed a peak force (59 N to 69 N) during maximum plantarflexion in the final moment of the lift off. During landing, the force developed by the LFTA reached its peak value (61 N to 70 N) at the first contact with the floor. After that, the LFTP developed a peak force (70 N to 118 N). This model allows the calculation of forces in lateral ankle ligaments. The information obtained in this study can serve as a basis for future research on ankle sprain and ankle sprain simulation.     

The Nogo receptor 2 is a novel substrate of Fbs1

Members of the Nogo66 receptor family (NgR) are closely associated with nerve growth inhibition and plasticity in the CNS. All three members, NgR1, NgR2 and NgR3, are GPI anchored and highly glycosylated proteins. The binding and signaling properties of NgR1 are well described, but largely unknown for NgR2. At present the only known ligands are myelin associated glycoprotein (MAG) and amyloid beta precursor protein (APP). Despite the requirement of co-receptors for signaling no other binding partner has been uncovered. To learn more about the interactome of NgR2 we performed pull down experiments and were able to identify F-box protein that recognizes sugar chain 1 (Fbs1) as binding partner. We confirmed this finding with co-immunoprecipitations and in vitro binding assays and showed that the binding is mediated by the substrate recognition domain of Fbs1. As a substrate recognition protein of the SCF complex, Fbs1 binding leads to polyubiquitination and finally degradation of its substrates. This is the first time a member of the Nogo receptor family has been connected with an intracellular degradation pathway, which has not only implications for its production, but also for amyloid deposition in Alzheimer's disease.     

Malaria parasite pre-erythrocytic infection: preparation meets opportunity

For those stricken with malaria, the classic clinical symptoms are caused by the parasite's cyclic infection of red blood cells. However, this erythrocytic phase of the parasite's life cycle initiates from an asymptomatic pre-erythrocytic phase: the injection of sporozoites via the bite of a parasite-carrying Anopheline mosquito, and the ensuing infection of the liver. With the increased capabilities of studying liver stages in mice, much progress has been made elucidating the cellular and molecular basis of the parasite's progression through this bottleneck of its life cycle. Here we review relevant findings on how sporozoites prepare for infection of the liver and factors crucial to liver stage development as well as key host/parasite interactions.     

Clinical and neurocognitive outcome in symptomatic isovaleric acidemia

Background

Approximately 20% of adrenoleukodystrophy (X-ALD) female carriers may develop clinical manifestations, typically consisting of progressive spastic gait, sensory deficits and bladder dysfunctions. A skewing in X Chromosome Inactivation (XCI), leading to the preferential expression of the X chromosome carrying the mutant ABCD1 allele, has been proposed as a mechanism influencing X-linked adrenoleukodystrophy (X-ALD) carrier phenotype, but reported data so far are conflicting.

Methods

To shed light into this topic we assessed the XCI pattern in peripheral blood mononuclear cells (PBMCs) of 30 X-ALD carriers. Since a frequent problem with XCI studies is the underestimation of skewing due to an incomplete sample digestion by restriction enzymes, leading to variable results, we developed a pyrosequencing assay to identify samples completely digested, on which to perform the XCI assay. Pyrosequencing was also used to quantify ABCD1 allele-specific expression. Moreover, very long-chain fatty acid (VLCFA) levels were determined in the same patients.

Results

We found severely (??90:10) or moderately (??75:25) skewed XCI in 23 out of 30 (77%) X-ALD carriers and proved that preferential XCI is mainly associated with the preferential expression of the mutant ABCD1 allele, irrespective of the manifestation of symptoms. The expression of mutant ABCD1 allele also correlates with plasma VLCFA concentrations.

Conclusions

Our results indicate that preferential XCI leads to the favored expression of the mutant ABCD1 allele. This emerges as a general phenomenon in X-ALD carriers not related to the presence of symptoms. Our data support the postulated growth advantage of cells with the preferential expression of the mutant ABCD1 allele, but argue against the use of XCI pattern, ABCD1 allele-specific expression pattern and VLCFA plasma concentration as biomarkers to predict the development of symptoms in X-ALD carriers.     

Designing and using RNA scaffolds to assemble proteins in vivo

RNA scaffolds are synthetic noncoding RNA molecules with engineered 3D folding harnessed to spatially organize proteins in vivo. Here we provide a protocol to design, express and characterize RNA scaffolds and their cognate proteins within 1 month. The RNA scaffold designs described here are based on either monomeric or multimeric units harboring RNA aptamers as protein docking sites. The scaffolds and proteins are cloned into inducible plasmids and expressed to form functional assemblies. RNA scaffolds find applications in many fields in which in vivo organization of biomolecules is of interest. RNA scaffolds provide extended flexibility compared with DNA or protein scaffolding strategies through programmed modulation of multiple protein stoichiometry and numbers, as well as the proteins' relative distances and spatial orientations. For synthetic biology, RNA scaffolds provide a new platform that can be used to increase yields of sequential metabolic pathways.     

Wood-inhabiting,polyporoid fungi in aspen-dominated forests managed for biomass in the U.S. Lake States

To better understand the potential long-term effects of biomass harvesting on biodiversity, the polyporoid fungi community was characterized from 120 plots in four aspen-dominated forests in Minnesota. Four deadwood variables (substratum species, substratum type, decay class and diameter class) were recorded for each polyporoid species occurrence. A total of 2 358 polyporoid occurrences, representing 86 species, were recorded on 16 tree species. Eight species (Trichaptum biforme, Bjerkandera adusta, Trametes hirsuta, Phellinus tremulae, Fomes fomentarius, Irpex lacteus, Fomitopsis ochracea and Antrodia serialis) made up 67 % of occurrences. Four polyporoid species (Funalia trogii, Pycnoporellus fulgens, Rigidoporus crocatus and Skeletocutis chrysella) are potentially rare and/or threatened in the Lake States. Non-metric multidimensional scaling and rarefaction curves demonstrated that small diameter substrata (especially those <5 cm) most strongly influenced polyporoid species occurrences. Aspen-dominated systems show great potential for biomass production, but these forests also support a species-rich community of polyporoid fungi, including potentially rare species.     

Automated and simultaneous two-dimensional micro-high-performance liquid chromatographic determination of proline and hydroxyproline enantiomers in mammals

A fully automated 2D-HPLC system employing a microbore-ODS column and a narrowbore-enantioselective column has been developed for the simultaneous enantiomer determination of proline, trans-4-hydroxyproline and cis-4-hydroxyproline in mammals. As a first dimension, a monolithic ODS column of 0.53 mm i.d. showed 3-6 times larger theoretical plate numbers than those of particle-packed ODS columns, and the best enantioseparations were obtained by a Chiralpak QN-2-AX column of 1.5 mm i.d. in the second dimension (separation factors: 1.44-1.83). The R.S.D. values for within-day and dayto-day precisions were less than 5.8%, and the lower limits of quantitation for the D-enantiomers were 1 fmol. The present method was successfully applied to the determination of proline and hydroxyproline enantiomers in the serum and collagen-rich skin tissue. Small amounts of D-proline were found both in the serum (1.57 +/- 0.19 nmol/mL) and in the skin (0.093 +/- 0.015 nmol/mg protein), while the amounts of D-hydroxyproline were smaller than the lower limit of quantitation.     

Identification and functional characterization of the 2-hydroxy fatty N-acyl-Delta3(E)-desaturase from Fusarium graminearum

Delta3(E)-unsaturated fatty acids are characteristic components of glycosylceramides from some fungi, including also human- and plant-pathogenic species. The function and genetic basis for this unsaturation is unknown. For Fusarium graminearum, which is pathogenic to grasses and cereals, we could show that the level of Delta3-unsaturation of glucosylceramide (GlcCer) was highest at low temperatures and decreased when the fungus was grown above 28 degrees C. With a bioinformatics approach, we identified a new family of polypeptides carrying the histidine box motifs characteristic for membrane-bound desaturases. One of the corresponding genes was functionally characterized as a sphingolipid-Delta3(E)-desaturase. Deletion of the candidate gene in F. graminearum resulted in loss of the Delta3(E)-double bond in the fatty acyl moiety of GlcCer. Heterologous expression of the corresponding cDNA from F. graminearum in the yeast Pichia pastoris led to the formation of Delta3(E)-unsaturated GlcCer.     

The third intracellular loop stabilizes the inactive state of the neuropeptide Y1 receptor

Constitutively active G-protein-coupled receptors (GPCRs) can signal even in the absence of ligand binding. Most Class I GPCRs are stabilized in the resting conformation by intramolecular interactions involving transmembrane domain (TM) 3 and TM6, particularly at loci 6.30 and 6.34 of TM6. Signaling by Gi/Go-coupled receptors such as the Neuropeptide Y1 receptor decreases already low basal metabolite levels. Thus, we examined constitutive activity using a biochemical assay mediated by a Gi/Gq chimeric protein and a more direct electrophysiological assay. Wild-type (WT-Y1) receptors express no measurable, agonist-independent activation, while mu-opioid receptors (MOR) and P2Y12 purinoceptors showed clear evidence of constitutive activation, especially in the electrophysiological assay. Neither point mutations at TM6 (T6.30A or N6.34A) nor substitution of the entire TM3 and TM6 regions from the MOR into the Y1 receptor increased basal WT-Y1 activation. By contrast, chimeric substitution of the third intracellular loop (ICL3) generated a constitutively active, Y1-ICL3-MOR chimera. Furthermore, the loss of stabilizing interactions from the native ICL3 enhanced the role of surrounding residues to permit basal receptor activation; because constitutive activity of the Y1-ICL3-MOR chimera was further increased by point mutation at locus 6.34, which did not alter WT-Y1 receptor activity. Our results indicate that the ICL3 stabilizes the Y1 receptor in the inactive state and confers structural properties critical for regulating Y receptor activation and signal transduction. These studies reveal the active participation of the ICL3 in the stabilization and activation of Class I GPCRs.