Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA

Toll‐like receptor (TLR) 13 and TLR2 are the major sensors of Gram‐positive bacteria in mice. TLR13 recognizes Sa19, a specific 23S ribosomal (r) RNA‐derived fragment and bacterial modification of Sa19 ablates binding to TLR13, and to antibiotics such as erythromycin. Similarly, RNase A‐treated Staphylococcus aureus activate human peripheral blood mononuclear cells (PBMCs) only via TLR2, implying single‐stranded (ss) RNA as major stimulant. Here, we identify human TLR8 as functional TLR13 equivalent that promiscuously senses ssRNA. Accordingly, Sa19 and mitochondrial (mt) 16S rRNA sequence‐derived oligoribonucleotides (ORNs) stimulate PBMCs in a MyD88‐dependent manner. These ORNs, as well as S. aureus‐, Escherichia coli‐, and mt‐RNA, also activate differentiated human monocytoid THP‐1 cells, provided they express TLR8. Moreover, Unc93b1 ^−/−‐ and Tlr8 ^−/−‐THP‐1 cells are refractory, while endogenous and ectopically expressed TLR8 confers responsiveness in a UR/URR RNA ligand consensus motif‐dependent manner. If TLR8 function is inhibited by suppression of lysosomal function, antibiotic treatment efficiently blocks bacteria‐driven inflammatory responses in infected human whole blood cultures. Sepsis therapy might thus benefit from interfering with TLR8 function.     

Shifts in plant functional community composition under hydrological stress strongly decelerate litter decomposition

Litter decomposition is a key process of nutrient and carbon cycling in terrestrial ecosystems. The decomposition process will likely be altered under ongoing climate change, both through direct effects on decomposer activity and through indirect effects caused by changes in litter quality. We studied how hydrological change indirectly affects decomposition via plant functional community restructuring caused by changes in plant species’ relative abundances (community‐weighted mean (CWM) traits and functional diversity). We further assessed how those indirect litter quality effects compare to direct effects. We set up a mesocosm experiment, in which sown grassland communities and natural turf pieces were subjected to different hydrological conditions (dryness and waterlogging) for two growing seasons. Species‐level mean traits were obtained from trait databases and combined with species’ relative abundances to assess functional community restructuring. We studied decomposition of mixed litter from these communities in a common “litterbed.” These indirect effects were compared to effects of different hydrological conditions on soil respiration and on decomposition of standard litter (direct effects). Dryness reduced biomass production in sown communities and natural turf pieces, while waterlogging only reduced biomass in sown communities. Hydrological stress caused profound shifts in species’ abundances and consequently in plant functional community composition. Hydrologically stressed communities had higher CMW leaf dry matter content, lower CMW leaf nitrogen content, and lower functional diversity. Lower CWM leaf N content and functional diversity were strongly related to slower decomposition. These indirect effects paralleled direct effects, but were larger and longer‐lasting. Species mean traits from trait databases had therefore considerable predictive power for decomposition. Our results show that stressful soil moisture conditions, that are likely to occur more frequently in the future, quickly shift species’ abundances. The resulting functional community restructuring will decelerate decomposition under hydrological stress.     

Structural and Mechanistic Studies Reveal the Functional Role of Bicovalent Flavinylation in Berberine Bridge Enzyme

Berberine bridge enzyme (BBE) is a member of the recently discovered family of bicovalently flavinylated proteins. In this group of enzymes, the FAD cofactor is linked via its 8α-methyl group and the C-6 atom to conserved histidine and cysteine residues, His-104 and Cys-166 for BBE, respectively. 6-S-Cysteinylation has recently been shown to have a significant influence on the redox potential of the flavin cofactor; however, 8α-histidylation evaded a closer characterization due to extremely low expression levels upon substitution. Co-overexpression of protein disulfide isomerase improved expression levels and allowed isolation and purification of the H104A protein variant. To gain more insight into the functional role of the unusual dual mode of cofactor attachment, we solved the x-ray crystal structures of two mutant proteins, H104A and C166A BBE, each lacking one of the covalent linkages. Information from a structure of wild type enzyme in complex with the product of the catalyzed reaction is combined with the kinetic and structural characterization of the protein variants to demonstrate the importance of the bicovalent linkage for substrate binding and efficient oxidation. In addition, the redox potential of the flavin cofactor is enhanced additively by the dual mode of cofactor attachment. The reduced level of expression for the H104A mutant protein and the difficulty of isolating even small amounts of the protein variant with both linkages removed (H104A-C166A) also points toward a possible role of covalent flavinylation during protein folding.Since the discovery of the first known example of a covalent bond between a flavin cofactor and an amino acid side chain occurring in enzymes in the 1950s (1), a number of different types of linkages have been identified: 8α-histidylation (either to N1 or to N3), 8α-O-tyrosylation, 8α-S-cysteinylation, and 6-S-cysteinylation. For current reviews relating to these modes of flavin attachment, see Refs. 2 and 3. Recently, another way of covalent tethering of FAD to proteins was discovered in x-ray crystallographic studies on glucooligosaccharide oxidase (GOOX)⁴ from Acremonium strictum (4). The mode of flavin linkage observed in this case employs both 8α-histidylation and 6-S-cysteinylation to form a bicovalently attached cofactor. Representative members of all these groups have been studied in detail, and several explanations for the role of the covalent flavinylation have been put forward. Some of the suggestions tend to be rather specific for the system being studied, e.g. prevention of cofactor inactivation at the C-6 position for trimethylamine dehydrogenase (5) or facilitation of electron transfer from the flavin to the cytochrome subunit for p-cresol methylhydroxylase (6). Other explanations including the increase of the flavin redox potential due to the covalent linkage (7–9) and the prevention of cofactor dissociation (10, 11) were found for several enzymes also harboring different types of cofactor attachments. Taking into account that protein stability (12) and optimal binding of substrate molecules (11, 13) are also positively influenced by covalent tethering of the flavin, one might speculate that no generally applicable explanation for the covalent attachment of flavins to proteins exists. Therefore, it seems likely that the large variety of systems operating with one of the above mentioned modes of cofactor tethering might have evolved to also adapt to a diversity of enzymatic challenges.Berberine bridge enzyme (BBE) from Eschscholzia californica is a plant enzyme involved in alkaloid biosynthesis, catalyzing the challenging oxidative cyclization of (S)-reticuline to (S)-scoulerine (Scheme 1). This enzyme was recently shown to belong to the group of flavoenzymes with a bicovalently attached FAD (14). After the discovery of this unusual mode of linkage in the crystal structure of GOOX (4), several members of this group, all belonging to the vanillyl-alcohol oxidase family (15), were identified by biochemical methods (16–18) and also structural studies (19). Because some of the suggested benefits of a covalent cofactor attachment can easily be brought about by a single linkage, e.g. prevention of cofactor dissociation or stabilization of the tertiary structure, the two amino acids attached to FAD might have different and individual functions as well as an additive effect on physicochemical properties such as redox potentials or substrate binding and oxidation. To elucidate the relative importance for the overall enzymatic functioning of members of this group, more detailed studies have been performed on GOOX (11), chito-oligosaccharide oxidase (ChitO) from Fusarium graminearum (17), and BBE (20). Common results of these analyses show that the bicovalent FAD has a redox potential of about +130 mV, which is among the highest potentials reported for flavoenzymes. Replacement of one of the amino acids involved in anchoring of the cofactor generally reduces the rate of cofactor reduction and the steady-state turnover rate, but whether this can be directly linked to reduced redox potentials of these mutant proteins has been under debate (11).Open in a separate windowSCHEME 1.Overall reaction catalyzed by BBE.To address these issues further, we report the expression of the H104A mutant protein of BBE. A biochemical characterization of this protein variant with respect to the redox potential, transient kinetics, and steady-state analysis is combined with the structural analysis of both the H104A and the C166A mutant proteins. In addition, a structure of wild type (WT) BBE in complex with the product of the enzyme-catalyzed reaction is presented, which provides further insights toward the involvement of active site amino acids during the course of the reaction. Together with the recently reported x-ray crystal structure of WT BBE with and without substrate bound (21) and the biochemical characterization of the C166A mutant protein (20), these results provide interesting insights into the role of bicovalent FAD attachment in enzymes.     

Retracted: Tusk shells in trouble? Physiology and behavior in response to changing temperature in a scaphopod (Mollusca: Scaphopoda: Dentaliida)

Scaphopods (tusk shells) are infaunal marine predators that occur at locally high densities in coastal and deep‐sea mud habitats, and as consumers of foraminifera they are important in carbon cycling. We investigated oxygen metabolism and burying behavior of the scaphopod Rhabdus rectius and its responses to altered temperatures. These are the first measurements of oxygen uptake rates for any member of this taxonomic class. In response to elevated temperatures, oxygen uptake rates increased, but the ability of animals to bury themselves in sediment was compromised. Female scaphopods were significantly larger than males and, when corrected for body mass, oxygen uptake rates were consistently higher for female individuals than for males. This is consistent with previous anecdotal observations of females in other scaphopod species being larger and potentially more active. In conditions of declining oxygen availability, individuals of Rhabdus rectius showed strong oxyregulatory ability by maintaining the same oxygen uptake rate displayed in normoxic conditions. The ability to maintain normal metabolic functioning even in conditions of oxygen limitation would benefit a species living in a benthic environment that may be prone to temporary or transient anoxic events. Yet the decrease in normal escape response in moderately elevated temperatures indicates these animals may be at risk from rising sea temperatures.     

Transient receptor potential channel A1 is directly gated by calcium ions

Members of the superfamily of transient receptor potential (TRP) channels are proposed to play important roles in sensory physiology. As an excitatory ion channel TRPA1 is robustly activated by pungent irritants in mustard and garlic and is suggested to mediate the inflammatory actions of environmental irritants and proalgesic agents. Here, we demonstrate that, in addition to pungent natural compounds, Ca(2+) directly gates heterologously expressed TRPA1 in whole-cell and excised-patch recordings with an apparent EC(50) of 905 nm. Pharmacological experiments and site-directed mutagenesis indicate that the N-terminal EF-hand calcium-binding domain of the channel is involved in Ca(2+)-dependent activation. Furthermore, we determine Ca(2+) as prerequisite for icilin activity on TRPA1.     

Molecular Genetics of Cystinuria: Identification of Four New Mutations and Seven Polymorphisms, and Evidence for Genetic Heterogeneity

A cystinuria disease gene (rBAT) has been recently identified, and some mutations causing the disease have been described. The frequency of these mutations has been investigated in a large sample of 51 Italian and Spanish cystinuric patients. In addition, to identify new mutated alleles, genomic DNA has been analyzed by an accurate and sensitive method able to detect nucleotide changes. Because of the lack of information available on the genomic structure of rBAT gene, the study was carried out using the sequence data so far obtained by us. More than 70% of the entire coding sequence and 8 intron-exon boundaries have been analyzed. Four new mutations and seven intragenic polymorphisms have been detected. All mutations so far identified in rBAT belong only to cystinuria type I alleles, accounting for ~44% of all type I cystinuric chromosomes. Mutation M467T is the most common mutated allele in the Italian and Spanish populations. After analysis of 70% of the rBAT coding region, we have detected normal sequences in cystinuria type II and type III chromosomes. The presence of rBAT mutated alleles only in type I chromosomes of homozygous (type I/I) and heterozygous (type I/III) patients provides evidence for genetic heterogeneity where rBAT would be responsible only for type I cystinuria and suggests a complementation mechanism to explain the intermediate type I/type III phenotype.     

Neuroprotective effects of Argon are mediated via an ERK‐1/2 dependent regulation of heme‐oxygenase‐1 in retinal ganglion cells

Retinal ischemia and reperfusion injuries (R‐IRI) damage neuronal tissue permanently. Recently, we demonstrated that Argon exerts anti‐apoptotic and protective properties. The molecular mechanism remains unclear. We hypothesized that Argon inhalation exert neuroprotective effects in rats retinal ganglion cells (RGC) via an ERK‐1/2 dependent regulation of heat‐shock proteins. Inhalation of Argon (75 Vol%) was performed after R‐IRI on the rats′ left eyes for 1 h immediately or with delay. Retinal tissue was harvested after 24 h to analyze mRNA and protein expression of heat‐shock proteins ?70, ?90 and heme‐oxygenase‐1, mitogen‐activated protein kinases (p38, JNK, ERK‐1/2) and histological changes. To analyze ERK dependent effects, the ERK inhibitor PD98059 was applicated prior to Argon inhalation. RGC count was analyzed 7 days after injury. Statistics were performed using anova . Argon significantly reduced the R‐IRI‐affected heat‐shock protein expression (p < 0.05). While Argon significantly induced ERK‐1/2 expression (p < 0.001), inhibition of ERK‐1/2 before Argon inhalation resulted in significantly lower vital RGCs (p < 0.01) and increase in heme‐oxygenase‐1 (p < 0.05). R‐IRI‐induced RGC loss was reduced by Argon inhalation (p < 0.001). Immunohistochemistry suggested ERK‐1/2 activation in Müller cells. We conclude, that Argon treatment protects R‐IRI‐induced apoptotic loss of RGC via an ERK‐1/2 dependent regulation of heme‐oxygenase‐1.

     

M234Glu is a component of the proton sponge in the reaction center from photosynthetic bacteria

Bacterial reaction centers use light energy to couple the uptake of protons to the successive semi-reduction of two quinones, namely Q_A and Q_B. These molecules are situated symmetrically in regard to a non-heme iron atom. Four histidines and one glutamic acid, M234Glu, constitute the five ligands of this atom. By flash-induced absorption spectroscopy and delayed fluorescence we have studied in the M234EH and M234EL variants the role played by this acidic residue on the energetic balance between the two quinones as well as in proton uptake. Delayed fluorescence from the P⁺Q_A^? state (P is the primary electron donor) and temperature dependence of the rate of P⁺Q_A^? charge recombination that are in good agreement show that in the two RC variants, both Q_A^? and Q_B^? are destabilized by about the same free energy amount: respectively ～ 100 ± 5 meV and 90 ± 5 meV for the M234EH and M234EL variants, as compared to the WT. Importantly, in the M234EH and M234EL variants we observe a collapse of the high pH band (present in the wild-type reaction center) of the proton uptake amplitudes associated with formation of Q_A^? and Q_B^?. This band has recently been shown to be a signature of a collective behaviour of an extended, multi-entry, proton uptake network. M234Glu seems to play a central role in the proton sponge-like system formed by the RC protein.