Structural characterization of intracellular C-terminal domains of group III metabotropic glutamate receptors

Metabotropic glutamate receptors (mGluRs) are regulated by interacting proteins that mostly bind to their intracellular C-termini. Here, we investigated if mGluR6, mGluR7a and mGluR8a C-termini form predefined binding surfaces or if they were rather unstructured. Limited tryptic digest of purified peptides argued against the formation of stable globular folds. Circular dichroism, ¹H NMR and ¹H¹⁵N HSQC spectra indicated the absence of rigid secondary structure elements. Furthermore, we localized short linear binding motifs in the unstructured receptor domains. Our data provide evidence that protein interactions of the analyzed mGluR C-termini are mediated rather by short linear motifs than by preformed folds.     

Proteins within the seminal fluid are crucial to keep sperm viable in the honeybee Apis mellifera

Seminal fluid is a biochemically complex mixture of glandular secretions that is transferred to the females sexual tract as part of the ejaculate. Seminal fluid has received increasing scientific interest in the fields of evolutionary and reproductive biology, as it seems a major determinant of male fertility/infertility and reproductive success. Here we used the honeybee Apis mellifera, where seminal fluid can be collected as part of a male's ejaculate, and performed a series of experiments to investigate the effects of seminal fluid and its components on sperm viability. We show that honeybee seminal fluid is highly potent in keeping sperm alive and this positive effect is present over a 24 h time span, comparable to the timing of the sperm storage process in the queen. We furthermore show that the presence of proteins within the seminal fluid and their structural integrity are crucial for this effect. Finally, we activated sperm using fructose and provide evidence that the positive effect of seminal fluid proteins on sperm survival cannot be replicated using generic protein substitutes. Our data provide experimental insights into the complex molecular interplay between sperm and seminal fluid defining male fertility and reproductive success.     

Peptides derived from a distinct region of GB virus C glycoprotein E2 mediate strain-specific HIV-1 entry inhibition

The nonpathogenic human GB virus C (GBV-C), a member of the Flaviviridae, is highly prevalent in individuals with HIV-1 infections or with parenteral and sexual risk factors. Long-term GBV-C viremia has been associated with better survival or improved diagnosis in several epidemiological studies. In a previous study we reported that the E2 glycoprotein of GBV-C interferes with HIV-1 entry in vitro. To address the question what region of the E2 protein is involved in suppression of HIV-1 replication, we performed an E2-derived peptide scanning and determined the HIV-inhibitory activity of each peptide in HIV replication assays. We demonstrate here that peptides representing the N-terminal part of the E2 protein from amino acids (aa) 29 to 72 are able to inhibit efficiently HIV-1 replication in vitro. In particular, the peptides P6-2 (representing the E2-region from aa 45 to 64) and P4762 (aa 37 to 64) showed the highest potency in HIV replication assays performed on TZM-bl cells with 50% inhibitory concentrations between 0.1 and 2 μM. However, primary HIV-1 isolates representing clades A to H showed a high variability in their sensitivity to E2 peptides. Pseudovirus inhibition assays revealed that the sensitivity is determined by the gp120/gp41 envelope proteins. Using HIV-1 BlaM-Vpr-based fusion assays, we demonstrate that the E2-derived peptides prevent HIV-1 binding or fusion, presumably via interaction with the HIV-1 particle. Together, these findings reveal a new mechanism of viral interference, suggesting that the envelope protein E2 of GBV-C target directly HIV-1 particles to avoid entry of these virions.     

Cytoplasmic and periplasmic proteomic signatures of exponentially growing cells of the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125

The psychrophilic model bacterium Pseudoalteromonas haloplanktis is characterized by remarkably fast growth rates under low-temperature conditions in a range from 5°C to 20°C. In this study the proteome of cellular compartments, the cytoplasm and periplasm, of P. haloplanktis strain TAC125 was analyzed under exponential growth conditions at a permissive temperature of 16°C. By means of two-dimensional protein gel electrophoresis and mass spectrometry, a first inventory of the most abundant cytoplasmic and periplasmic proteins expressed in a peptone-supplemented minimal medium was established. By this approach major enzymes of the amino acid catabolism of this marine bacterium could be functionally deduced. The cytoplasmic proteome showed a predominance of amino acid degradation pathways and tricarboxylic acid (TCA) cycle enzymes but also the protein synthesis machinery. Furthermore, high levels of cold acclimation and oxidative stress proteins could be detected at this moderate growth temperature. The periplasmic proteome was characterized by a significant abundance of transporters, especially of highly expressed putative TonB-dependent receptors. This high capacity for protein synthesis, efficient amino acid utilization, and substrate transport may contribute to the fast growth rates of the copiotrophic bacterium P. haloplanktis in its natural environments.     

Immunomodulatory effects of viral TLR ligands on experimental asthma depend on the additive effects of IL-12 and IL-10

Based on epidemiological data, the hygiene hypothesis associates poor hygienic living conditions during childhood with a lower risk for the development of allergic diseases such as bronchial asthma. The role of viral infections, and especially of viral TLR ligands, within this context remains to be clarified. Viral TLR ligands involve dsRNA and ssRNA which are recognized by TLR-3 or TLR-7, respectively. In this study, we evaluated the impact of TLR-3 or TLR-7 activation on experimental asthma in mice. Systemic application of the synthetic TLR-3 or TLR-7 ligands polycytidylic-polyinosinic acid (p(I:C)) or R-848, respectively, during the sensitization phase prevented the production of OVA-specific IgE and IgG1 Abs and subsequently abolished all features of experimental asthma including airway hyperresponsiveness and allergic airway inflammation. Furthermore, administration of p(I:C) or R-848 to animals with already established primary allergic responses revealed a markedly reduced secondary response following allergen aerosol rechallenges. In contrast to wild-type animals, application of p(I:C) or R-848 to IL-12p35(-/-) mice had no effect on airway inflammation, goblet cell hyperplasia, and airway hyperresponsiveness. However, in the absence of IL-12, the numbers of eosinophils and lymphocytes in bronchoalveolar lavage fluids were still significantly reduced. These partial effects could also be abolished by neutralizing anti-IL-10 Abs in IL-12p35(-/-) mice. These data indicate that TLR-3 or TLR-7 activation by viral TLR ligands has both preventive as well as suppressive effects on experimental asthma which is mediated by the additive effects of IL-12 and IL-10.     

Evidence for a novel binding site conformer of aldose reductase in ligand-bound state

Human aldose reductase (ALR2) has evolved as a promising therapeutic target for the treatment of diabetic long-term complications. The binding site of this enzyme possesses two main subpockets: the catalytic anion-binding site and the hydrophobic specificity pocket. The latter can be observed in the open or closed state, depending on the bound ligand. Thus, it exhibits a pronounced capability for induced-fit adaptations, whereas the catalytic pocket exhibits rigid properties throughout all known crystal structures. Here, we determined two ALR2 crystal structures at 1.55 and 1.65 A resolution, each complexed with an inhibitor of the recently described naphtho[1,2-d]isothiazole acetic acid series. In contrast to the original design hypothesis based on the binding mode of tolrestat (1), both inhibitors leave the specificity pocket in the closed state. Unexpectedly, the more potent ligand (2) extends the catalytic pocket by opening a novel subpocket. Access to this novel subpocket is mainly attributed to the rotation of an indole moiety of Trp 20 by about 35 degrees . The newly formed subpocket provides accommodation of the naphthyl portion of the ligand. The second inhibitor, 3, differs from 2 only by an extended glycolic ester functionality added to one of its carboxylic groups. However, despite this slight structural modification, the binding mode of 3 differs dramatically from that of the first inhibitor, but provokes less pronounced induced-fit adaptations of the binding cavity. Thus, a novel binding site conformation has been identified in a region where previous complex structures suggested only low adaptability of the binding pocket. Furthermore, the two ligand complexes represent an impressive example of how the slight change of a chemically extended side-chain at a given ligand scaffold can result in a dramatically altered binding mode. In addition, our study emphasizes the importance of crystal structure analysis for the translation of affinity data into structure-activity relationships.     

Efficiency and role of loss processes in light-driven water oxidation by PSII

Its superior quantum efficiency renders PSII a model for biomimetic systems. However, also in biological water oxidation by PSII, the efficiency is restricted by recombination losses. By laser-flash illumination, the secondary radical pair, P680(+)Q(-) (A) (where P680 is the primary Chl donor in PSII and Q(A), primary quinone acceptor of PSII), was formed in close to 100% of the PSII. Investigation of the quantum efficiency (or yield) of the subsequent steps by time-resolved delayed (10 micros to 60 ms) and prompt (70 micros to 700 ms) Chl fluorescence measurements on PSII membrane particles suggests that (1) the effective rate for P680(+) Q(-) (A) recombination is approximately 5 ms(-1) with an activation energy of approximately 0.34 eV, circumstantially confirming dominating losses by reformation of the primary radical pair followed by ground-state recombination. (2) Because of compensatory influences on recombination and forward reactions, the efficiency is only weakly temperature dependent. (3) Recombination losses are several-fold enhanced at lower pH. (4) Calculation based on delayed-fluorescence data suggests that the losses depend on the state of the water-oxidizing manganese complex, being low in the S(0)-->S(1) and S(1)-->S(2) transition, clearly higher in S(2)-->S(3) and S(3)-->S(4)-->S(0). (5) For the used artificial electron acceptor, the efficiency is limited by acceptor-side processes/S-state decay at high/low photon-absorption rates resulting in optimal efficiency at surprisingly low rates of approximately 0.15-15 photons s(-1) (per PSII). The pH and S-state dependence can be rationalized by the basic model of alternate electron-proton removal proposed elsewhere. A physiological function of the recombination losses could be limitation of the lifetime of the reactive donor-side tyrosine radical (Y(.) (Z)) in the case of low-pH blockage of water oxidation.