Characterization of the Semiquinone Radical Stabilized by the Cytochrome aa3-600 Menaquinol Oxidase of Bacillus subtilis

Cytochrome aa₃-600 is one of the principle respiratory oxidases from Bacillus subtilis and is a member of the heme-copper superfamily of oxygen reductases. This enzyme catalyzes the two-electron oxidation of menaquinol and the four-electron reduction of O₂ to 2H₂O. Cytochrome aa₃-600 is of interest because it is a very close homologue of the cytochrome bo₃ ubiquinol oxidase from Escherichia coli, except that it uses menaquinol instead of ubiquinol as a substrate. One question of interest is how the proteins differ in response to the differences in structure and electrochemical properties between ubiquinol and menaquinol. Cytochrome bo₃ has a high affinity binding site for ubiquinol that stabilizes a ubi-semiquinone. This has permitted the use of pulsed EPR techniques to investigate the protein interaction with the ubiquinone. The current work initiates studies to characterize the equivalent site in cytochrome aa₃-600. Cytochrome aa₃-600 has been cloned and expressed in a His-tagged form in B. subtilis. After isolation of the enzyme in dodecylmaltoside, it is shown that the pure enzyme contains 1 eq of menaquinone-7 and that the enzyme stabilizes a mena-semiquinone. Pulsed EPR studies have shown that there are both similarities as well as significant differences in the interactions of the mena-semiquinone with cytochrome aa₃-600 in comparison with the ubi-semiquinone in cytochrome bo₃. Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa₃-600 in comparison with ubiquinone in cytochrome bo₃. In addition, the electronic structure of the semiquinone cyt aa₃-600 is more shifted toward the anionic form from the neutral state in cyt bo₃.     

Differential Functions of ApoER2 and Very Low Density Lipoprotein Receptor in Reelin Signaling Depend on Differential Sorting of the Receptors

ApoER2 and very low density lipoprotein (VLDL) receptor transmit the Reelin signal into target cells of the central nervous system. To a certain extent, both receptors can compensate for each other, and only the loss of both receptors results in the reeler phenotype, which is characterized by a gross defect in the architecture of laminated brain structures. Nevertheless, both receptors also have specific distinct functions, as corroborated by analyses of the subtle phenotypes displayed in mice lacking either ApoER2 or VLDL receptor. The differences in their function(s), however, have not been defined at the cellular level. Here, using a panel of chimeric receptors, we demonstrate that endocytosis of Reelin and the fate of the individual receptors upon stimulation are linked to their specific sorting to raft versus non-raft domains of the plasma membrane. VLDL receptor residing in the non-raft domain endocytoses and destines Reelin for degradation via the clathrin-coated pit/clathrin-coated vesicle/endosome pathway without being degraded to a significant extent. Binding of Reelin to ApoER2, a resident of rafts, leads to the production of specific receptor fragments with specific functions of their own and to degradation of ApoER2 via lysosomes. These features contribute to a receptor-specific fine tuning of the Reelin signal, leading to a novel model that emphasizes negative feedback loops specifically mediated by ApoER2 and VLDL receptor, respectively.     

Further insight into the roles of the glycans attached to human blood protein C inhibitor

Activation of T-cells triggers store-operated Ca²⁺ entry, which begins a signaling cascade leading to induction of appropriate gene expression and eventually lymphocyte proliferation and differentiation. The simultaneous enhancement of Fas ligand gene expression in activated cells allows the immune response to be limited by committing the activated cells to apoptosis. In apoptotic cells the store-operated calcium entry is significantly inhibited. It has been documented that moderate activation of Fas receptor may cause reversible inhibition of store-operated channels by ceramide released from hydrolyzed sphingomyelin. Here we show that activation of Fas receptor in T-cells results in caspase-dependent decrease of cellular STIM1 and Orai1 protein content. This effect may be responsible for the substantial inhibition of Ca²⁺ entry into Jurkat cells undergoing apoptosis. In turn, this inhibition might prevent overloading of cells with calcium and protect them against necrosis.     

Alpha interferon potently enhances the anti-human immunodeficiency virus type 1 activity of APOBEC3G in resting primary CD4 T cells

The interferon (IFN) system, including various IFNs and IFN-inducible gene products, is well known for its potent innate immunity against wide-range viruses. Recently, a family of cytidine deaminases, functioning as another innate immunity against retroviral infection, has been identified. However, its regulation remains largely unknown. In this report, we demonstrate that through a regular IFN-alpha/beta signal transduction pathway, IFN-alpha can significantly enhance the expression of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) in human primary resting but not activated CD4 T cells and the amounts of APOBEC3G associated with a low molecular mass. Interestingly, short-time treatments of newly infected resting CD4 T cells with IFN-alpha will significantly inactivate human immunodeficiency virus type 1 (HIV-1) at its early stage. This inhibition can be counteracted by APOBEC3G-specific short interfering RNA, indicating that IFN-alpha-induced APOBEC3G plays a key role in mediating this anti-HIV-1 process. Our data suggest that APOBEC3G is also a member of the IFN system, at least in resting CD4 T cells. Given that the IFN-alpha/APOBEC3G pathway has potent anti-HIV-1 capability in resting CD4 T cells, augmentation of this innate immunity barrier could prevent residual HIV-1 replication in its native reservoir in the post-highly active antiretroviral therapy era.     

Plant-microbe interactions to probe regulation of plant carbon metabolism

Plant growth and development is dependent on coordinated assimilate production, distribution and allocation. Application of biochemical and molecular techniques substantially contributed to a better understanding of these processes, although the underlying regulatory mechanisms are still not fully elucidated and attempts to improve crop yield by modulating carbon partitioning were only partially successful. Plant pathogens also interfere with source–sink interaction. To this end they have evolved a wide range of sophisticated strategies to allow their systemic spread, suppression of plant defence and induction of sink function to support nutrient acquisition for their growth. Studying compatible interactions of plants and pathogens like viruses, bacteria and fungi can be exploited to investigate different levels of source–sink regulation. The identification of microbial factors and their host targets involved in regulation of plant primary metabolism may allow developing novel strategies to increase crop yield. Here we will discuss recent studies on plant–microbe interactions aimed at elucidating mechanisms of compatibility.     

Additional lack of iNOS attenuates diastolic dysfunction in aged ET-1 transgenic mice

Endothelin-1 (ET-1) exhibits potent proinflammatory and profibrotic properties. Moreover, inflammation is a potent stimulus for inducible NO synthase (iNOS), which has been shown to contribute to cardiac injury. We thus hypothesized that ET-1-induced cardiac injury is attenuated by concomitant lack of iNOS. We established crossbred animals of ET-1 transgenic mice (ET+/+) and iNOS knockout mice (iNOS-/-). At 13 months of age, mice were allocated according to their genotype to one of 4 study groups: wild type (WT) controls (n=8); ET-1 transgenic (ET+/+) mice (n=10); iNOS knockout (iNOS-/-) mice (n=7); and crossbred (ET+/+ iNOS-/-) mice (n=15). Left ventricular function was determined in vivo by using a tip catheter. Animals were subsequently euthanized and hearts were harvested for weight assessment and histologic evaluation. No cardiac hypertrophy was present, as evidenced by similar mean cardiac weight and myocyte diameter in all groups. Cardiac perivascular fibrosis was significantly increased in ET+/+ and iNOS-/- groups versus WT, whereas ET+/+ iNOS-/- mice did not differ from WT. Regarding left ventricular function, plasma B-type natriuretic peptide was elevated in ET+/+ and iNOS-/- mice, but again in crossbred animals this effect was blunted. Heart catheterization revealed a significantly increased stiffness constant in both ET-overexpressing groups versus WT, but this increase was significantly attenuated in the ET+/+iNOS-/- group versus the ET+/+ group. Parameters indicating systolic heart failure (EF, cardiac output), however, were not different between all study groups. Our study demonstrates that ET transgenic mice develop left ventricular stiffening with subsequent diastolic dysfunction in a slow, age-dependent manner. Additional knock out of iNOS significantly attenuates cardiac injury. We thus conclude that ET-1-induced cardiac injury is at least partially mediated by iNOS.