Differential {alpha}4(+)/(-){beta}2 Agonist Binding Site Contributions to {alpha}4{beta}2 Nicotinic Acetylcholine Receptor Function Within and Between Isoforms. [Neurobiology]

December 7th, 2015 by

Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries, and high vs. low agonist sensitivities, (HS & LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist binding sites in, and between, the isoforms have not been studied. We used a fully-linked subunit concatemeric nAChR approach to express uniform populations of HS- or LS-isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (-)face E-loop residues to their non-conserved α4 subunit counterparts, or vice-versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. ACh concentration-response curves and maximum function were measured using two-electrode voltage-clamp electrophysiology. Surface expression was measured with [125I]mAb 295 binding, and was used to define function/nAChR. If the α4(+)/(-)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly-differential outcomes within the HS-isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise-equivalent α4(+)/(-)β2 agonist sites modifies their contributions to nAChR activation, and that E-loop residues are an important contributor to this neighbor effect.
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Transferrin Receptor 1 Facilitates Poliovirus Permeation of Mouse Brain Capillary Endothelial Cells [Molecular Bases of Disease]

December 4th, 2015 by Mizutani, T., Ishizaka, A., Nihei, C.-i.

As a possible route for invasion of the central nervous system (CNS), circulating poliovirus (PV) in the blood is believed to traverse the blood-brain barrier (BBB), resulting in paralytic poliomyelitis. However, the underlying mechanism is poorly understood. In this study, we demonstrated that mouse transferrin receptor 1 (mTfR1) is responsible for PV attachment to the cell surface, allowing invasion into the CNS via BBB. PV interacts with the apical domain of mTfR1 on mouse brain capillary endothelial cells (MBEC4) in a dose-dependent manner, via its capsid protein (VP1). We found that F-G, G-H, and H-I loops in VP1 are important for this binding. However, C-D, D-E, and E-F loops in VP1-fused Venus proteins efficiently penetrate MBEC4 cells. These results imply that VP1 functional domain responsible for cell-attachment is different from that involved in viral permeation of the brain capillary endothelium. We observed that co-treatment of MBEC4 cells with excess PV particles but not dextran resulted in blockage of transferrin transport into cells. Using the transwell in vitro BBB model, transferrin co-treatment inhibited permeation of PV into MBEC4 cells and delayed further viral permeation via mTfR1 knockdown. Together, mTfR1 as a positive mediator of PV-host cell attachment and PV permeation of MBEC4 cells, our results indicate a novel role of TfR1 as a cellular receptor for human PV receptor (hPVR)/CD155-independent PV invasion of the CNS.

Munc13-4 is a Rab11-binding protein that regulates Rab11-positive vesicle trafficking and docking at the plasma membrane [Immunology]

December 4th, 2015 by

The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the upregulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium-binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis but the trafficking, upregulation and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane.

Structural basis of stereospecificity in the bacterial enzymatic cleavage of {beta}-aryl ether bonds in lignin [Protein Structure and Folding]

December 4th, 2015 by

Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second generation biofuels and the generation of valuable coproducts from lignins monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via betaaryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present X-ray crystal structures and biochemical characterization of the glutathione-dependent betaetherases, LigE and LigF from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathioneStransferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. As betaaryl ether bonds account for 50 to 70% of all inter-unit linkages in lignin, understanding the mechanism of enzymatic betaaryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.
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Severe Hypomyelination and Developmental Defects Are Caused in Mice Lacking Protein Arginine Methyltransferase 1 (PRMT1) in the Central Nervous System. [Developmental Biology]

December 4th, 2015 by

Protein arginine methyltransferase 1 (PRMT1) is involved in cell proliferation, DNA damage response, and transcriptional regulation. While PRMT1 is extensively expressed in the central nervous system (CNS) at embryonic and perinatal stages, the physiological role of PRMT1 was poorly understood. Here, to investigate the primary function of PRMT1 in the CNS, we generated CNS-specific PRMT1 knockout mice by Cre-loxP system. These mice exhibited post-natal growth retardation with tremors and most of them died in two weeks after birth. Brain histological analyses revealed the prominent cell reduction in the white matter tracts of the mutant mice. Furthermore, ultrastructural analysis demonstrated that myelin sheath was almost completely ablated in the CNS of these animals. In agreement with hypomyelination, we also observed that most major myelin proteins including MBP, CNPase, and MAG were dramatically decreased, although neuronal and astrocytic markers were preserved in the brain of CNS-specific PRMT1 knockout mice. These animals had reduced number of OLIG2+ oligodendrocyte lineage cells in the white matter. We found that expressions of transcription factors essential for oligodendrocyte specification and further maturation were significantly suppressed in the brain of the mutant mice. Our findings provide evidence that PRMT1 is required for CNS development, especially for oligodendrocyte maturation processes.
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Evidence for dual binding sites for DDT in Insect Sodium Channels [Computational Biology]

December 4th, 2015 by Du, Y., Nomura, Y., Zhorov, B. S., Dong, K.

DDT, the first organochlorine insecticide, and pyrethroid insecticides are sodium channel agonists. Although the use of DDT is banned in most of the world due to its detrimental impact on the ecosystem, indoor residual spraying of DDT is still recommended for malaria control in Africa. Development of resistance to DDT and pyrethroids is a serious global obstacle for managing disease vectors. Mapping DDT binding sites is necessary for understanding mechanisms of resistance and modulation of sodium channels by structurally different ligands. The pioneering model of the housefly sodium channel visualized the first receptor for pyrethroids, PyR1, in the II/III domain interface and suggested that DDT binds within PyR1. Previously we proposed the second pyrethroid receptor, PyR2, at the I/II domain interface. However, whether DDT binds to both PyRs remains unknown. Here, using computational docking of DDT into the Kv1.2-based mosquito sodium channel model, we predict that two DDT molecules can bind simultaneously within PyR1 and PyR2. The bulky trichloromethyl group of each DDT molecule fits snugly between four helices in the bent domain interface, while two p-chlorophenyl rings extend into two wings of the interface. Model-driven mutagenesis and electrophysiological analysis confirmed these propositions and revealed ten previously unknown DDT-sensing residues within PyR1 and PyR2. Our study proposes a dual DDT-receptor model and provides a structural background for rational development of new insecticides.

Mycobacterium tuberculosis WhiB3 responds to vacuolar pH- induced changes in mycothiol redox potential to modulate phagosomal maturation and virulence [Microbiology]

December 4th, 2015 by Mehta, M., Rajmani, R. S., Singh, A.

The ability of Mycobacterium tuberculosis (Mtb) to resist intraphagosomal stresses such as oxygen radicals and low pH is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major Mtb thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of MSH pathway subverted the expression of whiB3 along with other pH-specific genes in Mtb. Using a genetic biosensor of mycothiol redox potential (EMSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in EMSH of the Mtb population in a WhiB3-dependent manner. Data indicate that Mtb needs low pH as a signal to alter cytoplasmic EMSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependant production of polyketide lipids. Consistent with these observations, MtbΔwhiB3 displayed intramacrophage survival defect, which can be rescued by pharmacological inhibition of phagosomal acidification. Lastly, MtbΔwhiB3 exhibited marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in Mtb, and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in Mtb.
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Regulation of sphingolipid biosynthesis by the morphogenesis checkpoint kinase Swe1 [Enzymology]

December 3rd, 2015 by

Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1 regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle.

Interaction of the RcsB Response Regulator with Auxiliary Transcription Regulators in Eschericha coli [Microbiology]

December 3rd, 2015 by Pannen, D., Fabisch, M., Gausling, L., Schnetz, K.

The Rcs phosphorelay is a two-component signal transduction system that is induced by cell envelope stress. RcsB, the response regulator of this signaling system, is a pleiotropic transcription regulator, which is involved in the control of various stress responses, cell division, motility and biofilm formation. RcsB regulates transcription either as a homodimer or together with auxiliary regulators, such as RcsA, BglJ and GadE in Escherichia coli. In this study, we show that RcsB in addition forms heterodimers with MatA (=EcpR) and with DctR. Our data suggest that the MatA dependent transcription regulation is mediated by the MatA-RcsB heterodimer, and is independent of RcsB phosphorylation. Furthermore, we analyzed the relevance of amino acid residues of the active quintet that is coordinating phosphorylation, of conserved residues, as well as of surface exposed residues for activity of RcsB homo- and heterodimers. The data suggest that the activity of the phosphorylation-dependent dimers, such as RcsA-RcsB and RcsB-RcsB, is affected by mutation of residues in the vicinity of the phosphorylation site, suggesting that a phosphorylation-induced structural change modulates their activity. In contrast, the phosphorylation-independent heterodimers BglJ-RcsB and MatA-RcsB are affected by only very few mutations. Heterodimerization of RcsB with various auxiliary regulators and their differential dependence on phosphorylation thus adds an additional level of control to the Rcs system that is operating at the output level.

Biochemical and structural characterization of the interaction between the Siderocalin NGAL/LCN2 and the N-terminal domain of its endocytic receptor SLC22A17. [Cell Biology]

December 3rd, 2015 by

The neutrophil gelatinase associated lipocalin (NGAL, aslo known as LCN2) and its cellular receptor (LCN2-R) are involved in many physiological and pathological processes such as cell differentiation, apoptosis and inflammation. These pleiotropic functions mainly rely on NGALs siderophore mediated iron transport properties. However the molecular determinants underlying the interaction between NGAL and its cellular receptor remain largely unknown. Here, using solution-state biomolecular NMR in conjunction with other biophysical methods, we show that the N-terminal domain of LCN2-R is a soluble extracellular domain that is intrinsically disordered and interacts with NGAL preferentially in its apo-state to form a fuzzy complex. The relatively weak affinity (≈ 10μM) between hLCN2-R-NTD and apoNGAL suggests that the N-terminus on its own cannot account for the internalization of NGAL by LCN2-R. However, hLCN2-R-NTD could be involved in the fine-tuning of the interaction between NGAL and its cellular receptor, or in a biochemical mechanism allowing the receptor to discriminate between apo- and holo-NGAL.
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