Mitochondrial Calcium Uptake Modulates Synaptic Vesicle Endocytosis in Central Nerve Terminals [Cell Biology]

December 7th, 2015 by Marland, J. R. K., Hasel, P., Bonnycastle, K., Cousin, M. A.

Presynaptic calcium influx triggers synaptic vesicle (SV) exocytosis and modulates subsequent SV endocytosis. A number of calcium clearance mechanisms are present in central nerve terminals which regulate intracellular free calcium levels both during and after stimulation. During action potential stimulation mitochondria rapidly accumulate presynaptic calcium via the mitochondrial calcium uniporter (MCU). The role of mitochondrial calcium uptake in modulating SV recycling has been extensively debated; however a definitive conclusion has not been achieved. To directly address this question we manipulated the expression of the MCU channel subunit in primary cultures of neurons expressing a genetically-encoded reporter of SV turnover. Knockdown of MCU resulted in ablation of activity-dependent mitochondrial calcium uptake but had no effect on either the rate or extent of SV exocytosis. In contrast, the rate of SV endocytosis was increased in the absence of mitochondrial calcium uptake and slowed when MCU was overexpressed. MCU knockdown did not perturb activity-dependent increases in presynaptic free calcium, suggesting that SV endocytosis may be controlled by calcium accumulation and efflux from mitochondria in their immediate vicinity.

Global Kinetic Analysis of Mammalian E3 Reveals pH-dependent NAD+/NADH Regulation, Physiological Kinetic Reversibility, and Catalytic Optimum [Computational Biology]

December 7th, 2015 by Moxley, M. A., Beard, D. A., Bazil, J. N.

Mammalian E3 is an essential mitochondrial enzyme responsible for catalyzing the terminal reaction in the oxidative catabolism of several metabolites. E3 is a key regulator of metabolic fuel selection as a component of the pyruvate dehydrogenase complex (PDHc). E3 regulates PDHc activity by altering the affinity of pyruvate dehydrogenase kinase, an inhibitor of the enzyme complex, through changes in reduction and acetylation state of lipoamide moieties set by the NAD+/NADH ratio. Thus, an accurate kinetic model of E3 is needed to predict overall mammalian PDHc activity. Here we have combined numerous literature data sets and new equilibrium spectroscopic experiments with a multitude of independently collected forward and reverse steady-state kinetic assays using pig heart E3. The latter kinetic assays demonstrate a pH-dependent transition of NAD+ activation to inhibition, shown here, to our knowledge for the first time in a single consistent data set. Experimental data were analyzed to yield a thermodynamically constrained, four-redox-state model of E3 that simulates pH-dependent activation/inhibition and active site redox states for various conditions. The developed model was used to determine substrate/product conditions that give maximal E3 rates and show that, due to non-Michaelis-Menten behavior, the maximal flux is different compared to the classically defined kcat.
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Characterization of a novel intestinal glycerol-3-phosphate acyltransferase pathway and its role in lipid homeostasis [Metabolism]

December 7th, 2015 by

Dietary triglycerides (TG) are absorbed by the enterocytes of the small intestine following luminal hydrolysis into monacylglycerol and fatty acids. Prior to secretion on chylomicrons, these lipids are reesterified into TG, primarily through the monoacylglycerol pathway. However, targeted deletion of the primary murine monoacylglycerol acyltransferase does not quantitatively affect lipid absorption, suggesting the existence of alternative pathways. Therefore, we investigated the role of the glycerol-3-phosphate pathway in dietary lipid absorption. The expression of glycerol-3-phosphate acyltransferase (GPAT3) was examined throughout the small intestine. To evaluate the role for GPAT3 in lipid absorption, mice harboring a disrupted GPAT3 gene (Gpat3-/-) were subjected to an oral lipid challenge and fed western-type diet to characterize the role in lipid and cholesterol homeostasis. Additional mechanistic studies were performed in primary enterocytes. GPAT3 was abundantly expressed in the apical surface of enterocytes in the small intestine. Following an oral lipid bolus, Gpat3-/- mice exhibited attenuated plasma TG excursion and accumulated lipid in the enterocytes. Electron microscopy studies revealed a lack of lipids in the lamina propria and intercellular space in Gpat3-/- mice. Gpat3-/- enterocytes displayed a compensatory increase in the synthesis of phospholipid and cholesteryl ester. When fed a Western-type diet, hepatic TG and cholesteryl ester accumulation was significantly higher in Gpat3-/- mice compared to the wild-type mice, accompanied by elevated levels of alanine aminotransferase, a marker of liver injury. Dysregulation of bile acid metabolism was also evident in Gpat3-null mice. These studies identify GPAT3 as a novel enzyme involved in intestinal lipid metabolism.

Comparative Circadian Metabolomics reveal Differential Effects of Nutritional Challenge in the Serum and Liver [Molecular Bases of Disease]

December 7th, 2015 by

Diagnosis and therapeutic interventions in pathological conditions rely upon clinical monitoring of key metabolites in the serum. Recent studies show that a wide range of metabolic pathways is controlled by circadian rhythms whose oscillation is affected by nutritional challenges, underscoring the importance to assess a temporal window for clinical testing and thereby questioning the accuracy of the reading of critical pathological markers in circulation. We have been interested in studying the communication between peripheral tissues under metabolic homeostasis perturbation. Here we present a comparative circadian metabolomic analysis on serum and liver in mice under high fat diet. Our data reveal that the nutritional challenge induces a loss of serum metabolites rhythmicity compared to liver, indicating a circadian misalignment between the tissues analyzed. Importantly, our results show that the levels of serum metabolites do not reflect the circadian liver metabolic signature, nor the effect of nutritional challenge. This notion reveals the possibility that misleading reads of metabolites in circulation may result in misdiagnosis and improper treatments. Our findings also demonstrate a tissue-specific and time-dependent disruption of metabolic homeostasis in response to altered nutrition.

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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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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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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