Antigenic determinants of the bilobal cockroach allergen Bla g 2 [Immunology]

December 7th, 2015 by

Bla g 2 is a major indoor cockroach allergen associated with the development of asthma. Antigenic determinants on Bla g 2 were analyzed by mutagenesis based on the structure of the allergen alone and in complex with monoclonal antibodies that interfere with IgE antibody binding. The structural analysis revealed mechanisms of allergen-antibody recognition through cation-π interactions. Single and multiple Bla g 2 mutants were expressed in Pichia pastoris and purified. The triple mutant K132A-K251A-F162Y showed a ~100 reduced capacity to bind IgE, while preserving the native molecular fold, as proven by X-ray crystallography. This mutant was still able to induce mast cell release. T-cell responses were assessed by analyzing Th1/Th2 cytokine production and CD4+ T-cell phenotype in PBMC cultures. Whereas T-cell activating capacity was similar for the KKF mutant and Bla g 2 based on CD25 expression, the KKF mutant was a weaker inducer of the Th2 cytokine IL-13. Furthermore, this mutant induced IL-10 from a non-T-cell source at higher levels that those induced by Bla g 2. Our findings demonstrate that a rational design of site-directed mutagenesis was effective in producing a mutant with only 3 amino acid substitutions that maintained the same fold as wild type Bla g 2. These residues, which were involved in IgE antibody binding, endowed Bla g 2 with a T cell modulatory capacity. The antigenic analysis of Bla g 2 will be useful for the subsequent development of recombinant allergen vaccines.

Evidence for conservation of the calcitonin superfamily and activity-regulating mechanisms in the basal chordate Branchiostoma floridae: insight into the molecular and functional evolution in chordates [Cell Biology]

December 7th, 2015 by

The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH2. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell-surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R-Bf-RAMP-LP complexes resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenmedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs.
  • Posted in Journal of Biological Chemistry, Publications
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Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II [Enzymology]

December 7th, 2015 by

Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate:ubiquinone oxidoreductase, SQR). Aerobically the bacterium synthesizes SQR as part of its respiratory chain whereas under microaerophilic conditions the quinol:fumarate reductase (QFR) can be utilized. All complex II enzymes harbor a covalently bound FAD cofactor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond or how it binds the flavoprotein subunit of complex II remains unknown. Using photocrosslinking we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation.

Protein 4.1G Regulates Cell Adhesion, Spreading and Migration of Mouse Embryo Fibroblasts through {beta}1 Integrin Pathway [Membrane Biology]

December 7th, 2015 by

Protein 4.1G is a membrane skeletal protein that can serve as adapter between transmembrane proteins and the underlying membrane skeleton. The function of 4.1G remains largely unexplored. Here, using 4.1G-knockout mouse embryonic fibroblasts (4.1G-/-MEF) as a model system, we explored the function of 4.1G in motile cells. We show that the adhesion, spreading and migration of 4.1G-/-MEF cells are significantly impaired. We further show that although the total cellular expression of β1 integrin is unchanged, the surface expression of β1-integrin as well as its active form are significantly decreased in 4.1G-/-MEF cells. Moreover, the phosphorylation of focal adhesion kinase, a downstream component of integrin mediated signal transduction pathway, is suppressed in 4.1G-/-MEF cells. Co-immunoprecipitation experiments and in vitro binding assays showed that 4.1G directly binds to β1 integrin via its membrane-binding domain. These findings identified a novel role of 4.1G in cell adhesion, spreading and migration in MEF cells by modulating the surface expression of β1 integrin and subsequent downstream signal transduction.
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Agonistic Human Antibodies Binding To Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism [Lipids]

December 7th, 2015 by

Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed since the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouseTM platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT/27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35 % increase of the high density lipoprotein cholesterol that was observed up to 32 days post 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease.

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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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.