The nicotine metabolite, cotinine, alters the assembly and trafficking of a subset of nicotinic acetylcholine receptors [Molecular Biophysics]

August 12th, 2015 by Fox, A. M., Moonschi, F. H., Richards, C. I.

Exposure to nicotine alters the trafficking and assembly of nicotinic receptors (nAChRs) leading to their upregulation on the plasma membrane. While the mechanism is not fully understood, nicotine-induced upregulation is believed to contribute to nicotine addiction. The effect of cotinine, the primary metabolite of nicotine, on nAChR trafficking and assembly has not been extensively investigated. We utilize a pH sensitive variant of GFP, super ecliptic pHluorin, to differentiate between intracellular nAChRs and those expressed on the plasma membrane to quantify changes resulting from cotinine and nicotine exposure. Similar to nicotine, exposure to cotinine increases the number of α4β2 receptors on the plasma membrane and causes a redistribution of intracellular receptors. In contrast to this, cotinine exposure down regulates α6β2β3 receptors. We also used single molecule fluorescence studies to show that cotinine and nicotine both alter the assembly of α4β2 receptors to favor the high sensitivity (α4)2(β2)3 stoichiometry.
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The cAMP Signaling Pathway and Direct PKA Phosphorylation Regulate Polycystin-2 (TRPP2) Channel Function [Signal Transduction]

August 12th, 2015 by

Polycystin-2 (PC2) is a TRP-type, Ca2+-permeable non-selective cation channel that plays an important role in Ca2+ signaling in renal and non-renal cells. The effect(s) of the cAMP pathway and kinase mediated phosphorylation of PC2 seem to be relevant to PC2 trafficking and its interaction with polycystin-1. However, the role of PC2 phosphorylation in channel function is still poorly defined. Here we reconstituted apical membranes of term human syncytiotrophoblast (hST), containing endogenous PC2 (PC2hst), and in vitro translated channel protein (PC2iv). Addition of the catalytic subunit of PKA increased by 566% the spontaneous PC2hst channel activity in the presence of ATP. Interestingly, 8-Br-cAMP also stimulated spontaneous PC2hst channel activity in the absence of the exogenous kinase. Either stimulation was inhibited by addition of alkaline phosphatase, which in turn, was reversed by the phosphatase inhibitor vanadate. Neither maneuver modified the single channel conductance but instead increased channel mean open time. PKA directly phosphorylated PC2, which increased the mean open time but not the single channel conductance of the channel. PKA phosphorylation did not modify either R742X truncated or S829A-mutant PC2iv channel function. The data indicate that the cAMP pathway regulates PC2-mediated cation transport in the hST. The relevant PKA site for PC2 channel regulation centers on a single residue Serine 829, in the carboxy terminus.

Transcriptional regulation of the astrocytic excitatory amino acid transporter 1 (EAAT1) via NF-kB and Yin Yang 1 (YY1) [Gene Regulation]

August 12th, 2015 by Karki, P., Kim, C., Smith, K., Son, D.-S., Aschner, M., Lee, E.

Astrocytic glutamate transporter excitatory amino acid transporter (EAAT)1, also known as glutamate aspartate transporter (GLAST) in rodents, is one of two glial glutamate transporters that are responsible for removing excess glutamate from synaptic clefts to prevent excitotoxic neuronal death. Despite its important role in neurophysiological functions, the molecular mechanisms of EAAT1 regulation at the transcriptional level remain to be established. Here, we report that NF-kB is a main positive transcription factor for EAAT1, supported by 1) EAAT1 contains two consensus sites for NF-kB, 2) mutation of NF-kB binding sites decreased EAAT1 promoter activity, and 3) activation of NF-kB increased, while inhibition of NF-kB decreased EAAT1 promoter activity and mRNA/protein levels. Epidermal growth factor (EGF) increased EAAT1 mRNA/protein levels and glutamate uptake via NF-kB. The transcription factor yin yang 1 (YY1) plays as a critical negative regulator of EAAT1, supported by 1) the EAAT1 promoter contains multiple consensus sites for YY1, 2) overexpression of YY1 decreased EAAT1 promoter activity and mRNA/protein levels, and 3) knockdown of YY1 increased EAAT1 promoter activity and mRNA/protein levels. Manganese (Mn) decreased EAAT1 expression via YY1. Epigenetic modifiers histone deacetylases (HDACs) served as co-repressors of YY1 to further decrease EAAT1 promoter activity, whereas inhibition of HDACs reversed Mn-induced decrease of EAAT1 expression. Taken together, our findings suggest that NF-kB is a critical positive regulator of EAAT1, mediating the stimulatory effects of EGF, while YY1 is a negative regulator of EAAT1 with HDACs as co-repressors, mediating the inhibitory effects of Mn on EAAT1 regulation.

Probing the Catalytic Mechanism of Copper Amine Oxidase from Arthrobacter globiformis with Halide Ions [Enzymology]

August 11th, 2015 by

The catalytic reaction of copper amine oxidase proceeds through a ping-pong mechanism comprising two half-reactions. In the initial half-reaction, the substrate amine reduces the Tyr-derived cofactor, topa quinone (TPQ), to an aminoresorcinol form (TPQamr) that is in equilibrium with a semiquinone radical (TPQsq) via an intramolecular electron transfer to the active-site copper. We have analyzed this reductive half-reaction in crystals of the copper amine oxidase from Arthrobacter globiformis. Anaerobic soaking of the crystals with an amine substrate shifted the equilibrium toward TPQsq in an ′on-copper′ conformation, in which the 4-OH group ligated axially to the copper center, which was probably reduced to Cu(I). When the crystals were soaked with substrate in the presence of halide ions, which act as uncompetitive and noncompetitive inhibitors with respect to the amine substrate and dioxygen, respectively, the equilibrium in the crystals shifted toward the ′off-copper′ conformation of TPQamr. The halide ion was bound to the axial position of the copper center, thereby preventing TPQamr from adopting the on-copper conformation. Furthermore, transient kinetic analyses in the presence of viscogen (glycerol) revealed that only the rate constant in the step of TPQamr/TPQsq interconversion is markedly affected by the viscogen, which probably perturbs the conformational change. These findings unequivocally demonstrate that TPQ undergoes large conformational changes during the reductive half-reaction.

Nucling, a novel apoptosis-associated protein, controls mammary gland involution by regulating NF-{kappa}B and STAT3 [Developmental Biology]

August 11th, 2015 by

Postpartum mammary gland involution is the physiological process by which the lactating gland returns to its pre−pregnant state. In rodent models, the microenvironment of mammary gland involution is sufficient to induce enhanced tumor cell growth, local invasion, and metastasis. Therefore, a deeper understanding of the physiological regulation of involution may provide in−depth information on breast cancer therapy. We herein identified Nucling as an important regulator of involution of the mammary gland. A knockout mouse model was generated and revealed that postpartum involution were impaired in mice lacking Nucling. Involution is normally associated with an increase in the activation of NF−κB and STAT3, which is required for the organized regulation of involution, and was observed in WT glands, but not in the absence of Nucling. Furthermore, the loss of Nucling led to the suppression of calpain−1, IL−6 and C/EBPδ− factors, which are known to be essential for normal involution. The number of M2 macrophages, which are crucial for epithelial cell death and adipocyte repopulation after weaning, was also reduced in Nucling−KO glands. Taken together, the results of the present study demonstrated that Nucling played an important role in mammary gland involution by regulating NF−κB and STAT3 signaling pathways.

Amino Acid Proximities in Two Sup35 Prion Strains Revealed by Chemical Cross-Linking [Microbiology]

August 11th, 2015 by Wong, S.-H., King, C.-Y.

Strains of the yeast prion [PSI] are different folding patterns of the same Sup35 protein, which stacks up periodically to form a prion fiber. Chemical cross-linking is employed here to probe different fiber structures assembled with a mutant Sup35 fragment. The photo-reactive cross-linker, p-benzoyl-L-phenylalanine (pBpa), was biosynthetically incorporated into bacterially prepared recombinant Sup(1-61)-GFP, containing the first 61 residues of Sup35, followed by the green fluorescent protein. Four methionine (Met) substitutions and two alanine (Ala) substitutions were introduced at fixed positions in Sup(1-61) to allow cyanogen bromide cleavage to facilitate subsequent mass spectrometry analysis. Amyloid fibers of pBpa and Met/Ala substituted Sup(1-61)-GFP were nucleated from purified yeast prion particles of two different strains, namely VK and VL, and shown to faithfully transmit specific strain characteristics to yeast expressing the wild type Sup35 protein. Intra- and inter-molecular cross-linking were distinguished by tandem mass spectrometry analysis on fibers seeded from solutions containing equal amount of 14N and 15N-labeled protein. Fibers propagating the VL strain type exhibited intra-molecular cross-linking between amino acid residues 3 and 28, as well as intra- and inter-molecular linking between 32 and 55. Inter- and intra-molecular cross-linking between residues 32 and 55 were detected in fibers propagating the VK strain type. Adjacencies of amino acid residues in space revealed by cross-linking were used to constrain possible chain-folds of different [PSI] strains.

Connexin Type and Fluorescent Protein-fusion Tag Determine Structural Stability of Gap Junction Plaques [Neurobiology]

August 11th, 2015 by Stout, R. F., Snapp, E. L., Spray, D. C.

Gap junctions (GJs) are made up of plaques of laterally clustered intercellular channels and the membranes in which the channels are embedded. Arrangement of channels within a plaque determines subcellular distribution of connexin binding partners and sites of intercellular signaling. Here, we report the discovery that some connexin types form plaque structures with strikingly different degrees of fluidity in the arrangement of the GJ channel sub-components of the GJ plaque. We uncovered this property of GJs by applying fluorescence recovery after photobleaching (FRAP) to GJs formed from connexins fused with fluorescent protein tags. We found that connexin (Cx) 26 (Cx26) and Cx30 GJs readily diffuse within the plaque structures while Cx43 GJs remain persistently immobile for more than 2 min after bleaching. The cytoplasmic COOH-terminus of Cx43 was required for stability of Cx43 plaque arrangement. We provide evidence that these qualitative differences in GJ arrangement stability reflect endogenous characteristics, with the caveat that the sizes of the GJs examined were necessarily large for these measurements. We also uncovered an unrecognized effect of non-monomerized fluorescent protein on the dynamically arranged GJs and the organization of plaques composed of multiple connexin-types. Together, these findings redefine our understanding of the GJ plaque structure and should be considered in future studies using fluorescent protein tags to probe dynamics of highly ordered protein complexes.

Bacterial Rotary Export ATPases are Allosterically Regulated by the Nucleotide Second Messenger Cyclic-di-GMP [Signal Transduction]

August 11th, 2015 by

The widespread second messenger molecule cyclic-di-GMP (cdG) regulates the transition from motile and virulent lifestyles to sessile, biofilm-forming ones in a wide range of bacteria. Many pathogenic and commensal bacterial-host interactions are known to be controlled by cdG signalling. While the biochemistry of cyclic-dinucleotide metabolism is well understood, much remains to be discovered about the downstream signalling pathways that induce bacterial responses upon cdG binding. As part of our ongoing research into the role of cdG signalling in plant-associated Pseudomonas species, we carried out an affinity-capture screen for cdG binding proteins in the model organism P. fluorescens SBW25. The flagella export AAA+ ATPase FliI was identified as a result of this screen, and subsequently shown to bind specifically to the cdG molecule, with a KD in the low micromolar range. The interaction between FliI and cdG appears to be very widespread. In addition to FliI homologs from diverse bacterial species, high-affinity binding was also observed for the type-III secretion system homolog HrcN and the type-VI ATPase ClpB2. Addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro. Finally, a combination of site-specific mutagenesis, mass spectrometry and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits. Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins.

Enhanced Molecular Mobility of Ordinarily Structured Regions Drives Polyglutamine Disease [Molecular Bases of Disease]

August 10th, 2015 by

Polyglutamine expansion is a hallmark of nine neurodegenerative diseases, with protein aggregation intrinsically linked to disease progression. While polyglutamine expansion accelerates protein aggregation, the misfolding process is actually instigated by flanking domains. For example, polyglutamine expansion in ataxin-3 allosterically triggers the aggregation of the catalytic Josephin domain. The molecular mechanism that underpins this allosteric aggregation trigger remains to be determined. Here, we establish that polyglutamine expansion increases the molecular mobility of two juxtaposed helices critical to ataxin-3 deubiquitinase activity. Within one of these helices, we identified a highly amyloidogenic sequence motif that instigates aggregation and forms the core of the growing fibril. Critically, by mutating residues within this key region we decrease local structural fluctuations to slow ataxin-3 aggregation. This provides significant insight, down to the molecular level, into how polyglutamine expansion drives aggregation, and explains the positive correlation between polyglutamine tract length, protein aggregation, and disease severity.

Heterologous expression of moss LHCSR1: the Chlorophyll a-xanthophyll pigment-protein complex catalyzing Non-Photochemical Quenching, in Nicotiana sp. [Plant Biology]

August 10th, 2015 by

Oxygenic photosynthetic organisms evolved mechanisms for thermal dissipation of energy absorbed in excess to prevent formation of reactive oxygen species. The major and fastest component, called Non-Photochemical Quenching, occurs within Photosystem II antenna system by the action of two essential LHC-like proteins: PSBS in plants and LHCSR in green algae and diatoms. In the evolutionary intermediate Physcomitrella patens, a moss, both gene products are active. These proteins, present in low amounts, are difficult to purify, preventing structural and functional analysis. Here, we report on the overexpression of the LHCSR1 protein from P. patens in the heterologous systems, Nicotiana benthamiana and Nicotiana tabacum, using transient and stable nuclear transformation. We show that the protein accumulated in both heterologous systems is in its mature form, localizes in the chloroplast thylakoid membranes and is correctly folded with Chlorophyll a and xanthophylls but without Chlorophyll b, an essential chromophore for plants and algal LHC proteins. Finally, we show that recombinant LHCSR1 is active in quenching in vivo implying the recombinant protein obtained is a good material for future structural and functional studies.
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