Structural Basis for the ATP-Dependent Configuration of Adenylation Active Site in Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase [Protein Structure and Folding]

August 14th, 2015 by Chen, Y., Sun, Y., Song, H., Guo, Z.

o-Succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme targeted for development of novel antibiotics in the menaquinone biosynthesis. Using its crystal structures in a ligand-free form or in complex with nucleotides, a conserved pattern is identified in the interaction between ATP and adenylating enzymes of the family including acyl/aryl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and luciferases. It involves tight gripping interactions of the phosphate-binding loop (P-loop) with the ATP triphosphate moiety and an open-closed conformational change to form a compact adenylation active site. In MenE catalysis, this ATP-enzyme interaction creates a new binding site for the carboxylate substrate, allowing revelation of the determinants of substrate specificities and inline alignment of the two substrates for backside nucleophilic substitution reaction by molecular modeling. In addition, the ATP-enzyme interaction is suggested to play a crucial catalytic role by mutation of the P-loop residues hydrogen-bonded to ATP. Moreover, the ATP-enzyme interaction has also clarified the positioning and catalytic role of a conserved lysine residue in stabilization of the transition state. These findings provide new insights into the adenylation half-reaction in the domain alteration catalytic mechanism of the adenylate-forming enzymes.
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Unifying the DNA End Processing Roles of the Artemis Nuclease: Ku-Dependent Artemis Resection at Blunt DNA Ends [Enzymology]

August 14th, 2015 by Chang, H. H. Y., Watanabe, G., Lieber, M. R.

Artemis is a member of the metallo-β-lactamase protein family of nucleases. It is essential in vertebrates because during V(D)J recombination, the RAG complex generates hairpins when it creates the double-strand breaks at V, D, and J segments, and Artemis is required to open the hairpins so that they can be joined. Artemis is a diverse endo- and exonuclease, and a unified model for its wide range of nuclease properties has been challenging. Here we show that Artemis resects iteratively into blunt DNA ends with an efficiency that reflects the AT-richness of the DNA end. GC-rich ends are not cut by Artemis alone, due to a requirement for DNA end breathing (and confirmed using fixed pseudo-Y structures). All DNA ends are cut when both DNA-PKcs and Ku accompany Artemis, but not when Ku is omitted. This is the first biochemical data demonstrating a Ku-dependence of Artemis action on DNA ends of any configuration. The action of Artemis at blunt DNA ends is slower than at overhangs, consistent with a requirement for a slow DNA end breathing step preceding the cut. The AT-sequence dependence, the order of strand cutting, the length of the cuts, and the Ku-dependence of Artemis action at blunt ends can be reconciled with the other nucleolytic properties of both Artemis and Artemis:DNA-PKcs in a model incorporating DNA end breathing of blunt ends to form transient single- to double-strand boundaries that have structural similarities to hairpins and fixed 5′ and 3′ overhangs.

Mechanisms of Inhibition and Potentiation of {alpha}4{beta}2 Nicotinic Acetylcholine Receptors by Members of the Ly6 Protein Family [Neurobiology]

August 14th, 2015 by Wu, M., Puddifoot, C. A., Taylor, P., Joiner, W. J.

α4β2 nicotinic acetylcholine receptors (nAChRs) are abundantly expressed throughout the central nervous system and are thought to be the primary target of nicotine, the main addictive substance in cigarette smoking. Understanding the mechanisms by which these receptors are regulated may assist in developing compounds to selectively interfere with nicotine addiction. Here we report previously unrecognized modulatory properties of members of the Ly6 protein family on α4β2 nAChRs. Using a FRET-based Ca2+ flux assay, we found that the maximum response of α4β2 receptors to agonist was strongly inhibited by Ly6h and Lynx2 but potentiated by Ly6g6e. The mechanisms underlying these opposing effects appear to be fundamentally distinct. Receptor inhibition by Lynx2 was accompanied by suppression of α4β2 expression at the cell surface, even when assays were preceded by chronic exposure of cells to an established chaperone, nicotine. Receptor inhibition by Lynx2 also was resistant to pretreatment with extracellular phospholipase C, which cleaves lipid moieties like those that attach Ly6 proteins to the plasma membrane. In contrast, potentiation of α4β2 activity by Ly6g6e was readily reversible by pretreatment with phospholipase C. Potentiation was also accompanied by slowing of receptor desensitization and an increase in peak currents. Collectively our data support roles for Lynx2 and Ly6g6e in intracellular trafficking and allosteric potentiation of α4β2 nAChRs, respectively.
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The steroid hormone 20-hydroxyecdysone upregulates Ste-20 family serine/threonine kinase Hippo to induce programmed cell death [Gene Regulation]

August 13th, 2015 by Dong, D.-J., Jing, Y.-P., Liu, W., Wang, J.-X., Zhao, X.-F.

The steroid hormone 20-hydroxyecdysone (20E) and the serine/threonine Ste20-like kinase Hippo signal promote programmed cell death (PCD) during development, although the interaction between them remains unclear. Here, we present evidence that 20E upregulates Hippo to induce PCD during the metamorphic development of insects. We found that Hippo is involved in 20E-induced metamorphosis via promoting the phosphorylation and cytoplasmic retention of Yorkie (Yki), causing suppressed expression of the inhibitor of apoptosis (IAP), thereby releasing its inhibitory effect on caspase. Furthermore, we show that 20E induced the expression of Hippo at the transcriptional level through the ecdysone receptor (EcR), ultraspiracle protein (USP) and hormone receptor 3 (HR3). We also found that Hippo suppresses the binding of Yki complex to the HR3 promoter. In summary, 20E upregulates the transcription of Hippo via EcRB1, USP1 and HR3 to induce PCD, and Hippo has negative feedback effects on HR3 expression. These two signaling pathways coordinate PCD during insect metamorphosis.
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Genome-wide Mechanosensitive MicroRNA (MechanomiR) Screen Uncovers Dysregulation of their Regulatory Networks in the mdm Mouse Model of Muscular Dystrophy [Gene Regulation]

August 13th, 2015 by Mohamed, J. S., Hajira, A., Lopez, M. L., Boriek, A. M.

Muscular dystrophies (MDs) are a heterogeneous group of genetic and neuromuscular disorders, which result in severe loss of motor ability and skeletal muscle mass and function. Aberrant mechanotransduction and dysregulated-microRNA pathways are often associated with the progression of MD. Here, we hypothesized that dysregulation of mechanosensitive microRNAs (mechanomiRs) in dystrophic skeletal muscle play major roles in the progression of MD. To test our hypothesis, for the first time, we performed a genome-wide expression profile of anisotropically-regulated mechanomiRs and bioinformatically analyzed their target gene networks, and we assessed their roles in the advancement of MD using diaphragm muscles from wild-type and mdm (muscular dystrophy with myositis) mouse, an animal model of human tibial MD (titinopathy). We show that ex-vivo anisotropic mechanical stretch significantly alters the miRNA expression profile in diaphragm from WT and mdm mice, and as a result, some of the genes associated with MDs are dysregulated in mdm mice due to differential regulation of a distinct set of mechanomiRs. Interestingly, we found a contrasting expression pattern of the highly expressed let-7 family mechanomiRs let-7e-5p and miR-98-5p, and their target genes associated with extracellular matrix (ECM) and transforming growth factor-β signaling (TGF-β) pathways, respectively between WT and mdm mice. Gain- and loss-of-function analysis of let-7e-5p in myocytes isolated from the diaphragms of WT and mdm mice confirmed Col1a1, Col1a2, Col3a1, Col24a1, Col27a1, Itga1, Itga4, Scd1 and Thbs1 as target genes of let-7e-5p. Furthermore, we found that miR-98 negatively regulates myoblast differentiation. Our study therefore introducesanother biological player in the regulation of skeletal muscle structure and function that may contribute to unexplained disorders of MD.
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Allosteric Activation of Bacterial Swi2/Snf2 Protein RapA by RNA Polymerase: Biochemical and Structural Studies [Protein Structure and Folding]

August 13th, 2015 by

Members of the Swi2/Snf2 (switch/sucrose non-fermentable) family depend on their ATPase activity to mobilize nucleic acid-protein complexes for gene expression. In bacteria, RapA is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein that mediates RNAP recycling during transcription. It is known that the ATPase activity of RapA is stimulated by its interaction with RNAP. It is not known, however, how the RapA-RNAP interaction activates the enzyme. Previously, we determined the crystal structure of RapA. The structure revealed the dynamic nature of its N-terminal domain (Ntd), which prompted us to elucidate the solution structure and activity of both the full-length protein and its Ntd-truncated mutant (RapAΔN). Here, we report the ATPase activity of RapA and RapAΔN, in the absence or presence of RNAP, and the solution structures of RapA and RapAΔN, either ligand-free or in complex with RNAP. Determined by small-angle X-ray scattering, the solution structures reveal a new conformation of RapA, define the binding mode and binding site of RapA on RNAP, and show that the binding sites of RapA and σ70 on the surface of RNAP largely overlap. We conclude that the ATPase activity of RapA is inhibited by its Ntd but stimulated by RNAP in an allosteric fashion and that the conformational changes of RapA and its interaction with RNAP are essential for RNAP recycling. These and previous findings outline the functional cycle of RapA, which increases our understanding of the mechanism and regulation of Swi2/Snf2 proteins in general and of RapA in particular. The new structural information also leads to a hypothetical model of RapA in complex with RNAP immobilized during transcription.
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Degenerate CP1 Domain from Human Mitochondrial Leucyl-tRNA Synthetase [RNA]

August 13th, 2015 by

The connective polypeptide 1 (CP1) editing domain of leucyl-tRNA synthetase (LeuRS) from various species either harbors a conserved active site to exclude tRNA mis-charging with non-cognate amino acids or is evolutionarily truncated or lost because there is no requirement for high translational fidelity. However, human mitochondrial LeuRS (hmtLeuRS) contains a full-length but degenerate CP1 domain which has mutations in some residues important for post-transfer editing. The significance of such an inactive CP1 domain and a translational accuracy mechanism with different non-cognate amino acids are not completely understood. Herein, we identified the essential role of evolutionarily divergent CP1 domain in facilitating hmtLeuRS's catalytic efficiency and endowing enzyme with resistance to AN2690, broad-spectrum drug acting on LeuRSs. In addition, the canonical core of hmtLeuRS is not stringent for non-cognate norvaline (Nva) and valine (Val). HmtLeuRS has a very weak tRNA-independent pre-transfer editing activity for Nva, which is insufficient to remove mis-activated Nva. Besides, hmtLeuRS chimeras fused with a functional CP1 domain from LeuRSs of other species, regardless of origin, showed restored post-transfer editing activity, and acquired fidelity during aminoacylation. The present work offers a novel perspective on the role of the CP1 domain in optimizing aminoacylation efficiency.

The Cysteine Dioxygenase Homologue from Pseudomonas aeruginosa is a 3-Mercaptopropionate Dioxygenase [Enzymology]

August 13th, 2015 by

Thiol dioxygenation is the initial oxidation step that commits a thiol to important catabolic or biosynthetic pathways. The reaction is catalyzed by a family of specific non-heme mononuclear-iron proteins each of which is reported to react efficiently with only one substrate. This family of enzymes includes cysteine dioxygenase, cysteamine dioxygenase, mercaptosuccinate dioxygenase and 3-mercaptopropionate dioxygenase. Using sequence alignment to infer cysteine dioxygenase activity, a cysteine dioxygenase homologue from Pseudomonas aeruginosa (p3MDO) has been identified. Mass spectrometry of P. aeruginosa under standard growth conditions showed p3MDO is expressed in low levels suggesting that this metabolic pathway is available to the organism. Purified recombinant p3MDO is able to oxidize both cysteine and 3-mercaptopropionic acid (3-MPA) in vitro, with a marked preference for 3-MPA. We therefore describe this enzyme as a 3-mercaptopropionate dioxygenase. Mossbauer spectroscopy suggests that substrate binding to the ferrous iron is through the thiol but indicates each substrate could adopt different coordination geometries. Crystallographic comparison with mammalian cysteine dioxygenase shows that the overall active site geometry is conserved but suggests that the different substrate specificity can be related to replacement of an arginine by a glutamine in the active site.

A novel GLP-1 receptor interacting protein ATP6ap2 regulates insulin secretion in pancreatic beta cells [Metabolism]

August 13th, 2015 by

GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid (MYTH) assay to identify novel GLP1R interactors in both mouse and human islets. Among these, the V-type ATPase associated protein 6 (ATP6ap2), was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion, however, siRNA knockdown decreased both glucose stimulated- and GLP1-induced insulin secretion (GIIS). Decreases in GIIS were accompanied by attenuated GLP1 stimulated cAMP accumulation. Since ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knock-down but paradoxically increased proinsulin secretion. Importantly as GLP1R interactor, ATP6ap2 was required for GLP1 induced Ca2+ influx, in part explaining decreased insulin secretion in ATP6ap2 knock-down cells. Taken together our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signalling and insulin processing to affect insulin secretion.

Unliganded Fibroblast Growth Factor Receptor 1 Forms Density-Independent Dimers [Molecular Bases of Disease]

August 13th, 2015 by Comps-Agrar, L., Dunshee, D. R., Eaton, D. L., Sonoda, J.

Fibroblast growth factors receptors (FGFRs) are thought to initiate intracellular signaling cascades upon ligand-induced dimerization of the extracellular domain. Although the existence of unliganded FGFR1 dimers on the surface of living cells has been proposed, this notion remains rather controversial. Here, we employed time-resolved Forster resonance energy transfer (TR-FRET) combined with SNAP- and ACP-tag labeling in COS7 cells to monitor dimerization of full length FGFR1 at the cell-surface with or without the coreceptor βKlotho (hKLB). Using this approach, we observed homodimerization of unliganded FGFR1 that is independent of its surface density. The homo-interaction signal observed for FGFR1 was indeed as robust as that obtained for epidermal growth factor receptor (EGFR), and was further increased by addition of activating ligands or pathogenic mutations. Mutational analysis indicated that the kinase and the transmembrane domains, rather than the extracellular domain, mediate the ligand-independent FGFR1 dimerization. In addition, we observed a formation of a higher order ligand-independent complex by the c-spliced isoform of FGFR1 and KLB. Collectively, our approach provides novel insights into the assembly and dynamics of the full-length FGFRs on the cell surface.