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Updated: 4 years 31 weeks ago

Glucose–TOR signalling reprograms the transcriptome and activates meristems

Sun, 03/31/2013 - 00:00

Nature advance online publication 31 March 2013. doi:10.1038/nature12030

Authors: Yan Xiong, Matthew McCormack, Lei Li, Qi Hall, Chengbin Xiang & Jen Sheen

A pathogenic picornavirus acquires an envelope by hijacking cellular membranes

Sun, 03/31/2013 - 00:00

Nature advance online publication 31 March 2013. doi:10.1038/nature12029

Authors: Zongdi Feng, Lucinda Hensley, Kevin L. McKnight, Fengyu Hu, Victoria Madden, LiFang Ping, Sook-Hyang Jeong, Christopher Walker, Robert E. Lanford & Stanley M. Lemon

Animal viruses are broadly categorized structurally by the presence or absence of an envelope composed of a lipid-bilayer membrane, attributes that profoundly affect stability, transmission and immune recognition. Among those lacking an envelope, the Picornaviridae are a large and diverse family of positive-strand RNA viruses that includes hepatitis A virus (HAV), an ancient human pathogen that remains a common cause of enterically transmitted hepatitis. HAV infects in a stealth-like manner and replicates efficiently in the liver. Virus-specific antibodies appear only after 3–4 weeks of infection, and typically herald its resolution. Although unexplained mechanistically, both anti-HAV antibody and inactivated whole-virus vaccines prevent disease when administered as late as 2 weeks after exposure, when virus replication is well established in the liver. Here we show that HAV released from cells is cloaked in host-derived membranes, thereby protecting the virion from antibody-mediated neutralization. These enveloped viruses (‘eHAV’) resemble exosomes, small vesicles that are increasingly recognized to be important in intercellular communications. They are fully infectious, sensitive to extraction with chloroform, and circulate in the blood of infected humans. Their biogenesis is dependent on host proteins associated with endosomal-sorting complexes required for transport (ESCRT), namely VPS4B and ALIX. Whereas the hijacking of membranes by HAV facilitates escape from neutralizing antibodies and probably promotes virus spread within the liver, anti-capsid antibodies restrict replication after infection with eHAV, suggesting a possible explanation for prophylaxis after exposure. Membrane hijacking by HAV blurs the classic distinction between ‘enveloped’ and ‘non-enveloped’ viruses and has broad implications for mechanisms of viral egress from infected cells as well as host immune responses.

Manipulation of small Rho GTPases is a pathogen-induced process detected by NOD1

Sun, 03/31/2013 - 00:00

Nature advance online publication 31 March 2013. doi:10.1038/nature12025

Authors: A. Marijke Keestra, Maria G. Winter, Josef J. Auburger, Simon P. Fräßle, Mariana N. Xavier, Sebastian E. Winter, Anita Kim, Victor Poon, Mariëtta M. Ravesloot, Julian F. T. Waldenmaier, Renée M. Tsolis, Richard A. Eigenheer & Andreas J. Bäumler

Our innate immune system distinguishes microbes from self by detecting conserved pathogen-associated molecular patterns. However, these are produced by all microbes, regardless of their pathogenic potential. To distinguish virulent microbes from those with lower disease-causing potential the innate immune system detects conserved pathogen-induced processes, such as the presence of microbial products in the host cytosol, by mechanisms that are not fully resolved. Here we show that NOD1 senses cytosolic microbial products by monitoring the activation state of small Rho GTPases. Activation of RAC1 and CDC42 by bacterial delivery or ectopic expression of SopE, a virulence factor of the enteric pathogen Salmonella, triggered the NOD1 signalling pathway, with consequent RIP2 (also known as RIPK2)-mediated induction of NF-κB-dependent inflammatory responses. Similarly, activation of the NOD1 signalling pathway by peptidoglycan required RAC1 activity. Furthermore, constitutively active forms of RAC1, CDC42 and RHOA activated the NOD1 signalling pathway. Our data identify the activation of small Rho GTPases as a pathogen-induced process sensed through the NOD1 signalling pathway.

Crystal structure of a eukaryotic phosphate transporter

Sun, 03/31/2013 - 00:00

Nature advance online publication 31 March 2013. doi:10.1038/nature12042

Authors: Bjørn P. Pedersen, Hemant Kumar, Andrew B. Waight, Aaron J. Risenmay, Zygy Roe-Zurz, Bryant H. Chau, Avner Schlessinger, Massimiliano Bonomi, William Harries, Andrej Sali, Atul K. Johri & Robert M. Stroud

Phosphate is crucial for structural and metabolic needs, including nucleotide and lipid synthesis, signalling and chemical energy storage. Proton-coupled transporters of the major facilitator superfamily (MFS) are essential for phosphate uptake in plants and fungi, and also have a function in sensing external phosphate levels as transceptors. Here we report the 2.9 Å structure of a fungal (Piriformospora indica) high-affinity phosphate transporter, PiPT, in an inward-facing occluded state, with bound phosphate visible in the membrane-buried binding site. The structure indicates both proton and phosphate exit pathways and suggests a modified asymmetrical ‘rocker-switch’ mechanism of phosphate transport. PiPT is related to several human transporter families, most notably the organic cation and anion transporters of the solute carrier family (SLC22), which are implicated in cancer-drug resistance. We modelled representative cation and anion SLC22 transporters based on the PiPT structure to surmise the structural basis for substrate binding and charge selectivity in this important family. The PiPT structure demonstrates and expands on principles of substrate transport by the MFS transporters and illuminates principles of phosphate uptake in particular.

Techniques: 3D imaging of crystal defects

Wed, 03/27/2013 - 00:00

Nature advance online publication 27 March 2013. doi:10.1038/nature12089

Author: Patrick J. McNally

A clever combination of existing techniques has produced three-dimensional atomic images of individual platinum nanoparticles, disclosing the atomic structure of crystal defects within them.

Microbiology: Intraterrestrial lifestyles

Wed, 03/27/2013 - 00:00

Nature advance online publication 27 March 2013. doi:10.1038/nature12088

Author: David L. Valentine

Genome sequencing of cells plucked from marine sediments reveals metabolic details for two abundant lineages of Archaea. These microorganisms may play a key part in breaking down protein buried deep inside Earth.

Structures of protein–protein complexes involved in electron transfer

Wed, 03/27/2013 - 00:00

Nature advance online publication 27 March 2013. doi:10.1038/nature11996

Authors: Svetlana V. Antonyuk, Cong Han, Robert R. Eady & S. Samar Hasnain

Electron transfer reactions are essential for life because they underpin oxidative phosphorylation and photosynthesis, processes leading to the generation of ATP, and are involved in many reactions of intermediary metabolism. Key to these roles is the formation of transient inter-protein electron transfer complexes. The structural basis for the control of specificity between partner proteins is lacking because these weak transient complexes have remained largely intractable for crystallographic studies. Inter-protein electron transfer processes are central to all of the key steps of denitrification, an alternative form of respiration in which bacteria reduce nitrate or nitrite to N2 through the gaseous intermediates nitric oxide (NO) and nitrous oxide (N2O) when oxygen concentrations are limiting. The one-electron reduction of nitrite to NO, a precursor to N2O, is performed by either a haem- or copper-containing nitrite reductase (CuNiR) where they receive an electron from redox partner proteins a cupredoxin or a c-type cytochrome. Here we report the structures of the newly characterized three-domain haem-c-Cu nitrite reductase from Ralstonia pickettii (RpNiR) at 1.01 Å resolution and its M92A and P93A mutants. Very high resolution provides the first view of the atomic detail of the interface between the core trimeric cupredoxin structure of CuNiR and the tethered cytochrome c domain that allows the enzyme to function as an effective self-electron transfer system where the donor and acceptor proteins are fused together by genomic acquisition for functional advantage. Comparison of RpNiR with the binary complex of a CuNiR with a donor protein, AxNiR-cytc551 (ref. 6), and mutagenesis studies provide direct evidence for the importance of a hydrogen-bonded water at the interface in electron transfer. The structure also provides an explanation for the preferential binding of nitrite to the reduced copper ion at the active site in RpNiR, in contrast to other CuNiRs where reductive inactivation occurs, preventing substrate binding.

Proteolytic elimination of N-myristoyl modifications by the Shigella virulence factor IpaJ

Wed, 03/27/2013 - 00:00

Nature advance online publication 27 March 2013. doi:10.1038/nature12004

Authors: Nikolay Burnaevskiy, Thomas G. Fox, Daniel A. Plymire, James M. Ertelt, Bethany A. Weigele, Andrey S. Selyunin, Sing Sing Way, Steven M. Patrie & Neal M. Alto

Protein N-myristoylation is a 14-carbon fatty-acid modification that is conserved across eukaryotic species and occurs on nearly 1% of the cellular proteome. The ability of the myristoyl group to facilitate dynamic protein–protein and protein–membrane interactions (known as the myristoyl switch) makes it an essential feature of many signal transduction systems. Thus pathogenic strategies that facilitate protein demyristoylation would markedly alter the signalling landscape of infected host cells. Here we describe an irreversible mechanism of protein demyristoylation catalysed by invasion plasmid antigen J (IpaJ), a previously uncharacterized Shigella flexneri type III effector protein with cysteine protease activity. A yeast genetic screen for IpaJ substrates identified ADP-ribosylation factor (ARF)1p and ARF2p, small molecular mass GTPases that regulate cargo transport through the Golgi apparatus. Mass spectrometry showed that IpaJ cleaved the peptide bond between N-myristoylated glycine-2 and asparagine-3 of human ARF1, thereby providing a new mechanism for host secretory inhibition by a bacterial pathogen. We further demonstrate that IpaJ cleaves an array of N-myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function. Taken together, these findings show a previously unrecognized pathogenic mechanism for the site-specific elimination of N-myristoyl protein modification.

Three-dimensional imaging of dislocations in a nanoparticle at atomic resolution

Wed, 03/27/2013 - 00:00

Nature advance online publication 27 March 2013. doi:10.1038/nature12009

Authors: Chien-Chun Chen, Chun Zhu, Edward R. White, Chin-Yi Chiu, M. C. Scott, B. C. Regan, Laurence D. Marks, Yu Huang & Jianwei Miao

Dislocations and their interactions strongly influence many material properties, ranging from the strength of metals and alloys to the efficiency of light-emitting diodes and laser diodes. Several experimental methods can be used to visualize dislocations. Transmission electron microscopy (TEM) has long been used to image dislocations in materials, and high-resolution electron microscopy can reveal dislocation core structures in high detail, particularly in annular dark-field mode. A TEM image, however, represents a two-dimensional projection of a three-dimensional (3D) object (although stereo TEM provides limited information about 3D dislocations). X-ray topography can image dislocations in three dimensions, but with reduced resolution. Using weak-beam dark-field TEM and scanning TEM, electron tomography has been used to image 3D dislocations at a resolution of about five nanometres (refs 15, 16). Atom probe tomography can offer higher-resolution 3D characterization of dislocations, but requires needle-shaped samples and can detect only about 60 per cent of the atoms in a sample. Here we report 3D imaging of dislocations in materials at atomic resolution by electron tomography. By applying 3D Fourier filtering together with equal-slope tomographic reconstruction, we observe nearly all the atoms in a multiply twinned platinum nanoparticle. We observed atomic steps at 3D twin boundaries and imaged the 3D core structure of edge and screw dislocations at atomic resolution. These dislocations and the atomic steps at the twin boundaries, which appear to be stress-relief mechanisms, are not visible in conventional two-dimensional projections. The ability to image 3D disordered structures such as dislocations at atomic resolution is expected to find applications in materials science, nanoscience, solid-state physics and chemistry.

A solution to release twisted DNA during chromosome replication by coupled DNA polymerases

Wed, 03/27/2013 - 00:00

Nature advance online publication 27 March 2013. doi:10.1038/nature11988

Authors: Isabel Kurth, Roxana E. Georgescu & Mike E. O’Donnell

Chromosomal replication machines contain coupled DNA polymerases that simultaneously replicate the leading and lagging strands. However, coupled replication presents a largely unrecognized topological problem. Because DNA polymerase must travel a helical path during synthesis, the physical connection between leading- and lagging-strand polymerases causes the daughter strands to entwine, or produces extensive build-up of negative supercoils in the newly synthesized DNA. How DNA polymerases maintain their connection during coupled replication despite these topological challenges is unknown. Here we examine the dynamics of the Escherichia coli replisome, using ensemble and single-molecule methods, and show that the replisome may solve the topological problem independent of topoisomerases. We find that the lagging-strand polymerase frequently releases from an Okazaki fragment before completion, leaving single-strand gaps behind. Dissociation of the polymerase does not result in loss from the replisome because of its contact with the leading-strand polymerase. This behaviour, referred to as ‘signal release’, had been thought to require a protein, possibly primase, to pry polymerase from incompletely extended DNA fragments. However, we observe that signal release is independent of primase and does not seem to require a protein trigger at all. Instead, the lagging-strand polymerase is simply less processive in the context of a replisome. Interestingly, when the lagging-strand polymerase is supplied with primed DNA in trans, uncoupling it from the fork, high processivity is restored. Hence, we propose that coupled polymerases introduce topological changes, possibly by accumulation of superhelical tension in the newly synthesized DNA, that cause lower processivity and transient lagging-strand polymerase dissociation from DNA.

Predominant archaea in marine sediments degrade detrital proteins

Wed, 03/27/2013 - 00:00

Nature advance online publication 27 March 2013. doi:10.1038/nature12033

Authors: Karen G. Lloyd, Lars Schreiber, Dorthe G. Petersen, Kasper U. Kjeldsen, Mark A. Lever, Andrew D. Steen, Ramunas Stepanauskas, Michael Richter, Sara Kleindienst, Sabine Lenk, Andreas Schramm & Bo Barker Jørgensen

Half of the microbial cells in the Earth’s oceans are found in sediments. Many of these cells are members of the Archaea, single-celled prokaryotes in a domain of life separate from Bacteria and Eukaryota. However, most of these archaea lack cultured representatives, leaving their physiologies and placement on the tree of life uncertain. Here we show that the uncultured miscellaneous crenarchaeotal group (MCG) and marine benthic group-D (MBG-D) are among the most numerous archaea in the marine sub-sea floor. Single-cell genomic sequencing of one cell of MCG and three cells of MBG-D indicated that they form new branches basal to the archaeal phyla Thaumarchaeota and Aigarchaeota, for MCG, and the order Thermoplasmatales, for MBG-D. All four cells encoded extracellular protein-degrading enzymes such as gingipain and clostripain that are known to be effective in environments chemically similar to marine sediments. Furthermore, we found these two types of peptidase to be abundant and active in marine sediments, indicating that uncultured archaea may have a previously undiscovered role in protein remineralization in anoxic marine sediments.

Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway

Wed, 03/27/2013 - 00:00

Nature advance online publication 27 March 2013. doi:10.1038/nature12040

Authors: Jaekyoung Son, Costas A. Lyssiotis, Haoqiang Ying, Xiaoxu Wang, Sujun Hua, Matteo Ligorio, Rushika M. Perera, Cristina R. Ferrone, Edouard Mullarky, Ng Shyh-Chang, Ya’an Kang, Jason B. Fleming, Nabeel Bardeesy, John M. Asara, Marcia C. Haigis, Ronald A. DePinho, Lewis C. Cantley & Alec C. Kimmelman

Cancer cells have metabolic dependencies that distinguish them from their normal counterparts. Among these dependencies is an increased use of the amino acid glutamine to fuel anabolic processes. Indeed, the spectrum of glutamine-dependent tumours and the mechanisms whereby glutamine supports cancer metabolism remain areas of active investigation. Here we report the identification of a non-canonical pathway of glutamine use in human pancreatic ductal adenocarcinoma (PDAC) cells that is required for tumour growth. Whereas most cells use glutamate dehydrogenase (GLUD1) to convert glutamine-derived glutamate into α-ketoglutarate in the mitochondria to fuel the tricarboxylic acid cycle, PDAC relies on a distinct pathway in which glutamine-derived aspartate is transported into the cytoplasm where it can be converted into oxaloacetate by aspartate transaminase (GOT1). Subsequently, this oxaloacetate is converted into malate and then pyruvate, ostensibly increasing the NADPH/NADP+ ratio which can potentially maintain the cellular redox state. Importantly, PDAC cells are strongly dependent on this series of reactions, as glutamine deprivation or genetic inhibition of any enzyme in this pathway leads to an increase in reactive oxygen species and a reduction in reduced glutathione. Moreover, knockdown of any component enzyme in this series of reactions also results in a pronounced suppression of PDAC growth in vitro and in vivo. Furthermore, we establish that the reprogramming of glutamine metabolism is mediated by oncogenic KRAS, the signature genetic alteration in PDAC, through the transcriptional upregulation and repression of key metabolic enzymes in this pathway. The essentiality of this pathway in PDAC and the fact that it is dispensable in normal cells may provide novel therapeutic approaches to treat these refractory tumours.

Structural basis for the drug extrusion mechanism by a MATE multidrug transporter

Wed, 03/27/2013 - 00:00

Nature advance online publication 27 March 2013. doi:10.1038/nature12014

Authors: Yoshiki Tanaka, Christopher J. Hipolito, Andrés D. Maturana, Koichi Ito, Teruo Kuroda, Takashi Higuchi, Takayuki Katoh, Hideaki E. Kato, Motoyuki Hattori, Kaoru Kumazaki, Tomoya Tsukazaki, Ryuichiro Ishitani, Hiroaki Suga & Osamu Nureki

Multidrug and toxic compound extrusion (MATE) family transporters are conserved in the three primary domains of life (Archaea, Bacteria and Eukarya), and export xenobiotics using an electrochemical gradient of H+ or Na+ across the membrane. MATE transporters confer multidrug resistance to bacterial pathogens and cancer cells, thus causing critical reductions in the therapeutic efficacies of antibiotics and anti-cancer drugs, respectively. Therefore, the development of MATE inhibitors has long been awaited in the field of clinical medicine. Here we present the crystal structures of the H+-driven MATE transporter from Pyrococcus furiosus in two distinct apo-form conformations, and in complexes with a derivative of the antibacterial drug norfloxacin and three in vitro selected thioether-macrocyclic peptides, at 2.1–3.0 Å resolutions. The structures, combined with functional analyses, show that the protonation of Asp 41 on the amino (N)-terminal lobe induces the bending of TM1, which in turn collapses the N-lobe cavity, thereby extruding the substrate drug to the extracellular space. Moreover, the macrocyclic peptides bind the central cleft in distinct manners, which correlate with their inhibitory activities. The strongest inhibitory peptide that occupies the N-lobe cavity may pave the way towards the development of efficient inhibitors against MATE transporters.

Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation

Sun, 03/24/2013 - 00:00

Nature advance online publication 24 March 2013. doi:10.1038/nature12028

Authors: Jizeng Jia, Shancen Zhao, Xiuying Kong, Yingrui Li, Guangyao Zhao, Weiming He, Rudi Appels, Matthias Pfeifer, Yong Tao, Xueyong Zhang, Ruilian Jing, Chi Zhang, Youzhi Ma, Lifeng Gao, Chuan Gao, Manuel Spannagl, Klaus F. X. Mayer, Dong Li, Shengkai Pan, Fengya Zheng, Qun Hu, Xianchun Xia, Jianwen Li, Qinsi Liang, Jie Chen, Thomas Wicker, Caiyun Gou, Hanhui Kuang, Genyun He, Yadan Luo, Beat Keller, Qiuju Xia, Peng Lu, Junyi Wang, Hongfeng Zou, Rongzhi Zhang, Junyang Xu, Jinlong Gao, Christopher Middleton, Zhiwu Quan, Guangming Liu, Jian Wang, Huanming Yang, Xu Liu, Zhonghu He, Long Mao & Jun Wang

About 8,000 years ago in the Fertile Crescent, a spontaneous hybridization of the wild diploid grass Aegilops tauschii (2n = 14; DD) with the cultivated tetraploid wheat Triticum turgidum (2n = 4x = 28; AABB) resulted in hexaploid wheat (T. aestivum; 2n = 6x = 42; AABBDD). Wheat has since become a primary staple crop worldwide as a result of its enhanced adaptability to a wide range of climates and improved grain quality for the production of baker’s flour. Here we describe sequencing the Ae. tauschii genome and obtaining a roughly 90-fold depth of short reads from libraries with various insert sizes, to gain a better understanding of this genetically complex plant. The assembled scaffolds represented 83.4% of the genome, of which 65.9% comprised transposable elements. We generated comprehensive RNA-Seq data and used it to identify 43,150 protein-coding genes, of which 30,697 (71.1%) were uniquely anchored to chromosomes with an integrated high-density genetic map. Whole-genome analysis revealed gene family expansion in Ae. tauschii of agronomically relevant gene families that were associated with disease resistance, abiotic stress tolerance and grain quality. This draft genome sequence provides insight into the environmental adaptation of bread wheat and can aid in defining the large and complicated genomes of wheat species.

Draft genome of the wheat A-genome progenitor Triticum urartu

Sun, 03/24/2013 - 00:00

Nature advance online publication 24 March 2013. doi:10.1038/nature11997

Authors: Hong-Qing Ling, Shancen Zhao, Dongcheng Liu, Junyi Wang, Hua Sun, Chi Zhang, Huajie Fan, Dong Li, Lingli Dong, Yong Tao, Chuan Gao, Huilan Wu, Yiwen Li, Yan Cui, Xiaosen Guo, Shusong Zheng, Biao Wang, Kang Yu, Qinsi Liang, Wenlong Yang, Xueyuan Lou, Jie Chen, Mingji Feng, Jianbo Jian, Xiaofei Zhang, Guangbin Luo, Ying Jiang, Junjie Liu, Zhaobao Wang, Yuhui Sha, Bairu Zhang, Huajun Wu, Dingzhong Tang, Qianhua Shen, Pengya Xue, Shenhao Zou, Xiujie Wang, Xin Liu, Famin Wang, Yanping Yang, Xueli An, Zhenying Dong, Kunpu Zhang, Xiangqi Zhang, Ming-Cheng Luo, Jan Dvorak, Yiping Tong, Jian Wang, Huanming Yang, Zhensheng Li, Daowen Wang, Aimin Zhang & Jun Wang

Bread wheat (Triticum aestivum, AABBDD) is one of the most widely cultivated and consumed food crops in the world. However, the complex polyploid nature of its genome makes genetic and functional analyses extremely challenging. The A genome, as a basic genome of bread wheat and other polyploid wheats, for example, T. turgidum (AABB), T. timopheevii (AAGG) and T. zhukovskyi (AAGGAmAm), is central to wheat evolution, domestication and genetic improvement. The progenitor species of the A genome is the diploid wild einkorn wheat T. urartu, which resembles cultivated wheat more extensively than do Aegilops speltoides (the ancestor of the B genome) and Ae. tauschii (the donor of the D genome), especially in the morphology and development of spike and seed. Here we present the generation, assembly and analysis of a whole-genome shotgun draft sequence of the T. urartu genome. We identified protein-coding gene models, performed genome structure analyses and assessed its utility for analysing agronomically important genes and for developing molecular markers. Our T. urartu genome assembly provides a diploid reference for analysis of polyploid wheat genomes and is a valuable resource for the genetic improvement of wheat.

Succinate is an inflammatory signal that induces IL-1β through HIF-1α

Sun, 03/24/2013 - 00:00

Nature advance online publication 24 March 2013. doi:10.1038/nature11986

Authors: G. M. Tannahill, A. M. Curtis, J. Adamik, E. M. Palsson-McDermott, A. F. McGettrick, G. Goel, C. Frezza, N. J. Bernard, B. Kelly, N. H. Foley, L. Zheng, A. Gardet, Z. Tong, S. S. Jany, S. C. Corr, M. Haneklaus, B. E. Caffrey, K. Pierce, S. Walmsley, F. C. Beasley, E. Cummins, V. Nizet, M. Whyte, C. T. Taylor, H. Lin, S. L. Masters, E. Gottlieb, V. P. Kelly, C. Clish, P. E. Auron, R. J. Xavier & L. A. J. O’Neill

Macrophages activated by the Gram-negative bacterial product lipopolysaccharide switch their core metabolism from oxidative phosphorylation to glycolysis. Here we show that inhibition of glycolysis with 2-deoxyglucose suppresses lipopolysaccharide-induced interleukin-1β but not tumour-necrosis factor-α in mouse macrophages. A comprehensive metabolic map of lipopolysaccharide-activated macrophages shows upregulation of glycolytic and downregulation of mitochondrial genes, which correlates directly with the expression profiles of altered metabolites. Lipopolysaccharide strongly increases the levels of the tricarboxylic-acid cycle intermediate succinate. Glutamine-dependent anerplerosis is the principal source of succinate, although the ‘GABA (γ-aminobutyric acid) shunt’ pathway also has a role. Lipopolysaccharide-induced succinate stabilizes hypoxia-inducible factor-1α, an effect that is inhibited by 2-deoxyglucose, with interleukin-1β as an important target. Lipopolysaccharide also increases succinylation of several proteins. We therefore identify succinate as a metabolite in innate immune signalling, which enhances interleukin-1β production during inflammation.

Neuroscience: Anxiety is the sum of its parts

Wed, 03/20/2013 - 00:00

Nature advance online publication 20 March 2013. doi:10.1038/nature12087

Author: Joshua P. Johansen

Anxiety does not arise from a single neural circuit. An interplay between neighbouring, yet opposing, circuits produces anxiety, and outputs from these circuits regulate specific anxiety responses.

Cell biology: Receptor signals come in waves

Wed, 03/20/2013 - 00:00

Nature advance online publication 20 March 2013. doi:10.1038/nature12086

Authors: Martin J. Lohse & Davide Calebiro

The activity of G-protein-coupled receptors is not limited to the cell surface. Evidence from microscopy points to three temporally, spatially and perhaps functionally distinct waves of signalling by these receptors.

A syringe-like injection mechanism in Photorhabdus luminescens toxins

Wed, 03/20/2013 - 00:00

Nature advance online publication 20 March 2013. doi:10.1038/nature11987

Authors: Christos Gatsogiannis, Alexander E. Lang, Dominic Meusch, Vanda Pfaumann, Oliver Hofnagel, Roland Benz, Klaus Aktories & Stefan Raunser

Photorhabdus luminescens is an insect pathogenic bacterium that is symbiotic with entomopathogenic nematodes. On invasion of insect larvae, P. luminescens is released from the nematodes and kills the insect through the action of a variety of virulence factors including large tripartite ABC-type toxin complexes (Tcs). Tcs are typically composed of TcA, TcB and TcC proteins and are biologically active only when complete. Functioning as ADP-ribosyltransferases, TcC proteins were identified as the actual functional components that induce actin-clustering, defects in phagocytosis and cell death. However, little is known about the translocation of TcC into the cell by the TcA and TcB components. Here we show that TcA in P. luminescens (TcdA1) forms a transmembrane pore and report its structure in the prepore and pore state determined by cryoelectron microscopy. We find that the TcdA1 prepore assembles as a pentamer forming an α-helical, vuvuzela-shaped channel less than 1.5 nanometres in diameter surrounded by a large outer shell. Membrane insertion is triggered not only at low pH as expected, but also at high pH, explaining Tc action directly through the midgut of insects. Comparisons with structures of the TcdA1 pore inserted into a membrane and in complex with TcdB2 and TccC3 reveal large conformational changes during membrane insertion, suggesting a novel syringe-like mechanism of protein translocation. Our results demonstrate how ABC-type toxin complexes bridge a membrane to insert their lethal components into the cytoplasm of the host cell. We believe that the proposed mechanism is characteristic of the whole ABC-type toxin family. This explanation of toxin translocation is a step towards understanding the host–pathogen interaction and the complex life cycle of P. luminescens and other pathogens, including human pathogenic bacteria, and serves as a strong foundation for the development of biopesticides.

Conformational biosensors reveal GPCR signalling from endosomes

Wed, 03/20/2013 - 00:00

Nature advance online publication 20 March 2013. doi:10.1038/nature12000

Authors: Roshanak Irannejad, Jin C. Tomshine, Jon R. Tomshine, Michael Chevalier, Jacob P. Mahoney, Jan Steyaert, Søren G. F. Rasmussen, Roger K. Sunahara, Hana El-Samad, Bo Huang & Mark von Zastrow

A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute to the acute G-protein-mediated response. Evidence supporting these various latter hypotheses is indirect or subject to alternative interpretation, and it remains unknown if endosome-localized GPCRs are even present in an active form. Here we describe the application of conformation-specific single-domain antibodies (nanobodies) to directly probe activation of the β2-adrenoceptor, a prototypical GPCR, and its cognate G protein, Gs (ref. 12), in living mammalian cells. We show that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane, and suggest a versatile strategy for probing dynamic conformational change in vivo.