Differential sensing for the regio- and stereoselective identification and quantitation of glycerides
Katharine L. Diehl, Michelle Adams Ivy, Scott Rabidoux, Stefan Matthias Petry, Günter Müller, and Eric V. Anslyn
Lipid metabolism is a growing area of biochemical research because understanding these pathways could lead to treatments for metabolic disorders such as obesity and type 2 diabetes. To study lipid metabolism, researchers need tools to identify and quantitate glycerides, the main component of animal fat. However, it can be difficult to tell one glyceride apart from another subtly different glyceride using current analytical methods such as mass spectrometry. Thus, we developed a method of discriminating glycerides using an array of cross-reactive proteins in conjunction with pattern recognition algorithms. By incorporating an olefin metathesis pretreatment step, we were able to distinguish glyceride regio- and stereoisomers and to predict these structural features. Finally, we achieved quantitation of glycerides in mixtures. (See pp. E3977–E3986.)
Light-driven generation of hydrogen: New chromophore dyads for increased activity based on Bodipy dye and Pt(diimine)(dithiolate) complexes
Bo Zheng, Randy P. Sabatini, Wen-Fu Fu, Min-Sik Eum, William W. Brennessel, Lidong Wang, David W. McCamant, and Richard Eisenberg
The light-driven generation of H2, the reductive side of water splitting, requires a light absorber or photosensitizer (PS) for electron-hole creation and photoinduced electron transfer. To increase the effectiveness of charge transfer chromophores as PSs, this report describes the attachment of a strongly absorbing organic dye (dipyrromethene-BF2, commonly known as Bodipy) to Pt diimine dithiolate charge transfer chromophores and examination of systems containing these dyads for the light-driven generation of H2. The use of these dyads increases system activity under green light irradiation (530 nm) relative to systems with either chromophore alone, validating such an approach in designing artificial photosynthetic systems. One dyad system exhibits both high activity and substantial durability (40,000 turnovers relative to PSs over 12 d). (See pp. E3987–E3996.)
Reevaluating carbon fluxes in subduction zones, what goes down, mostly comes up
Peter B. Kelemen and Craig E. Manning
This paper reviews carbon fluxes into and out of subduction zones, using compiled data, calculations of carbon solubility in aqueous fluids, and estimates of carbon flux in metasedimentary diapirs. Upper-bound estimates suggest that most subducting carbon is transported into the mantle lithosphere and crust, whereas previous reviews suggested that about half is recycled into the convecting mantle. If upper-bound estimates are correct, and observed output from volcanoes and diffuse outgassing is smaller, then the mantle lithosphere is an important reservoir for carbon. If the subduction carbon cycle remains in balance, then outgassing from ridges and ocean islands is not balanced, so that the carbon content of the lithosphere + ocean + atmosphere has increased over Earth history. (See pp. E3997–E4006.)
Alleviation of off-target effects from vector-encoded shRNAs via codelivered RNA decoys
Stefan Mockenhaupt, Stefanie Grosse, Daniel Rupp, Ralf Bartenschlager, and Dirk Grimm
Induction of RNAi through shRNA expression holds great potential for biomedical research. Ideal shRNAs exhibit high specificity, potency, and safety, but options for rationally designing such shRNAs remain limited. One ensuing concern is unintended perturbation of gene expression by shRNA sense or antisense strands (off-targeting). Here, we report a novel solution that specifically counteracts adverse activity of shRNA sense strands via their sequestration by tough decoy RNAs (TuDs). Using hepatitis C virus as a clinically relevant example, we demonstrate how TuD coexpression from an Adeno-associated viral (AAV) gene therapy vector concurrently decreases shRNA off-targeting and enhances on-target inhibition. The versatility of TuDs and AAVs makes our new strategy useful for improving specificity and safety of a wide array of RNAi experiments. (See pp. E4007–E4016.)
Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy
Neil B. Blok, Dongyan Tan, Ray Yu-Ruei Wang, Pawel A. Penczek, David Baker, Frank DiMaio, Tom A. Rapoport, and Thomas Walz
Pex1 and Pex6 are members of the AAA family of ATPases, which contain two ATPase domains in a single polypeptide chain and form hexameric double rings. These two Pex proteins are involved in the biogenesis of peroxisomes, and mutations in them frequently cause diseases. Here, we determined structures of the Pex1/Pex6 complex by cryo-electron microscopy. Novel computational modeling methods allowed placement of Pex1/Pex6 domains into subnanometer density maps. Our results show that the peroxisomal Pex1/Pex6 ATPases form a unique double-ring structure in which the two proteins alternate around the ring. Our data shed light on the mechanism and function of this ATPase and suggest a role in peroxisomal protein import similar to that of p97 in ER-associated protein degradation. (See pp. E4017–E4025.)
Cloning, synthesis, and characterization of αO-conotoxin GeXIVA, a potent α9α10 nicotinic acetylcholine receptor antagonist
Sulan Luo, Dongting Zhangsun, Peta J. Harvey, Quentin Kaas, Yong Wu, Xiaopeng Zhu, Yuanyan Hu, Xiaodan Li, Victor I. Tsetlin (Цетлин), Sean Christensen, Haylie K. Romero, Melissa McIntyre, Cheryl Dowell, James C. Baxter, Keith S. Elmslie, David J. Craik, and J. Michael McIntosh
The α9α10 nicotinic AChR (nAChR) subtype is a recently identified target for the development of breast cancer chemotherapeutics and analgesics, particularly to treat neuropathic pain. Structure/function analyses of antagonists of this subtype are therefore essential for the development of specific therapeutic compounds. The Conus genus is a rich source of pharmacologically active peptides, and we report here that the αO-conotoxin GeXIVA is a potent and selective antagonist of the α9α10 nAChR subtype. GeXIVA displays unique structural properties among other Conus peptides and represents a previously unidentified template for molecules active against neuropathic pain. (See pp. E4026–E4035.)
Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1
Naotaka Sekiyama, Haribabu Arthanari, Evangelos Papadopoulos, Ricard A. Rodriguez-Mias, Gerhard Wagner, and Mélissa Léger-Abraham
Translation initiation governs many cellular processes, including cell proliferation, growth, and development. Central to this process is the translation initiation factor 4E (eIF4E), which recruits the small ribosomal subunit to the 5′ end of the mRNA through its interaction with the scaffold protein eIF4G. The eIF4E/eIF4G interaction is highly regulated by competitive binding of 4E-binding proteins (4E-BPs), which are at a convergence point of signaling pathways and act as tumor suppressors. The recently discovered eIF4E/eIF4G interaction inhibitor 1 (4EGI-1) dissociates eIF4G but enhances 4E-BP1 binding and has antitumor activity. Here, we elucidate the mechanism for the dual activity of 4EGI-1—it dissociates eIF4G from eIF4E but stabilizes the binding of 4E-BP1. (See pp. E4036–E4045.)
Mutation-induced protein interaction kinetics changes affect apoptotic network dynamic properties and facilitate oncogenesis
Linjie Zhao, Tanlin Sun, Jianfeng Pei, and Qi Ouyang
Cancer is highly correlated with somatic mutations. Understanding how mutation induces oncogenesis is an important task in cancer research. We proposed a strategy that combined network-based dynamics modeling with structural-based mutation analysis and molecular dynamics simulation to map cancer-related mutations to network dynamics changes via protein–protein interaction kinetics. This approach identifies the oncogenic role of mutations and subsequently determines the causal mechanism of mutation-induced oncogenesis. This work provides a framework for coupling mutation genotype to tumorigenesis phenotype. It is also a step toward seeking more effective anticancer drug targets in cellular pathways. (See pp. E4046–E4054.)
Dynamic epigenetic regulation of glioblastoma tumorigenicity through LSD1 modulation of MYC expression
David Kozono, Jie Li, Masayuki Nitta, Oltea Sampetrean, David Gonda, Deepa S. Kushwaha, Dmitry Merzon, Valya Ramakrishnan, Shan Zhu, Kaya Zhu, Hiroko Matsui, Olivier Harismendy, Wei Hua, Ying Mao, Chang-Hyuk Kwon, Hideyuki Saya, Ichiro Nakano, Donald P. Pizzo, Scott R. VandenBerg, and Clark C. Chen
Glioblastoma is the most common type of adult brain cancer, with near-uniform fatality within 2 y of diagnosis. Therapeutic failure is thought to be related to small subpopulations of cells that exhibit tumorigenicity, the cellular capacity to reconstitute the entire tumor mass. One fundamental issue is whether tumorigenicity exists within a static subpopulation of cells or whether the capacity is stochastically acquired. We provide evidence that tumorigenicity is a cellular property that is durable yet undergoes low-frequency stochastic changes. We showed that these changes are driven by lysine-specific demethylase 1 (LSD1)-mediated epigenetic (heritable non-DNA sequence-altering) modifications that impact expression of key transcription factors, which in turn govern transitions between tumorigenic states. These findings harbor implications for glioblastoma therapeutic development. (See pp. E4055–E4064.)
Climate change and decadal shifts in the phenology of larval fishes in the California Current ecosystem
Rebecca G. Asch
In terrestrial ecosystems, earlier phenology (i.e., seasonal timing) is a hallmark organismal response to global warming. Less is known about marine phenological responses to climate change, especially in Eastern Boundary Current Upwelling (EBCU) ecosystems that generate >20% of fish catch. The phenology of 43 EBCU fish species was examined over 58 years; 39% of phenological events occurred earlier in recent decades, with faster changes than many terrestrial ecosystems. Zooplankton did not shift their phenology synchronously with most fishes. Fishes that aren’t changing their phenology synchronously with zooplankton may be subject to mismatches with prey, potentially leading to reduced recruitment to fisheries. Adjusting the timing of seasonal management tactics (e.g., fishery closures, hatchery releases) may help ensure that management remains effective. (See pp. E4065–E4074.)
Evolutionary rewiring of gene regulatory network linkages at divergence of the echinoid subclasses
Eric M. Erkenbrack and Eric H. Davidson
This work provides direct evidence of evolutionary rewiring of gene-regulatory circuitry accompanying divergence of two subclasses of echinoderm, the cidaroid and euechinoid sea urchins. These forms descend from a known common Paleozoic ancestor, and their embryos develop differently, offering an opportunity to probe the basic evolutionary process by which clade divergence occurs at the gene-regulatory network (GRN) level. We carried out a systematic analysis of the use of particular genes participating in embryonic skeletogenic cell specification, building on an established euechinoid developmental GRN. This study revealed that the well-known and elegantly configured regulatory circuitry that underlies skeletogenic specification in modern sea urchins is largely a novel evolutionary invention. The results dramatically display extensive regulatory changes in a specific developmental GRN, underlying an incidence of cladistic divergence at the subclass level. (See pp. E4075–E4084.)
Rsu1 regulates ethanol consumption in Drosophila and humans
Shamsideen A. Ojelade, Tianye Jia, Aylin R. Rodan, Tao Chenyang, Julie L. Kadrmas, Anna Cattrell, Barbara Ruggeri, Pimphen Charoen, Hervé Lemaitre, Tobias Banaschewski, Christian Büchel, Arun L. W. Bokde, Fabiana Carvalho, Patricia J. Conrod, Herta Flor, Vincent Frouin, Jürgen Gallinat, Hugh Garavan, Penny A. Gowland, Andreas Heinz, Bernd Ittermann, Mark Lathrop, Steven Lubbe, Jean-Luc Martinot, Tomás Paus, Michael N. Smolka, Rainer Spanagel, Paul F. O'Reilly, Jaana Laitinen, Juha M. Veijola, Jianfeng Feng, Sylvane Desrivières, Marjo-Riitta Jarvelin, The IMAGEN Consortium, Gunter Schumann, and Adrian Rothenfluh
Genetic factors play a major role in the development of human addiction. Identifying these genes and understanding their molecular mechanisms are necessary first steps in the development of targeted therapeutic intervention. Here, we have isolated the gene encoding Ras suppressor 1 (Rsu1) in an unbiased genetic screen for altered ethanol responses in the vinegar fly, Drosophila melanogaster. Our behavioral, genetic, and biochemical experiments show that Rsu1 links signaling from the integrin cell adhesion molecule to the small GTPase Rac1 in adult neurons to regulate actin dynamics and alcohol consumption preference. We also show that variants in human RSU1 associate with altered drinking and brain activation during a reward prediction task, thereby validating the predictive power of our approach. (See pp. E4085–E4093.)
TACI deficiency leads to alternatively activated macrophage phenotype and susceptibility to Leishmania infection
Windy R. Allman, Ranadhir Dey, Lunhua Liu, Shafiuddin Siddiqui, Adam S. Coleman, Parna Bhattacharya, Masahide Yano, Kadriye Uslu, Kazuyo Takeda, Hira L. Nakhasi, and Mustafa Akkoyunlu
Here, we described a novel role for transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) in determining Mϕ phenotype, a molecule that is previously known to be important in B-cell responses. We found that TACI-deficient mouse Mϕs manifest an M2 phenotype. We also observed that, in WT mouse Mϕs, TACI mediates B-cell activating factor- and a proliferation inducing ligand-induced signals that favor M1 polarization and Leishmania clearance. In TACI-deficient mice, M2-polarized status of Mϕs was responsible for the diminished Th1 response and increased susceptibility to Leishmania infection. These findings have implications in explaining the propensity of infants to develop asthma and weak responses to vaccines because infant Mϕs are likely to be M2-skewed due to severely reduced expression of TACI. (See pp. E4094–E4103.)
Quantification of biological aging in young adults
Daniel W. Belsky, Avshalom Caspi, Renate Houts, Harvey J. Cohen, David L. Corcoran, Andrea Danese, HonaLee Harrington, Salomon Israel, Morgan E. Levine, Jonathan D. Schaefer, Karen Sugden, Ben Williams, Anatoli I. Yashin, Richie Poulton, and Terrie E. Moffitt
The global population is aging, driving up age-related disease morbidity. Antiaging interventions are needed to reduce the burden of disease and protect population productivity. Young people are the most attractive targets for therapies to extend healthspan (because it is still possible to prevent disease in the young). However, there is skepticism about whether aging processes can be detected in young adults who do not yet have chronic diseases. Our findings indicate that aging processes can be quantified in people still young enough for prevention of age-related disease, opening a new door for antiaging therapies. The science of healthspan extension may be focused on the wrong end of the lifespan; rather than only studying old humans, geroscience should also study the young. (See pp. E4104–E4110.)
Functional expression of sodium-glucose transporters in cancer
Claudio Scafoglio, Bruce A. Hirayama, Vladimir Kepe, Jie Liu (刘捷), Chiara Ghezzi, Nagichettiar Satyamurthy, Neda A. Moatamed, Jiaoti Huang, Hermann Koepsell, Jorge R. Barrio, and Ernest M. Wright
Cancers require high amounts of glucose to grow and survive, and dogma is that uptake is facilitated by passive glucose transporters (GLUTs). We have identified a new mechanism to import glucose into pancreatic and prostate cancer cells, namely active glucose transport mediated by sodium-dependent glucose transporters (SGLTs). This means that the specific radioactive imaging probe for SGLTs, α-methyl-4-deoxy-4-[18F]fluoro-d-glucopyranoside, may be used along with positron-emission tomography to diagnose and stage pancreatic and prostate cancers, tumors in which the GLUT probe 2-[18F]fluoro-2-deoxy-d-glucose has questionable utility. Moreover, we suggest, based on our results in mouse models, that Food and Drug Administration-approved SGLT2 inhibitors may be used to reduce the viability of pancreatic and prostate cancer cells in patients. (See pp. E4111–E4119.)
Limits and patterns of cytomegalovirus genomic diversity in humans
Nicholas Renzette, Cornelia Pokalyuk, Laura Gibson, Bornali Bhattacharjee, Mark R. Schleiss, Klaus Hamprecht, Aparecida Y. Yamamoto, Marisa M. Mussi-Pinhata, William J. Britt, Jeffrey D. Jensen, and Timothy F. Kowalik
Human cytomegalovirus (HCMV) is the leading cause of birth defects associated with infections and a leading cause of transplantation failure. This study reveals the patterns and limits of HCMV genomic diversity by performing a large-scale analysis of HCMV sequences sampled from human hosts, identifying the hot and cold spots of variability. We find that the diversity is unevenly distributed across three host compartments and show that HCMV populations of vascular compartments are genetically constrained while enriched for polymorphisms of glycoproteins and regulatory proteins. This work significantly advances our understanding of the genomic diversity of HCMV in humans and has clear implications for the development of therapeutics against HCMV. (See pp. E4120–E4128.)
The LGI1–ADAM22 protein complex directs synapse maturation through regulation of PSD-95 function
Kathryn L. Lovero, Yuko Fukata, Adam J. Granger, Masaki Fukata, and Roger A. Nicoll
The PSD-95 family proteins serve as central scaffolds of excitatory synapses. Their expression levels dictate synaptic strength, and the functions of many synaptic organizing molecules are dependent on interactions with these proteins. Yet, it is unclear what guides PSD-95 into maturing synapses, which occurs postnatally and is required for proper synapse development. Here, we establish that the secreted protein LGI1 controls the functional incorporation of PSD-95 through interactions with the transmembrane protein ADAM22. This process occurs in a paracrine fashion, with LGI1 released from the pre- and postsynaptic cell able to modulate postsynaptic strength. Our data illustrate a previously undescribed level of synaptic organization, identifying a critical role for the LGI1–ADAM22 complex in controlling the function of PSD-95 itself and, in turn, normal synapse development. (See pp. E4129–E4137.)
Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice
Chen-Ching Yuan, Priya Muthu, Katarzyna Kazmierczak, Jingsheng Liang, Wenrui Huang, Thomas C. Irving, Rosemeire M. Kanashiro-Takeuchi, Joshua M. Hare, and Danuta Szczesna-Cordary
Genetic hypertrophic cardiomyopathy (HCM) is a debilitating disease affecting 1 in 500 of the general population, and there is no effective therapy to reverse or prevent its development and/or progression to heart failure. To inhibit a detrimental HCM phenotype induced by the D166V mutation of cardiac myosin regulatory light chain (RLC) in mice that also show reduced phosphorylation of endogenous cardiac RLC, constitutively phosphorylated D166V mutant mice were produced and tested. Our in-depth investigation of heart morphology, structure, and function of S15D-D166V mice provided evidence for the pseudophosphorylation-elicited prevention of the progressive HCM-D166V phenotype. This study is significant for the field of HCM, and our findings may constitute a novel therapeutic modality to battle hypertrophic cardiomyopathy associated with RLC mutations. (See pp. E4138–E4146.)
Navigating natural variation in herbivory-induced secondary metabolism in coyote tobacco populations using MS/MS structural analysis
Dapeng Li, Ian T. Baldwin, and Emmanuel Gaquerel
The study of natural variation has profoundly advanced our understanding of plants’ phenotypic trait evolution. The analysis of intraspecific variations in metabolism, however, has lagged behind and frequently been biased toward central metabolism. To redress this bias, we present a metabolomics case study of leaf secondary metabolites of wild tobacco ecotypes subjected to simulated insect herbivory in which mass spectral maps are constructed. Navigating these maps revealed metabolic branch-specific variations and allowed the annotation of unknown metabolites of likely ecological importance. Although the profiling of entire plant metabolomes remains technically challenging due to their structural complexity, we predict that the workflow described here provides a significant advance in our ability to rapidly explore small molecules that mediate functionally important phenotypes. (See pp. E4147–E4155.)