Proceedings of the National Academy of Sciences (Proc Natl Acad Sci Unit States Am)

Publications in this journal

• Article: Formation of hybrid complex between Ca2+ and the ionophores Bromolasalocid and A23187

  M Deleers, M Gelbcke, WJ Malaisse
  Proceedings of the National Academy of Sciences 12/2013; 78(1981):279-282.
• Article: Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis

  Dahai Gao, Shishir Chundawat, Anurag Sethi, Venkatesh Balan, S Gnanakaran, Bruce Dale
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  ABSTRACT: Substrate binding is typically one of the rate-limiting steps preceding enzyme catalytic action during homogeneous reactions. However, interfacial-based enzyme catalysis on insoluble crystalline substrates, like cellulose, has additional bottlenecks of individual biopolymer chain decrystallization from the substrate interface followed by its processive depolymerization to soluble sugars. This additional decrystallization step has ramifications on the role of enzyme–substrate binding and its relationship to overall catalytic efficiency. We found that altering the crystalline structure of cellulose from its native allomorph Iβ to IIII results in 40–50% lower binding partition coefficient for fungal cellulases, but surprisingly, it enhanced hydrolytic activity on the latter allomorph. We developed a comprehensive kinetic model for processive cellulases acting on insoluble substrates to explain this anomalous finding. Our model predicts that a reduction in the effective binding affinity to the substrate coupled with an increase in the decrystallization procession rate of individual cellulose chains from the substrate surface into the enzyme active site can reproduce our anomalous experimental findings. Article is freely available online through the PNAS open access option.
  Proceedings of the National Academy of Sciences 06/2013;
• Article: IKK epsilon kinase is crucial for viral G protein-coupled receptor tumorigenesis.

  Yi Wang, Xiaolu Lu, Lining Zhu, Yan Shen, Shylet Chengedza, Hao Feng, Laiyee Wang, Jae U Jung, Julio S Gutkind, Pinghui Feng
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  ABSTRACT: G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that transmit diverse extracellular signals across a membrane. Herpesvirus genomes encode multiple GPCRs implicated in viral pathogenesis. Kaposi sarcoma-associated herpesvirus GPCR (kGPCR) activates proliferative pathways and, when expressed in endothelium in mice, sufficiently induces angiogenic tumor resembling human Kaposi's sarcoma. IKKε, an IκB kinase (IKK)-related kinase, is implicated in inflammation-driven tumorigenesis. We report here that IKKε is critically required for kGPCR tumorigenesis and links kGPCR to NF-κB activation. Using kGPCR-induced tumor models, we found that IKKε expression was drastically up-regulated in Kaposi sarcoma-like lesions and that loss of IKKε abolished tumor formation. Moreover, kGPCR interacted with and activated IKKε. Activated IKKε promoted NF-κB subunit RelA (also known as p65) phosphorylation, which correlated with NF-κB activation and inflammatory cytokine expression. The robust expression of IKKε and phosphorylated RelA was observed in human Kaposi sarcoma. Finally, a kinase-defective mutant of IKKε effectively abrogated NF-κB activation and tumorigenesis induced by kGPCR. Collectively, our findings uncover a critical IKKε in promoting NF-κB activation and tumorigenesis induced by a viral GPCR.
  Proceedings of the National Academy of Sciences 06/2013;
• Article: Mitochondrial calcium uptake.

  George S B Williams, Liron Boyman, Aristide C Chikando, Ramzi J Khairallah, W J Lederer
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  ABSTRACT: Calcium (Ca(2+)) uptake into the mitochondrial matrix is critically important to cellular function. As a regulator of matrix Ca(2+) levels, this flux influences energy production and can initiate cell death. If large, this flux could potentially alter intracellular Ca(2+) ([Ca(2+)]i) signals. Despite years of study, fundamental disagreements on the extent and speed of mitochondrial Ca(2+) uptake still exist. Here, we review and quantitatively analyze mitochondrial Ca(2+) uptake fluxes from different tissues and interpret the results with respect to the recently proposed mitochondrial Ca(2+) uniporter (MCU) candidate. This quantitative analysis yields four clear results: (i) under physiological conditions, Ca(2+) influx into the mitochondria via the MCU is small relative to other cytosolic Ca(2+) extrusion pathways; (ii) single MCU conductance is ∼6-7 pS (105 mM [Ca(2+)]), and MCU flux appears to be modulated by [Ca(2+)]i, suggesting Ca(2+) regulation of MCU open probability (PO); (iii) in the heart, two features are clear: the number of MCU channels per mitochondrion can be calculated, and MCU probability is low under normal conditions; and (iv) in skeletal muscle and liver cells, uptake per mitochondrion varies in magnitude but total uptake per cell still appears to be modest. Based on our analysis of available quantitative data, we conclude that although Ca(2+) critically regulates mitochondrial function, the mitochondria do not act as a significant dynamic buffer of cytosolic Ca(2+) under physiological conditions. Nevertheless, with prolonged (superphysiological) elevations of [Ca(2+)]i, mitochondrial Ca(2+) uptake can increase 10- to 1,000-fold and begin to shape [Ca(2+)]i dynamics.
  Proceedings of the National Academy of Sciences 06/2013;
• Article: miR-17~92 miRNA cluster promotes kidney cyst growth in polycystic kidney disease.

  Vishal Patel, Darren Williams, Sachin Hajarnis, Ryan Hunter, Marco Pontoglio, Stefan Somlo, Peter Igarashi
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  ABSTRACT: Polycystic kidney disease (PKD), the most common genetic cause of chronic kidney failure, is characterized by the presence of numerous, progressively enlarging fluid-filled cysts in the renal parenchyma. The cysts arise from renal tubules and are lined by abnormally functioning and hyperproliferative epithelial cells. Despite recent progress, no Food and Drug Administration-approved therapy is available to retard cyst growth. MicroRNAs (miRNAs) are short noncoding RNAs that inhibit posttranscriptional gene expression. Dysregulated miRNA expression is observed in PKD, but whether miRNAs are directly involved in kidney cyst formation and growth is not known. Here, we show that miR-17∼92, an oncogenic miRNA cluster, is up-regulated in mouse models of PKD. Kidney-specific transgenic overexpression of miR-17∼92 produces kidney cysts in mice. Conversely, kidney-specific inactivation of miR-17∼92 in a mouse model of PKD retards kidney cyst growth, improves renal function, and prolongs survival. miR-17∼92 may mediate these effects by promoting proliferation and through posttranscriptional repression of PKD genes Pkd1, Pkd2, and hepatocyte nuclear factor-1β. These studies demonstrate a pathogenic role of miRNAs in mouse models of PKD and identify miR-17∼92 as a therapeutic target in PKD. Our results also provide a unique hypothesis for disease progression in PKD involving miRNAs and regulation of PKD gene dosage.
  Proceedings of the National Academy of Sciences 06/2013;
• Article: Chimera states in mechanical oscillator networks.

  Erik Andreas Martens, Shashi Thutupalli, Antoine Fourrière, Oskar Hallatschek
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  ABSTRACT: The synchronization of coupled oscillators is a fascinating manifestation of self-organization that nature uses to orchestrate essential processes of life, such as the beating of the heart. Although it was long thought that synchrony and disorder were mutually exclusive steady states for a network of identical oscillators, numerous theoretical studies in recent years have revealed the intriguing possibility of "chimera states," in which the symmetry of the oscillator population is broken into a synchronous part and an asynchronous part. However, a striking lack of empirical evidence raises the question of whether chimeras are indeed characteristic of natural systems. This calls for a palpable realization of chimera states without any fine-tuning, from which physical mechanisms underlying their emergence can be uncovered. Here, we devise a simple experiment with mechanical oscillators coupled in a hierarchical network to show that chimeras emerge naturally from a competition between two antagonistic synchronization patterns. We identify a wide spectrum of complex states, encompassing and extending the set of previously described chimeras. Our mathematical model shows that the self-organization observed in our experiments is controlled by elementary dynamical equations from mechanics that are ubiquitous in many natural and technological systems. The symmetry-breaking mechanism revealed by our experiments may thus be prevalent in systems exhibiting collective behavior, such as power grids, optomechanical crystals, or cells communicating via quorum sensing in microbial populations.
  Proceedings of the National Academy of Sciences 06/2013;
• Article: Designed azurins show lower reorganization free energies for intraprotein electron transfer.

  Ole Farver, Nicholas M Marshall, Scot Wherland, Yi Lu, Israel Pecht
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  ABSTRACT: Low reorganization free energies are necessary for fast electron transfer (ET) reactions. Hence, rational design of redox proteins with lower reorganization free energies has been a long-standing challenge, promising to yield a deeper understanding of the underlying principles of ET reactivity and to enable potential applications in different energy conversion systems. Herein we report studies of the intramolecular ET from pulse radiolytically produced disulfide radicals to Cu(II) in rationally designed azurin mutants. In these mutants, the copper coordination sphere has been fine-tuned to span a wide range of reduction potentials while leaving the metal binding site effectively undisrupted. We find that the reorganization free energies of ET within the mutants are indeed lower than that of WT azurin, increasing the intramolecular ET rate constants almost 10-fold: changes that are correlated with increased flexibility of their copper sites. Moreover, the lower reorganization free energy results in the ET rate constants reaching a maximum value at higher driving forces, as predicted by the Marcus theory.
  Proceedings of the National Academy of Sciences 06/2013;
• Article: Francois Jacob memorial.

  Arthur B Pardee
  Proceedings of the National Academy of Sciences 06/2013;
• Article: Out of the tropics, but how? Fossils, bridge species, and thermal ranges in the dynamics of the marine latitudinal diversity gradient.

  David Jablonski, Christina L Belanger, Sarah K Berke, Shan Huang, Andrew Z Krug, Kaustuv Roy, Adam Tomasovych, James W Valentine
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  ABSTRACT: Latitudinal diversity gradients are underlain by complex combinations of origination, extinction, and shifts in geographic distribution and therefore are best analyzed by integrating paleontological and neontological data. The fossil record of marine bivalves shows, in three successive late Cenozoic time slices, that most clades (operationally here, genera) tend to originate in the tropics and then expand out of the tropics (OTT) to higher latitudes while retaining their tropical presence. This OTT pattern is robust both to assumptions on the preservation potential of taxa and to taxonomic revisions of extant and fossil species. Range expansion of clades may occur via "bridge species," which violate climate-niche conservatism to bridge the tropical-temperate boundary in most OTT genera. Substantial time lags (∼5 Myr) between the origins of tropical clades and their entry into the temperate zone suggest that OTT events are rare on a per-clade basis. Clades with higher diversification rates within the tropics are the most likely to expand OTT and the most likely to produce multiple bridge species, suggesting that high speciation rates promote the OTT dynamic. Although expansion of thermal tolerances is key to the OTT dynamic, most latitudinally widespread species instead achieve their broad ranges by tracking widespread, spatially-uniform temperatures within the tropics (yielding, via the nonlinear relation between temperature and latitude, a pattern opposite to Rapoport's rule). This decoupling of range size and temperature tolerance may also explain the differing roles of species and clade ranges in buffering species from background and mass extinctions.
  Proceedings of the National Academy of Sciences 06/2013;
• Article: Detection of an intermediary, protonated water cluster in photosynthetic oxygen evolution.

  Brandon C Polander, Bridgette A Barry
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  ABSTRACT: In photosynthesis, photosystem II evolves oxygen from water by the accumulation of photooxidizing equivalents at the oxygen-evolving complex (OEC). The OEC is a Mn4CaO5 cluster, and its sequentially oxidized states are termed the Sn states. The dark-stable state is S1, and oxygen is released during the transition from S3 to S0. In this study, a laser flash induces the S1 to S2 transition, which corresponds to the oxidation of Mn(III) to Mn(IV). A broad infrared band, at 2,880 cm(-1), is produced during this transition. Experiments using ammonia and (2)H2O assign this band to a cationic cluster of internal water molecules, termed "W5(+)." Observation of the W5(+) band is dependent on the presence of calcium, and flash dependence is observed. These data provide evidence that manganese oxidation during the S1 to S2 transition results in a coupled proton transfer to a substrate-containing, internal water cluster in the OEC hydrogen-bonded network.
  Proceedings of the National Academy of Sciences 06/2013;
• Article: Optimization criteria and biological process enrichment in homologous multiprotein modules.

  Luqman Hodgkinson, Richard M Karp
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  ABSTRACT: Biological process enrichment is a widely used metric for evaluating the quality of multiprotein modules. In this study, we examine possible optimization criteria for detecting homologous multiprotein modules and quantify their effects on biological process enrichment. We find that modularity, linear density, and module size are the most important criteria considered, complementary to each other, and that graph theoretical attributes account for 36% of the variance in biological process enrichment. Variations in protein interaction similarity within module pairs have only minor effects on biological process enrichment. As random modules increase in size, both biological process enrichment and modularity tend to improve, although modularity does not show this upward trend in modules with size at most 50 proteins. To adjust for these trends, we recommend a size correction based on random sampling of modules when using biological process enrichment or other attributes to evaluate module boundaries. Characteristics of homologous multiprotein modules optimized for each of the optimization criteria are examined.
  Proceedings of the National Academy of Sciences 06/2013;
• Article: Seeing sea spray by the seashore.

  Devin Powell
  Proceedings of the National Academy of Sciences 06/2013; 110(24):9617.
• Article: Neutron diffraction observations of interstitial protons in dense ice.

  Malcolm Guthrie, Reinhard Boehler, Christopher A Tulk, Jamie J Molaison, António M Dos Santos, Kuo Li, Russell J Hemley
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  ABSTRACT: The motif of distinct H2O molecules in H-bonded networks is believed to persist up to the densest molecular phase of ice. At even higher pressures, where the molecule dissociates, it is generally assumed that the proton remains localized within these same networks. We report neutron-diffraction measurements on D2O that reveal the location of the D atoms directly up to 52 GPa, a pressure regime not previously accessible to this technique. The data show the onset of a structural change at ∼13 GPa and cannot be described by the conventional network structure of ice VII above ∼26 GPa. Our measurements are consistent with substantial deuteron density in the octahedral, interstitial voids of the oxygen lattice. The observation of this "interstitial" ice VII form provides a framework for understanding the evolution of hydrogen bonding in ice that contrasts with the conventional picture. It may also be a precursor for the superionic phase reported at even higher pressure with important consequences for our understanding of dense matter and planetary interiors.
  Proceedings of the National Academy of Sciences 06/2013;
• Article: Molecular architecture of the uncleaved HIV-1 envelope glycoprotein trimer.

  Youdong Mao, Liping Wang, Christopher Gu, Alon Herschhorn, Anik Désormeaux, Andrés Finzi, Shi-Hua Xiang, Joseph G Sodroski
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  ABSTRACT: The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, a membrane-fusing machine, mediates virus entry into host cells and is the sole virus-specific target for neutralizing antibodies. Binding the receptors, CD4 and CCR5/CXCR4, triggers Env conformational changes from the metastable unliganded state to the fusion-active state. We used cryo-electron microscopy to obtain a 6-Å structure of the membrane-bound, heavily glycosylated HIV-1 Env trimer in its uncleaved and unliganded state. The spatial organization of secondary structure elements reveals that the unliganded conformations of both glycoprotein (gp)120 and gp41 subunits differ from those induced by receptor binding. The gp120 trimer association domains, which contribute to interprotomer contacts in the unliganded Env trimer, undergo rearrangement upon CD4 binding. In the unliganded Env, intersubunit interactions maintain the gp41 ectodomain helical bundles in a "spring-loaded" conformation distinct from the extended helical coils of the fusion-active state. Quaternary structure regulates the virus-neutralizing potency of antibodies targeting the conserved CD4-binding site on gp120. The Env trimer architecture provides mechanistic insights into the metastability of the unliganded state, receptor-induced conformational changes, and quaternary structure-based strategies for immune evasion.
  Proceedings of the National Academy of Sciences 06/2013;
• Article: Measuring the regulation of keratin filament network dynamics.

  Marcin Moch, Gerlind Herberich, Til Aach, Rudolf E Leube, Reinhard Windoffer
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  ABSTRACT: The organization of the keratin intermediate filament cytoskeleton is closely linked to epithelial function. To study keratin network plasticity and its regulation at different levels, tools are needed to localize and measure local network dynamics. In this paper, we present image analysis methods designed to determine the speed and direction of keratin filament motion and to identify locations of keratin filament polymerization and depolymerization at subcellular resolution. Using these methods, we have analyzed time-lapse fluorescence recordings of fluorescent keratin 13 in human vulva carcinoma-derived A431 cells. The fluorescent keratins integrated into the endogenous keratin cytoskeleton, and thereby served as reliable markers of keratin dynamics. We found that increased times after seeding correlated with down-regulation of inward-directed keratin filament movement. Bulk flow analyses further revealed that keratin filament polymerization in the cell periphery and keratin depolymerization in the more central cytoplasm were both reduced. Treating these cells and other human keratinocyte-derived cells with EGF reversed all these processes within a few minutes, coinciding with increased keratin phosphorylation. These results highlight the value of the newly developed tools for identifying modulators of keratin filament network dynamics and characterizing their mode of action, which, in turn, contributes to understanding the close link between keratin filament network plasticity and epithelial physiology.
  Proceedings of the National Academy of Sciences 06/2013;
• Article: Genetic circuitry of Survival motor neuron, the gene underlying spinal muscular atrophy.

  Anindya Sen, Douglas N Dimlich, K G Guruharsha, Mark W Kankel, Kazuya Hori, Takakazu Yokokura, Sophie Brachat, Delwood Richardson, Joseph Loureiro, Rajeev Sivasankaran, Daniel Curtis, Lance S Davidow, Lee L Rubin, Anne C Hart, David Van Vactor, Spyros Artavanis-Tsakonas
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  ABSTRACT: The clinical severity of the neurodegenerative disorder spinal muscular atrophy (SMA) is dependent on the levels of functional Survival Motor Neuron (SMN) protein. Consequently, current strategies for developing treatments for SMA generally focus on augmenting SMN levels. To identify additional potential therapeutic avenues and achieve a greater understanding of SMN, we applied in vivo, in vitro, and in silico approaches to identify genetic and biochemical interactors of the Drosophila SMN homolog. We identified more than 300 candidate genes that alter an Smn-dependent phenotype in vivo. Integrating the results from our genetic screens, large-scale protein interaction studies, and bioinformatic analysis, we define a unique interactome for SMN that provides a knowledge base for a better understanding of SMA.
  Proceedings of the National Academy of Sciences 06/2013;
• Article: Elasto-inertial turbulence.

  Devranjan Samanta, Yves Dubief, Markus Holzner, Christof Schäfer, Alexander N Morozov, Christian Wagner, Björn Hof
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  ABSTRACT: Turbulence is ubiquitous in nature, yet even for the case of ordinary Newtonian fluids like water, our understanding of this phenomenon is limited. Many liquids of practical importance are more complicated (e.g., blood, polymer melts, paints), however; they exhibit elastic as well as viscous characteristics, and the relation between stress and strain is nonlinear. We demonstrate here for a model system of such complex fluids that at high shear rates, turbulence is not simply modified as previously believed but is suppressed and replaced by a different type of disordered motion, elasto-inertial turbulence. Elasto-inertial turbulence is found to occur at much lower Reynolds numbers than Newtonian turbulence, and the dynamical properties differ significantly. The friction scaling observed coincides with the so-called "maximum drag reduction" asymptote, which is exhibited by a wide range of viscoelastic fluids.
  Proceedings of the National Academy of Sciences 06/2013;
• Article: Polyester synthesis genes associated with stress resistance are involved in an insect-bacterium symbiosis.

  Jiyeun Kate Kim, Yeo Jin Won, Naruo Nikoh, Hiroshi Nakayama, Sang Heum Han, Yoshitomo Kikuchi, Young Ha Rhee, Ha Young Park, Jeong Yun Kwon, Kenji Kurokawa, Naoshi Dohmae, Takema Fukatsu, Bok Luel Lee
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  ABSTRACT: Many bacteria accumulate granules of polyhydroxyalkanoate (PHA) within their cells, which confer resistance to nutritional depletion and other environmental stresses. Here, we report an unexpected involvement of the bacterial endocellular storage polymer, PHA, in an insect-bacterium symbiotic association. The bean bug Riptortus pedestris harbors a beneficial and specific gut symbiont of the β-proteobacterial genus Burkholderia, which is orally acquired by host nymphs from the environment every generation and easily cultivable and genetically manipulatable. Biochemical and cytological comparisons between symbiotic and cultured Burkholderia detected more PHA granules consisting of poly-3-hydroxybutyrate and associated phasin (PhaP) protein in the symbiotic Burkholderia. Among major PHA synthesis genes, phaB and phaC were disrupted by homologous recombination together with the phaP gene, whereby ΔphaB, ΔphaC, and ΔphaP mutants were generated. Both in culture and in symbiosis, accumulation of PHA granules was strongly suppressed in ΔphaB and ΔphaC, but only moderately in ΔphaP. In symbiosis, the host insects infected with ΔphaB and ΔphaC exhibited significantly lower symbiont densities and smaller body sizes. These deficient phenotypes associated with ΔphaB and ΔphaC were restored by complementation of the mutants with plasmids encoding a functional phaB/phaC gene. Retention analysis of the plasmids revealed positive selection acting on the functional phaB/phaC in symbiosis. These results indicate that the PHA synthesis genes of the Burkholderia symbiont are required for normal symbiotic association with the Riptortus host. In vitro culturing analyses confirmed vulnerability of the PHA gene mutants to environmental stresses, suggesting that PHA may play a role in resisting stress under symbiotic conditions.
  Proceedings of the National Academy of Sciences 06/2013;
• Article: Engineering of regulated stochastic cell fate determination.

  Min Wu, Ri-Qi Su, Xiaohui Li, Tom Ellis, Ying-Cheng Lai, Xiao Wang
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  ABSTRACT: Both microbes and multicellular organisms actively regulate their cell fate determination to cope with changing environments or to ensure proper development. Here, we use synthetic biology approaches to engineer bistable gene networks to demonstrate that stochastic and permanent cell fate determination can be achieved through initializing gene regulatory networks (GRNs) at the boundary between dynamic attractors. We realize this experimentally by linking a synthetic GRN to a natural output of galactose metabolism regulation in yeast. Combining mathematical modeling and flow cytometry, we show that our engineered systems are bistable and that inherent gene expression stochasticity does not induce spontaneous state transitioning at steady state. Mathematical analysis predicts that stochastic cell fate determination in this case can only be realized when gene expression fluctuation occurs on or near attractor basin boundaries (the points of instability). Guided by numerical simulations, experiments are designed and performed with quantitatively diverse gene networks to test model predictions, which are verified by both flow cytometry and single-cell microscopy. By interfacing rationally designed synthetic GRNs with background gene regulation mechanisms, this work investigates intricate properties of networks that illuminate possible regulatory mechanisms for cell differentiation and development that can be initiated from points of instability.
  Proceedings of the National Academy of Sciences 06/2013;