Crystal structure and mechanistic basis of a functional homolog of the antigen transporter TAP
Anne Nöll, Christoph Thomas, Valentina Herbring, Tina Zollmann, Katja Barth, Ahmad Reza Mehdipour, Thomas M. Tomasiak, Stefan Brüchert, Benesh Joseph, Rupert Abele, Vincent Oliéric, Meitian Wang, Kay Diederichs, Gerhard Hummer, Robert M. Stroud, Klaas M. Pos, and Robert Tampé
ABC transporters shuttle chemically diverse substances across membranes in an energy-dependent manner. They mediate multidrug resistance in microorganisms and cancer cells and can cause human pathologies when dysfunctional. Although important insights into ABC transporters have been gained in recent years, fundamental questions concerning their mechanism remain open. Here, we identify the protein complex TmrAB as a functional homolog of the antigenic peptide transporter TAP and present its high-resolution structure. The structure adopts an asymmetric conformational state and is characterized by C-terminal zipper helices that are essential for efficient substrate translocation. The structure, together with functional studies, enables us to outline the general conformational dynamics of heterodimeric ABC transporters and to establish TmrAB as a model system for TAP. (See pp. E438–E447.)
Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals
Colin J. McKinlay, Jessica R. Vargas, Timothy R. Blake, Jonathan W. Hardy, Masamitsu Kanada, Christopher H. Contag, Paul A. Wender, and Robert M. Waymouth
Protein expression using mRNA has the potential to transform many areas of life science research and affect the prevention, detection, and treatment of disease. However, realizing this potential requires the development of readily accessible, efficacious, and safe delivery systems that can functionally deliver mRNA to cells in culture and in vivo. A class of materials developed for mRNA delivery is described that operates through an unprecedented self-immolation mechanism. These materials are accessed in two steps through an organocatalytic oligomerization. They noncovalently complex, protect, deliver, and release mRNA with >99% transfection efficiency in cultured cells and with robust protein expression in mice using multiple routes of administration. This mRNA delivery technology should be broadly applicable to numerous research and therapeutic applications. (See pp. E448–E456.)
Content analysis of 150 years of British periodicals
Thomas Lansdall-Welfare, Saatviga Sudhahar, James Thompson, Justin Lewis, FindMyPast Newspaper Team, and Nello Cristianini
The use of large datasets has revolutionized the natural sciences and is widely believed to have the potential to do so with the social and human sciences. Many digitization efforts are underway, but the high-throughput methods of data production have not yet led to a comparable output in analysis. A notable exception has been the previous statistical analysis of the content of historical books, which started a debate about the limitations of using big data in this context. This study moves the debate forward using a large corpus of historical British newspapers and tools from artificial intelligence to extract macroscopic trends in history and culture, including gender bias, geographical focus, technology, and politics, along with accurate dates for specific events. (See pp. E457–E465.)
Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination
Gábor M. Harami, Yeonee Seol, Junghoon In, Veronika Ferencziová, Máté Martina, Máté Gyimesi, Kata Sarlós, Zoltán J. Kovács, Nikolett T. Nagy, Yuze Sun, Tibor Vellai, Keir C. Neuman, and Mihály Kovács
RecQ helicase and its eukaryotic homologs are thought to play crucial roles in the quality control of homologous recombination (HR)-based DNA repair. These enzymes have multiple functions in processes that can either promote or suppress HR. A major role suggested for RecQ is the selective inhibition of illegitimate recombination events that could lead to loss of genome integrity. How can RecQ enzymes perform an exceptionally wide range of activities and selectively inhibit potentially harmful recombination events? Here, we propose a model in which the conserved domain architecture of RecQ senses and responds to the geometry of DNA substrates to achieve HR quality control. (See pp. E466–E475.)
Analysis of 138 pathogenic mutations in presenilin-1 on the in vitro production of Aβ42 and Aβ40 peptides by γ-secretase
Linfeng Sun, Rui Zhou, Guanghui Yang, and Yigong Shi (施一公)
Alzheimer’s disease (AD) is the most common form of dementia, but the cause of AD remains poorly understood. Using highly purified recombinant γ-secretase, we examined the effect of 138 AD-derived presenilin-1 (PS1) mutations on the production of β-amyloid peptides (Aβ42 and Aβ40). These 138 mutations cover virtually all AD-targeted amino acids in PS1. Our results reveal no significant correlation between the Aβ42/Aβ40 ratio produced by a γ-secretase variant with a specific PS1 mutation and the mean age at onset of patients carrying this mutation. The comprehensive characterization of pathogenic PS1 mutations serves as a valuable resource for the analysis of γ-secretase activities and AD pathogenesis. (See pp. E476–E485.)
Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding
Patrick Koenig, Chingwei V. Lee, Benjamin T. Walters, Vasantharajan Janakiraman, Jeremy Stinson, Thomas W. Patapoff, and Germaine Fuh
On encountering antigens, antibody’s variable domains evolve and mature through multiple rounds of somatic mutation. By surveying the effects of all possible single mutations of an antibody’s variable domains on folding stability and antigen binding, we showed that even for a high-affinity antibody such as the one used in our study, beneficial mutations can be found both near and far from the antigen-binding site. Furthermore, our mutational scan revealed an antigen-distal framework position working as a structural switch whereby a mutation can allow the variable domains to sample different conformational spaces and thus potentially different binding functions. This understanding of the mechanism of somatic mutation in human antibodies illustrates how antibodies efficiently use somatic mutation for evolving diversity in immune recognition. (See pp. E486–E495.)
Regulatory module involving FGF13, miR-504, and p53 regulates ribosomal biogenesis and supports cancer cell survival
Débora R. Bublik, Slađana Bursać, Michal Sheffer, Ines Oršolić, Tali Shalit, Ohad Tarcic, Eran Kotler, Odelia Mouhadeb, Yonit Hoffman, Gilad Fuchs, Yishai Levin, Siniša Volarević, and Moshe Oren
MicroRNAs (miRNAs) can regulate the amounts of specific proteins by targeting their mRNA. miR-504, which targets the mRNA encoding the p53 tumor suppressor, resides within an intron of the fibroblast growth factor 13 (FGF13) gene. We show that expression of the FGF13/miR-504 locus is repressed by p53, defining an additional p53-regulatory feedback loop. Moreover, we report that the FGF13 protein, whose expression is upregulated in a subset of tumors, is essential for survival of cells derived from such tumors. Remarkably, FGF13 restricts the production of ribosomal RNA and attenuates protein synthesis. By tuning down protein synthesis, FGF13 upregulation might enable oncogene-driven cancer cells to avoid excessive accumulation of potentially toxic aberrant proteins, conferring a survival advantage. This work defines a unique vulnerability of cancer cells. (See pp. E496–E505.)
CD34+ mesenchymal cells are a major component of the intestinal stem cells niche at homeostasis and after injury
Igor Stzepourginski, Giulia Nigro, Jean-Marie Jacob, Sophie Dulauroy, Philippe J. Sansonetti, Gérard Eberl, and Lucie Peduto
Maintenance of stem cells in adult organs requires a specialized microenvironment called the niche, which provides structural cues and paracrine signals to ensure stemness. In the intestine, increasing evidence points toward a major role for the mesenchyme close to crypts to perform this function; however, such putative mesenchymal niche remains poorly characterized. Here, we identify nonmyofibroblastic CD34+ Gp38+ mesenchymal cells as a major component of the intestinal epithelial stem cells (IESCs) niche. We show that CD34+ Gp38+ mesenchymal cells develop after birth and contribute to the maintenance of IESCs at homeostasis and organization of intestinal inflammation after injury. (See pp. E506–E513.)
Inherited human IRAK-1 deficiency selectively impairs TLR signaling in fibroblasts
Erika Della Mina, Alessandro Borghesi, Hao Zhou, Salim Bougarn, Sabri Boughorbel, Laura Israel, Ilaria Meloni, Maya Chrabieh, Yun Ling, Yuval Itan, Alessandra Renieri, Iolanda Mazzucchelli, Sabrina Basso, Piero Pavone, Raffaele Falsaperla, Roberto Ciccone, Rosa Maria Cerbo, Mauro Stronati, Capucine Picard, Orsetta Zuffardi, Laurent Abel, Damien Chaussabel, Nico Marr, Xiaoxia Li, Jean-Laurent Casanova, and Anne Puel
We report the discovery of complete human interleukin-1 receptor (IL-1R)-associated kinase 1 (IRAK-1) deficiency resulting from a de novo Xq28 microdeletion encompassing MECP2 and IRAK1 in a boy. Like many boys with MECP2 defects, this patient died very early. IRAK-1 is a component of the Toll-like receptor (TLR)/IL-1R (TIR) signaling pathway. Unlike patients with autosomal-recessive complete deficiency of MyD88 or IRAK-4, two other components of the TIR pathway, this patient presented no invasive bacterial infections. We analyzed the impact of human IRAK-1 deficiency in fibroblasts and leukocytes. The role of IRAK-1 in signaling downstream from IL-1R and TLRs differed according to cell type. These findings reveal similarities and differences in the role of IRAK-1 in the TLR and IL-1R pathways between mice and humans. (See pp. E514–E523.)
Dual role of ALCAM in neuroinflammation and blood–brain barrier homeostasis
Marc-André Lécuyer, Olivia Saint-Laurent, Lyne Bourbonnière, Sandra Larouche, Catherine Larochelle, Laure Michel, Marc Charabati, Michael Abadier, Stephanie Zandee, Neda Haghayegh Jahromi, Elizabeth Gowing, Camille Pittet, Ruth Lyck, Britta Engelhardt, and Alexandre Prat
Multiple sclerosis (MS) is an inflammatory disorder characterized by multifocal lesions in the central nervous system. These lesions are caused by infiltrating leukocytes that take advantage and/or actively participate in the disruption of the blood–brain barrier (BBB). In this study, the specific role of the adhesion molecule ALCAM (activated leukocyte cell adhesion molecule) present on BBB endothelial cells was assessed. We demonstrated that ALCAM knockout mice develop a more severe experimental autoimmune encephalomyelitis, the mouse model of MS, due to an increased permeability of the BBB. This phenotypic change is caused by a dysregulation of junctional molecules with which ALCAM indirectly binds, suggesting that in addition to its role in leukocyte transmigration, ALCAM regulates and maintains tight junction stability by acting as an adaptor molecule. (See pp. E524–E533.)
Properdin binding to complement activating surfaces depends on initial C3b deposition
Morten Harboe, Christina Johnson, Stig Nymo, Karin Ekholt, Camilla Schjalm, Julie K. Lindstad, Anne Pharo, Bernt Christian Hellerud, Kristina Nilsson Ekdahl, Tom Eirik Mollnes, and Per H. Nilsson
The role of properdin in stabilization of the alternative pathway C3 convertase is indisputable, whereas its role as pattern recognition molecule remains controversial. Properdin lacks the structural homology shared by other pattern recognition molecules of the complement system, and has its major function in stabilizing the C3bBb convertase. We found that properdin binding was completely abolished by C3 inhibition after the exposure of human serum to myeloperoxidase, human umbilical vein endothelial cells, and Neisseria meningitidis, showing that properdin is not a pattern recognition molecule for these targets. We therefore challenge the view of properdin as a pattern recognition molecule, and argue that properdin typically binds a complement-activating surface subsequent to C3b to stabilize the alternative pathway C3 convertase. (See pp. E534–E539.)
C-type lectin receptor DCIR modulates immunity to tuberculosis by sustaining type I interferon signaling in dendritic cells
Anthony Troegeler, Ingrid Mercier, Céline Cougoule, Danilo Pietretti, André Colom, Carine Duval, Thien-Phong Vu Manh, Florence Capilla, Renaud Poincloux, Karine Pingris, Jérôme Nigou, Jörg Rademann, Marc Dalod, Frank A. W. Verreck, Talal Al Saati, Geanncarlo Lugo-Villarino, Bernd Lepenies, Denis Hudrisier, and Olivier Neyrolles
Tuberculosis (TB) is an immunopathology, mostly of the lung, due to an overexuberant immune response to the bacterial pathogen Mycobacterium tuberculosis. Here, we demonstrate in vitro and in vivo that dendritic cell (DC) immunoreceptor (DCIR), a C-type lectin receptor expressed by DCs, modulates immunity to TB by sustaining type I IFN signaling in DCs. These findings were generalized beyond TB, in a model of in vivo antigen-presentation assay unrelated to M. tuberculosis, suggesting that they may extend to other pathologies, such as viral infections or autoimmune disorders. Thus, modulating DCIR activity may help to develop type I IFN-targeting therapies for a large repertoire of inflammatory disorders, including TB. (See pp. E540–E549.)
Antiinflammatory actions of inorganic nitrate stabilize the atherosclerotic plaque
Rayomand S. Khambata, Suborno M. Ghosh, Krishnaraj S. Rathod, Tharssana Thevathasan, Federica Filomena, Qingzhong Xiao, and Amrita Ahluwalia
Reduced bioavailable nitric oxide generated within the vascular wall is a key pathogenic mechanism involved in the formation and rupture of an atheromatous plaque, the latter leading to myocardial infarction or stroke. We show that inorganic nitrate supplementation through delivery of nitric oxide reduces systemic leukocyte rolling and adherence, circulating neutrophil numbers, and monocyte activation through direct repression of neutrophil activation and up-regulation of interleukin-10–dependent antiinflammatory pathways in athero-prone mice. These antiinflammatory effects of inorganic nitrate result in reduced macrophage content of atherosclerotic plaques coupled with an elevation of smooth muscle content, resulting in a stable plaque phenotype. Our data suggest that inorganic nitrate supplementation has antiinflammatory effects, which may have clinical utility in the prophylaxis of atheroma development. (See pp. E550–E559.)
Transcription factor Xpp1 is a switch between primary and secondary fungal metabolism
Christian Derntl, Bernhard Kluger, Christoph Bueschl, Rainer Schuhmacher, Robert L. Mach, and Astrid R. Mach-Aigner
Fungi produce a vast number of different chemical compounds via secondary metabolism. These compounds are of great interest because of their potential applicability in medicine, pharmacy, and biotechnology. In addition, a number of such compounds are toxins that potentially represent severe threats to human and animal health. However, under standard cultivation conditions, fungal secondary metabolism remains largely inactive. Here, we show that the deletion of the regulator Xylanase promoter binding protein 1 (Xpp1) results in the production of significantly more secondary metabolites in terms of both number and concentration. Because homologs of Xpp1 exist in fungi with numerous bioactive secondary metabolites, our results can lead to the discovery of secondary metabolites. (See pp. E560–E569.)
A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection
Kapil Saxena, Lukas M. Simon, Xi-Lei Zeng, Sarah E. Blutt, Sue E. Crawford, Narayan P. Sastri, Umesh C. Karandikar, Nadim J. Ajami, Nicholas C. Zachos, Olga Kovbasnjuk, Mark Donowitz, Margaret E. Conner, Chad A. Shaw, and Mary K. Estes
Understanding host–enteric virus interactions has been limited by the inability to culture nontransformed small intestinal epithelial cells and to infect animal models with human viruses. We report epithelial responses in human small intestinal enteroid cultures from different individuals following infection with human rotavirus (HRV), a model enteric pathogen. RNA-sequencing and functional assays revealed type III IFN as the dominant transcriptional response that activates interferon-stimulated genes, but antagonism of the IFN response negates restriction of HRV replication. Exogenously added IFNs reduce HRV replication, with type I IFN being most effective. This highlights a paradox between the strong type III transcriptional response and the weaker functional role of type III IFN in human enteric viral restriction in human small intestinal cultures. (See pp. E570–E579.)
Redox crisis underlies conditional light–dark lethality in cyanobacterial mutants that lack the circadian regulator, RpaA
Spencer Diamond, Benjamin E. Rubin, Ryan K. Shultzaberger, You Chen, Chase D. Barber, and Susan S. Golden
The evolution of photosynthetic cyanobacteria under 24-h cycles of light and darkness selected for a robust circadian clock. Understanding how cyanobacteria integrate circadian clock signals with natural light–dark cycles to control metabolism is critical, because these organisms are central to global carbon cycling and hold promise for development of renewable energy. Here we assess how the circadian transcription factor regulator of phycobilisome association A (RpaA) influences metabolism as a cyanobacterium goes through a light-to-dark transition. The data show that RpaA plays a key role in maintaining metabolic stability during the night period. Additionally, RpaA is important in controlling redox balance, which in turn is very important for regulating metabolism at night. (See pp. E580–E589.)
Resistance to type 1 interferons is a major determinant of HIV-1 transmission fitness
Shilpa S. Iyer, Frederic Bibollet-Ruche, Scott Sherrill-Mix, Gerald H. Learn, Lindsey Plenderleith, Andrew G. Smith, Hannah J. Barbian, Ronnie M. Russell, Marcos V. P. Gondim, Catherine Y. Bahari, Christiana M. Shaw, Yingying Li, Timothy Decker, Barton F. Haynes, George M. Shaw, Paul M. Sharp, Persephone Borrow, and Beatrice H. Hahn
Effective prevention strategies are urgently needed to control the spread of HIV-1. A critical barrier to developing such strategies is the lack of understanding of the host antiviral defenses that control HIV-1 replication in the mucosa at the site of entry. Here, we characterized viruses from matched donor and recipient pairs to determine whether transmitted HIV-1 strains exhibit traits that increase their transmission fitness. Characterizing 300 limiting dilution-derived isolates, we identified several properties that enhance virus replication in the face of a vigorous innate immune response, of which resistance to type 1 IFNs is the most important. These results provide new insights into the HIV-1 transmission process and define possible new targets for AIDS prevention and therapy. (See pp. E590–E599.)
Task-specific reorganization of the auditory cortex in deaf humans
Łukasz Bola, Maria Zimmermann, Piotr Mostowski, Katarzyna Jednoróg, Artur Marchewka, Paweł Rutkowski, and Marcin Szwed
What principles govern large-scale reorganization of the brain? In the blind, several visual regions preserve their task specificity but switch to tactile or auditory input. It remains open whether this type of reorganization is unique to the visual cortex or is a general mechanism in the brain. Here we asked deaf and hearing adults to discriminate between temporally complex sequences of stimuli during an fMRI experiment. We show that the same auditory regions were activated when deaf subjects performed the visual version of the task and when hearing subjects performed the auditory version of the task. Thus, switching sensory input while preserving the task specificity of recruited areas might be a general principle that guides cortical reorganization in the brain. (See pp. E600–E609.)
Evidence for opposing roles of Celsr3 and Vangl2 in glutamatergic synapse formation
Sonal Thakar, Liqing Wang, Ting Yu, Mao Ye, Keisuke Onishi, John Scott, Jiaxuan Qi, Catarina Fernandes, Xuemei Han, John R. Yates III, Darwin K. Berg, and Yimin Zou
The signaling mechanisms mediating glutamatergic synapse assembly are fundamental to our understanding of neural circuit function, plasticity, and disorders, but have remained elusive. We provide direct evidence that two components of the conserved planar cell polarity signaling pathway, which assembles asymmetric cell–cell junctions, have opposing functions in glutamatergic synapse formation. Celsr3 promotes assembly whereas Vangl2 inhibits assembly, suggesting that this signaling mechanism is accessible for both positive and negative regulation and is also a candidate pathway for mediating synaptic plasticity. (See pp. E610–E618.)
Wnt5a is essential for hippocampal dendritic maintenance and spatial learning and memory in adult mice
Chih-Ming Chen, Lauren L. Orefice, Shu-Ling Chiu, Tara A. LeGates, Samer Hattar, Richard L. Huganir, Haiqing Zhao, Baoji Xu, and Rejji Kuruvilla
The maintenance of neuronal morphology in the adult brain is an understudied area. Here, using tissue-specific deletion in mice, we reveal Wnt5a, a member of the Wnt family of developmental morphogens, as an essential factor for the long-term stability of dendritic architecture in the adult hippocampus. Wnt5a influences synaptic plasticity and related cognitive functions in the mature hippocampus through CaMKII-mediated signaling, Rac1-dependent actin dynamics, and cyclic AMP-responsive element binding-mediated NMDA receptor biosynthesis. In the long-term, Wnt5a-mediated regulation of cytoskeletal signaling and excitatory synaptic transmission is responsible for the maintenance of dendritic arbors and spines in adult CA1 pyramidal neurons. These findings provide insight into the poorly understood structural maintenance mechanisms that exist in the adult brain. (See pp. E619–E628.)
Developmental pruning of excitatory synaptic inputs to parvalbumin interneurons in monkey prefrontal cortex
Daniel W. Chung, Zachary P. Wills, Kenneth N. Fish, and David A. Lewis
Synaptic pruning in primate prefrontal cortical circuitry has been proposed to contribute to working memory maturation. However, pruning of excitatory synapses has only been shown on pyramidal neurons despite the well-recognized role of parvalbumin (PV) interneurons in working memory. Moreover, in schizophrenia, working memory deficits are thought to result from disturbances in the maturation of PV interneurons. Here we demonstrate in the monkey prefrontal cortex that excitatory synapses on PV interneurons are pruned across adolescence, the remaining synapses are strengthened, and splicing of erb-b2 receptor tyrosine kinase 4 (ErbB4) may mediate these effects. These findings provide a developmental context for deficient excitatory synaptic inputs to PV interneurons in schizophrenia and implicate dysregulated ErbB4 splicing as a potential molecular mechanism underlying this process. (See pp. E629–E637.)
Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle
Carlo Manno, Lourdes C. Figueroa, Dirk Gillespie, Robert Fitts, ChulHee Kang, Clara Franzini-Armstrong, and Eduardo Rios
We show that calsequestrin, the main Ca2+ storing protein of muscle, is polymerized inside the sarcoplasmic reticulum (SR) and its mobility increases greatly upon SR depletion, indicating depolymerization. Deep depletion causes massive calsequestrin migration and radical SR remodeling, often accompanied by a surge in intra-SR free Ca2+. The changes in calsequestrin polymerization observed in aqueous solutions therefore also occur in vivo. These changes help explain some uniquely advantageous properties of the SR as a source of calcium for contractile activation. The results support untested hypotheses about additional calsequestrin roles in the control of channel gating and facilitation of calcium flux. They also provide insights on the consequences of calsequestrin mutations for functional competence and structural stability of skeletal and cardiac muscle. (See pp. E638–E647.)