De novo selection of oncogenes
Kelly M. Chacón, Lisa M. Petti, Elizabeth H. Scheideman, Valentina Pirazzoli, Katerina Politi, and Daniel DiMaio
Artificial proteins may have improved properties compared with proteins that arose during evolution, but approaches to construct active artificial proteins are cumbersome and often constrained by existing protein structures. Here (pp. E6–E14), we used mouse cells to select proteins that formed tumors from a library of small transmembrane proteins with randomized hydrophobic amino acid sequences. The resulting oncoproteins lack amino acid sequences from any known protein and function by activating a cellular growth factor receptor. This approach can be used to generate structures not observed in nature, create prototypes for research and possibly clinical uses, and provide insight into cell biology, protein–protein interactions, and evolution.
AFF1 is a ubiquitous P-TEFb partner to enable Tat extraction of P-TEFb from 7SK snRNP and formation of SECs for HIV transactivation
Huasong Lu, Zichong Li, Yuhua Xue, Ursula Schulze-Gahmen, Jeffrey R. Johnson, Nevan J. Krogan, Tom Alber, and Qiang Zhou
Transcriptional elongation by RNA polymerase II produces full-length RNA transcripts and plays a general and prominent role in regulating gene expression. The positive transcription elongation factor b (P-TEFb) is one of the most important transcription factors controlling this process. The core P-TEFb, consisting of cyclin-dependent kinase 9 and cyclin T (CycT), exists in a network of complexes that include the 7SK small nuclear ribonucleoprotein particle (7SK snRNP), the super elongation complexes (SECs), and the bromodomain protein 4 (Brd4)–P-TEFb complex. This study (pp. E15–E24) identifies AF4/FMR2 family member 1 (AFF1) as a ubiquitous binding partner of core P-TEFb throughout the entire P-TEFb network. By increasing the affinity of the HIV-encoded transactivating (Tat) protein for CycT1, AFF1 is required for Tat’s extraction of P-TEFb from 7SK snRNP and the formation of SECs for maximal HIV transcriptional activation.
A Cdc42- and Rac-interactive binding (CRIB) domain mediates functions of coronin
Karthic Swaminathan, Annette Müller-Taubenberger, Jan Faix, Francisco Rivero, and Angelika A. Noegel
Mammalian nonmuscle myosin II, a major player in cell migration and polarity, is regulated through heavy chain phosphorylation. The signaling mechanisms remain largely unknown. We implicate here (pp. E25–E33) coronin in this pathway. Coronin has a Cdc42- and Rac-interactive binding (CRIB) motif with which it binds preferentially to the GDP-loaded form of Rho GTPases. Loss of coronin in Dictyostelium amoebae leads to elevated levels of activated Rac, resulting in increased myosin II assembly through activation of p21-activated kinase a (PAKa). PAKa inactivates myosin heavy chain kinases that no longer can phosphorylate myosin II and prevent filament formation. By including myosin heavy chain kinase–deficient cells we were able to further map the signaling cascade regulating myosin II.
Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3
Kathrin Muda, Daniela Bertinetti, Frank Gesellchen, Jennifer Sarah Hermann, Felix von Zweydorf, Arie Geerlof, Anette Jacob, Marius Ueffing, Christian Johannes Gloeckner, and Friedrich W. Herberg
Leucine-rich repeat kinase 2 (LRRK2) is a multidomain protein implicated in Parkinson disease, and cAMP-dependent protein kinase (PKA) has been suggested to act as an upstream kinase phosphorylating LRRK2. Using a phosphoproteomics approach, we identified several novel PKA phosphorylation sites on LRRK2. We could demonstrate that one PKA phosphosite comprises the second most common mutation in LRRK2 (R1441C/G/H) attributed to Parkinson disease. Our findings (pp. E34–E43) reveal that this site is mandatory for subsequent 14-3-3 binding and affects LRRK2 kinase activity. These data provide a mechanistic insight into the regulation of LRRK2 kinase activity and its perturbation by disease-associated mutations.
Pch2 is a hexameric ring ATPase that remodels the chromosome axis protein Hop1
Cheng Chen, Ahmad Jomaa, Joaquin Ortega, and Eric E. Alani
The conserved PCH2 gene in baker’s yeast regulates meiotic double-strand break repair outcomes, helps establish a proper meiotic chromosome structure, and is important for the progression of meiotic recombination. Its mouse homolog is required for fertility. However, the molecular mechanism of how PCH2 regulates these diverse functions is not known. In this study (pp. E44–E53), we show that Pch2 is an AAA+ family ATPase (ATPases associated with diverse cellular activities) that oligomerizes into single hexameric rings. In the presence of ATP, Pch2 binds to and remodels Hop1, an important component of the synaptonemal complex, and displaces it from DNA. Based on these and previous observations, we suggest that Pch2 impacts meiotic chromosome organization by directly regulating Hop1 binding to DNA.
Structural and biochemical basis for the inhibition of cell death by APIP, a methionine salvage enzyme
Wonchull Kang, Se Hoon Hong, Hye Min Lee, Na Yeon Kim, Yun Chan Lim, Le Thi My Le, Bitna Lim, Hyun Chul Kim, Tae Yeon Kim, Hiroki Ashida, Akiho Yokota, Sang Soo Hah, Keun Ho Chun, Yong-Keun Jung, and Jin Kuk Yang
Apaf-1 interacting protein (APIP) inhibits two main types of programmed cell death: apoptosis and pyroptosis. In addition, APIP is a 5-methylthioribulose-1-phosphate dehydratase (MtnB) in the methionine salvage pathway. We verified its enzymatic activity directly through an enzyme assay and determined its high-resolution structure. Furthermore, we explored the relationship between two distinct functions of APIP/MtnB, cell death inhibition and methionine salvage, and determined that it functions as a cell death inhibitor independently of its MtnB enzyme activity for apoptosis, but dependently for caspase-1–induced pyroptosis. Our results (pp. E54–E61) provide groundwork for studies of the role of APIP/MtnB in development of cancers and inflammatory diseases.
Cooperative assembly of IFI16 filaments on dsDNA provides insights into host defense strategy
Seamus R. Morrone, Tao Wang, Leeza M. Constantoulakis, Richard M. Hooy, Michael J. Delannoy, and Jungsan Sohn
Human IFN-inducible protein-16 (IFI16) is an essential intracellular foreign DNA receptor of innate immunity and also implicated in several autoimmune disorders. However, little is known about molecular mechanisms that underlie its function. We show here (pp. E62–E71) that IFI16 cooperatively assembles into filaments on dsDNA. IFI16 thus oligomerizes even in the presence of excess DNA, and it is the non–DNA-binding pyrin domain of IFI16 that drives the filament assembly. These results provide unifying mechanistic explanations for several previous in vivo observations regarding IFI16 and also suggest that assembling filaments on foreign nucleic acids is a broad host defense strategy.
SuperBiHelix method for predicting the pleiotropic ensemble of G-protein–coupled receptor conformations
Jenelle K. Bray, Ravinder Abrol, William A. Goddard III, Bartosz Trzaskowski, and Caitlin E. Scott
It is known that G-protein–coupled receptors exhibit several distinct low-energy conformations, each of which might favor binding to different ligands and/or lead to different downstream functions. Understanding the function of such proteins requires knowledge of the ensemble of low-energy configurations that might play a role in this pleiotropic functionality. We present (pp. E72–E78) the SuperBiHelix methodology aimed at identifying all the low-energy structures that might play a role in binding, activation, and signaling. SuperBiHelix uses overall template information from all available experimental or theoretical structures and then does very complete (∼13 trillion configurations) sampling of the helix rotations and tilts to predict the ensemble of low-energy structures. We find that different mutations and ligands stabilize different conformations.
BK channel opening involves side-chain reorientation of multiple deep-pore residues
Xixi Chen, Jiusheng Yan, and Richard W. Aldrich
The BK calcium-activated potassium channels are important regulators of electrical and calcium signaling in many types of tissue. BK channel activation involves complex conformational changes. We investigated the molecular details of such conformational changes with scanning mutagenesis and electrophysiological measurements. We found multiple pore residues that reorient their side chains during channel opening. These findings (pp. E79–E88) bridge the gap between our knowledge about the static X-ray structure of ion channel pores and knowledge about their functional dynamics.
Interplay of mevalonate and Hippo pathways regulates RHAMM transcription via YAP to modulate breast cancer cell motility
Zhongyuan Wang, Yanping Wu, Haifeng Wang, Yangqing Zhang, Lin Mei, Xuexun Fang, Xudong Zhang, Fang Zhang, Hongbo Chen, Ying Liu, Yuyang Jiang, Shengnan Sun, Yi Zheng, Na Li, and Laiqiang Huang
How receptor for hyaluronan-mediated motility (RHAMM) expression is regulated, how statins exert anticancer effects, and what roles mevalonate and Hippo pathways play in tumors are important issues in cancer biology. We find (pp. E89–E98) that the two pathways converge onto Yes-associated protein (YAP)/TEAD to control RHAMM transcription leading to ERK activation and cancer metastasis, which is inhibited by simvastatin. YAP/TEAD binds RHAMM promoter at specific sites to activate RHAMM transcription, and mevalonate/simvastatin affects RHAMM transcription by modulating YAP phosphorylation and nuclear-cytoplasmic distribution. These in vitro and in vivo findings identify a mechanism regulating RHAMM expression and cancer metastasis wherein RHAMM is a downstream effector of mevalonate/Hippo pathways, and a YAP/TEAD-transcription and simvastatin-inhibition target, revealing interesting interplay of the pathways and potential targets for cancer therapeutic agents.
FMRP and Ataxin-2 function together in long-term olfactory habituation and neuronal translational control
Indulekha P. Sudhakaran, Jens Hillebrand, Adrian Dervan, Sudeshna Das, Eimear E. Holohan, Jörn Hülsmeier, Mihail Sarov, Roy Parker, K. VijayRaghavan, and Mani Ramaswami
This work explores the endogenous functions of two disease- and RNA-associated proteins, FMRP (fragile X protein) and Ataxin-2, in memory, synaptic plasticity, RNA regulation, and messenger ribo-nucleoprotein particle assembly. By documenting an array of common phenotypic consequences from loss of Atx2 or dFMR1, the results argue that both proteins have similar in vivo functions and that differences in spinocerebellar ataxia 2 and fragile X disease pathologies may arise from the distinct features of the respective disease causative mutations. This work (pp. E99–E108) provides insight into the in vivo mechanisms of long-term memory (LTM)-associated synaptic plasticity and into the roles of FMRP and Atx2 in neuronal translational control.
Trapping of naive lymphocytes triggers rapid growth and remodeling of the fibroblast network in reactive murine lymph nodes
Chen-Ying Yang, Tobias K. Vogt, Stéphanie Favre, Leonardo Scarpellino, Hsin-Ying Huang, Fabienne Tacchini-Cottier, and Sanjiv A. Luther
Lymph node swelling is a hallmark of adaptive immunity. Fibroblastic reticular cells form a fairly rigid scaffold throughout lymph nodes. They not only support organ structure and compartmentalization, but also guide lymphocyte trafficking. We describe how this rigid fibroblast network reacts to acute organ swelling. Rather than being disrupted or destroyed, the fibroblast network rapidly expands by proliferation and finally covers a much larger volume to accommodate many more lymphocytes. We identified (pp. E109–E118) naive lymphocyte trapping by innate triggers as an early fibroblast growth signal, with activated lymphocytes playing a role only in the later growth phase.
Effective functional maturation of invariant natural killer T cells is constrained by negative selection and T-cell antigen receptor affinity
Romain Bedel, Richard Berry, Thierry Mallevaey, Jennifer L. Matsuda, Jingjing Zhang, Dale I. Godfrey, Jamie Rossjohn, John W. Kappler, Philippa Marrack, and Laurent Gapin
Several different populations of T lymphocytes develop in the thymus from a common precursor. Each population plays a unique and critical role in the mounting and resolution of an immune response. The mechanisms responsible for the emergence of these different populations remain incompletely understood. We demonstrate that strict “Goldilocks” conditions of affinity for self-lipids by the T-cell antigen receptor expressed on T-cell precursors are necessary for imprinting the proper developmental program toward the invariant NK T-cell lineage. Our results (pp. E119–E128) establish a direct link between the affinity of the T-cell receptor for self-antigens and the proper development of a unique population of lymphocytes that has been implicated in the modulation of a multitude of immune responses in mice and humans.
Cytoglobin modulates myogenic progenitor cell viability and muscle regeneration
Sarvjeet Singh, Diana C. Canseco, Shilpa M. Manda, John M. Shelton, Rajendra R. Chirumamilla, Sean C. Goetsch, Qiu Ye, Robert D. Gerard, Jay W. Schneider, James A. Richardson, Beverly A. Rothermel, and Pradeep P. A. Mammen
Mammalian skeletal muscle is a dynamic and plastic tissue, capable of responding to physiological demands and pathophysiological stresses. This response relies on the muscle’s ability to activate myogenic progenitor cells (MPCs) resulting in myogenesis. In this study (pp. E129–E138), we demonstrate that cytoglobin, a stress-responsive hemoprotein abundantly expressed in MPCs, is capable of modulating MPCs’ viability and proliferative/differentiative capacity. Collectively, our data demonstrate that cytoglobin serves an important role in muscle regeneration. Thus, an enhanced understanding of cytoglobin’s role in myogenesis may enable the development of therapeutic approaches for treating patients with muscle injuries and other neuromuscular disorders.
Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate
Nicholas A. Bokulich, John H. Thorngate, Paul M. Richardson, and David A. Mills
We demonstrate (pp. E139–E148) that grape-associated microbial biogeography is nonrandomly associated with regional, varietal, and climatic factors across multiscale viticultural zones. This poses a paradigm shift in our understanding of food and agricultural systems beyond grape and wine production, wherein patterning of whole microbial communities associated with agricultural products may associate with downstream quality characteristics. Elucidating the relationship between production region, climate, and microbial patterns may enhance biological control within these systems, improving the supply, consumer acceptance, and economic value of important agricultural commodities.
Ghrelin triggers the synaptic incorporation of AMPA receptors in the hippocampus
Luís F. Ribeiro, Tatiana Catarino, Sandra D. Santos, Marion Benoist, J. Fiona van Leeuwen, José A. Esteban, and Ana Luísa Carvalho
Ghrelin is a peptide released from the empty stomach into circulation, and which acts on the brain to promote appetite. Recent studies showed that ghrelin also affects cognition, but the mechanism for this effect is unknown. We show (pp. E149–E158) that activation of ghrelin receptors in the hippocampus enhances synaptic signaling by glutamate, the major excitatory neurotransmitter in the brain. Ghrelin promotes the synaptic accumulation of glutamate receptors of the AMPA subtype, and increases long-term potentiation, one form of synaptic plasticity that is thought to underlie learning and memory. These molecular effects of ghrelin in the hippocampus may contribute to the cognition-enhancing role of ghrelin.
Origins of 
orientation dependence in gray and white matter
David A. Rudko, L. Martyn Klassen, Sonali N. de Chickera, Joseph S. Gati, Gregory A. Dekaban, and Ravi S. Menon
Differences in the apparent transverse relaxation rate (
) between tissues are exploited in numerous magnetic resonance imaging (MRI) techniques from functional MRI to susceptibility weighted imaging. Recent results show a surprising dependence of tissue 
on orientation. This study (pp. E159–E167) demonstrates that the orientation dependence of 
in both white and cortical gray matter has a sinusoidal dependence on tissue orientation and a linear dependence on the perturber volume fraction (measured by quantitative histology). A biophysical model is used to relate the observed orientation dependence to the local Larmor frequency shift and volume magnetic susceptibility of the tissue.
Dual role for Islet-1 in promoting striatonigral and repressing striatopallidal genetic programs to specify striatonigral cell identity
Kuan-Ming Lu, Sylvia M. Evans, Shinji Hirano, and Fu-Chin Liu
Basal ganglia circuits are engaged in controlling psychomotor function. The circuits consist of striatonigral and striatopallidal pathways. The opposing but balanced activity of these two neural pathways is important for regulating movement-related functions. How the cell types of striatonigral and striatopallidal neurons are specified to construct these two pathways during development remains elusive. We found that the LIM homeodomain transcription factor Islet-1 (Isl1) was specifically expressed in striatonigral neurons during development. Genetic inactivation of Isl1 resulted in increased apoptosis, abnormal differentiation, and aberrant axonal projections of striatonigral neurons. These findings (pp. E168–E177) suggest that Isl1 plays a key role in specification of striatonigral neurons. Our study provides insights into genetic mechanisms by which basal ganglia circuits are built to function.
Cortical neural populations can guide behavior by integrating inputs linearly, independent of synchrony
Mark H. Histed and John H. R. Maunsell
The brain performs computations by transforming sensory inputs to make decisions. We study these neuronal computations in the mouse, one of the smallest animals with a cerebral cortex, the part of the human brain that controls complex behavior. We find (pp. E178–E187) animals’ behavior can be insensitive to the timing of cortical inputs, depending only on total spike count, even though individual neurons are sensitive to timing. Thus, the cortex can integrate input linearly, or place equal weight on inputs regardless of their arrival time. This emergent linear network behavior may arise from fluctuations in membrane potential generated by background network activity. Brain diseases may arise from dysfunction of these network properties, perhaps by damaging the mechanisms that create this background noise.
Distinct cerebellar engrams in short-term and long-term motor learning
Wen Wang, Kazuhiko Nakadate, Miwako Masugi-Tokita, Fumihiro Shutoh, Wajeeha Aziz, Etsuko Tarusawa, Andrea Lorincz, Elek Molnár, Sebnem Kesaf, Yun-Qing Li, Yugo Fukazawa, Soichi Nagao, and Ryuichi Shigemoto
Long-term depression (LTD) of parallel fiber (PF) to Purkinje cell (PC) synapses has been postulated to cause cerebellar motor learning and extensively studied using in vitro preparations. However, there has been no in vivo evidence showing its occurrence after physiological learning, and much controversy on its role has persisted. We demonstrate that LTD as a form of AMPA receptor decrease does occur in PF–PC synapses after adaptation of horizontal optokinetic response. However, it lasted less than a day and was followed by elimination of these synapses. Our findings (pp. E188–E193) indicate distinct in vivo engrams for short-term and long-term memory in cerebellar motor learning, and open new mechanistic investigations of how the short-term memory is stabilized through structural reorganization of synaptic connections.
Distinct kinetics of synaptic structural plasticity, memory formation, and memory decay in massed and spaced learning
Wajeeha Aziz, Wen Wang, Sebnem Kesaf, Alsayed Abdelhamid Mohamed, Yugo Fukazawa, and Ryuichi Shigemoto
Learning with resting intervals induces longer-lasting memories. However, how structural alterations in neuronal synapses correlate with temporal regulation in learning with or without intervals remain unclear. We used a cerebellar motor learning paradigm in mice using massed or spaced training. Just after the training, we found similar acquisition and AMPA-type glutamate receptor reduction in parallel fiber–Purkinje cell synapses by both protocols. However, spacing induced quicker structural modifications with halving of the synapses and spines within 4 h, thus resulting in persistent long-term memory. We demonstrate (pp. E194–E202) the quick development of the synaptic plasticity as a structural basis for spacing effect.
Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis
Piyada Juntawong, Thomas Girke, Jérémie Bazin, and Julia Bailey-Serres
Plant survival in a highly varied environment requires flexibility in gene regulation. To capture dynamics of mRNA translation at the genome scale, we precisely mapped individual ribosomes to mRNAs of whole seedlings under control and low-oxygen conditions. The results (pp. E203–E212) demonstrate nearly 100-fold variation in the efficiency of translation of individual mRNAs under both conditions and provide unique insights into posttranscriptional and translational regulation modulated by low-energy stress in Arabidopsis thaliana.