The structured core domain of αB-crystallin can prevent amyloid fibrillation and associated toxicity
Georg K. A. Hochberg, Heath Ecroyd, Cong Liu, Dezerae Cox, Duilio Cascio, Michael R. Sawaya, Miranda P. Collier, James Stroud, John A. Carver, Andrew J. Baldwin, Carol V. Robinson, David S. Eisenberg, Justin L. P. Benesch, and Arthur Laganowsky
We find that the core domain of the human molecular chaperone αB-crystallin can function effectively in preventing protein aggregation and amyloid toxicity. The core domain represents only half the total sequence of the protein, but it is one of the most potent known inhibitors of the aggregation of amyloid-β, a process implicated in Alzheimer’s disease. We have determined high-resolution structures of this core domain and investigated its biophysical properties in solution. We find (pp. E1562–E1570) that the excised domain efficiently prevents amyloid aggregation and thereby reduces the toxicity of the resulting aggregates to cells. The structures of these domains that we present should represent useful scaffolds for the design of novel amyloid inhibitors.
Substrate-induced changes in the structural properties of LacY
Tetiana Serdiuk, M. Gregor Madej, Junichi Sugihara, Shiho Kawamura, Stefania A. Mari, H. Ronald Kaback, and Daniel J. Müller
Lactose permease of Escherichia coli (LacY), a model for the major facilitator superfamily, catalyzes galactopyranoside/H+ symport across the membrane by a mechanism in which large conformational changes expose the sugar-binding site in the middle of the molecule alternatively to either side of the membrane (an alternating access mechanism). Despite substantial progress with respect to static X-ray crystal structures of LacY, the dynamics of the transport mechanism are not fully understood. Here (pp. E1571–E1580) we use dynamic single-molecule force spectroscopy to quantify the structural properties that change upon substrate binding. The results reveal very significant changes in conformational, kinetic, energetic, and mechanical properties primarily in the N-terminal 6-helix bundle, while the C-terminal 6-helix bundle remains largely unaffected.
Integrative framework for identification of key cell identity genes uncovers determinants of ES cell identity and homeostasis
Senthilkumar Cinghu, Sailu Yellaboina, Johannes M. Freudenberg, Swati Ghosh, Xiaofeng Zheng, Andrew J. Oldfield, Brad L. Lackford, Dmitri V. Zaykin, Guang Hu, and Raja Jothi
A key step to understanding a phenotype of interest is the identification of genes defining that phenotype. We propose a computational framework for a systematic integration of published gene expression data to identify genes defining a cell identity of interest. We demonstrate the utility of the proposed approach by identifying genes essential for the maintenance of ES cell (ESC) identity. Follow-up functional studies on candidate gene Nucleolin (Ncl) reveal Ncl's essential role in the maintenance of ESC homeostasis. Ncl deficiency increases endogenous reactive oxygen species levels and induces p53 activity, resulting in p53-mediated suppression of Nanog and subsequent ESC differentiation. These studies (pp. E1581–E1590) uncover a previously unknown regulatory circuitry involving genes associated with traits in both ESCs and cancer.
The nature and extent of contributions by defective ribosome products to the HLA peptidome
Dmitry Bourdetsky, Christian E. H. Schmelzer, and Arie Admon
Rapid degradation of newly synthesized proteins, followed by presentation of the resulting peptides by the MHC molecules, serves as an early alert for the immune system during pathogen infection. This study (pp. E1591–E1599) defines the relative contribution to the MHC peptidome of defective ribosome products (DRiPs), which are newly synthesized and rapidly degraded proteins, vs. mature proteins, degraded at the end of their functional lifetimes (retirees). The rates of synthesis of the individual MHC peptides and their source proteins were followed using stable isotope labeling and quantitative proteomics and peptidomics analyses. We conclude that DRiPs are a significant source of MHC peptides. Many of these DRiPs are misassembled surplus subunits of protein complexes and therefore are degraded soon after synthesis.
p53 constrains progression to anaplastic thyroid carcinoma in a Braf-mutant mouse model of papillary thyroid cancer
David G. McFadden, Amanda Vernon, Philip M. Santiago, Raul Martinez-McFaline, Arjun Bhutkar, Denise M. Crowley, Martin McMahon, Peter M. Sadow, and Tyler Jacks
We generated a thyroid-specific CreER transgenic mouse and used this strain to model progression of v-raf murine sarcoma viral oncogene homolog B (BRAF)-mutant papillary thyroid cancer to anaplastic thyroid cancer (ATC). These murine tumors recapitulated the temporal progression and molecular hallmarks of human ATC. We demonstrated (pp. E1600–E1609) that combined mapk/Erk kinase (MEK) and BRAF inhibition resulted in enhanced antitumor activity vs. single-agent BRAF inhibitors in this preclinical model. This model represents a previously lacking mouse model of BRAF-mutant ATC and adds to the experimental armamentarium of a highly lethal disease in need of scientific advances. These data also suggest that potent inhibition of the MAPK pathway may improve outcomes in advanced thyroid cancers.
Bile salts act as effective protein-unfolding agents and instigators of disulfide stress in vivo
Claudia M. Cremers, Daniela Knoefler, Victor Vitvitsky, Ruma Banerjee, and Ursula Jakob
Bile salts are extremely abundant molecules in the mammalian intestine and play important roles in food digestion. Much less well known is the fact that bile salts are also highly antimicrobial and control bacterial growth in the gut. Here (pp. E1610–E1619), we report the discovery that bile salts affect bacterial growth by causing widespread unfolding and aggregation of cytosolic proteins in bacteria, while simultaneously triggering a prooxidizing shift in the cellular ratio of reduced to oxidized glutathione. Our studies therefore reveal an important and unrealized property of a set of compounds long known to be important in human physiology, demonstrate how bacteria such as Escherichia coli defend themselves against bile salts, and uncover perhaps the first physiological condition that causes disulfide stress in vivo.
Replication protein of tobacco mosaic virus cotranslationally binds the 5′ untranslated region of genomic RNA to enable viral replication
Kazue Kawamura-Nagaya, Kazuhiro Ishibashi, Ying-Ping Huang, Shuhei Miyashita, and Masayuki Ishikawa
Replication of many positive-strand RNA viruses is cis-preferential: i.e., viral replicase proteins replicate genomic RNA molecules that have served as translation templates for their own synthesis, but not the other molecules in the same cell. Here, we show that tobacco mosaic virus replicase cotranslationally binds the 5′ untranslated region of genomic RNA and that this binding inhibits further translation and leads to genomic RNA replication. Intriguingly, full-length replicase protein could not bind genomic RNA posttranslationally due to autoinhibition by the C-terminal domain. These results (pp. E1620–E1628) reveal an elegant viral strategy to enable cis-preferential replication and phase switching from translation to replication at once.
Degenerate target sites mediate rapid primed CRISPR adaptation
Peter C. Fineran, Matthias J. H. Gerritzen, María Suárez-Diez, Tim Künne, Jos Boekhorst, Sacha A. F. T. van Hijum, Raymond H. J. Staals, and Stan J. J. Brouns
Bacteria are constantly exposed to foreign elements, such as bacteriophages and plasmids. The CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR associated) adaptive immune systems provide heritable sequence-specific protection against these invaders. To develop immunity, bacteria add segments of foreign nucleic acid to their CRISPR memory. However, phage and plasmid mutants can evade CRISPR-Cas recognition by altering their targeted sequence. CRISPR-Cas responds to evasion by quickly generating immunity by acquiring new pieces of invader genome. We determined (pp. E1629–E1638) that this rapid generation of resistance is promiscuous, with recognition of highly diverged or related elements eliciting new immunity. Our results demonstrate that CRISPR-Cas systems are more robust than previously thought and, not only have a highly specific resistance memory, but also have a broad ability to identify divergent genetic elements.
Importance of positioning for microbial evolution
Wook Kim, Fernando Racimo, Jonas Schluter, Stuart B. Levy, and Kevin R. Foster
Microbes commonly form dense communities that are central to many diseases and bioremediation. Here we demonstrate a simple and general principle of living in dense communities: microbes will commonly compete to reach nutrients at the community edge, akin to plants competing for light. We follow the evolution of a highly competitive genotype in colonies of a soil bacterium. We show (pp. E1639–E1647) that the genotype gains its advantage not from its intrinsic growth rate but by positioning itself at the surface of the community, where it gains preferential access to oxygen. A large-scale genetic analysis reveals striking parallel evolution and evidence of strong natural selection. Our work suggests that positioning is a major basis for evolutionary competition in dense microbial communities.
Hypocretin (orexin) facilitates reward by attenuating the antireward effects of its cotransmitter dynorphin in ventral tegmental area
John W. Muschamp, Jonathan A. Hollander, Jennifer L. Thompson, George Voren, Linda C. Hassinger, Sara Onvani, Theodore M. Kamenecka, Stephanie L. Borgland, Paul J. Kenny, and William A. Carlezon, Jr.
Hypocretin (orexin) and dynorphin are neuromodulators that play an important role in regulating affect and motivation. Orexin is critical for reward and is implicated in drug seeking, whereas dynorphin mediates negative mood and is implicated in depressive-like states. Considering these opposing effects, reports that both peptides are expressed in the same neurons and are coreleased are counterintuitive. Here (pp. E1648–E1655), we demonstrate that orexin and dynorphin are coexpressed within the same synaptic vesicles and that this colocalization has a profound influence on reward, drug taking, and impulsive-like behavior. The fact that orexin occludes the depressive-like antireward effects of dynorphin significantly changes how we view the functional role of orexin in the brain.
Population receptive field analysis of the primary visual cortex complements perimetry in patients with homonymous visual field defects
Amalia Papanikolaou, Georgios A. Keliris, T. Dorina Papageorgiou, Yibin Shao, Elke Krapp, Eleni Papageorgiou, Katarina Stingl, Anna Bruckmann, Ulrich Schiefer, Nikos K. Logothetis, and Stelios M. Smirnakis
Partial damage of the primary visual cortex (V1), or damage to the white matter inputs to V1 (optic radiation), cause blindness in specific regions of the visual field. We use functional MRI to measure responses in individual patients with a localized, chronic V1 injury that resulted in blindness in a quarter of the visual field. The fMRI responses of patients and controls are generally similar, but in some patients differences from controls can be measured (pp. E1656–E1665). Importantly, responses in spared early visual cortex are not always congruent with visual perception. Understanding how the properties of early visual areas respond to injury will lead to better strategies for visual rehabilitation.
Brain galanin system genes interact with life stresses in depression-related phenotypes
Gabriella Juhasz, Gabor Hullam, Nora Eszlari, Xenia Gonda, Peter Antal, Ian Muir Anderson, Tomas G. M. Hökfelt, J. F. William Deakin, and Gyorgy Bagdy
Early and recent environmental stressors, such as maltreatment in childhood, or stressful life events in adulthood, are important risk factors for depression. Nevertheless, not all people who suffer from these will be depressed. The resilience or vulnerability to these stressors, and thus depression, is likely to reside in our genes. In the present study (pp. E1666–E1673), we used different statistical methods to demonstrate that variations in genes for galanin and its receptors increase the risk of depression only in heavily stress-exposed subjects. The work was predicated on the finding that galanin expression is strongly stimulated by stress in animal studies. In humans, variation in galanin function would appear to be important determinants of the outcome of psychosocial stress.