Quantitative conformational profiling of kinase inhibitors reveals origins of selectivity for Aurora kinase activation states
Eric W. Lake, Joseph M. Muretta, Andrew R. Thompson, Damien M. Rasmussen, Abir Majumdar, Erik B. Faber, Emily F. Ruff, David D. Thomas, and Nicholas M. Levinson
Many drugs trigger changes to the structure of their target receptor upon binding. These conformational effects are thought to be an essential part of molecular recognition but have proven challenging to quantify. Using a high-throughput method for tracking structural changes in a protein kinase in solution, we discovered that many clinically important cancer drugs trigger substantial structural changes to their target protein kinase Aurora A, and that these effects systematically account for the ability of the drugs to differentiate between different biochemical forms of Aurora A. The results provide insight into mechanisms of drug selectivity and suggest strategies for tailoring inhibitors to target certain cancers in which Aurora A has been dysregulated in different ways. (See pp. E11894–E11903.)
ALS mutations of FUS suppress protein translation and disrupt the regulation of nonsense-mediated decay
Marisa Kamelgarn, Jing Chen, Lisha Kuang, Huan Jin, Edward J. Kasarskis, and Haining Zhu
Mutations in fused in sarcoma (FUS) contribute to a subset of familial amyotrophic lateral sclerosis (ALS). ALS-linked mutations cause a liquid–liquid phase separation of FUS protein, induce cytoplasmic inclusions in cells, and interfere with RNA metabolism pathways. Our proteomic analysis shows that proteins sequestered into inclusions are enriched in translation and RNA quality surveillance pathways. This study demonstrates that ALS mutations in FUS indeed suppress protein translation and affect the mRNA nonsense-mediated decay (NMD) pathway. NMD-promoting factors UPF1 and UPF3b increased, whereas the negative regulator UPF3a decreased in the cells of patients with ALS. The disruption in NMD regulation and suppression of protein biosynthesis likely contribute to neurodegeneration in ALS. (See pp. E11904–E11913.)
Cooperative assembly of a four-molecule signaling complex formed upon T cell antigen receptor activation
Asit Manna, Huaying Zhao, Junya Wada, Lakshmi Balagopalan, Harichandra D. Tagad, Ettore Appella, Peter Schuck, and Lawrence E. Samelson
An early event during cellular activation in many biologic systems is recruitment of the cytosolic enzyme phospholipase-Cγ1 to the plasma membrane where, on activation, it cleaves its lipid substrates into functional second messengers. T cell antigen receptor engagement during the immune response leads to rapid formation of a multiprotein complex that brings phospholipase-Cγ1 to the plasma membrane with three adapter molecules. We reconstituted this quaternary complex in vitro and used biophysical techniques to determine stoichiometry and measure the affinities of the interacting proteins and the energetics of formation. We observe cooperative formation of a circular loop of interactions associated with a significant entropic penalty facilitating reversibility. Such instability might be a target of regulation during T cell activation. (See pp. E11914–E11923.)
Probing the mechanism of inhibition of amyloid-β(1–42)–induced neurotoxicity by the chaperonin GroEL
Marielle A. Wälti, Joseph Steiner, Fanjie Meng, Hoi Sung Chung, John M. Louis, Rodolfo Ghirlando, Vitali Tugarinov, Avindra Nath, and G. Marius Clore
Chaperones, including the chaperonin Hsp60, facilitate protein folding and prevent protein aggregation, thereby protecting cells from protein misfolding diseases, such as Alzheimer’s disease, a fatal neurodegenerative condition associated with the accumulation of amyloid-β plaques in the brain. We show that the bacterial homolog of human Hsp60, GroEL, protects neuronal cell cultures against morphological and electrophysiological damage induced by amyloid-β(1–42). Using a range of biophysical techniques, we demonstrate that transient interactions of GroEL with amyloid-β(1–42) monomers slow down the rate of appearance of protofibrils and fibrils, providing a mechanistic basis for the neuroprotective properties of Hsp60. (See pp. E11924–E11932.)
Coiled-coil 1-mediated fastening of the neck and motor domains for kinesin-3 autoinhibition
Jinqi Ren, Shuang Wang, Han Chen, Wenjuan Wang, Lin Huo, and Wei Feng
Kinesins are microtubule-based molecular motors that can drive long-range intracellular transport. When not transporting cargoes, processive kinesin motors often adopt an autoinhibited state. The autoinhibited conformation of kinesin-3 is predominantly mediated by a coiled-coil segment that follows the neck and motor domains. In this study, we found that this coiled-coil segment associates with both the neck and motor domains and fastens them together. With the aid of the motor domain (or, namely, head), the autoinhibitory coiled-coil segment tightly locks down the entire neck domain and inhibits both the neck domain-mediated dimerization and the ADP release from the motor head. This “head-aided lockdown of neck” mechanism mediated by the internal coiled-coil segment may represent a paradigm for kinesin autoinhibition. (See pp. E11933–E11942.)
Simple yet functional phosphate-loop proteins
Maria Luisa Romero Romero, Fan Yang, Yu-Ru Lin, Agnes Toth-Petroczy, Igor N. Berezovsky, Alexander Goncearenco, Wen Yang, Alon Wellner, Fanindra Kumar-Deshmukh, Michal Sharon, David Baker, Gabriele Varani, and Dan S. Tawfik
The complexity of modern proteins makes the understanding of how proteins evolved from simple beginnings a daunting challenge. The Walker-A motif is a phosphate-binding loop (P-loop) found in possibly the most ancient and abundant protein class, so-called P-loop NTPases. By combining phylogenetic analysis and computational protein design, we have generated simple proteins, of only 55 residues, that contain the P-loop and thereby confer binding of a range of phosphate-containing ligands—and even more avidly, RNA and single-strand DNA. Our results show that biochemical function can be implemented in small and simple proteins; they intriguingly suggest that the P-loop emerged as a polynucleotide binder and catalysis of phosphoryl transfer evolved later upon acquisition of higher sequence and structural complexity. (See pp. E11943–E11950.)
Microbiome interactions shape host fitness
Alison L. Gould, Vivian Zhang (张维嘉), Lisa Lamberti, Eric W. Jones, Benjamin Obadia, Nikolaos Korasidis, Alex Gavryushkin, Jean M. Carlson, Niko Beerenwinkel, and William B. Ludington
All animals have associated microbial communities called microbiomes that influence the physiology and fitness of their host. It is unclear to what extent individual microbial species versus interactions between them influence the host. Here, we mapped all possible interactions between individual species of bacteria against Drosophila melanogaster fruit fly fitness traits. Our approach revealed that the same bacterial interactions that shape microbiome abundances also shape host fitness traits. The fitness traits of lifespan and fecundity showed a life history tradeoff, where equal total fitness can be gotten by either high fecundity over a short life or low fecundity over a long life. The microbiome interactions are as important as the individual species in shaping these fundamental aspects of fly physiology. (See pp. E11951–E11960.)
Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection
Tai-Yuan Yu, Michael T. Kimble, and Lorraine S. Symington
Chromosomal double-strand breaks (DSBs) are cytotoxic forms of DNA damage that must be accurately repaired to maintain genome integrity. The conserved Mre11-Rad50-Xrs2NBS1 complex plays an important role in repair by functioning as a damage sensor and by regulating DNA end processing to ensure repair by the most appropriate mechanism. Yeast Sae2 is known to activate the Mre11 endonuclease to process DNA ends, and previous studies suggest an additional role to dampen checkpoint signaling. Here, we show Sae2 functions independently of the Mre11 nuclease to prevent Rad9 accumulation at DSBs. Excessive Rad9 binding inhibits DNA end processing by the Dna2-Sgs1 and Exo1 nucleases causing sensitivity of Sae2-deficient cells to DNA damaging agents. (See pp. E11961–E11969.)
Mother–child transmission of epigenetic information by tunable polymorphic imprinting
Brittany L. Carpenter, Wanding Zhou, Zachary Madaj, Ashley K. DeWitt, Jason P. Ross, Kirsten Grønbæk, Gangning Liang, Susan J. Clark, Peter L. Molloy, and Peter A. Jones
First, our work provides critical biological interpretation of intermediate DNA methylation readouts at the nc886 differentially methylated region (DMR). nc886 was identified in multiple large-scale epigenome-wide association studies (EWAS) that did not recognize that this region acts as a contiguous DMR imposed by genomic imprinting, highlighting the need to reexamine several 450k data sets. Second, strict control of genomic imprinting was thought to be required for organismal viability. Reports of polymorphic imprinting are limited to specific tissue types such as placenta and brain. In blood and somatic tissues, we show nc886 imprinting is mosaic in the population and influenced by maternal environment. (See pp. E11970–E11977.)
DPYSL3 modulates mitosis, migration, and epithelial-to-mesenchymal transition in claudin-low breast cancer
Ryoichi Matsunuma, Doug W. Chan, Beom-Jun Kim, Purba Singh, Airi Han, Alexander B. Saltzman, Chonghui Cheng, Jonathan T. Lei, Junkai Wang, Leonardo Roberto da Silva, Ergun Sahin, Mei Leng, Cheng Fan, Charles M. Perou, Anna Malovannaya, and Matthew J. Ellis
Mass spectrometry-based proteogenomics of patient-derived xenografts (PDXs) identified dihydropyrimidinase-like-3 (DPYSL3) as a multilevel (RNA/protein/phosphoprotein) expression outlier specific to a claudin-low (CLOW) PDX. DPYSL3 has established functions in neural cell migration and axon outgrowth but is understudied in breast cancer. Here, we demonstrate that loss of DPYSL3 promotes cell-cycle arrest, multinucleation, and collapse of the vimentin microfilament network associated with increased phospho-vimentin. DPYSL3 is also a negative regulator of p21-activated kinase (PAK) and suppresses epithelial-to-mesenchymal transition (EMT). In turn, EMT regulators induce DPYSL3, suggesting that DPYSL3 provides negative feedback on EMT. DPYSL3 therefore serves as a biomarker for CLOW tumors that exhibit PAK-dependent motility and EMT and is also susceptible to therapeutic approaches that promote vimentin phosphorylation during mitosis. (See pp. E11978–E11987.)
Estimating the proportion of bystander selection for antibiotic resistance among potentially pathogenic bacterial flora
Christine Tedijanto, Scott W. Olesen, Yonatan H. Grad, and Marc Lipsitch
Bystander selection, defined as the inadvertent pressure imposed by antibiotic treatment on microbes other than the targeted pathogen, is hypothesized to be a major factor in the propagation of antibiotic resistance, but its extent has not been characterized. We estimate the proportion of bystander exposures across a range of antibiotics and potential pathogens commonly found in the normal flora and describe factors driving variability of these proportions. Impact estimates for antibiotic resistance interventions, including vaccination, are often limited to effects on a target pathogen. However, the reduction of antibiotic treatment for conditions caused by one pathogen may have the broader potential to decrease bystander selection pressures for resistance on many other organisms. (See pp. E11988–E11995.)
Fiber-associated spirochetes are major agents of hemicellulose degradation in the hindgut of wood-feeding higher termites
Gaku Tokuda, Aram Mikaelyan, Chiho Fukui, Yu Matsuura, Hirofumi Watanabe, Masahiro Fujishima, and Andreas Brune
Xylan, the major hemicellulosic component of lignocellulose and the second most abundant polysaccharide after cellulose, contributes to the structural stability of wood and its recalcitrance to enzymatic digestion. The present study identifies Spirochaetes as primary agents of xylan degradation in the hindgut of wood-feeding higher termites, in contrast to the bovine rumen or the human colon, where Bacteroidetes are responsible for hydrolysis of xylan in grass or cereals. The presence of distinctive xylanases in Spirochaetes was so far undocumented to our knowledge. Their phylogenetic origin among gut bacteria of other phyla identifies horizontal gene transfer among the intestinal microbiota as an important driver in the evolutionary adaptation of higher termites to different lignocellulosic diets. (See pp. E11996–E12004.)
Mycoplasma promotes malignant transformation in vivo, and its DnaK, a bacterial chaperon protein, has broad oncogenic properties
Davide Zella, Sabrina Curreli, Francesca Benedetti, Selvi Krishnan, Fiorenza Cocchi, Olga S. Latinovic, Frank Denaro, Fabio Romerio, Muhammad Djavani, Man E. Charurat, Joseph L. Bryant, Hervé Tettelin, and Robert C. Gallo
We provide evidence here that (i) a strain of mycoplasma promotes lymphomagenesis in an in vivo mouse model; (ii) a bacterial chaperone protein, DnaK, is likely implicated in the transformation process and resistance to anticancer drugs by interfering with important pathways related to both DNA-damage control/repair and cell-cycle/apoptosis; and (iii) a very low copy number of DNA sequences of mycoplasma DnaK were found in some tumors of the infected mice. Other tumor-associated bacteria carry a similar DnaK protein. Our data suggest a common mechanism whereby bacteria can be involved in cellular transformation and resistance to anticancer drugs by a hit-and-hide/run mechanism. (See pp. E12005–E12014.)
Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories
Jeanette M. Criglar, Ramakrishnan Anish, Liya Hu, Sue E. Crawford, Banumathi Sankaran, B. V. Venkataram Prasad, and Mary K. Estes
Many DNA and RNA virus pathogens replicate in cytoplasmic compartments composed of viral and cellular proteins called virus factories or viroplasms. Using rotavirus (RV) as a model, we found that RV nonstructural protein NSP2 has autokinase activity. The cellular kinase CK1α additionally phosphorylates NSP2, triggering NSP2 octamers to form a lattice structure, and is required to build viroplasms. CK1α and viroplasm-specific NSP2 (vNSP2) are essential for RV nonstructural protein NSP5 hyperphosphorylation and association with vNSP2 for viroplasm formation. Protein regulation by phosphorylation is a common biological mechanism, raising the possibility that similar phosphorylation-dependent virus factory assembly mechanisms exist for other viral pathogens. (See pp. E12015–E12023.)
MRI demonstrates glutamine antagonist-mediated reversal of cerebral malaria pathology in mice
Brittany A. Riggle, Sanhita Sinharay, William Schreiber-Stainthorp, Jeeva P. Munasinghe, Dragan Maric, Eva Prchalova, Barbara S. Slusher, Jonathan D. Powell, Louis H. Miller, Susan K. Pierce, and Dima A. Hammoud
Cerebral malaria (CM) is the deadliest complication of Plasmodium falciparum infection, resulting in a 15 to 25% mortality rate in African children despite antimalarial chemotherapy. Tragically, nearly a fourth of pediatric CM survivors suffer long-term neurological sequelae. There is an urgent public health and humanitarian need for therapies for CM. In a mouse model of CM, we used magnetic resonance imaging (MRI) to monitor infected mice longitudinally as CM progressed and noninvasively demonstrate that the edema and blood–brain barrier dysfunction, which ultimately result in death, are rapidly reversed by treatment with the glutamine antagonist JHU-083. The similarities between CM MRI shown in mice and those reported in children and adults suggest that glutamine antagonists may be effective CM therapies. (See pp. E12024–E12033.)
Structural and effective brain connectivity underlying biological motion detection
Arseny A. Sokolov, Peter Zeidman, Michael Erb, Philippe Ryvlin, Karl J. Friston, and Marina A. Pavlova
Visual perception of body motion is of substantial value for social cognition and everyday life. By using an integrative approach to brain connectivity, the study sheds light on architecture and functional principles of the underlying cerebro-cerebellar network. This circuity is organized in a parallel rather than hierarchical fashion. This may explain why body-language reading is rather resilient to focal brain damage but severely affected in neuropsychiatric conditions with distributed network alterations. Furthermore, visual sensitivity to body motion is best predicted by specific top-down feedback to the early visual cortex, as well as functional communication (effective connectivity) and presence of white-matter pathways between the right fusiform gyrus and superior temporal sulcus. The findings allow better understanding of the social brain. (See pp. E12034–E12042.)
Ablation of α2δ-1 inhibits cell-surface trafficking of endogenous N-type calcium channels in the pain pathway in vivo
Manuela Nieto-Rostro, Krishma Ramgoolam, Wendy S. Pratt, Akos Kulik, and Annette C. Dolphin
Neuronal N-type (CaV2.2) voltage-gated calcium channels are important at the first synapse in the pain pathway. In this study, we have characterized a knockin mouse containing CaV2.2 with an extracellular HA tag to determine the localization of CaV2.2 in primary afferent pain pathways. These endogenous channels have been visualized at the plasma membrane and rigorously quantified in vivo. We examined the effect of ablation of the calcium channel auxiliary subunit α2δ-1 (the target of gabapentinoids) on CaV2.2 distribution. We found preferential cell-surface localization of CaV2.2 in DRG nociceptor neuron cell bodies was lost, accompanied by a dramatic reduction at dorsal horn terminals, but no effect on distribution of other spinal cord synaptic markers. (See pp. E12043–E12052.)
Synergistic neuroprotection by coffee components eicosanoyl-5-hydroxytryptamide and caffeine in models of Parkinson's disease and DLB
Run Yan, Jie Zhang, Hye-Jin Park, Eun S. Park, Stephanie Oh, Haiyan Zheng, Eunsung Junn, Michael Voronkov, Jeffry B. Stock, and M. Maral Mouradian
Coffee consumption is linked with reduced risk of Parkinson’s disease (PD), and caffeine is generally believed to be the protective agent. However, several lines of evidence suggest the presence of additional compound(s) in coffee that can be protective as well. Here we show that eicosanoyl-5-hydroxytryptamide, which we purified from coffee as an agent that leads to enhanced enzymatic activity of the specific phosphatase PP2A that dephosphorylates the pathogenic protein α-synuclein, works in synergy with caffeine in protecting against mouse models of PD and Dementia with Lewy bodies. The mechanism of this synergy is also through enhancing PP2A, which is dysregulated in the brains of individuals with these α-synucleinopathies. (See pp. E12053–E12062.)
Inflammation in the hippocampus affects IGF1 receptor signaling and contributes to neurological sequelae in rheumatoid arthritis
Karin M. E. Andersson, Caroline Wasén, Lina Juzokaite, Lovisa Leifsdottir, Malin C. Erlandsson, Sofia T. Silfverswärd, Anna Stokowska, Marcela Pekna, Milos Pekny, Kjell Olmarker, Rolf A. Heckemann, Marie Kalm, and Maria I. Bokarewa
Aberrant insulin-like growth factor 1 receptor (IGF1R)/insulin receptor signaling in brain has recently been linked to neurodegeneration in diabetes mellitus and in Alzheimer’s disease. In this study, we demonstrate that functional disability and pain in patients with rheumatoid arthritis (RA) and in experimental RA are associated with hippocampal inflammation and inhibition of IGF1R/insulin receptor substrate 1 (IRS1) signal, reproducing an IGF1/insulin-resistant state. This restricts formation of new neurons in the hippocampus, reduces hippocampal volume, and predisposes RA patients to develop neurological symptoms. Improving IRS1 function through down-regulation of IGF1R disinhibits neurogenesis and can potentially ameliorate neurological symptoms. This opens perspectives for drugs that revert IGF1/insulin resistance as an essential complement to the antirheumatic and antiinflammatory arsenal. (See pp. E12063–E12072.)
Two distinct profiles of fMRI and neurophysiological activity elicited by acetylcholine in visual cortex
Daniel Zaldivar, Alexander Rauch, Nikos K. Logothetis, and Jozien Goense
fMRI changes are typically assumed to be due to changes in neural activity, although whether this remains valid under the influence of neuromodulators is relatively unknown. Here, we found evidence that intracortical acetylcholine elicits distinct profiles of fMRI and electrophysiological activity in visual cortex. Two patterns of cholinergic activity were observed, depending on the distance to the injection site, although neurovascular coupling was preserved. Our results illustrate the effects of neuromodulators on fMRI and electrophysiological responses and show that these depend on neuromodulator concentration and kinetics. (See pp. E12073–E12082.)
GABA release selectively regulates synapse development at distinct inputs on direction-selective retinal ganglion cells
Adam Bleckert, Chi Zhang, Maxwell H. Turner, David Koren, David M. Berson, Silvia J. H. Park, Jonathan B. Demb, Fred Rieke, Wei Wei, and Rachel O. Wong
Output characteristics of a neuron are shaped by synaptic inhibition onto its soma and dendrites. These subcellular compartments of direction-selective retinal ganglion cells (DSGCs) receive input from different types of inhibitory neurons, and only dendritic inhibition generates the DSGC’s direction selectivity. Perturbing γ-aminobutyric acid (GABA) release either at all or at selected GABAergic inputs uncovered differential roles for GABAergic transmission on synaptic development at the DSGC’s soma and dendrites. Although dendritic GABAA receptor clustering is largely invariant to transmission loss, somatic receptor clusters can increase in number and size. These findings advance our understanding of the roles of activity-dependent and -independent mechanisms in the development of inhibitory inputs that play separate roles in controlling the output of an individual neuron. (See pp. E12083–E12090.)
Tissue-specific contributions of Tmem79 to atopic dermatitis and mast cell-mediated histaminergic itch
Joshua J. Emrick, Anubhav Mathur, Jessica Wei, Elena O. Gracheva, Karsten Gronert, Michael D. Rosenblum, and David Julius
Atopic dermatitis (AD) is a common skin disease affecting children, but the mechanisms underlying the accompanying itch remain poorly understood. Indeed, treatments to alleviate itch in AD and the substantial morbidity it represents for patients are often unsuccessful. Here, we shed light on a form of AD associated with the loss of Tmem79 and provide insights into cellular and molecular players in this genetically driven form of the disease that has been described in mice and humans. We propose a link between this inherited form of AD and the recognized contribution of oxidative stress to this disease. Our analysis may suggest pharmacological strategies for treating patients who suffer from AD and bear mutations in Tmem79. (See pp. E12091–E12100.)
Low-oxygen response is triggered by an ATP-dependent shift in oleoyl-CoA in Arabidopsis
Romy R. Schmidt, Martin Fulda, Melanie V. Paul, Max Anders, Frederic Plum, Daniel A. Weits, Monika Kosmacz, Tony R. Larson, Ian A. Graham, Gerrit T. S. Beemster, Francesco Licausi, Peter Geigenberger, Jos H. Schippers, and Joost T. van Dongen
To control adaptive responses to the ever-changing environment that plants are continuously exposed to, plant cells must integrate a multitude of information to make optimal decisions. Here, we reveal how plants can link information about the cellular energy status with the actual oxygen concentration of the cell to trigger a response reaction to low-oxygen stress. We reveal that oleoyl-CoA has a moonlighting function in an energy (ATP)-dependent signal transduction pathway in plants, and we provide a model that explains how diminishing oxygen availability can initiate adaptive responses when it coincides with a decreased energy status of the cell. (See pp. E12101–E12110.)
A unique ferredoxin acts as a player in the low-iron response of photosynthetic organisms
Michael Schorsch, Manuela Kramer, Tatjana Goss, Marion Eisenhut, Nigel Robinson, Deenah Osman, Annegret Wilde, Shamaila Sadaf, Hendrik Brückler, Lorenz Walder, Renate Scheibe, Toshiharu Hase, and Guy T. Hanke
Iron limits the growth of photosynthetic organisms, especially in marine environments. Understanding the response of photosynthetic organisms to changing iron concentrations is therefore important for agriculture and biotechnology. We have identified a protein that is essential for the correct response to changing iron concentrations in photosynthetic bacteria (cyanobacteria). This protein was previously annotated as an electron transfer component of photosynthesis, called Fed2, and contains an iron−sulfur cluster. We tested Fed2, and found that it cannot act in photosynthetic electron transport. The corresponding gene is essential, and is highly conserved between cyanobacteria, algae, and higher plants. By specifically perturbing its function, we could show that it is essential for the low-iron response at the posttranscriptional level. (See pp. E12111–E12120.)