Structural mechanisms of α7 nicotinic receptor allosteric modulation and activation.

The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that plays an important role in cholinergic signaling throughout the nervous system. Its unique physiological characteristics and implications in neurological disorders and inflammation make it a promising but challenging therapeutic target. Positive allosteric modulators overcome limitations of traditional α7 agonists, but their potentiation mechanisms remain unclear. Here, we present high-resolution structures of α7-modulator complexes, revealing partially overlapping binding sites but varying conformational states. Structure-guided functional and computational tests suggest that differences in modulator activity arise from the stable rotation of a channel gating residue out of the pore. We extend the study using a time-resolved cryoelectron microscopy (cryo-EM) approach to reveal asymmetric state transitions for this homomeric channel and also find that a modulator with allosteric agonist activity exploits a distinct channel-gating mechanism. These results define mechanisms of α7 allosteric modulation and activation with implications across the pentameric receptor superfamily.

Structural mechanisms of GABAA receptor autoimmune encephalitis.

Autoantibodies targeting neuronal membrane proteins can cause encephalitis, seizures, and severe behavioral abnormalities. While antibodies for several neuronal targets have been identified, structural details on how they regulate function are unknown. Here we determined cryo-electron microscopy structures of antibodies derived from an encephalitis patient bound to the γ-aminobutyric acid type A (GABAA) receptor. These antibodies induced severe encephalitis by directly inhibiting GABAA function, resulting in nervous-system hyperexcitability. The structures reveal mechanisms of GABAA inhibition and pathology. One antibody directly competes with a neurotransmitter and locks the receptor in a resting-like state. The second antibody targets the subunit interface involved in binding benzodiazepines and antagonizes diazepam potentiation. We identify key residues in these antibodies involved in specificity and affinity and confirm structure-based hypotheses for functional effects using electrophysiology. Together these studies define mechanisms of direct functional antagonism of neurotransmission underlying autoimmune encephalitis in a human patient.

Structure and gating mechanism of the α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor plays critical roles in the central nervous system and in the cholinergic inflammatory pathway. This ligand-gated ion channel assembles as a homopentamer, is exceptionally permeable to Ca2+, and desensitizes faster than any other Cys-loop receptor. The α7 receptor has served as a prototype for the Cys-loop superfamily yet has proven refractory to structural analysis. We present cryo-EM structures of the human α7 nicotinic receptor in a lipidic environment in resting, activated, and desensitized states, illuminating the principal steps in the gating cycle. The structures also reveal elements that contribute to its function, including a C-terminal latch that is permissive for channel opening, and an anionic ring in the extracellular vestibule that contributes to its high conductance and calcium permeability. Comparisons among the α7 structures provide a foundation for mapping the gating cycle and reveal divergence in gating mechanisms in the Cys-loop receptor superfamily.

Transaminase Inhibition by 2-Hydroxyglutarate Impairs Glutamate Biosynthesis and Redox Homeostasis in Glioma.

IDH1 mutations are common in low-grade gliomas and secondary glioblastomas and cause overproduction of (R)-2HG. (R)-2HG modulates the activity of many enzymes, including some that are linked to transformation and some that are probably bystanders. Although prior work on (R)-2HG targets focused on 2OG-dependent dioxygenases, we found that (R)-2HG potently inhibits the 2OG-dependent transaminases BCAT1 and BCAT2, likely as a bystander effect, thereby decreasing glutamate levels and increasing dependence on glutaminase for the biosynthesis of glutamate and one of its products, glutathione. Inhibiting glutaminase specifically sensitized IDH mutant glioma cells to oxidative stress in vitro and to radiation in vitro and in vivo. These findings highlight the complementary roles for BCATs and glutaminase in glutamate biosynthesis, explain the sensitivity of IDH mutant cells to glutaminase inhibitors, and suggest a strategy for maximizing the effectiveness of such inhibitors against IDH mutant gliomas.

Mating in the Closest Living Relatives of Animals Is Induced by a Bacterial Chondroitinase.

We serendipitously discovered that the marine bacterium Vibrio fischeri induces sexual reproduction in one of the closest living relatives of animals, the choanoflagellate Salpingoeca rosetta. Although bacteria influence everything from nutrition and metabolism to cell biology and development in eukaryotes, bacterial regulation of eukaryotic mating was unexpected. Here, we show that a single V. fischeri protein, the previously uncharacterized EroS, fully recapitulates the aphrodisiac-like activity of live V. fischeri. EroS is a chondroitin lyase; although its substrate, chondroitin sulfate, was previously thought to be an animal synapomorphy, we demonstrate that S. rosetta produces chondroitin sulfate and thus extend the ancestry of this important glycosaminoglycan to the premetazoan era. Finally, we show that V. fischeri, purified EroS, and other bacterial chondroitin lyases induce S. rosetta mating at environmentally relevant concentrations, suggesting that bacteria likely regulate choanoflagellate mating in nature.

Structural basis of sensory receptor evolution in octopus.

Chemotactile receptors (CRs) are a cephalopod-specific innovation that allow octopuses to explore the seafloor via 'taste by touch'1. CRs diverged from nicotinic acetylcholine receptors to mediate contact-dependent chemosensation of insoluble molecules that do not readily diffuse in marine environments. Here we exploit octopus CRs to probe the structural basis of sensory receptor evolution. We present the cryo-electron microscopy structure of an octopus CR and compare it with nicotinic receptors to determine features that enable environmental sensation versus neurotransmission. Evolutionary, structural and biophysical analyses show that the channel architecture involved in cation permeation and signal transduction is conserved. By contrast, the orthosteric ligand-binding site is subject to diversifying selection, thereby mediating the detection of new molecules. Serendipitous findings in the cryo-electron microscopy structure reveal that the octopus CR ligand-binding pocket is exceptionally hydrophobic, enabling sensation of greasy compounds versus the small polar molecules detected by canonical neurotransmitter receptors. These discoveries provide a structural framework for understanding connections between evolutionary adaptations at the atomic level and the emergence of new organismal behaviour.

Sensory specializations drive octopus and squid behaviour.

The evolution of new traits enables expansion into new ecological and behavioural niches. Nonetheless, demonstrated connections between divergence in protein structure, function and lineage-specific behaviours remain rare. Here we show that both octopus and squid use cephalopod-specific chemotactile receptors (CRs) to sense their respective marine environments, but structural adaptations in these receptors support the sensation of specific molecules suited to distinct physiological roles. We find that squid express ancient CRs that more closely resemble related nicotinic acetylcholine receptors, whereas octopuses exhibit a more recent expansion in CRs consistent with their elaborated 'taste by touch' sensory system. Using a combination of genetic profiling, physiology and behavioural analyses, we identify the founding member of squid CRs that detects soluble bitter molecules that are relevant in ambush predation. We present the cryo-electron microscopy structure of a squid CR and compare this with octopus CRs1 and nicotinic receptors2. These analyses demonstrate an evolutionary transition from an ancestral aromatic 'cage' that coordinates soluble neurotransmitters or tastants to a more recent octopus CR hydrophobic binding pocket that traps insoluble molecules to mediate contact-dependent chemosensation. Thus, our study provides a foundation for understanding how adaptation of protein structure drives the diversification of organismal traits and behaviour.

Deciphering the immunopeptidome in vivo reveals new tumour antigens.

Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I) molecules1-5. Current approaches to profiling of MHC-I-associated peptides, collectively known as the immunopeptidome, are limited to in vitro investigation or bulk tumour lysates, which limits our understanding of cancer-specific patterns of antigen presentation in vivo6. To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the KrasLSL-G12D/+Trp53fl/fl mouse model (KP/KbStrep)7. This approach enabled us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma and from lung adenocarcinoma (LUAD) in vivo. In addition, we profiled the LUAD immunopeptidome from the alveolar type 2 cell of origin up to late-stage disease. Differential peptide presentation in LUAD was not predictable by mRNA expression or translation efficiency and is probably driven by post-translational mechanisms. Vaccination with peptides presented by LUAD in vivo induced CD8+ T cell responses in naive mice and tumour-bearing mice. Many peptides specific to LUAD, including immunogenic peptides, exhibited minimal expression of the cognate mRNA, which prompts the reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance8. Beyond cancer, the KbStrep allele is compatible with other Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease and autoimmunity.

Context-Dependent Gene Regulation by Homeodomain Transcription Factor Complexes Revealed by Shape-Readout Deficient Proteins.

Eukaryotic transcription factors (TFs) form complexes with various partner proteins to recognize their genomic target sites. Yet, how the DNA sequence determines which TF complex forms at any given site is poorly understood. Here, we demonstrate that high-throughput in vitro DNA binding assays coupled with unbiased computational analysis provide unprecedented insight into how different DNA sequences select distinct compositions and configurations of homeodomain TF complexes. Using inferred knowledge about minor groove width readout, we design targeted protein mutations that destabilize homeodomain binding both in vitro and in vivo in a complex-specific manner. By performing parallel systematic evolution of ligands by exponential enrichment sequencing (SELEX-seq), chromatin immunoprecipitation sequencing (ChIP-seq), RNA sequencing (RNA-seq), and Hi-C assays, we not only classify the majority of in vivo binding events in terms of complex composition but also infer complex-specific functions by perturbing the gene regulatory network controlled by a single complex.

Candida albicans selection for human commensalism results in substantial within-host diversity without decreasing fitness for invasive disease.

Candida albicans is a frequent colonizer of human mucosal surfaces as well as an opportunistic pathogen. C. albicans is remarkably versatile in its ability to colonize diverse host sites with differences in oxygen and nutrient availability, pH, immune responses, and resident microbes, among other cues. It is unclear how the genetic background of a commensal colonizing population can influence the shift to pathogenicity. Therefore, we examined 910 commensal isolates from 35 healthy donors to identify host niche-specific adaptations. We demonstrate that healthy people are reservoirs for genotypically and phenotypically diverse C. albicans strains. Using limited diversity exploitation, we identified a single nucleotide change in the uncharacterized ZMS1 transcription factor that was sufficient to drive hyper invasion into agar. We found that SC5314 was significantly different from the majority of both commensal and bloodstream isolates in its ability to induce host cell death. However, our commensal strains retained the capacity to cause disease in the Galleria model of systemic infection, including outcompeting the SC5314 reference strain during systemic competition assays. This study provides a global view of commensal strain variation and within-host strain diversity of C. albicans and suggests that selection for commensalism in humans does not result in a fitness cost for invasive disease.

Structural variation in the sequencing era.

Identifying structural variation (SV) is essential for genome interpretation but has been historically difficult due to limitations inherent to available genome technologies. Detection methods that use ensemble algorithms and emerging sequencing technologies have enabled the discovery of thousands of SVs, uncovering information about their ubiquity, relationship to disease and possible effects on biological mechanisms. Given the variability in SV type and size, along with unique detection biases of emerging genomic platforms, multiplatform discovery is necessary to resolve the full spectrum of variation. Here, we review modern approaches for investigating SVs and proffer that, moving forwards, studies integrating biological information with detection will be necessary to comprehensively understand the impact of SV in the human genome.

Shared structural mechanisms of general anaesthetics and benzodiazepines.

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1-5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.

Interictal epileptiform discharges affect memory in an Alzheimer's disease mouse model.

Interictal epileptiform discharges (IEDs) are transient abnormal electrophysiological events commonly observed in epilepsy patients but are also present in other neurological diseases, such as Alzheimer's disease (AD). Understanding the role IEDs have on the hippocampal circuit is important for our understanding of the cognitive deficits seen in epilepsy and AD. We characterize and compare the IEDs of human epilepsy patients from microwire hippocampal recording with those of AD transgenic mice with implanted multilayer hippocampal silicon probes. Both the local field potential features and firing patterns of pyramidal cells and interneurons were similar in the mouse and human. We found that as IEDs emerged from the CA3-1 circuits, they recruited pyramidal cells and silenced interneurons, followed by post-IED suppression. IEDs suppressed the incidence and altered the properties of physiological sharp-wave ripples, altered their physiological properties, and interfered with the replay of place field sequences in a maze. In addition, IEDs in AD mice inversely correlated with daily memory performance. Together, our work implies that IEDs may present a common and epilepsy-independent phenomenon in neurodegenerative diseases that perturbs hippocampal-cortical communication and interferes with memory.

Physiological Functions of CRAC Channels.

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ signaling pathway that is evolutionarily conserved across eukaryotes. SOCE is triggered physiologically when the endoplasmic reticulum (ER) Ca2+ stores are emptied through activation of inositol 1,4,5-trisphosphate receptors. SOCE is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels, which are highly Ca2+ selective. Upon store depletion, the ER Ca2+-sensing STIM proteins aggregate and gain extended conformations spanning the ER-plasma membrane junctional space to bind and activate Orai, the pore-forming proteins of hexameric CRAC channels. In recent years, studies on STIM and Orai tissue-specific knockout mice and gain- and loss-of-function mutations in humans have shed light on the physiological functions of SOCE in various tissues. Here, we describe recent findings on the composition of native CRAC channels and their physiological functions in immune, muscle, secretory, and neuronal systems to draw lessons from transgenic mice and human diseases caused by altered CRAC channel activity.

Direct Structural Insights into GABAA Receptor Pharmacology.

GABAA receptors are pentameric ligand-gated ion channels that mediate most fast neuronal inhibition in the brain. In addition to their important physiological roles, they are noteworthy in their rich pharmacology; prominent drugs used for anxiety, insomnia, and general anesthesia act through positive modulation of GABAA receptors. Direct structural information for how these drugs work was absent until recently. Efforts in structural biology over the past few years have revealed how important drug classes and natural products interact with the GABAA receptor, providing a foundation for studies in dynamics and structure-guided drug design. Here, we review recent developments in GABAA receptor structural pharmacology, focusing on subunit assemblies of the receptor found at synapses.

Chlorthalidone vs. Hydrochlorothiazide for Hypertension-Cardiovascular Events.

BACKGROUND: Whether chlorthalidone is superior to hydrochlorothiazide for preventing major adverse cardiovascular events in patients with hypertension is unclear. METHODS: In a pragmatic trial, we randomly assigned adults 65 years of age or older who were patients in the Department of Veterans Affairs health system and had been receiving hydrochlorothiazide at a daily dose of 25 or 50 mg to continue therapy with hydrochlorothiazide or to switch to chlorthalidone at a daily dose of 12.5 or 25 mg. The primary outcome was a composite of nonfatal myocardial infarction, stroke, heart failure resulting in hospitalization, urgent coronary revascularization for unstable angina, and non-cancer-related death. Safety was also assessed. RESULTS: A total of 13,523 patients underwent randomization. The mean age was 72 years. At baseline, hydrochlorothiazide at a dose of 25 mg per day had been prescribed in 12,781 patients (94.5%). The mean baseline systolic blood pressure in each group was 139 mm Hg. At a median follow-up of 2.4 years, there was little difference in the occurrence of primary-outcome events between the chlorthalidone group (702 patients [10.4%]) and the hydrochlorothiazide group (675 patients [10.0%]) (hazard ratio, 1.04; 95% confidence interval, 0.94 to 1.16; P = 0.45). There were no between-group differences in the occurrence of any of the components of the primary outcome. The incidence of hypokalemia was higher in the chlorthalidone group than in the hydrochlorothiazide group (6.0% vs. 4.4%, P<0.001). CONCLUSIONS: In this large pragmatic trial of thiazide diuretics at doses commonly used in clinical practice, patients who received chlorthalidone did not have a lower occurrence of major cardiovascular outcome events or non-cancer-related deaths than patients who received hydrochlorothiazide. (Funded by the Veterans Affairs Cooperative Studies Program; ClinicalTrials.gov number, NCT02185417.).

Acetaminophen influences musculoskeletal signaling but not adaptations to endurance exercise training.

Acetaminophen (ACE) is a widely used analgesic and antipyretic drug with various applications, from pain relief to fever reduction. Recent studies have reported equivocal effects of habitual ACE intake on exercise performance, muscle growth, and risks to bone health. Thus, this study aimed to assess the impact of a 6-week, low-dose ACE regimen on muscle and bone adaptations in exercising and non-exercising rats. Nine-week-old Wistar rats (n = 40) were randomized to an exercise or control (no exercise) condition with ACE or without (placebo). For the exercise condition, rats ran 5 days per week for 6 weeks at a 5% incline for 2 min at 15 cm/s, 2 min at 20 cm/s, and 26 min at 25 cm/s. A human equivalent dose of ACE was administered (379 mg/kg body weight) in drinking water and adjusted each week based on body weight. Food, water intake, and body weight were measured daily. At the beginning of week 6, animals in the exercise group completed a maximal treadmill test. At the end of week 6, rats were euthanized, and muscle cross-sectional area (CSA), fiber type, and signaling pathways were measured. Additionally, three-point bending and microcomputer tomography were measured in the femur. Follow-up experiments in human primary muscle cells were used to explore supra-physiological effects of ACE. Data were analyzed using a two-way ANOVA for treatment (ACE or placebo) and condition (exercise or non-exercise) for all animal outcomes. Data for cell culture experiments were analyzed via ANOVA. If omnibus significance was found in either ANOVA, a post hoc analysis was completed, and a Tukey's adjustment was used. ACE did not alter body weight, water intake, food intake, or treadmill performance (p > .05). There was a treatment-by-condition effect for Young's Modulus where placebo exercise was significantly lower than placebo control (p < .05). There was no treatment by condition effects for microCT measures, muscle CSA, fiber type, or mRNA expression. Phosphorylated-AMPK was significantly increased with exercise (p < .05) and this was attenuated with ACE treatment. Furthermore, phospho-4EBP1 was depressed in the exercise group compared to the control (p < .05) and increased in the ACE control and ACE exercise group compared to placebo exercise (p < .05). A low dose of ACE did not influence chronic musculoskeletal adaptations in exercising rodents but acutely attenuated AMPK phosphorylation and 4EBP1 dephosphorylation post-exercise.

Natural mutagenesis of human genomes by endogenous retrotransposons.

Two abundant classes of mobile elements, namely Alu and L1 elements, continue to generate new retrotransposon insertions in human genomes. Estimates suggest that these elements have generated millions of new germline insertions in individual human genomes worldwide. Unfortunately, current technologies are not capable of detecting most of these young insertions, and the true extent of germline mutagenesis by endogenous human retrotransposons has been difficult to examine. Here, we describe technologies for detecting these young retrotransposon insertions and demonstrate that such insertions indeed are abundant in human populations. We also found that new somatic L1 insertions occur at high frequencies in human lung cancer genomes. Genome-wide analysis suggests that altered DNA methylation may be responsible for the high levels of L1 mobilization observed in these tumors. Our data indicate that transposon-mediated mutagenesis is extensive in human genomes and is likely to have a major impact on human biology and diseases.

The histone deacetylase Sirt6 regulates glucose homeostasis via Hif1alpha.

SIRT6 is a member of a highly conserved family of NAD(+)-dependent deacetylases with various roles in metabolism, stress resistance, and life span. SIRT6-deficient mice develop normally but succumb to a lethal hypoglycemia early in life; however, the mechanism underlying this hypoglycemia remained unclear. Here, we demonstrate that SIRT6 functions as a histone H3K9 deacetylase to control the expression of multiple glycolytic genes. Specifically, SIRT6 appears to function as a corepressor of the transcription factor Hif1alpha, a critical regulator of nutrient stress responses. Consistent with this notion, SIRT6-deficient cells exhibit increased Hif1alpha activity and show increased glucose uptake with upregulation of glycolysis and diminished mitochondrial respiration. Our studies uncover a role for the chromatin factor SIRT6 as a master regulator of glucose homeostasis and may provide the basis for novel therapeutic approaches against metabolic diseases, such as diabetes and obesity.

Differential interactions of resting, activated, and desensitized states of the α7 nicotinic acetylcholine receptor with lipidic modulators.

The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that modulates neuronal excitability, largely by allowing Ca2+ permeation. Agonist binding promotes transition from a resting state to an activated state, and then rapidly to a desensitized state. Recently, cryogenic electron microscopy (cryo-EM) structures of the human α7 receptor in nanodiscs were reported in multiple conformations. These were selectively stabilized by inhibitory, activating, or potentiating compounds. However, the functional annotation of these structures and their differential interactions with unresolved lipids and ligands remain incomplete. Here, we characterized their ion permeation, membrane interactions, and ligand binding using computational electrophysiology, free-energy calculations, and coarse-grained molecular dynamics. In contrast to nonconductive structures in apparent resting and desensitized states, the structure determined in the presence of the potentiator PNU-120596 was consistent with an activated state permeable to Ca2+. Transition to this state was associated with compression and rearrangement of the membrane, particularly in the vicinity of the peripheral MX helix. An intersubunit transmembrane site was implicated in selective binding of either PNU-120596 in the activated state or cholesterol in the desensitized state. This substantiates functional assignment of all three lipid-embedded α7-receptor structures with ion-permeation simulations. It also proposes testable models of their state-dependent interactions with lipophilic ligands, including a mechanism for allosteric modulation at the transmembrane subunit interface.