The E3 ubiquitin ligase RNF115 regulates phagosome maturation and host response to bacterial infection.

Phagocytosis is a key process in innate immunity and homeostasis. After particle uptake, newly formed phagosomes mature by acquisition of endolysosomal enzymes. Macrophage activation by interferon gamma (IFN-γ) increases microbicidal activity, but delays phagosomal maturation by an unknown mechanism. Using quantitative proteomics, we show that phagosomal proteins harbour high levels of typical and atypical ubiquitin chain types. Moreover, phagosomal ubiquitylation of vesicle trafficking proteins is substantially enhanced upon IFN-γ activation of macrophages, suggesting a role in regulating phagosomal functions. We identified the E3 ubiquitin ligase RNF115, which is enriched on phagosomes of IFN-γ activated macrophages, as an important regulator of phagosomal maturation. Loss of RNF115 protein or ligase activity enhanced phagosomal maturation and increased cytokine responses to bacterial infection, suggesting that both innate immune signalling from the phagosome and phagolysosomal trafficking are controlled through ubiquitylation. RNF115 knock-out mice show less tissue damage in response to S. aureus infection, indicating a role of RNF115 in inflammatory responses in vivo. In conclusion, RNF115 and phagosomal ubiquitylation are important regulators of innate immune functions during bacterial infections.

Fully Flexible Ligand Docking for the P2X7 Receptor Using ROSIE.

The availability of P2X7 receptor structures with allosteric antagonists bound enables us to predict specific interactions between receptor and antagonists at atomistic detail. In this chapter we outline how modern ligand docking techniques can be employed by the nonexpert to predict putative binding modes for known or hypothetical allosteric P2X7 antagonists.

Two-step nucleation in confined geometry: Phase diagram of finite particles on a lattice gas model.

We use a degenerated Ising model to describe nucleation and crystallization from solution in a confined two-component system. The free energy is calculated using metadynamics simulation with coordination numbers as the reaction coordinates. We deploy nudged elastic band simulation to determine the minimum energy path and give properties of the crystallization path. In this confined system, depletion effects, which could also be caused by slow material transport in the solution, prevent the post-critical cluster from further growth, and the crystalline state would only be stable at larger cluster sizes. Fluctuation of the higher coupling strength of the crystalline state enables further growth until the crystalline cluster is in equilibrium with the solvent, and this way, a second barrier is crossed. From the parameters and setup, we find necessary conditions for the occurrence of two-step nucleation in our system. These findings can be adapted to real systems as biomineralization, colloidal crystallization, and the solidification of metals.

Structural Basis of the Negative Allosteric Modulation of 5-BDBD at Human P2X4 Receptors.

The P2X4 receptor is a ligand-gated ion channel activated by extracellular ATP. P2X4 activity is associated with neuropathic pain, vasodilation, and pulmonary secretion and is therefore of therapeutic interest. The structure-activity relationship of P2X4 antagonists is poorly understood. Here we elucidate the structure-activity of 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) at human P2X4 by combining pharmacology, electrophysiology, molecular modeling, and medicinal chemistry. 5-BDBD antagonized P2X4 in a noncompetitive manner but lacked effect at human P2X2. Molecular modeling and site-directed mutagenesis suggested an allosteric binding site for 5-BDBD located between two subunits in the body region of P2X4, with M109, F178, Y300, and I312 on one subunit and R301 on the neighboring subunit as key residues involved in antagonist binding. The bromine group of 5-BDBD was redundant for the antagonist activity of 5-BDBD, although an interaction between the carbonyl group of 5-BDBD and R301 in P2X4 was associated with 5-BDBD activity. 5-BDBD could inhibit the closed channel but poorly inhibited the channel in the open/desensitizing state. We hypothesize that this is due to constriction of the allosteric site after transition from closed to open channel state. We propose that M109, F178, Y300, R301, and I312 are key residues for 5-BDBD binding; provide a structural explanation of how they contribute to 5-BDBD antagonism; and highlight that the limited action of 5-BDBD on open versus closed channels is due to a conformational change in the allosteric site. SIGNIFICANCE STATEMENT: Activity of P2X4 receptor is associated with neuropathic pain, inflammation, and vasodilatation. Molecular information regarding small-molecule interaction with P2X4 is very limited. Here, this study provides a structural explanation for the action of the small-molecule antagonist 5-BDBD at the human P2X4 receptor.

CRISPR/Cas9 directed to the Ube3a antisense transcript improves Angelman syndrome phenotype in mice.

Gene editing holds the potential to correct mutations and cure devastating genetic disorders. The technology has not yet proven efficacious for therapeutic use in CNS diseases with ubiquitous neuronal defects. Angelman syndrome (AS), a severe neurodevelopmental disorder, is caused by a lack of maternal expression of the UBE3A gene. Because of genomic imprinting, only neurons are affected. One therapeutic approach focuses on the intact paternal UBE3A copy in patients with AS that is silenced by an antisense transcript (UBE3A-ATS). We show here that gene editing of Ube3a-ATS in the mouse brain resulted in the formation of base pair insertions/deletions (indels) in neurons and the subsequent unsilencing of the paternal Ube3a allele in neurons, which partially corrected the behavioral phenotype of a murine AS model. This study provides compelling evidence to further investigate editing of the homologous region of the human UBE3A-ATS because this may provide a lasting therapeutic effect for patients with AS.

Chd8 haploinsufficiency impairs early brain development and protein homeostasis later in life.

BACKGROUND: Chromodomain helicase DNA-binding protein 8 (Chd8) is a high-confidence risk gene for autism spectrum disorder (ASD). However, how Chd8 haploinsufficiency impairs gene expression in the brain and impacts behavior at different stages of life is unknown. METHODS: We generated a mutant mouse line with an ASD-linked loss-of-function mutation in Chd8 (V986*; stop codon mutation). We examined the behavior of Chd8 mutant mice along with transcriptional changes in the cerebral cortex as a function of age, with a focus on one embryonic (E14.5) and three postnatal ages (1, 6, and 12 months). RESULTS: Chd8V986*/+ mutant mice displayed macrocephaly, reduced rearing responses and reduced center time in the open field, and enhanced social novelty preference. Behavioral phenotypes were more evident in Chd8V986*/+ mutant mice at 1 year of age. Pup survival was reduced in wild-type x Chd8V986*/+ crosses when the mutant parent was female. Transcriptomic analyses indicated that pathways associated with synaptic and neuronal projections and sodium channel activity were reduced in the cortex of embryonic Chd8V986*/+ mice and then equalized relative to wild-type mice in the postnatal period. At 12 months of age, expression of genes associated with endoplasmic reticulum (ER) stress, chaperone-mediated protein folding, and the unfolded protein response (UPR) were reduced in Chd8V986*/+ mice, whereas genes associated with the c-MET signaling pathway were increased in expression. LIMITATIONS: It is unclear whether the transcriptional changes observed with age in Chd8V986*/+ mice reflect a direct effect of CHD8-regulated gene expression, or if CHD8 indirectly affects the expression of UPR/ER stress genes in adult mice as a consequence of neurodevelopmental abnormalities. CONCLUSIONS: Collectively, these data suggest that UPR/ER stress pathways are reduced in the cerebral cortex of aged Chd8V986*/+ mice. Our study uncovers neurodevelopmental and age-related phenotypes in Chd8V986*/+ mice and highlights the importance of controlling for age when studying Chd8 haploinsufficient mice.

Adeno-Associated Virus-Induced Dorsal Root Ganglion Pathology.

The administration of adeno-associated virus (AAV) vectors to nonhuman primates (NHP) via the blood or cerebrospinal fluid (CSF) can lead to dorsal root ganglion (DRG) pathology. The pathology is minimal to moderate in most cases; clinically silent in affected animals; and characterized by mononuclear cell infiltrates, neuronal degeneration, and secondary axonopathy of central and peripheral axons on histopathological analysis. We aggregated data from 33 nonclinical studies in 256 NHP and performed a meta-analysis of the severity of DRG pathology to compare different routes of administration, dose, time course, study conduct, age of the animals, sex, capsid, promoter, capsid purification method, and transgene. DRG pathology was observed in 83% of NHP that were administered AAV through the CSF, and 32% of NHP that received an intravenous (IV) injection. We show that dose and age at injection significantly affected the severity whereas sex had no impact. DRG pathology was minimal at acute time points (i.e., <14 days), similar from one to 5 months post-injection, and was less severe after 6 months. Vector purification method had no impact, and all capsids and promoters that we tested resulted in some DRG pathology. The data presented here from five different capsids, five different promoters, and 20 different transgenes suggest that DRG pathology is almost universal after AAV gene therapy in nonclinical studies using NHP. None of the animals receiving a therapeutic transgene displayed any clinical signs. Incorporation of sensitive techniques such as nerve-conduction velocity testing can show alterations in a minority of animals that correlate with the severity of peripheral nerve axonopathy. Monitoring sensory neuropathies in human central nervous system and high-dose IV clinical studies seems prudent to determine the functional consequences of DRG pathology.

Discovery of a heme-binding domain in a neuronal voltage-gated potassium channel.

The EAG (ether-à-go-go) family of voltage-gated K+ channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer, and sudden cardiac death. A defining feature of EAG (Kv10-12) channels is a highly conserved domain on the N terminus, known as the eag domain, consisting of a Per-ARNT-Sim (PAS) domain capped by a short sequence containing an amphipathic helix (Cap domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pulldown assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole-cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in Escherichia coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells.

Author Correction: A transcriptomic analysis of the phylum Nematoda.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Discovery and Structure Relationships of Salicylanilide Derivatives as Potent, Non-acidic P2X1 Receptor Antagonists.

Antagonists for the ATP-gated ion channel receptor P2X1 have potential as antithrombotics and for treating hyperactive bladder and inflammation. In this study, salicylanilide derivatives were synthesized based on a screening hit. P2X1 antagonistic potency was assessed in 1321N1 astrocytoma cells stably transfected with the human P2X1 receptor by measuring inhibition of the ATP-induced calcium influx. Structure-activity relationships were analyzed, and selectivity versus other P2X receptor subtypes was assessed. The most potent compounds, N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (1, IC50 0.0192 µM) and N-[3,5-bis(trifluoromethyl)phenyl]-4-chloro-2-hydroxybenzamide (14, IC50 0.0231 µM), displayed >500-fold selectivity versus P2X2 and P2X3, and 10-fold selectivity versus P2X4 and P2X7 receptors, and inhibited collagen-induced platelet aggregation. They behaved as negative allosteric modulators, and molecular modeling studies suggested an extracellular binding site. Besides selective P2X1 antagonists, compounds with ancillary P2X4 and/or P2X7 receptor inhibition were discovered. These compounds represent the first potent, non-acidic, allosteric P2X1 receptor antagonists reported to date.

Identification of a distinct desensitisation gate in the ATP-gated P2X2 receptor.

P2X receptors are trimeric ATP-gated ion channels. In response to ATP binding, conformational changes lead to opening of the channel and ion flow. Current flow can decline during continued ATP binding in a process called desensitisation. The rate and extent of desensitisation is affected by multiple factors, for instance the T18A mutation in P2X2 makes the ion channel fast desensitising. We have used this mutation to investigate whether the gate restricting ion flow is different in the desensitised and the closed state, by combining molecular modelling and cysteine modification using MTSET (2-(Trimethylammonium)ethyl methanethiosulfonate). Homology modelling of the P2X2 receptor and negative space imaging of the channel suggested a movement of the restriction gate with residue T335 being solvent accessible in the desensitised, but not the closed state. This was confirmed experimentally by probing the accessibility of T335C in the P2X2 T18A/T335C (fast desensitisation) and T335C (slow desensitisation) mutants with MTSET which demonstrates that the barrier to ion flow is different in the closed and the desensitised states. To investigate the T18A induced switch in desensitisation we compared molecular dynamics simulations of the wild type and T18A P2X2 receptor which suggest that the differences in time course of desensitisation are due to structural destabilization of a hydrogen bond network of conserved residues in the proximity of T18.

Homology Modeling of P2X Receptors.

Since the X-ray structure of the zebra fish P2X4 receptor in the closed state was published in 2009 homology modeling has been used to generate structural models for P2X receptors. In this chapter, we outline how to use the MODELLER software to generate such structural models for P2X receptors whose structures have not been solved yet.

Mapping the Site of Action of Human P2X7 Receptor Antagonists AZ11645373, Brilliant Blue G, KN-62, Calmidazolium, and ZINC58368839 to the Intersubunit Allosteric Pocket.

The P2X7 receptor is a trimeric ligand-gated ion channel activated by ATP. It is implicated in the cellular response to trauma/disease and considered to have significant therapeutic potential. Using chimeras and point mutants we have mapped the binding site of the P2X7R-selective antagonist AZ11645373 to the known allosteric binding pocket at the interface between two subunits, in proximity to, but separated from the ATP binding site. Our structural model of AZ11645373 binding is consistent with effects of mutations on antagonist sensitivity, and the proposed binding mode explains variation in antagonist sensitivity between the human and rat P2X7 receptors. We have also determined the site of action for the P2X7R-selective antagonists ZINC58368839, brilliant blue G, KN-62, and calmidazolium. The effect of intersubunit allosteric pocket "signature mutants" F88A, T90V, D92A, F103A, and V312A on antagonist sensitivity suggests that ZINC58368839 comprises a binding mode similar to AZ11645373 and other previously characterized antagonists. For the larger antagonists, brilliant blue G, KN-62, and calmidazolium, our data imply an overlapping but distinct binding mode involving the central upper vestibule of the receptor in addition to the intersubunit allosteric pocket. Our work explains the site of action for a series of P2X7R antagonists and establishes "signature mutants" for P2X7R binding-mode characterization.

Cytosolic glucosylceramide regulates endolysosomal function in Niemann-Pick type C disease.

Niemann-Pick type C disease (NPCD) is a neurodegenerative disease associated with increases in cellular cholesterol and glycolipids and most commonly caused by defective NPC1, a late endosomal protein. Using ratiometric probes we find that NPCD cells show increased endolysosomal pH. In addition U18666A, an inhibitor of NPC1, was found to increase endolysosomal pH, and the number, size and heterogeneity of endolysosomal vesicles. NPCD fibroblasts and cells treated with U18666A also show disrupted targeting of fluorescent lipid BODIPY-LacCer to high pH vesicles. Inhibiting non-lysosomal glucocerebrosidase (GBA2) reversed increases in endolysosomal pH and restored disrupted BODIPY-LacCer trafficking in NPCD fibroblasts. GBA2 KO cells also show decreased endolysosomal pH. NPCD fibroblasts also show increased expression of a key subunit of the lysosomal proton pump vATPase on GBA2 inhibition. The results are consistent with a model where both endolysosomal pH and Golgi targeting of BODIPY-LacCer are dependent on adequate levels of cytosolic-facing GlcCer, which are reduced in NPC disease.

Lipid⁻Protein Interactions in Niemann⁻Pick Type C Disease: Insights from Molecular Modeling.

The accumulation of lipids in the late endosomes and lysosomes of Niemann⁻Pick type C disease (NPCD) cells is a consequence of the dysfunction of one protein (usually NPC1) but induces dysfunction in many proteins. We used molecular docking to propose (a) that NPC1 exports not just cholesterol, but also sphingosine, (b) that the cholesterol sensitivity of big potassium channel (BK) can be traced to a previously unappreciated site on the channel's voltage sensor, (c) that transient receptor potential mucolipin 1 (TRPML1) inhibition by sphingomyelin is likely an indirect effect, and (d) that phosphoinositides are responsible for both the mislocalization of annexin A2 (AnxA2) and a soluble NSF (N-ethylmaleimide Sensitive Fusion) protein attachment receptor (SNARE) recycling defect. These results are set in the context of existing knowledge of NPCD to sketch an account of the endolysosomal pathology key to this disease.

Stability of nanoparticles in solution: A statistical description of crystallization as a finite particle size effect in a lattice-gas model.

We employ the well-tempered parallel-bias metadynamics algorithm to study the stability of nanoparticles in a lattice gas for crystallization from solution. The model allows us to give a description for the transition from amorphous to crystalline nanoparticles by introducing parameters directly related to the surface tensions of the two phases and also the differences of the entropy per particle in each phase. By examining the parameter space, we find a critical cluster size of crystalline stability, whose temperature and size dependencies follow the Gibbs-Thomson equation. An additional melting point depression due to cluster surface fluctuations is observed, leading to a non-classical nucleation barrier of cluster growth.