Acetylcholine receptor based chemogenetics engineered for neuronal inhibition and seizure control assessed in mice.

Epilepsy is a prevalent disorder involving neuronal network hyperexcitability, yet existing therapeutic strategies often fail to provide optimal patient outcomes. Chemogenetic approaches, where exogenous receptors are expressed in defined brain areas and specifically activated by selective agonists, are appealing methods to constrain overactive neuronal activity. We developed BARNI (Bradanicline- and Acetylcholine-activated Receptor for Neuronal Inhibition), an engineered channel comprised of the α7 nicotinic acetylcholine receptor ligand-binding domain coupled to an α1 glycine receptor anion pore domain. Here we demonstrate that BARNI activation by the clinical stage α7 nicotinic acetylcholine receptor-selective agonist bradanicline effectively suppressed targeted neuronal activity, and controlled both acute and chronic seizures in male mice. Our results provide evidence for the use of an inhibitory acetylcholine-based engineered channel activatable by both exogenous and endogenous agonists as a potential therapeutic approach to treating epilepsy.

DNA sensor-associated type I interferon signaling is increased in ulcerative colitis and induces JAK-dependent inflammatory cell death in colonic organoids.

DNA sensor pathways can initiate inflammasome, cell death, and type I interferon (IFN) signaling in immune-mediated inflammatory diseases (IMIDs), including type I interferonopathies. We investigated the involvement of these pathways in the pathogenesis of ulcerative colitis (UC) by analyzing the expression of DNA sensor, inflammasome, and type I IFN biomarker genes in colonic mucosal biopsy tissue from control (n = 31), inactive UC (n = 31), active UC (n = 33), and a UC single-cell RNA-Seq dataset. The effects of type I IFN (IFN-ß), IFN-γ, and TNF-α on gene expression, cytokine production, and cell death were investigated in human colonic organoids. In organoids treated with cytokines alone, or in combination with NLR family pyrin domain-containing 3 (NLRP3), caspase, or JAK inhibitors, cell death was measured, and supernatants were assayed for IL-1ß/IL-18/CXCL10. The expression of DNA sensor pathway genes-PYHIN family members [absent in melanoma 2 (AIM2), IFI16, myeloid cell nuclear differentiation antigen (MNDA), and pyrin and HIN domain family member 1 (PYHIN1)- as well as Z-DNA-binding protein 1 (ZBP1), cyclic GMP-AMP synthase (cGAS), and DDX41 was increased in active UC and expressed in a cell type-restricted pattern. Inflammasome genes (CASP1, IL1B, and IL18), type I IFN inducers [stimulator of interferon response cGAMP interactor 1 (STING), TBK1, and IRF3), IFNB1, and type I IFN biomarker genes (OAS2, IFIT2, and MX2) were also increased in active UC. Cotreatment of organoids with IFN-ß or IFN-γ in combination with TNFα increased expression of IFI16, ZBP1, CASP1, cGAS, and STING induced cell death and IL-1ß/IL-18 secretion. This inflammatory cell death was blocked by the JAK inhibitor tofacitinib but not by inflammasome or caspase inhibitors. Increased type I IFN activity may drive elevated expression of DNA sensor genes and JAK-dependent but inflammasome-independent inflammatory cell death of colonic epithelial cells in UC.NEW & NOTEWORTHY This study found that patients with active UC have significantly increased colonic gene expression of cytosolic DNA sensor, inflammasome, STING, and type I IFN signaling pathways. The type I IFN, IFN-ß, in combination with TNF-α induced JAK-dependent but NLRP3 and inflammasome-independent inflammatory cell death of colonic organoids. This novel inflammatory cell death phenotype is relevant to UC immunopathology and may partially explain the efficacy of the JAKinibs tofacitinib and upadacitinib in patients with UC.

Pharmacological inhibition of P2RX7 ameliorates liver injury by reducing inflammation and fibrosis.

Extracellular adenosine triphosphate (eATP) released by damaged cells, and its purinergic receptors, comprise a crucial signaling network after injury. Purinergic receptor P2X7 (P2RX7), a major driver of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and IL-1ß processing, has been shown to play a role in liver injury in murine diet- and chemically-induced liver injury models. It is unclear, however, whether P2RX7 plays a role in non-alcoholic steatohepatitis (NASH) and which cell type is the main target of P2RX7 pharmacological inhibition. Here, we report that P2RX7 is expressed by infiltrating monocytes and resident Kupffer cells in livers from NASH-affected individuals. Using primary isolated human cells, we demonstrate that P2RX7 expression in CD14+ monocytes and Kupffer cells primarily mediates IL-1ß release. In addition, we show that pharmacological inhibition of P2RX7 in monocytes and Kupffer cells, blocks IL-1ß release, reducing hepatocyte caspase 3/7 activity, IL-1ß-mediated CCL2 and CCL5 chemokine gene expression and secretion, and hepatic stellate cell (HSC) procollagen secretion. Consequently, in a chemically-induced nonhuman primate model of liver fibrosis, treatment with a P2RX7 inhibitor improved histological characteristics of NASH, protecting from liver inflammation and fibrosis. Taken together, these findings underscore the critical role of P2RX7 in the pathogenesis of NASH and implicate P2RX7 as a promising therapeutic target for the management of this disease.

Stability of a receptor-binding active human immunodeficiency virus type 1 recombinant gp140 trimer conferred by intermonomer disulfide bonding of the V3 loop: differential effects of protein disulfide isomerase on CD4 and coreceptor binding.

Stable trimeric forms of human immunodeficiency virus recombinant gp140 (rgp140) are important templates for determining the structure of the glycoprotein to assist in our understanding of HIV infection and host immune response. Such information will aid the design of therapeutic drugs and vaccines. Here, we report the production of a highly stable and trimeric rgp140 derived from a HIV type 1 (HIV-1) subtype D isolate that may be suitable for structural studies. The rgp140 is functional in terms of binding to CD4 and three human monoclonal antibodies (17b, b12, and 2G12) that have broad neutralizing activities against a range of HIV-1 isolates from different subtypes. Treatment of rgp140 with protein disulfide isomerase (PDI) severely restricted 17b binding capabilities. The stable nature of the rgp140 was due to the lack of processing at the gp120/41 boundary and the presence of an intermonomer disulfide bond formed by the cysteines of the V3 loop. Further characterization showed the intermonomer disulfide bond to be a target for PDI processing. The relevance of these findings to the roles of the V3 domain and the timing of PDI action during the HIV infection process are discussed.

I222 crystal form of despentapeptide (B26-B30) insulin provides new insights into the properties of monomeric insulin.

Despentapeptide (des-B26-B30) insulin (DPI), an active modified insulin, has been crystallized in the presence of 20% acetic acid pH 2. A crystal structure analysis to 1.8 A spacing (space group I222) revealed that the DPI molecule, which is unable to make beta-strand interactions for physiological dimer formation and is apparently monomeric in solution, formed an alternative lattice-generated dimer. The formation of this dimer involved interactions between surfaces which included the B9-B19 alpha-helices (usually buried by the dimer-dimer contacts within the native hexamer). The two crystallographically independent molecules within the dimer were essentially identical and were similar in conformation to T-state insulin as seen in the T(6) insulin hexamer. An unusual feature of each molecule in the dimer was the presence of two independent conformations at the B-chain C-terminus (residues B20-B25). Both conformations were different from that of native insulin, involving a 3.5 A displacement of the B20-B23 beta-turn and a repositioning of residue PheB25 such that it made close van der Waals contact with the main body of the molecule, appearing to stabilize the B-chain C-terminus.

Protein flexibility: its role in structure and mechanism revealed by molecular simulations.

Computer simulations at the atomic level have arrived at a stage where they provide realistic modeling of flexibility in proteins (and the mobility of their associated solvent) that is important in understanding the nature of molecular motions. This can now be extended to the molecular and atomic motions that are associated with protein mechanisms. Moreover, the derived data agree reasonably accurately with experimental measurements of several kinetic and thermodynamic parameters. Fundamental insights emerge on the roles that this intrinsic flexibility plays in the thermodynamic characteristics of macromolecules in solution; these equip the investigator to probe the consequences of cognate interactions and ligand binding on entropy and enthalpy. Thus simulations can now provide a powerful tool for investigating protein mechanisms that complements the existing and the emerging experimental techniques.

Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants.

The crystallization of three catalytically inactive mutants of penicillin V acylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants will provide three-dimensional structures of precursor PVA forms, plus open a route to the study of enzyme-substrate complexes for this industrially important enzyme.

Crystallographic and solution studies of N-lithocholyl insulin: a new generation of prolonged-acting human insulins.

The addition of specific bulky hydrophobic groups to the insulin molecule provides it with affinity for circulating serum albumin and enables it to form soluble macromolecular complexes at the site of subcutaneous injection, thereby securing slow absorption of the insulin analogue into the blood stream and prolonging its half-life once there. N-Lithocholic acid acylated insulin [Lys(B29)-lithocholyl des-(B30) human insulin] has been crystallized and the structure determined by X-ray crystallography at 1.6 A resolution to explore the molecular basis of its assembly. The unit cell in the crystal consists of an insulin hexamer containing two zinc ions, with two m-cresol molecules bound at each dimer-dimer interface stabilizing an R(6) conformation. Six covalently bound lithocholyl groups are arranged symmetrically around the outside of the hexamer. These form specific van der Waals and hydrogen-bonding interactions at the interfaces between neighboring hexamers, possibly representing the kinds of interactions which occur in the soluble aggregates at the site of injection. Comparison with an equivalent nonderivatized native insulin hexamer shows that the addition of the lithocholyl group disrupts neither the important conformational features of the insulin molecule nor its hexamer-forming ability. Indeed, binding studies show that the affinity of N-lithocholyl insulin for the human insulin receptor is not significantly diminished.

Parity-violating electron deuteron scattering and the proton's neutral weak axial vector form factor.

We report on a new measurement of the parity-violating asymmetry in quasielastic electron scattering from the deuteron at backward angles at Q2=0.038 (GeV/c)2. This quantity provides a determination of the neutral weak axial vector form factor of the nucleon, which can potentially receive large electroweak corrections. The measured asymmetry A=-3.51+/-0.57 (stat)+/-0.58 (syst) ppm is consistent with theoretical predictions. We also report on updated results of the previous experiment at Q2=0.091 (GeV/c)2, which are also consistent with theoretical predictions.

The crystal structure of the globular domain of sheep prion protein.

The prion protein PrP is a naturally occurring polypeptide that becomes transformed from a normal conformation to that of an aggregated form, characteristic of pathological states in fatal transmissible spongiform conditions such as Creutzfeld-Jacob Disease and Bovine Spongiform Encephalopathy. We report the crystal structure, at 2 A resolution, of residues 123-230 of the C-terminal globular domain of the ARQ allele of sheep prion protein (PrP). The asymmetric unit contains a single molecule whose secondary structure and overall organisation correspond to those structures of PrPs from various mammalian species determined by NMR. The globular domain shows a close association of helix-1, the C-terminal portion of helix-2 and the N-terminal portion of helix-3, bounded by the intramolecular disulphide bond, 179-214. The loop 164-177, between beta2 and helix-2 is relatively well structured compared to the human PrP NMR structure. Analysis of the sheep PrP structure identifies two possible loci for the initiation of beta-sheet mediated polymerisation. One of these comprises the beta-strand, residues 129-131 that forms an intra-molecular beta-sheet with residues 161-163. This strand is involved in lattice contacts about a crystal dyad to generate a four-stranded intermolecular beta-sheet between neighbouring molecules. The second locus involves the region 188-204, which modelling suggests is able to undergo a partial alpha-->beta switch within the monomer. These loci provide sites within the PrPc monomer that could readily give rise to early intermediate species on the pathway to the formation of aggregated PrPSc containing additional intermolecular beta-structure.

Structure of the human S100A12-copper complex: implications for host-parasite defence.

S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins. Together with S100A8 and S100A9, it belongs to the calgranulin subfamily, i.e. it is mainly expressed in granulocytes, although there is an increasing body of evidence of expression in keratinocytes and psoriatic lesions. As well as being linked to inflammation, allergy and neuritogenesis, S100A12 is involved in host-parasite response, as are the other two calgranulins. Recent data suggest that the function of the S100-family proteins is modulated not only by calcium, but also by other metals such as zinc and copper. Previously, the structure of human S100A12 in low-calcium and high-calcium structural forms, crystallized in space groups R3 and P2(1), respectively, has been reported. Here, the structure of S100A12 in complex with copper (space group P2(1)2(1)2; unit-cell parameters a = 70.6, b = 119.0, c = 90.2 A) refined at 2.19 A resolution is reported. Comparison of anomalous difference electron-density maps calculated with data collected with radiation of wavelengths 1.37 and 1.65 A shows that each monomer binds a single copper ion. The copper binds at an equivalent site to that at which another S100 protein, S100A7, binds zinc. The results suggest that copper binding may be essential for the functional role of S100A12 and probably the other calgranulins in the early immune response.

Multiple structural states of S100A12: A key to its functional diversity.

S100A12 is a member of the S100 family of EF-hand calcium-binding proteins. Together with two other calgranulins, S100A8 and S100A9, it is mostly expressed in human granulocytes, although there is increasing evidence of expression in keratinocytes and psoriatic lesions. It is involved in host-parasite response, and linked to corneal autoimmune diseases connected with filarial parasite infestation. Interaction of S100A12 with a multiligand receptor for advanced glycation end products (RAGE) mediates inflammation. Human recombinant S100A12 was found to induce neuritogenesis of cultured hippocampal cells, similar to two other S100 proteins, S100B and S100A4. X-ray structure of S100A12 has been solved in two crystal forms: R3 and P2(1). In the R3 crystal form S100A12 is a dimer, and in the P2(1) crystal form the dimers are arranged as a hexamer. The hexameric form suggests its role in receptor oligomerisation. S100A12 binds copper at the predicted zinc/copper binding site, which is located close to the surface of the protein. We propose copper-mediated generation of reactive oxygen species by S100A12 as its function in host-parasite response.

Insulin at pH 2: structural analysis of the conditions promoting insulin fibre formation.

When insulin solutions are subjected to acid, heat and agitation, the normal pattern of insulin assembly (dimers-->tetramers-->hexamers) is disrupted; the molecule undergoes conformational changes allowing it to follow an alternative aggregation pathway (via a monomeric species) leading to the formation of insoluble amyloid fibres. To investigate the effect of acid pH on the conformation and aggregation state of the protein, the crystal structure of human insulin at pH 2.1 has been determined to 1.6 A resolution. The structure reveals that the native fold is maintained at low pH, and that the molecule is still capable of forming dimers similar to those found in hexameric insulin structures at higher pH. Sulphate ions are incorporated into the molecule and the crystal lattice where they neutralise positive charges on the protein, stabilising its structure and facilitating crystallisation. The sulphate interactions are associated with local deformations in the protein, which may indicate that the structure is more plastic at low pH. Transmission electron microscopy analysis of insulin fibres reveals that the appearance of the fibres is greatly influenced by the type of acid employed. Sulphuric acid produces distinctive highly bunched, truncated fibres, suggesting that the sulphate ions have a sophisticated role to play in fibre formation, rather as they do in the crystal structure. Analytical ultracentrifugation studies show that in the absence of heating, insulin is predominantly dimeric in mineral acids, whereas in acetic acid the equilibrium is shifted towards the monomer. Hence, the effect of acid on the aggregation state of insulin is also complex. These results suggest that acid conditions increase the susceptibility of the molecule to conformational change and dissociation, and enhance the rate of fibrillation by providing a charged environment in which the attractive forces between the protein molecules is increased.

The structure of substrate-free microbial ribonuclease binase and of its complexes with 3'GMP and sulfate ions.

The structures of Bacillus intermedius ribonuclease (binase), an extracellular 109-residue enzyme, and its complexes with 3'GMP and sulfate ions were solved at 1.65 and 2.0 A, respectively. The structures were refined using REFMAC. The crystal of free binase belongs to the space group C2, whereas the crystals of complexes belong to the space group P2(1)2(1)2(1). In both crystal lattices the asymmetric unit contains two molecules which form an identical dimer. The structure of the dimer is such that only one of its subunits can bind the nucleotide in the 3'GMP-binase complex, where the guanyl base is located in the recognition loop of the enzyme. In both binase complex structures the phosphate group of 3'GMP or one of the sulfate ions make an electrostatic interaction with the binase molecule at the catalytic site. A second phosphate-binding site was found in the structures of the complexes at the cleft formed by the loop 34-39, the main chain of Arg82 and the side chain of Trp34. Comparison of the complex and unliganded enzyme crystal structures shows that there are some small but distinct differences in the specificity loop (56-62) and in the loops 34-39 and 99-104 associated with the binding of the nucleotide and sulfate ions.

The structure of S100A12 in a hexameric form and its proposed role in receptor signalling.

S100A12 is a member of the S100 subfamily of EF-hand calcium-binding proteins; it has been shown to be one of the ligands of the 'receptor for advanced glycation end products' (RAGE) that belongs to the immunoglobulin superfamily and is involved in diabetes, Alzheimer's disease, inflammation and tumour invasion. The structure of the dimeric form of native S100A12 from human granulocytes in the presence of calcium in space group R3 has previously been reported. Here, the structure of a second crystal form in space group P2(1) (unit-cell parameters a = 53.9, b = 100.5, c = 112.7A, beta = 94.6 degrees) solved at 2.7A resolution by molecular replacement using the R3 structure as a search model is reported. Like most S100 proteins, S100A12 is a dimer. However, in the P2(1) crystal form dimers of S100A12 are arranged in a spherical hexameric assembly with an external diameter of about 55 A stabilized by calcium ions bound between adjacent dimers. The putative target-binding sites of S100A12 are located at the outer surface of the hexamer, making it possible for the hexamer to bind several targets. It is proposed that the S100A12 hexameric assembly might interact with three extracellular domains of the receptor, bringing them together into large trimeric assemblies.

Crystallization and preliminary crystallographic investigation of a low-pH native insulin monomer with flexible behaviour.

Insulin naturally aggregates as dimers and hexamers, whose structures have been extensively analysed by X-ray crystallography. Structural determination of the physiologically relevant insulin monomer, however, is an unusual challenge owing to the difficulty in finding solution conditions in which the concentration of insulin is high enough for crystallization yet the molecule remains monomeric. By utilizing solution conditions known to inhibit insulin assembly, namely 20% acetic acid, crystals of insulin in the monomeric state have been obtained. The crystals are strongly diffracting and a data set extending to 1.6 A has recently been collected. The crystals nominally belong to the space group I422, with unit-cell parameters a = b = 57.80, c = 54.61 A, giving rise to one molecule in the asymmetric unit. Preliminary electron-density maps show that whilst most of the insulin monomer is well ordered and similar in conformation to other insulin structures, parts of the B-chain C-terminus main chain adopt more than one conformation.

Crystal structure of the transcription elongation/anti-termination factor NusA from Mycobacterium tuberculosis at 1.7 A resolution.

Mycobacterium tuberculosis is the cause of tuberculosis in humans, a disease that affects over a one-third of the world's population. This slow-growing pathogen has only one ribosomal RNA operon, thus making its transcriptional apparatus a fundamentally interesting target for drug discovery. NusA binds to RNA polymerase and modulates several of the ribosomal RNA transcriptional processes. Here, we report the crystal structure of NusA, and reveal that the molecule consists of four domains. They are organised as two distinct entities. The N-terminal domain (residues 1 to 99) that resembles the B chain of the Rad50cd ATP binding cassette-ATPase (ABC-ATPase) and a C-terminal module (residues 108 to 329) consisting of a ribosomal S1 protein domain followed by two K homology domains. The S1 and KH domains are tightly integrated together to form an extensive RNA-binding structure, but are flexibly tethered to the N-terminal domain. The molecule's surfaces and architecture provide insights into RNA and polymerase interactions and the mechanism of pause site discrimination. They also allow us to rationalize certain termination-defective and cold shock-sensitive mutations in the nusA gene that have been studied in Escherichia coli.

Relativistic effects and two-body currents in (H)((-->)e(')p)n using out-of-plane detection.

Measurements of the (2)H((-->)e,e(')p)n reaction were performed with the out-of-plane magnetic spectrometers (OOPS) at the MIT-Bates Linear Accelerator. The longitudinal-transverse, f(LT) and f(')(LT), and the transverse-transverse, f(TT), interference responses at a missing momentum of 210 MeV/c were simultaneously extracted in the dip region at Q2 = 0.15 (GeV/c)(2). In comparison to models of deuteron electrodisintegration, the data clearly reveal strong effects of relativity and final-state interactions and the importance of two-body meson-exchange currents and isobar configurations. We demonstrate that such effects can be disentangled by extracting these responses using the novel out-of-plane technique.

Crystal structures of penicillin acylase enzyme-substrate complexes: structural insights into the catalytic mechanism.

The crystal structure of penicillin G acylase from Escherichia coli has been determined to a resolution of 1.3 A from a crystal form grown in the presence of ethylene glycol. To study aspects of the substrate specificity and catalytic mechanism of this key biotechnological enzyme, mutants were made to generate inactive protein useful for producing enzyme-substrate complexes. Owing to the intimate association of enzyme activity and precursor processing in this protein family (the Ntn hydrolases), most attempts to alter active-site residues lead to processing defects. Mutation of the invariant residue Arg B263 results in the accumulation of a protein precursor form. However, the mutation of Asn B241, a residue implicated in stabilisation of the tetrahedral intermediate during catalysis, inactivates the enzyme but does not prevent autocatalytic processing or the ability to bind substrates. The crystal structure of the Asn B241 Ala oxyanion hole mutant enzyme has been determined in its native form and in complex with penicillin G and penicillin G sulphoxide. We show that Asn B241 has an important role in maintaining the active site geometry and in productive substrate binding, hence the structure of the mutant protein is a poor model for the Michaelis complex. For this reason, we subsequently solved the structure of the wild-type protein in complex with the slowly processed substrate penicillin G sulphoxide. Analysis of this structure suggests that the reaction mechanism proceeds via direct nucleophilic attack of Ser B1 on the scissile amide and not as previously proposed via a tightly H-bonded water molecule acting as a "virtual" base.

Inhibition of human alpha-thrombin by a phosphonate tripeptide proceeds via a metastable pentacoordinated phosphorus intermediate.

X-ray crystallographic studies of human alpha-thrombin with a novel synthetic inhibitor, an acyl (alpha-aminoalkyl)phosphonate, reveal the existence of a pentacovalent phosphorus intermediate state. Crystal structures of the complex of alpha-thrombin with the phosphonate compound were determined independently using crystals of different ages. The first structure, solved from a crystal less than seven days old, showed a pentacoordinated phosphorus moiety. The second structure, determined from a crystal that was 12 weeks old, showed a tetracoordinated phosphorus moiety. In the first structure, a water molecule, made nucleophilic by coordination to His57 of alpha-thrombin, is bonded to the pentacoordinated phosphorus atom. Its position is approximately equivalent to that occupied by the water molecule responsible for hydrolytic deacylation during normal hydrolysis. The pentacoordinated phosphorus adduct collapses to give the expected pseudo tetrahedral complex, where the phosphorus atom is covalently bonded to Ser195 O(gamma). The crystallographic data presented here therefore suggest that the covalent bond formed between the inhibitor's phosphorus atom and O(gamma) of Ser195 proceeds via an addition-elimination mechanism, which involves the formation of a pentacoordinate intermediate.