Polymer-based open-channel blockers for the acetylcholine receptor: the effect of spacer length on blockade kinetics

Blocking open ion channels provides a promising way to modulate synaptic transmission. Using the muscle-type acetylcholine receptor (AChR) as a test system, we seek to develop blockers that have blockade kinetics tunable via structural modifications. Here we investigate whether the blockade kinetics can be modulated by specifying the length of a poly(ethylene glycol) (PEG) spacer incorporated into the blocker. Single-channel electrophysiological experiments show that simple bis(trimethylammonium) compounds ( 1a- 3) both activate the AChR and block the open channel. The blockade kinetics are found to depend on spacer length: both the association and dissociation rate constants decrease with increasing spacer length. The decrease in the association rate constant can be quantitatively explained by the entropic cost of polymer confinement in the transmembrane lumen, while the decrease in the dissociation rate constant is consistent with weak, additive noncovalent interactions between the channel and the spacer. These results provide useful insights into the future design of kinetically tunable open-channel blockers for the AChR.     

Near-atomic structure of the inner ring of the Saccharomyces cerevisiae nuclear pore complex

Nuclear pore complexes (NPCs) mediate bidirectional nucleocytoplasmic transport of substances in eukaryotic cells. However, the accurate molecular arrangement of NPCs remains enigmatic owing to their huge size and highly dynamic nature. Here we determined the structure of the asymmetric unit of the inner ring (IR monomer) at 3.73 Å resolution by single-particle cryo-electron microscopy, and created an atomic model of the intact IR consisting of 192 molecules of 8 nucleoporins. In each IR monomer, the Z-shaped Nup188–Nup192 complex in the middle layer is sandwiched by two approximately parallel rhomboidal structures in the inner and outer layers, while Nup188, Nup192 and Nic96 link all subunits to constitute a relatively stable IR monomer. In contrast, the intact IR is assembled by loose and instable interactions between IR monomers. These structures, together with previously reported structural information of IR, reveal two distinct interaction modes between IR monomers and extensive flexible connections in IR assembly, providing a structural basis for the stability and malleability of IR.Subject terms: Cryoelectron microscopy, Nuclear pore complex     

Conformational Changes during Pore Formation by the Perforin-Related Protein Pleurotolysin

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.     

Paraquat Induces Epithelial-Mesenchymal Transition-Like Cellular Response Resulting in Fibrogenesis and the Prevention of Apoptosis in Human Pulmonary Epithelial Cells

The aim of this study is to investigate the molecular mechanisms underlying delayed progressive pulmonary fibrosis, a characteristic of subacute paraquat (PQ) poisoning. Epithelial-mesenchymal transition (EMT) has been proposed as a cause of organ fibrosis, and transforming growth factor-β (TGF-β) is suggested to be a powerful mediator of EMT. We thus examined the possibility that EMT is involved in pulmonary fibrosis during PQ poisoning using A549 human alveolar epithelial cells in vitro. The cells were treated with various concentrations of PQ (0–500 μM) for 2–12 days. Short-term (2 days) high-dose (>100 μM) treatments with PQ induced cell death accompanied by the activation of caspase9 as well as a decrease in E-cadherin (an epithelial cell marker), suggesting apoptotic cell death with the features of anoikis (cell death due to the loss of cell-cell adhesion). In contrast, long-term (6–12 days) low-dose (30 μM) treatments with PQ resulted in a transformation into spindle-shaped mesenchymal-like cells with a decrease of E-cadherin as well as an increase of α-smooth muscle actin (α-SMA). The mesenchymal-like cells also secreted the extracellular matrix (ECM) protein fibronectin into the culture medium. The administration of a TGF-β1 receptor antagonist, SB431542, almost completely attenuated the mesenchymal transformation as well as fibronectin secretion, suggesting a crucial role of TGF-β1 in EMT-like cellular response and subsequent fibrogenesis. It is noteworthy that despite the suppression of EMT-fibrogenesis, apoptotic death was observed in cells treated with PQ+SB431542. EMT-like cellular response and subsequent fibrogenesis were also observed in normal human bronchial epithelial (NHBE) cells exposed to PQ in a TGF-β1-dependent manner. Taken together, our experimental model reflects well the etiology of PQ poisoning in human and shows the involvement of EMT-like cellular response in both fibrogenesis and resistance to cell death during subacute PQ poisoning of pulmonary epithelial cells.     

Higher Nucleoporin-Importinβ Affinity at the Nuclear Basket Increases Nucleocytoplasmic Import

Several in vitro studies have shown the presence of an affinity gradient in nuclear pore complex proteins for the import receptor Importinβ, at least partially contributing to nucleocytoplasmic transport, while others have historically argued against the presence of such a gradient. Nonetheless, the existence of an affinity gradient has remained an uncharacterized contributing factor. To shed light on the affinity gradient theory and better characterize how the existence of such an affinity gradient between the nuclear pore and the import receptor may influence the nucleocytoplasmic traffic, we have developed a general-purpose agent based modeling (ABM) framework that features a new method for relating rate constants to molecular binding and unbinding probabilities, and used our ABM approach to quantify the effects of a wide range of forward and reverse nucleoporin-Importinβ affinity gradients. Our results indicate that transport through the nuclear pore complex is maximized with an effective macroscopic affinity gradient of 2000 µM, 200 µM and 10 µM in the cytoplasmic, central channel and nuclear basket respectively. The transport rate at this gradient is approximately 10% higher than the transport rate for a comparable pore lacking any affinity gradient, which has a peak transport rate when all nucleoporins have an affinity of 200 µM for Importinβ. Furthermore, this optimal ratio of affinity gradients is representative of the ratio of affinities reported for the yeast nuclear pore complex – suggesting that the affinity gradient seen in vitro is highly optimized.     

Mechanisms of 8‐aminoquinoline induced haemolytic toxicity in a G6PDd humanized mouse model

Primaquine (PQ) and Tafenoquine (TQ) are clinically important 8‐aminoquinolines (8‐AQ) used for radical cure treatment of P. vivax infection, known to target hepatic hypnozoites. 8‐AQs can trigger haemolytic anaemia in individuals with glucose‐6‐phosphate dehydrogenase deficiency (G6PDd), yet the mechanisms of haemolytic toxicity remain unknown. To address this issue, we used a humanized mouse model known to predict haemolytic toxicity responses in G6PDd human red blood cells (huRBCs). To evaluate the markers of eryptosis, huRBCs were isolated from mice 24–48 h post‐treatment and analysed for effects on phosphatidylserine (PS), intracellular reactive oxygen species (ROS) and autofluorescence. Urinalysis was performed to evaluate the occurrence of intravascular and extravascular haemolysis. Spleen and liver tissue harvested at 24 h and 5–7 days post‐treatment were stained for the presence of CD169+ macrophages, F4/80+ macrophages, Ter119+ mouse RBCs, glycophorin A+ huRBCs and murine reticulocytes (muRetics). G6PDd‐huRBCs from PQ/TQ treated mice showed increased markers for eryptosis as early as 24 h post‐treatment. This coincided with an early rise in levels of muRetics. Urinalysis revealed concurrent intravascular and extravascular haemolysis in response to PQ/TQ. Splenic CD169+ macrophages, present in all groups at day 1 post‐dosing were eliminated by days 5–7 in PQ/TQ treated mice only, while liver F4/80 macrophages and iron deposits increased. Collectively, our data suggest 8‐AQ treated G6PDd‐huRBCs have early physiological responses to treatment, including increased markers for eryptosis indicative of oxidative stress, resulting in extramedullary haematopoiesis and loss of splenic CD169+ macrophages, prompting the liver to act as the primary site of clearance.     

Signalling through cerebral cavernous malformation protein networks

Cerebral cavernous malformations (CCMs) are neurovascular abnormalities characterized by thin, leaky blood vessels resulting in lesions that predispose to haemorrhages, stroke, epilepsy and focal neurological deficits. CCMs arise due to loss-of-function mutations in genes encoding one of three CCM complex proteins, KRIT1, CCM2 or CCM3. These widely expressed, multi-functional adaptor proteins can assemble into a CCM protein complex and (either alone or in complex) modulate signalling pathways that influence cell adhesion, cell contractility, cytoskeletal reorganization and gene expression. Recent advances, including analysis of the structures and interactions of CCM proteins, have allowed substantial progress towards understanding the molecular bases for CCM protein function and how their disruption leads to disease. Here, we review current knowledge of CCM protein signalling with a focus on three pathways which have generated the most interest—the RhoA–ROCK, MEKK3–MEK5–ERK5–KLF2/4 and cell junctional signalling pathways—but also consider ICAP1-β1 integrin and cdc42 signalling. We discuss emerging links between these pathways and the processes that drive disease pathology and highlight important open questions—key among them is the role of subcellular localization in the control of CCM protein activity.     

Laminin-induced Acetylcholine Receptor Clustering: An Alternative Pathway

The induction of acetylcholine receptor (AChR) clustering by neurally released agrin is a critical, early step in the formation of the neuromuscular junction. Laminin, a component of the muscle fiber basal lamina, also induces AChR clustering. We find that induction of AChR clustering in C2 myotubes is specific for laminin-1; neither laminin-2 (merosin) nor laminin-11 (a synapse-specific isoform) are active. Moreover, laminin-1 induces AChR clustering by a pathway that is independent of that used by neural agrin. The effects of laminin-1 and agrin are strictly additive and occur with different time courses. Most importantly, laminin- 1–induced clustering does not require MuSK, a receptor tyrosine kinase that is part of the receptor complex for agrin. Laminin-1 does not cause tyrosine phosphorylation of MuSK in C2 myotubes and induces AChR clustering in myotubes from MuSK−/− mice that do not respond to agrin. In contrast to agrin, laminin-1 also does not induce tyrosine phosphorylation of the AChR, demonstrating that AChR tyrosine phosphorylation is not required for clustering in myotubes. Laminin-1 thus acts by a mechanism that is independent of that used by agrin and may provide a supplemental pathway for AChR clustering during synaptogenesis.     

Loop C and the mechanism of acetylcholine receptor–channel gating

Agonist molecules at the two neuromuscular acetylcholine (ACh) receptor (AChR) transmitter-binding sites increase the probability of channel opening. In one hypothesis for AChR activation (“priming”), the capping of loop C at each binding site transfers energy independently to the distant gate over a discrete structural pathway. We used single-channel analyses to examine the experimental support for this proposal with regard to brief unliganded openings, the effects of loop-C modifications, the effects of mutations to residues either on or off the putative pathway, and state models for describing currents at low [ACh]. The results show that (a) diliganded and brief unliganded openings are generated by the same essential, global transition; (b) the radical manipulation of loop C does not prevent channel opening but impairs agonist binding; (c) both on- and off-pathway mutations alter gating by changing the relative stability of the open-channel conformation by local interactions rather than by perturbing a specific site–gate communication link; and (d) it is possible to estimate directly the rate constants for agonist dissociation from and association to both the low and high affinity forms of the AChR-binding site by using a cyclic kinetic model. We conclude that the mechanism of energy transfer between the binding sites and the gate remains an open question.     

Poisson-Nernst-Planck Models of Nonequilibrium Ion Electrodiffusion through a Protegrin Transmembrane Pore

Protegrin peptides are potent antimicrobial agents believed to act against a variety of pathogens by forming nonselective transmembrane pores in the bacterial cell membrane. We have employed 3D Poisson-Nernst-Planck (PNP) calculations to determine the steady-state ion conduction characteristics of such pores at applied voltages in the range of −100 to +100 mV in 0.1 M KCl bath solutions. We have tested a variety of pore structures extracted from molecular dynamics (MD) simulations based on an experimentally proposed octomeric pore structure. The computed single-channel conductance values were in the range of 290–680 pS. Better agreement with the experimental range of 40–360 pS was obtained using structures from the last 40 ns of the MD simulation, where conductance values range from 280 to 430 pS. We observed no significant variation of the conductance with applied voltage in any of the structures that we tested, suggesting that the voltage dependence observed experimentally is a result of voltage-dependent channel formation rather than an inherent feature of the open pore structure. We have found the pore to be highly selective for anions, with anionic to cationic current ratios (I_Cl−/I_K+) on the order of 10³. This is consistent with the highly cationic nature of the pore but surprisingly in disagreement with the experimental finding of only slight anionic selectivity. We have additionally tested the sensitivity of our PNP model to several parameters and found the ion diffusion coefficients to have a significant influence on conductance characteristics. The best agreement with experimental data was obtained using a diffusion coefficient for each ion set to 10% of the bulk literature value everywhere inside the channel, a scaling used by several other studies employing PNP calculations. Overall, this work presents a useful link between previous work focused on the structure of protegrin pores and experimental efforts aimed at investigating their conductance characteristics.     

BIREFRINGENCE OF SPERMATOZOA : I. Birefringence Melting of Squid, Bull, and Human Sperm Nucleoprotein

In experiments designed to determine the thermal stability and bonding strength of a natural nucleoprotein structure, the loss of birefringence as a function of time and temperature was investigated for both mammalian and nonmammalian sperm nuclei. At a constant temperature, this reaction was found to be first order for both types over a range of temperatures. The methods of chemical kinetics applied to results of these reactions, called birefringence melting reactions, produced values for the enthalpy and entropy of activation in the reactions, which gave some indication of the strength of binding in the nucleoprotein structure; and these results, plus those on the influence of chemicals on the structure, were consistent with the molecular structures which have been proposed by others for the nucleoprotein complex of sperm nuclei. For both bull and human sperm in ethylene glycol, the rate-limiting step in the melting reactions appeared to be the breakage of disulfide bonds. For squid sperm in ethylene glycol, and bull or squid sperm in ethylene glycol plus β-mercaptoethanol, the identity of this step was more ambiguous, but a possibility consistent with these and other results would be a cooperative breakage of ionic bonds.     

Mechanism of KCl enhancement in detection of nonionic polymers by nanopore sensors

The mechanisms of KCl-induced enhancement in identification of individual molecules of poly(ethylene glycol) using solitary α-hemolysin nanoscale pores are described. The interaction of single molecules with the nanopore causes changes in the ionic current flowing through the pore. We show that the on-rate constant of the process is several hundred times larger and that the off-rate is several hundred times smaller in 4 M KCl than in 1 M KCl. These shifts dramatically improve detection and make single molecule identification feasible. KCl also changes the solubility of poly(ethylene glycol) by the same order of magnitude as it changes the rate constants. In addition, the polymer-nanopore interaction is determined to be a strong non-monotonic function of voltage, indicating that the flexible, nonionic poly(ethylene glycol) acts as a charged molecule. Therefore, salting-out and Coulombic interactions are responsible for the KCl-induced enhancement. These results will advance the development of devices with sensor elements based on single nanopores.     

Early Hemoperfusion May Improve Survival of Severely Paraquat-Poisoned Patients

Background

Thousands of paraquat (PQ)-poisoned patients continue to die, particularly in developing countries. Although animal studies indicate that hemoperfusion (HP) within 2−4 h after intoxication effectively reduces mortality, the effect of early HP in humans remains unknown.

Methods

We analyzed the records of all PQ-poisoned patients admitted to 2 hospitals between 2000 and 2009. Patients were grouped according to early or late HP and high-dose (oral cyclophosphamide [CP] and intravenous dexamethasone [DX]) or repeated pulse (intravenous methylprednisolone [MP] and CP, followed by DX and repeated MP and/or CP) PQ therapy. Early HP was defined as HP <4 h, and late HP, as HP ≥4 h after PQ ingestion. We evaluated the associations between HP <4 h, <5 h, <6 h, and <7 h after PQ ingestion and the outcomes. Demographic, clinical, laboratory, and mortality data were analyzed.

Results

The study included 207 severely PQ-poisoned patients. Forward stepwise multivariate Cox hazard regression analysis showed that early HP <4 h (hazard ratio [HR] = 0.38, 95% confidence interval (CI) 0.16–0.86; P = 0.020) or HP <5 h (HR = 0.60, 95% CI: 0.39–0.92; P = 0.019) significantly decreased the mortality risk. Further analysis showed that early HP reduced the mortality risk only in patients treated with repeated pulse therapy (n = 136), but not high-dose therapy (n = 71). Forward stepwise multivariate Cox hazard regression analysis showed that HP <4.0 h (HR = 0.19, 95% CI: 0.05–0.79; P = 0.022) or <5.0 h (HR = 0.49, 95% CI: 0.24–0.98; P = 0.043) after PQ ingestion significantly decreased the mortality risk in repeated pulse therapy patients, after adjustment for relevant variables.

Conclusion

The results showed that early HP after PQ exposure might be effective in reducing mortality in severely poisoned patients, particularly in those treated with repeated pulse therapy.     

CLASP2-dependent microtubule capture at the neuromuscular junction membrane requires LL5β and actin for focal delivery of acetylcholine receptor vesicles

A hallmark of the neuromuscular junction (NMJ) is the high density of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane. The postsynaptic apparatus of the NMJ is organized by agrin secreted from motor neurons. The mechanisms that underlie the focal delivery of AChRs to the adult NMJ are not yet understood in detail. We previously showed that microtubule (MT) capture by the plus end–tracking protein CLASP2 regulates AChR density at agrin-induced AChR clusters in cultured myotubes via PI3 kinase acting through GSK3β. Here we show that knockdown of the CLASP2-interaction partner LL5β by RNAi and forced expression of a CLASP2 fragment blocking the CLASP2/LL5β interaction inhibit microtubule capture. The same treatments impair focal vesicle delivery to the clusters. Consistent with these findings, knockdown of LL5β at the NMJ in vivo reduces the density and insertion of AChRs into the postsynaptic membrane. MT capture and focal vesicle delivery to agrin-induced AChR clusters are also inhibited by microtubule- and actin-depolymerizing drugs, invoking both cytoskeletal systems in MT capture and in the fusion of AChR vesicles with the cluster membrane. Combined our data identify a transport system, organized by agrin through PI3 kinase, GSK3β, CLASP2, and LL5β, for precise delivery of AChR vesicles from the subsynaptic nuclei to the overlying synaptic membrane.     

The structure of the M2 channel-lining segment from the nicotinic acetylcholine receptor

The structures of functional peptides corresponding to the predicted channel-lining M2 segment of the nicotinic acetylcholine (AChR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. The AChR M2 peptide forms a straight transmembrane α-helix, with no kinks. M2 inserts in the lipid bilayer at an angle of 12° relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminus of the peptide, which is assigned to be intracellular. A molecular model of the AChR channel pore, constructed from the solid-state NMR 3-D structure of the AChR M2 helix in the membrane assuming a pentameric organization, results in a funnel-like architecture for the channel with the wide opening on the N-terminal intracellular side. A central narrow pore has a diameter ranging from about 3.0 Å at its narrowest, to 8.6 Å at its widest. Nonpolar residues are predominantly on the exterior of the bundle, while polar residues line the pore. This arrangement is in fair agreement with evidence collected from permeation, mutagenesis, affinity labeling and cysteine accessibility measurements. A pentameric M2 helical bundle may, therefore, represent the structural blueprint for the inner bundle that lines the channel of the nicotinic AChR.     

Crystal Structure of LGR4-Rspo1 Complex: INSIGHTS INTO THE DIVERGENT MECHANISMS OF LIGAND RECOGNITION BY LEUCINE-RICH REPEAT G-PROTEIN-COUPLED RECEPTORS (LGRs)*

Leucine-rich repeat G-protein-coupled receptors (LGRs) are a unique class of G-protein-coupled receptors characterized by a large extracellular domain to recognize ligands and regulate many important developmental processes. Among the three groups of LGRs, group B members (LGR4–6) recognize R-spondin family proteins (Rspo1–4) to stimulate Wnt signaling. In this study, we successfully utilized the “hybrid leucine-rich repeat technique,” which fused LGR4 with the hagfish VLR protein, to obtain two recombinant human LGR4 proteins, LGR4₁₅ and LGR4₉. We determined the crystal structures of ligand-free LGR4₁₅ and the LGR4₉-Rspo1 complex. LGR4 exhibits a twisted horseshoe-like structure. Rspo1 adopts a flat and β-fold architecture and is bound in the concave surface of LGR4 in the complex through electrostatic and hydrophobic interactions. All the Rspo1-binding residues are conserved in LGR4–6, suggesting that LGR4–6 bind R-spondins through an identical surface. Structural analysis of our LGR4-Rspo1 complex with the previously determined LGR4 and LGR5 structures revealed that the concave surface of LGR4 is the sole binding site for R-spondins, suggesting a one-site binding model of LGR4–6 in ligand recognition. The molecular mechanism of LGR4–6 is distinct from the two-step mechanism of group A receptors LGR1–3 and the multiple-interface binding model of group C receptors LGR7–8, suggesting LGRs utilize the divergent mechanisms for ligand recognition. Our structures, together with previous reports, provide a comprehensive understanding of the ligand recognition by LGRs.     

Crystal Structure of BamB Bound to a Periplasmic Domain Fragment of BamA,the Central Component of the β-Barrel Assembly Machine

The β-barrel assembly machinery (BAM) mediates folding and insertion of β-barrel outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. BAM is a five-protein complex consisting of the β-barrel OMP BamA and lipoproteins BamB, -C, -D, and -E. High resolution structures of all the individual BAM subunits and a BamD-BamC complex have been determined. However, the overall complex architecture remains elusive. BamA is the central component of BAM and consists of a membrane-embedded β-barrel and a periplasmic domain with five polypeptide translocation-associated (POTRA) motifs thought to interact with the accessory lipoproteins. Here we report the crystal structure of a fusion between BamB and a POTRA3–5 fragment of BamA. Extended loops 13 and 17 protruding from one end of the BamB β-propeller contact the face of the POTRA3 β-sheet in BamA. The interface is stabilized by several hydrophobic contacts, a network of hydrogen bonds, and a cation-π interaction between BamA Tyr-255 and BamB Arg-195. Disruption of BamA-BamB binding by BamA Y255A and probing of the interface by disulfide bond cross-linking validate the physiological relevance of the observed interface. Furthermore, the structure is consistent with previously published mutagenesis studies. The periplasmic five-POTRA domain of BamA is flexible in solution due to hinge motions in the POTRA2–3 linker. Modeling BamB in complex with full-length BamA shows BamB binding at the POTRA2–3 hinge, suggesting a role in modulation of BamA flexibility and the conformational changes associated with OMP folding and insertion.     

Acetylcholine Receptor (AChR) Clustering Is Regulated Both by Glycogen Synthase Kinase 3β (GSK3β)-dependent Phosphorylation and the Level of CLIP-associated Protein 2 (CLASP2) Mediating the Capture of Microtubule Plus-ends

The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2–9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2–8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends.     

Novel Transthyretin Amyloid Fibril Formation Inhibitors: Synthesis,Biological Evaluation,and X-Ray Structural Analysis

Transthyretin (TTR) is one of thirty non-homologous proteins whose misfolding, dissociation, aggregation, and deposition is linked to human amyloid diseases. Previous studies have identified that TTR amyloidogenesis can be inhibited through stabilization of the native tetramer state by small molecule binding to the thyroid hormone sites of TTR. We have evaluated a new series of β-aminoxypropionic acids (compounds 5–21), with a single aromatic moiety (aryl or fluorenyl) linked through a flexible oxime tether to a carboxylic acid. These compounds are structurally distinct from the native ligand thyroxine and typical halogenated biaryl NSAID-like inhibitors to avoid off-target hormonal or anti-inflammatory activity. Based on an in vitro fibril formation assay, five of these compounds showed significant inhibition of TTR amyloidogenesis, with two fluorenyl compounds displaying inhibitor efficacy comparable to the well-known TTR inhibitor diflunisal. Fluorenyl 15 is the most potent compound in this series and importantly does not show off-target anti-inflammatory activity. Crystal structures of the TTR∶inhibitor complexes, in agreement with molecular docking studies, revealed that the aromatic moiety, linked to the sp²-hybridized oxime carbon, specifically directed the ligand in either a forward or reverse binding mode. Compared to the aryl family members, the bulkier fluorenyl analogs achieved more extensive interactions with the binding pockets of TTR and demonstrated better inhibitory activity in the fibril formation assay. Preliminary optimization efforts are described that focused on replacement of the C-terminal acid in both the aryl and fluorenyl series (compounds 22–32). The compounds presented here constitute a new class of TTR inhibitors that may hold promise in treating amyloid diseases associated with TTR misfolding.