Essential role of coiled coils for aggregation and activity of Q/N-rich prions and PolyQ proteins.

The functional switch of glutamine/asparagine (Q/N)-rich prions and the neurotoxicity of polyQ-expanded proteins involve complex aggregation-prone structural transitions, commonly presumed to be forming ß sheets. By analyzing sequences of interaction partners of these proteins, we discovered a recurrent presence of coiled-coil domains both in the partners and in segments that flank or overlap Q/N-rich and polyQ domains. Since coiled coils can mediate protein interactions and multimerization, we studied their possible involvement in Q/N-rich and polyQ aggregations. Using circular dichroism and chemical crosslinking, we found that Q/N-rich and polyQ peptides form α-helical coiled coils in vitro and assemble into multimers. Using structure-guided mutagenesis, we found that coiled-coil domains modulate in vivo properties of two Q/N-rich prions and polyQ-expanded huntingtin. Mutations that disrupt coiled coils impair aggregation and activity, whereas mutations that enhance coiled-coil propensity promote aggregation. These findings support a coiled-coil model for the functional switch of Q/N-rich prions and for the pathogenesis of polyQ-expansion diseases.

Structural and functional analysis of a tandem repeat galacturonic acid-binding lectin from the sea hare Aplysia californica.

A d-galacturonic acid-specific lectin, named AcL, was purified from the sea hare Aplysia californica by galactose-agarose affinity chromatography. AcL has a molecular mass of 27.5 kDa determined by MALDI-TOF mass spectrometry. This lectin shows a good affinity for d-galacturonic acid and a lower affinity for galactosides: raffinose, melibiose, α and ß-lactose, and d-galactose. We determined the amino acid sequence of AcL by trypsin digestion and subsequent peptide analysis by mass spectrometry, resulting in a 238 amino acid protein with a theoretical molecular mass of 26.4 kDa. The difference between the theoretical and experimental values can be attributed to post-translational modifications. Thiol-disulfide quantification discerned five disulfide bonds and three free cysteines. The structure of Acl is mainly comprised of beta sheets, determined by circular dichroism, and predicted with AlphaFold. Theoretical models depict three nearly identical tandem domains consisting of two beta sheets each. From docking analysis, we identified AcL glycan-binding sites as multiple conserved motifs in each domain. Furthermore, phylogenetic analysis based on its structure and sequence showed that AcL and its closest homologues (GalULs) form a clear monophyletic group, distinct from other glycan-binding proteins with a jelly-roll fold: lectins of types F and H. GalULs possess four conserved sequence regions that distinguish them and are either ligand-binding motifs or stabilizing network hubs. We suggest that this new family should be referred to as GalUL or D-type, following the traditional naming of lectins; D standing for depilans, the epithet for the species (Aplysia depilans) from which a lectin of this family was first isolated and described.

Cryo-EM structure of the open high-conductance Ca2+-activated K+ channel.

The Ca2+-activated K+ channel, Slo1, has an unusually large conductance and contains a voltage sensor and multiple chemical sensors. Dual activation by membrane voltage and Ca2+ renders Slo1 central to processes that couple electrical signalling to Ca2+-mediated events such as muscle contraction and neuronal excitability. Here we present the cryo-electron microscopy structure of a full-length Slo1 channel from Aplysia californica in the presence of Ca2+ and Mg2+ at a resolution of 3.5 Å. The channel adopts an open conformation. Its voltage-sensor domain adopts a non-domain-swapped attachment to the pore and contacts the cytoplasmic Ca2+-binding domain from a neighbouring subunit. Unique structural features of the Slo1 voltage sensor suggest that it undergoes different conformational changes than other known voltage sensors. The structure reveals the molecular details of three distinct divalent cation-binding sites identified through electrophysiological studies of mutant Slo1 channels.

Structural basis for gating the high-conductance Ca2+-activated K+ channel.

The precise control of an ion channel gate by environmental stimuli is crucial for the fulfilment of its biological role. The gate in Slo1 K+ channels is regulated by two separate stimuli, intracellular Ca2+ concentration and membrane voltage. Slo1 is thus central to understanding the relationship between intracellular Ca2+ and membrane excitability. Here we present the Slo1 structure from Aplysia californica in the absence of Ca2+ and compare it with the Ca2+-bound channel. We show that Ca2+ binding at two unique binding sites per subunit stabilizes an expanded conformation of the Ca2+ sensor gating ring. These conformational changes are propagated from the gating ring to the pore through covalent linkers and through protein interfaces formed between the gating ring and the voltage sensors. The gating ring and the voltage sensors are directly connected through these interfaces, which allow membrane voltage to regulate gating of the pore by influencing the Ca2+ sensors.

Isolation and Synthesis of Azuriaplysins A and B, Bromoditerpenes with an α-Methylene Carbonyl from the Sea Hare Aplysia kurodai.

New bromoditerpenes having an α-methylene carbonyl structure, azuriaplysins A (1) and B (2), were isolated from the sea hare Aplysia kurodai. Their relative stereostructures were determined based on one- and two-dimensional NMR spectroscopic analysis. In addition, the absolute stereostructures were determined by the total synthesis of both enantiomers of azuriaplysins A (1) and B (2), the key points of which were bromocyclization of farnesol and optical resolution of a key intermediate. Azuriaplysin B (2) and its enantiomer exhibited moderate cytotoxicity against HeLa S3 cells.

Divergent CPEB prion-like domains reveal different assembly mechanisms for a generic amyloid-like fold.

BACKGROUND: Amyloids are ordered, insoluble protein aggregates, characterized by a cross-ß sheet quaternary structure in which molecules in a ß-strand conformation are stacked along the filament axis via intermolecular interactions. While amyloids are typically associated with pathological conditions, functional amyloids have also been identified and are present in a wide variety of organisms ranging from bacteria to humans. The cytoplasmic polyadenylation element-binding (CPEB) prion-like protein is an mRNA-binding translation regulator, whose neuronal isoforms undergo activity-dependent aggregation, a process that has emerged as a plausible biochemical substrate for memory maintenance. CPEB aggregation is driven by prion-like domains (PLD) that are divergent in sequence across species, and it remains unknown whether such divergent PLDs follow a similar aggregating assembly pathway. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD and compare them to those of the Drosophila ortholog, Orb2 PLD. RESULTS: Using in vitro single-molecule and bulk biophysical methods, we find transient oligomers and mature amyloid-like filaments that suggest similarities in the late stages of the assembly pathway for both ApCPEB and Orb2 PLDs. However, while prior to aggregation the Orb2 PLD monomer remains mainly as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising α-helical structures that evolve to coiled-coil species, indicating structural differences at the beginning of their amyloid assembly pathways. CONCLUSION: Our results indicate that divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.

Biphasic Liquid Microjunction Extraction for Profiling Neuronal RNA Modifications by Liquid Chromatography-Tandem Mass Spectrometry.

RNA modifications are emerging as critical players in the spatiotemporal regulation of gene expression. Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables the simultaneous quantification of numerous enzymatically modified RNAs in a biological sample, conventional RNA extraction and enzymatic digestion protocols that are employed prior to analysis have precluded the application of this technique for small-volume samples. In this study, a biphasic liquid microjunction (LMJ) extraction system using coaxial capillaries that direct and aspirate extraction solvents onto a ∼350 µm diameter sample spot was developed and applied for the extraction of RNA from individual cell clusters in the central nervous system of the marine mollusk Aplysia californica. To maximize RNA recoveries, optimized extraction solvents consisting of 10% methanol and chloroform were evaluated under dynamic and static extraction conditions. An MS-compatible RNA digestion buffer was developed to minimize the number of sample-transfer steps and facilitate the direct enzymatic digestion of extracted RNA within the sample collection tube. Compared to RNA extraction using a conventional phenol-chloroform method, the LMJ-based method provided a 3-fold greater coverage of the neuronal epitranscriptome for similar amounts of tissues and also produced mRNA of sufficient purity for reverse transcription polymerase chain reaction amplification. Using this approach, the expression of RNA-modifying enzymes in a given neuronal cell cluster can be characterized and simultaneously correlated with the LC-MS/MS analysis of RNA modifications within the same subset of neurons.

Discovery of peptide ligands through docking and virtual screening at nicotinic acetylcholine receptor homology models.

Venom peptide toxins such as conotoxins play a critical role in the characterization of nicotinic acetylcholine receptor (nAChR) structure and function and have potential as nervous system therapeutics as well. However, the lack of solved structures of conotoxins bound to nAChRs and the large size of these peptides are barriers to their computational docking and design. We addressed these challenges in the context of the α4ß2 nAChR, a widespread ligand-gated ion channel in the brain and a target for nicotine addiction therapy, and the 19-residue conotoxin α-GID that antagonizes it. We developed a docking algorithm, ToxDock, which used ensemble-docking and extensive conformational sampling to dock α-GID and its analogs to an α4ß2 nAChR homology model. Experimental testing demonstrated that a virtual screen with ToxDock correctly identified three bioactive α-GID mutants (α-GID[A10V], α-GID[V13I], and α-GID[V13Y]) and one inactive variant (α-GID[A10Q]). Two mutants, α-GID[A10V] and α-GID[V13Y], had substantially reduced potency at the human α7 nAChR relative to α-GID, a desirable feature for α-GID analogs. The general usefulness of the docking algorithm was highlighted by redocking of peptide toxins to two ion channels and a binding protein in which the peptide toxins successfully reverted back to near-native crystallographic poses after being perturbed. Our results demonstrate that ToxDock can overcome two fundamental challenges of docking large toxin peptides to ion channel homology models, as exemplified by the α-GID:α4ß2 nAChR complex, and is extendable to other toxin peptides and ion channels. ToxDock is freely available at rosie.rosettacommons.org/tox_dock.

Binding of Sulfoxaflor to Aplysia californica-AChBP: Computational Insights from Multiscale Approaches.

Structural features and binding properties of sulfoxaflor (SFX) with Ac-AChBP, the surrogate of the insect nAChR ligand binding domain (LBD), are reported herein using various complementary molecular modeling approaches (QM, molecular docking, molecular dynamics, and QM/QM'). The different SFX stereoisomers show distinct behaviors in terms of binding and interactions with Ac-AChBP. Molecular docking and Molecular Dynamics (MD) simulations highlight the specific intermolecular contacts involved in the binding of the different SFX isomers and the relative contribution of the SFX functional groups. QM/QM' calculations provide further insights and a significant refinement of the geometric and energetic contributions of the various residues leading to a preference for the SS and RR stereoisomers. Notable differences in terms of binding interactions are pointed out for the four stereoisomers. The results point out the induced fit of the Ac-AChBP binding site according to the SFX stereoisomer. In this process, the water molecules-mediated contacts play a key role, their energetic contribution being among the most important for the various stereoisomers. In all cases, the interaction with Trp147 is the major binding component, through CH···π and π···π interactions. This study provides a rationale for the binding of SFX to insect nAChR, in particular with respect to the new class of sulfoximine-based insect nAChR competitive modulators, and points out the requirements of various levels of theory for an accurate description of ligand-receptor interactions.

Anti-Inflammatory Effects of 5α,8α-Epidioxycholest-6-en-3ß-ol, a Steroidal Endoperoxide Isolated from Aplysia depilans, Based on Bioguided Fractionation and NMR Analysis.

Sea hares of Aplysia genus are recognized as a source of a diverse range of metabolites. 5α,8α-Endoperoxides belong to a group of oxidized sterols commonly found in marine organisms and display several bioactivities, including antimicrobial, anti-tumor, and immunomodulatory properties. Herein we report the isolation of 5α,8α-epidioxycholest-6-en-3ß-ol (EnP(5,8)) from Aplysia depilans Gmelin, based on bioguided fractionation and nuclear magnetic resonance (NMR) analysis, as well as the first disclosure of its anti-inflammatory properties. EnP(5,8) revealed capacity to decrease cellular nitric oxide (NO) levels in RAW 264.7 macrophages treated with lipopolysaccharide (LPS) by downregulation of the Nos2 (inducible nitric oxide synthase, iNOS) gene. Moreover, EnP(5,8) also inhibited the LPS-induced expression of cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) at the mRNA and protein levels. Mild selective inhibition of COX-2 enzyme activity was also evidenced. Our findings provide evidence of EnP(5,8) as a potential lead drug molecule for the development of new anti-inflammatory agents.

Discovery of leucokinin-like neuropeptides that modulate a specific parameter of feeding motor programs in the molluscan model, Aplysia.

A better understanding of neuromodulation in a behavioral system requires identification of active modulatory transmitters. Here, we used identifiable neurons in a neurobiological model system, the mollusc Aplysia, to study neuropeptides, a diverse class of neuromodulators. We took advantage of two types of feeding neurons, B48 and B1/B2, in the Aplysia buccal ganglion that might contain different neuropeptides. We performed a representational difference analysis (RDA) by subtraction of mRNAs in B48 versus mRNAs in B1/B2. The RDA identified an unusually long (2025 amino acids) peptide precursor encoding Aplysia leucokinin-like peptides (ALKs; e.g. ALK-1 and ALK-2). Northern blot analysis revealed that, compared with other ganglia (e.g. the pedal-pleural ganglion), ALK mRNA is predominantly present in the buccal ganglion, which controls feeding behavior. We then used in situ hybridization and immunohistochemistry to localize ALKs to specific neurons, including B48. MALDI-TOF MS on single buccal neurons revealed expression of 40 ALK precursor-derived peptides. Among these, ALK-1 and ALK-2 are active in the feeding network; they shortened the radula protraction phase of feeding motor programs triggered by a command-like neuron. We also found that this effect may be mediated by the ALK-stimulated enhancement of activity of an interneuron, which has previously been shown to terminate protraction. We conclude that our multipronged approach is effective for determining the structure and defining the diverse functions of leucokinin-like peptides. Notably, the ALK precursor is the first verified nonarthropod precursor for leucokinin-like peptides with a novel, marked modulatory effect on a specific parameter (protraction duration) of feeding motor programs.

Quantitative Reflection Imaging for the Morphology and Dynamics of Live Aplysia californica Pedal Ganglion Neurons Cultured on Nanostructured Plasmonic Crystals.

We describe a reflection imaging system that consists of a plasmonic crystal, a common laboratory microscope, and band-pass filters for use in the quantitative imaging and in situ monitoring of live cells and their substrate interactions. Surface plasmon resonance (SPR) provides a highly sensitive method to monitor changes in physicochemical properties occurring at metal-dielectric interfaces. Polyelectrolyte thin films deposited using the layer-by-layer (LBL) self-assembly method provide a reference system for calibrating the reflection contrast changes that occur when the polyelectrolyte film thickness changes and provide insight into the optical responses that originate from the multiple plasmonic features supported by this imaging system. Finite-difference time-domain (FDTD) simulations of the optical responses measured experimentally from the polyelectrolyte reference system are used to provide a calibration of the optical system for subsequent use in quantitative studies investigating live cell dynamics in cultures supported on a plasmonic crystal substrate. Live Aplysia californica pedal ganglion neurons cultured in artificial seawater were used as a model system through which to explore the utility of this plasmonic imaging technique. Here, the morphology of cellular peripheral structures ≲80 nm in thickness were quantitatively analyzed, and the dynamics of their trypsin-induced surface detachment were visualized. These results illustrate the capacities of this system for use in investigations of the dynamics of ultrathin cellular structures within complex bioanalytical environments.

A d-Amino Acid-Containing Neuropeptide Discovery Funnel.

A receptor binding class of d-amino acid-containing peptides (DAACPs) is formed in animals from an enzymatically mediated post-translational modification of ribosomally translated all-l-amino acid peptides. Although this modification can be required for biological actions, detecting it is challenging because DAACPs have the same mass as their all-l-amino acid counterparts. We developed a suite of mass spectrometry (MS) protocols for the nontargeted discovery of DAACPs and validated their effectiveness using neurons from Aplysia californica. The approach involves the following three steps, with each confirming and refining the hits found in the prior step. The first step is screening for peptides resistant to digestion by aminopeptidase M. The second verifies the presence of a chiral amino acid via acid hydrolysis in deuterium chloride, labeling with Marfey's reagent, and liquid chromatography-mass spectrometry to determine the chirality of each amino acid. The third involves synthesizing the putative DAACPs and comparing them to the endogenous standards. Advantages of the method, the d-amino acid-containing neuropeptide discovery funnel, are that it is capable of detecting the d-form of any common chiral amino acid, and the first two steps do not require peptide standards. Using these protocols, we report that two peptides from the Aplysia achatin-like neuropeptide precursor exist as GdYFD and SdYADSKDEESNAALSDFA. Interestingly, GdYFD was bioactive in the Aplysia feeding and locomotor circuits but SdYADSKDEESNAALSDFA was not. The discovery funnel provides an effective means to characterize DAACPs in the nervous systems of animals in a nontargeted manner.

Second-generation total synthesis of aplyronine A featuring Ni/Cr-mediated coupling reactions.

Second-generation total synthesis of aplyronine A, a potent antitumor marine macrolide, was achieved using Ni/Cr-mediated coupling reactions as key steps. The overall yield of the second-generation synthetic pathway of aplyronine A was 1.4%, obtained in 38 steps based on the longest linear sequence. Compared to our first-generation synthetic pathway of aplyronine A, the second-generation synthesis greatly improved both the yield and number of steps. In particular, we improved the stereoselectivity in the construction of the C13 stereogenic center and the C14-C15 (E)-trisubstituted double bond using the asymmetric Ni/Cr-mediated coupling reaction. Furthermore, we established efficient reaction conditions for the asymmetric Ni/Cr-mediated coupling reaction between the C21-C28 segment and C29-C34 segment. Thus, this coupling reaction proceeded with an equimolar ratio of each segment.

Analysis of the aplyronine A-induced protein-protein interaction between actin and tubulin by surface plasmon resonance.

The antitumor macrolide aplyronine A induces protein-protein interaction (PPI) between actin and tubulin to exert highly potent biological activities. The interactions and binding kinetics of these molecules were analyzed by the surface plasmon resonance with biotinylated aplyronines or tubulin as ligands. Strong binding was observed for tubulin and actin with immobilized aplyronine A. These PPIs were almost completely inhibited by one equivalent of either aplyronine A or C, or mycalolide B. In contrast, a non-competitive actin-depolymerizing agent, latrunculin A, highly accelerated their association. Significant binding was also observed for immobilized tubulin with an actin-aplyronine A complex, and the dissociation constant KD was 1.84µM. Our method could be used for the quantitative analysis of the PPIs between two polymerizing proteins stabilized with small agents.

Structure and dynamics of the fibronectin-III domains of Aplysia californica cell adhesion molecules.

Due to their homophilic and heterophilic binding properties, cell adhesion molecules (CAMs) such as integrin, cadherin and the immunoglobulin superfamily CAMs are of primary importance in cell-cell and cell-substrate interactions, signalling pathways and other crucial biological processes. We study the molecular structures and conformational dynamics of the two fibronectin type III (Fn-III) extracellular domains of the Aplysia californica CAM (apCAM) protein, by constructing and probing an atomically-detailed structural model based on apCAM's homology with other CAMs. The stability and dynamic properties of the Fn-III domains, individually and in tandem, are probed and analysed using all-atom explicit-solvent molecular dynamics (MD) simulations and normal mode analysis of their corresponding elastic network models. The refined structural model of the Fn-III tandem of apCAM reveals a specific pattern of amino acid interactions that controls the stability of the ß-sheet rich structure and could affect apCAM's response to physical or chemical changes of its environment. It also exposes the important role of several specific charged residues in modulating the structural properties of the linker segment connecting the two Fn-III domains, as well as of the inter-domain interface.

Dactylomelane diterpenes from the sea hare Aplysia depilans.

A chemical investigation of the organic extract of the sea hare Aplysia depilans, collected off Skyros Island, Greece, yielded eight new brominated diterpenes (1-8), featuring the rare dactylomelane skeleton, together with the previously reported luzodiol (9). The structure elucidation and the assignment of the relative configurations of the new natural products were based on extensive NMR spectroscopic and MS spectrometric analyses. Compounds 1-9 were evaluated for their cytotoxic activities against five human tumor cell lines, but were proven inactive.

Digestive Gland from Aplysia depilans Gmelin: Leads for Inflammation Treatment.

The exploitation of marine organisms for human nutritional and pharmaceutical purposes has revealed important chemical prototypes for the discovery of new drugs, stimulating compounds isolation and syntheses of new related compounds with biomedical application. Nowadays, it is well known that inflammatory processes are involved in many diseases and the interest in the search for marine natural products with anti-inflammatory potential has been increasing. The genus Aplysia belongs to the class Gastropoda, having a wide geographical distribution and including several species, commonly known as sea hares. Aplysia depilans Gmelin is usually found in the Mediterranean Sea and in the Atlantic Ocean, from West Africa to the French coast. In these marine organisms, most of the digestion and nutrient absorption occurs in the digestive gland. This work aimed to explore the chemical composition and bioactivity of the methanol extract from A. depilans digestive gland. Therefore, fatty acids and carotenoids were determined by GC-MS and HPLC-DAD, respectively. Twenty-two fatty acids and eight carotenoids were identified for the first time in this species. The A. depilans digestive gland revealed to be essentially composed by polyunsaturated fatty acids (PUFA) and xanthophylls. Regarding the anti-inflammatory potential in RAW 264.7 cells stimulated with lipopolysaccharide, it was observed that this matrix has capacity to reduce nitric oxide (NO) and L-citrulline levels, which suggests that its compounds may act by interference with inducible nitric oxide synthase. Taking into account the results obtained, A. depilans digestive gland may be a good source of nutraceuticals, due to their richness in health beneficial nutrients, such as carotenoids and long-chain PUFA.

Aplysiasecosterol†A: A 9,11-Secosteroid with an Unprecedented Tricyclic γ-Diketone Structure from the Sea Hare Aplysia kurodai.

A new 9,11-secosteroid having an unprecedented tricyclic γ-diketone structure, aplysiasecosterol A (1), was isolated from the sea hare Aplysia kurodai. The structure was determined by one- and two-dimensional NMR spectroscopic analysis, molecular modeling studies, a comparison of experimental and calculated ECD spectra, and a modified Mosher's method. Aplysiasecosterol A (1) exhibited cytotoxicity against human myelocytic leukemia HL-60 cells. A biosynthetic pathway for 1 from a known cholesterol was proposed and includes twice α-ketol rearrangements and an intramolecular acetalization.

Dactyloditerpenol acetate, a new prenylbisabolane-type diterpene from Aplysia dactylomela with significant in vitro anti-neuroinflammatory activity.

A new regular diterpene possessing an unusual 1,6-anti-3-methylcyclohex-2-en-1-ol ring system, dactyloditerpenol acetate (1), has been extracted from the tropical sea hare Aplysia dactylomela and its stereostructure elucidated by spectroscopic methods. The absolute configuration of 1 was determined as 1S, 6S, 7R, 10S, and 11R by application of Kishi's method for the assignment of absolute configuration of alcohols. The new diterpene potently inhibited in vitro thromboxane B2 (TXB2) (IC50 0.4µM) and superoxide anion (O2(-)) (IC50 1µM) generation from Escherichia coli lipopolysaccharide (LPS)-activated rat neonatal microglia, with concomitant low short-term toxicity.