Basic Residues at Position 11 of α-Conotoxin LvIA Influence Subtype Selectivity between α3ß2 and α3ß4 Nicotinic Receptors via an Electrostatic Mechanism.

Understanding the determinants of α-conotoxin (α-CTX) selectivity for different nicotinic acetylcholine receptor (nAChR) subtypes is a prerequisite for the design of tool compounds to study nAChRs. However, selectivity optimization of these small, disulfide-rich peptides is difficult not only because of an absence of α-CTX/nAChR co-structures but also because it is challenging to predict how a mutation to an α-CTX will alter its potency and selectivity. As a prototypical system to investigate selectivity, we employed the α-CTX LvIA that is 25-fold selective for the α3ß2 nAChR over the related α3ß4 nAChR subtype, which is a target for nicotine addiction. Using two-electrode voltage clamp electrophysiology, we identified LvIA[D11R] that is 2-fold selective for the α3ß4 nAChR, reversing the subtype preference. This effect is specifically due to the change in charge and not shape of LvIA[D11R], as substitution of D11 with citrulline retains selectivity for the α3ß2 nAChR. Furthermore, LvIA[D11K] shows a stronger reversal, with 4-fold selectivity for the α3ß4 nAChR. Motivated by these findings, using site-directed mutagenesis, we found that ß2[K79A] (I79 on ß4), but not ß2[K78A] (N78 on ß4), largely restores the potency of basic mutants at position 11. Finally, to understand the structural basis of this effect, we used AlphaFold2 to generate models of LvIA in complex with both nAChR subtypes. Both models confirm the plausibility of an electrostatic mechanism to explain the data and also reproduce a broad range of potency and selectivity structure-activity relationships for LvIA mutants, as measured using free energy perturbation simulations. Our work highlights how electrostatic interactions can drive α-CTX selectivity and may serve as a strategy for optimizing the selectivity of LvIA and other α-CTXs.

Collaborative Expression: Transcriptomics of Conus virgo Suggests Contribution of Multiple Secretory Glands to Venom Production.

Venomous marine gastropods of the family Conidae are among the most diversified predators in marine realm-in large due to their complex venoms. Besides being a valuable source of bioactive neuropeptides conotoxins, cone-snails venoms are an excellent model for molecular evolution studies, addressing origin of key innovations. However, these studies are handicapped by scarce current knowledge on the tissues involved in venom production, as it is generally assumed the sole prerogative of the venom gland (VG). The role of other secretory glands that are present in all Conus species (salivary gland, SG) or only in some species (accessory salivary gland, ASG) remains poorly understood. Here, for the first time, we carry out a detailed analysis of the VG, SG, and ASG transcriptomes in the vermivorous Conus virgo. We detect multiple transcripts clusters in both the SG and ASG, whose annotations imply venom-related functions. Despite the subsets of transcripts highly-expressed in the VG, SG, and ASG being very distinct, SG expresses an L-, and ASG-Cerm08-, and MEFRR- superfamily conotoxins, all previously considered specific for VG. We corroborate our results with the analysis of published SG and VG transcriptomes from unrelated fish-hunting C. geographus, and C. striatus, possibly fish-hunting C. rolani, and worm-hunting Conus quercinus. In spite of low expression levels of conotoxins, some other specific clusters of putative venom-related peptides are present and may be highly expressed in the SG of these species. Further functional studies are necessary to determine the role that these peptides play in envenomation. In the meantime, our results show importance of routine multi-tissue sampling both for accurate interpretation of tissue-specific venom composition in cone-snails, and for better understanding origin and evolution of venom peptides genes.

Systematic dissection of genomic features determining the vast diversity of conotoxins.

BACKGROUND: Conus, a highly diverse species of venomous predators, has attracted significant attention in neuroscience and new drug development due to their rich collection of neuroactive peptides called conotoxins. Recent advancements in transcriptome, proteome, and genome analyses have facilitated the identification of conotoxins within Conus' venom glands, providing insights into the genetic features and evolutionary patterns of conotoxin genes. However, the underlying mechanism behind the extraordinary hypervariability of conotoxins remains largely unknown. RESULTS: We analyzed the transcriptomes of 34 Conus species, examining various tissues such as the venom duct, venom bulb, and salivary gland, leading to the identification of conotoxin genes. Genetic variation analysis revealed that a subset of these genes (15.78% of the total) in Conus species underwent positive selection (Ka/Ks > 1, p < 0.01). Additionally, we reassembled and annotated the genome of C. betulinus, uncovering 221 conotoxin-encoding genes. These genes primarily consisted of three exons, with a significant portion showing high transcriptional activity in the venom ducts. Importantly, the flanking regions and adjacent introns of conotoxin genes exhibited a higher prevalence of transposon elements, suggesting their potential contribution to the extensive variability observed in conotoxins. Furthermore, we detected genome duplication in C. betulinus, which likely contributed to the expansion of conotoxin gene numbers. Interestingly, our study also provided evidence of introgression among Conus species, indicating that interspecies hybridization may have played a role in shaping the evolution of diverse conotoxin genes. CONCLUSIONS: This study highlights the impact of adaptive evolution and introgressive hybridization on the genetic diversity of conotoxin genes and the evolution of Conus. We also propose a hypothesis suggesting that transposable elements might significantly contribute to the remarkable diversity observed in conotoxins. These findings not only enhance our understanding of peptide genetic diversity but also present a novel approach for peptide bioengineering.

Research into the Bioengineering of a Novel α-Conotoxin from the Milked Venom of Conus obscurus.

The marine cone snail produces one of the fastest prey strikes in the animal kingdom. It injects highly efficacious venom, often causing prey paralysis and death within seconds. Each snail has hundreds of conotoxins, which serve as a source for discovering and utilizing novel analgesic peptide therapeutics. In this study, we discovered, isolated, and synthesized a novel α3/5-conotoxins derived from the milked venom of Conus obscurus (α-conotoxin OI) and identified the presence of α-conotoxin SI-like sequence previously found in the venom of Conus striatus. Five synthetic analogs of the native α-conotoxin OI were generated. These analogs incorporated single residue or double residue mutations. Three synthetic post-translational modifications (PTMs) were synthetically incorporated into these analogs: N-terminal truncation, proline hydroxylation, and tryptophan bromination. The native α-conotoxin OI demonstrated nanomolar potency in Poecilia reticulata and Homosapiens muscle-type nicotinic acetylcholine receptor (nAChR) isoforms. Moreover, the synthetic α-[P9K] conotoxin OI displayed enhanced potency in both bioassays, ranging from a 2.85 (LD50) to 18.4 (IC50) fold increase in comparative bioactivity. The successful incorporation of PTMs, with retention of both potency and nAChR isoform selectivity, ultimately pushes new boundaries of peptide bioengineering and the generation of novel α-conotoxin-like sequences.

Vexitoxins: conotoxin-like venom peptides from predatory gastropods of the genus Vexillum.

Venoms of predatory marine cone snails are intensely studied because of the biomedical applications of the neuropeptides that they contain, termed conotoxins. Meanwhile some gastropod lineages have independently acquired secretory glands strikingly similar to the venom gland of cone snails, suggesting that they possess similar venoms. Here we focus on the most diversified of these clades, the genus Vexillum. Based on the analysis of a multi-species proteo-transcriptomic dataset, we show that Vexillum species indeed produce complex venoms dominated by highly diversified short cysteine-rich peptides, vexitoxins. Vexitoxins possess the same precursor organization, display overlapping cysteine frameworks and share several common post-translational modifications with conotoxins. Some vexitoxins show sequence similarity to conotoxins and adopt similar domain conformations, including a pharmacologically relevant inhibitory cysteine knot motif. The Vexillum envenomation gland (gL) is a notably more recent evolutionary novelty than the conoidean venom gland. Thus, we hypothesize lower divergence between vexitoxin genes, and their ancestral 'somatic' counterparts compared to that in conotoxins, and we find support for this hypothesis in the evolution of the vexitoxin cluster V027. We use this example to discuss how future studies on vexitoxins can inform the origin of conotoxins, and how they may help to address outstanding questions in venom evolution.

Reconstructing the Origins of the Somatostatin and Allatostatin-C Signaling Systems Using the Accelerated Evolution of Biodiverse Cone Snail Toxins.

Somatostatin and its related peptides (SSRPs) form an important family of hormones with diverse physiological roles. The ubiquitous presence of SSRPs in vertebrates and several invertebrate deuterostomes suggests an ancient origin of the SSRP signaling system. However, the existence of SSRP genes outside of deuterostomes has not been established, and the evolutionary history of this signaling system remains poorly understood. Our recent discovery of SSRP-like toxins (consomatins) in venomous marine cone snails (Conus) suggested the presence of a related signaling system in mollusks and potentially other protostomes. Here, we identify the molluscan SSRP-like signaling gene that gave rise to the consomatin family. Following recruitment into venom, consomatin genes experienced strong positive selection and repeated gene duplications resulting in the formation of a hyperdiverse family of venom peptides. Intriguingly, the largest number of consomatins was found in worm-hunting species (>400 sequences), indicating a homologous system in annelids, another large protostome phylum. Consistent with this, comprehensive sequence mining enabled the identification of SSRP-like sequences (and their corresponding orphan receptor) in annelids and several other protostome phyla. These results established the existence of SSRP-like peptides in many major branches of bilaterians and challenge the prevailing hypothesis that deuterostome SSRPs and protostome allatostatin-C are orthologous peptide families. Finally, having a large set of predator-prey SSRP sequences available, we show that although the cone snail's signaling SSRP-like genes are under purifying selection, the venom consomatin genes experience rapid directional selection to target receptors in a changing mix of prey.

Venomics Reveals a Non-Compartmentalised Venom Gland in the Early Diverged Vermivorous Conus distans.

The defensive use of cone snail venom is hypothesised to have first arisen in ancestral worm-hunting snails and later repurposed in a compartmentalised venom duct to facilitate the dietary shift to molluscivory and piscivory. Consistent with its placement in a basal lineage, we demonstrate that the C. distans venom gland lacked distinct compartmentalisation. Transcriptomics revealed C. distans expressed a wide range of structural classes, with inhibitory cysteine knot (ICK)-containing peptides dominating. To better understand the evolution of the venom gland compartmentalisation, we compared C. distans to C. planorbis, the earliest diverging species from which a defence-evoked venom has been obtained, and fish-hunting C. geographus from the Gastridium subgenus that injects distinct defensive and predatory venoms. These comparisons support the hypothesis that venom gland compartmentalisation arose in worm-hunting species and enabled repurposing of venom peptides to facilitate the dietary shift from vermivory to molluscivory and piscivory in more recently diverged cone snail lineages.

Preparation and Functional Identification of a Novel Conotoxin QcMNCL-XIII0.1 from Conus quercinus.

Conotoxins are tools used by marine Conus snails to hunt and are a significant repository for marine drug research. Conotoxins highly selectively coordinate different subtypes of various ion channels, and a few have been used in pain management. Although more than 8000 conotoxin genes have been found, the biological activity and function of most have not yet been examined. In this report, we selected the toxin gene QcMNCL-XIII0.1 from our previous investigation and studied it in vitro. First, we successfully prepared active recombinant QcMNCL-XIII0.1 using a TrxA (Thioredoxin A)-assisted folding expression vector based on genetic engineering technology. Animal experiments showed that the recombinant QcMNCL-XIII0.1 exhibited nerve conduction inhibition similar to that of pethidine hydrochloride. With flow cytometry combined fluorescent probe Fluo-4 AM, we found that 10 ng/µL recombinant QcMNCL-XIII0.1 inhibited the fluorescence intensity by 31.07% in the 293T cell model transfected with Cav3.1, implying an interaction between α1G T-type calcium channel protein and recombinant QcMNCL-XIII0.1. This toxin could be an important drug in biomedical research and medicine for pain control.

A Combined Transcriptomics and Proteomics Approach Reveals the Differences in the Predatory and Defensive Venoms of the Molluscivorous Cone Snail Cylinder ammiralis (Caenogastropoda: Conidae).

Venoms are complex mixtures of proteins that have evolved repeatedly in the animal kingdom. Cone snail venoms represent one of the best studied venom systems. In nature, this venom can be dynamically adjusted depending on its final purpose, whether to deter predators or hunt prey. Here, the transcriptome of the venom gland and the proteomes of the predation-evoked and defensive venoms of the molluscivorous cone snail Cylinder ammiralis were catalogued. A total of 242 venom-related transcripts were annotated. The conotoxin superfamilies presenting more different peptides were O1, O2, T, and M, which also showed high expression levels (except T). The three precursors of the J superfamily were also highly expressed. The predation-evoked and defensive venoms showed a markedly distinct profile. A total of 217 different peptides were identified, with half of them being unique to one venom. A total of 59 peptides ascribed to 23 different protein families were found to be exclusive to the predatory venom, including the cono-insulin, which was, for the first time, identified in an injected venom. A total of 43 peptides from 20 protein families were exclusive to the defensive venom. Finally, comparisons of the relative abundance (in terms of number of peptides) of the different conotoxin precursor superfamilies showed that most of them present similar abundance regardless of the diet.

Potency- and Selectivity-Enhancing Mutations of Conotoxins for Nicotinic Acetylcholine Receptors Can Be Predicted Using Accurate Free-Energy Calculations.

Nicotinic acetylcholine receptor (nAChR) subtypes are key drug targets, but it is challenging to pharmacologically differentiate between them because of their highly similar sequence identities. Furthermore, α-conotoxins (α-CTXs) are naturally selective and competitive antagonists for nAChRs and hold great potential for treating nAChR disorders. Identifying selectivity-enhancing mutations is the chief aim of most α-CTX mutagenesis studies, although doing so with traditional docking methods is difficult due to the lack of α-CTX/nAChR crystal structures. Here, we use homology modeling to predict the structures of α-CTXs bound to two nearly identical nAChR subtypes, α3ß2 and α3ß4, and use free-energy perturbation (FEP) to re-predict the relative potency and selectivity of α-CTX mutants at these subtypes. First, we use three available crystal structures of the nAChR homologue, acetylcholine-binding protein (AChBP), and re-predict the relative affinities of twenty point mutations made to the α-CTXs LvIA, LsIA, and GIC, with an overall root mean square error (RMSE) of 1.08 ± 0.15 kcal/mol and an R2 of 0.62, equivalent to experimental uncertainty. We then use AChBP as a template for α3ß2 and α3ß4 nAChR homology models bound to the α-CTX LvIA and re-predict the potencies of eleven point mutations at both subtypes, with an overall RMSE of 0.85 ± 0.08 kcal/mol and an R2 of 0.49. This is significantly better than the widely used molecular mechanics-generalized born/surface area (MM-GB/SA) method, which gives an RMSE of 1.96 ± 0.24 kcal/mol and an R2 of 0.06 on the same test set. Next, we demonstrate that FEP accurately classifies α3ß2 nAChR selective LvIA mutants while MM-GB/SA does not. Finally, we use FEP to perform an exhaustive amino acid mutational scan of LvIA and predict fifty-two mutations of LvIA to have greater than 100X selectivity for the α3ß2 nAChR. Our results demonstrate the FEP is well-suited to accurately predict potency- and selectivity-enhancing mutations of α-CTXs for nAChRs and to identify alternative strategies for developing selective α-CTXs.

Venom duct origins of prey capture and defensive conotoxins in piscivorous Conus striatus.

The venom duct origins of predatory and defensive venoms has not been studied for hook-and-line fish hunting cone snails despite the pharmacological importance of their venoms. To better understand the biochemistry and evolution of injected predatory and defensive venoms, we compared distal, central and proximal venom duct sections across three specimens of C. striatus (Pionoconus) using proteomic and transcriptomic approaches. A total of 370 conotoxin precursors were identified from the whole venom duct transcriptome. Milked defensive venom was enriched with a potent cocktail of proximally expressed inhibitory α-, ω- and µ-conotoxins compared to milked predatory venom. In contrast, excitatory κA-conotoxins dominated both the predatory and defensive venoms despite their distal expression, suggesting this class of conotoxin can be selectively expressed from the same duct segment in response to either a predatory or defensive stimuli. Given the high abundance of κA-conotoxins in the Pionoconus clade, we hypothesise that the κA-conotoxins have evolved through adaptive evolution following their repurposing from ancestral inhibitory A superfamily conotoxins to facilitate the dietary shift to fish hunting and species radiation in this clade.

Diversity of Conopeptides and Conoenzymes from the Venom Duct of the Marine Cone Snail Conus bayani as Determined from Transcriptomic and Proteomic Analyses.

Marine cone snails are predatory gastropods characterized by a well-developed venom apparatus and highly evolved hunting strategies that utilize toxins to paralyze prey and defend against predators. The venom of each species of cone snail has a large number of pharmacologically active peptides known as conopeptides or conotoxins that are usually unique in each species. Nevertheless, venoms of only very few species have been characterized so far by transcriptomic approaches. In this study, we used transcriptome sequencing technologies and mass spectrometric methods to describe the diversity of venom components expressed by a worm-hunting species, Conus bayani. A total of 82 conotoxin sequences were retrieved from transcriptomic data that contain 54 validated conotoxin sequences clustered into 21 gene superfamilies including divergent gene family, 17 sequences clustered to 6 different conotoxin classes, and 11 conotoxins classified as unassigned gene family. Seven new conotoxin sequences showed unusual cysteine patterns. We were also able to identify 19 peptide sequences using mass spectrometry that completely overlapped with the conotoxin sequences obtained from transcriptome analysis. Importantly, herein we document the presence of 16 proteins that include five post-translational modifying enzymes obtained from transcriptomic data. Our results revealed diverse and novel conopeptides of an unexplored species that could be used extensively in biomedical research due to their therapeutic potentials.

Chemical Synthesis and NMR Solution Structure of Conotoxin GXIA from Conus geographus.

Conotoxins are disulfide-rich peptides found in the venom of cone snails. Due to their exquisite potency and high selectivity for a wide range of voltage and ligand gated ion channels they are attractive drug leads in neuropharmacology. Recently, cone snails were found to have the capability to rapidly switch between venom types with different proteome profiles in response to predatory or defensive stimuli. A novel conotoxin, GXIA (original name G117), belonging to the I3-subfamily was identified as the major component of the predatory venom of piscivorous Conus geographus. Using 2D solution NMR spectroscopy techniques, we resolved the 3D structure for GXIA, the first structure reported for the I3-subfamily and framework XI family. The 32 amino acid peptide is comprised of eight cysteine residues with the resultant disulfide connectivity forming an ICK+1 motif. With a triple stranded ß-sheet, the GXIA backbone shows striking similarity to several tarantula toxins targeting the voltage sensor of voltage gated potassium and sodium channels. Supported by an amphipathic surface, the structural evidence suggests that GXIA is able to embed in the membrane and bind to the voltage sensor domain of a putative ion channel target.

Structural and dynamical effects of targeted mutations on µO-Conotoxin MfVIA: Molecular simulation studies.

Conotoxins are a group of cysteine-rich, neurotoxic peptides isolated from the venom of marine cone snails. MfVIA is a member of the µO-conotoxin family, and acts as an inhibitor of subtype 1.8 voltage-gated sodium ion channels (NaV1.8). The unique selectivity of MfVIA as an inhibitor of NaV1.8 makes it an ideal peptide for elucidation of the physiological functions of this voltage-gated ion channel. Previous experimental studies of point mutations of MfVIA showed that the double mutant [E5K,E8K] exhibited greater activity at NaV1.8 relative to the wild-type toxin. The present study employs molecular dynamics (MD) simulations to examine the effects of various mutations at these key residues (E5 and E8) on the structure and dynamics of MfVIA. Five double mutants were studied, in which the positions 5 and 8 residues were mutated to amino acids with a range of different physicochemical properties, namely [E5A,E8A], [E5D,E8D], [E5F,E8F], [E5K,E8K], and [E5R,E8R]. Except for [E5D,E8D], all of the mutants tend to show decreased contacts at the N-terminus owing to the loss of the R1-E5 salt bridge relative to that of the wild-type, which subsequently lead to greater exposure and flexibility of the N-terminus for most of the mutant peptides studied, potentially rendering them more able to interact with other species, including NaV1.8. Molecular docking studies of the peptides to NaV1.8 via different binding mechanisms suggest that the [E5R, E8R] mutant may be especially worthy of further investigation owing to its predicted binding mode, which differs markedly from those of the other peptides in this study.

Diversity of Conopeptides and Their Precursor Genes of Conus Litteratus.

The venom of various Conus species is composed of a rich variety of unique bioactive peptides, commonly referred to as conotoxins (conopeptides). Most conopeptides have specific receptors or ion channels as physiologically relevant targets. In this paper, high-throughput transcriptome sequencing was performed to analyze putative conotoxin transcripts from the venom duct of a vermivorous cone snail species, Conus litteratus native to the South China Sea. A total of 128 putative conotoxins were identified, most of them belonging to 22 known superfamilies, with 43 conotoxins being regarded as belonging to new superfamilies. Notably, the M superfamily was the most abundant in conotoxins among the known superfamilies. A total of 15 known cysteine frameworks were also described. The largest proportion of cysteine frameworks were VI/VII (C-C-CC-C-C), IX (C-C-C-C-C-C) and XIV (C-C-C-C). In addition, five novel cysteine patterns were also discovered. Simple sequence repeat detection results showed that di-nucleotide was the major type of repetition, and the codon usage bias results indicated that the codon usage bias of the conotoxin genes was weak, but the M, O1, O2 superfamilies differed in codon preference. Gene cloning indicated that there was no intron in conotoxins of the B1- or J superfamily, one intron with 1273-1339 bp existed in a mature region of the F superfamily, which is different from the previously reported gene structure of conotoxins from other superfamilies. This study will enhance our understanding of conotoxin diversity, and the new conotoxins discovered in this paper will provide more potential candidates for the development of pharmacological probes and marine peptide drugs.

ConoMode, a database for conopeptide binding modes.

ConoMode is a database for complex three-dimensional (3D) structures of conopeptides binding with their target proteins. Conopeptides, a large family of peptides from the venom of marine snails of the Conus genus, have exceptionally diverse sequences, and their high specificity to block ion channels makes them crucial as drug leads and tools for physiological studies. ConoMode is a specialized archive for the collection of 3D coordinate data for the conopeptides and their binding target proteins from published literature and the Protein Data Bank. These 3D structures can be determined using experimental methods such as X-ray crystallography and electron microscopy and computational methods including docking, homology modeling and molecular dynamics simulations. The binding modes for the conopeptides determined using computational modeling must be validated based on experimental data. The 3D coordinate data from ConoMode can be searched, visualized, downloaded and uploaded. Currently, ConoMode manages 19 conopeptide sequences (from 10 Conus species), 15 protein sequences and 37 3D structures. ConoMode utilizes a modern technical framework to provide a good user experience on mobile devices with touch interaction features. Furthermore, the database is fully optimized for unstructured data and flexible data models. Database URL: http://conomode.qnlm.ac/conomode/conomode/index.

α-Conotoxin as Potential to α7-nAChR Recombinant Expressed in Escherichia coli.

α7 nicotinic acetylcholine receptors (nAChR) is an important nicotinic acetylcholine receptors subtype and closely associated with cognitive disorders, such as Alzheimer's and schizophrenia disease. The mutant ArIB (V11L, V16A) of α-conotoxin ArIB with 17-amino acid residues specifically targets α7 nAChR with no obvious effect on other nAChR subtypes. In the study, the synthetic gene encoding mature peptide of ArIB and mutant ArIB (V11L, V16A) carried a fusion protein Trx and 6 × His-tag was separately inserted in pET-32a (+) vector and transformed into Escherichia coli strain BL21(DE3) pLysS for expression. The expressions of Trx-ArIB-His6 and Trx-ArIB (V11L, V16A)-His6 were soluble in Escherichia coli, which were purified by Ni-NTA affinity chromatography column and cleaved by enterokinase to release rArIB and rArIB (V11L, V16A). Then, rArIB and rArIB (V11L, V16A) were purified by high-performance liquid chromatography (HPLC) and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Bioactivity of rArIB and rArIB (V11L, V16A) was assessed by two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing human nAChR subtypes. The results indicated that the yield of the fusion proteins was approximately 50 mg/L and rArIB (V11L, V16A) antagonized the α7 nAChR subtype selectively with 8-nM IC50. In summary, this study provides an efficient method to biosynthesize α-conotoxin ArIB and rArIB (V11L, V16A) in Escherichia coli, which could be economical to obtain massively bioactive disulfide-rich polypeptides at fast speed.

Pro-Inflammatory Signaling Upregulates a Neurotoxic Conotoxin-Like Protein Encrypted Within Human Endogenous Retrovirus-K.

Motor neuron degeneration and spinal cord demyelination are hallmark pathological events in Amyotrophic Lateral Sclerosis (ALS). Endogenous retrovirus-K (ERVK) expression has an established association with ALS neuropathology, with murine modeling pointing to a role for the ERVK envelope (env) gene in disease processes. Here, we describe a novel viral protein cryptically encoded within the ERVK env transcript, which resembles two distinct cysteine-rich neurotoxic proteins: conotoxin proteins found in marine snails and the Human Immunodeficiency Virus (HIV) Tat protein. Consistent with Nuclear factor-kappa B (NF-κB)-induced retrotransposon expression, the ERVK conotoxin-like protein (CTXLP) is induced by inflammatory signaling. CTXLP is found in the nucleus, impacting innate immune gene expression and NF-κB p65 activity. Using human autopsy specimens from patients with ALS, we further showcase CTXLP expression in degenerating motor cortex and spinal cord tissues, concomitant with inflammation linked pathways, including enhancement of necroptosis marker mixed lineage kinase domain-like (MLKL) protein and oligodendrocyte maturation/myelination inhibitor Nogo-A. These findings identify CTXLP as a novel ERVK protein product, which may act as an effector in ALS neuropathology.

Comparative transcriptomics of the venoms of continental and insular radiations of West African cones.

The transcriptomes of the venom glands of 13 closely related species of vermivorous cones endemic to West Africa from genera Africonus and Varioconus were sequenced and venom repertoires compared within a phylogenetic framework using one Kalloconus species as outgroup. The total number of conotoxin precursors per species varied between 108 and 221. Individuals of the same species shared about one-fourth of the total conotoxin precursors. The number of common sequences was drastically reduced in the pairwise comparisons between closely related species, and the phylogenetical signal was totally eroded at the inter-generic level (no sequence was identified as shared derived), due to the intrinsic high variability of these secreted peptides. A common set of four conotoxin precursor superfamilies (T, O1, O2 and M) was expanded in all studied cone species, and thus, they are considered the basic venom toolkit for hunting and defense in the West African vermivorous cone snails. Maximum-likelihood ancestral character reconstructions inferred shared conotoxin precursors preferentially at internal nodes close to the tips of the phylogeny (between individuals and between closely related species) as well as in the common ancestor of Varioconus. Besides the common toolkit, the two genera showed significantly distinct catalogues of conotoxin precursors in terms of type of superfamilies present and the abundance of members per superfamily, but had similar relative expression levels indicating functional convergence. Differential expression comparisons between vermivorous and piscivorous cones highlighted the importance of the A and S superfamilies for fish hunting and defense.

Critical residue properties for potency and selectivity of α-Conotoxin RgIA towards α9α10 nicotinic acetylcholine receptors.

The α9α10 nicotinic acetylcholine receptor (nAChR) has been characterized as an effective anti-pain target that functions through a non-opioid mechanism. However, as a pentameric ion channel comprised of two different subunits, the specific targeting of α9α10 nAChRs has proven challenging. Previously the 13-amino-acid peptide, RgIA, was shown to block α9α10 nAChRs with high potency and specificity. This peptide, characterized from the venom of the carnivorous marine snail, Conus regius, produced analgesia in several rodent models of chronic pain. Despite promising pre-clinical data in behavioral assays, the number of specific α9α10 nAChR antagonists remains small and the physiological mechanisms of analgesia remain cryptic. In this study, we implement amino-acid substitutions to definitively characterize the chemical properties of RgIA that contribute to its activity against α9α10 nAChRs. Using this mutational approach, we determined the vital role of biochemical side-chain properties and amino acids in the second loop that are amenable to substitutions to further engineer next-generation analogs for the blockade of α9α10 nAChRs.