A previously unrecognized superfamily of macro-conotoxins includes an inhibitor of the sensory neuron calcium channel Cav2.3.

Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, which defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and 2 additional conotoxin classes not previously described. The crystal structure of recombinant Mu8.1 displays a saposin-like fold and shows structural similarity with con-ikot-ikot. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of recombinantly expressed voltage-gated ion channels, Mu8.1 displayed the highest potency against the R-type (Cav2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K+ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study showcases the potential of uncovering novel structures and bioactivities within the largely unexplored group of macro-conotoxins.

High-resolution crystal structure of the Mu8.1 conotoxin from Conus mucronatus.

Marine cone snails produce a wealth of peptide toxins (conotoxins) that bind their molecular targets with high selectivity and potency. Therefore, conotoxins constitute valuable biomolecular tools with a variety of biomedical purposes. The Mu8.1 conotoxin from Conus mucronatus is the founding member of the newly identified saposin-like conotoxin class of conotoxins and has been shown to target Cav2.3, a voltage-gated calcium channel. Two crystal structures have recently been determined of Mu8.1 at 2.3 and 2.1 Šresolution. Here, a high-resolution crystal structure of Mu8.1 was determined at 1.67 Šresolution in the high-symmetry space group I4122. The asymmetric unit contained one molecule, with a symmetry-related molecule generating a dimer equivalent to that observed in the two previously determined structures. The high resolution allows a detailed atomic analysis of a water-filled cavity buried at the dimer interface, revealing a tightly coordinated network of waters that shield a lysine residue (Lys55) with a predicted unusually low side-chain pKa value. These findings are discussed in terms of a potential functional role of Lys55 in target interaction.

A single-chain variable fragment selected against a conformational epitope of a recombinantly produced snake toxin using phage display.

Phage display technology is a powerful tool for selecting monoclonal antibodies against a diverse set of antigens. Within toxinology, however, it remains challenging to generate monoclonal antibodies against many animal toxins, as they are difficult to obtain from venom. Recombinant toxins have been proposed as a solution to overcome this challenge, but so far, few have been used as antigens to generate neutralizing antibodies. Here, we describe the recombinant expression of α-cobratoxin in E. coli and its successful application as an antigen in a phage display selection campaign. From this campaign, an scFv (single-chain variable fragment) was isolated with similar binding affinity to a control scFv generated against the native toxin. The selected scFv recognizes a structural epitope, enabling it to inhibit the interaction between the acetylcholine receptor and the native toxin in vitro. This approach represents the first entirely in vitro antibody selection strategy for generating neutralizing monoclonal antibodies against a snake toxin.

Modern venomics-Current insights, novel methods, and future perspectives in biological and applied animal venom research.

Venoms have evolved >100 times in all major animal groups, and their components, known as toxins, have been fine-tuned over millions of years into highly effective biochemical weapons. There are many outstanding questions on the evolution of toxin arsenals, such as how venom genes originate, how venom contributes to the fitness of venomous species, and which modifications at the genomic, transcriptomic, and protein level drive their evolution. These questions have received particularly little attention outside of snakes, cone snails, spiders, and scorpions. Venom compounds have further become a source of inspiration for translational research using their diverse bioactivities for various applications. We highlight here recent advances and new strategies in modern venomics and discuss how recent technological innovations and multi-omic methods dramatically improve research on venomous animals. The study of genomes and their modifications through CRISPR and knockdown technologies will increase our understanding of how toxins evolve and which functions they have in the different ontogenetic stages during the development of venomous animals. Mass spectrometry imaging combined with spatial transcriptomics, in situ hybridization techniques, and modern computer tomography gives us further insights into the spatial distribution of toxins in the venom system and the function of the venom apparatus. All these evolutionary and biological insights contribute to more efficiently identify venom compounds, which can then be synthesized or produced in adapted expression systems to test their bioactivity. Finally, we critically discuss recent agrochemical, pharmaceutical, therapeutic, and diagnostic (so-called translational) aspects of venoms from which humans benefit.

Production of an Active, Human Membrane Protein in Saccharomyces cerevisiae: Full-Length FICD.

The human Fic domain-containing protein (FICD) is a type II endoplasmic reticulum (ER) membrane protein that is important for the maintenance of ER proteostasis. Structural and in vitro biochemical characterisation of FICD AMPylase and deAMPylase activity have been restricted to the soluble ER-luminal domain produced in Escherichia coli. Information about potentially important features, such as structural motifs, modulator binding sites or other regulatory elements, is therefore missing for the approximately 100 N-terminal residues including the transmembrane region of FICD. Expressing and purifying the required quantity and quality of membrane proteins is demanding because of the low yields and poor stability often observed. Here, we produce full-length FICD by combining a Saccharomyces cerevisiae-based platform with green fluorescent protein (GFP) tagging to optimise the conditions for expression, solubilisation and purification. We subsequently employ these conditions to purify milligram quantities of His-tagged FICD per litre of culture, and show that the purified, detergent-solubilised membrane protein is an active deAMPylating enzyme. Our work provides a straightforward methodology for producing not only full-length FICD, but also other membrane proteins in S. cerevisiae for structural and biochemical characterisation.

DisCoTune: versatile auxiliary plasmids for the production of disulphide-containing proteins and peptides in the E. coli T7 system.

Secreted proteins and peptides hold large potential both as therapeutics and as enzyme catalysts in biotechnology. The high stability of many secreted proteins helps maintain functional integrity in changing chemical environments and is a contributing factor to their commercial potential. Disulphide bonds constitute an important post-translational modification that stabilizes many of these proteins and thus preserves the active state under chemically stressful conditions. Despite their importance, the discovery and applications within this group of proteins and peptides are limited by the availability of synthetic biology tools and heterologous production systems that allow for efficient formation of disulphide bonds. Here, we refine the design of two DisCoTune (Disulphide bond formation in E. coli with tunable expression) plasmids that enable the formation of disulphides in the highly popular Escherichia coli T7 protein production system. We show that this new system promotes significantly higher yield and activity of an industrial protease and a conotoxin, which belongs to a group of disulphide-rich venom peptides from cone snails with strong potential as research tools and pharmacological agents.

Strategies for Heterologous Expression, Synthesis, and Purification of Animal Venom Toxins.

Animal venoms are complex mixtures containing peptides and proteins known as toxins, which are responsible for the deleterious effect of envenomations. Across the animal Kingdom, toxin diversity is enormous, and the ability to understand the biochemical mechanisms governing toxicity is not only relevant for the development of better envenomation therapies, but also for exploiting toxin bioactivities for therapeutic or biotechnological purposes. Most of toxinology research has relied on obtaining the toxins from crude venoms; however, some toxins are difficult to obtain because the venomous animal is endangered, does not thrive in captivity, produces only a small amount of venom, is difficult to milk, or only produces low amounts of the toxin of interest. Heterologous expression of toxins enables the production of sufficient amounts to unlock the biotechnological potential of these bioactive proteins. Moreover, heterologous expression ensures homogeneity, avoids cross-contamination with other venom components, and circumvents the use of crude venom. Heterologous expression is also not only restricted to natural toxins, but allows for the design of toxins with special properties or can take advantage of the increasing amount of transcriptomics and genomics data, enabling the expression of dormant toxin genes. The main challenge when producing toxins is obtaining properly folded proteins with a correct disulfide pattern that ensures the activity of the toxin of interest. This review presents the strategies that can be used to express toxins in bacteria, yeast, insect cells, or mammalian cells, as well as synthetic approaches that do not involve cells, such as cell-free biosynthesis and peptide synthesis. This is accompanied by an overview of the main advantages and drawbacks of these different systems for producing toxins, as well as a discussion of the biosafety considerations that need to be made when working with highly bioactive proteins.

Curses or Cures: A Review of the Numerous Benefits Versus the Biosecurity Concerns of Conotoxin Research.

Conotoxins form a diverse group of peptide toxins found in the venom of predatory marine cone snails. Decades of conotoxin research have provided numerous measurable scientific and societal benefits. These include their use as a drug, diagnostic agent, drug leads, and research tools in neuroscience, pharmacology, biochemistry, structural biology, and molecular evolution. Human envenomations by cone snails are rare but can be fatal. Death by envenomation is likely caused by a small set of toxins that induce muscle paralysis of the diaphragm, resulting in respiratory arrest. The potency of these toxins led to concerns regarding the potential development and use of conotoxins as biological weapons. To address this, various regulatory measures have been introduced that limit the use and access of conotoxins within the research community. Some of these regulations apply to all of the ≈200,000 conotoxins predicted to exist in nature of which less than 0.05% are estimated to have any significant toxicity in humans. In this review we provide an overview of the many benefits of conotoxin research, and contrast these to the perceived biosecurity concerns of conotoxins and research thereof.

Cellular functions and molecular mechanisms of non-lysine ubiquitination.

Protein ubiquitination is of great cellular importance through its central role in processes such as degradation, DNA repair, endocytosis and inflammation. Canonical ubiquitination takes place on lysine residues, but in the past 15 years non-lysine ubiquitination on serine, threonine and cysteine has been firmly established. With the emerging importance of non-lysine ubiquitination, it is crucial to identify the responsible molecular machinery and understand the mechanistic basis for non-lysine ubiquitination. Here, we first provide an overview of the literature that has documented non-lysine ubiquitination. Informed by these examples, we then discuss the molecular mechanisms and cellular implications of non-lysine ubiquitination, and conclude by outlining open questions and future perspectives in the field.

The three-dimensional structure of an H-superfamily conotoxin reveals a granulin fold arising from a common ICK cysteine framework.

Venomous marine cone snails produce peptide toxins (conotoxins) that bind ion channels and receptors with high specificity and therefore are important pharmacological tools. Conotoxins contain conserved cysteine residues that form disulfide bonds that stabilize their structures. To gain structural insight into the large, yet poorly characterized conotoxin H-superfamily, we used NMR and CD spectroscopy along with MS-based analyses to investigate H-Vc7.2 from Conus victoriae, a peptide with a VI/VII cysteine framework. This framework has CysI-CysIV/CysII-CysV/CysIII-CysVI connectivities, which have invariably been associated with the inhibitor cystine knot (ICK) fold. However, the solution structure of recombinantly expressed and purified H-Vc7.2 revealed that although it displays the expected cysteine connectivities, H-Vc7.2 adopts a different fold consisting of two stacked ß-hairpins with opposing ß-strands connected by two parallel disulfide bonds, a structure homologous to the N-terminal region of the human granulin protein. Using structural comparisons, we subsequently identified several toxins and nontoxin proteins with this "mini-granulin" fold. These findings raise fundamental questions concerning sequence-structure relationships within peptides and proteins and the key determinants that specify a given fold.

Ero1-Mediated Reoxidation of Protein Disulfide Isomerase Accelerates the Folding of Cone Snail Toxins.

Disulfide-rich peptides are highly abundant in nature and their study has provided fascinating insight into protein folding, structure and function. Venomous cone snails belong to a group of organisms that express one of the largest sets of disulfide-rich peptides (conotoxins) found in nature. The diversity of structural scaffolds found for conotoxins suggests that specialized molecular adaptations have evolved to ensure their efficient folding and secretion. We recently showed that canonical protein disulfide isomerase (PDI) and a conotoxin-specific PDI (csPDI) are ubiquitously expressed in the venom gland of cone snails and play a major role in conotoxin folding. Here, we identify cone snail endoplasmic reticulum oxidoreductin-1 (Conus Ero1) and investigate its role in the oxidative folding of conotoxins through reoxidation of cone snail PDI and csPDI. We show that Conus Ero1 preferentially reoxidizes PDI over csPDI, suggesting that the reoxidation of csPDI may rely on an Ero1-independent molecular pathway. Despite the preferential reoxidation of PDI over csPDI, the combinatorial effect of Ero1 and csPDI provides higher folding yields than Ero1 and PDI. We further demonstrate that the highest in vitro folding rates of two model conotoxins are achieved when all three enzymes are present, indicating that these enzymes may act synergistically. Our findings provide new insight into the generation of one of the most diverse classes of disulfide-rich peptides and may improve current in vitro approaches for the production of venom peptides for pharmacological studies.

How Are Proteins Reduced in the Endoplasmic Reticulum?

The reversal of thiol oxidation in proteins within the endoplasmic reticulum (ER) is crucial for protein folding, degradation, chaperone function, and the ER stress response. Our understanding of this process is generally poor but progress has been made. Enzymes performing the initial reduction of client proteins, as well as the ultimate electron donor in the pathway, have been identified. Most recently, a role for the cytosol in ER protein reduction has been revealed. Nevertheless, how reducing equivalents are transferred from the cytosol to the ER lumen remains an open question. We review here why proteins are reduced in the ER, discuss recent data on catalysis of steps in the pathway, and consider the implications for redox homeostasis within the early secretory pathway.

Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens.

The application of forward genetic screens to cultured human cells represents a powerful method to study gene function. The repurposing of the bacterial CRISPR/Cas9 system provides an effective method to disrupt gene function in mammalian cells, and has been applied to genome-wide screens. Here, we compare the efficacy of genome-wide CRISPR/Cas9-mediated forward genetic screens versus gene-trap mutagenesis screens in haploid human cells, which represent the existing 'gold standard' method. This head-to-head comparison aimed to identify genes required for the endoplasmic reticulum-associated degradation (ERAD) of MHC class I molecules. The two approaches show high concordance (>70%), successfully identifying the majority of the known components of the canonical glycoprotein ERAD pathway. Both screens also identify a role for the uncharacterized gene TXNDC11, which we show encodes an EDEM2/3-associated disulphide reductase. Genome-wide CRISPR/Cas9-mediated screens together with haploid genetic screens provide a powerful addition to the forward genetic toolbox.

Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides.

Formation of correct disulfide bonds in the endoplasmic reticulum is a crucial step for folding proteins destined for secretion. Protein disulfide isomerases (PDIs) play a central role in this process. We report a previously unidentified, hypervariable family of PDIs that represents the most diverse gene family of oxidoreductases described in a single genus to date. These enzymes are highly expressed specifically in the venom glands of predatory cone snails, animals that synthesize a remarkably diverse set of cysteine-rich peptide toxins (conotoxins). Enzymes in this PDI family, termed conotoxin-specific PDIs, significantly and differentially accelerate the kinetics of disulfide-bond formation of several conotoxins. Our results are consistent with a unique biological scenario associated with protein folding: The diversification of a family of foldases can be correlated with the rapid evolution of an unprecedented diversity of disulfide-rich structural domains expressed by venomous marine snails in the superfamily Conoidea.

Co- and Post-Translational Protein Folding in the ER.

The biophysical rules that govern folding of small, single-domain proteins in dilute solutions are now quite well understood. The mechanisms underlying co-translational folding of multidomain and membrane-spanning proteins in complex cellular environments are often less clear. The endoplasmic reticulum (ER) produces a plethora of membrane and secretory proteins, which must fold and assemble correctly before ER exit - if these processes fail, misfolded species accumulate in the ER or are degraded. The ER differs from other cellular organelles in terms of the physicochemical environment and the variety of ER-specific protein modifications. Here, we review chaperone-assisted co- and post-translational folding and assembly in the ER and underline the influence of protein modifications on these processes. We emphasize how method development has helped advance the field by allowing researchers to monitor the progression of folding as it occurs inside living cells, while at the same time probing the intricate relationship between protein modifications during folding.

Bioinformatics analysis identifies several intrinsically disordered human E3 ubiquitin-protein ligases.

The ubiquitin-proteasome system targets misfolded proteins for degradation. Since the accumulation of such proteins is potentially harmful for the cell, their prompt removal is important. E3 ubiquitin-protein ligases mediate substrate ubiquitination by bringing together the substrate with an E2 ubiquitin-conjugating enzyme, which transfers ubiquitin to the substrate. For misfolded proteins, substrate recognition is generally delegated to molecular chaperones that subsequently interact with specific E3 ligases. An important exception is San1, a yeast E3 ligase. San1 harbors extensive regions of intrinsic disorder, which provide both conformational flexibility and sites for direct recognition of misfolded targets of vastly different conformations. So far, no mammalian ortholog of San1 is known, nor is it clear whether other E3 ligases utilize disordered regions for substrate recognition. Here, we conduct a bioinformatics analysis to examine >600 human and S. cerevisiae E3 ligases to identify enzymes that are similar to San1 in terms of function and/or mechanism of substrate recognition. An initial sequence-based database search was found to detect candidates primarily based on the homology of their ordered regions, and did not capture the unique disorder patterns that encode the functional mechanism of San1. However, by searching specifically for key features of the San1 sequence, such as long regions of intrinsic disorder embedded with short stretches predicted to be suitable for substrate interaction, we identified several E3 ligases with these characteristics. Our initial analysis revealed that another remarkable trait of San1 is shared with several candidate E3 ligases: long stretches of complete lysine suppression, which in San1 limits auto-ubiquitination. We encode these characteristic features into a San1 similarity-score, and present a set of proteins that are plausible candidates as San1 counterparts in humans. In conclusion, our work indicates that San1 is not a unique case, and that several other yeast and human E3 ligases have sequence properties that may allow them to recognize substrates by a similar mechanism as San1.

Specialized insulin is used for chemical warfare by fish-hunting cone snails.

More than 100 species of venomous cone snails (genus Conus) are highly effective predators of fish. The vast majority of venom components identified and functionally characterized to date are neurotoxins specifically targeted to receptors, ion channels, and transporters in the nervous system of prey, predators, or competitors. Here we describe a venom component targeting energy metabolism, a radically different mechanism. Two fish-hunting cone snails, Conus geographus and Conus tulipa, have evolved specialized insulins that are expressed as major components of their venoms. These insulins are distinctive in having much greater similarity to fish insulins than to the molluscan hormone and are unique in that posttranslational modifications characteristic of conotoxins (hydroxyproline, γ-carboxyglutamate) are present. When injected into fish, the venom insulin elicits hypoglycemic shock, a condition characterized by dangerously low blood glucose. Our evidence suggests that insulin is specifically used as a weapon for prey capture by a subset of fish-hunting cone snails that use a net strategy to capture prey. Insulin appears to be a component of the nirvana cabal, a toxin combination in these venoms that is released into the water to disorient schools of small fish, making them easier to engulf with the snail's distended false mouth, which functions as a net. If an entire school of fish simultaneously experiences hypoglycemic shock, this should directly facilitate capture by the predatory snail.

The selenium metabolite methylselenol regulates the expression of ligands that trigger immune activation through the lymphocyte receptor NKG2D.

For decades, selenium research has been focused on the identification of active metabolites, which are crucial for selenium chemoprevention of cancer. In this context, the metabolite methylselenol (CH3SeH) is known for its action to selectively kill transformed cells through mechanisms that include increased formation of reactive oxygen species, induction of DNA damage, triggering of apoptosis, and inhibition of angiogenesis. Here we reveal that CH3SeH modulates the cell surface expression of NKG2D ligands. The expression of NKG2D ligands is induced by stress-associated pathways that occur early during malignant transformation and enable the recognition and elimination of tumors by activating the lymphocyte receptor NKG2D. CH3SeH regulated NKG2D ligands both on the transcriptional and the posttranscriptional levels. CH3SeH induced the transcription of MHC class I polypeptide-related sequence MICA/B and ULBP2 mRNA. However, the induction of cell surface expression was restricted to the ligands MICA/B. Remarkably, our studies showed that CH3SeH inhibited ULBP2 surface transport through inhibition of the autophagic transport pathway. Finally, we identified extracellular calcium as being essential for CH3SeH regulation of NKG2D ligands. A balanced cell surface expression of NKG2D ligands is considered to be an innate barrier against tumor development. Therefore, our work indicates that the application of selenium compounds that are metabolized to CH3SeH could improve NKG2D-based immune therapy.

GPx8 peroxidase prevents leakage of H2O2 from the endoplasmic reticulum.

Unbalanced endoplasmic reticulum (ER) homeostasis (ER stress) leads to increased generation of reactive oxygen species (ROS). Disulfide-bond formation in the ER by Ero1 family oxidases produces hydrogen peroxide (H2O2) and thereby constitutes one potential source of ER-stress-induced ROS. However, we demonstrate that Ero1α-derived H2O2 is rapidly cleared by glutathione peroxidase (GPx) 8. In 293 cells, GPx8 and reduced/activated forms of Ero1α co-reside in the rough ER subdomain. Loss of GPx8 causes ER stress, leakage of Ero1α-derived H2O2 to the cytosol, and cell death. In contrast, peroxiredoxin (Prx) IV, another H2O2-detoxifying rough ER enzyme, does not protect from Ero1α-mediated toxicity, as is currently proposed. Only when Ero1α-catalyzed H2O2 production is artificially maximized can PrxIV participate in its reduction. We conclude that the peroxidase activity of the described Ero1α-GPx8 complex prevents diffusion of Ero1α-derived H2O2 within and out of the rough ER. Along with the induction of GPX8 in ER-stressed cells, these findings question a ubiquitous role of Ero1α as a producer of cytoplasmic ROS under ER stress.

Biochemical evidence that regulation of Ero1ß activity in human cells does not involve the isoform-specific cysteine 262.

In the ER (endoplasmic reticulum) of human cells, disulfide bonds are predominantly generated by the two isoforms of Ero1 (ER oxidoreductin-1): Ero1α and Ero1ß. The activity of Ero1α is tightly regulated through the formation of intramolecular disulfide bonds to help ensure balanced ER redox conditions. Ero1ß is less tightly regulated, but the molecular details underlying control of activity are not as well characterized as for Ero1α. Ero1ß contains an additional cysteine residue (Cys262), which has been suggested to engage in an isoform-specific regulatory disulfide bond with Cys100 However, we show that the two regulatory disulfide bonds in Ero1α are likely conserved in Ero1ß (Cys90-Cys130 and Cys95-Cys100). Molecular modelling of the Ero1ß structure predicted that the side chain of Cys262 is completely buried. Indeed, we found this cysteine to be reduced and partially protected from alkylation in the ER of living cells. Furthermore, mutation of Cys100-but not of Cys262-rendered Ero1ß hyperactive in cells, as did mutation of Cys130 Ero1ß hyperactivity induced the UPR (unfolded protein response) and resulted in oxidative perturbation of the ER redox state. We propose that features other than a distinct pattern of regulatory disulfide bonds determine the loose redox regulation of Ero1ß relative to Ero1α.