Jason S. Biggs

Evolution of Conus peptide toxins: Analysis of Conus californicus Reeve, 1844

Conus species are characterized by their hyperdiverse toxins, encoded by a few gene superfamilies. Our phylogenies of the genus, based on mitochondrial genes, confirm previous results that C. californicus is highly divergent from all other species. Genetic and biochemical analysis of their venom peptides comprise the fifteen most abundant conopeptides and over 50 mature cDNA transcripts from the venom duct. Although C. californicus venom retains many of the general properties of other Conus species, they share only half of the toxin gene superfamilies found in other Conus species. Thus, in these two lineages, approximately half of the rapidly diversifying gene superfamilies originated after an early Tertiary split. Such results demonstrate that, unlike endogenously acting gene families, these genes are likely to be significantly more restricted in their phylogenetic distribution. In concordance with the evolutionary distance of C. californicus from other species, there are aspects of prey-capture behavior and prey preferences of this species that diverges significantly from all other Conus.

Α-Conopeptides specifically expressed in the salivary gland of Conus pulicarius

To date, studies conducted on cone snail venoms have attributed the origins of this complex mixture of neuroactive peptides entirely to gene expression by the secretory cells lining the lumen of the venom duct. However, specialized tissues such as the salivary glands also secrete their contents into the anterior gut and could potentially contribute some venom components injected into target animals; evidence supporting this possibility is reported here. Sequence analysis of a cDNA library created from a salivary gland of Conus pulicarius revealed the expression of two transcripts whose predicted gene products, after post-translational processing, strikingly resemble mature conopeptides belonging to the alpha-conotoxin family. These two transcripts, like alpha-conotoxin transcripts, putatively encode mature peptides containing the conserved A-superfamily cysteine pattern (CC-C-C) but the highly conserved A-superfamily signal sequences were not present. Analysis of A-superfamily members expressed in the venom duct of the same C. pulicarius specimens revealed three putative alpha-conotoxin sequences; the salivary gland transcripts were not found in the venom duct cDNA library, suggesting that these alpha-conotoxins are salivary gland specific. Therefore, expression of conotoxin-like gene products by the salivary gland could potentially add to the complexity of Conus venoms.

Veraguamides A–G, Cyclic Hexadepsipeptides from a Dolastatin 16-Producing Cyanobacterium Symploca cf. hydnoides from Guam

Cytotoxicity-directed purification of a Symploca cf. hydnoides sample from Cetti Bay, Guam, afforded seven new cyclic depsipeptides, veraguamides A-G (1-7), together with the known compound dolastatin 16. The planar structures of 1-7 were elucidated using NMR and MS experiments, while enantioselective HPLC and Moshers analysis of acid and base hydrolysates, respectively, were utilized to assign the absolute configurations of the stereocenters. Veraguamides A-G (1-7) are characterized by the presence of an invariant proline residue, multiple N-methylated amino acids, an α-hydroxy acid, and a C8-polyketide-derived β-hydroxy acid moiety with a characteristic terminus as either an alkynyl bromide, alkyne, or vinyl group. These compounds and a semisynthetic analogue (8) showed moderate to weak cytotoxic activity against HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cell lines. Preliminary structure-activity relationship analysis identified several sensitive positions in the veraguamide scaffold that affect the cytotoxic activity of this compound class. Dolastatin 16 showed only weak cytotoxic activity on both cell lines tested. The complete stereostructure of dolastatin 16 was proposed for the first time through degradation followed by a combination of advanced Marfeys analysis and modified Moshers analysis using phenylglycine methyl ester as a chiral anisotropic reagent.

Potent elastase inhibitors from cyanobacteria: structural basis and mechanisms mediating cytoprotective and anti-inflammatory effects in bronchial epithelial cells.

We discovered new structural diversity to a prevalent, yet medicinally underappreciated, cyanobacterial protease inhibitor scaffold and undertook comprehensive protease profiling to reveal potent and selective elastase inhibition. Structure-activity relationship (SAR) studies and X-ray cocrystal structure analysis allowed a detailed assessment of critical and tunable structural elements. To realize the therapeutic potential of these cyclodepsipeptides, we probed the cellular effects of a novel and representative family member, symplostatin 5 (1), which attenuated the downstream cellular effects of elastase in an epithelial lung airway model system, alleviating clinical hallmarks of chronic pulmonary diseases such as cell death, cell detachment, and inflammation. This compound attenuated the effects of elastase on receptor activation, proteolytic processing of the adhesion protein ICAM-1, NF-κB activation, and transcriptomic changes, including the expression of pro-inflammatory cytokines IL1A, IL1B, and IL8. Compound 1 exhibited activity comparable to the clinically approved elastase inhibitor sivelestat in short-term assays and demonstrated superior sustained activity in longer-term assays.

Microhabitats within venomous cone snails contain diverse actinobacteria.

ABSTRACT Actinomycetes can be symbionts in diverse organisms, including both plants and animals. Some actinomycetes benefit their host by producing small molecule secondary metabolites; the resulting symbioses are often developmentally complex. Actinomycetes associated with three cone snails were studied. Cone snails are venomous tropical marine gastropods which have been extensively examined because of their production of peptide-based neurological toxins, but no microbiological studies have been reported on these organisms. A microhabitat approach was used in which dissected tissue from each snail was treated as an individual sample in order to explore bacteria in the tissues separately. Our results revealed a diverse, novel, and highly culturable cone snail-associated actinomycete community, with some isolates showing promising bioactivity in a neurological assay. This suggests that cone snails may represent an underexplored reservoir of novel actinomycetes of potential interest for drug discovery.

Insights into the origins of fish hunting in venomous cone snails from studies of Conus tessulatus

Significance Only rarely is it possible to reconstruct molecular events that trigger the radiation of new lineages. Here we report key evidence that allows reconstruction of the transition from worm hunting to fish hunting among the species-rich family (Conidae) of marine cone snails (>700 species), which resulted in the emergence of multiple biodiverse piscivorous clades. A priori, the evolution of fish-hunting specialists would seem extremely improbable in a lineage of slowly moving snails that cannot swim, unlike their fish prey. The combination of results from molecular neuroscience, phylogenetic analysis, and chemical biology demonstrates that an ancestral cone snail venom peptide similar to δ-conotoxin TsVIA, a defensive venom component, preadapted a worm-hunting cone snail lineage, enabling the shift to a piscivorous lifestyle. Prey shifts in carnivorous predators are events that can initiate the accelerated generation of new biodiversity. However, it is seldom possible to reconstruct how the change in prey preference occurred. Here we describe an evolutionary “smoking gun” that illuminates the transition from worm hunting to fish hunting among marine cone snails, resulting in the adaptive radiation of fish-hunting lineages comprising ∼100 piscivorous Conus species. This smoking gun is δ-conotoxin TsVIA, a peptide from the venom of Conus tessulatus that delays inactivation of vertebrate voltage-gated sodium channels. C. tessulatus is a species in a worm-hunting clade, which is phylogenetically closely related to the fish-hunting cone snail specialists. The discovery of a δ-conotoxin that potently acts on vertebrate sodium channels in the venom of a worm-hunting cone snail suggests that a closely related ancestral toxin enabled the transition from worm hunting to fish hunting, as δ-conotoxins are highly conserved among fish hunters and critical to their mechanism of prey capture; this peptide, δ-conotoxin TsVIA, has striking sequence similarity to these δ-conotoxins from piscivorous cone snail venoms. Calcium-imaging studies on dissociated dorsal root ganglion (DRG) neurons revealed the peptide’s putative molecular target (voltage-gated sodium channels) and mechanism of action (inhibition of channel inactivation). The results were confirmed by electrophysiology. This work demonstrates how elucidating the specific interactions between toxins and receptors from phylogenetically well-defined lineages can uncover molecular mechanisms that underlie significant evolutionary transitions.

Complexity of Naturally Produced Polybrominated Diphenyl Ethers Revealed via Mass Spectrometry

Polybrominated diphenyl ethers (PBDEs) are persistent and bioaccumulative anthropogenic and natural chemicals that are broadly distributed in the marine environment. PBDEs are potentially toxic due to inhibition of various mammalian signaling pathways and enzymatic reactions. PBDE isoforms vary in toxicity in accordance with structural differences, primarily in the number and pattern of hydroxyl moieties afforded upon a conserved core structure. Over four decades of isolation and discovery-based efforts have established an impressive repertoire of natural PBDEs. Based on our recent reports describing the bacterial biosyntheses of PBDEs, we predicted the presence of additional classes of PBDEs to those previously identified from marine sources. Using mass spectrometry and NMR spectroscopy, we now establish the existence of new structural classes of PBDEs in marine sponges. Our findings expand the chemical space explored by naturally produced PBDEs, which may inform future environmental toxicology studies. Furthermore, we provide evidence for iodinated PBDEs and direct attention toward the contribution of promiscuous halogenating enzymes in further expanding the diversity of these polyhalogenated marine natural products.

Metagenomic discovery of polybrominated diphenyl ether biosynthesis by marine sponges

Naturally produced polybrominated diphenyl ethers (PBDEs) pervade the marine environment and structurally resemble toxic man-made brominated flame retardants. PBDEs bioaccumulate in marine animals and are likely transferred to the human food chain. However, the biogenic basis for PBDE production in one of their most prolific sources, marine sponges of the order Dysideidae, remains unidentified. Here, we report the discovery of PBDE biosynthetic gene clusters within sponge microbiome-associated cyanobacterial endosymbionts by employing an unbiased metagenome mining approach. By expression of PBDE biosynthetic genes in heterologous cyanobacterial hosts, we correlate the structural diversity of naturally produced PBDEs to modifications within PBDE biosynthetic gene clusters in multiple sponge holobionts. Our results establish the genetic and molecular foundation for the production of PBDEs in one of the most abundant natural sources of these molecules, further setting the stage for a metagenomic-based inventory of other PBDE sources in the marine environment.

Transcription factor binding to a putative double E-Box motif represses CYP3A4 expression in human lung cells

Two vital enzymes of the CYP3A subfamily, CYP3A4 and CYP3A5, are differentially expressed in the human lung. However, the molecular mechanisms that regulate tissue-selective expression of the genes are poorly understood. The ability of the 5′ upstream promoter region of these two genes to drive luciferase reporter activities in human lung A549 cells was dramatically different. The CYP3A5 promoter region activated luciferase gene expression by 10-fold over the promoterless construct, whereas the CYP3A4 promoter did not drive expression. Sequence comparisons of the promoters identified a 57-base pair insertion in the CYP3A4 promoter region (–71 to –127) that was absent in the CYP3A5 promoter. Deletion of the 57-bp motif from CYP3A4 or insertion into the CYP3A5 promoter, showed that this motif represses CYP3A4 expression in lung. EMSA analysis using nuclear extracts from either A549 cells or human lung tissues showed two specific protein/DNA complexes formed with the 32P-labeled CYP3A4 57-bp oligonucleotide. EMSA analyses identified two E-box motifs as the minimal specific cis-elements. Supershift assays with antibodies directed against known double- or single-E-box binding factors (TAL1, δEF1, E2A, HEB, etc.) failed to identify this factor as a previously characterized trans-acting double E-box binding protein. These results demonstrated that the 5′-upstream region of CYP3A4 contains an active putative double E-box repressor motif, not present in the 5′-upstream region of the CYP3A5 gene, that attenuates CYP3A4 expression in the human lung. We believe that this is the first documented case in which a cytochrome P450 gene is actively repressed in a tissue-specific manner.

Stenotrophomonas-like bacteria are widespread symbionts in cone snail venom ducts

ABSTRACT Cone snails are biomedically important sources of peptide drugs, but it is not known whether snail-associated bacteria affect venom chemistry. To begin to answer this question, we performed 16S rRNA gene amplicon sequencing of eight cone snail species, comparing their microbiomes with each other and with those from a variety of other marine invertebrates. We show that the cone snail microbiome is distinct from those in other marine invertebrates and conserved in specimens from around the world, including the Philippines, Guam, California, and Florida. We found that all venom ducts examined contain diverse 16S rRNA gene sequences bearing closest similarity to Stenotrophomonas bacteria. These sequences represent specific symbionts that live in the lumen of the venom duct, where bioactive venom peptides are synthesized. IMPORTANCE In animals, symbiotic bacteria contribute critically to metabolism. Cone snails are renowned for the production of venoms that are used as medicines and as probes for biological study. In principle, symbiotic bacterial metabolism could either degrade or synthesize active venom components, and previous publications show that bacteria do indeed contribute small molecules to some venoms. Therefore, understanding symbiosis in cone snails will contribute to further drug discovery efforts. Here, we describe an unexpected, specific symbiosis between bacteria and cone snails from around the world.