Short macrocyclic peptides in sponge genomes.

Sponges (Porifera) contain many peptide-specialized metabolites with potent biological activities and significant roles in shaping marine ecology. It is well established that symbiotic bacteria produce bioactive "sponge" peptides, both on the ribosome (RiPPs) and nonribosomally. Here, we demonstrate that sponges themselves also produce many bioactive macrocyclic peptides, such as phakellistatins and related proline-rich macrocyclic peptides (PRMPs). Using the Stylissa carteri sponge transcriptome, methods were developed to find sequences encoding 46 distinct RiPP-type core peptides, of which ten encoded previously identified PRMP sequences. With this basis set, the genome and transcriptome of the sponge Axinella corrugata was interrogated to find 35 PRMP precursor peptides encoding 31 unique core peptide sequences. At least 11 of these produced cyclic peptides that were present in the sponge and could be characterized by mass spectrometry, including stylissamides A-D and seven previously undescribed compounds. Precursor peptides were encoded in the A. corrugata genome, confirming their animal origin. The peptides contained signal peptide sequences and highly repetitive recognition sequence-core peptide elements with up to 25 PRMP copies in a single precursor. In comparison to sponges without PRMPs, PRMP sponges are incredibly enriched in potentially secreted polypeptides, with >23,000 individual signal peptide encoding genes found in a single transcriptome. The similarities between PRMP biosynthetic genes and neuropeptides in terms of their biosynthetic logic suggest a fundamental biology linked to circular peptides, possibly indicating a widespread and underappreciated diversity of signaling peptide post-translational modifications across the animal kingdom.

Salvianolic Acid B Ameliorated Chemotherapeutic Injury of Cardiac Myocytes through the Nrf2/ARE Signaling Pathway.

BACKGROUND: Cardiotoxicity has been corroborated to be the toxic influence of cisplatin (CDDP). Oxidative stress and cardiomyocyte apoptosis play a vital part in cardiotoxicity induced by CDDP. Salvianolic acid Salvianolic acid B (SalB) is a monomeric component of Salvia miltiorrhiza, which has antioxidant and anti-inflammatory influences. In this research, we explored the mechanism of SalB in cardiotoxicity induced by CDDP. METHOD: 36 Wistar rats were separated into sham subgroup, CDDP (10 mg/kg) subgroup, CDDP (10 mg/kg) + SalB (1 µM) subgroup at random, CDDP (10 mg/kg) + SalB (5 µM) subgroup and CDDP (10 mg/kg) + SalB (10 µM) subgroup, Nicotinic Acid Riboside (NAR, 5 µM), with 6 rats in each subgroup. The cardiac function of rats in each subgroup was estimated by echocardiography, and hematoxylin-eosin (HE) staining and Masson staining corroborated the pathological changes of cardiac tissue. Biochemical kits were utilized for detecting the lactate dehydrogenase (LDH), creatine kinase (CK), interleukin-1ß (IL-1ß), IL-18, and caspase-1 concentrations in serum, superoxide dismutase (SOD), and malondialdehyde (MDA) in myocardial tissue, TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, and flow cytometry were utilized for estimating the apoptosis level in myocardial tissue, western blot was used for estimating caspase-3, Bcl2-Associated X (Bax) levels in myocardial tissue and proteins levels related to Nuclear factor E2 related factor 2 (Nrf2) signal pathway. RESULTS: CDDP-induced cardiac dysfunction, myocardial injury, boosted LDH and CK levels in serum (p < 0.05), memorably increased oxidative stress level in myocardial tissue (p < 0.05), boosted inflammatory response (p < 0.05), boosted apoptosis rate of cardiomyocytes (p < 0.05), and declined the Nrf2, NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1) protein levels (p < 0.05). Interestingly, SalB remedy could alleviate the changes caused by CDDP in the above parameters, significantly decrease the level of myocardial oxidative stress and apoptosis (p < 0.05). CONCLUSIONS: SalB ameliorates the injury of cardiomyocytes induced by chemotherapy through oxidative stress mediated by the Nrf2/antioxidant response element (ARE) signal pathway.

The polyketide to fatty acid transition in the evolution of animal lipid metabolism.

Animals synthesize simple lipids using a distinct fatty acid synthase (FAS) related to the type I polyketide synthase (PKS) enzymes that produce complex specialized metabolites. The evolutionary origin of the animal FAS and its relationship to the diversity of PKSs remain unclear despite the critical role of lipid synthesis in cellular metabolism. Recently, an animal FAS-like PKS (AFPK) was identified in sacoglossan molluscs. Here, we explore the phylogenetic distribution of AFPKs and other PKS and FAS enzymes across the tree of life. We found AFPKs widely distributed in arthropods and molluscs (>6300 newly described AFPK sequences). The AFPKs form a clade with the animal FAS, providing an evolutionary link bridging the type I PKSs and the animal FAS. We found molluscan AFPK diversification correlated with shell loss, suggesting AFPKs provide a chemical defense. Arthropods have few or no PKSs, but our results indicate AFPKs contributed to their ecological and evolutionary success by facilitating branched hydrocarbon and pheromone biosynthesis. Although animal metabolism is well studied, surprising new metabolic enzyme classes such as AFPKs await discovery.

Resistance mechanisms for Gram-negative bacteria-specific lipopeptides, turnercyclamycins, differ from that of colistin.

IMPORTANCE: Bacterial resistance to antibiotics is a crisis. Acinetobacter baumannii is among the CDC urgent threat pathogens in part for this reason. Lipopeptides known as turnercyclamycins are produced by symbiotic bacteria that normally live in marine mollusks, where they may be involved in shaping their symbiotic niche. Turnercyclamycins killed Gram-negative pathogens including drug-resistant Acinetobacter, but how do the mechanisms of resistance compare to other lipopeptide drugs? Here, we define resistance from a truncation of MlaA, a protein involved in regulating bacterial membrane phospholipids. Intriguingly, this resistance mechanism only affected one turnercyclamycin variant, which differed only in two atoms in the lipid tail of the compounds. We could not obtain significant resistance to the second turnercyclamycin variant, which was also effective in an infection model. This study reveals an unexpected subtlety in resistance to lipopeptide antibiotics, which may be useful in the design and development of antibiotics to combat drug resistance.

Animal FAS-like polyketide synthases produce diverse polypropionates.

Animal cytoplasmic fatty acid synthase (FAS) represents a unique family of enzymes that are classically thought to be most closely related to fungal polyketide synthase (PKS). Recently, a widespread family of animal lipid metabolic enzymes has been described that bridges the gap between these two ubiquitous and important enzyme classes: the animal FAS-like PKSs (AFPKs). Although very similar in sequence to FAS enzymes that produce saturated lipids widely found in animals, AFPKs instead produce structurally diverse compounds that resemble bioactive polyketides. Little is known about the factors that bridge lipid and polyketide synthesis in the animals. Here, we describe the function of EcPKS2 from Elysia chlorotica, which synthesizes a complex polypropionate natural product found in this mollusc. EcPKS2 starter unit promiscuity potentially explains the high diversity of polyketides found in and among molluscan species. Biochemical comparison of EcPKS2 with the previously described EcPKS1 reveals molecular principles governing substrate selectivity that should apply to related enzymes encoded within the genomes of photosynthetic gastropods. Hybridization experiments combining EcPKS1 and EcPKS2 demonstrate the interactions between the ketoreductase and ketosynthase domains in governing the product outcomes. Overall, these findings enable an understanding of the molecular principles of structural diversity underlying the many molluscan polyketides likely produced by the diverse AFPK enzyme family.

Translating Marine Symbioses toward Drug Development.

Chemists have studied marine animals for the better part of a century because they contain a diverse array of bioactive compounds. Tens of thousands of compounds have been reported, many with elaborate structural motifs and biological mechanisms of action found nowhere else. The challenge holding back the field has long been that of supply. Compounds are sometimes obtained by cultivating marine animals or by wild harvest, but this often presents logistical and environmental challenges. Some of the most medically important marine animal compounds are supplied by synthesis, often through multistep procedures that delay drug development. A relatively small number of such agents have been approved by the U.S. Food and Drug Administration, often after a heroic effort. In a recent mBio paper, Uppal and coworkers (https://doi.org/10.1128/mBio.01524-22) address key hurdles underlying the supply issue, discovering an uncultivated new bacterial genus from a marine sponge and reconstituting the biosynthetic pathway for expression.

Contrasting modes of mitochondrial genome evolution in sister taxa of wood-eating marine bivalves (Teredinidae and Xylophagaidae).

The bivalve families Teredinidae and Xylophagaidae include voracious consumers of wood in shallow and deep-water marine environments, respectively. The taxa are sister clades whose members consume wood as food with the aid of intracellular cellulolytic endosymbionts housed in their gills. This combination of adaptations is found in no other group of animals and was likely present in the common ancestor of both families. Despite these commonalities, the two families have followed dramatically different evolutionary paths with respect to anatomy, life history and distribution. Here we present 42 new mitochondrial genome sequences from Teredinidae and Xylophagaidae and show that distinct trajectories have also occurred in the evolution and organization of their mitochondrial genomes. Teredinidae display significantly greater rates of amino acid substitution but absolute conservation of protein-coding gene order, whereas Xylophagaidae display significantly less amino acid change but have undergone numerous and diverse changes in genome organization since their divergence from a common ancestor. As with many bivalves, these mitochondrial genomes encode two ribosomal RNAs, 12 protein coding genes, and 22 tRNAs; atp8 was not detected. We further show that their phylogeny, as inferred from amino acid sequences of 12 concatenated mitochondrial protein-coding genes, is largely congruent with those inferred from their nuclear genomes based on 18S and 28S ribosomal RNA sequences. Our results provide a robust phylogenetic framework to explore the tempo and mode of mitochondrial genome evolution and offer directions for future phylogenetic and taxonomic studies of wood-boring bivalves.

Sea Urchin Polyketide Synthase SpPks1 Produces the Naphthalene Precursor to Echinoderm Pigments.

Nearly every animal species on Earth contains a unique polyketide synthase (PKS) encoded in its genome, yet no animal-clade PKS has been biochemically characterized, and even the chemical products of these ubiquitous enzymes are known in only a few cases. The earliest animal genome-encoded PKS gene to be identified was SpPks1 from sea urchins. Previous genetic knockdown experiments implicated SpPks1 in synthesis of the sea urchin pigment echinochrome. Here, we express and purify SpPks1, performing biochemical experiments to demonstrate that the sea urchin protein is responsible for the synthesis of 2-acetyl-1,3,6,8-tetrahydroxynaphthalene (ATHN). Since ATHN is a plausible precursor of echinochromes, this result defines a biosynthetic pathway to the ubiquitous echinoderm pigments and rewrites the previous hypothesis for echinochrome biosynthesis. Truncation experiments showed that, unlike other type I iterative PKSs so far characterized, SpPks1 produces the naphthalene core using solely ketoacylsynthase (KS), acyltransferase, and acyl carrier protein domains, delineating a unique class of animal nonreducing aromatic PKSs (aPKSs). A series of amino acids in the KS domain define the family and are likely crucial in cyclization activity. Phylogenetic analyses indicate that SpPks1 and its homologs are widespread in echinoderms and their closest relatives, the acorn worms, reinforcing their fundamental importance to echinoderm biology. While the animal microbiome is known to produce aromatic polyketides, this work provides biochemical evidence that animals themselves also harbor ancient, convergent, dedicated pathways to carbocyclic aromatic polyketides. More fundamentally, biochemical analysis of SpPks1 begins to define the vast and unexplored biosynthetic space of the ubiquitous animal PKS family.

Ancient defensive terpene biosynthetic gene clusters in the soft corals.

Diterpenes are major defensive small molecules that enable soft corals to survive without a tough exterior skeleton, and, until now, their biosynthetic origin has remained intractable. Furthermore, biomedical application of these molecules has been hampered by lack of supply. Here, we identify and characterize coral-encoded terpene cyclase genes that produce the eunicellane precursor of eleutherobin and cembrene, representative precursors for the >2,500 terpenes found in octocorals. Related genes are found in all sequenced octocorals and form their own clade, indicating a potential ancient origin concomitant with the split between the hard and soft corals. Eleutherobin biosynthetic genes are colocalized in a single chromosomal region. This demonstrates that, like plants and microbes, animals also harbor defensive biosynthetic gene clusters, supporting a recombinational model to explain why specialized or defensive metabolites are adjacently encoded in the genome.

Hsa_Circ_0001947/MiR-661/DOK7 Axis Restrains Non-Small Cell Lung Cancer Development.

Hsa_circ_0001947 is associated with multiple cancers, but its function in non-small cell lung cancer (NSCLC) is ambiguous and needs further research. The targeting relationship among circ_0001947, miR-661, and downstream of tyrosine kinase 7 (DOK7) was predicted by database and further verified by dual-luciferase reporter assay, while their expressions in cancer tissues and cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). After transfection, cell biological behaviors and expressions of miRNAs, miR-661 and DOK7 were determined by cell function experiments and qRT-PCR, respectively. Circ_0001947 was low-expressed in NSCLC tissues and cells. Circ_0001947 knockdown intensified cell viability and proliferation, induced cell cycle arrest at S phase, suppressed apoptosis and evidently enhanced miR-510, miR-587, miR-661 and miR-942 levels, while circ_0001947 overexpression did the opposite. MiR-661 was a target gene of circ_0001947 that participated in the regulation of circ_0001947 on cell biological behaviors. Furthermore, DOK7, the target gene of miR-661, partly participated in the regulation of miR-661 on cell viability. Hsa_circ_0001947 acts as a sponge of miR-661 to repress NSCLC development by elevating the expression of DOK7.

The Tunicate Metabolite 2-(3,5-Diiodo-4-methoxyphenyl)ethan-1-amine Targets Ion Channels of Vertebrate Sensory Neurons.

Marine tunicates produce defensive amino-acid-derived metabolites, including 2-(3,5-diiodo-4-methoxyphenyl)ethan-1-amine (DIMTA), but their mechanisms of action are rarely known. Using an assay-guided approach, we found that out of the many different sensory cells in the mouse dorsal root ganglion (DRG), DIMTA selectively affected low-threshold cold thermosensors. Whole-cell electrophysiology experiments using DRG cells, channels expressed in Xenopus oocytes, and human cell lines revealed that DIMTA blocks several potassium channels, reducing the magnitude of the afterhyperpolarization and increasing the baseline intracellular calcium concentration [Ca2+]i of low-threshold cold thermosensors. When injected into mice, DIMTA increased the threshold of cold sensation by >3 °C. DIMTA may thus serve as a lead in the further design of compounds that inhibit problems in the cold-sensory system, such as cold allodynia and other neuropathic pain conditions.

An Obligate Peptidyl Brominase Underlies the Discovery of Highly Distributed Biosynthetic Gene Clusters in Marine Sponge Microbiomes.

Marine sponges are prolific sources of bioactive natural products, several of which are produced by bacteria symbiotically associated with the sponge host. Bacteria-derived natural products, and the specialized bacterial symbionts that synthesize them, are not shared among phylogenetically distant sponge hosts. This is in contrast to nonsymbiotic culturable bacteria in which the conservation of natural products and natural product biosynthetic gene clusters (BGCs) is well established. Here, we demonstrate the widespread conservation of a BGC encoding a cryptic ribosomally synthesized and post-translationally modified peptide (RiPP) in microbiomes of phylogenetically and geographically dispersed sponges from the Pacific and Atlantic oceans. Detection of this BGC was enabled by mining for halogenating enzymes in sponge metagenomes, which, in turn, allowed for the description of a broad-spectrum regiospecific peptidyl tryptophan-6-brominase which possessed no chlorination activity. In addition, we demonstrate the cyclodehydrative installation of azoline heterocycles in proteusin RiPPs. This is the first demonstration of halogenation and cyclodehydration for proteusin RiPPs and the enzymes catalyzing these transformations were found to competently interact with other previously described proteusin substrate peptides. Within a sponge microbiome, many different generalized bacterial taxa harbored this BGC with often more than 50 copies of the BGC detected in individual sponge metagenomes. Moreover, the BGC was found in all sponges queried that possess high diversity microbiomes but it was not detected in other marine invertebrate microbiomes. These data shed light on conservation of cryptic natural product biosynthetic potential in marine sponges that was not detected by traditional natural product-to-BGC (meta)genome mining.

Nicotinic Acetylcholine Receptor Partial Antagonist Polyamides from Tunicates and Their Predatory Sea Slugs.

In our efforts to discover new drugs to treat pain, we identified molleamines A-E (1-5) as major neuroactive components of the sea slug, Pleurobranchus forskalii, and their prey, Didemnum molle, tunicates. The chemical structures of molleamines were elucidated by spectroscopy and confirmed by the total synthesis of molleamines A (1) and C (3). Synthetic 3 completely blocked acetylcholine-induced calcium flux in peptidergic nociceptors (PNs) in the somatosensory nervous system. Compound 3 affected neither the α7 nAChR nor the muscarinic acetylcholine receptors in calcium flux assays. In addition to nociceptors, 3 partially blocked the acetylcholine-induced calcium flux in the sympathetic nervous system, including neurons from the superior cervical ganglion. Electrophysiology revealed a block of α3ß4 (mouse) and α6/α3ß4 (rat) nicotinic acetylcholine receptors (nAChRs), with IC50 values of 1.4 and 3.1 µM, respectively. Molleamine C (3) is a partial antagonist, reaching a maximum block of 76-82% of the acetylcholine signal and showing no partial agonist response. Molleamine C (3) may thus provide a lead compound for the development of neuroactive compounds with unique biological properties.

Shipworm symbiosis ecology-guided discovery of an antibiotic that kills colistin-resistant Acinetobacter.

Teredinibacter turnerae is an intracellular bacterial symbiont in the gills of wood-eating shipworms, where it is proposed to use antibiotics to defend itself and its animal host. Several biosynthetic gene clusters are conserved in T. turnerae and their host shipworms around the world, implying that they encode defensive compounds. Here, we describe turnercyclamycins, lipopeptide antibiotics encoded in the genomes of all sequenced T. turnerae strains. Turnercyclamycins are bactericidal against challenging Gram-negative pathogens, including colistin-resistant Acinetobacter baumannii. Phenotypic screening identified the outer membrane as the likely target. Turnercyclamycins and colistin operate by similar cellular, although not necessarily molecular, mechanisms, but turnercyclamycins kill colistin-resistant A. baumannii, potentially filling an urgent clinical need. Thus, by exploring environments that select for the properties we require, we harvested the fruits of evolution to discover compounds with potential to target unmet health needs. Investigating the symbionts of shipworms is a powerful example of this principle.

Non-Peptidic Small Molecule Components from Cone Snail Venoms.

Venomous molluscs (Superfamily Conoidea) comprise a substantial fraction of tropical marine biodiversity (>15,000 species). Prior characterization of cone snail venoms established that bioactive venom components used to capture prey, defend against predators and for competitive interactions were relatively small, structured peptides (10-35 amino acids), most with multiple disulfide crosslinks. These venom components ("conotoxins, conopeptides") have been widely studied in many laboratories, leading to pharmaceutical agents and probes. In this review, we describe how it has recently become clear that to varying degrees, cone snail venoms also contain bioactive non-peptidic small molecule components. Since the initial discovery of genuanine as the first bioactive venom small molecule with an unprecedented structure, a broad set of cone snail venoms have been examined for non-peptidic bioactive components. In particular, a basal clade of cone snails (Stephanoconus) that prey on polychaetes produce genuanine and many other small molecules in their venoms, suggesting that this lineage may be a rich source of non-peptidic cone snail venom natural products. In contrast to standing dogma in the field that peptide and proteins are predominantly used for prey capture in cone snails, these small molecules also contribute to prey capture and push the molecular diversity of cone snails beyond peptides. The compounds so far characterized are active on neurons and thus may potentially serve as leads for neuronal diseases. Thus, in analogy to the incredible pharmacopeia resulting from studying venom peptides, these small molecules may provide a new resource of pharmacological agents.

Neuroactive Type-A γ-Aminobutyric Acid Receptor Allosteric Modulator Steroids from the Hypobranchial Gland of Marine Mollusk, Conus geographus.

In a program to identify pain treatments with low addiction potential, we isolated five steroids, conosteroids A-E (1-5), from the hypobranchial gland of the mollusk Conus geographus. Compounds 1-5 were active in a mouse dorsal root ganglion (DRG) assay that suggested that they might be analgesic. A synthetic analogue 6 was used for a detailed pharmacological study. Compound 6 significantly increased the pain threshold in mice in the hot-plate test at 2 and 50 mg/kg. Compound 6 at 500 nM antagonizes type-A γ-aminobutyric acid receptors (GABAARs). In a patch-clamp experiment, out of the six subunit combinations tested, 6 exhibited subtype selectivity, most strongly antagonizing α1ß1γ2 and α4ß3γ2 receptors (IC50 1.5 and 1.0 µM, respectively). Although the structures of 1-6 differ from those of known neuroactive steroids, they are cell-type-selective modulators of GABAARs, expanding the known chemical space of neuroactive steroids.

Salvianolic acid B attenuated cisplatin-induced cardiac injury and oxidative stress via modulating Nrf2 signal pathway.

Cardiovascular complications have been well documented as the downside to conventional cancer chemotherapy. As a notable side effect of cisplatin (CDDP), cardiotoxicity represents a major obstacle to the successful treatment of cancer. It has been reported that Salvianolic acid B (SalB) possesses cardioprotective quality. However, the effect of SalB on cardiac damage caused by conventional cancer chemotherapy remains unclear. In this study, we clarified the protective effect of SalB on cisplatin-induced heart injury. Furthermore, in H9c2 cells, SalB dramatically reduced cisplatin-induced apoptosis and oxidative stress by modulating the nuclear factor erythroid-2-related factor 2 (Nrf2) signaling pathway. In conclusion, SalB had great potential in mitigating cisplatin-induced cardiac injury. Furthermore, more attention should be placed on natural active compounds containing SalB with antioxidant effects for the treatment of cardiomyopathy.

Small-molecule mimicry hunting strategy in the imperial cone snail, Conus imperialis.

Venomous animals hunt using bioactive peptides, but relatively little is known about venom small molecules and the resulting complex hunting behaviors. Here, we explored the specialized metabolites from the venom of the worm-hunting cone snail, Conus imperialis Using the model polychaete worm Platynereis dumerilii, we demonstrate that C. imperialis venom contains small molecules that mimic natural polychaete mating pheromones, evoking the mating phenotype in worms. The specialized metabolites from different cone snails are species-specific and structurally diverse, suggesting that the cones may adopt many different prey-hunting strategies enabled by small molecules. Predators sometimes attract prey using the prey's own pheromones, in a strategy known as aggressive mimicry. Instead, C. imperialis uses metabolically stable mimics of those pheromones, indicating that, in biological mimicry, even the molecules themselves may be disguised, providing a twist on fake news in chemical ecology.