Lasso Peptides Research Articles

Discovery of phosphorylated lantibiotics with proimmune activity that regulate the oral microbiome

Lantibiotics are ribosomally synthesized and posttranslationally modified peptides (RiPPs) that are produced by bacteria. Interest in this group of natural products is increasing rapidly as alternatives to conventional antibiotics. Some human microbiome-derived commensals produce lantibiotics to impair pathogens' colonization and promote healthy microbiomes. Streptococcus salivarius is one of the first commensal microbes to colonize the human oral cavity and gastrointestinal tract, and its biosynthesis of RiPPs, called salivaricins, has been shown to inhibit the growth of oral pathogens. Herein, we report on a phosphorylated class of three related RiPPs, collectively referred to as salivaricin 10, that exhibit proimmune activity and targeted antimicrobial properties against known oral pathogens and multispecies biofilms. Strikingly, the immunomodulatory activities observed include upregulation of neutrophil-mediated phagocytosis, promotion of antiinflammatory M2 macrophage polarization, and stimulation of neutrophil chemotaxis-these activities have been attributed to the phosphorylation site identified on the N-terminal region of the peptides. Salivaricin 10 peptides were determined to be produced by S. salivarius strains found in healthy human subjects, and their dual bactericidal/antibiofilm and immunoregulatory activity may provide new means to effectively target infectious pathogens while maintaining important oral microbiota.

Genomic and phenotypic characterization of a red-pigmented strain of Massilia frigida isolated from an Antarctic microbial mat.

The McMurdo Dry Valleys of Antarctica experience a range of selective pressures, including extreme seasonal variation in temperature, water and nutrient availability, and UV radiation. Microbial mats in this ecosystem harbor dense concentrations of biomass in an otherwise desolate environment. Microbial inhabitants must mitigate these selective pressures via specialized enzymes, changes to the cellular envelope, and the production of secondary metabolites, such as pigments and osmoprotectants. Here, we describe the isolation and characterization of a Gram-negative, rod-shaped, motile, red-pigmented bacterium, strain DJPM01, from a microbial mat within the Don Juan Pond Basin of Wright Valley. Analysis of strain DJMP01's genome indicates it can be classified as a member of the Massilia frigida species. The genome contains several genes associated with cold and salt tolerance, including multiple RNA helicases, protein chaperones, and cation/proton antiporters. In addition, we identified 17 putative secondary metabolite gene clusters, including a number of nonribosomal peptides and ribosomally synthesized and post-translationally modified peptides (RiPPs), among others, and the biosynthesis pathway for the antimicrobial pigment prodigiosin. When cultivated on complex agar, multiple prodiginines, including the antibiotic prodigiosin, 2-methyl-3-propyl-prodiginine, 2-methyl-3-butyl-prodiginine, 2-methyl-3-heptyl-prodiginine, and cycloprodigiosin, were detected by LC-MS. Genome analyses of sequenced members of the Massilia genus indicates prodigiosin production is unique to Antarctic strains. UV-A radiation, an ecological stressor in the Antarctic, was found to significantly decrease the abundance of prodiginines produced by strain DJPM01. Genomic and phenotypic evidence indicates strain DJPM01 can respond to the ecological conditions of the DJP microbial mat, with prodiginines produced under a range of conditions, including extreme UV radiation.

SolS-catalyzed sulfoxidation of labionin to solabionin drives antibacterial activity of solabiomycins

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) with polar-functionalized fatty acyl groups are newly found lipopeptide-class natural products. We recently employed a combined approach of genome mining and stable isotope labeling and discovered solabiomycins as one of the polar-functionalized fatty-acylated RiPPs (PFARs) from Streptomyces lydicus NBRC13058. The solabiomycins contained a characteristic sulfoxide group in the labionin moiety referred to as the ‘solabionin’ structure for the RiPP moiety. A previous gene knockout experiment indicated that solS, which encodes a putative flavin adenine dinucleotide (FAD)-nicotinamide adenine dinucleotide (phosphate) (NAD(P))-binding protein, is involved in the sulfoxidation of an alkyl sulfide in the solabionin. In this study, we isolated deoxysolabiomycins A and B from ΔsolS mutant and fully determined the chemical structures using a series of NMR experiments. We also tested the bioactivity of deoxysolabiomycins against Gram-positive bacteria, including Mycolicibacterium smegmatis, and notably found that the sulfoxide is critical for the antibacterial activity. To characterize the catalytic activity of SolS, the recombinant protein was incubated with a putative substrate, deoxysolabiomycins, and the cofactors FAD and NADPH. In vitro reactions demonstrated that SolS catalyzes the sulfoxidation, converting deoxysolabiomycins to solabiomycins.

Development of a Streptomyces-based system for facile thioholgamide library generation and analysis

Derivatizing natural products (NPs) is essential in structure-activity relationship (SAR) studies, compound optimization, and drug development. Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent one of the major classes of natural products. Thioholgamide represents thioamitide – a recently emerged family of RiPPs with unique structures and great potential in anticancer drug development. Although the method for generating the RiPP library by codon substitutions in the precursor peptide gene is straightforward, the techniques to perform RiPP derivatization in Actinobacteria remain limited and time-consuming. Here, we report a facile system for producing a library of randomized thioholgamide derivatives utilizing an optimized Streptomyces host. This technique enabled us to access all possible amino acid substitutions of the thioholgamide molecule, one position at a time. Out of 152 potential derivatives, 85 were successfully detected, revealing the impact of amino acid substitutions on thioholgamide post-translational modifications (PTMs). Moreover, new PTMs were observed among thioholgamide derivatives: thiazoline heterocycles, which have not yet been reported for thioamitides, and S-methylmethionine, which is very rare in nature. The obtained library was subsequently used for thioholgamide SAR studies and stability assays.

Peptide Selenocysteine Substitutions Reveal Direct Substrate-Enzyme Interactions at Auxiliary Clusters in Radical S-Adenosyl-l-methionine Maturases.

Radical S-adenosyl-l-methionine (SAM) enzymes leverage the properties of one or more iron- and sulfide-containing metallocenters to catalyze complex and radical-mediated transformations. By far the most populous superfamily of radical SAM enzymes are those that, in addition to a 4Fe-4S cluster that binds and activates the SAM cofactor, also bind one or more additional auxiliary clusters (ACs) of largely unknown catalytic significance. In this report we examine the role of ACs in two RS enzymes, PapB and Tte1186, that catalyze formation of thioether cross-links in ribosomally synthesized and post-translationally modified peptides (RiPPs). Both enzymes catalyze a sulfur-to-carbon cross-link in a reaction that entails H atom transfer from an unactivated C-H to initiate catalysis, followed by formation of a C-S bond to yield the thioether. We show that both enzymes tolerate substitution of SeCys instead of Cys at the cross-linking site, allowing the systems to be subjected to Se K-edge X-ray spectroscopy. The EXAFS data show a direct interaction with the Fe of one of the ACs in the Michaelis complex, which is replaced with a Se-C interaction under reducing conditions that lead to the product complex. Site-directed deletion of the clusters in Tte1186 provide evidence for the identity of the AC. The implications of these observations in the context of the mechanism of these thioether cross-linking enzymes are discussed.

Macrocyclization and Backbone Rearrangement During RiPP Biosynthesis by a SAM-Dependent Domain-of-Unknown-Function 692.

The domain of unknown function 692 (DUF692) is an emerging family of post-translational modification enzymes involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. Members of this family are multinuclear iron-containing enzymes, and only two members have been functionally characterized to date: MbnB and TglH. Here, we used bioinformatics to select another member of the DUF692 family, ChrH, that is encoded in the genomes of the Chryseobacterium genus along with a partner protein ChrI. We structurally characterized the ChrH reaction product and show that the enzyme complex catalyzes an unprecedented chemical transformation that results in the formation of a macrocycle, an imidazolidinedione heterocycle, two thioaminals, and a thiomethyl group. Based on isotopic labeling studies, we propose a mechanism for the four-electron oxidation and methylation of the substrate peptide. This work identifies the first SAM-dependent reaction catalyzed by a DUF692 enzyme complex, further expanding the repertoire of remarkable reactions catalyzed by these enzymes. Based on the three currently characterized DUF692 family members, we suggest the family be called multinuclear non-heme iron dependent oxidative enzymes (MNIOs).

Establishing Heterologous Production of Microcins J25 and Y in Bacillus subtilis

Microcin J25 (MccJ25) and microcin Y (MccY) are lasso peptides and considered potential alternatives to antibiotics and harmful preservatives. The combination of these two microcins can provide a wide antimicrobial spectrum against food-borne Salmonella. Currently, MccJ25 and MccY are produced using Escherichia coli expression systems; however, the entire production process is accompanied by negative effects from endotoxins. In this study, we identified Bacillus subtilis as a suitable host for MccJ25 and MccY production. High-level production of microcins was achieved by promoter optimization, host strain selection, and recombinant expression. The engineered strains produced maximum yields of 2.827 μM MccJ25 and 1.481 μM MccY. This is the first study to demonstrate the expression of MccJ25 and MccY in B. subtilis, and it offers a few engineered strains that are without antibiotic resistance markers, inducer-free, sporulation-deficient, and free of the negative effects of endotoxins for antibacterial therapy and food preservation.

Genome mining unveils a class of ribosomal peptides with two amino termini

The era of inexpensive genome sequencing and improved bioinformatics tools has reenergized the study of natural products, including the ribosomally synthesized and post-translationally modified peptides (RiPPs). In recent years, RiPP discovery has challenged preconceptions about the scope of post-translational modification chemistry, but genome mining of new RiPP classes remains an unsolved challenge. Here, we report a RiPP class defined by an unusual (S)-N2,N2-dimethyl-1,2-propanediamine (Dmp)-modified C-terminus, which we term the daptides. Nearly 500 daptide biosynthetic gene clusters (BGCs) were identified by analyzing the RiPP Recognition Element (RRE), a common substrate-binding domain found in half of prokaryotic RiPP classes. A representative daptide BGC from Microbacterium paraoxydans DSM 15019 was selected for experimental characterization. Derived from a C-terminal threonine residue, the class-defining Dmp is installed over three steps by an oxidative decarboxylase, aminotransferase, and methyltransferase. Daptides uniquely harbor two positively charged termini, and thus we suspect this modification could aid in membrane targeting, as corroborated by hemolysis assays. Our studies further show that the oxidative decarboxylation step requires a functionally unannotated accessory protein. Fused to the C-terminus of the accessory protein is an RRE domain, which delivers the unmodified substrate peptide to the oxidative decarboxylase. This discovery of a class-defining post-translational modification in RiPPs may serve as a prototype for unveiling additional RiPP classes through genome mining.

Leader- and Terminal Residue Requirements for Circularin A Biosynthesis Probed by Systematic Mutational Analyses.

Circularin A is a circular bacteriocin belonging to a subgroup of the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily. The post-translational biosynthesis of circular bacteriocins primarily consists of leader cleavage, core peptide circularization, and bacteriocin secretion. However, none of these processes have been fully elucidated due to the complex biosynthesis of such bacteriocins and the lack of homology to the functions of other known biosynthetic enzymes. In this study, we investigated the leader- and terminal residue requirements for the biosynthesis of circularin A by systematic mutational analyses, including the mutational effects of variable leader lengths, as well as site-directed substitutions of residues at positions near the leader cleavage site and the circularization site. Results show that the leader with only one Met residue, the shortest leader possible, is sufficient to produce mature circularin A; helix-forming short-sidechain hydrophobic residues are required at positions Val1 and Ala2 of the N-terminus to form active peptide derivatives, indicating the possible steric hindrance effect at these two positions; and an aromatic residue is required at the C-terminal Tyr69 position to produce a mature circular derivative. However, the requirements for residues at position Ala68 are much more relaxed relative to the positions of Val1 and Ala2, since even substitution with the largest possible residue, i.e., tryptophan, still allows the generation of an active Ala68Trp derivative. Our findings provide new perspectives for the biosynthesis of this short-leader circular bacteriocin, which enables the application of circular bacteriocin biosynthesis in rational modified peptide engineering.

High-Throughput Screen Reveals the Structure-Activity Relationship of the Antimicrobial Lasso Peptide Ubonodin.

The Burkholderia cepacia complex (Bcc) is a group of bacteria including opportunistic human pathogens. Immunocompromised individuals and cystic fibrosis patients are especially vulnerable to serious infections by these bacteria, motivating the search for compounds with antimicrobial activity against the Bcc. Ubonodin is a lasso peptide with promising activity against Bcc species, working by inhibiting RNA polymerase in susceptible bacteria. We constructed a library of over 90 000 ubonodin variants with 2 amino acid substitutions and used a high-throughput screen and next-generation sequencing to examine the fitness of the entire library, generating the most comprehensive data set on lasso peptide activity so far. This screen revealed information regarding the structure-activity relationship of ubonodin over a large sequence space. Remarkably, the screen identified one variant with not only improved activity compared to wild-type ubonodin but also a submicromolar minimum inhibitory concentration (MIC) against a clinical isolate of the Bcc member Burkholderia cenocepacia. Ubonodin and several of the variants identified in this study had lower MICs against certain Bcc strains than those of many clinically approved antibiotics. Finally, the large library size enabled us to develop DeepLasso, a deep learning model that can predict the RNAP inhibitory activity of an ubonodin variant.

Structural and Biochemical Insights into Post-Translational Arginine-to-Ornithine Peptide Modifications by an Atypical Arginase.

Landornamide A is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product with antiviral activity. Its biosynthetic gene cluster encodes─among other maturases─the peptide arginase OspR, which converts arginine to ornithine units in an unusual post-translational modification. Peptide arginases are a recently discovered RiPP maturase family with few characterized representatives. They show little sequence similarity to conventional arginases, a well-characterized enzyme family catalyzing the hydrolysis of free arginine to ornithine and urea. Peptide arginases are highly promiscuous and accept a variety of substrate sequences. The molecular basis for binding the large peptide substrate and for the high promiscuity of peptide arginases remains unclear. Here, we report the first crystal structure of a peptide arginase at a resolution of 2.6 Å. The three-dimensional structure reveals common features and differences between conventional arginases and the peptide arginase: the binuclear metal cluster and the active-site environment strongly resemble each other, while the quaternary structures diverge. Kinetic analyses of OspR with various substrates provide new insights into the order of biosynthetic reactions during the post-translational maturation of landornamide A. These results provide the basis for pathway engineering to generate derivatives of landornamide A and for the general application of peptide arginases as biosynthetic tools for peptide engineering.

Characterization of Bacillus Strains from Natural Honeybee Products with High Keratinolytic Activity and Antimicrobial Potential

Two efficient feather-degrading bacteria were isolated from honeybee samples and identified as Bacillus sonorensis and Bacillus licheniformis based on 16S rRNA and genome sequencing. The strains were able to grow on chicken feathers as the sole carbon and nitrogen sources and degraded the feathers in a few days. The highest keratinase activity was detected by the B. licheniformis CG1 strain (3800 U × mL-1), followed by B. sonorensis AB7 (1450 U × mL-1). Keratinase from B. licheniformis CG1 was shown to be active across a wide range of pH, potentially making this strain advantageous for further industrial applications. All isolates displayed antimicrobial activity against Micrococcus luteus; however, only B. licheniformis CG1 was able to inhibit the growth of Mycobacterium smegmatis. In silico analysis using BAGEL and antiSMASH identified gene clusters associated with the synthesis of non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKSs) and/or ribosomally synthesized and post-translationally modified peptides (RiPPs) in most of the Bacillus isolates. B. licheniformis CG1, the only strain that inhibited the growth of the mycobacterial strain, contained sequences with 100% similarity to lichenysin (also present in the other isolates) and lichenicidin (only present in the CG1 strain). Both compounds have been described to display antimicrobial activity against distinct bacteria. In summary, in this work, we have isolated a strain (B. licheniformis CG1) with promising potential for use in different industrial applications, including animal nutrition, leather processing, detergent formulation and feather degradation.

Peptidase Activation by a Leader Peptide-Bound RiPP Recognition Element.

The RiPP precursor recognition element (RRE) is a conserved domain found in many prokaryotic ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic gene clusters (BGCs). RREs bind with high specificity and affinity to a recognition sequence within the N-terminal leader region of RiPP precursor peptides. Lasso peptide biosynthesis involves an RRE-dependent leader peptidase, which is discretely encoded or fused to the RRE as a di-domain protein. Here we leveraged thousands of predicted BGCs to define the RRE:leader peptidase interaction through evolutionary covariance analysis. Each interacting domain contributes a three-stranded β-sheet to form a hydrophobic β-sandwich-like interface. The bioinformatics-guided predictions were experimentally confirmed using proteins from discrete and fused lasso peptide BGC architectures. Support for the domain-domain interface derived from chemical shift perturbation, paramagnetic relaxation enhancement experiments, and rapid variant activity screening using cell-free biosynthesis. Further validation of selected variants was performed with purified proteins. We developed a p-nitroanilide-based leader peptidase assay to illuminate the role of RRE domains. Our data show that RRE domains play a dual function. RRE domains deliver the precursor peptide to the leader peptidase, and the rate is saturable as expected for a substrate. RRE domains also partially compose the elusive S2 proteolytic pocket that binds the penultimate threonine of lasso leader peptides. Because the RRE domain is required to form the active site, leader peptidase activity is greatly diminished when the RRE domain is supplied at substoichiometric levels. Full proteolytic activation requires RRE engagement with the recognition sequence-containing portion of the leader peptide. Together, our observations define a new mechanism for protease activity regulation.

Towards the Understanding of the Function of Lanthipeptide and TOMM-Related Genes in Haloferax mediterranei.

Research on secondary metabolites produced by Archaea such as ribosomally synthesized and post-translationally modified peptides (RiPPs) is limited. The genome of Haloferax mediterranei ATCC 33500 encodes lanthipeptide synthetases (medM1, medM2, and medM3) and a thiazole-forming cyclodehydratase (ycaO), possibly involved in the biosynthesis of lanthipeptides and the TOMMs haloazolisins, respectively. Lanthipeptides and TOMMs often have antimicrobial activity, and H. mediterranei has antagonistic activity towards haloarchaea shown to be independent of medM genes. This study investigated (i) the transcription of ycaO and medM genes, (ii) the involvement of YcaO in bioactivity, and (iii) the impact of YcaO and MedM-encoding genes' absence in the biomolecular profile of H. mediterranei. The assays were performed with biomass grown in agar and included RT-qPCR, the generation of knockout mutants, bioassays, and FTIR analysis. Results suggest that ycaO and medM genes are transcriptionally active, with the highest number of transcripts observed for medM2. The deletion of ycaO gene had no effect on H. mediterranei antihaloarchaea activity. FTIR analysis of medM and ycaO knockout mutants suggest that MedMs and YcaO activity might be directly or indirectly related t lipids, a novel perspective that deserves further investigation.

Unraveling the encrypted lipophilicity of disulfide bridges in the molecular architecture of proteins and peptides: A machine learning approach in structural bioinformatics.

Disulfide bridges can be considered the concertmaster in a molecular orchestra that directs folding or inhibits unfolding in the molecular architecture of proteins and peptides, so standing for key structural element in protein stability. Its formation requires that two side chain Sγ atoms of cysteine residues are spatially proximal forming the new cystine residues (CRs). Although cystine-containing biomolecules such as antimicrobial peptides (defensines and lantibiotics), toxins (snake venoms and conopeptides), hyperstable peptides (cyclotides and lasso peptides) are well represented in databases and a wide diversity of residue-based lipophilicity scales are reported in the literature, there are no experimental or theoretical studies that describe this fundamental property in CRs. Here, with the motivation to complement previous studies focused on the lipophilicity of amino acids, we have studied the conformational richness of CRs in the biomolecules mentioned above characterizing structural details such as chiralities of the disulfide bridge but also, computing the QM-based lipophilicity (logP), unraveling for the first time the encrypted lipophilicities of different conformations of CRs. Interestingly, we have found a very diverse lipophilicity profiles of CRs in the peptides and proteins studied, thus indicating a hydrophobic fingerprint that could explain molecular recognition events and the unique properties of these biomolecules. In addition, we have developed a machine learning model capable of recognizing whether a cystine residue is hydrophilic or hydrophobic using chiral descriptors of the disulfide bridge. Our results have the potential to contribute to a better understanding of the implications of CRs on the properties and functions of cystine-containing biomolecules.

Rational Optimizations of the Marine-Derived Peptide Sungsanpin as Novel Inhibitors of Cell Invasion.

Cancer metastasis, including cell invasion, is a major cause of poor clinical outcomes and death in numerous cancer patients. In recent years, many efforts have been made to develop potent therapeutic molecules from naturally derived peptides. Sungsanpin is a naturally derived lasso peptide that inhibits A549 cell invasion. We aimed to evaluate the potential of sungsanpin derivatives as candidates for anti-invasion drugs. We synthesized an analog of sungsanpin (Sun A) using a solid-phase peptide synthesis strategy (SPPS) and further modified its structure to improve its anti-invasion activity. All peptides were tested for their proliferative inhibition and anti-invasion activities in the A549 cell lines. Octapeptide S3 and cyclooctapeptide S4 upregulated the expression of TIMP-1 and TIMP-2 mRNA effectively and thus improved the inhibitory effect on the invasion of A549 cells. The two peptides can inhibit the invasion of A549 cells by up to 60 %, suggesting that they have potential as lead molecules for the development of peptide inhibitors.

Kinetics of Aspartimide Formation and Hydrolysis in Lasso Peptide Lihuanodin.

Aspartimides are notorious as undesired side products in solid-phase peptide synthesis and in pharmaceutical formulations. However, we have discovered several ribosomally synthesized and post-translationally modified peptides (RiPPs) in which aspartimide is installed intentionally via enzymatic activity of protein l-isoaspartyl methyltransferase (PIMT) homologues. In the case of the lasso peptide lihuanodin, the methyltransferase LihM recognizes the lassoed substrate pre-lihuanodin, specifically methylating the side chain of an l-Asp residue in the ring portion of the lasso peptide. The subsequent nucleophilic attack from the adjacent amide leads to the formation of an aspartimide. The resulting aspartimide hydrolyzes regioselectively to l-Asp in buffers above pH 7. Here we report the first Michaelis-Menten kinetic measurements of such a RiPP-associated PIMT homologue, LihM, acting on its cognate substrate pre-lihuanodin. Additionally, we measured the rate of aspartimide hydrolysis, which allowed us to deduce the kinetics of the entire reaction network. The relative magnitudes of these rates explain the accumulation and relative stability of aspartimide-containing lihuanodin. We also demonstrate that the residue C-terminal to the aspartimide controls the regioselectivity of hydrolysis and thus the threadedness of the peptide.

Cryptic Diversity of Black Band Disease Cyanobacteria in Siderastrea siderea Corals Revealed by Chemical Ecology and Comparative Genome-Resolved Metagenomics.

Black band disease is a globally distributed and easily recognizable coral disease. Despite years of study, the etiology of this coral disease, which impacts dozens of stony coral species, is not completely understood. Although black band disease mats are predominantly composed of the cyanobacterial species Roseofilum reptotaenium, other filamentous cyanobacterial strains and bacterial heterotrophs are readily detected. Through chemical ecology and metagenomic sequencing, we uncovered cryptic strains of Roseofilum species from Siderastrea siderea corals that differ from those on other corals in the Caribbean and Pacific. Isolation of metabolites from Siderastrea-derived Roseofilum revealed the prevalence of unique forms of looekeyolides, distinct from previously characterized Roseofilum reptotaenium strains. In addition, comparative genomics of Roseofilum strains showed that only Siderastrea-based Roseofilum strains have the genetic capacity to produce lasso peptides, a family of compounds with diverse biological activity. All nine Roseofilum strains examined here shared the genetic capacity to produce looekeyolides and malyngamides, suggesting these compounds support the ecology of this genus. Similar biosynthetic gene clusters are not found in other cyanobacterial genera associated with black band disease, which may suggest that looekeyolides and malyngamides contribute to disease etiology through yet unknown mechanisms.

The structure of MadC from Clostridium maddingley reveals new insights into class I lanthipeptide cyclases.

The rapid emergence of microbial multi-resistance against antibiotics has led to intense search for alternatives. One of these alternatives are ribosomally synthesized and post-translationally modified peptides (RiPPs), especially lantibiotics. They are active in a low nanomolar range and their high stability is due to the presence of characteristic (methyl-) lanthionine rings, which makes them promising candidates as bacteriocides. However, innate resistance against lantibiotics exists in nature, emphasizing the need for artificial or tailor-made lantibiotics. Obviously, such an approach requires an in-depth mechanistic understanding of the modification enzymes, which catalyze the formation of (methyl-)lanthionine rings. Here, we determined the structure of a class I cyclase (MadC), involved in the modification of maddinglicin (MadA) via X-ray crystallography at a resolution of 1.7 Å, revealing new insights about the structural composition of the catalytical site. These structural features and substrate binding were analyzed by mutational analyses of the leader peptide as well as of the cyclase, shedding light into the mode of action of MadC.

Thioalbamide inhibits FoF1-ATPase in breast cancer cells and reduces tumor proliferation and invasiveness in breast cancer in vivo models

ObjectiveThioalbamide is a ribosomally synthesized and post-translationally modified peptide (RiPP) belonging to the family of thioamitides, a rare class of microbial specialized metabolites with unusual post-translational modifications and promising biological activities. Recent studies have demonstrated the ability of thioalbamide to exert highly selective cytotoxic effects on tumor cells by affecting their energy metabolism, thus causing abnormal ROS production and triggering apoptosis. This study is aimed to investigate the molecular mechanisms underlying the antitumor activity of thioalbamide in order to identify its exact molecular target. MethodsWild type MCF-7 and MDA-MB-231 breast cancer cell lines as well as cancer cells deprived of mitochondrial DNA (ρ0 cells) were employed in order to assess thioalbamide effects on tumor bioenergetics. In this regard, metabolic profile was evaluated by a Seahorse XFe96 analyzer, and the activity of the enzyme complexes involved in oxidative phosphorylation was quantified by spectrophotometric assays. Thioalbamide effects on tumor invasiveness were assessed by gelatin zymography experiments and invasion assays. In vivo experiments were carried out on breast cancer xenograft and “experimental metastasis” mouse models. ResultsExperiments carried out on ρ0 breast cancer cells, together with Seahorse analysis and the application of spectrophotometric enzymatic assays, highlighted the ability of thioalbamide to affect the mitochondrial respiration process, and allowed to propose the FoF1-ATPase complex as its main molecular target in breast cancer cells. Additionally, thioalbamide-mediated OXPHOS inhibition was shown, for the first time, to reduce tumor invasiveness by inhibiting metalloproteinase-9 secretion. Furthermore, this study has confirmed the antitumor potential of thioalbamide in two different in vivo models. In particular, experiments on MCF-7 and MDA-MB-231 xenograft mouse models have confirmed in vivo its high anti-proliferative and pro-apoptotic activity, while experiments on MDA-MB-231 ″experimental metastasis” mouse models have highlighted its ability to inhibit breast cancer cell invasiveness. ConclusionsOverall, our results shed more light on the molecular mechanisms underlying the pharmacological potential of thioamidated peptides, thus reducing the gap that separates this rare class of microbial metabolites from clinical studies, which could validate them as effective tools for cancer treatment.