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Exploring the Diversity of Cysteine-Rich Natural Product Peptides via MS/MS Fingerprint Ions.

ABSTRACT: Natural product extracts present inherently complex matrices in which the identification of novel bioactive peptide species is challenged by low-abundance masses and significant structural and sequence diversity. Additionally, discovery efforts often result in the re-identification of known compounds, where modifications derived in vivo or during sample handling may obscure true sequence identity. Herein, we identify mass spectral (MS²) "fingerprint" ions characteristic of cyclotides, a diverse and biologically active family of botanical cysteine-rich peptides, based on regions of high sequence homology. We couple mass shift analysis with MS² spectral fingerprint ions cross referenced with CyBase-a cyclotide database-to discern unique mass species in Viola communis extracts from mass species that are likely already characterized and those with common modifications. The approach is extended to a related class of cysteine-rich peptides, the trypsin inhibitors, using the characterized botanical species Lagenaria siceraria. Coupling the observation of highly abundant MS² ions with mass shift analysis, we identify a new set of small, highly disulfide-bound cysteine-rich L. siceraria peptides.

SUBMITTER: Parsley NC

PROVIDER: S-EPMC7816094 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Exploring the diversity of cysteine-rich natural product peptides via MS/MS fingerprint ions

Project description:Stable and commonly bioactive cysteine-rich peptides (CRPs) have are typically discovered via mass shift analysis. However, the accurate assessment of unique CRP species in a given botanical species is often challenged by same mass species, post-translational modifications or modifications derived from sample handling, and incomplete MS2 fragmentation. Mass spectral fingerprint ions can be leveraged to gain additional information about a mass species prior to full sequence characterization and with only poor quality MS2 spectra. Herein we identify sets of mass spectral fingerprint ions characteristic of the CRP cyclotide family, which may indicate a mass belongs to a specific cyclotide subfamily, and “tell-tale” ions that are of importance when discriminating putative cyclotide species, including common oxidation and over-alkylation ions observed experimentally. Cyclotide-containing V. communis material is used as proof-of-principle, where experimental cyclotide fingerprint ions are explored. Fingerprint ions derived from a third type of CRP, the trypsin inhibitors, are assessed in the gourd L. siceraria. Combining mass shift analysis with the identification of prominent MS2 fingerprint ions is then used to identify three novel CRPs. We demonstrate that abundant mass spectral fingerprint ions can be used to quickly discern masses of interest in complex matrices and masses that are already characterized, aiding prioritization of the most promising novel mass species in a natural product sample for characterization.

2020-09-04 | PXD019501 | Pride

Cysteine-rich peptides promote interspecific genetic isolation in Arabidopsis

Project description:RNA was extracted from mature ovules of two samples (i.e., WT and myb98) and sequenced with an Illumina Hi-seq 2000 sequencer in the Biodynamic Optical Imaging Center (BIOPIC) of Peking University followed by the analysis on the High Performance Computing Platform of the Center for Life Science.

2019-06-13 | GSE128352 | GEO

Interactions of AsCy3 with cysteine-rich peptides.

Project description:There is great interest in fluorogenic compounds that tag biomolecules within cells. Biarsenicals are fluorogenic compounds that become fluorescent upon binding four proximal Cys thiols, a tetracysteine (Cys(4)) motif. This work details interactions between the biarsenical AsCy3 and Cys(4) peptides. Maximal affinity was observed when two Cys-Cys pairs were separated by at least 8 amino acids; the highest affinity ligand bound in the nanomolar concentration range (K(app) = 43 nM) and with a significant (3.2-fold) fluorescence enhancement.

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Urban park soil microbiomes are a rich reservoir of natural product biosynthetic diversity.

Project description:Numerous therapeutically relevant small molecules have been identified from the screening of natural products (NPs) produced by environmental bacteria. These discovery efforts have principally focused on culturing bacteria from natural environments rich in biodiversity. We sought to assess the biosynthetic capacity of urban soil environments using a phylogenetic analysis of conserved NP biosynthetic genes amplified directly from DNA isolated from New York City park soils. By sequencing genes involved in the biosynthesis of nonribosomal peptides and polyketides, we found that urban park soil microbiomes are both rich in biosynthetic diversity and distinct from nonurban samples in their biosynthetic gene composition. A comparison of sequences derived from New York City parks to genes involved in the biosynthesis of biomedically important NPs produced by bacteria originally collected from natural environments around the world suggests that bacteria producing these same families of clinically important antibiotics, antifungals, and anticancer agents are actually present in the soils of New York City. The identification of new bacterial NPs often centers on the systematic exploration of bacteria present in natural environments. Here, we find that the soil microbiomes found in large cities likely hold similar promise as rich unexplored sources of clinically relevant NPs.

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A high-throughput peptidomic strategy to decipher the molecular diversity of cyclic cysteine-rich peptides.

Project description:Cyclotides are plant cyclic cysteine-rich peptides (CRPs). The cyclic nature is reported to be gene-determined with a precursor containing a cyclization-competent domain which contains an essential C-terminal Asn/Asp (Asx) processing signal recognized by a cyclase. Linear forms of cyclotides are rare and are likely uncyclizable because they lack this essential C-terminal Asx signal (uncyclotide). Here we show that in the cyclotide-producing plant Clitoria ternatea, both cyclic and acyclic products, collectively named cliotides, can be bioprocessed from the same cyclization-competent precursor. Using an improved peptidomic strategy coupled with the novel Asx-specific endopeptidase butelase 2 to linearize cliotides at a biosynthetic ligation site for transcriptomic analysis, we characterized 272 cliotides derived from 38 genes. Several types of post-translational modifications of the processed cyclotides were observed, including deamidation, oxidation, hydroxylation, dehydration, glycosylation, methylation, and truncation. Taken together, our results suggest that cyclotide biosynthesis involves 'fuzzy' processing of precursors into both cyclic and linear forms as well as post-translational modifications to achieve molecular diversity, which is a commonly found trait of natural product biosynthesis.

| S-EPMC4786859 | biostudies-literature

Isolation of Cysteine-Rich Peptides from Citrullus colocynthis.

Project description:The plant Citrullus colocynthis, a member of the squash (Cucurbitaceae) family, has a long history in traditional medicine. Based on the ancient knowledge about the healing properties of herbal preparations, plant-derived small molecules, e.g., salicylic acid, or quinine, have been integral to modern drug discovery. Additionally, many plant families, such as Cucurbitaceae, are known as a rich source for cysteine-rich peptides, which are gaining importance as valuable pharmaceuticals. In this study, we characterized the C. colocynthis peptidome using chemical modification of cysteine residues, and mass shift analysis via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. We identified the presence of at least 23 cysteine-rich peptides in this plant, and eight novel peptides, named citcol-1 to -8, with a molecular weight between ~3650 and 4160 Da, were purified using reversed-phase high performance liquid chromatography (HPLC), and their amino acid sequences were determined by de novo assignment of b- and y-ion series of proteolytic peptide fragments. In silico analysis of citcol peptides revealed a high sequence similarity to trypsin inhibitor peptides from Cucumis sativus, Momordica cochinchinensis, Momordica macrophylla and Momordica sphaeroidea. Using genome/transcriptome mining it was possible to identify precursor sequences of this peptide family in related Cucurbitaceae species that cluster into trypsin inhibitor and antimicrobial peptides. Based on our analysis, the presence or absence of a crucial Arg/Lys residue at the putative P1 position may be used to classify these common cysteine-rich peptides by functional properties. Despite sequence homology and the common classification into the inhibitor cysteine knot family, these peptides appear to have diverse and additional bioactivities yet to be revealed.

| S-EPMC7565491 | biostudies-literature

Unfolding the fold of cyclic cysteine-rich peptides.

Project description:We propose a method to extensively characterize the native state ensemble of cyclic cysteine-rich peptides. The method uses minimal information, namely, amino acid sequence and cyclization, as a topological feature that characterizes the native state. The method does not assume a specific disulfide bond pairing for cysteines and allows the possibility of unpaired cysteines. A detailed view of the conformational space relevant for the native state is obtained through a hierarchic multi-resolution exploration. A crucial feature of the exploration is a geometric approach that efficiently generates a large number of distinct cyclic conformations independently of one another. A spatial and energetic analysis of the generated conformations associates a free-energy landscape to the explored conformational space. Application to three long cyclic peptides of different folds shows that the conformational ensembles and cysteine arrangements associated with free energy minima are fully consistent with available experimental data. The results provide a detailed analysis of the native state features of cyclic peptides that can be further tested in experiment.

| S-EPMC2248317 | biostudies-literature

Atypical DNA methylation of genes encoding cysteine-rich peptides in Arabidopsis thaliana

Project description:In plants, transposons and non-protein-coding repeats are epigenetically silenced by CG and non-CG methylation. This pattern of methylation is mediated in part by small RNAs and two specialized RNA polymerases, Pol IV and Pol V, in a process called RNA-directed DNA methylation. By contrast, many protein-coding genes transcribed by Pol II contain in their gene bodies exclusively CG methylation that is independent of small RNAs and Pol IV/Pol V activities. It is unclear how the different methylation machineries distinguish between transposons and genes. Here we report on a group of atypical genes that display in their coding region a transposon-like methylation pattern, which is associated with gene silencing in sporophytic tissues. We performed a methylation-sensitive amplification polymorphism analysis to search for targets of RNA-directed DNA methylation in Arabidopsis thaliana and identified several members of a gene family encoding cysteine-rich peptides (CRPs). We also examined small RNA abundance at individual CRP genes in the wild type plant, nrpd1, and rdr2 mutant plants.

2012-11-22 | E-GEOD-34207 | biostudies-arrayexpress

Atypical DNA methylation of genes encoding cysteine-rich peptides in Arabidopsis thaliana

2012-11-22 | GSE34207 | GEO

Amaranthus hypochondriacus seeds as a rich source of cysteine rich bioactive peptides.

Project description:Amaranthus hypochondriacus is a nutritious alternative grain native to Central and South America. Increased interest in the impact of A. hypochondriacus on the human body has driven characterization of bioactive secondary metabolites. The seeds are known to contain bioactive small molecules but little is known regarding endogenous peptides. Cysteine-rich peptides (CRPs) in foodstuffs are particularly relevant because they are stabilized by disulfide bonds enhancing resistance to digestion. Here, in silico predictions, proteomics, and simulated gastrointestinal digestions are leveraged to identify digestion resistant CRPs within A. hypochondriacus seeds. Thirteen in silico predicted CRPs were detected in a seed extract providing evidence for the translation of five CRP families. Mature forms of six CRPs were characterized via top-down proteomics revealing multiple post-translational modifications. All six peptides demonstrated resistance to simulated gastrointestinal digestion, suggesting that A. hypochondriacus CRPs may exhibit bioactivity after consumption and should be prioritized for further characterization.

| S-EPMC8821138 | biostudies-literature

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