Novel Cyclic Peptides from Lethal Amanita Mushrooms through a Genome-Guided Approach

Most species in the genus Amanita are ectomycorrhizal fungi comprising both edible and poisonous mushrooms. Some species produce potent cyclic peptide toxins, such as α-amanitin, which places them among the deadliest organisms known to mankind. These toxins and related cyclic peptides are encoded by genes of the “MSDIN” family (named after the first five amino acid residues of the precursor peptides), and it is largely unknown to what extent these genes are expressed in the basidiocarps. In the present study, Amanita rimosa and Amanita exitialis were sequenced through the PacBio and Illumina techniques. Together with our two previously sequenced genomes, Amanita subjunquillea and Amanita pallidorosea, in total, 46 previously unknown MSDIN genes were discovered. The expression of over 80% of the MSDIN genes was demonstrated in A. subjunquillea. Through a combination of genomics and mass spectrometry, 12 MSDIN genes were shown to produce novel cyclic peptides. To further confirm the results, three of the cyclic peptides were chemically synthesized. The tandem mass spectrometry (MS/MS) spectra of the natural and the synthetic peptides shared a majority of the fragment ions, demonstrating an identical structure between each peptide pair. Collectively, the results suggested that the genome-guided approach is reliable for identifying novel cyclic peptides in Amanita species and that there is a large peptide reservoir in these mushrooms.

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Novel cyclic peptides from lethal Amanita species through a genomic approach, and major peptide toxins in the ectomycorrhizal association

10.21203/rs.2.23821/v1 ◽

2020 ◽

Author(s):

Hong Luo ◽

Shengwen Zhou ◽

Xincan Li ◽

Xuan Li ◽

Zuo H. Chen ◽

...

Keyword(s):

Posttranslational Modifications ◽

Cyclic Peptides ◽

Cyclic Peptide ◽

Rna Seq ◽

The Past ◽

A Genome ◽

Mycorrhizal Association ◽

Genomic Approach ◽

Peptide Toxins ◽

Microbiome Profiling

Abstract Background: Most species in the genus Amanita are ectomycorrhizal fungi, and the cyclic peptide toxins that some species produce are notoriously deadly. In total, around 25 of these peptides were found in the fruiting bodies over the past 82 years, and whether any of them are present in the mycorrhizae is unknown. Reportedly, sequenced lethal Amanita genomes harbor a significant number of precursor genes of MSDIN family, indicating there could be a much larger capacity for cyclic peptide production in these mushrooms. However, it is largely unknown that to what extent these genes are transcribed, and further, translated into true cyclic peptides.Method: In this study, three poisonous Amanita species, A. rimosa, A. exitialis and A. subjunquillea, were sequenced through PacBio and Illumina techniques. For expression analysis, one strain of A. subjunquillea was sequenced through RNA-Seq. A genome-guided approach was adopted to identify cyclic peptides by coupling predicted toxin-biosynthetic genes with mass spectrometry (MS and MS/MS). To investigate whether any of the toxins were express in the microbiome, profiling of known major toxins was conducted on A. subjunquillea mycorrhizae via HRMS and gene cloning.Results: The resultant genomes showed significant potential to produce known and unknown cyclic peptides. Together with our 2 previously sequenced genomes, in total 37 unknown MSDIN genes were discovered. Expression of over 90% of the MSDIN genes was demonstrated in two strains of A. subjunquillea. Through the genome-guided approach, 12 MSDIN genes were found to produce true, novel cyclic peptides with no additional posttranslational modifications. When the ectomycorrhizae of A. subjunquillea were analyzed by MS, all major toxins were detected. The corresponding MSDINs for these cyclic peptides were successfully cloned directly from the mycorrhizae.Conclusions: The genome-guided approach provided a speedy method to identify cyclic peptides both in Amanita mushrooms and in the ectomycorrhizae. In this study, a significant number of novel MSDIN genes were discovered, most of which were found to be expressed in the tested species. The identification of the 12 novel cyclic peptides strongly suggests that Amanita species possess a much larger reservoir of these peptides than previously thought. This is the first report to demonstrate that the cyclic peptides in Amanita species are expressed in the mycorrhizal association. All four major toxins, α-amanitin, β-amanitin, phallacidin and phalloidin, are found to be present in the symbiosis, offering new clues to their biological function(s).

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Chemical Study of the Hepatotoxins from Microcystis Aeruginosa Collected in California

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879300500318 ◽

1993 ◽

Vol 5 (3) ◽

pp. 409-412 ◽

Cited By ~ 2

Author(s):

S. E. DeVries ◽

M. Namikoshi ◽

F. D. Galey ◽

J. E. Merritt ◽

K. L. Rinehart ◽

...

Keyword(s):

Mass Spectrometry ◽

Microcystis Aeruginosa ◽

High Performance ◽

Fast Atom Bombardment ◽

Cyclic Peptide ◽

Algal Cell ◽

Chemical Study ◽

Blue Green Alga ◽

Peptide Toxins ◽

Layer Chromatography

Four cyclic peptide toxins were purified and quantified from the aqueous extract of algal cell material utilizing high performance liquid chromatography, thin layer chromatography, and fast atom bombardment mass spectrometry. The cyclic peptide toxins appear to be similar structurally to hepatotoxins Iron previously identified blooms of the blue-green alga Microcystis aeruginosa.

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The Cyclic Peptide Toxins of Amanita and Other Poisonous Mushrooms

10.1007/978-3-319-76822-9 ◽

2018 ◽

Cited By ~ 6

Author(s):

Jonathan Walton

Keyword(s):

Cyclic Peptide ◽

Peptide Toxins ◽

Poisonous Mushrooms

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Dicarbonyl derived post-translational modifications: chemistry bridging biology and aging-related disease

Essays in Biochemistry ◽

10.1042/ebc20190057 ◽

2020 ◽

Vol 64 (1) ◽

pp. 97-110

Author(s):

Christian Sibbersen ◽

Mogens Johannsen

Keyword(s):

Mass Spectrometry ◽

Future Research ◽

Amino Acid Residues ◽

Post Translational Modification ◽

Dicarbonyl Compounds ◽

Protein Targets ◽

Post Translational Modifications ◽

Reactive Protein ◽

Glycation End Products ◽

Direct Mass Spectrometry

Abstract In living systems, nucleophilic amino acid residues are prone to non-enzymatic post-translational modification by electrophiles. α-Dicarbonyl compounds are a special type of electrophiles that can react irreversibly with lysine, arginine, and cysteine residues via complex mechanisms to form post-translational modifications known as advanced glycation end-products (AGEs). Glyoxal, methylglyoxal, and 3-deoxyglucosone are the major endogenous dicarbonyls, with methylglyoxal being the most well-studied. There are several routes that lead to the formation of dicarbonyl compounds, most originating from glucose and glucose metabolism, such as the non-enzymatic decomposition of glycolytic intermediates and fructosyl amines. Although dicarbonyls are removed continuously mainly via the glyoxalase system, several conditions lead to an increase in dicarbonyl concentration and thereby AGE formation. AGEs have been implicated in diabetes and aging-related diseases, and for this reason the elucidation of their structure as well as protein targets is of great interest. Though the dicarbonyls and reactive protein side chains are of relatively simple nature, the structures of the adducts as well as their mechanism of formation are not that trivial. Furthermore, detection of sites of modification can be demanding and current best practices rely on either direct mass spectrometry or various methods of enrichment based on antibodies or click chemistry followed by mass spectrometry. Future research into the structure of these adducts and protein targets of dicarbonyl compounds may improve the understanding of how the mechanisms of diabetes and aging-related physiological damage occur.

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A Target-Based Method for Designing Heterochiral Cyclic Peptide Binders: De Novo Inhibitors of the PD-1/PD-L1 Interaction

10.26434/chemrxiv.11663337.v2 ◽

2020 ◽

Author(s):

Salvador Guardiola ◽

Monica Varese ◽

Xavier Roig ◽

Jesús Garcia ◽

Ernest Giralt

Keyword(s):

Protein Interactions ◽

Cyclic Peptides ◽

General Framework ◽

Large Scale ◽

Cyclic Peptide ◽

De Novo ◽

Inhibitory Effect ◽

Original Text ◽

Retraction Notice ◽

Pharmaceutical Properties

NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above. ------------------------------------------------------------------------ Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-i3 and PD-i6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our de novo design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.

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Statistical Force-Field for Structural Modeling Using Chemical Cross-Linking/mass Spectrometry Distance Constraints

10.26434/chemrxiv.6030563 ◽

2018 ◽

Author(s):

Allan J. R. Ferrari ◽

Fabio C. Gozzo ◽

Leandro Martinez

Keyword(s):

Mass Spectrometry ◽

Amino Acid ◽

Force Field ◽

Protein Structures ◽

Protein Structure Determination ◽

Structural Modeling ◽

Cross Linking ◽

Amino Acid Residues ◽

Distance Constraints ◽

Chemical Cross Linking

<div>Chemical cross-linking/Mass Spectrometry (XLMS) is an experimental method to obtain distance constraints between amino acid residues, which can be applied to structural modeling of tertiary and quaternary biomolecular structures. These constraints provide, in principle, only upper limits to the distance between amino acid residues along the surface of the biomolecule. In practice, attempts to use of XLMS constraints for tertiary protein structure determination have not been widely successful. This indicates the need of specifically designed strategies for the representation of these constraints within modeling algorithms. Here, a force-field designed to represent XLMS-derived constraints is proposed. The potential energy functions are obtained by computing, in the database of known protein structures, the probability of satisfaction of a topological cross-linking distance as a function of the Euclidean distance between amino acid residues. The force-field can be easily incorporated into current modeling methods and software. In this work, the force-field was implemented within the Rosetta ab initio relax protocol. We show a significant improvement in the quality of the models obtained relative to current strategies for constraint representation. This force-field contributes to the long-desired goal of obtaining the tertiary structures of proteins using XLMS data. Force-field parameters and usage instructions are freely available at http://m3g.iqm.unicamp.br/topolink/xlff </div>

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Faculty Opinions recommendation of Self-assembling peptide nanotubes from enantiomeric pairs of cyclic peptides with alternating D and L amino acid residues.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1018033.206566 ◽

2004 ◽

Author(s):

James Nowick

Keyword(s):

Amino Acid ◽

Cyclic Peptides ◽

Amino Acid Residues ◽

Peptide Nanotubes ◽

Self Assembling

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Metastable fragment ions in multi-photon time-of-flight mass spectrometry: Decay channels of the benzene cation

International Journal of Mass Spectrometry and Ion Physics ◽

10.1016/0020-7381(83)85011-6 ◽

1983 ◽

Vol 51 (2-3) ◽

pp. 255-265 ◽

Cited By ~ 48

Author(s):

H. Kühlewind ◽

H.J. Neusser ◽

E.W. Schlag

Keyword(s):

Mass Spectrometry ◽

Time Of Flight ◽

Decay Channels ◽

Flight Mass Spectrometry ◽

Fragment Ions

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Chimera Spectrum Diagnostics for Peptides Using Two-Dimensional Partial Covariance Mass Spectrometry

Molecules ◽

10.3390/molecules26123728 ◽

2021 ◽

Vol 26 (12) ◽

pp. 3728

Author(s):

Taran Driver ◽

Nikhil Bachhawat ◽

Leszek J. Frasinski ◽

Jonathan P. Marangos ◽

Vitali Averbukh ◽

...

Keyword(s):

Mass Spectrometry ◽

Peptide Sequence ◽

Two Dimensional ◽

Peptide Ions ◽

Finite Mass ◽

Protein Database ◽

Spectra Analysis ◽

Fragment Ions ◽

Single Precursor ◽

The Common

The rate of successful identification of peptide sequences by tandem mass spectrometry (MS/MS) is adversely affected by the common occurrence of co-isolation and co-fragmentation of two or more isobaric or isomeric parent ions. This results in so-called `chimera spectra’, which feature peaks of the fragment ions from more than a single precursor ion. The totality of the fragment ion peaks in chimera spectra cannot be assigned to a single peptide sequence, which contradicts a fundamental assumption of the standard automated MS/MS spectra analysis tools, such as protein database search engines. This calls for a diagnostic method able to identify chimera spectra to single out the cases where this assumption is not valid. Here, we demonstrate that, within the recently developed two-dimensional partial covariance mass spectrometry (2D-PC-MS), it is possible to reliably identify chimera spectra directly from the two-dimensional fragment ion spectrum, irrespective of whether the co-isolated peptide ions are isobaric up to a finite mass accuracy or isomeric. We introduce ‘3-57 chimera tag’ technique for chimera spectrum diagnostics based on 2D-PC-MS and perform numerical simulations to examine its efficiency. We experimentally demonstrate the detection of a mixture of two isomeric parent ions, even under conditions when one isomeric peptide is at one five-hundredth of the molar concentration of the second isomer.

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