Chitin-binding hevein-like peptides (CB-HLPs) belong to a family of cysteine-rich peptides that play important roles in plant stress and defense mechanisms. CB-HLPs are ribosomally synthesized ...peptides that are known to be bioprocessed from the following two types of three-domain CB-HLP precursor architectures: cargo-carrying and non-cargo-carrying. Here, we report the identification and characterization of chenotides biosynthesized from the third type of precursors, which are cleavable hololectins of the quinoa (Chenopodium quinoa) family. Chenotides are 6-Cys-CB-HLPs of 29–31 amino acids, which have a third type of precursor architecture that encompasses a canonical chitin-binding domain that is involved in chitin binding and anti-fungal activities. Microbroth dilution assays and microscopic analyses showed that chenotides are effective against phyto-pathogenic fungi in the micromolar range. Structure determination revealed that chenotides are cystine knotted and highly compact, which could confer resistance against heat and proteolytic degradation. Importantly, chenotides are connected by a novel 18-residue Gly/Ala-rich linker that is a target for bioprocessing by cathepsin-like endopeptidases. Taken together, our findings reveal that chenotides are a new family of CB-HLPs from quinoa that are synthesized as a single multi-modular unit and bioprocessed to yield individual mature CB-HLPs. Importantly, such precursors constitute a new family of cleavable hololectins. This unusual feature could increase the biosynthetic efficiency of anti-fungal CB-HLPs, to provide an evolutionary advantage for plant survival and reproduction.
Species misidentification and adulteration are major concerns in authenticating herbal medicines. Radix Astragali (RA), the roots of
, is a traditional herbal medicine used for treating diabetes. ...However, it is often substituted by Radix Hedysarum (RH), the roots of
from the same plant family Fabaceae, which possesses different bioactivities. Current authentication methods, focusing on the chemical composition differences of herbal medicines based on small molecules, have limitations when these chemical markers are found in many species. Herein, we describe a rapid and general method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), coupled with multivariate analyses to differentiate herbal medicines. We used cysteine-rich peptide (CRP) fingerprinting, a method that exploits an underexplored chemical space between 2 to 6 kDa and which is populated by highly stable CRPs. To show the generality of the method, we screened 100 medicinal plant extracts and showed that CRP fingerprints are unique chemical markers. In addition, CRP fingerprinting was many-fold faster than the conventional authentication method using ultra-performance liquid chromatography (UPLC). Multivariate analyses showed that it has comparable classification accuracy as UPLC fingerprinting. Together, our findings revealed that CRP fingerprinting coupled with multivariate analyses is a rapid and general method for authentication and quality control for natural products in medicinal plants.
•Identified a novel cysteine-rich peptide carboxypeptidase inhibitor from wolfberry.•Data mining shows that β-lybatide is the smallest carboxypeptidase inhibitor.•β-lybatide is hyperstable bioactives ...against chemical and proteolytic degradation.•50 β-lybatide-like CPIs from 28 plant species, most of which are from staple food.
Hyperstable cysteine-rich peptides (CRPs) represent an underexplored superfamily of bioactives in functional foods. An example is wolfberry of the Lycium barbarum family. Previously, we discovered a CRP, designated α-lybatide, from L. barbarum bark. Herein, we report the discovery of β-lybatide, a novel carboxypeptidase inhibitor belonging to a different CRP family from the wolfberry plant. Proteomic and transcriptomic analyses showed that β-lybatide contains 36 amino acids with six cysteine residues. NMR spectroscopy revealed that β-lybatide displays a knottin-like structure that renders it highly resistant to thermal, chemical and enzymatic degradation, conditions important for keeping its structural integrity in gastrointestinal tract. Biochemical assays showed that β-lybatide is a potent carboxypeptidase inhibitor which could contribute to the wolfberry biological activities. Bioinformatics analysis revealed an additional 49 β-lybatide-like plant carboxypeptidase inhibitors. Together, our results show that β-lybatide is the first and the smallest plant-derived hyperstable carboxypeptidase inhibitor discovered from a functional food.
Mine tailings resulting from orogenic gold processing commonly form arsenic (As)-enriched environments. A result of this is that they draw environmental attention in modern gold mining settings and ...significant effort in long-term rehabilitation. The Alexander River mine processing site in the Reefton area of Westland provides an opportunity to examine the natural rehabilitation of As-enriched tailings that have been left undisturbed for >70 years in a humid environment. These tailings comprise a ∼1 m thick package of finely laminated (mm-scale) sediments that were manually accumulated on a terrace downstream of the gold extraction plant. The tailings sediments are As-rich (up to 5000 mg/kg) due to relict arsenopyrite and As-bearing pyrite. Diagenetic Ca-Fe-arsenate (tentatively identified as yukonite) and As-bearing iron oxyhydroxide have formed during localised oxidation. There was sufficient calcite in the tailings to maintain circumneutral pH in the tailings despite sulphide oxidation. The historical tailing impoundment is now covered by grass, ferns and shrubs, with beech and rimu forest covering several metres at the margins. Leaf analyses indicate that the vegetation is absorbing only minor As. Therefore, the tailings substrate has not halted natural vegetation re-colonisation of this As-rich historical site.
Heveins and hevein-containing (hev-) lectins play important roles in stress and pathogenic responses in plants but cause health concerns in humans. Hev-hololectins contain multiple modular ...hev-peptide domains and are abundantly present in cereals and pseudocereals. However, it is unclear why some cereal hev-hololectins are presented as different forms of proteolytically processed proteoforms. Here we show the precursor architectures of hev-hololectins lead to different processing mechanisms to give either hololectins or hevein-like peptides. We used mass spectrometry and datamining to screen hev-peptides from common cereals, and identified from the oat plant
nine novel hevein-like peptides, avenatide aV1-aV9. Bioinformatic analysis revealed that asparaginyl endopeptidase (AEP) can be responsible for the maturation of the highly homologous avenatides from five oat hev-hololectin precursors, each containing four tandemly repeating, hev-like avenatide domains connected by AEP-susceptible linkers with 13-16 residues in length. Further analysis of cereal hev-hololectins showed that the linker lengths provide a distinguishing feature between their cleavable and non-cleavable precursors, with the cleavables having considerably longer linkers (>13 amino acids) than the non-cleavables (<6 amino acids). A detailed study of avenatide aV1 revealed that it contains eight cysteine residues which form a structurally compact, metabolic-resistant cystine-knotted framework with a well-defined chitin-binding site. Antimicrobial assays showed that avenatide aV1 is anti-fungal and inhibits the growth of phyto-pathogenic fungi. Together, our findings of cleavable and non-cleavable hololectins found in cereals expand our knowledge to their biosynthesis and provide insights for hololectin-related health concerns in human.
Astragalus membranaceus root, Huang Qi in Chinese, is a popular medicinal herb traditionally used to regulate blood glucose. Herein, the identification and characterization of two families of ...cysteine-rich peptides (CRPs), designated α- and β-astratides, from A. membranaceus roots are reported. Proteomic analysis showed that α-astratide aM1 and β-astratide bM1 belong to two distinct CRP families. The six-cysteine-containing and proline-rich α-astratide aM1 displayed high sequence identity to Pea Albumin 1 Subunit b (PA1b), while the eight-cysteine-containing β-astratide bM1 showed sequence similarity to plant defensins. An antifungal assay revealed that bM1 possessed potent antifungal activity. In contrast, aM1 showed a cytotoxic effect against insect Sf9 cells. More importantly, aM1 decreased insulin secretion in mouse pancreatic β cells, suggesting it could interfere in glucose homeostasis, which accounts for the adaptogenic property of A. membranaceus. Phylogenetic clustering analysis suggested that the proline-rich aM1 is a putative prolyl oligopeptidase inhibitor and belongs to a novel subfamily of PA1b-like peptides, while bM1 belongs to a new subfamily of plant defensins. Together, the study reveals that astratides are multifunctional CRPs in plants, which expand the existing library of PA1b-like peptides and plant defensins and further our understanding of their roles in host-defense system and leads as peptidyl therapeutics.
A genetic mutation in the Vps35 subunit of retromer has recently been linked to late onset Parkinson's disease. We observed that the distribution and maturation of Vps35 D620N positive endosomes are ...altered. While Vps35 D620N containing retromer still binds CI‐M6PR, its trafficking is perturbed as shown by secretion of its ligand cathepsin D. As cathepsin D is involved in processing of α‐synuclein, a well‐established causative agent of Parkinson's disease, its altered trafficking may therefore represent the underlying cause of disease.
The retromer is a trimeric cargo‐recognition protein complex composed of Vps26, Vps29 and Vps35 associated with protein trafficking within endosomes. Recently, a pathogenic point mutation within the Vps35 subunit (D620N) was linked to the manifestation of Parkinson's disease (PD). Here, we investigated details underlying the molecular mechanism by which the D620N mutation in Vps35 modulates retromer function, including examination of retromer's subcellular localization and its capacity to sort cargo. We show that expression of the PD‐linked Vps35 D620N mutant redistributes retromer‐positive endosomes to a perinuclear subcellular localization and that these endosomes are enlarged in both model cell lines and fibroblasts isolated from a PD patient. Vps35 D620N is correctly folded and binds Vps29 and Vps26A with the same affinity as wild‐type Vps35. While PD‐linked point mutant Vps35 D620N interacts with the cation‐independent mannose‐6‐phosphate receptor (CI‐M6PR), a known retromer cargo, we find that its expression disrupts the trafficking of cathepsin D, a CI‐M6PR ligand and protease responsible for degradation of α‐synuclein, a causative agent of PD. In summary, we find that the expression of Vps35 D620N leads to endosomal alterations and trafficking defects that may partly explain its action in PD.
Quantification of halloysite and kaolinite in clay deposits from X-ray diffraction (XRD) commonly requires extensive sample preparation to differentiate the two phyllosilicates. When assessing ...hundreds of samples for mineral resource estimations, XRD analyses may become unfeasible due to time and expense. Fourier transform infrared (FTIR) analysis is a fast and cost-effective method to discriminate between kaolinite and halloysite; however, few efforts have been made to use this technique for quantified analysis of these minerals. In this study, we trained machine- and deep-learning models on XRD data to predict the abundance of kaolinite and halloysite from FTIR, chemical composition, and brightness data. The case study is from the Cloud Nine kaolinite–halloysite deposit, Noombenberry Project, Western Australia. The residual clay deposit is hosted in the saprolitic and transition zone of the weathering profile above the basement granite on the southwestern portion of the Archean Yilgarn Craton. Compared with XRD quantification, the predicted models have an R2 of 0.97 for kaolinite and 0.96 for halloysite, demonstrating an excellent fit. Based on these results, we demonstrate that our methodology provides a cost-effective alternative to XRD to quantify kaolinite and halloysite abundances.
Objective To determine if the chick chorioallantoic membrane (CAM) is a potential alternative that is capable of screening test substances for vasoactivity in terms of vessel diameter changes. The ...CAM was also evaluated as a tool for irritancy screening.
Methods Visual assessment of the CAM for irritancy after the application of the test substance or solvent to its surface was made. An imaging based‐in‐vivo CAM model was developed by imaging CAM blood vessels in a pre‐defined area using a semi‐automatic image processing and analysis technique to measure blood vessel diameters. Solvents and drugs such as 70% v/v ethanol, normal saline, 5% w/v glucose monohydrate, glycerin, glucagon, N‐methylpyrrolidone, nicotine, glyceryl trinitrate, glucagon, propranolol and caffeine were tested on the CAM.
Key findings Propranolol, nicotine and glycerin were irritants on CAM. Changes in the diameters of fine blood vessels were accurately measured by high resolution image analysis. Vasoconstriction was seen with 70% v/v ethanol while vasodilation was displayed with glucagon and caffeine. The results reflected expected trends with evidence of feedback mechanisms ensuring homeostasis.
Conclusion The CAM model can be applied to assess pharmaceutical and cosmetic formulations in early development work to gain useful insights to potential irritancy and biological effects of components and formulations.
The occurrence, concentration and geochemistry of REE-bearing minerals in beach sands along the West Coast of the South Island of New Zealand is reported as a proxy for the composition of adjacent ...uplifted Holocene marine terraces. The strandlines of 13 beaches along this coast contain REE-rich allanite (average: 15% REO), monazite (82% REO) and xenotime (83% REO), with allanite being most common and xenotime having a restricted range. Other REE-rich phases are apatite (average: 2259 ppm), zircon (1756ppm), epidote (761 ppm), titanite (699 ppm) and scheelite. Geographical trends to the distribution of these minerals are not entirely clear, but there is an influx of titanite and allanite north of the Grey River and an increase in apatite, allanite, monazite and epidote north of Little Wanganui River. In-situ trace elements and/or
87
Sr/
86
Sr isotopes indicate allanite, monazite and xenotime to be likely mainly derived from exhumed Paleozoic and Cretaceous granites, with minor amounts from the Alpine Schist metasedimentary rocks. The main source of the analysed apatite is likely also the granitoids, with contributions from the Greenland Group and Alpine Schist. When the REE abundances in the beach sands are compared to the size of the uplifted adjacent Holocene terraces, Waimangaroa terrace may contain the highest concentration of REE, followed by the Little Wanganui and Barrytown terraces.
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