RESUMEN
Legumes have evolved a nitrogen-fixing symbiotic interaction with rhizobia, and this association helps them to cope with the limited nitrogen conditions in soil. The compatible interaction between the host plant and rhizobia leads to the formation of root nodules, wherein internalization and transition of rhizobia into their symbiotic form, termed bacteroids, occur. Rhizobia in the nodules of the Inverted Repeat-Lacking Clade legumes, including Medicago truncatula, undergo terminal differentiation, resulting in elongated and endoreduplicated bacteroids. This transition of endocytosed rhizobia is mediated by a large gene family of host-produced nodule-specific cysteine-rich (NCR) peptides in M. truncatula. Few NCRs have been recently found to be essential for complete differentiation and persistence of bacteroids. Here, we show that a M. truncatula symbiotic mutant FN9285, defective in the complete transition of rhizobia, is deficient in a cluster of NCR genes. More specifically, we show that the loss of the duplicated genes NCR086 and NCR314 in the A17 genotype, found in a single copy in Medicago littoralis R108, is responsible for the ineffective symbiotic phenotype of FN9285. The NCR086 and NCR314 gene pair encodes the same mature peptide but their transcriptional activity varies considerably. Nevertheless, both genes can restore the effective symbiosis in FN9285 indicating that their complementation ability does not depend on the strength of their expression activity. The identification of the NCR086/NCR314 peptide, essential for complete bacteroid differentiation, has extended the list of peptides, from a gene family of several hundred members, that are essential for effective nitrogen-fixing symbiosis in M. truncatula.
Asunto(s)
Medicago truncatula , Familia de Multigenes , Proteínas de Plantas , Nódulos de las Raíces de las Plantas , Simbiosis , Medicago truncatula/microbiología , Medicago truncatula/genética , Medicago truncatula/fisiología , Nódulos de las Raíces de las Plantas/microbiología , Nódulos de las Raíces de las Plantas/genética , Simbiosis/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas , Rhizobium/fisiología , Rhizobium/genética , Fijación del Nitrógeno/genética , Péptidos/metabolismo , Péptidos/genética , Sinorhizobium meliloti/fisiología , Sinorhizobium meliloti/genética , Cisteína/metabolismoRESUMEN
Alfalfa (Medicago sativa L.) is one of the most important forage legumes in the world, including autotetraploid (M. sativa ssp. sativa) and diploid alfalfa (M. sativa ssp. caerulea, progenitor of autotetraploid alfalfa). Here, we reported a high-quality genome of ZW0012 (diploid alfalfa, 769 Mb, contig N50 = 5.5 Mb), which was grouped into the Northern group in population structure analysis, suggesting that our genome assembly filled a major gap among the members of M. sativa complex. During polyploidization, large phenotypic differences occurred between diploids and tetraploids, and the genetic information underlying its massive phenotypic variations remains largely unexplored. Extensive structural variations (SVs) were identified between ZW0012 and XinJiangDaYe (an autotetraploid alfalfa with released genome). We identified 71 ZW0012-specific PAV genes and 1296 XinJiangDaYe-specific PAV genes, mainly involved in defence response, cell growth, and photosynthesis. We have verified the positive roles of MsNCR1 (a XinJiangDaYe-specific PAV gene) in nodulation using an Agrobacterium rhizobia-mediated transgenic method. We also demonstrated that MsSKIP23_1 and MsFBL23_1 (two XinJiangDaYe-specific PAV genes) regulated leaf size by transient overexpression and virus-induced gene silencing analysis. Our study provides a high-quality reference genome of an important diploid alfalfa germplasm and a valuable resource of variation landscape between diploid and autotetraploid, which will facilitate the functional gene discovery and molecular-based breeding for the cultivars in the future.
Asunto(s)
Cromosomas de las Plantas , Diploidia , Genoma de Planta , Medicago sativa , Medicago sativa/genética , Genoma de Planta/genética , Cromosomas de las Plantas/genética , Variación GenéticaRESUMEN
Disulfides in peptides and proteins are essential for maintaining a properly folded structure. Their oxidative folding is invariably performed in an aqueous-buffered solution. However, this process is often slow and can lead to misfolded products. Here, we report a novel concept and strategy that is bio-inspired to mimic protein disulfide isomerase (PDI) by accelerating disulfide exchange rates many thousand-fold. The proposed strategy termed organic oxidative folding is performed under organic solvents to yield correctly folded cysteine-rich microproteins instantaneously without observable misfolded or dead-end products. Compared to conventional aqueous oxidative folding strategies, enormously large rate accelerations up to 113,200-fold were observed. The feasibility and generality of the organic oxidative folding strategy was successfully demonstrated on 15 cysteine-rich microproteins of different hydrophobicity, lengths (14 to 58 residues), and numbers of disulfides (2 to 5 disulfides), producing the native products in a second and in high yield.
Asunto(s)
Cisteína , Micropéptidos , Cisteína/metabolismo , Pliegue de Proteína , Biomimética , Péptidos/química , Proteína Disulfuro Isomerasas/metabolismo , Oxidación-Reducción , Solventes , Disulfuros/química , Estrés OxidativoRESUMEN
KEY MESSAGE: We analyzed the evolutionary pattern of cysteine-rich peptides (CRPs) to infer the relationship between CRP copy number and plant ecotype, and the origin of bi-domains CRPs. Plants produce cysteine-rich peptides (CRPs) that have long-lasting broad-spectrum antimicrobial activity to protect themselves from various groups of pathogens. We analyzed 240 plant genomes, ranging from algae to eudicots, and discovered that CRPs are widely distributed in plants. Our comparative genomics results revealed that CRP genes have been amplified through both whole genome and local tandem duplication. The copy number of these genes varied significantly across lineages and was associated with the plant ecotype. This may be due to their resistance to changing pathogenic environments. The conserved and lineage-specific CRP families contribute to diverse antimicrobial activities. Furthermore, we investigated the unique bi-domain CRPs that result from unequal crossover events. Our findings provide a unique evolutionary perspective on CRPs and insights into their antimicrobial and symbiosis characteristics.
Asunto(s)
Antiinfecciosos , Péptidos Antimicrobianos , Cisteína/genética , Plantas/genética , Péptidos/genética , Péptidos/farmacología , Antiinfecciosos/farmacología , Evolución Molecular , FilogeniaRESUMEN
Due to their advantages in structural stability and versatility, cysteine-rich peptides, which are secreted from the venom glands of venomous animals, constitute a naturally occurring pharmaceutical arsenal. However, the correct folding of disulfide bonds is a challenging task in the prokaryotic expression system like Escherichia coli due to the reducing environment. Here, a secretory expression plasmid pSE-G1M5-SUMO-HWTX-I for the spider neurotoxin huwentoxin-I (HWTX-I) with three disulfides as a model of cysteine-rich peptides was constructed. By utilizing the signal peptide G1M5, the fusion protein 6 × His-SUMO-HWTX-I was successfully secreted into extracellular medium of BL21(DE3). After enrichment using cation-exchange chromatography and purification utilizing the Ni-NTA column, 6 × His-SUMO-HWTX-I was digested via Ulp1 kinase to release recombinant HWTX-I (rHWTX-I), which was further purified utilizing RP-HPLC. Finally, both impurities with low and high molecular weights were completely removed. The molecular mass of rHWTX-I was identified as being 3750.8 Da, which was identical to natural HWTX-I with three disulfide bridges. Furthermore, by utilizing whole-cell patch clamp, the sodium currents of hNav1.7 could be inhibited by rHWTX-I and the IC50 value was 419 nmol/L.
Asunto(s)
Venenos de Araña , Arañas , Animales , Neurotoxinas/química , Neurotoxinas/farmacología , Cisteína/metabolismo , Arañas/química , Arañas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Venenos de Araña/genética , Venenos de Araña/química , Venenos de Araña/metabolismo , Péptidos/metabolismo , Disulfuros/metabolismoRESUMEN
Coffee processing generates a huge amount of waste that contains many natural products. Here, we report the discovery of a panel of novel cell-penetrating and metal ion-binding microproteins designated coffeetide cC1a-c and cL1-6 from the husk of two popular coffee plants, Coffea canephora and Coffea liberica, respectively. Combining sequence determination and a database search, we show that the prototypic coffeetide cC1a is a 37-residue, eight-cysteine microprotein with a hevein-like cysteine motif, but without a chitin-binding domain. NMR determination of cC1a reveals a compact structure that confers its resistance to heat and proteolytic degradation. Disulfide mapping together with chemical synthesis reveals that cC1a has a ginsentide-like, and not a hevein-like, disulfide connectivity. In addition, transcriptomic analysis showed that the 98-residue micrcoproten-like coffeetide precursor contains a three-domain arrangement, like ginsentide precursors. Molecular modeling, together with experimental validation, revealed a Mg2+ and Fe3+ binding pocket at the N-terminus formed by three glutamic acids. Importantly, cC1a is amphipathic with a continuous stretch of 19 apolar amino acids, which enables its cell penetration to target intracellular proteins, despite being highly negatively charged. Our findings suggest that coffee by-products could provide a source of ginsentide-like bioactive peptides that have the potential to target intracellular proteins.
Asunto(s)
Coffea , Café , Cisteína , Disulfuros , MicropéptidosRESUMEN
Acanthoscurria juruenicola is an Amazonian spider described for the first time almost a century ago. However, little is known about their venom composition. Here, we present a multiomics characterization of A. juruenicola venom by a combination of transcriptomics, proteomics, and peptidomics approaches. Transcriptomics of female venom glands resulted in 93,979 unique assembled mRNA transcript encoding proteins. A total of 92 proteins were identified in the venom by mass spectrometry, including 14 mature cysteine-rich peptides (CRPs). Quantitative analysis showed that CRPs, cysteine-rich secretory proteins, metalloproteases, carbonic anhydrases, and hyaluronidase comprise >90% of the venom proteome. Relative quantification of venom toxins was performed by DIA and DDA, revealing converging profiles of female and male specimens by both methods. Biochemical assays confirmed the presence of active hyaluronidases, phospholipases, and proteases in the venom. Moreover, the venom promoted in vivo paralytic activities in crickets, consistent with the high concentration of CRPs. Overall, we report a comprehensive analysis of the arsenal of toxins of A. juruenicola and highlight their potential biotechnological and pharmacological applications. Mass spectrometry data were deposited to the ProteomeXchange Consortium via the PRIDE repository with the dataset identifier PXD013149 and via the MassIVE repository with the dataset identifier MSV000087777.
Asunto(s)
Venenos de Araña , Arañas , Animales , Masculino , Femenino , Arañas/genética , Arañas/metabolismo , Venenos de Araña/genética , Venenos de Araña/química , Venenos de Araña/metabolismo , Cisteína/metabolismo , Proteómica/métodos , Espectrometría de Masas/métodos , Proteoma/genética , Proteoma/metabolismo , Péptidos/análisisRESUMEN
Plant cysteine-rich peptides (CRPs) represent a diverse group of molecules involved in different aspects of plant physiology. Antimicrobial peptides, which directly suppress the growth of pathogens, are regarded as promising templates for the development of next-generation pharmaceuticals and ecologically friendly plant disease control agents. Their oligopeptide fragments are even more promising because of their low production costs. The goal of this work was to explore the antimicrobial activity of nine short peptides derived from the γ-core-containing regions of tomato CRPs against important plant and human pathogens. We discovered antimicrobial activity in peptides derived from the defensin-like peptides, snakins, and MEG, which demonstrates the direct involvement of these CRPs in defense reactions in tomato. The CRP-derived short peptides appeared particularly active against the gram-positive bacterium Clavibacter michiganensis, which causes bacterial wilt-opening up new possibilities for their use in agriculture to control this dangerous disease. Furthermore, high inhibitory potency of short oligopeptides was demonstrated against the yeast Cryptococcus neoformans, which causes serious diseases in humans, making these peptide molecules promising candidates for the development of next-generation pharmaceuticals. Studies of the mode of action of the two most active peptides indicate fungal membrane permeabilization as a mechanism of antimicrobial action.
Asunto(s)
Péptidos Antimicrobianos/síntesis química , Péptidos Antimicrobianos/farmacología , Cisteína/química , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Solanum lycopersicum/química , Secuencia de Aminoácidos , Bacterias/efectos de los fármacos , Técnicas de Química Sintética , Relación Dosis-Respuesta a Droga , Solanum lycopersicum/inmunología , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Oligopéptidos/química , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Conformación Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-Actividad , Levaduras/efectos de los fármacosRESUMEN
Cysteine-rich peptides (CRPs) are small proteins of less than 100 amino acids in length characterized by the presence of disulfide bridges and common end-to-end macrocyclization. These properties confer hyperstability against high temperatures, salt concentration, serum presence, and protease degradation to CRPs. Moreover, their intercysteine domains (loops) are susceptible to residue hypervariability. CRPs have been successfully applied as stable scaffolds for molecular grafting, a protein engineering process in which cysteine-rich structures provide higher thermodynamic and metabolic stability to an epitope and acquire new biological function(s). This review describes the successes and limitations of seven cysteine-rich scaffolds, their bioactive epitopes, and the resulting grafted peptides.
Asunto(s)
Cisteína/química , Péptidos/metabolismo , Ingeniería de Proteínas , Animales , Ciclotidas/química , Ciclotidas/genética , Ciclotidas/metabolismo , Defensinas/química , Defensinas/genética , Defensinas/metabolismo , Péptidos/química , Péptidos/genética , Péptidos Cíclicos/química , Péptidos Cíclicos/genética , Péptidos Cíclicos/metabolismo , Estabilidad Proteica , Toxinas Biológicas/química , Toxinas Biológicas/genética , Toxinas Biológicas/metabolismoRESUMEN
There has been a spurt in the spread of microbial resistance to antibiotics due to indiscriminate use of antimicrobial agents in human medicine, agriculture, and animal husbandry. It has been realized that conventional antibiotic therapy would be less effective in the coming decades and more emphasis should be given for the development of novel antiinfective therapies. Cysteine rich peptides (CRPs) are broad-spectrum antimicrobial agents that modulate the innate immune system of different life forms such as bacteria, protozoans, fungi, plants, insects, and animals. These are also expressed in several plant tissues in response to invasion by pathogens, and play a crucial role in the regulation of plant growth and development. The present work explores the importance of CRPs as potent antimicrobial agents, which can supplement and/or replace the conventional antibiotics. Different plant parts of diverse plant species showed the presence of antimicrobial peptides (AMPs), which had significant structural and functional diversity. The plant-derived AMPs exhibited potent activity toward a range of plant and animal pathogens, protozoans, insects, and even against cancer cells. The cysteine-rich AMPs have opened new avenues for the use of plants as biofactories for the production of antimicrobials and can be considered as promising antimicrobial drugs in biotherapeutics.
Asunto(s)
Antiinfecciosos/farmacología , Cisteína/química , Plantas/química , Proteínas Citotóxicas Formadoras de Poros/farmacología , Secuencia de Aminoácidos , Animales , Bacterias/efectos de los fármacos , Humanos , Proteínas Citotóxicas Formadoras de Poros/química , Estructura Secundaria de ProteínaRESUMEN
Cysteine-rich peptides (CRPs) play an important role in plant physiology. However, their role in resistance induced by biogenic elicitors remains poorly understood. Using whole-genome transcriptome sequencing and our CRP search algorithm, we analyzed the repertoire of CRPs in tomato Solanum lycopersicum L. in response to Fusarium oxysporum infection and elicitors from F. sambucinum. We revealed 106 putative CRP transcripts belonging to different families of antimicrobial peptides (AMPs), signaling peptides (RALFs), and peptides with non-defense functions (Major pollen allergen of Olea europaea (Ole e 1 and 6), Maternally Expressed Gene (MEG), Epidermal Patterning Factor (EPF)), as well as pathogenesis-related proteins of families 1 and 4 (PR-1 and 4). We discovered a novel type of 10-Cys-containing hevein-like AMPs named SlHev1, which was up-regulated both by infection and elicitors. Transcript profiling showed that F. oxysporum infection and F. sambucinum elicitors changed the expression levels of different overlapping sets of CRP genes, suggesting the diversification of functions in CRP families. We showed that non-specific lipid transfer proteins (nsLTPs) and snakins mostly contribute to the response of tomato plants to the infection and the elicitors. The involvement of CRPs with non-defense function in stress reactions was also demonstrated. The results obtained shed light on the mode of action of F. sambucinum elicitors and the role of CRP families in the immune response in tomato.
Asunto(s)
Cisteína , Resistencia a la Enfermedad/genética , Péptidos/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Solanum lycopersicum/genética , Solanum lycopersicum/microbiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Biología Computacional/métodos , Secuencia Conservada , Cisteína/química , Cisteína/genética , Resistencia a la Enfermedad/inmunología , Perfilación de la Expresión Génica , Solanum lycopersicum/inmunología , Modelos Moleculares , Péptidos/química , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformación Proteica , TranscriptomaRESUMEN
The cross-strand disulfides (CSDs) found in ß-hairpin antimicrobial peptides (ß-AMPs) show a unique disulfide geometry that is characterized by unusual torsion angles and a short Cα-Cα distance. While the sequence and disulfide bond connectivity of disulfide-rich peptides is well studied, much less is known about the disulfide geometry found in CSDs and their role in the stability of ß-AMPs. To address this, we solved the nuclear magnetic resonance (NMR) structure of the ß-AMP gomesin (Gm) at 278, 298, and 310 K, examined the disulfide bond geometry of over 800 disulfide-rich peptides, and carried out extensive molecular dynamics (MD) simulation of the peptides Gm and protegrin. The NMR data suggests Cα-Cα distances characteristic for CSDs are independent of temperature. Analysis of disulfide-rich peptides from the Protein Data Bank revealed that right-handed and left-handed rotamers are equally likely in CSDs. The previously reported preference for right-handed rotamers was likely biased by restricting the analysis to peptides and proteins solved using X-ray crystallography. Furthermore, data from MD simulations showed that the short Cα-Cα distance is critical for the stability of these peptides. The unique disulfide geometry of CSDs poses a challenge to biomolecular force fields and to retain the stability of ß-hairpin fold over long simulation times, restraints on the torsion angles might be required.
Asunto(s)
Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Disulfuros/química , Animales , Antibacterianos/metabolismo , Péptidos Catiónicos Antimicrobianos/metabolismo , Disulfuros/metabolismo , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Arañas/química , Estereoisomerismo , Temperatura , TermodinámicaRESUMEN
The formation of symbiotic nodule cells in Medicago truncatula is driven by successive endoreduplication cycles and transcriptional reprogramming in different temporal waves including the activation of more than 600 cysteine-rich NCR genes expressed only in nodules. We show here that the transcriptional waves correlate with growing ploidy levels and have investigated how the epigenome changes during endoreduplication cycles. Differential DNA methylation was found in only a small subset of symbiotic nodule-specific genes, including more than half of the NCR genes, whereas in most genes DNA methylation was unaffected by the ploidy levels and was independent of the genes' active or repressed state. On the other hand, expression of nodule-specific genes correlated with ploidy-dependent opening of the chromatin as well as, in a subset of tested genes, with reduced H3K27me3 levels combined with enhanced H3K9ac levels. Our results suggest that endoreduplication-dependent epigenetic changes contribute to transcriptional reprogramming in the differentiation of symbiotic cells.
Asunto(s)
Epigenómica , Regulación de la Expresión Génica de las Plantas/fisiología , Genoma de Planta , Medicago truncatula/genética , Ploidias , Sinorhizobium/fisiología , Perfilación de la Expresión Génica , Medicago truncatula/metabolismo , Nódulos de las Raíces de las Plantas/metabolismo , SimbiosisRESUMEN
In legume nodules, rhizobia differentiate into nitrogen-fixing forms called bacteroids, which are enclosed by a plant membrane in an organelle-like structure called the symbiosome. In the Inverted Repeat-Lacking Clade (IRLC) of legumes, this differentiation is terminal due to irreversible loss of cell division ability and is associated with genome amplification and different morphologies of the bacteroids that can be swollen, elongated, spherical, and elongated-branched, depending on the host plant. In Medicago truncatula, this process is orchestrated by nodule-specific cysteine-rich peptides (NCRs) delivered into developing bacteroids. Here, we identified the predicted NCR proteins in 10 legumes representing different subclades of the IRLC with distinct bacteroid morphotypes. Analysis of their expression and predicted sequences establishes correlations between the composition of the NCR family and the morphotypes of bacteroids. Although NCRs have a single origin, their evolution has followed different routes in individual lineages, and enrichment and diversification of cationic peptides has resulted in the ability to impose major morphological changes on the endosymbionts. The wide range of effects provoked by NCRs such as cell enlargement, membrane alterations and permeabilization, and biofilm and vesicle formation is dependent on the amino acid composition and charge of the peptides. These effects are strongly influenced by the rhizobial surface polysaccharides that affect NCR-induced differentiation and survival of rhizobia in nodule cells.
Asunto(s)
Proteínas Bacterianas/metabolismo , Medicago truncatula/microbiología , Péptidos/metabolismo , Rhizobiaceae/metabolismo , Rizoma/microbiología , Simbiosis/fisiología , Proteínas Bacterianas/genética , Péptidos/genética , Rhizobiaceae/genéticaRESUMEN
Many of the small, cysteine-rich ion-channel modulatory peptides found in Cnidaria are distantly related to vertebrate defensins (of the trans-defensin superfamily). Transcriptomic and proteomic studies of the endemic Australian speckled sea anemone (Oulactis sp.) yielded homologous peptides to known defensin sequences. We extended these data using existing and custom-built hidden Markov models to extract defensin-like families from the transcriptomes of seven endemic Australian cnidarian species. Newly sequenced transcriptomes include three species of Actiniaria (true sea anemones); the speckled anemone (Oulactis sp.), Oulactis muscosa, Dofleinia cf. armata and a species of Corallimorpharia, Rhodactis sp. We analyzed these novel defensin-like sequences along with published homologues to study the evolution of their physico-chemical properties in vertebrate and invertebrate fauna. The cnidarian trans-defensins form a distinct cluster within the chemical space of the superfamily, with a unique set of motifs and biophysical properties. This cluster contains identifiable subgroups, whose distribution in chemical space also correlates with the divergent evolution of their structures. These sequences, currently restricted to cnidarians, form an evolutionarily distinct clade within the trans-defensin superfamily.
Asunto(s)
Defensinas/genética , Anémonas de Mar/genética , Transcriptoma , Animales , Cisteína/química , Cisteína/genética , Defensinas/química , Evolución Molecular , Modelos Moleculares , Filogenia , Conformación Proteica , Anémonas de Mar/químicaRESUMEN
Long-term cadmium intake can be very dangerous to human health due to its toxic effects. Although people can be contaminated with this element from different sources, contaminated food is probably the most important one. Foods such as vegetables and fruits can become contaminated with cadmium existing in soils, irrigation water, or chemical fertilizers. Some plants produce an excess of cysteine-rich peptides (CRp) when affected by high concentrations of heavy metals such as cadmium, thus indicating the presence of this type of contamination. Among these plants is tamarillo (Solanum betaceum), which is locally known as "tree tomato". This is a native plant widely consumed in the Ecuadorian Andes because of its abundance, low cost, and high content of vitamin C and fiber. The fact that Solanum betaceum produces CRp upon contamination with heavy metals means that this plant may be able to accumulate heavy metals. If this is the case, the plant can possibly be used as an indicator of metal pollution. The main goals of the present work were to evaluate the possibility of using Solanum betaceum as an indicator of metal contamination in plants and to examine its capability to accumulate metals. Both goals were met by determination of the amounts of CRp produced by Solanum betaceum cells cultivated in vitro in the laboratory under controlled conditions in the presence of different concentrations of cadmium. The CRp determination was carried out by means of electrogeneration of iodine in an iodide solution containing reduced glutathione as a biological thiol model. Solanum betaceum cells were grown in a Murashige and Skoog solution enriched with a 30 g L-1 sugar aqueous solution and 1 mg L-1 2,4-dichlorophenoxyacetic acid. The results of these experiments confirmed the following: (1) CRp production is a function of the amount of cadmium present as a contaminant up to a limiting value after which cell apoptosis occurs; (2) Solanum betaceum accumulates cadmium; (3) the analytical method used is appropriate for CRp determination; and (4) CRp determination is a valid alternative to detect contamination by heavy metals in plants.
Asunto(s)
Cadmio/análisis , Cadmio/metabolismo , Cisteína/análisis , Técnicas Electroquímicas , Péptidos/análisis , Solanum/química , Solanum/metabolismo , Metales Pesados/análisis , Metales Pesados/metabolismo , Células VegetalesRESUMEN
We report on two new electrochemical sensors which, coupled to differential pulse voltammetry, constitutes a useful tool for diagnosis of heavy metal pollution. The electrochemical sensors AgHgNf/Cu and the AgBiNf/Cu were obtained by deposition of bimetallic particles of AgHg or AgBi on copper electrodes covered with a Nafion (Nf) film, respectively. Micrographs of the electrode's surface showed evenly scattered bimetallic particles, with an approximate diameter of 150 nm, embedded in the Nafion (Nf) film. In order to test the electrodes, the hydrogen evolution signal according to the Brdicka reaction was measured for the determination of cysteine-rich peptides (CRp) produced by plants. To check the accuracy of the electrodes, real samples of Nicotiana tabacum cells exposed to cytotoxic levels of cadmium were tested. The AgHgNf/Cu electrode produced detection limits (DLs) of 0.088 µmol L-1 for Cysteine and 0.139µmol L-1 for Glutathione, while for the AgBiNf/Cu electrode DLs were 0.41 µmol L-1 for cysteine and 0.244 µmol L-1 for glutathione. Thus, the new electrodes could be a useful analytical electrochemical system very convenient for fieldwork. The electrodes were capable of direct, accurate, and sensitive detection of synthesized peptides, despite the complex matrix where the Nicotiana tabacum cells were grown.
Asunto(s)
Técnicas Biosensibles , Cobre , Electrodos , Nanopartículas del Metal , Péptidos , Cadmio/toxicidad , Cobre/química , Cisteína/química , Glutatión/análisis , Nanopartículas del Metal/química , Estructura Molecular , Péptidos/análisis , Péptidos/química , Nicotiana/química , Nicotiana/efectos de los fármacos , Nicotiana/metabolismoRESUMEN
Grafting a bioactive peptide onto a disulfide-rich scaffold is a promising approach to improve its structure and metabolic stability. The ginkgo plant-derived ß-ginkgotide ß-gB1 is a highly unusual molecule: Small, hyperdisulfide, and found only in selected ancient plants. It also contains a conserved 16-amino-acid core with three interlocking disulfides, as well as a six-amino-acid inter-cysteine loop 2 suitable for grafting peptide epitopes. However, very little is known about this recently-discovered family of molecules. Here, we report the biophysical and functional characterizations of the ß-ginkgotide ß-gB1 from G. biloba. A circular dichroism spectroscopy analysis at 90 °C and proteolytic treatments of ß-gB1 supported that it is hyperstable. Data mining revealed that the ß-gB1 loop 2 contains the canonical LC3 interacting region (LIR) motif crucial for selective autophagy. Cell-based assays and pull-down experiments showed that ß-gB1 is an adaptogen, able to maintain cellular homeostasis through induced autophagosomes formation and to protect cells by targeting intracellular proteins from stress-mediated damage against hypoxia and the hypoxia-reoxygenation of induced cell death. This is the first report of an LIR-containing peptide natural product. Together, our results suggest that the plant-derived ß-ginkgotide is cytoprotective, capable of targeting intracellular proteins, and holds promise as a hyperdisulfide scaffold for engineering peptidyl therapeutics with enhanced structural and metabolic stability.
Asunto(s)
Citoprotección/efectos de los fármacos , Ginkgo biloba/química , Péptidos , Proteínas de Plantas , Animales , Autofagosomas/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Línea Celular , Ratones , Estructura Molecular , Péptidos/química , Péptidos/farmacología , Proteínas de Plantas/química , Proteínas de Plantas/farmacología , RatasRESUMEN
BACKGROUND: Jasmintides jS1 and jS2 from Jasminum sambac were previously identified as a novel family of cysteine-rich peptides (CRPs) with an unusual disulfide connectivity. However, very little else is known about jasmintides, particularly their molecular diversity and functions. Here, we report the discovery and characterization of a novel suite of jasmintides from J. sambac using transcriptomic, peptidomic, structural and functional tools. RESULTS: Transcriptomic analysis of leaves, flowers and roots revealed 14 unique jasmintide precursors, all of which possess a three-domain architecture comprising a signal peptide, a pro-domain and a mature jasmintide domain. Peptidomic analysis, using fractionated mixtures of jasmintides and chemical derivatization of cysteine to pseudolysine, trypsin digestion and MS/MS sequencing, revealed an additional 86 jasmintides, some of which were post-translationally modified. NMR analysis showed that jasmintide jS3 has three anti-parallel ß-strands with a three-disulfide connectivity of CysI-CysV, CysII-CysIV and CysIII-CysVI, which is similar to jasmintide jS1. Jasmintide jS3 was able to withstand thermal, acidic and enzymatic degradation and, importantly, exhibited antifeedant activity against mealworm Tenebrio molitor. CONCLUSION: Together, this study expands the existing library of jasmintides and furthers our understanding of the molecular diversity and cystine framework of CRPs as scaffolds and tools for engineering peptides targeting pests.
Asunto(s)
Jasminum/metabolismo , Péptidos/fisiología , Proteínas de Plantas/fisiología , Flores/metabolismo , Perfilación de la Expresión Génica , Espectroscopía de Resonancia Magnética , Péptidos/química , Hojas de la Planta/metabolismo , Proteínas de Plantas/química , Raíces de Plantas/metabolismo , Precursores de Proteínas/metabolismoRESUMEN
Cone snails are predatory marine gastropods characterized by a sophisticated venom apparatus responsible for the biosynthesis and delivery of complex mixtures of cysteine-rich toxin peptides. These conotoxins fold into small highly structured frameworks, allowing them to potently and selectively interact with heterologous ion channels and receptors. Approximately 2,000 toxins from an estimated number of >70,000 bioactive peptides have been identified in the genus Conus to date. Here, we describe a high-resolution interrogation of the transcriptomes (available at www.ddbj.nig.ac.jp) and proteomes of the diverse compartments of the Conus episcopatus venom apparatus. Using biochemical and bioinformatic tools, we found the highest number of conopeptides yet discovered in a single Conus specimen, with 3,305 novel precursor toxin sequences classified into 9 known superfamilies (A, I1, I2, M, O1, O2, S, T, Z), and identified 16 new superfamilies showing unique signal peptide signatures. We were also able to depict the largest population of venom peptides containing the pharmacologically active C-C-CC-C-C inhibitor cystine knot and CC-C-C motifs (168 and 44 toxins, respectively), as well as 208 new conotoxins displaying odd numbers of cysteine residues derived from known conotoxin motifs. Importantly, six novel cysteine-rich frameworks were revealed which may have novel pharmacology. Finally, analyses of codon usage bias and RNA-editing processes of the conotoxin transcripts demonstrate a specific conservation of the cysteine skeleton at the nucleic acid level and provide new insights about the origin of sequence hypervariablity in mature toxin regions.