RESUMEN
The legume albumin-1 gene family, arising after nodulation, encodes linear a- and b-chain peptides for nutrient storage and defense. Intriguingly, in one prominent legume, Clitoria ternatea, the b-chains are replaced by domains producing ultra-stable cyclic peptides called cyclotides. The mechanism of this gene hijacking is until now unknown. Cyclotides require recruitment of ligase-type asparaginyl endopeptidases (AEPs) for maturation (cyclization), necessitating co-evolution of two gene families. Here we compare a chromosome-level C. ternatea genome with grain legumes to reveal an 8 to 40-fold expansion of the albumin-1 gene family, enabling the additional loci to undergo diversification. Iterative rounds of albumin-1 duplication and diversification create four albumin-1 enriched genomic islands encoding cyclotides, where they are physically grouped by similar pI and net charge values. We identify an ancestral hydrolytic AEP that exhibits neofunctionalization and multiple duplication events to yield two ligase-type AEPs. We propose cyclotides arise by convergence in C. ternatea where their presence enhances defense from biotic attack, thus increasing fitness compared to lineages with linear b-chains and ultimately driving the replacement of b-chains with cyclotides.
Asunto(s)
Proteínas de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Clitoria/metabolismo , Clitoria/genética , Ciclotidas/genética , Ciclotidas/química , Ciclotidas/metabolismo , Fijación del Nitrógeno/genética , Evolución Molecular , Ciclización , Filogenia , Familia de Multigenes , Duplicación de Gen , Fabaceae/genética , Fabaceae/metabolismo , Albúminas/metabolismo , Albúminas/genética , Genoma de Planta , Cisteína EndopeptidasasRESUMEN
The cyclic peptides, cyclotides, are identified mostly with 29-31-aa (amino acid residues) but rarely with ≥ 34-aa in plants. Viola philippica is a well-known medicinal plant but a rare metallophyte with cyclotides. A hypothesis was hence raised that the potential novel 34-aa cyclotide of Viola philippica would clearly broaden the structural and functional diversities of plant cyclotides. After homology-cloning the cyclotide precursor gene of VpCP5, a 34-aa cyclotide (viphi I) was identified to be larger than 22 other known cyclotides in V. philippica. It had a chimeric primary structure, due to its unusual loop structures (8 residues in loop 2 and 6 residues in loop 5) and aa composition (3 E and 5â¯R), by using phylogenetic analyses and an in-house cyclotide analysis tool, CyExcel_V1. A plasmid pCYC-viphi_I and a lab-used recombinant process were specially constructed for preparing viphi I. Typically, 0.12 or 0.25â¯mgâ¯ml-1 co-exposed viphi I could significantly remain cell activities with elevating Cd2+-exposed doses from 10-8 to 10-6 molâ¯l-1 in MCF7 cells. In the model nematode Caenorhabditis elegans, IC50 values of viphi I to inhibit adult ratios and to induce death ratios, were 184.7 and 585.9⯵gâ¯ml-1, respectively; the median lifespan of adult worms decreased from 14 to 2 d at viphi I doses ranging from 0.05 to 2â¯mgâ¯ml-1. Taken together, the newly identified viphi I exhibits functional potentials against cadmium and nematodes, providing new insights into structural and functional diversity of chimeric cyclotides in plants.
Asunto(s)
Cadmio , Ciclotidas , Viola , Animales , Ciclotidas/genética , Ciclotidas/química , Viola/genética , Viola/metabolismo , Secuencia de Aminoácidos , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Caenorhabditis elegans/genética , Caenorhabditis elegans/efectos de los fármacos , Nematodos/efectos de los fármacos , Nematodos/genéticaRESUMEN
Cyclotides are plant-derived disulfide-rich cyclic peptides that have a natural function in plant defense and potential for use as agricultural pesticides. Because of their highly constrained topology, they are highly resistant to thermal, chemical, or enzymatic degradation. However, the stability of cyclotides at alkaline pH for incubation times of longer than a few days is poorly studied but important since these conditions could be encountered in the environment, during storage or field application as insecticides. In this study, kalata B1 (kB1), the prototypical cyclotide, was engineered to improve its long-term stability and retain its insecticidal activity via point mutations. We found that substituting either Asn29 or Gly1 to lysine or leucine increased the stability of kB1 by twofold when incubated in an alkaline buffer (pH = 9.0) for 7 days, while retaining its insecticidal activity. In addition, when Gly1 was replaced with lysine or leucine, the mutants could be cyclized using an asparaginyl endopeptidase, in vitro with a yield of â¼90% within 5 min. These results demonstrate the potential to manufacture kB1 mutants with increased stability and insecticidal activity recombinantly or in planta. Overall, the discovery of mutants of kB1 that have enhanced stability could be useful in leading to longer term activity in the field as bioinsecticides.
Asunto(s)
Ciclotidas , Insecticidas , Oldenlandia , Ciclotidas/genética , Ciclotidas/farmacología , Ciclotidas/química , Insecticidas/química , Insecticidas/farmacología , Leucina , Lisina/genética , Mutagénesis , Proteínas de Plantas/metabolismo , Oldenlandia/química , Estabilidad Proteica , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacosRESUMEN
Multiple sclerosis (MS) is a debilitating disease that requires prolonged treatment with often severe side effects. One experimental MS therapeutic currently under development is a single amino acid mutant of a plant peptide termed kalata B1, of the cyclotide family. Like all cyclotides, the therapeutic candidate [T20K]kB1 is highly stable as it contains a cyclic backbone that is cross-linked by three disulfide bonds in a knot-like structure. This stability is much sought after for peptide drugs, which despite exquisite selectivity for their targets, are prone to rapid degradation in human serum. In preliminary investigations, it was found that [T20K]kB1 retains oral activity in experimental autoimmune encephalomyelitis, a model of MS in mice, thus opening up opportunities for oral dosing of the peptide. Although [T20K]kB1 can be synthetically produced, a recombinant production system provides advantages, specifically for reduced scale-up costs and reductions in chemical waste. In this study, we demonstrate the capacity of the Australian native Nicotiana benthamiana plant to produce a structurally identical [T20K]kB1 to that of the synthetic peptide. By optimizing the co-expressed cyclizing enzyme, precursor peptide arrangements, and transgene regulatory regions, we demonstrate a [T20K]kB1 yield in crude peptide extracts of ~ 0.3 mg/g dry mass) in whole plants and close to 1.0 mg/g dry mass in isolated infiltrated leaves. With large-scale plant production facilities coming on-line across the world, the sustainable and cost-effective production of cyclotide-based therapeutics is now within reach.
Asunto(s)
Ciclotidas , Esclerosis Múltiple , Ratones , Humanos , Animales , Ciclotidas/genética , Ciclotidas/química , Ciclotidas/metabolismo , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/genética , Australia , Nicotiana/genética , Nicotiana/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
Viola odorata L., known for its pharmacological properties, produces a plethora of structurally stable peptides called cyclotides. Cyclotides are macrocyclic peptides with a unique topology containing a cyclic cystine knot motif. The objective of the present study was to identify the precursor sequences and respective cyclotide domains from the petiole tissue of V. odorata. The study is based on the isolation, identification, and characterization of the cyclic peptides using LC-MS/MS followed by database searching and processing. Our study detected 47 precursor sequences encoded for 15 reported cyclotides, 4 putative novel cyclotides, and 3 acyclotides from the petiole tissue. The novel sequences identified were based on the hydrophobic nature, disulfide bonds, conserved cysteine residues, and presence of cyclic peptide backbone. Four putative novel and three acyclotides were also characterized for their sequence and subfamilies. A protein diversity wheel was used to reveal the variation in the amino acid sequence and cysteine residue conservation in the isolated cyclotides. The results provide information about the number of cyclotides and acyclotides from the petiole tissue and their sequence diversity, which may constitute novel tools for future research on this plant species.
Asunto(s)
Ciclotidas , Viola , Ciclotidas/genética , Viola/genética , Cromatografía Liquida , Cisteína , Espectrometría de Masas en TándemRESUMEN
Cyclotides have a wide range of bioactivities relevant for agricultural and pharmaceutical applications. This large family of naturally occurring macrocyclic peptides is divided into three subfamilies, with the bracelet subfamily being the largest and comprising the most potent cyclotides reported to date. However, attempts to harness the natural bioactivities of bracelet cyclotides and engineer-optimized analogs have been hindered by a lack of understanding of the structural and functional role of their constituent residues, which has been challenging because bracelet cyclotides are difficult to produce synthetically. We recently established a facile strategy to make the I11L mutant of cyclotide hyen D that is as active as the parent peptide, enabling the subsequent production of a series of variants. In the current study, we report an alanine mutagenesis structure-activity study of [I11L] hyen D to probe the role of individual residues on peptide folding using analytical chromatography, on molecular function using surface plasmon resonance, and on therapeutic potential using cytotoxicity assays. We found that Glu-6 and Thr-15 are critical for maintaining the structure of bracelet cyclotides and that hydrophobic residues in loops 2 and 3 are essential for membrane binding and cytotoxic activity, findings that are distinct from the structural and functional characteristics determined for other cyclotide subfamilies. In conclusion, this is the first report of a mutagenesis scan conducted on a bracelet cyclotide, offering insights into their function and supporting future efforts to engineer bracelet cyclotides for biotechnological applications.
Asunto(s)
Ciclotidas , Ciclotidas/química , Ciclotidas/genética , Ciclotidas/toxicidad , Interacciones Hidrofóbicas e Hidrofílicas , Mutagénesis , Unión Proteica/genéticaRESUMEN
Cyclotides are a class of ribosomally-synthesized plant peptides that function in plants as a defense against insects and fungal pathogens. Their unique structure comprises a cyclized peptide backbone threaded by three disulfide bonds, that imparts structural stability, a desirable quality for peptide-based therapeutics or insecticides. Producing these peptides synthetically is challenging due to the amount of chemical waste produced and inefficiency of folding certain cyclotides. Thus, it is desirable to develop a means to access cyclotide biosynthesis in their native hosts, cultured in defined conditions, at both laboratory and commercial scale. Here we developed suspension cell cultures from two species previously unexplored for cyclotide production in suspension cells, Clitoria ternatea L., Hybanthus enneaspermus F. Muell., as well as with Oldenlandia affinis (Roem. & Schult.) DC., a species reported previously to accumulate cyclotides in cell suspensions. We assessed the growth rate, cyclotide production and gene expression for the various species. We found that while many cyclotides had reduced expression in Oldenlandia affinis suspension cells when compared to plant organs, those in Clitoria ternatea and Hybanthus enneaspermus maintained or increased expression levels. The cyclotides that continued to be expressed in suspension cultures shared similar sequence and biophysical properties as a group, regardless of phylogenetic origin of the host. Of particular interest was the discovery of inducibility by NaCl of cyclotide expression in O. affinis, cycloviolacin O2 expression in O. affinis, and the scale up of cycloviolacin O2 production in H. enneaspermus. Together the results presented here highlight the utility of plant cell suspensions as modalities to produce macrocyclic peptides.
Asunto(s)
Ciclotidas , Secuencia de Aminoácidos , Ciclotidas/genética , Agricultura Molecular , Filogenia , Células Vegetales/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , SuspensionesRESUMEN
Legumains, also known as asparaginyl endopeptidases (AEPs), cleave peptide bonds after Asn/Asp (Asx) residues. In plants, certain legumains also have ligase activity that catalyzes biosynthesis of Asx-containing cyclic peptides. An example is the biosynthesis of MCoTI-I/II, a squash family-derived cyclic trypsin inhibitor, which involves splicing to remove the N-terminal prodomain and then N-to-C-terminal cyclization of the mature domain. To identify plant legumains responsible for the maturation of these cyclic peptides, we have isolated and characterized a legumain involved in splicing, McPAL1, from Momordica cochinchinensis (Cucurbitaceae) seeds. Functional studies show that recombinantly expressed McPAL1 displays a pH-dependent, trimodal enzymatic profile. At pH 4 to 6, McPAL1 selectively catalyzed Asp-ligation and Asn-cleavage, but at pH 6.5 to 8, Asn-ligation predominated. With peptide substrates containing N-terminal Asn and C-terminal Asp, such as is found in precursors of MCoTI-I/II, McPAL1 mediates proteolysis at the Asn site and then ligation at the Asp site at pH 5 to 6. Also, McPAL1 is an unusually stable legumain that is tolerant of heat and high pH. Together, our results support that McPAL1 is a splicing legumain at acidic pH that can mediate biosynthesis of MCoTI-I/II. We purport that the high thermal and pH stability of McPAL1 could have applications for protein engineering.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Momordica/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Ciclización , Ciclotidas/genética , Ciclotidas/metabolismo , Cisteína Endopeptidasas/análisis , Cisteína Endopeptidasas/genética , Modelos Moleculares , Momordica/química , Momordica/genética , Péptidos Cíclicos/genética , Péptidos Cíclicos/metabolismo , Proteínas de Plantas/análisis , Proteínas de Plantas/genética , Ingeniería de Proteínas , TranscriptomaRESUMEN
Cyclotides are cyclic peptides produced by plants. Due to their insecticidal properties, they are thought to be involved in host defense. Violets produce complex mixtures of cyclotides, that are characteristic for each species and variable in different environments. Herein, we utilized mass spectrometry (LC-MS, MALDI-MS), transcriptomics and biological assays to investigate the diversity, differences in cyclotide expression based on species and different environment, and antimicrobial activity of cyclotides found in violets from the Canary Islands. A wide range of different habitats can be found on these islands, from subtropical forests to dry volcano peaks at high altitudes. The islands are inhabited by the endemic Viola palmensis, V. cheiranthifolia, V. anagae and the common V. odorata. The number of cyclotides produced by a given species varied in plants from different environments. The highest diversity was noted in V. anagae which resides in subtropical forest and the lowest in V. cheiranthifolia from the Teide volcano. Transcriptome sequencing and LC-MS were used to identify 23 cyclotide sequences from V. anagae. Cyclotide extracts exhibited antifungal activities with the lowest minimal inhibitory concentrations noted for V. anagae (15.62 µg/ml against Fusarium culmorum). The analysis of the relative abundance of 30 selected cyclotides revealed patterns characteristic to both species and populations, which can be the result of genetic variability or environmental conditions in different habitats. The current study exemplifies how plants tailor their host defense peptides for various habitats, and the usefulness of cyclotides as markers for chemosystematics.
Asunto(s)
Ciclotidas/inmunología , Fusarium/inmunología , Proteínas de Plantas/inmunología , Viola/inmunología , Cromatografía Liquida , Ciclotidas/análisis , Ciclotidas/genética , Ciclotidas/metabolismo , Perfilación de la Expresión Génica , Geografía , Interacciones Huésped-Patógeno/inmunología , Pruebas de Sensibilidad Microbiana , Proteínas de Plantas/análisis , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , España , Especificidad de la Especie , Viola/clasificación , Viola/microbiologíaRESUMEN
Cyclotides are an extremely stable class of peptides, ubiquitously distributed in Violaceae. The aim of the present study was to investigate the presence of cyclotides in Sri Lankan Violaceae plants, using combined tools of transcriptomics and mass spectrometry. New cyclotides were discovered for the first time in the wild flora of Sri Lanka, within Viola betonicifolia, a plant used in traditional medicine as an antimicrobial. Plant extracts prepared in small scale from Viola betonicifolia were first subjected to LC-MS analysis. Subsequent transcriptome de novo sequencing of Viola betonicifolia uncovered 25 new (vibe 1-25) and three known (varv A/kalata S, viba 17, viba 11) peptide sequences from Möbius and bracelet cyclotide subfamilies as well as hybrid cyclotides. Among the transcripts, putative linear acyclotide sequences (vibe 4, vibe 10, vibe 11 and vibe 22) that lack a conserved asparagine or aspartic acid vital for cyclisation were also present. Four asparagine endopeptidases (AEPs), VbAEP1-4 were found within the Viola betonicifolia transcriptome, including a peptide asparaginyl ligase (PAL), potentially involved in cyclotide backbone cyclisation, showing >93% sequence homology to Viola yedoensis peptide asparaginyl ligases, VyPALs. In addition, we identified two protein disulfide isomerases (PDIs), VbPDI1-2, likely involved in cyclotide oxidative folding, having high sequence homology (>74%) with previously reported Rubiaceae and Violaceae PDIs. The current study highlights the ubiquity of cyclotides in Violaceae as well as the utility of transcriptomic analysis for cyclotides and their putative processing enzyme discovery. The high variability of cyclotide sequences in terms of loop sizes and residues in V. betonicifolia showcase the cyclotide structure as an adaptable scaffold as well as their importance as a combinatorial library, implicated in plant defense.
Asunto(s)
Ciclotidas , Viola , Secuencia de Aminoácidos , Ciclotidas/genética , Espectrometría de Masas , Proteínas de Plantas/metabolismo , Sri Lanka , Transcriptoma , Viola/genética , Viola/metabolismoRESUMEN
Cyclic disulfide-rich peptides have attracted significant interest in drug development and biotechnology. Here, we describe a protocol for producing cyclic peptide precursors in Pichia pastoris that undergo in vitro enzymatic maturation into cyclic peptides using recombinant asparaginyl endopeptidases (AEPs). Peptide precursors are expressed with a C-terminal His tag and secreted into the media, enabling facile purification by immobilized metal affinity chromatography. After AEP-mediated cyclization, cyclic peptides are purified by reverse-phase high-performance liquid chromatography and characterized by mass spectrometry, peptide mass fingerprinting, NMR spectroscopy, and activity assays. We demonstrate the broad applicability of this protocol by generating cyclic peptides from three distinct classes that are either naturally occurring or synthetically backbone cyclized, and range in size from 14 amino acids with one disulfide bond, to 34 amino acids with a cystine knot comprising three disulfide bonds. The protocol requires 14 d to identify and optimize a high-expressing Pichia clone in small-scale cultures (24 well plates or 50 mL tubes), after which large-scale production in a bioreactor and peptide purification can be completed in 10 d. We use the cyclotide Momordica cochinchinensis trypsin inhibitor II as an example. We also include a protocol for recombinant AEP production in Escherichia coli as AEPs are emerging tools for orthogonal peptide and protein ligation. We focus on two AEPs that preferentially cyclize different peptide precursors, namely an engineered AEP with improved catalytic efficiency [C247A]OaAEP1b and the plant-derived MCoAEP2. Rudimentary proficiency and equipment in molecular biology, protein biochemistry and analytical chemistry are needed.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Biosíntesis de Péptidos/efectos de los fármacos , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Biotecnología , Ciclización , Ciclotidas/química , Ciclotidas/genética , Ciclotidas/metabolismo , Cisteína Endopeptidasas/farmacología , Disulfuros , Modelos Moleculares , Péptidos/metabolismo , Péptidos Cíclicos/química , Saccharomycetales/metabolismoRESUMEN
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
Cyclotides are plant-derived mini-proteins of 28 - 37 amino acids. They have a characteristic head-to-tail cyclic backbone and three disulfide cross-linkages formed by six highly conserved cysteine residues, creating a unique knotted ring structure, known as a cyclic cystine knot (CCK) motif. The CCK topology confers immense stability to cyclotides with resistance to thermal and enzymatic degradation. Native cyclotides are of interest due to their multiple biological activities with several potential applications in agricultural (e.g. biopesticides, antifungal) and pharmaceutical (e.g. anti-HIV, cytotoxic to tumor cells) sectors. The most recent application of insecticidal activity of cyclotides is the commercially available biopesticidal spray known as 'Sero X' for cotton crops. Cyclotides have a general mode of action and their potency of bioactivity is determined through their binding ability, pore formation and disruption of the target biological membranes. Keeping in view the important potential applications of biological activities of cyclotides and the lack of an extensive and analytical compilation of bioactive cyclotides, the present review systematically describes eight major biological activities of the native cyclotides from four angiosperm families viz. Fabaceae, Poaceae, Rubiaceae, Violaceae. The bioactivities of 94 cytotoxic, 57 antibacterial, 44 hemolytic, 25 antifungal, 21 anti-HIV, 20 nematocidal, 10 insecticidal and 5 molluscicidal cyclotides have been comprehensively elaborated. Further, their distribution in angiosperm families, mode of action and future prospects have also been discussed.
Asunto(s)
Antiinfecciosos/química , Antineoplásicos/química , Ciclotidas/química , Proteínas de Plantas/química , Agricultura , Secuencia de Aminoácidos/genética , Antiinfecciosos/uso terapéutico , Antineoplásicos/uso terapéutico , Biofarmacia , Ciclotidas/genética , Ciclotidas/uso terapéutico , Humanos , Proteínas de Plantas/genética , Proteínas de Plantas/uso terapéuticoRESUMEN
MAIN CONCLUSION: We demonstrate the production of a structurally correct cyclotide in rice suspension cells with co-expression of a ligase-type AEP, which unlocks monocotyledons as production platforms to produce cyclotides. Cyclotides are a class of backbone-cyclic plant peptides that harbor a cystine knot composed of three disulfide bonds. These structural features make cyclotides particularly stable, and thus they have attracted significant attention for their use in biotechnological applications such as drug design. Currently, chemical synthesis is the predominant strategy to produce cyclotides for research purposes. However, synthetic production becomes costly both economically and environmentally at large scale. Plants offer an attractive alternative to chemical synthesis because of their lower cost and environmental footprint. In this study, rice suspension cells were engineered to produce the prototypical cyclotide, kalata B1 (kB1), a cyclotide with insecticidal properties from the African plant Oldenlandia affinis. Engineered rice cells produced structurally validated kB1 at yields of 64.21 µg/g (DW), which was dependent on the co-expression of a peptide ligase-competent asparaginyl endopeptidase OaAEP1b from O. affinis. Without co-expression, kB1 was predominantly produced as linear peptide. Through HPLC-MS co-elution, reduction, alkylation, enzymatic digestion, and proton NMR analysis, kB1 produced in rice was shown to be structurally identical to native kB1. This study reports the first example of an engineered plant suspension cell culture with the required molecular machinery for efficient production and cyclisation of a heterologous cyclotide.
Asunto(s)
Biotecnología , Ciclotidas , Oldenlandia , Oryza , Biotecnología/métodos , Ciclotidas/biosíntesis , Ciclotidas/genética , Oldenlandia/genética , Oryza/enzimología , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismoRESUMEN
Cyclotides are naturally occurring microproteins (≈30 residues long) present in several families of plants. All cyclotides share a unique head-to-tail circular knotted topology containing three disulfide bridges forming a cystine knot topology. Cyclotides possess high stability to chemical, physical, and biological degradation and have been reported to cross cellular membranes. In addition, naturally occurring and engineered cyclotides have shown to possess various pharmacologically relevant activities. These unique features make the cyclotide scaffold an excellent tool for the design of novel peptide-based therapeutics by using molecular evolution and/or peptide epitope grafting techniques. In this chapter, we provide protocols to recombinantly produce a natively folded cyclotide making use of a standard bacterial expression system in combination with an intein-mediated backbone cyclization with concomitant oxidative folding.
Asunto(s)
Clonación Molecular/métodos , Ciclotidas/biosíntesis , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/biosíntesis , Cromatografía de Afinidad/métodos , Cromatografía en Agarosa/métodos , Cromatografía Líquida de Alta Presión , Ciclización , Ciclotidas/química , Ciclotidas/genética , Ciclotidas/aislamiento & purificación , Cistina/química , Motivos Nodales de Cisteina , Disulfuros/química , Disulfuros/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Inteínas , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Cyclotides are a class of cyclic disulfide-rich peptides found in plants that have been adopted as a molecular scaffold for pharmaceutical applications due to their inherent stability and ability to penetrate cell membranes. For research purposes, they are usually produced and cyclized synthetically, but there are concerns around the cost and environmental impact of large-scale chemical synthesis. One strategy to improve this is to combine a recombinant production system with native enzyme-mediated cyclization. Asparaginyl endopeptidases (AEPs) are enzymes that can act as peptide ligases in certain plants to facilitate cyclotide maturation. One of these ligases, OaAEP1b, originates from the cyclotide-producing plant, Oldenlandia affinis, and can be produced recombinantly for use in vitro as an alternative to chemical cyclization of recombinant substrates. However, not all engineered cyclotides are compatible with AEP-mediated cyclization because new pharmaceutical epitopes often replace the most flexible region of the peptide, where the native cyclization site is located. Here we redesign a popular cyclotide grafting scaffold, MCoTI-II, to incorporate an AEP cyclization site located away from the usual grafting region. We demonstrate the incorporation of a bioactive peptide sequence in the most flexible region of MCoTI-II while maintaining AEP compatibility, where the two were previously mutually exclusive. We anticipate that our AEP-compatible scaffold, based on the most popular cyclotide for pharmaceutical applications, will be useful in designing bioactive cyclotides that are compatible with AEP-mediated cyclization and will therefore open up the possibility of larger scale enzyme-mediated production of recombinant or synthetic cyclotides alike.
Asunto(s)
Ciclotidas/química , Cisteína Endopeptidasas/química , Proteínas de Plantas/química , Secuencia de Aminoácidos , Ciclización , Ciclotidas/síntesis química , Ciclotidas/genética , Cisteína Endopeptidasas/genética , Escherichia coli/genética , Oldenlandia/enzimología , Proteínas de Plantas/síntesis química , Proteínas de Plantas/genética , Ingeniería de ProteínasRESUMEN
The cyclotides are the largest known family of cyclic proteins, which are found in several plant families including Violaceae. They are circular bioactive peptides consisting of 28-37 amino acids, which possess a cyclic cystine knot (CCK) motif and could be useful in biotechnology and drug design as scaffolds for peptide-based drugs. This study describes our finding of a potentially novel gene transcript from the petals of the Iranian Viola tricolor (V. tricolor) flowers. This study is based on the cDNA screening method employed for isolation of cyclotide precursor genes and in silico analysis. Our study resulted in the finding of a novel cyclotide-like precursor from V. tricolor, which is documented in the NCBI by GenBank accession number: KP065812. The in silico analysis revealed that there are lots of similar sequences in many other plant families and they all exhibit some different features from previously discovered cyclotide precursors. The differences occur particularly in the main cyclotide domain that exists without the usual CCK structure. All of these hypothetical precursors have a conserved ER-signal sequence, a Cysteine (C)-rich sequence forming two zinc finger motifs and a cyclotide-like region containing several conserved elements including two highly conserved C residues. In conclusion, using the cDNA screening method we found a potentially new cyclotide-like precursor gene and in silico studies revealed its significant characteristics that may open up a new research line on the distribution and evolution of cyclotides.
Asunto(s)
Ciclotidas/análisis , Proteínas de Plantas/análisis , Precursores de Proteínas/análisis , Viola/química , Secuencia de Aminoácidos , Ciclotidas/química , Ciclotidas/genética , Flores/química , Genes de Plantas , Irán , Hojas de la Planta/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Precursores de Proteínas/química , Precursores de Proteínas/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Molecular phylogenetics based on nucleotide sequence comparisons has profoundly influenced plant taxonomy. A comprehensive chemotaxonomical approach based on GC-MS and UHPLC-HRMS profiling was evaluated for its ability to characterize a large collection of plants all in the violet family Violaceae (nâ¯=â¯111) and thus decipher the taxonomy. A thorough identification of violets is challenging due to their natural hybridization and phenotypic variability. Phylogenetic inference performed on ribosomal internal transcribed spacer sequences using maximum likelihood and neighbor-joining distance methods allowed the clear identification of 58% of the collection. Metabolomic approaches with multivariate data analysis were performed on SPME/GC-MS chromatograms of volatile compounds emitted by fresh mature flowers and on UHPLC-HRMS/MS leaf extracts for non-volatile compounds. Interestingly, molecular and biochemical approaches provided separate classifications while highlighting several common clusters. The profiling of secondary metabolites was proved most suitable for the classification of hundreds of extracts. The combination of phylogenetic and chemotaxonomic approaches, allowed the classification of 96% of the entire collection. A correlation network revealed specific chemotaxonomic biomarkers, in particular flavonoids, coumarins and cyclotides. Overall, our pioneering approach could be useful to solve misclassification issues within collections of close plant species.
Asunto(s)
Cumarinas/análisis , Ciclotidas/genética , Flavonoides/genética , Viola/genética , Biomarcadores/análisis , Biomarcadores/metabolismo , Cromatografía Líquida de Alta Presión , Cumarinas/metabolismo , Ciclotidas/metabolismo , Flavonoides/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Espectrometría de Masas , Fenotipo , Filogenia , Viola/metabolismoRESUMEN
Cyclotides are fascinating microproteins (≈30-40 residues long) with a unique head-to-tail cyclized backbone, stabilized by three disulfide bonds forming a cystine knot. This unique topology makes them exceptionally stable to chemical, thermal and biological degradation compared to other peptides of similar size. Cyclotides have been also found to be highly tolerant to sequence variability, aside from the conserved residues forming the cystine knot, able to cross cellular membranes and modulate intracellular protein-protein interactions both in vitro and in vivo. These properties make them ideal scaffolds for many biotechnological applications. This article provides and overview of the properties of cyclotides and their applications as molecular imaging agents and peptide-based therapeutics.
Asunto(s)
Ciclotidas/química , Animales , Medios de Contraste/química , Ciclotidas/genética , Ciclotidas/metabolismo , Diseño de Fármacos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tomografía Computarizada por Tomografía de Emisión de Positrones , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/químicaRESUMEN
Cyclotides are fascinating microproteins (≈30 residues long) present in several families of plants that share a unique head-to-tail circular knotted topology of three disulfide bridges, with one disulfide penetrating through a macrocycle formed by the two other disulfides and inter-connecting peptide backbones, forming what is called a cystine knot topology. Naturally occurring cyclotides have shown to posses various pharmacologically relevant activities and have been reported to cross cell membranes. Altogether, these features make the cyclotide scaffold an excellent molecular framework for the design of novel peptide-based therapeutics, making them ideal substrates for molecular grafting of biological peptide epitopes. In this chapter we describe how to express a native folded cyclotide using intein-mediated protein trans-splicing in live Escherichia coli cells.