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
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
Cyclotides are a unique family of stable and cyclic mini-proteins found in plants that have nematicidal and anthelmintic activities. They are distributed across the Rubiaceae, Violaceae, Fabaceae, Cucurbitaceae, and Solanaceae plant families, where they are posited to act as protective agents against pests. In this study, we tested the nematicidal properties of extracts from four major cyclotide-producing plants, Oldenlandia affinis, Clitoria ternatea, Viola odorata, and Hybanthus enneaspermus, against the free-living model nematode Caenorhabditis elegans. We evaluated the nematicidal activity of the cyclotides kalata B1, cycloviolacin O2, and hyen D present in these extracts and found them to be active against the larvae of C. elegans. Both the plant extracts and isolated cyclotides exerted dose-dependent toxicity on the first-stage larvae of C. elegans. Isolated cyclotides caused death or damage upon interacting with the worms' mouth, pharynx, and midgut or membrane. Cycloviolacin O2 and hyen D produced bubble-like structures around the C. elegans membrane, termed blebs, implicating membrane disruption causing toxicity and death. All tested cyclotides lost their toxicity when the hydrophobic patches present on them were disrupted via a single-point mutation. The present results provide a facile assay design to measure and explore the nematicidal activities of plant extracts and purified cyclotides on C. elegans.
Asunto(s)
Ciclotidas , Fabaceae , Nematodos , Violaceae , Animales , Antinematodos/farmacología , Caenorhabditis elegans , Ciclotidas/farmacología , Ciclotidas/química , Fabaceae/química , Extractos Vegetales/química , Proteínas de Plantas/químicaRESUMEN
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
Upon wetting, chia (Salvia hispanica L.) nutlets produce a gel-like capsule of polysaccharides called mucilage that comprises a significant part of their dietary fibre content. Seed/nutlet mucilage is often used as a texture modifying hydrocolloid and bulking dietary fibre due to its water-binding ability, though the utility of mucilage from different sources is highly structure-function dependent. The composition and structure of chia nutlet mucilage is poorly defined, and a better understanding will aid in exploiting its dietary fibre functionality, particularly if, and how, it is utilised by gut microbiota. In this study, microscopy, chromatography, mass spectrometry and glycome profiling techniques showed that chia nutlet mucilage is highly complex, layered, and contains several polymer types. The mucilage comprises a novel xyloamylose containing both ß-linked-xylose and α-linked-glucose, a near-linear xylan that may be sparsely substituted, a modified cellulose domain, and abundant alcohol-soluble oligosaccharides. To assess the dietary fibre functionality of chia nutlet mucilage, an in vitro cumulative gas production technique was used to determine the fermentability of different chia nutlet preparations. The complex nature of chia nutlet mucilage led to poor fermentation where the oligosaccharides appeared to be the only fermentable substrate present in the mucilage. Of note, ground chia nutlets were better fermented than intact whole nutlets, as judged by short chain fatty acid production. Therefore, it is suggested that the benefits of eating chia as a "superfood", could be notably enhanced if the nutlets are ground rather than being consumed whole, improving the bioaccessibility of key nutrients including dietary fibre.
Asunto(s)
Mucílago de Planta , Salvia , Salvia hispanica , Fermentación , Salvia/química , Polisacáridos/química , Semillas/química , Oligosacáridos/análisis , Fibras de la Dieta/análisis , Mucílago de Planta/químicaRESUMEN
The stinging hairs of plants from the family Urticaceae inject compounds that inflict pain to deter herbivores. The sting of the New Zealand tree nettle (Urtica ferox) is among the most painful of these and can cause systemic symptoms that can even be life-threatening; however, the molecular species effecting this response have not been elucidated. Here we reveal that two classes of peptide toxin are responsible for the symptoms of U. ferox stings: Δ-Uf1a is a cytotoxic thionin that causes pain via disruption of cell membranes, while ß/δ-Uf2a defines a new class of neurotoxin that causes pain and systemic symptoms via modulation of voltage-gated sodium (NaV) channels. We demonstrate using whole-cell patch-clamp electrophysiology experiments that ß/δ-Uf2a is a potent modulator of human NaV1.5 (EC50: 55 nM), NaV1.6 (EC50: 0.86 nM), and NaV1.7 (EC50: 208 nM), where it shifts the activation threshold to more negative potentials and slows fast inactivation. We further found that both toxin classes are widespread among members of the Urticeae tribe within Urticaceae, suggesting that they are likely to be pain-causing agents underlying the stings of other Urtica species. Comparative analysis of nettles of Urtica, and the recently described pain-causing peptides from nettles of another genus, Dendrocnide, indicates that members of tribe Urticeae have developed a diverse arsenal of pain-causing peptides.
Asunto(s)
Neurotoxinas , Péptidos , Toxinas Biológicas , Urticaceae , Humanos , Neurotoxinas/química , Dolor , Técnicas de Placa-Clamp , Péptidos/química , Péptidos/toxicidad , Toxinas Biológicas/química , Urticaceae/química , Canales de Sodio Activados por Voltaje/efectos de los fármacosRESUMEN
Knottins are topologically complex peptides that are stabilised by a cystine knot and have exceptionally diverse functions, including protease inhibition. However, approaches for tuning their activity in situ are limited. Here, we demonstrate separate approaches for tuning the activity of knottin protease inhibitors using light or streptavidin. We show that the inhibitory activity and selectivity of an engineered knottin can be controlled with light by activating a second mode of action that switches the inhibitor ON against new targets. Guided by a knottin library screen, we also identify a position in the inhibitor's binding loop that permits insertion of a biotin tag without impairing activity. Using streptavidin, biotinylated knottins with nanomolar affinity can be switched OFF in activity assays, and the anticoagulant activity of a factor XIIa inhibitor can be rapidly switched OFF in human plasma. Our findings expand the scope of engineered knottins for precisely controlling protein function.
Asunto(s)
Miniproteínas Nodales de Cistina , Cistina , Miniproteínas Nodales de Cistina/metabolismo , Humanos , Péptidos/metabolismo , Péptidos/farmacología , Proteínas , EstreptavidinaRESUMEN
Chemoenzymatic protein and peptide modification is a powerful means of generating defined, homogeneous conjugates for a range of applications. However, the use of transpeptidases is limited by the need to prepare synthetic peptide conjugates to be ligated, bulky recognition tags remaining in the product, and inefficient substrate turnover. Here, we report a peptide/protein labeling strategy that utilizes a promiscuous, engineered transpeptidase to irreversibly incorporate diverse, commercially available amines at a C-terminal asparagine. To demonstrate the utility of this approach, we prepare a protein-drug conjugate, generate a genetically inaccessible C-to-C protein fusion, and site specifically label both termini of a single protein in sequential steps.
Asunto(s)
Aminas/química , Peptidil Transferasas/química , Ingeniería de Proteínas , Aminas/metabolismo , Modelos Moleculares , Peptidil Transferasas/metabolismoRESUMEN
Double-knotted peptides identified in venoms and synthetic bivalent peptide constructs targeting ion channels are emerging tools for the study of ion channel pharmacology and physiology. These highly complex and disulfide-rich peptides contain two individual cystine knots, each comprising six cysteines and three disulfide bonds. Until now, native double-knotted peptides, such as Hi1a and DkTx, have only been isolated from venom or produced recombinantly, whereas engineered double-knotted peptides have successfully been produced through enzymatic ligation using sortase A to form a seamless amide bond at the ligation site between two knotted toxins, and by alkyne/azide click chemistry, joining two peptide knots via a triazole linkage. To further pursue these double-knotted peptides as pharmacological tools or probes for therapeutically relevant ion channels, we sought to identify a robust methodology resulting in a high yield product that lends itself to rapid production and facile mutational studies. In this study, we evaluated the ligation efficiency of enzymatic (sortase A5°, butelase 1, wild-type OaAEP 1, C247A-OaAEP 1, and peptiligase) and mild chemical approaches (α-ketoacid-hydroxylamine, KAHA) for forming a native amide bond linking the toxins while maintaining the native disulfide connectivity of each pre-folded peptide. We used two NaV1.7 inhibitors: PaurTx3, a spider-derived gating modifier peptide, and KIIIA, a small cone snail-derived pore blocker peptide, which have previously been shown to increase affinity and inhibitory potency on hNaV1.7 when ligated together. Correctly folded peptides were successfully ligated in varying yields, without disulfide bond shuffling or reduction, with sortase A5° being the most efficient, resulting in 60% ligation conversion within 15 min. In addition, electrophysiology studies demonstrated that for these two peptides, the amino acid composition of the linker did not affect the activity of the double-knotted peptides. This study demonstrates the powerful application of enzymes in efficiently ligating complex disulfide-rich peptides, paving the way for facile production of double-knotted peptides.
Asunto(s)
DisulfurosRESUMEN
Enzyme-catalysed site-specific protein modifications enable the precision manufacture of conjugates for the study of protein function and/or for therapeutic or diagnostic applications. Asparaginyl ligases are a class of highly efficient transpeptidases with the capacity to modify proteins bearing only a tripeptide recognition motif. Herein, we review the types of protein modification that are accessible using these enzymes, including N- and C-terminal protein labelling, head-to-tail cyclisation, and protein-protein conjugation. We describe the progress that has been made to engineer highly efficient ligases as well as efforts to chemically manipulate the enzyme reaction to favour product formation. These enzymes are powerful additions to the protein engineer's toolbox.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Ingeniería de Proteínas , Procesamiento Proteico-PostraduccionalRESUMEN
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
The use of enzymes for the site-specific modification of proteins/peptides has become a highly accessible, widespread approach to study protein/peptide functions or to generate therapeutic conjugates. Asparaginyl endopeptidases (AEPs) that preferentially catalyze transpeptidation reactions (AEP ligases) have emerged as enticing alternatives to established approaches, such as bacterial sortases, due to their catalytic efficiency and short tripeptide recognition motifs. However, under standard conditions, a substantial excess of the nucleophile to be conjugated is needed to reach desirable yields. Herein we report a versatile approach to shift the AEP-catalyzed transpeptidation equilibrium toward product formation via selectively quenching the nucleophilicity of the competing leaving-group peptide. Our metal-complexation-based strategy enables efficient peptide/protein labeling at the N- or C-terminus with near-equimolar concentrations of nucleophile label. Furthermore, we show that this approach can enhance protein-protein ligation and facilitate the formation of transpeptidation products that are otherwise unattainable.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Péptidos/metabolismo , Secuencias de Aminoácidos , Biocatálisis , Cobre/química , Cobre/metabolismo , Humanos , Níquel/química , Níquel/metabolismo , Péptidos/química , Unión Proteica , Ingeniería de Proteínas , Albúmina Sérica/química , Albúmina Sérica/metabolismoRESUMEN
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
Stinging trees from Australasia produce remarkably persistent and painful stings upon contact of their stiff epidermal hairs, called trichomes, with mammalian skin. Dendrocnide-induced acute pain typically lasts for several hours, and intermittent painful flares can persist for days and weeks. Pharmacological activity has been attributed to small-molecule neurotransmitters and inflammatory mediators, but these compounds alone cannot explain the observed sensory effects. We show here that the venoms of Australian Dendrocnide species contain heretofore unknown pain-inducing peptides that potently activate mouse sensory neurons and delay inactivation of voltage-gated sodium channels. These neurotoxins localize specifically to the stinging hairs and are miniproteins of 4 kDa, whose 3D structure is stabilized in an inhibitory cystine knot motif, a characteristic shared with neurotoxins found in spider and cone snail venoms. Our results provide an intriguing example of inter-kingdom convergent evolution of animal and plant venoms with shared modes of delivery, molecular structure, and pharmacology.
RESUMEN
Ruthenium-catalysed azide-alkyne cycloaddition (RuAAC) provides access to 1,5-disubstituted 1,2,3-triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross-linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor-1. NMR and X-ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5â Å) of the triazole linkages compared to the parent disulfide molecules. The triazole-bridged peptides also displayed superior half-lives in liver S9 stability assays compared to disulfide-bridged peptides. This work establishes a foundation for the application of 1,5-disubstituted 1,2,3-triazoles as disulfide mimetics.
Asunto(s)
Disulfuros/química , Imitación Molecular , Péptidos Cíclicos/química , Triazoles/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Ciclización , Resonancia Magnética Nuclear Biomolecular , Rutenio/químicaRESUMEN
Asparaginyl endopeptidases (AEPs) catalyze the key backbone cyclization step during the biosynthesis of plant-derived cyclic peptides. Here, we report the identification of two AEPs from Momordica cochinchinensis and biochemically characterize MCoAEP2 that catalyzes the maturation of trypsin inhibitor cyclotides. Recombinantly produced MCoAEP2 catalyzes the backbone cyclization of a linear cyclotide precursor (MCoTI-II-NAL) with a kcat/Km of 620 mM-1 s-1, making it one of the fastest cyclases reported to date. We show that MCoAEP2 can mediate both the N-terminal excision and C-terminal cyclization of cyclotide precursors in vitro. The rate of cyclization/hydrolysis is primarily influenced by varying pH, which could potentially control the succession of AEP-mediated processing events in vivo. Furthermore, MCoAEP2 efficiently catalyzes the backbone cyclization of an engineered MCoTI-II analog with anti-angiogenic activity. MCoAEP2 provides enhanced synthetic access to structures previously inaccessible by direct chemistry approaches and enables the wider application of trypsin inhibitor cyclotides in biotechnology applications.
Asunto(s)
Biocatálisis , Cisteína Endopeptidasas/metabolismo , Inhibidores de Tripsina/metabolismo , Secuencia de Aminoácidos , Ciclización , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Proteínas de Plantas/metabolismo , Ingeniería de Proteínas , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
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
Chymase is a serine protease that is predominantly expressed by mast cells and has key roles in immune defense and the cardiovascular system. This enzyme has also emerged as a therapeutic target for cardiovascular disease due to its ability to remodel cardiac tissue and generate angiotensin II. Here, we used the nature-derived cyclic peptide sunflower trypsin inhibitor-1 (SFTI-1) as a template for designing novel chymase inhibitors. The key binding contacts of SFTI-1 were optimized by combining a peptide substrate library screen with structure-based design, which yielded several variants with potent activity. The lead variant was further modified by replacing the P1 Tyr residue with para-substituted Phe derivatives, generating new inhibitors with improved potency (Ki = 1.8 nM) and higher selectivity over closely related enzymes. Several variants were shown to block angiotensin I cleavage in vitro, highlighting their potential for further development and future evaluation as pharmaceutical leads.
Asunto(s)
Quimasas/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Sustitución de Aminoácidos , Angiotensina II/biosíntesis , Cristalografía por Rayos X , Diseño de Fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Fenilalanina/química , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Tirosina/químicaRESUMEN
Protein ligases of defined substrate specificity are versatile tools for protein engineering. Upon completion of the reaction, the products of currently reported protein ligases contain the amino acid sequence that is recognized by that same ligase, resulting in repeated cycles of ligation and hydrolysis as competing reactions. Thus, previous efforts to sequentially label proteins at distinct positions required ligases of orthogonal specificity. A recombinant Oldenlandia affinis asparaginyl endopeptidase, OaAEP1, is promiscuous for incoming nucleophiles. This promiscuity enabled us to define a nucleophile composed of natural amino acids that is ligated efficiently to the substrate yet yields a product that is poorly recognized by OaAEP1. Proteins modified with an efficient recognition module could be readily modified to yield a defined product bearing a cleavage-resistant motif, whereas proteins containing this inferior recognition motif remained essentially unmodified. We demonstrate the versatility of the N- or C-terminal protein modifications obtainable with this approach and modify the N- and C-termini of a single substrate protein in a sequential, site-specific manner in excellent yield.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Ingeniería de Proteínas/métodos , Proteínas/química , Secuencias de Aminoácidos , Catálisis , Cisteína Endopeptidasas/genética , Electroforesis en Gel de Poliacrilamida , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Oldenlandia/enzimología , Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticuerpos de Dominio Único/químicaRESUMEN
Extracellular vesicles (EVs) are membranous vesicles that are released by cells. In this study, the role of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in the biogenesis of yeast EVs was examined. Knockout of components of the ESCRT machinery altered the morphology and size of EVs as well as decreased the abundance of EVs. In contrast, strains with deletions in cell wall biosynthesis genes, produced more EVs than wildtype. Proteomic analysis highlighted the depletion of ESCRT components and enrichment of cell wall remodelling enzymes, glucan synthase subunit Fks1 and chitin synthase Chs3, in yeast EVs. Interestingly, EVs containing Fks1 and Chs3 rescued the yeast cells from antifungal molecules. However, EVs from fks1∆ or chs3∆ or the vps23∆chs3∆ double knockout strain were unable to rescue the yeast cells as compared to vps23∆ EVs. Overall, we have identified a potential role for yeast EVs in cell wall remodelling.