RESUMEN
The conformation with which natural agonistic peptides interact with G protein-coupled receptor(s) (GPCR(s)) partly results from intramolecular interactions such as hydrogen bridges or is induced by ligand-receptor interactions. The conformational freedom of a peptide can be constrained by intramolecular cross-links. Conformational constraints enhance the receptor specificity, may lead to biased activity and confer proteolytic resistance to peptidic GPCR agonists. Chemical synthesis allows to introduce a variety of cross-links into a peptide and is suitable for bulk production of relatively simple lead peptides. Lanthionines are thioether bridged alanines of which the two alanines can be introduced at different distances in chosen positions in a peptide. Thioether bridges are much more stable than disulfide bridges. Biosynthesis of lanthionine-constrained peptides exploiting engineered Gram-positive or Gram-negative bacteria that contain lanthionine-introducing enzymes constitutes a convenient method for discovery of lanthionine-stabilized GPCR agonists. The presence of an N-terminal leader peptide enables dehydratases to dehydrate serines and threonines in the peptide of interest after which a cyclase can couple the formed dehydroamino acids to cysteines forming (methyl)lanthionines. The leader peptide also guides the export of the formed lanthionine-containing precursor peptide out of Gram-positive bacteria via a lanthipeptide transporter. An engineered cleavage site in the C-terminus of the leader peptide allows to cleave off the leader peptide yielding the modified peptide of interest. Lanthipeptide GPCR agonists are an emerging class of therapeutics of which a few examples have demonstrated high efficacy in animal models of a variety of diseases. One lanthipeptide GPCR agonist has successfully passed clinical Phase Ia.
Asunto(s)
Alanina/análogos & derivados , Receptores Acoplados a Proteínas G/agonistas , Sulfuros/farmacología , Alanina/química , Alanina/farmacología , Animales , Antibacterianos/química , Proteínas Bacterianas/metabolismo , Disulfuros/química , Descubrimiento de Drogas , Proteínas de Unión al GTP/metabolismo , Bacterias Gramnegativas/metabolismo , Bacterias Grampositivas/metabolismo , Humanos , Elementos de la Serie de los Lantanoides/química , Proteínas de Transporte de Membrana , Ratones , Péptido Hidrolasas , Péptidos/química , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína/genética , Ratas , Especificidad por Sustrato , Sulfuros/químicaRESUMEN
Microbial lanthipeptides are formed by a two-step enzymatic introduction of (methyl)lanthionine rings. A dehydratase catalyzes the dehydration of serine and threonine residues, yielding dehydroalanine and dehydrobutyrine, respectively. Cyclase-catalyzed coupling of the formed dehydroresidues to cysteines forms (methyl)lanthionine rings in a peptide. Lanthipeptide biosynthetic systems allow discovery of target-specific, lanthionine-stabilized therapeutic peptides. However, the substrate specificity of existing modification enzymes impose limitations on installing lanthionines in non-natural substrates. The goal of the present study was to obtain a lanthipeptide dehydratase with the capacity to dehydrate substrates that are unsuitable for the nisin dehydratase NisB. We report high-throughput screening for tailored specificity of intracellular, genetically encoded NisB dehydratases. The principle is based on the screening of bacterially displayed lanthionine-constrained streptavidin ligands, which have a much higher affinity for streptavidin than linear ligands. The designed NisC-cyclizable high-affinity ligands can be formed via mutant NisB-catalyzed dehydration but less effectively via wild-type NisB activity. In Lactococcus lactis, a cell surface display precursor was designed comprising DSHPQFC. The Asp residue preceding the serine in this sequence disfavors its dehydration by wild-type NisB. The cell surface display vector was coexpressed with a mutant NisB library and NisTC. Subsequently, mutant NisB-containing bacteria that display cyclized strep ligands on the cell surface were selected via panning rounds with streptavidin-coupled magnetic beads. In this way, a NisB variant with a tailored capacity of dehydration was obtained, which was further evaluated with respect to its capacity to dehydrate nisin mutants. These results demonstrate a powerful method for selecting lanthipeptide modification enzymes with adapted substrate specificity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Lactococcus lactis/enzimología , Proteínas de la Membrana/metabolismo , Alanina/análogos & derivados , Alanina/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Ligandos , Proteínas de la Membrana/genética , Mutagénesis Sitio-Dirigida , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Estreptavidina/química , Estreptavidina/metabolismo , Especificidad por Sustrato , Sulfuros/químicaRESUMEN
Many therapeutic peptides can still be improved with respect to target specificity, target affinity, resistance to peptidases/proteases, physical stability, and capacity to pass through membranes required for oral delivery. Several modifications can improve the peptides' properties, in particular those that impose (a) conformational constraint(s). Screening of constrained peptides and the identification of hits is greatly facilitated by the generation of genetically encoded libraries. Recent breakthrough bacterial, phage, and yeast display screening systems of ribosomally synthesized post-translationally constrained peptides, particularly those of lanthipeptides, are earning special attention. Here we provide an overview of display systems for constrained, genetically encoded peptides and indicate prospects of constrained peptide-displaying phage and bacterial systems as such in vivo.
Asunto(s)
Biblioteca de Péptidos , Péptidos/metabolismo , Membrana Celular/metabolismo , Técnicas de Visualización de Superficie Celular , Pared Celular/metabolismo , Lactococcus lactis/genética , Péptidos/genética , Procesamiento Proteico-Postraduccional , Levaduras/genéticaRESUMEN
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products with drug-like properties. To fully exploit the potential of RiPPs as peptide drug candidates, tools for their systematic engineering are required. Here we report the engineering of lanthipeptides, a subclass of RiPPs characterized by multiple thioether cycles that are enzymatically introduced in a regio- and stereospecific manner, by phage display. This was achieved by heterologous co-expression of linear lanthipeptide precursors fused to the widely neglected C-terminus of the bacteriophage M13 minor coat protein pIII, rather than the conventionally used N-terminus, along with the modifying enzymes from distantly related bacteria. We observe that C-terminal precursor peptide fusions to pIII are enzymatically modified in the cytoplasm of the producing cell and subsequently displayed as mature cyclic peptides on the phage surface. Biopanning of large C-terminal display libraries readily identifies artificial lanthipeptide ligands specific to urokinase plasminogen activator (uPA) and streptavidin.
Asunto(s)
Bacteriófago M13/genética , Proteínas de la Cápside/genética , Biblioteca de Péptidos , Péptidos/genética , Secuencia de Aminoácidos , Bacteriófago M13/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Modelos Genéticos , Péptidos/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Some modified glucagon-like-peptide-1 (GLP-1) analogs are highly important for treating type 2 diabetes. Here we investigated whether GLP-1 analogs expressed in Lactococcus lactis could be substrates for modification and export by the nisin dehydratase and transporter enzyme. Subsequently we introduced a lysinoalanine by coupling a formed dehydroalanine with a lysine and investigated the structure and activity of the formed lysinoalanine-bridged GLP-1 analog. Our data show: (i) GLP-1 fused to the nisin leader peptide is very well exported via the nisin transporter NisT, (ii) production of leader-GLP-1 via NisT is higher than via the SEC system, (iii) leader-GLP-1 exported via NisT was more efficiently dehydrated by the nisin dehydratase NisB than when exported via the SEC system, (iv) individual serines and threonines in GLP-1 are dehydrated by NisB to a significantly different extent, (v) an introduced Ser30 is well dehydrated and can be coupled to Lys34 to form a lysinoalanine-bridged GLP-1 analog, (vi) a lysinoalanine(30-34) variant's conformation shifts in the presence of 25% trifluoroethanol towards a higher alpha helix content than observed for wild type GLP-1 under identical condition, (vii) a lysinoalanine(30-34) GLP-1 variant has retained significant activity. Taken together the data extend knowledge on the substrate specificities of NisT and NisB and their combined activity relative to export via the Sec system, and demonstrate that introducing a lysinoalanine bridge is an option for modifying therapeutic peptides.
Asunto(s)
Proteínas Bacterianas/metabolismo , Péptido 1 Similar al Glucagón/análogos & derivados , Péptido 1 Similar al Glucagón/metabolismo , Lactococcus lactis/metabolismo , Lisinoalanina/química , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Alanina/análogos & derivados , Alanina/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Péptido 1 Similar al Glucagón/biosíntesis , Péptido 1 Similar al Glucagón/genética , Hidroliasas/química , Lactococcus lactis/genética , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Procesamiento Proteico-Postraduccional , Canales de Translocación SEC/metabolismo , Serina/química , Especificidad por Sustrato , Treonina/químicaRESUMEN
The lantibiotic nisin is produced by Lactococcus lactis as a precursor peptide comprising a 23 amino acid leader peptide and a 34 amino acid post-translationally modifiable core peptide. We previously demonstrated that the conserved FNLD part of the leader is essential for intracellular enzyme-catalyzed introduction of lanthionines in the core peptide and also for transporter-mediated export, whereas other positions are subject to large mutational freedom. We here demonstrate that, in the absence of the extracellular leader peptidase, NisP, export of precursor nisin via the modification and transporter enzymes, NisBTC, is strongly affected by multiple substitutions of the leader residue at position -2, but not by substitution of positions in the vicinity of this site. Export levels of precursor nisin increased by more than 70% for position -2 mutants Asp, Thr, Ser, Trp, Lys, Val and decreased more than 70% for Cys, His, Met. In a strain with leader peptidase, the Pro-2Lys and Pro-2Asp precursor nisins were less efficiently cleaved by NisP than wild type precursor nisin. Taken together, the wild type precursor nisin with a proline at position -2 allows balanced export and cleavage efficiencies by precursor nisin's transporter and leader peptidase.
Asunto(s)
Lactococcus lactis/genética , Nisina/genética , Prolina/genética , Señales de Clasificación de Proteína/genética , Secuencia de Aminoácidos , Antibacterianos/metabolismo , Proteínas Bacterianas/genética , Clonación Molecular , Lactococcus lactis/metabolismo , Proteínas de la Membrana/genética , Mutagénesis , Nisina/química , Nisina/metabolismo , Prolina/metabolismo , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Serina Endopeptidasas/genética , Subtilisinas/genéticaRESUMEN
Stimulation of MAS oncogene receptor (MAS) or angiotensin (Ang) receptor type 2 (AT2) may be novel therapeutic options for neonatal chronic lung disease (CLD) by counterbalancing the adverse effects of the potent vasoconstrictor angiotensin II, consisting of arterial hypertension (PAH)-induced right ventricular hypertrophy (RVH) and pulmonary inflammation. We determined the cardiopulmonary effects in neonatal rats with CLD of daily treatment during continuous exposure to 100% oxygen for 10 days with specific ligands for MAS [cyclic Ang-(1-7); 10-50 µg·kg(-1)·day(-1)] and AT2 [dKcAng-(1-7); 5-20 µg·kg(-1)·day(-1)]. Parameters investigated included lung and heart histopathology, fibrin deposition, vascular leakage, and differential mRNA expression in the lungs of key genes involved in the renin-angiotensin system, inflammation, coagulation, and alveolar development. We investigated the role of nitric oxide synthase inhibition with N(ω)-nitro-l-arginine methyl ester (25 mg·kg(-1)·day(-1)) during AT2 agonist treatment. Prophylactic treatment with agonists for MAS or AT2 for 10 days diminished cardiopulmonary injury by reducing alveolar septum thickness and medial wall thickness of small arterioles and preventing RVH. Both agonists attenuated the pulmonary influx of inflammatory cells, including macrophages (via AT2) and neutrophils (via MAS) but did not reduce alveolar enlargement and vascular alveolar leakage. The AT2 agonist attenuated hyperoxia-induced fibrin deposition. In conclusion, stimulation of MAS or AT2 attenuates cardiopulmonary injury by reducing pulmonary inflammation and preventing PAH-induced RVH but does not affect alveolar and vascular development in neonatal rats with experimental CLD. The beneficial effects of AT2 activation on experimental CLD were mediated via a NOS-independent mechanism.
Asunto(s)
Angiotensina I/farmacología , Hiperoxia/complicaciones , Hipertensión Pulmonar/prevención & control , Hipertrofia Ventricular Derecha/prevención & control , Lesión Pulmonar/etiología , Fragmentos de Péptidos/farmacología , Neumonía/prevención & control , Proteínas Proto-Oncogénicas/agonistas , Receptor de Angiotensina Tipo 2/agonistas , Receptores Acoplados a Proteínas G/agonistas , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Western Blotting , Lavado Broncoalveolar , Proliferación Celular/efectos de los fármacos , Hiperoxia/patología , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/metabolismo , Hipertrofia Ventricular Derecha/etiología , Hipertrofia Ventricular Derecha/metabolismo , Lesión Pulmonar/patología , Masculino , Oxígeno/metabolismo , Neumonía/etiología , Neumonía/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/genética , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Angiotensina Tipo 2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sistema Renina-Angiotensina/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Lanthipeptides are ribosomally synthesized and posttranslationally modified peptides produced by microorganisms. The name lanthipeptide is derived from lanthionine, a thioether-bridged amino acid installed by dedicated modification enzymes. Serines and threonines are dehydrated and subsequently coupled to cysteines, thus forming intramolecular lanthionine rings. A well-known subclass of lanthipeptides are lantibiotics: lanthipeptides with antimicrobial activity. The lantibiotic nisin is applied worldwide in the food industry to prevent food spoilage. This review focuses on lanthipeptide leader peptides, which have a crucial and central role in lanthipeptide biosynthesis. Lanthipeptide leader peptides are present at the N-terminus within precursor lanthipeptides. Intriguingly, a single leader peptide can interact with highly different modifying enzyme(s) (domains) and furthermore induce export out of the cell via a dedicated export protein. Eventually the leader peptide is cleaved off by a leader peptidase, either extracellularly or intracellularly as part of the transporter. Recent exciting mechanistic and engineering studies ignited the unraveling of the fascinating interactions of lanthipeptide leader peptides with the lanthipeptide modification enzymes and transporters. The biosynthesis of at least some lanthipeptides is performed by a highly flexible enzyme system. Novel lantibiotics can be synthesized by fusing lanthipeptide leader peptides to completely different silent lantibiotics obtained by genome mining. Moreover, the fusion of leader peptides to the N-terminus of medically and economically important therapeutic peptides has resulted in lanthioninestabilized therapeutics with enhanced bioavailability and optimized receptor interaction.
Asunto(s)
Bacteriocinas/metabolismo , Señales de Clasificación de Proteína , Secuencia de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriocinas/genética , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Procesamiento Proteico-PostraduccionalRESUMEN
The use of mushroom extracts has been common practice in traditional medicine for centuries, including the treatment of cancer. Proteins called hydrophobins are very abundant in mushrooms. Here, it was examined whether they have antitumor activity. Hydrophobin SC3 of Schizophyllum commune was injected daily intraperitoneally starting 1 day after tumor induction in two tumor mouse models (sarcoma and melanoma). SC3 reduced the size and weight of the melanoma significantly, but the sarcoma seemed not affected. However, microscopic analysis of the tumors 12 days after induction revealed a strong antitumor effect of SC3 on both tumors. The mitotic activity of the tumor decreased 1.6- (melanoma) to 2.3-fold (sarcoma), while the vital mass decreased 2.3- (melanoma) to 4.3-fold (sarcoma) compared to the control. Treatment did not cause any signs of toxicity. Behavior, animal growth, and weight of organs were similar to animals injected with vehicle, and no histological abnormalities were found in the organs. In vitro cell culture studies revealed no direct cytotoxic effect of SC3 towards sarcoma cells, while cytotoxic activity was observed towards melanoma cells at a high SC3 concentration. Daily treatment with SC3 did not result in detectable levels of anti-SC3 antibodies in the plasma. Instead, a cellular immune response was observed. Incubation of spleen cells with SC3 resulted in a 1.5- to 2.5-fold increase in interleukin-10 and TNF-α mRNA levels. In conclusion, the nontoxic fungal hydrophobin SC3 showed tumor-suppressive activity possibly via immunomodulation and may be of benefit as adjuvant in combination with chemotherapy and radiation.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Fúngicas/farmacología , Animales , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Schizophyllum/químicaRESUMEN
Modulation of renin-angiotensin system (RAS) by angiotensin-(1-7) (Ang-(1-7)) is an attractive approach to combat the detrimental consequences of myocardial infarction (MI). However Ang-(1-7) has limited clinical potential due to its unfavorable pharmacokinetic profile. We investigated effects of a stabilized, thioether-bridged analogue of Ang-(1-7) called cyclic Ang-(1-7) in rat model of myocardial infarction. Rats underwent coronary ligation or sham surgery. Two weeks thereafter infusion with 0.24 or 2.4 µg/kg/h cAng-(1-7) or saline was started for 8 weeks. Thereafter, cardiac morphometric and hemodynamic variables as wells as aortic endothelial function were measured. The average infarct size was 13.8% and was not changed by cAng-(1-7) treatment. MI increased heart weight and myocyte size, which was restored by cAng-(1-7) to sham levels. In addition, cAng-(1-7) lowered left ventricular end-diastolic pressure and improved endothelial function. The results suggest that cAng-(1-7) is a promising new agent in treatment of myocardial infarction and warrant further research.
RESUMEN
A major hurdle in the application of therapeutic peptides is their rapid degradation by peptidases. Thioether bridges effectively protect therapeutic peptides against breakdown, thereby strongly increasing bioavailability, enabling oral and pulmonary delivery and potentially significantly optimizing the receptor interaction of selected variants. To efficiently select optimal variants, a library of DNA-coupled thioether-bridged peptides is highly desirable. Here, we present a unique cell surface display system of thioether-bridged peptides and successfully demonstrate highly selective screening. Peptides are posttranslationally modified by thioether bridge-installing enzymes in Lactococcus lactis, followed by export and sortase-mediated covalent coupling to the lactococcal cell wall. This allows the combinatorial optimization and selection of medically and economically highly important therapeutic peptides with strongly enhanced therapeutic potential.
Asunto(s)
Pared Celular/metabolismo , Lactococcus lactis/química , Lactococcus lactis/metabolismo , Tamizaje Masivo/métodos , Péptidos/química , Péptidos/metabolismo , Sulfuros/metabolismo , Pared Celular/química , Lactococcus lactis/genética , Péptidos/genética , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Transporte de Proteínas , Sulfuros/químicaRESUMEN
Nisin A is a pentacyclic peptide antibiotic produced by Lactococcus lactis. The leader peptide of prenisin keeps nisin inactive and has a role in inducing NisB- and NisC-catalyzed modifications of the propeptide and NisT-mediated export. The highly specific NisP cleaves off the leader peptide from fully modified and exported prenisin. We present here a detailed mutagenesis analysis of the nisin leader peptide. For alternative cleavage, we successfully introduced a putative NisP autocleavage site and sites for thrombin, enterokinase, Glu-C, and factor Xa in the C-terminal part of the leader peptide. Replacing residue F-18 with Trp or Thr strongly reduced production. On the other hand, D-19A, F-18H, F-18M, L-16D, L-16K, and L-16A enhanced production. Substitutions within and outside the FNLD box enhanced or reduced the transport efficiency. None of the above substitutions nor even an internal 6His tag from positions -13 to -8 had any effect on the capacity of the leader peptide to induce NisB and NisC modifications. Therefore, these data demonstrate a large mutational freedom. However, simultaneous replacement of the FNLD amino acids by four alanines strongly reduced export and even led to a complete loss of the capacity to induce modifications. Reducing the leader peptide to MSTKDFNLDLR led to 3- or 4-fold dehydration. Taken together, the FNLD box is crucial for inducing posttranslational modifications.
Asunto(s)
Antibacterianos/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Nisina/metabolismo , Señales de Clasificación de Proteína , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Antibacterianos/farmacología , Análisis Mutacional de ADN , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Datos de Secuencia Molecular , Nisina/genética , Nisina/farmacología , Ingeniería de Proteínas , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Recombinación GenéticaRESUMEN
Instability and proteolytic degradation limit the delivery options and in vivo efficacy of many therapeutic peptides. We previously generated a thioether stabilized angiotensin-(1-7) analog, cAng-(1-7), which is resistant against proteolytic degradation in the circulation. We here investigated oral and pulmonary delivery of this compound. In a first step we investigated the in vitro stability of the peptide under conditions that mimic those that will be met after oral administration. We demonstrated that cAng-(1-7) is stable at pH 2.0, a pH value close to that of the stomach, has enhanced resistance to breakdown by proteases from pancreas at pH 7.4, and is resistant to breakdown by proteases from liver at the lysosomal pH 5.0. We subsequently demonstrated that, in the absence of any delivery system or formulation, cAng-(1-7) can be delivered orally and via the lung, with bioavailabilities of 0.28+/-0.05% and 28+/-5%, whereas drug uptake was maximal after subcutaneous administration (bioavailability of 98+/-6%). Therapeutic concentrations could be reached via all three routes of administration. The data prove that introduction of a thioether bridge in peptides opens novel delivery options for medically important peptides.
Asunto(s)
Administración Oral , Angiotensina I/administración & dosificación , Angiotensina I/farmacocinética , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/farmacocinética , Administración Cutánea , Animales , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Vías de Administración de Medicamentos , Masculino , Espectrometría de Masas , Ratas , Ratas Sprague-Dawley , Tráquea/metabolismoRESUMEN
This minireview focuses on the use of bacteria to introduce dehydroresidues and (methyl)lanthionines in (poly)peptides. It mainly describes the broad exploitation of bacteria containing lantibiotic enzymes for the engineering of these residues in a wide variety of peptides in particular in peptides unrelated to lantibiotics. Lantibiotic dehydratases dehydrate serines and threonines present in peptides preceded by a lantibiotic leader peptide thus forming dehydroalanine and dehydrobutyrine, respectively. These dehydroresidues can be coupled to cysteines thus forming (methyl)lanthionines. This coupling is catalysed by lantibiotic cyclases. The design, synthesis, and export of microbially engineered dehydroresidue and or lanthionine-containing peptides in non-lantibiotic peptides are reviewed, illustrated by some examples which demonstrate the high relevance of these special residues. This minireview is the first with special focus on the microbial engineering of nonlantibiotic peptides by exploiting lantibiotic enzymes.
Asunto(s)
Alanina/análogos & derivados , Aminoácidos/biosíntesis , Bacterias/metabolismo , Vías Biosintéticas/genética , Ingeniería Genética , Biosíntesis de Proteínas , Alanina/biosíntesis , Bacterias/enzimología , Bacterias/genética , SulfurosRESUMEN
Recent studies showed that the nisin modification machinery can successfully dehydrate serines and threonines and introduce lanthionine rings in small peptides that are fused to the nisin leader sequence. This opens up exciting possibilities to produce and engineer larger antimicrobial peptides in vivo. Here we demonstrate the exploitation of the class I nisin production machinery to generate, modify, and secrete biologically active, previously not-yet-isolated and -characterized class II two-component lantibiotics that have no sequence homology to nisin. The nisin synthesis machinery, composed of the modification enzymes NisB and NisC and the transporter NisT, was used to modify and secrete a putative two-component lantibiotic of Streptococcus pneumoniae. This was achieved by genetically fusing the propeptide-encoding sequences of the spr1765 (pneA1) and spr1766 (pneA2) genes to the nisin leader-encoding sequence. The chimeric prepeptides were secreted out of Lactococcus lactis, purified by cation exchange fast protein liquid chromatography, and further characterized. Mass spectrometry analyses demonstrated the presence and partial localization of multiple dehydrated serines and/or threonines and (methyl)lanthionines in both peptides. Moreover, after cleavage of the leader peptide from the prepeptides, both modified propeptides displayed antimicrobial activity against Micrococcus flavus. These results demonstrate that the nisin synthetase machinery can be successfully used to modify and produce otherwise difficult to obtain antimicrobially active lantibiotics.
Asunto(s)
Bacteriocinas/biosíntesis , Streptococcus pneumoniae/metabolismo , Secuencia de Aminoácidos , Antibacterianos/biosíntesis , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriocinas/química , Bacteriocinas/genética , Bacteriocinas/farmacología , Genes Bacterianos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Familia de Multigenes , Nisina/biosíntesis , Ingeniería de Proteínas/métodos , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Homología de Secuencia de Aminoácido , Streptococcus pneumoniae/genéticaRESUMEN
The poor water solubility of many drugs requires a specific formulation to achieve a sufficient bioavailability after oral administration. Suspensions of small drug particles can be used to improve the bioavailability. We here show that the fungal hydrophobin SC3 can be used to make suspensions of water insoluble drugs. Bioavailability of two of these drugs, nifedipine and cyclosporine A (CyA), was tested when administered as a SC3-based suspension. SC3 (in a 1:2 (w/w) drug:SC3 ratio) or 100% PEG400 increased the bioavailability of nifedipine to a similar degree (6+/-2- and 4+/-3-fold, respectively) compared to nifedipine powder without additives. Moreover, SC3 (in a 7:1 (w/w) drug:hydrophobin ratio) was as effective as a 20-fold diluted Neoral formulation by increasing bioavailability of CyA 2.3+/-0.3-fold compared to CyA in water. Interestingly, using SC3 in the CyA formulation resulted in a slower uptake (p<0.001 in T(max)) of the drug, with a lower peak concentration (C(max) 1.8 mg ml(-1)) at a later time point (T(max) 9+/-2 h) compared to Neoral (C(max) 2.2 mg ml(-1); T(max) 3.2+/-0.2). Consequently, SC3 will result in a more constant, longer lasting drug level in the body. Taken together, hydrophobins are attractive candidates to formulate hydrophobic drugs.
Asunto(s)
Composición de Medicamentos , Interacciones Hidrofóbicas e Hidrofílicas , Preparaciones Farmacéuticas/administración & dosificación , Agua/química , Administración Oral , Animales , Disponibilidad Biológica , Ciclosporina/sangre , Masculino , Nifedipino/administración & dosificación , Tamaño de la Partícula , Ratas , Ratas Wistar , Solubilidad , SolucionesRESUMEN
Class I hydrophobins function in fungal growth and development by self-assembling at hydrophobic-hydrophilic interfaces into amyloid-like fibrils. SC3 of the mushroom-forming fungus Schizophyllum commune is the best studied class I hydrophobin. This protein spontaneously adopts the amyloid state at the water-air interface. In contrast, SC3 is arrested in an intermediate conformation at the interface between water and a hydrophobic solid such as polytetrafluoroethylene (PTFE; Teflon). This finding prompted us to study conditions that promote assembly of SC3 into amyloid fibrils. Here, we show that SC3 adopts the amyloid state at the water-PTFE interface at high concentration (300 microg ml(-1)) and prolonged incubation (16 h). Moreover, we show that amyloid formation at both the water-air and water-PTFE interfaces is promoted by the cell wall components schizophyllan (beta(1-3),beta(1-6)-glucan) and beta(1-3)-glucan. Hydrophobin concentration and cell wall polysaccharides thus contribute to the role of SC3 in formation of aerial hyphae and in hyphal attachment.
Asunto(s)
Amiloide/metabolismo , Proteínas Fúngicas/metabolismo , Polisacáridos/metabolismo , Schizophyllum/metabolismo , Amiloide/química , Pared Celular/química , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/química , Glucanos/química , Glucanos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Polisacáridos/química , Politetrafluoroetileno/química , Estructura Secundaria de Proteína , Agua/químicaRESUMEN
This study demonstrates for the first time that a thioether-containing peptide, an azurin fragment, can be translocated via the Sec pathway. This methyl-lanthionine was introduced by the nisin modification enzymes. The Sec pathway can therefore be a successful alternative for those cyclized peptides that are inefficiently transported via NisT.
Asunto(s)
Azurina/metabolismo , Proteínas Bacterianas/metabolismo , Lactococcus lactis/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Fragmentos de Péptidos/metabolismo , Transporte de ProteínasRESUMEN
The in vivo efficacy of many therapeutic peptides is hampered by their rapid proteolytic degradation. Cyclization of these therapeutic peptides is an excellent way to render them more resistant against breakdown. Here, we describe the enzymatic introduction of a thioether ring in angiotensin [Ang-(1-7)], a heptapeptide that plays a pivotal role in the renin-angiotensin system and possesses important therapeutic activities. The lactic acid bacterium Lactococcus lactis, equipped with the plasmid-based nisin modification machinery, was used to produce thioether-bridged Ang-(1-7). The resulting cyclized Ang-(1-7) is fully resistant against purified angiotensin-converting enzyme, has significantly increased stability in homogenates of different organs and in plasma derived from pig, and displays a strongly (34-fold) enhanced survival in Sprague-Dawley (SD) rats in vivo. With respect to functional activity, cyclized Ang-(1-7) induces relaxation of precontracted SD rat aorta rings in vitro. The magnitude of this effect is 2-fold larger than that obtained for natural Ang-(1-7). The Ang-(1-7) receptor antagonist D-Pro(7)-Ang-(1-7), which completely inhibits the activity of natural Ang-(1-7), also abolishes the vasodilation by cyclized Ang-(1-7), providing evidence that cyclized Ang-(1-7) also interacts with the Ang-(1-7) receptor. Taken together, applying a highly innovative enzymatic peptide stabilization method, we generated a stable Ang-(1-7) analog with strongly enhanced therapeutic potential.
Asunto(s)
Angiotensinas/química , Fragmentos de Péptidos/química , Peptidil-Dipeptidasa A/metabolismo , Sulfuros/análisis , Angiotensinas/sangre , Angiotensinas/metabolismo , Angiotensinas/farmacología , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Estabilidad de Medicamentos , Infusiones Intravenosas , Corteza Renal/metabolismo , Lactococcus lactis/enzimología , Hígado/metabolismo , Masculino , Tasa de Depuración Metabólica , Oligopéptidos/química , Oligopéptidos/metabolismo , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , PorcinosRESUMEN
The thioether rings in the lantibiotics lacticin 3147 and nisin are posttranslationally introduced by dehydration of serines and threonines, followed by coupling of these dehydrated residues to cysteines. The prepeptides of the two-component lantibiotic lacticin 3147, LtnA1 and LtnA2, are dehydrated and cyclized by two corresponding bifunctional enzymes, LtnM1 and LtnM2, and are subsequently processed and exported via one bifunctional enzyme, LtnT. In the nisin synthetase complex, the enzymes NisB, NisC, NisT, and NisP dehydrate, cyclize, export, and process prenisin, respectively. Here, we demonstrate that the combination of LtnM2 and LtnT can modify, process, and transport peptides entirely different from LtnA2 and that LtnT can process and transport unmodified LtnA2 and unrelated peptides. Furthermore, we demonstrate a higher extent of NisB-mediated dehydration in the absence of thioether rings. Thioether rings apparently inhibited dehydration, which implies alternating actions of NisB and NisC. Furthermore, certain (but not all) NisC-cyclized peptides were exported with higher efficiency as a result of their conformation. Taken together, these data provide further insight into the applicability of Lactococcus lactis strains containing lantibiotic enzymes for the design and production of modified peptides.