RESUMEN
In order to recapitulate complex eukaryotic compartmentalization, synthetic biology aims to recreate cellular membrane-lined compartments from the bottom-up. Many important cellular organelles and cell-produced extracellular vesicles are in the size range of several hundreds of nanometers. Although attaining a fundamental characterization and mimicry of their cellular functions is a compelling goal, the lack of methods for controlled vesicle formation in this size range has hindered full understanding. Here, we show the optimization of a simple and efficient protocol for the production of large unilamellar vesicles (LUVs) with a median diameter in the range of 450-550 nm with high purity. Importantly, we rely on commercial reagents and common laboratory equipment. We thoroughly characterize the influence of different experimental parameters on the concentration and size of the resulting vesicles and assess changes in their lipid composition and surface charge. We provide guidance for researchers to optimize LUV production further to suit specific applications.
Asunto(s)
Liposomas , Liposomas UnilamelaresRESUMEN
The ongoing COVID-19 pandemic has been brought on by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike glycoprotein (S), which decorates the viral envelope forming a corona, is responsible for the binding to the angiotensin-converting enzyme 2 (ACE2) receptor and initiating the infection. In comparison to previous variants, Omicron S presents additional binding sites as well as a more positive surface charge. These changes hint at additional molecular targets for interactions between virus and cell, such as the cell membrane or proteoglycans on the cell surface. Herein, bottom-up assembled synthetic SARS-CoV-2 miniviruses (MiniVs), with a lipid composition similar to that of infectious particles, are implemented to study and compare the binding properties of Omicron and Alpha variants. Toward this end, a systematic functional screening is performed to study the binding ability of Omicron and Alpha S proteins to ACE2-functionalized and nonfunctionalized planar supported lipid bilayers. Moreover, giant unilamellar vesicles are used as a cell membrane model to perform competitive interaction assays of the two variants. Finally, two cell lines with and without presentation of the ACE2 receptor are used to confirm the binding properties of the Omicron and Alpha MiniVs to the cellular membrane. Altogether, the results reveal a significantly higher affinity of Omicron S toward both the lipid membrane and ACE2 receptor. The research presented here highlights the advantages of creating and using bottom-up assembled SARS-CoV-2 viruses to understand the impact of changes in the affinity of S for ACE2 in infection studies.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Enzima Convertidora de Angiotensina 2 , Pandemias , Glicoproteína de la Espiga del Coronavirus , Membrana Dobles de Lípidos , Proteoglicanos , Unión ProteicaRESUMEN
The genomes of anaerobic ammonium-oxidizing (anammox) bacteria contain a gene cluster comprising genes of unusual fatty acid biosynthesis enzymes that were suggested to be involved in the synthesis of the unique "ladderane" lipids produced by these organisms. This cluster encodes an acyl carrier protein (denoted as "amxACP") and a variant of FabZ, an ACP-3-hydroxyacyl dehydratase. In this study, we characterize this enzyme, which we call anammox-specific FabZ ("amxFabZ"), to investigate the unresolved biosynthetic pathway of ladderane lipids. We find that amxFabZ displays distinct sequence differences to "canonical" FabZ, such as a bulky, apolar residue on the inside of the substrate-binding tunnel, where the canonical enzyme has a glycine. Additionally, substrate screens suggest that amxFabZ efficiently converts substrates with acyl chain lengths of up to eight carbons, whereas longer substrates are converted much more slowly under the conditions used. We also present crystal structures of amxFabZs, mutational studies and the structure of a complex between amxFabZ and amxACP, which show that the structures alone cannot explain the apparent differences from canonical FabZ. Moreover, we find that while amxFabZ does dehydrate substrates bound to amxACP, it does not convert substrates bound to canonical ACP of the same anammox organism. We discuss the possible functional relevance of these observations in the light of proposals for the mechanism for ladderane biosynthesis.
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Proteína Transportadora de Acilo , Hidroliasas , Hidroliasas/metabolismo , Lípidos , Enoil-CoA Hidratasa/metabolismoRESUMEN
Coenzyme A (CoA) is one of the central cofactors of metabolism, yet a method for measuring its concentration in living cells is missing. Here we introduce the first biosensor for measuring CoA levels in different organelles of mammalian cells. The semisynthetic biosensor is generated through the specific labeling of an engineered GFP-HaloTag fusion protein with a fluorescent ligand. Its readout is based on CoA-dependent changes in Förster resonance energy transfer efficiency between GFP and the fluorescent ligand. Using this biosensor, we probe the role of numerous proteins involved in CoA biosynthesis and transport in mammalian cells. On the basis of these studies, we propose a cellular map of CoA biosynthesis that suggests how pools of cytosolic and mitochondrial CoA are maintained.
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Técnicas Biosensibles , Proteínas , Animales , Ligandos , Colorantes , Homeostasis , Técnicas Biosensibles/métodos , Coenzima A , MamíferosRESUMEN
Sactipeptides are ribosomally synthesized peptides containing a unique sulfur to α-carbon crosslink. Catalyzed by sactisynthases, this thioether pattern endows sactipeptides with enhanced structural, thermal, and proteolytic stability, which makes them attractive scaffolds for the development of novel biotherapeutics. Herein, we report the in-depth study on the substrate tolerance of the sactisynthase AlbA to catalyze the formation of thioether bridges in sactipeptides. We identified a possible modification site within the sactipeptide subtilosin A allowing for peptide engineering without compromising formation of thioether bridges. A panel of natural and hybrid sactipeptides was produced to study the AlbA-mediated formation of thioether bridges, which were identified mass-spectrometrically. In a proof-of-principle study, we re-engineered subtilosin A to a thioether-bridged, specific streptavidin targeting peptide, opening the door for the functional engineering of sactipeptides.
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Péptidos , Sulfuros , Sulfuros/química , Péptidos/químicaRESUMEN
SARS-CoV-2 infection is a major global public health concern with incompletely understood pathogenesis. The SARS-CoV-2 spike (S) glycoprotein comprises a highly conserved free fatty acid binding pocket (FABP) with unknown function and evolutionary selection advantage1,2. Deciphering FABP impact on COVID-19 progression is challenged by the heterogenous nature and large molecular variability of live virus. Here we create synthetic minimal virions (MiniVs) of wild-type and mutant SARS-CoV-2 with precise molecular composition and programmable complexity by bottom-up assembly. MiniV-based systematic assessment of S free fatty acid (FFA) binding reveals that FABP functions as an allosteric regulatory site enabling adaptation of SARS-CoV-2 immunogenicity to inflammation states via binding of pro-inflammatory FFAs. This is achieved by regulation of the S open-to-close equilibrium and the exposure of both, the receptor binding domain (RBD) and the SARS-CoV-2 RGD motif that is responsible for integrin co-receptor engagement. We find that the FDA-approved drugs vitamin K and dexamethasone modulate S-based cell binding in an FABP-like manner. In inflammatory FFA environments, neutralizing immunoglobulins from human convalescent COVID-19 donors lose neutralization activity. Empowered by our MiniV technology, we suggest a conserved mechanism by which SARS-CoV-2 dynamically couples its immunogenicity to the host immune response.
Asunto(s)
COVID-19/inmunología , Ácidos Grasos/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Virión/inmunología , Células A549 , Sitio Alostérico/genética , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Sitios de Unión/genética , COVID-19/metabolismo , COVID-19/virología , Células Cultivadas , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Proteínas de Unión a Ácidos Grasos/inmunología , Proteínas de Unión a Ácidos Grasos/metabolismo , Ácidos Grasos/metabolismo , Humanos , Células MCF-7 , Microscopía Confocal/métodos , Unión Proteica , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiología , Homología de Secuencia de Aminoácido , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Virión/metabolismo , Virión/ultraestructuraRESUMEN
Extracellular vesicles (EVs) are fundamental for intercellular communication and influence nearly every process in cell physiology. However, because of their intricate molecular complexity, quantitative knowledge on their signaling mechanisms is missing, particularly impeding their therapeutic application. We used a complementary and quantitative engineering approach based on sequential synthetic bottom-up assembly of fully functional EVs with precisely controlled lipid, protein, and RNA composition. We show that the functionalities of synthetic EVs are analogous to natural EVs and demonstrate their programmable therapeutic administration for wound healing and neovascularization therapy. We apply transcriptome profiling to systematically decode synergistic effects between individual EV constituents, enabling analytical dissection and a fundamental understanding of EV signaling.
RESUMEN
Lipid-based vesicles have found widespread applications in the life sciences, allowing for fundamental insights into membrane-based processes in cell biology and as carrier systems for drug delivery purposes. So far, mostly small unilamellar vesicles (SUVs) with diameters of ~100 nm have been applied as carrier systems for biomedical applications. Despite this progress, several systematic limitations have arisen due to SUV dimensions, e.g., the size and total amount of applicable cargo is limited. Giant unilamellar vesicles (GUVs) might offer a pragmatic alternative for efficient cargo delivery. However, due to the lack of reliable high-throughput production technologies for GUV-carrier systems, only little is known about their interaction with cells. Here we present a microfluidic-based mechanical droplet-splitting pipeline for the production of carrier-GUVs with diameters of ~2 µm. The technology developed allows for highly efficient cargo loading and unprecedented control over the biological and physicochemical properties of GUV membranes. By generating differently charged (between -31 and + 28 mV), bioligand-conjugated (e.g. with E-cadherin, NrCam and antibodies) and PEG-conjugated GUVs, we performed a detailed investigation of attractive and repulsive GUV-cell interactions. Fine-tuning of these interactions allowed for targeted cellular GUV delivery. Moreover, we evaluated strategies for intracellular GUV cargo release by lysosomal escape mediated by the pH sensitive lipid DOBAQ, enabling cytoplasmic transmission. The presented GUV delivery technology and the systematic characterization of associated GUV-cell interactions could provide a means for more efficient drug administration and will pave the way for hitherto impossible approaches towards a targeted delivery of advanced cargo such as microparticles, viruses or macromolecular DNA-robots.
Asunto(s)
Microfluídica , Liposomas Unilamelares , LípidosRESUMEN
The cellular level of nicotinamide adenine dinucleotide (NAD+), through its different functions, affects cellular metabolism and signalling1-3. A decrease in the NAD+ content has been associated with various pathologies and physiological aging4,5, while strategies to boost cellular NAD+ levels have been shown to be effective against age-related diseases in many animal models6. The link between decreased NAD+ levels and numerous pathologies and physiological aging has triggered the need for a simple quantification method for NAD+, ideally applicable at the point of care. Here, we introduce a bioluminescent biosensor for the rapid quantification of NAD+ levels in biological samples, which can be used either in laboratories or at the point of care. The biosensor is a semisynthetic, light-emitting sensor protein that changes the colour of emitted light from blue to red on binding of NAD+. This NAD+-dependent colour change enables the use of the biosensor in paper-based assays in which NAD+ is quantified by measuring the colour of the emitted light by using either a simple digital camera or a plate reader. We used the approach to quantify NAD+ levels in cell culture, tissue and blood samples, yielding results that agreed with those from standard testing methods. The same biosensor furthermore allows the quantification of NAD+-dependent enzymatic activities in blood samples, thus expanding its utility as a tool for point-of-care diagnostics.
Asunto(s)
Técnicas Biosensibles , NAD/metabolismo , Sistemas de Atención de Punto , Animales , Células Cultivadas , Color , Diseño de Equipo , Biblioteca de Genes , Humanos , Cinética , Hígado/química , Luminiscencia , Masculino , Ratones , Ratones Endogámicos C57BL , NAD/análisis , NAD/sangre , Pruebas en el Punto de AtenciónRESUMEN
Monitoring metabolites at the point of care could improve the diagnosis and management of numerous diseases. Yet for most metabolites, such assays are not available. We introduce semisynthetic, light-emitting sensor proteins for use in paper-based metabolic assays. The metabolite is oxidized by nicotinamide adenine dinucleotide phosphate, and the sensor changes color in the presence of the reduced cofactor, enabling metabolite quantification with the use of a digital camera. The approach makes any metabolite that can be oxidized by the cofactor a candidate for quantitative point-of-care assays, as shown for phenylalanine, glucose, and glutamate. Phenylalanine blood levels of phenylketonuria patients were analyzed at the point of care within minutes with only 0.5 microliters of blood. Results were within 15% of those obtained with standard testing methods.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia , Técnicas Biosensibles , Proteínas de Escherichia coli/química , Monitoreo Fisiológico/métodos , Pruebas en el Punto de Atención , Tetrahidrofolato Deshidrogenasa/química , Glucemia/análisis , Proteínas de Escherichia coli/genética , Ácido Glutámico/sangre , Humanos , NADP/metabolismo , Oxidación-Reducción , Fenilalanina/sangre , Fenilcetonurias/sangre , Fenilcetonurias/diagnóstico , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
Differential mobility spectrometry (DMS) is capable of separating stereoisomeric molecular ions based on their mobility in an oscillating electrical field with an asymmetric waveform. Thus, it is an "orthogonal" technique to chromatography and (tandem) mass spectrometry. Bioactive lipids, particularly of the eicosanoid and docosanoid class feature numerous stereoisomers, which exhibit a highly specific structure-activity relationship. Moreover, the geometry of these compounds also reflects their biochemical origin. Therefore, the unambiguous characterization of related isomers of the eicosanoid and docosanoid classes is of fundamental importance to the understanding of their origin and function in many biological processes. Here we show, that SelexION DMS technology coupled to µLC-MS/MS is capable of differentiating at least five closely related leukotrienes partially coeluting and (almost) unresolvable using LC-MS/MS only. We applied the developed method to the separation of LTB4 and its coeluting isomer 5S,12S-diHETE in murine peritoneal exudate cells, showing that LTB4 is present only after zymosan A injection while its isomer 5S,12S-diHETE is produced after saline (PBS) administration. Additionally, we show that the SelexION technology can also be applied to the separation of PD1 and PDX (10S,17S-diHDHA), two isomeric protectins.
Asunto(s)
Antígenos CD59/aislamiento & purificación , Leucotrienos/aislamiento & purificación , Análisis Espectral/métodos , Animales , Antígenos CD59/química , Cromatografía Liquida , Isomerismo , Leucotrienos/química , Ratones , Espectrometría de Masas en TándemRESUMEN
Over the last decades the field of pharmaceutically relevant peptides has enormously expanded. Among them, several peptide families exist that contain three or more disulfide bonds. In this context, elucidation of the disulfide patterns is extremely important as these motifs are often prerequisites for folding, stability, and activity. An example of this structure-determining pattern is a cystine knot which comprises three constrained disulfide bonds and represents a core element in a vast number of mechanically interlocked peptidic structures possessing different biological activities. Herein, we present our studies on disulfide pattern determination and structure elucidation of cystine-knot miniproteins derived from Momordica cochinchinensis peptide MCoTI-II, which act as potent inhibitors of human matriptase-1. A top-down mass spectrometric analysis of the oxidised and bioactive peptides is described. Following the detailed sequencing of the peptide backbone, interpretation of the MS(3) spectra allowed for the verification of the knotted topology of the examined miniproteins. Moreover, we found that the fragmentation pattern depends on the knottin's folding state, hence, tertiary structure, which to our knowledge has not been described for a top-down MS approach before.
Asunto(s)
Ciclotidas/química , Miniproteínas Nodales de Cistina/química , Disulfuros/análisis , Momordica/química , Proteínas de Plantas/química , Secuencia de Aminoácidos , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
Cystine-knot miniproteins define a class of bioactive molecules with several thousand natural members. Their eponymous motif comprises a rigid structured core formed by six disulfide-connected cysteine residues, which accounts for its exceptional stability towards thermic or proteolytic degradation. Since they display a remarkable sequence tolerance within their disulfide-connected loops, these molecules are considered promising frameworks for peptide-based pharmaceuticals. Natural open-chain cystine-knot trypsin inhibitors of the MCoTI (Momordica cochinchinensis trypsin inhibitor) and SOTI (Spinacia oleracea trypsin inhibitor) families served as starting points for the generation of inhibitors of matriptase-1, a type II transmembrane serine protease with possible clinical relevance in cancer and arthritic therapy. Yeast surface-displayed libraries of miniproteins were used to select unique and potent matriptase-1 inhibitors. To this end, a knowledge-based library design was applied that makes use of detailed information on binding and folding behavior of cystine-knot peptides. Five inhibitor variants, four of the MCoTI family and one of the SOTI family, were identified, chemically synthesized and oxidatively folded towards the bioactive conformation. Enzyme assays revealed inhibition constants in the low nanomolar range for all candidates. One subnanomolar binder (Ki = 0.83 nM) with an inverted selectivity towards trypsin and matriptase-1 was identified.
Asunto(s)
Miniproteínas Nodales de Cistina/química , Miniproteínas Nodales de Cistina/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Serina Endopeptidasas/metabolismo , Línea Celular , Miniproteínas Nodales de Cistina/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Modelos Moleculares , Biblioteca de Péptidos , Unión Proteica , Conformación Proteica , Inhibidores de Tripsina/química , Inhibidores de Tripsina/farmacología , Técnicas del Sistema de Dos Híbridos , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidoresRESUMEN
Small, compact, and highly symmetric cube-octameric silsesquioxanes have recently attracted increased attention as scaffolds for tailor-made bioconjugates. The expanded arsenal of effective conjugation methods (CuAAC, TEC, oxime ligation) allows one to decorate these nanoparticles bearing up to eight addressable organic substituents, with a wide range of biorelevant ligands, among them carbohydrates, peptides, miniproteins, reporter molecules, and rare-earth chelates. Low toxicity of COSS-based molecules combined with solubility in aqueous systems and half-life sufficient for in vivo studies make these structures attractive targets for a number of applications, among them drug delivery, tumor diagnostics and therapy.
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Portadores de Fármacos/química , Modelos Moleculares , Compuestos Organometálicos/química , Dióxido de Silicio/química , Silicio/química , Animales , Carbohidratos/química , Portadores de Fármacos/metabolismo , Portadores de Fármacos/farmacología , Humanos , Compuestos Organometálicos/metabolismo , Péptidos/química , Péptidos/metabolismo , Siloxanos/químicaRESUMEN
Cube octameric silsesquioxanes (COSS) are among the smallest nanoparticles known to date with a diameter of only 0.7 nm. We describe a COSS-based delivery system which allows for the drug targeting in human cells. It comprises a siloxane core with seven pendant aminopropyl groups and a fluorescently labeled peptidic ligand attached to one cage corner via a reversible disulfide bond to ensure its intracellular release. Bimodal amplitude-modulated atomic force microscopy (AFM) experiments revealed the formation of dendritic COSS structures by a self-assembly of single particles on negatively charged surfaces. Nuclear targeting was demonstrated in HeLa cells by selective binding of released p21(Cip1/Waf1)-derived cargo peptide to PCNA, a protein involved in DNA replication and repair.
Asunto(s)
Sistemas de Liberación de Medicamentos , Compuestos de Organosilicio/química , Péptidos/metabolismo , Compuestos de Amonio Cuaternario/química , Cromatografía Líquida de Alta Presión , Células HeLa , Humanos , Microscopía de Fuerza Atómica , Estructura Molecular , Nanopartículas/química , Tamaño de la Partícula , Péptidos/química , Péptidos/genética , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismoRESUMEN
Bioactive peptides often contain several disulfide bonds that provide the main contribution to conformational rigidity and structural, thermal, or biological stability. Among them, cystine-knot peptides-commonly named "knottins"-make up a subclass with several thousand natural members. Hence, they are considered promising frameworks for peptide-based pharmaceuticals. Although cystine-knot peptides are available through chemical and recombinant synthetic routes, oxidative folding to afford the bioactive isomers still remains a crucial step. We therefore investigated the oxidative folding of ten protease-inhibiting peptides from two knottin families, as well as that of an HIV entry inhibitor and of aprotinin, under two conventional sets of folding conditions and by a newly developed procedure. Kinetic studies identified folding conditions that resulted in correctly folded miniproteins with high rates of conversion even for highly hydrophobic and aggregation-prone peptides in concentrated solutions.
Asunto(s)
Cistina/química , Fragmentos de Péptidos/química , Pliegue de Proteína , Acetonitrilos/química , Secuencia de Aminoácidos , Aprotinina/química , Tampones (Química) , Dimetilsulfóxido/química , Disulfuros/química , Guanidina/química , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Trifluoroetanol/químicaRESUMEN
A method for the detection of haloalkane conversion to the corresponding alcohols by haloalkane dehalogenases is described. It is based on a multistage enzyme reaction which allows for the analysis of alkyl halides in buffered systems. Irreversible hydrolytic dehalogenation catalyzed by haloalkane dehalogenase DhaA from Rhodococcus erythropolis transfers an alkyl halide into a corresponding alcohol that is further oxidized by alcohol oxidase AOX from Pichia pastoris yielding a respective aldehyde and hydrogen peroxide easily detectable via the horseradish peroxidase catalyzed oxidation of chromogenic molecules. Due to its high sensitivity (0.025 mM, 0.43 ppm for 1,3-dibromopropane), low expenditure and the ability of handling a large number of samples in parallel, this method is an attractive alternative to existing procedures for the monitoring of both haloalkanes and dehalogenases.
RESUMEN
Polyhedral silsesquioxanes are considered valuable conjugation scaffolds. Nevertheless, only a few examples of silsesquioxane-assembled peptide oligomers have been reported to date. We developed a new bioorthogonal cube-octameric silsesquioxane (COSS) scaffold bearing eight aminooxy coupling sites allowing for the conjugation of diverse peptides via oxime ligation. We found that the coupling efficacy depends on the ligand in view of steric hindrance and electrostatic repulsion. For the first time scaffold-based conjugation of cystine-knot miniproteins having a backbone of about thirty amino acids was successfully accomplished without loss of bioactivity. Atomic force microscopy (AFM) provided further knowledge on the size of COSS verifying them as picoscaffolds growing upon bioconjugation to nano-dimension.
Asunto(s)
Nanopartículas/química , Compuestos de Organosilicio/química , Péptidos/química , Aldehídos/química , Secuencia de Aminoácidos , Microscopía de Fuerza Atómica , Modelos Moleculares , Datos de Secuencia Molecular , Nanopartículas/ultraestructura , Oximas/químicaRESUMEN
Cube-octameric silsesquioxane (POSS) based conjugation scaffolds for copper catalysed azide-alkyne [3+2] cycloaddition are reported. The synthetic route to octaazido and octaalkyno functionalised POSS templates without cage rearrangements is described. A set of click couplings is conducted including the first effective conjugation with a fully unprotected functional peptide towards a POSS assembled peptide octamer.