RESUMEN
New methods to handle membrane bound proteins, e.g. G-protein coupled receptors (GPCRs), are highly desirable. Recently, apoliprotein A1 (ApoA1) based lipoprotein particles have emerged as a new platform for studying membrane proteins, and it has been shown that they can self-assemble in combination with phospholipids to form discoidal shaped particles that can stabilize membrane proteins. In the present study, we have investigated an ApoA1 mimetic peptide with respect to its solution structure when in complex with phospholipids. This was achieved using a powerful combination of small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) supported by coarse-grained molecular dynamics simulations. The detailed structure of the discs was determined in unprecedented detail and it was found that they adopt a discoidal structure very similar to the ApoA1 based nanodiscs. We furthermore show that, like the ApoA1 and derived nanodiscs, these peptide discs can accommodate and stabilize a membrane protein. Finally, we exploit their dynamic properties and show that the 18A discs may be used for transferring membrane proteins and associated phospholipids directly and gently into phospholipid nanodiscs.
Asunto(s)
Apolipoproteína A-I/química , Proteínas de la Membrana/química , Nanotubos de Péptidos/química , Péptidos/química , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Fosfolípidos/química , Estabilidad ProteicaRESUMEN
Carbohydrates are considered as promising templates for the display of multiple copies of antimicrobial peptides. Herein, we describe the design and synthesis of chimeric structures containing two or four copies of the antimicrobial peptides KKLFKKILKYL-NH(2) (BP100) and KKLfKKILKYL-NH(2) (BP143) attached to the carbohydrate template cyclodithioerythritol (cDTE) or α-D-galactopyranoside (Galp). The synthesis involved the preparation of the corresponding peptide aldehyde followed by coupling to an aminooxy-functionalized carbohydrate template. After purification, the multivalent display systems were obtained in high purities (90-98%) and in good yields (42-64%). These compounds were tested against plant and human pathogenic bacteria and screened for their cytotoxicity on eukaryotic cells. They showed lower MIC values than the parent peptides against the bacteria analyzed. In particular, the carbopeptides derived from cDTE and Galp, which contained two or four copies of BP100, respectively, were 2- to 8-fold more active than the monomeric peptide against the phytopathogenic bacteria. These results suggest that preassembling antimicrobial peptides to multimeric structures is not always associated with a significant improvement of the activity. In contrast, the carbopeptides synthesized were active against human red blood cells pointing out that peptide preassembly is critical for the hemolytic activity. Notably, peptide preassembly resulted in an enhanced bactericidal effect.
RESUMEN
Glioblastoma (GBM) is a devastating cancer with basically no curative treatment. Even with aggressive treatment, the median survival is disappointing 14 months. Surgery remains the key treatment and the postoperative survival is determined by the extent of resection. Unfortunately, the invasive growth with irregular infiltrating margins complicates an optimal surgical resection. Precise intraoperative tumor visualization is therefore highly needed and molecular targeted near-infrared (NIR) fluorescence imaging potentially constitutes such a tool. The urokinase-type Plasminogen Activator Receptor (uPAR) is expressed in most solid cancers primarily at the invading front and the adjacent activated peritumoral stroma making it an attractive target for targeted fluorescence imaging. The purpose of this study was to develop and evaluate a new uPAR-targeted optical probe, IRDye800CW-AE344, for fluorescence guided surgery (FGS). Methods: In the present study we characterized the fluorescent probe with regard to binding affinity, optical properties, and plasma stability. Further, in vivo imaging characterization was performed in nude mice with orthotopic human patient derived glioblastoma xenografts, and we performed head-to-head comparison within FGS between our probe and the traditional procedure using 5-ALA. Finally, the blood-brain barrier (BBB) penetration was characterized in a 3D BBB spheroid model. Results: The probe effectively visualized GBM in vivo with a tumor-to-background ratio (TBR) above 4.5 between 1 to 12 h post injection and could be used for FGS of orthotopic human glioblastoma xenografts in mice where it was superior to 5-ALA. The probe showed a favorable safety profile with no evidence of any acute toxicity. Finally, the 3D BBB model showed uptake of the probe into the spheroids indicating that the probe crosses the BBB. Conclusion: IRDye800CW-AE344 is a promising uPAR-targeted optical probe for FGS and a candidate for translation into human use.
Asunto(s)
Glioblastoma , Indoles , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentales , Imagen Óptica , Péptidos , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Animales , Línea Celular Tumoral , Glioblastoma/diagnóstico por imagen , Glioblastoma/metabolismo , Glioblastoma/cirugía , Xenoinjertos , Humanos , Indoles/química , Indoles/farmacología , Ratones , Trasplante de Neoplasias , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/cirugía , Péptidos/química , Péptidos/farmacologíaRESUMEN
Plant metabolism depends on cascade reactions mediated by dynamic enzyme assemblies known as metabolons. In this context, the cytochrome P450 (P450) superfamily catalyze key reactions underpinning the unique diversity of bioactive compounds. In contrast to their soluble bacterial counterparts, eukaryotic P450s are anchored to the endoplasmic reticulum membrane and serve as metabolon nucleation sites. Hence, membrane anchoring appears to play a pivotal role in the evolution of complex biosynthetic pathways. Here, a model membrane assay enabled characterization of membrane anchor dynamics by single molecule microscopy. As a model system, we reconstituted the membrane anchor of cytochrome P450 oxidoreductase (POR), the ubiquitous electron donor to all microsomal P450s. The transmembrane segment in the membrane anchor of POR is relatively conserved, corroborating its functional importance. We observe dynamic colocalization of the POR anchors in our assay suggesting that membrane anchoring might promote intermolecular interactions and in this way impact assembly of metabolic multienzyme complexes.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Plantas/enzimología , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Oxidación-ReducciónRESUMEN
Biomarker microarrays are becoming valuable tools for serological screening of disease-associated autoantibodies. Post-translational modifications (PTMs) such as glycosylation extend the range of protein function, and a variety of glycosylated proteins are known to be altered in disease progression. Here, we have developed a synthetic screening microarray platform for facile display of O-glycosylated peptides (O-PTMs). By introduction of a capping step during chemical solid-phase glycopeptide synthesis, selective enrichment of N-terminal glycopeptide end products was achieved on an amine-reactive hydrogel-coated microarray glass surface, allowing high-throughput display of large numbers of glycopeptides. Utilizing a repertoire of recombinant glycosyltransferases enabled further diversification of the array libraries in situ and display of a new level of potential biomarker candidates for serological screening. As proof-of-concept, we have demonstrated that MUC1 glycopeptides could be assembled and used to detect autoantibodies in vaccine-induced disease-free breast cancer patients and in patients with confirmed disease at time of diagnosis.
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Autoanticuerpos/sangre , Biomarcadores de Tumor/sangre , Glicopéptidos/análisis , Proteómica/métodos , Autoanticuerpos/inmunología , Biomarcadores de Tumor/inmunología , Neoplasias de la Mama/sangre , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/inmunología , Vacunas contra el Cáncer/inmunología , Femenino , Glicopéptidos/inmunología , Glicopéptidos/metabolismo , Glicosilación , Glicosiltransferasas/metabolismo , Humanos , Mucina-1/análisis , Mucina-1/inmunología , Mucina-1/metabolismo , Péptidos/análisis , Péptidos/inmunología , Péptidos/metabolismoRESUMEN
Inhaled antibiotic treatment of cystic fibrosis-related bacterial biofilm infections is challenging because of the pathological environment of the lungs. Here, we present an "environment-adaptive" nanoparticle composed of a solid poly lactic-co-glycolic acid (PLGA) core and a mucus-inert, enzymatically cleavable shell of d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) for the site-specific delivery of antibiotics to bacterial biofilms via aerosol administration. The hybrid nanoparticles with ultrasmall size were self-assembled via a nanoprecipitation process by using a facile microfluidic method. The interactions of the nanoparticles with the biological barriers were comprehensively investigated by using cutting-edge techniques (e.g., quartz crystal microbalance with dissipation monitoring, total internal reflection fluorescence microscopy-based particle tracking, in vitro biofilm model cultured in a flow-chamber system, and quantitative imaging analysis). Our results suggest that the mucus-inert, enzymatically cleavable TPGS shell enables the nanoparticles to penetrate through the mucus, accumulate in the deeper layer of the biofilms, and serve as sustained release depot, thereby improving the killing efficacy of azithromycin (a macrolide antibiotic) against biofilm-forming Pseudomonas aeruginosa. In conclusion, the ultrasmall TPGS-PLGA hybrid nanoparticles represent an efficient delivery system to overcome the multiple barriers and release antibiotics in a sustained manner in the vicinity of the biofilm-forming bacteria.
Asunto(s)
Antibacterianos/química , Biopelículas/efectos de los fármacos , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Nanopartículas/química , Ácido Poliglicólico/administración & dosificación , Ácido Poliglicólico/química , Pseudomonas aeruginosa/efectos de los fármacos , Administración por Inhalación , Antibacterianos/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
Recognition of Nod factors by LysM receptors is crucial for nitrogen-fixing symbiosis in most legumes. The large families of LysM receptors in legumes suggest concerted functions, yet only NFR1 and NFR5 and their closest homologs are known to be required. Here we show that an epidermal LysM receptor (NFRe), ensures robust signalling in L. japonicus. Mutants of Nfre react to Nod factors with increased calcium spiking interval, reduced transcriptional response and fewer nodules in the presence of rhizobia. NFRe has an active kinase capable of phosphorylating NFR5, which in turn, controls NFRe downstream signalling. Our findings provide evidence for a more complex Nod factor signalling mechanism than previously anticipated. The spatio-temporal interplay between Nfre and Nfr1, and their divergent signalling through distinct kinases suggests the presence of an NFRe-mediated idling state keeping the epidermal cells of the expanding root system attuned to rhizobia.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Lipopolisacáridos/genética , Lotus/metabolismo , Proteínas de Plantas/genética , Receptores de Superficie Celular/genética , Rhizobium/metabolismo , Nódulos de las Raíces de las Plantas/metabolismo , Calcio/metabolismo , Lipopolisacáridos/metabolismo , Lotus/genética , Lotus/microbiología , Mutación , Fijación del Nitrógeno/fisiología , Fosforilación , Células Vegetales/metabolismo , Células Vegetales/microbiología , Proteínas de Plantas/metabolismo , Nodulación de la Raíz de la Planta/genética , Receptores de Superficie Celular/metabolismo , Rhizobium/genética , Nódulos de las Raíces de las Plantas/genética , Nódulos de las Raíces de las Plantas/microbiología , Transducción de Señal , Simbiosis/fisiologíaRESUMEN
Morphogens provide positional information and their concentration is key to the organized development of multicellular organisms. Nitrogen-fixing root nodules are unique organs induced by Nod factor-producing bacteria. Localized production of Nod factors establishes a developmental field within the root where plant cells are reprogrammed to form infection threads and primordia. We found that regulation of Nod factor levels by Lotus japonicus is required for the formation of nitrogen-fixing organs, determining the fate of this induced developmental program. Our analysis of plant and bacterial mutants shows that a host chitinase modulates Nod factor levels possibly in a structure-dependent manner. In Lotus, this is required for maintaining Nod factor signalling in parallel with the elongation of infection threads within the nodule cortex, while root hair infection and primordia formation are not influenced. Our study shows that infected nodules require balanced levels of Nod factors for completing their transition to functional, nitrogen-fixing organs.