RESUMEN
Antibodies not only play a major role in clinical diagnostics and biopharmaceutical analysis but also are a class of drugs that are regularly used to treat numerous diseases. The identification of antibody-epitope binding sites is then of great interest to many emerging medical and bioanalytical applications, particularly to design monoclonal antibodies (mAb) mimics taking advantage of amino acid residues involved in the binding. Among relevant antibodies, the monoclonal antibody rituximab has received significant attention as it is exploited to treat several cancers including non-Hodgkin's lymphoma and chronic lymphocytic leukemia, as well as some autoimmune disorders such as rheumatoid arthritis. The binding of rituximab to the targeted cells occurs via the recognition of the CD20 epitope. A crystallographic study has shown that the binding area, named paratope, is located at the surface of rituximab. Combining the SPOT method and the complementary surface plasmon resonance technique allowed us to detect an extended recognition domain buried in the pocket of the rituximab Fab formed by four ß-sheets. More generally, the present study offers a comprehensive approach to identify antibody-epitope binding sites.
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Antígenos CD20 , Resonancia por Plasmón de Superficie , Anticuerpos Monoclonales de Origen Murino , Sitios de Unión , Epítopos , RituximabRESUMEN
Recently, we designed novel amphipathic cell-penetrating peptides, called WRAP, able to transfer efficiently siRNA molecules into cells. In order to gain more information about the relationship between amino acid composition, nanoparticle formation and cellular internalization of these peptides composed of only three amino acids (leucine, arginine and tryptophan), we performed a structure-activity relationship (SAR) study. First, we compared our WRAP1 and WRAP5 peptides with the C6M1 peptide also composed of the same three amino acids and showing similar behaviors in siRNA transfection. Afterwards, to further define the main determinants in the WRAP activity, we synthesized 13 new WRAP analogues harboring different modifications like the number and location of leucine and arginine residues, the relative location of tryptophan residues, as well as the role of the α-helix formation upon proline insertions within the native WRAP sequence. After having compared the ability of these peptides to form peptide-based nanoparticles (PBNs) using different biophysical methods and to induce a targeted gene silencing in cells, we established the main sequential requirements of the amino acid composition of the WRAP peptide. In addition, upon measuring the WRAP-based siRNA transfection ability into cells compared to several non-peptide transfection agents available on the markets, we confirmed that WRAP peptides induced an equivalent level of targeted gene silencing but in most of the cases with lower cell toxicity as clearly shown in clonogenic assays.
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Vendajes de Compresión , Lípidos/química , Nanopartículas/química , ARN Interferente Pequeño/química , Transfección , Secuencia de Aminoácidos , Línea Celular Tumoral , Péptidos de Penetración Celular , Sistemas de Liberación de Medicamentos , Silenciador del Gen/efectos de los fármacos , Glioblastoma , Humanos , Indicadores y Reactivos/química , ARN Interferente Pequeño/farmacología , Relación Estructura-ActividadRESUMEN
Delivery of small interfering RNA (siRNA) as a therapeutic tool is limited due to critical obstacles such as the cellular barrier, the negative charges of the siRNA molecule, and its instability in serum. Several siRNA delivery systems have been constructed using cell-penetrating peptides (CPPs) since the CPPs have shown a high potential for oligonucleotide delivery into the cells, especially by forming nanoparticles. In this study, we have developed a new family of short (15mer or 16mer) tryptophan-(W) and arginine-(R) rich Amphipathic Peptides (WRAP) able to form stable nanoparticles and to enroll siRNA molecules into cells. The lead peptides, WRAP1 and WRAP5, form defined nanoparticles smaller than 100 nm as characterized by biophysical methods. Furthermore, they have several benefits as oligonucleotide delivery tools such as the rapid encapsulation of the siRNA, the efficient siRNA delivery in several cell types, and the high gene silencing activity, even in the presence of serum. In conclusion, we have designed a new family of CPPs specifically dedicated for siRNA delivery through nanoparticle formation. Our results indicate that the WRAP family has significant potential for the safe, efficient, and rapid delivery of siRNA for diverse applications.
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Péptidos de Penetración Celular/química , Nanopartículas/química , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Secuencia de Aminoácidos , Línea Celular Tumoral , Humanos , Modelos Moleculares , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
BACKGROUND: Small interfering RNAs (siRNAs) are powerful tools to control gene expression. However, due to their poor cellular permeability and stability, their therapeutic development requires a specific delivery system. Among them, cell-penetrating peptides (CPP) have been shown to transfer efficiently siRNA inside the cells. Recently we developed amphipathic peptides able to self-assemble with siRNAs as peptide-based nanoparticles and to transfect them into cells. However, despite the great potential of these drug delivery systems, most of them display a low resistance to proteases. RESULTS: Here, we report the development and characterization of a new CPP named RICK corresponding to the retro-inverso form of the CADY-K peptide. We show that RICK conserves the main biophysical features of its L-parental homologue and keeps the ability to associate with siRNA in stable peptide-based nanoparticles. Moreover the RICK:siRNA self-assembly prevents siRNA degradation and induces inhibition of gene expression. CONCLUSIONS: This new approach consists in a promising strategy for future in vivo application, especially for targeted anticancer treatment (e.g. knock-down of cell cycle proteins). Graphical abstract RICK-based nanoparticles: RICK peptides and siRNA self-assemble in peptide-based nanoparticles to penetrate into the cells and to induce target protein knock-down.
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Péptidos de Penetración Celular/química , Nanopartículas/química , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Transfección , Línea Celular Tumoral , Péptidos de Penetración Celular/metabolismo , Genes Reporteros , Humanos , Nanopartículas/metabolismo , Nanopartículas/ultraestructura , Estabilidad del ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
The established correlation between neurodegenerative disorders and intracerebral deposition of polyglutamine aggregates motivates attempts to better understand their fibrillar structure. We designed polyglutamines with a few lysines inserted to overcome the hindrance of extreme insolubility and two D-lysines to limit the lengths of ß-strands. One is 33 amino acids long (PolyQKd-33) and the other has one fewer glutamine (PolyQKd-32). Both form well-dispersed fibrils suitable for analysis by electron microscopy. Electron diffraction confirmed cross-ß structures in both fibrils. Remarkably, the deletion of just one glutamine residue from the middle of the peptide leads to substantially different amyloid structures. PolyQKd-32 fibrils are consistently 10-20% wider than PolyQKd-33, as measured by negative staining, cryo-electron microscopy, and scanning transmission electron microscopy. Scanning transmission electron microscopy analysis revealed that the PolyQKd-32 fibrils have 50% higher mass-per-length than PolyQKd-33. This distinction can be explained by a superpleated ß-structure model for PolyQKd-33 and a model with two ß-solenoid protofibrils for PolyQKd-32. These data provide evidence for ß-arch-containing structures in polyglutamine fibrils and open future possibilities for structure-based drug design.
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Sustitución de Aminoácidos , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Péptidos , Multimerización de Proteína , Concentración de Iones de Hidrógeno , Estructura Secundaria de ProteínaRESUMEN
Alternative splicing is an important means of generating the protein diversity necessary for cellular functions. Hence, there is a growing interest in assessing the structural and functional impact of alternative protein isoforms. Typically, experimental studies are used to determine the structures of the canonical proteins ignoring the other isoforms. Therefore, there is still a large gap between abundant sequence information and meager structural data on these isoforms. During the last decade, significant progress has been achieved in the development of bioinformatics tools for structural and functional annotations of proteins. Moreover, the appearance of the AlphaFold program opened up the possibility to model a large number of high-confidence structures of the isoforms. In this study, using state-of-the-art tools, we performed in silico analysis of 58 eukaryotic proteomes. The evaluated structural states included structured domains, intrinsically disordered regions, aggregation-prone regions, and tandem repeats. Among other things, we found that the isoforms have fewer signal peptides, transmembrane regions, or tandem repeat regions in comparison with their canonical counterparts. This could change protein function and/or cellular localization. The AlphaFold modeling demonstrated that frequently isoforms, having differences with the canonical sequences, still can fold in similar structures though with significant structural rearrangements which can lead to changes of their functions. Based on the modeling, we suggested classification of the structural differences between canonical proteins and isoforms. Altogether, we can conclude that a majority of isoforms, similarly to the canonical proteins are under selective pressure for the functional roles.
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Biología Computacional , Proteoma , Proteoma/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/química , Empalme AlternativoRESUMEN
During the last two decades, delivery has become a major challenge for the development of new therapeutic molecules for the clinic. Although, several strategies either viral or non viral have been proposed to favor cellular uptake and targeting of therapeutics, only few of them have reach preclinical evaluation. Amongst them, cell-penetrating peptide (CPP) constitutes one of the most promising strategy and has applied for systemic in vivo delivery of a variety of therapeutic molecules. Two CPP-strategies have been described; using peptide carriers either covalently-linked to the cargo or forming non-covalent stable complexes with cargo. Peptide-based nanoparticle delivery system corresponds to small amphipathic peptides able to form stable nanoparticles with either proteins/peptides or nucleic acids and to enter the cell independently of the endosomal pathway. Three families of peptide-based nanoparticle systems; MPG, PEP and CADY have been successfully used for the delivery of various biologically active cargoes both ex vivo and in vivo in several animal models. This review will focus on the mechanism of the peptide-based nanoparticles; PEP, MPG and CADY in a structural and biophysical context. It will also highlight the major parameters associated to particle formation/stabilization and the impact of the carrier structural polymorphism in triggering cellular uptake.
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Péptidos de Penetración Celular , Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/química , Animales , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacología , Humanos , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
Delivery of siRNA remains a major limitation to their clinical application, and several technologies have been proposed to improve their cellular uptake. We recently described a peptide-based nanoparticle system for efficient delivery of siRNA into primary cell lines: CADY. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA and improves their cellular uptake independently of the endosomal pathway. In the present work, we have combined molecular modeling, spectroscopy, and membrane interaction approaches in order to gain further insight into CADY/siRNA particle mechanism of interaction with biological membrane. We demonstrate that CADY forms stable complexes with siRNA and binds phospholipids tightly, mainly through electrostatic interactions. Binding to siRNA or phospholipids triggers a conformational transition of CADY from an unfolded state to an alpha-helical structure, thereby stabilizing CADY/siRNA complexes and improving their interactions with cell membranes. Therefore, we propose that CADY cellular membrane interaction is driven by its structural polymorphism which enables stabilization of both electrostatic and hydrophobic contacts with surface membrane proteoglycan and phospholipids.
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Péptidos/química , ARN Interferente Pequeño/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Línea Celular , Membrana Celular/metabolismo , Dicroismo Circular , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Liposomas/química , Liposomas/metabolismo , Micelas , Modelos Moleculares , Datos de Secuencia Molecular , Distribución Normal , Oligorribonucleótidos/química , Péptidos/síntesis química , Péptidos/metabolismo , Fosfolípidos/química , Fosfolípidos/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Gene silencing mediated by double-stranded small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for a variety of diseases and, indeed, the first therapeutic siRNA was approved by the FDA in 2018. As an alternative to the traditional delivery systems for nucleic acids, peptide-based nanoparticles (PBNs) have been applied successfully for siRNA delivery. Recently, we have developed amphipathic cell-penetrating peptides (CPPs), called WRAP allowing a rapid and efficient siRNA delivery into several cell lines at low doses (20 to 50 nM). In this study, using a highly specific gene silencing system, we aimed to elucidate the cellular uptake mechanism of WRAP:siRNA nanoparticles by combining biophysical, biological, confocal and electron microscopy approaches. We demonstrated that WRAP:siRNA complexes remain fully active in the presence of chemical inhibitors of different endosomal pathways suggesting a direct cell membrane translocation mechanism. Leakage studies on lipid vesicles indicated membrane destabilization properties of the nanoparticles and this was supported by the measurement of WRAP:siRNA internalization in dynamin triple-KO cells. However, we also observed some evidences for an endocytosis-dependent cellular internalization. Indeed, nanoparticles co-localized with transferrin, siRNA silencing was inhibited by the scavenger receptor A inhibitor Poly I and nanoparticles encapsulated in vesicles were observed by electron microscopy in U87 cells. In conclusion, we demonstrate here that the efficiency of WRAP:siRNA nanoparticles is mainly based on the use of multiple internalization mechanisms including direct translocation as well as endocytosis-dependent pathways.
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Péptidos de Penetración Celular/uso terapéutico , Sistemas de Liberación de Medicamentos/métodos , Endocitosis , Nanopartículas/química , ARN Interferente Pequeño/metabolismo , Animales , Línea Celular , Péptidos de Penetración Celular/metabolismo , Silenciador del Gen , HumanosAsunto(s)
Péptidos de Penetración Celular/química , Portadores de Fármacos/química , Nanopartículas/química , Péptidos/química , ARN Interferente Pequeño/química , Animales , Línea Celular , Permeabilidad de la Membrana Celular , Humanos , Células Jurkat , Nanopartículas/administración & dosificación , ARN Interferente Pequeño/administración & dosificaciónRESUMEN
Small interfering RNAs (siRNAs) present a strong therapeutic potential because of their ability to inhibit the expression of any desired protein. Recently, we developed the retro-inverso amphipathic RICK peptide as novel non-covalent siRNA carrier. This peptide is able to form nanoparticles (NPs) by self-assembling with the siRNA resulting in the fully siRNA protection based on its protease resistant peptide sequence. With regard to an in vivo application, we investigated here the influence of the polyethylene glycol (PEG) grafting to RICK NPs on their in vitro and in vivo siRNA delivery properties. A detailed structural study shows that PEGylation did not alter the NP formation (only decrease in zeta potential) regardless of the used PEGylation rates. Compared to the native RICK:siRNA NPs, low PEGylation rates (≤20%) of the NPs did not influence their cellular internalization capacity as well as their knock-down specificity (over-expressed or endogenous system) in vitro. Because the behavior of PEGylated NPs could differ in their in vivo application, we analyzed the repartition of fluorescent labeled NPs injected at the one-cell stage in zebrafish embryos as well as their pharmacokinetic (PK) profile after administration to mice. After an intra-cardiac injection of the PEGylated NPs, we could clearly determine that 20% PEG-RICK NPs reduce significantly liver and kidney accumulation. NPs with 20% PEGylation constitutes a modular, easy-to-handle drug delivery system which could be adapted to other types of functional moieties to develop safe and biocompatible delivery systems for the clinical application of RNAi-based cancer therapeutics.
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Péptidos de Penetración Celular/administración & dosificación , Nanopartículas/administración & dosificación , Polietilenglicoles/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/administración & dosificación , Animales , Péptidos de Penetración Celular/química , Cisteína/administración & dosificación , Cisteína/química , Embrión no Mamífero , Luciferasas/genética , Masculino , Ratones Endogámicos C57BL , Nanopartículas/química , Polietilenglicoles/química , ARN Interferente Pequeño/química , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/química , Propiedades de Superficie , Pez CebraRESUMEN
RNA interference provides a powerful technology for specific gene silencing. Therapeutic applications of small interfering RNA (siRNA) however require efficient vehicles for stable complexation and intracellular delivery. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. In this context, the secondary amphipathic peptide CADY has shown to form stable siRNA complexes and to improve their cellular uptake independent of the endosomal pathway. In the present work, we have described the parameters influencing CADY nanoparticle formation (buffers, excipients, presence of serum, etc.), and have followed in details the CPP:siRNA self-assembly. Once optimal conditions were determined, we have compared the ability of seven different CADY analogues to form siRNA-loaded nanoparticles compared to CADY:siRNA. First of all, we were able to show by biophysical methods that structural polymorphism (α-helix) is an important prerequisite for stable nanoparticle formation independently of occurring sequence mutations. Luciferase assays revealed that siRNA complexed to CADY-K (shorter version) shows better knock-down efficiency on Neuro2a-Luc(+) and B16-F10-Luc(+) cells compared to CADY:siRNA. Altogether, CADY-K is an ideal candidate for further application especially with regards to ex vivo or in vivo applications.
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Péptidos de Penetración Celular/química , Animales , Línea Celular Tumoral , Portadores de Fármacos/química , Silenciador del Gen/efectos de los fármacos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Nanopartículas/química , Interferencia de ARN/fisiología , ARN Interferente Pequeño/metabolismoRESUMEN
Previous studies have highlighted the potential physiopathological and diagnostic role of N- and C-terminally truncated amyloid-ß (Aß) peptides in Alzheimer's disease. However, our knowledge about their production remains incomplete, in part due to the lack of very specific and sensitive tools for their detection. We thus developed specific monoclonal antibodies that target either Aß11-x or Aß17-x species, which result from the combined cleavages by ß/γ- or α/γ-secretases, respectively. The presence of Aß peptides truncated at residue 11 and 17 peptides was qualitatively and quantitatively assessed, using surface enhanced laser desorption ionization-time of flight mass spectrometry and xMAP (Multi-Analyte Profiling) immunoassays, in the supernatant of HEK293 cells that overexpress wild type or mutant Aß protein precursor or in which α- and ß-secretase activities had been modulated. Our results show a differential secretion of Aß11-40 and Aß17-40 species by these HEK293 cell lines. Finally, Aß11-40 concentration in human cerebrospinal fluid (measured with the new xMAP immunoassays) from a first pilot study was higher in cerebrospinal fluid samples from patients with Alzheimer's disease than in samples from patients with other types of dementia.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Demencia/metabolismo , Fragmentos de Péptidos/metabolismo , Anciano , Péptidos beta-Amiloides/líquido cefalorraquídeo , Humanos , Inmunoensayo , Persona de Mediana Edad , Fragmentos de Péptidos/líquido cefalorraquídeo , Fosforilación , Proyectos Piloto , Proteínas tau/líquido cefalorraquídeoRESUMEN
Due to the poor permeability of the plasma membrane, several strategies are designed to enhance the transfer of therapeutics into cells. Over the last 20 years, small peptides called Cell-Penetrating Peptides (CPPs) have been widely developed to improve the cellular delivery of biomolecules. These small peptides derive from protein transduction domains, chimerical constructs, or model sequences. Several CPPs are primary or secondary amphipathic peptides, depending on whether the distribution of their hydrophobic and hydrophilic domains occurs from their amino-acid sequence or through α-helical folding. Most of the CPPs are able to deliver different therapeutics such as nucleic acids or proteins in vitro and in vivo. Although their mechanisms of internalization are varied and controversial, the understanding of the intrinsic features of CPPs is essential for future developments. This chapter describes several protocols for the investigation of biophysical properties of amphipathic CPPs. Surface physics approaches are specifically applied to characterize the interactions of amphipathic peptides with model membranes. Circular dichroism and infra-red spectroscopy allow the identification of their structural state. These methods are exemplified by the analyses of the main biophysical features of the cell-penetrating peptides MPG, Pep-1, and CADY.
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Biofisica/métodos , Membrana Celular/metabolismo , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Aire , Secuencia de Aminoácidos , Membrana Celular/química , Dicroismo Circular , Cisteamina/análogos & derivados , Cisteamina/química , Cisteamina/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Fosfolípidos/química , Fosfolípidos/metabolismo , Unión Proteica , Espectroscopía Infrarroja por Transformada de Fourier , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismo , Agua/químicaRESUMEN
Cell penetrating peptides constitute a potent approach to overcome the limitations of in vivo siRNA delivery. We recently proposed a peptide-based nanoparticle system, CADY, for efficient delivery of siRNA into numerous cell lines. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA thereby improving both their cellular uptake and biological response. With the aim of understanding the cellular uptake mechanism of CADY:siRNA complexes, we have combined biochemical, confocal and electron microscopy approaches. In the present work, we provide evidence that the major route for CADY:siRNA cellular uptake involves direct translocation through the membrane but not the endosomal pathway. We have demonstrated that CADY:siRNA complexes do not colocalize with most endosomal markers and remain fully active in the presence of inhibitors of the endosomal pathway. Moreover, neither electrostatic interactions with cell surface heparan sulphates nor membrane potential are essential for CADY:siRNA cell entry. In contrast, we have shown that CADY:siRNA complexes clearly induce a transient cell membrane permeabilization, which is rapidly restored by cell membrane fluidity. Therefore, we propose that direct translocation is the major gate for cell entry of CADY:siRNA complexes. Membrane perturbation and uptake are driven mainly by the ability of CADY to interact with phospholipids within the cell membrane, followed by rapid localization of the complex in the cytoplasm, without affecting cell integrity or viability.