RESUMEN
The excessive deposition of fibrillar collagens is a hallmark of fibrosis. Collagen fibril formation requires proteolytic maturations by Procollagen N- and C-proteinases (PNPs and PCPs) to remove the N- and C-propeptides which maintain procollagens in the soluble form. Procollagen C-Proteinase Enhancer-1 (PCPE-1, a glycoprotein composed of two CUB domains and one NTR domain) is a regulatory protein that activates the C-terminal processing of procollagens by the main PCPs. It is often up-regulated in fibrotic diseases and represents a promising target for the development of novel anti-fibrotic strategies. Here, our objective was to develop the first antagonists of PCPE-1, based on the nanobody scaffold. Using both an in vivo selection through the immunization of a llama and an in vitro selection with a synthetic library, we generated 18 nanobodies directed against the CUB domains of PCPE1, which carry its enhancing activity. Among them, I5 from the immune library and H4 from the synthetic library have a high affinity for PCPE-1 and inhibit its interaction with procollagens. The crystal structure of the complex formed by PCPE-1, H4 and I5 showed that they have distinct epitopes and enabled the design of a biparatopic fusion, the diabody diab-D1. Diab-D1 has a sub-nanomolar affinity for PCPE-1 and is a potent antagonist of its activity, preventing the stimulation of procollagen cleavage in vitro. Moreover, Diab-D1 is also effective in reducing the proteolytic maturation of procollagen I in cultures of human dermal fibroblasts and hence holds great promise as a tool to modulate collagen deposition in fibrotic conditions.
Asunto(s)
Proteolisis , Anticuerpos de Dominio Único , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/inmunología , Animales , Humanos , Camélidos del Nuevo Mundo , Procolágeno/metabolismo , Procolágeno/química , Dominios Proteicos , Modelos Moleculares , Colágenos Fibrilares/metabolismo , Colágenos Fibrilares/química , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Cristalografía por Rayos XRESUMEN
BMP-1/tolloid-like proteinases (BTPs) are major players in tissue morphogenesis, growth and repair. They act by promoting the deposition of structural extracellular matrix proteins and by controlling the activity of matricellular proteins and TGF-ß superfamily growth factors. They have also been implicated in several pathological conditions such as fibrosis, cancer, metabolic disorders and bone diseases. Despite this broad range of pathophysiological functions, the putative existence of a specific endogenous inhibitor capable of controlling their activities could never be confirmed. Here, we show that procollagen C-proteinase enhancer-2 (PCPE-2), a protein previously reported to bind fibrillar collagens and to promote their BTP-dependent maturation, is primarily a potent and specific inhibitor of BTPs which can counteract their proteolytic activities through direct binding. PCPE-2 therefore differs from the cognate PCPE-1 protein and extends the possibilities to fine-tune BTP activities, both in physiological conditions and in therapeutic settings.
Asunto(s)
Glicoproteínas , Péptido Hidrolasas , Humanos , Glicoproteínas/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Morfogénesis , Péptidos y Proteínas de Señalización IntercelularRESUMEN
The correct balance between collagen synthesis and degradation is essential for almost every aspect of life, from development to healthy aging, reproduction and wound healing. When this balance is compromised by external or internal stress signals, it very often leads to disease as is the case in fibrotic conditions. Fibrosis occurs in the context of defective tissue repair and is characterized by the excessive, aberrant and debilitating deposition of fibril-forming collagens. Therefore, the numerous proteins involved in the biosynthesis of fibrillar collagens represent a potential and still underexploited source of therapeutic targets to prevent fibrosis. One such target is procollagen C-proteinase enhancer-1 (PCPE-1) which has the unique ability to accelerate procollagen maturation by BMP-1/tolloid-like proteinases (BTPs) and contributes to trigger collagen fibrillogenesis, without interfering with other BTP functions or the activities of other extracellular metalloproteinases. This role is achieved through a fine-tuned mechanism of action that is close to being elucidated and offers promising perspectives for drug design. Finally, the in vivo data accumulated in recent years also confirm that PCPE-1 overexpression is a general feature and early marker of fibrosis. In this review, we describe the results which presently support the driving role of PCPE-1 in fibrosis and discuss the questions that remain to be solved to validate its use as a biomarker or therapeutic target.
RESUMEN
Ribosome display is a powerful method for selection and molecular evolution of proteins and peptides from large libraries. Displayed proteins are recovered from target molecules in multiple rounds of selection in order to enrich specific binders with the desired properties. Nowadays, ribosome display has become one of the most widely-used display technologies thanks to its advantages over cell-display as phage display. Ribosome display is an in vitro method, in which a stable ternary complex is formed between the mRNA, the ribosome and the nascent protein. A selection cycle can be performed in a few days and bacterial transformation is not necessary. Ribosome display has been used to screen and select peptides, proteins or molecular scaffolds in order to increase their affinity, specificity, catalytic activity or stability. In this review, ribosome display systems and their applications in selection and evolution of proteins are described.
TITLE: La présentation sur ribosome - Évolution et sélection acellulaire de banques moléculaires. ABSTRACT: La présentation sur ribosome (en anglais, ribosome display) est une méthode d'évolution moléculaire et de sélection de banques peptidiques et protéiques. Le ribosome display est réalisé in vitro dans un milieu acellulaire et repose sur la formation d'un complexe ternaire ribonucléoprotéique entre l'ARN, le ribosome et la protéine. Le ribosome display est devenu de nos jours l'une des méthodes de présentation les plus utilisées. Elle a notamment permis le criblage et la sélection de peptides, de protéines, d'échafaudages moléculaires afin d'améliorer leur affinité, leur spécificité, leur activité catalytique ou même leur stabilité. Cette revue présente la mise en Åuvre du ribosome display et les applications qui découlent de l'utilisation de cette technologie.
Asunto(s)
Sistema Libre de Células/metabolismo , Evolución Molecular Dirigida/métodos , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Animales , Sitios de Unión/genética , Sistema Libre de Células/química , Perfilación de la Expresión Génica/tendencias , Humanos , Unión Proteica/genética , Proteínas Ribosómicas/metabolismoRESUMEN
CTX-M15 is one of the most widespread, extended spectrum ß-lactamases, a major determinant of antibiotic resistance representing urgent public health threats, among enterobacterial strains infecting humans and animals. Here we describe the selection of binders to CTX-M15 from a combinatorial affibody library displayed on ribosomes. Upon three increasingly selective ribosome display iterations, selected variants were identified by next generation sequencing (NGS). Nine affibody variants with high relative abundance bearing QRP and QLH amino acid motifs at residues 9-11 were produced and characterized in terms of stability, affinity and specificity. All affibodies were correctly folded, with affinities ranging from 0.04 to 2 µM towards CTX-M15, and successfully recognized CTX-M15 in bacterial lysates, culture supernatants and on whole bacteria. It was further demonstrated that the binding of affibody molecules to CTX-M15 modulated the enzyme's kinetic parameters. This work provides an approach using ribosome display coupled to NGS for the rapid generation of protein ligands of interest in diagnostic and research applications.
Asunto(s)
Ribosomas/metabolismo , beta-Lactamasas/metabolismo , Escherichia coli/enzimología , Klebsiella pneumoniae/enzimología , beta-Lactamasas/aislamiento & purificaciónRESUMEN
In the past decades protein nanoparticles have successfully been used for vaccine applications. Their particulate nature and dense repetitive subunit organization makes them perfect carriers for antigen surface display and confers high immunogenicity. Nanoparticles have emerged as excellent candidates for vectorization of biological and immunostimulating molecules. Nanoparticles and biomolecular nanostructures such as ferritins or virus like particles have been used as diagnostic and therapeutic delivery systems, in vaccine development, as nanoreactors, etc. Recently, a new class of bacterial protein compartment has been discovered referred to as encapsulin nanocompartment. These compartments have been used for targeted diagnostics, as therapeutic delivery systems and as nanoreactors. Their biological origin makes them conveniently biocompatible and allows genetic functionalization. The aim of our study was to implement encapsulin nanocompartements for simultaneous epitope surface display and heterologous protein loading for rational vaccine design. For this proof-of-concept-study, we produced Thermotoga maritima encapsulin nanoparticles in E. coli. We demonstrated the ability of simultaneous display in our system by inserting Matrix protein 2 ectodomain (M2e) of influenza A virus at the nanoparticle surface and by packaging of a fluorescent reporter protein (GFP) into the internal cavity. Characterization of the nanoparticles by electronic microscopy confirmed homogenously shaped particles of 24â¯nm diameter in average. The results further show that engineering of the particle surface improved the loading capacity of the heterologous reporter protein suggesting that surface display may induce a critical elastic deformation resulting in improved stiffness. In Balb/c mice, nanoparticle immunization elicited antibody responses against both the surface epitope and the loaded cargo protein. These results confirm the potential of encapsulin nanocompartments for customized vaccine design and antigen delivery.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Proteínas Bacterianas/genética , Nanopartículas/química , Plásmidos/inmunología , Vacunas de ADN/genética , Proteínas de la Matriz Viral/genética , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Inmunización , Ratones , Ratones Endogámicos BALB C , Nanopartículas/administración & dosificación , Nanopartículas/ultraestructura , Tamaño de la Partícula , Plásmidos/administración & dosificación , Plásmidos/química , Thermotoga maritima/genética , Thermotoga maritima/metabolismo , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Proteínas de la Matriz Viral/inmunologíaRESUMEN
Codon usage distribution has been soundly used by nature to fine tune protein biogenesis. Alteration of the mRNA structure or sequential scheduling of codons can profoundly affect translation, thus altering protein yield, functionality, solubility, and proper folding. Building on these observations, here, we present an evaluation of different recently designed algorithms of sequence adaptation based on Codon Adaptation Index (CAI) profiling. The first algorithm globally harmonizes synonymous codons in the original sequence in full respect to the heterologous expression host codon usage. The second recodes the sequence in accordance with the native sequence CAI profile. Our data, generated on three model proteins, highlights the importance to consider gene recoding as a parameter itself for recombinant protein expression improvement.
Asunto(s)
Codón/genética , Biología Computacional/métodos , Regulación de la Expresión Génica , Algoritmos , Secuencia de Bases , Biosíntesis de Proteínas , SolubilidadRESUMEN
Virus-like particles (VLPs) are promising molecular structures for the design and construction of novel vaccines, diagnostic tools, and gene therapy vectors. Size, oligomer assembly and repetitiveness of epitopes are optimal features to induce strong immune responses. Several VLP-based vaccines are currently licensed and commercialized, and many vaccine candidates are now under preclinical and clinical studies. In recent years, the development of genetically engineered recombinant VLPs has accelerated the need for new, improved downstream processes. In particular, a rapid low cost purification process has been identified as a remaining key challenge in manufacturing process development. In the present study we set up a size-exclusion chromatography-based, scalable purification protocol for the purification of a VLP-based influenza A vaccine produced in Escherichia coli. Recombinant VLPs derived from the RNA bacteriophage MS2 displaying an epitope from the ectodomain of Matrix 2 protein from influenza A virus were produced and purified. The 3 steps purification protocol uses a recently developed multimodal size-exclusion chromatography medium (Capto™ Core 700) in combination with detergent extraction and size-exclusion polishing to reach a 89% VLP purity with a 19% yield. The combination of this downstream strategy following production in E. coli would be suited for production of VLP-based veterinary vaccines targeting livestock and companion animals where large amounts of doses must be produced at an affordable price.