ABSTRACT
Odorant binding proteins (OBPs) are small soluble proteins found in olfactory systems that are capable of binding several types of odorant molecules. Cantilevers based on polycrystalline diamond surfaces are very promising as chemical transducers. Here two methods were investigated for chemically grafting porcine OBPs on polycrystalline diamond surfaces for biosensor development. The first approach resulted in random orientation of the immobilized proteins over the surface. The second approach based on complexing a histidine-tag located on the protein with nickel allowed control of the proteins' orientation. Evidence confirming protein grafting was obtained using electrochemical impedance spectroscopy, fluorescence imaging and X-ray photoelectron spectroscopy. The chemical sensing performances of these OBP modified transducers were assessed. The second grafting method led to typically 20% more sensitive sensors, as a result of better access of ligands to the proteins active sites and also perhaps a better yield of protein immobilization. This new grafting method appears to be highly promising for further investigation of the ligand binding properties of OBPs in general and for the development of arrays of non-specific biosensors for artificial olfaction applications.
Subject(s)
Biomimetic Materials , Diamond/chemistry , Dielectric Spectroscopy/instrumentation , Micro-Electrical-Mechanical Systems/instrumentation , Odorants/analysis , Receptors, Odorant/chemistry , Smell , Equipment Design , Equipment Failure Analysis , Protein Binding , Protein Interaction Mapping/instrumentation , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Atopic dermatitis (AD) is a multifactorial chronic inflammatory disease mainly stemming from a genetic predisposition that leads to hypersensitivity to environmental factors and a common involvement of Staphylococcus aureus (SA) colonization. The aim of this work was to propose a new non-invasive approach to enumerate the genes coding for the toxins of SA in atopic skin samples. In parallel, the study aimed to evaluate the change in AD through 3 markers of the inflammatory response: IL-8, IL-1RA/IL-1alpha and IL-18. These methods were tested on 31 patients with AD, and finally on a group of 19 subjects for whom clinical improvement had been reported after various treatments. The study revealed the presence of a large number of genes encoding toxins in atopic samples, indicating a high rate of SA colonization, and also an increase in the level of all cytokine markers in atopic skin compared to the skin of healthy subjects. Finally, we found a positive correlation between increases in the SCORAD (Scoring Atopic Dermatitis Index) value after treatment and the corresponding evolution of the SA density. These methods provide a means to clinically evaluate the course of AD, and may help in the development of potential treatments.
Subject(s)
Bacterial Toxins/genetics , Dermatitis, Atopic/genetics , Polymerase Chain Reaction/methods , Staphylococcus aureus/genetics , Bacterial Toxins/isolation & purification , Case-Control Studies , Child , Child, Preschool , DNA, Bacterial/isolation & purification , Dermatitis, Atopic/microbiology , Genetic Predisposition to Disease , Genotype , Humans , Infant , Inflammation/genetics , Inflammation/microbiology , Interleukins/metabolism , Oligonucleotide Array Sequence Analysis/methods , Severity of Illness Index , Staphylococcal Skin Infections/genetics , Staphylococcal Skin Infections/microbiology , Staphylococcus aureus/isolation & purification , Treatment OutcomeSubject(s)
Chromosomes, Human, Pair 2 , Keratoconus/genetics , Adolescent , Adult , Child , Chromosome Mapping , Family , France , Guadeloupe , Humans , SpainABSTRACT
Nucleolin is an abundant nucleolar protein involved in several steps of ribosome biogenesis. The protein is highly conserved through evolution and possesses four RNA-binding domains (RBD), which are likely to determine its RNA binding specificity. Previous studies have shown that nucleolin interacts with two different RNA targets. The first is a small stem-loop structure, the nucleolin recognition element (NRE), found all along the pre-ribosomal RNA. The second is a short single-stranded RNA sequence, the evolutionary conserved motif (ECM), located five nucleotides downstream of the first processing site in the pre-ribosomal RNA 5' external transcribed spacer. Biochemical, genetic, and structural studies have shown that the first two RBD of nucleolin are necessary and sufficient for the specific interaction of nucleolin with the NRE motif. In this work, we have studied the interaction of nucleolin with the ECM sequence. Deletion and mutational analyses showed that all four RBDs of hamster nucleolin were required for the interaction with the ECM sequence. This RNA binding specificity is conserved between hamster and Xenopus laevis, whereas the Xenopus protein does not interact with the NRE. Nucleolin is the first example of a protein that requires four RBDs for its interaction with an RNA target, demonstrating that a single protein can use different combinations of RBD to interact specifically with several RNA sequences.
Subject(s)
Phosphoproteins/chemistry , Phosphoproteins/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , RNA/metabolism , Animals , Base Sequence , Conserved Sequence , Cricetinae , DNA Mutational Analysis , Dose-Response Relationship, Drug , Gene Deletion , Kinetics , Molecular Sequence Data , Mutation , Phosphoproteins/genetics , Plasmids/metabolism , Protein Binding , Protein Conformation , Protein Structure, Tertiary , RNA, Ribosomal/metabolism , RNA-Binding Proteins/genetics , Recombinant Proteins/metabolism , Transcription, Genetic , Ultraviolet Rays , Xenopus , NucleolinABSTRACT
The first processing event of the precursor ribosomal RNA (pre-rRNA) takes place within the 5' external transcribed spacer. This primary processing requires conserved cis-acting RNA sequence downstream from the cleavage site and several nucleic acids (small nucleolar RNAs) and proteins trans-acting factors including nucleolin, a major nucleolar protein. The specific interaction of nucleolin with the pre-rRNA is required for processing in vitro. Xenopus laevis and hamster nucleolin interact with the same pre-rRNA site and stimulate the processing activity of a mouse cell extract. A highly conserved 11-nucleotide sequence located 5-6 nucleotides after the processing site is required for the interaction of nucleolin and processing. In vitro selection experiments with nucleolin have identified an RNA sequence that contains the UCGA motif present in the 11-nucleotide conserved sequence. The interaction of nucleolin with pre-rRNA is required for the formation of an active processing complex. Our findings demonstrate that nucleolin is a key factor for the assembly and maturation of pre-ribosomal ribonucleoparticles.
Subject(s)
Phosphoproteins/metabolism , RNA, Ribosomal/metabolism , RNA-Binding Proteins/metabolism , Animals , CHO Cells , Cricetinae , Extracellular Matrix/metabolism , Mice , Protein Binding , Protein Processing, Post-Translational , RNA, Ribosomal/genetics , Recombinant Proteins/metabolism , Xenopus laevis , NucleolinABSTRACT
Nucleolin is an abundant protein of the nucleolus. Nucleolar proteins structurally related to nucleolin are found in organisms ranging from yeast to plants and mammals. The association of several structural domains in nucleolin allows the interaction of nucleolin with different proteins and RNA sequences. Nucleolin has been implicated in chromatin structure, rDNA transcription, rRNA maturation, ribosome assembly and nucleo-cytoplasmic transport. Studies of nucleolin over the last 25 years have revealed a fascinating role for nucleolin in ribosome biogenesis. The involvement of nucleolin at multiple steps of this biosynthetic pathway suggests that it could play a key role in this highly integrated process.
Subject(s)
Cell Nucleolus/physiology , Phosphoproteins/chemistry , Phosphoproteins/physiology , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/physiology , Animals , Cell Nucleolus/ultrastructure , Chromatin/physiology , Chromatin/ultrastructure , Humans , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/physiology , Phosphoproteins/genetics , RNA-Binding Proteins/genetics , Ribosomes/physiology , NucleolinABSTRACT
The first processing step of precursor ribosomal RNA (pre-rRNA) involves a cleavage within the 5' external transcribed spacer. This processing requires sequences downstream of the cleavage site which are perfectly conserved among human, mouse and Xenopus and also several small nucleolar RNAs (snoRNAs): U3, U14, U17 and E3. In this study, we show that nucleolin, one of the major RNA-binding proteins of the nucleolus, is involved in the early cleavage of pre-rRNA. Nucleolin interacts with the pre-rRNA substrate, and we demonstrate that this interaction is required for the processing reaction in vitro. Furthermore, we show that nucleolin interacts with the U3 snoRNP. Increased levels of nucleolin, in the presence of the U3 snoRNA, activate the processing activity of a S100 cell extract. Our results suggest that the interaction of nucleolin with the pre-rRNA substrate might be a limiting step in the primary processing reaction. Nucleolin is the first identified metazoan proteinaceous factor that interacts directly with the rRNA substrate and that is required for the processing reaction. Potential roles for nucleolin in the primary processing reaction and in ribosome biogenesis are discussed.
