Effets de peptides vecteurs sur le cytosquelette d'actine après internalisation dans les cellules tumorales

Trois peptides vecteurs ont été criblés pour leur capacité à réorganiser le cytosquelette d actine dans des fibroblastes tumoraux (cellules EF). Les peptides vecteurs sont des peptides chargés positivement capables de traverser les membranes plasmiques des cellules. Dans un premier temps, une étiquette stable permettant la quantification de l internalisation et l étude du processus de dégradation de peptides vecteurs via une méthode de quantification du peptide basée sur la spectrométrie de masse MALDI-TOF a été synthétisée. Ainsi, un nouveau peptide vecteur : msr(R/W)9 est capable d entrer et peut s accumuler dans des cellules CHO. Dans une deuxième étude, nous avons testé trois peptides vecteurs sur des fibroblastes tumoraux qui n expriment pas la Zyxine, entraînant alors une désorganisation du cytosquelette d actine. Leurs effets potentiels sur la réorganisation du cytosquelette d actine et la ré-expression de la Zyxine ont été évalués à partir d études de la polymérisation d actine in vitro dans des lysats cellulaires par anisotropie de fluorescence, de la morphologie des fibroblastes par immunomarquage de l actine, de la motilité cellulaire par vidéomicroscopie et enfin de la capacité à croître en indépendance d ancrage. Les résultats obtenus montrent que ces trois peptides semblent réverser le phénotype tumoral des cellules EF alors qu ils sont inactifs sur les fibroblastes sains. Enfin, des expériences de pontage chimique entre ces trois peptides et l actine, nous ont permis de mettre en évidence une zone d interactions, identifiée et caractérisée par spectrométrie de masse, entre ces différents partenaires.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Dosage et pistage de peptides Troyens dans les cellules

Les peptides Troyens ou vecteurs sont capables de passer les membranes biologiques et de véhiculer des principes actifs dans le cytoplasme ou le noyau des cellules dans lesquelles ils sont entrés. Les méthodes indirectes utilisées jusqu'à présent pour détecter ces peptides dans les cellules n'ont pas permis d'établir de manière univoque le(s) mécanisme(s) de leur internalisation. La méthode de quantification, basée sur la spectrométrie de masse MALDI-TOF, que nous avons mise au point pour quantifier ces peptides Troyens dans les cellules est développée dans cette revue

Cell-penetrating Peptides with Intracellular Actin-remodeling Activity in Malignant Fibroblasts*

Cell-penetrating peptides can cross cell membranes and are commonly seen as biologically inert molecules. However, we found that some cell-penetrating peptides could remodel actin cytoskeleton in oncogene-transformed NIH3T3/EWS-Fli cells. These cells have profound actin disorganization related to their tumoral transformation. These arginine- and/or tryptophan-rich peptides could cross cell membrane and induce stress fiber formation in these malignant cells, whereas they had no perceptible effect in non-tumoral fibroblasts. In addition, motility (migration speed, random motility coefficient, wound healing) of the tumor cells could be decreased by the cell-permeant peptides. Although the peptides differently influenced actin polymerization in vitro, they could directly bind monomeric actin as determined by NMR and calorimetry studies. Therefore, cell-penetrating peptides might interact with intracellular protein partners, such as actin. In addition, the fact that they could reverse the tumoral phenotype is of interest for therapeutic purposes

A20 Inhibits β-Cell Apoptosis by Multiple Mechanisms and Predicts Residual β-Cell Function in Type 1 Diabetes

Activation of the transcription factor nuclear factor kappa B (NFkB) contributes to beta-cell death in type 1 diabetes (T1D). Genome-wide association studies have identified the gene TNF-induced protein 3 (TNFAIP3), encoding for the zinc finger protein A20, as a susceptibility locus for T1D. A20 restricts NF-kappa B signaling and has strong antiapoptotic activities in beta-cells. Although the role of A20 on NF-kappa B inhibition is well characterized, its other antiapoptotic functions are largely unknown. By studying INS-1E cells and rat dispersed islet cells knocked down or overexpressing A20 and islets isolated from the beta-cell-specific A20 knockout mice, we presently demonstrate that A20 has broader effects in beta-cells that are not restricted to inhibition of NF-kappa B. These involves, suppression of the proapoptotic mitogen-activated protein kinase c-Jun N-terminal kinase (JNK), activation of survival signaling via v-akt murine thymoma viral oncogene homolog (Akt) and consequently inhibition of the intrinsic apoptotic pathway. Finally, in a cohort of T1D children, we observed that the risk allele of the rs2327832 single nucleotide polymorphism of TNFAIP3 predicted lower C-peptide and higher hemoglobin A1c (HbA1c) levels 12 months after disease onset, indicating reduced residual beta-cell function and impaired glycemic control. In conclusion, our results indicate a critical role for A20 in the regulation of beta-cell survival and unveil novel mechanisms by which A20 controls beta-cell fate. Moreover, we identify the single nucleotide polymorphism rs2327832 of TNFAIP3 as a possible prognostic marker for diabetes outcome in children with T1D

Translocation and Endocytosis for Cell-penetrating Peptide Internalization

Cell-penetrating peptides (CPPs) share the property of cellular internalization. The question of how these peptides reach the cytoplasm of cells is still widely debated. Herein, we have used a mass spectrometry-based method that enables quantification of internalized and membrane-bound peptides. Internalization of the most used CPP was studied at 37 °C (endocytosis and translocation) and 4 °C (translocation) in wild type and proteoglycan-deficient Chinese hamster ovary cells. Both translocation and endocytosis are internalization pathways used by CPP. The choice of one pathway versus the other depends on the peptide sequence (not the number of positive changes), the extracellular peptide concentration, and the membrane components. There is no relationship between the high affinity of these peptides for the cell membrane and their internalization efficacy. Translocation occurs at low extracellular peptide concentration, whereas endocytosis, a saturable and cooperative phenomenon, is activated at higher concentrations. Translocation operates in a narrow time window, which implies a specific lipid/peptide co-import in cells

CSF biomarker variability in the Alzheimer's Association quality control program

Item does not contain fulltextBACKGROUND: The cerebrospinal fluid (CSF) biomarkers amyloid beta 1-42, total tau, and phosphorylated tau are used increasingly for Alzheimer's disease (AD) research and patient management. However, there are large variations in biomarker measurements among and within laboratories. METHODS: Data from the first nine rounds of the Alzheimer's Association quality control program was used to define the extent and sources of analytical variability. In each round, three CSF samples prepared at the Clinical Neurochemistry Laboratory (Molndal, Sweden) were analyzed by single-analyte enzyme-linked immunosorbent assay (ELISA), a multiplexing xMAP assay, or an immunoassay with electrochemoluminescence detection. RESULTS: A total of 84 laboratories participated. Coefficients of variation (CVs) between laboratories were around 20% to 30%; within-run CVs, less than 5% to 10%; and longitudinal within-laboratory CVs, 5% to 19%. Interestingly, longitudinal within-laboratory CV differed between biomarkers at individual laboratories, suggesting that a component of it was assay dependent. Variability between kit lots and between laboratories both had a major influence on amyloid beta 1-42 measurements, but for total tau and phosphorylated tau, between-kit lot effects were much less than between-laboratory effects. Despite the measurement variability, the between-laboratory consistency in classification of samples (using prehoc-derived cutoffs for AD) was high (>90% in 15 of 18 samples for ELISA and in 12 of 18 samples for xMAP). CONCLUSIONS: The overall variability remains too high to allow assignment of universal biomarker cutoff values for a specific intended use. Each laboratory must ensure longitudinal stability in its measurements and use internally qualified cutoff levels. Further standardization of laboratory procedures and improvement of kit performance will likely increase the usefulness of CSF AD biomarkers for researchers and clinicians