Peptides derived from the knuckle epitope of BMP-9 induce the cholinergic differentiation and inactivate GSk3beta in human SH-SY5Y neuroblastoma cells

The incidence of brain degenerative disorders like Alzheimer's disease (AD) will increase as the world population ages. While there is presently no known cure for AD and current treatments having only a transient effect, an increasing number of publications indicate that growth factors (GF) may be used to treat AD. GFs like the bone morphogenetic proteins (BMPs), especially BMP-9, affect many aspects of AD. However, BMP-9 is a big protein that cannot readily cross the blood-brain barrier. We have therefore studied the effects of two small peptides derived from BMP-9 (pBMP-9 and SpBMP-9). We investigated their capacity to differentiate SH-SY5Y human neuroblastoma cells into neurons with or without retinoic acid (RA). Both peptides induced Smad 1/5 phosphorylation and their nuclear translocation. They increased the number and length of neurites and the expression of neuronal markers MAP-2, NeuN and NSE better than did BMP-9. They also promoted differentiation to the cholinergic phenotype more actively than BMP-9, SpBMP-9 being the most effective as shown by increases in intracellular acetylcholine, ChAT and VAchT. Finally, both peptides activated the PI3K/Akt pathway and inhibited GSK3beta, a current AD therapeutic target. BMP-9-derived peptides, especially SpBMP-9, with or without RA, are promising molecules that warrant further investigation

Évolution dirigée de la néocarzinostatine (ingénierie d une ossature protéique alternative aux anticorps)

Les anticorps monoclonaux sont des outils moléculaires remarquables utilisés dans des applications thérapeutiques, diagnostiques et biotechnologiques. Néanmoins, certains inconvénients, liés à leur structure moléculaire, entraînent des coûts élevés de développement et de production. Malgré d importants efforts de recherche ces limitations n ont pu être dépassées. C est pourquoi plusieurs laboratoires développent des ossatures alternatives aux anticorps monoclonaux. Il s agit d effectuer par évolution dirigée un remodelage fonctionnel sur une ossature protéique différente des anticorps et présentant des propriétés plus favorables. Les travaux décrits dans ce manuscrit concernent différents aspects du remodelage fonctionnel de la néocarzinostatine (NCS). Le chapitre I porte sur la résolution de structures de complexe NCS-ligand. Ces mutants de NCS sont capables de reconnaître de façon spécifique la testostérone, un ligand non naturel. Le chapitre II présente les résultats obtenus pour un projet d optimisation de l ossature NCS visant à supprimer ses ponts disulfure pour disposer d une ossature fonctionnelle dans le compartiment cytoplasmique. Ce travail a permis d isoler des mutants sans pont disulfure, exprimés sous forme soluble et repliée dans le cytoplasme bactérien. Ces mutants devraient permettre de développer des applications intracellulaires de type interférences à protéine. Le chapitre III concerne le remodelage des boucles exposées de la NCS dans l objectif de créer des interactions protéine-protéine entre la NCS et des cibles d intérêt. Ce chapitre décrit les outils développés pour construire une bibliothèque de mutants optimisée pour le repliement.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica.

Background : Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS.[br/] Results : Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement).[br/] Conclusions : Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (10(5) strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development

New Glycosidase Substrates for Droplet-Based Microfluidic Screening

Droplet-based microfluidics is a powerful technique allowing ultra-high-throughput screening of large libraries of enzymes or microorganisms for the selection of the most efficient variants. Most applications in droplet microfluidic screening systems use fluorogenic substrates to measure enzymatic activities with fluorescence readout. It is important, however, that there is little or no fluorophore exchange between droplets, a condition not met with most commonly employed substrates. Here we report the synthesis of fluorogenic substrates for glycosidases based on a sulfonated 7-hydroxycoumarin scaffold. We found that the presence of the sulfonate group effectively prevents leakage of the coumarin from droplets, no exchange of the sulfonated coumarins being detected over 24 h at 30 °C. The fluorescence properties of these substrates were characterized over a wide pH range, and their specificity was studied on a panel of relevant glycosidases (cellulases and xylanases) in microtiter plates. Finally, the β-d-cellobioside-6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate substrate was used to assay cellobiohydrolase activity on model bacterial strains (<i>Escherichia coli</i> and <i>Bacillus subtilis</i>) in a droplet-based microfluidic format. These new substrates can be used to assay glycosidase activities in a wide pH range (4–11) and with incubation times of up to 24 h in droplet-based microfluidic systems