Glycogen Synthase Kinase 3Beta as Target for Neurodegenerative Disease Drug Discovery: Proteomic Approaches to Characterize its Activity in Vitro

The work described in this thesis was performed in order to develop advanced analytical methods suitable to select and characterize GS- 3β inhibitors in vitro. GSK-3β is recognized as a key target for the development of new therapeutic agents for Alzheimer disease (AD). We validated an UHPLC-UV-Vis diode arrays detector (DAD) method for the very fast identification (resolution in less than 2 min) and determination of ADP and ATP in enzyme-based assay containing GSM-S syntetic peptide, ATP and GSK-3β. By using this method, selected inhibition hits will be characterized by defining their competitive mode of action with the substrate rather than with the ATP cofactor, in view of the discovery of compounds endowed of an increased GSK-3β selectivity over other protein-kinases. Next we hypothesized that GSK-3β could be directly involved in the regulation of histone acetylation through HDAC protein. Our hypothesis accounts that inhibition of GSK-3β, which leads to reduced HDAC activity, could restores the acetylation level in histones, protecting against neurodegeneration as both aging and AD pathology are associated with loss of histone acetylation (H4/H3) at N-terminus. Therefore, quantification of histone modifications on individual lysine residues is of crucial importance to understand their role in cell biology. We developed a targeted liquid chromatography mass spectrometry (LC-MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 from murine macrophage-like cell line RAW 264.7, with the perspective to apply the method on neuronal cells upon administration of GSK-3β inhibitors. The analytical strategy we developed shows that careful optimization of chemical derivatization steps at the protein and at the peptide level, combined with a more extensive digestion using chymotrypsin and trypsin, allows to differentiate between acetylation levels of each lysine residues

Site-specific quantification of lysine acetylation in the N-terminal tail of histone H4 using a double-labelling, targeted UHPLC MS/MS approach

We developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 by combining chemical derivatization at the protein and peptide levels with digestion using chymotrypsin and trypsin. Unmodified epsilon-amino groups were first modified with propionic acid anhydride and the derivatized protein digested with trypsin and chymotrypsin. The newly formed peptide N-termini were subjected to a second derivatization step with d(6)- (heavy) or d(0)- (light) acetic acid anhydride. Samples were mixed at different ratios and peptides monitored by multiple reaction monitoring (MRM) LC-MS/MS. The method was validated in terms of linearity (R (2) a parts per thousand yenaEuro parts per thousand 0.94), precision (RSD a parts per thousand currency signaEuro parts per thousand 10 %), and accuracy (a parts per thousand currency sign27 %) and used to assess the effect of the histone deacetylase (HDAC) inhibitors SAHA and MS-275 in the murine macrophage-like cell line RAW 264.7. SAHA and MS-275 showed site-specific effects on the acetylation levels of K5 and K8 with the K5(Ac)-K8 and K5-K8(Ac) peptides increasing 2.5-fold and 5-fold upon treatment with SAHA and MS-275, respectively. Assessing lysine acetylation in a site-specific manner is important for gaining a better understanding of the effects of HDAC inhibitors and for clarifying disease mechanisms where lysine acetylation plays a role

HspB8 interacts with BAG3 in a "native-like" conformation forming a complex that displays chaperone-like activity

The HspB8-BAG3 complex plays an important role in the protein quality control acting alone or within multi-components complexes. To clarify the mechanism underlying its activity, in this work we used biochemical and biophysical approaches to study the tendency of both proteins to auto-assemble and to form the complex. Solubility and Thioflavin T assays, Fourier transform infrared spectroscopy and atomic force microscopy analyses clearly showed the tendency of HspB8 to self-assemble at high concentration and to form oligomers in a "native-like" conformation; otherwise, BAG3 aggregates poorly. Noteworthy, also HspB8 and BAG3 associate in a "native-like" conformation, forming a stable complex. Furthermore, the high difference between dissociation constant values of HspB8-HspB8 interaction with respect to the binding to BAG3 obtained by surface plasmon resonance confirms that HspB8 is an obligated partner of BAG3 in vivo. Lastly, both proteins alone or in the complex are able to bind and affect the aggregation of the Josephin domain, the structured domain that triggers the ataxin-3 fibrillation. In particular, the complex displayed higher activity than HspB8 alone. All this considered, we can assert that the two proteins form a stable assembly with chaperone-like activity that could contribute to the physiological role of the complex in vivo

Direct determination of GSK-3β activity and inhibition by UHPLC-UV-vis diode arrays detector (DAD)

Altered GSK-3β activity can contribute to a number of pathological processes including Alzheimer's disease (AD). Indeed, GSK-3β catalyzes the hyperphosphorylation of tau protein by transferring a phosphate moiety from ATP to the protein substrate serine residue causing the formation of the toxic insoluble neurofibrillary tangles; for this reason it represents a key target for the development of new therapeutic agents for AD treatment. Herein we describe a new selective UHPLC methodology developed for the direct characterization of GSK-3β kinase activity and for the determination of its inhibition, which could be crucial in AD drug discovery. The UHPLC-UV (DAD) based method was validated for the very fast determination of ATP as reactant and ADP as product, and applied for the analysis of the enzymatic reaction between a phosphate primed peptide substrate (GSM), resembling tau protein sequence, ATP and GSK-3β, with/without inhibitors. Analysis time was ten times improved, when compared with previously published chromatographic methods. The method was also validated by determining enzyme reaction kinetic constants (KM and vmax) for GSM and ATP and by analyzing well known GSK-3β inhibitors. Inhibition potency (IC50) values for SB-415286 (81 ± 6 nM) and for Tideglusib (251 ± 17 nM), found by the newly developed UHPLC method, were in good agreement with the luminescence method taken as independent reference method. Further on, the UHPLC method was applied to the elucidation of Tideglusib mechanism of action by determining its inhibition constants (Ki). In agreement with literature data, Tideglusib resulted a GSM competitive inhibitor, whereas SB-415286 was found inhibiting GSK-3β in an ATP competitive manner. This method was applied to the determination of the potency of a new lead compound and was found potentially scalable to inhibitor screening of large compounds collections

Application of an ESI-QTOF method for the detailed characterization of GSK-3\u3b2 inhibitors

The crucial role of Glycogen Synthase Kinase 3 (GSK-3\u3b2) as a pivotal player in Alzheimer's Disease (AD) has recently inspired significant attempts to design and synthesize potent kinase inhibitors. In fact GSK-3\u3b2 is considered the main kinase which catalyzes the microtubule-associated protein tau hyper-phosphorylation and the neurofibrillary tangles (NFT) in vitro and in vivo, The first classes of GSK-3\u3b2 inhibitors were classified as ATP-competitive and, therefore, they lack of an efficient degree of selectivity over other kinases. In light of this consideration, many efforts are devoted to characterize new non ATP-competitive GSK-3\u3b2 inhibitors, endowed with high selectivity. In parallel, there is an urgent need to develop new analytical methodologies for the hit selection (highthroughput screening) and ligand binding characterization in terms of potency, affinity and mechanism of action. The new methodology for GSK-3\u3b2 enzymatic activity determination can be adopted as a realistic alternative to the currently used radioactive, luminescence and fluorescence detection methods, each showing limitations in terms of safety and interferences. Herein, we propose an alternative and selective electrospray ionization quadrupole time-of-flight (ESI-QTOF) method, based on the direct quantification of phosphorylated substrate muscle glycogen synthase GSM, a peptide resembling the high affinity sequence of natural substrate muscle glycogen synthase 1, for the detailed characterization of GSK-3\u3b2 inhibitors. The method was validated in terms of accuracy and reproducibility of GSM signal intensity with a relative standard deviation RSD% value of 3.55%; Limit of Detection (LOD): 0.006\u3bcM; Lower Limit of Quantification (LLOQ): 0.02\u3bcM; linearity r 2 0.9951. The kinetic constants (K M and v max) of the GSK-3\u3b2 catalyzed kinase reaction and the inhibitory potency of known ligands (IC50), were determined. All the obtained results were in agreement with those reported in literature or obtained in house by the standard reference luminometric approach. The proposed method was applied to the elucidation of well known inhibitors mechanism of action by the construction of a Lineweaver-Burk plot and the K i determination. Furthermore, the potency, affinity and mechanism of action of a new non ATP-competitive compound were established. We demonstrated the ESI-QTOF method to be more feasible than the classic kinase assays since it avoids drawbacks inherently connected with radioisotope labeling or the indirect detection of kinase activity, so far. It is also scalable to the screening of large library collections and suitable for pharmaceutical industries purposes

Characterization of the Genome of the Dairy Lactobacillus helveticus Bacteriophage ΦAQ113

The complete genomic sequence of the dairy Lactobacillus helveticus bacteriophage ΦAQ113 was determined. Phage ΦAQ113 is a Myoviridae bacteriophage with an isometric capsid and a contractile tail. The final assembled consensus sequence revealed a linear, circularly permuted, double-stranded DNA genome with a size of 36,566 bp and a G+C content of 37%. Fifty-six open reading frames (ORFs) were predicted, and a putative function was assigned to approximately 90% of them. The ΦAQ113 genome shows functionally related genes clustered together in a genome structure composed of modules for DNA replication/regulation, DNA packaging, head and tail morphogenesis, cell lysis, and lysogeny. The identification of genes involved in the establishment of lysogeny indicates that it may have originated as a temperate phage, even if it was isolated from natural cheese whey starters as a virulent phage, because it is able to propagate in a sensitive host strain. Additionally, we discovered that the ΦAQ113 phage genome is closely related to Lactobacillus gasseri phage KC5a and Lactobacillus johnsonii phage Lj771 genomes. The phylogenetic similarities between L. helveticus phage ΦAQ113 and two phages that belong to gut species confirm a possible common ancestral origin and support the increasing consideration of L. helveticus as a health-promoting organism

Assessing local building cultures for resilience & development: A practical guide for community-based assessment

International audienceThe development of a set of tools to enhance appreciation of local practices developed by communities regarding settlements and risks is an initiative that aims to promote context-based strategies for responding efficiently and adequately to habitat improvement and vulnerability reduction needs. This guide offers a methodological and operational support for decision-making and practices towards approaches and actions deeply rooted in local contexts. It is a practical tool that provides detailed explanation on planning, preparing and undertaking field assessments of local practices related to habitat and risks. It refers to a participatory approach suitable for, and adapted to, various geographical, cultural and risk-prone areas. By supporting habitat assessment in all its different aspects, it also fosters links between programmes, providing clues and keys to define and implement coherent projects including income generating activities, livelihood, health and other related sectors. The necessary investment to be taken into account in project planning to achieve the basic step described in this booklet will result in huge savings, as logistical issues will be drastically reduced during the project implementation. It is a worthwhile investment that will lead to decisions ensuring more benefits to the affected communities, including a long-term enhancement of their resilience capacityCet ouvrage s'adresse aux responsables de projets et aux preneurs de décisions. L'auteur explique tout d'abord la démarche de projet respectueuse des cultures constructives. L'auteur présente ensuite une méthode d'évaluation des cultures constructives locales et expose l'importance de celle-ci dans un proje