Alpha-synuclein (AS) is a natively unfolded protein whose structure is extremely sensitive to its environment. The hallmark of Parkinson’s disease (PD) is aggregation and deposition of AS in inclusion bodies. Formation of misfolded AS monomers which are partially folded is the first and critical stage in fibrillation of AS and is a good target for designing therapeutic strategies. Characterization the biochemical properties of partially folded intermediates induced by fibrillization and anti- fibrillization agents will help to design drugs as new inhibitors of AS misfolding and aggregation. Nanopore analysis is an emerging technique for studying the molecular mechanism of protein misfolding. This technique was used to characterize the conformational change of AS in the presence of two groups of chemicals; anti-parkinsonian small molecules (dopamine and nicotine) and Parkinson’s developing toxin (Cu(II) and methamphetamine). Other biophysical techniques such as NMR spectroscopy and isothermal titration calorimentry (ITC) were able to confirm the nanopore analysis results and also to study other biophysical properties of the partially folded intermediates such as the binding constant of the interaction and the secondary structure content. The results from nanopore analysis showed that both groups of ligands shifted the blockade current peak of AS (centered at -86 pA) to lower blockade currents but in a different manner. Anti-parkinsonian drugs shifted the blockade current of AS to intermediate peaks between -40 to -80 pA but Parkinson developing toxins shifted the peak to a lower blockade current centered at -25 pA which suggests a more compact conformation. Thus nanopore analysis distinguished the different conformation induced by different ligands. Furthermore nanopore analysis with AS fragments showed that these ligands bind to different regions of AS. NMR spectroscopy of AS in the presence of dopamine and nicotine isomers was in agreement with the nanopore analysis and showed conformational changes of AS in a concentration dependent manner. CD spectroscopy results showed that the secondary structure of AS alone and in the presence of ligands was mostly random coil and suggests a loop formation model for the interaction of ligands with AS. The results of this thesis showed the application of nanopore analysis as a real-time and label-free technique to screen a library of ligands for designing misfolding inhibitors for PD treatment. The result of a synergic experiment with nicotine and caffeine showed that combination of these anti-parkinsonian small molecules would be a promising new drug for treatment of PD.

Les virus Nipah (NiV) et Hendra (HeV) appartiennent au genre Henipavirus au sein de la famille des Paramyxoviridae. Cette famille comporte de nombreux pathogènes tel que le virus de la rougeole (MeV). Les paramyxovirus possèdent un génome de type ARN simple brin encapsidé par la nucléoprotéine (N) au sein d'une nucléocapside hélicoïdale. N interagit avec la phosphoprotéine (P) et cette dernière recrute la polymérase (L) qui assure la transcription et la réplication du génome viral. L'objectif de mon projet de thèse était de caractériser les protéines N et P ainsi que les interactions qui existent entre elles chez les trois virus, NiV, HeV et MeV. A la différence du MeV, qui a été intensivement étudié au cours des dernières années, les données moléculaires et structurales sur les Henipavirus étaient très limitées. A l'aide d'analyses computationnelles, nous avons pu déchiffrer l'organisation modulaire de N et de P, et nous avons montré que les régions, C-terminale de N (NTAIL) et N-terminale de P (PNT), sont prédites comme intrinsèquement désordonnées (RIDs). Les RIDs sont des régions fonctionnelles dépourvues de structures secondaires et tertiaires stables dans des conditions physiologiques. En utilisant des approches biochimiques et biophysiques, nous avons confirmé que NTAIL et PNT sont désordonnées. Elles conservent toutefois des structures secondaires transitoires qui pourraient correspondre à des éléments de reconnaissance moléculaire (ou MoREs) impliqués dans de transitions structurales en présence d'un partenaire. / The Paramyxoviridae family includes many important human and animal pathogens, such as measles virus (MeV), a morbillivirus, and the emerging Nipah (NiV) and Hendra (HeV) viruses, members of the Henipavirus genus. Paramyxoviruses possess a negative-strand RNA genome that is encapsidated by the nucleoprotein (N) into a helical nucleocapsid. N interacts with the phosphoprotein (P), and this latter recruits the polymerase that ensures genome replication and transcription. My PhD project has mainly focused on the characterization of the N and P proteins and on the interactions between these two proteins from the three cognate viruses, namely NiV, HeV and MeV. While MeV has been extensively studied through the past years, structural and molecular information on Henipavirus N and P proteins were rather scarce. Using computational analyses, we deciphered the modular organization of Henipavirus N and P. Intrinsically disordered regions (IDRs) were predicted within these proteins, notably at the C-terminus of N (referred to as NTAIL), and at the N-terminus of P (referred to as PNT). IDRs are functional despite they lack of a well-defined 3-D structure under physiological conditions. Biochemical and biophysical approaches pointed out a mostly disordered state for both NTAIL and PNT, although they were shown to contain short-order prone segments (i.e. molecular recognition elements, MoREs). These latter are involved in partner recognition and in disorder-to-order transitions. The C-terminal domains of the P proteins (referred to as PXD) were found to bind to NTAIL and to induce an α-helical transition thereof.

The group 2 late embryogenesis abundant (LEA) proteins, also known as the dehydrins, are intrinsically disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. In this work, we study the potential roles that dehydrins may have in stabilizing membranes and actin microfilaments during cold stress. We have cloned and expressed in E. coli two dehydrins from Thellungiella salsuginea, denoted TsDHN-1 (acidic) and TsDHN-2 (basic). These proteins were expressed as SUMO-fusion proteins for in vitro phosphorylation by casein kinase II (CKII), and for structural analysis by CD and Fourier transform infrared (FTIR) spectroscopy. We show using transmission-FTIR spectroscopy that ordered secondary structure is induced and stabilized in these proteins by association with large unilamellar vesicles emulating the lipid compositions of plant plasma and organellar membranes. The increase in secondary structure by membrane association is further facilitated by the presence of Zn2+. Lipid composition and temperature have synergistic effects on the secondary structure. Our single molecule force spectroscopy studies also suggest tertiary folding of both TsDHN-1 and TsDHN-2 induced by association with lipids. From Langmuir-Blodgett monolayer compression studies, and from topographic studies using atomic force microscopy at variable temperature, we conclude that TsDHN-1 stabilizes the membrane at lower temperatures. Finally, we show that the conformations of TsDHN-1 and TsDHN-2 are affected by pH, interactions with cations and membranes, and phosphorylation. Actin assembly by these dehydrins was assessed by sedimentation assays, and viewed by transmission electron and atomic force microscopy. Phosphorylation enabled both dehydrins to polymerize actin filaments, a phenomenon that may occur in the cytosols of plant cells undergoing environmental stress. These results support the hypothesis that dehydrins stabilize plant organellar membranes and/or the cytoskeleton in conditions of stress, and further that phosphorylation may be an important feature of this stabilization. / NSERC