The oxidative folding pathway of the disulfide containing protein bovine pancreatic trypsin inhibitor (BPTI) was one of the first to be elucidated and has served as a basis for understanding the folding pathways of other proteins. During the oxidative folding of reduced BPTI, two intermediates (N' and N*) accumulate in significant amounts and act as kinetic traps. Both N' and N* bury their two remaining free thiols in their hydrophobic cores, which inhibits further oxidation. Historically, the rate limiting step was considered to be the intramolecular rearrangements of N' and N* to another intermediate with two free thiols, NSH. The two free thiols in NSH are solvent-exposed and easily oxidized to a disulfide, producing native protein (N). Nevertheless, our research using reduced BPTI indicated that the folding rate of N* to N was proportional to the concentration of added glutathione disulfide (GSSG), inconsistent with the slow intramolecular rearrangement of N* to NSH. To confirm our initial results, the intermediate N* was purified and refolded in the presence of GSSG. The conversion of N* to N was dependent upon the disulfide concentration and singly mixed disulfide N*(SG) was observed during folding. These results emphasize that the folding of N* can proceed via a growth type pathway, direct oxidation of the two remaining thiols in N* by an exogenous small molecule disulfide, such as GSSG, to form N. Folding of reduced BPTI via N* was performed under changing concentrations of GSSG and GSH as a function of time. The folding was improved dramatically in terms of rate and yield. Aromatic disulfides and thiols have been demonstrated to improve the folding efficiency of disulfide containing proteins including ribonuclease A (RNase A) and lysozyme. Herein, N* and N' were refolded in the presence of aromatic disulfides. Folding of the two kinetic traps with aromatic disulfides indicated that folding proceed via a growth type pathway. The singly and doubly mixed disulfide intermediates were observed during most folding reactions. The oxidative folding of reduced BPTI with aromatic disulfides and thiols were also investigated. Reduced BPTI can be folded to disulfide intermediates rapidly.

Un G-quadruplex (G4) est une structure non-canonique d’acides nucléiques formée par des séquences riches en guanines. Certains G4s sont polymorphiques, une même séquence peut former desG4s de différentes topologies. Les G4s sont proposés comme régulateurs de processus biologiques car ils sont trouvés dans des régions génomiques clés telles que dans des promoteurs de gènes et au niveau des télomères. Stabiliser ces G4s par rapport à la forme duplexe est une stratégie proposée pour combattre le cancer. Pour ce faire, des ligands spécifiques et affins sont utilisés. Le design de ces ligands implique habituellement de larges plans aromatiques, optimisés pour se lier par des interactions π-π sur les Gquartets extérieurs. Cependant, si ce type d’interaction était le seul mode de liaison, tous les ligands auraient des affinités similaires pour tous les G4s.Afin de caractériser les structures ciblées et de quelle manière les ligands vont interagir avec celles-ci, nous avons utilisé la spectrométrie de masse de type native (MS). D’abord, nous avons développé une méthode de préparation d’échantillons en conditions KCl pour former les G4s dans des conditions biologiquement pertinentes. Ensuite, nous avons caractérisé les équilibres de liaison du K+ aux G4s et caractérisé leur mécanisme de repliement. Ce mécanisme implique la présence d’une impasse constituée de G4s antiparallèles à 1- et 2-K+ qui sont formés rapidement. Enfin, nos études de liaison de ligands ont montré que certains des ligands les plus affins pouvaient influencer la structure des G4s comme observé par le nombre d’ions potassium liés. Les ligands Phen-DC3, 360A et PDS sont capables de déplacer les équilibres vers la forme à 1-K+ antiparallèle. La structure antiparallèle à 2-K+ est favorisée par la liaison coopérative de deux ligands Cu-ttpy. Ces résultats démontrent l’importance de la caractérisation des stoechiométries de complexes ternaires (G4:ligand:K+), obtenue par la spectrométrie de masse native. / A G-quadruplex (G4) is a non-canonical nucleic acids structure formed by guanine-rich sequences. Some G4s are polymorphic, a given sequence can form G4s of different topologies. G4s are proposed to be biological regulators because they are found in key regions of the genome, for example, ingene promoters or at the telomeres. Stabilizing G4s formed in those regions as compared to the duplex form is a strategy to fight cancer. To do so, specific and affine ligands are used. Ligand design usually implies the optimization of large aromatic planes to π-π stack on external G-quartets. However, if this was the only binding mode, all ligands would bind with similar affinities to all G4s.To characterize which structures should be targeted and how the ligands interact with these structures, we used native mass spectrometry (MS).First, we developed a MS-compatible sample preparation method in KCl conditions in which G4s are folded with similar topologies as compared to those obtained in biologically relevant conditions. Then, we characterized the K+ binding equilibria and G4s folding pathways. This folding pathway involves the presence of a dead-end constituted by antiparallel G4s with either 1- or 2-K+ cations that are folded first. Finally, our ligand binding studies showed that some of the most affine ligands can influence G4’sstructures, as probed by the number of K+ ions bound. Ligands Phen-DC3, 360A and PDS are able to shift the equilibria towards the 1-K+ antiparallel G4s. The formation of antiparallel with 2-K+ complexes is induced by the cooperative binding of two Cu-ttpy ligands. Our results demonstrate the importance to characterize ternary complex stoichiometries (G4:ligand:K+) as obtained from native mass spectrometry.

La mise en conformation de l’α et β tubulines en hétérodimeres polymérisables nécessite l’intervention de cinq protéines « Tubulin Binding Cofactors » (TBCA a TBCE) dont TBCC qui joue un rôle indispensable. Dans des cellules humaines d’adénocarcinome mammaire, nous avons modifié le niveau d’expression de TBCC et nous avons montre que ceci avait un impact sur le contenu des fractions de tubuline, la dynamique des microtubules ainsi que sur le phénotype et chimiosensibilité des cellules. La distribution en cycle cellulaire et les durées de la mitose et de la phase S ont été altérées. La modification de TBCC avait un faible effet sur la vitesse de prolifération in vitro par contre les cellules présentaient des différences significatives de croissance tumorale in vivo. Les réponses aux agents antimicrotubulaires et à la gemcitabine ont montrées une chimiosensibilité dépendante de la distribution en cycle cellulaire. Tous ces résultats montrent l’importance de la régulation du contenu en tubulines et l’impact de ceci sur le comportement de la cellule en général et vis-à-vis des traitements / The proper folding pathway of α and β-tubulin into the α/β-tubulin heterodimers involve five Tubulin Binding Cofactors (TBCA to TBCE). TBCC plays a crucial role in the formation of polymerization-competent the α/β-tubulin heterodimers. To evaluate the impact of microtubule mass and dynamics on the phenotype and chemosensitivity of breast cancer cells, we targeted TBCC in human breast adenocarcinoma and developed variants of breast cancer cells with modified content of TBCC. We have shown that the modifications in TBCC expression level influenced tubulin fraction distribution and microtubule dynamics. Cell cycle distribution and the durations of mitosis and S-phase were altered. The proliferation rate in vitro was slightly modified whereas in vivo the TBCC variants presented major differences in tumor growth capacity. Chemosensitivity to antimicrotubule agents (paclitaxel and vinorelbine) as well as to gemcitabine was observed to be dependent on the cell cycle distribution of the TBCC variants. These results underline the essential role of fine tuned regulation of tubulin content in tumor cells and the major impact of dysregulation of tubulin dimer content on tumor cell phenotype, cell cycle progression and response to chemotherapy. A better understanding of how the microtubule cytoskeleton is dysregulated in cancer cells would greatly contribute to a better understanding of tumor cell biology and characterization of resistant phenotypes