La mort cellulaire programmée, ou MCP, est un processus dynamique où les cellules ont la capacité de déclencher et de contrôler leur propre mort. Elle est essentielle et présente chez tous les organismes multicellulaires. Chez les plantes, la MCP permet le développement optimal (ex : formation du xylème, morphologie des feuilles, etc.) de la plante tout en protégeant contre divers stress biotiques et abiotiques (ex : la sécheresse, la chaleur, les UV, etc.). Malgré les avancées de ces dernières années, peu de gènes impliqués dans la mise en place de la MCP induite par les UV-C ont été identifiés chez les plantes. L'objectif était donc de caractériser ces gènes chez Arabidopsis thaliana. L'un des candidats est le gène BI-1, pour Bax Inhibitor-1. Il code pour un facteur anti-apoptotique de la MCP animale également retrouvé chez Arabidopsis thaliana. BI-1 régule aussi le taux de calcium du réticulum endoplasmique, ainsi que la formation des espèces réactives de l'oxygène (ROS), permettant l'activation de la réponse de la cellule au stress. L'expression de ce gène suite à une exposition aux UV-C est augmentée, faisant de lui un candidat pour la réponse aux UV-C. Une fusion du promoteur du gène AtBI-1 au gène rapporteur de la luciférase (pAtBI-1::luciférase) a été insérée dans le génome d'Arabidopsis. Une plante homozygote pour l'insertion de l'ADN-T, 5PL20E, a été isolée. Cette lignée parentale a été ensuite mutagénéisée, et un crible effectué sur la descendance exposée aux UV-C a permis d'identifier un mutant, 2017, dont l'expression de la luciférase était modifiée. Le premier objectif de ce projet était de caractériser ce mutant et de déterminer le lien potentiel avec la régulation de BI-1. Dans une deuxième partie, nous nous sommes intéressés aux rôles des thiorédoxines (TRX) dans la MCP végétale. Ces protéines sont présentes chez tous les organismes vivants. Elles régulent les activités de beaucoup d'enzymes en réduisant leurs ponts disulfures. Peu représentées chez l'homme (deux gènes), elles ont néanmoins un rôle de protection dans la voie de signalisation de l'apoptose. Par contre, chez Arabidopsis, une vingtaine de gènes codant pour les thiorédoxines sont connus. Suite à un autre crible aux UV-C d'une banque de mutants d'insertion, un nouveau mutant pour lequel l'insertion de l'ADN-T était dans le promoteur du gène codant pour la thiorédoxine f1 (AtTRX f1) a été identifié. Chez Arabidopsis, deux gènes codent pour les TRX f. Ces protéines ont d’abord été étudiées en tant que régulateur d’enzymes du cycle de Calvin. Plus récemment, d’autres fonctions ont été attribuées, surtout à la TRX f1. Nous avons obtenu de simples mutants pour les deux gènes et produit par croisement des doubles mutants, pour étudier l'implication des TRX f dans la MCP végétale induite par les UV-C (ultraviolets de type C), le MV (methyl viologen) et l'ABA (acide abscissique). En parallèle, des surexpresseurs de la TRX f1 ont été analysés pour ces mêmes stress abiotiques. L'objectif était de déterminer s'il y a une redondance de fonction entre les deux gènes, puisque ces deux protéines, de la même sous-famille, sont localisées au niveau des chloroplastes. Suite à une induction de la MCP par l'un des stress abiotiques, les réponses observées chez les simples mutants sont similaires à celles des plantes sauvages. Par contre, les doubles mutants paraissent plus résistants aux divers stress, alors que les surexpresseurs semblent nettement plus sensibles que les plantes sauvages. Enfin, un dernier axe a été développé sur le rôle des TRX f dans le contrôle des voies de synthèse et de dégradation de l'amidon pour l'ensemble des plantes de notre essai. Il s'avère que les surexpresseurs produisent une plus grande quantité d'amidon que les plantes sauvages. À l'opposé, les simples mutants ont moins d'amidon au niveau des différents tissus étudiés que les plantes sauvages, et les doubles mutants n'en présentent quasiment pas. La présence d'une quantité plus importante d'amidon chez les surexpresseurs de TRX f influence la sensibilité de ces plantes aux stress abiotiques. À l'inverse, les doubles mutants sont plus résitants à ces mêmes stress, où cette quantité d'amidon est fortement diminuée pour ces plantes.

Native states of globular proteins typically show stabilization in the range of 5 to 15 kcal/mol with respect to their unfolded states. There has been a considerable progress in the area of protein stability and folding in recent years, but increasing protein stability through rationally designed mutations has remained a challenging task. Current ability to predict protein structure from the amino acid sequence is also limited due to the lack of quantitative understanding of various factors that defines the single lowest energy fold or native state. The most important factors, which are considered primarily responsible for the structure and stability of the biological active form of proteins, are hydrophobic interactions, hydrogen bonding and electrostatic interactions such as salt bridges as well as packing interactions. Several studies have been carried out to decipher the importance of each these factors in protein stability and structure via rationally designed mutant proteins. The limited success of previous studies emphasizes the need for comprehensive studies on various aspect of protein stability. An integrated approach involving thermodynamic and structural analysis of a protein is very useful in understanding this particular phenomenon. This approach is very useful in relating the thermodynamic stability with the structure of a protein. A survey of the current literature on thermodynamic stability of protein indicates that the majority of the model proteins which have been used for understanding the determinants of protein stability are small, monomeric, single domain globular proteins like RNase A, Lysozyme and Myoglobin. On the other hand large proteins often show complex unfolding transition profiles that are rarely reversible. The major part of this thesis is focused on studying potential stabilizing/destabilizing interactions in small and large globular proteins. These interactions have been identified and characterized by exploring the effects of various rationally designed mutations on protein stability. Spectroscopic, molecular biological and calorimetric techniques were employed to understand the relationships between protein sequence, structure and stability. The experimental systems used are Leucine binding proteins, Leucine isoleucine valine binding protein (LIVBP), Maltose binding protein (MBP), Ribose binding protein (RBP) and Thioredoxin (Trx). The last section of the thesis discusses thermodynamic properties of molten globule states of the periplasmic protein LBP, LIVBP, MBP and RBP. The amino acid Pro is unique among all the twenty naturally occurring amino acids. In the case of proline, the Cδ of the side chain is covalently linked with the main chain nitrogen atom in a five membered ring. Therefore, Pro lacks amide hydrogen and it is not able to form a main chain hydrogen bond with a carbonyl oxygen. Hence Pro is typically not found in the hydrogen bonded, interior region of α-helix. There have been several studies which showed that introduction of the Pro residue into the interior of an α-helix is destabilizing. Although, it is not common to find Pro residue in the interiors of an α-helix, it has been reported that it occurs with appreciable frequency (14%). The thermodynamic effects of replacements of Pro residue in helix interiors of MBP were investigated in Chapter 2 of this thesis. Unlike many other small proteins, MBP contains 21 Pro residues distributed throughout the structure. It contains three residues in the interiors of α-helices, at positions 48, 133 and 159. These Pro residues were replaced with an alanine and serine amino acids using site directed mutagenesis. Stabilities of all the mutant and wild type proteins have been studied via isothermal chemical denaturation at pH 7.4 and thermal denaturation as a function of pH ranging from pH 6.5 to 10.4. It has been observed that replacement of a proline residue in the middle of an α-helix does not always stabilize a protein. It can be stabilizing if the carbonyl oxygen of residue (i-3) or (i-4) is well positioned to form a hydrogen bond with the ith (mutated) residue and the position of mutation is not buried or conserved in the protein. Partially exposed position have the ability to form main chain hydrogen bonds and Ala seems to be a better choice to substitute Pro than Ser. Unlike other amino acids, the pyrolidine ring of Pro residue imposes rigid constraints on the rotation about the N---Cα bond in the peptide backbone. This causes conformational restriction of the φ dihedral angle of Pro to -63±15º in polypeptides. Therefore, introduction of a rigid Pro residue into an appropriate position in a protein sequence is expected to decrease the conformational entropy of the denatured state and consequently lead to protein stabilization. In Chapter 3 of this thesis, the thermodynamic effects of Pro introduction on protein stability has been investigated in LIVBP, MBP, RBP and Trx. Thirteen single and two double mutants have been generated in the above four proteins. Three of the MBP mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, CD spectroscopy was used to show the absence of structural changes. Stability of all the mutant and wild type proteins was studied via isothermal chemical denaturation at neutral pH and thermal denaturation as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, six of the thirteen single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, two mutants showed no change and five were destabilized. In five of the six cases, the stabilization was because of a reduced entropy of unfolding. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were non-additive. In addition to the hydrogen bond, hydrophobic and electrostatic interactions, other interactions like cation-π and aromatic-aromatic interactions etc. are also considered to make important contributions to protein stability. The relevance of cation-π interaction in biological systems has been recognized in recent years. It has been reported that positively charged amino acids (Lys, Arg and His) are often located within 6 Å of the ring centroids of aromatic amino acids (Phe, Tyr and Trp). The importance of cation-π interaction in protein stability has been suggested by previous theoretical and experimental studies. We have attempted to determine the magnitude of cation-π interactions of Lys with aromatic amino acids in four different proteins (LIVBP, MBP, RBP and Trx) in Chapter 4 of the thesis. Cation-π pairs have been identified by using the program CaPTURE. We have found thirteen cation-π pairs in five different proteins (PDB ID’s 2liv, 1omp, 1anf, 1urp and 2trx). Five cation-π pairs were selected for the study. In each pair, Lys was replaced with Gln and Met. In a separate series of experiments, the aromatic amino acid in each cation-π pair was replaced by Leu. Stabilities of wild type (WT) and mutant proteins were characterized by similar methods, to those discussed in previous chapters. Gln and Aromatic → Leu mutants were consistently less stable than the corresponding Met mutants reflecting the non-isosteric nature of these substitutions. The strength of the cation-π interaction was assessed by the value of the change in the free energy of unfolding (ΔΔG0=ΔG0 (Met) - ΔG0(WT)). This ranged from +1.1 to –1.9 kcal/mol (average value – 0.4 kcal/mol) at 298 K and +0.7 to –2.6 kcal/mol (average value –1.1 kcal/mol) at the Tm of each WT. It therefore appears that the strength of cation-π interactions increases with temperature. In addition, the experimentally measured values are appreciably smaller in magnitude than the calculated values with an average difference |ΔG0expt -ΔG0calc|avg of 2.9 kcal/mol. At room temperature, the data indicate that cation-π interactions are at best weakly stabilizing and in some cases are clearly destabilizing. However at elevated temperatures, close to typical Tm’s, cation-π interactions are generally stabilizing. In Chapter 5, we have attempted to characterize molten globule states for the periplasmic proteins LBP, LIVBP, MBP and RBP. It was observed that all these proteins form molten globule states at acidic pH (3 - 3.4). All these molten globule states showed cooperative thermal transitions and bound with their ligand comparable to (LBP and LIVBP) or with lower (MBP and RBP) affinity than the corresponding native states. Trp, ANS fluorescence and near-UV CD spectra for ligand bound and free forms of molten globule states were found to be very similar. This shows that molten globule states of these proteins have the ability to bind to their corresponding ligand without conversion to the native state. All four molten globule states showed destabilization relative to the native state. ΔCp values indicate that these molten globule states contain approximately 29-67% of tertiary structure relative to the native state. All four proteins lack prosthetic groups and disulfide bonds. Therefore, it is likely that molten globule states of these proteins are stabilized via hydrophobic and hydrogen bonding interactions.

The redox state in the cell plays a major role in determining vital functions and its major imbalance can lead to severe cell injury or death. Redox active proteins and cytokines involved in this process includes thioredoxin (Trx), protein disulfide isomerase (PDI), and tumor necrosis factor (TNF) superfamilies. Trx is a multipotent protein and key regulator of cellular redox balance operating in synergy with Trx reductase and NADPH (the Trx system). Trx has gene regulatory activity of several transcription factors. It also controls in a fascinating way redox-sensitive “on-off” decisions for apoptotic or hypertrophic pathways. Trx protects against H2O2 and TNFmediated cytotoxicity, a pathway in which TNF receptor-binding generates ROS. TNF is an autocrine growth factor and survival factor in vitro and in vivo for B-type of chronic lymphocytic leukemia (B-CLL) cells. The overall aim of this study was to investigate the importance of redox active proteins and cytokines in inflammation and cancer. We focused on: i) the role of Trx, TrxR, and selenium in carcinogenesis and in resistant cancer cells. ii) the importance of Trx in cancer cells and the redox regulation of TNF and its receptors TNFR1 and TNFR2. iii) the potential role of Trx as a key regulator in cellular redox balance, in the pathogenesis of cardiac dysfunction; its relationship to stress response parameters. iv) whether unmutated CLL (UCLL) responses to PKC and ROS pathways were different from mutated CLL (M-CLL) responses. Our results demonstrate pronounced selective selenium-mediated apoptosis in therapy resistant cells and suggest that redox regulation through the Trx system is an important target for cancer therapy. Trx was strikingly elevated in heart failure cases compared with controls signifying an adaptive stress response that is higher the more severe the disease. TNF autocrine release was redox modulated and the TNF receptors interacted at the cell surface membrane with the redox-active PDI, which excerted a stringent redox-control of the TNFR signaling. The proliferative response as well as increase of autocrine TNF and Trx were higher in U-CLL than in M-CLL. The overall conclusion of the four papers included in this thesis is that redox-active proteins and cytokines plays an important role in control and regulation of cancer and inflammation. Furthermore, redox regulation via thioredoxin by selenium, may offer novel treatment possibilities for resistant tumors disease.

No description available.

No description available.