<p>Functional genomic analyses provide information that allows hypotheses to be formulated on protein function. These hypotheses, however, need to be validated using reverse genetic approaches, which are difficult to perform on a large scale and in diploid organisms. To address this problem, we developed a genetic screen to rapidly isolate lariat peptides that function as trans dominant inhibitors of protein function.</p> <p>We engineered intein proteins to genetically produce lariats. A lariat consists of a lactone peptide covalently attached to a linear peptide. Cyclizing peptides with a lactone bond imposes a constraint even within the reducing environment found inside of cells. The covalently attached linear peptide provides a site for fusing protein moieties. We fused a transcriptional activation domain to a combinatorial lactone peptide, which allowed combinatorial lariat libraries to be screened for protein interactions using the yeast two-hybrid assay.</p> <p>We confirmed that the intein processed in yeast using Western blot analysis. A chemoselective ring opening of the lactone bond with heavy water, followed by mass spectrometry analysis showed that ~ 44% of purified lariat contained an intact lactone bond. To improve the stability of the lactone bond, we introduced mutations into the engineered intein and analyzed their processing and stability by mass spectrometery. Several mutations were identified that increased the amount of intact lariat.</p> <p>Combinatorial libraries of lactone peptides were generated and screened using the yeast-two-hybrid interaction trap. Lactone cyclic peptides that bound to a number of different targets including LexA, Jak2, and Riz1 were isolated. A lactone cyclic peptide isolated against the bacterial repressor protein LexA was characterized. LexA regulates bacterial SOS response and LexA mutants that cannot undergo autoproteolyis make bacteria more sensitive to, and inhibit resistance against cytotoxic reagents. The anti-LexA lariat interacted with LexA with a dissociation constant of 37 µM by surface plasmon resonance. The lactone constraint was determined to be required for the interaction of the anti-LexA L2 lariat with LexA in the yeast-two-hybrid assay. Alanine scanning showed that only two amino acids (G8 and E9) in the anti-LexA L2 sequence (1-SRSWDLPGEY-10) were not required for the interaction with LexA. The interaction of the anti-LexA lariat with LexA in vivo was confirmed by chromatin precipitation of the lactone peptide-LexA-DNA complex. The anti-microbial properties of the anti-LexA lariat were also characterized. The anti-LexA lariat potentiated the activity of a DNA damaging agent mitomycin C and inhibited the cleavage of LexA, preventing the SOS response pathway from being activated.</p> <p>In summary, lariats possess desired traits for characterizing the function and therapeutic potential of proteins. The ability to genetically and chemically synthesize lariats allows the lariat transcription activation domain to be replaced by other peptide and chemical moieties such as affinity tags, fluorescent molecules, localization sequences, et cetera, which give them advantages over head to tail cyclized peptides, which have no free end to attach moieties.</p>

<p>Functional genomic analyses provide information that allows hypotheses to be formulated on protein function. These hypotheses, however, need to be validated using reverse genetic approaches, which are difficult to perform on a large scale and in diploid organisms. To address this problem, we developed a genetic screen to rapidly isolate lariat peptides that function as trans dominant inhibitors of protein function.</p> <p>We engineered intein proteins to genetically produce lariats. A lariat consists of a lactone peptide covalently attached to a linear peptide. Cyclizing peptides with a lactone bond imposes a constraint even within the reducing environment found inside of cells. The covalently attached linear peptide provides a site for fusing protein moieties. We fused a transcriptional activation domain to a combinatorial lactone peptide, which allowed combinatorial lariat libraries to be screened for protein interactions using the yeast two-hybrid assay.</p> <p>We confirmed that the intein processed in yeast using Western blot analysis. A chemoselective ring opening of the lactone bond with heavy water, followed by mass spectrometry analysis showed that ~ 44% of purified lariat contained an intact lactone bond. To improve the stability of the lactone bond, we introduced mutations into the engineered intein and analyzed their processing and stability by mass spectrometery. Several mutations were identified that increased the amount of intact lariat.</p> <p>Combinatorial libraries of lactone peptides were generated and screened using the yeast-two-hybrid interaction trap. Lactone cyclic peptides that bound to a number of different targets including LexA, Jak2, and Riz1 were isolated. A lactone cyclic peptide isolated against the bacterial repressor protein LexA was characterized. LexA regulates bacterial SOS response and LexA mutants that cannot undergo autoproteolyis make bacteria more sensitive to, and inhibit resistance against cytotoxic reagents. The anti-LexA lariat interacted with LexA with a dissociation constant of 37 µM by surface plasmon resonance. The lactone constraint was determined to be required for the interaction of the anti-LexA L2 lariat with LexA in the yeast-two-hybrid assay. Alanine scanning showed that only two amino acids (G8 and E9) in the anti-LexA L2 sequence (1-SRSWDLPGEY-10) were not required for the interaction with LexA. The interaction of the anti-LexA lariat with LexA in vivo was confirmed by chromatin precipitation of the lactone peptide-LexA-DNA complex. The anti-microbial properties of the anti-LexA lariat were also characterized. The anti-LexA lariat potentiated the activity of a DNA damaging agent mitomycin C and inhibited the cleavage of LexA, preventing the SOS response pathway from being activated.</p> <p>In summary, lariats possess desired traits for characterizing the function and therapeutic potential of proteins. The ability to genetically and chemically synthesize lariats allows the lariat transcription activation domain to be replaced by other peptide and chemical moieties such as affinity tags, fluorescent molecules, localization sequences, et cetera, which give them advantages over head to tail cyclized peptides, which have no free end to attach moieties.</p>

No description available.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Die aberrante Aktivierung des NOTCH Signalweges trägt entscheidend zu verschiedensten malignen Erkrankungen im Menschen bei. Basierend auf der Analyse von hochdimensionalen Microarray-Datensätzen von klassischen Hodgkin Lymphoma Fällen und nicht-Hodgkin Fällen, haben wir eine Hodgkin Lymphoma-spezifische NOTCH Signatur identifiziert. Diese wird von dem essentiellen NOTCH-Koaktivator Mastermindlike 2 (MAML2) signifikant dominiert. Auf der Grundlage dieses Resultates haben wir die Rolle von MAML2 im Kontext des Hodgkin Lymphoma-spezifischen, aberrant regulierten NOTCH Signalweges weiter untersucht. Die signifikante Überexpression von MAML2 im Hodgkin Lymphom konnte in verschiedenen Hodgkin Lymphom Zelllinien und auch durch die immunhistochemische Analyse von primären Hodgkin Lymphom Fällen verifiziert werden. Mit Hilfe des Knockdowns von MAML2 bzw. der Inhibition des NOTCH Signalweges durch die Verwendung einer kompetitiv, dominant-negativ wirkenden, trunkierten Variante von MAML1 konnte daraufhin gezeigt werden, dass die Überexpression von MAML2 der limitierende Faktor für die Hodgkin Lymphomaspezifische, pathologische Deregulation des NOTCH Signalweges ist. Die MAML2- vermittelte Überaktivierung des NOTCH Signalweges ist darüber hinaus essentiell für die Proliferation von Hodgkin Lymphom Zelllinien und die aberrante Expression der NOTCH Zielgene HES7 und HEY1. Das konstitutive Vorhandensein von aktiviertem, intrazellulären NOTCH1 in Hodgkin Lymphom Zelllinien impliziert darüber hinaus,dass der Signalweg im Hodgkin Lymphom zellautonom aktiviert ist. In dieser Arbeit wird damit ein neuer, pathologisch hochwirksamer Mechanismus der NOTCH Signalweg-Deregulation aufgedeckt. / Inappropriate activation of the NOTCH signaling pathway, e.g. by activating mutations, contributes to the pathogenesis of various human malignancies. Using a bottom up approach based on the acquisition of high–dimensional microarray data of classical Hodgkin lymphoma (cHL) and non-Hodgkin B cell lymphomas as control, we identify a cHL specific NOTCH gene-expression signature dominated by the NOTCH co-activator Mastermind-like 2 (MAML2). This set the basis for demonstrating that aberrant expression of the essential NOTCH co-activator MAML2 provides an alternative mechanism to activate NOTCH signaling in human lymphoma cells. Using immunohistochemistry we detected high-level MAML2 expression in several B cell-derived lymphoma types, including cHL cells, whereas in normal B cells no staining for MAML2 was detectable. Inhibition of MAML protein activity by a dominant negative form of MAML or by shRNAs targeting MAML2 in cHL cells resulted in down-regulation of the NOTCH target genes HES7 and HEY1, which we identified as overexpressed in cHL cells, and in reduced proliferation. In order to target the NOTCH transcriptional complex directly we developed short peptide constructs that competitively inhibit NOTCH dependent transcriptional activity as demonstrated by NOTCH reporter assays and EMSA analyses. We conclude that NOTCH signaling is aberrantly activated in a cell autonomous manner in cHL. This is mediated by high-level expression of the essential NOTCH coactivator MAML2, a protein that is only weakly expressed in B cells from healthy donors. Using short peptide constructs we moreover show, that this approach is promising in regard to the development of NOTCH pathway inhibitors that will also work in NOTCH associated malignancies that are resistant to -secretase inhibition.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Le cancer de la prostate est le cancer le plus fréquent chez les hommes et la capacité des tumeurs à développer une résistance face aux thérapies anti-androgéniques vient souvent compromettre le pronostic des patients. Le développement de nouvelles approches thérapeutiques afin de circonvenir à la progression de ces tumeurs représente un besoin important la gestion de ce type de cancer. Plusieurs démonstrations récentes établissent l’implication de la famille des proprotéines convertases dans la progression tumorale. Ces enzymes ont pour fonctions biologiques de cliver une variété de précurseurs protéiques jouant des rôles importants dans la tumorigénèse. Dans le cancer de la prostate, la proprotéine convertase PACE4 est fortement surexprimée dans les cellules cancéreuses et joue un rôle dans la prolifération et la capacité à former des tumeurs, ce qui en fait une cible thérapeutique d’intérêt. En ce sens, des inhibiteurs peptidomimétiques ont été développés dans l’optique de la thérapie ciblée contre la PACE4. Toutefois, dans le but de développer une approche thérapeutique optimale, il convient néanmoins de comprendre le niveau de redondance fonctionnelle entre les différents membres de la famille des convertases, qui sont connus pour partager plusieurs de leurs substrats, ainsi que les mécanismes moléculaires régissant l’activité de la PACE4 et de ses substrats sous-jacents. L’utilisation d’une approche de répression génique stable envers les différentes convertases a permis de mettre en lumière les fonctions uniques de la PACE4 dans la progression tumorale. De plus, grâce à une approche de protéomique comparative, le premier substrat de la PACE4 dans le cancer de la prostate; le growth differenciation factor 15, a été découvert. Ce substrat permet de commencer à dresser l’implication de PACE4 dans le paysage moléculaire du cancer de la prostate. Grâce à des modalités d’imagerie moléculaire, l’emploi de versions radiomarquées des inhibiteurs peptidiques a également permis de démontrer que les composés s’accumulent dans les cellules cancéreuses en fonction des niveaux de PACE4 présents, et ce, tant in cellulo qu’in vivo. Ces données suggèrent un potentiel pour le développement d’un examen théranostique pour prédire la réponse tumorale à la pharmacothérapie anti-PACE4. Finalement, l’analyse de l’épissage alternatif de l’ARNm de PACE4 a permis l’élucidation des caractéristiques biochimiques et des fonctions spécifiques d’une nouvelle isoforme; la PACE4-altCT, qui est exprimée chez les cellules cancéreuses de la prostate, mais aussi d’autres types de cancer. Cette découverte a permis de redéfinir le modèle de travail en intégrant le concept de la rétention intracellulaire de cette isoforme qui semble médier la plupart de l’activité pro-proliférative reliée à l’activité PACE4, ce qui en fait la cible pharmacologique principale des inhibiteurs peptidiques dans le cancer de la prostate, mais aussi un biomarqueur potentiel. / Abstract: Prostate cancer is the most common cancer among men. The capabilities of tumors to adapt and overcome antiandrogenic therapy is persistently worsening patient’s prognostic and the development of novel therapeutic approaches to circumvent tumor progression therefore represents an unmet need. Many reports now demonstrate the implication of the enzymes from the proprotein convertase family in the progression of tumor from many cancer types. These enzymes are responsible for the processing of various protein precursors playing important roles in tumorigenesis. In prostate cancer, the proprotein convertase PACE4 is strongly overexpressed in cancer cells and plays a role in cell proliferation and tumor formation thus making a strong case for its use as a pharmacological target. For this reason, PACE4 peptidomimetic inhibitors were generated to develop PACE4-targeted therapies. However, to develop an optimal therapeutic approach regarding the inhibition of this enzyme, a complete understanding of the level of functional redundancy between the different convertases in prostate cancer is needed. Moreover, understanding the molecular mechanisms both upstream and downstream of PACE4 in prostate cancer cells would allow a better understanding of the considerations underneath such a therapeutic strategy. Using a stable gene silencing approach to knockdown all co-expressed member of the convertase family in prostate cancer cells, the roles of PACE4 in tumor progression were found to be unique and non-redundant among the other family member. Through a comparative proteomic approach, the first PACE4-specific substrate in prostate cancer; growth and differentiation factor 15, was identified. With this substrate growth factor, it is now possible to initiate the dissection of PACE4 biochemical functions in the prostate cancer molecular landscape. Using a radiolabelled version of the PACE4 peptide inhibitors, it was possible to demonstrate using molecular imaging that when applied in cellulo and in vivo, the compound is uptaken by cancer cells as well as by tissues according to their PACE4 expression levels. These data suggest that such PACE4 molecular imaging with pharmacological inhibitor could be developed as a theranostic assay to predict which tumor could be treated by PACE4-targetted therapy. Lastly, PACE4 mRNA alternative splicing analysis permitted the discovery of a new PACE4 isoform; named PACE4-altCT, which is strongly overexpressed by prostate cancer cells as well as other cancer types. As this isoform displays specific biochemical features and functions, notably being intracellularly retained and mediating most of the PACE4-associated cell growth capabilities, this discovery further redefined our working model, pointing to PACE4-altCT as the pharmacological target of inhibitory peptides in prostate cancer as well as a potential biomarker.

This thesis describes different aspects of protein interactions. Initially the function of peptides and their conjugates with small molecule inhibitors on the surface of Human Carbonic Anhydrase isoenzyme II (HCAII) is evaluated. The affinities for HCAII of the flexible, synthetic helix-loop-helix motif conjugated with a series of spacered inhibitors were measured by fluorescence spectroscopy and found in the best cases to be in the low nM range. Dissociation constants show considerable dependence on linker length and vary from 3000 nM for the shortest spacer to 40 nM for the longest with a minimum of 5 nM for a spacer with an intermediate length. A rationale for binding differences based on cooperativity is presented and supported by affinities as determined by fluorescence spectroscopy. Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance (HSQC) spectroscopic experiments with 15N-labeled HCAII were used for the determination of the site of interaction. The influence of peptide charge and hydrophobicity was evaluated by surface plasmon resonance experiments. Hydrophobic sidechain branching and, more pronounced, peptide charge was demonstrated to modulate peptide – HCAII binding interactions in a cooperative manner, with affinities spanning almost two orders of magnitude. Detailed synthesis of small molecule inhibitors in a general lead discovery library as well as a targeted library for inhibition of α-thrombin is described. For the lead discovery library 160 members emanate from two N4-aryl-piperazine-2-carboxylic acid scaffolds derivatized in two dimensions employing a combinatorial approach on solid support. The targeted library was based on peptidomimetics of the D-Phe-Pro-Arg showing the scaffolds cyclopropane-1R,2R-dicarboxylic acid and (4-amino-3-oxo-morpholin-2-yl)- acetic acid as proline isosters. Employing 4-aminomethyl-benzamidine as arginine mimic and different hydrophobic amines and electrophiles as D-phenylalanine mimics resulted in 34 compounds showing IC50 values for α-thrombin ranging more than three orders of magnitude with the best inhibitor showing an IC50 of 130 nM. Interestingly, the best inhibitors showed reversed stereochemistry in comparison with a previously reported series employing a 3-oxo-morpholin-2-yl-acetic acid scaffold.

Le RXP470.1 est l’un des premiers inhibiteurs puissants de la MMP-12, une métalloprotéase à zinc impliquée dans de nombreuses pathologies comme l’athéroclérose et la bronchopneumopathie obstructive chronique (BPCO). Pour comprendre les bases moléculaires contrôlant l’interaction de cet inhibiteur avec sa cible, des approches pluridisciplinaires associant des relations structure-activité, avec des études de cristallographie de complexes enzymes inhibiteurs et d’études de microcalorimétrie, décrivant les contributions enthalpiques et entropiques impliquées dans la formation des complexes, ont été réalisées dans ce travail de thèse. Les affinités de trois analogues du RXP470.1 ont été tout d’abord déterminées. Puis quatre structures cristallographiques de complexes enzyme/inhibiteur décrivant le mode d’interaction duRXP470.1 et de ces trois analogues ont été obtenues avec des résolutions de 1.15 Å, 1.50 Å, 1.50Å et 1.30 Å, respectivement. Parallèlement les études de microcalorimétrie ont été menées pour étudier les facteurs énergétiques contrôlant l’interaction du RXP470.1 avec la MMP-12. Les résultats indiquent que la présence d’une chaîne latérale très longue et hydrophobe en position P1’de l’inhibiteur s’insérant dans la cavité S1’ de la MMP-12 est essentielle à la très bonne affinité de cet inhibiteur pour la MMP-12. Cette interaction met essentiellement en jeu un effet entropique très important de - 4 kcal/mol. L’interaction du RXP470.1 est aussi essentiellement dirigée par une forte augmentation d’entropie (-TDS= -10 kal/mol) et une composante enthalpique beaucoup plus faible (DH= -2.5 kcal/mol), et ce malgré l’observation dans le cristal de nombreuses interactions entre l’inhibiteur et le site actif de la MMP-12. L’étude de microcalorimétrie met aussi en lumière la prise d’un proton au cours de la formation du complexe enzyme inhibiteur impliquant deux résidus chargés négativement en solution, le résidu catalytique Glu219 et le groupe phosphoryle chélatant du zinc dans l’inhibiteur. Cette étude révèle aussi que si le groupe phosphoryle est considéré comme un chélatant faible de l’atome de zinc, il impose néanmoins des contraintes directionnelles très importantes qui ont un impact sur le positionnement des autres parties de l’inhibiteur dans le site actif de l’enzyme. Ce dernier effet pourrait expliquer pourquoi un certain nombre d’interactions entre l’inhibiteur et l’enzyme ne sont pas optimisées et pourquoi la variation d’enthalpie pour former le complexe reste relativement faible. Cette étude ouvre maintenant la voie à d’autres études en plaçant au centre des futurs travaux le rôle du groupe chélatant dans la conception des inhibiteurs de MMP, ainsi de nouveaux inhibiteurs puissants et sélectifs d’autres MMP devraient voir le jour grâce à ce travail et aux résultats obtenus. / RXP470.1 is one of the first highly potent and selective inhibitor of MMP-12, a zinc protease involved in several human diseases such as atherosclerosis and chronic obstructive pulmonary disease (COPD). To understand the molecular determinants controlling the interaction of RXP470.1 with MMP-12 active site, a multidisciplinary approach combining structure-activity data, crystallography and microcalorimetry have been performed on RXP470.1 and its three analogues. The affinities of the three RXP470.1 analogues have been determined. Then, fourcrystal structures of MMP-12 in interaction with these inhibitors have beendetermined at high resolution, 1.15 Å, 1.50 Å, 1.50 Å et 1.30 Å, respectively. These data have indicated that the presence of a long hydrophobic side chain in the P1’ position of the RXP470.1, which enters deeply inside the S1’ cavity of MMP-12, is playing a key role in the inhibitor affinity. The contribution of this side chain is mostly entropic (-TDS - 4 kcal/mol). The interaction of RXP470.1 with MMP-12 is also mostly driven by a sizeable entropy increase (-TDS= -10 kal/mol) and a more modest enthalpy contribution (DH= -2.5 kcal/mol), despite the observation in the crystal structure of several contacts between inhibitor and MMP-12 active site. Furthermore, this study reveals that the binding of RXP470.1 to MMP-12 is linked to a proton uptake involving two negatively charged residues, the catalytic Glu219 and the phosphoryl group of the inhibitor. Furthermore, despite that the phosphoryl group is considered as a weak zincbinding group, this study highlights that the interactions of this group with the active site zinc atom involved strong directionality between these two groups. This effect has strong impact on the positioning of the other parts of the inhibitor in the MMP-12 active site. This last effect could be responsible for the modest enthalpy increase associated with the binding of RXP470.1 to MMP-12, by preventing the optimization of several interactions between the inhibitor and the enzyme. The results indicate that the role of the zinc-binding group should be better consider in the future. Finally this study opens a new vision in this field and should allow the design of new selective inhibitors of other MMPs.