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No description available.

No description available.

Genome sequencing has resulted in the identification of >55,000 cytochrome P450 enzymes, many of which have an unknown function and regulation. In mammals, CYP3 genes appear in only one subfamily (CYP3A), which metabolize >50% of pharmaceuticals and some steroids in humans. Unlike mammals, fish contain genes in the CYP3A, CYP3B, CYP3C and CYP3D subfamilies. While it is commonly assumed that fish and mammalian CYP3A are functional similar, the function and regulation of fish CYP3 remains largely unknown. In this thesis, the receptors and compounds that regulate CYP3C genes in zebrafish were assessed. The induction of CYP3C genes in response to the aryl hydrocarbon (AHR) and estrogen receptor (ER) ligands, β-naphthoflavone and 17β-estradiol, was measured using quantitative PCR in intestine, liver and gonads. Zebrafish CYP3C genes were inducible by β-naphthoflavone and 17β-estradiol, implicating the aryl hydrocarbon and estrogen receptor in CYP3C gene regulation and suggesting that regulation of CYP3 genes in fish differs from that in mammals. To define the function of zebrafish CYP3A65 and CYP3C1, fluorogenic compounds which are specific markers of CYP1 and CYP3A activity in humans, were screened for metabolism by CYP3A65 and CYP3C1. Both CYP3A65 and CYP3C1 had the capacity to metabolize several of these compounds and the substrate profile overlapped with zebrafish CYP1A, suggesting that these compounds are not specific in fish. A high throughput approach was employed to screen ~4000 small biologically and pharmacologically active compounds for metabolism by CYP3A65 and CYP3C1, using NADPH consumption to assess catalytic activity. The substrate profiles of CYP3A65 and CYP3C1 largely overlapped and were different than mammalian CYP3A4. CYP3A65 and CYP3C1 appeared to have a bias for quinone-based compounds but further studies are required to confirm quinones as substrates and to assess a strong structure-activity relationship. Overall, this study provides insight on the regulation, function and evolution on CYP3 genes in fish. / Dissertation / Doctor of Philosophy (PhD) / Cytochrome P450 (CYP) enzymes break down compounds such as hormones and pharmaceuticals. While mammals have genes in the CYP3A subfamily, fish have unique subfamilies not found in mammals. The function and regulation of the CYP3 family in fish is unknown, but commonly assumed to be like human CYP3. The purpose of this thesis was to identify what receptors and compounds regulate CYP3C enzymes in zebrafish. We found that regulation of CYP3C enzymes in zebrafish is different than humans. Zebrafish CYP3C genes are regulated by the aryl hydrocarbon receptor and estrogen receptor, while human CYP3A is regulated by the pregnane-x-receptor. I used a high throughput approach to screen thousands of compounds to identify the function of CYP3A65 and CYP3C1 from zebrafish. CYP3A65 and CYP3C1 metabolize several plant-based and pharmaceutical compounds. CYP3A65 and CYP3C1 are more functionally similar to each other than to CYP3A in humans.

RNAs have gained significant attention in recent years because they can fold into well-defined secondary or tertiary structures. These three dimensional architectures provide interfaces for specific RNA-RNA or RNA-protein interactions that are essential for biological processes in a living system. These discoveries greatly increased interest in RNA as a potential drug target for the treatment of diseases. Two of the most studied RNA based regulatory systems are HIV-1 trans-activating response element (TAR)/Tat replication pathway and Rev response element (RRE)/Rev export pathway. To efficiently target TAR and RRE RNA, we designed and synthesized three generations of branched peptide libraries that resulted in medium sized molecules. The first generation of BPs were discovered from screening a one-bead one-compound library (4,096 compounds) against HIV-1 TAR RNA. One peptide FL4 displayed a binding affinity of 600 nM to TAR RNA, which is tighter than its native protein counterpart, Tat. Biophysical characterization of these BP demonstrated that "branches" in BPs impart multivalency, and they are cell permeable and non-toxic. The second generation peptides were discovered from an on-bead high-throughput screening of a 3.3.4 branched peptide boronic acids (BPBAs) library that bind selectively to the tertiary structure of RRE IIB. The library comprised of 46,656 unique sequences. We demonstrate that our highest affinity BPBA (BPBA1) selectively binds RRE IIB in the presence of competitor tRNAs as well as against six RRE IIB structural variants. Further, we show that the boronic acid moieties afford a novel binding mode towards RNA that is tunable; their Lewis acidity has critical effects on binding affinity. In addition, biophysical characterizations provide evidence that "branching" in these peptides is a key structural motif for multivalent interactions with the target RNA. Finally, RNA footprinting studies revealed that the BPBA1 binding site encompasses a large surface area that spans both the upper stem as well as the internal loop regions of RRE IIB. BPBA1 is cell permeable and non-toxic. In the next generation of branched peptides, a 3.3.4 branched peptide library composed of 4,096 unique sequences that featured boronic acid and acridine moieties was designed. We chose acridine as the amino acid side chain due to its potential for π-stacking interaction that provides high binding affinity to RNA target. The library was screened against HIV-1 RRE IIB RNA. Fifteen peptides were sequenced and four contained acridine alone and/or in conjunction with boronic acid moieties displayed dissociation constants lower than 100 nM. The ribonuclease protection assays of A7, a sequence that contains both boronic acid and acridine residues, showed a similar protection pattern compared to previous peptide BPBA1, suggesting that the 3.3.4 branched peptides shared similar structural elements and contacted comparable regions of the RRE IIB RNA. The results from this research indicated that "branching" in peptides imparts multivalent interactions to the RNA, and that functional groups such as boronic acid and acridine are key structural features for efficient binding and selectivity for the folded RNA target. We demonstrated that the branched peptides are cell permeable and non-toxic. / Ph. D.

Covalent modifications of histone tails play essential roles in mediating chromatin structure and epigenetic regulation. JmjD3 is a JumonjiC domain containing histone demethylase, belongs to the KDM6 subfamily, and catalyses the removal of methyl groups on methylated lysine 27 on histone 3 (H3K27), a critical mark to promote polycomb mediated repression and gene silencing. The importance of JmjD3 has been implicated in development, cancer biology and immunology. In this thesis, I report the recombinant production of active human JmjD3, development of two in vitro screening assays, a cell-based assay, and structural determination of JmjD3 in complex with the inhibitor 8-hydroxy-5-carboxyquinoline (8HQ). A highly selective and potent small molecule inhibitor GSK-J1 was subsequently identified. The inhibitor is active in HeLa cells and promotes a dose-dependent increase of global H3K27 methylation. The inhibitor GSK-J1 was used in two different cell assay systems related to inflammation and differentiation, to understand how H3K27 demethylation controls cellular functions. By inhibiting H3K27me3 demethylation, it is demonstrated that tumor necrosis factor (TNF) and other pro-inflammatory cytokines are regulated by H3K27 demethylase inhibition in M1- type macrophages derived from healthy volunteers and rheumatoid arthritis patients. It is also shown that inhibition of H3K27me3 demethylation abrogates cellular fusion of M2- type macrophages. During RANKL induced osteoclast differentiation, JmjD3 is up-regulated and promotes the expression of the key transcription factor NFATc1. By inhibiting JmjD3, NFATc1 expression is reduced and osteoclastogenesis is inhibited. This mechanism demonstrates a novel anti-resorptive principle of potential utility in conditions of excess bone resorption such as osteoporosis, bone erosion in inflammatory arthritis or cancer of the bone. These experiments further resolve the ambiguity between scaffold and catalytic function associ- ated with the H3K27 demethylase in these biological systems, and demonstrate that its enzymatic activity is crucial for epigenetic regulation of macrophage and osteoclast function.

Les leucémies myéloïdes aigües résultent d’un dérèglement du processus de l’hématopoïèse et regroupent des maladies hétérogènes qui présentent des profils cliniques et génétiques variés. La compréhension des processus cellulaires responsables de l’initiation et du maintien de ces cancers permettrait de développer des outils thérapeutiques efficaces et ciblés. Au cours des dernières années, une quantité croissante d’anomalies génétiques reliées au développement de leucémies ont été corrélées à une expression anormale des gènes HOX et de leurs cofacteurs MEIS et PBX. Des modèles expérimentaux murins ont confirmé le rôle direct joué par ces protéines dans le développement de leucémies. En effet, la protéine MEIS1 collabore avec HOXA9 dans la leucémogenèse et requiert pour ce faire trois domaines distincts. Deux de ces domaines sont conservés chez PREP1, un membre de la même classe d’homéoprotéine que MEIS1. En utilisant une approche de gain-de-fonction, j’ai confirmé l’importance du rôle joué par le domaine C-terminal de MEIS1 dans l’accélération des leucémies induites par HOXA9. J’ai également montré que l’activité de ce domaine était corrélée avec une signature transcriptionnelle associée à la prolifération cellulaire. J’ai ensuite réalisé un criblage à haut débit afin d’identifier des antagonistes de l’interaction MEIS-PBX, également essentielle à l’accélération des leucémies HOX. À cette fin, j’ai développé un essai de transfert d’énergie de résonance de bioluminescence (BRET) permettant de détecter la dimérisation MEIS-PBX dans les cellules vivantes. Plus de 115 000 composés chimiques ont été testés et suite à une confirmation par un essai orthogonal, une vingtaine de molécules ont été identifiées comme inhibiteurs potentiels. Ces composés pourront être rapidement testés sur la prolifération de cellules leucémiques primaires dans un contexte d’étude préclinique. Finalement, deux approches protéomiques complémentaires ont permis d’identifier des partenaires potentiels de MEIS1 et PREP1. La catégorisation fonctionnelle de ces candidats suggère un nouveau rôle pour ces homéoprotéines dans l’épissage de l’ARN et dans la reconnaissance de l’ADN méthylé. / Acute myeloid leukemias are the result of a perturbed hematopoietic process and regroup heterogeneous diseases with distinct clinical and genetic profiles. Identifying and understanding the faulty cellular processes would allow the development of targeted and efficient therapeutic tools. Over the last 15 years, a growing number of disease-linked genetic anomalies have been correlated with abnormal expression levels of HOX genes and their cofactors MEIS and PBX. Mouse model experimentations revealed a direct role for these proteins in leukemogenesis. Indeed, the protein MEIS1 collaborates with HOXA9 in the acceleration of leukemia development. This specific function requires the presence of three different domains, two of which are highly conserved in PREP1, another member of the MEIS class of homeoproteins. Using a gain-of-function approach, I confirmed the importance of the C-terminal domain of MEIS1 in the acceleration of HOXA9-induced leukemias. I also correlated the activity of this domain with a transcriptional signature related to cell proliferation. Furthermore, I performed a high-throughput screen to identify antagonists of the MEIS-PBX interaction, also required for acceleration of HOX-induced leukemogenesis. In this regard I developed an assay that exploits bioluminescence resonance energy transfer (BRET) to monitor the MEIS-PBX dimerization in living cells. More than 115 000 compounds were tested and upon confirmation of their activity using an orthogonal assay, 20 small molecules were identified as potential inhibitors. These compounds will be rapidly tested on proliferation of primary leukemic cells in a preclinical setting. Finally two complementary proteomic approaches allowed the identification of new potential partners of MEIS1 and PREP1. The functional clustering of these candidates suggests a new role for homeoproteins in mRNA splicing and methylated DNA recognition.

Aqueous droplets submerged in an oil-lipid mixture become enclosed by a lipid monolayer. The droplets can be connected to form robust networks of droplet interface bilayers (DIBs) with functions such as a biobattery and a light sensor. The discovery and characterization of an engineered nanopore with diode-like properties is enabling the construction of DIB networks capable of biochemical computing. Moreover, DIB networks might be used as model systems for the study of membrane-based biological phenomena. We develop and experimentally validate an electrical modeling approach for DIB networks. Electrical circuit simulations will be important in guiding the development of increasingly complex DIB networks. In cell membranes, the lipid compositions of the inner and outer leaflets differ. Therefore, a robust model system that enables single-channel electrical recording with asymmetric bilayers would be very useful. Towards this end, we incorporate lipid vesicles of different compositions into aqueous droplets and immerse them in an oil bath to form asymmetric DIBs (a-DIBs). Both α-helical and β-barrel membrane proteins insert readily into a-DIBs, and their activity can be measured by single-channel electrical recording. We show that the gating behavior of outer membrane protein G (OmpG) from Escherichia coli differs depending on the side of insertion in an asymmetric DIB with a positively charged leaflet opposing a negatively charged leaflet. The a-DIB system provides a general platform for studying the effects of bilayer leaflet composition on the behavior of ion channels and pores. Even with the small volumes (~100 nL) that can be used to form DIBs, the separation between two adjacent bilayers in a DIB network is typically still hundreds of microns. In contrast, dual-membrane spanning proteins require the bilayer separation to be much smaller; for example, the bilayer separation for gap junctions must be less than 5 nm. We designed a double bilayer system that consists of two monolayer-coated aqueous spheres brought into contact with each side of a water film submerged in an oil-lipid solution. The spheres could be brought close enough together such that they physically deflected without rupturing the double bilayer. Future work on quantifying the bilayer separation and studying dual-membrane spanning proteins with the double bilayer platform is planned.

This thesis explores roles of 2-oxoglutarate-dependent (2OG) oxygenases as interfaces that modulate steps in the flow of genetic information in cells in response to oxygen availability. Chapter 1 introduces mechanistic, biochemical and physiological aspects of major subfamilies of 2OG oxygenases, and their established regulatory roles in cells. In addition, structural and functional aspects of the ribosome and the translation process are discussed, with a focus on post-translational ribosome modifications. Chapter 2 investigates histone demethylases, which mediate chromatin-dependent regulation of gene expression and provides proof-of-concept for the rational, structure-guided design of small-molecules for selective inhibition of 2OG oxygenases with roles in cancer and inflammatory disease. Chapter 3 suggests regulatory roles for ten-eleven-translocation (TET)- catalysed DNA hydroxylation; calorimetric and thermal analyses reveal a duplex-stabilizing effect of the epigenetic 5-methylcytosine mark that is reversed upon conversion to 5- hydroxymethylcytosine (also termed the ‘sixth’ DNA base), raising the possibility that 2OG oxygenase catalysis might affect transcription via biophysical effects. Chapter 4 investigates fluoride release assays as a technology to enable medicinal chemistry studies on 2OG oxygenases with roles in fat mass regulation and obesity, cancer and inflammation; studies on the ALKBH5 enzyme show that it is a hypoxically upregulated 2OG oxygenase with a substrate preference distinct from previously characterized ALKBH enzymes. Chapter 5 identifies OGFOD1 as a 2OG-dependent ribosomal protein hydroxylase. OGFOD1 catalysis is conserved from yeast to humans. OGFOD1 catalyses formation of trans-3- hydroxy-L-proline in a highly conserved loop of ribosomal protein S23 proximal to the ribosomal decoding centre, possibly to modulate the interactions of eukaryotic ribosomes with tRNA, mRNA and translation factors in an oxygen-dependent manner. OGFOD1 is the functionally most well-conserved protein-modifying 2OG oxygenase; likewise, ribosomal protein S23 hydroxylation is the most well-conserved post-translational ribosome modification in eukaryotes. Some cell lines require OGFOD1 for proliferation, and scaffolds for OGFOD1- selective inhibitors are developed for use as potential antiproliferative agents and probes for cellular function. Chapter 6 shows the development of assays to investigate whether OGFOD1 catalysis affects ribosome assembly and function, including processivity, accuracy of initiation, elongation and termination, in yeast and mammalian cell lines. Chapter 7 concludes that ribosome hydroxylation might present an additional layer of regulatory complexity by which 2OG oxygenases could enable cells to respond to fluctuating oxygen levels.