The function of the Escherichia coli Ner like protein (Nlp) was investigated by identifying genes which Nlp may regulate. To identify these genes, lac Z gene fusions were generated in a strain of E. coli whose own nlp gene had been interrupted by a luxAB tet insertion. Each clone within this gene fusion collection had the lac Z gene integrated into a random location on the E. coli chromosome. Clones in which the lac Z gene had inserted within a gene regulated by Nlp were predicted to have a different lac Z phenotype on X-gal plates depending on the presence or absence of Nlp expressed from a plasmid borne copy of the nlp gene. Of the 3360 clones screened, 5 were found to have an altered lac phenotype in the presence of Nlp. Three of these clones were found to have lac Z expression induced by Nlp, one was found to be repressed by Nlp, and one clone died upon Nlp expression. These putative Nlp-responsive genes were subcloned into pBR322 to facilitate their identification. Restriction map analysis, double stranded DNA sequencing and single stranded DNA sequencing were used to identify the Nlp responsive genes. Genes that responded to Nlp overexpression were: o80 ('glycosol hydrol F5'), plsB (which encodes a glycerol-3-phosphate acyltransferase), cscB (which encodes a sucrose permease), pgi (which encodes glucose-6-phosphate isomerase), and nuoF (which encodes the F subunit of NADH dehydrogenase). (Abstract shortened by UMI.)

The process of kidney regeneration recapitulates many aspect of development; it involves cell migration, proliferation and differentiation. Our previous studies point to the involvement of HCaRG "Hypertension-related calcium-regulated gene" in two major processes contributing to kidney repair, i.e. control of cell proliferation and differentiation. We extended our studies on the cellular function of HCaRG by comparing cell migration of two kidney cell lines. HCaRG expressing HEK293 cells, which undergo lower proliferation, migrated faster than control cells and presented greater adhesiveness to the extracellular matrix. Faster migration was also observed for the MDKC-C7 cells, after stably transfecting them with HCaRG cDNA. Screening of a human kidney cDNA library with HCaRG as bait revealed its interaction with several ionic transporters, among them Na+,K+,2Cl- cotransporter (NKCC) and Na,K-ATPase (NK pump). HCaRG overexpression induced major morphological changes of HEK293 cells including the formation of lamellipodia. An interaction and a co-localization were further found between HCaRG and actin at the leading edge of migrating cells. Increased activities of the NK pump and NKCC were observed in MDCK-C7 cells expressing HCaRG. These cells displayed higher content of intracellular Na+, water, and total proteins. Ouabain and bumetanide dose-dependently suppressed cells migration with keeping a higher migratory potential for the HCaRG-expressing HEK293 cells. Expression microarrays of HCaRG clones cells resulted in a profile of differential regulation of molecules involved in cell proliferation, differentiation and migration as well as molecules involved in morphogenesis and cytoskeleton organization. Among the quantitatively most up-regulated genes was the transforming growth factor-alpha (TGF-a) which has been associated with normal renal development and recovery. HCaRG-expressing cells exhibited augmented synthesis and release of activated TGF-a and / Le processus de la régénération rénale récapitule beaucoup d'aspects du développement; Il comporte la migration, la prolifération et la différentiation cellulaire. Nos études précédentes démontrent l'implication de HCaRG "Hypertension-related calcium-regulated gene" dans 2 processus principaux contribuant à la réparation des reins; le contrôle de la prolifération et de la différentiation cellulaires. Nous avons prolongé nos études sur la fonction cellulaire de HCaRG en comparant la migration de deux lignées de cellules rénales. Les cellules HEK293 qui expriment HCaRG, et qui se caractérisent par une prolifération réduite, ont migré plus rapidement que les cellules contrôles et ont présenté une plus grande adhérence à la matrice extracellulaire. On a également observé une migration plus rapide pour les cellules MDKC-C7, après la transfection stable avec l’ADNc de HCaRG. Le criblage d'une banque d'ADNc humaine de rein avec HCaRG indiquait son interaction avec plusieurs transporteurs ioniques, parmi eux le co-transporteur Na+, K+, 2Cl- (NKCC) et la pompe Na+,K+-Atpase. L'expression de HCaRG a induit des changements morphologiques majeurs des cellules HEK293, ceci comprend la formation des lamellipodes. Une interaction et une co-localisation entre HCaRG et actine ont ensuite été retrouvées au bord des cellules en migration. On a observé une augmentation de l'activité de la pompe et du co-transporteur dans les cellules MDCK-C7 qui expriment HCaRG. Ces cellules ont montré une teneur plus élevée de Na+ intracellulaire, d'eau, et de protéines. La Ouabaïne et le bumétanide ont supprimé d'une façon dose dépendante la migration de cellules en gardant un potentiel migrateur plus élevé pour les cellules HEK293 qui expriment HCaRG. L'analyse des "microarrays" des clones qui expriment HCaRG a indiqué un profil de régulation différentiel des molécules impliquées dans la prolifération, la diff

Recently, NF-kappaB activation has been shown to be directly influenced by the cytoskeletal environment. In an attempt to better understand and characterize cytoskeletal regulation of NF-kappaB a series of experiments were designed to determine whether the microtubule (MT) stabilizing agent taxol could affect NF-kappaB activation in the presence of different NF-kappaB inducers. Pretreatment of murine NIH 3T3 and human 293 cells with 5 muM taxol resulted in complete abolition of phorbol, 12 myristate, 13-acetate (PMA) mediated NF-kappaB activation including loss of DNA binding potential and reduced CAT reporter activity. Phosphorylation and turnover of IkappaBalpha was effectively abrogated in taxol pretreated COS-7 cells. However, taxol was not capable of preventing TNF-alpha induced NF-kappaB activation nor could taxol suppress IkappaBalpha inducible phosphorylation in TNF-alpha treated cells, suggesting TNF-alpha may function through a microtubule-independent pathway. In vitro kinase assays of PMA stimulated cells established that taxol could reduce activation of protein kinase C by 30%, establishing loss of PKC activity as a possible regulatory step in taxol-mediated suppression of NF-kappaB transactivation. Indirect immunofluorescence analysis revealed that PMA treated COS-7 cells underwent dramatic changes in cell morphology as well as depolymerization of MTs. These observations were similar to that seen for nocodazole treated cells, a known MT depolymerizing agent. In contrast, taxol blocked both nocodazole induced effects as well as PMA induced morphological changes. These findings establish a potential mechanism for taxol-mediated stability of MTs and inhibition of NF-kappaB activity, suggesting a link between the state of microtubule integrity and gene regulation.

Studying an organelle with traditional biochemistry, histology, or microscopy techniques allows the determination of the presence of up to three proteins simultaneously. Mass spectrometry based proteomics has changed the study of organelles; for the first time it is possible to investigate the whole protein complement of a subcellular compartment. In this work I demonstrate our ability to use redundant peptide counting as a quantitative technique to compare the relative abundance of different proteins in a complex sample, specifically, enriched organellar preparations. I present the pipeline that we use to isolate, characterize, and prepare samples for mass spectrometric analysis, followed by the automatic mass spectrometric data acquisition and data processing that result in the output of a list of protein identifications. A highly involved and time consuming manual annotation effort is applied to this preliminary list in order to generate a final set of tables where standardized functional categories and assigned names are applied to every protein identified in order facilitate the application of redundant peptide counting. The organelles of the early secretory pathway are processed by the pipeline, and after rigorous manual verification of the data, the proteomes of the rough microsomes, smooth microsomes, Golgi apparatus, and Golgi derived COPI GTP and COPI GTP?S vesicles are determined. The focus of this thesis is on the proteomes of the Golgi and Golgi derived vesicles. The characteristics and most abundant proteins of the proteomes of the Golgi apparatus, COPI GTP, and COPI GTP?S vesicles are described in detail. The hypothesis of cisternal maturation, a theory describing secretory cargo progress through the Golgi apparatus, is tested and eventually supported by our proteomics data. Finally, outlines of the abundant proteins of unknown function of the Golgi, COPI GTP and COPI GTP?S vesicles are presented. / Les techniques traditionnelles utilisées en biochimie, en histologie ainsi qu'en microscopie permettent la détermination d'un maximum de trois protéines à la fois dans l'étude d'une organelle. La mise en œuvre de la spectrométrie de masse en protéomique a complètement changé le panorama d'investigation des organelles. Pour la première fois, il est possible d'étudier le panel entier de protéines présent dans un compartiment sub-cellulaire. Dans cette étude, je démontre dans un premier temps que l'utilisation du dénombrement de peptides redondants permet la quantification des protéines et donc la capacité de comparer l'abondance relative de différentes protéines dans un échantillon complexe tel qu'une préparation d'organelle. Je présenterai par la suite le pipeline que nous utilisons pour isoler, caractériser et préparer les échantillons avant leur analyse par acquisition automatique par spectrométrie de masse laquelle est suivie par le traitement des données dont le résultat consiste à l'identification d'une liste de protéines. Un effort manuel important d'annotation est appliqué à cette liste préliminaire afin de générer un tableau final où sont assignées à la fois la fonction dans laquelle chaque protéine identifiée est impliquée, ainsi que l'attribution de la nomenclature la plus appropriée. Ce travail laborieux facilite par conséquent le dénombrement et l'attribution des peptides redondants aux protéines. Les organelles de la voie précoce de sécrétion sont analysées par le pipeline et après une vérification manuelle rigoureuse des données, les protéomes des microsomes rugueux, des microsomes lisses, de l'appareil de Golgi, et des vésicules dérivées du Golgi COPI GTP et COPI GTP?S sont déterminés. L'objectif de cette étude porte sur les protéomes du Golgi et des vésicules dérivées du Golgi dont les protéines les plus abondantes ainsi que leurs caractéristiques sont décrites en détail. L'hypoth

A WW domain containing transcriptional coactivator, TAZ (transcriptional coactivator with PDZ-binding motif) plays an important role in transcriptional regulation and perhaps tumorigenesis. Recent studies of TAZ knockout mice reveal a novel function of TAZ in kidney development, polycystic kidney disease, and pulmonary emphysema. As a binding partner of the F-box protein β-TRCP, TAZ regulates kidney homeostasis. It has been shown that as a transcriptional coactivator, TAZ is modulated by the Hippo pathway through phosphorylation and 14-3-3 binding. However, the regulatory mechanism of TAZ interaction with β-TRCP is not well understood. To gain insights into this regulation and the functional consequences, I sought to study the phosphorylation of TAZ and elucidate how it regulates the β-TRCP binding. From this work, I discover that a key component of the Hippo pathway, the large tumor suppressor kinase 2 (LATS2), as well as cAMP-dependent protein kinase (also known as protein kinase A or PKA), phosphorylates TAZ at serine 306 and promotes β-TRCP2 binding. This finding not only poses a novel post-translational modification model for TAZ, but also suggests a new function of the Hippo pathway in kidney homeostasis. / TAZ (transcriptional coactivator with PDZ-binding motif) est un coactivateur de la transcription qui a un rôle bien important dans le développement animal ainsi que dans la tumorogénèse. Des études récentes de souris knockout de TAZ lui ont attribué une nouvelle function. Puisque TAZ est le partenaire de la protéine F-box β-TRCP, il régule l'homeostasie rénale. De plus, la phosphorylation de TAZ est régulée par le réseau de signalisation cellulaire Hippo et de par sa la liaison aux protéines 14-3-3. Le mécanisme qui régule l'interaction entre TAZ et β-TRCP est peu connu. Par conséquent, le but de cette thèse est de démontrer comment la phosphorylation de TAZ peut réguler son interaction à β-TRCP. Ces études ont démontré que la kinase LATS2 (large tumor suppressor kinase 2), une composante de la voie de signalisation Hippo, ainsi que la protéine kinase A (PKA) phosphorylent TAZ à la sérine 306 ce qui induit son interaction à β-TRCP2. Donc, ces résultats démontrent non seulement un nouveau modèle de modification post-traductionnelle de TAZ mais suggèrent une nouvelle fonction de la voie de signalisation Hippo dans homeostasie rénale.

Oxytocin (OT) is a neurohypophyseal hormone that has an important role in mediating uterine contractions during parturition. As preterm labor is a major cause of perinatal mortality, the importance of elucidating the mechanisms involved in parturition become evident. Recently, our lab has discovered a novel mechanism of a non-genomic steroid-induced inhibition of OT receptor (OTR) function (1). It was shown that the steroid hormone progesterone specifically inhibits the rat OTR functions, not the human, via direct binding. Conversely, the human OTR is inhibited by a progesterone metabolite, 5beta-dihydroprogesterone. As there are only a limited number of amino acids that differ between the rat and human OTRs, site directed mutagenesis was used to determine those residues involved in mediating the specific steroid effects, by exchanging selected human-specific residues for the corresponding rat-specific residues. Whereas mutations L51F and A159G of the human OTR did not have any significant effects, the T218A mutant interacted with progesterone and not 5beta-dihydroprogesterone. The corresponding mutation in the rat OTR (A218T) induced an equivalent opposite change of its phenotype. Other mutations such as A84R, 1201V, V313A and A84-T218A were unable to bind OT, suggesting that these residues may be involved directly or indirectly in ligand binding. The data indicate that the 218th residue is critically involved in mediating the steroid specificity.

In this work, we characterized the novel mouse orphan receptor, Estrogen-Related Receptor gamma (ERRgamma). ERRgamma is closely related to the other ERR family members. ERRalpha and ERRbeta. ERRgamma binds to both an ERR response element (ERRE) and an estrogen response element (ERE) and displays constitutive transcriptional activity which is enhanced when ERRgamma is fused to the VP16 activation domain. ERRalpha, ERRbeta, and ERRgamma heterodimerize both on an ERE and in solution in absence of DNA. This may allow for a more versatile mode of gene regulation. Furthermore, the ligand-binding domain (LBD) of ERRgamma interacts with coactivators and corepressors in vitro. Expression of ERRgamma in mouse embryogenesis starts at 7.5 until 10.5 day stage and is observed in the ventricular zones of the brain and the spinal cord. This suggests a role for ERRgamma in the developing nervous system. Although ERRgamma is more closely related to ERRbeta than ERRalpha, the expression pattern of ERRgamma is similar to ERRalpha and distinct to ERRbeta, suggesting a unique role for ERRgamma in development.

Saccharomyces cerevisiae has become a model for studying various biological processes in eukaryotic cells, including human cancer cells and fungal pathogens. Here we endeavoured to study a class of transcriptional regulators, the zinc cluster proteins, and their roles in mediating pleiotropic drug resistance. Because of the presence of functional redundancy within the yeast genome, the role of these proteins has been difficult to unmask. In this study, double knockouts of genes encoding regulators believed to play a role in multidrug resistance were made and assayed using a panel of drugs in order to gain further insight into the functional relationships among these transcriptional regulators. Our phenotypic analysis allowed us to infer new possible functional relationships among zinc cluster transcription factors, as well as confirm and dispute the existence of previously described relationships. / Saccharomyces cerevisiae est maintenant un organisme modèle pour l'étude de la multi-résistance aux médicaments chez les cellules eucaryotes, incluant les cellules de cancer chez les humains et les levures pathogéniques. Au cours de ce projet, nous avons étudié un groupe de régulateurs de transcription, les protéines du type Zn(II)2Cys6, et leur rôle dans la multi-résistance aux médicaments. La redondance fonctionnelle est commune dans la séquence génétique de S. cerevisiae, ce qui rend l'étude de ces protéines difficile. En raison de cette redondance fonctionnelle, nous avons généré des souches comportant des double deletions pour les gènes codant pour les protéines Zn(II)2Cys6 responsables pour la multi-résistance aux médicaments. Ces déletions ont été étudiées phénotypiquement en les exposants à différents médicaments. Ces expériences nous ont aidé à obtenir davantage d'informations sur les relations fonctionnelles entre les membres de ce groupe de protéines et à confirmer ou infirmer l'existence de relations décrites auparavant.

Tumor protein p53 binding protein 1 (53BP1) is a cell cycle checkpoint protein that is important in the early DNA double strand break (DSB) response signal pathway. Aberrant reduction or lack of 53BP1 is found in significant proportions of carcinomas. 53BP1 is recruited to DSB sites and forms foci through its tandem Tudor domain by recognizing dimethylated lysines in histones. The 53BP1 tandem Tudor (53BP1TT) domain consists of two tightly packed Tudor domains follow by a C-terminal alpha helix, and actively binds to methylated histone lysines H4K20 and H3K79. I hypothesized that 53BP1TT domain can potentially interact with non-histone targets, which contain methylated residues, and may be involved in the maintenance of genomic stability. The primary goal of the work presented in this thesis is to identify the proteins that interact with the 53BP1TT domain. I performed a proteomic screen by employing in vitro transcription/translation coupled reactions on pools of cDNA plasmids and identified two putative 53BP1TT targets, brahma-related gene 1 (BRG1), which is a chromatin remodeling catalytic subunit that has helicase and ATPase activities, and is thought to regulate transcription by altering the chromatin structure, and checkpoint kinase 1 (CHK1), which mediates DNA damage signal to downstream damage responsive proteins and initiates cell cycle checkpoint arrest. I demonstrated that both endogenous BRG1 and CHK1 interacted with the 53BP1TT domain in glutathione-S-transferase pulldown assays. Co-immunoprecipitation between ectopically expressed BRG1 and 53BP1 was observed. Interestingly, the interaction between endogenous BRG1 and 53BP1 was observed only after DNA dama / La protéine 53BP1 (p53 binding protein 1) (53BP1) est une protéine impliquée dans la surveillance du cycle cellulaire (checkpoint) activé par les brisures d'ADN double-brin (double-strand break ou DSB). L'absence ou la réduction d'expression de 53BP1 est une caractéristique retrouvée dans la majorité de carcinomes. 53BP1 est recrutée rapidement aux sites de DSB par ses domaines Tudor tandem qui reconnaissent les résidus de lysines dimethylées des histones. Les domaines 53BP1 Tudor tandem (53BP1TT) est comprennent deux domaines Tudor suivi par une hélice alpha au C-terminal et ces domaines ont une affinité spécifique pour les lysines methylées H4K20 et H3K79 des histones. Étant donné que les domaines Tudor tandem sont généralement caractérisés par leur interaction avec le des résidus methylés, j'ai émis l'hypothèse que les 53BP1TT pourraient d'interagir avec des protéines autres que les histones contenant des résidus methylés, ce qui révèlerait un rôle important dans le maintien de la stabilité génomique. Donc, le principal objectif du travail présenté dans cette thèse est l'identification de protéines interagissant avec 53BP1TT. Pour ce faire, j'ai employé la réaction couplée de transcription et traduction in vitro sur une banque d'ADNc. Ceci m'a permis d'identifier deux cibles putatives de 53BP1TT, soit CHK1 (checkpoint kinase 1) et BRG1 (brahma-related gene 1). Chk1 est connu de jouer un rôle clé dans la cascade de signalisation des brisures d'ADN double-brin et pour son interaction avec les homologues de levure de 53BP1, tandis que BRG1 est la sous-unité ATPase des complexes SWI/SNF impliques dans le remodelage de la chroma

The Slc11a family of integral, proton-coupled divalent metal transporters exhibits a high degree of conservation among phylogenetically distinct organisms and contributes to a variety of pleotropic effects in humans. The topology of mammalian Slc11a family members remains unclear and was investigated by insertion of hemagglutinin (HA) epitopes in the predicted hydrophilic segments of Slc11a2 isoform I, followed by cation transport assays to ensure proper protein function and targeting at the plasma membrane. Immunofluorescence, corroborated by a surface labeling assay, on stably transfected intact and permeabilized LLC-PK1 cells indicated that both termini and the intervening segments separating predicted transmembrane domains 4/5, 6/7, and 10/11 are intracellular, while those linking predicted TMDs 5/6, 7/8, and 11/12 correspond to extracellular regions. Epitope insertion in any of the first three predicted hydrophilic loops of the N-terminus abrogated cation transport activity. These results are consistent with a topological model for mammalian Slc11a2 having 12 TMDs and intracellular termini. / La famille de transporteurs membranaires d'ions métalliques divalents couplés aux protons, Slc11a, présente un haut niveau de conservation parmi les organismes phylogénétiquement distincts et contribue à des effets pléiotropiques chez l'humain. La topologie des membres de la famille Slc11a de mammifère n'est pas clairement élucidée. Elle a été investiguée par l'insertion d'épitopes d'hémagglutinine (HA) dans les segments prédits hydrophiles de l'isoforme I de la protéine Slc11a2. Des essais de transport de cations ont été effectués afin de s'assurer du bon fonctionnement de la protéine et de sa localisation à la membrane plasmique. La technique d'immunofluorescence, effectuée sur des cellules intactes et perméabilisées LLC-PK1 exprimant de façon stable ces constructions, a indiqué que les deux extrémités de la protéine ainsi que les segments séparant les domaines transmembranaires prédits 4/5, 6/7 et 10/11 sont intracellulaires, alors que ceux séparant les domaines transmembranaires prédits 5/6, 7/8 et 11/12 correspondent à des régions extracellulaires. Données, qui ont été validées par un essai de marquage de surface. L'insertion d'épitopes dans l'une ou l'autre des trois premières boucles hydrophiles prédites de la région N-terminale a résulté en la perte d'activité de transport de cations de la protéine. Ces résultats sont en accord avec le modèle topologique existant pour la protéine Slc11a2 qui suggère 12 domaines transmembranaires et des extrémités intracellulaires.