The ultimate goal of retroviral infection is to transfer the RNA genome from one cell to another. This requires that the viral genome be assembled within the structure of an infectious particle. The Gag precursor in HIV-1 is alone sufficient to produce viral particles. In order to obtain interactions required for assembly, the Gag molecules must first be concentrated at a cellular site. Membrane and RNA have been proposed to act as intracellular scaffolds for aligning Gag molecules and facilitating their interaction with each other. In vitro studies using truncated Gag molecules have indicated that RNA is important in facilitating a membrane-independent interaction between Gag molecules, and that this RNA need not be viral. During HIV-1 assembly, Gag-Pol is also incorporated into the virus. Gag-Pol has also been shown also responsible for the selective packaging of tRNALys3, the primer for reverse transcriptase. The factors facilitating the interaction between Gag and Gag-Pol, as well as the role of Gag-Po1 in the assembly process have been less studied. It has been assumed, however, that these molecules interact with each other through similar sequences involved in Gag/Gag interactions. Evidence for this includes the fact that unmyristylated Gag or Gag-Pol molecules can be rescued into assembly complexes by myristylated Gag. / In this work, we used COS7 cells transfected with wild type and mutant HIV-1 proviral DNA to investigate the role of Gag-Pol in viral assembly. Experiments conducted on lysates of transfected cells revealed an RNA requirement for Gag/Gag-Pol interaction. Analysis of the effect of mutations within the nucleocapsid region in either Gag or Gag-Pol precursors indicated that RNA-facilitated Gag polymerization is required for Gag/Gag-Pol interaction, and not a direct association of Gag-Pol with RNA. We further examined the selective packaging of tRNALys3 into viral particles during viral assembly. The effects of various mutations in Pr160gag-pol upon the incorporation of tRNALys were studied. We determined that a 54 amino acid sequence in the thumb region of RT within Gag-Pol is required for tRNA Lys incorporation into the virus, and may contribute to the tRNA Lys binding site in Pr160gag-pol. These results demonstrate an important role for Gag-Pol in the assembly process.

Urokinase plasminogen activator (uPA) promotes tumor invasion and metastasis in several malignancies including breast and prostate cancer. High levels of uPA are associated with invasive and metastatic characteristics of cancer. I examined the differential regulation of uPA expression by DNA methylation in normal, hormone responsive and hormone insensitive human breast and prostate cancer cell lines. Lack of uPA expression in normal and hormone responsive cells is due to methylation of uPA promoter. Treatment of these cells with a demethylating agent resulted in induction of uPA, higher invasive capacity of the cells, and significant increase in tumor volume. These studies demonstrated that DNA methylation is the molecular mechanism responsible for uPA gene silencing in normal and hormone responsive cancer cells. Since levels of uPA production is already a reliable prognostic marker of disease progression, determination of uPA promoter methylation status can potentially serve as a reliable early marker for induction of uPA and thus progression of cancer into metastatic stages. I therefore examined the methylation status of uPA promoter in surgical biopsy samples from patients with breast cancer of different grades and found that hypomethylation of uPA promoter is in fact associated with induction of uPA expression and poor grade in these patients. Determination of uPA promoter methylation status can therefore serve as an early reliable indicator of uPA production in breast cancer patients. Since uPA expression and its hypomethylated state is strongly correlated with the malignant phenotype, I utilized uPA gene in the highly invasive human breast cancer cells as a model system to test the hypothesis that pharmacological reversal of the uPA hypomethylation can result in its silencing and inhibition of metastasis. I found that treatment of these cells with the methylating agent SAM significantly inhibits uPA expression, tumor cell invasion, t

The human genome is composed of very few genes (the DNA which encodes proteins), and the remaining 98% of all our DNA is made up of repetitive sequences or "junk" DNA with little or no known function. Our complexity, and the cause of many genetic diseases such as cancer, is brought about by the differential expression of that genetic material, in part due to shuffling and movement of chromosomes within the cell nucleus. A major threat to the integrity of the genome is the occurrence of a DNA double-strand break (DSB). These DSBs occur frequently in every cell, and if repaired improperly or left unattended can lead to genomic rearrangements and cell death. With the publication of the human genome sequence, we were able to establish two systems to investigate the possibility that DNA fragments can insert into breaks sites during DSB repair in human cells. / We find that both foreign and human genomic DNA can insert into extrachromosomal and chromosomal DSBs. Genomic instability syndromes, like those which result from deficiencies in repair proteins, still permit this DSB insertional repair process, however the spectrum of source material provided can differ. The deregulation of replication origins, such as the amplification of sequences flanking viral integration sites, can lead to the spread and further gene amplification of DNA by this insertion mechanism. / Our demonstration that DSB insertional repair takes place in human cells provides a mechanism for reshuffling genomic DNA, and acquiring new sequences. We propose that a selective advantage is conferred upon a cell able to insert DNA at a DSB, providing a complexity of gene content and interaction which could explain the origins and evolutionary role of the vast majority of the human genome.

In eukaryotes, control of protein synthesis or translation is critical for maintenance of cellular function and adaptation to environmental stimuli or stresses. The entire process of producing a functional protein molecule from an mRNA template is elaborately controlled and involves several integrated phases. It is the initiation phase of recruiting the protein synthesis machinery to an mRNA that is rate-limiting for translation. Importantly, a major regulatory mechanism of translation initiation is performed by the eIF4E-binding proteins (4E-BPs). These small proteins interact with the mRNA 5' cap-binding protein, eIF4E, and inhibit translation by preventing it from forming a complex that promotes translation. This is accomplished by the competition of the 4E-BPs with eIF4G for association to eIF4E. This competition is determined by the degree of stimulus-induced 4E-BP phosphorylation: whereas hypophophorylated 4E-BPs bind tightly to eIF4E, hyperphosphorylation causes their dissociation from eIF4E and permits translation. In the nervous system, translational control is obligatory for learning and memory. 4E-BP2, the predominant mammalian 4E-BP in the brain, is required to ensure normal functioning of translation-dependent memory processes. This thesis describes the identification of asparagine deamidation as a novel posttranslational modification of 4E-BP2 in the brain. Deamidation is the spontaneous conversion of asparagines to aspartates. Deamidated 4E-BP2 exhibits increased binding to the mammalian Target of Rapamycin (mTOR)-binding protein, Raptor. Furthemore, 4E-BP2 deamidation, which occurs during postnatal development, alters neuronal activity. It is conceivable that this modification of 4E-BP2 compensates for its attenuated phosphorylation in the brain. 4E-BP2 is also identified here, by virtue of its propensity to deamidate, as a novel substrate for the enzyme Protein L-Isoaspartyl Methyltransferase (PIMT). As a whole, this thesis describes a posttransl / Le contrôle de la synthèse protéique ou traduction chez les eucaryotes est d'une importance capitale dans le maintien de l'homéostasie cellulaire et dans l'adaptation aux stimuli et stress environnementaux. La production de protéines fonctionnelles à partir d'ARN messagers est soumise à un fin contrôle et consiste en une succession d'étapes intégrées. C'est au cours de la première étape de la traduction, l'initiation, que la machinerie traductionnelle est recrutée au niveau de l'ARN messager. L'initiation est l'étape limitante du processus de synthèse. Au coeur du processus de régulation de l'initiation de la traduction se trouve les facteurs liant eIF4E, les 4E-BP. Ces protéines de faible poids moléculaire interagissent avec le facteur eIF4E qui lie le 5'-cap des ARN messagers et inhibe ainsi la traduction en empêchant eIF4E de participer à la formation du complexe d'initiation. Cette inhibition est causée par la compétition entre les 4E-BP et eIF4G pour lier eIF4E. Le niveau de compétition est déterminé par le degré de phosphorylation de 4E-BP induit par les stimuli extracellulaire. Les formes hypophosphorylées de 4E-BP lient eIF4E avec une forte affinité, tandis que les formes hyperphosphorylées relâchent eIF4E, ce qui stimule la traduction. Dans le système nerveux, le contrôle de la traduction est nécessaire pour l'apprentissage et la mémoire. 4E-BP2, le facteur liant eIF4E dont l'abondance est prédominante dans le système nerveux des mammifères, est requis afin d'assurer un fonctionnement normal des processus de mémoire qui dépendent de la traduction. Cette thèse décrit la déamidation d'asparagine en tant que nouvelle modification post-traductionnelle du facteur 4E-BP2 dans le cerveau. Cet événement de déamidation consiste en la conversion spontanée de résidu asparagine en résidu acide aspartique. La forme déamidée de 4E-BP2 présente une affinité accrue pour la protéine Raptor, un facteur associé au comp

Orexins (OX-A, OX-B) are involved in the regulation of sleep, feeding and reward. The action of these peptides is governed by Orexin Receptors 1 and 2 (OX1R, OX2R). In aim to understand the mechanisms involved upon activation of these receptors, we have identified the dynein light chains 1 and 3 (Dynlt1/3) as novel partners. We hypothesize that Dynlt1/3 are important for orexin receptor intracellular regulation. After identification of a strong interaction between OX1R and Dynlt1 and the importance of the OX1R C-terminal domain residues, the functional implication of this novel interaction was assessed. Ligand-induced internalization of OX1R was not altered by modification of Dynlt1/3 expression, yet its transit in early endosomes was accelerated by Dynlt1 over-expression. In conclusion, these data suggest that Dynlt1 promotes the exit of OX1R from early endosomes following ligand-induced internalization, in association with an accelerated signal termination as measured by the phosphorylation levels of ERK1/2. / Les orexines (OX-A, OX-B) sont impliquées dans le sommeil, l'alimentation et la récompense. Leur action est médiée par les récepteurs aux orexines 1 et 2 (OX1R, OX2R). Pour comprendre les mécanismes découlant de leur activation, nous avons identifié les chaînes légères de la dynéine 1 et 3 (Dynlt1/3) comme partenaires de ces récepteurs. Notre hypothèse est que les Dynlt1/3 sont importantes pour réguler les récepteurs. Après avoir identifié une forte interaction entre OX1R et Dynlt1 et l'importance du domaine C-terminal d'OX1R, l'importance fonctionnelle de cette nouvelle interaction a été caractérisée. Le départ d'OX1R de la membrane n'est pas affecté par une modification de l'expression des Dynlt1/3, mais sa transition dans les endosomes a été accélérée par la surexpression de Dynlt1. En conclusion, nos données suggèrent que Dynlt1 favorise la sortie d'OX1R des endosomes après l'internalisation, accélèrant la fin de signalisation du récepteur (mesurée par les niveaux de phosphorylation d'ERK1/2).

The poly(A) binding protein (PABP) is an essential translation factor that enhances protein synthesis and protects mRNAs from degradation. PABP is composed of four RNA recognition motifs (RRM) at its N-terminal and a peptide-interacting C-terminal domain. PABP-interacting proteins 1 (Paip1) and 2 (Paip2) were found to respectively stimulate or inhibit translation. Both Paip 1 and 2 contain two PABP interacting regions (PAM1 and PAM2). In this study, NMR was used to elucidate the mechanism by which Paip2 down-regulates translation through disruption of PABP's binding to poly(A) RNA. A fragment consisting of PABP RRM 2 and 3 was isotopically labeled and backbone NMR assignments were determined. From NMR titration with PABP RRM2-3 and the Paip2 PAM1 region, we determined that Paip2 binds in a positively charged cleft on the beta-sheet surface of RRM2-3 which is evolutionarily conserved. These results suggest a mechanism of how Paip2 displaces the mRNAs from PABP leading to translation inhibition.

Transforming Growth Factor Beta (TGF-beta) is a multifunctional cytokine with a broad spectrum of effects including cellular proliferation, differentiation, and extracellular matrix production. Dysregulation of its action has been implicated in a wide variety of conditions including hypertrophic scarring. CD109 is a novel TGF-P accessory receptor that inhibits TGF-beta1 signaling and cellular responses in an isoform specific manner. Modulation of signaling via CD109 may have the potential to abrogate scarring by altering pro-scarring TGF-beta ligand isoform ratios. The objectives of the current study were to (1) map the TGF-beta1 binding domain of CD 109 as a first step towards the development of a small molecular antagonist based on that domain, and (2) test whether such a molecule inhibits TGF-beta1 binding to its signaling receptors. / We cloned a series of partial CD109 constructs into a GST-containing vector, and used it to synthesize and purify GST-fusion proteins spanning the entire CD109 molecule. We then used these constructs to test for their ability to inhibit TGF-beta1 binding to its receptors in an in vitro affinity labeling assay using HaCat and HEK293 cell lines. Of the six constructs tested, three, encompassing regions spanning amino acids 606-766, 640-720, and 640-1045 successfully bound TGF-beta1 and sequestered it away from its signaling receptors. The current study reports the development and synthesis of a novel 80 amino acid peptide capable of binding to and sequestering TGF-beta1 ligand and demonstrating that a TGF-beta1 binding domain on CD 109 resides between amino acids 640-720.

Previous studies have shown that folate is a determinant of male reproductive health but the underlying molecular mechanisms are unknown. The objective of this study was to determine the impact of a low folate diet, during embryonic development and into adulthood on histone methylation, DNA integrity, spermatogenesis, fertility and offspring health. C57/BL6 females were fed either a folate-sufficient (FS, 2 mg of folate/kg of diet) or a folate-deficient diet (FD, 0.3 mg of folate/kg of diet) two weeks prior to breeding, through pregnancy and lactation. Weaned male pups received the same diet as their mother until sacrifice. Histological analysis of postnatal testis revealed a delay in meiotic onset in FD males. While germ cells were affected by reduced folate, Sertoli and Leydig cells were not. In breeding trials, FD males had reduced fertility in comparison to FS males. Remarkably, increased DNA double strand breaks were found in pachytene spermatocytes from FD males. However, these DNA breaks were repaired in later stages of spermatogenesis, as no difference of DNA breaks was detected by COMET assay in spermatozoa. Epigenetic programming was disturbed in the sperm of FD mice with a reduction in histone H3-lysine4 mono-methylation and in histone H3-lysine9 mono-methylation. The pregnancy outcomes were compromised by folate deficiency, as evidenced by increased occurrence of resorption and abnormalities of placental and embryonic development in the litters sired by FD males. These results suggest adequate folate intake is required for normal spermatogenesis, histone methylation and offspring health. / Les recherches précédentes ont montrée que la folacine est un élément important requis dans la santé reproductive des mâles. Pourtant, les mécanismes moléculaires restent inconnus. L'objectif de cette recherche était de déterminer l'influence exercée par un régime alimentaire faible en folacine sur la méthylation de l'histone, l'intégration d'ADN, la spermatogenèse, la fertilité et la santé des progénitures pendant le développement embryonnaire jusqu'à l'âge adulte. Les femelles C57/BL6 ont été offertes une diète contenant de la folacine suffisante (FS, 2 mg folacine/kg), ou de la folacine déficiente (FD, 0.3 mg folacine/kg), durant les deux dernières semaines avant l'accouplement, pendant toute la période de la gestation et ainsi que la lactation. Les progénitures mâles sevrés ont été nourris d'un régime semblable à celui de leurs mères jusqu'à leurs sacrifices. L'analyse histologique des testicules postnatals indique un retard dans le processus de la méiose dans les mâles FD. Tandis que les cellules germinales ont été influées par la réduction de la folacine, celles de Sertoli et Leydig ne l'étaient pas. Selon les essais d e reproduction, les mâles FS, avaient une baisse de fertilité en comparaison à des mâles FD. Remarquablement, une augmentation dans les cassures double brins d'ADN ont étaient trouvées dans les pachytènes des spermatocytes dans les mâles FD. Mais ces cassures double brin d'ADN ont été réparés dans les spermatozoïdes car aucun différence n'avait été détecté dans les cassures double brins d'ADN par l'analyse de COMET. La programmation épigénétique a été perturbée dans les spermes des souris FD accompagner par une réduction de l'histone H3-lysine 4 mono-méthylation et l'histone H3-lysine 9 mono-méthylation. Les résultats des grossesses ont été compromis par une déficience de folacine comme démontré par l'augmentation dans le taux de la résorption et des anomalies da

Although Growth hormone (GH) therapy can effectively reduce adiposity in some types of obesity, GH treatments of idiopathic obesity have little/no effect. We hypothesized that this may be due to altered GH receptor (GHR) expression in adipose tissues. We first studied the mRNA levels of GHR in fat tissues from a cohort of women ranging from lean to obese and found a significant reduction.To understand the mechanism(s) involved, we analyzed the effects of three obesity-associated factors (TNFα, HIF-1α and glucocorticoids) on GHR gene expression. GHR mRNA levels and promoter activities were significantly inhibited by TNFα, stimulated by HIF-1α while dexamethasone had biphasic effects.Our results suggest that the increased activity of TNFα, HIF-1α and glucocorticoids in obese adipose tissues could alter GHR expression and that TNFα may be involved in the development of GH resistance. / Bien que la thérapie aux hormones de croissance (GH) puisse réduire l'adiposité efficacement dans certaines formes d'obésité, les traitements aux GH de l'obésité idiopathique n'ont peu voire pas d'effet. Nous pensons que cela pouurait être dû à une modification dans l'expression du récepteur aux hormones de croissance (GHR) dans les tissus adipeux. En premier lieu, nous avons évalué les niveaux de GHR dans les tissus adipeux provenant d'une cohorte de femmes allant de maigres à obèses et trouvé une réduction significative du récepteur.Afin de comprendre les mécanismes impliqués, nous avons analysé les effets de trois facteurs associés à l'obesité (TNFα, HIF-1α et glucocorticoïdes) sur l'expression du gène GHR. Les niveaux d'ARNm du GHR et les activités du promoteur sont significativement inhibés par TNFα, stimulés par HIF-1α alors que la dexaméthasone a des effets biphasiques.Nos résultats suggèrent qu'une augmentation de l'activité de TNFα, d'HIF-1α et des glucocorticoïdes dans les tissus adipeux d'individus obèses pourrait modifier l'expression du GHR et que TNFα pourrait être impliqué dans le développement de la résistance aux GH.

Regulators of G-protein Signaling (RGSs) are proteins which attenuate G-Protein coupled receptor (GPCR) signaling by acting as GAPs (GTPase activating proteins) for the Galpha subunit of the heterotrimeric G protein. Although RGSs have been clearly shown to bind the Galpha subunit of heterotrimeric G-proteins, recent studies have shown that RGS specificity occurs via receptor association. To examine possible RGS/GPCR interactions, we constructed a somatostatin 5 (SST5) receptor deletion mutant lacking most of its intracellular C-tail. Here we show that the activation of a GPCR-responsive FUS1 -LacZ reporter gene in yeast strains expressing full length WT SST5 receptor as well as the C-terminally truncated mutant were both inhibited by RGSs 1, 2, 5 and 16, suggesting that the C-tail does not play an integral role in RGS function. As an alternative approach to examine possible RGS/GPCR interactions, RGS function was analyzed via halo assay in yeast cells expressing different RGSs as well the C-tail of different GPCRs including mouse LPA 1, LPA4, Cbeta1, 5HT2A, beta 2AR and human SST5 receptors as well as the third intracellular loop of human SST5. The C-tails and the i3-loop were constructed as GFP fusions. Western blot analysis confirmed that the fusions were expressed in yeast. Of all the combinations of GPCR-C-tail-GFP fusions and RGSs expressed in yeast, only LPA4-GFP was able to interfere with RGS2 function. RGS function was also not inhibited by the expression of SST5-i3-GFP. This suggests that there is a high degree of specificity involved in dictating the interaction between RGSs and GPCRs. In a second study, we wanted to further characterize an immunoreactive RGS5 protein band which was detected from western blot analysis of extract from a yeast strain expressing RGS5 and that was double the size of RGS5. To examine the possibility that this band represents an RGS5 dimer, we examined the molecular weight of RGS5 protein in yeast cells expressing an RGS5-GFP fusion. Western blot analysis of yeast extract expressing GFP-tagged RGS5 detected a band at approximately 50 kDa (representing RGS5-GFP) and a second band at 100 kDa. This suggests that RGS5, like GPCRs, are capable of forming dimers.