No description available.

No description available.

The growth and survival of all cells require functional ribosomes that are capable of protein synthesis. The disruption of the steps required for the function of ribosomes represents a potential future target for pharmacological anti-cancer therapy. ABCE1 is an essential Fe-S protein involved in ribosomal function and is vital for protein synthesis and cell survival. Thus, ABCE1 is potentially a great therapeutic target for cancer treatment. Previously, cell biological, genetic, and structural studies uncovered the general importance of ABCE1, although the exact function of the Fe-S clusters was previously unclear, only a simple structural role was suggested. Additionally, due to the essential nature of ABCE1, its function in ribosome biogenesis, ribosome recycling, and the presence of Fe-S within ABCE1, the protein has been hypothesized to be a target for oxidative degradation by ROS and critically impact cellular function. In an effort to better understand the function of ABCE1 and its associated Fe-S cofactors, the goal of this research was to achieve a better biochemical understanding of the Fe-S clusters of ABCE1. The kinetics of the ATPase activity for the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe-S clusters) and holo- (with full complement of Fe-S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe-S clusters and Mg2+. Typically, ATPases require Mg2+, as is true for PabABCE1, but Mg2+ also acts as a unusual negative allosteric effector that modulates ATP affinity of PabABCE1. Comparative kinetic analysis of Mg2+ inhibition shows differences in the degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent Km for ATP of apo- iv PabABCE1 increases >30 fold from ~30 µM to over 1 mM when in the presence of physiologically relevant concentrations of Mg2+. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge () to being dependent on  with cellular [Mg2+]. The effect of ROS on the Fe-S clusters within ABCE1 from Saccharomyces cerevisiae was studied by in vivo 55Fe labeling. A dose and time dependent depletion of ABCE1 bound 55Fe after exposure to H2O2 was discovered, suggesting the progressive degradation of Fe-S clusters under oxidative stress conditions. Furthermore, our experiments show growth recovery, upon removal of the H2O2, reaching a growth rate close to that of untreated cells after ~8 hrs. Additionally, a corresponding increase (~88% recovery) in the ABCE1 bound 55Fe (Fe-S) was demonstrated. Observations presented in this work demonstrate that the majority of growth inhibition, induced by oxidative stress, can be explained by a comparable decrease in ABCE1 bound 55Fe and likely loss of ABCE1 activity that is necessary for normal ribosomal activity. The regulatory roles of the Fe-S clusters with ABCE1 provide the cell a way to modulate the activity of ABCE1 and effectively regulate translation based on both cellular energy charge and the redox state of the cell. Intricate overlapping effects by both [Mg2+] and the status of Fe-S clusters regulate ABCE1’s ATPase activity and suggest a regulatory mechanism, where under oxidative stress conditions, the translational activity of ABCE1 can be inhibited by oxidative degradation of the Fe-S clusters. These findings uncover the regulatory function of the Fe-S clusters with v ABCE1, providing important clues needed for the development of pharmacological agents toward ABCE1 targeted anti-cancer therapy.

No description available.

No description available.

<p>Ribosome biogenesis is a major metabolic expense of bacteria and a promising target for antibacterial drug discovery. <em>Trans-</em>acting proteins, called ribosome biogenesis factors, aid this complex and cooperative process. EngA (YfgK, Der) is a widely distributed bacterial GTPase that is shown here to be important for normal ribosome biogenesis. EngA is an attractive antibacterial target because it is essential for viability in bacteria but is absent in humans.</p> <p>The GTPase activity and cellular function of EngA was investigated in <em>Escherichia coli</em>. Depletion of EngA caused accumulation of 30S and 50S ribosomal subunits at the expense of 70S ribosomes, showing for the first time that EngA is important for normal ribosome biogenesis. Mutation of either of the tandem GTPase domains of EngA led to abnormal ribosome profiles, cell death and loss of GTPase activity, revealing that the two GTPase domains act cooperatively to carry out an essential function. EngA bound the 50S subunit of the ribosome in cells and <em>in vitro</em>. Depletion of EngA resulted in sensitization to aminoglycoside antibiotics, which bind at the aminoacyl-tRNA binding site of ribosomes. To search for an inhibitor of ribosome biogenesis, a high-throughput screen of the GTPase activity of EngA was developed. A specific inhibitor was not identified, however, this robust screen can be extended to other compound libraries. Thus, we showed that the GTPase domains of EngA have a cooperative function in ribosome biogenesis, probably in maturation of the 50S subunit, and that EngA is an amenable target for further inhibitor screens.</p> / Doctor of Philosophy (PhD)

<p>Using the model organism <em>Escherichia coli</em>, we discuss herein two novel antimicrobial targets: namely, the protein YjeE and the process of ribosome assembly.</p> <p>YjeE is essential for viability and widely conserved amongst bacterial pathogens and has no human homologue. We searched for a small molecule probe of the function of YjeE to help circumvent the inadequate genetic tools that are available for studying this protein. Sensitive methods for detecting ligand binding were optimized; however, this effort yielded no inhibitors. A second approach to studying the function of YjeE was the development of a reporter using a promoter that is directly upstream of <em>yjeE </em>in <em>E. coli</em>. The activity of this promoter was tested in the presence of small molecules of known function and in diverse gene deletion backgrounds. YjeE found to be linked to the inhibition of DNA and protein translation as well as central metabolism and respiration. These interactions prompted experiments that revealed YjeE to be dispensable under anaerobic conditions.</p> <p>Many antibiotics target ribosomal protein synthesis; however, no current antibiotics target the process of ribosome biogenesis. In order to identify new biogenesis factors, the non-essential fraction of the <em>E. coli </em>genome was screened for deletions that gave rise to cold-sensitive growth. We found that genes associated with ribosome function were the most represented cold sensitive factors amongst the genes of known function. We identified and present here two new putative ribosome biogenesis factors, <em>prfC</em> and <em>ychF</em>, which had phenotypes associated with ribosome assembly defects.</p> / Doctor of Philosophy (PhD)

The DExD/H-box protein Prp43 is essential for two biological processes: nucleoplasmic pre-mRNA splicing and nucleolar rRNA maturation. The biological basis for the temporal and spatial regulation of Prp43 remains elusive. The Spp382/Ntr1, Sqs1/Pfa1 and Pxr1/Gno1 G-patch proteins bind to and activate the Prp43 DExD/H box-helicase in pre-mRNA splicing (Spp382) and rRNA processing (Sqs1, Pxr1). These Prp43-interacting proteins each contain the G-patch domain, a conserved sequence of ~48 amino acids that includes 6 highly conserved glycine (G) residues. Five annotated G-patch proteins in baker’s yeast (i.e., Spp382, Pxr1, Spp2, Sqs1 and Ylr271) and with the possible exception of the uncharacterized Ylr271 protein, all are associated with ribonucleoprotein (RNP) complexes. Understanding the role of G-patch proteins in modulating the DExD/H box protein Prp43 biological function was the motivation of this thesis. The G-patch domain has been proposed as a protein-protein or a protein-RNA interaction module for RNP proteins. This study found that the three Prp43-associated G-patch domains interact with Prp43 in a yeast 2 hybrid (Y2H) assay but differ in apparent relative affinities. Using a systemic Y2H analysis, I identified the conserved Winged-helix (WH) domain in Prp43 as a major binding site for G-patch motif. Intriguingly, removal of the non-essential N-terminal domain (NTD) of Prp43 (amino acids 2-94), greatly improves G-patch binding, suggesting that the NTD may play a role in modulating enzyme activity by the G-patch effectors. I identify a second site within the Pxr1 that strongly binds Prp43 but, unlike the G-patch, is dispensable for Pxr1 function in vivo. By constructing chimeric proteins, I demonstrated that individual G-patch peptides differ in the ability to reconstitute Spp382 and Pxr1 function in support of pre-mRNA splicing and rRNA biogenesis, respectively. Through amino acid sequence comparisons and selective mutagenesis I identified several residues within the G-patch motif critical for Prp43-stimulated pre-mRNA splicing without greatly altering its ability to bind Prp43. These data lead me to propose that the G-patch motif is not a simple Prp43 binding interface but may contribute more directly to substrate selection or Prp43 enzyme activation in the biologically distinct pre-mRNA splicing and rRNA processing pathways.

La réponse stringente est un processus mis en place lors d'une carence nutritionnelle qui permet l'arrêt coordonné de la croissance. Cette réponse essentielle à la survie des bactéries est très conservée. Elle se caractérise par la production et l'accumulation de guanosine tretra- et pentaphosphate (ppGpp). Le ppGpp, en se fixant sur l'ARN polymérase modifie ses propriétés cinétiques et affecte ainsi de manière globale la transcription de très nombreux gènes. Principalement, l'accumulation de ppGpp inhibe la biosynthèse des ARNs stables (ARNr et ARNt) et en conséquence inhibe la biogenèse des ribosomes. Chez Escherichia coli, le niveau de ppGpp est régulé par les deux enzymes RelA et SpoT. Lors d'une carence en acides aminés, RelA fixée au ribosome détecte le blocage de la machinerie traductionnelle causée par la fixation d'un ARNt déacylé au site A du ribosome, et synthétise du ppGpp. SpoT quant à elle serait capable de détecter et de synthétiser le ppGpp en réponse à d'autres carences nutritionnelles notamment en source de carbone, mais les mécanismes et les signaux détectés sont inconnus. Il a été proposé que la protéine CgtA serait impliquée dans le contrôle de la réponse stringente, en interagissant avec SpoT au niveau des ribosomes. CgtA est une GTPase conservée et essentielle de la famille Obg, mais sa fonction précise est inconnue. Elle a été impliquée à la fois dans la maturation des ribosomes et dans la ségrégation des chromosomes et la division. Le gène cgtA est situé en aval des gènes rplU, rpmA, et yhbE codant respectivement pour les protéines L21 et L27 de la sous-unité 50S du ribosome et pour une protéine intégrale de membrane interne de fonction inconnue. / The stringent response is a physiological process that occurs when bacterial cells encounter nutritional stresses, and allowing coordinated growth arrest. This conserved response is characterized by the accumulation of tetra- and pentaphosphate guanosine (ppGpp). ppGpp bind to RNA polymerase and modifies its kinetic properties, thereby affecting the transcription of many genes. Prinicpaly, ppGpp accumulation inhibits stable RNAs (rRNA and tRNA) biosynthesis, which in consequence inhibits ribosome biogenesis. Escherichia coli contains two enzymes involved in ppGpp metabolism, RelA and SpoT. During amino acid starvation, RelA bound to ribosomes produces ppGpp in response to the presence of uncharged tRNA in the ribosomal A-site. In contrast, SpoT produces ppGpp in response to other types of nutrient limitations, such as carbon starvation, but the detected signals and mechanism involved are still unknown. It has been proposed that the CgtA protein is involved in the stringent response control by interacting with SpoT at the ribosome. CgtA is a conserved and essential small GTPase of the Obg family. CgtA has also been implicated in ribosome maturation, chromosome segregation and division, but its precise function remains unknown. The cgtA gene is located downstream of rplU, rpmA and yhbE genes coding respectively for L21 and L27 proteins of the 50S subunit of the ribosome, and an integral inner membrane protein of unknown function. This genetic proximity with rplU and rpmA genes is highly conserved in bacteria. My thesis work was therefore organized around three questions. First, understanding the role of CgtA in growth control and in the stringent response.

Cell replication and division are central to the proliferation of life, and have implications for normal growth and development as well as disease state. Assembly of a complete picture of the systems which control this process requires identification of individual genetic components, but the identity and complete sequence of events that trigger initiation of cell division, at a point called START in yeast, remain unknown. Here, we evaluated panels of non-essential single gene deletion strains and tested the effects of FDA-approved drugs on cell-cycle progression, using flow cytometry to detect altered DNA content. Previous studies relied mainly on cell size changes to systematically identify genes required for the timely completion of START. This analysis revealed that most gene deletions that altered cell-cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell-cycle control mechanisms. We found that cystathionine-beta-synthase (CBS) advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function which does not require that activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Screening chemical libraries to identify compounds that affect overall cell proliferation is common. However, it is generally not known whether the compounds tested alter the timing of particular cell-cycle transitions. Our approach revealed strong cell-cycle effects of several commonly used pharmaceuticals. We show that the antilipemic gemfibrozil delays initiation of DNA replication, while cells treated with the antidepressant fluoxetine severely delay progression through mitosis. We discovered a strong suppressive interaction between gemfibrozil and fluoxetine. The novel interaction between gemfibrozil and fluoxetine suggests that identifying and combining drugs that show cell-cycle effects might streamline identification of drug combinations with a pronounced impact on cell proliferation. Our studies not only transform our view of START, but also expand the repertoire of genetic and chemical means to modulate the eukaryotic cell cycle.