Poly(ADP)ribose (PAR) metabolism is essential to many cellular functions, including the maintenance of genomic integrity, the regulation of cell death mechanisms, as well as the regulation of gene expression. Recent work has uncovered many new players in the expanding effort to understand PAR metabolism and its cellular impact. PARP-1, the prototypical poly(ADP)ribose polymerase, was the first to be discovered, and has since been shown to be vital in the cellular response to DNA damage. Indeed, one report demonstrating that PARP-1 activation is required for apoptosis-inducing factor (AIF) release from mitochondria uncovered a novel link between DNA damage and signaling for cell death. The events following PARP activation, leading to signaling for AIF release, however, are still poorly understood. Based on our observations, we have developed a model to explain the nuclear/mitochondrial crosstalk that occurs following PARP activation. The work presented here answers several important questions regarding the relationship between ADP-ribose metabolism and mitochondria, including the role of PAR in signaling for the release of AIF, the presence of ADP-ribose metabolism protein members in mitochondria, and mitochondrial transcriptional effects following PARP activation. This work presents several novel findings, including the first report of a mitochondrial matrix isoform of poly(ADP-ribose) glycohydrolase (PARG) as well as direct evidence of mitochondria-associated PARP activity. Furthermore, it provides evidence for a novel effect of PARP-1 activation, in the specific transcriptional upregulation of the mitochondrial gene, NADH dehydrogenase, subunit 1 (ND1). Our data is consistent with the hypothesis that uncontrolled PARP activity results in energy metabolism dysfunction and cell death. Furthermore, it supports a model in which PARP activity is required for normal transcriptional responses in mitochondria following DNA damage. In total, this report adds to the body of work outlining the roles of PARP following DNA damage recognition and activation, demonstrating that ADP-ribose metabolism plays an important role in cell death regulation by both direct and indirect means.

Thèse (M.Sc.)--Université Laval, 2003. / Bibliogr.: f. 89-104. Publié aussi en version électronique.

The ADP-ribose elongation catalyzed by poly(ADP-ribose) polymerase (PARP) [EC 2.2.2.30] has been partially characterized utilizing mono (ADP-ribosyl)ated polyamines. Arginine methyl ester (AME)-(ADP-ribose) and agmatine (AGMT)-(ADP-ribose) were synthesized enzymatically with a eukarytic mono(ADP-ribosyl) transferase and cholera toxin, respectively.

La poly(ADP-ribosyl)ation est une modification post-traductionnelle qui est stimulée en réponse à des dommages à l'ADN. Les poly(ADP-ribose) polymerases (PARPs) synthétisent des polymères branchés de poly(ADP-ribose) (PAR) qui peuvent se lier de manière covalente et non-covalente à des protéines jouant ainsi un rôle dans des processus tels que la progression du cycle cellulaire, la réparation de l'ADN, la stabilité de l'intégrité génomique et l'apoptose. Le cycle de dégradation du PAR induit aussi une variation des réserves en nucleotides comme le NAD+, l'ATP et l'AMP, influençant les voies énergétiques des cellules. Les techniques de « Matrix Assisted Laser Desorption Ionisation » (MALDI) et de quantification de nucleotides par colorimétrie, fluorométrie et HPLC ont été utilisées pour déterminer de nouveaux partenaires protéiques interagissant avec le polymère d'ADPr, par l'entremise d'interaction directe, covalente et non-covalente, ou par l'entremise d'interaction indirecte, via le cycle de synthèse/dégradation du PAR qui induit d'importants changements métaboliques cellulaires.

No description available.

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition to its principal virulent factors, Toxin A and Toxin B, some C. difficile strains produce a binary toxin (CDT) composed of two subunits namely CDTa and CDTb that are produced and secreted from the cell as two separate polypeptides. Once in the gut, these fragments have the potential to combine to form a potent cytotoxin whose role in the pathogenesis of CDI is presently unclear. This thesis is a step towards understanding structural and functional aspects of the binary toxin produced by C. difficile. The first half of this thesis (chapter I and II) provides a brief introduction to the method of structure determination of proteins molecules, i. e. X-ray crystallography and a detailed overview of C. difficile and the three known toxins from C. difficile namely – Toxin A, Toxin B and the binary toxin. Chapter II further focuses on C. difficile binary toxin and other related toxins. These toxins, known as the ADP-ribosylating toxins (ADPRTs) form a big family of potent toxins which includes Cholera, Pertussis and Diphtheria toxins and are capable of transferring the ADP-ribose part of NAD/NADPH to a varity of substrates in the target cell which ultimately results in cell death. The second half of the thesis comprises of experimental procedures that were carried out during the course of this study and their results. Cloning and expression methods for recombinant CDTa and CDTb in bacterial system followed by their purification are described with the abnormal behaviour exhibited by CDTb (chapter III). We show for the first time that purified CDTa and CDTb can combine to form an active CDT which is cytotoxic to Vero cells (Chapter IV). The purification processes described yielded milligram quantities of binary toxin fragments of high purity that led to the successful crystallisation of the proteins (chapter IV) for further functional and structural studies. High resolution crystal structures of CDTa in its native form (at pH 4.0, 8.5 and 9.0) and in complex with the ADP ribose donors -NAD and NADPH (at pH 9.0) have been determined (chapter V). The crystal structures of the native protein show ‘pronounced conformational flexibility’ confined to the active site region of the protein and ‘enhanced’ disorder at low pH while the complex structures highlight significant differences in ‘ligand specificity’ compared with the enzymatic subunit of a close homologue, Clostridium perfringens Iota toxin (Ia). These structural data provide the first detailed information on protein-donor substrate complex stabilisation in CDTa which may have implications in understanding CDT recognition. Crystallisation of CDTb yielded preliminary crystals. The optimisation of these crystallisation conditions is underway. The thesis concludes with some thoughts and discussion on future directions of this research.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

RNA interference (RNAi) is an evolutionarily conserved process that regulates gene expression. Host cells and viruses interact in many ways, including through miRNAs and RNAi. Viral miRNAs are encoded when viruses, specially including the the polyoma and herpes families, are transcribed in the nucleus. Some viral miRNAs function to regulate host or viral gene expression. Most viral miRNAs’ functions are not known, however, in great detail. A miRNA can be encoded late during infection, as it is by SV40, a model polyomavirus. This downregulates early viral gene expression by directing mRNA RISC-mediated cleavage. As more polyomaviruses are discovered that are associated with human disease, it becomes more important to understand their function and to uncover whether these emerging viruses encode miRNAs. The work presented here shows the discovery of several viral miRNAs in human polyomaviruses—JCV, BKV, and MCV. In addition, I found that viral miRNAs have the evolutionarily conserved function of negatively regulating viral early gene transcripts at a late stage in the infection. During viral replication, viruses utilize the miRNA components of RNAi. However, in invertebrate organisms RNAi also actively defends against viral infection. It is still being debated, though, whether RNAi plays an antiviral role in mammalian cells. Should it be true that RNAi is an antiviral response in mammalian cells, then what is predicted by such a scenario is inconsistent with my studies. I have found that RNAi is strongly inhibited in the early stages after viral infection. Studies with a chemical mimic of viral infection (poly I:C) imply that the innate cellular immune response is responsible for this inhibition. I investigated the molecular changes, in response to viral infection, (e.g. poly ADP-ribosylation of Ago2) in the RNA-induced silencing complex (RISC). I determined that the inhibition of RNAi is brought about by components of the innate response. Completion of this study details a previously unknown “cross talk” between RNAi and the host innate immune response in mammalian cells. Furthermore, I found mir-17 family attenuates a subclass of interferon-stimulated genes. An understanding of viral miRNA and RNAi offers a clue as to we can use molecular intervention for viral infections. / text

Thesis (Ph. D.)--Cornell University, May, 2007. / Vita. Includes bibliographical references (leaves 79-89).