This work presents the development of a highly sensitive and selective chemical assay for mono(ADP-ribose) residues covalently bound to proteins in vivo. An extensive review of the literature is presented in the introduction of this work. The physiological.functions of mono(ADP-ribosyl)transferase activities associated with certain bacterial toxins (e.g., diphtheria, cholera and pertussis toxins) are well established. However, the roles of endogenous vertebrate transferases are unknown. The elucidation of the roles of these cellular transferases will likely require identification of the physiologically relevant target proteins. Toward this end, it will also be important to identify the types of (ADP-ribose)-protein linkages present in vivo. ADP-ribosylation reactions catalyzed by the different bacterial and vertebrate transferases are specific for different amino acid acceptors in vitro. However, the vertebrate transferases that have been characterized thus far are NAD:arginine mono(ADP-ribosyl)transferases. The work presented here describes the development of a chemical assay for the detection of in vivo modified, ADP-ribosylated proteins containing N-glycosylic linkages to arginine. The assay was applied to the analysis of ADP-ribose residues in adult rat liver. The strategy employed for detection of protein-bound ADP-ribose residues eliminated potential artifacts arising from trapped nucleotides (or their degradation products), since the acid-insoluble material was completely dissolved in a strongly denaturing solution and freed of non-covalently bound nucleotides prior to chemical release from proteins. Thus, the studies presented here demonstrate the unambiguous detection and quantification of protein-bound ADP-ribose residues in adult rat liver. "Arginine-linked" mono(ADP-ribose) residues (31.8 pmol/mg protein) were present in vivo at a level almost 400-fold higher than poly(ADP-ribose). A minor fraction (23%) of the ADP—ribose residues detected were bound via a second more labile linkage with chemical properties very similar to those described previously for carboxlylate esterlinked ADP-ribose. After fractionation of rat liver proteins by gel filtration HPLC, the major peak of "arginine-linked" ADP-ribose residues eluted in the 40-60 kDa region. The later result is consistent with previous suggestions that G-proteins (40-50 kDa) of the adenylate cyclase complex, which are targets for toxin-catalyzed ADP-ribosylation, may also represent target proteins for endogenous transferases.

In these studies, a procedure which allowed the in vivo labeling and detection of poly(ADP-ribose) was combined with nuclear fractionation techniques to analyze the nuclear distribution of ADP-ribose polymers. The results from these studies suggest the occurrence of poly(ADP-ribose) metabolism in two compartments of chromatin; one that is nuclear matrix-associated and one that is not. The biological significance of this compartmentalization is conceptualization in a model. This model postulates that, under some physiological conditions, poly(ADP-ribose) metabolism accomplishes the reversible targeting of specific regions of chromatin to the nuclear matrix domain by modulating DNA-protein and or protein-protein interactions.

La poly(ADP-ribosyl)ation est une modification post-traductionnelle créée par l'ajout successif d'unités ADP-ribose sur une protéine acceptrice pour former un polymère (pADPr) hétérogène branché. La caractérisation biochimique de tous les membres de la famille des PARPs est incomplète mais un constat important peut être dégagé par l'analyse de cette famille élargie : les PARPs présentent une grande diversité de domaines protéiques fonctionnels. Conséquemment, il est logique de croire que cette étonnante diversité sera responsable de fonctions variées dans plusieurs sentiers de signalisation ou événements cellulaires. La poly(ADP-ribosyl)ation, bien qu'étant une modification cruciale impliquée dans la régulation de l'intégrité génomique et la survie cellulaire, ne se limite plus aux seules fonctions nucléaires mais se présente de plus en plus comme un événement pouvant se dérouler dans un contexte physiologique extra-nucléaire. De plus, la liaison noncovalente de plusieurs protéines au pADPr libre ou à d'autres protéines poly(ADP-ribosyl)ées est un phénomène dont nous commençons à mieux mesurer les impacts fonctionnels. Contrairement aux PARPs, lesquelles sont exprimées par une superfamille de gènes apparentés, la majorité de l'activité de dégradation du pADPr est attribuable à l'expression d'un seul gène chez les mammifères : la poly(ADP-ribose) glycohydrolase (PARG). Un volet important de mon projet de recherche visait à identifier des partenaires de la PARG dans le but de reconnaître les sentiers biochimiques qui pourraient inclure une composante de poly(ADP-ribosylation) dans leur régulation et définir des interactions fonctionnellement pertinentes. Dans une optique complémentaire, les protéines associées au pADPr, que ce soit de manière covalente, noncovalente ou en association avec des complexes protéiques, ont été ciblées par des approches protéomiques. Enfin, des études protéomiques ont permis d'aborder l'état de phosphorylation de la PARP-1 et de la PARG

No description available.

Thèse (M.Sc.)--Université Laval, 2003. / Bibliogr.: f. 77-93. Publié aussi en version électronique.

The work presented here included the development of a highly sensitive method to estimate the size and complexity of poly(ADP-ribose). This involved radiolabeling of the precursor pools, purification of polymers using a boronate resin, polymer fractionation according to size by molecular sieve chromatography and analysis of polymer complexity by enzymatic digestion to nucleotides which were quantified by strong anion exchange chromatography.

La poly(ADP-ribose) polymerases consistant en une population hétérogène de polymères formés à partir du NAD. La poly(ADP-ribose) glycohydrolase est responsable de la dégradation du poly(ADP-ribose). Les activités enzymatiques de ces enzymes constituent un système de régulation pour différents sentiers métaboliques. En effet, l'interaction démontrée entre le pADPr et de multiples protéines a permis de confirmer un rôle de modulateur de nombreuses voies de signalisation tel que la réparation de l'ADN, apoptose, cycle cellulaire, surveillance de l'intégrité du génome, transcription et modulation de la chromatine. Ainsi, nous avons formulé l'hypothèse que le pADPr pourrait coordonner la réparation des lésions à l'ADN et la progression du cycle cellulaire avec la signalisation d'événement apoptotiques. Une approche efficace constituerait à identifier et caractériser les protéines (intermédiaires) associées au pADPr selon une logique temporelle. Les diverses actions du pADPr sur les processus biologiques dépendent (dans le cas de la majorité du pADPr, soit celui métabolisé par PARP-1) de la gravité des dommages induits à l'ADN. Par conséquent, il existe probablement des points de seuil faisant basculer les voies de signalisation de la réparation vers la mort cellulaire. Une approche réductionniste, dans ce type de problème, ne peut apporter des réponses satisfaisantes. L'utilisation de la protéomique quantitative semble être une approche plus appropriée. Un volet du travail présenté dans cette thèse visait à identifier des partenaires des PARP-1, PARP-2 et PARG dans le but de reconnaître les sentiers biochimiques qui pourraient inclure une composante de poly(ADP-ribosylation) dans leur régulation et définir des interactions fonctionnellement pertinentes. Par la suite, nous avons établi un réseau dynamique des complexes associés au pADPr en fonction du temps suivant un dommage alkylant induit par un stress génotoxique. Ainsi, certains événements modulés par le pADPr ont été analysés et cartographies. De plus, nous avons caractérisé un rôle novateur du pADPr dans la formation des granules de stress suite à un stress génotoxique. En conséquence, nos résultats ont permis d'édifier les premières bases pour la biologie des systèmes de la poly(ADP-ribosyl)ation en fournissant un répertoire d'interactions protéique exhaustif.

Poly(ADP-ribosyl)ation is a reversible post-translational modification of histones and nuclear proteins rapidly stimulated by DNA damage. Its homeostasis is a dynamic process regulated by the synthesizing enzymes poly(ADP-ribose) polymerases (PARPs) and the degrading enzyme poly(ADP-ribose) glycohydrolase (PARG). PARP-1, the first-discovered and major PARP, has been the focus of many studies aimed at clarifying the biological function of poly(ADP-ribose) (PAR). This abundant nuclear enzyme plays key roles in a variety of cellular processes, including the regulation of chromatin structure, transcription and genomic integrity. Its multifunctionality has made it an attractive and potential target for therapy, as evidenced by the numerous PARP-1 inhibitors currently undergoing clinical trials. The transient nature of PAR, explained by the close coordination between PARP-1 and PARG, has also highlighted the potential of targeting PARG for diseases of inappropriate cell death. A number of obstacles, however, have prevented PARG from being studied as extensively as PARP-1. The extreme sensitivity of PARG to proteases and its insolubility at high concentrations have limited structure-activity relationship analyses and structural studies of PARG, and the unavailability of high-throughput activity assays has stalled the discovery and development of specific and cell permeable PARG inhibitors, subsequently slowing down the validation of PARG as a therapeutic target. The work presented in this dissertation describes in detail strategies devised to overcome these difficulties. First, a novel colorimetric high-throughput assay for PARG was evaluated and its sensitivity and precision were compared to a widely-used radiolabelling assay. Second, several expression and purification systems were constructed in order to obtain high quantities of soluble human PARG protein adequate for in vitrostructural studies. The efficacy of these strategies was demonstrated in structure-activity analyses of PARG which led to the identification of a regulatory segment far removed linearly from the catalytic site of PARG. This region, necessary for catalytic activity, corresponds with a recently identified mitochondrial targeting sequence (MTS) and was thus named the ‘regulatory segment/MTS’ (REG/MTS). Finally, based on structural data obtained, secondary structure predictions were made to provide insight into the molecular composition of the different domains of PARG, whose structures still remain to be determined.

Polymers of ADP-ribose (PAR) are rapidly synthesized by poly(ADPribose) polymerases (PARPs) and rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG) following genotoxic stress. Since PAR metabolism plays an important role in cell fate determination following genotoxic stress, enzymes involved in PAR metabolism potentially represent promising therapeutic targets for modulating diseases of inappropriate cell proliferation or death. PARP-1 has been well validated and several PARP-1 inhibitors are currently being evaluated in clinical trials for cancer and ischemia treatment. In contrast, the biological function of PARG is still poorly understood. Due to low abundance of protein levels in mammalian cells and its unique substrate, PARG potentially represents another attractive target for pathological conditions mentioned above. PARG-Δ2,3 cells derived from homozygous PARG-Δ2,3 mice with targeted disruption of exons 2 and 3 of the PARG gene are used in this dissertation. The nuclear isoform PARG60 in PARG-Δ2,3 cells lacks the putative regulatory domain A compared to the nuclear isoform PARG110 in wild type cells. We report in this dissertation that PARG-Δ2,3 cells accumulate less PAR in spite of more rapid depletion of NAD following treatment with N-methyl- N’- Nitro-N-Nitrosoguanidine (MNNG). The estimation of PARP and PARG activity in intact cells shows increased activity of both enzymes in PARG-Δ2,3 cells following MNNG treatment, indicating the important role of domain A in the regulation of PARG and PARP activity under these conditions. Following MNNG treatment, PARG-Δ2,3 cells show reduced formation of XRCC1 foci, decreased H2AX phosphorylation, decreased DNA break intermediates during repair, and increased cell death. The altered PAR metabolism and defective cellular responses related to DNA repair in PARG-Δ2,3 cells may contribute to increased sensitivity of these cells to MNNG. Studies presented in this dissertation clearly demonstrate the important role of PARG110 in PAR metabolism and cellular responses to genotoxic stress, and thus provide supportive data for the validation of PARG as a promising potential therapeutic target.

Yes / Here, we report the biochemical characterization of the mono-ADP-ribosyltransferase 2,3,7,8-tetrachlorodibenzo-p-dioxin poly-ADP-ribose polymerase (TIPARP/ARTD14/PARP7), which is known to repress aryl hydrocarbon receptor (AHR)-dependent transcription. We found that the nuclear localization of TIPARP was dependent on a short N-terminal sequence and its zinc finger domain. Deletion and in vitro ADP-ribosylation studies identified amino acids 400–657 as the minimum catalytically active region, which retained its ability to mono-ADP-ribosylate AHR. However, the ability of TIPARP to ADP-ribosylate and repress AHR in cells was dependent on both its catalytic activity and zinc finger domain. The catalytic activity of TIPARP was resistant to meta-iodobenzylguanidine but sensitive to iodoacetamide and hydroxylamine, implicating cysteines and acidic side chains as ADP-ribosylated target residues. Mass spectrometry identified multiple ADP-ribosylated peptides in TIPARP and AHR. Electron transfer dissociation analysis of the TIPARP peptide 33ITPLKTCFK41 revealed cysteine 39 as a site for mono-ADP-ribosylation. Mutation of cysteine 39 to alanine resulted in a small, but significant, reduction in TIPARP autoribosylation activity, suggesting that additional amino acid residues are modified, but loss of cysteine 39 did not prevent its ability to repress AHR. Our findings characterize the subcellular localization and mono-ADP-ribosyltransferase activity of TIPARP, identify cysteine as a mono-ADP-ribosylated residue targeted by this enzyme, and confirm the TIPARP-dependent mono-ADP-ribosylation of other protein targets, such as AHR. / This work was supported by Canadian Institutes of Health Research (CIHR) operating grants [MOP-494265 and MOP-125919]; CIHR New Investigator Award; an Early Researcher Award from the Ontario Ministry of Innovation [ER10-07-028]; the Johan Throne Holst Foundation; Novo Nordic Foundation; and the Norwegian Cancer Society to J.M. This work was also funded by grants from the Johan Throne Holst Foundation; and the Novo Nordic Foundation to H.I.N.