Poly(ADP-ribosyl)ation, a covalent modification of proteins catalyzed by poly(ADP-ribose) polymerases (PARPs), plays a crucial role in regulating DNA repair, DNA replication, and cell death. Poly(ADP-ribose) Polymerase-1 (PARP-1) is a nuclear zinc-finger DNA-binding protein that is the most extensively studied member of the PARP family. The activation of PARP-1 depends on the N-terminal DNA-binding domain, which consists of two unusually long zinc finger-like motifs (termed FI and FII) of the form CX₂CX₂₈/₃₀HX₂C and a newly discovered zinc-ribbon motif (FIII). Though zinc is indispensible for PARP-1 activity, the metal binding affinities of the unusual zinc fingers of PARP-1 is not yet known. In this dissertation, the second zinc finger of PARP-1 was used as a model peptide to study the binding properties of several divalent metal ions (Co²⁺, Cd²⁺, Zn²⁺, and Pb²⁺). Metal-induced protein folding was investigated by circular dichroism, and the effects of the metal ions on PARP-1 activity were investigated by poly(ADP-ribosyl)ation activity assays. This study represents the first detailed biochemical characterization of the PARP zinc fingers. The functional role of each zinc finger in DNA damage recognition is critical for understanding how PARP-1 is involved in DNA repair. Thus, we constructed a series of PARP-1 zinc finger variant proteins and investigated their DNA binding properties and their effects on PARP activity. Using a combination of southwestern blotting and activity assays, we demonstrated that FII is more important for DNA binding, while FI and FIII seem to facilitate PARP activity. The DNA sequence-independent binding properties of PARP-1 were further characterized using DNA probes bearing defined secondary structures. Together, our results indicate that the zinc fingers help position the enzyme at specific DNA damage sites, and also help to activate the catalytic domain upon DNA binding. PARP-1 is involved in caspase-independent apoptosis, and the translocation of apoptosis inducing factor (AIF) out of the mitochondrial matrix has been shown to require PARP-1 activity. However, it is not readily apparent how the catalytic activity of PARP-1 (a nuclear protein) triggers the release of AIF from the mitochondrial matrix. In an attempt to understand the relationship between PARP-1 activity and caspase-independent apoptosis, we demonstrate here that AIF is an in vitro protein substrate for PARP-1. The possible implications of this finding will be discussed. / text

Le traumatisme crânien (TC) représente un des problèmes majeurs de santé publique, pour lequel à l’heure actuelle il n’existe aucun traitement. Le TC induit une neuro-inflammation délétère qui pourrait contribuer à l’apparition des lésions de la substance blanche (SB). Ces dernières sont à l’origine de lourdes conséquences neurologiques chez les patients victimes de TC. Néanmoins, très peu d’études se sont intéressées à ces lésions bien que plus sévères que les lésions de la substance grise. Ainsi une meilleure connaissance de leur évolution et des causes devient indispensable. L’hyperactivation de la poly(ADP ribose)polymérase (PARP) joue un rôle délétère dans les conséquences post-traumatiques, notamment sur la neuro-inflammation. Ainsi son inhibition pourrait être bénéfique le développement des lésions de la SB. Dans ce contexte, l’objectif de notre travail a été d’évaluer le rôle de la PARP dans les lésions de la SB dans un modèle expérimental de TC induit par impact cortical contrôlé chez la souris. Dans une première partie, nous avons étudié l’évolution de la démyélinisation dans le corps calleux, une structure riche en SB, entre 6 heures et 3 mois post-TC. Parallèlement, les évolutions de la lésion cérébrale, des déficits sensorimoteurs, de la neuro-inflammation et de l’œdème cérébral ont été étudiées. Le TC induit (1) une démyélinisation dès 7 jours et au moins jusqu’à 3 mois post-TC, précédée par (2) une lésion cérébrale entre 24 et 72 heures suivie par une cicatrisation, (3) une neuro-inflammation entre 6 heures et 7 jours et (4) un œdème cérébral entre 6 et 72 heures post-TC. De plus, le TC induit des déficits sensorimoteurs à 6 heures et 3 mois. Ces résultats montrent que ce modèle est adapté pour étudier les lésions de la SB post-TC, et que la neuro-inflammation et l’œdème cérébral pourrait être impliqués dans la démyélinisation. Dans une deuxième partie, nous avons étudié le rôle de la PARP dans les lésions de la SB suite à TC à l’aide de souris knockout (KO) et wild-type (WT) pour le gène de la PARP. Nous avons mis en évidence que les souris KO ne présentent pas de démyélinisation bilatérale du corps calleux après un TC par rapport aux souris WT à 7 jours post-TC, démontrant pour la première fois l’implication de cette enzyme dans les lésions de la SB consécutives à un TC. De plus, nous avons constaté que les souris KO non traumatisées présentent une diminution de myélinisation comparativement aux souris WT non traumatisées, suggérant un rôle de la PARP dans le processus physiologique de la myélinisation.En conclusion, l’ensemble de ce travail expérimental a permis (1) une meilleure caractérisation de la démyélinisation post-TC et des mécanismes potentiellement impliqués dans cette dernière, et (2) de démontrer pour la première fois le rôle délétère de la PARP dans la démyélinisation induite par un TC. Nos travaux suggèrent le potentiel de l’inhibition de la PARP comme stratégie thérapeutique pour la prévention des lésions de la SB post-traumatiques. / Traumatic brain injury (TBI) is a leading cause of death and disability for which there is no neuroprotective treatment up to date. It results in neuroinflammation that may participate in lasting motor and cognitive impairments accompanied by changes in white matter (WM) tracts. WM lesions, evidenced by demyelination, are associated with neurological disorders and in clinical studies are common consequences in patients with chronic TBI. Several studies suggest a contribution of an overactivation of the poly(ADP-ribose) polymerase (PARP) to the neuroinflammatory response which may lead to demyelination. The first part of this study was dedicated to a detailed in vivo assessment of the evolution over time of neurological disorders, cerebral lesion and edema, neuroinflammation and white matter injury induced by controlled cortical impact (CCI) between 6 hours and 12 weeks post-TBI. Notably in the corpus callosum, a significant demyelination starting at 7 days appeared to be a major consequence to post-traumatic neuroinflammation associated with motor dysfunctions. The second part of this study was dedicated to the evaluation of PARP’s role in WM lesions post-TBI, using PARP knockout (KO) mice. Our main findings reveal a diminished demyelination in the corpus callosum of TBI PARP KO as opposed to TBI PARP wildtype specimens. Hence, these data suggest for the first time PARP’s deleterious role in post-traumatic demyelination. In conclusion, taken together these data give an overall view of motor/sensorimotor deficits, neuroinflammation and demyelination in a CCI model of TBI that could help to validate pharmacological strategy for preventing post-traumatic WM injury. Notably, PARP’s inhibition seems to be a valid candidate as this enzyme participates in the establishment of a demyelinating process.

Les accidents vasculaires cérébraux (AVC) constituent la 3e cause de mortalité dans les pays industrialisés, et sont à 80% de type ischémique (AVCi). A l’heure actuelle, le seul traitement disponible est l’activateur tissulaire du plasminogène recombinant (rt-PA), dont l’utilisation est très limitée, en raison d’une fenêtre thérapeutique étroite et de l’augmentation du risque de transformations hémorragiques (TH). Après un AVCi, les cliniciens sont confrontés, entre autres, à 3 objectifs d’ordre vasculaire: (1) reperfuser les tissus ischémiés, (2) éviter les TH, ainsi que (3) les ré-occlusions précoces ou tardives. Les travaux du laboratoire ont précédemment établi qu’après une ischémie cérébrale (IC), l’hyperactivation de la poly(ADP-ribose)polymérase (PARP), une enzyme nucléaire, est (1) neurotoxique et (2) contribue aux TH spontanées ou induites par le rt-PA. Par ailleurs, des études suggèrent que les inhibiteurs de PARP pourraient également réduire les phénomènes de ré-occlusion, en inhibant l’activation/agrégation plaquettaires, et ceci via 2 mécanismes : (1) « PARP-indépendant », lié à une analogie structurale de certains inhibiteurs de PARP avec des agonistes plaquettaires, comme l’ADP, et (2) « PARP-dépendant », lié à leur effet anti-inflammatoire. Cependant, à l’heure actuelle, il n’existe aucune donnée dans l’IC. Dans ce contexte, ce travail a consisté à évaluer les effets de plusieurs inhibiteurs de PARP sur l’activation et l’agrégation plaquettaire. Il nous est notamment apparu nécessaire de rechercher si la réduction des TH par les inhibiteurs de PARP pourrait être liée, au moins en partie, à une activité pro-agrégante, qui compromettrait leur association avec le rt-PA. A l’inverse, une activité anti-agrégante, bien que favorisant les hémorragies, pourrait améliorer la reperfusion ou diminuer les risques de ré-occlusion. Dans la 1ère partie, nos résultats montrent in vitro que deux inhibiteurs de PARP (PJ34 et minocycline) sont anti-agrégants plaquettaires, et que cet effet serait « PARP-indépendant », puisque deux autres inhibiteurs de PARP, le 3-aminobenzamide et l’INO-1001, n’ont pas modifié l’agrégation. De plus, sur du sang humain, mais pas murin, le PJ34 exerce un effet anti-agrégant, qui pourrait être lié à un antagonisme du récepteur à l’ADP, P2Y12. La 2nde partie a été réalisé sur des modèles in vivo chez la souris. L’utilisation de 3 tests d’exploration des fonctions plaquettaires (temps de saignement, modèles de thromboembolie pulmonaire et de thrombose carotidienne par le FeCl3) a mis en évidence l’absence d’effet du PJ34 et de la minocycline sur les fonctions plaquettaires, et notamment, pas d’effet pro-agrégant pouvant expliquer la réduction des TH. Dans un modèle de thrombose de l’artère cérébrale moyenne par le FeCl3, le PJ34 n’entrave pas la thrombolyse par le rt-PA, mais au contraire, pourrait tendre à l’améliorer. Parallèlement, dans un modèle d’IC chez la souris, nos travaux ont mis en évidence une augmentation cérébrale de l’adhésion des plaquettes et de l’expression d’ICAM-1. La suite de cette étude sera d’étudier si les inhibiteurs de PARP, en protégeant la paroi vasculaire, pourraient réduire les phénomènes de ré-occlusion. L’ensemble de ce travail s’inscrit dans une thématique plus globale de notre laboratoire qui vise à identifier l’intérêt d’associer un inhibiteur de PARP au rt-PA pour une meilleure prise en charge de la thrombolyse post-AVCi. / Stroke is the 3rd leading cause of death in industrialized countries and 80% are ischemic. The recombinant tissue-plasminogen activator (rt-PA) is currently the only available treatment but its use remains very limited due to a narrow therapeutic window and an increased risk of hemorrhagic transformation (HT). After ischemic stroke, the key vascular objectives of clinicians are : (1) to reperfuse ischemic tissue, (2) to avoid both HT and (3) early or late reocclusions. Our laboratory previously established that after cerebral ischemia (CI), the overactivation of poly(ADP-ribose)polymerase (PARP), a nuclear enzyme, is (1) neurotoxic and (2) contributes to spontaneous or rt-PA-induced HT. Moreover, studies suggest that PARP inhibitors could also reduce the risk of reocclusion by inhibiting platelet activation/aggregation via two mechanisms : (1) one is "PARP-independent" and linked to a structural analogy of certain PARP inhibitors with platelet agonists such as ADP, and (2) the second one is "PARP-dependent" and due to their anti-inflammatory effect. However, so far, there is no data in CI.In this context, the aim of this work was to evaluate the effects of several PARP inhibitors on platelet activation and aggregation. In particular, it appeared necessary to examine whether the reduction of HT by PARP inhibitors could be related, at least in part, to a pro-aggregatory activity, which would then compromise their association with rt-PA. By contrast, an anti-aggregatory activity could improve reperfusion or reduce the risk of reocclusion, although it would also contribute to hemorrhage. In the 1st part, our results show that, in vitro, two PARP inhibitors (PJ34 and minocycline) are antiplatelet agents and that this effect is "PARP-independent" since two other PARP inhibitors, 3-aminobenzamide and INO-1001 did not alter the aggregation. Moreover, in human blood but not in murine one, PJ34 exerts an anti-aggregatory effect which may be related to the antagonism of the ADP receptor P2Y12. The 2nd part was performed on in vivo models in mice. The use of three tests of platelet function exploration (bleeding time and models of pulmonary thromboembolism and FeCl3-induced carotid thrombosis) showed no effect of minocycline and PJ34 on platelet function and in particular, no pro-aggregatory effect which may explain the reduction of HT. In a thrombosis model of the middle cerebral artery by FeCl3, PJ34 does not impede the thrombolysis induced by rt-PA, but even tends to improve it. Meanwhile, in a CI model in mice, our work shows an increase of platelet adhesion and ICAM-1 expression in the brain. The next step will be to investigate whether PARP inhibitors could reduce reocclusions by protecting the vascular wall. All this work is part of a broader topic of our laboratory aims to identify the interest of combining a PARP inhibitor with rt-PA for a better management of post-ischemic thrombolysis.

No description available.

Previous studies have shown extensive cellular damage can activate poly(ADP-ribose) polymerase-1 (PARP-1) and cause a rapid decrease in the levels of NAD+ and ATP, thereby preventing apoptosis and promoting necrosis and inflammation. The purpose of this study was to extend previous observations that inhibitors of PARP-1 could alter acetaminophen and carbon tetrachloride-induced hepatotoxicity. Bromobenzene (BB) a glutathione dependent hepatotoxicant was tested. Groups of male mice were treated with a single dosage of 112mg/kg (0.075 ml/kg) BB by the intraperitoneal (ip) route. All animals were maintained in a controlled environment and provided food and water ad libitum. This dosage of BB resulted in hepatotoxicity as measured by an increase in serum alanine transferase (ALT). BB treatment resulted in a 5-fold increase in ALT. Moderate hepatotoxicity was detected with this treatment regime. Subsequently, another group of mice were treated with three treatments of nicotinamide at 0.5, 1 and 2 hours following BB treatment. Serum ALT elevations were reduced by 90% at 24 hours following BB and nicotinamide treatments. BB-induced liver pathology was also blocked by nicotinamide. Mortality among BB treated animals was also significantly reduced by nicotinamide treatment. Mortality among mice treated with BB and nicotinamide was near control. The model was verified with a more potent and specific inhibitor, Phen. BB treatment was keep at the same level as in the previous study, and Phen was administered concomitantly. Serum ALT elevations were reduced by 75%. Phen also blocked BB-induced liver pathology. Mortality among mice treated with BB and Phen was reduced 75%. PARP-1 inhibitors appear to alter chemical-induced hepatotoxicity that has either a glutathione dependent or independent mechanism.

The specific interactions of macromolecules along with the activity of enzymes are central to all aspects of biology. It is well recognized that when the relative concentration or activity of macromolecules is perturbed, it can lead to human diseases. Thus, the development of simple methods for the detection of macromolecules and the activity of enzymes in complex environments is important for understanding biology. Moreover, the development of methods for measuring interactions allows for the testing of inhibitors that can be used as tools or drugs for improving human health. Towards this goal, a promising new method has been developed, which is the focus of this thesis, called split-protein reassembly or protein fragment complementation. In this method, a protein reporter, such as the green fluorescent protein or firefly luciferase, is dissected into two fragments, which are attached to designed adaptor proteins. The designed split-protein systems only produce a measurable signal, either fluorescence or luminescence, when a specific macromolecular interaction or activity is present. In this thesis, I have extended previous research on the direct detection of DNA using split-protein sensors utilizing a red fluorescent protein, dsRED from Discosoma that allows for multiplexed DNA detection. I have designed a new split-luciferase based sensor for detection of poly (ADP-ribose) or PAR, which plays a key role in the response to DNA damage and have applied it for monitoring the activity of poly (ADP-ribose) glycohydrolase that controls PAR levels in the cell. Furthermore, I have significantly expanded upon a three-hybrid split-luciferase system for identifying protein kinase inhibitors. I have designed and tested two orthogonal peptide based chemical inducers of dimerization based on BAD and p53mt conjugates. I have studied these chemically induced dimerization systems in detail in order to begin to provide a theoretical basis for the observed experimental results. Finally, in a less related area, I have developed methods for producing water soluble semiconductor nanoparticles called Quantum Dots (QDs), with potential application in biological imaging. I have developed methods for functionalizing the QDs with orthogonal peptides, which can be potentially used for the assembly of high affinity non-covalent QD targeted proteins.

Thèse (Ph. D.)--Université Laval, 2008. / Bibliogr.: f. 250-258. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Pharmacology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

A method for assaying poly (ADP-ribose) polymerase (PADPRP) activity in lymphocytes of systemic lupus erythematosus (SLE) patients has been developed. Using this method, PADPRP activity has been studied in lymphocytes from 15 patients and 13 controls. The mean activity in SLE lymphocytes was significantly lower than that in controls and 60% of the SLE patients demonstrated activities below the minimum of the control population. Possible mechanisms for this altered metabolism were investigated. The Km app of PADPRP for NAD; size distribution, branch frequency, and rates of turnover of polymers; competition for substrate; and number of PADPRP molecules were studied. The data demonstrated that SLE lymphocytes have a decreased synthetic capacity rather than alterations in the substrate or in turnover of the product.

No description available.