Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear protein that catalyzes the cleavage of NAD⁺ into nicotinamide and ADP-ribose moiety, the latter of which may be covalently attached as a branched polymer of poly(ADP-ribose) to PARP-1 itself (automodification) or to other nuclear acceptor proteins (transmodification). PARP-1 plays pivotal roles in many fundamental biological processes, including DNA repair, gene expression, cell death and cell cycle regulation. The multiple functions of PARP-1 in various cellular events correlate well to its roles in carcinogenesis, inflammatory response, neural function, and aging. PARP-1 has a modular organization comprising six independent domains (domain A-F). Each domain has its own characteristic function in PARP-1 enzymatic catalysis. In this dissertation, the solution structure of domain C was determined by multi-dimensional NMR spectroscopy. To complement the structural results, the requirement of domain C for PARP-1 catalysis was demonstrated using activity assays. This structure-function relationship study will help to unveil the mechanism of the PARP-1 reaction, and should provide valuable information for the design of more potent and selective PARP-1 inhibitors. The determination of poly(ADP-ribosyl)ation sites is critical for understanding the biological roles of this modification. However, the identification of poly(ADPribosyl)ation sites has countered some daunting technical limitations due to the difficulties resulting from the heterogenous nature of this modification. In this dissertation, a methodology based on mass spectrometry is developed and used to identify ADP-ribosylation sites within the automodification domain (domain D) of PARP-1. Using this method, we were able to unambiguously localize three ADPribosylation sites on domain D. This method can be readily applied to study the transmodification of other substrates as well as PARP-1 automodification. As many as seventeen PARP homologues exist in the human proteome. The functional redundancy of the multiple PARP proteins has complicated the analysis of mammalian PARP-1 function in vivo. We have probed the biological roles of PARP-1 using an artificial PARP-1 pathway in yeast, an organism lacking the endogenous PARP-1. Our data suggest the heterologously expressed human PARP-1 in yeast retains some similar functions as it does in mammalian cells. Furthermore, a new function of PARP-1 in ribosome biogenesis was proposed. / text

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Adenosine diphosphate (ADP) ribosylation is a post-translational modification dependent on the transfer of ADPr units from nicotinamide adenine dinucleotide (NAD+) on to a plethora of biomolecules (i.e., proteins, DNA, RNA, etc.) in response to physiological stressors (i.e., nutrient deprivation, oxidative stress, DNA strand breaks). Poly-ADP-ribosylation (PARylation) is primarily mediated by the family of poly(ADP-ribose) polymerases (PARPs) and enzymatically degraded (dePARylation) by hydrolases such as poly(ADP-ribose) glycohydrolase (PARG). This thesis characterizes the role of poly(ADP-ribose) polymerase 1 (PARP1) and PARG in the skeletal muscle of healthy mature mice under normal physiological conditions. Specifically, we validate the deletion of Parp1 and Parg in inducible skeletal muscle-specific KO mouse models followed by performing general phenotyping of both male and female mice. The thesis concludes that under normal physiological conditions the activity of Parp1 or Parg in (de)PARylation is dispensable for maintaining skeletal muscle mass, function, and homeostasis in healthy mature mice.

Introdução: A sepse é causada por uma resposta desregulada a uma infecção cujo tratamento é de suporte, inexistindo alternativas imunomoduladoras. Linfócitos T reguladores são responsáveis por limitar a inflamação, mas podem causar imunossupressão e os Th17 são pró-inflamatórios e responsáveis pela imunidade de mucosas; ambos apresentam-se elevados nos pacientes com sepse. A PARP é uma enzima sensor de dano ao DNA que é continuamente ativada na sepse, sendo importante também na diferenciação¸ dos linfócitos T reguladores e como coativador de NF-kB. Neste estudo, avaliamos se o tratamento com inibidor de PARP é capaz de manter os linfócitos Th17 e T reguladores próximos aos valores basais, impedindo que ocorra a resposta exacerbada causada por estas células e servindo, portanto, como opção de tratamento imunomodulador. Métodos: Camundongos machos da linhagem C57Bl/6 com 7 semanas de idade e pesando entre 20-25 gramas foram submetidos à ligadura e punção cecal e receberam tratamento com olaparibe (10mg/Kg) após 30 minutos e após 8 horas da cirurgia. Baço, timo e sangue foram coletados e utilizados para análise das populações de linfócitos T reguladores e Th17, citocinas e miRNAs. Resultados: O modelo de ligadura e punção cecal foi capaz de mimetizar a linfopenia encontrada em pacientes e o aumento de linfócitos T reguladores e Th17. O tratamento com olaparibe reduziu os linfócitos T reguladores no baço tanto em porcentagem quanto em quantidade de células. Tanto o nível de IL-10 quanto a expressão do miRNA 146a-5p caíram em ambos os grupos CLP, sugerindo menor atividade supressora destes linfócitos. No sangue houve aumento dos linfócitos T reguladores, mas apenas o grupo não tratado apresentou alta de IL-10, sugerindo que o tratamento conteve o perfil supressor. No timo o tratamento parece agir por uma forma diferente; embora ocorra aumento dos linfócitos T reguladores, o grupo tratado teve aumento da expressão do miRNA 17a-5p, que reduz a atividade supressora desses linfócitos, mostrando que as células produzidas tem sua atividade supressora alterada, o que é corroborado pelo não aumento de IL-10 nesse grupo. Os linfócitos Th17, que são pró-inflamatórios, foram controlados com o tratamento no baço e no sangue. Isso possivelmente ocorreu pela ação da PARP que impediu o aumento de citocinas como IL-1beta, IL-6, TNF-alfa, IL-17A, INF-y, que estavam elevadas apenas no grupo não tratado. Além disso, a relação entre linfócitos Th17 e T reguladores foi controlada, sugerindo melhora no desfecho clínico. Conclusões: O tratamento com olaparibe se mostrou eficiente em reduzir as respostas inflamatória (causada pelo Th17) e supressora (causada pelo Treg) neste modelo, talvez pela alteração de citocinas e da expressão dos miRNA 17a-5p e 146a-5p / Introduction: Sepsis is caused by a dysregulated response to an infection whose treatment is supportive, and there are no immunomodulatory alternatives. Regulatory T lymphocytes are responsible for limiting inflammation but may cause immunosuppression and Th17 are proinflammatory and responsible for mucosal immunity; both are elevated in patients with sepsis. PARP is a DNAdamaging enzyme that is continuously activated in sepsis, also important in the di_erentiation of regulatory T lymphocytes and as a cofactor of NF-kB. In this study, we evaluated whether treatment with PARP inhibitor is able to keep the T regulatory and Th17 lymphocytes close to the baseline values, preventing the exacerbated response caused by these cells and therefore serving as an option for immunomodulatory treatment. Methods: C57Bl male mice at 7 weeks of age weighing between 20-25 grams were submited at cecal binding and puncture and received treatment with olaparib (10mg/kg) after 30 minutes and after 8 hours of the surgery. Spleen, thymus and blood cells have been used for analysis of T regulatory and Th17 lymphocytes populations, cytokines and miRNA. Results: The cecal ligation and puncture model was able to mimic the lymphopenia found in patients and the increase of T regulatory and Th17 lymphocytes. Treatment with olaparib reduced the T regulatory lymphocytes in the spleen in both percentage and number of cells. Both the IL-10 level and the 146a-5p miRNA expression fell in both CLP groups, suggesting lower suppressor activity of these lymphocytes. In the blood there was an increase in the T regulatory lymphocytes, but only the untreated group showed high IL-10, suggesting that the treatment contained the suppressor profile. In the thymus the treatment seems to act in a di_erent way; although there is an increase in the T regulatory lymphocytes, the treated group had increased expression of 17a-5p miRNA, which reduces the suppressive activity of these lymphocytes, showing that the cells produced have their supressor activity altered, which is corroborated by the non-increase of IL- 10 in this group. Th17 lymphocytes, which are proinflammatory, were controlled with treatment in the spleen and blood. This was possibly due to the action of PARP which prevented the increase of cytokines such as IL-1beta, IL-6, TNF-alpha, IL-17A, INF-y which were raised only in the group not treated. In addition, the ratio between Th17 and T regulatory lymphocytes was controlled, suggesting improvement in clinical outcome. Conclusions: The treatment with olaparib was e_cient in reducing inflammatory responses (caused by Th17) and suppressor (caused by Treg) in this model, perhaps due to the alteration of cytokines and the expression of miRNAs 17a-5p and 146a-5p

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

Cardiovascular diseases are one of the leading causes of mortality. A common denominator across most of the cardiovascular diseases like diabetic cardiomyopathy, hypertrophic cardiomyopathy, myocardial infarction and dilated cardiomyopathy is the pathological remodelling of heart leading to fibrosis. Cardiac fibrosis is characterized by the excessive production and deposition of extracellular matrix components due to unwarranted proliferation of fibroblasts. Under normal conditions, following cardiac remodelling, my fibroblasts undergo programmed cell death. However, this does not happen under pathological conditions ultimately leading to fibrosis. Although the molecular events and signalling pathways that contribute to the development of cardiac fibrosis is well established, there are limited studies which try to understand the mechanisms by which fibroblasts persist and resist programmed cell death. Here we demonstrate that SIRT6, one of the members of sirtuin family of histone deacetylases, plays an important role in regulating my fibroblast cell death. When we analysed the mice hearts and isolated fibroblasts deficient in SIRT6, we observed increased expression of my fibroblast markers, suggesting that SIRT6 deficient hearts might have a high proportion of resident my fibroblasts. Also, when SIRT6 deficient fibroblasts were subjected to genotoxic stress, they showed reduced cell death and impaired mitochondrial to nuclear AIF translocation as compared to WT controls. An important regulator of AIF mediated cell death is the protein PARP-1. When we checked the expression levels of this protein under SIRT6 deficient conditions, it was found to be low. PARP-1 was also found to degrade faster under SIRT6 deficient conditions. Further qPCR analysis revealed that the transcript levels of PARP-1 were unaffected by SIRT6 suggesting that the regulation might not be at the transcriptional level. When we studied the acetylation of PARP-1 under SIRT6 deficient conditions we found the protein to be hypo-acetylated indicating a more complex mechanism of regulation.

No description available.

L’ostéonécrose (ON) et les fractures (FR) sont des complications qui prennent de plus en plus place dans le traitement pédiatrique de la leucémie aiguë lymphoblastique (LAL). L’ON peut être causée par différents facteurs, dont principalement l’utilisation de glucocorticoïdes. Les glucocorticoïdes sont administrés lors du traitement de la leucémie dans le but d’initier l’apoptose des cellules malignes tout en ayant un effet anti-inflammatoire. Cependant, l’utilisation de ces corticostéroïdes comprend des effets secondaires sérieux, notamment le développement d’ostéonécrose. Des variantes génétiques peuvent mettre certains patients plus à risque que d’autres. Plusieurs gènes ont déjà été signalés comme régulés par les actions glucocorticoïdes (GC). Les variations génétiques présentes dans les régions régulatrices de ces gènes peuvent affecter leur fonctionnement normal et, en fin de compte, de déterminer un risque accru de développer l’ON associé au traitement contre la leucémie. Pour cette raison, plusieurs polymorphismes ont été identifiés et étudiés dans la cohorte QcALL de Ste-Justine, concernant les gènes suivants : ABCB1, ACP1, BCL2L11, NFKB1, PARP1, et SHMT1. Ces gènes jouent majoritairement un rôle dans les mécanismes d’action des glucocorticoïdes, mais quelques-uns ont plutôt un effet direct sur le développement d’ostéonécrose. Nos recherches ont démontré une corrélation entre ces polymorphismes et l’apparition d’ostéonécrose chez les patients de la cohorte QcALL, traités aux glucocorticoïdes. L'incidence cumulative de l'ostéonécrose a été évaluée rétrospectivement chez 305 enfants atteints de la leucémie qui ont subi un traitement à l’hôpital Ste-Justine selon les protocoles DFCI de Boston (87-01, 91-01, 95-01 et 2000-01). Parmi les huit polymorphismes de BCL2L11 étudiés, les 891T> G (rs2241843) et 29201C> T (rs724710) ont été significativement associés à ON (p = 0.01 et p = 0.03, respectivement). L'association du polymorphisme 891T> G a été modulée par le type de corticostéroïde (CS), l’âge, le sexe et le groupe à risque (p ≤ 0,05). Le polymorphisme 29201C> T était particulièrement apparent chez les patients à haut risque (p = 0,003). La même étude était conduite en parallèle sur des patients de la cohorte DFCI de Boston (N = 192), et montrait des résultats significatifs pour les polymorphismes étudiés. En conclusion, les résultats de cette étude permettront de confirmer l’association de ces polymorphismes au développement d’ON chez les patients de LLA traités aux GC. / Osteonecrosis (ON) and fractures (FR) are complications that take place in the treatment of children acute lymphoblastic leukemia (cALL). They can be caused by various factors, mainly using glucocorticoids. The corticosteroids, dexamethasone (DXM) and prednisone (PDN) are administered during the treatment of leukemia to initiate apoptosis of malignant cells; while having an anti-inflammatory effect. However, the use of these corticosteroids has severe side effects, including the development of osteonecrosis. Moreover, some patients develop resistance to treatment, and are at risk of developing side effects. The genetic variants predispose some patients at higher risk than others. Several genes have been previously reported as up- or down regulated by the GCs actions. The genetic variations present in gene coding or regulatory regions can affect their function and ultimately determine an increased risk of developing ON associated to ALL therapy. Therefore, we investigated the association between several single nucleotide polymorphisms (SNPs) in six candidate genes: BCL2L11, NFKB1, PARP1, ABCB1, ACP1, and SHMT1. These genes play a role in the mechanisms of action of glucocorticoids, but some have more of a direct effect on the development of osteonecrosis. Our research has shown a correlation between these polymorphisms and the occurrence of osteonecrosis in patients in the QCALL cohort, treated with glucocorticoids. Cumulative incidence of osteonecrosis was assessed retrospectively in 305 children with ALL who underwent treatment with DFCI protocols (87-01, 91-01, 95-01 and 2000-01) in childhood ALL cohort from Quebec (QcALL). Among the eight tag BCL2L11 polymorphisms studied the 891T>G (rs2241843) and 29201C>T (rs724710) were significantly associated with ON (p = 0.01 and p = 0.03, respectively). Association of 891T>G polymorphism was modulated by type of corticosteroid (CS), age, sex and risk group (p ≤ 0.05 and that of 29201C>T was particularly apparent among high risk (p = 0.003) patients. These polymorphisms have shown significant ON association in several QcALL risk groups, mainly in corticosteroid groups, age < 10 years, and high risk (HR) group. Furthermore, the same study was conducted in parallel with patients in the replication (DFCI) cohort (N = 192), and we showed significant genetic association results for all studied polymorphisms. In conclusion, this study identifies that some ALL children have a high incidence of ON during the treatment that is highly associated with polymorphisms in different genes regulated by corticosteroids and ALL prognostic factors.