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.

Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in β-adrenergic receptor (β-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). β-AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed β-AR-stimulated increases in MMP-2 protein-levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited β-AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with β1-integrins after β-AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of β1-integrin signaling using laminin inhibited the increased association of MMP-2 with β1- integrins. β-AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) β-AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits β-AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with β1 integrins and poly-ADP-ribose-polymerase cleavage.

Acrolein is an electrophilic α, β-unsaturated aldehyde. Additionally, acrolein is a metabolite of the antineoplastic alkylating agent cyclophosphamide and is implicated in off-target effects, including to bladder hemorrhagic cystitis and cyclophosphamide-induced cardiotoxicity, both of which have led to serious secondary iatrogenic injury during and following chemotherapy. At low concentrations acrolein inhibits cell proliferation without inducing apoptosis, while at high concentrations may result in secondary apoptosis promotion. This investigation assessed the role of the enzyme poly (ADP-ribose) polymerase (PARP) in acrolein induced toxicity using the established toxicological H9c2 (2-1) cardiomyoblast in vitro model. H9c2 (2-1) cells were plated in 24-well plates at 75,000 cells per well three days prior to testing, followed by acrolein dosing at concentrations between 10 µM and 1000µM for either 30 or 55 minutes. PARP activity was quantitatively measured in total cell lysates using a biotin-avidin-conjugated horseradish peroxidase-TMB reporter system in a 96-well microplate formate. The lowest effective dose of toxicity at 30 minute dosing was found at 25 μM (PARP Activity 1.65-fold control) which returned to baseline at 100 μM; concentrations at or above 250 μM results in significant PARP activity reductions (≤ 0.46-fold control). Biomarkers were further characterized for cytotoxicity (AST presence), and viability (MTT reduction) in order to facilitate mechanistic characterization of PARP-mediated acrolein cardiotoxicity. Investigation of a PARP inhibitor was assessed to explore the intervention for acrolein induced cardiac tissue damage.

Drug-induced cardiotoxicity has resulted in a thorough evaluation of patient doses, treatments, and rehabilitation. One of the most commonly prescribed chemotherapeutic agents is cyclophosphamide. The active metabolite, acrolein, is one of the most potent inducers of cardiomyopathy. In this study, research was conducted on the H9c2 (2-1) cardiomyocyte cell line derived from the embryonic myocardium of rattus norvegicus to assess its competency for evaluation of the change in poly (ADP-ribose) polymerase (PARP) activity. The application of this model to study the effects of acrolein on PARP activation was chosen as an ideal determinant of cell damage produced by nitrogen mustards. To verify the legitimacy of this model, cardiomyocytes were exposed to acrolein in varying concentrations and time durations with a subsequent protein concentration measurement determined through the BCA Protein Assay. After the normalization of samples through volume adjustments and verification of sufficient protein, other aliquots were subjected to a PARP Assay in order to measure PARP activity. PARP was activated at exposure concentrations of 75 μM in all trials, with an average detection of 0.00569 ± 0.001 mU/200ng protein. Other concentrations showed varying degrees of PARP activation, verifying the model’s competency. PARP activation implies the potential use of this model for further research into targeted molecular therapy of PARP inhibition. Therefore, this model has the ability to be used as an assessment tool for the combined use of PARP inhibitors and chemotherapeutic agents; it can be useful for future research investigating the use and efficacy of PARP inhibitors in reducing drug-induced cardiotoxicity.

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.

No description available.

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.

No description available.