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No / Flavonoids, of which flavones are a sub-group, are plant secondary metabolites found in a variety of natural food sources (e.g., vegetables) and wines. They elicit beneficial roles in health and disease through their antioxidant activity, but some of them have also now been found to exert specific effects on cell signalling. We recently showed that methoxylation of the flavone structure at the 4ʹ position, or additionally at the 3ʹ position, to produce 4ʹ-methoxyflavone (4MF) and 3ʹ,4ʹ-dimethoxyflavone (DMF), respectively, significantly enhanced activity against the cell death (“parthanatos”) mediated by poly (ADP-ribose) polymerase (PARP). We report here our attempt to correlate the antioxidant and parthanatos-inhibitory activities of these methoxylated flavones with those of the hydroxylated flavonoids. Cultures of HeLa and HaCaT cells were exposed to MNNG (50µM, up to 25min), which induces parthanatos, and the oxidant hydrogen peroxide (100µM – 2mM, up to 24h). The effects (up to 20µM) of the methoxylated flavones 4MF and DMF, the hydroxylated flavone luteolin (LN), and the non-flavone flavonoids quercetin (QE), naringin (NG) and epigallocatechin gallate (EGCG) on the reduction in viability (indicative of cell death) and morphological changes induced by MNNG or peroxide were then investigated. Both alamar blue and MTT assays were used to quantify viability. MNNG induced significant reduction in cell viability, which was not affected by the pan-caspase inhibitor Z-VAD-fmk but significantly blocked by DPQ, a PARP-1 inhibitor, consistent with the biochemical profile of parthanatos. Hydrogen peroxide also elicited a significant decrease in cell viability, with partial or no protection afforded by either Z-VAD-fmk or DPQ (dependent on peroxide concentration and treatment duration). 4MF and DMF demonstrated significant protection against MNNG-induced cell death but LN, QE, NG and EGCG showed little or no protection. On the other hand, 4MF and DMF elicited mostly negligible effects against hydrogen peroxide, whereas LN, QE, NG and EGCG elicited various levels of protection against it. We conclude that methoxylation at the 4ʹ or 3ʹ, 4ʹ positions of flavones favours anti-parthanatos but not antioxidant activity, whereas hydroxylation enhances antioxidant but not anti-parthanatos activity. / Abstract of conference paper.

Transforming growth factor-β (TGF-β) signaling is initiated when the ligand binds to type II and type I serine/threonine kinase receptors at the cell surface. Activated TGF-β type I receptors phosphorylate R-Smads which relocate, together with co-Smads, to the cell nucleus and regulate transcription. Enhancement or repression of Smad-specific gene targets leads to intracellular protein compositions which organize functional complexes and thus govern cellular processes such as proliferation, migration and differentiation. TGF-β/Smad signaling relays are regulated by various post-translational modifications. From receptors to gene promoters, intricate interplays between phosphorylation, acetylation, ubiquitination and numerous other modifications, control Smad signaling initiation and duration. However, many steps in the cascade, including receptor internalization, Smad nuclear shuttling and transcriptional termination, still remain elusive. The open gaps in our understanding of these mechanisms most likely involve additional post-translational regulations. Thus, the aim of the present investigation was to identify novel modulators of TGF-β/Smad signaling. In the first part of this thesis, we show the importance of ADP-ribosylation in Smad-mediated transcription. We identified poly(ADP-ribose) polymerase 1 (PARP-1) as a Smad interacting protein. Our work revealed that PARP-1 forms direct interactions with Smad3/4, and PARylates residues in their MH1 domains. This modification restricts Smads from binding to DNA and attenuates Smad-activated transcription. PARylation is reversed by the glycohydrolase PARG. We provide evidence that PARG can de-ADP-ribosylate Smads, which enhances Smad-promoted gene regulation. In the second part, we examine a Smad-dependent gene target of TGF-β signaling, salt inducible kinase 1 (SIK). After induction, SIK cooperates with Smad7 and Smurf2 to downregulate the TGF-β type I receptor. The mechanism relies on both the kinase and UBA domain of SIK as well as the E3-ligase activity of Smurf2. In summary, we have unveiled two enzyme-dependent TGF-β/Smad modulatory mechanisms; SIK promoted receptor turnover and PARP-1/PARG-regulated Smad signaling.

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.

La structure de la chromatine et sa compaction sont modulées par la substitution des histones conventionnelles par des variants d'histones. MacroH2A est l'un de ces variants et se singularise par sa grande taille. De nombreuses données suggèrent que macroH2A pourrait participer à l'inactivation de la transcription.<br />Par immunofluorescence, cette protéine est retrouvée accumulée sur le territoire du chromosome X inactif (Xi) chez les mammifères femelles. Néanmoins, cette association préférentielle pourrait simplement refléter la forte concentration en nucléosomes de cette région. Pour aborder le rôle de macroH2A dans le phénomène de l'inactivation du chromosome X, notre principale approche a consisté en des expériences de «ChIP-on-CHIP» sur de la chromatine native. Nos résultats montrent un enrichissement global et modeste de macroH2A sur le chromosome X femelle, excepté sur la plupart des gènes échappant à l'inactivation. <br />Nous avons souhaité nous intéresser également au rôle potentiel de macroH2A dans le mécanisme de réparation de l'ADN. En effet, il a été montré que le domaine macro est capable de lier l'ADP-ribose, un nucléotide déterminant dans de nombreux processus biologiques tels que la transcription ou la réparation. Plusieurs expériences nous ont permis de démontrer que les nucléosomes macroH2A sont associés in vivo à l'enzyme PARP-1, protéine clef de la réparation des cassures simple brin de l'ADN. La PARP-1 associée au nucléosome variant est inactive, et le traitement par H2O2 va induire son relâchement et son activation. L'absence de macroH2A conduit à une sur-activation de PARP-1, ce qui compromet sévèrement la réparation de l'ADN endommagé.