Methods for Purification of Proteins Associated with Cellular Poly(ADP-Ribose) and PARP-Specific Poly(ADP-Ribose)

available in PMC 2012 November 09Poly(ADP-ribose) (pADPr) is a posttranslational modification that regulates protein function through two major mechanisms: covalent modification of acceptor proteins and noncovalent binding of proteins to pADPr. pADPr is synthesized by a family of enzymes called poly(ADP-ribose) polymerases (PARPs) that are themselves major targets of pADPr modification. Here, we outline two methods for the purification of pADPr-binding proteins via pADPr purification under native conditions: purification of cellular pADPr and pADPr covalently linked to specific PARPs. Together, these methods provide complementary approaches to the identification of noncovalent pADPr–protein interactions in the cell

Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions

BACKGROUND: The adaptation to hypoxia is mainly controlled by the HIF transcription factors. Increased expression/activity of HIF-1α correlates with poor prognosis in cancer patients. PARP-1 inhibitors are used in the clinic to treat BRCAness breast/ovarian cancer and have been shown to regulate the hypoxic response; therefore, their use could be expanded. METHODS: In this work by integrating molecular/cell biology approaches, genome-wide ChIP-seq, and patient samples, we elucidate the extent to which PARP-1 exerts control over HIF-1-regulated genes. RESULTS: In human melanoma, PARP-1 and HIF-1α expression are strongly associated. In response to a hypoxic challenge poly(ADP-ribose) (PAR) is synthesized, HIF-1α is post-transcriptionally modified (PTM) and stabilized by PARylation at specific K/R residues located at its C-terminus. Using an unbiased ChIP-seq approach we demonstrate that PARP-1 dictates hypoxia-dependent HIF-recruitment to chromatin in a range of HIF-regulated genes while analysis of HIF-binding motifs (RCGTG) reveals a restriction on the recognition of hypoxia responsive elements in the absence of PARP-1. Consequently, the cells are poorly adapted to hypoxia, showing a reduced fitness during hypoxic induction. CONCLUSIONS: These data characterize the fine-tuning regulation by PARP-1/PARylation of HIF activation and suggest that PARP inhibitors might have therapeutic potential against cancer types displaying HIF-1α over-activation

Modeling Pharmacodynamic Response to the Poly(ADP-Ribose) Polymerase Inhibitor ABT-888 in Human Peripheral Blood Mononuclear Cells

Poly(ADP-ribose) polymerase (PARP) facilitates DNA repair and PARP inhibitors may potentiate the effect of DNA-damaging chemotherapeutic agents in patients with cancer. Collection of peripheral blood mononuclear cells (PBMCs) as a surrogate tissue to monitor PARP inhibitor pharmacodynamic effects has several advantages over tumor biopsy collection, including minimally invasive sample collection and the ability to collect multiple samples for longitudinal assessment of drug effect.Using our previously validated immunoassay for measuring poly(ADP-ribose) (PAR), a product of PARP, in tumor biopsies, we validated a method to quantify PAR levels in PBMCs to monitor the pharmacodynamic effects of the PARP inhibitor ABT-888 in clinical trials. The inter-individual variation in PAR levels was large. No significant difference (P = 0.67) was measured between median baseline PAR levels in 144 healthy volunteers (131.7 pg/1×10(7) PBMCs [interquartile range, 79.5-241.6]) and 49 patients with cancer (149.2 pg/1×10(7) PBMCs [interquartile range, 83.2-249.3]). In addition, PAR levels monitored in healthy volunteers over 3 weeks had considerable intra- and inter-individual variation (range, 44-1073 pg PAR/1×10(7) PBMCs). As a pharmacodynamic model, we quantified changes in PAR levels in human PBMCs treated ex vivo with clinically relevant concentrations of ABT-888. Of 40 healthy volunteer PBMC samples treated with ABT-888, 47.5% had greater than 50% PAR reduction compared to vehicle-treated controls. Considerable inter-sample heterogeneity in PAR levels was measured, and several ABT-888-insensitive samples were identified.Our results emphasize the importance of using a validated method to measure PAR levels, and support further investigation into the role of PARP in PBMCs. To this end, the PAR immunoassay has been validated for use with PBMCs and incorporated into clinical trials to assess PBMCs as a potential pharmacodynamic surrogate for tumor biopsies in clinical trials of PARP inhibitors

Collective foraging decision in a gregarious insect

Group foraging by eusocial insects implies sophisticated recruitment processes that often result in collective decisions to exploit the most profitable sources. These advanced levels of cooperation, however, remain limited to a small range of species, and we still know little about the mechanisms underlying group foraging behaviours in the great mass of animals exhibiting lower levels of social complexity. In this paper, we report, for the first time in a gregarious insect, the cockroach Blattella germanica (L.), a collective foraging decision whereby the selection of food sources is reached without requiring active recruitment. Groups of cockroaches given a binary choice between identical food sources exhibited exploitation asymmetries whose amplitude increases with group size. By coupling behavioural observations to computer simulations, we demonstrate that selection of food sources relies uniquely on a retention effect of feeding individuals on newcomers without comparison between available opportunities. This self-organised pattern presents similarities with the foraging dynamics of eusocial species, thus stressing the generic dimension of collective decision-making mechanisms based on social amplification rules despite fundamental differences in recruitment processes. We hypothesise that such parsimony could apply to a wide range of species and help understand the emergence of collective behaviours in simple social systems. © 2010 Springer-Verlag.SCOPUS: ar.jinfo:eu-repo/semantics/publishe