Michèle Rouleau

PARP inhibition: PARP1 and beyond

Recent findings have thrust poly(ADP-ribose) polymerases (PARPs) into the limelight as potential chemotherapeutic targets. To provide a framework for understanding these recent observations, we review what is known about the structures and functions of the family of PARP enzymes, and then outline a series of questions that should be addressed to guide the rational development of PARP inhibitors as anticancer agents.

PARP-1, a determinant of cell survival in response to DNA damage

Poly(ADP-ribose) polymerase-1 (PARP-1) plays a primary role in the process of poly(ADP-ribosyl)ation. This posttranslational modification of nuclear proteins is activated in response to DNA damage. Having been studied for more than 30 years, PARP-1 is now known to be implicated in several crucial cellular processes: DNA replication, transcription, DNA repair, apoptosis, and genome stability. In this review, we focus on recent findings suggesting that PARP-1 participates in DNA damage signaling in cell death. Of clinical relevance is its role in cancer therapy, irradiation, and chemotherapy, all of which may cause DNA damage and overactivate PARP-1, resulting in inflammation caused by necrosis. Therefore, we will discuss how inhibition of PARP-1 may enhance the efficiency of cancer therapy.

PARP-3 and APLF Function Together to Accelerate Nonhomologous End-Joining

PARP-3 is a member of the ADP-ribosyl transferase superfamily of unknown function. We show that PARP-3 is stimulated by DNA double-strand breaks (DSBs) in vitro and functions in the same pathway as the poly (ADP-ribose)-binding protein APLF to accelerate chromosomal DNA DSB repair. We implicate PARP-3 in the accumulation of APLF at DSBs and demonstrate that APLF promotes the retention of XRCC4/DNA ligase IV complex in chromatin, suggesting that PARP-3 and APLF accelerate DNA ligation during nonhomologous end-joining (NHEJ). Consistent with this, we show that class switch recombination in Aplf(-/-) B cells is biased toward microhomology-mediated end-joining, a pathway that operates in the absence of XRCC4/DNA ligase IV, and that the requirement for PARP-3 and APLF for NHEJ is circumvented by overexpression of XRCC4/DNA ligase IV. These data identify molecular roles for PARP-3 and APLF in chromosomal DNA double-strand break repair reactions.

Inactivation of Brassinosteroid Biological Activity by a Salicylate-inducible Steroid Sulfotransferase from Brassica napus

Recent discoveries from brassinosteroid-deficient mutants led to the recognition that plants, like animals, use steroids to regulate their growth and development. We describe the characterization of one member of a Brassica napussulfotransferase gene family coding for an enzyme that catalyzes theO-sulfonation of brassinosteroids and of mammalian estrogenic steroids. The enzyme is specific for the hydroxyl group at position 22 of brassinosteroids with a preference for 24-epicathasterone, an intermediate in the biosynthesis of 24-epibrassinolide. Enzymatic sulfonation of 24-epibrassinolide abolishes its biological activity in the bean second internode bioassay. This mechanism of hormone inactivation by sulfonation is similar to the modulation of estrogen biological activity observed in mammals. Furthermore, the expression of the B. napussteroid sulfotransferase genes was found to be induced by salicylic acid, a signal molecule in the plant defense response. This pattern of expression suggests that, in addition to an increased synthesis of proteins having antimicrobial properties, plants respond to pathogen infection by modulating steroid-dependent growth and developmental processes.

Sulfation and sulfotransferases 6: Biochemistry and molecular biology of plant sulfotransferases.

It is now well established that, in mammals, sulfate conjugation constitutes an important reaction in the transformation of xenobiotics and in the modulation of the biological activity of steroid hormones and neurotransmitter (1,2). The presence of a sulfate group on some molecules can also be a prerequisite for their biological function. For example, it is well known that the sulfate groups are directly involved in the molecular interaction between heparin and antithrombin III (3). In plants, sulfation also seems to play an important role in the intermolecular recognition and signaling processes, as indicated by the requirement of a sulfate moiety for the biological activity of gallic acid glucoside sulfate in the seismonastic and gravitropic movements of plants (4), and of Nod RM1 in the cortical cell division during early nodule initiation in Rhizobium meliloti‐alfalfainteraction (5). In addition, recent studies indicate that flavonoid conjugates, including the sulfate esters, may play a role in the regulation of plant growth by strongly binding the naphthylphthalamic acid receptor, thus blocking the quercetin‐stimulated accumulation of the auxin phytohormone (6). Although several sulfated metabolites are known to accumulate in a variety of plant species (7), the study of enzymes that catalyze the sulfation reaction in plants lagged considerably compared to those conducted with their mammalian homologs. This apparent lack of interest may have been because the function of plant‐sulfated metabolites is difficult to predict, since their accumulation is often restricted to a limited number of species. Despite this limitation, several plant sulfotransferases (STs) have been characterized at the biochemical level, and the cDNA clones encoding six plant STs have been isolated. Based on sequence homology, the plant ST coding sequences are grouped under the SULT3 family, also known as the flavonol ST family. This review summarizes our current knowledge of the plant STs and focuses on the functional significance of the sulfate conjugation in plant growth, development, and adaptation to stress.—Varin, L., Marsolais, F., Richard, M., Rouleau, M. Biochemistry and molecular biology of plant sulfotransferases. FASEB J. 11, 517–525 (1997)

PARP‐3 associates with polycomb group bodies and with components of the DNA damage repair machinery

Poly(ADP‐ribose) polymerase 3 (PARP‐3) is a novel member of the PARP family of enzymes that synthesize poly(ADP‐ribose) on themselves and other acceptor proteins. Very little is known about this PARP, which is closely related to PARP‐1 and PARP‐2. By sequence analysis, we find that PARP‐3 may be expressed in two isoforms which we studied in more detail to gain insight into their possible functions. We find that both PARP‐3 isoforms, transiently expressed as GFP or FLAG fusions, are nuclear. Detection of endogenous PARP‐3 with a specific antibody also shows a widespread nuclear distribution, appearing in numerous small foci and a small number of larger foci. Through co‐localization experiments and immunoprecipitations, the larger nuclear foci were identified as Polycomb group bodies (PcG bodies) and we found that PARP‐3 is part of Polycomb group protein complexes. Furthermore, using a proteomics approach, we determined that both PARP‐3 isoforms are part of complexes comprising DNA‐PKcs, PARP‐1, DNA ligase III, DNA ligase IV, Ku70, and Ku80. Our findings suggest that PARP‐3 is a nuclear protein involved in transcriptional silencing and in the cellular response to DNA damage. J. Cell. Biochem. 100: 385–401, 2007.

Investigation of PARP-1, PARP-2, and PARG interactomes by affinity-purification mass spectrometry

BackgroundPoly(ADP-ribose) polymerases (PARPs) catalyze the formation of poly(ADP-ribose) (pADPr), a post-translational modification involved in several important biological processes, namely surveillance of genome integrity, cell cycle progression, initiation of the DNA damage response, apoptosis, and regulation of transcription. Poly(ADP-ribose) glycohydrolase (PARG), on the other hand, catabolizes pADPr and thereby accounts for the transient nature of poly(ADP-ribosyl)ation. Our investigation of the interactomes of PARP-1, PARP-2, and PARG by affinity-purification mass spectrometry (AP-MS) aimed, on the one hand, to confirm current knowledge on these interactomes and, on the other hand, to discover new protein partners which could offer insights into PARPs and PARG functions.ResultsPARP-1, PARP-2, and PARG were immunoprecipitated from human cells, and pulled-down proteins were separated by gel electrophoresis prior to in-gel trypsin digestion. Peptides were identified by tandem mass spectrometry. Our AP-MS experiments resulted in the identifications of 179 interactions, 139 of which are novel interactions. Gene Ontology analysis of the identified protein interactors points to five biological processes in which PARP-1, PARP-2 and PARG may be involved: RNA metabolism for PARP-1, PARP-2 and PARG; DNA repair and apoptosis for PARP-1 and PARP-2; and glycolysis and cell cycle for PARP-1.ConclusionsThis study reveals several novel protein partners for PARP-1, PARP-2 and PARG. It provides a global view of the interactomes of these proteins as well as a roadmap to establish the systems biology of poly(ADP-ribose) metabolism.

Reprogramming cellular events by poly(ADP-ribose)-binding proteins

Abstract Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions.

PARP activation regulates the RNA-binding protein NONO in the DNA damage response to DNA double-strand breaks

After the generation of DNA double-strand breaks (DSBs), poly(ADP-ribose) polymerase-1 (PARP-1) is one of the first proteins to be recruited and activated through its binding to the free DNA ends. Upon activation, PARP-1 uses NAD+ to generate large amounts of poly(ADP-ribose) (PAR), which facilitates the recruitment of DNA repair factors. Here, we identify the RNA-binding protein NONO, a partner protein of SFPQ, as a novel PAR-binding protein. The protein motif being primarily responsible for PAR-binding is the RNA recognition motif 1 (RRM1), which is also crucial for RNA-binding, highlighting a competition between RNA and PAR as they share the same binding site. Strikingly, the in vivo recruitment of NONO to DNA damage sites completely depends on PAR, generated by activated PARP-1. Furthermore, we show that upon PAR-dependent recruitment, NONO stimulates nonhomologous end joining (NHEJ) and represses homologous recombination (HR) in vivo. Our results therefore place NONO after PARP activation in the context of DNA DSB repair pathway decision. Understanding the mechanism of action of proteins that act in the same pathway as PARP-1 is crucial to shed more light onto the effect of interference on PAR-mediated pathways with PARP inhibitors, which have already reached phase III clinical trials but are until date poorly understood.

Pharmacogenomics of Human Uridine Diphospho-Glucuronosyltransferases and Clinical Implications

Glucuronidation by uridine diphospho‐glucuronosyltransferase enzymes (UGTs) is a major phase II biotransformation pathway and, complementary to phase I metabolism and membrane transport, one of the most important cellular defense mechanisms responsible for the inactivation of therapeutic drugs, other xenobiotics, and endogenous molecules. Interindividual variability in UGT pathways is significant and may have profound pharmacological and toxicological implications. Several genetic and genomic processes underlie this variability and are discussed in relation to drug metabolism and diseases such as cancer.