Arnaud Droit

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.

Comparative proteome analysis of human epithelial ovarian cancer

BackgroundEpithelial ovarian cancer is a devastating disease associated with low survival prognosis mainly because of the lack of early detection markers and the asymptomatic nature of the cancer until late stage. Using two complementary proteomics approaches, a differential protein expression profile was carried out between low and highly transformed epithelial ovarian cancer cell lines which realistically mimic the phenotypic changes observed during evolution of a tumour metastasis. This investigation was aimed at a better understanding of the molecular mechanisms underlying differentiation, proliferation and neoplastic progression of ovarian cancer.ResultsThe quantitative profiling of epithelial ovarian cancer model cell lines TOV-81D and TOV-112D generated using iTRAQ analysis and two-dimensional electrophoresis coupled to liquid chromatography tandem mass spectrometry revealed some proteins with altered expression levels. Several of these proteins have been the object of interest in cancer research but others were unrecognized as differentially expressed in a context of ovarian cancer. Among these, series of proteins involved in transcriptional activity, cellular metabolism, cell adhesion or motility and cytoskeleton organization were identified, suggesting their possible role in the emergence of oncogenic pathways leading to aggressive cellular behavior.ConclusionThe differential protein expression profile generated by the two proteomics approaches combined to complementary characterizations studies will open the way to more exhaustive and systematic representation of the disease and will provide valuable information that may be helpful to uncover the molecular mechanisms related to epithelial ovarian cancer.

Proteomic investigation of phosphorylation sites in poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase.

Phosphorylation is a very common post-translational modification event known to modulate a wide range of biological responses. Beyond the regulation of protein activity, the interrelation of phosphorylation with other post-translational mechanisms is responsible for the control of diverse signaling pathways. Several observations suggest that phosphorylation of poly(ADP-ribose) polymerase-1 (PARP-1) regulates its activity. There is also accumulating evidence to suggest the establishment of phosphorylation-dependent assembly of PARP-1-associated multiprotein complexes. Although it is relatively straightforward to demonstrate phosphorylation of a defined target, identification of the actual amino acids involved still represents a technical challenge for many laboratories. With the use of a combination of bioinformatics-based predictions tools for generic and kinase-specific phosphorylation sites, in vitro phosphorylation assays and mass spectrometry analysis, we investigated the phosphorylation profile of PARP-1 and poly(ADP-ribose) glycohydrolase (PARG), two major enzymes responsible for poly(ADP-ribose) turnover. Mass spectrometry analysis revealed the phosphorylation of several serine/threonine residues within important regulatory domains and motifs of both enzymes. With the use of in vivo microirradiation-induced DNA damage, we show that altered phosphorylation at specific sites can modify the dynamics of assembly and disassembly of PARP-1 at sites of DNA damage. By documenting and annotating a collection of known and newly identified phosphorylation sites, this targeted proteomics study significantly advances our understanding of the roles of phosphorylation in the regulation of PARP-1 and PARG.

Proteome profiling of human epithelial ovarian cancer cell line TOV-112D

A proteome profiling of the epithelial ovarian cancer cell line TOV-112D was initiated as a protein expression reference in the study of ovarian cancer. Two complementary proteomic approaches were used in order to maximise protein identification: two-dimensional gel electrophoresis (2DE) protein separation coupled to matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and one-dimensional gel electrophoresis (1DE) coupled to liquid-chromatography tandem mass spectrometry (LC MS/MS). One hundred and seventy-two proteins have been identified among 288 spots selected on two-dimensional gels and a total of 579 proteins were identified with the 1DE LC MS/MS approach. This proteome profiling covers a wide range of protein expression and identifies several proteins known for their oncogenic properties. Bioinformatics tools were used to mine databases in order to determine whether the identified proteins have previously been implicated in pathways associated with carcinogenesis or cell proliferation. Indeed, several of the proteins have been reported to be specific ovarian cancer markers while others are common to many tumorigenic tissues or proliferating cells. The diversity of proteins found and their association with known oncogenic pathways validate this proteomic approach. The proteome 2D map of the TOV-112D cell line will provide a valuable resource in studies on differential protein expression of human ovarian carcinomas while the 1DE LC MS/MS approach gives a picture of the actual protein profile of the TOV-112D cell line. This work represents one of the most complete ovarian protein expression analysis reports to date and the first comparative study of gene expression profiling and proteomic patterns in ovarian cancer.

Quantitative proteomic analysis of amphotericin B resistance in Leishmania infantum

Graphical abstract

An Integrated Pipeline for the Genome-Wide Analysis of Transcription Factor Binding Sites from ChIP-Seq

ChIP-Seq has become the standard method for genome-wide profiling DNA association of transcription factors. To simplify analyzing and interpreting ChIP-Seq data, which typically involves using multiple applications, we describe an integrated, open source, R-based analysis pipeline. The pipeline addresses data input, peak detection, sequence and motif analysis, visualization, and data export, and can readily be extended via other R and Bioconductor packages. Using a standard multicore computer, it can be used with datasets consisting of tens of thousands of enriched regions. We demonstrate its effectiveness on published human ChIP-Seq datasets for FOXA1, ER, CTCF and STAT1, where it detected co-occurring motifs that were consistent with the literature but not detected by other methods. Our pipeline provides the first complete set of Bioconductor tools for sequence and motif analysis of ChIP-Seq and ChIP-chip data.

Integration of High-Resolution Methylome and Transcriptome Analyses to Dissect Epigenomic Changes in Childhood Acute Lymphoblastic Leukemia

B-cell precursor acute lymphoblastic leukemia (pre-B ALL) is the most common pediatric cancer. Although the genetic determinants underlying disease onset remain unclear, epigenetic modifications including DNA methylation are suggested to contribute significantly to leukemogenesis. Using the Illumina 450K array, we assessed DNA methylation in matched tumor-normal samples of 46 childhood patients with pre-B ALL, extending single CpG-site resolution analysis of the pre-B ALL methylome beyond CpG-islands (CGI). Unsupervised hierarchical clustering of CpG-site neighborhood, gene, or microRNA (miRNA) gene-associated methylation levels separated the tumor cohort according to major pre-B ALL subtypes, and methylation in CGIs, CGI shores, and in regions around the transcription start site was found to significantly correlate with transcript expression. Focusing on samples carrying the t(12;21) ETV6-RUNX1 fusion, we identified 119 subtype-specific high-confidence marker CpG-loci. Pathway analyses linked the CpG-loci-associated genes with hematopoiesis and cancer. Further integration with whole-transcriptome data showed the effects of methylation on expression of 17 potential drivers of leukemogenesis. Independent validation of array methylation and sequencing-derived transcript expression with Sequenom Epityper technology and real-time quantitative reverse transcriptase PCR, respectively, indicates more than 80% empirical accuracy of our genome-wide findings. In summary, genome-wide DNA methylation profiling enabled us to separate pre-B ALL according to major subtypes, to map epigenetic biomarkers specific for the t(12;21) subtype, and through a combined methylome and transcriptome approach to identify downstream effects on candidate drivers of leukemogenesis.

Proteomic and Genomic Analyses of Antimony Resistant Leishmania infantum Mutant

Background Antimonials remain the primary antileishmanial drugs in most developing countries. However, drug resistance to these compounds is increasing and our understanding of resistance mechanisms is partial. Methods/Principal Findings In the present study, quantitative proteomics using stable isotope labelling of amino acids in cell culture (SILAC) and genome next generation sequencing were used in order to better characterize in vitro generated Leishmania infantum antimony resistant mutant (Sb2000.1). Using the proteomic method, 58 proteins were found to be differentially regulated in Sb2000.1. The ABC transporter MRPA (ABCC3), a known marker of antimony resistance, was observed for the first time in a proteomic screen. Furthermore, transfection of its gene conferred antimony resistance in wild-type cells. Next generation sequencing revealed aneuploidy for 8 chromosomes in Sb2000.1. Moreover, specific amplified regions derived from chromosomes 17 and 23 were observed in Sb2000.1 and a single nucleotide polymorphism (SNP) was detected in a protein kinase (LinJ.33.1810-E629K). Conclusion/Significance Our results suggest that differentially expressed proteins, chromosome number variations (CNVs), specific gene amplification and SNPs are important features of antimony resistance in Leishmania.

Quantitative site-specific ADP-ribosylation profiling of DNA-dependent PARPs

An important feature of poly(ADP-ribose) polymerases (PARPs) is their ability to readily undergo automodification upon activation. Although a growing number of substrates were found to be poly(ADP-ribosyl)ated, including histones and several DNA damage response factors, PARPs themselves are still considered as the main acceptors of poly(ADP-ribose). By monitoring spectral counts of specific hydroxamic acid signatures generated after the conversion of the ADP-ribose modification onto peptides by hydroxylamine hydrolysis, we undertook a thorough mass spectrometry mapping of the glutamate and aspartate ADP-ribosylation sites onto automodified PARP-1, PARP-2 and PARP-3. Thousands of hydroxamic acid-conjugated peptides were identified with high confidence and ranked based on their spectral count. This semi-quantitative approach allowed us to locate the preferentially targeted residues in DNA-dependent PARPs. In contrast to what has been reported in the literature, automodification of PARP-1 is not predominantly targeted towards its BRCT domain. Our results show that interdomain linker regions that connect the BRCT to the WGR module and the WGR to the PRD domain undergo prominent ADP-ribosylation during PARP-1 automodification. We also found that PARP-1 efficiently automodifies the D-loop structure within its own catalytic fold. Interestingly, additional major ADP-ribosylation sites were identified in functional domains of PARP-1, including all three zinc fingers. Similar to PARP-1, specific residues located within the catalytic sites of PARP-2 and PARP-3 are major targets of automodification following their DNA-dependent activation. Together our results suggest that poly(ADP-ribosyl)ation hot spots make a dominant contribution to the overall automodification process.

Investigation of male infertility using quantitative comparative proteomics.

Male factors account for 40% of infertility cases. The identification of differentially expressed proteins on spermatozoa from fertile and infertile men can help in the elucidation of the molecular basis of male infertility. The aim of this study was to compare sperm proteomes from 3 different groups: fertile men, normozoospermic men consulting for infertility, and normozoospermic men with an impaired capacity for fertilization (IVF-failure). We used differential proteomics with isobaric tags for relative and absolute quantitation (iTRAQ) labeling, and LC-MS analysis to identify proteins that are differentially expressed. A total of 348 unique proteins were identified and quantified. The analysis identified 33 proteins that were differentially expressed in the IVF-failure group vs the fertile group. Comparison of the infertile and fertile groups revealed that 18 proteins appeared to be differentially expressed. Four proteins were similarly altered in the IVF-failure and infertile groups: semenogelin 1 (SEMG1), prolactin-induced protein (PIP), glyceraldehyde-3-phosphate dehydrogenase (GAPDHS), and phosphoglycerate kinase 2 (PGK2). These protein markers were selected for validation using multiple reactions monitoring mass spectrometry (MRM-MS) and further confirmed by Western blot analysis. Overall, these results suggest that a panel of proteins may be used as biomarkers for future studies of infertility.