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.
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).
Our results suggest that differentially expressed proteins, chromosome number variations (CNVs), specific gene amplification and SNPs are important features of antimony resistance in Leishmania.
Glutamate acting on N-methyl-D-aspartate (NMDA) receptors plays an important role in neurodegenerative diseases and neuronal injury following stroke, through activation of poly(ADP-ribose) polymerase-1 and generation of the death molecule poly(ADP-ribose) (PAR) polymer. Here we identify Iduna, a novel NMDA receptor-induced survival gene that is neuroprotective against glutamate NMDA receptor mediated excitotoxicity both in vitro and in vivo and against stroke through interfering with PAR polymer induced cell death (parthanatos). Iduna’s protective effects are independent and downstream of PARP-1 activity. Iduna is a PAR polymer binding protein and mutations at the PAR polymer binding site abolishes the PAR binding activity of Iduna and attenuates its protective actions. Iduna is protective in vivo against NMDA-induced excitotoxicity and middle cerebral artery occlusion (MCAO)-induced stroke in mice. These results define Iduna as the first endogenous inhibitor of parthanatos. Interfering with PAR polymer signaling offers a new therapeutic strategy for the treatment of neurologic disorders.
A lysosomal pathway, characterized by partial rupture or labilization of lysosomal membranes and cathepsin activation, is evoked during camptothecin-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. In this study, comparative and quantitative proteome analyses were performed to identify early changes in lysosomal protein expression/localization from U-937 cells undergoing apoptosis. In 2 independent experiments, among a total of more than 538 proteins putatively identified and quantitated by iTRAQ isobaric labeling and LC-ESI-MS/MS, 18 proteins were found to be upregulated and 9 downregulated in lysosomes purified from early apoptotic compared to control cells. Protein expression was validated by Western blotting on enriched lysosome fractions, and protein localization confirmed by fluorescence confocal microscopy of representative protein candidates, whose functions are associated with lysosomal membrane fluidity and dynamics. These include sterol-4-alpha-carboxylate 3-dehydrogenase (NSDHL), prosaposin (PSAP) and protein kinase C delta (PKC-δ). This comparative proteome analysis provides the basis for novel hypothesis and rationale functional experimentation, where the 3 validated candidate proteins are associated with lysosomal membrane fluidity and dynamics, particularly cholesterol, sphingolipid and glycosphingolipid metabolism.
PMID: 19393779 CAMSID: cams3168
Apoptosis; Camptothecin; Lysosome; iTRAQ reagent; LC-ESI-MS/MS; Confocal Microscopy
Poly(ADP-ribose) polymerase (PARP) inhibitors are undergoing extensive clinical testing for their single-agent activity in homologous recombination- (HR-) deficient tumors and ability to enhance the action of certain DNA damaging agents. Compared to other PARP inhibitors in development, iniparib (4-iodo-3-nitrobenzamide) is notable for its simple structure and the reported ability of its intracellular metabolite 4-iodo-3-nitrosobenzamide to covalently inhibit PARP1 under cell-free conditions. The present preclinical studies were performed to compare the actions iniparib with the more extensively characterized PARP inhibitors olaparib and veliparib.
The abilities of iniparib, olaparib and veliparib to i) selectively induce apoptosis or inhibit colony formation in HR-deficient cell lines, ii) selectively sensitize HR-proficient cells to topoisomerase I poisons and iii) inhibit formation of poly(ADP-ribose) polymer in intact cells were compared.
Consistent with earlier reports, olaparib and veliparib selectively induced apoptosis and inhibited colony formation in cells lacking BRCA2 or ATM. Moreover, like earlier-generation PARP inhibitors, olaparib and veliparib sensitized cells to the topoisomerase I poisons campto-thecin and topotecan. Finally, olaparib and veliparib inhibited formation of poly(ADP-ribose) polymer in intact cells. In contrast, iniparib exhibited little or no ability to selectively kill HR-deficient cells, sensitize cells to topoisomerase I poisons, or inhibit poly(ADP-ribose) polymer formation in situ. In further experiments, iniparib also failed to sensitize cells to cisplatin, gemcitabine or paclitaxel.
While iniparib kills normal and neoplastic cells at high (>40 µM) concentrations, its effects are unlikely to reflect PARP inhibition and should not be used to guide decisions about other PARP inhibitors.
Poly(ADP-ribose) polymerase; enzyme inhibitor; nicotinamide; topoisomerase; homologous recombination
Poly(ADP-ribose) polymerase-1 (PARP-1) is widely involved in cell death responses. Depending on the degree of injury and on cell type, PARP activation may lead to autophagy, apoptosis or necrosis. In HEK293 cells exposed to the alkylating agent N-methyl-N’-nitro-N’-nitrosoguanine (MNNG), we show that PARP-1 activation triggers a necrotic cell death response. The massive poly(ADP-ribose) (PAR) synthesis following PARP-1 activation leads to the modulation of mTORC1 pathway. Shortly after MNNG exposure, NAD+ and ATP levels decrease, while AMP levels drastically increase. We characterized at the molecular level the consequences of these altered nucleotide levels. First, AMP-activated protein kinase (AMPK) is activated and the mTORC1 pathway is inhibited by the phosphorylation of Raptor, in an attempt to preserve cellular energy. Phosphorylation of the mTORC1 target S6 is decreased as well as the phosphorylation of the mTORC2 component Rictor on Thr1135. Finally, Akt phosphorylation on Ser473 is lost and then, cell death by necrosis occurs. Inhibition of PARP-1 with the potent PARP inhibitor AG14361 prevents all of these events. Moreover, the antioxidant N-acetyl-L-cysteine (NAC) can also abrogate all the signaling events caused by MNNG exposure suggesting that reactive oxygen species (ROS) production is involved in PARP-1 activation and modulation of mTOR signaling. In this study, we show that PARP-1 activation and PAR synthesis affect the energetic status of cells, inhibit the mTORC1 signaling pathway and possibly modulate the mTORC2 complex affecting cell fate. These results provide new evidence that cell death by necrosis is orchestrated by the balance between several signaling pathways, and that PARP-1 and PAR take part in these events.
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.
Upon DNA damage induction, DNA-dependent poly(ADP-ribose) polymerases (PARPs) synthesize an anionic poly(ADP-ribose) (pADPr) scaffold to which several proteins bind with the subsequent formation of pADPr-associated multiprotein complexes. We have used a combination of affinity-purification methods and proteomics approaches to isolate these complexes and assess protein dynamics with respect to pADPr metabolism. As a first approach, we developed a substrate trapping strategy by which we demonstrate that a catalytically inactive Poly(ADP-ribose) glycohydrolase (PARG) mutant can act as a physiologically selective bait for the isolation of specific pADPr-binding proteins through its macrodomain-like domain. In addition to antibody-mediated affinity-purification methods, we used a pADPr macrodomain affinity resin to recover pADPr-binding proteins and their complexes. Second, we designed a time course experiment to explore the changes in the composition of pADPr-containing multiprotein complexes in response to alkylating DNA damage-mediated PARP activation. Spectral count clustering based on GeLC-MS/MS analysis was complemented with further analyses using high precision quantitative proteomics through isobaric tag for relative and absolute quantitation (iTRAQ)- and Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics. Here, we present a valuable resource in the interpretation of systems biology of the DNA damage response network in the context of poly(ADP-ribosyl)ation and provide a basis for subsequent investigations of pADPr-binding protein candidates.
The mitochondrial protein apoptosis-inducing factor (AIF) plays a pivotal role in poly(ADP-ribose) polymerase-1 (PARP-1)-mediated cell death (parthanatos), during which it is released from the mitochondria and translocates to the nucleus. Here, we show that AIF is a high affinity poly(ADP-ribose) (PAR)–binding protein and that PAR binding to AIF is required for parthanatos both in vitro and in vivo. AIF bound PAR at a site distinct from AIF’s DNA binding site and this interaction triggered AIF release from the cytosolic side of the mitochondrial outer membrane. Mutation of the PAR binding site in AIF did not affect its NADH oxidase activity, its ability to bind FAD or DNA, or its ability to induce nuclear condensation. However, this AIF mutant was not released from mitochondria and did not translocate to the nucleus or mediate cell death following PARP-1 activation. These results suggest a mechanism for PARP-1 to initiate AIF-mediated cell death and indicate that AIF’s bioenergetic cell survival-promoting functions are separate from its effects as a mitochondrially-derived death effector. Interference with the PAR-AIF interaction or PAR signaling may provide unique opportunities for preventing cell death following activation of PARP-1.
Polycomb group (PcG) proteins are involved in epigenetic silencing where they function as major determinants of cell identity, stem cell pluripotency and the epigenetic gene silencing involved in cancer development. Recently numerous PcG proteins, including CBX4, have been shown to accumulate at sites of DNA damage. However, it remains unclear whether or not CBX4 or its E3 sumo ligase activity is directly involved in the DNA damage response (DDR). Here we define a novel role for CBX4 as an early DDR protein that mediates SUMO conjugation at sites of DNA lesions. DNA damage stimulates sumoylation of BMI1 by CBX4 at lysine 88, which is required for the accumulation of BMI1 at DNA damage sites. Moreover, we establish that CBX4 recruitment to the sites of laser micro-irradiation-induced DNA damage requires PARP activity but does not require H2AX, RNF8, BMI1 nor PI-3-related kinases. The importance of CBX4 in the DDR was confirmed by the depletion of CBX4, which resulted in decreased cellular resistance to ionizing radiation. Our results reveal a direct role for CBX4 in the DDR pathway.
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.
Mass spectrometers can produce a large number of tandem mass spectra. They are unfortunately noise-contaminated. Noises can affect the quality of tandem mass spectra and thus increase the false positives and false negatives in the peptide identification. Therefore, it is appealing to develop an approach to denoising tandem mass spectra.
We propose a novel approach to denoising tandem mass spectra. The proposed approach consists of two modules: spectral peak intensity adjustment and intensity local maximum extraction. In the spectral peak intensity adjustment module, we introduce five features to describe the quality of each peak. Based on these features, a score is calculated for each peak and is used to adjust its intensity. As a result, the intensity will be adjusted to a local maximum if a peak is a signal peak, and it will be decreased if the peak is a noisy one. The second module uses a morphological reconstruction filter to remove the peaks whose intensities are not the local maxima of the spectrum. Experiments have been conducted on two ion trap tandem mass spectral datasets: ISB and TOV. Experimental results show that our algorithm can remove about 69% of the peaks of a spectrum. At the same time, the number of spectra that can be identified by Mascot algorithm increases by 31.23% and 14.12% for the two tandem mass spectra datasets, respectively.
The proposed denoising algorithm can be integrated into current popular peptide identification algorithms such as Mascot to improve the reliability of assigning peptides to spectra.
Availability of the software
The software created from this work is available upon request.
Tandem mass spectrometry has become particularly useful for the rapid identification and characterization of protein components of complex biological mixtures. Powerful database search methods have been developed for the peptide identification, such as SEQUEST and MASCOT, which are implemented by comparing the mass spectra obtained from unknown proteins or peptides with theoretically predicted spectra derived from protein databases. However, the majority of spectra generated from a mass spectrometry experiment are of too poor quality to be interpreted while some of spectra with high quality cannot be interpreted by one method but perhaps by others. Hence a filtering algorithm that removes those spectra with poor quality prior to the database search is appealing.
This paper proposes a support vector machine (SVM) based approach to assess the quality of tandem mass spectra. Each mass spectrum is mapping into the 16 proposed features to describe its quality. Based the results from SEQUEST, four SVM classifiers with the input of the 16 features are trained and tested on ISB data and TOV data, respectively. The superior performance of the proposed SVM classifiers is illustrated both by the comparison with the existing classifiers and by the validation in terms of MASCOT search results.
The proposed method can be employed to effectively remove the poor quality spectra before the spectral searching, and also to find the more peptides or post-translational peptides from spectra with high quality using different search engines or de novo method.
Poly(ADP-ribose) (pADPr) is a polymer assembled from the enzymatic polymerization of the ADP-ribosyl moiety of NAD by poly(ADP-ribose) polymerases (PARPs). The dynamic turnover of pADPr within the cell is essential for a number of cellular processes including progression through the cell cycle, DNA repair and the maintenance of genomic integrity, and apoptosis. In spite of the considerable advances in the knowledge of the physiological conditions modulated by poly(ADP-ribosyl)ation reactions, and notwithstanding the fact that pADPr can play a role of mediator in a wide spectrum of biological processes, few pADPr binding proteins have been identified so far. In this study, refined in silico prediction of pADPr binding proteins and large-scale mass spectrometry-based proteome analysis of pADPr binding proteins were used to establish a comprehensive repertoire of pADPr-associated proteins. Visualization and modeling of these pADPr-associated proteins in networks not only reflect the widespread involvement of poly(ADP-ribosyl)ation in several pathways but also identify protein targets that could shed new light on the regulatory functions of pADPr in normal physiological conditions as well as after exposure to genotoxic stimuli.
Due to the limited coding capacity of their small genomes, human papillomaviruses (HPV) rely extensively on host factors for the completion of their life cycles. Accordingly, most HPV proteins, including the replicative helicase E1, engage in multiple protein interactions. The fact that conserved regions of E1 have not yet been ascribed a function prompted us to use tandem affinity protein purification (TAP) coupled to mass spectrometry to identify novel targets of this helicase. This method led to the discovery of a novel interaction between the N-terminal 40 amino acids of HPV type 11 (HPV11) E1 and the cellular WD repeat protein p80 (WDR48). We found that interaction with p80 is conserved among E1 proteins from anogenital HPV but not among cutaneous or animal types. Colocalization studies showed that E1 can redistribute p80 from the cytoplasm to the nucleus in a manner that is dependent on the E1 nuclear localization signal. Three amino acid substitutions in E1 proteins from HPV11 and -31 were identified that abrogate binding to p80 and its relocalization to the nucleus. In HPV31 E1, these substitutions reduced but did not completely abolish transient viral DNA replication. HPV31 genomes encoding two of the mutant E1 proteins were not maintained as episomes in immortalized primary keratinocytes, whereas one encoding the third mutant protein was maintained at a very low copy number. These findings suggest that the interaction of E1 with p80 is required for efficient maintenance of the viral episome in undifferentiated keratinocytes.
Tandem mass spectrometry has emerged as a cornerstone of high throughput proteomic studies owing in part to various high throughput search engines which are used to interpret these tandem mass spectra. However, majority of experimental tandem mass spectra cannot be interpreted by any existing methods. There are many reasons why this happens. However, one of the most important reasons is that majority of experimental spectra are of too poor quality to be interpretable. It wastes time to interpret these "uninterpretable" spectra by any methods. On the other hand, some spectra of high quality are not able to get a score high enough to be interpreted by existing search engines because there are many similar peptides in the searched database. However, such spectra may be good enough to be interpreted by de novo methods or manually verifying methods. Therefore, it is worth in developing a method for assessing spectral quality, which can used for filtering the spectra of poor quality before any interpretation attempts or for finding the most potential candidates for de novo methods or manually verifying methods.
This paper develops a novel method to assess the quality of tandem mass spectra, which can eliminate majority of poor quality spectra while losing very minority of high quality spectra. First, a number of features are proposed to describe the quality of tandem mass spectra. The proposed method maps each tandem spectrum into a feature vector. Then Fisher linear discriminant analysis (FLDA) is employed to construct the classifier (the filter) which discriminates the high quality spectra from the poor quality ones. The proposed method has been tested on two tandem mass spectra datasets acquired by ion trap mass spectrometers.
Computational experiments illustrate that the proposed method outperforms the existing ones. The proposed method is generic, and is expected to be applicable to assessing the quality of spectra acquired by instruments other than ion trap mass spectrometers.
Translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus can play a major role in neuronal death elicited by oxidant stress. The time course of nuclear translocation of AIF after experimental stroke may vary with the severity of injury and may be accelerated by oxidant stress associated with reperfusion and nitric oxide (NO) production. Western immunoblots of AIF on nuclear fractions of ischemic hemisphere of male mice showed no significant increase with 1 hour of middle cerebral artery occlusion and no reperfusion, whereas increases were detectable after 6 and 24 hours of permanent ischemia. However, as little as 20 minutes of reperfusion after 1 hour of middle cerebral artery occlusion resulted in an increase in nuclear AIF coincident with an increase in poly(ADP-ribose) polymer (PAR) formation. Further nuclear AIF accumulation was seen at 6 and 24 hours of reperfusion. In contrast, 20 minutes of reperfusion after 2 hours of occlusion did not increase nuclear AIF. In this case, nuclear AIF became detectable at 6 and 24 hours of reperfusion. With brief occlusion of 30 minute duration, nuclear AIF remained undetectable at both 20 minutes and 6 hours and became evident only after 24 hours of reperfusion. Inhibition of neuronal NO synthase attenuated formation of PAR and nuclear AIF accumulation. Gene deletion of neuronal NO synthase also attenuated nuclear AIF accumulation. Therefore, reperfusion accelerates AIF translocation to the nucleus when focal ischemia is of moderate duration (1 hour), but is markedly delayed after brief ischemia (30 minutes). Nuclear translocation of AIF eventually occurs with prolonged focal ischemia with or without reperfusion. Neuronally-derived NO is a major factor contributing to nuclear AIF accumulation after stroke.
apoptosis; middle cerebral artery; mouse; poly(ADP-ribose); reperfusion; stroke
In the "post-genome" era, mass spectrometry (MS) has become an important method for the analysis of proteins and the rapid advancement of this technique, in combination with other proteomics methods, results in an increasing amount of proteome data. This data must be archived and analysed using specialized bioinformatics tools.
We herein describe "PARPs database," a data analysis and management pipeline for liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics. PARPs database is a web-based tool whose features include experiment annotation, protein database searching, protein sequence management, as well as data-mining of the peptides and proteins identified.
Using this pipeline, we have successfully identified several interactions of biological significance between PARP-1 and other proteins, namely RFC-1, 2, 3, 4 and 5.
Poly(ADP-ribose) polymerases (PARPs) are members of a family of enzymes that utilize NAD+ as substrate to form large ADP-ribose polymers (PAR) in the nucleus. PAR has a very short half life due to its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). PARP-1 mediates acute neuronal cell death induced by a variety of insults including cerebral ischemia, MPTP-induced Parkinsonism, and CNS trauma. While PARP-1 is localized to the nucleus, PARG resides in both the nucleus and cytoplasm. Surprisingly, there appears to be only one gene encoding PARG activity, which has been characterized in vitro to generate different splice variants, in contrast to the growing family of PARPs. Little is known regarding the spatial and functional relationships of PARG and PARP-1. Here we evaluate PARG expression in the brain and its cellular and subcellular distribution in relation to PARP-1. Anti-PARG (α–PARG) antibodies raised in rabbits using a purified 30 kDa C-terminal fragment of murine PARG recognize a single band at 111 kDa in the brain. Western blot analysis also shows that PARG and PARP-1 are evenly distributed throughout the brain. Immunohistochemical studies using α-PARG antibodies reveal punctate cytosolic staining, whereas anti-PARP-1 (α–PARP-1) antibodies demonstrate nuclear staining. PARG is enriched in the mitochondrial fraction together with manganese superoxide dismutase (MnSOD) and cytochrome C (Cyt C) following whole brain subcellular fractionation and Western blot analysis. Confocal microscopy confirms the co-localization of PARG and Cyt C. Finally, PARG translocation to the nucleus is triggered by NMDA-induced PARP-1 activation. Therefore, the subcellular segregation of PARG in the mitochondria and PARP-1 in the nucleus suggests that PARG translocation is necessary for their functional interaction. This translocation is PARP-1 dependent, further demonstrating a functional interaction of PARP-1 and PARG in the brain.
NMDA; mitochondria; nucleus; immunostaining; subcellular fractionation
Some polychlorinated biphenyl (PCB) congeners are CYP1A1 inducers, and induction of this enzyme in the placenta has been linked to adverse effects on fetal development. The objective of this study was to determine if the body burden of PCBs is related to placental CYP1A1 activity in Inuit women from Nunavik (northern Québec), a population highly exposed to organochlorines. Placenta and cord blood samples were obtained from 35 Inuit women and 30 women from a southern Québec community exposed to background levels of organochlorines. We measured PCB concentrations in all cord plasma samples and in a subset of placenta samples from the Nunavik group and assessed CYP1A1 activity (ethoxyresorufin-O-deethylase; EROD) in placental microsomes from all participants. Concentrations of PCBs in cord plasma were strongly correlated to those in placenta (Pearson's r) = 0.77-0.97, p < 0.001) and were on average 4-fold higher in Inuit women than in southern Québec women [for PCB 153, the geometric means (geometric SDs) were 83.3 (1.9) ng/g lipid vs. 16.9 (1.6) ng/g lipid, respectively]. Despite this difference in PCB body burden, both study groups had similar EROD activities when data were stratified according to tobacco smoking. Although simple correlation analysis first showed that placental EROD activity was correlated with PCB 153 plasma concentration in the Nunavik group, a multivariate analysis failed to demonstrate a significant contribution of PCBs to EROD activity when tobacco smoking was included in the analysis. We conclude that dietary exposure to PCBs in Inuit women from Nunavik does not significantly influence EROD activity in the placenta, implicating tobacco smoking as the major modulating factor.
Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is activated by binding to DNA breaks induced by ionizing radiation or through repair of altered bases in DNA by base excision repair. Mice lacking PARP-1 and, in certain cases, the cells derived from these mice exhibit hypersensitivity to ionizing radiation and alkylating agents. In this study we investigated base excision repair in cells lacking PARP-1 in order to elucidate whether their augmented sensitivity to DNA damaging agents is due to an impairment of the base excision repair pathway. Extracts prepared from wild-type cells or cells lacking PARP-1 were similar in their ability to repair plasmid DNA damaged by either X-rays (single-strand DNA breaks) or by N-methyl-N′-nitro-N-nitrosoguanidine (methylated bases). In addition, we demonstrated in vivo that PARP-1-deficient cells treated with N-methyl-N′-nitro-N-nitrosoguanidine repaired their genomic DNA as efficiently as wild-type cells. Therefore, we conclude that cells lacking PARP-1 have a normal capacity to repair single-strand DNA breaks inflicted by X-irradiation or breaks formed during the repair of modified bases. We propose that the hypersensitivity of PARP-1 null mutant cells to γ-irradiation and alkylating agents is not directly due to a defect in DNA repair itself, but rather results from greatly reduced poly(ADP-ribose) formation during base excision repair in these cells.
Proteases are now firmly established as major regulators of the “execution” phase of apoptosis. Here, we examine the role of proteases and their relationship to ceramide, a proposed mediator of apoptosis, in the tumor necrosis factor-α (TNF-α)–induced pathway of cell death. Ceramide induced activation of prICE, the protease that cleaves the death substrate poly(ADP-ribose) polymerase. Bcl-2 inhibited ceramide-induced death, but not ceramide generation. In contrast, Cytokine response modifier A (CrmA), a potent inhibitor of Interleukin-1β converting enzyme and related proteases, inhibited ceramide generation and prevented TNF-α–induced death. Exogenous ceramide could overcome the CrmA block to cell death, but not the Bcl-2 block. CrmA, however, did not inhibit the activation of nuclear factor (NF)-κB by TNF-α, demonstrating that other signaling functions of TNF-α remain intact and that ceramide does not play a role in the activation of NF-κB. These studies support a distinct role for proteases in the signaling/activation phase of apoptosis acting upstream of ceramide formation.
The PARP family member poly(ADP-ribose) polymerase 3 (PARP3) is structurally related to the well characterized PARP1 that orchestrates cellular responses to DNA strand breaks and cell death by the synthesis of poly(ADP-ribose). In contrast to PARP1 and PARP2, the functions of PARP3 are undefined. Here, we reveal critical functions for PARP3 during vertebrate development.
We have used several in vitro and in vivo approaches to examine the possible functions of PARP3 as a transcriptional regulator, a function suggested from its previously reported association with several Polycomb group (PcG) proteins. We demonstrate that PARP3 gene occupancy in the human neuroblastoma cell line SK-N-SH occurs preferentially with developmental genes regulating cell fate specification, tissue patterning, craniofacial development and neurogenesis. Addressing the significance of this association during zebrafish development, we show that morpholino oligonucleotide-directed inhibition of parp3 expression in zebrafish impairs the expression of the neural crest cell specifier sox9a and of dlx3b/dlx4b, the formation of cranial sensory placodes, inner ears and pectoral fins. It delays pigmentation and severely impedes the development of the median fin fold and tail bud.
Our findings demonstrate that Parp3 is crucial in the early stages of zebrafish development, possibly by exerting its transcriptional regulatory functions as early as during the specification of the neural plate border.
Poly(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.
PARP-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.
This 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.
Epithelial 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.
The 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.
The 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.