PARP1 parylation promotes silent locus transmission in the nucleolus: the suspicion confirmed.

Parylation modulates various processes, including transcription. In this issue of Molecular Cell, Guetg et al. (2012) show that, in the nucleolus, PARP1-mediated parylation of TIP5 promotes the silencing of rDNA chromatin during replication, uncovering the mechanism by which PARP1 ensures that silent rDNA regions are properly inherited.

Quantitative proteomics and dynamic imaging reveal that G3BP-mediated stress granule assembly is poly(ADP-ribose)-dependent following exposure to MNNG-induced DNA alkylation.

Poly(ADP-ribose) (pADPr) is a heterogenic molecule synthesised from NAD by poly(ADP-ribose) polymerases (PARPs). Many cellular functions from genome integrity surveillance, cell cycle progression and DNA repair to apoptosis are affected by pADPr through its network of associated proteins. Using quantitative proteomics, we established a temporal map of pADPr-associated complexes upon genotoxic stress. Results suggested a strong pADPr association to many proteins involved in stress granule formation, notably the ras-GAP SH3-binding protein G3BP, as well as in the later phases of alkylation-stress-induced responses. Further investigation with dynamic imaging clearly demonstrated a pADPr-dependent initiation of stress granule assembly originating from the nucleus. The co-transfection of G3BP with poly(ADP-ribose) glycohydrolase (PARG) indicates that pADPr is involved in modulating the nuclear translocation of G3BP. Moreover, a peptide pADPr blot assay of G3BP revealed that pADPr binds to the glycine-arginine-rich domain of G3BP. Thereafter, we established a comprehensive G3BP interactome in the presence of pADPr. Our findings establish a novel function for pADPr in the formation of G3BP-induced stress granules upon genotoxic stress.

Quantitative proteomics profiling of the poly(ADP-ribose)-related response to genotoxic stress.

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.

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis.

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes.

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.

Effect of PON1 polymorphism on HDL antioxidant potential is blunted with aging.

Paraoxonase1 is a HDL-associated enzyme, which is responsible for their antioxidant property. This study was aimed to investigate the effect of PON1 [Q192R] and [L55M] genotypes on susceptibility of LDL and HDL to lipid peroxidation and on antioxidant activity of HDL as a function of aging. Seventy-eight healthy subjects distributed in two age groups, young (20-30 years) and elderly (60-89 years) were recruited. PON1 activities and genotype polymorphisms were determined for each subject. LDL and HDL susceptibility to lipid peroxidation was evaluated by the measure of lag-phase (LP) for conjugated diene formation. HDL antioxidant property was evaluated by the measure of their capacity to protect LDL against lipid peroxidation. Our results show that LP for LDL and HDL peroxidation decreased with age of donors. Moreover, PON1 genotypes affect significantly the susceptibility of LDL and HDL to lipid peroxidation. Furthermore, basal- and salt-stimulated paraoxonase as well arylesterase activities were significantly reduced in elderly compared to young subjects. These results show a beneficial effect of PON1 towards susceptibility of HDL to oxidation as well to their antioxidant effect. However, this PON1 protective effect seems to be blunted with advancing age. Altogether our results suggest that the decrease in the PON1 protective effect with aging may contribute to the acceleration of the atherosclerosis process in elderly.

Comparative proteome analysis of human epithelial ovarian cancer.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Phenolic-extract from argan oil (Argania spinosa L.) inhibits human low-density lipoprotein (LDL) oxidation and enhances cholesterol efflux from human THP-1 macrophages.

Argan oil is rich in unsaturated fatty acids, tocopherol and phenolic compounds. These protective molecules make further study of its cardiovascular diseases (CVDs) action interesting. Furthermore, no previous study has explored the antioxidant activity of argan oil in comparison with olive oil. The present study was conducted to evaluate the beneficial properties of Virgin argan oil phenolic extracts (VAO-PE) towards CVD by: (A) protecting human (low-density lipoprotein, LDL) against lipid peroxidation and (B) promoting high-density lipoprotein (HDL)-mediated cholesterol efflux. Human LDLs were oxidized by incubation with CuSO(4) in the presence of different concentrations of VAO-PE (0-320mug/ml). LDL lipid peroxidation was evaluated by conjugated diene and MDA formation as well as Vitamin E disappearance. Incubation of LDL with VAO-PE significantly prolonged the lag-phase and lowered the progression rate of lipid peroxidation (P<0.01) and reduced the disappearance of Vitamin E in a concentration-dependent manner. Incubation of HDL with VAO-PE significantly increased the fluidity of the HDL phospholipidic bilayer (P=0.0004) and HDL-mediated cholesterol efflux from THP-1 macrophages. These results suggest that Virgin argan oil provides a source of dietary phenolic antioxidants, which prevent cardiovascular diseases by inhibiting LDL-oxidation and enhancing reverse cholesterol transport. These properties increase the anti-atherogenic potential of HDL.

Age-related decrease in high-density lipoproteins antioxidant activity is due to an alteration in the PON1's free sulfhydryl groups.

The aim of this study was to evaluate the antioxidant activity of HDL with aging and to investigate the implication of PON1 in this process. The study involved 54 healthy subjects distributed in two age groups, young (20-25 years) and elderly (65-85 years). Lipid peroxidation was induced by *OH and O2*- oxygen free radicals produced by gamma-radiolysis of water. LDL oxidation was followed by the measurement of conjugated diene (CD), lipid peroxide (LP) and malondialdehyde (MDA) formation. PON1 was purified separately from young (Y-PON1) and elderly subjects (E-PON1). PON1 activity and structure was followed by measurement of PON1 paraoxonase (p.ase) activity, titration of the SH groups, and electrophoretic mobility by SDS-PAGE. Our results show a significant decrease in the HDL antioxidant activity: percentage of protection against CD formation=27.70% (p<0.01) for E-HDL versus 73.08% (p<0.001) for Y-HDL. Moreover, E-PON1 showed a lower antioxidant activity when compared to Y-PON1 47.08% versus 78.14%, respectively (p<0.0001). Exposition of PON1 to *OH and O2*- oxygen free radicals induced a significant decrease in PON1 p.ase activity as well as a reduction in the number of PON1's free sulfhydryl groups. Moreover, our results show a close association between PON1's free sulfhydryl groups and its capacity to protect LDL against lipid peroxidation. There was a significant decrease in the number of free sulfhydryls between Y-PON1 and E-PON1 with respect to cysteine-284 amino acid residues (p<0.0092).

Marrubium vulgare extract inhibits human-LDL oxidation and enhances HDL-mediated cholesterol efflux in THP-1 macrophage.

The objective of the present study was to elucidate the beneficial properties of aqueous extracts of Marrubium vulgare (AEM) towards cardiovascular disease by protecting human-LDL against lipid peroxidation and promoting HDL-mediated cholesterol efflux. Human-LDL were oxidised by incubation with CuSO(4) in the presence of increased concentrations of AEM (0-100 microg/ml). LDL lipid peroxidation was evaluated by conjugated diene formation, vitamin E disappearance as well as LDL-electrophoretic mobility. HDL-mediated cholesterol efflux assay was carried out in human THP-1 macrophages. Incubation of LDL with AEM significantly prolonged the lag phase (P=0.014), lowered the progression rate of lipid peroxidation (P=0.004), reduced the disappearance of vitamin E and the electrophoretic mobility in a dose-dependent manner. Also, incubation of HDL with AEM significantly increased HDL-mediated cholesterol efflux from THP-1 macrophages implicating an independent ATP binding cassette A1 (ABCA1) pathways. Our findings suggest that M. vulgare provides a source of natural antioxidants, which inhibit LDL oxidation and enhance reverse cholesterol transport and thus can prevent cardiovascular diseases development. These antioxidant properties increase the anti-atherogenic potential of HDL.

Protective effects of Peganum harmala L. extract, harmine and harmaline against human low-density lipoprotein oxidation.

Oxidative modification of low-density lipoprotein (LDL) particles has been implicated in the process of atherogenesis. Antioxidants that prevent LDL from oxidation may reduce atherosclerosis. We have investigated the protective effect of Peganum harmala-extract (P-extract) and the two major alkaloids (harmine and harmaline) from the seeds of P. harmala against CuSO4-induced LDL oxidation. Through determination of the formation of malondialdehyde (MDA) and conjugated diene as well as the lag phase, the extract (P-extract) and compounds were found to possess an inhibitory effect. Moreover, harmaline and harmine reduced the rate of vitamin E disappearance and exhibited a significant free radical scavenging capacity (DPPH*). However, harmaline had a markedly higher antioxidant capacity than harmine in scavenging or preventive capacity against free radicals as well as inhibiting the aggregation of the LDL protein moiety (apolipoprotein B) induced by oxidation. The results suggested that P. harmala compounds could be a major source of compounds that inhibit LDL oxidative modification induced by copper.

A rapid and sensitive UPLC-MS/MS method for the simultaneous quantification of serum androsterone glucuronide, etiocholanolone glucuronide, and androstan-3α, 17ß diol 17-glucuronide in postmenopausal women.

Quantification of steroidal glucuronide conjugates by the indirect methods of immunoassay and GC-MS/MS may underestimate some conjugates since hydrolysis is needed in sample processing. In the present work, a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous direct quantification of androsterone glucuronide, etiocholanolone glucuronide, and androstan-3α, 17ß diol 17-glucuronide in postmenopausal women's serum. The quantification limits are 0.1ng/mL for 3α-diol-17G and 4ng/mL for both ADT-G and Etio-G, respectively, with an extraction from 200µL serum while the total run time is less than 6min for all three glucuronides. In this method, solid phase extraction is used for sample preparation. The assay has been validated in compliance with EndoCeutics SOPs and FDA guidelines for bioanalytical method development and validation. The recovery of glucuronides in stripped serum is consistent with that in unstripped serum, where the average difference in stripped and unstripped is less than 10%. A linear regression model fits well the standard curves of all three compounds with R≥0.99 where the weighting factor is 1/X. Interday accuracy and CV for all levels of QCs are within the range of 15% in both stripped and unstripped serum while all calibration curves are within the range of 6% except for LLOQs, which are within the range of 9%. Other parameters have also been assessed such as selectivity, matrix, lipemic and hemolysis effects as well as stabilities in solution and matrix. Incurred sample reanalysis has been performed with a result of over 93% within 20% of the original values. This reliable, sensitive and fast method is ready for large-scale clinical sample assays.

Validated LC-MS/MS simultaneous assay of five sex steroid/neurosteroid-related sulfates in human serum.

Conventionally, the concentration of steroidal sulfates was estimated by indirect or immuno­based assays before the use of liquid-chromatography tandem mass spectrometry (LC-MS/MS). In the present study, a validated LC-MS/MS method is described for the simultaneous quantification of dehydroepiandrosterone sulfate (DHEA-S), estrone sulfate (E1­S), androsterone sulfate (ADT­S), pregnenolone sulfate (Preg­S) and allopregnanolone sulfate (Allopreg­S). E1­S binding to serum proteins was observed, especially for the high concentration quality control serum samples, leading to -10 to -15% bias using a polymer-based SPE. This protein binding can be efficiently eliminated using a Waters Oasis™ WAX following the same extraction procedure. Most likely, the E1­S binding elimination on Oasis™ WAX can be attributed to its different sorbent structure, where the benzeno group of E1-S can interact with the benzene of the backbone of Oasis™ WAX. With this improvement, the method has been fully validated according to the FDA guidelines. The low quantification limits (LLOQs) are 40ng/mL, 40pg/mL, 5ng/mL, 1.5ng/mL and 0.25ng/mL for DHEA­S, E1-S, ADT­S, Preg­S and Allopreg-S, respectively. A good linearity is obtained with R>0.99 for all compounds within the appropriate calibration range. Accuracies of all levels of QCs are within the range of 10% for DHEA-S, E1­S, ADT­S and Preg­S while for Allopreg­S, the accuracy is within the 15% range. The interday coefficient variance is 5.5-9.5% for the low limits of quantification of all five compounds while values of 1.3-9.9% are found for higher levels of QCs of all five compounds. Recovery of the five compounds in stripped serum is equivalent to that in unstripped serum. The average recovery difference is less than 5% between stripped and unstripped serum for each compound. All results of other test parameters such as matrix, hemolysis and lipemic effects as well as stabilities meet the acceptance criteria of EndoCeutics SOPs and FDA guidelines.

The anti-inflammatory effect of paraoxonase 1 against oxidized lipids depends on its association with high density lipoproteins.

AIM: The aims of this study were to investigate whether purified PON1 can reduce the pro-inflammatory effect of oxidized phospholipids and whether the effect depended on its association with HDL. MAIN METHODS: Lipid peroxidation was induced by copper ions and was measured using the conjugated diene method. Lysophosphatidylcholine (lyso-PC) formation was measured by HPLC with evaporative light scattering detection (ELSD) and ICAM-1 expression on Ea.hy926 endothelial cells was analyzed by flow cytometry. KEY FINDINGS: Purified PON1 significantly inhibited copper-induced oxidation of LDL and HDL, causing a 60.5% and 77.7% decrease in conjugated diene formation, respectively. Incubating PON1 with oxLDL caused a significant increase in lyso-PC levels, while oxHDL caused a significant decrease. PON1 (12.5 to 50 µg/mL) had a pro-inflammatory effect in the presence of oxLDL, increasing ICAM-1 levels in Ea.hy926 cells by 33.0% and 40.6% (p<0.001) respectively, and had an anti-inflammatory effect in the presence of oxHDL, causing a 3-fold reduction in ICAM-1 levels. PON1 also caused a significant decrease in TNFα and purified lyso-PC-induced ICAM-1 expression. The results obtained with reconstituted HDL as well as LCAT and PAF-AH inhibitors suggested that the anti-inflammatory effect of PON1 against oxidized lipids is dependent on its association with HDL. SIGNIFICANCE: Our results clearly showed that PON1 is involved in the anti-inflammatory effect of HDL and that the effect appears to depend on its association with HDL.

Age-related impairment of HDL-mediated cholesterol efflux.

Our aim in this study was to investigate the effect of aging on the capacity of HDLs to promote reverse cholesterol transport. HDLs were isolated from plasma of young (Y-HDL) and elderly (E-HDL) subjects. HDL-mediated cholesterol efflux was studied using THP-1 and J774 macrophages. Our results show that E-HDLs present a lower capacity to promote cholesterol efflux than Y-HDLs (41.7 +/- 1.4% vs. 49.0 +/- 2.2%, respectively; P = 0.013). Reduction in the HDL-mediated cholesterol efflux capacity with aging was more significant with HDL(3) than HDL(2) (Y-HDL(3), 57.3 +/- 1% vs. E-HDL(3), 50.9 +/- 2%; P = 0.012). Moreover, our results show that ABCA1-mediated cholesterol efflux is the more affected pathway in terms of cholesterol-removing capacity. Interestingly, the composition and structure of HDL revealed a reduction in the phosphatidylcholine-sphingomyelin ratio (E-HDL, 32.7 +/- 2.7 vs. Y-HDL, 40.0 +/- 1.9; P = 0.029) and in the phospholipidic layer membrane fluidity in E-HDL compared with Y-HDL as well as an alteration in the apolipoprotein A-I structure and charge. In conclusion, our results shown that E-HDLs present a reduced capacity to promote cholesterol efflux, principally through the ABCA1 pathway, and this may explain the increase of the incidence of cardiovascular diseases observed during aging.

Antiatherogenic activity of extracts of Argania spinosa L. pericarp: beneficial effects on lipid peroxidation and cholesterol homeostasis.

Prevention of lipoprotein oxidation by natural compounds may prevent atherosclerosis via reducing early atherogenesis. In this study, we investigated for the first time the beneficial properties of methanolic extract of argania pericarp (MEAP) towards atherogenesis by protecting human low-density lipoprotein (LDL) against oxidation while promoting high-density lipoprotein (HDL)-mediated cholesterol efflux. By measuring the formation of malondialdehyde (MDA) and conjugated diene as well as the lag phase and the progression rate of lipid peroxidation, the MEAP was found to possess an inhibitory effect. In addition, MEAP reduced the rate of disappearance of alpha-tocopherol as well as the apoB electrophoretic mobility in a dose-dependent manner. These effects are related to the free radical scavenging and copper-chelating effects of MEAP. In terms of cell viability, MEAP has shown a cytotoxic effect (0-40 microg/mL). Incubation of 3H-cholesterol-loaded J774 macrophages with HDL in the presence of increasing concentrations of MEAP enhanced HDL-mediated cholesterol efflux independently of ABCA1 receptor pathways. Our findings suggest that argania seed pericarp provides a source of natural antioxidants that inhibit LDL oxidation and enhance cholesterol efflux and thus can prevent development of cardiovascular diseases.