The Streptococcus pyogenes NAD+ glycohydrolase modulates epithelial cell PARylation and HMGB1 release

Streptococcus pyogenes uses the cytolysin streptolysin O (SLO) to translocate an enzyme, the S. pyogenes NAD⁺ glycohydrolase (SPN), into the host cell cytosol. However, the function of SPN in this compartment is not known. As a complication, many S. pyogenes strains express a SPN variant lacking NAD⁺ glycohydrolase (NADase) activity. Here, we show that SPN modifies several SLO‐ and NAD⁺‐dependent host cell responses in patterns that correlate with NADase activity. SLO pore formation results in hyperactivation of the cellular enzyme poly‐ADP‐ribose polymerase‐1 (PARP‐1) and production of polymers of poly‐ADP‐ribose (PAR). However, while SPN NADase activity moderates PARP‐1 activation and blocks accumulation of PAR, these processes continued unabated in the presence of NADase‐inactive SPN. Temporal analyses revealed that while PAR production is initially independent of NADase activity, PAR rapidly disappears in the presence of NADase‐active SPN, host cell ATP is depleted and the pro‐inflammatory mediator high‐mobility group box‐1 (HMGB1) protein is released from the nucleus by a PARP‐1‐dependent mechanism. In contrast, HMGB1 is not released in response to NADase‐inactive SPN and instead the cells release elevated levels of interleukin‐8 and tumour necrosis factor‐α. Thus, SPN and SLO combine to induce cellular responses subsequently influenced by the presence or absence of NADase activity.     

Hydrogen sulfide is a novel potential virulence factor of Mycoplasma pneumoniae: characterization of the unusual cysteine desulfurase/desulfhydrase HapE

Mycoplasma pneumoniae is a human pathogen causing atypical pneumonia with a minimalized and highly streamlined genome. So far, hydrogen peroxide production, cytadherence, and the ADP‐ribosylating CARDS toxin have been identified as pathogenicity determinants. We have studied haemolysis caused by M. pneumoniae, and discovered that hydrogen peroxide is responsible for the oxidation of heme, but not for lysis of erythrocytes. This feature could be attributed to hydrogen sulfide, a compound that has previously not been identified as virulence factor in lung pathogens. Indeed, we observed hydrogen sulfide production by M. pneumoniae. The search for a hydrogen sulfide‐producing enzyme identified HapE, a protein with similarity to cysteine desulfurases. In contrast to typical cysteine desulfurases, HapE is a bifunctional enzyme: it has both the cysteine desulfurase activity to produce alanine and the cysteine desulfhydrase activity to produce pyruvate and hydrogen sulfide. Experiments with purified HapE showed that the enzymatic activity of the protein is responsible for haemolysis, demonstrating that HapE is a novel potential virulence factor of M. pneumoniae.     

Deficiency of terminal ADP‐ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease

Adenosine diphosphate (ADP)‐ribosylation is a post‐translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP‐ribosylation reactions are the poly(ADP‐ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP‐ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP‐ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP‐ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP‐interacting protein that removes mono(ADP‐ribosyl)ation on glutamate amino acid residues in PARP‐modified proteins. X‐ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl‐(ADP‐ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP‐ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans.     

Reactive oxygen species‐induced parthanatos of immunocytes by human cytomegalovirus‐associated substance

Previous studies have examined various immune evasion strategies of human cytomegalovirus (HCMV) to gain understanding of its pathogenesis. Although the mechanism that underlies immunocyte destruction near HCMV‐infected lesions has yet to be established, it is here shown that substances produced by HCMV‐infected cells induce death in several types of immunocytes, but not in fibroblasts or astrocytomas. These substances contain HCMV proteins and were termed HCMV‐associated insoluble substance (HCMVAIS). The mechanism by which HCMVAIS induces cell death was characterized to improve understanding the death of immunocytes near HCMV‐infected lesions. HCMVAIS were found to trigger production of intracellular nicotinamide adenine dinucleotide phosphate oxidase‐derived reactive oxygen species (ROS), resulting in cell death, this effect being reversed following treatment with ROS inhibitors. Cell death was not induced in splenocytes from NOX‐2 knockout mice. It was hypothesized that DNA damage induced by oxidative stress initiates poly ADP‐ribose polymerase‐1 (PARP‐1)‐mediated cell death, or parthanatos. HCMVAIS‐induced cell death is accompanied by PARP‐1 activation in a caspase‐independent manner, nuclear translocation of apoptosis‐inducing factor (AIF), and DNA fragmentation, which are typical features of parthanatos. Treatment with an AIF inhibitor decreased the rate of HCMVAIS‐induced cell death, this being confirmed by hematoxylin and eosin staining; cell death in most HCMV‐positive foci in serial section samples of a large intestine with HCMV infection was TUNEL‐positive, cleaved caspase 3‐negative and CD45‐positive. Taken together, these data suggest that HCMV inhibits local immune responses via direct killing of immunocytes near HCMV‐infected cells through ROS‐induced parthanatos by HCMVAIS.     

NAMPT-derived NAD+ fuels PARP1 to promote skin inflammation through parthanatos cell death

Several studies have revealed a correlation between chronic inflammation and nicotinamide adenine dinucleotide (NAD⁺) metabolism, but the precise mechanism involved is unknown. Here, we report that the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the salvage pathway of NAD⁺ biosynthesis, reduced oxidative stress, inflammation, and keratinocyte DNA damage, hyperproliferation, and cell death in zebrafish models of chronic skin inflammation, while all these effects were reversed by NAD⁺ supplementation. Similarly, genetic and pharmacological inhibition of poly(ADP-ribose) (PAR) polymerase 1 (Parp1), overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, inhibition of NADPH oxidases, and reactive oxygen species (ROS) scavenging all phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH oxidases/NAMPT/PARP/AIFM1 axis decreased the expression of pathology-associated genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant induction of NAMPT and PARP activity, together with AIFM1 nuclear translocation, was observed in lesional skin from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD⁺, mediates skin inflammation through parthanatos cell death.

A study of chronic skin inflammation reveals that hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation via parthanatos cell death, identifying NAMPT, PARP1 and AIFM1 as novel therapeutic targets for psoriasis.

Highlights

• Nicotinamide phosphoribosyltransferase (NAMPT) inhibition alleviates inflammation in zebrafish and human organotypic 3D skin models of psoriasis.
• NADPH oxidase–derived reactive oxygen species (ROS) mediate keratinocyte DNA damage and poly(ADP-ribose) polymerase 1 (PARP1) overactivation.
• Inhibition of parthanatos cell death phenocopies the effects of NAMPT inhibition in zebrafish and human psoriasis models.
• NAMPT and poly(ADP-ribose) (PAR) metabolism is altered in psoriasis patients.

     

IDO induction in IFN-γ activated astroglia: A role in improving cell viability during oxidative stress

Abstract

In the central nervous system (CNS), astrocytes play an integral role in the maintenance of neuronal viability and function. Inflammation within the CNS increases the concentration of oxidative metabolites and, therefore, the potential for NAD depletion through increased poly-(ADP-ribose) polymerase (PARP) activity. However, the activity of indoleamine 2,3-dioxygenase (IDO), the rate limiting enzyme for de novo NAD synthesis, is also markedly increased in astrocytes during inflammation. This study investigated the role of IDO induction in the maintenance of intracellular NAD and its relationship to improved cell viability under conditions of increased oxidative stress in the human astroglioma cell line, HTB-138. Treatment with the pro-inflammatory cytokine IFN-γ increased IDO activity in these cells. Intracellular NAD levels also increased significantly after treatment with IFN-γ in the presence of a PARP inhibitor. Pretreatment of astroglial cells with IFN-γ significantly moderated both the drop in intracellular NAD concentration and cell death following exposure to hydrogen peroxide. These results suggest that induction of IDO and subsequent de novo NAD synthesis may contribute to the maintenance of intracellular NAD levels and cell viability under conditions of increased oxidative stress.     

Poly (ADP‐ribose) polymerase inhibition protects against myocardial ischaemia/reperfusion injury via suppressing mitophagy

Myocardial ischaemia/reperfusion (I/R) injury attenuates the beneficial effects of reperfusion therapy. Poly(ADP‐ribose) polymerase (PARP) is overactivated during myocardial I/R injury. Mitophagy plays a critical role in the development of myocardial I/R injury. However, the effect of PARP activation on mitophagy in cardiomyocytes is unknown. In this study, we found that I/R induced PARP activation and mitophagy in mouse hearts. Poly(ADP‐ribose) polymerase inhibition reduced the infarct size and suppressed mitophagy after myocardial I/R injury. In vitro, hypoxia/reoxygenation (H/R) activated PARP, promoted mitophagy and induced cell apoptosis in cardiomyocytes. Poly(ADP‐ribose) polymerase inhibition suppressed H/R‐induced mitophagy and cell apoptosis. Parkin knockdown with lentivirus vectors inhibited mitophagy and prevented cell apoptosis in H/R‐treated cells. Poly(ADP‐ribose) polymerase inhibition prevented the loss of the mitochondrial membrane potential (ΔΨm). Cyclosporin A maintained ΔΨm and suppressed mitophagy but FCCP reduced the effect of PARP inhibition on ΔΨm and promoted mitophagy, indicating the critical role of ΔΨm in H/R‐induced mitophagy. Furthermore, reactive oxygen species (ROS) and poly(ADP‐ribosylation) of CypD and TSPO might contribute to the regulation of ΔΨm by PARP. Our findings thus suggest that PARP inhibition protects against I/R‐induced cell apoptosis by suppressing excessive mitophagy via the ΔΨm/Parkin pathway.     

Critical role of poly(ADP‐ribose) polymerase‐1 in modulating the mode of cell death caused by continuous oxidative stress

Continuously generated hydrogen peroxide (H₂O₂) inhibits typical apoptosis and instead initiates a caspase‐independent, apoptosis‐inducing factor (AIF)‐mediated pyknotic cell death. This may be related to H₂O₂‐mediated DNA damage and subsequent ATP depletion, although the exact mechanisms by which the mode of cell death is decided after H₂O₂ exposure are still unclear. Accumulated evidence and our previous data led us to hypothesize that continuously generated H₂O₂, not an H₂O₂ bolus, induces severe DNA damage, signaling poly(ADP‐ribose) polymerase‐1 (PARP‐1) activation, ATP depletion, and eventually caspase‐independent cell death. Results from the present study support that H₂O₂ generated continuously by glucose oxidase causes excessive DNA damage and PARP‐1 activation. Blockage of PARP‐1 by a siRNA transfection or by pharmacological inhibitor resulted in the significant inhibition of ATP depletion, loss of mitochondrial membrane potential, nuclear translocation of AIF and endonuclease G, and eventually conversion to caspase‐dependent apoptosis. Overall, the current study demonstrates the different roles of PARP‐1 inhibition in modulation of cell death according to the method of H₂O₂ exposure, that is, continuous generation versus a direct addition. J. Cell. Biochem. 108: 989–997, 2009. © 2009 Wiley‐Liss, Inc.     

Molecular mechanisms of apoptosis induced by ajoene in 3T3-L1 adipocytes

Objective : Determine the biochemical pathways involved in induction of apoptosis by ajoene, an organosulfur compound from garlic. Research Methods and Procedures : Mature 3T3‐L1 adipocytes were incubated with ajoene at concentrations up to 200 μM. Viability and apoptosis were quantified using an MTS‐based cell viability assay and an enzyme‐linked immunosorbent assay for single‐stranded DNA (ssDNA), respectively. Intracellular reactive oxygen species (ROS) production was measured based on production of the fluorescent dye, dichlorofluorescein. Activation of the mitogen‐activated protein kinases extracellular signal‐regulating kinase 1/2 (ERK) and c‐Jun‐N‐terminal kinase (JNK) was shown by Western blot. Western blot was also used to show activation of caspase‐3, translocation of apoptosis‐inducing factor (AIF) from mitochondria to nucleus, and cleavage of 116‐kDa poly(ADP‐ribose) polymerase (PARP)‐1. Results : Ajoene induced apoptosis of 3T3‐L1 adipocytes in a dose‐ and time‐dependent manner. Ajoene treatment resulted in activation of JNK and ERK, translocation of AIF from mitochondria to nucleus, and cleavage of 116‐kDa PARP‐1 in a caspase‐independent manner. Ajoene treatment also induced an increase in intracellular ROS level. Furthermore, the antioxidant N‐acetyl‐l ‐cysteine effectively blocked ajoene‐mediated ROS generation, activation of JNK and ERK, translocation of AIF, and degradation of PARP‐1. Discussion : These results indicate that ajoene‐induced apoptosis in 3T3‐L1 adipocytes is initiated by the generation of hydrogen peroxide, which leads to activation of mitogen‐activated protein kinases, degradation of PARP‐1, translocation of AIF, and fragmentation of DNA. Ajoene can, thus, influence the regulation of fat cell number through the induction of apoptosis and may be a new therapeutic agent for the treatment of obesity.     

Thrombin or Ca++-Ionophore-Mediated Fall in Endothelial ATP Levels Independent of Poly(ADP-Ribose) Polymerase Activity and NAD Levels - Comparison with the Effects of Hydrogen Peroxide

To test the hypothesis that a fall in cellular ATP following stimulation of endothelial cells with thrombin is secondary to a decrease in NAD levels caused by poly(ADP-Ribose)polymerase (PARP), we measured the levels of NAD and ATP in endothelial cells after treatment with thrombin, the Ca⁺⁺-ionophore A23187, or hydrogen peroxide (H₂O₂), and compared the effects of inhibitors of PARP, NAD synthesis, and ADP-ribose breakdown on these responses. Neither thrombin nor A23187 caused a reduction in endothelial NAD levels and A23187 affected ATP levels independently of NAD levels or PARP activity. H₂O₂ induced lowering of NAD caused modest lowering of ATP but marked additional ATP-lowering, independent of PARP and NAD, was also demonstrated. We conclude that in endothelial cells ATP levels are largely independent of NAD and PARP, which do not play a role in thrombin or Ca⁺⁺-ionophore-mediated lowering of ATP. H₂O₂ caused ATP lowering through a similar mechanism as thrombin and A23187 but, additionally, caused a further ATP lowering through its intense stimulation of PARP and marked lowering of NAD.     

PARP1 inhibition affects pleural mesothelioma cell viability and uncouples AKT/mTOR axis via SIRT1

Malignant Pleural Mesothelioma (MMe) is a rare but increasingly prevalent, highly aggressive cancer with poor prognosis. The aetiology of MMe is essentially a function of previous exposure to asbestos fibres, which are considered to be an early‐stage carcinogen. Asbestos is toxic to human mesothelial cells (HMCs), that activate the nuclear enzyme poly(ADP‐ribose) polymerase‐1 (PARP1) to repair DNA. The targeting of PARP1 is showing considerable potential for delivering selective tumour cell kill while sparing normal cells, and offers a scientifically rational clinical application. We investigated PARP1 expression in normal mesothelial and MMe tissues samples. Immunohistochemical analysis revealed low PARP1 staining in peritumoural mesothelium. As opposite, a progressive increase in epithelioid and in the most aggressive sarcomatoid MMe tissues was evident. In MMe cell lines, we correlated increased PARP1 expression to sensitivity to its inhibitor CO‐338 and demonstrated that CO‐338 significantly reduced cell viability as single agent and was synergistic with cis‐platin. Interestingly, we described a new correlation between PARP1 and the AKT/mTOR axis regulated by SIRT1. SIRT1 has a role in the modulation of AKT activation and PARP1 has been described to be a gatekeeper for SIRT1 activity by limiting NAD+ availability. Here, we firstly demonstrate an inverse correlation between AKT acetylation and phosphorylation modulated by SIRT1 in MMe cells treated with CO‐338. In conclusion, this study demonstrates that PARP1 overexpression defines increased responsiveness to its inhibition, then these results imply that a substantial fraction of patients could be candidates for therapy with PARP inhibitors.     

Intact Cell Evidence for the Early Synthesis, and Subsequent Late Apopain-Mediated Suppression, of Poly(ADP-ribose) during Apoptosis

Poly(ADP-ribose) polymerase (PARP), which is catalytically activated by DNA strand breaks, has been implicated in apoptosis, or programmed cell death. A protease (CPP32) responsible for the cleavage of PARP and necessary for apoptosis was recently purified and characterized. The coordinated sequence of events related to PARP activation and cleavage in apoptosis has now been examined in individual cells. Apoptosis was studied in a human osteosarcoma cell line that undergoes a slow (8 to 10 days), spontaneous, and reproducible death program in culture. Changes in the abundance of intact PARP, poly(ADP-ribose) (PAR), and a proteolytic cleavage product of PARP that contains the DNA-binding domain were examined during apoptosis in the context of individual, whole cells by immunofluorescence with specific antibodies. The synthesis of PAR from NAD increased early, within 2 days of cell plating for apoptosis, prior to the appearance of internucleosomal DNA cleavage and before the cells become irreversibly committed to apoptosis, since replating yields viable, nonapoptotic cells. Strong expression of full-length PARP was also detected, by immunofluorescence as well as by Western analysis, during this same time period. However, after ∼4 days in culture, the abundance of both full-length PARP and PAR decreased markedly. After 6 days, a proteolytic cleavage product containing the DNA-binding domain of PARP was detected immunocytochemically and confirmed by Western analysis, both in the nuclei and in the cytoplasm of cells. A recombinant peptide spanning the DNA-binding domain of PARP was expressed, purified, and biotinylated, and was then used as a probe for DNA strand breaks. Fluorescence microscopy with this probe revealed extensive DNA fragmentation during the later stages of apoptosis. This is the first report, using individual,intact cells,demonstrating that poly(ADP-ribosyl)ation of nuclear proteins occurs prior to the commitment to apoptosis, that inactivation and cleavage of PARP begin shortly thereafter, and that very little PAR per se is present during the later stages of apoptosis, despite the presence of a very large number of DNA strand breaks. These results suggest a negative regulatory role for PARP during apoptosis, which in turn may reflect the requirement for adequate NAD and ATP during the later stages of programmed cell death.     

Possible Involvement of Poly(ADP-Ribosyl) Polymerase in Triggering Stress-Induced Apoptosis

U937 human myeloid leukemia cells respond to mild treatment with hydrogen peroxide and hyperthermia by undergoing apoptosis, an active mode of cell suicide. Higher concentrations of hydrogen peroxide, or longer incubation at the hyperthermic temperature, change the mode of cell death from apoptosis to the passive necrosis. Stress treatments cause a severe drop in the intracellular NAD concentration. 3-Aminobenzamide (3-ABA), a specific inhibitor of poly(ADP-ribosyl) polymerase (PARP), a nuclear enzyme which is activated by breaks in DNA to catabolize intracellular NAD, is capable of relieving such a drop. This suggests that breaks in DNA have been induced by both oxidative stress and heat shock, thereby activating PARP. Upon stress, NAD concentration has a first initial sharp drop; then, for mild stress treatments, it recovers, just when apoptosis begins to be detectable (8 h of recovery). At 20 h, when the apoptotic ladder-like pattern of DNA is visible, NAD concentration has dropped again, probably because of a second PARP activation due to the extensive DNA degradation that accompanies apoptosis. The presence of 3-ABA, concomitantly with the preservation of the intracellular NAD content, reduces the extent of apoptosis upon oxidative stress and strongly enhances cell survival, thus suggesting a role for PARP in triggering stress-induced apoptosis. All apoptotic U937 cells have a reduced NAD content, independently of the inducing agent; however, upon treatments which do not cause immediate DNA breaks, the drop in NAD concentration occurs only after the apoptotic ladder is detectable and can be ascribed to the activation of PARP by the free ends of DNA formed during the endonucleolitic degradation. Moreover, in these instances the inhibition of PARP, although effective in blocking the drop in NAD concentration, has no effect on apoptosis, thus being only circumstantial.     

Poly(ADP-ribose) signaling in cell death

Poly(ADP-ribosyl)ation (PARylation) is a reversible protein modification carried out by the concerted actions of poly(ADP-ribose) polymerase (PARP) enzymes and poly(ADP-ribose) (PAR) decomposing enzymes such as PAR glycohydrolase (PARG) and ADP-ribosyl hydrolase 3 (ARH3). Reversible PARylation is a pleiotropic regulator of various cellular functions but uncontrolled PARP activation may also lead to cell death. The cellular demise pathway mediated by PARylation in oxidatively stressed cells has been described almost thirty years ago. However, the underlying molecular mechanisms have only begun to emerge relatively recently. PARylation has been implicated in necroptosis, autophagic cell death but its role in extrinsic and intrinsic apoptosis appears to be less predominant and depends largely on the cellular model used. Currently, three major pathways have been made responsible for PARP-mediated necroptotic cell death: (1) compromised cellular energetics mainly due to depletion of NAD, the substrate of PARPs; (2) PAR mediated translocation of apoptosis inducing factor (AIF) from mitochondria to nucleus (parthanatos) and (3) a mostly elusive crosstalk between PARylation and cell death/survival kinases and phosphatases. Here we review how these PARP-mediated necroptotic pathways are intertwined, how PARylation may contribute to extrinsic and intrinsic apoptosis and discuss recent developments on the role of PARylation in autophagy and autophagic cell death.     

Comprehensive ADP‐ribosylome analysis identifies tyrosine as an ADP‐ribose acceptor site

Despite recent mass spectrometry (MS)‐based breakthroughs, comprehensive ADP‐ribose (ADPr)‐acceptor amino acid identification and ADPr‐site localization remain challenging. Here, we report the establishment of an unbiased, multistep ADP‐ribosylome data analysis workflow that led to the identification of tyrosine as a novel ARTD1/PARP1‐dependent in vivo ADPr‐acceptor amino acid. MS analyses of in vitro ADP‐ribosylated proteins confirmed tyrosine as an ADPr‐acceptor amino acid in RPS3A (Y155) and HPF1 (Y238) and demonstrated that trans‐modification of RPS3A is dependent on HPF1. We provide an ADPr‐site Localization Spectra Database (ADPr‐LSD), which contains 288 high‐quality ADPr‐modified peptide spectra, to serve as ADPr spectral references for correct ADPr‐site localizations.     

Poly(ADP-ribose) polymerase 1 is involved in glucose toxicity through SIRT1 modulation in HepG2 hepatocytes

Accelerated glucose metabolism leads to oxidative stress and DNA damage in cells; these effects are related to glucose toxicity. The precise mechanisms of glucose toxicity are still unclear. The aim of this work was to investigate the mechanism of poly(ADP‐ribose) polymerase 1 (PARP1), which is a DNA repair enzyme activated by high‐glucose‐induced oxidative stress, and its effect on glucose toxicity in HepG2 hepatocytes. HepG2 cells were cultured under normal (5.5 mM) or high (30 mM) glucose conditions for 4 days. PJ34, which is an inhibitor of PARP1, was used to determine the downstream effects of PARP1 activation. PARP1 activity in 30 mM‐glucose‐treated cells was more than that in 5.5 mM‐glucose‐treated cells, and the activity correlated with the increase in ROS generation and DNA damage. PJ34 suppressed PARP1 activation and prevented the high‐glucose‐induced suppression of SIRT1 and AMP‐activated protein kinase (AMPK) activity, which was similar to its effect on the restoration of intracellular nicotinamide adenine dinucleotide (NAD) content. Further, the phosphorylation of insulin receptor was attenuated in response to insulin stimulation under high glucose conditions, and PJ34 could reverse this effect. The results of transfection of HepG2 cells with PARP1 small interfering RNA were similar to those obtained by treatment of the cells with PARP1 inhibitor PJ34. These data suggest that high‐glucose‐induced PARP1 activation might play a role in glucose toxicity by down‐regulating SIRT1 and AMPK activity through NAD depletion and resulting in insulin insensitivity. J. Cell. Biochem. 112: 299–306, 2011. © 2010 Wiley‐Liss, Inc.     

Molecular mechanism of poly(ADP-ribosyl)ation by PARP1 and identification of lysine residues as ADP-ribose acceptor sites

Poly(ADP-ribose) polymerase 1 (PARP1) synthesizes poly(ADP-ribose) (PAR) using nicotinamide adenine dinucleotide (NAD) as a substrate. Despite intensive research on the cellular functions of PARP1, the molecular mechanism of PAR formation has not been comprehensively understood. In this study, we elucidate the molecular mechanisms of poly(ADP-ribosyl)ation and identify PAR acceptor sites. Generation of different chimera proteins revealed that the amino-terminal domains of PARP1, PARP2 and PARP3 cooperate tightly with their corresponding catalytic domains. The DNA-dependent interaction between the amino-terminal DNA-binding domain and the catalytic domain of PARP1 increased V_max and decreased the K_m for NAD. Furthermore, we show that glutamic acid residues in the auto-modification domain of PARP1 are not required for PAR formation. Instead, we identify individual lysine residues as acceptor sites for ADP-ribosylation. Together, our findings provide novel mechanistic insights into PAR synthesis with significant relevance for the different biological functions of PARP family members.