Posttranscriptional regulation of PARG mRNA by HuR facilitates DNA repair and resistance to PARP inhibitors

The majority of pancreatic ductal adenocarcinomas (PDAC) rely on the mRNA stability factor HuR (ELAV-L1) to drive cancer growth and progression. Here, we show that CRISPR-Cas9–mediated silencing of the HuR locus increases the relative sensitivity of PDAC cells to PARP inhibitors (PARPi). PDAC cells treated with PARPi stimulated translocation of HuR from the nucleus to the cytoplasm, specifically promoting stabilization of a new target, poly (ADP-ribose) glycohydrolase (PARG) mRNA, by binding a unique sequence embedded in its 30 untranslated region. HuR-dependent upregulation of PARG expression facilitated DNA repair via hydrolysis of polyADP-ribose on related repair proteins. Accordingly, strategies to inhibit HuR directly promoted DNA damage accumulation, inefficient PAR removal, and persistent PARP-1 residency on chromatin (PARP-1 trapping). Immunoprecipitation assays demonstrated that the PARP-1 protein binds and posttranslationally modifies HuR in PARPi-treated PDAC cells. In a mouse xenograft model of human PDAC, PARPi monotherapy combined with targeted silencing of HuR significantly reduced tumor growth compared with PARPi therapy alone. Our results highlight the HuR–PARG axis as an opportunity to enhance PARPi-based therapies. ©2017 AACR

Giardia Cyst Wall Protein 1 Is a Lectin That Binds to Curled Fibrils of the GalNAc Homopolymer

The infectious and diagnostic stage of Giardia lamblia (also known as G. intestinalis or G. duodenalis) is the cyst. The Giardia cyst wall contains fibrils of a unique β-1,3-linked N-acetylgalactosamine (GalNAc) homopolymer and at least three cyst wall proteins (CWPs) composed of Leu-rich repeats (CWPLRR) and a C-terminal conserved Cys-rich region (CWPCRR). Our goals were to dissect the structure of the cyst wall and determine how it is disrupted during excystation. The intact Giardia cyst wall is thin (~400 nm), easily fractured by sonication, and impermeable to small molecules. Curled fibrils of the GalNAc homopolymer are restricted to a narrow plane and are coated with linear arrays of oval-shaped protein complex. In contrast, cyst walls of Giardia treated with hot alkali to deproteinate fibrils of the GalNAc homopolymer are thick (~1.2 µm), resistant to sonication, and permeable. The deproteinated GalNAc homopolymer, which forms a loose lattice of curled fibrils, is bound by native CWP1 and CWP2, as well as by maltose-binding protein (MBP)-fusions containing the full-length CWP1 or CWP1LRR. In contrast, neither MBP alone nor MBP fused to CWP1CRR bind to the GalNAc homopolymer. Recombinant CWP1 binds to the GalNAc homopolymer within secretory vesicles of Giardia encysting in vitro. Fibrils of the GalNAc homopolymer are exposed during excystation or by treatment of heat-killed cysts with chymotrypsin, while deproteinated fibrils of the GalNAc homopolymer are degraded by extracts of Giardia cysts but not trophozoites. These results show the Leu-rich repeat domain of CWP1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. During excystation, host and Giardia proteases appear to degrade bound CWPs, exposing fibrils of the GalNAc homopolymer that are digested by a stage-specific glycohydrolase. Author SummaryWhile the walls of plants and fungi contain numerous sugar homopolymers (cellulose, chitin, and β-1,3-glucans) and dozens of proteins, the cyst wall of Giardia is relatively simple. The Giardia wall contains a unique homopolymer of β-1,3-linked N-acetylgalactosamine (GalNAc) and at least three cyst wall proteins (CWPs), each of which is composed of Leu-rich repeats and a C-terminal Cys-rich region. The three major discoveries here are: 1) Fibrils of the GalNAc homopolymer are curled and form a lattice that is compressed into a narrow plane by bound protein in intact cyst walls. 2) Leu-rich repeats of CWP1 form a novel lectin domain that is specific for fibrils of the GalNAc homopolymer, which can be isolated by methods used to deproteinate fungal walls. 3) A cyst-specific glycohydrolase is able to degrade deproteinated fibrils of the GalNAc homopolymer. We incorporate these findings into a new curled fiber and lectin model of the intact Giardia cyst wall and a protease and glycohydrolase model of excystation.National Institutes of Health (AI048082, AI44070, GM31318, RR1088

Poly(ADP-Ribose) Polymerase 1 Accelerates Single-Strand Break Repair in Concert with Poly(ADP-Ribose) Glycohydrolase

Single-strand breaks are the commonest lesions arising in cells, and defects in their repair are implicated in neurodegenerative disease. One of the earliest events during single-strand break repair (SSBR) is the rapid synthesis of poly(ADP-ribose) (PAR) by poly(ADP-ribose) polymerase (PARP), followed by its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). While the synthesis of poly(ADP-ribose) is important for rapid rates of chromosomal SSBR, the relative importance of poly(ADP-ribose) polymerase 1 (PARP-1) and PARP-2 and of the subsequent degradation of PAR by PARG is unclear. Here we have quantified SSBR rates in human A549 cells depleted of PARP-1, PARP-2, and PARG, both separately and in combination. We report that whereas PARP-1 is critical for rapid global rates of SSBR in human A549 cells, depletion of PARP-2 has only a minor impact, even in the presence of depleted levels of PARP-1. Moreover, we identify PARG as a novel and critical component of SSBR that accelerates this process in concert with PARP-1

APLF (C2orf13) is a novel component of poly(ADP-ribose) signaling in mammalian cells

APLF is a novel protein of unknown function that accumulates at sites of chromosomal DNA strand breakage via forkhead-associated (FHA) domain-mediated interactions with XRCC1 and XRCC4. APLF can also accumulate at sites of chromosomal DNA strand breaks independently of the FHA domain via an unidentified mechanism that requires a highly conserved C-terminal tandem zinc finger domain. Here, we show that the zinc finger domain binds tightly to poly(ADP-ribose), a polymeric posttranslational modification synthesized transiently at sites of chromosomal damage to accelerate DNA strand break repair reactions. Protein poly(ADP-ribosyl)ation is tightly regulated and defects in either its synthesis or degradation slow global rates of chromosomal single-strand break repair. Interestingly, APLF negatively affects poly(ADP-ribosyl)ation in vitro, and this activity is dependent on its capacity to bind the polymer. In addition, transient overexpression in human A549 cells of full-length APLF or a C-terminal fragment encoding the tandem zinc finger domain greatly suppresses the appearance of poly(ADP-ribose), in a zinc finger-dependent manner. We conclude that APLF can accumulate at sites of chromosomal damage via zinc finger-mediated binding to poly(ADP-ribose) and is a novel component of poly(ADP-ribose) signaling in mammalian cells

Helicoverpa armigera nucleopolyhedrovirus occlusion-derived virus-associated protein, HA100, affects oral infectivity in vivo but not virus replication in vitro

ORF100 (ha100) of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) has been reported as one of the unique genes of group II alphabaculoviruses encoding a protein located in the occlusion-derived virus (ODV) envelope and nucleocapsid. The protein consists of 510 aa with a predicted mass of 58.1 kDa and is a homologue of poly(ADP–ribose) glycohydrolase in eukaryotes. Western blot analysis detected a 60 kDa band in HearNPV-infected HzAM1 cells starting at 18 h post-infection. Transient expression of GFP-fused HA100 in HzAM1 cells resulted in cytoplasmic localization of the protein, but after superinfection with HearNPV, GFPfused HA100 was localized in the nucleus. To study the function of HA100 further, an ha100-null virus was constructed using bacmid technology. Viral one-step growth curve analyses showed that the ha100-null virus had similar budded virus production kinetics to that of the parental virus. Electron microscopy revealed that deletion of HA100 did not alter the morphology of ODVs or occlusion bodies (OBs). However, bioassays in larvae showed that the 50% lethal concentration (LC50) value of HA100-null OBs was significantly higher than that of parental OBs; the median lethal time (LT50) of ha100-null OBs was about 24 h later than control virus. These results indicate that HA100 is not essential for virus replication in vitro. However, it significantly affects the oral infectivity of OBs in host insects, suggesting that the association HA100 with the ODV contributes to the infectivity of OBs in vivo

Methylation status of Dnmt1 promoter depends on poly(ADP-ribosy)lation

Research is focused on CpG islands and on the mechanism that poly(ADP-ribosyl)ation uses to defend the unmethylated state of these important DNA sequences which are located in the promoter regions of the housekeeping genes having a role of transcription regulators. Data here reported show that inhibition of PARP activity allows the diffuse insertion of methyl groups onto some CpG islands and in particular on the CpG island which is located in the promoter region of Dnmt1 gene. Hence, following inhibition of PARPs activity, this promoter loses its protection against methylation becoming silenced through methylation as shown by analyses with Methylation Sensitive PCR (MS-PCR) and sequencing after bisulphite treatment. Analyses of Western Blotting, RT-PCR and Real-time RT-PCR confirm that the gene has undergone silencing. The role of ADP-ribose polymers in silencing Dnmt1 has been demonstrated by additional experiments in which overexpression of poly(ADP-ribose) glycohydrolase leads to reduction of ADP-ribose polymers in nuclei associated to a sharp decrease of Dnmt1 level respect to control. A parallel genome-wide methyl-sensitive restriction assay demonstrates that the variation of Dnmt1 level is followed by a bimodal alteration of DNA methylation pattern. In fact, the inhibition of poly(ADP-ribosyl)ation initially causes an increase in methyl-group insertion onto DNA while this phenomenon is reversed after prolonged treatments and demethylation is detected within Alu sequences. Considering the important role played by Dnmt1 in the epigenetic scenario, these data lead us to think about what happens in tumor cells where both anomalous methylation of some CpG islands and diffuse hypomethylation are present. These findings open up a new path into epigenetic research in tumors. What is remarkable is that the demethylated pattern found in Alu sequences after treatment of cells with 3-ABA for 96 hours is very similar to the one found on DNA from cells treated with 5-AZA for the same time. The discovery of a DNA demethylating activity dependent on the use of inhibitors of poly(ADP-ribosyl)ation process increases the knowledge of mechanism by which these inhibitors enhance the cytotoxicity of other anticancer agents

Methylation status of Dnmt1 promoter depends on poly(ADP-ribosy)lation

Research is focused on CpG islands and on the mechanism that poly(ADP-ribosyl)ation uses to defend the unmethylated state of these important DNA sequences which are located in the promoter regions of the housekeeping genes having a role of transcription regulators. Data here reported show that inhibition of PARP activity allows the diffuse insertion of methyl groups onto some CpG islands and in particular on the CpG island which is located in the promoter region of Dnmt1 gene. Hence, following inhibition of PARPs activity, this promoter loses its protection against methylation becoming silenced through methylation as shown by analyses with Methylation Sensitive PCR (MS-PCR) and sequencing after bisulphite treatment. Analyses of Western Blotting, RT-PCR and Real-time RT-PCR confirm that the gene has undergone silencing. The role of ADP-ribose polymers in silencing Dnmt1 has been demonstrated by additional experiments in which overexpression of poly(ADP-ribose) glycohydrolase leads to reduction of ADP-ribose polymers in nuclei associated to a sharp decrease of Dnmt1 level respect to control. A parallel genome-wide methyl-sensitive restriction assay demonstrates that the variation of Dnmt1 level is followed by a bimodal alteration of DNA methylation pattern. In fact, the inhibition of poly(ADP-ribosyl)ation initially causes an increase in methyl-group insertion onto DNA while this phenomenon is reversed after prolonged treatments and demethylation is detected within Alu sequences. Considering the important role played by Dnmt1 in the epigenetic scenario, these data lead us to think about what happens in tumor cells where both anomalous methylation of some CpG islands and diffuse hypomethylation are present. These findings open up a new path into epigenetic research in tumors. What is remarkable is that the demethylated pattern found in Alu sequences after treatment of cells with 3-ABA for 96 hours is very similar to the one found on DNA from cells treated with 5-AZA for the same time. The discovery of a DNA demethylating activity dependent on the use of inhibitors of poly(ADP-ribosyl)ation process increases the knowledge of mechanism by which these inhibitors enhance the cytotoxicity of other anticancer agents

Visualization of poly(ADP-ribose) bound to PARG reveals inherent balance between exo- and endo-glycohydrolase activities

Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios

The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD.

Mycobacterium tuberculosis (Mtb) induces necrosis of infected cells to evade immune responses. Recently, we found that Mtb uses the protein CpnT to kill human macrophages by secreting its C-terminal domain, named tuberculosis necrotizing toxin (TNT), which induces necrosis by an unknown mechanism. Here we show that TNT gains access to the cytosol of Mtb-infected macrophages, where it hydrolyzes the essential coenzyme NAD(+). Expression or injection of a noncatalytic TNT mutant showed no cytotoxicity in macrophages or in zebrafish zygotes, respectively, thus demonstrating that the NAD(+) glycohydrolase activity is required for TNT-induced cell death. To prevent self-poisoning, Mtb produces an immunity factor for TNT (IFT) that binds TNT and inhibits its activity. The crystal structure of the TNT-IFT complex revealed a new NAD(+) glycohydrolase fold of TNT, the founding member of a toxin family widespread in pathogenic microorganisms