ABSTRACT
PARP1 plays a pivotal role in DNA repair within the base excision pathway, making it a promising therapeutic target for cancers involving BRCA mutations. Current study is focused on the discovery of PARP inhibitors with enhanced selectivity for PARP1. Concurrent inhibition of PARP1 with PARP2 and PARP3 affects cellular functions, potentially causing DNA damage accumulation and disrupting immune responses. In step 1, a virtual library of 593 million compounds has been screened using a shape-based screening approach to narrow down the promising scaffolds. In step 2, hierarchical docking approach embedded in Schrödinger suite was employed to select compounds with good dock score, drug-likeness and MMGBSA score. Analysis supplemented with decomposition energy, molecular dynamics (MD) simulations and hydrogen bond frequency analysis, pinpointed that active site residues; H862, G863, R878, M890, Y896 and F897 are crucial for specific binding of ZINC001258189808 and ZINC000092332196 with PARP1 as compared to PARP2 and PARP3. The binding of ZINC000656130962, ZINC000762230673, ZINC001332491123, and ZINC000579446675 also revealed interaction involving two additional active site residues of PARP1, namely N767 and E988. Weaker or no interaction was observed for these residues with PARP2 and PARP3. This approach advances our understanding of PARP-1 specific inhibitors and their mechanisms of action, facilitating the development of targeted therapeutics.
Subject(s)
Antineoplastic Agents , Drug Design , Molecular Dynamics Simulation , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerase Inhibitors , Humans , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors , Poly (ADP-Ribose) Polymerase-1/chemistry , Poly(ADP-ribose) Polymerase Inhibitors/chemistry , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Molecular Docking Simulation , Catalytic Domain , Poly(ADP-ribose) Polymerases/metabolism , Poly(ADP-ribose) Polymerases/chemistry , Hydrogen BondingABSTRACT
SignificancePARP is an important target in the treatment of cancers, particularly in patients with breast, ovarian, or prostate cancer that have compromised homologous recombination repair (i.e., BRCA-/-). This review about inhibitors of PARP (PARPi) is for readers interested in the development of next-generation drugs for the treatment of cancer, providing insights into structure-activity relationships, in vitro vs. in vivo potency, PARP trapping, and synthetic lethality.
Subject(s)
Poly(ADP-ribose) Polymerase Inhibitors/chemistry , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , BRCA1 Protein/genetics , BRCA2 Protein/genetics , DNA Repair , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Humans , Models, Molecular , Molecular Structure , Mutation , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Structure-Activity Relationship , Synthetic Lethal MutationsABSTRACT
Poly (ADP-ribose) polymerases (PARP) 1-3 are well-known multi-domain enzymes, catalysing the covalent modification of proteins, DNA, and themselves. They attach mono- or poly-ADP-ribose to targets using NAD+ as a substrate. Poly-ADP-ribosylation (PARylation) is central to the important functions of PARP enzymes in the DNA damage response and nucleosome remodelling. Activation of PARP happens through DNA binding via zinc fingers and/or the WGR domain. Modulation of their activity using PARP inhibitors occupying the NAD+ binding site has proven successful in cancer therapies. For decades, studies set out to elucidate their full-length molecular structure and activation mechanism. In the last five years, significant advances have progressed the structural and functional understanding of PARP1-3, such as understanding allosteric activation via inter-domain contacts, how PARP senses damaged DNA in the crowded nucleus, and the complementary role of histone PARylation factor 1 in modulating the active site of PARP. Here, we review these advances together with the versatility of PARP domains involved in DNA binding, the targets and shape of PARylation and the role of PARPs in nucleosome remodelling.
Subject(s)
Cell Cycle Proteins/chemistry , Nucleosomes/metabolism , Poly (ADP-Ribose) Polymerase-1/chemistry , Poly(ADP-ribose) Polymerases/chemistry , Allosteric Regulation/drug effects , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , DNA Repair , Humans , Models, Molecular , Nuclear Proteins/metabolism , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerases/metabolism , Protein Domains/drug effectsABSTRACT
Rosemary extract is used in food additives and traditional medicine and has been observed to contain anti-tumor activity. In this study, rosemary extract is hypothesized to induce synthetic lethality in BRCA2 deficient cells by PARP inhibition. Chinese hamster lung V79 cells and its mutant cell lines, V-C8 (BRCA2 deficient) and V-C8 with BRCA2 gene correction were used. Rosemary extract and its major constituent chemicals were tested for their cytotoxicity by colony formation assay in cells of different BRCA2 status. The latter chemicals were tested for inhibitory effect of poly (ADP-ribose) polymerase (PARP) activity in vitro and in vivo. Rosemary has shown selective cytotoxicity against V-C8 cells (IC50 17 µg/ml) compared to V79 cells (IC50 26 µg/ml). Among tested chemicals, gallic acid and carnosic acid showed selective cytotoxicity to V-C8 cells along with PARP inhibitory effects. Carnosol showed comparative PARP inhibitory effects at 100 µM compared to carnosic acid and gallic acid, but the selective cytotoxicity was not observed. In conclusion, we predict that within rosemary extract two specific constituent components; gallic acid and carnosic acid were the cause for the synthetic lethality.
Subject(s)
BRCA2 Protein/genetics , Plant Extracts/chemistry , Poly(ADP-ribose) Polymerase Inhibitors/chemistry , Rosmarinus/chemistry , Abietanes/chemistry , Abietanes/isolation & purification , Abietanes/pharmacology , Animals , BRCA2 Protein/deficiency , CHO Cells , Cell Survival/drug effects , Cinnamates/chemistry , Cinnamates/isolation & purification , Cinnamates/pharmacology , Cricetinae , Cricetulus , DNA Damage/drug effects , Depsides/chemistry , Depsides/isolation & purification , Depsides/pharmacology , Plant Extracts/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Rosmarinus/metabolism , Rosmarinic AcidABSTRACT
ABSTACT Artemisia nilagirica (Clarke) is a widely used medicinal herb in Indian traditional system of medicine. Therefore, the present study was designed to evaluate the effects of A. nilagirica extracts/fractions on inhibition of proliferation and apoptosis in a human monocytic leukemia (THP-1) cell line. The crude extracts (A. nilagirica ethyl acetate extract [ANE] and A. nilagirica methanolic extract [ANA]) showed cytotoxic activity toward THP-1 cells with the IC50 values of 38.21 ± 7.37 and 132.41 ± 7.19 µg/ml, respectively. However, the cytotoxic activity of active fractions (ANE-B and ANM-9) obtained after column chromatography was found to be much more pronounced than their parent extracts. The IC50 values of ANE-B and ANM-9 were found to be 27.04 ± 2.54 µg/ml and 12.70 ± 4.79 µg/ml, respectively, suggesting greater susceptibility of the malignant cells. Cell cycle analysis and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay revealed that inhibition of cell growth by A. nilagirica fractions on THP-1 cells was mediated by apoptosis. Active fractions of A. nilagirica increased the expression levels of caspase-3, -7, and poly-ADP-ribose polymerase (PARP), a critical member of the apoptotic pathway. These results suggested that active fractions of A. nilagirica may play a promising role in growth suppression by inducing apoptosis in human monocytic leukemic cells via mitochondria-dependent and death receptor-dependent apoptotic pathways.
Subject(s)
Anticarcinogenic Agents/isolation & purification , Antineoplastic Agents, Phytogenic/isolation & purification , Apoptosis/drug effects , Artemisia/chemistry , Leukemia, Monocytic, Acute/drug therapy , Macrophages, Peritoneal/drug effects , Animals , Anticarcinogenic Agents/adverse effects , Anticarcinogenic Agents/chemistry , Anticarcinogenic Agents/pharmacology , Antineoplastic Agents, Phytogenic/adverse effects , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Biological Assay , Caspase 3/chemistry , Caspase 3/genetics , Caspase 3/metabolism , Caspase 7/chemistry , Caspase 7/genetics , Caspase 7/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , India , Inhibitory Concentration 50 , Leukemia, Monocytic, Acute/metabolism , Leukemia, Monocytic, Acute/pathology , Macrophages, Peritoneal/cytology , Mice, Inbred BALB C , Neoplasm Proteins/agonists , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Plant Extracts/adverse effects , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , THP-1 CellsABSTRACT
In the present study, we report the delivery of anti-cancer drug curcumin to cancer cells using mesoporous silica materials. A series of mesoporous silica material based drug delivery systems (S2, S4 and S6) were first designed and developed through the amine functionalization of KIT-6, MSU-2 and MCM-41 followed by the loading of curcumin. The curcumin loaded materials were characterized with several physico-chemical techniques and thoroughly screened on cancer cells to evaluate their in vitro drug delivery efficacy. All the curcumin loaded silica materials exhibited higher cellular uptake and inhibition of cancer cell viability compared to pristine curcumin. The effective internalization of curcumin in cancer cells through the mesoporous silica materials initiated the generation of intracellular reactive oxygen species and the down regulation of poly ADP ribose polymerase (PARP) enzyme levels compared to free curcumin leading to the activation of apoptosis. This study shows that the anti-cancer activity of curcumin can be potentiated by loading onto mesoporous silica materials. Therefore, we strongly believe that mesoporous silica based curcumin loaded drug delivery systems may have future potential applications for the treatment of cancers.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Curcumin/chemistry , Curcumin/pharmacology , Drug Delivery Systems , Nanoparticles/chemistry , Poly(ADP-ribose) Polymerases/chemistry , Silicon Dioxide/chemistry , Silicon Dioxide/pharmacology , Apoptosis/drug effects , Cell Survival/drug effects , Humans , Poly(ADP-ribose) Polymerases/metabolism , PorosityABSTRACT
Nectandra megapotamica (Spreng.) Mez. (Lauraceae) is a well-known Brazilian medicinal plant that has been used in folk medicine to treat several diseases. In continuation of our ongoing efforts to discover new bioactive natural products from the Brazilian flora, this study describes the identification of cytotoxic compounds from the MeOH extract of N. megapotamica (Lauraceae) leaves using bioactivity-guided fractionation. This approach resulted in the isolation and characterization of eight tetrahydrofuran neolignans: calopeptin (1), machilin-G (2), machilin-I (3), aristolignin (4), nectandrin A (5), veraguensin (6), ganschisandrin (7), and galgravin (8). Different assays were conducted to evaluate their cytotoxic activities and to determine the possible mechanism(s) related to the activity displayed against human leukemia cells. The most active compounds 4, 5 and 8 gave IC50 values of 14.2 ± 0.7, 16.9 ± 0.8 and 16.5 ± 0.8 µg/mL, respectively, against human leukemia (HL-60) tumor cells. Moreover, these compounds induced specific apoptotic hallmarks, such as plasma membrane bleb formation, nuclear DNA condensation, specific chromatin fragmentation, phosphatidyl-serine exposure on the external leaflet of the plasma membrane, cleavage of PARP as well as mitochondrial damage, which as a whole could be related to the intrinsic apoptotic pathway.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Cytotoxins/pharmacology , Lauraceae/chemistry , Lignans/pharmacology , Plant Leaves/chemistry , Animals , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Apoptosis/drug effects , Brazil , Cell Membrane/drug effects , Cell Membrane/ultrastructure , Cell Survival/drug effects , Cytotoxins/chemistry , Cytotoxins/isolation & purification , DNA Fragmentation/drug effects , HL-60 Cells , HeLa Cells , Humans , Inhibitory Concentration 50 , Lignans/chemistry , Lignans/isolation & purification , MCF-7 Cells , Melanoma, Experimental , Mice , Organ Specificity , Phosphatidylserines/chemistry , Phosphatidylserines/metabolism , Plant Extracts/chemistry , Plants, Medicinal , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Proteolysis , Structure-Activity RelationshipABSTRACT
AIMS: A component of the base excision repair pathway, poly(ADP-ribose) polymerase-1 (PARP1) functions in multiple cellular processes, including DNA repair and programmed cell death. We previously showed that Salidroside, a phenylpropanoid glycoside isolated from medicinal plants, prevented the loss of hematopoietic stem cells (HSCs) in native mice and rescued HSCs repopulating in transplanted recipients under oxidative stress. The aim of this study was to investigate the mechanism by which PARP1 activation by Salidroside maintains HSCs under oxidative stress. RESULTS: We found that although there were no spontaneous defects in hematopoiesis in Parp1(-/-) mice, oxidative stress compromised the repopulating capacity of Parp1(-/-) HSCs in transplanted recipient mice. A biochemical study using truncated proteins lacking the defined functional domains of PARP1 showed that the tryptophan-glycine-arginine-rich (WGR) domain of PARP1 was critical for Salidroside binding and subsequent PARP1 activation under oxidative stress. Functionally, complementation of Parp1(-/-) HSCs with full-length PARP1WT, but not the PARP1R591K mutant in WGR domain restored Salidroside-stimulated PARP1 activation in vitro. Mechanistically, activated PARP1 by Salidroside enhanced the repopulating capacity of the stressed HSCs by accelerating oxidative DNA damage repair. INNOVATIONS AND CONCLUSION: Our findings reveal the action of mechanism for Salidroside in PARP1 stimulation and a novel role of PARP1 activation in maintaining HSC function under oxidative stress.
Subject(s)
Glucosides/pharmacology , Hematopoietic Stem Cells/drug effects , Oxidative Stress/drug effects , Phenols/pharmacology , Poly(ADP-ribose) Polymerases/metabolism , Amino Acid Sequence , Animals , Enzyme Activation , Glucosides/metabolism , Hematopoietic Stem Cells/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Phenols/metabolism , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/genetics , Rats , Sequence Homology, Amino AcidABSTRACT
Proteolysis is a key regulatory event that controls intracellular and extracellular signaling through irreversible changes in a protein's structure that greatly alters its function. Here we describe a platform for profiling caspase substrates which encompasses two highly complementary proteomic techniques--the first is a differential gel based approach termed Global Analyzer of SILAC-derived Substrates of Proteolysis (GASSP) and the second involves affinity enrichment of peptides containing a C-terminal aspartic acid residue. In combination, these techniques have enabled the profiling of a large cellular pool of apoptotic-mediated proteolytic events across a wide dynamic range. By applying this integrated proteomic work flow to analyze proteolytic events resulting from the induction of intrinsic apoptosis in Jurkat cells via etoposide treatment, 3346 proteins were quantified, of which 360 proteins were identified as etoposide-induced proteolytic substrates, including 160 previously assigned caspase substrates. In addition to global profiling, a targeted approach using BAX HCT116 isogenic cell lines was utilized to dissect pre- and post-mitochondrial extrinsic apoptotic cleavage events. By employing apoptotic activation with a pro-apoptotic receptor agonist (PARA), a limited set of apoptotic substrates including known caspase substrates such as BH3 interacting-domain death agonist (BID) and Poly (ADP-ribose) polymerase (PARP)-1, and novel substrates such as Basic Transcription Factor 3, TRK-fused gene protein (TFG), and p62/Sequestosome were also identified.
Subject(s)
Apoptosis/drug effects , Proteolysis , Proteomics/methods , Adaptor Proteins, Signal Transducing/chemistry , Aspartic Acid/chemistry , BH3 Interacting Domain Death Agonist Protein/chemistry , Caspases/chemistry , Computational Biology , Etoposide/pharmacology , HCT116 Cells , Humans , Jurkat Cells , Nuclear Proteins/chemistry , Peptides/chemistry , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/chemistry , Proteins/chemistry , RNA-Binding Proteins/chemistry , Sequestosome-1 Protein , Substrate Specificity , Transcription Factors/chemistryABSTRACT
Poly(ADP-ribose) polymerase (PARP) plays a pivotal role in the repair of DNA strand breaks. However, excessive activation of PARP causes a rapid depletion of intracellular energy, leading to cell death. PARP inhibitors may have potential therapeutic benefit in the treatment of myocardial ischemia, stroke, and neurodegenerative disease. With these emerging medicinal interests, various screening programs have identified small molecules that inhibit PARP with reasonable potencies. However, the increasing numbers of diverse small molecules generated through combinatorial chemistry necessitate the use of robust assays with good sensitivity and specificity for use as a high-throughput screening (HTS) program. Here, we report the development and the validation of a nonisotopic PARP-1 assay suitable for HTS by converting a biotinylated NAD-based colorimetric assay to a miniaturized 384-well plate format. Comparing with the conventional methods, this miniaturized PARP-1 inhibition assay was equally sensitive with excellent reproducibility and cost-effectiveness. Because nonisotopic PARP-1 inhibition assays are widely used, the methodology described in this article can expand the feasibility of this assay as a high-throughput assay for screening of PARP-1 inhibitors from a random chemical library.
Subject(s)
Drug Evaluation, Preclinical/methods , Poly(ADP-ribose) Polymerase Inhibitors , Biotinylation , Colorimetry , Drug Design , Enzyme Inhibitors/chemistry , Inhibitory Concentration 50 , Miniaturization , NAD/chemistry , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/chemistryABSTRACT
Oxidative stress results from an oxidant/antioxidant imbalance, an excess of oxidants, and/or a depletion of antioxidants. A considerable body of recent evidence suggests that oxidative stress and exaggerated production of reactive oxygen species play a major role in several aspects ischemia and reperfusion. Hypericum perforatum is a medicinal plant species containing many polyphenolic compounds, namely flavonoids and phenolic acids. Because polyphenolic compounds have high antioxidant potential, in this study we evaluated the effect of H. perforatum extract on splanchnic artery occlusion (SAO) shock-mediated injury. SAO shock was induced in rats by clamping the superior mesenteric artery and the celiac trunk for 45 min. After 1 h of reperfusion, SAO-shocked rats developed a significant fall in mean arterial blood pressure. Treatment of rats with H. perforatum extract (applied at 25 mg/kg 15 min before reperfusion) significantly reduced a significant fall in mean arterial blood pressure and the migration of polymorphonuclear cells caused by SAO-shock. H. perforatum extract also attenuated the ileum injury (histology) as well as the increase in the tissue levels of myeloperoxidase and malondialdehyde caused by SAO shock in the ileum. Immunohistochemical analysis for nitrotyrosine and for poly ADP-ribosylated proteins revealed a positive staining in ileum from SAO-shocked rats. The degree of staining for nitrotyrosine and poly ADP-ribosylated proteins was markedly reduced in tissue sections obtained from SAO-shocked rats that had received H. perforatum extract. Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin and for intercellular adhesion molecule-1 in the vascular endothelial cells. H. perforatum extract treatment markedly reduced the intensity and degree of P-selectin and intercellular adhesion molecule-1 in tissue section from SAO-shocked rats. H. perforatum extract treatment significantly improved survival. In conclusion, this study demonstrates that H. perforatum extract exerts multiple protective effects in splanchnic artery occlusion-reperfusion shock and suggests that H. perforatum extract may be a candidate for consideration as a therapeutic intervention for ischemia-reperfusion injury.
Subject(s)
Hypericum/metabolism , Phytotherapy/methods , Plant Extracts/pharmacology , Reperfusion Injury/drug therapy , Animals , Antioxidants/chemistry , Blood Pressure , Cytokines/metabolism , Densitometry , Flavonoids/chemistry , Hydroxybenzoates/chemistry , Immunohistochemistry , Intercellular Adhesion Molecule-1/metabolism , Lipid Peroxidation , Male , Malondialdehyde/metabolism , Mesenteric Artery, Superior/pathology , Neutrophils/metabolism , P-Selectin/chemistry , P-Selectin/metabolism , Peroxidase/metabolism , Phenols/chemistry , Poly(ADP-ribose) Polymerases/chemistry , Polyphenols , Random Allocation , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species , Shock , Time Factors , Treatment Outcome , Tyrosine/analogs & derivatives , Tyrosine/chemistryABSTRACT
Nicotinamide mononucleotide adenylyltransferase (NMNAT) is the central enzyme of the NAD biosynthetic pathway. Three human NMNAT isoforms have recently been identified, but isoform-specific functions are presently unknown, although a tissue-specific role has been suggested. Analyses of the subcellular localization confirmed NMNAT1 to be a nuclear protein, whereas NMNAT2 and -3 were localized to the Golgi complex and the mitochondria, respectively. This differential subcellular localization points to an organelle-specific, nonredundant function of each of the three proteins. Comparison of the kinetic properties showed that particularly NMNAT3 exhibits a high tolerance toward substrate modifications. Moreover, as opposed to preferred NAD+ synthesis by NMNAT1, the other two isoforms could also form NADH directly from the reduced nicotinamide mononucleotide, supporting a hitherto unknown pathway of NAD generation. A variety of physiological intermediates was tested and exerted only minor influence on the catalytic activities of the NMNATs. However, gallotannin was found to be a potent inhibitor, thereby compromising its use as a specific inhibitor of poly-ADP-ribose glycohydrolase. The presence of substrate-specific and independent nuclear, mitochondrial, and Golgi-specific NAD biosynthetic pathways is opposed to the assumption of a general cellular NAD pool. Their existence appears to be consistent with important compartment-specific functions rather than to reflect simple functional redundance.
Subject(s)
Golgi Apparatus/metabolism , Nicotinamide-Nucleotide Adenylyltransferase/chemistry , Adenosine Triphosphate/chemistry , Catalysis , Catalytic Domain , Cell Line , Cell Nucleus/metabolism , Cloning, Molecular , DNA, Complementary/metabolism , Glycoside Hydrolases/chemistry , HeLa Cells , Humans , Hydrolyzable Tannins/chemistry , Kinetics , Nicotinamide-Nucleotide Adenylyltransferase/physiology , Poly(ADP-ribose) Polymerases/chemistry , Protein Conformation , Protein Isoforms , Substrate Specificity , Time Factors , Tissue DistributionABSTRACT
Sexual reproduction in many angiosperm plants involves self-incompatibility (SI), which is one of the most important mechanisms to prevent inbreeding. SI is genetically controlled by the S-locus, and involves highly specific interactions during pollination between pollen and the pistil on which it lands. This results in the rejection of incompatible ('self') pollen, whereas compatible ('non-self') pollen is allowed to fertilize the plant. In Papaver rhoeas, S-proteins encoded by the stigma component of the S-locus interact with incompatible pollen, triggering a Ca2+-dependent signalling network, resulting in the inhibition of pollen-tube growth. Programmed cell death (PCD) is a mechanism used by many organisms to destroy unwanted cells in a precisely regulated manner. Here we show that PCD is triggered by SI in an S-specific manner in incompatible pollen. This provides a demonstration of a SI system using PCD, revealing a novel mechanism to prevent self-fertilization. Furthermore, our data reveal that the response is biphasic; rapid inhibition of pollen-tube growth is followed by PCD, which is involved in a later 'decision-making' phase, making inhibition irreversible.
Subject(s)
Apoptosis , Papaver/cytology , Papaver/physiology , Pollen/cytology , Pollen/physiology , Animals , Apoptosis/drug effects , Calcium Signaling , Caspase Inhibitors , Caspases/metabolism , Cattle , Cytochromes c/metabolism , Cytosol/metabolism , DNA Fragmentation/drug effects , Fertilization/drug effects , Fertilization/genetics , Fertilization/physiology , Flowers/genetics , Flowers/physiology , Genes, Plant/genetics , Papaver/drug effects , Papaver/genetics , Pollen/drug effects , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Species SpecificityABSTRACT
Poly(ADP-ribose) polymerase-1 (PARP-1) is the canonical member of the PARP family of enzymes and modulates many crucial nuclear functions. PARP-1 is involved in apoptosis and is the substrate of caspase-3, a protease that cleaves PARP-1 at the conserved sequence 211DEVD214. To generate a caspase-3-uncleavable PARP-1, we introduced an amino acid substitution D214-->A214 at the site of cleavage. We observed that following over-expression in bacteria, the mutant protein HIS-PARP-1D214A was expressed several-fold more than a unmutated copy, HIS-PARP-1. The specific activity of HIS-PARP-1 enzyme in total bacterial extracts was 6.94 U/mg and 4.61 U/mg for HIS-PARP-1D214A. This approach should provide new avenues for crystallographic study of PARP-1 as well as new information for drug design targeting PARP-1.
Subject(s)
Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Amino Acid Sequence , Amino Acids/chemistry , Apoptosis , Base Sequence , Blotting, Western , Caspase 3 , Caspases/metabolism , Cloning, Molecular , Crystallography, X-Ray , DNA, Complementary/metabolism , Densitometry , Escherichia coli/metabolism , Humans , Molecular Sequence Data , Mutation , Point Mutation , Poly(ADP-ribose) Polymerases/genetics , Protein Binding , Protein Processing, Post-Translational , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Stem CellsABSTRACT
The effects of lemon pure essential oils on the heat shock-induced apoptosis in human astrocytes cell line CCF-STTG1 were examined. In previous studies, heat shock has been reported to induce the apoptosis or programmed cell death through the activation of caspase-3. Treatment of heat shock on CCF-STTG1 cells markedly induced apoptotic cell death as determined by flow cytometry. Interestingly, pre-treatment with lemon pure essential oils on CCF-STTG1 cells inhibited the heat shock-induced apoptosis. Lemon oil also inhibited the heat shock-induced apoptosis in primary cultured rat astrocytes. To determine whether lemon oil inhibits the heat shock-induced activation of the apoptotic proteases, activation of caspase-3 was assessed by Western blotting. DNA fragmentation, giemsa staining, and heat shock-induced activation of caspase-3 were blocked by lemon pure essential oil, which is consistent with flow cytometry. Poly-ADP-ribose polymerase (PARP), the cysteine protease substrate, was fragmented as a consequence of apoptosis by heat shock. Lemon oil inhibited the PARP fragmentation. These results suggest that lemon pure essential oils may modulate the apoptosis through the activation of the interleukin-1 beta -converting enzyme-like caspases.
Subject(s)
Apoptosis/drug effects , Astrocytes/enzymology , Citrus/chemistry , Hot Temperature , Plant Oils/pharmacology , Animals , Astrocytes/cytology , Astrocytes/drug effects , Blotting, Western , Brain/cytology , Brain/enzymology , Caspase 3 , Caspases/metabolism , Cell Line , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Chromatin/metabolism , DNA/biosynthesis , DNA/chemistry , DNA Fragmentation/drug effects , Depression, Chemical , Electrophoresis, Agar Gel , Enzyme Activation , Flow Cytometry , Humans , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/metabolism , RatsABSTRACT
BACKGROUND: Brain astrocytes play a pivotal role in neuronal activities. METHODS: An investigation was undertaken to determine whether juniper oil inhibits heat shock-induced apoptosis of astrocytes. RESULTS: Juniper oil inhibited the heat shock-induced apoptosis in human astrocyte CCF-STTG1 cells. Pretreatment of the cells with juniper oil inhibited the heat shock-induced DNA fragmentation and condensation of nuclear chromatin. Juniper oil alone did not affect the apoptosis. Juniper oil inhibited the heat shock-induced caspase-3 activation and poly-ADP-ribose polymerase (PARP) fragmentation in the human astrocytes. CONCLUSIONS: Juniper oil may inhibit the apoptosis of astrocytes by preventing the caspase-3 activation.
Subject(s)
Apoptosis/drug effects , Astrocytes/enzymology , Caspases/metabolism , Hot Temperature/adverse effects , Juniperus/chemistry , Plant Oils/pharmacology , Shock/pathology , Astrocytes/drug effects , Blotting, Western , Brain/cytology , Brain/enzymology , Caspase 3 , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/ultrastructure , Chromatin/chemistry , Chromatin/metabolism , DNA/biosynthesis , DNA/chemistry , DNA Fragmentation/drug effects , Depression, Chemical , Enzyme Activation/drug effects , Flow Cytometry , Humans , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/metabolismABSTRACT
A human homologue of the rodent T cell mono(ADP-ribosyl)transferase RT6 mRNA was identified by a systematic partial sequencing of human testis transcripts. This messenger encodes for a precursor protein of 367 aa (MW: 41.5 kDa) which exhibits a peptide signal, consensus domains for mono(ADP-ribosyl)transferase and a C terminal part which contains three repeated motives (GEKNQKLEDH) and a region characteristic of glycophosphatidyl inositol anchored proteins. This mRNA is transcribed from a gene localized in 4q13-q21. Surprisingly, it is not expressed in human white blood cells but it exhibits a very specific testis expression in which it is likely to correspond to a new ADP-ribosyl transferase.
Subject(s)
ADP Ribose Transferases , Poly(ADP-ribose) Polymerases/genetics , Proteins/genetics , Testis/enzymology , Amino Acid Sequence , Animals , Base Sequence , Chromosomes, Human, Pair 4 , Cloning, Molecular , DNA, Complementary , GPI-Linked Proteins , Gene Expression , Humans , Male , Mice , Molecular Sequence Data , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Proteins/chemistry , Proteins/metabolism , RNA, Messenger , Rabbits , Rats , Sequence Homology, Amino AcidABSTRACT
Poly(ADP-ribose) polymerase cDNAs have been isolated from different classes of animals. Cloning of genes from lower eukaryotes has allowed us to investigate directly the biological functions of poly(ADP-ribosyl)ation in vivo. The conservation of specific regions among mammals, chicken, Xenopus laevis, and Drosophila melanogaster reveals the essential structural elements required for recognition of breaks in DNA and for catalytic activity. Cys, His and basic residues in the zinc-finger consensus region are conserved. The carboxyl terminal region corresponding to an NAD-binding domain is strongly conserved. The dinucleotide-binding consensus sequence and beta 1-alpha A-beta 2, Rossmann fold structure, and beta-sheet structures are completely conserved from mammals to insect. In Drosophila, a putative leucine-zipper motif has been identified, and other poly(ADP-ribose) polymerases also contain an alpha-helical, amphipathic structure in the auto-modification domain. In this article, we review the recent structural analyses of the functional domains of poly(ADP-ribose) polymerase in phylogenetically divergent species, and discuss the implications of structural conservation for its biological functions.
Subject(s)
Biological Evolution , Poly(ADP-ribose) Polymerases/chemistry , Amino Acid Sequence , Animals , Cattle , Chickens , DNA, Complementary/isolation & purification , Drosophila melanogaster , Humans , Mice , Molecular Sequence Data , Poly(ADP-ribose) Polymerases/genetics , Xenopus laevisABSTRACT
We show that poly(ADP-ribose) polymerase is present in maize, pea and wheat nuclei. We have identified the enzyme product as poly(ADP-ribose) by purification and electrophoresis on a DNA sequencing gel. This reveals a polymer ladder consisting of up to 45 residues. The polymer product from maize, after digestion with snake venom phosphodiesterase, gave only 5'-AMP and (phosphoribosyl)-AMP; the mean chain length of the polymer was 5 and 11 residues in two separate experiments. The optimum pH of the plant enzyme is greater than pH 7.0 in pea, wheat and maize; the optimum temperature for enzyme activity is approximately 15 degrees C. The Km for NAD+ for the enzyme from maize is estimated to be approximately 50 microM under optimal conditions. Several compounds (nicotinamide, deoxythymidine, 3-aminobenzamide, 3-methoxybenzamide and 5-bromodeoxyuridine) that specifically inhibit the animal enzyme also inhibit the enzyme from plants. The ratio of the IC50 for 5-bromodeoxyuridine to the IC50 for 3-aminobenzamide in maize is similar to that of the animal enzyme indicating that the enzyme involved is poly(ADP-ribose) polymerase and not mono(ADP-ribosyl) transferase. SDS gel electrophoresis and gel filtration analysis of a crude extract of maize nuclei indicate a molecular mass for poly(ADP-ribose) polymerase of approximately 114 kDa.