Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this paper, we review our recent work on poly(ADP-ribosyl)ation and its relationships with DNA amplification and with the life span of different mammalian species. Poly(ADP-ribosyl)ation is a eukaryotic posttranslational protein modification catalyzed by poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30). This enzyme is strongly activated by DNA strand breaks and apparently plays a role in DNA repair and other cellular responses to DNA damage. Our data from two different cell culture systems for inducible DNA amplification strongly suggest that poly(ADP-ribosyl)ation acts as a negative regulatory factor in the DNA amplification induced by carcinogens. Furthermore, we could show a strong positive correlation between directly stimulated PARP activities in mononuclear leukocytes of 13 mammalian species and the species' maximal life spans. The hypothesis is raised that a higher poly(ADP-ribosyl)ation capacity of long-lived species might contribute to the efficient maintenance of genome integrity and stability over their longer life span. Finally, we could show that the selectively overexpressed PARP DNA-binding domain efficiently inhibits poly(ADP-ribosyl)ation in a transdominant manner. This molecular genetic approach should permit further interventional studies on biological role(s) of poly(ADP-ribosyl)ation without application of low-molecular-weight PARP inhibitors, thus avoiding any of their possible side effects.
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PMID:Poly(ADP-ribosyl)ation: its role in inducible DNA amplification, and its correlation with the longevity of mammalian species. 130 44

Addition of the ionic detergent N-lauroylsarcosine (Sarkosyl) affects the efficiency of transcription of genes of the protozoan Trypanosoma brucei in nuclear run-on assays. Transcription of the PARP (procyclin or procyclic acidic repetitive protein), variant cell surface glycoprotein (VSG) and ribosomal RNA (rRNA) genes was resistant or increased after addition of Sarkosyl. In contrast, the transcription of seven protein coding house keeping genes and the mini-exon donor RNA (medRNA) genes was completely abolished by the addition of Sarkosyl, while the transcription of the 5S rRNA genes showed an intermediate sensitivity. We conclude that Sarkosyl can be used to discriminate between the different types of trypanosome transcription units. The PARP and VSG protein coding genes had previously been postulated to be transcribed by an RNA polymerase I-like enzyme on the basis of their resistance to the RNA polymerase II inhibitor alpha-amanitin. This model is now supported by their resistance to the addition of Sarkosyl.
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PMID:The PARP and VSG genes of Trypanosoma brucei do not resemble RNA polymerase II transcription units in sensitivity to Sarkosyl in nuclear run-on assays. 137 45

The African trypanosome, Trypanosoma brucei, expresses two abundant stage-specific glycosylphosphatidylinositol (GPI)-anchored glycoproteins, the procyclic acidic repetitive protein (PARP or procyclin) in the procyclic form, and the variant surface glycoprotein (VSG) in the mammalian bloodstream form. The GPI anchor of VSG can be readily cleaved by phosphatidylinositol (PI)-specific phospholipase C (PI-PLC), whereas that of PARP cannot, due to the presence of a fatty acid esterified to the inositol. In the bloodstream form trypanosome, a number of GPIs which are structurally related to the VSG GPI anchor have been identified. In addition, several structurally homologous GPIs have been described, both in vivo and in vitro, that contain acyl-inositol. In vivo the procyclic stage trypanosome synthesizes a GPI that is structurally homologous to the PARP GPI anchor, i.e. contains acyl-inositol. No PI-PLC-sensitive GPIs have been detected in the procyclic form. Using a membrane preparation from procyclic trypanosomes which is capable of synthesizing GPI lipids upon the addition of nucleotide sugars we find that intermediate glycolipids are predominantly of the acyl-inositol type, and the mature ethanolamine-phosphate-containing precursors are exclusively acylated. We suggest that the differences between the bloodstream and procyclic form GPI biosynthetic intermediates can be accounted for by the developmental regulation of an inositol acylhydrolase, which is active only in the bloodstream form, and a glyceride fatty acid remodeling system, which is only partially functional in the procyclic form.
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PMID:Developmental variation of glycosylphosphatidylinositol membrane anchors in Trypanosoma brucei. In vitro biosynthesis of intermediates in the construction of the GPI anchor of the major procyclic surface glycoprotein. 137 98

Poly(ADP-ribose)polymerase (PARP)-activity was assessed in vitro from the incorporation of the adenosine-diphosphate-ribose moiety of 14C-NAD+ in the acid-insoluble cell fraction. When compared to mammalian (rat) cells, chicken embryo cells exhibit an almost three- to fourfold higher constitutive PARP-activity and an about two- to threefold lower chromatin compactness as evidenced by viscometry of alkaline cell lysates and nucleoid sedimentation. X-irradiation, bleomycin and H2O2 activated PARP. Hyperthermia (43 degrees C), doxorubicin, ethidium bromide and novobiocin resulted in an inhibition of the enzyme activity. Even at the highest doses used, UV-light, monofunctionally alkylating agents and the bisbenzimide Hoechst 33258 remained without significant effects. It is suggested that, with respect to DNA-and/or chromatin-interactive agents, the chicken embryo PARP-test may be complementary to the results of morphological and biochemical studies.
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PMID:Poly(ADP-ribose)polymerase-activity of chicken embryo cells exposed to nucleotoxic agents. 146 59

Poly(ADP-ribosyl)ation is a eukaryotic posttranslational modification of proteins that is strongly induced by the presence of DNA strand breaks and plays a role in DNA repair and the recovery of cells from DNA damage. We compared poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) activities in Percoll gradient-purified, permeabilized mononuclear leukocytes from mammalian species of different maximal life span. Saturating concentrations of a double-stranded octameric oligonucleotide were applied to provide a direct and maximal stimulation of PARP. Our results on 132 individuals from 13 different species yield a strong positive correlation between PARP activity and life span (r = 0.84; P << 0.001), with human cells displaying approximately 5 times the activity of rat cells. Intraspecies comparisons with both rat and human cells from donors of all age groups revealed some decline of PARP activity with advancing age, but it was only weakly correlated. No significant polymer degradation was detectable under our assay conditions, ruling out any interference by poly(ADP-ribose) glycohydrolase activity. By Western blot analysis of mononuclear leukocytes from 11 species, using a crossreactive antiserum directed against the extremely well-conserved NAD-binding domain, no correlation between the amount of PARP protein and the species' life spans was found, suggesting a greater specific enzyme activity in longer-lived species. We propose that a higher poly(ADP-ribosyl)ation capacity in cells from long-lived species might contribute to the efficient maintenance of genome integrity and stability over their longer life span.
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PMID:Poly(ADP-ribose) polymerase activity in mononuclear leukocytes of 13 mammalian species correlates with species-specific life span. 146 94

Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is a zinc finger DNA-binding protein involved in DNA repair processes in eukaryotes. By deletion and extensive site-directed mutagenesis, its DNA-binding domain fused to the N-terminus of beta-galactosidase was shown to contain a nuclear localization signal (NLS) of the form KRK-X(11)-KKKSKK (residues 207-226). In vitro, both the DNA-binding capacity and the polymerizing activity of PARP are independent of the nuclear location function. Each basic cluster is essential but not sufficient on its own for this function, while both motifs together are. Crucial basic amino acids (K207, R208 and K222) in each of these two motifs are required for nuclear homing. The results presented here support the concept that the human PARP NLS is an autonomous functional element and belongs to the class of bipartite NLSs. We show that the linear distance between the two basic clusters is not crucial. Insertional mutation analysis leading to a partial reversion of the cytoplasmic phenotype displayed by the mutant K222I highlights the crucial positioning of this lysine. The structure-function relationship of the second cluster of basic residues is discussed.
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PMID:The human poly(ADP-ribose) polymerase nuclear localization signal is a bipartite element functionally separate from DNA binding and catalytic activity. 150 17

The poly ADP-ribosylation of proteins catalyzed by poly(ADP-ribose)polymerase (PARP) is involved in a number of important cellular metabolic activities. We evaluated various analogs of deoxythymidine and deoxyuridine as inhibitors of PARP. Most of these compounds have antiviral and/or anticancer activities. The structural requirements for these nucleoside analogs to be inhibitors of PARP were determined. The compounds evaluated had various substitutions on the 2-, 4- and/or 5-position of the pyrimidine ring, as well as on the 2'-, 3'- and/or 5'-position of the pentose moiety. Inhibition of PARP was strongly dependent on the size of the alkyl or halogen substituent on the 5-position of the pyrimidine ring. Whereas the 5-position of the pyrimidine ring could be varied, alteration of the 2- or 4-position drastically decreased the inhibition of PARP. Kinetic analysis was performed with concentrations of 1-10 microM NAD+. The Ki values for many compounds were five to seven times lower than the Ki for 3-aminobenzamide, a previously described potent inhibitor of PARP. Compounds with combined substituents at both the 5-position of the pyrimidine ring and the 3'- or 5'-position of deoxyribose generally were potent inhibitors of PARP, as for example 3'-amino-2', 3'-dideoxy-(E)-5-(2-bromovinyl)uridine (Ki = 0.7 microM), or 5'-azido-2',5'-dideoxy-5-ethyluridine (Ki = 0.8 microM). The 5-halogenated analogs had Ki values of 18, 35, 110 and greater than 1000 microM for 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, 5-chloro-2'-deoxyuridine, and 5-fluoro-2'-deoxyuridine, respectively, and the 5-alkyl analogs had Ki values of 45, 2.2, 7, 16 and 180 microM for 5-methyl-2'-deoxyuridine, 5-ethyl-2'-deoxyuridine, 5-propyl-2'-deoxyuridine, 5-butyl-2'-deoxyuridine and 5-pentyl-2'-deoxyuridine, respectively. Two other compounds with substituents in the 5-position of the pyrimidine moiety also had potent activities: (E)-5-(2-bromovinyl)-2'-deoxyuridine (Ki = 6 microM) and 5-trifluoromethyl-2'-deoxyuridine (Ki = 1.6 microM). Compounds substituted in the 2'-, 3'- and/or 5'-position of the deoxyribose moiety were investigated and 5'-azido-5'-deoxythymidine, 5'-amino-5'-deoxythymidine, 3'-azido-3'-deoxythymidine and 3'-deoxythymidine (d2T) and Ki values of 12, 16, 18 and 30 microM, respectively.
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PMID:Inhibition of poly(ADP-ribose)polymerase activity by nucleoside analogs of thymidine. 153 Jun 62

We have overproduced the full-length human poly(ADP-ribose) polymerase (PARP) in Spodoptera frugiperda (Sf9) cells using a baculovirus expression vector system. Approx. 20 mg of purified protein from 5 x 10(8) Sf9 cells were obtained by a simple three-step purification procedure including 3-aminobenzamide affinity chromatography. The recombinant protein (rePARP), which migrates as a unique 116-kDa band on SDS-polyacrylamide gels, was identified as PARP by Western blotting using either polyclonal or monoclonal antibodies raised against the purified human and calf thymus enzymes. Furthermore, rePARP is a functional protein, as demonstrated by its ability to specifically bind Zn2+ and DNA, and to recognize single-strand breaks in DNA. The purified enzyme has the same affinity for NAD+ and turnover number as the human placental PARP. Thus, rePARP produced in insect cells is biologically active and suitable for functional analysis. The reproducibility of the overproduction and the simplicity of the purification protocol, as well as the yield of the produced protein, should greatly facilitate physicochemical and structural studies.
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PMID:Overproduction and large-scale purification of the human poly(ADP-ribose) polymerase using a baculovirus expression system. 160 10

The influence of poly(ADP-ribose) polymerase (PARP) on the replication of DNA containing the SV40 origin of replication has been examined. Extensive replication of SV40 DNA can be carried out in the presence of T antigen, topoisomerase I, the multimeric human single strand DNA-binding protein (HSSB), and DNA polymerase alpha-DNA primase (pol alpha-primase) complex (the monopolymerase system). In the monopolymerase system, both small products (Okazaki fragments), arising from lagging strand synthesis, and long products, arising from leading strand synthesis, are formed. The synthesis of long products requires the presence of relatively high levels of pol alpha-primase complex. In the presence of PARP, the synthesis of long products was blocked and only small Okazaki fragments accumulated, arising from the replication of the lagging strand template. The inhibition of leading strand synthesis by PARP can be effectively reversed by supplementing the monopolymerase system with the multimeric activator 1 protein (A1), the proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta (the dipolymerase system). The inhibition of leading strand synthesis in the monopolymerase system was caused by the binding of PARP to the ends of DNA chains, which blocked their further extension by pol alpha. The selective accumulation of Okazaki fragments was shown to be due to the coupled synthesis of primers by DNA primase and their immediate extension by pol alpha complexed to primase. PARP had little effect on this coupled reaction, but did inhibit the subsequent elongation of products, presumably after pol alpha dissociated from the 3'-end of the DNA fragments. PARP inhibited several other enzymatic reactions which required free ends of DNA chains. PARP inhibited exonuclease III, DNA ligase, the 5' to 3' exonuclease, and the elongation of primed DNA templates by pol alpha. In contrast, PARP only partly competed with the elongation of primed DNA templates by the pol delta elongation system which required SSB, A1, and PCNA. These results suggest that the binding of PARP at the ends of nascent DNA chains can be displaced by the binding of A1 and PCNA to primer ends. HSSB can be poly(ADP-ribosylated) in vivo as well as in vitro. However, the selective effect of PARP in blocking leading strand synthesis in the monopolymerase system was shown to depend primarily on its DNA binding property rather than on its ability to synthesize poly(ADP-ribose).
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PMID:Influence of poly(ADP-ribose) polymerase on the enzymatic synthesis of SV40 DNA. 167 70

At least one of the procyclic acidic repetitive protein (PARP or procyclin) loci of Trypanosoma brucei is a small (5- to 6-kilobase) polycistronic transcription unit which is transcribed in an alpha-amanitin-resistant manner. Its single promoter, as mapped by run-on transcription analysis and UV inactivation of transcription, is located immediately upstream of the first alpha-PARP gene. Transcription termination occurs in a region approximately 3 kilobases downstream of the beta-PARP gene. The location of the promoter was confirmed by its ability to direct transcription of the bacterial chloramphenicol acetyltransferase gene in insect-form (procyclic) T. brucei. The putative PARP promoter is located in the region between the 3' splice acceptor site (nucleotide position 0) and nucleotide position -196 upstream of the alpha-PARP genes. Regulatory regions influencing the levels of PARP expression may be located further upstream. We conclude that a single promoter, which is located very close to the 3' splice acceptor site of the alpha-PARP genes, directs the transcription of a small, polycistronic, and alpha-amanitin-resistant transcription unit.
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PMID:Procyclic acidic repetitive protein (PARP) genes located in an unusually small alpha-amanitin-resistant transcription unit: PARP promoter activity assayed by transient DNA transfection of Trypanosoma brucei. 169 12


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