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)

We have used site-directed insertion and point mutagenesis in an attempt to increase the cytotoxic potency and receptor-binding affinity of the diphtheria-toxin-related interleukin-2 (IL-2) fusion toxins. Previous studies have demonstrated that both the DAB486-IL-2 and DAB389-IL-2 forms of the fusion toxin consist of three functional domains: the N-terminal fragment-A-associated ADP-ribosyltransferase, the hydrophobic-membrane-associating domains, and the C-terminal receptor-binding domain of human IL-2. By insertion mutagenesis we have increased the apparent flexibility of the polypeptide chain between the membrane-associating domains and the receptor-binding domain of this fusion toxin. In comparison to DAB486-IL-2, the cytotoxic potency of the insertion mutants was increased by approximately 17-fold for high-affinity IL-2-receptor-bearing cell lines in vitro. Moreover, competitive displacement experiments using [125I]rIL-2 demonstrate that the increase in cytotoxic potency correlates with an increase in receptor-binding affinity for both the high and intermediate forms of the IL-2 receptor.
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PMID:Protein engineering of diphtheria-toxin-related interleukin-2 fusion toxins to increase cytotoxic potency for high-affinity IL-2-receptor-bearing target cells. 188 72

We have used cassette and deletion mutagenesis to analyze the structural features of fragment B-related sequences in the fusion toxin DAB486-IL-2 (where IL-2 represents interleukin-2) that are necessary for the efficient delivery of fragment A to the cytosol of target cells. We demonstrate that whereas an intact disulfide bond between Cys461 and Cys471 may be required for the cytotoxic action of native diphtheria toxin, this bond is not required for the cytotoxic action of DAB486-IL-2. The in-frame deletion of the 97 amino acids from Thr387 to His485 of DAB486-IL-2 increases both the potency and the apparent dissociation constant (Kd) of the resulting fusion toxin for high affinity interleukin-2 receptor-bearing target cells. In contrast, the inframe deletion of either the 191 amino acids between Asp291 and Gly483 or the 85 amino acids between Asn204 and Ile290 results in a 1000-fold loss in potency. These regions contain the putative membrane-spanning regions and the amphipathic membrane surface-associating regions of fragment B, respectively. These results indicate that the efficient delivery of the ADP-ribosyltransferase from DAB486-IL-2 to the cytosol requires the membrane-associating domains of fragment B. This function has been postulated to play a role in the diphtherial intoxication of eukaryotic cells. However, unlike native diphtheria toxin, fragment B sequences distal to Thr387 do not enhance the potency of DAB486-IL-2.
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PMID:Structure/function analysis of interleukin-2-toxin (DAB486-IL-2). Fragment B sequences required for the delivery of fragment A to the cytosol of target cells. 219 27

DAB486IL-2 is a novel fusion toxin in which the ADP-ribosyltransferase and membrane-translocating domains of diphtheria toxin have been combined with the interleukin-2 (IL-2) gene, creating a recombinant protein capable of selectively intoxicating cells bearing the high-affinity IL-2 receptor. Clinical activity has been documented in Hodgkin disease and the non-Hodgkin lymphomas; toxicities have been minimal and include mild hepatic transaminitis, proteinuria, and hypersensitivity reactions. In this report, a patient with tumor-stage cutaneous T-cell lymphoma developed clinical adrenal failure with bilateral adrenal hemorrhage and necrosis 7 weeks after completing a 5-day course of treatment with DAB486IL-2. The relationship of fusion toxin therapy to the development of this unusual toxicity is discussed.
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PMID:Bilateral adrenal hemorrhage and adrenal insufficiency in a patient with lymphomatous adrenal infiltration following administration of a fusion toxin (DAB486 interleukin-2). 783 23

Cassette and deletion mutagenesis were used to analyze the function of the amphipathic alpha-helices in the transmembrane domain of DAB389-interleukin-2 (IL-2), a fusion protein which is targeted to the interleukin-2 receptor. We demonstrate that the in-frame deletion of 60 amino acids, from Asn204 to Glu263 in DAB389-IL-2, results in complete loss of cytotoxic activity, whereas when the amphipathic regions from Asp208 to Ser220 and Ala244 to His258 are replaced with idealized amphipathic helices composed of repeating Glu, Lys, and Leu residues, the mutant fusion toxin has low but detectable activity. DAB389-IL-2 and both variants form channels in artificial phospholipid bilayers with conductances identical to those formed by diphtheria toxin. Both mutant fusion toxins bind to the high affinity IL-2 receptor with affinities similar to that of DAB389-IL-2. The fact that these mutants have markedly reduced or absent cytotoxic activity, but possess "wild type" catalytic activity, binding affinities, and channel conductances, suggests the existence of a step in the intoxication pathway, defective in the mutants, which occurs after DAB389-IL-2 binds to the IL-2 receptor. It is unknown whether this step occurs prior or subsequent to channel formation, but it is essential for the efficient delivery of the ADP-ribosyltransferase from DAB389-IL-2 to the cytosol of target cells.
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PMID:Structure/function analysis of the transmembrane domain of DAB389-interleukin-2, an interleukin-2 receptor-targeted fusion toxin. The amphipathic helical region of the transmembrane domain is essential for the efficient delivery of the catalytic domain to the cytosol of target cells. 850 30

Induction of potent apoptosis is required in cancer therapy. We examined the combination effect of interleukin-2-activated lymphocytes (LAK cells) and anticancer drugs or gamma (gamma)-rays on the induction of apoptosis in an established oral squamous cell carcinoma cell line (OSC-3 cells). By pretreatment of OSC-3 cells with (137)Cs (5 Gy), 5-fluorouracil (5-FU) (0.5 microg/ml) or cis-dichlorodiammine-platinum (CDDP) (5 microg/ml), the activation of bid and caspase-3 by LAK cells was strongly increased and associated with an enhanced degradation of poly-(ADP-ribose) polymerase (PARP) and/or nuclear mitotic apparatus protein (NuMA) and the increased fragmentation of DNA. The LAK cell-enhanced caspase-3 activity in the pretreated OSC-3 cells was decreased to approximately 70% and 40% of the control by the addition of Z-AAD-CMK (a granzyme B inhibitor) and neutralising monoclonal antibody to Fas antigen (alphaFas-IgG), respectively. The combined treatment-induced DNA fragmentation was suppressed by approximately 20% and 30% of the control by the addition of Z-AAD-CMK and alpha Fas-IgG, respectively, in the co-culture system. While Ac-DEVD-CHO (a caspase-3 inhibitor) suppressed the DNA fragmentation levels to approximately half and this was similar to the amount of suppression that was obtained by the addition of both alpha Fas-IgG and Z-AAD-CMK. In addition, LAK cell-activated bid may have increased the intracellular reactive oxygen intermediates (ROI) level and induced a decrease of mitochondrial membrane potential. These influences by LAK cells were enhanced when OSC-3 cells were pretreated with each anticancer drug or (137)Cs. Furthermore, the increase of ROI by LAK cells was suppressed by alpha Fas-IgG and Z-AAD-CMK to approximately half the level of the control. These results indicate that anticancer drugs and gamma-rays prime squamous cell carcinoma cells to be susceptible to apoptosis by LAK cells, that LAK cell-induced apoptosis largely depends on the activation of caspase-3 by the Fas/Fas-ligand signal and granzyme B, and that LAK cells induce ROI in the target cells, which is largely mediated by Fas and granzyme B.
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PMID:Enhanced apoptosis of squamous cell carcinoma cells by interleukin-2-activated cytotoxic lymphocytes combined with radiation and anticancer drugs. 1100 May 84

Fusion proteins are recombinant molecules that combine a targeting mechanism to a cytocidal moiety. DAB(389)IL-2 (denileukin diftitox; ONTAK), with a unique mechanism of action, is the first genetically constructed fusion protein to reach the clinic. In this molecule, the interleukin-2 (IL-2) gene is genetically fused to the enzymatically active and translocating domains of diphtheria toxin. DAB(389)IL-2 is internalized into IL-2 receptor-bearing cells by endocytosis. The ADP-ribosyltransferase activity of diphtheria toxin is cleaved in the endosome and is translocated into the cytosol where it inhibits protein synthesis, leading to apoptosis. DAB(389)IL-2 and its predecessor, DAB(486)IL-2, have shown clinical activity in a variety of diseases, including B-cell non-Hodgkin's lymphoma, cutaneous T-cell lymphoma (CTCL), Hodgkin's disease, psoriasis, rheumatoid arthritis, and HIV infection. The highest response rates were observed in CTCL, and this became the focus of clinical trials leading to its subsequent approval by the United States Food and Drug Administration for this disease. The potential applications of DAB(389)IL-2 in lymphomas are reviewed.
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PMID:DAB(389)IL-2 (ONTAK): a novel fusion toxin therapy for lymphoma. 1170 18

Treatment of patients with adult T-cell leukemia-lymphoma (ATLL) using conventional chemotherapy has limited benefit because human T-cell leukemia virus type 1 (HTLV-1) cells are resistant to most apoptosis-inducing agents. The recent report that arsenic trioxide induces apoptosis in HTLV-1-transformed cells prompted investigation of the mechanism of action of this drug in HTLV-1 and HTLV-2 interleukin-2-independent T cells and in HTLV-1-immortalized cells or in ex vivo ATLL samples. Fluorescence-activated cell sorter analysis, fluorescence microscopy, and measures of mitochondrial membrane potential (Delta Psi m) demonstrated that arsenic trioxide alone was sufficient to induce programmed cell death in all HTLV-1 and -2 cells tested and in ATLL patient samples. I kappa B-alpha phosphorylation strongly decreased, and NF-kappa B translocation to the nucleus was abrogated. Expression of the antiapoptotic protein Bcl-X(L), whose promoter is NF-kappa B dependent, was down-regulated. The collapse of Delta Psi m and the release of cytochrome c to the cytosol resulted in the activation of caspase-3, as demonstrated by the cleavage of PARP. A specific caspase-3 inhibitor (Ac-DEVD-CHO) could reverse this phenotype. The antiapoptotic factor Bcl-2 was then cleaved, converting it to a Bax-like death effector. These results demonstrated that arsenic trioxide induces apoptosis in HTLV-1- and -2-infected cells through activation of the caspase pathway.
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PMID:Arsenic trioxide induces apoptosis in human T-cell leukemia virus type 1- and type 2-infected cells by a caspase-3-dependent mechanism involving Bcl-2 cleavage. 1173 84

1. In the presence of genotoxic stress poly(ADP-ribose) polymerase-1 (PARP-1) leads to NAD(+) and ATP depletion, participating in the pathogenesis of several disorders including inflammation. Accordingly, chemical inhibitors of PARP-1 are efficacious anti-inflammatories, albeit the underlying molecular mechanisms are still under debate. 2. This study investigated the effect of the PARP-1 inhibitors 6(5H)-phenanthridinone and benzamide as well as that of benzoic acid, an inactive analogue of benzamide, on development of experimental allergic encephalomyelitis (EAE) in rats. Both 6(5H)-phenanthridinone and benzamide attenuated development of EAE, reducing clinical score, neuroimmune infiltration and expression of inflammatory mediators such as inducible nitric oxide synthase, interleukin-1beta and -2, cyclooxygenase-2, tumour necrosis factor-alpha and interferon-gamma in the spinal cord of myelin-immunized rats. Importantly, no evidence of NAD(+) and ATP depletion as well as poly(ADP-ribose) formation was detected in the spinal cord. 3. By contrast, a robust formation of poly(ADP-ribose) occurred in B- and T-cell areas in lymph nodes of myelin-immunized rats and was suppressed by the treatment with 6(5H)-phenanthridinone and benzamide. In cultures of activated rat lymphocytes, 6(5H)-phenanthridinone and benzamide reduced the DNA-binding activity of NF-kappaB and AP-1 and transcription of pro-inflammatory cytokines such as interleukin-2, interferon-gamma and tumour necrosis factor-alpha. 4. Notably, benzoic acid did not reproduce the in vivo and in vitro effects of its parent compound. 5. These findings indicate that PARP-1 promotes transcriptional activation in lymphocytes and inhibitors of its enzymatic activity are useful for the treatment of autoimmune disorders of the central nervous system.
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PMID:Inhibitors of poly(ADP-ribose) polymerase-1 suppress transcriptional activation in lymphocytes and ameliorate autoimmune encephalomyelitis in rats. 1241 6

DAB(389)IL-2 (ONTAK) is a fusion protein consisting of the ADP-ribosyltransferase and membrane translocating domains of native diphtheria toxin and the full-length sequence for interleukin-2 (IL-2) gene. In vitro data demonstrates that DAB(389)IL-2 is cytotoxic to cells expressing the high affinity IL-2 receptor (IL-2R). In Phases I and II clinical trials of patients whose tumor cells express a component of the IL-2R, the response rates were 18% for B-cell non-Hodgkin lymphoma (NHL) and 30% for cutaneous T-cell lymphoma (CTCL). In this study, we examined the effects of arginine butyrate on IL-2R expression and susceptibility of leukemia cells to intoxication by DAB(389)IL-2. We demonstrate that the p75 subunit of the IL-2R (IL-2Rbeta) is upregulated in the presence of low concentrations of arginine butyrate (0.06mM) which had no direct growth inhibitory effect on the cells. To explore mechanisms of this upregulation, we examined the effect of 0.06mM arginine butyrate on relevant transcriptional elements and on histone deacetylase and found activation of cAMP response element (CRE) but not NFAT or NFKB, as well as inhibition of histone deacetylase (HDAC). Our results suggest that the effects of physiologically achievable concentrations of butyrate on IL-2R expression could be exploited to enhance the susceptibility of intermediate and low-affinity IL-2R expressing leukemia cells to DAB(389)IL-2.
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PMID:Arginine butyrate increases the cytotoxicity of DAB(389)IL-2 in leukemia and lymphoma cells by upregulation of IL-2Rbeta gene. 1244 77

ADP-ribosylation is a reversible posttranslational modification mediated by poly-ADP-ribose polymerase (PARP). The results of recent studies demonstrate that ADP-ribosylation contributes to transcription regulation. Here, we report that transcription factor NFAT binds to and is ADP-ribosylated by PARP-1 in an activation-dependent manner. Mechanistically, ADP-ribosylation increases NFAT DNA binding. Functionally, NFAT-mediated interleukin-2 (IL-2) expression was reduced in T cells upon genetic ablation or pharmacological inhibition of PARP-1. Parp-1(-/-) T cells also exhibit reduced expression of other NFAT-dependent cytokines, such as IL-4. Together, these results demonstrate that ADP-ribosylation mediated by PARP-1 provides a molecular switch to positively regulate NFAT-dependent cytokine gene transcription. These results also imply that, similar to the effect of calcineurin inhibition, PARP-1 inhibition may be beneficial in modulating immune functions.
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PMID:Regulation of transcription factor NFAT by ADP-ribosylation. 1829 89


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