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:6.5.1.2 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA ligase from the hyperthermophilic marine archaeon Pyrococcus furiosus (Pfu DNA ligase) synthesizes adenosine 5'-tetraphosphate (p4A) and dinucleoside polyphosphates by displacement of the adenosine 5'-monophosphate (AMP) from the Pfu DNA ligase-AMP (E-AMP) complex with tripolyphosphate (P3), nucleoside triphosphates (NTP), or nucleoside diphosphates (NDP). The experiments were performed in the presence of 1-2 microM [alpha-32P]ATP and millimolar concentrations of NTP or NDP. Relative rates of synthesis (%) of the following adenosine(5')tetraphospho(5')nucleosides (Ap4N) were observed: Ap4guanosine (Ap4G) (from GTP, 100); Ap4deoxythymidine (Ap4dT) (from dTTP, 95); Ap4xanthosine (Ap4X) (from XTP, 94); Ap4deoxycytidine (Ap4dC) (from dCTP, 64); Ap4cytidine (Ap4C) (from CTP, 60); Ap4deoxyguanosine (Ap4dG) (from dGTP, 58); Ap4uridine (Ap4U) (from UTP, <3). The relative rate of synthesis (%) of adenosine(5')triphospho(5')nucleosides (Ap3N) were: Ap3guanosine (Ap3G) (from GDP, 100); Ap3xanthosine (Ap3X) (from XDP, 110); Ap3cytidine (Ap3C) (from CDP, 42); Ap3adenosine (Ap3A) (from ADP, <1). In general, the rate of synthesis of Ap4N was double that of the corresponding Ap3N. The enzyme presented optimum activity at a pH value of 7.2-7.5, in the presence of 4 mM Mg2+, and at 70 degrees C. The apparent Km values for ATP and GTP in the synthesis of Ap4G were about 0.001 and 0.4mM, respectively, lower values than those described for other DNA or RNA ligases. Pfu DNA ligase is used in the ligase chain reaction (LCR) and some of the reactions here reported [in particular the synthesis of Ap4adenosine (Ap4A)] could take place during the course of that reaction.
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PMID:Thermostable Pyrococcus furiosus DNA ligase catalyzes the synthesis of (di)nucleoside polyphosphates. 1187 61

Previous studies have shown that the liver is the first organ to display signs of injury during hemorrhagic shock. We examined the mechanism by which pyruvate can prevent liver damage during hemorrhagic shock in swine anesthetized with halothane. Thirty minutes after the induction of a 240-min controlled arterial hemorrhage targeted at 40 mmHg, hypertonic sodium pyruvate (0.5 g. kg(-1). h(-1)) was infused to achieve an arterial concentration of 5 mM. The volume and osmolality effects of pyruvate were matched with 10% saline (HTS) and 0.9% saline (NS). Although the peak hemorrhage volume increased significantly in both the pyruvate and HTS group, only the pyruvate treatment was effective in delaying cardiovascular decompensation. In addition, pyruvate effectively maintained the NADH/NAD redox state, as evidenced by increased microdialysate pyruvate levels and a significantly lower lactate-to-pyruvate ratio. Pyruvate also prevented the loss of intracellular antioxidants (GSH) and a reduction in the GSH-to-GSSG ratio. These beneficial effects on the redox environment decreased hepatic cellular death by apoptosis. Pyruvate significantly increased the ratio of Bcl-Xl (antiapoptotic molecule)/Bax (proapoptotic molecule), prevented the release of cytochrome c from mitochondria, and decreased the fragmentation of caspase 3 and poly(ADP ribose) polymerase (DNA repair enzyme). These beneficial findings indicate that pyruvate infused 30 min after the onset of severe hemorrhagic shock is effective in maintaining the redox environment, preventing the loss of the key antioxidant GSH, and decreasing early apoptosis indicators.
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PMID:Pyruvate improves redox status and decreases indicators of hepatic apoptosis during hemorrhagic shock in swine. 1223 18

Resveratrol, an edible polyphenolic stilbene, has been reported to possess substantial antileukemic activities in different leukemia cell lines. We investigated whether resveratrol is active against fresh acute myeloid leukemia (AML) cells and its mechanism of action. Because interleukin 1beta(IL-1beta) plays a key role in proliferation of AML cells, we first tested the effect of resveratrol on the AML cell lines OCIM2 and OCI/AML3, both of which produce IL-1beta and proliferate in response to it. Resveratrol inhibited proliferation of both cell lines in a dose-dependent fashion (5-75 microM) by arresting the cells at S phase, thus preventing their progression through the cell cycle; IL-1beta partially reversed this inhibitory effect. Resveratrol significantly reduced production of IL-1beta in OCIM2 cells. It also suppressed the IL-1beta-induced activation of transcription factor nuclear factor kappaB (NF-kappaB), which modulates an array of signals controlling cellular survival, proliferation, and cytokine production. Indeed, incubation of OCIM2 cells with resveratrol resulted in apoptotic cell death. Because caspase inhibitors Ac-DEVD-CHO or z-DEVD-FMK partially reversed the antiproliferative effect of resveratrol, we tested its effect on the caspase pathway and found that resveratrol induced the activation of the cysteine protease caspase 3 and subsequent cleavage of the DNA repair enzyme poly (adenosine diphosphate [ADP]-ribose) polymerase. Finally, resveratrol suppressed colony-forming cell proliferation of fresh AML marrow cells from 5 patients with newly diagnosed AML in a dose-dependent fashion. Taken together, our data showing that resveratrol is an effective in vitro inhibitor of AML cells suggest that this compound may have a role in future therapies for AML.
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PMID:Resveratrol blocks interleukin-1beta-induced activation of the nuclear transcription factor NF-kappaB, inhibits proliferation, causes S-phase arrest, and induces apoptosis of acute myeloid leukemia cells. 1268 43

The repair of DNA single-strand breaks in mammalian cells is mediated by poly(ADP-ribose) polymerase 1 (PARP-1), DNA ligase IIIalpha, and XRCC1. Since these proteins are not found in lower eukaryotes, this DNA repair pathway plays a unique role in maintaining genome stability in more complex organisms. XRCC1 not only forms a stable complex with DNA ligase IIIalpha but also interacts with several other DNA repair factors. Here we have used affinity chromatography to identify proteins that associate with DNA ligase III. PARP-1 binds directly to an N-terminal region of DNA ligase III immediately adjacent to its zinc finger. In further studies, we have shown that DNA ligase III also binds directly to poly(ADP-ribose) and preferentially associates with poly(ADP-ribosyl)ated PARP-1 in vitro and in vivo. Our biochemical studies have revealed that the zinc finger of DNA ligase III increases DNA joining in the presence of either poly(ADP-ribosyl)ated PARP-1 or poly(ADP-ribose). This provides a mechanism for the recruitment of the DNA ligase IIIalpha-XRCC1 complex to in vivo DNA single-strand breaks and suggests that the zinc finger of DNA ligase III enables this complex and associated repair factors to locate the strand break in the presence of the negatively charged poly(ADP-ribose) polymer.
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PMID:Physical and functional interaction between DNA ligase IIIalpha and poly(ADP-Ribose) polymerase 1 in DNA single-strand break repair. 1289 60

A gene encoding a putative ATP-dependent DNA ligase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (accession no. APE1094) from A. pernix encoding a 69-kDa protein showed a 39-61% identity with other ATP-dependent DNA ligases from the archaea. Normally DNA ligase is activated by NAD(+) or ATP. There has been no report about the other activators for DNA ligase. The recombinant ligase was a monomeric protein and catalyzed strand joining on a singly nicked DNA substrate in the presence of ADP and a divalent cation (Mg(2+), Mn(2+), Ca(2+) and Co(2+)) at high temperature. The optimum temperature and pH for nick-closing activity were above 70 degrees C and 7.5 degrees C, respectively. The ligase remained stable for 60 min of treatment at 100 degrees C, and the half-life was about 25 min at 110 degrees C. This is the first report of a novel hyperthermostable DNA ligase that can utilize ADP to activate the enzyme.
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PMID:A novel ADP-dependent DNA ligase from Aeropyrum pernix K1. 1293 88

Deposition of beta-amyloid protein in the brain is a neuropathological hallmark of Alzheimer's disease. An additional feature of this disease is an upregulation of the lysosomal system, however, the role of lysosomal proteins in the pathogenesis of this neurodegenerative condition is unclear. In this study, we demonstrate that Abeta increases activity of the lysosomal protease, cathepsin-L, and promotes a transient increase in cytosolic expression of cathepsin-L in cultured cortical neurones. The increase in cathepsin-L activity and concentration in the cytosol is evident 6 h following beta-amyloid treatment. The proclivity of beta-amyloid to induce apoptotic changes, such as activation of caspase-3, cleavage of the DNA repair enzyme, poly-ADP ribose polymerase, and DNA fragmentation, were prevented by the selective cathepsin-L inhibitor Z-FF-FMK. In contrast, beta-amyloid had no effect on expression levels or cellular distribution of cathepsin-D and the cathepsin-D inhibitor peptide failed to protect cortical neurones from beta-amyloid-induced apoptosis. Thus, the results from this study demonstrate that beta-amyloid impacts on cathepsin-L as an upstream event in the neurodegenerative process and this result highlights the potential role of lysosomal components in the pathogenesis of Alzheimer's disease.
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PMID:Abeta-mediated activation of the apoptotic cascade in cultured cortical neurones: a role for cathepsin-L. 1467 34

Aging may pose a challenge to the central nervous system, increasing its susceptibility to apoptotic events. Recent findings indicate that caloric restriction (CR) may have a profound effect on brain function and vulnerability to injury and diseases, by enhancing neuroprotection, stimulating the production of new neurons, and increasing synaptic plasticity. Apoptosis and apoptotic regulatory proteins in the brain frontal cortex of 6-month-old ad libitum fed (6AD), 26-month-old ad libitum fed (26AD), and 26-month-old caloric-restricted (26CR) male Fischer 344 rats (40% restriction compared to ad libitum fed) were investigated. Levels of Poly-ADP ribose polymerase (PARP-DNA repair enzyme; its cleaved 89 kDA fragment is a marker of apoptosis), cytoplasmic histone-associated DNA fragments, and X chromosome-linked inhibitor of apoptosis (XIAP--an endogenous apoptosis inhibitor) were determined. A significant age-associated increase in PARP was found, which was ameliorated in the frontal cortices of the CR rats. No significant differences in cytoplasmic histone-associated DNA fragments with age or with CR were observed. XIAP levels significantly increased with age in the brains of the ad libitum animals, while CR animals exhibited the highest levels of this inhibitor compared to all groups. Our findings suggest that caloric restriction may provide neuroprotection to the aging brain by preserving DNA repair enzymes in their intact form, and/or upregulating specific antiapoptotic proteins involved in neuronal cell death.
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PMID:Effects of age and caloric restriction on brain neuronal cell death/survival. 1524

Multiple myeloma (MM) accounts for 1 % of all cancer deaths. Although treated aggressively, almost all myelomas eventually recur and become resistant to treatment. Atiprimod (2-(3-Diethylaminopropyl)-8,8-dipropyl-2-azaspiro[4,5] decane dimaleate) has exerted anti-inflammatory activities and inhibited oeteoclast-induced bone resorption in animal models and been well tolerated in patients with rheumatoid arthritis in phase I clinical trials. Therefore, we investigated its activity in MM cells and its mechanism of action. We found that Atiprimod inhibited proliferation of the myeloma cell lines U266-B1, OCI-MY5, MM-1, and MM-1R in a time- and dose-dependent manner. Atiprimod blocked U266-B1 myeloma cells in the G(0)/G(1) phase, preventing cell cycle progression. Furthermore, Atiprimod inhibited signal transducer and activator of transcription (STAT) 3 activation, blocking the signalling pathway of interleukin-6, which contributes to myeloma cell proliferation and survival, and downregulated the antiapoptotic proteins Bcl-2, Bcl-X(L), and Mcl-1. Incubation of U266-B1 myeloma cells with Atiprimod induced apoptosis through the activation of caspase 3 and subsequent cleavage of the DNA repair enzyme poly(adenosine diphosphate-ribose) polymerase. Finally, Atiprimod suppressed myeloma colony-forming cell proliferation in fresh marrow cells from five patients with newly diagnosed MM in a dose-dependent fashion. These data suggest that Atiprimod has a role in future therapies for MM.
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PMID:Atiprimod blocks STAT3 phosphorylation and induces apoptosis in multiple myeloma cells. 1597 Sep 28

Protein interactions critical to DNA repair and cell cycle control systems are often coordinated by modules that belong to a superfamily of structurally conserved BRCT domains. Because the mechanisms of BRCT interactions and their significance are not well understood, we sought to define the affinity and specificity of those BRCT modules that orchestrate base excision repair and single-strand break repair. Common to these pathways is the essential XRCC1 DNA repair protein, which interacts with at least nine other proteins and DNA. Here, we characterized the interactions of four purified BRCT domains, two from XRCC1 and their two partners from DNA ligase IIIalpha and poly(ADP-ribosyl) polymerase 1. A monoclonal antibody was selected that recognizes the ligase IIIalpha BRCT domain, but not the other BRCT domains, and was used to capture the relevant ligase IIIalpha BRCT complex. To examine the assembly states of isolated BRCT domains and pairwise domain complexes, we used size-exclusion chromatography coupled with on-line light scattering. This analysis indicated that isolated BRCT domains form homo-oligomers and that the BRCT complex between the C-terminal XRCC1 domain and the ligase IIIalpha domain is a heterotetramer with 2:2 stoichiometry. Using affinity capture and surface plasmon resonance methods, we determined that specific heteromeric interactions with high nanomolar dissociation constants occur between pairs of cognate BRCT domains. A structural model for a XRCC1 x DNA ligase IIIalpha heterotetramer is proposed as a core base excision repair complex, which constitutes a scaffold for higher order complexes to which other repair proteins and DNA are brought into proximity.
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PMID:Specificity of protein interactions mediated by BRCT domains of the XRCC1 DNA repair protein. 1598 76

Cytokine stimulation induces proliferation and growth of acute myeloid leukemia (AML) blasts and high levels of cytokines have been associated with poor prognosis in AML. The Jak-Stat pathway constitutes a major mediator of cytokine activity. We investigated whether WP-1034, a novel Jak-Stat inhibitor, is active against AML blasts. OCIM2 and fresh AML cells were incubated with 1 to 6 microM WP-1034 to determine its effect on proliferation. WP-1034 effectively inhibited proliferation of OCIM2 cells and fresh AML samples. We then analyzed the expressions of Stat 1, 3, and 5, as well as Phospho-Stat 1, 3, and 5 by Western immunoblotting after incubation of OCIM2 cells without and with 1 to 10 microM WP-1034 for 2 hours, and at 5 microM from 20 minutes up to 4 hours and found that WP-1034 blocked Stat 3 and 5 activation. Analysis of cell cycle status by PI staining and flow cytometry showed that WP-1034 caused cell cycle arrest of OCIM2 cells in sub-Go phase. We then evaluated the induction of apoptosis of OCIM2 cells following incubation with WP-1034 at 3 to 6 microM by annexin V-CY5 assay and analyzed caspase 3 and PARP cleavage using Western immunoblotting. We found that WP-1034 induced apoptosis of OCIM2 cells and that induction of apoptosis involved cleavage of caspase 3 and the DNA repair enzyme poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP). Taken together, our data suggest that WP-1034 is a potent inhibitor of AML cell proliferation by inhibition of Stat 3 and 5 and induction of caspase-dependent apoptosis.
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PMID:WP-1034, a novel JAK-STAT inhibitor, with proapoptotic and antileukemic activity in acute myeloid leukemia (AML). 1615 16


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