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Molecular Pharmacology Nov 1966
Topics: Adenine; Alkylation; Animals; Cattle; Chromatography, Paper; DNA; DNA, Bacterial; Escherichia coli; Guanine; Hot Temperature; Hydrogen-Ion Concentration; Mutation; Purines; RNA; Sarcina; Thymus Gland
PubMed: 5337732
DOI: No ID Found -
The FEBS Journal Oct 2012Ricin A-chain (RTA) depurinates the sarcin-ricin loop of 28S ribosomal RNA and inhibits protein synthesis in mammalian cells. In yeast, the ribosomal stalk facilitates...
Ricin A-chain (RTA) depurinates the sarcin-ricin loop of 28S ribosomal RNA and inhibits protein synthesis in mammalian cells. In yeast, the ribosomal stalk facilitates the interaction of RTA with the ribosome and subsequent depurination. Despite homology between the stalk structures from yeast and humans, there are notable differences. The human ribosomal stalk contains two identical heterodimers of P1 and P2 bound to P0, whereas the yeast stalk consists of two different heterodimers, P1α-P2β and P2α-P1β, bound to P0. RTA exhibits higher activity towards mammalian ribosomes than towards ribosomes from other organisms, suggesting that the mode of interaction with ribosomes may vary. Here, we examined whether the human ribosomal stalk proteins facilitate the interaction of RTA with human ribosomes and subsequent depurination of the sarcin-ricin loop. Using small interfering RNA-mediated knockdown of P1/P2 expression in human cells, we demonstrated that the depurination activity of RTA is lower when P1 and P2 levels are reduced. Biacore analysis showed that ribosomes from P1/P2-depleted cells have a reduced ability to bind RTA, which correlates with reduced depurination activity both in vitro and inside cells. RTA interacts directly with recombinant human P1-P2 dimer, further demonstrating the importance of human P1 and P2 in enabling RTA to bind and depurinate human ribosomes.
Topics: HEK293 Cells; Humans; Immunoblotting; Immunoprecipitation; Phosphoproteins; Protein Binding; Protein Multimerization; Purines; RNA Interference; RNA, Ribosomal, 28S; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Proteins; Ribosomes; Ricin
PubMed: 22909382
DOI: 10.1111/j.1742-4658.2012.08752.x -
The Journal of Biological Chemistry Nov 2002Pokeweed antiviral protein (PAP), a single chain ribosome-inactivating protein (RIP) isolated from pokeweed plants (Phytolacca americana), removes specific adenine and...
Pokeweed antiviral protein regulates the stability of its own mRNA by a mechanism that requires depurination but can be separated from depurination of the alpha-sarcin/ricin loop of rRNA.
Pokeweed antiviral protein (PAP), a single chain ribosome-inactivating protein (RIP) isolated from pokeweed plants (Phytolacca americana), removes specific adenine and guanine residues from the highly conserved, alpha-sarcin/ricin loop in the large rRNA, resulting in inhibition of protein synthesis. We recently demonstrated that PAP could also inhibit translation of mRNAs and viral RNAs that are capped by binding to the cap structure and depurinating the RNAs downstream of the cap. Cell growth is inhibited when PAP cDNA is expressed in the yeast Saccharomyces cerevisiae under the control of the galactose-inducible GAL1 promoter. Here, we show that overexpression of wild type PAP in yeast leads to a decrease in PAP mRNA abundance. The decrease in mRNA levels is not observed with an active site mutant, indicating that it is due to the N-glycosidase activity of the protein. PAP expression had no effect on steady state levels of mRNA from four different endogenous yeast genes examined, indicating specificity. We demonstrate that PAP can depurinate the rRNA in trans in a translation-independent manner. When rRNA is depurinated and translation is inhibited, the steady state levels of PAP mRNA increase dramatically relative to the U3 snoRNA. Using a PAP variant which depurinates rRNA, inhibits translation but does not destabilize its mRNA, we demonstrate that PAP mRNA is destabilized after its levels are up-regulated by a mechanism that occurs independently of rRNA depurination and translation. We quantify the extent of rRNA depurination in vivo using a novel primer extension assay and show that the temporal pattern of rRNA depurination is similar to the pattern of PAP mRNA destabilization, suggesting that they may occur by a common mechanism. These results provide the first in vivo evidence that a single chain RIP targets not only the large rRNA but also its own mRNA. These findings have implications for understanding the biological function of RIPs.
Topics: Antiviral Agents; Endoribonucleases; Fungal Proteins; Galactose; N-Glycosyl Hydrolases; Plant Proteins; Protein Biosynthesis; Purines; RNA Stability; RNA, Messenger; RNA, Plant; RNA, Ribosomal; Ribosome Inactivating Proteins, Type 1; Ribosomes; Ricin; Saccharomyces cerevisiae
PubMed: 12171922
DOI: 10.1074/jbc.M205463200 -
Molecular Plant Pathology Nov 2010Ribosome inactivating proteins are glycosidases synthesized by many plants and have been hypothesized to serve in defence against pathogens. These enzymes catalytically...
Ribosome inactivating proteins are glycosidases synthesized by many plants and have been hypothesized to serve in defence against pathogens. These enzymes catalytically remove a conserved purine from the sarcin/ricin loop of the large ribosomal RNA, which has been shown in vitro to limit protein synthesis. The resulting toxicity suggests that plants may possess a mechanism to protect their ribosomes from depurination during the synthesis of these enzymes. For example, pokeweed antiviral protein (PAP) is cotranslationally inserted into the lumen of the endoplasmic reticulum and travels via the endomembrane system to be stored in the cell wall. However, some PAP may retrotranslocate across the endoplasmic reticulum membrane to be released back into the cytosol, thereby exposing ribosomes to depurination. In this work, we isolated and characterized a complexed form of the enzyme that exhibits substantially reduced activity. We showed that this complex is a homodimer of PAP and that dimerization involves a peptide that contains a conserved aromatic amino acid, tyrosine 123, located in the active site of the enzyme. Bimolecular fluorescence complementation demonstrated that the homodimer may form in vivo and that dimerization is prevented by the substitution of tyrosine 123 for alanine. The homodimer is a minor form of PAP, observed only in the cytosol of cells and not in the apoplast. Taken together, these data support a novel mechanism for the limitation of depurination of autologous ribosomes by molecules of the protein that escape transport to the cell wall by the endomembrane system.
Topics: Immunoblotting; Mass Spectrometry; Phytolacca americana; Plant Proteins; Protein Multimerization; Ribosome Inactivating Proteins, Type 1; Ribosomes
PubMed: 21029321
DOI: 10.1111/j.1364-3703.2010.00640.x -
Advances in Clinical and Experimental... 2012Estrogens are female sex hormones, belonging to a group of steroid hormones, derivatives of cholesterol. These hormones can be divided in terms of origin: natural and... (Review)
Review
Estrogens are female sex hormones, belonging to a group of steroid hormones, derivatives of cholesterol. These hormones can be divided in terms of origin: natural and synthetic. Biologically, the most active is estradiol (E2). Estrogens are responsible for the development of tertiary sexual characteristics and a number of metabolic processes. In our environment there are many substances, metals and toxins which can mimic the biological functions of estrogens. Due to its characteristics, it can induce cell proliferation and stimulate tumor development. Estrogens are subject to a complex metabolism that, inadequately controlled, can lead to toxic derivatives. Catecholestrogens quinones (CE-Q) interact with DNA and form depurinating adducts disturbing cellular processes. By affecting cell proliferation, it can stimulate the formation of mutation and carcinogenesis, by stimulating the production of free radicals exhibiting genotoxicity. Estrogens are mostly used in hormone replacement therapy and hormonal contraception. There are three main sources of administration of preparations containing estrogen: oral, vaginal and percutaneous. The latter two are characterized by a lack of a so-called "first pass effect". Numerous studies carried out on estrogens (both natural and synthetic) demonstrate the possibility of their detrimental function on the human body. Through its impact on clotting factors, it increases the risk of thrombosis and is considered to participate in the formation of cancer of the breast and uterus. That is why the introduction of estrogen therapy should always be preceded by a careful assessment of individual circumstances and the balance between potential benefits and risks.
Topics: Breast Neoplasms; Cell Transformation, Neoplastic; Estrogens; Humans
PubMed: 23240460
DOI: No ID Found -
OncoTargets and Therapy 2019Breast cancer is the most common malignancy among women worldwide. Various studies indicate that prolonged exposure to elevated levels of estrogens is associated with... (Review)
Review
Breast cancer is the most common malignancy among women worldwide. Various studies indicate that prolonged exposure to elevated levels of estrogens is associated with development of breast cancer. Both estrogen receptor-dependent and independent mechanisms can contribute to the carcinogenic effects of estrogens. Among them, the oxidative metabolism of estrogens plays a key role in the initiation of estradiol-induced breast cancer by generation of reactive estrogen quinones as well as the associated formation of oxygen free radicals. These genotoxic metabolites can react with DNA to form unstable DNA adducts which generate mutations leading to the initiation of breast cancer. A variety of endogenous and exogenous factors can alter estrogen homeostasis and generate genotoxic metabolites. The use of specific phytochemicals and dietary supplements can inhibit the risk of breast cancer not only by the modulation of several estrogen-activating enzymes (CYP19, CYP1B1) but also through the induction of various cytoprotective enzymes (eg, SOD3, NQO1, glutathione S-transferases, OGG-1, catechol--methyltransferases, CYP1B1A, etc.) that reestablish the homeostatic balance of estrogen metabolism via nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent and independent mechanisms.
PubMed: 31388303
DOI: 10.2147/OTT.S183192 -
Molecular Medicine Reports Aug 2017Excessive exposure to estrogen is associated with increased risk of breast cancer. The mechanisms of carcinogenesis in the breast caused by estrogen metabolism include... (Review)
Review
Excessive exposure to estrogen is associated with increased risk of breast cancer. The mechanisms of carcinogenesis in the breast caused by estrogen metabolism include formation of depurinating adducts which are released from DNA to generate apurinic sites, and production of reactive oxygen species (ROS). Excess ROS not only exerts genotoxicity by indirectly increasing genomic instability, but also stimulates progression of mammary carcinogenicity by inducing a redox‑associated signaling pathway. Estrogen metabolism enzymes serve an important role in estrogen metabolism. Alterations in the expression and activity of estrogen metabolism enzymes may influence estrogen metabolism homeostasis. The present review discusses the process of estrogen metabolism, the role of estrogen metabolites and ROS in breast carcinogenesis, and the effect of metabolism enzyme polymorphisms on generation of pro‑carcinogens and breast cancer susceptibility.
Topics: Animals; Breast Neoplasms; Carcinogenesis; Estrogens; Female; Humans; Polymorphism, Genetic; Reactive Oxygen Species
PubMed: 28627646
DOI: 10.3892/mmr.2017.6738 -
FEBS Letters Dec 1995Peroxynitrite is a strong oxidant formed by reaction of nitric oxide with superoxide in inflamed tissues. We have demonstrated that 8-nitroguanine is formed...
Peroxynitrite is a strong oxidant formed by reaction of nitric oxide with superoxide in inflamed tissues. We have demonstrated that 8-nitroguanine is formed dose-dependently in calf thymus DNA incubated with low concentrations of peroxynitrite in vitro. 8-Nitroguanine in acid-hydrolyzed DNA was chemically reduced into 8-aminoguanine, which was analyzed using high performance liquid chromatography with electrochemical detection. Only peroxynitrite, but not nitrite, tetranitromethane nor NO-releasing compounds, formed 8-nitroguanine. Antioxidants and desferrioxamine inhibited the reaction. 8-Nitroguanine was depurinated from DNA incubated at pH 7.4, 37 degrees C (t1/2 = approximately 4 h). Peroxynitrite did not increase 8-oxoguanine levels in DNA.
Topics: Animals; Antioxidants; Apurinic Acid; Cattle; DNA; Deferoxamine; Free Radicals; Guanine; Nitrates
PubMed: 7498543
DOI: 10.1016/0014-5793(95)01281-6 -
Free Radical Biology & Medicine Aug 2012Growing evidence indicates that RNA oxidation is correlated with a number of age-related neurodegenerative diseases, and RNA oxidation has also been shown to induce...
Growing evidence indicates that RNA oxidation is correlated with a number of age-related neurodegenerative diseases, and RNA oxidation has also been shown to induce dysfunction in protein synthesis. Here we study in vitro RNA oxidation catalyzed by cytochrome c (cyt c)/H(2)O(2) or by the Fe(II)/ascorbate/H(2)O(2) system. Our results reveal that the products of RNA oxidation vary with the oxidant used. Guanosine residues are preferentially oxidized by cyt c/H(2)O(2) relative to the Fe(II)/ascorbate/H(2)O(2) system. GC/MS and LC/MS analyses demonstrated that the guanine base was not only oxidized but also depurinated to form an abasic sugar moiety. Results from gel electrophoresis and HPLC analyses show that RNA formed a cross-linked complex with cyt c in an H(2)O(2) concentration-dependent manner. Furthermore, when cyt c was associated with liposomes composed of cardiolipin/phosphatidylcholine, and incubated with RNA and H(2)O(2), it was found cross-linked with the oxidized RNA and dissociated from the liposome. Results of the quantitative analysis indicate that the release of the cyt c from the liposome is facilitated by the formation of an RNA-cyt c cross-linked complex. Thus, RNA oxidation may facilitate the release of cyt c from the mitochondrial membrane to induce apoptosis in response to oxidative stress.
Topics: Animals; Antioxidants; Apurinic Acid; Ascorbic Acid; Cattle; Chick Embryo; Cytochromes c; Guanosine; Hydrogen Peroxide; Hydrolysis; Liposomes; Mitochondrial Proteins; Oxidants; Oxidation-Reduction; Oxidative Stress; RNA
PubMed: 22683603
DOI: 10.1016/j.freeradbiomed.2012.05.044 -
Environmental Science & Technology Mar 2013Polyvinyl pyrrolidone polymer (PVP) has been widely applied in biological and medical fields. A few in vitro studies indicated that PVP might cause toxicity. However,...
Polyvinyl pyrrolidone polymer (PVP) has been widely applied in biological and medical fields. A few in vitro studies indicated that PVP might cause toxicity. However, the underlying mechanism is poorly understood. In this work, we found that PVP directly induced strand breakages of various DNA molecules, implicating a cleavage activity. Moreover, reactive oxygen species (ROS) scavenging analysis shows that DNA cleavage activity of PVP is not related to ROS-induced oxidation. As revealed by gel electrophoresis and liquid chromatography/mass spectrometry analysis, the major cleavage products of DNA were identified as two purine bases, guanine and adenine, suggesting that PVPs have a novel depurination activity. The selective depurination and DNA cleavage activity of PVPs were further confirmed by studying the interaction of PVP with four nucleosides and four well-designed oligodeoxynucleotides probes containing specific nucleotides. This study may provide insights into PVP-DNA interactions and resultant genotoxicity and may also open a new way for DNA study.
Topics: Base Sequence; DNA; DNA Cleavage; Models, Molecular; Mutagens; Povidone; Purines; Reactive Oxygen Species
PubMed: 23425130
DOI: 10.1021/es3046229