145 research outputs found
Direct Observation of Multiple Tautomers of Oxythiamine and their Recognition by the Thiamine Pyrophosphate Riboswitch
Structural diversification of canonical nucleic acid bases and nucleotide analogues by tautomerism has been proposed to be a powerful on/off switching mechanism allowing regulation of many biological processes mediated by RNA enzymes and aptamers. Despite the suspected biological importance of tautomerism, attempts to observe minor tautomeric forms in nucleic acid or hybrid nucleic acid-ligand complexes have met with challenges due to the lack of sensitive methods. Here, a combination of spectroscopic, biochemical, and computational tools probed tautomerism in the context of an RNA aptamer-ligand complex; studies involved a model ligand, oxythiamine pyrophosphate (OxyTPP), bound to the thiamine pyrophosphate (TPP) riboswitch (an RNA aptamer) as well as its unbound nonphosphorylated form, oxythiamine (OxyT). OxyTPP, similarly to canonical heteroaromatic nucleic acid bases, has a pyrimidine ring that forms hydrogen bonding interactions with the riboswitch. Tautomerism was established using two-dimensional infrared (2D IR) spectroscopy, variable temperature FTIR and NMR spectroscopies, binding isotope effects (BIEs), and computational methods. All three possible tautomers of OxyT, including the minor enol tautomer, were directly identified, and their distributions were quantitated. In the bound form, BIE data suggested that OxyTPP existed as a 4β²-keto tautomer that was likely protonated at the N1β²-position. These results also provide a mechanistic framework for understanding the activation of riboswitch in response to deamination of the active form of vitamin B1 (or TPP). The combination of methods reported here revealing the fine details of tautomerism can be applied to other systems where the importance of tautomerism is suspected.National Institutes of Health (U.S.) (Grant CA080024)National Institutes of Health (U.S.) (Grant CA26731)National Institutes of Health (U.S.) (Grant ES002109)National Institutes of Health (U.S.) (Grant ES007020)National Science Foundation (U.S.) (Grant CHE-1212557)Massachusetts Institute of Technology. Center for Environmental Health Sciences (National Institutes of Health (U.S.) Center Grant P30-ES002109)Massachusetts Institute of Technology. Laser Biomedical Research Center (National Institutes of Health (U.S.) Center Grant P41-EB015871)National Institutes of Health (U.S.) (Traineeship T32 ES007020)Massachusetts Institute of Technology (Poitras Pre-Doctoral Fellowship
Mechanism of Repair of Acrolein- and Malondialdehyde-Derived Exocyclic Guanine Adducts by the Ξ±-Ketoglutarate/Fe(II) Dioxygenase AlkB
The structurally related exocyclic guanine adducts Ξ±-hydroxypropano-dG (Ξ±-OH-PdG), Ξ³-hydroxypropano-dG (Ξ³-OH-PdG), and M[subscript 1]dG are formed when DNA is exposed to the reactive aldehydes acrolein and malondialdehyde (MDA). These lesions are believed to form the basis for the observed cytotoxicity and mutagenicity of acrolein and MDA. In an effort to understand the enzymatic pathways and chemical mechanisms that are involved in the repair of acrolein- and MDA-induced DNA damage, we investigated the ability of the DNA repair enzyme AlkB, an Ξ±-ketoglutarate/Fe(II) dependent dioxygenase, to process Ξ±-OH-PdG, Ξ³-OH-PdG, and M[subscript 1]dG in both single- and double-stranded DNA contexts. By monitoring the repair reactions using quadrupole time-of-flight (Q-TOF) mass spectrometry, it was established that AlkB can oxidatively dealkylate Ξ³-OH-PdG most efficiently, followed by M[subscript 1]dG and Ξ±-OH-PdG. The AlkB repair mechanism involved multiple intermediates and complex, overlapping repair pathways. For example, the three exocyclic guanine adducts were shown to be in equilibrium with open-ring aldehydic forms, which were trapped using (pentafluorobenzyl)hydroxylamine (PFBHA) or NaBH[subscript 4]. AlkB repaired the trapped open-ring form of Ξ³-OH-PdG but not the trapped open-ring of Ξ±-OH-PdG. Taken together, this study provides a detailed mechanism by which three-carbon bridge exocyclic guanine adducts can be processed by AlkB and suggests an important role for the AlkB family of dioxygenases in protecting against the deleterious biological consequences of acrolein and MDA.National Institutes of Health (U.S.) (Grant R01 CA080024)National Institutes of Health (U.S.) (Grant R01 CA26731)National Institutes of Health (U.S.) (Center Grant P30 ES02109)National Institutes of Health (U.S.) (Training Grant T32 ES007020
Mutational spectra of aflatoxin B
Aflatoxin Bβ (AFBβ) and/or hepatitis B and C viruses are risk factors for human hepatocellular carcinoma (HCC). Available evidence supports the interpretation that formation of AFBβ-DNA adducts in hepatocytes seeds a population of mutations, mainly G:CβT:A, and viral processes synergize to accelerate tumorigenesis, perhaps via inflammation. Responding to a need for early-onset evidence predicting disease development, highly accurate duplex sequencing was used to monitor acquisition of high-resolution mutational spectra (HRMS) during the process of hepatocarcinogenesis. Four-day-old male mice were treated with AFBβ using a regimen that induced HCC within 72 wk. For analysis, livers were separated into tumor and adjacent cellular fractions. HRMS of cells surrounding the tumors revealed predominantly G:CβT:A mutations characteristic of AFBβ exposure. Importantly, 25% of all mutations were GβT in one trinucleotide context (CGC; the underlined G is the position of the mutation), which is also a hotspot mutation in human liver tumors whose incidence correlates with AFBβ exposure. The technology proved sufficiently sensitive that the same distinctive spectrum was detected as early as 10 wk after dosing, well before evidence of neoplasia. Additionally, analysis of tumor tissue revealed a more complex pattern than observed in surrounding hepatocytes; tumor HRMS were a composite of the 10-wk spectrum and a more heterogeneous set of mutations that emerged during tumor outgrowth. We propose that the 10-wk HRMS reflects a short-term mutational response to AFBβ, and, as such, is an early detection metric for AFBβ-induced liver cancer in this mouse model that will be a useful tool to reconstruct the molecular etiology of human hepatocarcinogenesis.National Institutes of Health (U.S.) (Grant R01-ES016313)National Institutes of Health (U.S.) (Grant P30-ES002109)National Institutes of Health (U.S.) (Grant T32-ES007020)National Institutes of Health (U.S.) (Grant R01-CA080024
Tautomerism provides a molecular explanation for the mutagenic properties of the anti-HIV nucleoside 5-aza-5,6-dihydro-2'-deoxycytidine
Exocyclic Carbons Adjacent to the N6 of Adenine are Targets for Oxidation by the Escherichia coli Adaptive Response Protein AlkB
The DNA and RNA repair protein AlkB removes alkyl groups from nucleic acids by a unique iron- and Ξ±-ketoglutarate-dependent oxidation strategy. When alkylated adenines are used as AlkB targets, earlier work suggests that the initial target of oxidation can be the alkyl carbon adjacent to N1. Such may be the case with ethano-adenine (EA), a DNA adduct formed by an important anticancer drug, BCNU, whereby an initial oxidation would occur at the carbon adjacent to N1. In a previous study, several intermediates were observed suggesting a pathway involving adduct restructuring to a form that would not hinder replication, which would match biological data showing that AlkB almost completely reverses EA toxicity in vivo. The present study uses more sensitive spectroscopic methodology to reveal the complete conversion of EA to adenine; the nature of observed additional putative intermediates indicates that AlkB conducts a second oxidation event in order to release the two-carbon unit completely. The second oxidation event occurs at the exocyclic carbon adjacent to the N[superscript 6] atom of adenine. The observation of oxidation of a carbon at N[superscript 6] in EA prompted us to evaluate N[superscript 6]-methyladenine (m6A), an important epigenetic signal for DNA replication and many other cellular processes, as an AlkB substrate in DNA. Here we show that m6A is indeed a substrate for AlkB and that it is converted to adenine via its 6-hydroxymethyl derivative. The observation that AlkB can demethylate m6A in vitro suggests a role for AlkB in regulation of important cellular functions in vivo.National Institutes of Health (U.S.) (Grant number CA080024)National Institutes of Health (U.S.) (Grant number CA26731)National Institutes of Health (U.S.) (Grant number ES02109
The Selectivity of Austocystin D Arises from Cell-Line-Specific Drug Activation by Cytochrome P450 Enzymes
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Diversity in Expression of Phosphorus (P) Responsive Genes in Cucumis melo L
Phosphorus (P) is a major limiting nutrient for plant growth in many soils. Studies in model species have identified genes involved in plant adaptations to low soil P availability. However, little information is available on the genetic bases of these adaptations in vegetable crops. In this respect, sequence data for melon now makes it possible to identify melon orthologues of candidate P responsive genes, and the expression of these genes can be used to explain the diversity in the root system adaptation to low P availability, recently observed in this species
Flip-Flop of Phospholipids in Proteoliposomes Reconstituted from Detergent Extract of Chloroplast Membranes: Kinetics and Phospholipid Specificity
Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6Β±1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents
Mutation of HIV-1 Genomes in a Clinical Population Treated with the Mutagenic Nucleoside KP1461
The deoxycytidine analog KP1212, and its prodrug KP1461, are prototypes of a new class of antiretroviral drugs designed to increase viral mutation rates, with the goal of eventually causing the collapse of the viral population. Here we present an extensive analysis of viral sequences from HIV-1 infected volunteers from the first βmechanism validationβ phase II clinical trial of a mutagenic base analog in which individuals previously treated with antiviral drugs received 1600 mg of KP1461 twice per day for 124 days. Plasma viral loads were not reduced, and overall levels of viral mutation were not increased during this short-term study, however, the mutation spectrum of HIV was altered. A large number (N = 105 per sample) of sequences were analyzed, each derived from individual HIV-1 RNA templates, after 0, 56 and 124 days of therapy from 10 treated and 10 untreated control individuals (>7.1 million base pairs of unique viral templates were sequenced). We found that private mutations, those not found in more than one viral sequence and likely to have occurred in the most recent rounds of replication, increased in treated individuals relative to controls after 56 (p = 0.038) and 124 (p = 0.002) days of drug treatment. The spectrum of mutations observed in the treated group showed an excess of A to G and G to A mutations (p = 0.01), and to a lesser extent T to C and C to T mutations (p = 0.09), as predicted by the mechanism of action of the drug. These results validate the proposed mechanism of action in humans and should spur development of this novel antiretroviral approach.Koronis Pharmaceutical
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