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Targeted Imaging of VCAM-1 mRNA in a Mouse Model of Laser-Induced Choroidal Neovascularization Using Antisense Hairpin-DNA-Functionalized Gold-Nanoparticles.
Uddin MI, Kilburn TC, Yang R, McCollum GW, Wright DW, Penn JS
(2018) Mol Pharm 15: 5514-5520
MeSH Terms: Animals, Biomarkers, Choroid, Choroidal Neovascularization, Disease Models, Animal, Fluorescent Dyes, Gold, Humans, Intravital Microscopy, Lasers, Male, Metal Nanoparticles, Mice, Mice, Inbred C57BL, Molecular Imaging, Molecular Probes, Oligodeoxyribonucleotides, Antisense, Optical Imaging, RNA, Messenger, Vascular Cell Adhesion Molecule-1, Wet Macular Degeneration
Show Abstract · Added April 10, 2019
Mouse laser-induced choroidal neovascularization (mouse LCNV) recapitulates the "wet" form of human age-related macular degeneration (AMD). Vascular cell adhesion molecule-1 (VCAM-1) is a known inflammatory biomarker, and it increases in the choroidal neovascular tissues characteristic of this experimental model. We have designed and constructed gold nanoparticles (AuNPs) functionalized with hairpin-DNA that incorporates an antisense sequence complementary to VCAM-1 mRNA (AS-VCAM-1 hAuNPs) and tested them as optical imaging probes. The 3' end of the hairpin is coupled to a near-infrared fluorophore that is quenched by the AuNP surface via Förster resonance energy transfer (FRET). Hybridization of the antisense sequence to VCAM-1 mRNA displaces the fluorophore away from the AuNP surface, inducing fluorescent activity. In vitro testing showed that hAuNPs hybridize to an exogenous complementary oligonucleotide within a pH range of 4.5-7.4, and that they are stable at reduced pH. LCNV mice received tail-vein injections of AS-VCAM-1 hAuNPs. Hyperspectral imaging revealed the delivery of AS-VCAM-1 hAuNPs to excised choroidal tissues. Fluorescent images of CNV lesions were obtained, presumably in response to the hybridization of AS-hAuNPs to LCNV-induced VCAM-1 mRNA. This is the first demonstration of systemic delivery of hAuNPs to ocular tissues to facilitate mRNA imaging of any target.
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21 MeSH Terms
Metformin reduces liver glucose production by inhibition of fructose-1-6-bisphosphatase.
Hunter RW, Hughey CC, Lantier L, Sundelin EI, Peggie M, Zeqiraj E, Sicheri F, Jessen N, Wasserman DH, Sakamoto K
(2018) Nat Med 24: 1395-1406
MeSH Terms: Adenosine Monophosphate, Aminoimidazole Carboxamide, Animals, Base Sequence, Chickens, Disease Models, Animal, Fructose-Bisphosphatase, Glucose, Glucose Intolerance, Homeostasis, Humans, Hypoglycemia, Liver, Metformin, Mice, Inbred C57BL, Mutation, Obesity, Prodrugs, Ribonucleotides
Show Abstract · Added March 26, 2019
Metformin is a first-line drug for the treatment of individuals with type 2 diabetes, yet its precise mechanism of action remains unclear. Metformin exerts its antihyperglycemic action primarily through lowering hepatic glucose production (HGP). This suppression is thought to be mediated through inhibition of mitochondrial respiratory complex I, and thus elevation of 5'-adenosine monophosphate (AMP) levels and the activation of AMP-activated protein kinase (AMPK), though this proposition has been challenged given results in mice lacking hepatic AMPK. Here we report that the AMP-inhibited enzyme fructose-1,6-bisphosphatase-1 (FBP1), a rate-controlling enzyme in gluconeogenesis, functions as a major contributor to the therapeutic action of metformin. We identified a point mutation in FBP1 that renders it insensitive to AMP while sparing regulation by fructose-2,6-bisphosphate (F-2,6-P), and knock-in (KI) of this mutant in mice significantly reduces their response to metformin treatment. We observe this during a metformin tolerance test and in a metformin-euglycemic clamp that we have developed. The antihyperglycemic effect of metformin in high-fat diet-fed diabetic FBP1-KI mice was also significantly blunted compared to wild-type controls. Collectively, we show a new mechanism of action for metformin and provide further evidence that molecular targeting of FBP1 can have antihyperglycemic effects.
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19 MeSH Terms
Human DNA polymerase η accommodates RNA for strand extension.
Su Y, Egli M, Guengerich FP
(2017) J Biol Chem 292: 18044-18051
MeSH Terms: Base Pair Mismatch, DNA Primers, DNA Replication, DNA-Directed DNA Polymerase, Deoxyguanosine, Electrophoretic Mobility Shift Assay, Humans, Kinetics, Nucleic Acid Heteroduplexes, Nucleic Acid Hybridization, Oligodeoxyribonucleotides, Oligoribonucleotides, Pyrimidine Dimers, RNA, Recombinant Proteins, Reverse Transcription, Substrate Specificity, Transcription Elongation, Genetic
Show Abstract · Added March 14, 2018
Ribonucleotides are the natural analogs of deoxyribonucleotides, which can be misinserted by DNA polymerases, leading to the most abundant DNA lesions in genomes. During replication, DNA polymerases tolerate patches of ribonucleotides on the parental strands to different extents. The majority of human DNA polymerases have been reported to misinsert ribonucleotides into genomes. However, only PrimPol, DNA polymerase α, telomerase, and the mitochondrial human DNA polymerase (hpol) γ have been shown to tolerate an entire RNA strand. Y-family hpol η is known for translesion synthesis opposite the UV-induced DNA lesion cyclobutane pyrimidine dimer and was recently found to incorporate ribonucleotides into DNA. Here, we report that hpol η is able to bind DNA/DNA, RNA/DNA, and DNA/RNA duplexes with similar affinities. In addition, hpol η, as well as another Y-family DNA polymerase, hpol κ, accommodates RNA as one of the two strands during primer extension, mainly by inserting dNMPs opposite unmodified templates or DNA lesions, such as 8-oxo-2'-deoxyguanosine or cyclobutane pyrimidine dimer, even in the presence of an equal amount of the DNA/DNA substrate. The discovery of this RNA-accommodating ability of hpol η redefines the traditional concept of human DNA polymerases and indicates potential new functions of hpol η .
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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18 MeSH Terms
Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η.
Su Y, Egli M, Guengerich FP
(2016) J Biol Chem 291: 3747-56
MeSH Terms: Crystallography, X-Ray, DNA, DNA-Directed DNA Polymerase, Humans, Protein Structure, Tertiary, Ribonucleotides
Show Abstract · Added March 14, 2018
Ribonucleotides and 2'-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2'-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2'-OH group. Y-family human DNA polymerase η (hpol η) is of interest because of its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol η maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2'-deoxyguanosine and cyclobutane pyrimidine dimer but with rates that are 10(3)-fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that the hpol η scaffolds the incoming rNTP to pair with the template base (dG) or 7,8-dihydro-8-oxo-2'-deoxyguanosine with a significant propeller twist. As a result, the 2'-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and Pα of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18. This is the first crystal structure of a DNA polymerase with an incoming rNTP opposite a DNA lesion.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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6 MeSH Terms
Polymerase Bypass of N(6)-Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene.
Kotapati S, Wickramaratne S, Esades A, Boldry EJ, Quirk Dorr D, Pence MG, Guengerich FP, Tretyakova NY
(2015) Chem Res Toxicol 28: 1496-507
MeSH Terms: Butadienes, Chromatography, High Pressure Liquid, DNA, DNA Adducts, DNA Primers, DNA Replication, DNA-Directed DNA Polymerase, Deoxyadenosines, Epoxy Compounds, Humans, Kinetics, Oligodeoxyribonucleotides, Spectrometry, Mass, Electrospray Ionization
Show Abstract · Added March 14, 2018
N(6)-(2-Hydroxy-3-buten-1-yl)-2'-deoxyadenosine (N(6)-HB-dA I) and N(6),N(6)-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N(6),N(6)-DHB-dA) are exocyclic DNA adducts formed upon alkylation of the N(6) position of adenine in DNA by epoxide metabolites of 1,3-butadiene (BD), a common industrial and environmental chemical classified as a human and animal carcinogen. Since the N(6)-H atom of adenine is required for Watson-Crick hydrogen bonding with thymine, N(6)-alkylation can prevent adenine from normal pairing with thymine, potentially compromising the accuracy of DNA replication. To evaluate the ability of BD-derived N(6)-alkyladenine lesions to induce mutations, synthetic oligodeoxynucleotides containing site-specific (S)-N(6)-HB-dA I and (R,R)-N(6),N(6)-DHB-dA adducts were subjected to in vitro translesion synthesis in the presence of human DNA polymerases β, η, ι, and κ. While (S)-N(6)-HB-dA I was readily bypassed by all four enzymes, only polymerases η and κ were able to carry out DNA synthesis past (R,R)-N(6),N(6)-DHB-dA. Steady-state kinetic analyses indicated that all four DNA polymerases preferentially incorporated the correct base (T) opposite (S)-N(6)-HB-dA I. In contrast, hPol β was completely blocked by (R,R)-N(6),N(6)-DHB-dA, while hPol η and κ inserted A, G, C, or T opposite the adduct with similar frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed that while translesion synthesis past (S)-N(6)-HB-dA I was mostly error-free, replication of DNA containing (R,R)-N(6),N(6)-DHB-dA induced significant numbers of A, C, and G insertions and small deletions. These results indicate that singly substituted (S)-N(6)-HB-dA I lesions are not miscoding, but that exocyclic (R,R)-N(6),N(6)-DHB-dA adducts are strongly mispairing, probably due to their inability to form stable Watson-Crick pairs with dT.
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13 MeSH Terms
Structural basis for 2'-5'-oligoadenylate binding and enzyme activity of a viral RNase L antagonist.
Ogden KM, Hu L, Jha BK, Sankaran B, Weiss SR, Silverman RH, Patton JT, Prasad BV
(2015) J Virol 89: 6633-45
MeSH Terms: Adenine Nucleotides, Capsid Proteins, Catalytic Domain, Crystallography, X-Ray, Exoribonucleases, Oligoribonucleotides, Protein Binding, Protein Conformation, Rotavirus
Show Abstract · Added April 26, 2017
UNLABELLED - Synthesis of 2'-5'-oligoadenylates (2-5A) by oligoadenylate synthetase (OAS) is an important innate cellular response that limits viral replication by activating the latent cellular RNase, RNase L, to degrade single-stranded RNA. Some rotaviruses and coronaviruses antagonize the OAS/RNase L pathway through the activity of an encoded 2H phosphoesterase domain that cleaves 2-5A. These viral 2H phosphoesterases are phylogenetically related to the cellular A kinase anchoring protein 7 (AKAP7) and share a core structure and an active site that contains two well-defined HΦ(S/T)Φ (where Φ is a hydrophobic residue) motifs, but their mechanism of substrate binding is unknown. Here, we report the structures of a viral 2H phosphoesterase, the C-terminal domain (CTD) of the group A rotavirus (RVA) VP3 protein, both alone and in complex with 2-5A. The domain forms a compact fold, with a concave β-sheet that contains the catalytic cleft, but it lacks two α-helical regions and two β-strands observed in AKAP7 and other 2H phosphoesterases. The cocrystal structure shows significant conformational changes in the R loop upon ligand binding. Bioinformatics and biochemical analyses reveal that conserved residues and residues required for catalytic activity and substrate binding comprise the catalytic motifs and a region on one side of the binding cleft. We demonstrate that the VP3 CTD of group B rotavirus, but not that of group G, cleaves 2-5A. These findings suggest that the VP3 CTD is a streamlined version of a 2H phosphoesterase with a ligand-binding mechanism that is shared among 2H phosphodiesterases that cleave 2-5A.
IMPORTANCE - The C-terminal domain (CTD) of rotavirus VP3 is a 2H phosphoesterase that cleaves 2'-5'-oligoadenylates (2-5A), potent activators of an important innate cellular antiviral pathway. 2H phosphoesterase superfamily proteins contain two conserved catalytic motifs and a proposed core structure. Here, we present structures of a viral 2H phosphoesterase, the rotavirus VP3 CTD, alone and in complex with its substrate, 2-5A. The domain lacks two α-helical regions and β-strands present in other 2H phosphoesterases. A loop of the protein undergoes significant structural changes upon substrate binding. Together with our bioinformatics and biochemical findings, the crystal structures suggest that the RVA VP3 CTD domain is a streamlined version of a cellular enzyme that shares a ligand-binding mechanism with other 2H phosphodiesterases that cleave 2-5A but differs from those of 2H phosphodiesterases that cleave other substrates. These findings may aid in the future design of antivirals targeting viral phosphodiesterases with cleavage specificity for 2-5A.
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9 MeSH Terms
Synthesis of G-N2-(CH2)3-N2-G Trimethylene DNA Interstrand Cross-Links.
Gruppi F, Johnson Salyard TL, Rizzo CJ
(2014) Curr Protoc Nucleic Acid Chem 56: 5.14.1-15
MeSH Terms: Cross-Linking Reagents, Oligodeoxyribonucleotides
Show Abstract · Added January 7, 2016
The synthesis of G-N(2)-(CH(2))(3)-N(2)-G trimethylene DNA interstrand cross-links (ICLs) in a 5'-CG-3' and 5'-GC-3' sequence from oligodeoxynucleotides containing N(2)-(3-aminopropyl)-2'-deoxyguanosine and 2-fluoro-O(6)-(trimethylsilylethyl)inosine is presented. Automated solid-phase DNA synthesis was used for unmodified bases and modified nucleotides were incorporated via their corresponding phosphoramidite reagent by a manual coupling protocol. The preparation of the phosphoramidite reagents for incorporation of N(2)-(3-aminopropyl)-2'-deoxyguanosine is reported. The high-purity trimethylene DNA interstrand cross-link product is obtained through a nucleophilic aromatic substitution reaction between the N(2)-(3-aminopropyl)-2'-deoxyguanosine- and 2-fluoro-O(6)-(trimethylsilylethyl)inosine-containing oligodeoxynucleotides.
Copyright © 2014 John Wiley & Sons, Inc.
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2 MeSH Terms
Prior AICAR stimulation increases insulin sensitivity in mouse skeletal muscle in an AMPK-dependent manner.
Kjøbsted R, Treebak JT, Fentz J, Lantier L, Viollet B, Birk JB, Schjerling P, Björnholm M, Zierath JR, Wojtaszewski JF
(2015) Diabetes 64: 2042-55
MeSH Terms: AMP-Activated Protein Kinases, Aminoimidazole Carboxamide, Animals, Biological Transport, Blotting, Western, Electrophoresis, Polyacrylamide Gel, GTPase-Activating Proteins, Insulin, Mice, Muscle, Skeletal, Phosphorylation, Ribonucleotides
Show Abstract · Added May 16, 2019
An acute bout of exercise increases glucose uptake in skeletal muscle by an insulin-independent mechanism. In the period after exercise, insulin sensitivity to increased glucose uptake is enhanced. The molecular mechanisms underpinning this phenomenon are poorly understood but appear to involve an increased cell surface abundance of GLUT4. While increased proximal insulin signaling does not seem to mediate this effect, elevated phosphorylation of TBC1D4, a downstream target of both insulin (Akt) and exercise (AMPK) signaling, appears to play a role. The main purpose of this study was to determine whether AMPK activation increases skeletal muscle insulin sensitivity. We found that prior AICAR stimulation of wild-type mouse muscle increases insulin sensitivity to stimulate glucose uptake. However, this was not observed in mice with reduced or ablated AMPK activity in skeletal muscle. Furthermore, prior AICAR stimulation enhanced insulin-stimulated phosphorylation of TBC1D4 at Thr(649) and Ser(711) in wild-type muscle only. These phosphorylation events were positively correlated with glucose uptake. Our results provide evidence to support that AMPK activation is sufficient to increase skeletal muscle insulin sensitivity. Moreover, TBC1D4 phosphorylation may facilitate the effect of prior AMPK activation to enhance glucose uptake in response to insulin.
© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.
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MeSH Terms
Anti-microRNA-21 oligonucleotides prevent Alport nephropathy progression by stimulating metabolic pathways.
Gomez IG, MacKenna DA, Johnson BG, Kaimal V, Roach AM, Ren S, Nakagawa N, Xin C, Newitt R, Pandya S, Xia TH, Liu X, Borza DB, Grafals M, Shankland SJ, Himmelfarb J, Portilla D, Liu S, Chau BN, Duffield JS
(2015) J Clin Invest 125: 141-56
MeSH Terms: Animals, Autoantigens, Collagen Type IV, Disease Progression, Fibrosis, Kidney, Metabolic Networks and Pathways, Mice, 129 Strain, MicroRNAs, Nephritis, Hereditary, Oligoribonucleotides, Antisense, Reactive Oxygen Species, Transcriptome, Up-Regulation
Show Abstract · Added December 2, 2016
MicroRNA-21 (miR-21) contributes to the pathogenesis of fibrogenic diseases in multiple organs, including the kidneys, potentially by silencing metabolic pathways that are critical for cellular ATP generation, ROS production, and inflammatory signaling. Here, we developed highly specific oligonucleotides that distribute to the kidney and inhibit miR-21 function when administered subcutaneously and evaluated the therapeutic potential of these anti-miR-21 oligonucleotides in chronic kidney disease. In a murine model of Alport nephropathy, miR-21 silencing did not produce any adverse effects and resulted in substantially milder kidney disease, with minimal albuminuria and dysfunction, compared with vehicle-treated mice. miR-21 silencing dramatically improved survival of Alport mice and reduced histological end points, including glomerulosclerosis, interstitial fibrosis, tubular injury, and inflammation. Anti-miR-21 enhanced PPARα/retinoid X receptor (PPARα/RXR) activity and downstream signaling pathways in glomerular, tubular, and interstitial cells. Moreover, miR-21 silencing enhanced mitochondrial function, which reduced mitochondrial ROS production and thus preserved tubular functions. Inhibition of miR-21 was protective against TGF-β-induced fibrogenesis and inflammation in glomerular and interstitial cells, likely as the result of enhanced PPARα/RXR activity and improved mitochondrial function. Together, these results demonstrate that inhibition of miR-21 represents a potential therapeutic strategy for chronic kidney diseases including Alport nephropathy.
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14 MeSH Terms
Major groove orientation of the (2S)-N(6)-(2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine DNA adduct induced by 1,2-epoxy-3-butene.
Kowal EA, Wickramaratne S, Kotapati S, Turo M, Tretyakova N, Stone MP
(2014) Chem Res Toxicol 27: 1675-86
MeSH Terms: Alkylation, Butadienes, DNA, DNA Adducts, Deoxyadenosines, Epoxy Compounds, Humans, Molecular Dynamics Simulation, Nuclear Magnetic Resonance, Biomolecular, Nucleic Acid Conformation, Nucleic Acid Denaturation, Oligodeoxyribonucleotides, Stereoisomerism, Transition Temperature, ras Proteins
Show Abstract · Added January 20, 2015
1,3-Butadiene (BD) is an environmental and occupational toxicant classified as a human carcinogen. It is oxidized by cytochrome P450 monooxygenases to 1,2-epoxy-3-butene (EB), which alkylates DNA. BD exposures lead to large numbers of mutations at A:T base pairs even though alkylation of guanines is more prevalent, suggesting that one or more adenine adducts of BD play a role in BD-mediated genotoxicity. However, the etiology of BD-mediated genotoxicity at adenine remains poorly understood. EB alkylates the N(6) exocyclic nitrogen of adenine to form N(6)-(hydroxy-3-buten-1-yl)-2'-dA ((2S)-N(6)-HB-dA) adducts ( Tretyakova , N. , Lin , Y. , Sangaiah , R. , Upton , P. B. , and Swenberg , J. A. ( 1997 ) Carcinogenesis 18 , 137 - 147 ). The structure of the (2S)-N(6)-HB-dA adduct has been determined in the 5'-d(C(1)G(2)G(3)A(4)C(5)Y(6)A(7)G(8)A(9)A(10)G(11))-3':5'-d(C(12)T(13)T(14)C(15)T(16)T(17)G(18)T(19) C(20)C(21)G(22))-3' duplex [Y = (2S)-N(6)-HB-dA] containing codon 61 (underlined) of the human N-ras protooncogene, from NMR spectroscopy. The (2S)-N(6)-HB-dA adduct was positioned in the major groove, such that the butadiene moiety was oriented in the 3' direction. At the Cα carbon, the methylene protons of the modified nucleobase Y(6) faced the 5' direction, which placed the Cβ carbon in the 3' direction. The Cβ hydroxyl group faced toward the solvent, as did carbons Cγ and Cδ. The Cβ hydroxyl group did not form hydrogen bonds with either T(16) O(4) or T(17) O(4). The (2S)-N(6)-HB-dA nucleoside maintained the anti conformation about the glycosyl bond, and the modified base retained Watson-Crick base pairing with the complementary base (T(17)). The adduct perturbed stacking interactions at base pairs C(5):G(18), Y(6):T(17), and A(7):T(16) such that the Y(6) base did not stack with its 5' neighbor C(5), but it did with its 3' neighbor A(7). The complementary thymine T(17) stacked well with both 5' and 3' neighbors T(16) and G(18). The presence of the (2S)-N(6)-HB-dA resulted in a 5 °C reduction in the Tm of the duplex, which is attributed to less favorable stacking interactions and adduct accommodation in the major groove.
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15 MeSH Terms