The Experts below are selected from a list of 288 Experts worldwide ranked by ideXlab platform
Donald H. Burke - One of the best experts on this subject based on the ideXlab platform.
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Selective Inactivation of Functional RNAs by Ribozyme-Catalyzed Covalent Modification
ACS synthetic biology, 2016Co-Authors: Raghav R. Poudyal, Malak Benslimane, Melissa P. Lokugamage, Mackenzie K. Callaway, Seth Staller, Donald H. BurkeAbstract:The diverse functions of RNA provide numerous opportunities for programming biological circuits. We describe a new strategy that uses ribozyme K28min to Covalently tag a specific nucleobase within an RNA or DNA target strand to regulate and selectively inactivate those nucleic acids. K28min variants with appropriately reprogrammed internal guide sequences efficiently tagged multiple sites from an mRNA and from aptamer and ribozyme targets. Upon Covalent Modification by the corresponding K28min variant, an ATP-binding aptamer lost all affinity for ATP, and the fluorogenic Mango aptamer lost its ability to activate fluorescence of its dye ligand. Modifying a hammerhead ribozyme near the catalytic core led to loss of almost all of its substrate-cleaving activity, but modifying the same hammerhead ribozyme within a tertiary stabilizing element that reduces magnesium dependence only impaired substrate cleavage at low magnesium concentration. Thus, ribozyme-mediated Covalent Modification can be used both to sel...
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Selective Inactivation of Functional RNAs by Ribozyme-Catalyzed Covalent Modification
2016Co-Authors: Raghav R. Poudyal, Malak Benslimane, Melissa P. Lokugamage, Mackenzie K. Callaway, Seth Staller, Donald H. BurkeAbstract:The diverse functions of RNA provide numerous opportunities for programming biological circuits. We describe a new strategy that uses ribozyme K28min to Covalently tag a specific nucleobase within an RNA or DNA target strand to regulate and selectively inactivate those nucleic acids. K28min variants with appropriately reprogrammed internal guide sequences efficiently tagged multiple sites from an mRNA and from aptamer and ribozyme targets. Upon Covalent Modification by the corresponding K28min variant, an ATP-binding aptamer lost all affinity for ATP, and the fluorogenic Mango aptamer lost its ability to activate fluorescence of its dye ligand. Modifying a hammerhead ribozyme near the catalytic core led to loss of almost all of its substrate-cleaving activity, but modifying the same hammerhead ribozyme within a tertiary stabilizing element that reduces magnesium dependence only impaired substrate cleavage at low magnesium concentration. Thus, ribozyme-mediated Covalent Modification can be used both to selectively inactivate and to fine-tune the activities of targeted functional RNAs, analogous to the effects of post-translational Modifications of proteins. Ribozyme-catalyzed Covalent Modification could therefore be developed to regulate nucleic acids components of synthetic and natural circuits
Shigeki Sasaki - One of the best experts on this subject based on the ideXlab platform.
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Site-specific Covalent Modification of RNA guided by functionality-transfer oligodeoxynucleotides.
Bioconjugate chemistry, 2009Co-Authors: Kazumitsu Onizuka, Yosuke Taniguchi, Shigeki SasakiAbstract:Efficient methods for the Covalent Modification of large RNA molecules should find significance utility as innovative biological tools as well as therapeutic methods. In this study, the development of a general method for site-specific RNA Modification guided by the functional ODN template has been investigated. The ODN probe containing 6-thioguanosine was modified by the methylenediketone derivative to form the S-functionalized ODN. Site-specific and cytosine-selective RNA Modifications were achieved by the functionality-transfer reaction from the sulfur atom of the functionalized probe to the amino group of the cytosine base of the target strand. It was shown that the base and site selectivity were due to the close proximity of the reactants in the DNA-RNA duplexes.
Kazumitsu Onizuka - One of the best experts on this subject based on the ideXlab platform.
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Site-specific Covalent Modification of RNA guided by functionality-transfer oligodeoxynucleotides.
Bioconjugate chemistry, 2009Co-Authors: Kazumitsu Onizuka, Yosuke Taniguchi, Shigeki SasakiAbstract:Efficient methods for the Covalent Modification of large RNA molecules should find significance utility as innovative biological tools as well as therapeutic methods. In this study, the development of a general method for site-specific RNA Modification guided by the functional ODN template has been investigated. The ODN probe containing 6-thioguanosine was modified by the methylenediketone derivative to form the S-functionalized ODN. Site-specific and cytosine-selective RNA Modifications were achieved by the functionality-transfer reaction from the sulfur atom of the functionalized probe to the amino group of the cytosine base of the target strand. It was shown that the base and site selectivity were due to the close proximity of the reactants in the DNA-RNA duplexes.
Lijuan Chen - One of the best experts on this subject based on the ideXlab platform.
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Covalent Modification of cys 239 in β tubulin by small molecules as a strategy to promote tubulin heterodimer degradation
Journal of Biological Chemistry, 2019Co-Authors: Jianhong Yang, Wei Yan, Qiang Qiu, Lijuan ChenAbstract:Clinical microtubule-targeting drugs are functionally divided into microtubule-destabilizing and microtubule-stabilizing agents. Drugs from both classes achieve microtubule inhibition by binding different sites on tubulin and inhibiting or promoting polymerization with no concomitant effects on the protein levels of tubulin heterodimers. Here, we have identified a series of small molecules with diverse structures potentially representing a third class of novel tubulin inhibitors that promote degradation by Covalent binding to Cys-239 of β-tubulin. The small molecules highlighted in this study include T0070907 (a peroxisome proliferator-activated receptor γ inhibitor), T007-1 (a T0070907 derivative), T138067, N,N′-ethylene-bis(iodoacetamide) (EBI), and allyl isothiocyanate (AITC). Label-free quantitative proteomic analysis revealed that T007-1 promotes tubulin degradation with high selectivity. Mass spectrometry findings showed Covalent binding of both T0070907 and T007-01 to Cys-239 of β-tubulin. Furthermore, T007-1 exerted a degradative effect on tubulin isoforms possessing Cys-239 (β2, β4, and β5(β)) but not those containing Ser-239 (β3, β6) or mutant β-tubulin with a C239S substitution. Three small molecules (T138067, EBI, and AITC) also reported to bind Covalently to Cys-239 of β-tubulin similarly induced tubulin degradation. Our results strongly suggest that Covalent Modification of Cys-239 of β-tubulin by small molecules could serve as a novel strategy to promote tubulin heterodimer degradation. We propose that these small molecules represent a third novel class of tubulin inhibitor agents that exert their effects through degradation activity.
Gregory R. J. Thatcher - One of the best experts on this subject based on the ideXlab platform.
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dimethyl fumarate inhibits the nuclear factor κb pathway in breast cancer cells by Covalent Modification of p65 protein
Journal of Biological Chemistry, 2016Co-Authors: Irida Kastrati, Gregory R. J. Thatcher, Marton I Siklos, Esther L Calderongierszal, Lamiaa Elshennawy, Gergana Georgieva, Emily N Thayer, Jonna FrasorAbstract:In breast tumors, activation of the nuclear factor κB (NFκB) pathway promotes survival, migration, invasion, angiogenesis, stem cell-like properties, and resistance to therapy--all phenotypes of aggressive disease where therapy options remain limited. Adding an anti-inflammatory/anti-NFκB agent to breast cancer treatment would be beneficial, but no such drug is approved as either a monotherapy or adjuvant therapy. To address this need, we examined whether dimethyl fumarate (DMF), an anti-inflammatory drug already in clinical use for multiple sclerosis, can inhibit the NFκB pathway. We found that DMF effectively blocks NFκB activity in multiple breast cancer cell lines and abrogates NFκB-dependent mammosphere formation, indicating that DMF has anti-cancer stem cell properties. In addition, DMF inhibits cell proliferation and significantly impairs xenograft tumor growth. Mechanistically, DMF prevents p65 nuclear translocation and attenuates its DNA binding activity but has no effect on upstream proteins in the NFκB pathway. Dimethyl succinate, the inactive analog of DMF that lacks the electrophilic double bond of fumarate, is unable to inhibit NFκB activity. Also, the cell-permeable thiol N-acetyl l-cysteine, reverses DMF inhibition of the NFκB pathway, supporting the notion that the electrophile, DMF, acts via Covalent Modification. To determine whether DMF interacts directly with p65, we synthesized and used a novel chemical probe of DMF by incorporating an alkyne functionality and found that DMF Covalently modifies p65, with cysteine 38 being essential for the activity of DMF. These results establish DMF as an NFκB inhibitor with anti-tumor activity that may add therapeutic value in the treatment of aggressive breast cancers.
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Analysis of protein Covalent Modification by xenobiotics using a covert oxidatively activated tag: raloxifene proof-of-principle study.
Chemical research in toxicology, 2005Co-Authors: Ju Liu, Xiaofeng Yang, Richard B. Van Breemen, Judy L. Bolton, Gregory R. J. ThatcherAbstract:Numerous xenobiotics, including therapeutics agents, are substrates for bioactivation to electrophilic reactive intermediates that may Covalently modify biomolecules. Selective estrogen receptor modulators (SERMs) are in clinical use for long-term therapy of postmenopausal syndromes and chemoprevention and provide a potential alternative for hormone replacement therapy (HRT). Raloxifene, in common with many SERMs and other xenobiotics, is a polyaromatic phenol that has been shown to be metabolically bioactivated to electrophilic and redox active quinoids. Nucleic acid and glutathione adduct formation have been reported, but little is known about protein Covalent Modification. A novel COATag (covert oxidatively activated tag) was synthesized in which raloxifene was linked to biotin. The COATag was reactive toward a model protein, human glutathione-S-transferase P1-1, in the presence but not the absence of monooxygenase. The Covalent Modification of proteins in rat liver microsomal incubations was NADPH-dependent implicating cytochrome P450 oxidase. The COATag facilitated isolation and identification of Covalently modified microsomal proteins: cytosolic glucose regulated protein (GRP78/BiP), three protein disulfide isomerases, and microsomal glutathione S-transferase 1. Oxidative metabolism of raloxifene produces reactive intermediates of sufficient lifetimes to Covalently modify proteins in tissue microsomes, behavior anticipated for other polyaromatic phenol xenobiotics that can be tested by the COATag methodology. The combined use of a COATag with a simple biotin-linked electrophile (such as an iodoacetamide tag) is a new technique that allows quantification of protein Covalent Modification via alkylation vs oxidation in response to xenobiotic reactive intermediates. The identification of modified proteins is important for defining pathways that might lead alternatively to either cytotoxicity or cytoprotection.
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Covalent Modification of Proteins and Peptides by the Quinone Methide from 2-tert-Butyl-4,6-dimethylphenol: Selectivity and Reactivity with Respect to Competitive Hydration
The Journal of Organic Chemistry, 1997Co-Authors: Paul G. Mccracken, Judy L. Bolton, Gregory R. J. ThatcherAbstract:Covalent Modification of cellular nucleophiles by electrophilic metabolites has been shown to be an important pathway in the toxicological activity of many xenobiotic compounds. The p-quinone methi...
