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Regulation of tumor necrosis factor alpha (TNFα) mRNA by NUP-475, a novel zinc binding protein
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Journal of Inorganic Biochemistry 96 (2003)
Regulation of Tumor Necrosis Factor Alpha (TNFa) mRNA by NUP-475, a Novel Zinc Binding Protein Sarah LJ Michel, The Johns Hopkins University School of Medicine, UnitedStates Barbara T Amann, The Johns Hopkins University School of Medicine, United States Anthony L Guerrerio, The Johns Hopkins University School of Medicine, United States Jeremy M Berg, The Johns Hopkins University School of Medicine, United States Zinc-binding domains have been found to mediate a wide range of macromolecular interactions including the regulation of gene expression. The protein NUP-475 (also named Tristetraprolin and Tisll) belongs to a newly discovered family of zinc binding proteins with tandem CCCH repeats. NUP-475 binds to the AU-rich sequence elements (AF&) of certain mRNA molecules, such as tumor necrosis factor alpha (TNF a), and favors the degradation ofthese mRNAs. As part of an effort to understand the macromolecular implications of sequence and the role of these novel zinc binding domains in RNA regulation, we are studying peptides that correspond to the zinc binding domains ofNUP-475. To this end, we have expressed and synthesized a series of peptides that correspond to the zinc binding domains of NUP-475 and have begun to identify the domains required for nucleic acid binding using a combination of fluorescence anisotropy and NMR spectroscopy. Although earlier studies on NUP-475 using gel shift assaysdetermined that both zinc binding domains are necessary for nucleic acid binding, using our more sensitive fluorescence anisotropy based assay,we have found that a single CCCH domain is capable of binding single-stranded RNA with considerable affinity and selectivity. These results, as well as the peptides’ metal binding, folding capabilities and structural studies will be presented.
Metallocenter characterization of AlkB, a mononuclear non-heme Fe(I1) DNA repair enzyme Robert P Hausinger, Michigan State University, United States John McCracken, Michigan State University, United States Timothy F Henshaw, Michigan State University, United States Alkylation of Escherichia coli DNA produces an array of modifications that are reversed by DNA repair enzymes. AlkB, an a-ketoglutarate (aKG)-dependent non-heme Fe oxygenase, directly repairs lesions involving 1-methyl-deoxyadenine and 3-methyl-deoxycytosine by hydroxylation of the methyl group followed by spontaneous loss of formaldehyde. Under anaerobic conditions, the metallocenter of Fe(II)AlkB exhibits no discernable electronic or EPR spectrum. Addition of aKG results in a weak absorption (h -500 nm, E -200 M-r cm-‘) attributed to an MLCT transition, as observed for other members of the aKG-dependent dioxygenase superfamily. Subsequent exposure to oxygen produces a blue chromophore (h -590 nm, E 640 Ml cm” as a lower limit) resulting from self-hydroxylation of an aromatic side chain and binding to the oxidized metal ion. Mass spectrometric investigation revealed the presence of OH-Trp at position 178, which likely coordinates the Fe(II1) to produce the observed chromophore. When exposed to the oxygen analogue NO, anaerobic Fe(II)AlkB develops an S = 312 EPR signal. This signal is perturbed by the addition of aKG and is further altered by inclusion of DNA. The precise nature of this perturbation is dependent on the methylation status of the included DNA. Further Cytosine characterization of the NO-FeAlkB complex with methylated and control DNA will be described.
Journal of Inorganic Biochemistry 96 (2003)
Regulation of Tumor Necrosis Factor Alpha (TNFa) mRNA by NUP-475, a Novel Zinc Binding Protein Sarah LJ Michel, The Johns Hopkins University School of Medicine, UnitedStates Barbara T Amann, The Johns Hopkins University School of Medicine, United States Anthony L Guerrerio, The Johns Hopkins University School of Medicine, United States Jeremy M Berg, The Johns Hopkins University School of Medicine, United States Zinc-binding domains have been found to mediate a wide range of macromolecular interactions including the regulation of gene expression. The protein NUP-475 (also named Tristetraprolin and Tisll) belongs to a newly discovered family of zinc binding proteins with tandem CCCH repeats. NUP-475 binds to the AU-rich sequence elements (AF&) of certain mRNA molecules, such as tumor necrosis factor alpha (TNF a), and favors the degradation ofthese mRNAs. As part of an effort to understand the macromolecular implications of sequence and the role of these novel zinc binding domains in RNA regulation, we are studying peptides that correspond to the zinc binding domains ofNUP-475. To this end, we have expressed and synthesized a series of peptides that correspond to the zinc binding domains of NUP-475 and have begun to identify the domains required for nucleic acid binding using a combination of fluorescence anisotropy and NMR spectroscopy. Although earlier studies on NUP-475 using gel shift assaysdetermined that both zinc binding domains are necessary for nucleic acid binding, using our more sensitive fluorescence anisotropy based assay,we have found that a single CCCH domain is capable of binding single-stranded RNA with considerable affinity and selectivity. These results, as well as the peptides’ metal binding, folding capabilities and structural studies will be presented.
Metallocenter characterization of AlkB, a mononuclear non-heme Fe(I1) DNA repair enzyme Robert P Hausinger, Michigan State University, United States John McCracken, Michigan State University, United States Timothy F Henshaw, Michigan State University, United States Alkylation of Escherichia coli DNA produces an array of modifications that are reversed by DNA repair enzymes. AlkB, an a-ketoglutarate (aKG)-dependent non-heme Fe oxygenase, directly repairs lesions involving 1-methyl-deoxyadenine and 3-methyl-deoxycytosine by hydroxylation of the methyl group followed by spontaneous loss of formaldehyde. Under anaerobic conditions, the metallocenter of Fe(II)AlkB exhibits no discernable electronic or EPR spectrum. Addition of aKG results in a weak absorption (h -500 nm, E -200 M-r cm-‘) attributed to an MLCT transition, as observed for other members of the aKG-dependent dioxygenase superfamily. Subsequent exposure to oxygen produces a blue chromophore (h -590 nm, E 640 Ml cm” as a lower limit) resulting from self-hydroxylation of an aromatic side chain and binding to the oxidized metal ion. Mass spectrometric investigation revealed the presence of OH-Trp at position 178, which likely coordinates the Fe(II1) to produce the observed chromophore. When exposed to the oxygen analogue NO, anaerobic Fe(II)AlkB develops an S = 312 EPR signal. This signal is perturbed by the addition of aKG and is further altered by inclusion of DNA. The precise nature of this perturbation is dependent on the methylation status of the included DNA. Further Cytosine characterization of the NO-FeAlkB complex with methylated and control DNA will be described.