Abstract
It has become increasingly evident that nitric oxide exerts its effects, in part, by S-nitrosylation of cysteine residues. We tested in vitro whether nitric oxide may indirectly control p53 by S-nitrosylation and inactivation of the p53 negative regulator, Hdm2. Treatment of Hdm2 with a nitric oxide donor inhibits Hdm2-p53 binding, a critical step in Hdm2 regulation of p53. The presence of excess amounts of cysteine or dithiothreitol blocks this inhibition of binding. Moreover, nitric oxide inhibition of Hdm2-p53 binding was found to be reversible. Sulfhydryl sensitivity and reversibility are consistent with nitrosylation. Finally, we have identified a critical cysteine residue that nitric oxide modifies to disrupt Hdm2-p53 binding. This cysteine is proximal to the Hdm2-p53 binding interface and is conserved across species from zebrafish to humans. Mutation of this residue from a cysteine to an alanine does not interfere with binding but rather eliminates the sensitivity of Hdm2 to nitric oxide inactivation.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Binding Sites
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Cysteine / chemistry
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Dithiothreitol / pharmacology
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Enzyme-Linked Immunosorbent Assay
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Glutathione / chemistry
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Glutathione / metabolism
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Glutathione Transferase / genetics
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Glutathione Transferase / metabolism
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Humans
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Mutation
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Neoplasm Proteins / genetics
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Neoplasm Proteins / metabolism*
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Nuclear Proteins*
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Protein Binding / drug effects*
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Protein Conformation
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism*
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Proto-Oncogene Proteins c-mdm2
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Recombinant Fusion Proteins / metabolism
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Sequence Homology, Amino Acid
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Triazenes / pharmacology*
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Tumor Suppressor Protein p53 / metabolism*
Substances
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1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene
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Neoplasm Proteins
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Nuclear Proteins
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Proto-Oncogene Proteins
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Recombinant Fusion Proteins
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Triazenes
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Tumor Suppressor Protein p53
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MDM2 protein, human
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Proto-Oncogene Proteins c-mdm2
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Glutathione Transferase
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Glutathione
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Cysteine
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Dithiothreitol