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Tumor suppressor p53 regulates the expression of p53-induced genes (PIG) that trigger apoptosis. PIG3 or TP53I3 is the only known member of the medium chain dehydrogenase/reductase superfamily induced by p53 and is used as a proapoptotic marker. Although the participation of PIG3 in the apoptotic pathway is proven, the protein and its mechanism of action were never characterized. We analyzed human PIG3 enzymatic function and found NADPH-dependent reductase activity with ortho-quinones, which is consistent with the classification of PIG3 in the quinone oxidoreductase family. However, the activity is much lower than that of zeta-crystallin, a better known quinone oxidoreductase. In addition, we report the crystallographic structure of PIG3, which allowed the identification of substrate- and cofactor-binding sites, with residues fully conserved from bacteria to human. Tyr-59 in zeta-crystallin (Tyr-51 in PIG3) was suggested to participate in the catalysis of quinone reduction. However, kinetics of Tyr/Phe and Tyr/Ala mutants of both enzymes demonstrated that the active site Tyr is not catalytic but may participate in substrate binding, consistent with a mechanism based on propinquity effects. It has been proposed that PIG3 contribution to apoptosis would be through oxidative stress generation. We found that in vitro activity and in vivo overexpression of PIG3 accumulate reactive oxygen species. Accordingly, an inactive PIG3 mutant (S151V) did not produce reactive oxygen species in cells, indicating that enzymatically active protein is necessary for this function. This supports that PIG3 action is through oxidative stress produced by its enzymatic activity and provides essential knowledge for eventual control of apoptosis.

Original publication

DOI

10.1074/jbc.M109.001800

Type

Journal article

Journal

J Biol Chem

Publication Date

19/06/2009

Volume

284

Pages

17194 - 17205

Keywords

Amino Acid Sequence, Apoptosis, Binding Sites, Catalytic Domain, Crystallography, X-Ray, Humans, In Vitro Techniques, Intracellular Signaling Peptides and Proteins, Kinetics, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, NADP, Phylogeny, Protein Structure, Quaternary, Proto-Oncogene Proteins, Reactive Oxygen Species, Recombinant Proteins, Sequence Homology, Amino Acid, Tumor Suppressor Protein p53, Tyrosine