Three-dimensional Structure and Enzymatic Function of Proapoptotic Human p53-inducible Quinone Oxidoreductase PIG3

Abstract

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 -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 -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.

p53-dependent upregulation of PIG3 transcription by γ-ray irradiation and its interaction with KAP1 in responding to DNA damage

“…Recent research reported that overexpression of PIG3 accumulates ROS in vivo and in vitro; while an inactive PIG3 mutant (Ser151Val) does not [3]. Our result, H 2 O 2 stimulating PIG3 expression, implied that ROS may also contribute to PIG3 induction by ionizing radiation via the oxidative DNA damage mechanism.…”

“…As consistent with its classification in the quinine oxidoreductase (QOR) family, PIG3 was confirmed an activity of NADPH dependent reductase with orthoquinones [3]. In addition, in vitro activation and in vivo overexpression of PIG3 was demonstrated to accumulate reactive oxygen species (ROS), while an inactive PIG3 mutant (Ser151Val) did not produce ROS in cells, indicating that enzymatic active protein is necessary for its function [3]. PIG3 was also recently demonstrated to contribute to early cellular response to DNA damage induced by UV radiation and the DNA damaging-chemical neocarzinostatin through recruiting 53BP1, Mre11, Rad50 and Nbs1 to the sites of DNA break lesions and modulating intra-S and G2/M phase checkpoint [4].…”

“…The closest relative of PIG3 in mammals is zeta-Crystallin, an NADPH-quinone oxidoreductase and a potent generator of reactive oxygen species (ROS) [2]. As consistent with its classification in the quinine oxidoreductase (QOR) family, PIG3 was confirmed an activity of NADPH dependent reductase with orthoquinones [3]. In addition, in vitro activation and in vivo overexpression of PIG3 was demonstrated to accumulate reactive oxygen species (ROS), while an inactive PIG3 mutant (Ser151Val) did not produce ROS in cells, indicating that enzymatic active protein is necessary for its function [3].…”

p53-Mediated Oxidative Stress and Tubular Injury in Rats with Glycerol-Induced Acute Kidney Injury

“…Polyak et al [7] showed that redox active transcripts (PIGs) were markedly increased in p53-expressing cells. Recently, the enzymatic function of PIG3 was characterized as a prooxidant oxido-reductase and PIG3-induced ROS generation was detected in human colon cancer cells infected with virus carrying the PIG3 gene [20]. Sablina et al [21] also showed an increased ROS generation in lung carcinoma cells overexpressing the PIG3 gene, but they showed an even higher ROS production after infection with the PUMA gene.…”

The p53-inducible gene 3 (PIG3) contributes to early cellular response to DNA damage

“…Activated p53 increases cellular ROS levels by enhancing the transcription of proapoptotic genes, such as PIGs and Bax (Polyak et al, 1997;Kirkland et al, 2002). PIG3 shows sequence similarity with NQO, and influences the production of intracellular ROS (Porte et al, 2009). Thus, although PIG3 expression alone is insufficient to induce apoptosis (Polyak et al, 1997), PIG3 may be one of the factors involved in p53-induced apoptosis through ROS generation.…”