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

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 ζ-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 ζ-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. 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 ζ-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 ζ-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 is an essential transcription factor in the control of the cell cycle, inducing cell cycle arrest or apoptosis, through the control of the expression of distinct genes (1Levine A.J. Momand J. Finlay C.A. Nature. 1991; 351: 453-456Crossref PubMed Scopus (3665) Google Scholar). TP53I3 (tumor protein p53-inducible protein 3), also called PIG3 (p53-inducible gene 3), is one of the p53 protein targets and is used as a long lived proapoptotic marker. PIG3 was discovered along with 12 other proteins in a serial analysis of gene expression studies designed to determine genes induced by p53 before the apoptosis onset (2Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2246) Google Scholar). In addition, PIG3 expression can also be elicited by p63 and p73 (3Bergamaschi D. Samuels Y. Jin B. Duraisingham S. Crook T. Lu X. Mol. Cell. Biol. 2004; 24: 1341-1350Crossref PubMed Scopus (208) Google Scholar), which are able to induce apoptosis in a p53-independent manner (4Moll U.M. Slade N. Mol. Cancer Res. 2004; 2: 371-386PubMed Google Scholar) and which are implicated in the induction of PIG3 through flavonoid exposure (5Zhang Q. Zhao X.H. Wang Z.J. Food Chem. Toxicol. 2008; 46: 2042-2053Crossref PubMed Scopus (177) Google Scholar). PIG3 expression is largely regulated through its promoter (6Contente A. Dittmer A. Koch M.C. Roth J. Dobbelstein M. Nat. Genet. 2002; 30: 315-320Crossref PubMed Scopus (198) Google Scholar), and polymorphisms within this site have been associated with invasive bladder cancer (7Ito M. Nishiyama H. Watanabe J. Kawanishi H. Takahashi T. Kamoto T. Habuchi T. Ogawa O. Jpn. J. Clin. Oncol. 2006; 36: 116-120Crossref PubMed Scopus (11) Google Scholar) and leukemia (8Nomdedéu J.F. Perea G. Estivill C. Badell I. Lasa A. Aventín A. Leuk. Res. 2008; 32: 186-188Crossref PubMed Scopus (4) Google Scholar). Some p53 mutants, which are able to induce cell cycle arrest but not apoptosis, could not induce PIG3 expression (9Campomenosi P. Monti P. Aprile A. Abbondandolo A. Frebourg T. Gold B. Crook T. Inga A. Resnick M.A. Iggo R. Fronza G. Oncogene. 2001; 20: 3573-3579Crossref PubMed Scopus (120) Google Scholar). Moreover, it has been recently reported that PIG3 expression is also regulated by a new transcription factor (hCAS/CSE1L), which associates with a subset of p53 target genes increasing apoptosis. The factor binds to the PIG3 promoter region, whereas hCAS/CSE1L silencing leads to decreased PIG3 transcription and to increased methylation of histone H3Lys-27 found within the PIG3 gene, finally resulting in decreased apoptosis (10Tanaka T. Ohkubo S. Tatsuno I. Prives C. Cell. 2007; 130: 638-650Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Despite its clear relationship with apoptosis, the mechanism of the PIG3 action has not been established. The protein shares significant sequence identity with the quinone oxidoreductase (QOR) 2The abbreviations used are: QORquinone oxidoreductaseROSreactive oxygen speciesDCFH-DA2′,7′-dichlorodihydrofluorescein diacetatePBSphosphate-buffered salinePDBProtein Data BankLICligation independent cloningMDRmedium chain dehydrogenases/reductaseAKRaldo-keto reductaseDCIP2,6-dichloroindophenol. family, which led us to propose a possible role in apoptosis through a putative QOR function. QORs catalyze quinone reduction, generating hydroxyquinone (11Rao P.V. Krishna C.M. Zigler Jr., J.S. J. Biol. Chem. 1992; 267: 96-102Abstract Full Text PDF PubMed Google Scholar, 12Schlegel B.P. Ratnam K. Penning T.M. Biochemistry. 1998; 37: 11003-11011Crossref PubMed Scopus (43) Google Scholar, 13Oppermann U. Annu. Rev. Pharmacol. Toxicol. 2007; 47: 293-322Crossref PubMed Scopus (174) Google Scholar), which is reoxidized in the presence of O2 leading to the formation of reactive oxygen species (ROS) (Scheme 1). On the basis of this mechanism, it has been proposed that PIG3 may induce oxidative stress, like other p53 targets (14Liang X.Q. Cao E.H. Zhang Y. Qin J.F. FEBS Lett. 2004; 569: 94-98Crossref PubMed Scopus (19) Google Scholar). In support of this notion, oxidative stress has been proposed as a proapoptotic mechanism (15Johnson T.M. Yu Z.X. Ferrans V.J. Lowenstein R.A. Finkel T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11848-11852Crossref PubMed Scopus (525) Google Scholar), and PIG3 expression precedes the appearance of ROS in p53-induced apoptosis. However, evidence that supports an apoptotic role of PIG3 by increasing oxidative stress through a QOR function is only indirect because its enzymatic activity has never been reported, and only preliminary information exists on the role of PIG3 in ROS production in vivo (2Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2246) Google Scholar, 16Flatt P.M. Polyak K. Tang L.J. Scatena C.D. Westfall M.D. Rubinstein L.A. Yu J. Kinzler K.W. Vogelstein B. Hill D.E. Pietenpol J.A. Cancer Lett. 2000; 156: 63-72Crossref PubMed Scopus (71) Google Scholar). quinone oxidoreductase reactive oxygen species 2′,7′-dichlorodihydrofluorescein diacetate phosphate-buffered saline Protein Data Bank ligation independent cloning medium chain dehydrogenases/reductase aldo-keto reductase 2,6-dichloroindophenol. QOR is a poorly studied protein family within the medium chain dehydrogenases/reductases (MDR). There is no report on the involvement of any QOR in ROS increase or in vivo quinone oxidation. In QORs the enzymatic activity has been only in the ζ-crystallin and proteins in S. N. J. J.A. X. Cell. Mol. Sci. 2007; PubMed Scopus Google Scholar) and bacteria A. Y. K. K. H. T. PubMed Scopus Google Scholar). In were as proteins found in in and P. Zigler Jr., J.S. Res. PubMed Scopus Google Scholar), and recently function as and proteins has been reported S. N. J. J.A. X. Cell. Mol. Sci. 2007; PubMed Scopus Google Scholar, A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, N. Res. 2006; PubMed Scopus Google Scholar). However, the role of any QOR has not been and proposed for in and of by of in H. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar), have no relationship with enzymatic In this we have the structure and analyzed the enzymatic of PIG3 and its role in ROS generation. The has been by the of ζ-crystallin, a active protein which as a and which allowed the of and the enzymatic mechanism of The identification of PIG3 and structure provides a target for the control of apoptosis. PIG3 genes were in the and both and were In addition, the PIG3 protein sequence was by the which also the and human PIG3 proteins from and were Zhang J. Zhang Res. 1997; PubMed Scopus Google Scholar) in a PIG3 the protein analysis and were Scholar). sequence were the Res. PubMed Scopus Google Scholar) with and were this expression were PIG3 was from PIG3 was by the cloning PIG3 expression or expression in human cells, PIG3 was by for in the and in the The resulting was or for PIG3 expression in the PIG3 was by a expression with an and an PIG3 mutants were by the PIG3 as a on the we designed for as and The ζ-crystallin were ζ-crystallin as a S. N. J. J.A. X. Cell. Mol. Sci. 2007; PubMed Scopus Google Scholar) and for were in a with were with for to the The resulting were to was to that were PIG3 and ζ-crystallin were as and by S. N. J. J.A. X. Cell. Mol. Sci. 2007; PubMed Scopus Google Scholar). with was in medium for Protein expression was induced by the of and were for cell by the was a with in the was with a in the Protein were and was with an The protein was in PIG3 production was with a PIG3 and in medium for Protein expression was induced by and for PIG3 was by with phosphate-buffered saline the protein was by of for was with a and by a in was to in a PIG3 production expression and of protein were by and protein was to and Protein was by by as as an of Protein was by a as a PubMed Scopus Google Scholar) or a The structure of human PIG3 was by a and an 1). were by of from a protein and from the Data were the from were also by of of a protein with of the and Data were the a with were and and 1997; PubMed Scopus Google Scholar). was by A. A. Biol. 2000; PubMed Scopus Google Scholar) for the with one protein in the as a the from QOR a protein sequence identity with PIG3 However, of this structure that were only the by as a the PIG3 structure was Biol. PubMed Scopus Google Scholar) and with the M. A. 1991; 47: PubMed Scopus Google Scholar). were the in the The resulting PIG3 were in the with and for the and 1). The of the PIG3 was and were with and of to to to to and are the and structure has the as for a of of the The is in of in in allowed in allowed in of of and are the and structure has the as for a of of the The is in in a new sequence are from are from protein are from reductase are from are from oxidoreductase are from are from are from Data are from S. N. J. J.A. X. Cell. Mol. Sci. 2007; PubMed Scopus Google Data are from A. Y. K. K. H. T. PubMed Scopus Google Data are from Z.J. R. S. J. Mol. Biol. 2008; PubMed Scopus Google Data are from T. T. Takahashi T. T. Y. Y. T. J. Biol. Chem. Full Text PDF PubMed Google Data are from J. J. A. D. S. K. J. 2000; 267: PubMed Scopus Google Data are from J. Y. S. K. K. K. D. S. K. 2002; PubMed Scopus Google in a new to PIG3 was by the in protein by a was in The of in to which from were to with PIG3 and of for the control was by and by the were reported as The for to PIG3 was as R.A. J. PubMed Scopus Google Scholar, C. X. I. J.A. J. Mol. Biol. 2004; PubMed Scopus (43) Google Scholar). were in in the presence of with substrate quinone were in resulting in lower than in the was a from R. and P. of The enzymatic activity was in a by the of The activity was by the The enzymatic was as O. S. J. X. J. 2006; PubMed Scopus Google Scholar). substrate or were The with the of the the were in with substrate and the were the reported are as the of independent was as a of the The or with and of a of the or a of the was to the The of was with of to the in a of with to produce The were on and was a by and were by of and were on in with was in a were with to the medium was and were The is based on 2′,7′-dichlorodihydrofluorescein diacetate a for ROS in J.A. H. PubMed Scopus Google Scholar, D. A. M. M.A. 1997; PubMed Scopus Google Scholar). is by generating a which could be by ROS and were with and for that been with for were used as a control of ROS The were in of with for in the were with and finally in which be from were analyzed by cell within the of and the Data were as was by the PIG3 was from was from In the PIG3 gene is found in of the from to 1). it is in and and and this a PIG3 gene, a has not been found in and In it is in analysis the presence of the only in PIG3 that p53 was A. H. A. Dobbelstein M. Cancer Res. Google Scholar). In addition, PIG3 are found in bacteria and other bacteria and indicating a in of the from of the PIG3 an increasing to and within and In the identity is than in with the human the proteins to PIG3, from which information is of are proteins as and of can be as in the reductase family, and and oxidoreductase both in the reductase family. as as the and are known to enzymatic activity within the QOR family. We have PIG3 NADPH-dependent reductase activity with reported ζ-crystallin with for and with PIG3 enzymatic activity with 3), with a for the the the is the only substrate able to the but the substrate for in of is inactive with activity was also for the with PIG3, it did not the In was not a substrate for a used as in oxidoreductase K. C. M. PubMed Scopus Google Scholar), as the the of and were not activity with ζ-crystallin but was inactive reductase of PIG3 and with are from are from Data are from S. N. J. J.A. X. Cell. Mol. 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Protein PubMed Scopus Google Scholar), the PIG3 is of as the catalytic to and to and the cofactor-binding to are by a which The catalytic is by a whereas the cofactor-binding of which are the However, is an which was also in the human reductase Z.J. R. S. J. Mol. Biol. 2008; PubMed Scopus Google Scholar). and an of of the cofactor-binding are in the are through with the of the In the the is the the was in the the catalytic and the is in an with an of the and the A. L.J. The Scholar). of residues implicated in are fully conserved the PIG3 1). The of the to enzymes has been to a sequence In PIG3, the is as which is to the and found in other QORs J. 1996; PubMed Scopus Google Scholar). The from the PIG3 with the chain oxygen and from the and a is in that as in other QOR a the to the in PIG3, the presence of an or a the of the in the is A. J. Mol. Biol. 1992; PubMed Scopus Google Scholar). This is by a in the enzymes A. X. I. J. J. Mol. Biol. 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The of a conserved in PIG3 proteins but in other QOR enzymes is from the and by a with residues and In addition, from and to be to other through a in the of quinone a is this in the sequence a is this in the sequence in a new of that the substrate the PIG3 putative with the oxygen in the to with the and with the of In in the the oxygen not the which would in the could not be the PIG3 in to the presence of in with the of activity this In evidence supports a Tyr as an Z.J. R. S. J. Mol. Biol. 2008; PubMed Scopus Google Scholar, T. T. H. M. G. M. T. T. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, B. C. C. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, S. A.J. J. Mol. Biol. PubMed Scopus Google Scholar). The crystallographic that in PIG3 and Tyr-59 in ζ-crystallin are to this catalytic function. the mutant were and were In both proteins the of Tyr to did not but in a significant increase of was This that the active site Tyr is not the catalytic of The increase in catalytic of the Tyr/Phe mutants the is to The of Tyr to not the activity the the of PIG3 with which could not the The ζ-crystallin mutant as of the The activity by ζ-crystallin leads to the production of and (11Rao P.V. Krishna C.M. Zigler Jr., J.S. J. Biol. Chem. 1992; 267: 96-102Abstract Full Text PDF PubMed Google Scholar). we ROS as a of PIG3 activity of is in the which is consistent with a significant in the of a of and the However, ROS production is much in the presence of the It can be that the of by PIG3 a mechanism as found with In PIG3 of not We have also the overexpression of active PIG3 in the production of ROS in vivo a control we an inactive that the in the The PIG3 mutant was to and was of enzymatic the mutant a expression and and demonstrated a structure indicating a were with the and of was by a The of the increased with the The was than that of the with the inactive which did not the inactive mutant did not ROS in vitro the we PIG3 binds in In to which significant S. N. J. J.A. X. Cell. Mol. Sci. 2007; PubMed Scopus Google Scholar), PIG3 not any We that PIG3, found in and can could be for the substrate found for lower activity is with the which with the for reported for other QORs S. N. J. J.A. X. Cell. Mol. 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