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• W2101821132 abstract "•p53 is downregulated in tumorigenesis-associated iron/heme excess•The C terminus of p53 specifically binds to heme•Heme excess destabilizes p53 by promoting its nuclear export and cytosolic degradation•Tumor suppression by iron deprivation relies on wild-type p53 signaling Iron excess is closely associated with tumorigenesis in multiple types of human cancers, with underlying mechanisms yet unclear. Recently, iron deprivation has emerged as a major strategy for chemotherapy, but it exerts tumor suppression only on select human malignancies. Here, we report that the tumor suppressor protein p53 is downregulated during iron excess. Strikingly, the iron polyporphyrin heme binds to p53 protein, interferes with p53-DNA interactions, and triggers both nuclear export and cytosolic degradation of p53. Moreover, in a tumorigenicity assay, iron deprivation suppressed wild-type p53-dependent tumor growth, suggesting that upregulation of wild-type p53 signaling underlies the selective efficacy of iron deprivation. Our findings thus identify a direct link between iron/heme homeostasis and the regulation of p53 signaling, which not only provides mechanistic insights into iron-excess-associated tumorigenesis but may also help predict and improve outcomes in iron-deprivation-based chemotherapy. Iron excess is closely associated with tumorigenesis in multiple types of human cancers, with underlying mechanisms yet unclear. Recently, iron deprivation has emerged as a major strategy for chemotherapy, but it exerts tumor suppression only on select human malignancies. Here, we report that the tumor suppressor protein p53 is downregulated during iron excess. Strikingly, the iron polyporphyrin heme binds to p53 protein, interferes with p53-DNA interactions, and triggers both nuclear export and cytosolic degradation of p53. Moreover, in a tumorigenicity assay, iron deprivation suppressed wild-type p53-dependent tumor growth, suggesting that upregulation of wild-type p53 signaling underlies the selective efficacy of iron deprivation. Our findings thus identify a direct link between iron/heme homeostasis and the regulation of p53 signaling, which not only provides mechanistic insights into iron-excess-associated tumorigenesis but may also help predict and improve outcomes in iron-deprivation-based chemotherapy. Iron is essential for cell survival, proliferation, and metabolism, a fact highlighted by the association of dysregulated iron metabolism with a myriad of human disorders including cancer and diabetes (Andrews, 2008Andrews N.C. Forging a field: the golden age of iron biology.Blood. 2008; 112: 219-230Crossref PubMed Scopus (486) Google Scholar, Fleming and Ponka, 2012Fleming R.E. Ponka P. Iron overload in human disease.N. Engl. J. Med. 2012; 366: 348-359Crossref PubMed Scopus (431) Google Scholar, Rouault, 2005Rouault T.A. Linking physiological functions of iron.Nat. Chem. Biol. 2005; 1: 193-194Crossref PubMed Scopus (10) Google Scholar, Simcox and McClain, 2013Simcox J.A. McClain D.A. Iron and diabetes risk.Cell Metab. 2013; 17: 329-341Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). In particular, decades of epidemiological and experimental studies have established that iron excess, due to either genetic factors or excessive dietary intake, is implicated in multiple types of human cancers (Torti and Torti, 2013Torti S.V. Torti F.M. Iron and cancer: more ore to be mined.Nat. Rev. Cancer. 2013; 13: 342-355Crossref PubMed Scopus (992) Google Scholar, Toyokuni, 2009Toyokuni S. Role of iron in carcinogenesis: cancer as a ferrotoxic disease.Cancer Sci. 2009; 100: 9-16Crossref PubMed Scopus (411) Google Scholar). Hereditary hemochromatosis (HH) is a genetic disorder of iron overload, with clinical complications including liver cirrhosis and a 20- to 200-fold increased risk for hepatocellular carcinoma (Elmberg et al., 2003Elmberg M. Hultcrantz R. Ekbom A. Brandt L. Olsson S. Olsson R. Lindgren S. Lööf L. Stål P. Wallerstedt S. et al.Cancer risk in patients with hereditary hemochromatosis and in their first-degree relatives.Gastroenterology. 2003; 125: 1733-1741Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, Niederau et al., 1985Niederau C. Fischer R. Sonnenberg A. Stremmel W. Trampisch H.J. Strohmeyer G. Survival and causes of death in cirrhotic and in noncirrhotic patients with primary hemochromatosis.N. Engl. J. Med. 1985; 313: 1256-1262Crossref PubMed Scopus (978) Google Scholar) or other cancer types (Osborne et al., 2010Osborne N.J. Gurrin L.C. Allen K.J. Constantine C.C. Delatycki M.B. McLaren C.E. Gertig D.M. Anderson G.J. Southey M.C. Olynyk J.K. et al.HFE C282Y homozygotes are at increased risk of breast and colorectal cancer.Hepatology. 2010; 51: 1311-1318Crossref PubMed Scopus (110) Google Scholar, Pietrangelo, 2010Pietrangelo A. Hereditary hemochromatosis: pathogenesis, diagnosis, and treatment.Gastroenterology. 2010; 139: 393-408Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, Radulescu et al., 2012Radulescu S. Brookes M.J. Salgueiro P. Ridgway R.A. McGhee E. Anderson K. Ford S.J. Stones D.H. Iqbal T.H. Tselepis C. Sansom O.J. Luminal iron levels govern intestinal tumorigenesis after Apc loss in vivo.Cell Rep. 2012; 2: 270-282Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Meanwhile, tumors reprogram iron metabolism to achieve a growth advantage or metastasis, resulting in the emergence of iron deprivation, via iron chelation or application of transferrin receptor-neutralizing antibodies, as a major chemotherapeutic strategy. However, preclinic and clinical studies have demonstrated that iron deprivation only suppresses select human malignancies while having no effect on other cancer types, with underlying mechanisms of the selectivity yet elusive (Buss et al., 2004Buss J.L. Greene B.T. Turner J. Torti F.M. Torti S.V. Iron chelators in cancer chemotherapy.Curr. Top. Med. Chem. 2004; 4: 1623-1635Crossref PubMed Scopus (176) Google Scholar, Yamasaki et al., 2011Yamasaki T. Terai S. Sakaida I. Deferoxamine for advanced hepatocellular carcinoma.N. Engl. J. Med. 2011; 365: 576-578Crossref PubMed Scopus (116) Google Scholar). Therefore, a thorough interrogation of the mechanisms of how iron excess contributes to tumorigenesis and the molecular basis of the selective efficacy of iron deprivation would not only further our understanding of cancer biology but also improve the design of targeted chemotherapy for better clinical outcomes. Heme is an iron polyporphyrin that constitutes the prosthetic group for proteins functioning in a myriad of fundamental biological processes, including respiration, energetic homeostasis, signal transduction, xenobiotic detoxification, iron metabolism, mRNA processing, and control of circadian rhythm (Boon et al., 2005Boon E.M. Huang S.H. Marletta M.A. A molecular basis for NO selectivity in soluble guanylate cyclase.Nat. Chem. Biol. 2005; 1: 53-59Crossref PubMed Scopus (170) Google Scholar, Dioum et al., 2002Dioum E.M. Rutter J. Tuckerman J.R. Gonzalez G. Gilles-Gonzalez M.A. McKnight S.L. NPAS2: a gas-responsive transcription factor.Science. 2002; 298: 2385-2387Crossref PubMed Scopus (381) Google Scholar, Faller et al., 2007Faller M. Matsunaga M. Yin S. Loo J.A. Guo F. Heme is involved in microRNA processing.Nat. Struct. Mol. Biol. 2007; 14: 23-29Crossref PubMed Scopus (226) Google Scholar, Gilles-Gonzalez and Gonzalez, 2005Gilles-Gonzalez M.A. Gonzalez G. Heme-based sensors: defining characteristics, recent developments, and regulatory hypotheses.J. Inorg. Biochem. 2005; 99: 1-22Crossref PubMed Scopus (302) Google Scholar, Hu et al., 2008Hu R.G. Wang H. Xia Z. Varshavsky A. The N-end rule pathway is a sensor of heme.Proc. Natl. Acad. Sci. USA. 2008; 105: 76-81Crossref PubMed Scopus (93) Google Scholar, Ishikawa et al., 2005Ishikawa H. Kato M. Hori H. Ishimori K. Kirisako T. Tokunaga F. Iwai K. Involvement of heme regulatory motif in heme-mediated ubiquitination and degradation of IRP2.Mol. Cell. 2005; 19: 171-181Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, Rajagopal et al., 2008Rajagopal A. Rao A.U. Amigo J. Tian M. Upadhyay S.K. Hall C. Uhm S. Mathew M.K. Fleming M.D. Paw B.H. et al.Haem homeostasis is regulated by the conserved and concerted functions of HRG-1 proteins.Nature. 2008; 453: 1127-1131Crossref PubMed Scopus (226) Google Scholar, Yang et al., 2010Yang F. Xia X.A. Lei H.Y. Wang E.D. Hemin binds to human cytoplasmic arginyl-tRNA synthetase and inhibits its catalytic activity.J. Biol. Chem. 2010; 285: 39437-39446Crossref PubMed Scopus (29) Google Scholar). The switch between ferrous (Fe2+) and ferric (Fe3+) states of iron in the metallo-polyporphyrin, termed as heme and hemin, respectively, underlies its unique roles in transducing redox and gas signaling in vivo. Heme was identified as a ligand for transcriptional factors such as NPAS2 (Dioum et al., 2002Dioum E.M. Rutter J. Tuckerman J.R. Gonzalez G. Gilles-Gonzalez M.A. McKnight S.L. NPAS2: a gas-responsive transcription factor.Science. 2002; 298: 2385-2387Crossref PubMed Scopus (381) Google Scholar), E75 (Reinking et al., 2005Reinking J. Lam M.M. Pardee K. Sampson H.M. Liu S. Yang P. Williams S. White W. Lajoie G. Edwards A. Krause H.M. The Drosophila nuclear receptor e75 contains heme and is gas responsive.Cell. 2005; 122: 195-207Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), and Rev-erb α (Yin et al., 2007Yin L. Wu N. Curtin J.C. Qatanani M. Szwergold N.R. Reid R.A. Waitt G.M. Parks D.J. Pearce K.H. Wisely G.B. Lazar M.A. Rev-erbalpha, a heme sensor that coordinates metabolic and circadian pathways.Science. 2007; 318: 1786-1789Crossref PubMed Scopus (575) Google Scholar), where heme was shown to play a regulatory role by modulating the respective protein functionalities. We have also found that arginyl-tRNA protein transferase (Ate1), a key component of the N-end rule pathway in the ubiquitin (Ub)-proteasome system, binds to heme and allows the N-end rule pathway to act as a sensor of heme and redox state (Hu et al., 2005Hu R.G. Sheng J. Qi X. Xu Z. Takahashi T.T. Varshavsky A. The N-end rule pathway as a nitric oxide sensor controlling the levels of multiple regulators.Nature. 2005; 437: 981-986Crossref PubMed Scopus (243) Google Scholar, Hu et al., 2008Hu R.G. Wang H. Xia Z. Varshavsky A. The N-end rule pathway is a sensor of heme.Proc. Natl. Acad. Sci. USA. 2008; 105: 76-81Crossref PubMed Scopus (93) Google Scholar, Kwon et al., 2002Kwon Y.T. Kashina A.S. Davydov I.V. Hu R.G. An J.Y. Seo J.W. Du F. Varshavsky A. An essential role of N-terminal arginylation in cardiovascular development.Science. 2002; 297: 96-99Crossref PubMed Scopus (269) Google Scholar, Varshavsky, 2012Varshavsky A. The ubiquitin system, an immense realm.Annu. Rev. Biochem. 2012; 81: 167-176Crossref PubMed Scopus (222) Google Scholar). Because heme is identified as a prosthetic group in an expanding body of proteins in multiple pathophysiological processes, it is conceivable that we might be still at an early stage in understanding the regulatory roles of heme. Tumor suppressor p53 suppresses tumorigenesis and regulates DNA-damage repair, cell-cycle arrest, and tumor responses to chemotherapy (Baker et al., 1989Baker S.J. Fearon E.R. Nigro J.M. Hamilton S.R. Preisinger A.C. Jessup J.M. vanTuinen P. Ledbetter D.H. Barker D.F. Nakamura Y. et al.Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas.Science. 1989; 244: 217-221Crossref PubMed Scopus (1712) Google Scholar, Espinosa et al., 2003Espinosa J.M. Verdun R.E. Emerson B.M. p53 functions through stress- and promoter-specific recruitment of transcription initiation components before and after DNA damage.Mol. Cell. 2003; 12: 1015-1027Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, Kastan et al., 1991Kastan M.B. Onyekwere O. Sidransky D. Vogelstein B. Craig R.W. Participation of p53 protein in the cellular response to DNA damage.Cancer Res. 1991; 51: 6304-6311PubMed Google Scholar, Liu et al., 2011Liu J. Xia H. Kim M. Xu L. Li Y. Zhang L. Cai Y. Norberg H.V. Zhang T. Furuya T. et al.Beclin1 controls the levels of p53 by regulating the deubiquitination activity of USP10 and USP13.Cell. 2011; 147: 223-234Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, Vogelstein et al., 2000Vogelstein B. Lane D. Levine A.J. Surfing the p53 network.Nature. 2000; 408: 307-310Crossref PubMed Scopus (5850) Google Scholar, Vousden and Prives, 2009Vousden K.H. Prives C. Blinded by the light: the growing complexity of p53.Cell. 2009; 137: 413-431Abstract Full Text Full Text PDF PubMed Scopus (2355) Google Scholar). Recent work also identified p53 as a cellular hub in regulating and responding to cell metabolism (Jiang et al., 2013Jiang P. Du W. Mancuso A. Wellen K.E. Yang X. Reciprocal regulation of p53 and malic enzymes modulates metabolism and senescence.Nature. 2013; 493: 689-693Crossref PubMed Scopus (339) Google Scholar, Maddocks and Vousden, 2011Maddocks O.D. Vousden K.H. Metabolic regulation by p53.J. Mol. Med. (Berl.). 2011; 89: 237-245Crossref PubMed Scopus (246) Google Scholar). p53 protein is also a major regulator of cellular responses to redox signaling. Thus far, a few small molecules, such as NAD+ and ADP, have been identified as physiological ligands for p53 protein, modulating the transcription of a set of p53 target genes in response to changes in cell redox state and energy metabolism (McLure et al., 2004McLure K.G. Takagi M. Kastan M.B. NAD+ modulates p53 DNA binding specificity and function.Mol. Cell. Biol. 2004; 24: 9958-9967Crossref PubMed Scopus (71) Google Scholar). It is yet unknown whether p53 might bind to any other cellular small molecules, either metabolites or signaling messengers, and directly sense cellular redox signaling. Given the prominent roles of tumor suppressor p53 in regulating tumorigenesis and cellular responses to genotoxic stresses, we set out to examine whether and how iron excess might affect p53 signaling and thus contribute to tumorigenesis associated with iron overload. We also used cell and animal models to investigate the molecular determinants underlying the selective efficacy of iron deprivation-based chemotherapy. Patients with HH have defects in the control of iron entry into circulation, which allows for the toxic accumulation of iron in parenchymal cells of vital organs (Pietrangelo, 2010Pietrangelo A. Hereditary hemochromatosis: pathogenesis, diagnosis, and treatment.Gastroenterology. 2010; 139: 393-408Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, Weiss, 2010Weiss G. Genetic mechanisms and modifying factors in hereditary hemochromatosis.Nat. Rev. Gastroenterol. Hepatol. 2010; 7: 50-58Crossref PubMed Scopus (68) Google Scholar). The known genetic factors for human HH are mutations in the genes encoding the human hemochromatosis protein (Hfe), transferrin receptor 2 (Tfr2), hemojuvelin (Hjv), ferroportin (Fpn), and more rarely hepcidin (Hamp). It was estimated that 10% of the white population worldwide carries disease-associated mutations in Hfe, with disease penetrance of 2%–38% among male and 1%–10% among female carriers (Fleming and Ponka, 2012Fleming R.E. Ponka P. Iron overload in human disease.N. Engl. J. Med. 2012; 366: 348-359Crossref PubMed Scopus (431) Google Scholar). A 20- to 200-fold higher risk for hepatocellular carcinoma or many other types of cancer was reported for patients with hemochromatosis with iron overload, highlighting a strong association between increased iron metabolism and human malignancy (Torti and Torti, 2013Torti S.V. Torti F.M. Iron and cancer: more ore to be mined.Nat. Rev. Cancer. 2013; 13: 342-355Crossref PubMed Scopus (992) Google Scholar). However, the underlying mechanism remains unclear. Mice with homozygous deletion of the human hemochromatosis (Hfe) gene (Hfe−/−) faithfully recapitulate most human HH symptoms, including significant liver iron overload as assessed by Perls’ Prussian Blue staining (Zhou et al., 1998Zhou X.Y. Tomatsu S. Fleming R.E. Parkkila S. Waheed A. Jiang J. Fei Y. Brunt E.M. Ruddy D.A. Prass C.E. et al.HFE gene knockout produces mouse model of hereditary hemochromatosis.Proc. Natl. Acad. Sci. USA. 1998; 95: 2492-2497Crossref PubMed Scopus (495) Google Scholar) (Figure 1A). Using a 3,3′,5,5′-tetramethylbenzidine (TMB) assay to quantitatively assess liver heme content, we found that Hfe−/− mouse liver lysate had 9-fold more heme than lysate from wild-type livers (3.71 ± 0.4 nmol/mg protein versus 0.41 ± 0.12 nmol/mg protein) (Figure 1B). We next assessed liver p53 protein content and found that total Hfe−/− liver lysates had significantly lower endogenous p53 protein levels than wild-type livers (Figure 1C). The level of p53 protein in primary hepatocytes from Hfe−/− mice was also markedly lower than that in wild-type hepatocytes (Figure S1A). Moreover, compared to wild-type mice on a normal diet, wild-type mice fed with a high iron diet had considerably lower p53 protein levels (Figures 1D and 1E). Thus, iron overload in mice, due to either a genetic perturbation of iron metabolism or a high iron diet, correlates with a significant reduction in p53 protein levels. We next demonstrated that hemin treatment could downregulate endogenous p53 protein in both mouse primary hepatocytes and human hepatocarcinoma HepG2 cells in a hemin dose-dependent manner (Figures S1B and 2A ). Treatment with ferric ammonium citrate (FAC; 100 μg/ml, 6 hr) apparently also reduced p53 levels (Figure S1C). The p53 reduction seemed to be independent of transcription because hemin also decreased exogenously expressed p53 protein in the presence of the protein translation inhibitor cycloheximide (CHX) (Figure 2B). Furthermore, inhibition of heme biogenesis in HepG2 cells with succinylacetone, a 5-aminolevulinate synthase (ALAS) inhibitor, led to increased p53 (Figure 2C). This effect could be reversed by hemin, but not FAC, suggesting that hemin may directly downregulate endogenous p53 protein. Treatment of HepG2 cells with the iron chelator deferoxamine (DFO) significantly increased the steady-state level of endogenous p53 protein in a manner independent of protein synthesis (Figures 2D and 2E). The HepG2 cells were cultured in normal media that contained serum iron, and hemin could reverse the stabilizing effect of DFO treatment on endogenous p53 protein (Figure 2D). When the cells were adapted to serum- and iron-free media (VP-SFM), DFO itself could no longer stabilize endogenous p53 protein, but hemin treatment still efficiently destabilized p53 protein in a manner that could not be reversed by iron chelation (Figure 2F). This strongly suggests that hemin directly modulates the stability of endogenous p53 protein, without involving iron ions that may be released from heme-oxygenase-catabolized heme. A previous study has shown that DFO may promote p53 transcription through stabilizing hypoxia-inducible factor 1 α (HIF-1α) (An et al., 1998An W.G. Kanekal M. Simon M.C. Maltepe E. Blagosklonny M.V. Neckers L.M. Stabilization of wild-type p53 by hypoxia-inducible factor 1alpha.Nature. 1998; 392: 405-408Crossref PubMed Scopus (656) Google Scholar). To test if we are observing such a phenomena, we probed for p53 protein levels in human renal cancer 786-O cells that are null in both vHL (von Hippel-Lindau tumor suppressor) and HIF-1α (Kaelin, 2004Kaelin Jr., W.G. The von Hippel-Lindau tumor suppressor gene and kidney cancer.Clin. Cancer Res. 2004; 10: 6290S-6295SCrossref PubMed Scopus (264) Google Scholar) and found that hemin destabilized endogenous p53 protein and iron chelation stabilized p53 protein in a hemin treatment-reversible manner (Figure 2G). These data collectively suggested that the effect of hemin or iron chelation on the homeostasis of p53 protein involves posttranslational mechanisms that do not significantly involve the transcription factor HIF-1α. An examination of the p53 amino acid sequences across several species revealed that all of them bear three putative heme regulatory motifs (HRMs), consisting of Cys-Pro (CP) sequences that occur in a subset of heme-binding proteins (Zhang and Guarente, 1995Zhang L. Guarente L. Heme binds to a short sequence that serves a regulatory function in diverse proteins.EMBO J. 1995; 14: 313-320Crossref PubMed Scopus (255) Google Scholar) (Figures 3A and 3B ). A TMB assay verified that the His6-tagged p53 protein, freshly purified from bacteria, contained heme (Figure 3C), whereas a similarly tagged human Ub did not (data not shown). Matrix-assisted laser desorption-ionization (MALDI) mass spectra analysis further confirmed that free heme (m/z 616 ± 2 Da) was associated with freshly purified p53 protein (Figure S2A). In addition, hemin immobilized on agarose beads, but not agarose beads alone, recovered endogenous mouse and human p53 proteins from multiple cell types (Figure S2C). However, p53 protein fractions lost heme during dialysis (Figure S2E), suggesting that heme might associate with p53 noncovalently. p53 proteins, as well as ATE1, bound to heme with a dissociation constant (KD) in the low micromolar range and lost their associated heme at a rate faster than BSA, whose affinity to heme is at nanomolar range (Figure S2E). Under the same conditions, lysozyme, which does not bind to heme, readily lost heme even more quickly (Figure S2E). The rates of heme loss from the heme-binding proteins were thus conversely related to how tightly the proteins might bind to heme. We next incubated heme with purified human p53 protein (see Figure 3D for purity of the protein) to reconstitute the heme-p53 complex in vitro, using gel filtration in size-exclusion chromatography (SEC) to remove unbound heme. UV-visible (UV-Vis) spectra analysis of the recovered heme-p53 complexes manifested a Soret peak at 413 nm, diagnostic of heme-protein binding (Ponka, 1999Ponka P. Cell biology of heme.Am. J. Med. Sci. 1999; 318: 241-256Crossref PubMed Google Scholar) (Figure 3D). The TMB assay again confirmed the presence of heme in the post-SEC heme-p53 complexes. A tryptophan fluorescence quenching assay further indicated that the p53 bound to heme at a 1:1 molecular ratio, with a KD of ∼1.20 μM (Figure 3E), suggesting a fairly strong interaction between heme and p53 protein. Consistent results were obtained by surface plasmon resonance analysis (Figure S2D). A previous study has demonstrated that hemoglobin, NPAS2, E75, or other heme-binding proteins gain gas-sensing properties only upon complexing with heme (Reinking et al., 2005Reinking J. Lam M.M. Pardee K. Sampson H.M. Liu S. Yang P. Williams S. White W. Lajoie G. Edwards A. Krause H.M. The Drosophila nuclear receptor e75 contains heme and is gas responsive.Cell. 2005; 122: 195-207Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). To test whether heme-p53 complexes also respond to gas, the solution containing the preformed hemin-p53 complex was degassed and Argon purged to maximally remove dissolved oxygen (O2). Subsequent addition of dithionite solution and carbon monoxide (CO) infusion led to substantial blue shifts in the UV-Vis spectra: with a λmax of Soret peak I shifting from 423 to 414 nm and a λmax of Soret peak II from 475 to 535 nm (Figure 3F). Interestingly, nitric oxide (NO) infusion induced distinct changes in the UV spectra of p53-heme, whereas O2 infusion caused little or no change. These data suggest that the heme-binding property of p53 protein might confer gas responsiveness to p53, with specificity comparable to that observed with many other heme-binding proteins (Dioum et al., 2002Dioum E.M. Rutter J. Tuckerman J.R. Gonzalez G. Gilles-Gonzalez M.A. McKnight S.L. NPAS2: a gas-responsive transcription factor.Science. 2002; 298: 2385-2387Crossref PubMed Scopus (381) Google Scholar, Reinking et al., 2005Reinking J. Lam M.M. Pardee K. Sampson H.M. Liu S. Yang P. Williams S. White W. Lajoie G. Edwards A. Krause H.M. The Drosophila nuclear receptor e75 contains heme and is gas responsive.Cell. 2005; 122: 195-207Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). To further dissect the interaction between p53 and heme, we examined the three HRM-like CP motifs that are conserved in p53 (Figures 3A and 3B). Among them, the C-terminal CP motif (Cys277Pro278 in human p53) resides within a redox-sensitive CXC motif at the C terminus of the highly conserved DNA-binding domain (DBD) of p53 protein (Cho et al., 1994Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations.Science. 1994; 265: 346-355Crossref PubMed Scopus (2158) Google Scholar, Friedman et al., 1993Friedman P.N. Chen X. Bargonetti J. Prives C. The p53 protein is an unusually shaped tetramer that binds directly to DNA.Proc. Natl. Acad. Sci. USA. 1993; 90: 3319-3323Crossref PubMed Scopus (224) Google Scholar, Hainaut and Milner, 1993Hainaut P. Milner J. Redox modulation of p53 conformation and sequence-specific DNA binding in vitro.Cancer Res. 1993; 53: 4469-4473PubMed Google Scholar) (Figure 3B). We performed alanine (Ala) substitutions at Cys141-Pro142, Cys176-Pro177, and/or Cys277-Pro278 to generate single or joint mutants of the CP motifs in p53. Both the TMB assay and UV spectra analyses of postgel filtration heme-protein complexes found no significant differences between the wild-type and mutant p53s, regardless of single or joint mutation in any of the three CP motifs (data not shown). Interestingly, Ala substitution of the Cys residues in the C275AC277P stretch manifested in substantially lower heme affinity than wild-type p53 (Figures 4A and 4B ). A fluorescence quenching assay further indicated that the corresponding KD value for p53C275, 277A-heme binding rose to ∼15.7 μM, over 10-fold higher than that of wild-type p53 (KD ∼1.2 μM) (Figure 3E). Meanwhile, mass spectra analysis showed that little or no heme could be detected in association with p53C275, 277A, which was expressed and freshly recovered from E. coli (Figure S2B). This again suggested a weaker binding of the p53C275, 277A mutant to heme that is naturally available in E. coli culture. Altogether, we have identified Cys275 and Cys277 in human p53 protein as two key residues required for p53-heme interaction. Interestingly, sporadic mutations at Cys275 and Cys277 were found in human patients with cancer (Frebourg et al., 1995Frebourg T. Barbier N. Yan Y.X. Garber J.E. Dreyfus M. Fraumeni Jr., J. Li F.P. Friend S.H. Germ-line p53 mutations in 15 families with Li-Fraumeni syndrome.Am. J. Hum. Genet. 1995; 56: 608-615PubMed Google Scholar) and caused a loss of expression of a p53 transcriptional reporter (Figure S3). We next sought to investigate whether heme-binding would directly affect p53-DNA interactions by assaying the impact of hemin on an electrophoretic mobility shift assay (EMSA) of recombinant p53 protein and double-stranded DNA probes containing the consensus p53-responsive element (p53RE) (Jayaraman and Prives, 1995Jayaraman J. Prives C. Activation of p53 sequence-specific DNA binding by short single strands of DNA requires the p53 C-terminus.Cell. 1995; 81: 1021-1029Abstract Full Text PDF PubMed Scopus (353) Google Scholar). Hemin interfered with p53-p53RE interactions in a dose-dependent manner, with 20 μM hemin almost completely abolishing the gel shift (Figure 4D). Hemin, when applied at 10 μM for 6 hr, furthermore appeared to significantly interfere with p53-mediated transcription of luciferase reporters under the control of p21 or Bax promoter-derived p53RE sequences (Figure 4E). Immunoblotting indicated that hemin treatment also led to a reduced expression of endogenous p21 and Bax proteins in a p53-dependent manner (Figure 6B), showing that heme-p53 interactions may potentially play a biological role. Because we found that the homeostatic level of endogenous p53 protein decreased in a hemin dose-dependent manner (Figures 2A and S1B), we further investigated the potential functional consequences of the heme-p53 interaction in a CHX-chase experiment to examine the stability of endogenous p53 in cells with or without hemin treatment. To this end, the hepatoma cell line HepG2 was first adapted to growth in VP-SFM (8 hr) and subsequently treated with hemin at concentrations comparable to that in previous work (Hu et al., 2008Hu R.G. Wang H. Xia Z. Varshavsky A. The N-end rule pathway is a sensor of heme.Proc. Natl. Acad. Sci. USA. 2008; 105: 76-81Crossref PubMed Scopus (93) Google Scholar, Wu et al., 2009Wu N. Yin L. Hanniman E.A. Joshi S. Lazar M.A. Negative feedback maintenance of heme homeostasis by its receptor, Rev-erbalpha.Genes Dev. 2009; 23: 2201-2209Crossref PubMed Scopus (94) Google Scholar). In a 3 hr time course experiment, the p53 protein half-life in hemin-treated cells was about 2-fold shorter than that in untreated controls, indicating that hemin treatment accelerated the degradation of p53 in cells (Figures 5A and S4). In addition, hemin-triggered destabilization of p53 protein was concurrent with increased p53 ubiquitylation and could be efficiently blocked by the proteasome inhibitor MG132, but not by the autophagy inhibitor bafilomycin A (BAF), suggesting that hemin-induced p53 degradation occurs mainly through the Ub-proteasome system (Figures 5B and S4). Subcellular localization of p53 protein is critical for regulating its stability because degradation of p53 protein largely takes place in the cytosol (Brooks and Gu, 2011Brooks C.L. Gu W. p53 regulation by ubiquitin.FEBS Lett. 2011; 585: 2803-2809Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, Liu et al., 2011Liu J. Xia H. Kim M. Xu L. Li Y. Zhang L. Cai Y. Norberg H.V. Zhang T. Furuya T. et al.Beclin1 controls the levels of p53 by regulating the deubiquitination activity of U" @default.
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