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Iron metabolism regulates p53 signaling through direct heme-p53 interaction and modulation of p53 localization, stability, and function.

Shen J, Sheng X, Chang Z, Wu Q, Wang S, Xuan Z, Li D, Wu Y, Shang Y, Kong X, Yu L, Li L, Ruan K, Hu H, Huang Y, Hui L, Xie D, Wang F, Hu R - Cell Rep (2014)

Bottom Line: Iron excess is closely associated with tumorigenesis in multiple types of human cancers, with underlying mechanisms yet unclear.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular Biology, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China; University of Chinese Academy of Sciences, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China.

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Tumor Suppressor p53 Protein Directly Interacts with Heme(A) Schematic domain structure of human p53 protein, with putative HRMs marked by their amino acid sequences. TAD, transcription activation domain; TET, tetramerization domain.(B) Sequence alignment of the CACP motif in p53 proteins across species.(C) Gel filtration of p53 protein. BSA was used as a positive control of heme binding and lysozyme as the negative control.(D) UV-Vis spectrum of heme-p53 complexes (after gel filtration). Inset shows the purity of the p53 protein.(E) A tryptophan fluorescence quenching assay of hemin with wild-type and mutant p53 determined that heme associates with wild-type human p53 protein at a KD of ~1.20 μM and with p53C275,277A at a KD of ~15.70 μM.(F) UV-Vis spectra analyses were performed with p53-heme complexes before (red line) and after infusion of CO. Arrow indicates the spectral shift upon infusion of increasing CO until saturation (cyan line). Inset panel zooms in on the 450–625 nm range.
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Figure 3: Tumor Suppressor p53 Protein Directly Interacts with Heme(A) Schematic domain structure of human p53 protein, with putative HRMs marked by their amino acid sequences. TAD, transcription activation domain; TET, tetramerization domain.(B) Sequence alignment of the CACP motif in p53 proteins across species.(C) Gel filtration of p53 protein. BSA was used as a positive control of heme binding and lysozyme as the negative control.(D) UV-Vis spectrum of heme-p53 complexes (after gel filtration). Inset shows the purity of the p53 protein.(E) A tryptophan fluorescence quenching assay of hemin with wild-type and mutant p53 determined that heme associates with wild-type human p53 protein at a KD of ~1.20 μM and with p53C275,277A at a KD of ~15.70 μM.(F) UV-Vis spectra analyses were performed with p53-heme complexes before (red line) and after infusion of CO. Arrow indicates the spectral shift upon infusion of increasing CO until saturation (cyan line). Inset panel zooms in on the 450–625 nm range.

Mentions: 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, 1995) (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 non-covalently. 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.


Iron metabolism regulates p53 signaling through direct heme-p53 interaction and modulation of p53 localization, stability, and function.

Shen J, Sheng X, Chang Z, Wu Q, Wang S, Xuan Z, Li D, Wu Y, Shang Y, Kong X, Yu L, Li L, Ruan K, Hu H, Huang Y, Hui L, Xie D, Wang F, Hu R - Cell Rep (2014)

Tumor Suppressor p53 Protein Directly Interacts with Heme(A) Schematic domain structure of human p53 protein, with putative HRMs marked by their amino acid sequences. TAD, transcription activation domain; TET, tetramerization domain.(B) Sequence alignment of the CACP motif in p53 proteins across species.(C) Gel filtration of p53 protein. BSA was used as a positive control of heme binding and lysozyme as the negative control.(D) UV-Vis spectrum of heme-p53 complexes (after gel filtration). Inset shows the purity of the p53 protein.(E) A tryptophan fluorescence quenching assay of hemin with wild-type and mutant p53 determined that heme associates with wild-type human p53 protein at a KD of ~1.20 μM and with p53C275,277A at a KD of ~15.70 μM.(F) UV-Vis spectra analyses were performed with p53-heme complexes before (red line) and after infusion of CO. Arrow indicates the spectral shift upon infusion of increasing CO until saturation (cyan line). Inset panel zooms in on the 450–625 nm range.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4219651&req=5

Figure 3: Tumor Suppressor p53 Protein Directly Interacts with Heme(A) Schematic domain structure of human p53 protein, with putative HRMs marked by their amino acid sequences. TAD, transcription activation domain; TET, tetramerization domain.(B) Sequence alignment of the CACP motif in p53 proteins across species.(C) Gel filtration of p53 protein. BSA was used as a positive control of heme binding and lysozyme as the negative control.(D) UV-Vis spectrum of heme-p53 complexes (after gel filtration). Inset shows the purity of the p53 protein.(E) A tryptophan fluorescence quenching assay of hemin with wild-type and mutant p53 determined that heme associates with wild-type human p53 protein at a KD of ~1.20 μM and with p53C275,277A at a KD of ~15.70 μM.(F) UV-Vis spectra analyses were performed with p53-heme complexes before (red line) and after infusion of CO. Arrow indicates the spectral shift upon infusion of increasing CO until saturation (cyan line). Inset panel zooms in on the 450–625 nm range.
Mentions: 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, 1995) (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 non-covalently. 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.

Bottom Line: Iron excess is closely associated with tumorigenesis in multiple types of human cancers, with underlying mechanisms yet unclear.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular Biology, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China; University of Chinese Academy of Sciences, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China.

Show MeSH
Related in: MedlinePlus