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Ferritin is a ubiquitous intracellular iron storage protein that consists of 24 subunits of the H and L type. The ability to sequester iron from participation in oxygen free radical formation is consistent with a cytoprotective role for ferritin. Here we demonstrate that ferritins H and L are induced in cells treated with ␤-napthoflavone (␤-NF) and chemopreventive dithiolethiones. Induction of ferritin H by ␤-NF and the dithiolethiones oltipraz and 1,2-dithiole-3-thione (D3T) occurs via a transcriptional mechanism that is mediated by the ferritin H electrophile/antioxidant-responsive element (EpRE/ARE). The murine ferritin H gene contains five potential xenobiotic-responsive element (XRE) sequences in its 5-promoter region. However, deletion analysis demonstrates that these XRE sequences are not functional in inducing ferritin H in response to ␤-NF.Electrophoretic mobility shift assays demonstrate that the ferritin H EpRE/ARE binds Nrf2. Transfection of chimeric ferritin H reporter genes with Nrf2 expression vectors and Nrf2 dominant-negative mutants indicate that Nrf2 functions at the EpRE/ARE to mediate transcriptional activation of ferritin H. Induction of ferritin H and L was not seen in Nrf2 knockout cells, demonstrating that this transcription factor is required for the induction of ferritin in response to polycyclic aromatic xenobiotics and chemopreventive agents. Nrf2 may also play a role in basal transcription of both ferritin H and L. These results provide a mechanistic link between regulation of the iron storage protein ferritin and the cancer chemopreventive response.

Genotoxic stress triggers a rapid translocation of p53 to the mitochondria, contributing to apoptosis in a transcription-independent manner. Using immunopurification protocols and mass spectrometry, we previously identified the proapoptotic protein BAK as a mitochondrial p53-binding protein and showed that recombinant p53 directly binds to BAK and can induce its oligomerization, leading to cytochrome c release. In this work we describe a combination of molecular modeling, electrostatic analysis, and site-directed mutagenesis to define contact residues between BAK and p53. Our data indicate that three regions within the core DNA binding domain of p53 make contact with BAK; these are the conserved H2 ␣-helix and the L1 and L3 loop. Notably, point mutations in these regions markedly impair the ability of p53 to oligomerize BAK and to induce transcription-independent cell death. We present a model whereby positively charged residues within the H2 helix and L1 loop of p53 interact with an electronegative domain on the N-terminal ␣-helix of BAK; the latter is known to undergo conformational changes upon BAK activation. We show that mutation of acidic residues in the N-terminal helix impair the ability of BAK to bind to p53. Interestingly, many of the p53 contact residues predicted by our model are also direct DNA contact residues, suggesting that p53 interacts with BAK in a manner analogous to DNA. The combined data point to the H2 helix and L1 and L3 loops of p53 as novel functional domains contributing to transcription-independent apoptosis by this tumor suppressor protein.The tumor suppressor p53 is vital in maintaining cellular genomic integrity and controlled cell growth (1, 2). The best understood function of p53 is as a sequence-specific DNAbinding protein and transcription factor. Although the mechanism for p53-mediated cell cycle arrest is thought to be entirely dependent on the transactivation function of p53 (3-5), the role of p53 in apoptosis induction is considerably more complex.Transactivation of pro-apoptotic genes (such as the Bcl-2 family members BAX, Puma, and Noxa) and trans-repression of anti-apoptotic genes (such as survivin and Bcl-2) clearly contribute to the apoptotic response (6 -10). Additionally, p53 has an apoptotic activity that is independent of its transcriptional functions (11-13). In response to an oncogenic or genotoxic stimulus, this protein rapidly translocates to mitochondria, inducing cytochrome c release from this organelle and ensuing cell death (14 -18). Targeting of p53 exclusively to the mitochondrial compartment is sufficient to induce apoptosis (15, 18) and renders p53 capable of functioning efficiently as a tumor suppressor (19,20).Previous studies from our laboratory indicated that a single amino acid change in human p53 resulting from a polymorphism at codon 72 markedly alters the apoptotic potential of this protein along with its ability to localize to mitochondria (18). This result prompted us to use affinity chromatography and mass spectrometry to identify p53-intera...

In addition to a well-defined transcriptional activity that is necessary for efficient apoptosis induction, the p53 tumor suppressor also has a direct apoptogenic role at the mitochondria. This direct role in cell death is mediated at least in part by interaction of p53 with BCL2 family members, including the pro-apoptotic protein BAK. Whereas it is currently accepted that the mitochondrial function of p53 contributes to its tumor suppressive role, the regulation of p53 function at this organelle is poorly understood. In this manuscript we examine the role of p53 oligomerization in the regulation of its pro-apoptotic function at the mitochondria, specifically in regard to its ability to induce BAK oligomerization. We find that deletion or mutation of p53's oligomerization domain markedly impairs the ability of this protein to oligomerize BAK. Along these lines, cross-linking studies indicate that the majority of p53 localized to mitochondria is in dimeric or higher-order oligomeric form. In support of the importance of the p53-BAK interaction in the localization of p53 to mitochondria, we find that mouse embryo fibroblasts from the BAK null mouse have greatly reduced mitochondrial p53 compared to wild type fibroblasts. These data indicate that pro-apoptotic BAK, unlike other BCL2 family members, may serve as a major receptor for p53 on the mitochondria.

Highlights d Crystal structure of the malaria parasite lipocalin d Comparative analysis of lipocalin superfamily members in alveolate genomes d Localization of PfLipocalin to the parasitophorous vacuole and food vacuole d Reverse genetics reveal PfLipocalin function in oxidative damage control