Regulation of iron homeostasis mediated by the heme-binding protein Dap1 (damage resistance protein 1) via the P450 protein Erg11/Cyp51.

Fungal infections arise frequently in immunocompromised patients, and sterol synthesis is a primary pathway targeted by antifungal drugs. In particular, the P450 protein Erg11/Cyp51 catalyzes a critical step in ergosterol synthesis, and the azole class of antifungal drugs inhibits Erg11. Dap1 is a heme-binding protein related to cytochrome b5 that activates Erg11, so that cells lacking Dap1 accumulate the Erg11 substrate and are hypersensitive to Erg11 inhibitors. Heme binding by Dap1 is crucial for its function, and point mutants in its heme-binding domain render Dap1 inactive for sterol biosynthesis and DNA damage resistance. Like Dap1, the human homologue, PGRMC1/Hpr6, also regulates sterol synthesis and DNA damage resistance. In the present study, we demonstrate that the Dap1 heme-1 domain is required for growth under conditions of low iron availability. Loss of Dap1 is suppressed by elevated levels of Erg11 but not by increased heme biosynthesis. Dap1 localizes to punctate cytoplasmic structures that co-fractionate with endosomes, and Dap1 contributes to the integrity of the vacuole. The results suggest that Saccharomyces cerevisiae Dap1 stimulates a P450-catalyzed step in sterol synthesis via a distinct localization from its homologues in Schizosaccharomyces pombe and mammals and that this function regulates iron metabolism.

A novel group of genes regulates susceptibility to antineoplastic drugs in highly tumorigenic breast cancer cells.

Doxorubicin is an anthracycline antibiotic used for cancer chemotherapy. The utility of doxorubicin is limited by its inability to kill all of the cells within a tumor and by resistant cells emerging from the treated population. We have screened for genes that regulate doxorubicin susceptibility in highly tumorigenic breast cancer cells by cDNA microarray and RNA interference (RNAi) analysis, and we have identified genes associated with both proliferation and cell cycle arrest after doxorubicin treatment. We confirmed that MDA-MB-231 cells treated with doxorubicin induce the expression of carbonic anhydrase II (CAII), inhibitor of differentiation/DNA binding 2 (Id2), activating transcription factor 3 (Atf3), and the phosphatidylinositol 3-kinase 55-kDa regulatory subunit p55PIK. These genes were induced at different times and with varying specificities to different chemotherapeutic drugs. In addition to being induced at the transcriptional level, the CAII and clusterin proteins were elevated after doxorubicin treatment. CAII, Id2, p55PIK, and clusterin were not altered by doxorubicin in MCF-7 cells, a weakly tumorigenic cell line used in previous studies of doxorubicin-regulated gene expression. By inhibiting gene expression using RNAi, we found that CAII and clusterin increase cell survival after doxorubicin treatment, whereas Id2 increases susceptibility to doxorubicin. Our results support a model in which highly tumorigenic breast cancer cells induce a transcriptional response to doxorubicin that is distinct from less malignant cells. The induced genes regulate drug susceptibility positively and negatively and may be novel targets for therapeutic intervention.

Dap1p, a heme-binding protein that regulates the cytochrome P450 protein Erg11p/Cyp51p in Saccharomyces cerevisiae.

Alkylating agents chemically modify DNA and cause mutations that lead to cancer. In the budding yeast Saccharomyces cerevisiae, resistance to the alkylating agent methyl methanesulfonate (MMS) is mediated in part by Dap1p (damage resistance protein 1). Dap1p is related to cytochrome b5, which activates cytochrome P450 proteins, elevating the metabolism of lipids and xenobiotic compounds. We have found that Dap1p, like cytochrome b5, binds to heme and that Dap1p targets the cytochrome P450 protein Erg11p/Cyp51p. Genetic analysis indicates that Erg11p acts downstream of Dap1p. Furthermore, Dap1p regulates the stability of Erg11p, and Erg11p is stabilized in dap1Delta cells by the addition of heme. Thus, Dap1p utilizes heme to stabilize Erg11p, which in turn regulates ergosterol synthesis and MMS resistance. Dap1p homologues have been identified in numerous eukaryotes, including mammals, suggesting that the Dap1p-cytochrome P450 protein pathway is broadly conserved in eukaryotic species.

Amino acid changes in Xrs2p, Dun1p, and Rfa2p that remove the preferred targets of the ATM family of protein kinases do not affect DNA repair or telomere length in Saccharomyces cerevisiae.

In eukaryotes, mutations in a number of genes that affect DNA damage checkpoints or DNA replication also affect telomere length [Curr. Opin. Cell Biol. 13 (2001) 281]. Saccharomyces cerevisae strains with mutations in the TEL1 gene (encoding an ATM-like protein kinase) have very short telomeres, as do strains with mutations in XRS2, RAD50, or MRE11 (encoding members of a trimeric complex). Xrs2p and Mre11p are phosphorylated in a Tel1p-dependent manner in response to DNA damage [Genes Dev. 15 (2001) 2238; Mol. Cell 7 (2001) 1255]. We found that Xrs2p, but not Mre11p or Rad50p, is efficiently phosphorylated in vitro by immunopreciptated Tel1p. Strains with mutations eliminating all SQ and TQ motifs in Xrs2p (preferred targets of the ATM kinase family) had wild-type length telomeres and wild-type sensitivity to DNA damaging agents. We also showed that Rfa2p (a subunit of RPA) and the Dun1p checkpoint kinase, which are required for DNA damage repair and which are phosphorylated in response to DNA damage in vivo, are in vitro substrates of the Tel1p and Mec1p kinases. In addition, Dun1p substrates with no SQ or TQ motifs are phosphorylated by Mec1p in vitro very inefficiently, but retain most of their ability to be phosphorylated by Tel1p. We demonstrated that null alleles of DUN1 and certain mutant alleles of RFA2 result in short telomeres. As observed with Xrs2p, however, strains with mutations of DUN1 or RFA2 that eliminate SQ motifs have no effect on telomere length or DNA damage sensitivity.