BRCA1 deficiency enhances the aggressiveness of breast cancer cells expressing HPV16 oncoproteins.

BACKGROUND INFORMATION: The precise etiology of breast cancer is not completely understood, although women with BRCA1 gene mutations have a significantly increased risk of developing the disease. In addition, sporadic breast cancer is frequently associated with decreased BRCA1 gene expression. Growing evidence of Human papillomaviruses (HPVs) infections in breast tumors has raised the possibility of the involvement of HPVs in the pathogenesis of breast cancer. We investigated whether the effects of HPV oncoproteins E6 and E7 were influenced by the expression levels of BRCA1. HPV16E6E7 (prototype or E6D25E/E7N29S Asian variant type) were stably expressed in MDA-MB231 breast cancer cells, wild type for BRCA1, or with BRCA1 knocked down. RESULTS: Expression of HPV16E6E7 oncogenes did not affect BRCA1 levels and the abundance of HPV16E6E7 was not altered by BRCA1 knockdown. BRCA1 levels did not alter HPV16E6E7-dependent degradation of G1-S cell cycle proteins p53 and pRb. However, we found that the expression of G2-M cell cycle protein cyclin B1 enhanced by HPV16E6E7 was impacted by BRCA1 levels. Especially, we found the correlation between BRCA1 and cyclin B1 expression and this was also confirmed in breast cancer samples from a Thai cohort. We further demonstrated that the combination of HPV oncoproteins and low levels of BRCA1 protein appears to enhance proliferation and invasion. Transactivation activities of HPV16E6E7 on genes regulating cell proliferation and invasion (TGF-ß and vimentin) were significantly increased in BRCA1-deficient cells. CONCLUSIONS: Our results indicate that a deficiency of BRCA1 promotes the transactivation activity of HPV16E6E7 leading to increase of cell proliferation and invasion. SIGNIFICANCE: HPV infection appears to have the potential to enhance the aggressiveness of breast cancers, especially those deficient in BRCA1.

Chemical-Genetic Interactions of Bacopa monnieri Constituents in Cells Deficient for the DNA Repair Endonuclease RAD1 Appear Linked to Vacuolar Disruption.

Colorectal cancer is a common cancer worldwide and reduced expression of the DNA repair endonuclease XPF (xeroderma pigmentosum complementation group F) is associated with colorectal cancer. Bacopa monnieri extracts were previously found to exhibit chemical-genetic synthetic lethal effects in a Saccharomyces cerevisiae model of colorectal cancer lacking Rad1p, a structural and functional homologue of human XPF. However, the mechanisms for B. monnieri extracts to limit proliferation and promote an apoptosis-like event in RAD1 deleted yeast was not elucidated. Our current analysis has revealed that B. monnieri extracts have the capacity to promote mutations in rad1∆ cells. In addition, the effects of B. monnieri extracts on rad1∆ yeast is linked to disruption of the vacuole, similar to the mammalian lysosome. The absence of RAD1 in yeast sensitizes cells to the effects of vacuole disruption and the release of proteases. The combined effect of increased DNA mutations and release of vacuolar contents appears to induce an apoptosis-like event that is dependent on the meta-caspase Yca1p. The toxicity of B. monnieri extracts is linked to sterol content, suggesting saponins may be involved in limiting the proliferation of yeast cells. Analysis of major constituents from B. monnieri identified a chemical-genetic interaction between bacopasaponin C and rad1∆ yeast. Bacopasaponin C may have potential as a drug candidate or serve as a model for the development of analogs for the treatment of colorectal cancer.

Disruption in iron homeostasis and impaired activity of iron-sulfur cluster containing proteins in the yeast model of Shwachman-Diamond syndrome.

BACKGROUND: Shwachman-Diamond syndrome (SDS) is a congenital disease that affects the bone marrow, skeletal system, and pancreas. The majority of patients with SDS have mutations in the SBDS gene, involved in ribosome biogenesis as well as other processes. A Saccharomyces cerevisiae model of SDS, lacking Sdo1p the yeast orthologue of SBDS, was utilized to better understand the molecular pathogenesis in the development of this disease. RESULTS: Deletion of SDO1 resulted in a three-fold over-accumulation of intracellular iron. Phenotypes associated with impaired iron-sulfur (ISC) assembly, up-regulation of the high affinity iron uptake pathway, and reduced activities of ISC containing enzymes aconitase and succinate dehydrogenase, were observed in sdo1∆ yeast. In cells lacking Sdo1p, elevated levels of reactive oxygen species (ROS) and protein oxidation were reduced with iron chelation, using a cell impermeable iron chelator. In addition, the low activity of manganese superoxide dismutase (Sod2p) seen in sdo1∆ cells was improved with iron chelation, consistent with the presence of reactive iron from the ISC assembly pathway. In yeast lacking Sdo1p, the mitochondrial voltage-dependent anion channel (VDAC) Por1p is over-expressed and its deletion limits iron accumulation and increases activity of aconitase and succinate dehydrogenase. CONCLUSIONS: We propose that oxidative stress from POR1 over-expression, resulting in impaired activity of ISC containing proteins and disruptions in iron homeostasis, may play a role in disease pathogenesis in SDS patients.

Possible Role of the Ca2+/Mn2+ P-Type ATPase Pmr1p on Artemisinin Toxicity through an Induction of Intracellular Oxidative Stress.

Artemisinins are widely used to treat Plasmodium infections due to their high clinical efficacy; however, the antimalarial mechanism of artemisinin remains unresolved. Mutations in P. falciparum ATPase6 (PfATP6), a sarcoplasmic endoplasmic reticulum Ca2+-transporting ATPase, are associated with increased tolerance to artemisinin. We utilized Saccharomyces cerevisiae as a model to examine the involvement of Pmr1p, a functional homolog of PfATP6, on the toxicity of artemisinin. Our analysis demonstrated that cells lacking Pmr1p are less susceptible to growth inhibition from artemisinin and its derivatives. No association between sensitivity to artemisinin and altered trafficking of the drug efflux pump Pdr5p, calcium homeostasis, or protein glycosylation was found in pmr1∆ yeast. Basal ROS levels are elevated in pmr1∆ yeast and artemisinin exposure does not enhance ROS accumulation. This is in contrast to WT cells that exhibit a significant increase in ROS production following treatment with artemisinin. Yeast deleted for PMR1 are known to accumulate excess manganese ions that can function as ROS-scavenging molecules, but no correlation between manganese content and artemisinin resistance was observed. We propose that loss of function mutations in Pmr1p in yeast cells and PfATP6 in P. falciparum are protective against artemisinin toxicity due to reduced intracellular oxidative damage.

Decreased accumulation of superoxide dismutase 2 within mitochondria in the yeast model of Shwachman-Diamond syndrome.

Mutations in the human SBDS gene is the most common cause of Shwachman-Diamond syndrome (SDS). The SBDS protein participates in ribosome biogenesis; however, effects beyond reduced translation efficiency are thought to be involved in SDS progression. Impaired mitochondrial function has been reported for cells lacking either SBDS or Sdo1p, the Saccharomyces cerevisiae SBDS ortholog. To better understand how the loss of SBDS/Sdo1p leads to mitochondria damage, we utilized the S. cerevisiae model of SDS. Yeast deleted for SDO1 show increased oxidative damage to mitochondrial proteins and a marked decrease in protein levels and activity of mitochondrial superoxide dismutase 2 (Sod2p), a key enzyme involved in defense against oxidants. Immature forms of Sod2p are observed in sdo1∆ cells suggesting a defect in proteolysis of the presequence. Yeast deleted for CYM1, encoding a presequence protease, display a similar reduction in Sod2p activity as sdo1∆ cells, as well as elevated oxidative damage, to mitochondrial proteins. Sod2p protein levels and activity are largely restored in a por1∆ sdo1∆ strain, lacking the major mitochondrial voltage-dependent anion channel. Together these results indicate that mitochondrial insufficiency in sdo1∆ cells may be linked to the accumulation of immature presequence containing proteins and this effect is a consequence, at least in part, from loss of counter-regulation of Por1p by Sdo1p.

Interrogation of ethnomedicinal plants for synthetic lethality effects in combination with deficiency in the DNA repair endonuclease RAD1 using a yeast cell-based assay.

ETHNOPHARMACOLOGICAL RELEVANCE: Plant materials used in this study were selected based on the ethnobotanical literature. Plants have either been utilized by Thai practitioners as alternative treatments for cancer or identified to exhibit anti-cancer properties. AIM OF THE STUDY: To screen ethnomedicinal plants using a yeast cell-based assay for synthetic lethal interactions with cells deleted for RAD1, the yeast homologue of human ERCC4 (XPF) MATERIALS AND METHODS: Ethanolic extracts from thirty-two species of medicinal plants utilized in Thai traditional medicine were screened for synthetic lethal/sick interactions using a yeast cell-based assay. Cell growth was compared between the parental strain and rad1∆ yeast following exposure to select for specific toxicity of plant extracts. Candidate extracts were further examined for the mode of action using genetic and biochemical approaches. RESULTS: Screening a library of ethanolic extracts from medicinal plants identified Bacopa monnieri and Colubrina asiatica as having synthetic lethal effects in the rad1∆ cells but not the parental strain. Synthetic lethal effects for B. monneiri extracts were more apparent and this plant was examined further. Genetic analysis indicates that pro-oxidant activities and defective excision repair pathways do not significantly contribute to enhanced sensitivity to B. monneiri extracts. Exposure to B. monneiri extracts resulted in nuclear fragmentation and elevated levels of ethidium bromide staining in rad1∆ yeast suggesting promotion of an apoptosis-like event. Growth inhibition also observed in the human Caco-2 cell line suggesting the effects of B. monnieri extracts on both yeast and human cells may be similar. CONCLUSIONS: B. monneiri extracts may have utility in treatment of colorectal cancers that exhibit deficiency in ERCC4 (XPF).

Deletion of Mitochondrial Porin Alleviates Stress Sensitivity in the Yeast Model of Shwachman-Diamond Syndrome.

Shwachman-Diamond syndrome (SDS) is a multi-system disorder characterized by bone marrow failure, pancreatic insufficiency, skeletal abnormalities, and increased risk of leukemic transformation. Most patients with SDS contain mutations in the Shwachman-Bodian-Diamond syndrome gene (SBDS), encoding a highly conserved protein that has been implicated in ribosome biogenesis. Emerging evidence also suggests a distinct role of SBDS beyond protein translation. Using the yeast model of SDS, we examined the underlying mechanisms that cause cells lacking Sdo1p, the yeast SBDS ortholog, to exhibit reduced tolerance to various stress conditions. Our analysis indicates that the environmental stress response (ESR), heat shock response (HSR), and endoplasmic reticulum unfolded protein response (UPR) of sdo1Δ cells are functional and that defects in these pathways do not produce the phenotypes observed in sdo1Δ yeast. Depletion of mitochondrial DNA (mtDNA) was observed in sdo1Δ cells, and this is a probable cause of the mitochondrial insufficiency in SDS. Prior disruption of POR1, encoding the mitochondrial voltage dependent anion channel (VDAC), abrogated the effects of SDO1 deletion and substantially restored resistance to environmental stressors and protected against damage to mtDNA. Conversely, wild-type cells over-expressing POR1 exhibited growth impairment and increased stress sensitivity similar to that seen in sdo1Δ cells. Overall, our results suggest that specific VDAC inhibitors may have therapeutic benefits for SDS patients.

Screening of SLC25A13 mutation in the Thai population.

AIM: To determine the prevalence of SLC25A13 mutations in the Thai population. METHODS: A total of 1537 subjects representing the Thai population were screened for a novel pathologic allele p.Met1? (c.2T > C) and six previously known common SLC25A13 mutations: [I] (c.851_854delGTAT), [II] (g.IVS11 + 1G > A), [III] (c.1638_1660dup), [IV] (p.S225X), [V] (IVS13 + 1G > A), and [XIX] (g.IVS16ins3kb) using a newly developed TaqMan and established HybProbe assay, respectively. Sanger sequencing was employed for specimens showing an aberrant peak to confirm the targeted mutation as well as the unknown aberrant peaks detected. Frequencies of the mutations identified were compared in each region. Carrier frequency and disease prevalence of citrin deficiency caused by SCL25A13 mutations were estimated. RESULTS: p.Met1? was identified in the heterozygous state in 85 individuals, giving a carrier frequency of 1/18, which suggests possible selective advantage of this variant. The question of p.Met1? homozygote lethality remains unanswered which may serve as an explanation as to why this homozygote has yet to be identified in patients/controls even with high allele frequency. The p.Met1? mutation has rarely been studied in populations other than Thai and Chinese; therefore, may have been overlooked. Development of the TaqMan assay in the present study would allow a simple, rapid, and cost-effective method for mass screening. Heterozygous mutations: [XIX] and [I] were identified in 17 individuals, giving a carrier rate of 1/90 and a calculated homozygote rate of 1/33000. Two novel variants, g.IVS11 + 17C > G and c.1311C > T, of unknown clinical significance were identified at low frequency. CONCLUSION: This study highlighted the current underestimation of citrin deficiency and suggests the possible selective advantage of the p.Met1? allele.

A novel giant peroxisomal superoxide dismutase motif-containing protein.

Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for neuronal cell death by oxidative stress. In this model, extracellular glutamate blocks cystine uptake via the glutamate/cystine antiporter system x(c)(), eventually leading to depletion of the antioxidant glutathione and cell death. We used subtractive suppression hybridization and a screening procedure using various HT22 sublines to identify transcripts relevantly upregulated in resistance to oxidative glutamate toxicity. One of these coded for a novel protein of 3440 amino acids comprising a superoxide dismutase (SOD) motif, which we named TIGR for "transcript increased in glutamate resistance." TIGR is mainly expressed in the nervous system in cortical pyramidal and hippocampal neurons. Intracellularly, TIGR colocalizes with catalase, strongly suggesting a peroxisomal localization. Overexpression of TIGR but not of a mutant lacking two conserved histidine residues in the SOD motif increased SOD activity and protected against oxidative stress in mammalian cells, but had no direct SOD activity in yeast. We conclude that this novel giant peroxisomal protein is implicated in resistance to oxidative stress. Despite the presence of a SOD motif, which is necessary for protection in mammalian cells, the protein is not a functional SOD, but might be involved in SOD activity.

The interaction of mitochondrial iron with manganese superoxide dismutase.

Superoxide dismutase 2 (SOD2) is one of the rare mitochondrial enzymes evolved to use manganese as a cofactor over the more abundant element iron. Although mitochondrial iron does not normally bind SOD2, iron will misincorporate into Saccharomyces cerevisiae Sod2p when cells are starved for manganese or when mitochondrial iron homeostasis is disrupted by mutations in yeast grx5, ssq1, and mtm1. We report here that such changes in mitochondrial manganese and iron similarly affect cofactor selection in a heterologously expressed Escherichia coli Mn-SOD, but not a highly homologous Fe-SOD. By x-ray absorption near edge structure and extended x-ray absorption fine structure analyses of isolated mitochondria, we find that misincorporation of iron into yeast Sod2p does not correlate with significant changes in the average oxidation state or coordination chemistry of bulk mitochondrial iron. Instead, small changes in mitochondrial iron are likely to promote iron-SOD2 interactions. Iron binds Sod2p in yeast mutants blocking late stages of iron-sulfur cluster biogenesis (grx5, ssq1, and atm1), but not in mutants defective in the upstream Isu proteins that serve as scaffolds for iron-sulfur biosynthesis. In fact, we observed a requirement for the Isu proteins in iron inactivation of yeast Sod2p. Sod2p activity was restored in mtm1 and grx5 mutants by depleting cells of Isu proteins or using a dominant negative Isu1p predicted to stabilize iron binding to Isu1p. In all cases where disruptions in iron homeostasis inactivated Sod2p, we observed an increase in mitochondrial Isu proteins. These studies indicate that the Isu proteins and the iron-sulfur pathway can donate iron to Sod2p.

The overlapping roles of manganese and Cu/Zn SOD in oxidative stress protection.

In various organisms, high intracellular manganese provides protection against oxidative damage through unknown pathways. Herein we use a genetic approach in Saccharomyces cerevisiae to analyze factors that promote manganese as an antioxidant in cells lacking Cu/Zn superoxide dismutase (sod1 Delta). Unlike certain bacterial systems, oxygen resistance in yeast correlates with high intracellular manganese without a lowering of iron. This manganese for antioxidant protection is provided by the Nramp transporters Smf1p and Smf2p, with Smf1p playing a major role. In fact, loss of manganese transport by Smf1p together with loss of the Pmr1p manganese pump is lethal to sod1 Delta cells despite normal manganese SOD2 activity. Manganese-phosphate complexes are excellent superoxide dismutase mimics in vitro, yet through genetic disruption of phosphate transport and storage, we observed no requirement for phosphate in manganese suppression of oxidative damage. If anything, elevated phosphate correlated with profound oxidative stress in sod1 Delta mutants. The efficacy of manganese as an antioxidant was drastically reduced in cells that hyperaccumulate phosphate without effects on Mn SOD activity. Non-SOD manganese can provide a critical backup for Cu/Zn SOD1, but only under appropriate physiologic conditions.

Instability of superoxide dismutase 1 of Drosophila in mutants deficient for its cognate copper chaperone.

Copper,zinc superoxide dismutase (SOD1) in mammals is activated principally via a copper chaperone (CCS) and to a lesser degree by a CCS-independent pathway of unknown nature. In this study, we have characterized the requirement for CCS in activating SOD1 from Drosophila. A CCS-null mutant (Ccs(n)(29)(E)) of Drosophila was created and found to phenotypically resemble Drosophila SOD1-null mutants in terms of reduced adult life span, hypersensitivity to oxidative stress, and loss of cytosolic aconitase activity. However, the phenotypes of CCS-null flies were less severe, consistent with some CCS-independent activation of Drosophila SOD1 (dSOD1). Yet SOD1 activity was not detectable in Ccs(n)(29)(E) flies, due largely to a striking loss of SOD1 protein. In contrast, human SOD1 expressed in CCS-null flies is robustly active and rescues the deficits in adult life span and sensitivity to oxidative stress. The dependence of dSOD1 on CCS was also observed in a yeast expression system where the dSOD1 polypeptide exhibited unusual instability in CCS-null (ccs1Delta) yeast. The residual dSOD1 polypeptide in ccs1Delta yeast was nevertheless active, consistent with CCS-independent activation. Stability of dSOD1 in ccs1Delta cells was readily restored by expression of either yeast or Drosophila CCS, and this required copper insertion into the enzyme. The yeast expression system also revealed some species specificity for CCS. Yeast SOD1 exhibits preference for yeast CCS over Drosophila CCS, whereas dSOD1 is fully activated with either CCS molecule. Such variation in mechanisms of copper activation of SOD1 could reflect evolutionary responses to unique oxygen and/or copper environments faced by divergent species.

Activation of CuZn superoxide dismutases from Caenorhabditis elegans does not require the copper chaperone CCS.

Reactive oxygen species are produced as the direct result of aerobic metabolism and can cause damage to DNA, proteins, and lipids. A principal defense against reactive oxygen species involves the superoxide dismutases (SOD) that act to detoxify superoxide anions. Activation of CuZn-SODs in eukaryotic cells occurs post-translationally and is generally dependent on the copper chaperone for SOD1 (CCS), which inserts the catalytic copper cofactor and catalyzes the oxidation of a conserved disulfide bond that is essential for activity. In contrast to other eukaryotes, the nematode Caenorhabditis elegans does not contain an obvious CCS homologue, and we have found that the C. elegans intracellular CuZn-SODs (wSOD-1 and wSOD-5) are not dependent on CCS for activation when expressed in Saccharomyces cerevisiae. CCS-independent activation of CuZn-SODs is not unique to C. elegans; however, this is the first organism identified that appears to exclusively use this alternative pathway. As was found for mammalian SOD1, wSOD-1 exhibits a requirement for reduced glutathione in CCS-independent activation. Unexpectedly, wSOD-1 was inactive even in the presence of CCS when glutathione was depleted. Our investigation of the cysteine residues that form the disulfide bond in wSOD-1 suggests that the ability of wSODs to readily form this disulfide bond may be the key to obtaining high levels of activation through the CCS-independent pathway. Overall, these studies demonstrate that the CuZn-SODs of C. elegans have uniquely evolved to acquire copper without the copper chaperone and this may reflect the lifestyle of this organism.

Manganese activation of superoxide dismutase 2 in the mitochondria of Saccharomyces cerevisiae.

Manganese-dependent superoxide dismutase 2 (SOD2) in the mitochondria plays a key role in protection against oxidative stress. Here we probed the pathway by which SOD2 acquires its manganese catalytic cofactor. We found that a mitochondrial localization is essential. A cytosolic version of Saccharomyces cerevisiae Sod2p is largely apo for manganese and is only efficiently activated when cells accumulate toxic levels of manganese. Furthermore, Candida albicans naturally produces a cytosolic manganese SOD (Ca SOD3), yet when expressed in the cytosol of S. cerevisiae, a large fraction of Ca SOD3 also remained manganese-deficient. The cytosol of S. cerevisae cannot readily support activation of Mn-SOD molecules. By monitoring the kinetics for metalation of S. cerevisiae Sod2p in vivo, we found that prefolded Sod2p in the mitochondria cannot be activated by manganese. Manganese insertion is only possible with a newly synthesized polypeptide. Furthermore, Sod2p synthesis appears closely coupled to Sod2p import. By reversibly blocking mitochondrial import in vivo, we noted that newly synthesized Sod2p can enter mitochondria but not a Sod2p polypeptide that was allowed to accumulate in the cytosol. We propose a model in which the insertion of manganese into eukaryotic SOD2 molecules is driven by the protein unfolding process associated with mitochondrial import.

Manganese toxicity and Saccharomyces cerevisiae Mam3p, a member of the ACDP (ancient conserved domain protein) family.

Manganese is an essential, but potentially toxic, trace metal in biological systems. Overexposure to manganese is known to cause neurological deficits in humans, but the pathways that lead to manganese toxicity are largely unknown. We have employed the bakers' yeast Saccharomyces cerevisiae as a model system to identify genes that contribute to manganese-related damage. In a genetic screen for yeast manganese-resistance mutants, we identified S. cerevisiae MAM3 as a gene which, when deleted, would increase cellular tolerance to toxic levels of manganese and also increased the cell's resistance towards cobalt and zinc. By sequence analysis, Mam3p shares strong similarity with the mammalian ACDP (ancient conserved domain protein) family of polypeptides. Mutations in human ACDP1 have been associated with urofacial (Ochoa) syndrome. However, the functions of eukaryotic ACDPs remain unknown. We show here that S. cerevisiae MAM3 encodes an integral membrane protein of the yeast vacuole whose expression levels directly correlate with the degree of manganese toxicity. Surprisingly, Mam3p contributes to manganese toxicity without any obvious changes in vacuolar accumulation of metals. Furthermore, through genetic epistasis studies, we demonstrate that MAM3 operates independently of the well-established manganese-trafficking pathways in yeast, involving the manganese transporters Pmr1p, Smf2p and Pho84p. This is the first report of a eukaryotic ACDP family protein involved in metal homoeostasis.

Mutations in Saccharomyces cerevisiae iron-sulfur cluster assembly genes and oxidative stress relevant to Cu,Zn superoxide dismutase.

Saccharomyces cerevisiae lacking Cu,Zn superoxide dismutase (SOD1) show several metabolic defects including aerobic blockages in methionine and lysine biosynthesis. We have previously shown that mutations in genes implicated in the formation of iron-sulfur clusters, designated seo (suppressors of endogenous oxidation), reverse the oxygen-dependent methionine and lysine auxotrophies of a sod1Delta strain. We now report the surprising finding that seo mutants do not reduce oxidative damage as shown by the lack of reduction of EPR-detectable "free" iron, which is characteristic of sod1Delta mutants. In fact, they exhibit increased oxidative damage as evidenced by increased accumulation of protein carbonyls. The seo class of mutants overaccumulates mitochondrial iron, and this iron accumulation is critical for suppression of the sod1Delta biosynthetic defects. Blocking overaccumulation of mitochondrial iron abolished the ability of the seo mutants to suppress the sod1Delta auxotrophies. By contrast, increasing the mitochondrial iron content of sod1Delta yeast using high copy MMT1, which encodes a mitochondrial iron transporter, was sufficient to mimic the seo mutants. Our studies indicated that suppression of the sod1Delta methionine auxotrophy was dependent on the pentose phosphate pathway, which is a major source of NADPH production. By comparison, the sod1Delta lysine auxotrophy appears to be reversed in the seo mutants by increased expression of genes in the lysine biosynthetic pathway, perhaps through sensing of mitochondrial damage by the retrograde response.

Characterization of human soluble high and low activity catechol-O-methyltransferase catalyzed catechol estrogen methylation.

The major detoxification pathway of the carcinogenic catechol estrogens is methylation by catechol- -methyltransferase (COMT). It has been hypothesized that the enzyme encoded by the low-activity allele (COMT(L) ) has a lower catalytic activity for catechol estrogen methylation than that encoded by the high activity allele (COMT(H) ). We expressed and purified human soluble (S)-COMT(H) and S-COMT(L) in and characterized the methylation of 2- and 4-hydroxyestradiol (2- and 4-OH-E2). There were no differences between the kinetic parameters for COMT(H) and COMT(L). The kinetic parameters for S-adenosylmethionine (SAM), the methyl donor in these reactions, also did not differ for COMT(H) and COMT(L). S-adenosylhomocysteine, the demethylated SAM metabolite, inhibited methylation of the catechol estrogens in a non-competitive manner similarly for COMT(H) and COMT(L). Each COMT substrate tested inhibited the methylation of other substrates in a mixed competitive and non-competitive fashion similarly for COMT(H) and COMT(L). Furthermore, in cytosolic fractions of COMT(HH)(MCF-10A and ZR-75-1) and COMT(LL)(MCF-7 and T47D) human breast epithelial cell lines, no differences were detected between the kinetic parameters of COMT with respect to 2- and 4-OH-E2 methylation; nor were COMT protein levels associated with the COMT genotype. These data suggest that the decreased COMT enzymatic activity that has been detected in human tissue in association with the COMT(L) allele is not reflected by differences in the affinity or capacity of COMT(H) and COMT(L) for catechol estrogen methylation. These results raise the question of what accounts for the difference in COMT activity associated with the COMT(HH) and COMT(LL) genotypes in human tissue.

Regulation of Saccharomyces cerevisiae FET4 by oxygen and iron.

Saccharomyces cerevisiae expresses two distinct iron transport systems under aerobic and anaerobic conditions. The high affinity transporters, Ftr1p and Fet3p, are primarily expressed in oxygenated cultures, whereas anaerobic conditions induce the low affinity iron transporter, Fet4p. The oxygen regulation of FET4 was found to involve the Rox1p transcriptional repressor. The physiological significance of this control by Rox1p is twofold. First, FET4 repression by Rox1p under oxygenated conditions helps minimize metal toxicity. Sensitivity towards cadmium was high in either anaerobically grown wild-type yeast or in oxygenated rox1Delta strains, and in both cases cadmium toxicity was reversed by FET4 mutations. Secondly, the loss of Rox1p repression under anaerobic conditions serves to induce FET4 and facilitate continual accumulation of iron. We noted that fet4 mutants accumulate lower levels of iron under anaerobic conditions. Regulation of FET4 was examined using FET4-lacZ reporters. We found that FET4 contains a complex promoter regulated both by oxygen and iron status. The region surrounding approximately -960 to -490 contains two consensus Rox1p binding sites and mediates Rox1p, but not iron control of FET4. Sequences downstream of -490 harbor a consensus binding site for the iron regulatory factor Aft1p that is essential for iron regulation in wild-type strains. In addition, a secondary mode of iron regulation becomes evident in strains lacking AFT1. The induction by iron limitation in conjunction with low oxygen is more than additive, suggesting that these activities are synergistic. Fet4p is not the only metal transporter that is negatively regulated by oxygen; we find that Rox1p also represses S. cerevisiae SMF3, proposed to function in vacuolar iron transport. This oxygen control of iron transporter gene expression is part of an adaptation response to changes in the redox state of transition metals.

The distinct methods by which manganese and iron regulate the Nramp transporters in yeast.

The bakers yeast Saccharomyces cerevisiae expresses three Smf metal transport proteins that are differentially regulated by metal ions. Smf1p and Smf2p are regulated at the post-translational level by manganese, whereas Smf3p is regulated by iron through a mechanism that, up until now, was unknown. Through promoter and protein-domain swapping experiments, we now demonstrate that the manganese regulation of Smf1p involves an internal protein-coding region that is separate from the N-terminal domain of this transporter. By comparison, iron regulation of Smf3p involves the upstream non-coding region of the gene. Using SMF3-lacZ reporter constructs, we identified two distinct regions of the SMF3 promoter that contribute to iron regulation: (1) approx. nt -435 to -350 that contain dual consensus recognition sites for the Aft1 iron transcription factor; and (2) nt -348 to -247 that do not contain obvious Aft1 binding sites. The -348 to -247 region by itself can confer strong iron regulation to the heterologous CYC1 core promoter, and therefore harbours a putative upstream activating sequence for iron. Iron regulation of SMF3 was dramatically reduced, but not completely eliminated, in strains lacking both the AFT1 and AFT2 iron regulatory factors. Together with the promoter mapping studies, these results suggest that both Aft-dependent and Aft-independent pathways may contribute to iron regulation of SMF3.